Concentration-dependent HAT/ET mechanism of the reaction of phenols with 2,2-diphenyl-1-picrylhydrazyl (dpph˙) in methanol.

The reaction of a 2,2-diphenyl-1-picrylhydrazyl radical (dpph˙) with phenols carried out in alcohols is a frequently used assay for estimation of the antiradical activity of phenolic compounds. The rates of reactions of dpph˙ with five phenols (ArOH: unsubstituted phenol, 4-hydroxyacetophenone, two calix[4]resorcinarenes and baicalein) measured in methanol indicate the different kinetics of the process for very diluted phenols compared to their non-diluted solutions. This effect was explained as dependent on the ratio [ArO-]/[ArOH] and for diluted ArOH corresponds to an increased contribution of much faster electron transfer (ET, ArO-/dpph˙) over the Hydrogen Atom Transfer (HAT, ArOH/dpph˙). Simplified analysis of the reaction kinetics resulted in estimation of k ET/k HAT ratios for each studied ArOH, and in calculation of the rate constants k ET. Described results are cautionary examples of how the concentration of a phenol might change the reaction mechanism and the overall kinetics of the observed process.

Chemically Tuned, Reversible Fluorogenic Electrophile for Live Cell Nanoscopy.

We report a chemically tuned fluorogenic electrophile designed to conduct live-cell super-resolution imaging by exploiting its stochastic reversible alkylation reaction with cellular nucleophiles. Consisting of a lipophilic BODIPY fluorophore tethered to an electrophilic cyanoacrylate warhead, the new probe cyanoAcroB remains nonemissive due to internal conversion along the cyanoacrylate moiety. Intermittent fluorescence occurs following thiolate Michael addition to the probe, followed by retro-Michael reaction, tuned by the cyano moiety in the acrylate warhead and BODIPY decoration. This design enables long-term super-resolved imaging of live cells by preventing fluorescent product accumulation and background increase, while preserving the pool of the probe. We demonstrate the imaging capabilities of cyanoAcroB via two methods: (i) single-molecule localization microscopy imaging with nanometer accuracy by stochastic chemical activation and (ii) super-resolution radial fluctuation. The latter tolerates higher probe concentrations and low imaging powers, as it exploits the stochastic adduct dissociation. Super-resolved imaging with cyanoAcroB reveals that electrophile alkylation is prevalent in mitochondria and endoplasmic reticulum. The 2D dynamics of these organelles within a single cell are unraveled with tens of nanometers spatial and sub-second temporal resolution through continuous imaging of cyanoAcroB extending for tens of minutes. Our work underscores the opportunities that reversible fluorogenic probes with bioinspired warheads bring toward illuminating chemical reactions with super-resolved features in live cells.

Antiradical Activity of Dopamine, L-DOPA, Adrenaline, and Noradrenaline in Water/Methanol and in Liposomal Systems.

Catecholamines play a crucial role in signal transduction and are also expected to act as endogeneous antioxidants, but the mechanism of their antioxidant action is not fully understood. Here, we describe the impact of pH on the kinetics of reaction of four catecholamines (L-DOPA, dopamine, adrenaline, and noradrenaline) with model 2,2-diphenyl-1-picrylhydrazyl radical (dpph•) in methanol/water. The increase in pH from 5.5 to 7.4 is followed by a 2 order of magnitude increase in the rate constant, e.g., for dopamine (DA) kpH5.5 = 1,200 M-1 s-1 versus kpH7.4 = 170,000 M-1 s-1, and such rate acceleration is attributed to a fast electron transfer from the DA anion to dpph•. We also proved that at pH 7.0 DA breaks the peroxidation chain of methyl linoleate in liposomes assembled from neutral and negatively charged phospholipids. In contrast to no inhibitory effect during peroxidation in non-ionic emulsions, in bilayers one molecule of DA traps approximately four peroxyl radicals, with a rate constant kinh >103 M-1 s-1. Our results from a homogeneous system and bilayers prove that catecholamines act as effective, radical trapping antioxidants with activity depending on the ionization status of the catechol moiety, as well as microenvironment: organization of the lipid system (emulsions vs bilayers) and interactions of catecholamines with the biomembrane.

Antioxidant activity of dopamine and L-DOPA in lipid micelles and their cooperation with an analogue of α-tocopherol.

Oxidative stress contributes to the progression of neurodegenerative diseases and considerable attention has been given to the development of new antioxidant-based therapies aimed at limiting neuronal cell damage. Structural analysis of catecholamine neurotransmitters indicates that these molecules can exhibit antioxidant activity due to the presence of a catechol moiety. This hypothesis is confirmed in cell culture experiments but the mechanism of antioxidant action of catecholamines is not described. Herein, we present quantitative kinetic studies on the effect of dopamine (DA) and L-3,4-dihydroxyphenylalanine (L-DOPA) on the peroxidation of methyl linoleate dispersed in Triton X-100 micelles as a model heterogeneous lipid system. Experiments were performed at extended pH range 4.0-10.0 in order to study how protonation/deprotonation of catecholamine affect its antioxidant activity. At pH 4.0-7.0, the activity of catecholamines is limited to retardation of lipid peroxidation (caused by the reaction of catecholamines with initiating radicals in the aqueous phase). The effective suppression of lipid peroxidation can be achieved by applying catecholamines together with an analogue of α-tocopherol (2,2,5,7,8-pentamethyl-6-hydroxychroman, PMHC). For example, a mixture of 1 µM PMHC with 10 µM L-DOPA causes 18-fold elongation of suppression time as compared to 1 µM PMHC used alone. We suggest that catecholamines together with α-tocopherol efficiently enhance the protection of biological systems from oxidative stress. At pH above 8.0 a prooxidative effect caused by reaction of semiquinone radical anions with molecular oxygen is observed. However, this toxic action can be completely suppressed by PMHC acting as an agent removing the potentially harmful semiquinone radicals from the reaction environment.

First experimental evidence of dopamine interactions with negatively charged model biomembranes.

Dopamine is essential for receptor-related signal transduction in mammalian central and peripheral nervous systems. Weak interactions between the neurotransmitter and neuronal membranes have been suggested to modulate synaptic transmission; however, binding forces between dopamine and neuronal membranes have not yet been quantitatively described. Herein, for the first time, we have explained the nature of dopamine interactions with model lipid membranes assembled from neutral 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), negatively charged 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG), and the mixture of these two lipids using isothermal titration calorimetry and differential scanning calorimetry. Dopamine binding to anionic membranes is a thermodynamically favored process with negative enthalpy and positive entropy, quantitatively described by the mole ratio partition coefficient, K. K increases with membrane charge to reach its maximal value, 705.4 ± 60.4 M(-1), for membrane composed from pure DMPG. The contribution of hydrophobic effects to the binding process is expressed by the intrinsic partition coefficient, K(0). The value of K(0) = 74.7 ± 6.4 M(-1) for dopamine/DMPG interactions clearly indicates that hydrophobic effects are 10 times weaker than electrostatic forces in this system. The presence of dopamine decreases the main transition temperature of DMPG, but no similar effect has been observed for DMPC. Basing on these results, we propose a simple electrostatic model of dopamine interactions with anionic membranes with the hydrophobic contribution expressed by K(0). We suggest that dopamine interacts superficially with phospholipid membranes without penetrating into the bilayer hydrocarbon core. The model is physiologically important, since neuronal membranes contain a large (even 20%) fraction of anionic lipids.