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.

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.

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.

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.

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.

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.

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.

Synthesis of substrates for periodate-coupled assay of phospholipases C and sphingomyelinases.

A series of 4-nitrophenyl (pNP) and 4-methylumbelliferyl (4MU) substrate analogues of phosphatidyl choline (PC) and phosphatidic acid (PA) were synthesized from 4-bromo-1-butene by ether formation, olefin epoxidation and ring opening with the phosphate head group. The pNP PC analogue, 4-(4-nitrophenoxy)-2-hydroxy-butyl-1-phosphoryl choline (1) was evaluated in assays of fungal sphingomyelinases, also displaying phospholipase C activity. Reactions were terminated with a periodate-containing stop solution, leading to liberation of pNP, quantified spectrophotometrically in an end-point measurement. A kinetic evaluation of sphingomyelinases from Kionochaeta sp. and Penicillium emersonii showed relatively high KM and low kcat values for this substrate, limiting its practical applicability in assays with low sphingomyelinase concentrations.

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.

Substituent effects on the properties of photochromic hybrid diarylethenes with a naphthalene moiety.

Four new unsymmetrical photochromic diarylethenes bearing both naphthalene and thiophene moieties were synthesized, and the structures of two diarylethenes were determined by single-crystal X-ray diffraction analysis. The naphthalene ring was connected directly to the central perfluorocyclopentene ring as an aryl moiety and available to participate in photoisomerization reaction. All the diarylethenes exhibited favorable photochromism and functioned as fluorescence switches in both solution and poly(methyl methacrylate) films. The electron-withdrawing substituent significantly shifted the absorption maxima to a longer wavelength and evidently suppressed the cycloreversion quantum yield, whereas the electron-donating substituents enhanced the fluorescence quantum yield of diarylethenes with a naphthalene moiety. Furthermore, cyclic voltammograms suggested that the oxidation onsets and band-gaps of the open-ring isomers were much bigger than those of the closed-ring isomers. The results indicated that the substituents at the 5-position of thiophene ring could availably modulate their optical and electrochemical behaviors.

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.

In vitro assay for protease activity of proprotein convertase subtilisin kexins (PCSKs): an overall review of existing and new methodologies.

The mammalian proprotein convertase subtilisin kexins (PCSKs) previously called proprotein or prohormone convertases (PCs) are a family of Ca(+2)-dependent endoproteases in the subtilisin family. These proteolytic enzymes exert their many crucial physiological and biological functions in vivo via their ability to cleave larger inactive precursor proteins into their biologically active mature forms. This event takes place in a highly efficient and selective manner. Such actions of PCSKs either alone or in combination to cleave specific protein bonds are the hallmark events that not only define the normal functions and metabolism of the body but also may lead to a variety of diseases or disorders with associated conditions. These include among others, diabetes, obesity, cancer, cardiovascular diseases, reproduction abnormalities as well as viral bacterial infections. These conditions were the direct consequences of an enhanced level of enzymatic activity of one or more PCSKs except only PCSK9, whose protease activity in relation to its physiological substrate has yet to be characterized. Owing to this finding, a large number of research studies have been exclusively devoted to develop rapid, efficient and reliable in vitro methods for examining the protease activity of these enzymes. Several assays have been developed to monitor PCSK activity and these are widely used in chemical, biochemical, cellular and animal studies. This review will cover various methodologies and protocols that are currently available in the literature for PCSK activity assays. These include liquid phase methods using fluorogenic, chromogenic and intramolecularly quenched fluorescent substrates as well as a newly developed novel solid phase fluorescence method. This review will also highlight the usefulness of these methodologies and finally a comparative analysis has been made to examine their merits and demerits with some key examples.

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.

A selective chromogenic chemosensor for carboxylate salt recognition.

A new heteroditopic chromogenic chemosensor bearing a crown ether substituted at the intraannular position with a nitrophenylthiourea moiety has been synthesized. The binding behavior of this sensor was investigated by (1)H NMR spectroscopy and UV-vis spectroscopy. The receptor binds in a cooperative fashion to both a potassium cation and a carboxylate anion whereas a sodium cation sequesters an anion from the anion-receptor complex. The binding events are confirmed by selective color changes of the chemosensor solution.

A highly selective and sensitive colorimetric chemosensor for Fe(2+) based on fluoran dye.

A highly selective chemosensor based on fluoran dye for Fe(2+), 2'-anilino-3'-methyl-6'-dibuthylamino-N-((2'-(2''-ethylimino) methyl) naphthalen-2-ol) iso-indolin-1-one-fluoran (5), was designed and synthesized. The chemical structures of all the intermediates and the fluoran dye 5 are characterized by (1)H NMR, (13)C NMR, Ms and elemental analysis. Upon addition of Fe(2+), the fluoran dye 5 shows a new peak around 658nm in its absorption spectra, and the color of solution changed from colorless to greenish black. Whereas other ions including Mg(2+), Pb(2+), Ni(2+), Hg(2+), Cd(2+), Fe(3+), Cu(2+), Zn(2+) and Al(3+) and so on induced basically no spectral change, which constituted a Fe(2+) highly sensitive and selective colorimetric chemosensor by "naked eyes".

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.

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.