Fluorescence quenching and bonding properties of some hydroxamic acid derivatives by iron(III) and manganese(II)

Spectrophotometric investigations of highly fluorescent metal chelating molecules are of relevance due to their potential application in novel, selective fluorescence‐based sensors. Benzene and naphthalene chromophores are highly fluorescent while hydroxamic acids are widely used as ligands for complexation of transition metals. In order to develop fluorescence probes, several phenyl derivatives of N‐phenylbenzohydroxamic acid and an aminodihydroxamic acid linked with a naphthalene chromophore were synthesized and their selective ionophoric properties towards iron(III) and manganese(II) ions were investigated using fluorescence and absorption spectroscopy. Both methods confirm the formation of 1:1 and 1:2 complexes for iron(III) and a 1:1 complex for manganese(II). The complex that is formed depends on the concentration of the ligand and pH of the medium. The amino dihydroxamic acid exhibits a prominent selectivity towards iron(III) with a two‐step 1:1 and 1:2 quenching mechanism at pH 3 and towards manganese(II) with a 1:1 quenching mechanism at a probe concentration of 1 × 10^?5 mol dm^?3 at pH 9.5 The logarithm of overall formation constants of 1:1 and 1:2 complexes of iron(III) were estimated as 3.30 and 9.05, respectively. Copyright © 2008 John Wiley & Sons, Ltd.     

On the nature of the energised state of submitochondrial particles; Investigations with N-aryl naphthalene sulphonate probes

1. A further investigation has been made of the way in which the fluorescent probes 1-anilino-naphthalene-8-sulphonate and 2-(N-methyl-anilino)naphthalene-6-sulphonate report on the energised state of bovine heart submitochondrial particles.2. A comparison of the probe responses to energisation with ATP or to a potassium diffusion potential has been made. The fluorescence enhancements seen in these two cases have different characteristics, and in view of this it is questioned whether a substrate generated energised state of a submitochondrial particle can be equated with a trans-membrane potassium diffusion potential.3. Substitution of ITP for ATP reduces the rate at which either of the probes respond to energisation. In contrast reducing the ATPase activity of the particles by treatment with the covalent ATPase inhibitors 4-chloro-7-nitrobenzofurazan or N,N′-dicyclohexyl-carbodiimide has no effect on this rate. This finding that the rate of the fluorescence changes is directly sensitive to events at the level of the ATPase, but not to the total ATPase activity, suggests that this rate may not be controlled by a delocalised energised state. Reduction of ATPase activity decreases the extent of the fluorescence enhancement and a relationship between the change in probe fluorescence and ATPase activity is given.4. The results in this paper are discussed in the context of the mechanisms which have been proposed to account for the fluorescence enhancements of N-aryl naphthalene sulphonate probes upon energisation of submitochondrial particles.     

Synthesis and biological evaluation of N-difluoromethyl-1,2-dihydropyrid-2-one acetic acid regioisomers: Dual Inhibitors of cyclooxygenases and 5-lipoxygenase

A new group of acetic acid (7a–c, R¹ = H), and propionic acid (7d–f, R¹ = Me), regioisomers wherein a N-difluoromethyl-1,2-dihydropyrid-2-one moiety is attached via its C-3, C-4, and C-5 position was synthesized. This group of compounds exhibited a more potent inhibition, and hence selectivity, for the cyclooxygenase-2 (COX-2) relative to the COX-1 isozyme. Attachment of the N-difluoromethyl-1,2-dihydropyrid-2-one ring system to an acetic acid, or propionic acid, moiety confers potent 5-LOX inhibitory activity, that is, absent in traditional arylacetic acid NSAIDs. 2-(1-Difluoromethyl-2-oxo-1,2-dihydropyridin-5-yl)acetic acid (7c) exhibited the best combination of dual COX-2 and 5-LOX inhibitory activities. Molecular modeling (docking) studies showed that the highly electronegative CHF₂ substituent present in 7c, that showed a modest selectivity for the COX-2 isozyme, is oriented within the secondary pocket (Val523) present in COX-2 similar to the sulfonamide (SO₂NH₂) COX-2 pharmacophore present in celecoxib, and that the N-difluoromethyl-1,2-dihydropyrid-2-one pharmacophore is oriented close to the region containing the LOX enzyme catalytic iron (His361, His366, and His545). Accordingly, the N-difluoromethyl-1,2-dihyrdopyrid-2-one moiety possesses properties suitable for the design of dual COX-2/5-LOX inhibitory drugs.     

Copper(I)-imine complexes: Synthesis and catalytic activity in olefin cyclopropanation

Different imine-type ligands, prepared by the condensation of anilines or of α-methylbenzylamine with 2-pyridinecarboxaldehyde (pyim^1,2) or 2-quinolinecarboxaldehyde (quim^1,2) were prepared. These species act as N,N′-bidentate, chelating ligands upon coordination to Cu(I): treatment of [Cu(PPh₃)₃Cl] with an equimolar amount of the ligands resulted in the displacement of two molecules of PPh₃, giving rise to the formation of [Cu(pyim^1,2)(PPh₃)Cl] (1-2) and [Cu(quim^1,2)(PPh₃)Cl] (3-4), respectively. The copper derivatives 1-4 proved to be highly active catalysts in olefin cyclopropanation in the presence of ethyl diazoacetate, even using deactivated olefins (namely, 2-cyclohexen-1-one) as substrate. The X-ray structure of complex 2, [Cu(pyim²)(PPh₃)Cl], is also reported.     

Interactions of phenytoin with rat brain synaptosomes examined by fluorescent fatty acid probes

Phenytoin (PHT) modified the fluorescent characteristics of anthroyloxy-fatty acids in synaptosomal membranes. Association of PHT with synaptosomal membranes caused the greatest change when the fluorescent probe was located at the 6-carbon position of N-(anthroyloxy)stearic acid and was incorporated into the membranes. Phenytoin and 6-(anthroyloxy)stearic acid compete for high affinity binding regions which are probably lipid domains within the membrane. Phenytoin has a weaker association with the sites than the fluorescent fatty acids. Divalent cations, e.g. Mg²⁺ or Ca²⁺, are required to observe maximal change in polarization of fluorescence of fatty acid probes in the presence of PHT. It is proposed that the membrane lipid bilayer reorganizes to accommodate exogenous compounds, such as phenytoin or the fatty acid probe in order to permit the most efficient packing of lipids. This reorganization of the lipid bilayer may influence membrane enzyme activities and ion channels.     

Effect of translocation on topology and conformation of anticodon and D loops of tRNAPhe

Steady-state fluorescence and fluorescence anisotropy measurements have been carried out on isolated complexes of fluorescent derivatives of N-AcPhe-tRNA^Phe with 70 S ribosomes from Escherichia coli. As a fluorescent probe, proflavine was inserted into either the anticodon loop or the D loop.Upon binding to the A site of poly(U)-programmed ribosomes, the probe in the anticodon loop is highly immobilized and effectively shielded against solvent access in a hydrophobic binding site. Elongation factor G-dependent translocation to the P site does not change any of the fluorescence parameters. These observations indicate that in both sites the environment of the probe with respect to hydrophobicity and shielding against solvent access is rather similar. Moreover, substantial conformational changes of the anticodon loop upon translocation are made unlikely.In contrast to the anticodon loop, the D loop is fully exposed to the solvent in both A and P sites, indicating that the variable region in the middle of the D loop is oriented away from the ribosomal surface.On the other hand, depolarization measurements show that the D loop is strongly immobilized in the A site, possibly by binding interactions of invariant bases of the loop. Upon translocation, the D loop gains considerable flexibility, indicating that in the P site it is neither fixed by contacts with the ribosome nor by intramolecular base-pairing with the T loop.In the absence of poly(U) or in the presence of poly(C), the fluorescence parameters of the probes in the anticodon loop and, more significantly, in the D loop, differ from those observed in the presence of poly(U). These differences are best explained by assuming a codon-induced conformational change of the anticodon loop, which in turn is transmitted to the D loop.When the non-aminoacylated tRNA^Phe derivatives are studied, spectroscopic differences as compared to the respective N-AcPhe-tRNA^Phe derivatives are observed only for the A site complexes. It appears that the aminoacylation influences the binding of transfer RNA in the A site, but not in the P site.     

Fluorescence from abnormally sterile pollen of the Japanese apricot

We observed trees of the Japanese apricot, Prunus mume ‘Nanko’ (Rosaceae), bearing two types of flowers: 34% had blue fluorescent pollen under UV irradiation, and 66% had non-fluorescent pollen. The fluorescent pollen grains were abnormally crushed, sterile, and devoid of intine and pollenkitt. The development of microspores within anthers was investigated: in the abnormally developed anthers, tapetal cells were vacuolated at the unicellular microspore stage, and fluorescent pollen was produced. Compounds responsible for the blue fluorescence of pollen were identified as chlorogenic acid and 1-O-feruloyl-β-D-glucose. The anthers with fluorescent pollen contained 6.7-fold higher and 3.8-fold lower amounts of chlorogenic acid and N¹,N⁵,N¹⁰-tri-p-coumaroylspermidine, respectively, compared to those with non-fluorescent pollen. The tapetal vacuolization, highly accumulated chlorogenic acid, and deficiency of N¹,N⁵,N¹⁰-tri-p-coumaroylspermidine imply that low-temperature stress during the early unicellular microspore stage caused a failure in microsporogenesis. Furthermore, potential effects of the visual difference on the bee behavior were also discussed through the colorimetry. The sterility, likely induced by low-temperature stress, and the preference of honeybees for fluorescence may reduce the pollination efficiency of P. mume.     

A novel optical probe based on d‐penicillamine‐functionalized graphene quantum dots: Preparation and application as signal amplification element to minoring of ions in human biofluid

In this study, d ‐penicillamine‐functionalized graphene quantum dots (DPA‐GQD) has been synthesized, which significantly increases the fluorescence intensity of GQD. We used this simple fluorescent probe for metal ions detection in human plasma samples. Designed DPA‐GQD respond to Hg²⁺, Cu²⁺, Au²⁺, Ag⁺, Co²⁺, Zn²⁺, and Pb²⁺ with high sensitivity. The fluorescence intensity of this probe decreased significantly in the presence of metal ions such as, Hg²⁺, Cu²⁺, Au²⁺, Ag⁺, Co²⁺, Zn²⁺, and Pb²⁺. In this work, a promising probe for ions monitoring was introduced. Moreover, DPA‐GQD probe has been tested in plasma samples. The functionalized DPA‐GQDs exhibits great promise as an alternative to previous fluorescent probes for bio‐labeling, sensing, and other biomedical applications in aqueous solution.     

Sequence selective formation of 1,N6-ethenoadenine in DNA by furan-conjugated probe

1,N⁶-Ethenoadenosine derivatives have been applied as fluorescence probes in various fields of biochemistry and molecular biology. We developed a 1,N⁶-ethenoadenosine-forming reaction at a target adenine in DNA duplex and applied it to a mutation diagnosis. Furan-derivatized oligodeoxyribonucleotides were synthesized and fluorescence properties were studied in the presence of complementary strand under oxidative conditions. Strong emissions at 430 nm were observed in the presence of the complementary strand with an adenine in front of furan moiety.     

“Chelatable iron pool”: inositol 1,2,3-trisphosphate fulfils the conditions required to be a safe cellular iron ligand

Mammalian cells contain a pool of iron that is not strongly bound to proteins, which can be detected with fluorescent chelating probes. The cellular ligands of this biologically important “chelatable”, “labile” or “transit” iron are not known. Proposed ligands are problematic, because they are saturated by magnesium under cellular conditions and/or because they are not “safe”, i.e. they allow iron to catalyse hydroxyl radical formation. Among small cellular molecules, certain inositol phosphates (InsPs) excel at complexing Fe³⁺ in such a “safe” manner in vitro. However, we previously calculated that the most abundant InsP, inositol hexakisphosphate, cannot interact with Fe³⁺ in the presence of cellular concentrations of Mg²⁺. In this work, we study the metal complexation behaviour of inositol 1,2,3-trisphosphate [Ins(1,2,3)P ₃], a cellular constituent of unknown function and the simplest InsP to display high-affinity, “safe”, iron complexation. We report thermodynamic constants for the interaction of Ins(1,2,3)P ₃ with Na⁺, K⁺, Mg²⁺, Ca²⁺, Cu²⁺, Fe²⁺ and Fe³⁺. Our calculations indicate that Ins(1,2,3)P ₃ can be expected to complex all available Fe³⁺ in a quantitative, 1:1 reaction, both in cytosol/nucleus and in acidic compartments, in which an important labile iron subpool is thought to exist. In addition, we calculate that the fluorescent iron probe calcein would strip Fe³⁺ from Ins(1,2,3)P ₃ under cellular conditions, and hence labile iron detected using this probe may include iron bound to Ins(1,2,3)P ₃. Therefore Ins(1,2,3)P ₃ is the first viable proposal for a transit iron ligand. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A novel colorimetric fluorescence sensor for Fe3+ based on quinoline Schiff base

A novel fluorescent sensor bearing a quinoline and an anisidine moiety has been developed for highly selective detection of Fe³⁺, which shows photo‐induced electron transfer (PET) behavior induced by Fe³⁺. Binding of Fe³⁺ to the sensor induced the electron of C = N group transfer from quinoline to iron, the result exhibits fluorescent enhancement. With the features of easy synthesis, simple structural skeleton and excellent sensing ability, the newly synthesized chemosensor also applied as a highly selective fluorescent probe in complex samples containing various competitive metal ions. The probe could fulfill various needs in biological and environmental fields.     

Rational design of an iminocoumarin-based fluorescence probe for peroxynitrite with high signal-to-noise ratio

As a high reactive oxygen species (ROS) and a reactive nitrogen species (RNS), peroxynitrite anion (ONOO⁻) is widely present in organisms and plays influential roles in physiological and pathological processes. It is of great significance to develop effective fluorescent probes for imaging peroxynitrite variation in living systems. Herein we present a novel fluorescent probe TQC0 for monitoring ONOO⁻ based on the iminocoumarin platform, and this probe was synthesized by the knoevenagel condensation between a dihydropyridine-salicylaldehyde derivative and 2-benzothiazole-acetonitrile, and subsequently masked with the boronate moiety. The obtained probe TQC0 exhibited a high signal-to-noise ratio (206-fold) and a quick ‘turn-on’ response (about 10 min) with great selectivity and sensitivity. Furthermore, the probe TQC0 was successfully applied for imaging ONOO⁻ in living cells with low cytotoxicity.     

Design and applications of methods for fluorescence detection of iron in biological systems

Fluorescence metalosensors provide a means to detect iron in biological systems that is versatile, economical, sensitive and of a high-throughput nature. They rely on relatively high-affinity iron-binding carriers conjugated to highly fluorescent probes that undergo quenching after metal complexation. Metal specificity is determined by probes containing either an iron-binding moiety of high affinity (type A) or of relatively lower affinity (type B) used in combination with a strong specific iron chelator. Due to the heterogeneous nature of biological systems, the apparent metal-binding affinity and complexation stoichiometry ought to be specifically defined. Fluoresceinated moieties coupled to metal-binding cores detect Fe at sub-micromolar concentrations and even sub-microlitre volumes (i.e. cells). Although an ideal probe should also be specific for a particular oxidation state of iron, in physiological conditions that property might be difficult to attain. Quantification of labile iron in cells has relied on the ability of permeant iron chelators to restore the fluorescence of probes quenched by intracellular Fe. Modern design of probes aims to (a) improve probe targeting to specific cell compartments and (b) create probes that respond to metal binding by signal enhancement.     

Detection of cyclin D1 mRNA by hybridization sensitive NIC–oligonucleotide probe

A large group of fluorescent hybridization probes, includes intercalating dyes for example thiazole orange (TO). Usually TO is coupled to nucleic acids post-synthetically which severely limits its use. Here, we have developed a phosphoramidite monomer, 10, and prepared a 2′-OMe-RNA probe, labeled with 5-(trans-N-hexen-1-yl-)-TO-2′-deoxy-uridine nucleoside, dU^TO, (Nucleoside bearing an Inter-Calating moiety, NIC), for selective mRNA detection. We investigated a series of 15-mer 2′-OMe-RNA probes, targeting the cyclin D1 mRNA, containing one or several dU^TO at various positions. dU^TO-2′-OMe-RNA exhibited up to 7-fold enhancement of TO emission intensity upon hybridization with the complementary RNA versus that of the oligomer alone. This NIC-probe was applied for the specific detection of a very small amount of a breast cancer marker, cyclin D1 mRNA, in total RNA extract from cancerous cells (250 ng/μl). Furthermore, this NIC-probe was found to be superior to our related NIF (Nucleoside with Intrinsic Fluorescence)-probe which could detect cyclin D1 mRNA target only at high concentrations (1840 ng/μl). Additionally, dU^T can be used as a monomer in solid-phase oligonucleotide synthesis, thus avoiding the need for post-synthetic modification of oligonucleotide probes. Hence, we propose dU^TO oligonucleotides, as hybridization probes for the detection of specific RNA in homogeneous solutions and for the diagnosis of breast cancer.     

Multimodality PET/MRI agents targeted to activated macrophages

The recent emergence of multimodality imaging, particularly the combination of PET and MRI, has led to excitement over the prospect of improving detection of disease. Iron oxide nanoparticles have become a popular platform for the fabrication of PET/MRI probes owing to their advantages of high MRI detection sensitivity, biocompatibility, and biodegradability. In this article, we report the synthesis of dextran-coated iron oxide nanoparticles (DIO) labeled with the positron emitter ⁶⁴Cu to generate a PET/MRI probe, and modified with maleic anhydride to increase the negative surface charge. The modified nanoparticulate PET/MRI probe (MDIO-⁶⁴Cu-DOTA) bears repetitive anionic charges on the surface that facilitate recognition by scavenger receptor type A (SR-A), a ligand receptor found on activated macrophages but not on normal vessel walls. MDIO-⁶⁴Cu-DOTA has an average iron oxide core size of 7–8 nm, an average hydrodynamic diameter of 62.7 nm, an r ₁ relaxivity of 16.8 mM^?1 s^?1, and an r ₂ relaxivity of 83.9 mM^?1 s^?1 (37 °C, 1.4 T). Cell studies confirmed that the probe was nontoxic and was specifically taken up by macrophages via SR-A. In comparison with the nonmodified analog, the accumulation of MDIO in macrophages was substantially improved. These characteristics demonstrate the promise of MDIO-⁶⁴Cu-DOTA for identification of vulnerable atherosclerotic plaques via the targeting of macrophages.     

Identification of single-point mutations in mycobacterial 16S rRNA sequences by confocal single-molecule fluorescence spectroscopy

We demonstrate the specific identification of single nucleotide polymorphism (SNP) responsible for rifampicin resistance of Mycobacterium tuberculosis applying fluorescently labeled DNA-hairpin structures (smart probes) in combination with single-molecule fluorescence spectroscopy. Smart probes are singly labeled hairpin-shaped oligonucleotides bearing a fluorescent dye at the 5′ end that is quenched by guanosine residues in the complementary stem. Upon hybridization to target sequences, a conformational change occurs, reflected in a strong increase in fluorescence intensity. An excess of unlabeled (‘cold’) oligonucleotides was used to prevent the formation of secondary structures in the target sequence and thus facilitates hybridization of smart probes. Applying standard ensemble fluorescence spectroscopy we demonstrate the identification of SNPs in PCR amplicons of mycobacterial rpoB gene fragments with a detection sensitivity of 10⁻⁸ M. To increase the detection sensitivity, confocal fluorescence microscopy was used to observe fluorescence bursts of individual smart probes freely diffusing through the detection volume. By measuring burst size, burst duration and fluorescence lifetime for each fluorescence burst the discrimination accuracy between closed and open (hybridized) smart probes could be substantially increased. The developed technique enables the identification of SNPs in 10⁻¹¹ M solutions of PCR amplicons from M.tuberculosis in only 100 s.     

Development of a convenient and supersensitive high-throughput screening system for genetically encoded fluorescent probes of small molecules using a confocal microscope

Monitoring the dynamic patterns of intracellular signaling molecules, such as inositol 1,4,5-trisphosphate (IP₃) and Ca²⁺, that control many diverse cellular processes, provides us significant information to understand the regulatory mechanism of cellular functions. For searching more sensitive and higher dynamic range probes for signaling molecules, convenient and supersensitive high throughput screening systems are required. Here we show the optimal “in Escherichia coli (E. coli) colony” screening method based on the twin-arginine translocase (Tat) pathway and introduce a novel application of a confocal microscope as a supersensitive detection system to measure changes in the fluorescence intensity of fluorescent probes in E. coli grown on an agar plate. To verify the performance of the novel detection system, we compared the changes detected in the fluorescent intensity of genetically encoded Ca²⁺ indicator after Ca²⁺ exposure to two kinds of conventional fluorescence detection systems (luminescent image analyzer and fluorescence stereomicroscope). The rate of fluorescence change between Ca²⁺ binding and unbinding detected by novel supersensitive detection system was almost double than those measured by conventional detection systems. We also confirmed that the Tat pathway-based screening method is applicable to the development of genetically encoded probes for IP₃. Our convenient and supersensitive screening system improves the speed of developing florescent probes for small molecules.     

ICPBCZin: a red emitting ratiometric fluorescent indicator with nanomolar affinity for Zn2+ ions

A new fluorescent Zn²⁺ indicator, namely, ICPBCZin was synthesized and the spectral profile of its free and Zn²⁺ bound forms was studied. The newly synthesized zinc indicator incorporates as chromophore the chromeno [3′,2′:3,4]pyrido[1,2a] [1,3]benzimidazole moiety and belongs to the dicarboxylate-type of zinc probes. The compound is excited with visible light, exhibits high selectivity for zinc in the presence of calcium and other common biological ions, and its Zn²⁺ dissociation constant is 4.0 nM. Fluorescence spectra studies of ICPBCZin indicated a clear shift in its emission wavelength maxima upon Zn²⁺ binding, as it belongs to the class of Photoinduced Charge Transfer (PCT) indicators, along with changes in fluorescence intensity that enable the compound to be used as a ratiometric, visible-excitable Zn²⁺ probe.     

Toward preparation of antibody-based imaging probe libraries for dual-modality positron emission tomography and fluorescence imaging

Two novel imaging agents trastuzumab-Cy5.5-CHX-A″ 1 and cetuximab-Cy7-CHX-A″ 2, bearing both a chelating moiety (CHX-A″) for sequestering metallic radionuclides (⁸⁶Y or ¹¹¹In) and the near infrared dye Cy5.5/Cy7, were prepared by a novel modular synthetic strategy as examples of dual-labeled, antibody-based imaging probe library. Fluorescent microscopy illustrated that 1 and 2 strongly bind to HER2-expressing cancer cells (e.g., NIH3T3–HER2⁺, SKOV-3) and to EGFR-expressing cancer cells (e.g., A431), respectively, thereby demonstrating that the functionality of the targeting moiety is conserved. Hence, the described novel synthesis strategy can be applied to engineer other tumor-targeted monoclonal antibody based probes for multimodality imaging.     

Structural and fluorescent study of zinc complexes of dansyl aminoquinoline

We have shown previously that 8-(5′-N,N-dimethylamino-1′-naphthalene)-sulfonamidoquinoline (DANQUIN) demonstrated a remarkable selectivity and sensitivity for the Zn(II) ion. In this work, the crystal structures of DANQUIN, Cu(DANQUIN)₂ and Cu(DANPY)₂ (DANPY, N-2-picolyl-(5′-N,N-dimethylamino-1′-naphthalene)-sulfonamide) are reported and compared with the simulated structure of Zn(DANQUIN)₂, which is important for the understanding of the factors that govern the fluorescence of DANQUIN. Free DANQUIN mainly displays the fluorescence of the dansyl group at 547 nm while the Zn(II)-DANQUIN complex mainly shows the enhanced fluorescence of aminoquinoline at 469 nm, while the emission of the dansyl group shifted to 517 nm with an almost constant intensity. This result demonstrates the advantage of this hybrid fluorescent chemosensor for Zn(II), and also makes it a potential candidate for ratiometric Zn(II) detection.