A Dansyl-Modified Sphingosine Kinase Inhibitor DPF-543 Enhanced De Novo Ceramide Generation.

Sphingosine-1-phosphate (S1P) synthesized by sphingosine kinase (SPHK) is a signaling molecule, involved in cell proliferation, growth, differentiation, and survival. Indeed, a sharp increase of S1P is linked to a pathological outcome with inflammation, cancer metastasis, or angiogenesis, etc. In this regard, SPHK/S1P axis regulation has been a specific issue in the anticancer strategy to turn accumulated sphingosine (SPN) into cytotoxic ceramides (Cers). For these purposes, there have been numerous chemicals synthesized for SPHK inhibition. In this study, we investigated the comparative efficiency of dansylated PF-543 (DPF-543) on the Cers synthesis along with PF-543. DPF-543 deserved attention in strong cytotoxicity, due to the cytotoxic Cers accumulation by ceramide synthase (CerSs). DPF-543 exhibited dual actions on Cers synthesis by enhancing serine palmitoyltransferase (SPT) activity, and by inhibiting SPHKs, which eventually induced an unusual environment with a high amount of 3-ketosphinganine and sphinganine (SPA). SPA in turn was consumed to synthesize Cers via de novo pathway. Interestingly, PF-543 increased only the SPN level, but not for SPA. In addition, DPF-543 mildly activates acid sphingomyelinase (aSMase), which contributes a partial increase in Cers. Collectively, a dansyl-modified DPF-543 relatively enhanced Cers accumulation via de novo pathway which was not observed in PF-543. Our results demonstrated that the structural modification on SPHK inhibitors is still an attractive anticancer strategy by regulating sphingolipid metabolism.

Design, synthesis and cell imaging of a simple peptide-based probe for the selective detection of RNA.

Here we present a novel peptide-based fluorescent "turn-on" molecule P1 for detecting RNA, in a double or single strand, AU-rich or CG-rich. Both computational and experimental studies indicate that the detection efficiency depends on the binding affinity of P1 and conformational changes. P1 could be applied for cell imaging without any additional transfection vectors. Selective detection of RNA in cells was determined by RNase digestion. Successful application of P1 for RNA imaging in cell mitosis reveals that it may have broad applications in research, biotechnology and medical science.

A simple and effective dansyl acid based "turn-on" fluorescent probe for detecting labile ferrous iron in physiological saline and live cells.

Labile ferrous iron (Fe2+) plays important biochemical functions in many physiologically essential processes. It is very important to find an effective method to detect Fe2+. Herein, a simple and effective Fe2+ fluorescent probe (FeP1) has been constructed via a unique strategy of Fe2+-induced reducing reaction. As expected, FeP1 exhibited a 'turn-on' fluorescence response toward Fe2+ over various small analytes, with high selectivity and excellent sensitivity (DL = 18 nM) for the detection of Fe2+ in Tris-DMSO (4:1, pH = 7.4, v/v) solution. Moreover, the probe can act in different real samples, such as physiological saline and living cells.

Liquid chromatography-diode array-mass spectrometric analysis of amino and mercapto compounds coupled with chloroimino derivatization reagent.

A new derivatization reagent, N-(naphthalen-1-yl)-2-oxopropanehydrazonoyl chloride (UOSA54), was prepared and coupled with four drugs, bearing primary amino, secondary amino or mercapto functional groups. Glucosamine sulfate (GLU), cysteine (CYS), captopril (CAP), and vildagliptin (VIL), were used as representative reactive analytes. The prepared reagent was successfully coupled with the targeted analytes in the presence of triethylamine (TEA) as hydrochloride acceptor and acetonitrile as solvent. The resulting reaction products were separated by high-performance liquid chromatography and monitored simultaneously by diode array and triple quad mass spectrometry detectors. Enhanced DAD and electrospray ionization-MS (ESI-MS) responses were observed for the derivatized products. Complete derivatization of VIL was achieved after heating at 65 ± 3 °C for 4 min, while other analytes were derivatized instantaneously at room temperature. Both, the ESI-ionization suppression, due to the excess reagent, and matrix effect, due to co-eluted biogenic plasma constituents, were negligible. The derivatized GLU, CYS, CAP, and VIL showed a maximum absorption wavelength at 376, 417, 340, and 376 nm, with MS-limit of quantification value of 250.0, 2.0, 2.5, and 3.0 pg/µL, respectively. The relative ESI-MS response of UOSA54 derivatization products was within the range of 0.6-4.1 compared with dansylated products. The method was optimized and validated for optimal reaction product stability, sensitivity, linearity, range, precision, and accuracy. The percentage recovery was exceeding 97.2%, with an RSD value of less than 4.0%. The limit of quantification of targeted analytes was ranged from 80.0 to 0.7 pg/µL.

Parallel derivatization strategy coupled with liquid chromatography-mass spectrometry for broad coverage of steroid hormones.

Steroid hormones are a type of crucial substances that mediate numerous vital physiological functions. The comprehensive detection of steroid hormones can help understand the physiopathologic mechanism of steroid hormone-related diseases. It is very difficult to determine steroid hormones in biological samples due to their low endogenous concentrations and poor ionization efficiency. In this study, an efficient and sensitive approach was developed for profiling steroid hormones by combining liquid-liquid extraction and parallel derivatization with liquid chromatography-tandem mass spectrometry. Methoxyamine and dansyl chloride were used to derivatize steroid hormones containing carbonyl and phenolic hydroxyl groups, respectively. Our established method achieved simultaneous analysis of carbonyl and phenolic hydroxyl-containing steroid hormones and could cover estrogens, androgens, corticoids and progestogens. Twenty-nine steroid hormones were detected at pg/mL levels with the sensitivity enhanced by three orders of magnitude after derivatization. The linearity (with linear range of 2-4 orders of magnitude), precision (less than 15%) and recovery (71.1-128.7%) were satisfactory for quantitative analysis of steroid hormones. Finally, the established method was successfully employed to the determination of steroid hormones in serum samples of healthy males and females as well as ovarian cancer patients. The results showed that this approach was suitable and reliable for routine test of steroid hormones containing carbonyl and phenolic hydroxyl groups.

A novel fluorescent chemosensor for detection of mercury(II) ions based on dansyl-peptide and its application in real water samples and living LNcap cells.

Mercury is one of the most hazardous pollutants, and mercury pollution is a serious hazard to our environment. Herein, we designed and synthesized a new peptide-based fluorescent chemosensor (L) based on a Fmoc-Lys (Fmoc)-OH backbone conjugated with two Serines and dansyl groups using solid phase peptide synthesis (SPPS) technology. L exhibited highly selective and excellent sensitive detection of Hg2+ ions in 100% aqueous solutions through fluorescence quenching. The chemosensor L forms a 2:1 stoichiometry with high binding constants (4.89×106M-1) and the detection limit for Hg2+ ions of the proposed assay was 7.59nM. In addition, the recovery test results of Hg2+ concentration in actual water samples showed that the quantitative detection of Hg2+ ions can be realized in two water samples. Moreover, L showed low cytotoxicity and excellent membrane permeability in HK2 cells, which has been successfully applied for monitoring Hg2+ ions in living LNCaP cells.

Gender-Related Differences on Polyamine Metabolome in Liquid Biopsies by a Simple and Sensitive Two-Step Liquid-Liquid Extraction and LC-MS/MS.

Polyamines are involved in the regulation of many cellular functions and are promising biomarkers of numerous physiological conditions. Since the concentrations of these compounds in biological fluids are low, sample extraction is one of the most critical steps of their analysis. Here, we developed a comprehensive, sensitive, robust, and high-throughput LC-MS/MS stable-isotope dilution method for the simultaneous determination of 19 metabolites related to polyamine metabolism, including polyamines, acetylated and diacetylated polyamines, precursors, and catabolites from liquid biopsies. The sample extraction was optimized to remove interfering compounds and to reduce matrix effects, thus being useful for large clinical studies. The method consists of two-step liquid-liquid extraction with a Folch extraction and ethyl acetate partitioning combined with dansyl chloride derivatization. The developed method was applied to a small gender-related trial concerning human serum and urine samples from 40 obese subjects. Sex differences were found for cadaverine, putrescine, 1,3-diaminopropane, γ-aminobutyric acid, N8-acetylspermidine, and N-acetylcadaverine in urine; N1-acetylspermine in serum; and spermine in both serum and urine. The results demonstrate that the developed method can be used to analyze biological samples for the study of polyamine metabolism and its association with human diseases.

Two-Photon Fluorescence Probe for Selective Monitoring of Superoxide in Live Cells and Tissues.

The abnormal location or generation of superoxide radical anion (O2•-) are implicated in many diseases, including cancers; thus, development of an efficient method to detect O2•- is of great importance. Inspired by the fluorophore-governed selective manner to O2•- and peroxynitrite (ONOO-) of previously reported phosphinate-based fluorescence probes, in this contribution, a phosphinothioate-containing probe, TPP, was designed. The probe exhibited easy accessibility through a one-step sequence and good photostability and biocompatibility. Interestingly, TPP showed high specificity and sensitivity to O2•- over other reactive oxygen species/nitrogen species including ONOO-. Furthermore, with the assistance of two-photon microscopy, TPP was successfully applied for imaging endogenous O2•- in live cells and tissues.

Crystal structure of zinc-α2-glycoprotein in complex with a fatty acid reveals multiple different modes of protein-lipid binding.

Human zinc-α2-glycoprotein (ZAG) is a 42 kDa adipokine which regulates body fat mass and is associated with cachexia and obesity. ZAG belongs to the major histocompatibility complex class I protein family and binds long-chain polyunsaturated fatty acids in its groove formed from the α1 and α2 domains. To identify the molecular basis of its lipid-binding function, we determined the first crystal structure at 2.49 Šresolution for fatty acid-bound ZAG, where the ligand was the fluorescent 11-(dansylamino)undecanoic acid (DAUDA). The 192 kDa crystallographic asymmetric unit contained six ZAG and eight fatty acid molecules in unique conformations. Six fatty acid molecules were localised to the ZAG grooves, where their tails were bound in two distinct conformations. The carboxylate groups of three fatty acids projected out of the groove, while the fourth was hydrogen bonded with R73 inside the groove. Other ligand-residue contacts were primarily hydrophobic. A new fatty acid site was revealed for two further DAUDA molecules at the ZAG α3 domains. Following conformational changes from unbound ZAG, the α3 domains formed tetrameric ß-barrel structures lined by fatty acid molecules that doubled the binding capacity of ZAG. Analytical ultracentrifugation revealed that ZAG in solution was a monomer in the absence of DAUDA, but formed small amounts of tetramers with DAUDA. By showing that ZAG binds fatty acids in different locations, we demonstrate an augmented mechanism for fatty acid binding in ZAG that is distinct from other known fatty acid binding proteins, and may be relevant to cachexia.

Development of dansyl based copper(ii) complex to detect hydrogen sulfide in hypoxia.

Hydrogen sulfide (H2S) has been reported as a gaseous signaling molecule in cells. H2S modulation is dependent on the partial pressure of oxygen in cells, which means hypoxia can induce H2S production under various pathophysiological conditions. Hypoxia is a common condition in solid tumors and can lead to malignant tumors that may become aggressive and result in worse prognosis. We designed and synthesized probe Cu-CD for H2S detection under hypoxia conditions. It is selective and sensitive toward various biological thiols, reactive nitrogen species (RNS), and reactive oxygen species (ROS). The fluorescence intensity of Cu-CD in the cytoplasms of HeLa and EMT6 cells was enhanced in proportion to the concentration of exogenous/endogenous H2S. Moreover, Cu-CD can be able to detect endogenous H2S production accompanied by expression of HIF-1α. Therefore, Cu-CD can be a key tool to explore how H2S contributes to neovascularization and growth of solid tumor tissues in pathophysiological or hypoxic conditions.

A novel fluorescent chemosensor for detection of Zn(II) ions based on dansyl-appended dipeptide in two different living cells.

This paper describes a new fluorescent chemosensor (DSH) based on dipeptide conjugated with dansyl group, which was synthesized by solid phase peptide synthesis (SPPS) technology. DSH exhibited a highly selective and sensitive toward Zn2+ ions by "turn-on" response based on generation of monomer-excimer mechanism in aqueous solutions, and the detection limit was calculated at 11.2 nM. In addition, the reversible of DSH-Zn with Na2EDTA establishes the reuse of DSH, and the circulation effect was very good. Moreover, DSH had good water solubility, and was successfully applied to bioimage intracellular Zn2+ ions and Na2EDTA in two different living cells with exciting cellular permeability and low cytotoxicity, which indicated that DSH had great potential in the application of biological imaging.

Targeting amine- and phenol-containing metabolites in urine by dansylation isotope labeling and liquid chromatography mass spectrometry for evaluation of bladder cancer biomarkers.

Metabolomics is considered an effective approach for understanding metabolic responses in complex biological systems. Accordingly, it has attracted increasing attention for biomarker discovery, especially in cancer. In this study, we used a non-invasive method to evaluate four urine metabolite biomarker candidates-o-phosphoethanolamine, 3-amio-2-piperidone, uridine and 5-hydroxyindoleactic acid-for their potential as bladder cancer diagnostic biomarkers. To analyze these targeted amine- and phenol-containing metabolites, we used differential 12C2-/13C2-dansylation labeling coupled with liquid chromatography/tandem mass spectrometry, which has previously been demonstrated to exhibit high sensitivity and reproducibility. Specifically, we used ultra-performance liquid chromatography (UPLC) coupled with high-resolution Fourier transform ion-cyclotron resonance MS system (LC-FT/MS) and an ion trap MS with MRM function (LC-HCT/MS) for targeted quantification. The urinary metabolites of interest were well separated and quantified using this approach. To apply this approach to clinical urine specimens, we spiked samples with 13C2-dansylatedsynthetic compounds, which served as standards for targeted quantification of 12C2-dansylated urinary endogenous metabolites using LC-FT/MS as well as LC-HCT/MS with MRM mode. These analyses revealed significant differences in two of the four metabolites of interest-o-phosphoethanolamine and uridine-between bladder cancer and non-cancer groups. O-phosphoethanolamine was the most promising single biomarker, with an area-under-the-curve (AUC) value of 0.709 for bladder cancer diagnosis. Diagnostic performance was improved by combining uridine and o-phosphoethanolamine in a marker panel, yielding an AUC value of 0.726. This study confirmed discovery-phase features of the urine metabolome of bladder cancer patients and verified their importance for further study.

One-Component Array Based on a Dansyl-Modified Polylysine: Generation of Differential Fluorescent Signatures for the Discrimination of Human Cells.

A one-component array-based sensor consisting of a dansyl-modified polylysine (PLL-Dnc) is capable of discriminating the types and compositional ratios of human cells using varying buffer conditions. The PLL-Dnc sensor array, which affords turn-on fluorescence responses against analyte cells that depend on the pH value and the ionic strength, generates differential fluorescence signatures of cells and successfully discriminates eight types of human cell lines (2.0 × 104 cells/mL) with 100% accuracy using multivariate analysis. The array also allows differentiation of the composition of the cell mixtures that contain cells with the same tissue origin but of different subtypes. The good discrimination ability and simple platform of our "one-component"-based array allows an easy and rapid sensing of cells without requiring any information on specific biomarkers.

Fluorescent indomethacin-dansyl conjugates utilize the membrane-binding domain of cyclooxygenase-2 to block the opening to the active site.

Many indomethacin amides and esters are cyclooxygenase-2 (COX-2)-selective inhibitors, providing a framework for the design of COX-2-targeted imaging and cancer chemotherapeutic agents. Although previous studies have suggested that the amide or ester moiety of these inhibitors binds in the lobby region, a spacious alcove within the enzyme's membrane-binding domain, structural details have been lacking. Here, we present observations on the crystal complexes of COX-2 with two indomethacin-dansyl conjugates (compounds 1 and 2) at 2.22-Å resolution. Both compounds are COX-2-selective inhibitors with IC50 values of 0.76 and 0.17 µm, respectively. Our results confirmed that the dansyl moiety is localized in and establishes hydrophobic interactions and several hydrogen bonds with the lobby of the membrane-binding domain. We noted that in both crystal structures, the linker tethering indomethacin to the dansyl moiety passes through the constriction at the mouth of the COX-2 active site, resulting in displacement and disorder of Arg-120, located at the opening to the active site. Both compounds exhibited higher inhibitory potency against a COX-2 R120A variant than against the WT enzyme. Inhibition kinetics of compound 2 were similar to those of the indomethacin parent compound against WT COX-2, and the R120A substitution reduced the time dependence of COX inhibition. These results provide a structural basis for the further design and optimization of conjugated COX reagents for imaging of malignant or inflammatory tissues containing high COX-2 levels.

Enhancement of liquid chromatography-ion trap mass spectrometry analysis of 4(5)-methylimidazole in biscuits through derivatization with dansyl chloride.

The presence of potential carcinogen 4(5)-Methylimidazole (4-MeI) in foods causes much concern, stressing the need of a sensitive determination means. Here, we proposed a high-sensitivity method to determine 4-MeI in biscuits using dansyl chloride derivatization and disperse liquid-liquid micro-extraction (DLLME) followed by a liquid chromatography-ion trap mass spectrometry (LC-IT-MS) analysis. This developed method was subsequently compared to the solid phase extraction combining with liquid chromatography-triple quadrupole mass spectrometry (SPE-LC-QqQ-MS) method. The optimized derivatization conditions were 30 °C and 10 min. Results suggested that the column retention time (RT) was significantly extended, and the MS signal response of 4-MeI-dansyl derivative was also amplified. The developed DLLME-LC-IT-MS method in 4-MeI determination provided satisfactory linearity in a range of 0.5-300 ng/mL (R2 > 0.9991), inter-day accuracy (87-102%) and precision (%RSD ≤ 13.6%) with the quantification limit of 0.2 ng/mL, which obtained similar results comparing to the conventional SPE-LC-QqQ-MS method.

Use of a deoxynojirimycin-fluorophore conjugate as a cell-specific imaging probe targeting α-glucosidase on cell membranes.

Molecules designed for cell-specific imaging were studied, taking advantage of an enzyme-inhibitor interaction. 1-Deoxynojirimycin (DNJ) can be actively captured by cells which express the surface membrane protein α-glucosidase. New probes composed of DNJ for recognition linked to a fluorophore signal portion were prepared (DNJ-CF31, DNJ-Dans 2 and DNJ-DEAC 3). Docking simulations revealed that the inhibitors acarbose and miglitol and the inhibitor portion of the probes bind at the same position in the pocket of α-glucosidase (human-derived PDB: 3TON). The ability of probes 1-3 to detect the difference between HeLa cells (from human cervical cancer tissue), Neuro-2a cells (from a mouse neuroblastoma C1300 tumor), N1E-115 cells (from a mouse brain neuroblastoma C1300 tumor), A1 cells (from the astrocyte of a newborn mouse brain), and Caco-2 cells (from a human colon carcinoma) was evaluated, and cell-specific fluorescence imaging was possible for conjugate probes 1 and 2. Caco-2 cells treated with probes 1 and 2 showed blue and green fluorescence, respectively, from the cell membrane, and did not stain the Caco-2 cells inside. These results show that DNJ-CF31 and DNJ-Dans 2 recognize an α-glucosidase protein on the surface of Caco-2 cells. Probes 1 and 2 did not stain any part of the other cells. This cell-specific imaging strategy is applicable for a variety of therapeutic agents for many diseases.

A colorimetric/fluorescence dual-channel probe for highly discriminating detection of cysteine.

In this work, a novel fluorescence (FL) probe for selective and sensitive detection of Cys with colorimetric and FL dual signal changes was reported. The probe was synthesized by two step of sulfonamide reaction coupling between a sulfonyl benzoxadiazole (SBD) dye and dansyl chloride linked with rigid piperazine group. The probe showed a specific off-on response to Cys in aqueous solution with nanomolar LOD, and without interference by a range of amino acids and several competing analytes. Upon addition of Cys, the probe will undergo sequential substitution and intramolecular rearrangement reactions, yielding a 4-amino SBD derivative, which results in generation of strong yellow fluorescence emission at 575 nm accompanied by a two-step red shift in the absorption spectral. Moreover, it can be used for imaging of endogenous Cys in living cells.

In chemico assessment of potential sensitizers: Stability and direct peptide reactivity of 24 fragrance ingredients.

Twenty-four pure fragrance ingredients of concern as potential skin sensitizers were previously subjected to degradation studies and evaluated using the high throughput with dansyl cysteamine (HTS-DCYA) method. The experimental results showed that two-thirds of the 24 fragrance ingredients underwent chemical degradation. In some cases, such degradation was accompanied by an increase in thio-reactivity. These results prompted us to investigate the reactivity of the same ingredients using the direct peptide reactivity assay (DPRA). In the present work, the 24 chemicals were subjected to forced degradation for 150 days, and evaluated with both DPRA and HTS-DCYA methods. At the end of the study, four and eight compounds remained non-reactive in the DPRA and DCYA assay, respectively. Coumarin, benzyl salicylate, benzyl cinnamate and hexyl cinnamal were found unreactive in both assays, while cinnamal, cinnamyl alcohol, hydroxycitronellal and lilial were found negative in the DCYA but positive in the DPRA method. The incongruity in reactivity of these four compounds was attributed to a possible role of pro-oxidants formed upon degradation, resulting in depletion of peptide without formation of apparent covalent adducts with the test chemical. To validate this hypothesis, the effect of hydrogen peroxide as model pro-oxidant on both lysine- and cysteine-heptapeptide depletion in the DPRA method was thus investigated. The obtained results showed little effect of oxidative conditions on lysine depletion, while cysteine depletion was significantly affected by concentrations above 1.1 mg/L of hydrogen peroxide. Overall, both in chemico methods confirmed chemical instability should be considered when assessing the skin sensitization potential of (un)known chemicals with alternative methods.

Labeling of Nanofiber-Forming Peptides by Site-Directed Bioconjugation: Effect of Spacer Length on Self-Assembly.

BACKGROUND: The conjugation of small organic molecules to self-assembling peptides is a versatile tool to decorate nanostructures with original functionalities. Labeling with chromophores or fluorophores, for example, creates optically active fibers with potential interest in photonic devices. AIM AND OBJECTIVE: In this work, we present a rapid and effective labeling procedure for a self-assembling peptide able to form nanofibers. Rapid periodate oxidation of the N-terminal serine residue of the peptide and subsequent conjugation with dansyl moiety generated fluorophore-decorated peptides. RESULTS: Three dansyl-conjugated self-assembling peptides with variable spacer-length were synthesized and characterized and the role of the size of the linker between fluorophore and peptide in self-assembling was investigated. Our results show that a short linker can alter the self-assembly in nanofibers of the peptide. CONCLUSIONS: Herein we report on an alternative strategy for creating functionalized nanofibrils, able to expand the toolkit of chemoselective bioconjugation strategies to be used in site-specific decoration of self-assembling peptides.

Liquid chromatography-time-of-flight high-resolution mass spectrometry study and determination of the dansylated products of estrogens and their hydroxylated metabolites in water and wastewater.

A method combining liquid chromatography with a dual-probe ultraspray electrospray ionization (ESI) source and time-of-flight high-resolution mass spectrometry (LC-ESI-TOF/MS) was developed for the simultaneous determination of four steroidal sex hormones, estrone (E1), 17ß-estradiol (E2), 17α-ethinyl estradiol (EE2), and estriol (E3), as well as five of their hydroxylated metabolites, 2-hydroxyestrone (2-OHE1), 4-hydroxyestrone (4-OHE1), 16α-hydroxyestrone (16-OHE1), 2-hydroxyestradiol (2-OHE2), and 4-hydroxyestradiol (4-OHE2), in water samples in a short chromatographic run of 10 min. Derivatization of the analytes was optimized using dansyl chloride as the derivatizing agent. Under optimal positive ionization conditions, the following signals, which had not been previously reported, were observed (with theoretical values of m/z 377.1373 for 2- and 4-OHE1 and 378.1452 for 2- and 4-OHE2), corresponding to doubly derivatized catechol estrogens in the form of [M+2H]2+. These mass spectrometric signals were more abundant than those reported previously for the [M+H]+ forms of these hydroxylated metabolites. Solid-phase extraction (SPE) with an octadecyl-endcapped sorbent was used to pretreat tap water and effluent from a wastewater treatment plant (WWTP) in Santiago, Chile. The method achieved the simple, fast, and sensitive measurement of nine estrogens with quantitative recoveries (higher than 85.4%). Detection and quantification limits were between 1 and 17 ng L-1 and between 3 and 58 ng L-1, respectively, for all compounds in water. The estrogens E1 and E2 were found in WWTP effluent at concentrations of 7 ± 1 and 41 ± 1 ng L-1, respectively, and EE2 was detected at a concentration below the limit of quantitation. This study shows that the proposed method is suitable for the accurate, rapid, and selective determination of all these analytes at trace levels. Graphical abstract ᅟ.