Development of a Fluorescent Pb2+ Sensor

A real turn‐on : The emission intensity of heterocycle 1 increases upon binding to Pb²⁺. Thus, 1 acts as a small‐molecule “turn‐on” fluorescent sensor for lead. The sensor is highly selective and is functional over a wide range of pH values.

     

A Sensitive and Selective Fluorescent Thiol Probe in Water Based on the Conjugate 1,4‐Addition of Thiols to α,β‐Unsaturated Ketones

Shedding light on thiol detection : A compound (see scheme) was developed as a novel, highly sensitive and selective fluorescent thiol probe, which also features suitable water solubility, functions rapidly under neutral conditions, and has excitation and emission in the visible region. Thus, it may be useful for potential biological applications.

     

A Dual‐Response Fluorescent Probe Reveals the H2O2‐Induced H2S Biogenesis through a Cystathionine β‐Synthase Pathway

The two signaling molecules H₂S and H₂O₂ play key roles in maintaining intracellular redox homeostasis. The biological relationship between H₂O₂ and H₂S remains largely unknown in redox biology. In this study, we rationally designed and synthesized single‐ and dual‐response fluorescent probes for detecting both H₂O₂ and H₂S in living cells. The dual‐response probe was shown to be capable of mono‐ and dual‐detection of H₂O₂ and H₂S selectively and sensitively. Detailed bioimaging studies based on the probes revealed that both exogenous and endogenous H₂O₂ could induce H₂S biogenesis in living cells. By using gene‐knockdown techniques with bioimaging, the H₂S biogenesis was found to be majorly cystathionine β‐synthase (CBS)‐dependent. Our finding shows the first direct evidence on the biological communication between H₂O₂ (ROS) and H₂S (RSS) in vivo.     

A New 3,5‐Bisporphyrinylpyridine Derivative as a Fluorescent Ratiometric Probe for Zinc Ions

A new 3,5‐disubstituted pyridine with two porphyrin moieties was prepared through an efficient synthetic approach involving 2‐formyl‐5,10,15,20‐tetraphenylporphyrin ( 1 ), piperidine, and catalytic amounts of [La(OTf)₃]. 3,5‐Bis(5,10,15,20‐tetraphenylporphyrin‐2‐ylmethyl)pyridine ( 2 ) was fully characterized and its sensing ability towards Zn²⁺, Cu²⁺, Hg²⁺, Cd²⁺, and Ag⁺ was evaluated in solution by absorption and fluorescence spectroscopy and in gas phase by using matrix‐assisted laser desorption/ionization (MALDI)‐TOF mass spectrometry. Strong changes in the ground and excited state were detected in the case of the soft metal ions Zn²⁺, Cd²⁺, Hg²⁺, and Cu²⁺. A three‐metal‐per‐ligand molar ratio was obtained in all cases and a significant ratiometric behavior was observed in the presence of Zn²⁺ with the appearance of a new band at 608 nm, which can be assigned to a metal‐to‐ligand charge transfer. The system was able to quantify 79 ppb of Zn²⁺ and the theoretical calculations are in accordance with the stoichiometry observed in solution. The gas‐phase sensorial ability of compound 2 towards all metal ions was confirmed by using MALDI‐TOF MS and in solid state by using polymeric films of polymethylmethacrylate (PMMA) doped with ligand 2 . The results showed that compound 2 can be analytically used to develop new colorimetric molecular devices that are able to discriminate between Hg²⁺ and Zn²⁺ in solid phase. The crystal structure of Zn^II complex of 3,5‐bisporphyrinylpyridine was unequivocally elucidated by using single‐crystal X‐ray diffraction studies.     

Selective Ablation of β‐Galactosidase‐Expressing Cells with a Rationally Designed Activatable Photosensitizer

We have developed an activatable photosensitizer capable of specifically inducing the death of β‐galactosidase‐expressing cells in response to photoirradiation. By using a selenium‐substituted rhodol scaffold bearing β‐galactoside as a targeting substituent, we designed and synthesized HMDESeR‐βGal, which has a non‐phototoxic spirocyclic structure owing to the presence of the galactoside moiety. However, β‐galactosidase efficiently converted HMDESeR‐βGal into phototoxic HMDESeR, which exists predominantly in the open xanthene form. This structural change resulted in drastic recovery of visible‐wavelength absorption and the ability to generate singlet oxygen (¹O₂). When HMDESeR‐βGal was applied to larval Drosophila melanogaster wing disks, which express β‐galactosidase only in the posterior region, photoirradiation induced cell death in the β‐galactosidase‐expressing region with high specificity.     

A Covalent Reporter of β‐Lactamase Activity for Fluorescent Imaging and Rapid Screening of Antibiotic‐Resistant Bacteria

Bacterial resistance to antibiotics poses a great clinical challenge in fighting serious infectious diseases due to complicated resistant mechanisms and time‐consuming testing methods. Chemical reaction‐directed covalent labeling of resistance‐associated bacterial proteins in the context of a complicated environment offers great opportunity for the in‐depth understanding of the biological basis conferring drug resistance, and for the development of effective diagnostic approaches. In the present study, three fluorogenic reagents LRBL1–3 for resistant bacteria labeling have been designed and prepared on the basis of fluorescence resonance energy transfer (FRET). The hydrolyzed probes could act as reactive electrophiles to attach the enzyme, β‐lactamase, and thus facilitated the covalent labeling of drug resistant bacterial strains. SDS electrophoresis and MALDI‐TOF mass spectrometry characterization confirmed that these probes were sensitive and specific to β‐lactamase and could therefore serve for covalent and localized fluorescence labeling of the enzyme structure. Moreover, this β‐lactamase‐induced covalent labeling provides quantitative analysis of the resistant bacterial population (down to 5 %) by high resolution flow cytometry, and allows single‐cell detection and direct observation of bacterial enzyme activity in resistant pathogenic species. This approach offers great promise for clinical investigations and microbiological research.     

A Cell‐Targeted Non‐Cytotoxic Fluorescent Nanogel Thermometer Created with an Imidazolium‐Containing Cationic Radical Initiator

A cationic fluorescent nanogel thermometer based on thermo‐responsive N‐isopropylacrylamide and environment‐sensitive benzothiadiazole was developed with a new azo compound bearing imidazolium rings as the first cationic radical initiator. This cationic fluorescent nanogel thermometer showed an excellent ability to enter live mammalian cells in a short incubation period (10 min), a high sensitivity to temperature variations in live cells (temperature resolution of 0.02–0.84 °C in the range 20–40 °C), and remarkable non‐cytotoxicity, which permitted ordinary cell proliferation and even differentiation of primary cultured cells.     

A Highly Selective Mitochondria‐Targeting Fluorescent K+ Sensor

Regulation of intracellular potassium (K⁺) concentration plays a key role in metabolic processes. So far, only a few intracellular K⁺ sensors have been developed. The highly selective fluorescent K⁺ sensor KS6 for monitoring K⁺ ion dynamics in mitochondria was produced by coupling triphenylphosphonium, borondipyrromethene (BODIPY), and triazacryptand (TAC). KS6 shows a good response to K⁺ in the range 30–500 mM , a large dynamic range (F_max/F₀≈130), high brightness (?_f=14.4 % at 150 mM of K⁺), and insensitivity to both pH in the range 5.5–9.0 and other metal ions under physiological conditions. Colocalization tests of KS6 with MitoTracker Green confirmed its predominant localization in the mitochondria of HeLa and U87MG cells. K⁺ efflux/influx in the mitochondria was observed upon stimulation with ionophores, nigericin, or ionomycin. KS6 is thus a highly selective semiquantitative K⁺ sensor suitable for the study of mitochondrial potassium flux in live cells.     

Mono‐6A‐(4‐methoxybutylamino)‐6A‐β‐cyclodextrin as a chiral selector for enantiomeric separation

A new member of the family of methoxylalkylamino monosubstituted β‐cyclodextrins, mono‐6^A‐(4‐methoxybutylamino)‐6^A‐β‐cyclodextrin, has been developed as a chiral selector for enantioseparation in capillary electrophoresis. This amino cyclodextrin exhibited good enantioselectivities for 16 model acidic racemates including three dansyl amino acids at an optimum pH of 6.0. Excellent chiral resolutions over six were obtained for α‐hydroxy acids and 2‐phenoxypropionic acids with 3.0 mM chiral selector. The good chiral recognition for α‐hydroxyl acids was attributed to inclusion complexation, electrostatic interactions, and hydrogen bonding. The hydrogen‐bonding‐enhanced chiral recognition was revealed by NMR spectroscopy. The chiral separation of acidic racemates was further improved with the addition of methanol (≤10 vol%) as an organic additive.     

A cationic β‐cyclodextrin as a dynamic coating for the separation of proteins in capillary electrophoresis

A cationic cyclodextrin was used as dynamic coating for the capillary electrophoresis of a model mixture of proteins (i.e., ubiquitin, α‐lactoglobulin, cytochrome‐c, and myoglobin) as positively charged species in a fused silica capillary. An interesting feature of the coating is that by simple adjustment of the concentration of cyclodextrin added into the background electrolyte, a neutral or positively charged surface, which was beneficial in preventing protein adsorption at the inner capillary wall surface, was obtained. This is the first demonstration of a dynamic coating that yielded a neutral surface for protein separations in capillary electrophoresis. Based on electro‐osmotic flow measurements, addition of 0.05 to 0.10 mg/mL quaternary β‐cyclodextrin in a low pH electrolyte resulted in a neutral or positive surface (undetectable to very slow anodic electro‐osmotic flow). The coating approach afforded the electrophoretic separation of the mixture of proteins at positive polarity with good repeatability and separation performance.     

Photoirradiation surface molecularly imprinted polymers for the separation of 6‐O‐α‐d‐maltosyl‐β‐cyclodextrin

Photoirradiation surface molecularly imprinted polymers for the separation of 6‐O‐α‐d ‐maltosyl‐β‐cyclodextrin were synthesized using functionalized silica as a matrix, 4‐(phenyldiazenyl)phenol as a light‐sensitive monomer, and 6‐O‐α‐d ‐maltosyl‐β‐cyclodextrin as a template. Fourier transform infrared spectroscopy results indicated that 4‐(phenyldiazenyl)phenol was grafted onto the surface of functionalized silica. The obtained imprinted polymers exhibited specific recognition toward 6‐O‐α‐d ‐maltosyl‐β‐cyclodextrin. Equilibrium binding experiments showed that the photoirradiation surface molecularly imprinted polymers obtained the maximum adsorption amount of 6‐O‐α‐d ‐maltosyl‐β‐cyclodextrin at 20.5 mg/g. In binding kinetic experiments, the adsorption reached saturation within 2 h with binding capacity of 72.8%. The experimental results showed that the adsorption capacity and selectivity of imprinted polymers were effective for the separation of 6‐O‐α‐d ‐maltosyl‐β‐cyclodextrin, indicating that imprinted polymers could be used to isolate 6‐O‐α‐d ‐maltosyl‐β‐cyclodextrin from a conversion mixture containing β‐cyclodextrin and maltose. The results showed that the imprinted polymers prepared by this method were very promising for the selective separation of 6‐O‐α‐d ‐maltosyl‐β‐cyclodextrin.     

Two‐Photon Probes for Zn2+ Ions with Various Dissociation Constants. Detection of Zn2+ Ions in Live Cells and Tissues by Two‐Photon Microscopy

A series of Zn²⁺‐selective two‐photon fluorescent probes (AZnM1−AZnN) that had a wide range of dissociation constants (K_d^TP=8 nm‐ 12 μM ) were synthesized. These probes showed appreciable water solubility (>3 μM ), cell permeability, high photostability, pH insensitivity at pH>7, significant two‐photon action cross‐sections (86–110 GM) upon complexation with Zn²⁺, and can detect the Zn²⁺ ions in HeLa cells and in living tissue slices of rat hippocampal at a depth of >80 μm without mistargeting and photobleaching problems. These probes can potentially find application in the detection of various amounts of Zn²⁺ ions in live cells and intact tissues.     

Sequestration of a β‐Hairpin for Control of α‐Synuclein Aggregation

The misfolding and aggregation of the protein α‐synuclein (α‐syn), which results in the formation of amyloid fibrils, is involved in the pathogenesis of Parkinson’s disease and other synucleinopathies. The emergence of amyloid toxicity is associated with the formation of partially folded aggregation intermediates. Here, we engineered a class of binding proteins termed β‐wrapins (β‐wrap proteins) with affinity for α‐synuclein (α‐syn). The NMR structure of an α‐syn:β‐wrapin complex reveals a β‐hairpin of α‐syn comprising the sequence region α‐syn(37–54). The β‐wrapin inhibits α‐syn aggregation and toxicity at substoichiometric concentrations, demonstrating that it interferes with the nucleation of aggregation.     

Rational Design of a Solvatochromic Fluorescent Uracil Analogue with a Dual‐Band Ratiometric Response Based on 3‐Hydroxychromone

Fluorescent nucleoside analogues with strong and informative responses to their local environment are in urgent need for DNA research. In this work, the design, synthesis and investigation of a new solvatochromic ratiometric fluorophore compiled from 3‐hydroxychromones (3HCs) and uracil fragments are reported. 3HC dyes are a class of multi‐parametric, environment‐sensitive fluorophores providing a ratiometric response due to the presence of two well‐resolved bands in their emission spectra. The synthesized conjugate demonstrates not only the preservation but also the improvement of these properties. The absorption and fluorescence spectra are shifted to longer wavelengths together with an increase of brightness. Moreover, the two fluorescence bands are better resolved and provide ratiometric responses across a broader range of solvent polarities. To understand the photophysical properties of this new fluorophore, a series of model compounds were synthesized and comparatively investigated. The obtained data indicate that uracil and 3HC fragments of this derivative are coupled into an electronic conjugated system, which on excitation attains strong charge‐transfer character. The developed fluorophore is a prospective label for nucleic acids. Abstract in Ukrainian: .     

Enantioseparation of acetyltropic acid by countercurrent chromatography with sulfobutyl ether‐β‐cyclodextrin as chiral selector

Acetyltropic acid is an important synthetic intermediate for preparation of tropane alkaloid derivatives, which can be used as anticholinergic drugs, deliriants, and stimulants. In the present work, acetyltropic acid was successfully enantioseparated by countercurrent chromatography using sulfobutyl ether‐β‐cyclodextrin as chiral selector. A biphasic solvent system composed of n‐butyl acetate/n‐hexane/0.1 mol/L citrate buffer at pH = 2.2 containing 0.1 mol/L of sulfobutyl ether‐β‐cyclodextrin (7:3:10, v/v) was selected, which produced a suitable distribution ratio D_S = 1.14, D_R = 2.31 and a high enantioseparation factor α = 2.03. Baseline separation was achieved for preparative enantioseparation of 50 mg of racemic acetyltropic acid. A method for chiral analysis of acetyltropic acid by conventional reverse phase liquid chromatography with hydroxylpropyl‐β‐cyclodextrin as mobile phase additive was established, and formation constants of inclusion complex were determined. It was found that different substituted β‐cyclodextrin should be selected for enantioseparation of acetyltropic acid by countercurrent chromatography and reverse phase liquid chromatography.     

Separation of epimeric aromatic acid (−)‐menthol esters by countercurrent chromatography using hydroxypropyl‐β‐cyclodextrin as an additive

The separation of ten epimeric aromatic acid (−)‐menthol esters by countercurrent chromatography with hydroxypropyl‐β‐cyclodextrin as the mobile phase additive was investigated, and methods for the analysis of all the epimeric esters by reversed‐phase high‐performance liquid chromatography were established. A biphasic solvent system composed of n‐hexane/20–70% methanol containing 50 mmol/L of hydroxypropyl‐β‐cyclodextrin (1:1, v/v) was selected, which provided high separation factors for five of the epimeric esters, and successful separations by countercurrent chromatography were achieved. The complete separation of five pairs of epimeric ester was obtained with the purity being over 98% for each peak fractions, as determined by high‐performance liquid chromatography. The recovery of each analyte from the eluted fractions reached around 80–88%.     

Enantioseparation of pheniramine enantiomers by high‐speed countercurrent chromatography using β‐cyclodextrin derivatives as a chiral selector

The enantioselective separation of pheniramine was studied by a high‐speed countercurrent chromatography method using β‐cyclodextrin derivatives as a chiral selector. Several key variables, for instance, type of organic solvent and chiral selector, concentration of chiral selector, pH value of aqueous phase, and temperature on the enantioselectivity, were investigated systematically by liquid–liquid extraction experiments. Combining the results of extraction experiments and high‐speed countercurrent chromatography, the most suitable conditions for separation of pheniramine enantiomers were obtained with the two‐phase system that consisted of isobutyl acetate/aqueous phase, containing 0.02 mol/L carboxymethyl‐β‐cyclodextrin, pH 8.50 at 278.15 K. Under the optimal conditions, pheniramine enantiomer was successfully resolved after four cycles of high‐speed countercurrent chromatography. By using high‐performance liquid chromatography to analyze the fractions, the purities of both (+)‐pheniramine and (–)‐pheniramine were over 99% and the recovery of this method was up to 85–90%.     

Synthesis of meso‐Pyrrole‐Substituted 22‐Oxacorroles by a “3+2” Approach

Unsymmetrical 22‐oxacorrole containing two aryl groups and one pyrrole group at the meso position was synthesized by condensing one equivalent of 16‐oxatripyrrane with one equivalent of meso aryl dipyromethane under mild acid‐catalyzed conditions followed by oxidation with 2,3‐dichloro‐5,6‐dicyano‐1,4‐benzoquinone (DDQ). This [3+2] condensation approach was expected to yield meso‐free 25‐oxasmaragdyrin but unexpectedly afforded unsymmetrical meso‐pyrrole‐substituted 22‐oxacorrole. We demonstrated the versatility of the reaction by synthesizing four new meso‐pyrrole‐substituted 22‐oxacorroles. The reactivity of α‐position of meso‐pyrrole was tested by carrying out various functionalization reactions such as bromination, formylation, and nitration and obtained the functionalized meso‐pyrrole‐substituted 22‐oxacorroles in decent yields. The X‐ray structure obtained for one of the functionalized meso‐pyrrole substituted 22‐oxacorrole revealed that the macrocycle was nearly planar and the meso‐pyrrole was in the perpendicular orientation with respect to the macrocyclic plane. The meso‐pyrrole‐substituted 22‐oxacorroles absorb strongly in 400–700 nm region with one strong Soret band and four weak Q bands. The 22‐oxacorroles are strongly fluorescent and showed emission maxima at ≈650 nm with decent quantum yields and singlet‐state lifetimes. The 22‐oxacorroles are redox‐active and exhibited three irreversible oxidations and one or two reversible reduction(s). A preliminary biological study indicated that meso‐pyrrole corroles are biocompatible.     

Fluorescent Light‐up Probe for the Detection of Protein Aggregates

A fluorescent dye was decorated with water‐soluble pyridinium groups in order to be applied in the detection of cyclodextrins or DNA. The dye displays an enhancement of its emission intensity when the internal rotations are restricted due to the formation of an inclusion complex with cyclodextrins or upon interaction with DNA. In vivo, the fluorescent probe can stain protein aggregates with a selectivity comparable to the widely used Proteostat®.     

A Triazatruxene‐Based Glycocluster as a Fluorescent Sensor for Concanavalin A

A new triazatruxene‐based fluorescent glycocluster has been designed, synthesized, and fully characterized by NMR spectroscopy and mass spectrometry. Furthermore, its specific and selective binding properties with concanavalin A (Con A) have been investigated by fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and turbidity assay. The obtained results showed that the multivalent mannose‐modified triazatruxene exhibited specific binding with Con A, but no binding to peanut agglutinin (PNA) lectin or bovine serum albumin (BSA), corresponding to a two‐orders‐of‐magnitude higher affinity than that of monovalent mannose ligands. Most interestingly, a fluorescence enhancement of the triazatruxene‐based glycocluster was observed upon binding with Con A because of hydrophobic interactions involving sites close to the triazatruxene moiety. Furthermore, the inhibitory ability of the triazatruxene‐based glycocluster against ORN178‐ induced haemagglutination has been investigated by haemagglutination inhibition assay. The results indicated selective binding with ORN178.