Modular and stereoselective synthesis of tetrasubstituted vinyl sulfides leading to a library of AIEgens.

Tetraarylethylenes exhibit intriguing photophysical properties and sulfur atom frequently play a vital role in organic photoelectric materials and biologically active compounds. Tetrasubstituted vinyl sulfides, which include both sulfur atom and tetrasubstituted alkenes motifs, might be a suitable skeleton for the discovery of the new material molecules and drug with unique functions and properties. However, how to modular synthesis these kinds of compounds is still challenging. Herein, a chemo- and stereo-selective Rh(II)-catalyzed [1,4]-acyl rearrangements of α-diazo carbonyl compounds and thioesters has been developed, providing a modular strategy to a library of 63 tetrasubstituted vinyl sulfides. In this transformation, the yield is up to 95% and the turnover number is up to 3650. The mechanism of this reaction is investigated by combining experiments and density functional theory calculation. Moreover, the "aggregation-induced emission" effect of tetrasubstituted vinyl sulfides were also investigated, which might useful in functional material, biological imaging and chemicalnsing via structural modification.

A Toxic RNA Templates the Synthesis of Its Own Fluorogenic Inhibitor by Using a Bio-orthogonal Tetrazine Ligation in Cells and Tissues.

Expanded RNA repeats cause more than 30 incurable diseases. One approach to mitigate their toxicity is by using small molecules that assemble into potent, oligomeric species upon binding to the disease-causing RNA in cells. Herein, we show that the expanded repeat [r(CUG)exp] that causes myotonic dystrophy type 1 (DM1) catalyzes the in situ synthesis of its own inhibitor using an RNA-templated tetrazine ligation in DM1 patient-derived cells. The compound synthesized on-site improved DM1-associated defects at picomolar concentrations, enhancing potency by 10 000-fold, compared to its parent compounds that cannot undergo oligomerization. A fluorogenic reaction is also described where r(CUG)exp templates the synthesis of its own imaging probe to enable visualization of the repeat in its native context in live cells and muscle tissue.

HaloTag-Based Hybrid Targetable and Ratiometric Sensors for Intracellular Zinc.

We report a new series of small molecule-protein hybrid zinc sensors that combine genetic targetability with the spectroscopic profile of synthetic fluorophores. We functionalized the zinc sensor ZinPyr-1 (ZP1) with a chloroalkane linker (ZP1-12Cl) that reacts specifically with the engineered protein HaloTag. The resulting construct, ZP1-HaloTag, binds zinc ions with a threefold fluorescence enhancement. Through exploitation of the protein synthesis machinery of live cells, the HaloTag protein component was expressed, and the ZP1-HaloTag hybrid was assembled upon bath application of ZP1-12Cl. After fusion of HaloTag with targeting peptides or proteins, the resulting hybrid sensor could be directed to specific subcellular locales, including the nucleus, mitochondrial outer membrane, and endoplasmic reticulum. Furthermore, HaloTag was linked with the red fluorescent protein mCherry, permitting formation of a two-fluorophore system that provides not only targetable but also ratiometric sensing of cellular zinc. This system reversibly detects both exogenous and endogenous mobile Zn2+ in response to reactive nitrogen species in live HeLa cells. HaloTag-based hybrid zinc sensors offer new opportunities for visualizing and quantifying biological mobile zinc at discrete subcellular compartments.

A multiple fluorescein-based turn-on fluorophore (FHCS) identified for simultaneous determination and living imaging of toxic Al3+ and Zn2+ by improved Stokes shift.

A multiple turn-on fluorophore (FHCS), combining fluorescein, hydrazone, cyanuric chloride and salicylaldehyde chromone into a molecule, was identified and developed based on density functional theoretical calculation. It was expected that FHCS could express exclusive fluorescent signals and improved Stokes shifts when chelating Al3+ or Zn2+. After it was synthesized and characterized in detail, it was noted that FHCS could turn-on fluorescently discriminate trace Al3+ and Zn2+ under the optimized conditions, i.e., from no-fluorescence to strong blue fluorescence for Al3+ and to green fluorescence for Zn2+ with low detection limits of 5.37 × 10-8 M and 7.90 × 10-8 M respectively. Owing to its low toxicity, FHCS was successfully applied for quantitative determination of Al3+ and Zn2+ in natural aqueous samples and toxicity evaluation of Al3+ and Zn2+ in living cells and bio-tissues with excellent linear relationships. The action mechanisms for FHCS with Al3+ and Zn2+ were confirmed to form stable 5-member-co-6-member condensed rings between Al3+/Zn2+ and N/O atoms in FHCS by both theoretic and experimental methods, which resulted in turn-on fluorescence with different dipolar moments and improved Stokes shifts.

F-containing initiatior for ultrasensitive fluorescent detection of lung cancer DNA via atom transfer radical polymerization.

An ultrasensitive fluorescence method for early diagnosis of lung cancer via Nafion-initiated atom transfer radical polymerization (ATRP) is reported, in this paper. In the proposed method, thiolated peptide nucleic acid (PNA) is modified to amino magnetic beads (MBs) via a cross-linking agent to specifically capture target DNA (tDNA), and the initiator (Nafion) of ATRP is attached to PNA/DNA heteroduplexes based on the phosphate groups of the tDNA and sulfonate groups of Nafion via phosphate-Zr4+-sulfonate chemistry. Nafion as a macroinitiator of ATRP possesses multiple C-F active sites to initiate polymerization, and numerous polymeric chains that significantly amplify the fluorescent signal are formed. Under optimal conditions, a good linear relationship is obtained in the range of 0.1 nM-0.1 fM with correlation coefficients of 0.9975, and the detection limit is as low as 35.5 aM (∼214 molecules). The proposed strategy has several advantages of simplicity, cost-effectiveness, selectivity and sensitivity. More importantly, the anti-interference results demonstrate that the proposed Nafion-initiated ATRP strategy has great potential in bioanalytical applications.

Evaluation of linker length effects on a BET bromodomain probe.

Fueled by the therapeutic potential of the epigenetic machinery, BET bromodomains have seen high interest as drug targets. Herein, we introduce different linkers to a BET bromodomain benzodiazepine ligand (I-BET762) to gauge its implications in the development of hybrid drugs, imaging probes and small molecule drug conjugates. Biophysical studies confirmed minimal disruption to binding of the BRD4 cavity by the synthesized entities, which includes imaging probes. Target engagement was confirmed in a cellular context, but poor membrane diffusion was found despite efficient localization in the nuclei after membrane disruption. Our study highlights challenges and opportunities for the successful design of benzodiazepine-derived drug-delivery systems.

Highly selective and wash-free visualization of resistant bacteria with a relebactam-derived fluorogenic probe.

Reported herein is a relebactam-derived fluorogenic reagent for covalent labeling of serine ß-lactamases (SBLs), which are the major causes of bacterial resistance to ß-lactam antibiotics. This highly selective imaging reagent generates over 300-fold stronger near-infrared fluorescence signals upon covalently bonding to SBLs, allowing wash-free visualization of live antimicrobial-resistant bacteria.

An in cellulo-activated multicolor cell labeling approach used to image dying cell clearance.

Dying cell clearance is critical for myriad biological processes such as tissue homeostasis. We herein report an enzyme-activated fluorescence cell labeling approach and its use for multicolor imaging of dying cell clearance. Diacetylated 4-hydroxymandelic acid (DHA)-conjugated dyes give rise to reactive quinone methides upon deacetylation in live cells, which in turn covalently labels cellular proteins. With partner cells tagged with distinct fluorescence, apoptotic cell clearance by Raw 264.7 macrophages and epithelial HeLa cells was captured by confocal microscopy, showing the potential of DHA-based cell labeling for investigating cell-cell interactions.

A near-infrared biothiol-specific fluorescent probe for cancer cell recognition.

Cancer is a global health issue and a leading cause of death. The discrimination of cancer cells from normal cells is of significant importance for the early diagnosis of cancers. As one of the useful biomarkers for developing cancer diagnosis and chemotherapy resistance systems, biothiols not only play an essential role in physiological and pathological processes but also exhibit cytoprotective effects in the susceptibility to carcinogenesis. It would be highly desirable to explore near-infrared biothiol-specific fluorescent probes for cancer diagnosis with outstanding specificity. In this study, a novel near-infrared fluorescent probe BPO-THAZ decorated with thiazole as a recognition site was presented for sensitive and selective detection of endogenous biothiols. BPO-THAZ can be used to not only evaluate the biothiol level in living HeLa cells upon treatment with H2O2 or anti-cancer drugs but also assess endogenous biothiols in stem cells. Furthermore, BPO-THAZ was successfully utilized to discriminate cancer cells from normal cells showing great promise for cancer diagnosis.

A dual-responsive probe for detecting cellular hypoxia using 19F magnetic resonance and fluorescence.

We report the first dual-responsive 19F MRI and fluorescence imaging probe for cellular hypoxia. The Cu2+-based probe exhibits no 19F MR signal and reduced fluorescence signal due to paramagnetic quenching; however, the probe turns-on in both modes following reduction to Cu+. This bimodal agent can differentiate hypoxic and normoxic cells in both modalities.

Synthesis of Sulfonated Carbofluoresceins for Voltage Imaging.

We present the design, synthesis, and applications of a new class of voltage-sensitive fluorescent indicators built on a modified carbofluorescein scaffold. Carbofluoresceins are an attractive target for responsive probes because they maintain oxygen substitution patterns at the 3' and 6' positions, similar to fluorescein, while simultaneously possessing excitation and emission profiles red-shifted nearly 50 nm compared to fluorescein. However, the high p Ka of carbofluorescein dyes, coupled with their tendency to cyclize to nonfluorescent configurations, precludes their use in voltage-imaging applications. Here, we overcome the limitations of carbofluoresceins via chlorination to lower the p Ka by 2 units to 5.2 and sulfonation to prevent cyclization to the nonabsorbing form. To achieve this, we devise a synthetic route to halogenated sulfonated carbofluoresceins from readily available, inexpensive starting materials. New, chlorinated sulfone carbofluoresceins have low p Ka values (5.2) and can be incorporated into phenylenevinylene molecular wire scaffolds to create carboVoltage-sensitive fluorophores (carboVF dyes). The best of the new carboVF dyes, carboVF2.1(OMe).Cl, possesses excitation and emission profiles of >560 nm, displays high voltage sensitivity (>30% Δ F/ F per 100 mV), and can be used in the presence of other blue-excited fluorophores such as green fluorescent protein. Because carboVF2.1(OMe).Cl contains a phenolic oxygen, it can be incorporated into fluorogenic labeling strategies. Alkylation with a sterically bulky cyclopropylmethyl-derived acetoxymethyl ether renders carboVF weakly fluorescent; we show that fluorescence can be restored by the action of porcine liver esterase both in vitro and on the surface of living cells and neurons. Together, these results suggest chlorinated sulfone carbofluoresceins can be promising candidates for hybrid chemical-genetic voltage imaging at wavelengths beyond typical fluorescein excitation and emission.

Excellent Temperature-Control Based on Reversible Thermochromic Materials for Light-Driven Phase Change Materials System.

Light-driven phase change materials (PCMs) have received significant attention due to their capacity to convert visible light into thermal energy, storing it as latent heat. However, continuous photo-thermal conversion can cause the PCMs to reach high thermal equilibrium temperatures after phase transition. In our study, a novel light-driven phase change material system with temperature-control properties was constructed using a thermochromic compound. Thermochromic phase change materials (TC-PCMs) were prepared by introducing 2-anilino-6-dibutylamino-3-methylfluoran (ODB-2) and bisphenol A (BPA) into 1-hexadecanol (1-HD) in various proportions. Photo-thermal conversion performance was investigated with solar radiation (low power of 0.09 W/cm2) and a xenon lamp (at a high power of 0.14 W/cm2). The TC-PCMs showed a low equilibrium temperature due to variations in absorbance. Specifically, the temperature of TC-PCM180 (ODB-2, bisphenol A and 1-HD ratio 1:2:180) could stabilize at 54 °C approximately. TC-PCMs exhibited reversibility and repeatability after 20 irradiation and cooling cycles.

Fluorogenic approach to evaluating prodrug hydrolysis and stability in live cells.

Fluorescein diester which is conjugated with cell membrane permeable Arg9 peptide was proposed as probe for ester prodrug stability and drug release study in living cells. α-Amino protected d-Val and l-Ala which bear differently hindered side chains were used to afford model diesters of 5-maleimide-fluorescein. Such fluorescein diesters were further conjugated with a Cys containing cell membrane permeable Arg9 peptide via thiol-ene crosslink reaction. The resulted conjugates of fluorescein diester and Arg9 peptide were purified with HPLC and characterized with MALDI-TOF MS. Upon incubation with cultured cells, the fluorescein diesters were delivered into the cells, the following hydrolysis of fluorescein diesters and release of fluorescein inside living cells were observed by monitoring the fluorescence accumulation. Fluorescence microscopic imaging studies of HeLa cells treated with fluorescein l-Ala diester show strong fluorescence accumulation in 30 min indicating fast hydrolysis of fluorescein diester and fluorescein release; in contrast d-Val diester remains stable inside cells evidenced by margin fluorescence formation. Further flowcytometry studies on the fluorescein diester-Arg9 conjugate treated cells show that the hydrolysis t1/2 for l-Ala diester is 15 min. The results also show that Arg9 peptide not only transports the ester probes into cell efficiently but also can retain and concentrate hydrolytic product fluorescein inside cells so that the accumulated fluorescence can be accurately quantified. This fluorogenic probe approach provides feasible applications in dynamic studies on ester prodrug hydrolysis and release, facilitating screening and optimization of prodrug structures in living cell settings.

Investigation of the Assembly Behavior of an Amphiphilic Lipopeptide at the Liquid Crystal-Aqueous Interface.

In this article, we designed an amphiphilic lipopeptide molecule, 5(6)-carboxyfluorescein-KKKKKKSKTK-Cys(C12H25)-OMe (FAM-lipopeptide-C12), and studied its assembly behavior at the 4-cyano-4'-pentylbiphenyl (5CB)-aqueous interface. The ordering transitions of liquid crystals (LCs) revealed that FAM-lipopeptide-C12 can assemble at the LC-aqueous interface (both planar and curved interfaces). The assembly can be destroyed by adding trypsin, which catalyzes the hydrolysis of lipopeptides. Fluorescence measurements further confirmed the assembly and deassembly behavior of FAM-lipopeptide-C12 at the LC-aqueous interface. Overall, our work provides a general method for the construction of a biointerface by directly assembling amphiphilic lipopeptides at the LC-aqueous interface, which can potentially be used in selectively detecting the activity of specific enzymes and other biomolecular interactions.

A FRET-based ratiometric two-photon fluorescent probe for superoxide anion detection and imaging in living cells and tissues.

The superoxide anion (O2˙-) plays a crucial role in several physiological processes and many human diseases. Developing new methods for O2˙- detection in biological systems is very important. A FRET-based two-photon (TP) fluorescent probe with a ratiometric signal, TFR-O, was developed. A naphthalene derivative based TP fluorescent group was selected as the energy donor group, and a rhodol fluorescent group was chosen as the energy acceptor; the trifluoromethanesulfonate group was chosen as the recognition moiety. After reacting with O2˙-, the recognition moiety was removed and the fluorophore was released, leading to a fluorescence intensity decrease at the wavelength of 425 nm and a significant enhancement of the fluorescence intensity at 550 nm. The fluorescence intensity ratio between 550 and 425 nm (I550/I425) varied from 0.15 to 6.72, with the O2˙- concentration increasing from 0 to 50 µM. The detection limit of the TFR-O was 83 nM. Moreover, TFR-O was applied for detecting and imaging O2˙- in cells and liver tissues.

Spying on Neuronal Membrane Potential with Genetically Targetable Voltage Indicators.

Methods for optical measurement of voltage dynamics in living cells are attractive because they provide spatial resolution surpassing traditional electrode-based measurements and temporal resolution exceeding that of widely used Ca2+ imaging. Chemically synthesized voltage-sensitive dyes that use photoinduced electron transfer as a voltage-sensing trigger offer high voltage sensitivity and fast-response kinetics, but targeting chemical indicators to specific cells remains an outstanding challenge. Here, we present a new family of readily functionalizable, fluorescein-based voltage-sensitive fluorescent dyes (sarcosine-VoltageFluors) that can be covalently attached to a genetically encoded cell surface receptor to achieve voltage imaging from genetically defined neurons. We synthesized four new VoltageFluor derivatives that possess carboxylic acid functionality for simple conjugation to flexible tethers. The best of this new group of dyes was conjugated via a polyethylene glycol (PEG) linker to a small peptide (SpyTag, 13 amino acids) that directs binding and formation of a covalent bond with its binding partner, SpyCatcher (15 kDa). The new VoltageSpy dyes effectively label cells expressing cell-surface SpyCatcher, display good voltage sensitivity, and maintain fast-response kinetics. In cultured neurons, VoltageSpy dyes enable robust, single-trial optical detection of action potentials at neuronal soma with sensitivity exceeding genetically encoded voltage indicators. Importantly, genetic targeting of chemically synthesized dyes enables VoltageSpy to report on action potentials in axons and dendrites in single trials, tens to hundreds of micrometers away from the cell body. Genetic targeting of synthetic voltage indicators with VoltageSpy enables voltage imaging with low nanomolar dye concentration and offers a promising method for allying the speed and sensitivity of synthetic indicators with the enhanced cellular resolution of genetically encoded probes.

ß-cyclodextrin based dual-responsive multifunctional nanotheranostics for cancer cell targeting and dual drug delivery.

Multifunctional nanoconjugates possessing an assortment of key functionalities such as magnetism, florescence, cell-targeting, pH and thermo-responsive features were developed for dual drug delivery. The novelty lies in careful conjugation of each of the functionality with magnetic Fe3O4 nanoparticles by virtue of urethane linkages instead of silica in a simple one pot synthesis. Further ß-cyclodextrin (CD) was utilized to carry hydrophobic as well as hydrophilic drug. Superlative release of DOX could be obtained under acidic pH conditions and elevated temperature, which coincides with the tumor microenvironment. Mathematical modelling studies revealed that the drug release kinetics followed diffusion mechanism for both hydrophobic drug and hydrophilic drug. A number of fluorophores onto a single nanoparticle produced a strong fluorescence signal to optically track the nanoconjugates. Enhanced internalization due to folate specificity could be observed by fluorescence imaging. Further their accumulation driven by magnet near tumor site led to magnetic hyperthermia. in vitro studies confirmed the nontoxicity and hemocompatibility of the nanoconjugates. Remarkable cell death was observed with drug-loaded nanoconjugates at very low concentrations in cancer cells. The internalization and cellular uptake of poor bioavailable anticancer agent curcumin were found to be remarkably enhanced on dosing the drug loaded nanoconjugates as compared to free curcumin. Site specific drug delivery due to folate conjugation and subsequent significant suppression in tumor growth was demonstrated by in vivo studies.

Ratiometric DNA sensing with a host-guest FRET pair.

A supramolecular host-guest FRET pair based on a carboxyfluorescein-labelled cucurbit[7]uril (CB7-CF, as acceptor) and the fluorescent dye 4',6-diamidino-2-phenylindole (DAPI, as donor) is developed for sensing of DNA. In comparison to the commercial DNA staining dye SYBR Green I, the new chemosensing ensemble offers dual-emission signals, which allows a linear ratiometric response over a wide concentration range.

Fluorescent Benzothiazinone Analogues Efficiently and Selectively Label Dpre1 in Mycobacteria and Actinobacteria.

Benzothiazinones (BTZ) are highly potent bactericidal inhibitors of mycobacteria and the lead compound, BTZ043, and the optimized drug candidate, PBTZ169, have potential for the treatment of tuberculosis. Here, we exploited the tractability of the BTZ scaffold by attaching a range of fluorophores to the 2-substituent of the BTZ ring via short linkers. We show by means of fluorescence imaging that the most advanced derivative, JN108, is capable of efficiently labeling its target, the essential flavoenzyme DprE1, both in cell-free extracts and after purification as well as in growing cells of different actinobacterial species. DprE1 displays a polar localization in Mycobacterium tuberculosis, M. marinum, M. smegmatis, and Nocardia farcinica but not in Corynebacterium glutamicum. Finally, mutation of the cysteine residue in DprE1 in these species, to which BTZ covalently binds, abolishes completely the interaction with JN108, thereby highlighting the specificity of this fluorescent probe.

Novel π-extended hybrid xanthene dyes with two spirolactone rings for optoelectronic and biological applications.

The design, synthesis, and photophysical properties of organic fluorophores have attracted considerable research interest due to the utility of these compounds for various optoelectronic, analytical, and biological applications. In this study, we synthesized two novel π-extended red-emitting hybrid xanthene dyes, each of which has two spirolactone rings and combines a seminaphthofluorescein moiety and a seminaphthorhodafluor moiety in a single molecule. The photophysical properties of the dyes in methanol in the presence of acid, base, and metal cations were investigated. Mono-ring-opened seminaphthofluorescein and seminaphthorhodafluor forms of the dyes could be obtained by the addition of OH- or H+, respectively. Owing to the changes in the absorbance spectra of the mono-ring-opened forms induced by addition of H+ and OH-, the dyes could perform simultaneously the functions of an XOR gate and an INHIBIT gate, with the absorbances at 510 and 560 nm as outputs, respectively, and could act as half-subtractors with H+ and OH- as inputs. Furthermore, stepwise ring-opening could be induced by Hg2+ ions in methanol. In water, the dyes existed in double-ring-opened forms that emitted deep-red fluorescence and were mitochondria-targetable, suggesting that these chromophores might be useful as fluorescence tracers in biological applications. Because the absorption and fluorescence properties of these fluorophores can be regulated via their two spirolactone rings, we expect that these compounds will find utility in various optoelectronic, analytical, and biological applications.