Synthesis and evaluation of new NIR-fluorescent probes for cathepsin B: ICT versus FRET as a turn-ON mode-of-action

Recent years have seen tremendous progress in the design and study of molecular imaging geared towards biological and biomedical applications. The expression or activity of specific enzymes including proteases can be monitored by cutting edge molecular imaging techniques. Cathepsin B plays key roles in tumor progression via controlled degradation of extracellular matrix. Consequently, this protease has been attracting significant attention in cancer research, and many imaging probes targeting its activity have been developed. Here, we describe the design, synthesis and evaluation of two novel near infrared (NIR) fluorescent probes for detection of cathepsin B activity with different turn-ON mechanisms. One probe is based on an ICT activation mechanism of a donor-two-acceptor π-electron dye system, while the other is based on the FRET mechanism obtained by a fluorescent dye and a quencher. The two probes exhibit significant fluorescent turn-ON response upon cleavage by cathepsin B. The NIR fluorescence of the ICT probe in its OFF state was significantly lower than that of the FRET-based probe. This effect results in a higher signal-to-noise ratio and consequently increased sensitivity and better image contrast.     

Studies of monomeric and homodimeric oxazolo[4,5-b]pyridinium cyanine dyes as fluorescent probes for nucleic acids visualization

The series of recently synthesized monomeric and homodimeric cyanine dyes based on monomethine cyanine chromophore with oxazolo[4,5-b]pyridinium and quinoline end groups [Vassilev A, Deligeorgiev T, Gadjev N, Drexhage K-H. Synthesis of novel monomeric and homodimeric cyanine dyes based on oxazolo[4,5-b]pyridinium and quinolinium end groups for nucleic acid detection, Dyes Pigm 2005;66:135-142] were studied as possible fluorescent probes for nucleic acids detection. Significant fluorescence enhancement and intensity level (quantum yield up to 0.75) was observed for all the dyes in the presence of DNA. The oxazolo[4,5-b]pyridinium cyanines demonstrated high sensitivity as fluorescent stains for post-electrophoretic visualization of nucleic acids in agarose gels upon both VIS and UV transillumination, and the visualized band contained 0.8 ng of dsDNA.     

Lymphocyte stimulation by concanavalin a studied by the fluorescent probe acridine orange

Summary Viable mouse thymocytes or spleen leucocytes stained with acridine orange (AO) were divided into one part used for stimulation, and the other part for control. Analysis of cellular green-fluorescence emission enabled physicochemical changes in lymphocytes to be detected after 30 min stimulation with the mitogens concanavalin A (Con A) and pokeweed mitogen (PWM). No change in fluorescence was observed with the nonmitogenic reagent wheat germ lectin (WGL) or with allogeneic cell stimulation (MLR). When green fluorescence intensity of individual cells was monitored by microfluorimetry, 30 min stimulation with Con A induced an increase, whereas PWM induced a decrease. When analysed by fluorescence spectrophotometry, Con A induced a 2 nm blue shift in emission maximum and a decrease in polarization values.Supported by grants from the Anti-Cancer Council of Victoria. We are grateful to Dr. H.A. Ward for helpful discussion     

Trimethine cyanine dyes as fluorescent probes for amyloid fibrils: The effect of N,N′-substituents

The effect of various N,N′-substituents in the molecule of benzothiazole trimethine cyanine dye on its ability to sense the amyloid aggregates of protein was studied. The dyes are low fluorescent when free and in the presence of monomeric proteins, but their emission intensity sharply increases in complexes with aggregated insulin and lysozyme, with the fluorescence quantum yield reaching up to 0.42.     

Early changes in concanavalin A-stimulated lymphocytes detected by the fluorescent probe N-phenyl-1-naphthylamine

Summary The hydrophobic fluorescent cell-membrane probe N-phenyl-1-naphthylamine (NPN) is a useful investigative tool for studies of early lymphocyte activation. NPN-labelled mouse thymus cells incubated with 5 g/ml concanavalin A (Con A) for 30 min at 37° C gave a reproducible increase in mean cell-fluorescence intensity measured by microfluorimetry on 100 single cells. The dose-response curve was similar to that obtained by ³H-thymidine assay.Increased fluorescence was not observed in the presence of 10 mM -methyl mannoside, 5mM sodium azide, 10^–5 M cytochalasin B, or Ca²⁺-free culture medium.However, incubation with 10^–5 M colchicine did not alter the probe response. Fluorescence change was also shown by spleen cells from a normal mouse but not from an athymic mouse, indicating T cell dependence of the response.Comparison with other lectins showed that increased fluorescence followed incubation with phytohaemagglutinin, and the non-mitogenic wheat germ lectin, but there was no change with succinyl-Con A, and decreased fluorescence with pokeweed mitogen. Use of fluorescent-labelled lectins showed that the NPN fluorescence change did not correlate with surface receptor patching and capping. Increased phospholipid-fatty acid turnover and subsequent increased membrane fluidity with alteration of molecular polarity are suggested as likely explanations of increased NPN fluorescence.Supported by a grant from the Anti-Cancer Council of VictoriaWe are grateful to Miss R. Jenkins and Mr. R. McGready for preparations of succinyl-Con A, to Dr. H.A. Ward for helpful discussion, and to Dr. M. Hohnes of the Walter and Eliza Hall Institute for providing BALB/c.nu mice     

Characterization of a series of far-red-absorbing thiobarbituric acid oxonol derivatives as fluorescent probes for biological applications

Chromophores that absorb in the far-red region of the spectrum are increasingly being utilized for applications in the biosciences. We have synthesized and evaluated a novel series of fluorescent oxonols based on thiobarbituric acids containing aryl and heteroaryl substituents. The novel chromophores possess narrow absorption spectra ( approximately 40-nm bandwidths), reasonable Stokes shifts ( approximately 25 nm), and quantum yields of up to 0.67 in organic solvents and 0.3 in aqueous solvents, with absorption wavelength maxima at 620-640 nm. The spectral properties of the compounds are sensitive to base and exhibit a loss of far-red absorbance that is concentration and time dependent. Derivatives have been synthesized that can be used for the labeling of macromolecules such as proteins and DNA. The probes show environment sensitivity and the oligonucleotide conjugates sense the formation of duplex DNA. These novel far-red fluorophores have potential applications in diagnostic and research applications.     

A new fluorescent pH probe for imaging lysosomes in living cells

A new rhodamine B-based pH fluorescent probe has been synthesized and characterized. The probe responds to acidic pH with short response time, high selectivity and sensitivity, and exhibits a more than 20-fold increase in fluorescence intensity within the pH range of 7.5–4.1 with the pK_a value of 5.72, which is valuable to study acidic organelles in living cells. Also, it has been successfully applied to HeLa cells, for its low cytotoxicity, brilliant photostability, good membrane permeability and no ‘alkalizing effect’ on lysosomes. The results demonstrate that this probe is a lysosome-specific probe, which can selectively stain lysosomes and monitor lysosomal pH changes in living cells.     

Interference of calmidazolium with measurement of mitochondrial membrane potential using the tetraphenylphosphonium electrode or the fluorescent probe rhodamine 123

Calmidazolium (CMZ) is a positively charged, hydrophobic compound used as a calmodulin antagonist. It may cause unspecific effects in mitochondria, e.g., a decrease in membrane potential (deltapsi), swelling, and uncoupling. Several groups have advised against use of CMZ in studying signal transduction in mitochondria. We report here that it interferes with measurement of deltapsi in rat liver mitochondria (RLM) when using the tetraphenyl phosphonium (TPP+) electrode. We also found that CMZ reduces the signal, indicating an apparent drop in deltapsi. CMZ itself gave a signal with the TPP+ electrode in the absence of RLM. At high concentrations, > 10 microM, it also reduced the fluorescence quenching of the probe rhodamine 123. This may be due to an interference with mitochondrial uptake and binding of this positively charged probe or to an uncoupling effect. It is concluded that CMZ and similar positively charged calmodulin antagonists such as trifluoperazine may be used in mitochondria if these interferences are controlled and calibration is carried out under the experimental conditions used.     

Development of a fluorescent cardiomyocyte specific binding probe

Cardiomyocytes are the major component of the heart. Their dysfunction or damage could lead to serious cardiovascular diseases, which have claimed numerous lives around the world. A molecule able to recognize cardiomyocytes would have significant value in diagnosis and treatment. Recently a novel peptide termed myocyte targeting peptide (MTP), with three residues of a non-natural amino acid biphenylalanine (Bip), showed good affinity to cardiomyocytes. Its selectivity towards cardiac tissues was concluded to be due to the ability of Bip to bind cardiac troponin I. With the aim of optimizing the affinity and the specificity towards cardiac myocytes and to better understand structure–activity relationship, a library of MTP derivatives was designed. Exploiting a fluorescent tag, the selectivity of the MTP analogs to myocardium over skeletal and stomach muscle tissues was assayed by fluorescence imaging. Among the tested sequences, the peptide probe Bip2, H-Lys(FITC)-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Gly-Ser-Gly-Ser-Bip-Bip-NH₂, displayed the best selectivity for cardiomyocytes.     

A novel nitro-substituted benzothiadiazole as fluorescent probe for tumor cells under hypoxic condition

Most of solid tumor cells are hypoxic and hard to trace and measure. A new compound, 4,7-bis(4-dodecylthiophen-2-yl)-5,6-dinitrobenzo[c][1,2,5]thiadiazole (BTTD-NO₂), was synthesized for labeling the hypoxic cells specially in this paper. BTTD-NO₂ showed no cytotoxicity to MG63 cells by MTT method. When MG63 cells were cultured with BTTD-NO₂ under hypoxic condition for 24 h, strong red fluorescence distribution in cytoplasm was observed. Flow cytometry results showed that 65% of MG63 cells were labeled with strong red fluorescence in hypoxic condition while only 2.4% in oxic condition. Furthermore, Real time RT-PCR proved that BTTD-NO₂ could stimulate high gene expression of the nitroreductase in the cells which could improve the conversion rate of BTTD-NO₂ to BTTD-NH₂ in turn. It proved that the fluorescence of BTTD-NO₂ was quenched by its two nitro groups, however, strong red fluorescence could emit in the cytoplasm after the reduction of its nitro groups to amino groups in the tumor cells under hypoxic condition. These results suggested that BTTD-NO₂ had the potential as a superior fluorescent probe for tumor detection.     

Novel sequence-responding fluorescent oligoDNA probe bearing a silylated pyrene molecule

A novel fluorescent phosphoramidite derivative of dimethylsilylated pyrene was prepared and incorporated into oligoDNA. The fluorescent oligoDNA exhibited marked fluorescent signal upon binding to the fully matched complementary DNA strand, however, the signal was strongly quenched in the single-stranded form as well as in the duplex having mismatched base pair at the terminus of the duplex-forming region.     

Light-dependent proton transport into mesophyll vacuoles of leaves of C3 plants as revealed by pH-indicating fluorescent dyes: a reappraisal

Esculin, a pH-sensitive fluorescent dye, was used to indicate light-dependent pH changes in leaves of Spinacia oleracea L. and Pelargonium zonale L. Shortly after its introduction into the leaves via the transpiration stream, esculin was localized mainly in the symplasm. An increase in its blue fluorescence on illumination with red actinic light indicated that the cytosolic pH had increased. A similar light-dependent alkalinization was seen when the green fluorescence of pyranine was used to monitor changes in the cytosolic pH. After esculin had been transferred into the vacuoles, a light-dependent vacuolar acidification was indicated by a decrease in its blue fluorescence. Since the pK of esculin is close to neutrality, it is suitable as an indicator of proton transport into vacuoles provided the vacuolar sap is only moderately acidic. In leaf cells with very acidic vacuoles, esculin therefore responds only to cytosolic pH changes as long as it remains in the cytosol. The observations made with esculin after it had entered the vacuoles confirmed earlier conclusions on light-dependent proton transport into the vacuoles of mesophyll cells. Previous measurements had been made with 5-carboxy-2,7-dichlorofluoresceine (CDCF), which has a pK of 4.8. In contrast to esculin, CDCF can, in principle, record pH changes in very acidic vacuoles. However, earlier conclusions made on the basis of observed CDCF fluorescence are now recognized to have no unambiguous basis because new measurements, reported here, show that CDCF fluorescence is influenced not only by pH changes but also by changes in light scattering. The latter are, like pH changes, light-dependent and originate from the thylakoid system of chloroplasts. They indicate both the formation of a large transthylakoid proton gradient and the dissipation of excess light energy as heat. Decreased green fluorescence of leaves which had been fed CDCF may therefore, depending on conditions, indicate vacuolar acidification or the dissipation of excess light energy absorbed by the pigment system of chloroplasts, or both. Pyranine fluorescence was found to be much less influenced by light scattering than CDCF fluorescence.Abbreviations CDCF 5-(and 6-)carboxy-2,7-dichlorofluoresceine - P₇₀₀ primary donor of PS I - PFD photon flux density - Q_A primary quinone acceptor of PS II - Q_P, Q_N photochemical, non-photochemical quenching of chlorophyll fluorescence, respectively This work was supported by the Deutsche Forschungsgemeinschaft within the framework of the research of the Sonderforschungsbereich 251 of the University of Würzburg. We are grateful to Drs. U. Schreiber and K.-J. Dietz and to Mrs. B. Hollenbach (all from our Institute) for discussions.     

Organelle-selective energy transfer: a fluorescent indicator of intracellular environment

A dye cassette fluoresces green (ca 520 nm) in the cytoplasm, endoplasmic reticulum (ER), and lysosomes, but red in mitochondria, that is, it illustrates ‘organelle specific energy transfer’. This phenomenon may open new horizons in intracellular imaging.     

A highly sensitive,cell-membrane-permeable fluorescent probe for glutathione

Glutathione (GSH) is a primary intracellular antioxidant. Here, we developed a novel, highly sensitive fluorescent probe for GSH, designated DNs-HMRG, whose fluorescence is regulated by two distinct switching mechanisms, intramolecular spirocyclization and photo-induced electron transfer (PeT). DNs-HMRG showed good cell permeability, and a rapid increase in fluorescence intensity was observed when it was applied to living cells. Further, taking advantage of the fact that the intracellular GSH level in tumor tissue is higher than that in normal tissue, we employed this probe for rapid (within a few tens of seconds) in vivo detection of tiny tumor nodules (less than 1 mm in diameter) in tumor-bearing mice. This probe is expected be a powerful tool in various biological applications, especially studies on redox status.     

Design and evaluation of a novel fluorescent CB2 ligand as probe for receptor visualization in immune cells

Cannabinoid CB2 receptor has emerged as a very promising target over the last decades. We have synthesized and evaluated a new fluorescent probe designated NMP6 based on 6-methoxyisatin scaffold, which exhibited selectivity and K(i) value at hCB2 of 387 nM. We have demonstrated its ability to be an effective probe for visualization of CB2 receptor binding using confocal microscopy and a flow cytometry probe for the analysis of CB2 protein expression. Furthermore, NMP6 was easily obtained in two chemical steps from commercially available building blocks.     

The lysosomotropic agent monodansylcadaverine also acts as a solvent polarity probe.

The autofluorescent substance monodansylcadaverine has recently been reported as a specific in vivo marker for autophagic vacuoles. However, the mechanism for this specific labeling remained unclear. Our results reveal that the common model of ion trapping in acidic compartments cannot completely account for the observed autophagic vacuole staining. Because autophagic vacuoles are characterized by myelin-like membrane inclusions, we tested whether this lipid-rich environment is responsible for the staining properties of monodansylcadaverine. In in vitro experiments using either liposomes or solvents of different polarity, monodansylcadaverine showed an increased relative fluorescence intensity in a hydrophobic environment as well as a Stokes shift dependent on the solvent polarity. To test the effect of autophagic vacuoles or autophagic vacuole lipids on monodansylcadaverine fluorescence, we isolated autophagic vacuoles and purified autophagic vacuole lipids depleted of proteins. Entire autophagic vacuoles and autophagic vacuole lipids had the same effect on monodansylcadaverine fluorescence properties, suggesting lipids as the responsible component. Our results suggest that the in vivo fluorescence properties of monodansylcadaverine do not depend exclusively on accumulation in acidic compartments by ion trapping but also on an effective interaction of this molecule with autophagic vacuole membrane lipids. (J Histochem Cytochem 48:251-258, 2000)     

Coumarin-suberoylanilide hydroxamic acid as a fluorescent probe for determining binding affinities and off-rates of histone deacetylase inhibitors

Histone deacetylases (HDACs) are intimately involved in epigenetic regulation and, thus, are one of the key therapeutic targets for cancer, and two HDAC inhibitors, namely suberoylanilide hydroxamic acid (SAHA) and romidepsin, have been recently approved for cancer treatment. Because the screening and detailed characterization of HDAC inhibitors has been time-consuming, we synthesized coumarin-SAHA (c-SAHA) as a fluorescent probe for determining the binding affinities (K_d) and the dissociation off-rates (k_off) of the enzyme–inhibitor complexes. The determination of the above parameters relies on the changes in the fluorescence emission intensity (λ_ex = 325 nm, λ_em = 400 nm) of c-SAHA due to its competitive binding against other HDAC inhibitors, and such determination neither requires employment of polarization accessories nor is dependent on the fluorescence energy transfer from the enzyme’s tryptophan residues to the probe. Our highly sensitive and robust analytical protocol presented here is applicable to most of the HDAC isozymes, and it can be easily adopted in a high-throughput mode for screening the HDAC inhibitors as well as for quantitatively determining their K_d and k_off values.     

Highly sensitive fluorescence imaging of cancer with avidin-protease probe conjugate

It is a long-term goal of cancer diagnosis to develop tumor-imaging techniques that have sufficient specificity and sensitivity to detect small tumor nodules during surgery or endoscopic surgery. Here, we introduce an avidin-conjugated fluorescence probe, Avidin-Leu-HMRG, which consists of a cancer-targeting macromolecule (avidin) and a protease-activatable probe. The conjugate has a high affinity for lectin on cancer cells and undergoes endocytosis, followed by irreversible fluorescence activation due to cleavage by lysosomal leucine aminopeptidase. In a mouse model of peritoneal ovarian metastases, the probe could detect submillimeter-sized tumor nodules with a high S/N ratio at 1 h after intraperitoneal injection.     

Advanced functional fluorescent probes for cell plasma membranes

Imaging the plasma membrane (PM) by fluorescence techniques using molecular fluorescent probes enable cell segmentation, studying membrane organization and dynamics, formation, and tracking of vesicles. Rational molecular design brings fluorescent PM probes to a new level, providing PM probes with new functions beyond basic PM staining and imaging. We herein review the latest advances in fluorescent PM probes for chemical and biophysical sensing as well as for super-resolution imaging.     

Ratiometric assay of CARM1 activity using a FRET-based fluorescent probe

One of the regulatory mechanisms of epigenetic gene expression is the post-translational methylation of arginine residues, which is catalyzed by protein arginine methyltransferases (PRMTs). Abnormal expression of PRMT4/CARM1, one of the PRMTs, is associated with various diseases, including cancers. Here, we designed and synthesized a Förster resonance energy transfer (FRET)-based probe, FRC, which contains coumarin and fluorescein fluorophores at the N-terminus and C-terminus of a peptide containing an arginine residue within an appropriate amino acid sequence to serve as a substrate of CARM1; the two fluorophores act as a FRET donor and a FRET acceptor, respectively. Since trypsin specifically hydrolyzes the arginine residue, but not a monomethylarginine or dimethylarginine residue, CARM1 activity can be evaluated from the change of the coumarin/fluorescein fluorescence ratio of FRC in the presence of trypsin.