A selective activity-based probe for the papain family cysteine protease dipeptidyl peptidase I/cathepsin C

Dipeptidyl peptidase I is involved in the activation of a number of disease-related proteases by removal of N-terminal prodipeptides. We here report a selective activity-based probe for monitoring dipeptidyl peptidase I activity in whole proteomes as well as in intact cells, without labeling of closely related enzyme family members.     

A unique paradigm for a Turn-ON near-infrared cyanine-based probe: noninvasive intravital optical imaging of hydrogen peroxide

The development of highly sensitive fluorescent probes in combination with innovative optical techniques is a promising strategy for intravital noninvasive quantitative imaging. Cyanine fluorochromes belong to a superfamily of dyes that have attracted substantial attention in probe design for molecular imaging. We have developed a novel paradigm to introduce a Turn-ON mechanism in cyanine molecules, based on a distinctive change in their π-electrons system. Our new cyanine fluorochrome is synthesized through a simple two-step procedure and has an unprecedented high fluorescence quantum yield of 16% and large extinction coefficient of 52,000 M(-1)cm(-1). The synthetic strategy allows one to prepare probes for various analytes by introducing a specific triggering group on the probe molecule. The probe was equipped with a corresponding trigger and demonstrated efficient imaging of endogenous hydrogen peroxide, produced in an acute lipopolysaccharide-induced inflammation model in mice. This approach provides, for the first time, an available methodology to prepare modular molecular Turn-ON probes that can release an active cyanine fluorophore upon reaction with specific analyte.     

A ratiometric optical imaging probe for intracellular pH based on modulation of europium emission

A set of three pH-responsive ratiometric Eu(III)complexes has been synthesised incorporating a coordinated azathioxanthone sensitiser and a pH dependent alkylsulfonamide moiety. Emission properties, anion binding affinities, pH response curves and protein binding constants were studied in detail in aqueous media, and solutions containing various concentrations of interfering anions and protein were also examined. The complex, [EuL3] exhibited some interference from protein and endogenous anions, e.g. lactate and hydrogen carbonate, but possessed a protonation constant of 7.2 in human serum solution. A suitable calibration curve was obtained and was used to determine the local pH using a 680/589 nm intensity ratio vs. pH plot. Confocal fluorescence microscopy images revealed fast uptake of the complex and a well distributed localisation within the cell; fast egress also occurred. Ribosomal localisation, with a high concentration within the protein-dense nucleoli was observed, in a similar manner to structurally related complexes bearing the same coordinated sensitising moiety. An IC(50) value of 67 (+/-20) microM was estimated using an MTT assay. Selected emission band ratio versus pH plots allow pH measurement in the range 6 to 8, enabling intracellular pH to be measured by microscopy. A value of 7.4 was estimated for NIH 3T3 cells in the protein rich regions of the nucleolus and ribosomes.     

An activity-based protein profiling probe for the nicotinic acetylcholine receptor

Activity-based protein profiling (ABPP) has been used extensively to characterize the physiological functions of enzymes but has not yet been extended to ion channels. We have synthesized a state-dependent photoaffinity probe for the nicotinic acetylcholine receptor (nAChR) as a proof of concept for the development of ion channel directed ABPP probes. The candidate probe BPyneTEA comprises an nAChR binding moiety, a benzophenone moiety for photolabeling, and an alkyne moiety for biotinylation via "click chemistry". Single-molecule current measurements show that BPyneTEA blocks both the closed and open (i.e., nondesensitized) conformations of the nAChR with similar kinetics. In living cells, BPyneTEA photolabels the closed state selectively over the inactive desensitized state. BPyneTEA thus shows promise as a probe for nondesensitized nAChRs and may be useful in studying the molecular physiology of desensitization. The structure and reactivity of ion channel pores in general suggest that they will be a broadly useful target for ABPP probes.     

An optimized activity-based probe for the study of caspase-6 activation

Although significant efforts have been made to understand the mechanisms of caspase activation during apoptosis, many questions remain regarding how and when executioner caspases get activated. We describe the design and synthesis of an activity-based probe that labels caspase-3/-6/-7, allowing direct monitoring of all executioner caspases simultaneously. This probe has enhanced in vivo properties and reduced cross-reactivity compared to our previously reported probe, AB50. Using this probe, we find that caspase-6 undergoes a conformational change and can bind substrates even in the absence of cleavage of the proenzyme. We also demonstrate that caspase-6 activation does not require active caspase-3/-7, suggesting that it may autoactivate or be cleaved by other proteases. Together, our results suggest that caspase-6 activation proceeds through a unique mechanism that may be important for its diverse biological functions.     

An activity-based fluorogenic probe for sensitive and selective monoamine oxidase-B detection

We report the design and synthesis of a new class of fluorogenic probes based on monoamine oxidase-triggered oxidative C-O bond cleavage. The selectivity of probe P1 towards MAO-B was 22-fold higher than that towards MAO-A.     

New directions of activity-based sensing for in vivo NIR imaging

In vivo imaging is a powerful approach to study biological processes. Beyond cellular methods, in vivo studies allow for biological stimuli (small molecules or proteins) to be studied in their native environment. This has the potential to aid in the discovery of new biology and guide the development of diagnostics and therapies for diseases. To ensure selectivity and an observable readout, the probe development field is shifting towards activity-based sensing (ABS) approaches and near-infrared (NIR) imaging modalities. This perspective will highlight recent in vivo ABS applications that utilize NIR imaging platforms.

In vivo imaging is a powerful approach to study biological processes.     

A TICS-fluorophore based probe for dual-color GSH imaging

Cascading reactions in fluorophores accompanied by the replacement of different fluorescence wavelengths can be used to develop luminescent materials and reactive fluorescent probes. Based on multiple signal channels, the selectivity of probes can be improved and the range of response to guest molecule recognition can be expanded. By regulating the position, number, and activity of active sites in fluorophores, fluorescent probes that successively react with thiol and amino groups in cysteine (Cys), homocysteine (Hcy) have been developed, which can only react with the thiol group of GSH. In this paper, we report the first probe capable of cascading nucleophilic substitution reaction with the thiol group and amino group of GSH at a single reaction site, and showed the dual-color recognition of GSH, which improved the selectivity of GSH also was an extension of GSH probes. The probe Rho-DEA was based on a TICS fluorophore, and the intramolecular cascade nucleophilic substitution reaction occurs with Cys/Hcy. The thiol substitution of the first step reaction with Cys/Hcy was quenched due to intersystem crossing to triplet state, so GSH can be selectively recognized from the fluorescence signal. Rho-DEA has the ability of mitochondrial localization, and finally realized in situ dual-color fluorescence recognition of GSH in mitochondria.     

A specific probe for two-photon fluorescence lysosomal imaging

Lysosomes are vital organelles in physiological processes, as they receive and degrade macromolecules from the secretory and endocytic procedures. Evidences have shown that lysosomes were related to oncogenic activation and cancer progression, so lysosomes targeting and imaging probes make them convenient to be observed. In this study, a lysosome specific probe W-7 was designed and synthesized via convenient one-pot reaction and Heck reaction. This probe was derived from Tröger's base with a dimethylaminomethyl end group. The optical properties of this compound were measured. W-7 also showed two-photon absorption (TPA) effect by using laser excitation at the wavelength of infrared light. In vivo experiment, W-7 showed high specificity and selectivity for lysosomes in living cells (HeLa cells, MRC-5 cells and NRK cells), compared with LT Red, GT Red and MT Red (R = 0.96). Two-photon fluorescence images of HeLa cells stained by W-7 were obtained. And high resolution 3D reconstruction of lysosomes in one HeLa cell was provided by using two-photon confocal microscopy. The anantioseparation of racemic W-7 was carried out by chiral-HPLC, and the two enantiomers showed no significant difference in lysosomes imaging.     

A molecular probe for the optical detection of biogenic amines

A coumarin derivative was employed for the detection of biogenic amines in buffered aqueous solution by UV-Vis or fluorescence spectroscopy. Incorporated in a polymeric matrix, the dye can also be used for the optical detection of gaseous amines.     

A GSH-responsive PET-based fluorescent probe for cancer cells imaging

An efficient PET-based probe, in which the ferrocene quencher and the naphthalimide fluorophore are linked by a disulfide bond, has been developed. This probe can be activated by GSH with fluorescence a turn-on response for blocking the PET process. In addition, it was successfully applied for distinguishing cancer cells from normal cells     

A fluorescent probe for the detection of myeloperoxidase activity in atherosclerosis-associated macrophages

The myeloperoxidase (MPO)-derived oxidant hypochlorous acid (HOCl/OCl(-)) is implicated in the pathogenesis of atherosclerosis and other inflammatory states. We have synthesized an imaging probe, sulfonaphthoaminophenyl fluorescein (SNAPF), that selectively reacts with HOCl. SNAPF detects HOCl produced by stimulated MPO-expressing cells cultured from human whole blood, as well as HOCl from bone marrow (BM)-derived macrophages isolated from transgenic mice that express human MPO. Two lines of evidence indicate that SNAPF permits the in vivo imaging of HOCl production. First, we used this approach to demonstrate HOCl production by neutrophils in experimental murine peritonitis. Second, we detected HOCl production by MPO expressing cells in human atherosclerotic arteries. Thus, fluorescence reflectance imaging by SNAPF may provide a valuable noninvasive molecular imaging tool for implicating HOCl and MPO in the damage of inflamed tissues.     

A wide-angle secondary ion probe for organic ion imaging

A secondary ion source has been developed for an organic ion microprobe capable of imaging samples up to 2 em in diameter. The source uses a focused 5 keY Cs⁺ ion beam which is rastered across the sample surface, and secondary ions from each point on the sample are collected and formed into a low energy beam to be analyzed by a quadrupole mass filter. Dynamic emittance matching is employed to deflect ions from off-axis points on the sample back onto the mass analyzer axis. Rastering and dynamic emittance matching are rapidly controlled by assembly language programs using an IBM/AT (80286) type computer. A low energy ion monitor was used to tune and evaluate the secondary ion source by providing a magnified cross-sectional image of the ion beam at the source exit aperture. A well-focused and centered secondary ion beam was obtained from each point on the sample, indicating that large-scale dynamic emittance matching with high collection efficiency is possible. Mass resolved images of grids and glycerol samples are shown to demonstrate the performance of the integrated secondary ion source mass analyzer and control system.     

A bestatin-based fl uorescent probe for aminopeptidase N cell imaging

Aminopeptidase N (APN) is an important drug target and biomarker for various tumors. The current work characterizes a novel APN-targeted fluorescent probe (Bes-Green, 2) that manifests comparable inhibitory activity with Bestatin. This probe has capacity of tightly binding to the APN for imaging endogenous APN in living human ovarian clear cell carcinoma cells (ES-2) and has potential application in biological study of cellular APN.     

Radiolabeled peptide probe for tumor imaging

Radionuclide imaging is now the premier imaging method in clinical practice for its high sensitivity and tomographic capability. Current clinically available radio imaging methods mostly use positron-emission tomography (PET) and single-photon emission computed tomography (SPECT) to detect anatomic abnormalities that conventional imaging techniques typically have challenges for visualizing. Contrast agents are indispensable for radionuclide imaging, and the radionuclide is always attached to a suitable vector that achieves targeted delivery. Nowadays, peptides have attracted increasing interest in targeting vectors of contrast agents, mainly due to their high specificity for target receptors at nanomolar concentrations and low toxicity. Radiolabeled peptide probes as kinds of PET/SPECT tracers had become essential tools for clinical radionuclide diagnosis. This review mainly summarizes radiolabeled peptide probes for bioimaging, including fundamental concepts of radiolabeled peptide probe design, some typical peptide analogs radiocontrast agents for PET, SPECT, and the combination imaging.     

A pH‐responsive self‐fluorescent polymeric micelle as a potential optical imaging probe

A series of methoxypolyethylene glycol‐terminated self‐fluorescent polyurethane multi‐block copolymers with excellent pH‐responsivity, self‐fluorescence, and biocompatibility are designed and synthesized. In our design, 1, 4‐bis (hydroxyethyl) piperazine is chosen as a pH‐responsive segment which can donate or accept protons in response to the change of environmental pH, and fluorescein isothiocyanate is used as a fluorescent dye conjugated into the micelles to offer self‐fluorescence. The chemical structure of the polyurethane multi‐block copolymers is characterized by nuclear magnetic resonance and Fourier transform infrared spectroscopy. The results of the acid‐based titration, the fluorescence spectrometry, and the ultraviolet visible spectroscopy indicate that the polyurethane multi‐block copolymers own an excellent pH‐buffering capacity responded to the change of pH values and the favorable self‐fluorescence property in an aqueous solution. And the ultraviolet absorption peaks of samples are strengthened with increasing of pH values, indicating that methoxypolyethylene glycol‐terminated self‐fluorescent polyurethane multi‐block copolymer can be a pH‐dependent fluorescent probe in a broad pH range. In addition, the in vitro cytotoxicity test showed that the polyurethane multi‐block copolymer has low cytotoxicity and good biocompatibility, which make it a promising nanoplatform for molecular imaging, diagnosis, and treatment of disease.     

A dansyl-based optical probe for detection of singly and doubly charged anions

This paper reports the synthesis of a new molecule, 5-(dimethylamino)naphthalene-1-sulfonic acid 2,3-bis(3-phenylureido)phenyl ester (1), as an optical probe for anions. The effect of the presence of various anions on the spectroscopic properties of 1 was examined using UV–vis absorption spectroscopy, fluorescence and ¹H NMR titration experiments. Strong binding of 1 to carboxylate, oxalate, cyanide and dihydrogen phosphate anions resulted in an increase in the emission of 1 and changes in its ¹H NMR chemical shifts. Binding constants of 1 to anions were also calculated based on the binding isotherms derived from the spectroscopic titrations.