Polymeric fluorescent dyes for labeling of proteins and nucleic acids

In order to increase the sensitivity of fluorescence labeling in biochemical reactions and diagnostic procedures a labeling technique with polymeric fluorescence dyes was established and tested for its applicability. The fluorescence dye is based on the fluorophor coumarine and was covalently linked to the model proteins strepavidine and IgG. The dye was synthesized by radical polymerization of three different types of functional monomers to ensure water solubility, covalent coupling to proteins, and fluorescence. The molecular weight range was between 20 and 200 kDa. Fractions of narrow molecular weight distribution were prepared by gel filtration on Superdex 200. The relationship between size and charge of the different fractions was analyzed by gel electrophoresis. Covalent conjugation to proteins was carried out by formation of a peptide bond between a carboxylic group of the functional monomers and an amino group of the protein mediated by 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide (EDC). A novel type of gel electrophoresis was developed in order to analyze and optimize the conjugation reaction; the results were in agreement with those from analytical ultracentrifugation with fluorescence detection. Hydrodynamic studies of the uncoupled dye and the protein-dye conjugates exhibited a drastic decrease of Stokes radius of the dye due to the coupling to the protein. Under optimum conditions the fluorescence intensity of a protein-polymeric dye conjugate was enhanced 40-fold compared to a monomeric dye. Biotin binding to the protein streptavidin was not affected significantly by the conjugation with the polymeric dye. At present, the applicability of the polymeric dye in biochemical and diagnostic reactions seems to be limited due to strong but unspecific hydrophobic interactions which might be overcome by using fluoresceine as monomeric dye.     

High-contrast and real-time visualization of membrane proteins in live cells with malachite green-based fluorogenic probes

Imaging dynamics of membrane proteins of live cells in a wash-free and real-time manner has been a challenging task. Herein, we report unprecedented applications of malachite green(MG), an organic dye widely used in pigment industry, as a switchable fluorophore to monitor membrane enzymes or noncatalytic proteins in live cells. Conformationally flexible MG is non-fluorescent in aqueous solution, yet covalent binding with endogenous proteins of cells significantly enhances its fluorescence at 670...     

Puromycin Analogues Capable of Multiplexed Imaging and Profiling of Protein Synthesis and Dynamics in Live Cells and Neurons

Newly synthesized proteins constitute an important subset of the proteome involved in every cellular process, yet existing chemical tools used to study them have major shortcomings. Herein we report a suite of cell‐permeable puromycin analogues capable of being metabolically incorporated into newly synthesized proteins in different mammalian cells, including neuronal cells. Subsequent labeling with suitable bioorthogonal reporters, in both fixed and live cells, enabled direct imaging and enrichment of these proteins. By taking advantage of the mutually orthogonal reactivity of these analogues, we showed multiplexed labeling of different protein populations, as well as quantitative measurements of protein dynamics by fluorescence correlation spectroscopy, could be achieved in live‐cell environments.     

Preparation and properties of vesicles from condensable amphiphilic amino acids

Three double‐chain amphiphiles with amino acid groups as hydrophilic moiety were synthesized. These amphiphiles can be easily dispersed in buffer solution to form transparent dispersion. Examination of the dispersion by transmission electron microscopy (TEM) showed the formation of stable vesicular aggregates, which was also confirmed by the ability to encapsulate water‐soluble dyes. Since amino acid groups are located on the surface of the vesicles, water‐soluble carbodiimide can induce the condensation of these groups to form peptide. The phase transition temperatures of these vesicles were estimated by differential scanning calorimetry (DSC), and a decrease of phase transition temperature was observed after polycondensation due to the disturbance of the ordered arrangement of the hydrophobic chains. The leakage rate of the vesicles before and after condensation was studied by monitoring the increase of fluorescence intensity of water‐soluble dye. These vesicles belong to the least permeable ones and the leakage rate can be controlled by varying the degree of condensation or the temperature.     

Unprecedented binary Cu(I)/Cu(II) catalyzed one-pot, three-component synthesis and evaluation of luminescent property of 2-amino-3-iminoethenylidene-2-indolones: a new class of merocyanine dye analogues

A facile and efficient binary Cu(I)/Cu(II) catalyzed one-pot, three-component synthesis of 2-amino-3-iminoethenylidene-2-indolones in excellent yield has been achieved. Remarkably, these newly synthesized, stable merocyanine dye analogues showed strong luminescence in the blue region with large Stokes shifts.     

Highly sensitive and selective cyanide detection via Cu2+ complex ligand exchange

A new type of fluorescent probe (1) with two triazole groups that are conjugated with a carbazole moiety was synthesized by a Cu(I)-catalyzed alkyne-azide click reaction for the selective and sensitive detection of cyanide via fluorescence enhancement by ligand exchange and metal ion removal.     

Exceptional time response,stability and selectivity in doubly-activated phenyl selenium-based glutathione-selective platform

A phenyl-selenium-substituted coumarin probe was synthesized for the purpose of achieving highly selective and extremely rapid detection of glutathione (GSH) over cysteine (Cys)/homocysteine (Hcy) without background fluorescence. The fluorescence intensity of the probe with GSH shows a ∼100-fold fluorescent enhancement compared with the signal generated for other closely related amino acids, including Cys and Hcy. Importantly, the substitution reaction with the sulfhydryl group of GSH at the 4-position of the probe, which is doubly-activated by two carbonyl groups, occurs extremely fast, showing subsecond maximum fluorescence intensity attainment; equilibrium was reached within 100 ms (UV-vis). The probe selectivity for GSH was confirmed in Hep3B cells by confocal microscopy imaging.     

Fluorimetric studies and noncovalent labeling of protein with the near-infrared dye HITCI for analysis by CE-LIF

1,1',3,3,3',3'-Hexamethylindotricarbocyanine iodide (HITCI) is a commercially available, positively charged, indocarbocyanine dye used typically as a laser dye in the near infrared (NIR). The absorbance and fluorescence properties of HITCI in a variety of solvent systems were determined. Results indicate that the fluorescence of HITCI is not significantly affected by the pH. Titration of HITCI with human serum albumin (HSA) and trypsinogen was carried out to investigate the interactions between this dye and proteins. These studies revealed that the absorbance and fluorescence properties of the dye change upon binding to protein in a wide range of solution pH's. The potential use of HITCI as a noncovalent protein labeling probe, therefore, was explored. Determination and separation of HITCI and HITCI-protein complexes was performed by capillary electrophoresis with diode-laser induced fluorescence detection (CE-LIF). Both pre-column and on-column noncovalent labeling methods are demonstrated.     

Lighting‐Up of the Dye Malachite Green with Mercury(II)–DNA and Its Application for Fluorescence Turn‐Off Detection of Cysteine and Glutathione

This work describes a novel strategy for the highly sensitive and selective detection of cysteine (Cys) and glutathione (GSH) based on the Hg²⁺–AGRO100–malachite green (MG) complex system. The dye MG, which has a very low quantum yield in aqueous solution by itself, can bind with the thymine‐rich DNA AGRO100 in the presence of Hg²⁺ ions to generate a striking fluorescence intensity enhancement of 1000‐fold. As sulfur‐containing amino acids, Cys and GSH effectively sequester Hg²⁺ ions from the Hg²⁺–AGRO100–MG complex structure to switch the ‘lit‐up’ chemosensor to the ‘off’ state (about a 50‐fold fluorescence intensity decrease), thus providing a facile, but effective, method to probe for Cys/GSH. The fluorescence titration, UV absorption, CD, and Raman spectra provide some insight into the structural and chemical basis for the enhancement effect. The formation of the Hg²⁺–AGRO100–MG complex significantly affects the electronic structure and conformation of the MG molecule by leading to an extended π system, which is the likely origin of the observed striking fluorescence intensity enhancement. Notably, the proposed sensing platform exhibits exquisite selectivity and sensitivity toward Cys/GSH with limits of detection of 5 nM for Cys and 10 nM for GSH, respectively. Furthermore, the straightforward assay design avoids labeling of the probe, uses only commercially available materials, and still displays comparable sensitivity and excellent selectivity.     

Specific and stable fluorescence labeling of histidine-tagged proteins for dissecting multi-protein complex formation

Labeling of proteins with fluorescent dyes offers powerful means for monitoring protein interactions in vitro and in live cells. Only a few techniques for noncovalent fluorescence labeling with well-defined localization of the attached dye are currently available. Here, we present an efficient method for site-specific and stable noncovalent fluorescence labeling of histidine-tagged proteins. Different fluorophores were conjugated to a chemical recognition unit bearing three NTA moieties (tris-NTA). In contrast to the transient binding of conventional mono-NTA, the multivalent interaction of tris-NTA conjugated fluorophores with oligohistidine-tagged proteins resulted in complex lifetimes of more than an hour. The high selectivity of tris-NTA toward cumulated histidines enabled selective labeling of proteins in cell lysates and on the surface of live cells. Fluorescence labeling by tris-NTA conjugates was applied for the analysis of a ternary protein complex in solution and on surfaces. Formation of the complex and its stoichiometry was studied by analytical size exclusion chromatography and fluorescence quenching. The individual interactions were dissected on solid supports by using simultaneous mass-sensitive and multicolor fluorescence detection. Using these techniques, formation of a 1:1:1 stoichiometry by independent interactions of the receptor subunits with the ligand was shown. The incorporation of transition metal ions into the labeled proteins upon labeling with tris-NTA fluorophore conjugates provided an additional sensitive spectroscopic reporter for detecting and monitoring protein-protein interactions in real time. A broad application of these fluorescence conjugates for protein interaction analysis can be envisaged.     

新型吲嗪三联吡啶的合成及其荧光性能

以醋酸钯为催化剂,三环己基膦为配体,碳酸铯为碱,甲苯为溶剂,4-溴-三联吡啶和2-甲酸甲酯-吲嗪经Heck偶联反应合成了一个新型的吲嗪三联吡啶(3),其结构经¹H NMR, ¹³C NMR和HR-MS表征。并研究了3的荧光性能。结果表明：溶剂极性增加,3的荧光强度降低,最大发射波长红移;Cu²⁺可完全淬灭3的荧光;3的Cu²⁺, Cd²⁺, Zn²⁺配合物对部分氨基酸有荧光响应。     

Study on the new fluorescence enhancement system of Tb-1,10-bis(2′-carboxylphenyl)-1,4,7,10-tetraoxadecane in silver chloride collosol and its analytical application

A new salicylic-based open-chain crown ether ligand, 1,10-bis(2′-carboxylphenyl)-1,4,7,10-tetraoxadecane (BCPTD) was synthesized. Solutions of its complex with Tb³⁺ can emit the intrinsic fluorescence of Tb³⁺. The fluorescence intensity of the complex in KCl solution was enhanced by the addition of silver(I), leading to a new fluorescence enhancement phenomenon. The spectrofluorimetric determination of traces of silver(I) based on the above phenomenon was carried out. The excitation and emission wavelengths are 298 and 545 nm, respectively. Under optimal conditions, the differential value of fluorescence intensity in the absence and presence of Ag⁺ was proportional to the concentration of silver(I) in the range 0.5-20 μg ml⁻¹. The method was applied to the determination of silver(I) in a standard ore sample. The analytical performance is investigated in detail by using common aromatic carboxylic acids or synthetic analogues of BCPTD as ligands to replace BCPTD. It was found that Tb-aromatic acid complexes did not result in fluorescence enhancement of Tb³⁺ in AgCl collosol. The phenomenon was only observed in Tb(III) with BCPTD or its open-chain crown ether analogues solutions.In addition, the enhancement of the fluorescence intensity of terbium(III) in these complexes depends on the extent of formation of the AgCl collosol.     

Part-per-trillion level detection of estradiol by competitive fluorescence immunoassay using DNA/dye conjugate as antibody multiple labels

Fluorescent organic dyes are currently the standard signal-generating labels used in microarray quantification. However, new labeling strategies are needed to meet the demand for high sensitivity in the detection of low-abundance proteins and small molecules. In this report, a long-chain DNA/dye conjugate was used to attach multiple fluorescence labels on antibodies to improve signal intensity and immunoassay sensitivity. Compared with the 30 base-pair (bp) oligonucleotide used in our previous work [Q. Zhang, L.-H. Guo, Bioconjugate Chem. 18 (2007) 1668-1672], conjugation of a 219 bp DNA in solution with a fluorescent DNA binder SYBR Green I resulted in more than sixfold increase in signal intensity, consistent with the increase in bp number. In a direct immunoassay for the detection of goat anti-mouse IgG in a mouse IgG-coated 96-well plate, the long DNA conjugate label also produced higher fluorescence than the short one, accompanied by about 15-fold improvement in the detection limit. To demonstrate its advantage in real applications, the DNA/dye conjugate was employed in the competitive immunoassay of 17β-estradiol, a clinically and environmentally important analyte. The biotin-terminated DNA was attached to biotinylated anti-estradiol antibody through the biotin/streptavidin/biotin bridge after the immuno-reaction was completed, followed by conjugation with SYBR Green I. The limit of detection for 17β-estradiol is 1.9 pg mL⁻¹, which is 200-fold lower than the assay using fluorescein-labeled antibodies. The new multiple labeling strategy uses readily available reagents, and is also compatible with current biochip platform. It has great potential in the sensitive detection of protein and antibody microarrays.     

A novel fluorescent sensor for metal cations and protons based of bis-1,8-naphthalimide

A newly synthesized bis-1,8-naphthalimide aimed to increase its fluorescence intensity in the presence of protons or metal cations has been investigated. Its spectral photophysical characteristics in acetonitrile and chloroform solutions are described. The influence of metal cations (Zn(2+), Ni(2+), Ce(3+), Co(2+), Cu(2+) and Ag(+)) and protons on the fluorescence intensity has been investigate with regard to obtain fluorescence sensors for this ions in the environment.     

Metabolically stable isotope labeling prior to electrophoretic protein separation reveals differences in fractional synthesis rates between mitochondrial aldehyde dehydrogenase isoforms

Living mice were subjected to whole body labeling by intravenous infusion of [(13)C]glucose as the sole carbon source. After 10 h infusion the mice were sacrificed, and liver proteins were separated by two-dimensional polyacrylamide gel electrophoresis. Five spots were found to contain mitochondrial aldehyde dehydrogenase (ALDH2) by matrix assisted time of flight mass spectrometry protein identification. By measuring the isotopologue mass distributions of peptide ions, and modeling the (13)C content of the precursor amino acid pool, the fractional synthesis rate of ALDH2 molecules synthesized during the labeling period was determined. One of the five spots was observed to have a five-fold higher fraction of (13)C-containing newly synthesized ALDH2 than the spot with the highest ALDH2 content, and contained more than 60% of newly synthesized ALDH2 although it accounted for less than 20% of the total ALDH2 detected. The total range in the fraction of (13)C-containing proteins between different ALDH2 spots approached 50-fold. The ability to quantitatively characterize different protein isoforms of biological origin for ALDH2 and other proteins from living animals provides new avenues for the exploration of protein function.     

One-pot labeling and purification of peptides and proteins with fluorescein maleimide

Fluorescein labeling of peptides and proteins is required for numerous biophysical or biological experiments such as fluorescence microscopy, fluorescence resonance energy transfer (FRET) or fluorescence imaging. The commonly used strategy relied on the coupling of the dye reagent followed by a gel filtration to recover the labeled molecule. Here we report a simplified method for the labeling of peptides and proteins on a cysteine residue and their purification. The method is based on the precipitation of peptides and proteins in acetone, fluorescein maleimide being soluble in this solvent. The excess of dye is fully eliminated after a couple of acetone washes and the precipitated peptide or protein is readily recovered.     

Determination of dissolved oxygen in water based on its quenching effect on the fluorescent intensity of bis(2,2'-bipyridine)-5-amino-1,10-phenanthroline ruthenium complex

A water-soluble fluorescence dye, bis(2,2'-bipyridine)-5-amino-1,10-phenanthroline ruthenium complex (Ru(bpy)(2)(5-NH(2)-1,10-phen)), was synthesized and used as a fluorescence probe for detecting dissolved oxygen in water. The fluorescence intensity of the probe in different dissolved-oxygen concentrations was investigated. The sensitivity of the probe was evaluated in terms of the ratio I(N(2))/I(O(2)), where I(N(2)) and I(O(2)) correspond to the detected fluorescence intensity of nitrogen and oxygen-saturated solutions, respectively. The experimental results showed that the probe yielded a linear Stern-Volmer plot, and had a I(N(2))/I(O(2)) ratio of about 5.2. The detection limit, defined as three-times the standard deviation, was 8.6 × 10(-7) mol L(-1) after eleven determinations of nitrogen-saturated blank solutions. Additionally, the probe was pH-insensitive and ionic strength-independent with good characteristics of practicality and selectivity.     

新型水溶性不对称五甲川菁染料的合成、光稳定性及蛋白质的荧光标记

合成并表征了系列水溶性五甲川菁染料, 研究了其在不同溶剂中的光谱性能. 结果表明, 染料在水中的最大吸收和荧光光谱在647~665 nm波长范围内, 荧光量子产率达到0.1左右. 考察了N位取代基对染料水溶液光稳定性的影响, 结果表明, 在N原子上引入带有苯环结构和大体积的磺酸基, 可以提高染料的光稳定性. 高效液相色谱(HPLC)分析结果表明, 染料4a的N-羟基琥珀酰亚胺(NHS)活性酯标记牛血清白蛋白(BSA)的检测限为1.2×10^-8 mol/L, 与紫外检测相比, 检测灵敏度提高了近2个数量级.     

Design,Synthesis, Spectroscopic Characterisation and In Vitro Cytostatic Evaluation of Novel Bis(coumarin-1,2,3-triazolyl)benzenes and Hybrid Coumarin-1,2,3-triazolyl-aryl Derivatives

In this work, a series of novel 1,2,3-triazolyl-coumarin hybrid systems were designed as potential antitumour agents. The structural modification of the coumarin ring was carried out by Cu(I)-catalysed Huisgen 1,3-dipolar cycloaddition of 7-azido-4-methylcoumarin and terminal aromatic alkynes to obtain 1,4-disubstituted 1,2,3-triazolyl-coumarin conjugates 2a–g, bis(1,2,3-triazolyl-coumarin)benzenes 2h–i and coumarin-1,2,3-triazolyl-benzazole hybrids 4a–b. The newly synthesised hybrid molecules were investigated for in vitro antitumour activity against five human cancer cell lines, colon carcinoma HCT116, breast carcinoma MCF-7, lung carcinoma H 460, human T-lymphocyte cells CEM, cervix carcinoma cells HeLa, as well as human dermal microvascular endothelial cells (HMEC-1). Most of these compounds showed moderate to pronounced cytotoxic activity, especially towards MCF-7 cell lines with IC₅₀ = 0.3–32 μM. In addition, compounds 2a–i and 4a–b were studied by UV-Vis absorption and fluorescence spectroscopy and their basic photophysical parameters were determined.     

Fluorescent nitric oxide detection by copper complexes bearing anthracenyl and dansyl fluorophore ligands

Anthrancenyl and dansyl fluorophore ligands [AnCH2pipCS(2)K (1), Ds-Hen (2), Ds-HAMP (3), Ds-HAQ (4), and Ds-HAPP (5)] were prepared for coordination to Cu(II). Five Cu complexes, [Cu(AnCH2pipCS2)2] (6), [Cu(Ds-en)2] (7), [Cu(Ds-AMP)2] (8), [Cu(Ds-AQ)2] (9), and [Cu(Ds-APP)(OTf)] (10), were synthesized as candidates for detecting nitric oxide (NO) by fluorescence and characterized by X-ray crystallography. A decrease in fluorescence compared to that of the free ligands (1-5) was measured following the formation of the corresponding five Cu(II) complexes 6-10. Fluorescence was restored in the presence of NO in CH3OH/CH2Cl2 solutions of the compounds. Complexes 7, 8, and 10 exhibited a fluorescence response to NO in pH 7.0 or 9.0 buffered aqueous solutions. Spectroscopic studies revealed that NO-induced fluorescence enhancement in these Cu(II) complexes occurs by reduction to Cu(I). The present studies demonstrate that Cu(II) complexes are effective as fluorescent probes for detecting NO in both organic and aqueous environments.