RESUMEN
Isatoic anhydride (IA) has been shown to be a useful platform for quantifiable bioconjugation. The elaboration of a water-soluble isatoic anhydride-based platform with biotin offers readily quantifiable biotinylation reagents through nondestructive methods of quantification. The incorporation of functionality is directly quantified using the reagent's unique absorbance or fluorescence signature, located outside the biological window. Several biotinylation reagents are prepared with various linker lengths, and the quantification of biotinylated proteins is demonstrated and compared to results from the traditional HABA assay.
Asunto(s)
Oxazinas/química , Agua/química , Biotinilación , SolubilidadRESUMEN
Due to the mounting evidence that RNA structure plays a critical role in regulating almost any physiological as well as pathological process, being able to accurately define the folding of RNA molecules within living cells has become a crucial need. We introduce here 2-aminopyridine-3-carboxylic acid imidazolide (2A3), as a general probe for the interrogation of RNA structures in vivo. 2A3 shows moderate improvements with respect to the state-of-the-art selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) reagent NAI on naked RNA under in vitro conditions, but it significantly outperforms NAI when probing RNA structure in vivo, particularly in bacteria, underlining its increased ability to permeate biological membranes. When used as a restraint to drive RNA structure prediction, data derived by SHAPE-MaP with 2A3 yields more accurate predictions than NAI-derived data. Due to its extreme efficiency and accuracy, we can anticipate that 2A3 will rapidly take over conventional SHAPE reagents for probing RNA structures both in vitro and in vivo.
Asunto(s)
ARN/química , Células HEK293 , Humanos , Indicadores y Reactivos , Conformación de Ácido Nucleico , ADN Polimerasa Dirigida por ARNRESUMEN
1-Methyl-7-nitroisatoic anhydride (1M7) and 2-methylnicotinic acid imidazolide (NAI) are two of the most commonly applied RNA-SHAPE electrophiles; 1M7 due to its high reactivity and NAI for its solubility and cell permeability. While the addition of a nitro group yields desirable activation of the reagent, it also leads to poorer water solubility. This limited solubility has motivated the development of water-soluble reagents. We present alternative, isatoic anhydride-based reagents possessing variable reactivities that are simultaneously water-soluble. Solubility is gained by using a quaternary ammonium, while modulation of the reactivity is obtained by functionalization of the aryl ring. The syntheses of the reagents are discussed, and the electrophiles are demonstrated to be suitable for use for an in vitro RNA SHAPE experiment when directly compared to 1M7.
Asunto(s)
Oxazinas/química , ARN/química , Agua/química , Acilación , Secuencia de Bases , Radical Hidroxilo/química , ARN/genética , SolubilidadRESUMEN
N-(3-Iodopropyl)isatoic anhydride (IPIA) has been demonstrated to serve as an efficient substrate for the development of an extended bioconjugation platform. Derivatives of IPIA are water-soluble and adaptable and share a common chromophore, rendering them easily quantifiable. We demonstrate the preparation of the readily diversified bioconjugation platform technology and application of the reagents in RNA-SHAPE analysis.
Asunto(s)
Oxazinas/química , Proteínas/química , ARN/química , Estructura Molecular , Solubilidad , Agua/químicaRESUMEN
Despite the potential of silver to mediate synthetically valuable cross-coupling reactions, the operating mechanisms have remained unknown. Here, we use a combination of rapid-injection NMR spectroscopy, electrospray-ionization mass spectrometry, and quantum chemical calculations to demonstrate that these transformations involve argentate(i) and (iii) complexes as key intermediates.
RESUMEN
BACKGROUND: Pancreatic ductal adenocarcinoma (PDA) remains the most aggressive cancers with a 5-year survival below 10%. Systemic delivery of chemotherapy drugs has severe side effects in patients with PDA and does not significantly improve overall survival rate. It is highly desirable to advance the therapeutic efficacy of chemotherapeutic drugs by targeting their delivery and increasing accumulation at the tumor site. MUC1 is a membrane-tethered glycoprotein that is aberrantly overexpressed in > 80% of PDA thus making it an attractive antigenic target. METHODS: Poly lactic-co-glycolic acid nanoparticles (PLGA NPs) conjugated to a tumor specific MUC1 antibody, TAB004, was used as a nanocarrier for targeted delivery into human PDA cell lines in vitro and in PDA tumors in vivo. The PLGA NPs were loaded with fluorescent imaging agents, fluorescein diacetate (FDA) and Nile Red (NR) or isocyanine green (ICG) for in vitro and in vivo imaging respectively or with a chemotherapeutic drug, paclitaxel (PTX) for in vitro cytotoxicity assays. Confocal microscopy was used to visualize internalization of the nanocarrier in vitro in PDA cells with high and low MUC1 expression. The in vivo imaging system (IVIS) was used to visualize in vivo tumor targeting of the nanocarrier. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay was used to determine in vitro cell survival of cells treated with PTX-loaded nanocarrier. One-sided t-test comparing treatment groups at each concentration and two-way ANOVAs comparing internalization of antibody and PLGA nanoparticles. RESULTS: In vitro, TAB004-conjugated ICG-nanocarriers were significantly better at internalizing in PDA cells than its non-conjugated counterpart. Similarly, TAB004-conjugated PTX-nanocarriers were significantly more cytotoxic in vitro against PDA cells than its non-conjugated counterpart. In vivo, TAB004-conjugated ICG-nanocarriers showed increased accumulation in the PDA tumor compared to the non-conjugated nanocarrier while sparing normal organs. CONCLUSIONS: The study provides promising data for future development of a novel MUC1-targeted nanocarrier for direct delivery of imaging agents or drugs into the tumor microenvironment.
Asunto(s)
Antígenos de Neoplasias/inmunología , Antineoplásicos Inmunológicos/administración & dosificación , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/inmunología , Nanopartículas , Paclitaxel/administración & dosificación , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/inmunología , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Antineoplásicos Inmunológicos/química , Antineoplásicos Inmunológicos/farmacocinética , Biomarcadores de Tumor , Carcinoma Ductal Pancreático/mortalidad , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Supervivencia Celular , Modelos Animales de Enfermedad , Liberación de Fármacos , Endocitosis , Femenino , Expresión Génica , Humanos , Ratones , Terapia Molecular Dirigida , Mucina-1/inmunología , Nanopartículas/química , Nanopartículas/ultraestructura , Paclitaxel/química , Paclitaxel/farmacocinética , Neoplasias Pancreáticas/patología , Polietilenglicoles/química , Ensayos Antitumor por Modelo de Xenoinjerto , Neoplasias PancreáticasRESUMEN
Isatoic anhydride is elaborated to water soluble bioconjugation reagents that gives functionality and water solubility in one self-cleaning step. This new platform offers high atom economy with carbon dioxide being the only byproduct, and is shown to very quickly and efficiently label proteins in bicarbonate buffered solutions.
Asunto(s)
Materiales Biomiméticos/química , Oxazinas/química , Rayos Ultravioleta , Agua/química , Materiales Biomiméticos/síntesis química , Dióxido de Carbono/química , Estructura Molecular , Oxazinas/síntesis química , SolubilidadRESUMEN
An amido cuprate formed from CuCN and LDA allows a general deconjugative α-alkylation of cyclic alkenenitriles. Deprotonating cyclic alkenenitriles with LDA-CuCN avoids polymerization that otherwise plagues these alkylations and generates a reactive metalated nitrile for alkylations with a range of carbon and heteroatom electrophiles. The strategy provides an effective synthesis of quaternary 5-, 6-, and 7-membered cycloalk-1-enecarbonitriles substituted on the nitrile-bearing carbon.
RESUMEN
Grow slow: The usual direct treatment of MeLi and CuSPh did not yield X-ray quality crystals of MeCu(SPh)Li. An indirect method starting from Me2CuLiâ LiSPh and chalcone afforded the desired crystals by the slow reaction of the intermediate π-complex (see scheme). This strategy produced the first X-ray crystal structure of a Posner cuprate. A complementary NMR study showed that the contact ion pair was also the main species in solution.
RESUMEN
Typical aldehydes and ketones form π complexes with Me2CuLi at low temperatures in tetrahydrofuran. They range in stability from fleeting intermediates at -100 °C to entities that persist up to -20 °C. Three subsequent reaction pathways have been identified.
Asunto(s)
Aldehídos/química , Cobre/química , Furanos/química , Cetonas/química , Litio/química , Compuestos Organometálicos/química , Estructura Molecular , TemperaturaRESUMEN
Rapid injection of pyruvonitrile or benzoyl cyanide into solutions of Me2CuLi in THF-d8 at -100 °C gave complexes that were stable at this temperature. 1D NMR with multiply labelled substrates ((13)C/(15)N) and 2D NMR ((1)H/(13)C) identified them as the first cuprate-carbonyl π-complexes.
RESUMEN
By using rapid injection NMR, it has now been possible to prepare and characterize the η(3) 'π-allyl' copper(III) intermediate that has been proposed for addition reactions of organocopper(I) reagents and α,ß-unsaturated carbonyl compounds.
RESUMEN
They "know" where to go: a powerful orientation effect has been observed in complexes of mixed organocuprates [R(T)R(NT)CuLi] and substrates with C-C, C-N, and C-S double bonds (see scheme; Th=thienyl). The preferred geometry of the intermediate complex sets up the facile addition of R(T) to the double bond, rather than addition of the "dummy ligand", R(NT) .
RESUMEN
Upon rapid injection, a variety of thiocarbonyl compounds react with the Gilman reagent Me(2)CuLi at -100 degrees C inside the probe of an NMR spectrometer to give high yields of complexes. Typical examples of substrates include carbon disulfide, methyl dithioacetate, methyl dithiobenzoate, thiobenzophenone, ethylene trithiocarbonate, and phenyl isothiocyanate. Evidence suggesting the formal oxidation state of copper in these complexes to be Cu(III) is presented. The last example was particularly interesting, since it involved a transient intermediate that was identified as a complex with a C-N double bond. Methyl isothiocyanate gave a stable C-N double-bond complex.
RESUMEN
Lithium tetramethylcuprate(III), Me(4)CuLi, the Cu(III) analog of the Gilman reagent, has been prepared in high yield from halo-Gilman reagents Me(2)CuLi.LiX (X = Cl, Br, I) and 2,3-dichloropropene and found to have surprising thermal stability. The cyano-Gilman reagent (X = CN) follows a different pathway.
RESUMEN
Lithium tetramethylcuprate(III) 1 reacts readily at -100 degrees C with appropriate sources of H(+) or X(+) (X = Br, I) to remove a methyl and in some cases incorporate the counterion (e.g., arylthio or cyano) to give stable complexes. These derivatives can in turn serve as starting materials for other Cu(III) complexes.
RESUMEN
The title compound, C(18)H(19)NO(4), was prepared as an indole derivative with possible anti-mitotic properties. The planes of the indole and trimethoxy-phenyl rings make a dihedral angle of 45.35â (5)° with one another. In the crystal, mol-ecules related by a twofold screw axis exhibit arene C-Hâ¯arene-π inter-actions which are 3.035â (1)â Å in length.
RESUMEN
The title compound, C(44)H(60)Si, was prepared as an inter-nal standard for diffusion-ordered NMR spectroscopy. The Si atom lies on a special position with site symmetry.
RESUMEN
The title compound, C(12)H(13)NO(4), was prepared as a precursor to an indole derivative with possible anti-mitotic properties. The mol-ecule is very nearly planar; the maximum deviation of any non-H atom from the mean plane of the indole ring is 0.120â (3)â Å for each of two meth-oxy C atoms. The pairs of mol-ecules related by the inversion centre at (0,0,) are connected by two symmetry-equivalent N-Hâ¯O hydrogen bonds, while the pairs of mol-ecules related by the inversion centre at (0,0,0) exhibit a π-stacking inter-action of the indole rings, with an inter-planar separation of 3.39â (3)â Å.