RESUMEN
Dendritic cells (DCs) are highly effective antigen-presenting cells that shape immune responses. Vaccines that deliver antigen to the DCs can harness their power. DC surface lectins recognize glycans not typically present on host tissue to facilitate antigen uptake and presentation. Vaccines that target these surface lectins should offer improved antigen delivery, but their efficacy will depend on how lectin targeting influences the T cell subtypes that result. We examined how antigen structure influences uptake and signaling from the C-type lectin DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin or CD209). Virus-like particles (VLPs) were engineered from bacteriophage Qß to present an array of mannoside ligands. The VLPs were taken up by DCs and efficiently trafficked to endosomes. The signaling that ensued depended on the ligand displayed on the VLP: only those particles densely functionalized with an aryl mannoside, Qß-Man540, elicited DC maturation and induced the expression of the proinflammatory cytokines characteristic of a T helper type 1 (TH1)-like immune response. This effect was traced to differential binding to DC-SIGN at the acidic pH of the endosome. Mice immunized with a VLP bearing the aryl mannoside, and a peptide antigen (Qß-Ova-Man540) had antigen-specific responses, including the production of CD4+ T cells producing the activating cytokines interferon-γ and tumor necrosis factor-α. A TH1 response is critical for intracellular pathogens (e.g., viruses) and cancer; thus, our data highlight the value of targeting DC lectins for antigen delivery and validate the utility of DC-targeted VLPs as vaccine vehicles that induce cellular immunity.
Asunto(s)
Células Dendríticas , Polisacáridos , Animales , Antígenos , Inmunidad Celular , Ratones , Linfocitos TRESUMEN
Mucin-1 (MUC1) is a highly attractive antigenic target for anticancer vaccines. Naturally existing MUC1 can contain multiple types of O-linked glycans, including the Thomsen-Friedenreich (Tf) antigen and the Sialyl Thomsen-nouveau (STn) antigen. In order to target these antigens as potential anticancer vaccines, MUC1 glycopeptides SAPDT*RPAP (T* is the glycosylation site) bearing the Tf and the STn antigen, respectively, have been synthesized. The bacteriophage Qß carrier is a powerful carrier for antigen delivery. The conjugates of MUC1-Tf and -STn glycopeptides with Qß were utilized to immunize immune-tolerant human MUC1 transgenic (MUC1.Tg) mice, which elicited superior levels of anti-MUC1 IgG antibodies with titers reaching over 2 million units. The IgG antibodies recognized a wide range of MUC1 glycopeptides bearing diverse glycans. Antibodies induced by Qß-MUC1-Tf showed strongest binding, with MUC1-expressing melanoma B16-MUC1 cells, and effectively killed these cells in vitro. Vaccination with Qß-MUC1-Tf first followed by tumor challenge in a lung metastasis model showed significant reductions of the number of tumor foci in the lungs of immunized mice as compared to those in control mice. This was the first time that a MUC1-Tf-based vaccine has shown in vivo efficacy in a tumor model. As such, Qß-MUC1 glycopeptide conjugates have great potential as anticancer vaccines.
Asunto(s)
Vacunas contra el Cáncer/uso terapéutico , Glicopéptidos/uso terapéutico , Inmunoconjugados/uso terapéutico , Mucina-1/inmunología , Fragmentos de Péptidos/uso terapéutico , Proteínas Virales/uso terapéutico , Allolevivirus/química , Secuencia de Aminoácidos , Animales , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Vacunas contra el Cáncer/síntesis química , Vacunas contra el Cáncer/inmunología , Línea Celular Tumoral , Femenino , Glicopéptidos/síntesis química , Glicopéptidos/inmunología , Humanos , Inmunoconjugados/inmunología , Inmunoglobulina G/inmunología , Neoplasias Pulmonares/terapia , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/inmunología , Proteínas Virales/síntesis química , Proteínas Virales/inmunologíaAsunto(s)
Biomarcadores de Tumor/análisis , Perfilación de la Expresión Génica , Neoplasias de las Glándulas Salivales/genética , Ensayos Clínicos como Asunto , Terapia Combinada , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias de las Glándulas Salivales/patología , Neoplasias de las Glándulas Salivales/terapiaRESUMEN
Human mucin-1 (MUC1) is a highly attractive antigen for the development of anticancer vaccines. However, in human clinical trials of multiple MUC1 based vaccines, despite the generation of anti-MUC1 antibodies, the antibodies often failed to exhibit much binding to tumor presumably due to the challenges in inducing protective immune responses in the immunotolerant environment. To design effective MUC1 based vaccines functioning in immunotolerant hosts, vaccine constructs were first synthesized by covalently linking the powerful bacteriophage Qß carrier with MUC1 glycopeptides containing 20-22 amino acid residues covering one full length of the tandem repeat region of MUC1. However, IgG antibodies elicited by these first generation constructs in tolerant human MUC1 transgenic (Tg) mice did not bind tumor cells strongly. To overcome this, a peptide array has been synthesized. By profiling binding selectivities of antibodies, the long MUC1 glycopeptide was found to contain immunodominant but nonprotective epitopes. Critical insights were obtained into the identity of the key protective epitope. Redesign of the vaccine focusing on the protective epitope led to a new Qß-MUC1 construct, which was capable of inducing higher levels of anti-MUC1 IgG antibodies in MUC1.Tg mice to react strongly with and kill a wide range of tumor cells compared to the construct containing the gold standard protein carrier, i.e., keyhole limpet hemocyanin. Vaccination with this new Qß-MUC1 conjugate led to significant protection of MUC1.Tg mice in both metastatic and solid tumor models. The antibodies exhibited remarkable selectivities toward human breast cancer tissues, suggesting its high translational potential.
Asunto(s)
Neoplasias de la Mama/terapia , Vacunas contra el Cáncer/uso terapéutico , Epítopos/inmunología , Mucina-1/inmunología , Allolevivirus/química , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Anticuerpos/metabolismo , Neoplasias de la Mama/inmunología , Vacunas contra el Cáncer/inmunología , Femenino , Gastrópodos/química , Hemocianinas/síntesis química , Hemocianinas/química , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Mucina-1/química , Mucina-1/genética , Metástasis de la Neoplasia/inmunología , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Proteínas Virales/síntesis química , Proteínas Virales/químicaRESUMEN
Protein-protein interactions are integral to host-virus interactions and can contribute significantly to enzyme regulation by changing the localization of both host and viral enzymes within the cell, inducing conformational change relevant to enzyme activity or recruiting other additional proteins to form functional complexes. Identifying the interactors of active enzymes using an activity-based protein profiling probe has allowed us to characterize both normal enzyme activation mechanisms and the manner by which these mechanisms are hijacked and altered by the hepatitis C virus (HCV). Here, we report use of a novel activity-based probe, PIKBPyne, which labels phosphatidylinositol kinases (PIKs) in an activity-based manner, to investigate HCV-dependent changes in protein-protein interactions for PI4KB. Herein, we report the synthesis of new variations on PIKBPyne, compare their ability to label the interacting partners of PI4KB, and demonstrate the utility of our approach in characterizing virus-mediated changes to host function.
Asunto(s)
Hepacivirus/fisiología , Hepatitis C/metabolismo , Hepatitis C/virología , Interacciones Huésped-Patógeno , Mapeo de Interacción de Proteínas , Proteínas no Estructurales Virales/metabolismo , Replicación Viral , 1-Fosfatidilinositol 4-Quinasa/genética , 1-Fosfatidilinositol 4-Quinasa/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Humanos , Unión Proteica , Mapeo de Interacción de Proteínas/métodos , Interferencia de ARN , ARN Interferente Pequeño/genética , Replicación Viral/efectos de los fármacosRESUMEN
Secreted and surface-displayed carbohydrates are essential for virulence and viability of many parasites, including for immune system evasion. We have identified the α-Gal trisaccharide epitope on the surface of the protozoan parasites Leishmania infantum and Leishmania amazonensis, the etiological agents of visceral and cutaneous leishmaniasis, respectively, with the latter bearing larger amounts of α-Gal than the former. A polyvalent α-Gal conjugate on the immunogenic Qß virus-like particle was tested as a vaccine against Leishmania infection in a C57BL/6 α-galactosyltransferase knockout mouse model, which mimics human hosts in producing high titers of anti-α-Gal antibodies. As expected, α-Gal-T knockout mice infected with promastigotes of both Leishmania species showed significantly lower parasite load in the liver and slightly decreased levels in the spleen, compared with wild-type mice. Vaccination with Qß-α-Gal nanoparticles protected the knockout mice against Leishmania challenge, eliminating the infection and proliferation of parasites in the liver and spleen as probed by qPCR. The α-Gal epitope may therefore be considered as a vaccine candidate to block human cutaneous and visceral leishmaniasis.
RESUMEN
Vaccines targeting glycan structures at the surface of pathogenic microbes must overcome the inherent T cell-independent nature of immune responses against glycans. Carbohydrate conjugate vaccines achieve this by coupling bacterial polysaccharides to a carrier protein that recruits heterologous CD4 T cells to help B cell maturation. Yet they most often produce low- to medium-affinity immune responses of limited duration in immunologically fit individuals and disappointing results in the elderly and immunocompromised patients. Here, we hypothesized that these limitations result from suboptimal T cell help. To produce the next generation of more efficacious conjugate vaccines, we have explored a synthetic design aimed at focusing both B cell and T cell recognition to a single short glycan displayed at the surface of a virus-like particle. We tested and established the proof of concept of this approach for 2 serotypes of Streptococcus pneumoniae. In both cases, these vaccines elicited serotype-specific, protective, and long-lasting IgG antibodies of nanomolar affinity against the target glycans in mice. We further identified a requirement for CD4 T cells in the anti-glycan antibody response. Our findings establish the design principles for improved glycan conjugate vaccines. We surmise that the same approach can be used for any microbial glycan of interest.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Afinidad de Anticuerpos , Linfocitos T CD4-Positivos/inmunología , Infecciones Neumocócicas/inmunología , Polisacáridos Bacterianos/inmunología , Adulto , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos/química , Linfocitos B/inmunología , Proteínas Bacterianas/inmunología , Niño , Cristalografía por Rayos X , Femenino , Glicopéptidos/inmunología , Humanos , Hibridomas , Inmunoglobulina G/sangre , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Modelos Moleculares , Infecciones Neumocócicas/prevención & control , Vacunas Neumococicas/química , Vacunas Neumococicas/inmunología , Polisacáridos Bacterianos/química , Unión Proteica , Streptococcus pneumoniae/inmunología , Vacunación , Potencia de la VacunaRESUMEN
The α-Gal antigen [Galα(1,3)Galß(1,4)GlcNAcα] is an immunodominant epitope displayed by infective trypomastigote forms of Trypanosoma cruzi, the causative agent of Chagas disease. A virus-like particle displaying a high density of α-Gal was found to be a superior reagent for the ELISA-based serological diagnosis of Chagas disease and the assessment of treatment effectiveness. A panel of sera from patients chronically infected with T. cruzi, both untreated and benznidazole-treated, was compared with sera from patients with leishmaniasis and from healthy donors. The nanoparticle-α-Gal construct allowed for perfect discrimination between Chagas patients and the others, avoiding false negative and false positive results obtained with current state-of-the-art reagents. As previously reported with purified α-Gal-containing glycosylphosphatidylinositol-anchored mucins, the current study also showed concentrations of anti-α-Gal IgG to decrease substantially in patients receiving treatment with benznidazole, suggesting that the semiquantitative assessment of serum levels of this highly abundant type of antibody can report on disease status in individual patients.
Asunto(s)
Enfermedad de Chagas/diagnóstico , Trisacáridos/análisis , Trypanosoma cruzi/aislamiento & purificación , Anticuerpos Antiprotozoarios/análisis , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/análisis , Antígenos de Protozoos/inmunología , Enfermedad de Chagas/parasitología , Ensayo de Inmunoadsorción Enzimática , Humanos , Trisacáridos/inmunología , Trypanosoma cruzi/genética , Trypanosoma cruzi/inmunología , Virus/genética , Virus/metabolismoRESUMEN
Unusually fast rates of nucleophilic catalysis of hydrazone ligation were observed when polyvalent anthranilic acid catalysts operating on polyvalent aldehyde substrates were used with PAMAM dendrimers as the common platform. When presented in this way, the catalyst has a strong accelerating effect at concentrations 40-400 times lower than those required for similar monovalent catalysts and displays unique kinetic parameters. We attribute these properties to polyvalent engagement between the dendrimer surface groups, and a potential "rolling" effect leading to fast interparticle kinetic turnover. The phenomenon is sensitive to the density of functional groups on each dendrimer, and insensitive to factors that promote or inhibit nonspecific particle aggregation. These findings constitute a rare experimental example of an underappreciated phenomenon in biological and chemical systems that are organized on interacting surfaces.
RESUMEN
The anaphylaxis response is frequently associated with food allergies, representing a significant public health hazard. Recently, exposure to tick bites and production of specific IgE against α-galactosyl (α-Gal)-containing epitopes has been correlated to red meat allergy. However, this association and the source of terminal, non-reducing α-Gal-containing epitopes have not previously been established in Brazil. Here, we employed the α-1,3-galactosyltransferase knockout mouse (α1,3-GalT-KO) model and bacteriophage Qß-virus like particles (Qß-VLPs) displaying Galα1,3Galß1,4GlcNAc (Galα3LN) epitopes to investigate the presence of α-Gal-containing epitopes in the saliva of Amblyomma sculptum, a species of the Amblyomma cajennense complex, which represents the main tick that infests humans in Brazil. We confirmed that the α-1,3-galactosyltransferase knockout animals produce significant levels of anti-α-Gal antibodies against the Galα1,3Galß1,4GlcNAc epitopes displayed on Qß-virus like particles. The injection of A. sculptum saliva or exposure to feeding ticks was also found to induce both IgG and IgE anti-α-Gal antibodies in α-1,3-galactosyltransferase knockout mice, thus indicating the presence of α-Gal-containing epitopes in the tick saliva. The presence of α-Gal-containing epitopes was confirmed by ELISA and immunoblotting following removal of terminal α-Gal epitopes by α-galactosidase treatment. These results suggest for the first known time that bites from the A. sculptum tick may be associated with the unknown etiology of allergic reactions to red meat in Brazil.
Asunto(s)
Hipersensibilidad a los Alimentos/inmunología , Carne Roja/efectos adversos , Saliva/inmunología , Garrapatas/inmunología , Animales , Brasil , Mapeo Epitopo , Epítopos/química , Epítopos/inmunología , Femenino , Hipersensibilidad a los Alimentos/etiología , Galactosiltransferasas/genética , Galactosiltransferasas/inmunología , Humanos , Inmunoglobulina E/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Carne Roja/análisis , Saliva/química , Garrapatas/químicaRESUMEN
The development of carbohydrate-based antitumor vaccines is an attractive approach towards tumor prevention and treatment. Herein, we focused on the ganglioside GM2 tumor-associated carbohydrate antigen (TACA), which is overexpressed in a wide range of tumor cells. GM2 was synthesized chemically and conjugated with a virus-like particle derived from bacteriophage Qß. Although the copper-catalyzed azide-alkyne cycloaddition reaction efficiently introduced 237 copies of GM2 per Qß, this construct failed to induce significant amounts of anti-GM2 antibodies compared to the Qß control. In contrast, GM2 immobilized on Qß through a thiourea linker elicited high titers of IgG antibodies that recognized GM2-positive tumor cells and effectively induced cell lysis through complement-mediated cytotoxicity. Thus, bacteriophage Qß is a suitable platform to boost antibody responses towards GM2, a representative member of an important class of TACA: the ganglioside.
Asunto(s)
Allolevivirus/química , Anticuerpos Monoclonales , Gangliósido G(M2)/química , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/uso terapéutico , Vacunas contra el Cáncer/síntesis química , Vacunas contra el Cáncer/química , Secuencia de Carbohidratos , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática , Gangliósido G(M2)/síntesis química , Gangliósido G(M2)/uso terapéutico , Ratones , Datos de Secuencia Molecular , Neoplasias/tratamiento farmacológicoRESUMEN
Development of an effective vaccine targeting tumor associated carbohydrate antigens (TACAs) is an appealing approach toward tumor immunotherapy. While much emphasis has been typically placed on generating high antibody titers against the immunizing antigen, the impact of immunogen design on the diversity of TACA-specific antibodies elicited has been overlooked. Herein, we report that the immunogen structure can significantly impact the breadth and the magnitude of humoral responses. Vaccine constructs that induced diverse TACA-binding antibodies provided much stronger recognition of a variety of Tn positive tumor cells. Optimization of the breadth of the antibody response led to a vaccine construct that demonstrated long lasting efficacy in a mouse tumor model. After challenged with the highly aggressive TA3Ha cells, mice immunized with the new construct exhibited a statistically significant improvement in survival relative to controls (0% vs 50% survival; p < 0.0001). Furthermore, the surviving mice developed long-term immunity against TA3Ha. Thus, both the magnitude and the breadth of antibody reactivity should be considered when designing TACA-based antitumor vaccines.
Asunto(s)
Anticuerpos/sangre , Anticuerpos/clasificación , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Vacunas contra el Cáncer/química , Vacunas contra el Cáncer/inmunología , Animales , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Inmunoterapia , Ratones , Ratones Endogámicos C57BLAsunto(s)
Enzimas/química , Células Eucariotas/enzimología , Interacciones Huésped-Patógeno , Sondas Moleculares/química , beta-Lactamas/química , Línea Celular Tumoral , Enzimas/metabolismo , Células Eucariotas/metabolismo , Células Eucariotas/microbiología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Conformación Molecular , Sondas Moleculares/síntesis química , Sondas Moleculares/metabolismo , beta-Lactamas/síntesis química , beta-Lactamas/metabolismoRESUMEN
The selective chemical modification of biological molecules drives a good portion of modern drug development and fundamental biological research. While a few early examples of reactions that engage amine and thiol groups on proteins helped establish the value of such processes, the development of reactions that avoid most biological molecules so as to achieve selectivity in desired bond-forming events has revolutionized the field. We provide an update on recent developments in bioorthogonal chemistry that highlights key advances in reaction rates, biocompatibility, and applications. While not exhaustive, we hope this summary allows the reader to appreciate the rich continuing development of good chemistry that operates in the biological setting.
Asunto(s)
Química Clic , Proteínas/química , Aldehídos/química , Aminas/química , Aminoácidos/química , Hidróxidos/química , Cetonas/química , Compuestos Organometálicos/química , Profármacos/química , Profármacos/metabolismo , Proteínas/metabolismoRESUMEN
Phosphatidylinositol kinases (PIKs) are key enzymatic regulators of membrane phospholipids and membrane environments that control many aspects of cellular function, from signal transduction to secretion, through the Golgi apparatus. Here, we have developed a photoreactive "clickable" probe, PIK-BPyne, to report the activity of PIKs. We investigated the selectivity and efficiency of the probe to both inhibit and label PIKs, and we compared PIK-BPyne to a wortmannin activity-based probe also known to target PIKs. We found that PIK-BPyne can act as an effective in situ activity-based probe, and for the first time, report changes in PI4K-IIIß activity induced by the hepatitis C virus. These results establish the utility of PIK-BPyne for activity-based protein profiling studies of PIK function in native biological systems.
Asunto(s)
Alquinos/farmacología , Benzofenonas/farmacología , Colorantes Fluorescentes/farmacología , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Alquinos/química , Benzofenonas/química , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Colorantes Fluorescentes/química , Células HEK293 , Humanos , Estructura Molecular , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Relación Estructura-ActividadRESUMEN
Highly strained biarylazacyclooctynone (BARAC) and analogous bioconjugation reagents were shown to undergo novel rearrangement and addition reactions leading to tetracyclic products. This may limit their practical applicability as bioorthogonal reporters for imaging biomolecules within living systems.
Asunto(s)
Compuestos Aza/química , Química Clic , Óxidos de Nitrógeno/química , Cobre/química , Ciclización , Espectroscopía de Resonancia Magnética , Estructura MolecularRESUMEN
Strain-promoted cycloadditions of cyclic nitrones with biaryl-aza-cyclooctynone (BARAC) proceed with rate constants up to 47.3 M(-1) s(-1), this corresponds to a 47-fold rate enhancement relative to reaction of BARAC with benzyl azide and a 14-fold enhancement over previously reported strain promoted alkyne-nitrone cycloadditions (SPANC). Studies of the SPANC reaction using the linear free energy relationship defined by the Hammett equation demonstrated that the cycloaddition reaction has a rho value of 0.25 ± 0.04, indicating that reaction is not sensitive to substituents and thus should have broad applicability.
Asunto(s)
Alquinos/química , Compuestos Aza/química , Óxidos de Nitrógeno/química , Azidas/química , Cinética , Espectroscopía de Resonancia Magnética , TermodinámicaRESUMEN
Virologists have benefited from large-scale profiling methods to discover new host-virus interactions and to learn about the mechanisms of pathogenesis. One such technique, referred to as activity-based protein profiling (ABPP), uses active site-directed probes to monitor the functional state of enzymes, taking into account post-translational interactions and modifications. ABPP gives insight into the catalytic activity of enzyme families that does not necessarily correlate with protein abundance. ABPP has been used to investigate several viruses and their interactions with their hosts. Differential enzymatic activity induced by viruses has been monitored using ABPP. In this review, we present recent advances and trends involving the use of ABPP methods in understanding host-virus interactions and in identifying novel targets for diagnostic and therapeutic applications.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Interacciones Huésped-Patógeno , Proteoma/análisis , Virología/métodos , Virosis/patología , Virosis/virología , Virus/patogenicidadRESUMEN
Copper toxicity is a critical issue in the development of copper-based catalysts for copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reactions for applications in living systems. The effects and related toxicity of copper on mammalian cells are dependent on the ligand environment. Copper complexes can be highly toxic, can induce changes in cellular metabolism, and can be rapidly taken up by cells, all of which can affect their ability to function as catalysts for CuAAC in living systems. Herein, we have evaluated the effects of a number of copper complexes that are typically used to catalyze CuAAC reactions on four human cell lines by measuring mitochondrial activity based on the metabolism of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to study toxicity, inductively coupled plasma mass spectrometry to study cellular uptake, and coherent anti-Stokes Raman scattering (CARS) microscopy to study effects on lipid metabolism. We find that ligand environment around copper influences all three parameters. Interestingly, for the Cu(II)-bis-L-histidine complex (Cu(his)(2)), cellular uptake and metabolic changes are observed with no toxicity after 72 h at micromolar concentrations. Furthermore, we show that under conditions where other copper complexes kill human hepatoma cells, Cu(I)-L-histidine is an effective catalyst for CuAAC labeling of live cells following metabolic incorporation of an alkyne-labeled sugar (Ac(4)ManNAl) into glycosylated proteins expressed on the cell surface. This result suggests that Cu(his)(2) or derivatives thereof have potential for in vivo applications where toxicity as well as catalytic activity are critical factors for successful bioconjugation reactions.
Asunto(s)
Cobre/química , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Compuestos Organometálicos/química , Compuestos Organometálicos/metabolismo , Catálisis , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Colorantes Fluorescentes/farmacología , Células HEK293 , Células HeLa , Humanos , Microscopía Fluorescente , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Estructura Molecular , Compuestos Organometálicos/farmacología , Relación Estructura-ActividadRESUMEN
Strain-promoted cycloadditions of cyclic nitrones with cyclooctynes proceed with rate constants up to 3.38 ± 0.31 M(-1) s(-1) in CD(3)CN, or 59 times faster than the analogous reaction of azides. This highly specific modular labeling strategy can be applied for direct labeling of proteins and for live cell imaging of cancer cells.