Adrenergic Modulation With Photochromic Ligands.

Adrenoceptors are ubiquitous and mediate important autonomic functions as well as modulating arousal, cognition, and pain on a central level. Understanding these physiological processes and their underlying neural circuits requires manipulating adrenergic neurotransmission with high spatio-temporal precision. Here we present a first generation of photochromic ligands (adrenoswitches) obtained via azologization of a class of cyclic amidines related to the known ligand clonidine. Their pharmacology, photochromism, bioavailability, and lack of toxicity allow for broad biological applications, as demonstrated by controlling locomotion in zebrafish and pupillary responses in mice.

Synthesis, Characterization, Photoluminescence, Molecular Docking and Bioactivity of Zinc (II) Compounds Based on Different Substituents.

Six new zinc(II) complexes were prepared by the reaction of ZnBr2 or ZnI2 with 4'-(substituted-phenyl)-2,2':6',2''-terpyridine compounds, bearing p-methylsulfonyl (L1), p-methoxy (L2) and p-methyl (L3), which were characterized by elemental analysis, FT-IR, NMR and single crystal X-ray diffraction. The antiproliferative properties against Eca-109, A549 and Bel-7402 cell lines and the cytotoxicity test on RAW-264.7 of these compounds were monitored using a CCK-8 assay, and the studies indicate that the complexes show higher antiproliferative activities than cisplatin. The interactions of these complexes with CT-DNA and proteins (BSA) were studied by UV-Vis, circular dichroism (CD) and fluorescent spectroscopy, respectively. The results indicate that the interaction of these zinc(II) complexes with CT-DNA is achieved through intercalative binding, and their strong binding affinity to BSA is fulfilled through a static quenching mechanism. The simulation of the complexes with the CT-DNA fragment and BSA was studied by using molecular docking software. It further validates that the complexes interact with DNA through intercalative binding mode and that they have a strong interaction with BSA.

Detection of esterase activity by chromogenic and fluorogenic probe based on an O-nitrobenzoxadiazole (O-NBD) unit.

We designed a conjugated molecule bearing an O-nitrobenzoxadiazole (O-NBD) unit and an acetylated trimethyl lock as a chromogenic and fluorogenic probe for the detection of esterase activity. The designed molecule was briefly synthesized from a commercially available compound in two steps. Several experiments revealed that the conjugated molecule serves as a sensitive chromogenic and fluorogenic probe for the detection of porcine liver esterase activity. Mechanistic studies indicated that an intramolecular O- to N-NBD migration is involved in the chromogenic/fluorogenic phenomena. The results here would be helpful for designing other O-NBD-based chromogenic/fluorogenic probes in future.

Covalently deposited dyes: a new chromogen paradigm that facilitates analysis of multiple biomarkers in situ.

Multiplexed analysis of multiple biomarkers in a tissue sample requires use of reporter dyes with specific spectral properties that enable discrimination of signals. Conventional chromogens with broad absorbance spectra, widely used in immunohistochemistry (IHC), offer limited utility for multiplexed detection. Many dyes with narrow absorbance spectra, eg rhodamines, fluoresceins, and cyanines, potentially useful for multiplexed detection are well-characterized; however, generation of a chromogenic reagent useful for IHC analysis has not been demonstrated. Studies reported herein demonstrate utility of tyramine-chemistry for synthesis of a wide variety of new chromogenic dye conjugates useful for multiplexed in situ analysis using conventional light microscopes. The dyes, useful individually or in blends to generate new colors, provide signal sensitivity and dynamic range similar to conventional DAB chromogen, while enabling analysis of co-localized biomarkers. It is anticipated that this new paradigm will enable generation of a wide variety of new chromogens, useful for both research and clinical biomarker analysis that will benefit clinicians and patients.

Exploiting sp2 -Hybridisation in the Development of Potent 1,5-α-l-Arabinanase Inhibitors.

The synthesis of potent inhibitors of GH93 arabinanases as well as a synthesis of a chromogenic substrate to measure GH93 arabinanase activity are described. An insight into the reasons behind the potency of the inhibitors was gained through X-ray crystallographic analysis of the arabinanase Arb93A from Fusarium graminearum. These compounds lay a foundation for future inhibitor development as well as for the use of the chromogenic substrate in biochemical studies of GH93 arabinanases.

A Dispersion-Dominated Chromogenic Strategy for Colorimetric Sensing of Glutathione at the Nanomolar Level Using Gold Nanoparticles.

A dispersion-dominated chromogenic strategy for glutathione sensing is developed. Glutathione prevents the aggregation of arginine-modified gold nanoparticles via mercury-thiol interaction, which allows for glutathione sensing at the nanomolar level (10.9 × 10(-9) m) with facile operation and naked-eye readout.

Synthesis and application of resorufin ß-D-glucuronide, a low-cost chromogenic substrate for detecting Escherichia coli in drinking water.

The development of low-cost tests for Escherichia coli is hampered by the expense and limited choice of enzyme substrates. Most chromogenic substrates are required in costly amounts, while fluorogenic substrates require an additional apparatus (e.g., an ultraviolet lamp) to be detected. Herein, we propose an alternative chromogenic substrate, resorufin ß-d-glucuronide (REG), which is exceptionally sensitive and may be employed in very small amounts. We show that REG can be produced similarly to other simple glucuronides and should therefore be no more expensive. The compound is used by both healthy and injured E. coli, resulting in a pronounced color change from orange to a bright pink. Because the released dye (resorufin) has a high extinction coefficient, substantially lower amounts are needed than for commercially available substrates. The potential of this substrate is demonstrated by a presence/absence test requiring just 0.1 mg of REG/100 mL of water sample, one hundredth of the quantity needed for common chromogenic substrates, with an estimated bulk cost of ≤0.1 U.S. cents/test. REG shows promise as a chromogenic substrate for E. coli detection and should be considered in the development of new water tests, especially for low-income settings.

Chromogenic substrate from 4-nitro-1-naphthol for hydrolytic enzyme of neutral or slightly acidic optimum pH: 4-nitro-1-naphthyl-ß-D-galactopyranoside as an example.

At pH from 5.5 to 7.6, absorptivity of 4-nitro-1-naphthol at 450 nm is over 2.1-fold of that of para-nitrophenol at 405 nm and over 9.6-fold of that of ortho-nitrophenol at 415 nm. On 4-nitro-1-naphthyl-ß-D-galactopyranoside at pH 7.4, catalytic efficiency of Escherichia coli ß-D-galactosidase is 3-fold of that on para-nitrophenyl-ß-D-galactopyranoside and about 40% of that on ortho-nitrophenyl-ß-D-galactopyranoside, and produces a lower quantification limit of penicillin G by enzyme-linked-immunoabsorbent-assay. Hence, 4-nitro-1-naphthol is favorable to prepare chromogenic substrates of hydrolytic enzymes of neutral or slightly acidic optimum pH.

Design, synthesis, and characterization of chromogenic substrates of coagulation factor XIIIa.

A series of Glu(pNA)-containing peptides was designed to determine the activity of the transglutaminase factor XIIIa at 405 nm due to p-nitroaniline release. The most suitable substrate properties were found for peptides containing the Glu(pNA) residue in the second position from the N terminus. For the best substrate 12 (H-Tyr-Glu(pNA)-Val-Lys-Val-Ile-Gly-NH(2)), a k(cat)/K(m) value of 3531 s(-1)M(-1) was found. Although the k(cat)/K(m) values of the Glu(pNA) peptides are more than 100-fold reduced compared with the previously reported cleavage of natural glutamine-containing substrates such as α(2)-antiplasmin and ß-casein, these chromogenic substrates can be useful tools for convenient determination of FXIII-A(2)* activity e.g., for in vitro inhibitor screening. As an example, peptide 12 was used to characterize the inhibition of FXIII-A(2)* by the well-known irreversible inhibitor iodoacetic acid.

Synthesis of alanine-based colorimetric sensors and enantioselective recognition of aspartate and malate anions.

Two chiral colorimetric sensors (1,2) were synthesized and characterized by spectroscopic techniques and their enantioselective recognition of chiral dicarboxylic anions (D/L-aspartate and D/L-malate) was examined by UV-vis and (1)H NMR spectroscopy. Interaction of the receptors 1 and 2 with the enantiomers of aspartate or malate caused different color changes, and they act as optical chemosensors for the recognition of D-aspartate vs. L-aspartate and d-malate vs.l-malate. Receptor 1 exhibits high enantioselective binding for aspartate anions [K(A(D))/K(A(L)) = 12.15].

Development of [Ile4°]HTLV-I protease inhibition assay using novel fluorogenic and chromogenic substrate.

HTLV-I is a debilitating and/or lethal retrovirus that causes HTLV-I-associated myelopathy/tropical spastic paraparesis, adult T-cell leukemia and several inflammatory diseases. HTLV-I protease is an aspartic retropepsin involved in HTLV-I replication and its inhibition could treatHTLV-I infection. A recombinant L40I mutant HTLV-I protease was designed and obtained from Escherichia coli, self-processingand purification by ion-exchange chromatography. The protease was refolded by a one-step dialysis and recovered activity. The cleavage efficiency of the [Ile4°]HTLV-I protease was at least 300 times higher for a fluorescent substratethan that of our previously reported recombinant His-tagged non-mutated HTLV-I protease. In addition, we designed and synthesized a substrate containing a highly fluorescent Mca moiety in the fragment before the scissile bond, and a chromogenic p-nitrophenylalanine moiety after the scissile bond that greatly amplified spectrometry detection and improved the HTLV-I protease inhibition potency assay. The HTLV-I protease inhibition assay with the [Ile4°]HTLV-I protease and fluorogenic substrate requires distinctively less protease, substrate, inhibitor and assay time than our previous methods. This means our new assay is more cost-effective and more time-efficient while being reproducible and less labor-intensive.

Design and synthesis new colorimetric receptors for naked-eye detection of biologically important fluoride and acetate anions in organic and arsenite in aqueous medium based on ICT mechanism: DFT study and test strip application.

Novel three colorimetric anion receptors R1, R2 and R3 have been designed and synthesized via condensation reaction and characterized using IR, MS, and NMR spectroscopic techniques. Anion sensing properties were studied using colorimetric, UV-vis titration, 1H NMR titration, and Cyclic Voltammetric Studies. Comparing the UV-visible titration data of the receptors R1 and R2, R2 showed high redshift (∆λmax) in the mixed competitive solution (DMSO: H2O, 9: 1; v/v) of about 155 nm, 157 nm, 169 nm for Na+F-, Na+AcO-, and Na+AsO2- ions with LOD of 0.23 ppm, 0.18 ppm, and 0.30 ppm, respectively. The observed spectral change of receptor R2 is due to the anion-induced deprotonation of the OH proton, which is confirmed by UV-vis titration, 1HNMR titration, and cyclic voltammetric studies. Theoretical studies via DFT calculation were carried for R1 and R2 to optimize the structure and to explain the anion-binding mechanism. The application of designed receptor R2 was successfully demonstrated for the detection of F- and AsO2- ions using a test strip. The receptors R1 and R2 proved itself to be potentially useful for real-life application by sensing F- and AcO- ions in real samples like toothpaste, mouthwash, vinegar and seawater in a complete aqueous medium.

ß-Lactamase triggered visual detection of bacteria using cephalosporin functionalized biomaterials.

We report the conjugation of a chromogenic cephalosporin ß-lactamase (ßL) substrate to polymers and integration into biomaterials for facile, visual ßL detection. Identification of these bacterial enzymes, which are a leading cause of antibiotic resistance, is critical in the treatment of infectious diseases. The ßL substrate polymer conjugate undergoes a clear to deep yellow color change upon incubation with common pathogenic Gram-positive and Gram-negative bacteria species. We have demonstrated the feasibility of formulating hydrogels with the ßL substrate covalently tethered to a poly(ethylene glycol) (PEG) polymer matrix, exhibiting a visible color change in the presence of ßLs. This approach has the potential to be used in diagnostic biomaterials for point-of-care detection of ßL-producing bacteria, helping combat the spread of drug resistant microbes.

Detection of creatinine by a designed receptor.

An artificial receptor has been designed to bind creatinine with a color change (chromogenic response) caused by proton transfer from one end of the receptor to the other. The receptor was synthesized and found to extract creatinine from water into chlorocarbon solvents. The color change in the organic layer is specific for creatinine relative to other organic solutes, and it is selective for creatinine relative to sodium, potassium, and ammonium ions. The chromogenic mechanism is revealed by x-ray crystal structures of creatinine, the free receptor, and the complex, showing "induced fit" binding resulting from electronic complementarity between host and guest.

Hg2+ -selective chromogenic and fluorogenic chemodosimeter based on thiocoumarins.

The highly selective chemodosimetric behavior of thiocoumarins toward Hg(2+) ions was investigated; significant chromogenic and fluorogenic signaling of Hg(2+) ions occurs from the transformation of thiocoumarin to coumarin by Hg(2+)-induced selective desulfurization.

A selective redox and chromogenic probe for Hg(II) in aqueous environment based on a ferrocene-azaquinoxaline dyad.

A new chemosensor molecule 4 based on a ferrocene-azaquinoxaline dyad effectively recognizes Hg(2+) in an aqueous environment as well as Pb(2+) and Zn(2+) metal cations in CH(3)CN solution through three different channels. Upon recognition, an anodic shift of the ferrocene/ferrocenium oxidation peaks and a progressive red shift (Deltalambda = 112-40 nm) of the low energy band, in their absorption spectra, is produced. These changes in the absorption spectra are accompanied by color changes from orange to deep green, for Hg(2+), and to purple in the cases of Pb(2+) and Zn(2+). Remarkably, the redox and colorimetric responses toward Hg(2+) are preserved in the presence of water (CH(3)CN/H(2)O, 3/7). The emission spectrum of 4 in CH(3)CN (lambda(exc) = 270 nm) undergoes important chelation enhancement of fluorescence (CHEF) in the presence of Hg(2+) (CHEF = 204), Pb(2+) (CHEF = 90), and Zn(2+) (CHEF = 184) metal cations. Along with the spectroscopic data, the combined (1)H NMR data of the complexes and the theoretical calculation suggest the proposed bridging coordination modes.

A ditopic colorimetric sensor for fluoride ion based on thiourea mercury complex.

A novel ditopic chromogenic receptor, N-5-(8-hydroxy)quinoline-N'-4'-nitro-phenyl thiourea (1), was synthesized. The metal complex 1-Hg(2+) showed sensitive and highly selective responses to F(-) over other anions such as CH(3)CO(2)(-), H(2)PO(4)(-), HSO(4)(-) and Cl(-). 1-Hg(2+)-F(-) complex formed, which promoted the intramolecular charge transfer and led to a dramatic spectral change. The color of 1-Hg(2+) solution changed from colorless to red upon addition of F(-). Thus, a colorimetric assay of F(-) was developed in acetonitrile by naked-eye detection. F(-) behaved linearly in the 8.0 x 10(-6) to 2.0 x 10(-5) mol L(-1) concentration range with LOD as 1.4 x 10(-6) mol L(-1).

Trimethyl lock: a stable chromogenic substrate for esterases.

p-Nitrophenyl acetate is the most commonly used substrate for detecting the catalytic activity of esterases, including those that activate prodrugs in human cells. This substrate is unstable in aqueous solution, limiting its utility. Here, a stable chromogenic substrate for esterases is produced by the structural isolation of an acetyl ester and p-nitroaniline group using a trimethyl lock moiety. Upon ester hydrolysis, unfavorable steric interactions between the three methyl groups of this o-hydroxycinnamic acid derivative encourage rapid lactonization to form a hydrocoumarin and release p-nitroaniline. This "prochromophore" could find use in a variety of assays.

Preparation and characterization of thiacalix[4]arene coated water-soluble CdSe/ZnS quantum dots as a fluorescent probe for Cu2+ ions.

Highly fluorescent water-soluble CdSe/ZnS (core/shell) quantum dots (QDs) as a fluorescent Cu2+ ion probe were synthesized using thiacalix[4]arene carboxylic acid (TCC) as a surface coating agent. Hydrophobic trioctylphosphine oxide (TOPO) capped CdSe/ZnS QDs were overcoated with TCC in tetrahydrofuran at room temperature, and deprotonation of the carboxyl groups of TCC resulted in the formation of water-soluble QDs. The surface structure of the QDs was characterized by using transmission electron microscopy (TEM) and fluorescence correlation spectroscopy (FCS). TEM images showed that TCC-coated QDs were monodispersed with the particle size (core-shell moiety) of approximately 5 nm. Hydrodynamic diameter of the TCC-coated QDs was determined to be 8.9 nm by FCS, showing that the thickness of the surface organic layer of the QDs was approximately 2 nm. These results indicate that the surface layer of TCC-coated QDs forms a bilayer structure consisting of TOPO and TCC molecules. TCC-coated CdSe/ZnS QDs were highly fluorescent (quantum yield, 0.21) compared to the QDs surface-modified with mercaptoacetic acid and mercaptoundecanoic acid. Fluorescence of the TCC-coated QDs was effectively quenched by Cu2+ ions even in the presence of other transition metal ions such as Cd2+, Zn2+, Co2+, Fe2+, and Fe3+ ions in the same solution. The Stern-Volmer plot for the fluorescence quenching by Cu2+ ions showed a linear relationship up to 30 microM of Cu2+ ions. The ion selectivity of TCC-coated QDs was determined by measurements of fluorescence responses towards biologically important transition metal ions (50 microM) including Fe2+, Fe3+, Co2+>Zn2+, Cd2+. The fluorescence of TCC-coated QDs was almost insensitive to other biologically important ions such as Na+, K+, Mg2+, and Ca2+, suggesting that TCC-coated QDs can be used as a fluorescent Cu2+ ion probe for biological samples. A possible quenching mechanism by Cu2+ ions was also discussed on the basis of a Langmuir-type adsorption isotherm.

Selection of new chromogenic substrates of serine proteinases using combinatorial chemistry methods.

Chemical synthesis, physicochemical characterization and kinetic investigations of a tetrapeptide library of chromogenic substrates containing the amide of 5-amino-2-nitrobenzoic acid (Anb(5,2)-NH(2)) at their C-termini are reported. Anb(5,2)-NH(2) served as a chromophore released upon enzymatic action. The library consisting of 9567 peptides was synthesized using the portioning-mixing method and was screened against bovine a-chymotrypsin and human leukocyte elastase in solution applying an iterative approach. The selected chromogenic substrates were resynthesized and further modified at their N- and C-termini. Finally, two sequences, Z-Phe-Ala-Thr-Tyr-Anb(5,2)-NH(2) and Z-Phe-Phe-Pro-Val-Anb(5,2)-NH(2), were obtained as highly specific substrates for bovine alpha-chymotrypsin and human leukocyte elastase, respectively. The method of synthesis and selection of chromogenic substrates of serine proteinases described herein is straightforward and can be applied to design substrates for other proteases.