A Rhodamine-coumarin Triazole Conjugate as a Fluorescent Chemodosimeter for Cu(II) Detection and its Application in Live Cell Bioimaging.

A rhodamine-triazole fluorescent probe bearing a coumarin moiety RTC was synthesized using the Cu(I)-catalyzed click reaction. The rhodamine-triazole conjugate was highly selective to Cu2+ among other metal ions, including Ca2+, Co2+, Cu2+, Cd2+, Mg2+, Fe2+, Fe3+, Hg2+, Zn2+, Ni2+, Pd2+ and Pb2+ in physiological conditions. Upon the addition of Cu2+, the colorless RTC solution turned pink and exhibited a significant fluorescence emission centered at 578 nm. The binding of Cu2+ induced a hydrolysis reaction, leading to a release of the coumarin unit from the rhodamine probe, as confirmed by mass spectrometric data. From the fluorescence titration, the detection limit of RTC for Cu2+ was determined to be 21 nM (1.3 ppb). The sensor was responsive to Cu2+ in a wide pH range and successfully applied to monitor Cu2+ in HEK293T cells by confocal fluorescence imaging.

A Rhodamine-based Fluorescent Chemodosimeter for Au3+ in Aqueous Solution and Living Cells.

A highly selective rhodamine hydrazide-based fluorescent chemosensor for Au3+ detection was developed. The aqueous solution of rhodamine N-hydroxysemicarbazide (RHS), in the presence of Au3+, exhibited a significant 55-fold turn-on fluorescence response at 591 nm and a colorimetric change from colorless to pink. Other interested ions including Li+, Na+, K+, Cs+, Mg2+, Ca2+, Ba2+, Pb2+, Mn2+, Co2+, Ni2+, Ag+, Cd2+, Cu2+, Hg2+, Zn2+, Sn2+, Fe2+, Fe3+, Cr3+, Ce3+ did not induce any distinct color/spectral changes. The irreversible detection mechanism occurred via Au3+-promoted 5-exo-trig ring closure to yield 1,3,4-oxadiazole-2-one product. The RHS probe is non-responsive to other biologically relevant metal ions and the limit of detection for Au3+ was calculated to be 0.5 µM with a linear range of 0 to 90 µM. Fluorescence bioimaging of Au3+ in HepG2 cells was also successfully demonstrated.

A highly sensitive and selective rhodamine-semicarbazide based fluorescent sensor for Cu2+ detection in real water samples and fluorescence bioimaging in HepG2 cells.

A colorimetric and fluorescent sensor, selective for Cu2+ ions, was synthesized in two steps using a rhodamine-based compound attached to the semicarbazide-picolylamine moiety (RBP). Spectroscopic measurements, including UV-Vis absorption and fluorescence emission, were conducted in the semi-aqueous medium containing acetonitrile/4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, denoted as MeCN/HEPES buffer (2:8, v/v, pH 7.0). The sensor exhibited high selectivity towards Cu2+ ions compared to other cations and demonstrated remarkable sensitivity towards Cu2+ ions, with a limit of detection at the nanomolar level. The calculated transitions indicated a 1:1 stoichiometric binding of RBP to Cu2+ ions based on a 4-coordination mode involving additional chelation in the semi-aqueous medium. The sensing mechanism for the detection of Cu2+ ions was investigated using high-resolution mass spectroscopy. The sensor could be employed as a real-time chemosensor for monitoring Cu2+ ions. Furthermore, the sensor has the potential for utilization in the detection of Cu2+ ions in actual water samples with the high precision and accuracy, as indicated by the small relative standard derivation values. The 50th percentile cytotoxicity concentration of RBP was found to be 22.92 µM. Additionally, the fluorescence bioimaging capability of RBP was demonstrated for the detection of Cu2+ ions in human hepatocellular carcinoma (HepG2) cells.

Structural and mutational analysis of glycoside hydrolase family 1 Br2 ß-glucosidase derived from bovine rumen metagenome.

Ruminant animals rely on the activities of ß-glucosidases from residential microbes to convert feed fibers into glucose for further metabolic uses. In this report, we determined the structures of Br2, which is a glycoside hydrolase family 1 ß-glucosidase from the bovine rumen metagenome. Br2 folds into a classical (ß/α)8-TIM barrel domain but displays unique structural features at loop ß5→α5 and α-helix 5, resulting in different positive subsites from those of other GH1 enzymes. Br2 exhibited the highest specificity toward laminaritriose, suggesting its involvement in ß-glucan hydrolysis in digested feed. We then substituted the residues at subsites +1 and + 2 of Br2 with those of Halothermothrix orenii ß-glucosidase. The C170E and C221T mutations provided favorable interactions with glucooligosaccharide substrates at subsite +2, while the A219N mutation probably improved the substrate preference for cellobiose and gentiobiose relative to laminaribiose at subsite +1. The N407Y mutation increased the affinity toward cellooligosaccharides. These results give further insights into the molecular determinants responsible for substrate specificity in GH1 ß-glucosidases and may provide a basis for future enzyme engineering applications.