Reaction‐Based and Single Fluorescent Emitter Decorated Ratiometric Nanoprobe to Detect Hydrogen Peroxide

A novel reaction‐based cross‐linked polymeric nanoprobe with a self‐calibrating ratiometric fluorescence readout to selectively detect H₂O₂ is reported. The polymeric nanoprobe is fabricated by using hydrophobic H₂O₂‐reactive boronic ester groups, crosslinker units, and environmentally sensitive 3‐hydroxyflavone fluorophores through a miniemulsion polymerization. On treatment with H₂O₂, the boronic esters in the polymer are cleaved to form hydrophilic alcohols and subsequently lead to a hydrophobic–hydrophilic transition. Covalently linked 3‐hydroxyflavones manifest the change in polarity as a ratiometric transition from green to blue, accompanied by a 500‐fold increase in volume. Furthermore, this nanoprobe has been used for ratiometric sensing of glucose by monitoring the H₂O₂ generated during the oxidation of glucose by glucose oxidase, and thus successfully distinguished between normal and pathological levels of glucose.     

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: .     

A Ratiometric Fluorescent Probe for Hypochlorite Based on a Deoximation Reaction

The first ratiometric fluorescent probe for hypochlorite has been developed through regulation of the electron‐withdrawing ability of the electron acceptor in an intramolecular charge‐transfer (ICT) system by a deoximation reaction (see figure; EWG=electron‐withdrawing group).

     

A Native‐Chemical‐Ligation‐Mechanism‐Based Ratiometric Fluorescent Probe for Aminothiols

Thiol‐containing amino acids (aminothiols) such as cysteine (Cys) and homocysteine (Hcy) play a key role in various biological processes including maintaining the homeostasis of biological thiols. However, abnormal levels of aminothiols are associated with a variety of diseases. The native chemical ligation (NCL) reaction has attracted great attention in the fields of chemistry and biology. NCL of peptide segments involves cascade reactions between a peptide‐α‐thioester and an N‐terminal cysteine peptide. In this work, we employed the NCL reaction mechanism to formulate a Förster resonance energy transfer (FRET) strategy for the design of ratiometric fluorescent probes that were selective toward aminothiols. On the basis of this new strategy, the ratiometric fluorescent probe 1 for aminothiols was judiciously designed. The new probe is highly selective toward aminothiols over other thiols and exhibits a very large variation (up to 160‐fold) in its fluorescence ratio (I₄₅₈/I₆₀₃). The new fluorescent probe is capable of ratiometric detection of aminothiols in newborn calf and human serum samples and is also suitable for ratiometric fluorescent imaging of aminothiols in living cells.     

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.     

Benzothiazole‐Based Neutral Ratiometric Fluorescence Sensor for Amyloid Fibrils

Early detection of amyloid fibrils is very important for the timely diagnosis of several neurological diseases. Thioflavin‐T (ThT) is a gold standard fluorescent probe for amyloid fibrils and has been used for the last few decades. However, due to its positive charge, ThT is incapable of crossing the blood–brain barrier and cannot be used for in vivo imaging of fibrils. In the present work, we synthesized a neutral ThT derivative, 2‐[2’‐Me,4’‐(dimethylamino)phenyl]benzothiazole (2Me‐DABT), which showed a strong affinity towards the amyloid fibrils. On association with the amyloid fibrils, 2Me‐DABT not only showed a large increase in its emission intensity, but also, unlike ThT, a large blueshift in its emission spectrum was observed. Thus, unlike ThT, 2Me‐DABT is a potential candidate for the ratiometric sensor of the amyloid fibrils. Detailed photophysical properties of 2Me‐DABT in amyloid fibrils and different solvent media were studied to understand its sensory activity. Fluorescence resonance energy transfer (FRET) studies suggested that the sites of localization for ThT and 2Me‐DABT in amyloid fibrils are not same and their average distance of separation in amyloid fibrils was determined. The experimental data was nicely supported by molecular docking studies, which confirmed the binding of 2Me‐DABT in the inner core of the amyloid fibrils.     

Construction of a Near‐Infrared Fluorescent Turn‐On Probe for Selenol and Its Bioimaging Application in Living Animals

As selenocysteine (Sec) carries out the majority of the functions of the various Se‐containing species in vivo, it is of high importance to develop reliable and rapid assays with biocompatibility to detect Sec. Herein, an NIR fluorescent turn‐on probe for highly selective detection of selenol was designed and synthesized. The probe exhibits large turn‐on signal upon treatment with selenocysteine (R‐SeH), and it was further demonstrated that the new NIR fluorescent probe can be employed to image selenol in living animals.     

Construction of Fluorescent Probes Via Protection/Deprotection of Functional Groups: A Ratiometric Fluorescent Probe for Cu2+

Herein a ratiometric fluorescent Cu²⁺ probe was rationally constructed in a straightforward manner with the concept of aldehyde group protection/deprotection. The probe showed a ratiometric fluorescent response to Cu²⁺ with a large emission wavelength shift (>100 nm) and displayed high selectivity for Cu²⁺ over other metal ions due to distinct deprotection conditions. In addition, a Cu²⁺‐promoted dethioacetalization mechanism was proposed.     

Design of Ratiometric Fluorescent Probes Based on Arene–Metal‐Ion Interactions and Their Application to CdII and Hydrogen Sulfide Imaging in Living Cells

Non‐coordinative interactions between a metal ion and the aromatic ring of a fluorophore can act as a versatile sensing mechanism for the detection of metal ions with a large emission change of fluorophores. We report the design of fluorescent probes based on arene–metal‐ion interactions and their biological applications. This study found that various probes having different fluorophores and metal binding units displayed significant emission redshift upon complexation with metal ions, such as Ag^I, Cd^II, Hg^II, and Pb^II. X‐ray crystallography of the complexes confirmed that the metal ions were held in close proximity to the fluorophore to form an arene–metal‐ion interaction. Electronic structure calculations based on TDDFT offered a theoretical basis for the sensing mechanism, thus showing that metal ions electrostatically modulate the energy levels of the molecular orbitals of the fluorophore. A fluorescent probe was successfully applied to the ratiometric detection of the uptake of Cd^II ions and hydrogen sulfide (H₂S) in living cells. These results highlight the utility of interactions between arene groups and metal ions in biological analyses.     

Near‐IR Core‐Substituted Naphthalenediimide Fluorescent Chemosensors for Zinc Ions: Ligand Effects on PET and ICT Channels

Near‐IR (NIR) emission can offer distinct advantages for both in vitro and in vivo biological applications. Two NIR fluorescent turn‐on sensors N,N′‐di‐n‐butyl‐2‐(N‐{2‐[bis(pyridin‐2‐ylmethyl)amino]ethyl})‐6‐(N‐piperidinyl)naphthalene‐1,4,5,8‐tetracarboxylic acid bisimide and N,N′‐di‐ n‐butyl‐2‐[N,N,N′‐tri(pyridin‐2‐ylmethyl)amino]ethyl‐6‐(N‐piperidinyl)naphthalene‐1,4,5,8‐tetracarboxylic acid bisimide (PND and PNT) for Zn²⁺ based on naphthalenediimide fluorophore are reported. Our strategy was to choose core‐substituted naphthalenediimide (NDI) as a novel NIR fluorophore and N,N‐di(pyridin‐2‐ylmethyl)ethane‐1,2‐diamine (DPEA) or N,N,N′‐tri(pyridin‐2‐ylmethyl)ethane‐1,2‐diamine (TPEA) as the receptor, respectively, so as to improve the selectivity to Zn²⁺. In the case of PND, the negligible shift in absorption and emission spectra is strongly suggestive that the secondary nitrogen atom (directly connected to the NDI moiety, N¹) is little disturbed with Zn²⁺. The fluorescence enhancement of PND with Zn²⁺ titration is dominated with a typical photoinduced electron‐transfer (PET) process. In contrast, the N¹ atom for PNT can participate in the coordination of Zn²⁺ ion, diminishing the electron delocalization of the NDI moiety and resulting in intramolecular charge‐transfer (ICT) disturbance. For PNT, the distinct blueshift in both absorbance and fluorescence is indicative of a combination of PET and ICT processes, which unexpectedly decreases the sensitivity to Zn²⁺. Due to the differential binding mode caused by the ligand effect, PND shows excellent selectivity to Zn²⁺ over other metal ions, with a larger fluorescent enhancement centered at 650 nm. Also both PND and PNT were successfully used to image intracellular Zn²⁺ ions in the living KB cells.     

Phosphono Bisbenzguanidines as Irreversible Dipeptidomimetic Inhibitors and Activity‐Based Probes of Matriptase‐2

Matriptase‐2, a type II transmembrane serine protease, plays a key role in human iron homeostasis. Inhibition of matriptase‐2 is considered as an attractive strategy for the treatment of iron‐overload diseases, such as hemochromatosis and β‐thalassemia. In the present study, synthetic routes to nine dipeptidomimetic inactivators were developed. Five active compounds ( 41 – 45 ) were identified and characterized kinetically as irreversible inhibitors of matriptase‐2. In addition to a phosphonate warhead, these dipeptides possess two benzguanidine moieties as arginine mimetics to provide affinity for matriptase‐2 by binding to the S1 and S3/S4 subpockets, respectively. This binding mode was strongly supported by covalent docking analysis. Compounds 41 – 45 were obtained as mixtures of two diastereomers and were therefore separated into the single epimers. Compound 45 A , with S configuration at the N‐terminal amino acid and R configuration at the phosphonate carbon atom, was the most potent matriptase‐2 inactivator with a rate constant of inactivation of 2790 m ^?1 s^?1 and abolished the activity of membrane‐bound matriptase‐2 on the surface of intact cells. Based on the chemotyp of phosphono bisbenzguanidines, the design and synthesis of a fluorescent probe ( 51 A ) by insertion of a coumarin label is described. The in‐gel fluorescence detection of matriptase‐2 was demonstrated by applying 51 A as the first activity‐based probe for this enzyme.     

Asymmetric Rhodamine‐Based Fluorescent Probe for Multicolour In Vivo Imaging

To achieve rapid and sensitive detection of cancer, activatable fluorescent probes targeting proteases that are overexpressed in various types of cancer have been developed, based on the hydroxymethyl rhodamine green (HMRG) scaffold. However, to visualize altered activities of multiple enzymes in cancer sites, other scaffolds with distinct fluorescence properties from those of HMRG are needed. A novel asymmetrically modified rhodamine with suitable absorption/emission, brightness and equilibrium constant of intramolecular spirocyclization, working in the yellow/orange region, is introduced. As a proof of concept, a probe targeting γ‐glutamyl transpeptidase (gGlu‐HMJCR) was developed on the basis of the new scaffold. Simultaneous visualization and discrimination of tumours expressing γ‐glutamyl transpeptidase (with gGlu‐HMJCR) and cathepsins (with Z‐Phe‐Arg‐HMRG) by colour were achieved in a mouse model in vivo.     

Fluorescent Detection of Hypochlorous Acid from Turn‐On to FRET‐Based Ratiometry by a HOCl‐Mediated Cyclization Reaction

Hypochlorous acid (HOCl), a reactive oxygen species (ROS), plays a significant biological role in living systems. However, abnormal levels of HOCl are implicated in many inflammation‐associated diseases. Therefore, the detection of HOCl is of great importance. In this work, we describe the HOCl‐promoted cyclization of rhodamine‐thiosemicarbazides to rhodamine‐oxadiazoles, which is then exploited as a novel design strategy for the development of a new fluorescence turn‐on HOCl probe 2 . On the basis of the fluorescence resonance energy transfer (FRET) signaling mechanism, 2 was further converted into 1 a and 1 b , which represent the first paradigm of FRET‐based ratiometric fluorescent HOCl probes. The outstanding features of 1 a and 1 b include well‐resolved emission peaks, high sensitivity, high selectivity, good functionality at physiological pH, rapid response, low cytotoxicity, and good cell‐membrane permeability. Furthermore, these excellent attributes enable us to demonstrate, for the first time, the ratiometric imaging of endogenously produced HOCl in living cells by using these novel ratiometric probes. We expect that 1 a and 1 b will be useful molecular tools for studies of HOCl biology. In addition, the HOCl‐promoted cyclization reaction of rhodamine‐thiosemicarbazides to rhodamine‐oxadiazoles should be widely applicable for the development of different types of fluorescent HOCl probes.     

Water‐Soluble Polymer Functionalized CdTe/ZnS Quantum Dots: A Facile Ratiometric Fluorescent Probe for Sensitive and Selective Detection of Nitroaromatic Explosives

A ratiometric fluorescent probe based on dual luminescence QD/CPL for selective sensing of the nitroaromatic explosive picric acid (PA) was constructed. The observed ratiometric fluorescence intensity change allows the quantitative detection of PA with a detection limit of 9 nM .     

Development of an AND Logic‐Gate‐Type Fluorescent Probe for Ratiometric Imaging of Autolysosome in Cell Autophagy

The concomitant detection of two biological events facilitates the highly selective and sensitive analysis of specific biological functions. In this article, we report an AND logic‐gate‐type fluorescent probe that can concurrently sense two biological events in living cells: H₂O₂ accumulation and acidification. The probe exhibits a unique fluorescence sensing mechanism, in which a xanthene fluorophore is oxidatively transformed to a xanthone derivative by H₂O₂, thereby resulting in a clear dual‐emission change. This transformation is significantly accelerated under weak acidic conditions, which enables the selective and sensitive detection of H₂O₂ production in an acidic cellular compartment. This unique sensing property was successfully applied to the ratiometric fluorescence imaging of autolysosome formation in selective mitochondrial autophagy (mitophagy), which highlights the utility of this novel probe in autophagy research.     

pH‐Dependent Optical Properties of Synthetic Fluorescent Imidazoles

New pH‐sensitive probe design : A peek into the structure of fluorescent proteins led to the synthesis of fluorescent imidazoles. The prepared compounds demonstrated an array of remarkable pH‐dependent optical properties including at least two types of excited‐state charge transfers (see picture).