Multifunctional fluorescence probe for simultaneous detection of viscosity, polarity, and ONOO- and its bioimaging in vitro and vivo.

Intracellular microenvironment (viscosity and polarity) and peroxynitrite ions (ONOO-) are involved in maintaining cell morphology, cell function, and signaling so that it is crucial to explore their level changes in vitro and vivo. In this work, we designed and synthesized a mitochondria-targeted fluorescence probe XBL for monitoring the dynamic changes of viscosity, polarity, and ONOO- based on TICT and ICT mechanism. The fluorescence spectra showed obvious changes for polarity at 500 nm as well as ONOO- and viscosity at 660 nm, respectively. The XBL can image simultaneously viscosity, polarity, and ONOO- in cells, and the results showed excess ONOO- leaded to the increase of viscosity in mitochondrial. The ferroptosis process was accompanied by increase of intracellular viscosity and ONOO- levels (or decrease of polarity), which allowed us to better understand the relevant physiological and pathological processes. The XBL can distinguish normal cells and cancerous cells by the fluorescence intensity changes in green and red channels, and image viscosity in inflamed mice. Thus, XBL can provided the chemical tool to understand the physiological and pathological mechanisms of disease by simultaneous detection of viscosity, polarity and ONOO-.

Hydroxycinnamic acids as DNA-cleaving agents in the presence of Cu(II) ions: mechanism, structure-activity relationship, and biological implications.

The effectiveness of hydroxycinnamic acids (HCAs), that is, caffeic acid (CaA), chlorogenic acid (ChA), sinapic acid (SA), ferulic acid (FA), 3-hydroxycinnamic acid (3-HCA), and 4-hydroxycinnamic acid (4-HCA), as pBR322 plasmid DNA-cleaving agents in the presence of Cu(II) ions was investigated. Compounds bearing o-hydroxy or 3,5-dimethoxy groups on phenolic rings (CaA, SA, and ChA) were remarkably more effective at causing DNA damage than the compounds bearing no such groups; furthermore, CaA was the most active among the HCAs examined. The involvement of reactive oxygen species (ROS) and Cu(I) ions in the DNA damage was affirmed by the inhibition of the DNA breakage by using specific scavengers of ROS and a Cu(I) chelator. The interaction between CaA and Cu(II) ions and the influence of ethylenediaminetetraacetic acid (EDTA), the solvent, and pH value on the interaction were also studied to help elucidate the detailed prooxidant mechanism by using UV/Vis spectroscopic analysis. On the basis of these observations, it is proposed that it is the CaA phenolate anion, instead of the parent molecule, that chelates with the Cu(II) ion as a bidentate ligand, hence facilitating the intramolecular electron transfer to form the corresponding CaA semiquinone radical intermediate. The latter undergoes a second electron transfer with oxygen to form the corresponding o-quinone and a superoxide, which play a pivotal role in the DNA damage. The intermediacy of the semiquinone radical was supported by isolation of its dimer from the Cu(II)-mediated oxidation products. Intriguingly, CaA was also the most cytotoxic compound among the HCAs toward human promyelocytic leukemia (HL-60) cell proliferation. Addition of exogenous Cu(II) ions resulted in an effect dichotomy on cell viability depending on the concentration of CaA; that is, low concentrations of CaA enhanced the cell viability and, conversely, high concentrations of CaA almost completely inhibited the cell proliferation. On the other hand, when superoxide dismutase was added before, the two stimulation effects of exogenous Cu(II) ions were significantly ameliorated, thus clearly indicating that the oxidative-stress level regulates cell proliferation and death. These findings provide direct evidence for the antioxidant/prooxidant mechanism of cancer chemoprevention.

Radical-scavenging activity and mechanism of resveratrol-oriented analogues: influence of the solvent, radical, and substitution.

Resveratrol (3,5,4'-trihydroxy-trans-stilbene, 3,5,4'-THS) is a well-known natural antioxidant and cancer chemopreventive agent that has attracted much interest in the past decade. To find a more active antioxidant and investigate the antioxidative mechanism with resveratrol as the lead compound, we synthesized 3,5-dihydroxy-trans-stilbene (3,5-DHS), 4-hydroxy-trans-stilbene (4-HS) 3,4-dihydroxy-trans-stilbene (3,4-DHS), 4,4'-dihydroxy-trans-stilbene (4,4'-DHS), 4-hydroxy-3-methoxy-trans-stilbene (3-MeO-4-HS), 4-hydroxy-4'-methoxy-trans-stilbene (4'-MeO-4-HS), 4-hydroxy-4'-methyl-trans-stilbene (4'-Me-4-HS), 4-hydroxy-4'-nitro-trans-stilbene (4'-NO(2)-4-HS), and 4-hydroxy-4'-trifluoromethyl-trans-stilbene (4'-CF(3)-4-HS). The radical-scavenging activity and detailed mechanism of resveratrol and its analogues (ArOHs) were investigated by the reaction kinetics with galvinoxyl (GO(*)) and 2,2-diphenyl-1-picrylhydrazyl (DPPH(*)) radicals in ethanol and ethyl acetate at 25 degrees C, using UV-vis spectroscopy. It was found that the reaction rates increase with increasing the electron-rich environment in the molecules, and the compound bearing o-dihydroxyl groups (3,4-DHS) is the most reactive one among the examined resveratrol analogues. The effect of added acetic acid on the measured rate constant for GO(*)-scavenging reaction reveals that in ethanol that supports ionization solvent besides hydrogen atom transfer (HAT), the kinetics of the process is partially governed by sequential proton loss electron transfer (SPLET). In contrast to GO(*), DPPH(*) has a relatively high reduction potential and therefore enhances the proportion of SPLET in ethanol. The relatively low rate constants for the reactions of ArOHs with GO(*) or DPPH(*) in ethyl acetate compared with the rate constants in ethanol prove that in ethyl acetate these reactions occur primarily by the HAT mechanism. The contribution of SPLET and HAT mechanism depends on the ability of the solvent to ionize ArOH and the reduction potential of the free radical involved. Furthermore, the fate of the ArOH-derived radicals, i.e., the phenoxyl radicals, was investigated by the oxidative product analysis of ArOHs and GO(*) in ethanol. The major products were dihydrofuran dimers in the case of resveratrol, 4,4'-DHS, and 4-HS and a dioxane-like dimer in the case of 3,4-DHS. It is suggested from the oxidative products of these ArOHs that the hydroxyl group at the 4-position is much easier to subject to oxidation than other hydroxyl groups, and the dioxane-like dimer is formed via an o-quinone intermediate.

4,4'-Dihydroxy-trans-stilbene, a resveratrol analogue, exhibited enhanced antioxidant activity and cytotoxicity.

Resveratrol (3,5,4'-trans-trihydroxystibene) is a natural phytoalexin present in grapes and red wine, which possesses a variety of biological activities including antioxidant activity. In order to find more active antioxidant with resveratrol as the lead compound we synthesized 4,4'-dihydroxy-trans-stilbene (4,4'-DHS). The antioxidant activities of resveratrol and 4,4'-DHS were evaluated by the reaction kinetics with galvinoxyl radical or Cu(II) ions, and the inhibition effects against free-radical-induced peroxidation of human erythrocyte ghosts. It was found that 4,4'-DHS exhibits remarkably higher antioxidant activity than resveratrol. The oxidative products of resveratrol and 4,4'-DHS in the presence of Cu(II) in acetonitrile were identified as the dihydrofuran dimers by spectroscopic method, and the antioxidant mechanism for 4,4'-DHS was proposed. In addition, 4,4'-DHS exhibits remarkably higher cytotoxicity against human promyelocytic leukemia (HL-60) cells than resveratrol.