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Cyanine (Cy) dyes show a general propensity to localize in polarized mitochondria. This mitochondriotropism was used to perform a copper-free click reaction in the mitochondria of living cells. The in organello reaction of dyes Cy3 and Cy5 led to a product that was easily traceable by Förster resonance energy transfer (FRET). As determined by confocal laser scanning microscopy, the Cy3-Cy5 conjugate showed enhanced retention in mitochondria, relative to that of the starting compounds. This enhancement of a favorable property can be achieved by synthesis in organello, but not outside mitochondria.

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A fundamental mechanism of the innate immune system is the recognition, via extra- and intracellular pattern-recognition receptors, of pathogen-associated molecular patterns. A prominent example is represented by foreign nucleic acids, triggering the activation of several signaling pathways. Among these, the endosomal toll-like receptor 7 (TLR7) is known to be activated by single-stranded RNA (ssRNA), which can be specifically influenced through elements of sequence structure and posttranscriptional modifications. Furthermore, small molecules TLR7 agonists (smTLRa) are applied as boosting adjuvants in vaccination processes. In this context, covalent conjugations between adjuvant and vaccines have been reported to exhibit synergistic effects. Here, we describe a concept to chemically combine three therapeutic functions in one RNA bioconjugate. This consists in the simultaneous TLR7 stimulation by ssRNA and smTLRa as well as the therapeutic function of the RNA itself, e.g., as a vaccinating or knockdown agent. We have hence synthesized bioconjugates of mRNA and siRNA containing covalently attached smTLRa and tested their function in TLR7 stimulation. Strikingly, the bioconjugates displayed decreased rather than synergistically increased stimulation. The decrease was distinct from the antagonistic action of an siRNA bearing a Gm motive, as observed by direct comparison of the effects in the presence of otherwise stimulatory RNA. In summary, these investigations showed that TRL7 activation can be impeded by bioconjugation of small molecules to RNA.

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We describe the synthesis and characterization of a novel bioconjugate, consisting of an octaarginine cell-penetrating peptide and a highly DNA-affine doxorubicin dimer. The linkage between the two components is composed of a cleavable disulfide bond, which enables the efficient intracellular delivery of the cytotoxic payload within the reductive environment of the cytosol, mediated through glutathione. To determine the DNA-binding affinity of the dimeric drug molecule, microscale thermophoresis was applied. This is the first utilization of this method to assess the binding interactions of an anthracycline drug with nucleic acids. The cytotoxic effect of the peptide-drug conjugate, studied with drug-sensitive and doxorubicin-resistant cancer cells, demonstrates that the bioconjugate can successfully overcome drug resistance in neuroblastoma cells.

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This review focuses on the various approaches to covalently attach a chromophore to a biomolecule of interest in site-specific manner. Novel methods like inverse electron-demand Diels-Alder reaction, Pictet-Spengler ligation and enzyme tags like SNAP and Halo-tags are critically discussed and compared to established techniques like copper-free click reaction and native chemical ligation. Selected examples in which the tags have been exploited for in vitro or in vivo imaging are reviewed and evaluated.

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Biocompatible organic dyes emitting in the near-infrared are highly desirable in fluorescence imaging techniques. Herein we report a synthetic approach for building novel small peri-guanidine-fused naphthalene monoimide and perylene monoimide chromophores. The presented structures possess near-infrared absorption and emission, high photostability, and good water solubility. After a fast cellular uptake, they selectively stain mitochondria with a low background in live and fixed cells. They can be additionally modified in a one-step reaction with functional groups for covalent labeling of proteins. The low cytotoxicity allows a long time exposure of live cells to the dyes without the necessity of washing. Successful application in localization super-resolution microscopy was demonstrated in phosphate-buffered saline without any reducing or oxidizing additives.

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Although recent methods for targeted drug delivery have addressed many of the existing problems of cancer therapy associated with undesirable side effects, significant challenges remain that have to be met before they find significant clinical relevance. One such area is the delicate chemical bond that is applied to connect a cytotoxic drug with targeting moieties like antibodies or peptides. Here we describe a novel platform that can be utilized for the preparation of drug-carrier conjugates in a site-specific manner, which provides excellent versatility and enables triggered release inside cancer cells. Its key feature is a cleavable doxorubicin-octreotide bioconjugate that targets overexpressed somatostatin receptors on tumor cells, where the coupling between the two components was achieved through the first cleavable disulfide-intercalating linker. The tumor targeting ability and suppression of adrenocorticotropic hormone secretion in AtT-20 cells by both octreotide and the doxorubicin hybrid were determined via a specific radioimmunoassay. Both substances reduced the hormone secretion to a similar extent, which demonstrated that the tumor homing peptide is able to interact with the relevant cell surface receptors after the attachment of the drug. Effective drug release was quickly accomplished in the presence of the physiological reducing agent glutathione. We also demonstrate the relevance of this scaffold in biological context in cytotoxicity assays with pituitary, pancreatic, and breast cancer cell lines.

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The overproduction of free radicals and reactive oxygen species (ROS) has been proved as a basic damage mechanism and cause for oxidative stress. Their measurement is often hindered by the low signal. This could be resolved with the application of luminescent probes (lophines, luminol, lucigenin, etc.). The focus of this study is to synthesize and describe the spectral properties and physicochemical characteristics of lophine and its derivatives as new chemiluminescent in vitro activators. The prepared luminophores are analogues of lophine. Their absorption maxima are in the range 329-340 nm, with good-to-high extinction coefficients. Their spectral properties are measured in methanol and buffer solutions with pH 3.5, 7.4 and 8.5. Same conditions were applied in the systems for chemiluminescent assay in vitro: (1) Fenton's (Fe(2+)+H2O2) for the generation of ·OH and -OH species, (2) Hydrogen peroxide (H2O2), (3) Iron (II) sulfate (FeSO4), (4) Glutathione-peroxidase, monitoring the deactivation of H2O2, (5) Ascorbic acid-Fenton's reagent: Vit.C appears a strong oxidant, generating free-radical products when applied in higher than physiological concentrations, (6) Reduced α-nicotinamide adenine dinucleotide (NADH)-phenazine methosulfate-for the generation of superoxide radicals (O2 (·-)). Lophine and all novel compounds do not alter the kinetics, except of the dimethyl amino substituted derivative (4-(3a,11b-dihydro-1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)-N,N-dimethylaniline) in the glutathione-peroxidase system, at pH 8.5. Same derivative showed a comparable or higher activity than Lucigenin and Rhodamine 6G. In neutral and acidic medium, in the Fenton's system, Rhodamine 6G was the most appropriate probe. In alkaline pH and oxidant H2O2, Lucigenin induced a signal twice as strong as the signal compared to all other activators.

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Innovations in labeling techniques and in the design and synthesis of dye structures are closely related to the development of service equipment such as light sources and detection methods. Novel styryl homodimers and monomethine cyanine dyes were synthesized and their staining abilities for discrimination between live and dead lactic acid bacterial cells were investigated. The dyes were combined in pairs based on their excitation and emission maxima and the capacity to penetrate through cell membranes of viable bacterial cells. The absorption maxima in the same region and the large Stocks shifts of the styryl derivatives allowed viability analysis to be done with epifluorescent microscope with a very basic configuration - one light source about 480nm and one filter for the fluorescent emissions. A staining protocol was developed and applied for live/dead analysis of Bulgarian yoghurt starters. The live cells quantification by the fluorescence dyes coincided well with the results of the much more time-consuming tests by plate counting. Thus, the proposed dye combinations are appropriate for rapid viability estimation in small laboratories that may have conventional equipment.

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In this study, we describe the synthesis of novel functional non-nucleoside adenylyl cyclase inhibitors, which can be easily modified with thiol containing biomolecules such as tumour targeting structures. The linkage between inhibitor and biomolecule contains cleavable bonds to enable efficient intracellular delivery in the reductive milieu of the cytosol as well as in the acidic environment within endosomes and lysosomes. The suitability of this synthetic approach was shown by the successful bioconjugation of a poor cell-permeable inhibitor with a cell-penetrating peptide. Additionally, we have demonstrated the excellent inhibitory effect of the compounds presented here in a live-cell Förster resonance energy transfer-based assay in human embryonic kidney cells.

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Fluorescent microscopy and fluorescent imaging by flow cytometry are two of the fastest growing areas in the medical and biological research. Innovations in fluorescent chemistry and synthesis of new dye probes are closely related to the development of service equipment such as light sources, and detection techniques. Among compounds known as fluorescent labels, the cyanine-based dyes have become widely used since they have high excitation coefficients, narrow emission bands and high fluorescence upon binding to nucleic acids. The key methods for evaluation of apoptosis and cell cycle allow measuring DNA content by several flow cytometric techniques. We have synthesized new monomethine cyanine dyes and have characterized their applicability for staining of live and/or apoptotic cells. Imaging experiments by flow cytometry and confocal laser scanning microscopy (CLSM) have been also performed. Two of the dyes have shown high-affinity binding to the nuclei at high dilutions, up to 10(-9)M. Flow cytometry and CLSM have confirmed that these dyes labeled selectively non-living, e.g. ethanol-fixed cells that makes them appropriate for estimations of cell viability and apoptosis. The novel structures proved to be appropriate also for analysis of the cell cycle.

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In this study, an improved, rapid, high yield synthesis of N,N'-4,4'-bis(benzyl-2-boronic acid)-bipyridinium dibromide (o-BBV) is described. The obtained o-BVV is applied in a two-component saccharide sensing system (complex) where it serves as a fluorescence quencher and a saccharide receptor. This system was applied to different natural oligosaccharides isolated from molluscan Rapana venosa (RvH1-a) and arthropodan Carcinus aestuarii (CaeH) hemocyanins (Hcs) and cyclodextrins (CDs). The carbohydrate contents of both Hcs were calculated in our previous work to be 1,6 % and 7 % for CaeH and RvH1-a, respectively. We propose that the difference in fluorescence increase of the native CaeH and RvH1-a when titrating them with the complex is due to the fact that the carbohydrate content of CaeH is lower and the carbohydrate chains are buried in between the structural subunits of the native molecule, while the glycans of the functional unit RvH1-a are exposed on the surface of the molecule leading to a 4-fold fluorescence's intensity change.

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A series of 2-alkylthio derivatives of hetaryl thiols were synthesized by selective S-alkylation with alkyl halides (bromides and iodides) with ultrasonic irradiation at room temperature. The reaction was found to be generally applicable to hetaryl thiol derivatives with different substituents in the aromatic nucleus and various alkyl halides. The reaction gives high to excellent yields of products with high purity.

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The 4,4,4-trifluoro-1-(biphenyl-4-yl)butane-1,3-dione(HL) has been synthesized and its complexation properties in solution was examined. Mixed ligand chelate extraction of light trivalent lanthanoids (La÷Gd) from chloride medium at constant ionic strength µ = 0.1 into C6H6 with HL in combination with one of the three phosphine oxide compounds trioctylphosphine oxide(TOPO), tributylphosphine oxide(TBPO) or triphenyphosphine oxide(TPPO) was studied. The composition of the extracted species was established as LnL3 with HL alone and as LnL3 2S in the presence of TOPO and TBPO or LnL3 S with the mixture of HL-TPPO. The 28 values of the overall equilibrium constants were calculated. A synergic effect up to 103-104 was observed for the extraction of the above-mentioned lanthanoid ions with binary mixtures of extractants. The change of the synergistic agent causes a significant increase of the KL,S values in the order TBPO < TPPO < TOPO. The parameters of the extraction process were determined and the separation factors between two adjacent Ln(III) were calculated.