Development of fluorescent dual-FRET probe for simultaneous detection of caspase-8 and caspase-9 activities and their relative quantification.

A multi-FRET three-fluorophore probe containing coumarin, fluorescein and rhodamine B with two enzymatically cleavable linkers has been synthesized and optimized for the simultaneous activity detection and relative quantification of two proteases - caspase-8 and caspase-9. The probe designed as a ratiometric single-excitation triple-emission system shows specific change in fluorescence intensities upon enzymatic cleavage of individual linkers in model mixtures as well as in a cell lysate. The activation of caspase-8 and caspase-9 is responsible for initiation of extrinsic or intrinsic apoptotic pathway, respectively, and the probe was proposed as a single chemical tool which could help to decipher a mechanism of cell death induced by various stimuli. The main advantage of this probe is the simplicity of its preparation using conventional organic synthesis, easy application for measurement and evaluation of the results.

AminoBODIPY Conjugates for Targeted Drug Delivery Systems and Real-Time Monitoring of Drug Release.

In this work, we report two concepts of drug delivery based on small-molecule drug conjugates with the ability of specific targeting and drug release monitoring via ratiometric fluorescence. The functionality of these concepts has been verified by two model systems consisting of three parts: (i) fluorescent aminoBODIPY for real-time detection of conjugate cleavage, (ii) a c(RGDfK) peptide specific for αvß3 integrin receptors targeting angiogenesis in most solid tumors or redBODIPY for conjugate cleavage monitoring via FRET, and (iii) pegylated-2-phenyl-3-hydroxy-4(1H)-quinolinone (3HQ) as a model drug. The model drug release is based on a self-immolative disulfide linker sensitive to environments containing thiols, especially glutathione, which is overexpressed in cancer cells. The results show effective thiol-mediated cleavage of the fluorescent reporter and the subsequent liberation of the drug in a tube. The conjugate with c(RGDfK) was confirmed to penetrate the cells via interaction with integrin receptors. Drug release from this conjugate is possible to monitor inside the cells. Further, the synthetic approach to the conjugates and the method of fluorescence monitoring of the drug release have also been described.

1,3-Dipolar Cycloaddition in the Preparation of New Fused Heterocyclic Compounds via Thermal Initiation.

This paper describes the synthesis of precursors with a benzo[b]furan skeleton for the intramolecular 1,3-dipolar cycloaddition of azomethine ylides prepared from N-substituted 3-allyl-aminobenzo[b]furan-2-aldehydes and secondary amines derived from α-amino acid esters. Reactions were initiated by heating. The products consisted of four fused rings with three stereogenic centers. Their structure and stereochemistry were determined by NMR spectra and X-ray measurements.

Near-infrared pH-switchable BODIPY photosensitizers for dual biotin/cRGD targeted photodynamic therapy.

Photodynamic therapy (PDT) is a clinically-approved cancer treatment that is based on production of cytotoxic reactive oxygen species to induce cell death. However, its efficiency depends on distribution of photosensitizer (PS) and depth of light penetration through the tissues. Tendency of pathological cancer tissues to exhibit lower pH than healthy tissues inspired us to explore dual-targeted pH-activatable photosensitizers based on tunable near-infrared (NIR) boron-dipyrromethene (BODIPY) dyes. Our BODIPY PSs were designed to carry three main attributes: (i) biotin or cRGD peptide as an effective cancer cell targeting unit, (ii) amino moiety that is protonated in acidic (pH <6.5) conditions for pH-activation of the PS based on photoinduced electron transfer (PET) and (iii) hydrophilic groups enhancing the water solubility of very hydrophobic BODIPY dyes. Illumination of such compounds with suitable light (>640nm) allowed for high phototoxicity against HeLa (αvß3 integrin and biotin receptor positive) and A549 (biotin receptor positive) cells compared to healthy MRC-5 (biotin negative) cells. Moreover, no dark toxicity was observed on selected cell lines (>10 µM) providing promising photosensitizers for tumour-targeted photodynamic therapy.

Cytotoxicity of Amino-BODIPY Modulated via Conjugation with 2-Phenyl-3-Hydroxy-4(1H)-Quinolinones.

The combination of cytotoxic amino-BODIPY dye and 2-phenyl-3-hydroxy-4(1H)-quinolinone (3-HQ) derivatives into one molecule gave rise to selective activity against lymphoblastic or myeloid leukemia and the simultaneous disappearance of the cytotoxicity against normal cells. Both species' conjugation can be realized via a disulfide linker cleavable in the presence of glutathione characteristic for cancer cells. The cleavage liberating the free amino-BODIPY dye and 3-HQ derivative can be monitored by ratiometric fluorescence or by the OFF-ON effect of the amino-BODIPY dye. A similar cytotoxic activity is observed when the amino-BODIPY dye and 3-HQ derivative are connected through a non-cleavable maleimide linker. The work reports the synthesis of several conjugates, the study of their cleavage inside cells, and cytotoxic screening.

Amino-BODIPY as the ratiometric fluorescent sensor for monitoring drug release or "power supply" selector for molecular electronics.

The glutathione cleavable conjugates of amino-BODIPY dye with model drugs have been tested for monitoring the drug release via ratiometric fluorescence based on two excitation and one emission wavelength. As a self-immolative linker was used for the construction of conjugates, free amino-BODIPY was released with the drug. Different excitation profiles of the dye before and after conjugate cleavage and similar emission wavelengths that enabled monitoring the release of the drug via the OFF-ON effect were successfully tested inside the cancer cells. UV/Vis spectrometry could be used in the quantification of the conjugate/drug in an analyte irrespective of the cleavage grade. As the system functionality was based only on the altered acylamino-BODIPY present in the conjugate to amino-BODIPY released during the cleavage, the method could be applied as a ratiometric fluorescence theranostic system to other non-fluorescent drugs. Moreover, the present conjugates demonstrated their potential application in molecular electronics as a "power supply" selector enabling the application of two power sources for one "bulb" to maintain its light intensity.

A novel three-fluorophore system as a ratiometric sensor for multiple protease detection.

A synthetic three-fluorophore system with two enzymatically cleavable linkers has been developed for the simultaneous detection of two proteases in a mixture. The probe was designed to afford single excitation/triple emission ratiometric detection through a fluorescence change during the cleavage of a peptide linker. The developed assays were verified for trypsin and chymotrypsin as the model enzymes.