Design and synthesis of a novel fluorescence probe for Zn based on the spirolactam ring-opening process of rhodamine derivatives

The spirolactam ring-opening process of rhodamine derivative is one of the most useful mechanisms for controlling fluorescence properties. However, the open/closed equilibrium reaction of rhodamine spirolactam has not been well characterized. Therefore, we examined the relationship between the spirolactam ring-opening process of rhodamine derivatives and the structure of the xanthene moiety. Based on the results of this investigation, we selected a candidate xanthene moiety for a Zn²⁺ sensor, and successfully developed a new fluorescence probe for Zn²⁺.     

A triazole‐based fluorescence probe for detecting Hg2+ ions and its biological application

A new compound, ethyl 5‐phenyl‐2‐(p‐tolyl)‐2H‐1,2,3‐triazole‐4‐carboxylate was successfully introduced and synthesized as a novel rhodamine B derivative named REPPC, and characterized by ¹H nuclear magnetic resonance (NMR), ¹³C NMR, and high resolution mass spectrometry (HRMS). It showed an obvious fluorescence and UV–visible light absorption enhancement towards Hg²⁺ ion without interference from common metal ions in N,N‐dimethylformamide–H₂O (pH 7.4). The spirolactam ring moiety of rhodamine in REPPC was converted to the open‐ring form generating a 1:1 complex with the intervention of a mercury ion, verified by electrospray ionization‐mass spectroscopy testing and density functional theory calculation. REPPC was used to visualize the level of mercury ions in living HeLa cells with encouraging results.     

Rhodamine-based ‘turn-on’ fluorescent probe for Cu(II) and its fluorescence imaging in living cells

A novel rhodamine spirolactam derivative 3′,6′-Bis(diethylamino)-2-(2-hydroxyethylamino) spiro[isoindoline-1,9′-xanthen]-3-one (RO1) was synthesized, and characterized by high-resolution mass spectrometry (HRMS), X-ray crystallography, Infrared spectroscopy (IR), and ¹H NMR and ¹³C NMR spectroscopy. RO1 exhibited highly sensitive and exclusively selective fluorescence response toward Cu²⁺ over other metal ions with a detection limit of 0.56 ppb in mixed aqueous solution. The fluorescence was pH-independent in the wide range pH 3.1–11.6. The turn-on fluorescence enhancement of the probe is based on Cu²⁺ induced ring-opening mechanism of the rhodamine spirolactam. Moreover, by means of fluorescence microscopy experiments, it was demonstrated that RO1 could monitor trace Cu²⁺ changes by live cell imaging.     

A tricorn‐rhodamine fluorescent chemosensor for detection of Co2+ ions

A novel chemosensor TrisRh based on tris(2‐aminoethyl)amine and rhodamine 6G is designed and synthesized as a fluorescence turn‐on probe for Co²⁺ ions that is paramagnetic with a property of quenching fluorescence. Rhodamine spirolactam forms are nonfluorescent, whereas, ring‐opening of corresponding spirocyclic induced by Co²⁺ results in strong fluorescence emission. Upon the addition of Co²⁺ ions, TrisRh can display significant enhancements in absorbance and fluorescence intensity as well as evident colorific transformation, which can be perceived by the naked eye. The association stoichiometry of TrisRh to Co²⁺ ions was inferred to be 1:1 through Job's plot and electrospray ionization mass spectrometry analysis. The binding model was speculated from Fourier transform infrared spectra and ¹H–nuclear magnetic resonance technologies. Significantly, the limit of detection was determined to be as low as 1.22 nmol. Furthermore, TrisRh can exhibit robust anti‐jamming ability against other interference metal ions.     

Thyroid hormone conjugates with rhodamine B as fluorescent ligands of human plasma transport proteins]

Conjugates of thyroxine (T4) and triiodothyronine (T3) with rhodamine B in which the hormone and the fluorescent dye are linked via a thiourea bond have been synthesized. These conjugates possess an ability to inhibit in a competitive manner the binding of [125I]T4 to three protein preparations: T4-binding globulin (TBG), apolipoprotein A-I (ApoA-I), and high density lipoprotein particles (ApoA-I-HDL) isolated from human serum by T4-Sepharose 4B chromatography and further purified. The following values of association constants have been estimated: for the T4 derivative-3 x 10(7) M-1 (TBG), 4.1 x 10(5) M-1 (ApoA-I), and 4.2 x 10(5) M-1 (ApoA-I-HDL); for the T3 derivative-1.6 x 10(7) M-1 (TBG), 5.3 x 10(5) M-1 (ApoA-I), and 5.4 x 10(5) M-1 (ApoA-I-HDL). The binding of rhodamine B-labeled thyroid hormones to TBG or ApoA-I do not alter significantly the parameters of rhodamine B chromophore absorption and fluorescence. The interaction of the conjugates with ApoI-HDL leads to a significant enhancement of the absorption intensity and a 3 nm blue shift in the absorption maximum as well as to a 1.5-fold increase in the fluorescence band amplitude at 586 nm. Biological and fluorescent properties of T4 and T3 derivatives suggest that these compounds may be a useful tool in fluorescence studies of plasma binding protein-driven transport of thyroid hormones in model biological systems.     

Fluorescent somatostatin receptor probes for the intraoperative detection of tumor tissue with long-wavelength visible light

Targeted fluorescent dyes are of substantial value for the intraoperative delineation of primary tumors and metastatic lesions. For this purpose long-wavelength red light (lambda=550-650 nm) offers advantages because of good tissue penetration and direct visibility. Since somatostatin receptors (SSTR) are overexpressed in a number of tumors, a series of potentially tumor-selective peptide-dye conjugates were synthesized by solid-phase peptide synthesis (SPPS). The octapeptides octreotate, Tyr(3)-octreotate and Tyr(3)-octreotide were employed and exhibited high affinity for somatostatin receptors (SSTR). The fluorescent dyes rhodamine 101, sulforhodamine B acid chloride, sulforhodamine 101 or rhodamine B isothiocyanate were conjugated either directly or via spacers, for example the peptidase-labile pentapeptide sequence Ala-Leu-Ala-Leu-Ala. The conjugates were completely assembled on the solid support: Fmoc-SPPS, cyclization via a disulfide linkage, N-terminal attachment of a spacer, and linkage to the fluorescent dye. An in vitro competition assay revealed that the conjugates bind to SSTRs with IC(50) values between 0.7 and 89 nM. The conjugates were generally stable to hydrolysis at pH 7-8 in buffer or serum. However, the rhodamine 101 conjugates revealed a loss of absorption at alkaline pH due to conversion to a neutral spirolactam form, as characterized by NMR.     

Rapid assessment of bacterial viability and vitality by rhodamine 123 and flow cytometry

A.S. KAPRELYANTS AND D.B. KELL. 1992. The fluorescent dye rhodamine 123 (Rh 123) is concentrated by microbial cells in an uncoupler-sensitive fashion. Steady-state fluorescence measurements with Micrococcus luteus indicated that provided the added dye concentration is below approximately 1 mmol/1, uptake is fully uncoupler-sensitive and is not accompanied by significant self-quenching of the fluorescence of accumulated dye molecules. 'Viable' and 'non-viable' cells are easily and quantitatively distinguished in a flow cytometer by the extent to which they accumulate the dye. The viability of a very slowly growing chemostat culture of Mic. luteus is apparently only about40–50%, as judged by plate counts, but most of the 'non-viable' cells can be resuscitated by incubation of the culture in nutrient medium before plating. The extent to which individual cells accumulate rhodamine 123 can be rapidly assessed by flow cytometry, and reflects the three distinguishable physiological states exhibited by the culture ('non-viable', 'viable' and 'non-viable-but-resuscitable'). Gram-negative bacteria do not accumulate rhodamine 123 significantly because their outer membrane is not permeable to it; a simple treatment overcomes this. Flow cytometry using rhodamine 123 should prove of general utility for the rapid assessment of microbial viability and vitality.     

Dynamics of single dye molecules observed by confocal imaging and spectroscopy.

The fluorescence emission of single rhodamine dye molecules (rhodamine 6G and rhodamine 630) at room temperature was analyzed by using scanning confocal laser microscopy in conjunction with polarization analysis, fluorescence spectroscopy, time-resolved detection (minutes to microseconds), and excitation saturation. Results are presented and discussed 1) for samples with dye molecules at the glass-air interface and 2) covered with an additional thin protective polymer film (polyvinylbutyral). Under the polymer layer, the single-molecule fluorescence was more stable than the glass-air interface. This result may be explained by fewer spontaneous variations of the fluorescence rate, polarization changes, spectral shifts, and longer photochemical lifetimes.     

Ratiometric fluorescent detection of Cu2+ based on dual‐emission ZIF‐8@rhodamine‐B nanocomposites

Zeolitic imidazolate framework‐8 (ZIF‐8) loading rhodamine‐B (ZIF‐8@rhodamine‐B) nanocomposites was proposed and used as ratiometric fluorescent sensor to detect copper(II) ion (Cu²⁺). Scanning electron microscopy, Fourier transform infrared spectroscopy, X‐ray powder diffraction, nitrogen adsorption/desorption isotherms and fluorescence emission spectroscopy were employed to characterize the ZIF‐8@rhodamine‐B nanocomposites. The results showed the rhodamine‐B was successfully assembled on ZIF‐8 based on the π‐π interaction and the hydrogen bond between the nitrogen atom of ZIF‐8 and –COOH of rhodamine‐B. The as‐obtained ZIF‐8@rhodamine‐B nanocomposites were octahedron with size about 150–200 nm, had good water dispersion, and exhibited the characteristic fluorescence emission of ZIF‐8 at 335 nm and rhodamine‐B at 575 nm. The Cu²⁺ could quench fluorescence of ZIF‐8 rather than rhodamine‐B. The ZIF‐8 not only acted as the template to assemble rhodamine‐B, but also was employed as the signal fluorescence together with the fluorescence of rhodamine‐B as the reference to construct a novel ratiometric fluorescent sensor to detect Cu²⁺. The resulted ZIF‐8@rhodamine‐B nanocomposite fluorescence probe showed good linear range (68.4 nM to 125 μM) with a low detection limit (22.8 nM) for Cu²⁺ combined with good sensitivity and selectivity. The work also provides a better way to design ratiometric fluorescent sensors from ZIF‐8 and other fluorescent molecules.     

Bifluorophoric molecules as fluorescent beacons for antibody-antigen binding

The quenching of fluorescence (up to 98%) by anti-fluorescein antibodies is well documented in the literature. Here we report a system where, instead of quenching, bifluorophoric molecules are designed to increase in fluorescence upon binding by an anti-fluorescein antibody. Bifluorophoric molecules are made of fluorescein (F) linked to tetramethylrhodamine (T) via varying numbers of methylene units, denoted as F-(CH(2))(n)-T. These F-(CH(2))(n)-T conjugates are almost nonfluorescent when free in solution due to intramolecular dimerization and stacking. Upon binding to an anti-fluorescein antibody, however, up to 110-fold increase in fluorescence was observed from the rhodamine moiety. This increase is believed to result from intramolecular dimer dissociation that dequenches the rhodamine fluorescence. Fluorescein fluorescence, on the other hand, remains quenched due to binding and intramolecular resonance energy transfer. Moreover, the excitation wavelength was at the absorption maxima of fluorescein, giving a Stoke's shift of about 90 nm. This system couples directly molecular recognition with a concurrent increase in fluorescence emission, obviating wash and incubation steps required by most assays. It is an important molecular reporter system for developing homogeneous assays.     

A new rhodamine‐based fluorescent chemodosimeter for mercuric ions in water media

A new rhodamine–ethylenediamine–nitrothiourea conjugate (RT) was synthesized and its sensing property as a fluorescent chemodosimeter toward metal ions was explored in water media. Analytical results from absorption and fluorescence spectra revealed that the addition of Hg²⁺ ions to the aqueous solution of the chemodosimeter RT caused a distinct fluorescence OFF–ON response with a remarkable visual color change from colorless to pink; however, no clear spectral and color changes were observed from other metal ions including: Zn²⁺, Cu²⁺, Cd²⁺, Pb²⁺, Ag⁺, Fe²⁺, Cr³⁺, Co³⁺, Ni²⁺, Ca²⁺, Mg²⁺, K⁺ and Na⁺. The sensing results and the molecular structure suggested that a Hg²⁺‐induced a desulfurization reaction and cyclic guanylation of the thiourea moiety followed by ring‐opening of the rhodamine spirolactam in RT are responsible for a distinct fluorescence turn‐on signal, indicating that RT is a remarkably sensitive and selective chemodosimeter for Hg²⁺ ions in aqueous solution. Hg²⁺ within a concentration range from 0.1 to 25 μM can be determined using RT as a chemodosimeter and a detection limit of 0.04 μM is achieved. Copyright © 2014 John Wiley & Sons, Ltd.     

Rhodamine B as a mitochondrial probe for measurement and monitoring of mitochondrial membrane potential in drug-sensitive and -resistant cells

In order to get more insight into the energetic state of multidrug-resistance (MDR) cell compared with its corresponding sensitive cell, a noninvasive fluorescence method for determining and monitoring the mitochondrial membrane potential (DeltaPsi(m)), using rhodamine B and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was established. Rhodamine B distributes across biological membranes in response to the electrical transmembrane potential. P-glycoprotein- and MRP1-protein-mediated efflux do not create a concentration gradient, leading the cell-rhodamine B system to reach a steady state, where the ratio of cytosolic to extracellular rhodamine B was equal to 1. The mitochondrial matrix rhodamine B concentration was precisely determined as a decrease of rhodamine B fluorescence in the presence of formazan, a rhodamine B fluorescence quencher, which locally accumulates in the matrix of mitochondria. The kinetics of decrease in rhodamine B fluorescence (V(i)) can be used to estimate DeltaPsi(m) using the Nernst equation: DeltaPsi(m)=-61.54 log V(i)-258.46. The DeltaPsi(m) values determined were -160+/-4 mV for K562 cell, -146+/-6 mV for K562/adr cell, -161+/-10 mV for GLC4 cell and -168+/-2 mV for GLC4/adr cell. An increase or a decrease in DeltaPsi(m) consequently followed an increase or a decrease in the cellular ATP contents. An increase ATP content in the two MDR cell lines can protect cells from cytotoxicity induced by pirarubicin.     

Identification by high throughput screening of small compounds inhibiting the nucleic acid destabilization activity of the HIV-1 nucleocapsid protein

Due to its highly conserved zinc fingers and its nucleic acid chaperone properties which are critical for HIV-1 replication, the nucleocapsid protein (NC) constitutes a major target in AIDS therapy. Different families of molecules targeting NC zinc fingers and/or inhibiting the binding of NC with its target nucleic acids have been developed. However, their limited specificity and their cellular toxicity prompted us to develop a screening assay to target molecules able to inhibit NC chaperone properties, and more specifically the initial NC-promoted destabilization of the nucleic acid secondary structure. Since this destabilization is critically dependent on the properly folded fingers, the developed assay is thought to be highly specific. The assay was based on the use of cTAR DNA, a stem–loop sequence complementary to the transactivation response element, doubly labelled at its 5′ and 3′ ends by a rhodamine 6G fluorophore and a fluorescence quencher, respectively. Addition of NC(12-55), a peptide corresponding to the zinc finger domain of NC, to this doubly-labelled cTAR, led to a partial melting of the cTAR stem, which increases the distance between the two labels and thus, restores the rhodamine 6G fluorescence. Thus, positive hits were detected through the decrease of rhodamine 6G fluorescence. An “in-house” chemical library of 4800 molecules was screened and five compounds with IC₅₀ values in the micromolar range have been selected. The hits were shown by mass spectrometry and fluorescence anisotropy titration to prevent binding of NC(12-55) to cTAR through direct interaction with the NC folded fingers, but without promoting zinc ejection. These non-zinc ejecting NC binders are a new series of anti-NC molecules that could be used to rationally design molecules with potential anti-viral activities.     

Confocal quantification of cis-regulatory reporter gene expression in living sea urchin

Quantification of GFP reporter gene expression at single cell level in living sea urchin embryos can now be accomplished by a new method of confocal laser scanning microscopy (CLSM). Eggs injected with a tissue-specific GFP reporter DNA construct were grown to gastrula stage and their fluorescence recorded as a series of contiguous Z-section slices that spanned the entire embryo. To measure the depth-dependent signal decay seen in the successive slices of an image stack, the eggs were coinjected with a freely diffusible internal fluorescent standard, rhodamine dextran. The measured rhodamine fluorescence was used to generate a computational correction for the depth-dependent loss of GFP fluorescence per slice. The intensity of GFP fluorescence was converted to the number of GFP molecules using a conversion constant derived from CLSM imaging of eggs injected with a measured quantity of GFP protein. The outcome is a validated method for accurately counting GFP molecules in given cells in reporter gene transfer experiments, as we demonstrate by use of an expression construct expressed exclusively in skeletogenic cells.     

Molecular interactions of peptides with phospholipid vesicle membranes as studied by fluorescence correlation spectroscopy

Interactions of the peptides melittin and magainin with phospholipid vesicle membranes have been studied using fluorescence correlation spectroscopy. Molecular interactions of melittin and magainin with phospholipid membranes are performed in rhodamine-entrapped vesicles (REV) and in rhodamine-labelled phospholipid vesicles (RLV), which did not entrap free rhodamine inside. The results demonstrate that melittin makes channels into vesicle membranes since exposure of melittin to vesicles causes rhodamine release only from REV but not from RLV. It is obvious that rhodamine can not be released from RLV because the inside of RLV is free of dye molecules. In contrast, magainin breaks vesicles since addition of magainin to vesicles results in rhodamine release from both REV and RLV. As the inside of RLV is free of rhodamine, the appearance of rhodamine in solution confirms that these vesicles are broken into rhodamine-labelled phospholipid fragments after addition of magainin. This study is of pharmaceutical significance since it will provide insights that fluorescence correlation spectroscopy can be used as a rapid protocol to test incorporation and release of drugs by vesicles.     

Fluorescent probes for monitoring virus fusion kinetics: comparative evaluation of reliability

Fluorescence assays for viral membrane fusion employ lipidic probes whose kinetics of fluorescence dequenching should mimic the actual kinetics of membrane merging. We examined the fusion of influenza virus with CEM cells, erythrocyte ghosts or liposomes by monitoring the fluorescence dequenching of each one of the three probes, octadecylrhodamine B chloride (R18), N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (Rh-PE), or rac-2,3-dioleoylglycerol ester of rhodamine B (DORh-B), inserted into the virus membrane. Experimental conditions were designed to allow a clear distinction between membrane mixing and non-specific probe transfer. Fluorescence dequenching observed with Rh-PE was much slower than with R18, unless a particular experimental procedure was used. Using liposomes as a target membrane, the kinetics and extent of the decrease in resonance energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (NBD-PE) and Rh-PE, initially embedded in the liposome membrane, were matched by that of the dequenching of viral R18, but not of viral Rh-PE. DORh-B was found not to be appropriate to follow membrane merging. Our results indicate that on a time scale of several minutes R18 more accurately reflects the kinetics of membrane fusion. Nevertheless, control experiments should be performed to evaluate non-specific probe transfer of R18 molecules, whose contribution to fluorescence dequenching can become significant after long incubation times.     

Self-association of glucagon as measured by the optical properties of rhodamine 6G

The fluorescence of rhodamine 6G is completely quenched in glucagon solutions in 0.6 M K2HOP4 at pH 10.6. The absorption of rhodamine 6G is red-shifted by the same reaction. A single rhodamine 6G molecule appears to be bound to a hydrophobic patch in the center of the trimer of glucagon. Since the glucagon monomer has almost no organized structure this site exists only in the associated trimer form of glucagon. The self-association of glucagon to the trimer has been determined from the variation in rhodamine 6G fluorescence and absorption measured over a 60-fold range of dye concentration. The self-association constant agrees with values determined by other methods in the absence of dye. The binding isotherms of rhodamine 6G to glucagon shift with glucagon concentration and exhibit negative cooperativity.     

Fluorescence quenching competitive immunoassay in micro droplets

A fluorescence quenching competitive immunoassay in micro droplets was applied to the sensitive detection of the pyrethroid insecticide, esfenvalerate. Laser induced fluorescence from rhodamine dye was used as a marker. The competitive immunoreaction was performed in micro droplets generated by a vibrating orifice aerosol generator system with a 10-microm diameter orifice. Fluorescence that was emitted from the droplets was detected by a 1/8 m imaging spectrograph with a 512 x 512 thermoelectrically cooled, charged-coupled device camera. The conjugate of esfenvalerate with rhodamine exhibited similar fluorescence to that of pure rhodamine 6G. When anti-esfenvalerate antibodies were added to the droplets, the fluorescence decreased. The reduction in emission was due to a strong quenching effect that arises from the interaction between the protein and rhodamine molecules following the antigen-antibody reaction. When a sample of esfenvalerate was added to the droplets, the release of the conjugated rhodamine from the antigen-antibody complex allowed the fluorescence signal to recover. An assay in a picoliter droplet sample was shown to enable detection down to approximately 0.1 nM. A very small mass of analyte could be detected with this method. A sample of river water was used to gauge the impact of matrix effects and was shown to give rise to negligible interference with the immunoassay.     

Phenothiazine–rhodamine‐based colorimetric and fluorogenic ‘turn‐on' sensor for Zn2+ and bioimaging studies in live cells

A phenothiazine–rhodamine (PTRH) fluorescent dyad was synthesized and its ability to selectively sense Zn²⁺ ions in solution and in in vitro cell lines was tested using various techniques. When compared with other competing metal ions, the PTRH probe showed the high selectivity for Zn²⁺ ions that was supported by electronic and emission spectral analyses. The emission band at 528 nm for the PTRH probe indicated the ring closed form of PTRH, as for Zn²⁺ ion binding to PTRH, the λ_em get shift to 608 nm was accompanied by a pale yellow to pink colour (under visible light) and green to pinkish red fluorescence emission (under UV light) due to ring opening of the spirolactam moiety in the PTRH ligand. Spectral overlap of the donor emission band and the absorption band of the ring opened form of the acceptor moiety contributed towards the fluorescence resonance energy transfer ON mechanism for Zn²⁺ ion detection. The PTRH sensor had the lowest detection limit for Zn²⁺, found to be 2.89 × 10^?8 M. The sensor also demonstrated good sensing application with minimum toxicity for in vitro analyses using HeLa cells.     

Proximity of lectin receptors on the cell surface measured by fluorescence energy transfer in a flow system.

Molecules of the lectin concanavalin A have been labeled separately with the fluorescein and rhodamine chromophores and jointly bound to the surface of transformed Friend erythroleukemia cells. The two dyes constitute an ideal donor-acceptor pair for fluorescence resonance energy transfer thereby permitting the determination of the proximity relationships between bound ligand molecules and the corresponding surface receptors. The transfer efficiency at saturation (about 57%) was measured in a multiparameter flow system using laser excitation at 488 nm and detection of fluorescein and rhodamine emission intensities as well as the emission anisotropy of the rhodamine fluorescence for each cell. The degree of energy transfer was estimated from the quenching of donor emission, the sensitization of acceptor emission, and the depolarization of acceptor fluorescence. The system has been modeled according to a formalism developed by Gennis and Cantor (Biochemistry 11: 2509, 1972). We estimate the separation between the surfaces of bound lectin molecules at saturation to be 0-40 A, a range possibly characteristic for micropatches induced by ligand binding.