Germán Günther

Singlet Oxygen Reactions with Flavonoids. A Theoretical – Experimental Study

Detection of singlet oxygen emission, λmax = 1270 nm, following laser excitation and steady-state methods were employed to measure the total reaction rate constant, kT, and the reactive reaction rate constant, kr, for the reaction between singlet oxygen and several flavonoids. Values of kT determined in deuterated water, ranging from 2.4×107 M−1s−1 to 13.4×107 M−1s−1, for rutin and morin, respectively, and the values measured for kr, ranging from 2.8×105 M−1s−1 to 65.7×105 M−1s−1 for kaempferol and morin, respectively, being epicatechin and catechin chemically unreactive. These results indicate that all the studied flavonoids are good quenchers of singlet oxygen and could be valuable antioxidants in systems under oxidative stress, in particular if a flavonoid-rich diet was previously consumed. Analysis of the dependence of rate constant values with molecular structure in terms of global descriptors and condensed Fukui functions, resulting from electronic structure calculations, supports the formation of a charge transfer exciplex in all studied reactions. The fraction of exciplex giving reaction products evolves through a hydroperoxide and/or an endoperoxide intermediate produced by singlet oxygen attack on the double bond of the ring C of the flavonoid.

Methyl-β-Cyclodextrins Preferentially Remove Cholesterol from the Liquid Disordered Phase in Giant Unilamellar Vesicles

Methyl-β-cyclodextrins (MβCDs) are molecules that are extensively used to remove and to load cholesterol (Chol) from artificial and natural membranes; however, the mechanism of Chol extraction by MβCD from pure lipids or from complex mixtures is not fully understood. One of the outstanding questions in this field is the capability of MβCD to remove Chol from lipid domains having different packing. Here, we investigated the specificity of MβCD to remove Chol from coexisting macrodomains with different lipid packing. We used giant unilamellar vesicles (GUVs) made of 1,2-dioleoylphosphatidylcholine:1,2-dipalmitoylphatidylcholine:free cholesterol, 1:1:1 molar ratio at 27°C. Under these conditions, individual GUVs present Chol distributed into lo and ld phases. The two phases can be distinguished and visualized using Laurdan generalized polarization and two-photon excitation fluorescence microscopy. Our data indicate that MβCD removes Chol preferentially from the more disordered phase. The process of selective Chol removal is dependent on the MβCD concentration. At high concentrations, MβCD also removes phospholipids.

Solubilization of lipid bilayers by myristyl sucrose ester: effect of cholesterol and phospholipid head group size

The solubilization of biological membranes by detergents has been used as a major method for the isolation and purification of membrane proteins and other constituents. Considerable interest in this field has resulted from the finding that different components can be solubilized selectively. Certain membrane constituents are incorporated into small micelles, whereas others remain in the so-called detergent-resistant membrane domains that are large enough to be separated by centrifugation. The detergent-resistant fractions contain an elevated percentage of cholesterol, and thus its interaction with specific lipids and proteins may be key for membrane organization and regulation of cellular signaling events. This report focuses on the solubilization process induced by the sucrose monoester of myristic acid, beta-D-fructofuranosyl-6-O-myristyl-alpha-D-glucopyranoside (MMS), a nonionic detergent. We studied the effect of the head group and the cholesterol content on the process. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and dioctadecyl-dimethyl-ammonium chloride (DODAC) vesicles were used, and the solubilization process was followed using Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) generalized polarization (GP) measurements, carried out in the cuvette and in the 2-photon microscope. Our results indicate that: (i) localization of the MMS moieties in the lipid bilayer depends on the characteristics of the lipid polar head group and influences the solubilization process. (ii) Insertion of cholesterol molecules into the lipid bilayer protects it from solubilizaton and (iii) the microscopic mechanism of solubilization by MMS implies the decrease in size of the individual liposomes.

Sucrose Monoester Micelles Size Determined by Fluorescence Correlation Spectroscopy (FCS)

One of the several uses of sucrose detergents, as well as other micelle forming detergents, is the solubilization of different membrane proteins. Accurate knowledge of the micelle properties, including size and shape, are needed to optimize the surfactant conditions for protein purification and membrane characterization. We synthesized sucrose esters having different numbers of methylene subunits on the substituent to correlate the number of methylene groups with the size of the corresponding micelles. We used Fluorescence Correlation Spectroscopy (FCS) and two photon excitation to determine the translational D of the micelles and calculate their corresponding hydrodynamic radius, Rh. As a fluorescent probe we used LAURDAN (6-dodecanoyl-2-dimethylaminonaphthalene), a dye highly fluorescent when integrated in the micelle and non-fluorescent in aqueous media. We found a linear correlation between the size of the tail and the hydrodynamic radius of the micelle for the series of detergents measured.

Photophysics and photochemistry of naphthoxazinone derivatives

The photophysics and photochemistry of a series of naphthoxazinones have been studied using a combination of methods ranging from steady-state and time-resolved spectroscopic techniques to product analysis. The photophysics of naphthoxazinone derivatives is very dependent on the structure: phenanthrene-like compounds exhibit higher fluorescence quantum yield than the less aromatic anthracene-like homologous. The latter, exhibit a substantial degree of charge transfer in the excited singlet state. These compounds are fairly photostable in the absence of additives, yielding a single photoproduct arising from the triplet state. The presence of electron donors such as amines increases the photoconsumption quantum yield and changes the product distribution, the primary photoproduct being a dihydronaphthoxazinone that photoreacts further yielding ultimately an oxazoline derivative.

Sensitized Photooxidation of Thyroidal Hormones. Evidence for Heavy Atom Effect on Singlet Molecular Oxygen [O2(1Δg)]-mediated Photoreactions¶

Thyronine derivatives are essential indicators of thyroid gland diseases in clinical diagnosis and are currently used as standards for developing ordinary biochemical assays. Photooxidation of gland hormones of the thyronine (TN) family and structurally related compounds (TN, 3,5‐diiodo‐thyronine,3,3′,5‐triiodothyronine and 3,3′,5,5′‐tetraiodothyronine or thyroxine) was studied using rose bengal, eosin and perinaphthenone (PN) as dye sensitizers. Tyrosine (Tyr) and two iodinated derivatives (3‐iodotyrosine and 3,5‐diiodotyrosine) were also included in the study for comparative purposes. Irradiation of aqueous solutions of substrates containing xanthene dyes with visible light triggers a complex series of competitive interactions, which include the triplet excited state of the dye (3Xdye*) and singlet molecular oxygen [O2(1Δg)]‐mediated and superoxide ion‐mediated reactions. Rate constants for interaction with the 3Xdye*, attributed to an electron transfer process, are in the order of 108‐109M−1 s−1 depending on the dye and the particular substrate. The photosensitization using PN follows a pure Type‐II (O2(1Δg) mediated) mechanism. The presence of the phenolic group in Tyr, TN and iodinated derivatives dominates the kinetics of photooxidation of these compounds. The reactive rate constants, kr, and the quotient between reactive and overall rate constants (krlkt values, in the range of 0.7–0.06) behave in an opposite fashion compared with the overall rate constants and oxidation potentials. This apparent inconsistency was interpreted on the basis of an internal heavy atom effect, favoring the intersystem‐crossing deactivation route within the encounter complex with the concomitant reduction of effective photooxidation.

Sensitized photoxygenation of piroxicam in neat solvents and solvent mixtures

Detection of O(2)(1Delta(g)) phosphorescence emission, lambda(max)=1270 nm, following laser excitation and steady state methods were employed to determine the total rate constant, k(T), for the reaction between the non-steroidal anti-inflammatory drug piroxicam (PRX) and singlet oxygen in several solvents. Values of k(T) ranged from 0.048+/-0.003 x 10(6) M(-1) s(-1) in chloroform to 71.2+/-2.2 x 10(6) M(-1) s(-1) in N,N-dimethylformamide. The chemical reaction rate constant, k(R), was determined by using thermal decomposition of 1,4-dimethylnaphthalene endoperoxide as the singlet oxygen source. In acetonitrile, the k(R) value is equal to 5.0+/-0.4 x 10(6) M(-1) s(-1), very close to the k(T) value. This result indicates that, in this solvent, the chemical reaction corresponds to the main reaction path. Dependence of total rate constant on the solvent parameters pi* and beta can be explained in terms of a reaction mechanism that involves the formation of a perepoxide intermediate. Rearrangement of the perepoxide to dioxetane followed by ring cleavage and transacylation accounts for the formation of N-methylsaccharine and N-(2-pyridyl)oxamic acid, the main reaction products. Data obtained in dioxane-water (pH 4) mixtures with neutral enolic and zwitterionic tautomers of piroxicam in equilibrium show that the zwitterionic tautomer reacts with singlet oxygen faster than the enolic tautomer.

A kinetic study of the reaction between boldine and singlet oxygen[O2(1ωg)]

The sensitized photo-oxidation of boldine and its permethylated derivative, glaucine, has been studied in several solvents using steady-state kinetic methods. Values of kB, the total rate constant for the reaction between boldine and O2(1ωg), vary from 3.44 × 107M-1 s-1 in benzene to 150.00 × 107M-1 s-1 in water. For glaucine, similar values were determined. Quenching is faster in solvents with high capacity to stabilize a charge or dipole (higher π* parameter values). From these results, a charge-transfer quenching mechanism, to account for the interaction between boldine and O2(1Δg), is proposed. Rate constants for the chemical reaction are at least two orders of magnitude smaller than the total rate constants, which also show a large dependence on the π* parameter. This last result can be explained in terms of a more polar transition state leading to products.

Dual Emission of a Novel (P,N) ReI Complex: A Computational and Experimental Study on [P,N-{(C6H5)2(C5H4N)P}Re(CO)3Br]

The spectroscopic, electrochemical, and photophysical properties of the new complex [P,N-{(C6H5)2(C5H4N)P}Re(CO)3Br] are reported. The UV-vis spectrum in dichloromethane shows an absorption maximum centered at 315 nm and a shoulder at 350 nm. These absorption bands have been characterized to have MLCT character. Excitation at both wavelengths (maximum and shoulder) leads to an emission band centered at 550 nm. Cyclic voltammetry experiments show two ill-defined irreversible oxidation waves around +1.50 and 1.80 V that are assigned to Re(I)/Re(II) and Re(II)/Re(III) couples whereas an irreversible reduction signal centered at -1.80 V is likewise assigned to a ligand reduction process. These results support the proposal of the MLCT nature of the states implied by the emission of the complex. The luminescent decay fits to a biexponential function, where the lifetimes and emission quantum yields are dependent on the solvent polarity. DFT calculations suggest that dπ → π*pyridine and dπ → π*phenyl excited states may account for the existence of two decay lifetimes.

Boldo prevents UV light and nitric oxide-mediated plasmid DNA damage and reduces the expression of Hsp70 protein in melanoma cancer cells

Objectives  This study was designed to investigate the potential protective effect of a methanolic extract of Peumus boldus leaves on UV light and nitric oxide (NO)‐mediated DNA damage. In addition, we investigated the growth inhibitory activity of this natural product against human melanoma cells (M14).