carbocyanines and maleimide

Various types of albumin-binding molecules have been conjugated to anticancer drugs, and these modified prodrugs could be effective in cancer treatments compared to free anticancer drugs. However, the tumor targeting of albumin-binding prodrugs has not been clearly investigated. Herein, we examined the in vitro and in vivo tumor-targeting efficiency of three different albumin-binding molecules including albumin-binding peptide (DICLPRWGCLW: PEP), fatty acid (palmitic acid: PA), and maleimide (MI), respectively. In order to characterize the different targeting efficiency of albumin-binding molecules, PEP, PA, or MI was chemically labeled with near-infrared fluorescence (NIRF) dye, Cy5.5, in resulting PEP-Cy5.5, PA-Cy5.5, and MI-Cy5.5. These NIRF dye-labeled albumin-binding molecules were physically or chemically bound to albumin via gentle incubation in aqueous conditions in vitro. Notably, PA-Cy5.5 with reversible and multivalent binding affinities formed stable albumin complexes, compared to PEP-Cy5.5 and MI-Cy5.5, confirmed via surface plasmon resonance measurement, gel electrophoresis assay, and albumin-bound column-binding test. In tumor-bearing mice model, the different albumin-binding affinities of PA-Cy5.5, PEP-Cy5.5, and MI-Cy5.5 greatly contributed to their tumor-targeting ability. Even though the binding affinity of PEP-Cy5.5 and MI-Cy5.5 to albumin is higher than that of PA-Cy5.5 in vitro, intravenous PA-Cy5.5 showed a higher tumor-targeting efficiency in tumor-bearing mice compared to that of PEP-Cy5.5 and MI-Cy5.5. The reversible and multivalent affinities of albumin-binding molecules to native serum albumin greatly increased the pharmacokinetics and tumor-targeting efficiency in vivo.

Topics: Amino Acid Sequence; Animals; Antineoplastic Agents; Carbocyanines; Drug Delivery Systems; Humans; Maleimides; Mice; Palmitic Acid; Peptides; Prodrugs; Protein Binding; Serum Albumin

Two 2'-deoxyuridines as new building blocks for automated DNA synthesis carry a small aryltetrazole as "photoclickable" group at their 5-positions. The postsynthetic "photoclick" labeling of such presynthesized DNA using a maleimide-modified Cy3 dye shows an up to 17-fold fluorogenicity due to an energy transfer between the pyrazoline moiety and the Cy3 fluorophore in the DNA products. This concept is also applicable to other maleimide-modified dyes.

Topics: Base Sequence; Carbocyanines; Cycloaddition Reaction; Deoxyuridine; DNA; Fluorescent Dyes; Maleimides; Photochemical Processes; Staining and Labeling

Polymeric nanoparticle micelles provide a possible platform for theranostic delivery, combining the role of therapeutics and diagnostics in one vehicle. To explore dual-functional micelles, the amphiphilic copolymer of poly(d,l-lactide-co-2-methyl-2-carboxytrimethylene carbonate)-graft-poly(ethylene glycol)-X (P(LA-co-TMCC)-g-PEG-X) was self-assembled to form micelles, with X representing either azide or furan. Micelles of P(LA-co-TMCC)-g-PEG-azide and P(LA-co-TMCC)-g-PEG-furan terminal functional groups were used to conjugate dibenzylcyclooctyne and maleimide-modified probes, respectively, taking advantage of orthogonal coupling chemistry. To verify the utility of the dual-functional micelles, trastuzumab-maleimide antibodies and FLAG-dibenzylcyclooctyne peptides were covalently bound by sequential click chemistry reactions. SKOV-3luc cells that were treated with the dual-functionalized micelles showed colocalization of the antibodies and peptides by confocal imaging, demonstrating the promise of this approach.

Topics: Alkynes; Antibodies, Monoclonal, Humanized; Azides; Carbocyanines; Carbonates; Cell Line, Tumor; Click Chemistry; Furans; Humans; Hydrazines; Maleimides; Micelles; Oligopeptides; Peptides; Polyesters; Polyethylene Glycols; Trastuzumab

Protein S-nitrosation is a reversible post-translation modification critical for redox-sensitive cell signaling that is typically studied using the Biotin Switch method. This method and subsequent modifications usually require avidin binding or Western blot analysis to detect biotin labeled proteins. We describe here a modification of the Biotin Switch assay that eliminates the need for Western blot or avidin enrichment protocols and allows direct comparison of the S-nitrosation state proteins from two different samples in the same gel lane or on the same 2D gel. This S-FLOS method offers detection, identification and quantification of S-nitrosated proteins, with the potential for site-specific identification of nitrosation events.

Topics: Animals; Biotin; Brain Chemistry; Carbocyanines; Databases, Protein; Fluorescence; Maleimides; Mice; Nitric Oxide; Nitric Oxide Synthase; Nitrosation; Oxidation-Reduction; Protein Processing, Post-Translational; Proteins; Proteomics; S-Nitrosothiols; Silver Staining; Tandem Mass Spectrometry

We report enhancement in the fluorescent signal of the carbocyanine dye Cy5 by using an engineered virus as a scaffold to attach >40 Cy5 reporter molecules at fixed locations on the viral capsid. Although cyanine dye loading is often accompanied by fluorescence quenching, our results demonstrate that organized spatial distribution of Cy5 reporter molecules on the capsid obviates this commonly encountered problem. In addition, we observe energy transfer from the virus to adducted dye molecules, resulting in a highly fluorescent viral nanoparticle. We have used this enhanced fluorescence for the detection of DNA-DNA hybridization. When compared with the most often used detection methods in a microarray-based genotyping assay for Vibrio cholerae O139, these viral nanoparticles markedly increased assay sensitivity, thus demonstrating their applicability for existing DNA microarray protocols.

Topics: Avidin; Carbocyanines; Comovirus; DNA; DNA Probes; DNA, Viral; Fluorescent Dyes; Genetic Engineering; Maleimides; Models, Molecular; Nanoparticles; Rhodamines; Spectrometry, Fluorescence; Vibrio cholerae

Barley thioredoxin h isoforms HvTrxh1 and HvTrxh2 differ in temporal and spatial distribution and in kinetic properties. Target proteins of HvTrxh1 and HvTrxh2 were identified in mature seeds and in seeds after 72 h of germination. Improvement of the established method for identification of thioredoxin-targeted proteins based on two-dimensional electrophoresis and fluorescence labelling of thiol groups was achieved by application of a highly sensitive Cy5 maleimide dye and large-format two-dimensional gels, resulting in a 10-fold increase in the observed number of labelled protein spots. The technique also provided information about accessible thiol groups in the proteins identified in the barley seed proteome. In total, 16 different putative target proteins were identified from 26 spots using tryptic in-gel digestion, matrix-assisted laser-desorption ionization-time-of-flight MS and database search. HvTrxh1 and HvTrxh2 were shown to have similar target specificity. Barley alpha-amylase/subtilisin inhibitor, previously demonstrated to be reduced by both HvTrxh1 and HvTrxh2, was among the identified target proteins, confirming the suitability of the method. Several alpha-amylase/trypsin inhibitors, some of which are already known as target proteins of thioredoxin h, and cyclophilin known as a target protein of m-type thioredoxin were also identified. Lipid transfer protein, embryospecific protein, three chitinase isoenzymes, a single-domain glyoxalase-like protein and superoxide dismutase were novel identifications of putative target proteins, suggesting new physiological roles of thioredoxin h in barley seeds.

Topics: Bridged Bicyclo Compounds; Carbocyanines; Electrophoresis, Gel, Two-Dimensional; Fluorescent Dyes; Hordeum; Maleimides; Plant Extracts; Plant Proteins; Protein Isoforms; Proteome; Seeds; Sulfhydryl Compounds; Thioredoxin h; Thioredoxins

Topics: Animals; Caenorhabditis elegans; Carbocyanines; Fluorescent Dyes; Green Fluorescent Proteins; Kinesins; Luminescent Proteins; Male; Maleimides; Microscopy, Fluorescence; Microscopy, Video; Molecular Motor Proteins; Movement; Mutagenesis, Site-Directed; Protein Biosynthesis; Protein Isoforms; Recombinant Fusion Proteins; Sea Urchins; Sperm Tail