Nitrobenzoxadiazole Research Articles

Discovery of EGFR-Targeted Environment-Sensitive fluorescent probes for cell imaging and efficient tumor detection

Overexpression of human epidermal growth factor receptor (EGFR) plays an important role in several signaling pathways inside and outside the cell, especially in the processes of cell proliferation, differentiation, and death in various cancers. Due to the complexity of the structure and function of EGFR, research on the fluorescence visualization of EGFR protein visualization has proved challenging. One possible strategy for designing a receptor-targeting fluorescent probe with a switching mechanism is to introduce an environment-sensitive fluorophore into the drug ligand. Based on this strategic molecular design, we introduced two environment-sensitive small molecular fluorophores, dansyl chloride (DNS) and nitrobenzoxadiazole (NBD), to replace the morpholine group of gefitinib, achieving a series of fluorescent molecular probes bearing a switching mechanism. The GN probes exhibited prominent environment sensitivity, suggesting good performance as turn-on EGFR-targeting fluorescent ligands. The representative probe GN3 specifically responded to tumor cells overexpressing EGFR, which was validated with live-cell fluorescence imaging and in vivo xenograft tumor imaging. Ligand-induced EGFR phosphorylation in A431 cells was considerably inhibited by probe GN3, demonstrating that this probe still functions as an EGFR inhibitor. Owing to the turn-on response of GN3 to EGFR in tumor cells, and the competitive replacement behavior to the EGFR inhibitor gefitinib, these probes have the potential to be used for fluorescence imaging of cells overexpressing EGFR.

Cytotoxic and other bioactivities of a novel and known sesquiterpene lactones isolated from Vernonia leopoldi (Sch. Bip. ex Walp.) Vatke in breast cancer cell lines

Vernonia leopoldi (Sch. Bip. ex Walp.) Vatke (Asteraceae) is one of the widely used anti-cancer traditional medicinal plants in Ethiopia, despite the lack of data to support its therapeutic efficacy. Here we describe the isolation of compounds from the plant and the investigation of their cytotoxicity and other bioactivities. We identified the novel sesquiterpene lactone (SL) 11ß,13-dihydrovernodalol along with the three other SLs (vernomenin, vernolepin, and 11ß,13-dihydrovernodalin) and three flavonoids (apigenin, eriodyctiol, and luteolin) isolated from this plant for the first time. The structures of all the compounds were established based on extensive analysis of nuclear magnetic resonance spectroscopic data and confirmed by high-resolution electrospray ionization mass spectrometry. We then studied the biological activities of the SLs and found that all were cytotoxic at low μM ranges against MCF-7 and JIMT-1 breast cancer cells as well as against the normal-like MCF-10A breast epithelial cells evaluated in a spectrophotometric assay. All the SLs significantly reduced JIMT-1 cell migration after 72 h of treatment with 2 μM concentrations in a wound healing assay. Treatment with all SLs reduced the aldehyde dehydrogenase expressing cancer stem cell sub-population of the JIMT-1 cells significantly, evaluated by flow cytometry. Only 11ß,13-dihydrovernodalin resulted in a significant inhibition of tumor necrosis factor-α-induced translocation of nuclear factor κB to the cell nucleus. In addition, we show that the reporter fluorophore nitrobenzoxadiazole (NBD) can successfully be conjugated with an SL and that this SL-NBD conjugate is taken up efficiently in JIMT-1 cells. Therefore, the overall bioactivities of the SL compounds and specifically their effects against the stemness of breast cancer cells make them prime candidates for further in-depth investigation.

Abstract P112: A Simple, Rapid, And Sensitive Fluorescence-based Method To Assess Triacylglycerol Hydrolase Activities

Introduction: Lipases constitute an important class of water-soluble enzymes that catalyze the hydrolysis of hydrophobic triacylglycerol (TAG). Their activity is usually measured after isolation and quantification of the hydrolyzed free fatty acids. We have used NBD-labeled lipids previously to develop TAG, cholesteryl ester, and phospholipid transfer assays for microsomal triglyceride transfer protein. To date, however, NBD-labeled TAG (NBD-TAG) has not been used to assess the enzymatic activities of different lipases. Methods: We developed methods to measure lipase activities using adipose triglyceride lipase (ATGL), lipoprotein lipase (LpL), and pancreatic triglyceride lipase (PNLIP) as model lipases. In these assays, we incubated a source of ATGL, LpL, or PNLIP with substrate emulsions containing nitrobenzoxadiazole (NBD)-labeled TAG in phosphatidylcholine (PC) and phosphatidylinositol (PI), measured increases in NBD fluorescence with time, and calculated enzyme activities. Results: Incorporation of NBD-TAG into PC vesicles resulted in some hydrolysis; but required product isolation step to measure NBD fluorescence. However, incorporation of PI into these NBD-TAG/PC vesicles significantly increased substrate hydrolysis in time, protein, and substrate concentration dependent manner negating the need to isolate products to measure lipase activities. Further, increasing the ratio of NBD-TAG to PC enhanced substrate hydrolysis. Next, we tested specific lipase inhibitors and found that orlistat inhibits all three enzymes indicating that it is a pan-lipase inhibitor. Conclusions: We describe a simple, rapid fluorescence-based TAG hydrolysis assay to assess three major TAG hydrolases: ATGL localized intracellularly, LpL localized at the extracellular endothelium, and PNLIP produced and secreted from the pancreas into the intestinal lumen. The major advantages of this method are its speed, simplicity, and avoidance of a product isolation step. This assay is potentially applicable to a wide range of lipases and is amenable to high-throughput screening to discover novel modulators of triacylglycerol hydrolases, and in the diagnosis of diseases associated with increases in plasma lipase activities.

Dual-emission ratiometric fluorescent sensor based molecularly imprinted nanoparticles for visual detection of okadaic acid in seawater and sediment

A dual-emitting ratiometric fluorescence nanosensor based on novel molecularly imprinted polymers (MIPs), which possesses self-referencing ability by nitrobenzoxadiazole (NBD) as recognition signal and new carbon dots (CDs) as interior label, has been prepared for sensitive and visual detection of okadaic acid (OA) in seawater and sediment samples. The analyte can rapidly enhance the fluorescence intensity of NBD at 549 nm, but will not change the CDs’ fluorescence signal at 667 nm. The nanosensor has a linear fluorescence enhancement response in the range of 100∼1770 ng L−1 with a limit of detection of 25 ng L−1, and excellent selective recognition for OA over its interferences. The ratiometric fluorescence nanosensor has been successfully applied to detect OA in seawater and sediment samples. The results show that the nanosensor has high recoveries at four spiking levels of OA ranged from 90.0 % to 107.0 % with the relative standard deviation less than 4.1 % (n = 3). More importantly, a reliable visual sense change with two distinct color tonality from bright pink to yellow green can be easily differentiated by the naked eyes during fluorescence analysis, which demonstrates promising potential for the applications of molecularly imprinted sensors in trace analysis of marine environmental matrix.

Enantiomer Separation of Chiral Amines and Amino Acid Esters on Polysaccharide Phenylcarbamates Derived Chiral Stationary Phases

We have developed an efficient chiral HPLC method for enantiomeric resolution of some chiral amines and α- amino acid esters as nitrobenzoxadiazole (NBD) derivatives on amylose and cellulose phenylcarbamates derived chiral stationary phases (CSPs) under simultaneous ultraviolet (UV) and fluorescence (FL) detection. The observed enantioselectivities were affected by the type of the analyte (amines or α-amino acid esters), backbone structure (cellulose or amylose) of the CSP used, and substituents of phenyl moiety on the chiral selector of CSP. Further, for same chiral selectors, coated type CSPs showed better enantiomeric separation than those observed for the covalently bonded type CSPs. In general, for the enantiomeric resolution of chiral amines, Chiralpak IE and Chiralcel OD-H columns showed the highest enantioselectivities. However, for resolution of α-amino acid esters, the performances of Chiralpak IA and Chiralpak AD-H (or Lux Amylose-1) columns were superior to those of other CSPs used. In general, the degrees of enantioselectivity for α-amino acid ethyl esters are greater than those for chiral amines. The developed analytical method was applied to determine the enantiomeric purity of two commercially available chiral amine and α-amino acid ester; the enantiomeric impurities were observed to be 0.35 and 0.05% for (S)-α-methylbenzylamine and (S)-leucine ethyl ester, respectively. The validation data obtained from intra- and inter-day accuracy assays for (S)-3,3-dimethyl-2-butylamine and (S)-leucine ethyl ester were 99.92-101.46 and 100.61-101.78%; and 100.05-102.12 and 101.25-102.73%, respectively; the intra- and interday precision assays in terms of relative standard deviation (% RSD) were found to be 0.96-1.57 and 0.81- 1.91%; and 0.24-1.01% and 0.65-1.35%, respectively. Owing to the high accuracy and precision results, the developed analytical method can prove to be useful for the enantiomeric separation of various chiral amines and amino acid esters as NBD derivatives using polysaccharide derived CSPs.

Creation of Fluorescent RXR Antagonists Based on CBTF-EE and Application to a Fluorescence Polarization Binding Assay.

Retinoid X receptor (RXR) ligands often bind in modes in which the carboxy group forms a hydrogen bond inside the ligand-binding pocket (LBP). However, our previously reported RXR antagonist, CBTF-EE (4a), binds with its carboxy group directed outside the LBP and its alkoxy side chain located inside the LBP. Here, we examined the binding modes of 4b and 4c bearing a nitrobenzoxadiazole (NBD) or boron-dipyrromethene (BODIPY) fluorophore, respectively, at the end of the alkoxy chain of 4a. Both compounds function as RXR antagonists. 4c, but not 4b, was available for a fluorescence polarization binding assay, indicating that rotation of BODIPY, but not NBD, is restricted in the bound state. The fluorescence findings, supported by docking simulations, suggest the fluorophores are located outside the LBP, so that the binding mode of 4b and 4c is different from that of 4a. The assay results were highly correlated with those of a [3H]9-cis-retinoic acid assay.

Construction of ultrasensitive devices for visualization and quantification of phosgene based on FRET-mediated two-photon chemosensor

As a widely used but highly toxic chemical species, phosgene is threatening to human health because of possible accidental leakage during industrial manufacture. Herein, we exploited a turn-on fluorescent sensor F671 for phosgene detection. The exact distance of nitro benzoxadiazole (NBD) and boron dipyrromethene (BODIPY) in F671 was changed from 5.027 Å to 15.535 Å after reaction with phosgene, accompanied with obvious fluorescence enhancement, which is attributed to the inhibition of Förster resonance energy transfer (FRET) process. The FRET-associated strategy possesses the capacity of detecting phosgene with a rapid response time (<6 s), ultra-sensitivity (LOD = 0.36 nM in solution and 0.14 ppm in gas phase) and remarkable selectivity to phosgene in solutions and in gas phase. Furthermore, the F671-embedded degreasing cotton, has been fabricated to serves as a specific gaseous phosgene device, for facile, selective and visual detection of phosgene gas. Confocal imaging of F671-embedded degreasing cotton displayed increasing emission intensity in the presence of phosgene in both of one-photon and two-photon patterns. It is envisioned that the developed convenient device is a promising tool for phosgene gas detection and quantification in real industrial and environmental applications.

Theoretical studies on triplet formations in nitrobenzoxadiazole (NBD) derivatives: The impact of donor group and heteroatom substitution

Heavy-atom-free triplet photosensitizers (TPs) have attracted much interest due to the applications in triplet–triplet annihilation (TTA) and photodynamic therapy. Recently, several nitrobenzoxadiazole (NBD) derivatives have been reported to generate singlet oxygens. However, the mechanistic understanding of the intersystem crossing (ISC) process has not been studied, resulting in a lack of rational design strategies for creating effective TPs. Based on DFT and TD-DFT calculations, we found that the replacement of donor group (methylamino → methoxyl) and the heteroatom (oxadiazole → triazole) could induce a significant change in the nature of triplet excited states in comparison to that of the single excited states, and thus enable substantial spin-orbital couplings for efficient triplet conversions. We expect that modifying the electronic properties of the triplet excited states through donor/heteroatom substitution is a promising method for creating heavy-atom-free TPs in many chemical families of fluorophores.

A simple, rapid, and sensitive fluorescence-based method to assess triacylglycerol hydrolase activity

Lipases constitute an important class of water-soluble enzymes that catalyze the hydrolysis of hydrophobic triacylglycerol (TAG). Their enzymatic activity is typically measured using multistep procedures involving isolation and quantification of the hydrolyzed products. We report here a new fluorescence method to measure lipase activity in real time that does not require the separation of substrates from products. We developed this method using adipose triglyceride lipase (ATGL) and lipoprotein lipase (LpL) as model lipases. We first incubated a source of ATGL or LpL with substrate vesicles containing nitrobenzoxadiazole (NBD)-labeled TAG, then measured increases in NBD fluorescence, and calculated enzyme activities. Incorporation of NBD-TAG into phosphatidylcholine (PC) vesicles resulted in some hydrolysis; however, incorporation of phosphatidylinositol into these NBD-TAG/PC vesicles and increasing the ratio of NBD-TAG to PC greatly enhanced substrate hydrolysis. This assay was also useful in measuring the activity of pancreatic lipase and hormone-sensitive lipase. Next, we tested several small-molecule lipase inhibitors and found that orlistat inhibits all lipases, indicating that it is a pan-lipase inhibitor. In short, we describe a simple, rapid, fluorescence-based triacylglycerol hydrolysis assay to assess four major TAG hydrolases: intracellular ATGL and hormone-sensitive lipase, LpL localized at the extracellular endothelium, and pancreatic lipase present in the intestinal lumen. The major advantages of this method are its speed, simplicity, and elimination of product isolation. This assay is potentially applicable to a wide range of lipases, is amenable to high-throughput screening to discover novel modulators of triacylglycerol hydrolases, and can be used for diagnostic purposes.

NBD-based synthetic probes for sensing small molecules and proteins: design, sensing mechanisms and biological applications.

Compounds with a nitrobenzoxadiazole (NBD) skeleton exhibit prominent useful properties including environmental sensitivity, high reactivity toward amines and biothiols (including H2S) accompanied by distinct colorimetric and fluorescent changes, fluorescence-quenching ability, and small size, all of which facilitate biomolecular sensing and self-assembly. Amines are important biological nucleophiles, and the unique activity of NBD ethers with amines has allowed for site-specific protein labelling and for the detection of enzyme activities. Both H2S and biothiols are involved in a wide range of physiological processes in mammals, and misregulation of these small molecules is associated with numerous diseases including cancers. In this review, we focus on NBD-based synthetic probes as advanced chemical tools for biomolecular sensing. Specifically, we discuss the sensing mechanisms and selectivity of the probes, the design strategies for multi-reactable multi-quenching probes, and the associated biological applications of these important constructs. We also highlight self-assembled NBD-based probes and outline future directions for NBD-based chemosensors. We hope that this comprehensive review will facilitate the development of future probes for investigating and understanding different biological processes and aid the development of potential theranostic agents.

Molecular Origins of Heteroatom Engineering on the Emission Wavelength Tuning, Quantum Yield Variations and Fluorogenicity of NBD-like SCOTfluors.

Molecular engineering of fluorophore scaffolds, especially heteroatom replacement, is a promising method to yield novel fluorophores with tailored properties for various applications. Yet, molecular origins of the distinct fluorescent properties in newly developed SCOTfluors, i. e., varied emission wavelengths, distinct quantum yields, and fluorogenicity, remain elusive. Such understanding, however, is critical for the rational molecular engineering of high-performance fluorophores. Herein, we employed quantum chemical calculations to understand the structure-property relationships of nitrobenzoxadiazole (NBD)-like SCOTfluors. Our findings are important not only for the rational deployment of SCOTfluors, but also for the effective modifications of other fluorophore scaffolds, for satisfying the increasingly diversified requirements of bioimaging and biosensing applications.

Fluorescent turn-on assay of C-type natriuretic peptide using a molecularly imprinted ratiometric fluorescent probe with high selectivity and sensitivity.

Anovel molecularly imprinted ratiometric fluorescent probe was fabricated by simple sol-gel polymerization for selective and sensitive assay of C-type natriuretic peptide(CNP) in biosamples. Both the nitrobenzoxadiazole (NBD) and carbon dots (CDs) were located on the surface of silica, used as the detection signal and reference signal, respectively. For the turn-on-based probe, the fluorescence intensity of NBD could be quantitatively enhanced by CNP based on the strategy of photo-induced electron transfer (PET), while the fluorescence of CDs remained unchanged. The obtained probe exhibited excellent recognition selectivity and fast kinetics to CNP templates, and also showed good stability. The linear range of CNP determination was 5-80pgmL-1 with a low detection limit of 2.87pgmL-1. Finally, the probe was successfully applied to determine CNP in human serum samples and attained high recoveries between 97.3 and 104% with precisions below 4.7%. The result indicates that the proposed method has promising potential for the assay of trace peptides incomplex matrices. Schematic illustration for the formation and determination mechanism of the probe.

Desuccinylation-Triggered Peptide Self-Assembly: Live Cell Imaging of SIRT5 Activity and Mitochondrial Activity Modulation.

Mimicking nature's ability to orchestrate molecular self-assembly in living cells is important yet challenging. Molecular self-assembly has found wide applications in cellular activity control, drug delivery, biomarker imaging, etc. Nonetheless, examples of suborganelle-confined supramolecular self-assembly are quite rare and research in this area remains challenging. Herein, we have presented a new strategy to program supramolecular self-assembly specifically in mitochondria by leveraging on a unique enzyme SIRT5. SIRT5 is a mitochondria-localized enzyme belonging to a family of NAD+-dependent histone deacetylases. Accumulating studies suggest that SIRT5 is involved in regulating diverse biological processes, such as reactive oxygen defense, fatty acid metabolism, and apoptosis. In this study, we designed a novel class of succinylated peptide precursors that can be transformed into self-assembling building blocks through SIRT5 catalysis, leading to the formation of supramolecular nanofibers in vitro and in living cells. The increased hydrophobicity arising from self-assembly remarkably enhanced the fluorescence of nitrobenzoxadiazole (NBD) in the nanofibers. With this approach, we have enabled activity-based imaging of SIRT5 in living cells for the first time. Moreover, SIRT5-mediated peptide self-assembly was found to depolarize mitochondria membrane potential and promote ROS formation. Coincubation of the peptide with three different chemotherapeutic agents significantly boosted the anticancer activities of these drugs. Our work has thus illustrated a new way of mitochondria-confined peptide self-assembly for SIRT5 imaging and potential anticancer treatment.

Two-photon small molecular fluorogenic probe visualizing biothiols and sulfides in living cells, mice brain slices and zebrafish

Biothiols and sulfides, especially glutathione (GSH), cysteine (Cys), homocysteine (Hcy) and hydrogen sulfide (H2S) play important roles in an extensive range of biological processes and functions. Due to their reductivity, they are deemed as second messengers of oxidative stress, and the imbalance of these biothiols are closely associated to a variety of diseases. Herein, we report a two-photon (TP) turn-on probe DT-01 that can detect biothiols and sulfides both in vitro and in vivo. The probe was synthesized by nitrobenzoxadiazole (NBD) derivative (NBD-Cl) and pyrimidine-based TP fluorophore (FL-01), the fluorescence of DT-01 is quenched due to the photoinduced charge transfer (PET) from FL-01 moiety to NBD moiety. Upon substitution reaction by nucleophilic biothiols, TP fluorophore released and exhibited turn-on fluorescence at 550 nm. We further demonstrated that DT-01 can detect biothiols and H2S in living cells, mice brain slices and zebrafish. More importantly, by real-time detecting the total content of biothiols and H2S, we have successively measured the changes of oxidative stress levels in HeLa cells.

Design, Synthesis and Evaluation of Fluorescent Analogues of Abscisic Acid

AbstractA fluorescent analogue of abscisic acid has been prepared by combining (S)‐abscisic acid (ABA) with nitrobenzoxadiazole (NBD) fluorophore using ethanol amine as a linker. Isomerization of the double bond at the side chain of abscisic acid happened during the synthesis. The resulting fluorophore analogues derived from both isomeric compounds entered cells suggesting a wide applicability of the ABA‐NBD conjugates as fluorescent probes to study ABA mechanism of action. The functional properties of the isomeric ABA‐NBD conjugates were tested in vitro, by measuring TNFα expression and nitrite concentration in LPS‐stimulated macrophages and compared with their non‐fluorescent ABA isomers. Rationalization of the results is given as supported by docking studies of the compounds with LANCL2 and PPARγ targets.

Selective detection of phospholipids using molecularly imprinted fluorescent sensory core-shell particles

Sphingosine-1-phosphate (S1P) is a bioactive sphingo-lipid with a broad range of activities coupled to its role in G-protein coupled receptor signalling. Monitoring of both intra and extra cellular levels of this lipid is challenging due to its low abundance and lack of robust affinity assays or sensors. We here report on fluorescent sensory core-shell molecularly imprinted polymer (MIP) particles responsive to near physiologically relevant levels of S1P and the S1P receptor modulator fingolimod phosphate (FP) in spiked human serum samples. Imprinting was achieved using the tetrabutylammonium (TBA) salt of FP or phosphatidic acid (DPPA·Na) as templates in combination with a polymerizable nitrobenzoxadiazole (NBD)-urea monomer with the dual role of capturing the phospho-anion and signalling its presence. The monomers were grafted from ca 300 nm RAFT-modified silica core particles using ethyleneglycol dimethacrylate (EGDMA) as crosslinker resulting in 10–20 nm thick shells displaying selective fluorescence response to the targeted lipids S1P and DPPA in aqueous buffered media. Potential use of the sensory particles for monitoring S1P in serum was demonstrated on spiked serum samples, proving a linear range of 18–60 µM and a detection limit of 5.6 µM, a value in the same range as the plasma concentration of the biomarker.

Controllable Cleavage of C-N Bond-Based Fluorescent and Photoacoustic Dual-Modal Probes for the Detection of H2S in Living Mice.

Hydrogen sulfide (H2S) has been recognized to influence a wide range of physiological and pathological processes. Its underlying molecular events, however, are still poorly understood. An activatable H2S probe for photoacoustic (PA) imaging is desirable to further explore the role of H2S in vivo. Nevertheless, only a few activatable PA probes for H2S detection have been reported. In particular, examples of dual-modal H2S probes with the combined advantages of fluorescence (high sensitivity and resolution) and PA imaging (deep penetration) are very rare. Herein the controllable cleavage of the C-N bond in nitrobenzoxadiazole (NBD) amine derivatives by H2S is presented for the first time. The cleavage reactivity was found to be accelerated by the introduction of an electron-withdrawing group. Through this strategy, a series of fluorescent and PA dual-modal probes (1-3) were developed for H2S detection. Among them, probe 3 shows a high fluorescence on-off response rate (k2 = 4.04 M-1 s-1) and excellent selectivity for H2S over other biothiols. Moreover, probe 3 can also work as an activatable PA H2S probe because of the significant shift of its absorption peak from 468 to 532 nm in the H2S reaction. Importantly, probe 3 demonstrates its capability for fluorescence and PA imaging of H2S in living cells and mice. These results indicate that the controllable cleavage of the C-N bond can serve as an efficient strategy for designing fluorescent and PA dual-modal H2S probes.

Evaluation of a Novel Boron-Containing α-D-Mannopyranoside for BNCT.

Boron neutron capture therapy (BNCT) is a unique anticancer technology that has demonstrated its efficacy in numerous phase I/II clinical trials with boronophenylalanine (BPA) and sodium borocaptate (BSH) used as 10B delivery agents. However, continuous drug administration at high concentrations is needed to maintain sufficient 10B concentration within tumors. To address the issue of 10B accumulation and retention in tumor tissue, we developed MMT1242, a novel boron-containing α-d-mannopyranoside. We evaluated the uptake, intracellular distribution, and retention of MMT1242 in cultured cells and analyzed biodistribution, tumor-to-normal tissue ratio and toxicity in vivo. Fluorescence imaging using nitrobenzoxadiazole (NBD)-labeled MMT1242 and inductively coupled mass spectrometry (ICP-MS) were performed. The effectiveness of BNCT using MMT1242 was assessed in animal irradiation studies at the Kyoto University Research Reactor. MMT1242 showed a high uptake and broad intracellular distribution in vitro, longer tumor retention compared to BSH and BPA, and adequate tumor-to-normal tissue accumulation ratio and low toxicity in vivo. A neutron irradiation study with MMT1242 in a subcutaneous murine tumor model revealed a significant tumor inhibiting effect if injected 24 h before irradiation. We therefore report that 10B-MMT1242 is a candidate for further clinical BNCT studies.

A Membrane Tubule Bilayer Assay for Curvature Sorting of Phosphatidic Acid

Phosphatidic acid (PA) is a signaling lipid that is thought to support membrane fusion by stabilizing negative curvature. PA is the product of phospholipase D, an enzyme that is essential for exocytosis. Using supported lipid bilayers with tubules to induce curvature, we identify to what extent PA localizes to curvature. Liposomes containing POPC, pegylated DOPE, and dye-labeled lipids with varying concentrations of NaCl (20 mM to 1 M) were deposited on glass to form bilayers with connected tubules. Tubules were then imaged using TIRF or confocal microscopy. The fluorescence intensity is related to the amount of the lipid present, meaning tubules appear brighter than flat regions. Using nitrobenzoxadiazole(NBD)-labeled lipids and its quencher, dithionite, we then selectively quench the outer leaflet of the bilayer, allowing us to determine which curvature a lipid localizes to; the outer leaflet is positively curved and the inner leaflet is negatively curved. Melittin was then added to form pores in the membrane, allowing dithionite to quench the inner leaflet. Current data suggests that PA localizes to the inner leaflet of tubules (negative curvature) in bilayers and that PA could be a way that the curvature observed during membrane fusion is stabilized.

A ratiometric and near-infrared fluorescent probe for discrimination of Cys/Hcy/GSH in aqueous solution and its intracellular imaging in living cells

In this work, a unique ratiometric and NIR fluorescent probe, Hecy-NBD, was designed and synthesized to detect cysteine/homocysteine/glutathione in aqueous solution. Hemicyanine skeleton was chosen as the major fluorophore and nitro benzoxadiazole (NBD) chromophore was selected as the recognition unit to construct a ratiometric fluorescent probe. The fluorescence of probe Hecy-NBD was very weak due to the introduction of quenching group NBD. Upon the addition of 100 μM GSH to 10 μM Hecy-NBD, the fluorescence intensity at 702 nm was very strong with the releasing of compound Hecy-OH. While upon the addition of 100 μM Cys or 100 μM Hcy, probe exhibited distinct fluorescence enhancement at 540 nm and 702 nm, which resulted from the formation of compound NBD-Cys and NBD-Hcy through the 5/6-membered cyclic transition and the fluorescence releasing of compound Hecy-OH. The proposed mechanism based on SNAr reaction and molecular rearrangement, which were confirmed by HRMS spectra. The detection limit of these three biothiols was also calculated (GSH, 50 nM; Cys, 81.0 nM; Hcy, 130 nM). Moreover, probe Hecy-NBD could function well at wide pH conditions from 4 to 10. Besides that, probe Hecy-NBD with low cell toxicity had been successfully applied to dual-channel fluorescence imaging in living cells. Considering the good characteristics, Hecy-NBD had great potential to detect biothiols-associated diseases in the future.