Exploring Mannosylpurines as Copper Chelators and Cholinesterase Inhibitors with Potential for Alzheimer's Disease.

Alzheimer's Disease (AD) is characterized by a progressive cholinergic neurotransmission imbalance, with a decrease of acetylcholinesterase (AChE) activity followed by a significant increase of butyrylcholinesterase (BChE) in the later AD stages. BChE activity is also crucial for the development of Aß plaques, the main hallmarks of this pathology. Moreover, systemic copper dyshomeostasis alters neurotransmission leading to AD. In the search for structures targeting both events, a set of novel 6-benzamide purine nucleosides was synthesized, differing in glycone configuration and N7/N9 linkage to the purine. Their AChE/BChE inhibitory activity and metal ion chelating properties were evaluated. Selectivity for human BChE inhibition required N9-linked 6-deoxy-α-d-mannosylpurine structure, while all three tested ß-d-derivatives appeared as non-selective inhibitors. The N9-linked l-nucleosides were cholinesterase inhibitors except the one embodying either the acetylated sugar or the N-benzyl-protected nucleobase. These findings highlight that sugar-enriched molecular entities can tune bioactivity and selectivity against cholinesterases. In addition, selective copper chelating properties over zinc, aluminum, and iron were found for the benzyl and acetyl-protected 6-deoxy-α-l-mannosyl N9-linked purine nucleosides. Computational studies highlight molecular conformations and the chelating molecular site. The first dual target compounds were disclosed with the perspective of generating drug candidates by improving water solubility.

Ionic Liquids and Water: Hydrophobicity vs. Hydrophilicity.

Many chemical processes rely extensively on organic solvents posing safety and environmental concerns. For a successful transfer of some of those chemical processes and reactions to aqueous media, agents acting as solubilizers, or phase-modifiers, are of central importance. In the present work, the structure of aqueous solutions of several ionic liquid systems capable of forming multiple solubilizing environments were modeled by molecular dynamics simulations. The effect of small aliphatic chains on solutions of hydrophobic 1-alkyl-3-methylimidazolium bis(trifluoromethyl)sulfonylimide ionic liquids (with alkyl = propyl [C3C1im][NTf2], butyl [C4C1im][NTf2] and isobutyl [iC4C1im][NTf2]) are covered first. Next, we focus on the interactions of sulphonate- and carboxylate-based anions with different hydrogenated and perfluorinated alkyl side chains in solutions of [C2C1im][CnF2n+1SO3], [C2C1im][CnH2n+1SO3], [C2C1im][CF3CO2] and [C2C1im][CH3CO2] (n = 1, 4, 8). The last system considered is an ionic liquid completely miscible with water that combines the cation N-methyl-N,N,N-tris(2-hydroxyethyl)ammonium [N1 2OH 2OH 2OH]+, with high hydrogen-bonding capability, and the hydrophobic anion [NTf2]-. The interplay between short- and long-range interactions, clustering of alkyl and perfluoroalkyl tails, and hydrogen bonding enables a wealth of possibilities in tailoring an ionic liquid solution according to the needs.

Ground and excited state properties of furanoflavylium derivatives.

The comparison of the ground-state reactivity of anthocyanins and aurone model compounds (i.e. with and without the furano bridge) has shown that the kinetic paradigm does not depend on the bridge but only on the hydroxyl substituent pattern, independently of the presence of the bridge: (i) bell shaped kinetics for those with two hydroxyl substituents in position 4' and 7, and (ii) four distinct kinetic steps for the mono substituted compounds with a hydroxyl in position 4'. The excited state proton transfer (ESPT) properties of these compounds were also investigated using steady-state and time-resolved spectroscopic techniques. It was found that the ESPT efficiency is significantly higher for the bridged compounds. Interestingly, pH-dependent steady-state fluorescence emission experiments show that in 4',7-dihydroxyfuranoflavylium the hydroxyl group in position 7 is the more acidic one in the excited state, while 1H NMR titration curves indicate a higher acidity constant in the ground state for the proton at the hydroxyl group in position 4'. Differently, the fluorescence emission spectrum of the quinoidal base deprotonated at position 7 is only observed upon excitation of the flavylium cation while the one from the base deprotonated at 4' is observed upon direct excitation.

Triplet Excited States and Singlet Oxygen Production by Analogs of Red Wine Pyranoanthocyanins.

During the maturation of red wines, the anthocyanins of grapes are transformed into pyranoanthocyanins, which possess a pyranoflavylium cation as their basic chromophore. Photophysical properties of the singlet and triplet excited states of a series of synthetic pyranoflavylium cations were determined at room temperature in acetonitrile solution acidified with 0.10 mol dm-3 trifluoroacetic acid (TFA, to inhibit competitive excited state proton transfer) and at 77 K in a rigid TFA-acidified isopropanol glass. In solution, the triplet states of these pyranoflavylium cations are efficiently quenched by molecular oxygen, resulting in sensitized formation of singlet oxygen, as confirmed by direct detection of the triplet-state decay by laser flash photolysis and of singlet oxygen monomol emission in the near infrared. The strong visible light absorption, the relatively small singlet-triplet energy differences, the excited state redox potentials and the reasonably long lifetimes of pyranoflavylium triplet states in the absence of molecular oxygen suggest that they might be useful as triplet sensitizers and/or as cationic redox initiators in polar aprotic solvents like acetonitrile.

Ground- and Excited-State Acidity of Analogs of Red Wine Pyranoanthocyanins.

Pyranoflavylium cations are synthetic analogs containing the same basic chromophore as the pyranoanthocyanins that form in red wine during maturation and are responsible for its final color. Determination of the ground- and excited-state acidities for a series of eight substituted hydroxy pyranoflavylium cations shows that they are weak acids in the ground state (pKa ranging from 3.4 to 4.4 in aqueous buffer solution), but substantially more acidic in the first excited singlet state (pKa * ranging from ca. 0.2 to 0.7 in 30% methanol-water). Unlike the ground-state acidities, which show no obvious trend with electronic effects of the substituents, the excited-state pKa * values correlate well with Hammett sigma parameters for the substituents on the pyranoflavylium chromophore. This difference in the transmission of electronic effects between ground and excited state is reflected in the localization of the HOMO of the cation and conjugate base in distinct regions of the chromophore as compared to delocalization of the LUMO over the entire molecule. The current results provide further support for the conclusion that excited-state proton transfer is the dominant deactivation pathway for the pyranoflavylium cation excited singlet state in aqueous or aqueous-organic media and presumably for pyranoanthocyanins as well.

Structural and aggregate analyses of (Li salt + glyme) mixtures: the complex nature of solvate ionic liquids.

The structure and interactions of different (Li salt + glyme) mixtures, namely equimolar mixtures of lithium bis(trifluoromethylsulfonyl)imide, nitrate or trifluoroacetate salts combined with either triglyme or tetraglyme molecules, are probed using Molecular Dynamics simulations. structure factor functions, calculated from the MD trajectories, confirmed the presence of different amounts of lithium-glyme solvates in the aforementioned systems. The MD results are corroborated by S(q) functions derived from diffraction and scattering data (HEXRD and SAXS/WAXS). The competition between the glyme molecules and the salt anions for the coordination to the lithium cations is quantified by comprehensive aggregate analyses. Lithium-glyme solvates are dominant in the lithium bis(trifluoromethylsulfonyl)imide systems and much less so in systems based on the other two salts. The aggregation studies also emphasize the existence of complex coordination patterns between the different species (cations, anions, glyme molecules) present in the studied fluid media. The analysis of such complex behavior is extended to the conformational landscape of the anions and glyme molecules and to the dynamics (solvate diffusion) of the bis(trifluoromethylsulfonyl)imide plus triglyme system.

Femtosecond and temperature-dependent picosecond dynamics of ultrafast excited-state proton transfer in water-dioxane mixtures.

Synthetic flavylium salts like the 7-hydroxy-4-methylflavylium (HMF) cation have been used as prototypes to study the chemistry and photochemistry of anthocyanins, the major group of water-soluble pigments in the plant kingdom. In this work, a combination of fluorescence upconversion with femtosecond time resolution and time-correlated single photon counting (TCSPC) with picosecond time resolution have been employed to investigate in details the excited-state proton transfer (ESPT) of HMF in water and in binary water/1,4-dioxane mixtures. TCSPC measurements as a function of temperature provide activation parameters for all of the individual rate constants involved in the proton transfer, including those for dissociation and recombination of the geminate excited base-proton pair (A*···H(+)) that can be detected in the water/dioxane mixtures (but not in water). Unlike the other rate constants, the deprotonation rate constant kd shows a non-Arrhenius dependence on temperature in both water and water/dioxane mixtures. At low temperatures kd is close to the dielectric relaxation rate of the solvent with a barrier of ca. 8 kJ mol(-1), suggesting that the solvent reorganization is the rate-limiting step. At higher temperatures (>30 °C) the proton transfer process is nearly barrierless and solvent-dependent. Fluorescence upconversion results in H2O, D2O, and water/dioxane mixtures confirm the two-step model for the ESPT of HMF and provide additional details of the early events prior to the onset of proton transfer, attributed to conformational relaxation and solvent reaccommodation around the initially formed excited state. The results are consistent with DFT calculations that indicate that charge redistribution occurs after rather than prior to the onset of the ESPT process.

Improved analysis of excited state proton transfer kinetics by the combination of standard and convolution methods.

We wish to report a powerful methodology for analysis and interpretation of complex fluorescence decays and other kinetic data, based on the addition of convolution analysis to standard (individual and global) analysis. The method was applied to cases involving excited state proton transfer of flavylium salts in water, in water-1,4-dioxane mixtures and in micellar solutions of sodium dodecyl sulfate (SDS). The results clearly support the proposal of the intermediacy of a proton-base geminate pair in environments other than water.

Picosecond dynamics of proton transfer of a 7-hydroxyflavylium salt in aqueous-organic solvent mixtures.

The intermediacy of the geminate base-proton pair (A*···H(+)) in excited-state proton-transfer (ESPT) reactions (two-step mechanism) has been investigated employing the synthetic flavylium salt 7-hydroxy-4-methyl-flavylium chloride (HMF). In aqueous solution, the ESPT mechanism involves solely the excited acid AH(+)* and base A* forms of HMF as indicated by the fluorescence spectra and double-exponential fluorescence decays (two species, two decay times). However, upon addition of either 1,4-dioxane or 1,2-propylene glycol, the decays become triple-exponential with a term consistent with the presence of the geminate base-proton pair A*···H(+). The geminate pair becomes detectable because of the increase in the recombination rate constant, k(rec), of (A*···H(+)) with increasing the mole fraction of added organic cosolvent. Because the two-step ESPT mechanism splits the intrinsic prototropic reaction rates (deprotonation of AH(+)*, k(d), and recombination, k(rec), of A*···H(+)) from the diffusion controlled rates (dissociation, k(diss), and formation, k(diff)[H(+)], of A*···H(+)), the experimental detection of the geminate pair provides a wealth of information on the proton-transfer reaction (k(d) and k(rec)) as well as on proton diffusion/migration (k(diss) and k(diff)).

Polymeric scaffolds for enhanced stability of melanin incorporated in liposomes.

The use of melanin in bioinspired applications is mostly limited by its poor stability in solid films. This problem has been addressed here by incorporating melanin into dipalmitoyl phosphatidyl glycerol (DPPG) liposomes, which were then immobilized onto a solid substrate as an LbL film. Results from steady-state and time-resolved fluorescence indicated an increased stability for melanin incorporated into DPPG liposomes. If not protected by liposomes, melanin looses completely its fluorescence properties in LbL films. The thickness of the liposome-melanin layer obtained from neutron reflectivity data was 4.1+/-0.2 nm, consistent with the value estimated for the phospholipid bilayer of the liposomes, an evidence of the collapse of most liposomes. On the other hand, the final roughness indicated that some of the liposomes had their structure preserved. In summary, liposomes were proven excellent for encapsulation, thus providing a suitable environment, closer to the physiological conditions without using organic solvents or high pHs.

Picosecond dynamics of the prototropic reactions of 7-hydroxyflavylium photoacids anchored at an anionic micellar surface.

Three water-insoluble, micelle-anchored flavylium salts, 7-hydroxy-3-octyl-flavylium chloride, 4'-hexyl-7-hydroxyflavylium chloride, and 6-hexyl-7-hydroxy-4-methyl-flavylium chloride, have been employed to probe excited-state prototropic reactions in micellar sodium dodecyl sulfate (SDS). In SDS micelles, the fluorescence decays of these three flavylium salts are tetraexponential functions in the pH range from 1.0 to 4.6 at temperatures from 293 to 318 K. The four components of the decays are assigned to four kinetically coupled excited species in the micelle: specifically, promptly deprotonable (AH(+)*) and nonpromptly deprotonable (AH(h)(+)*) orientations of the acid in the micelle, the base-proton geminate pair (A*...H(+)), and the free conjugate base (A*). The initial prompt deprotonation to form the geminate pair occurs at essentially the same rate (k(d) approximately 6-7 x 10(10) s(-1)) for all three photoacids. Recombination of the geminate pair is approximately 3-fold faster than the rate of proton escape from the pair (k(rec) approximately 3 x 10(10) s(-1) and k(diss) approximately 1 x 10(10) s(-1)), corresponding to an intrinsic recombination efficiency of the pair of approximately 75%. Finally, the reprotonation of the short-lived free A* (200-350 ps, depending on the photoacid) has two components, only one of which depends on the proton concentration in the intermicellar aqueous phase. Ultrafast transfer of the proton to water and substantial compartmentalization of the photogenerated proton at the micelle surface on the picosecond time scale strongly suggest preferential transfer of the proton to preformed hydrogen-bonded water bridges between the photoacid and the anionic headgroups. This localizes the proton in the vicinity of the excited base much more efficiently than in bulk water, resulting in the predominance of geminate reprotonation at the micelle surface.

Ultrafast internal conversion in a model anthocyanin-polyphenol complex: implications for the biological role of anthocyanins in vegetative tissues of plants.

The red flavylium cations of anthocyanins form ground-state charge-transfer complexes with several naturally occurring electron-donor copigments, such as hydroxylated flavones and hydroxycinnamic or benzoic acids. Excitation of the 7-methoxy-4-methyl-flavylium-protocatechuic acid complex results in ultrafast (240 fs) internal conversion to the ground state of the complex by way of a low-lying charge-transfer state. Thus, both uncomplexed anthocyanins, whose excited state decays by fast (5-20 ps) excited-state proton transfer, and anthocyanin-copigment complexes have highly efficient mechanisms of deactivation that are consistent with the proposed protective role of anthocyanins against excess solar radiation in the vegetative tissues of plants.

Acid-base equilibria and dynamics in sodium dodecyl sulfate micelles: geminate recombination and effect of charge stabilization.

The synthetic flavylium salt 4-carboxy-7-hydroxy-4'-methoxyflavylium chloride (CHMF) exhibits two acid-base equilibria in the range of pH 1-8 in both aqueous and micellar sodium dodecyl sulfate (SDS) solutions. The values of pK(a1) and pK(a2) for the cation-zwitterion (AH(2)(+) <--> Z + H(+)) and the zwitterion-base (Z <--> A(-) + H(+)) equilibria increase from 0.73 and 4.84 in water to 2.77 and 5.64 in SDS micelles, respectively. The kinetic study of the Z <--> A(-) + H(+) ground-state reactions in SDS points to the diffusion-controlled protonation of A(-) in the aqueous phase (k(p2w) = 4.2 x 10(10) M(-)(1) s(-)(1)) and in the micelle (k(p2m) = 2.3 x 10(11) M(-)(1) s(-)(1)). The deprotonation rate of Z did not significantly change upon going from water (k(d2) = 6.3 x 10(5) s(-)(1)) to SDS (k(d2) = 5.2 x 10(5) s(-)(1)), in contrast with the behavior of ordinary cationic flavylium salts, for which k(d2) strongly decreases in SDS micelles. These results suggest that deprotonation of the zwitterionic acid is not substantially perturbed by the micellar charge. Electronic excitation of the Z form of CHMF induces fast adiabatic deprotonation of the hydroxyl group of Z() (2.9 x 10(10) s(-)(1) in water and 8.4 x 10(9) s(-)(1) in 0.1 M SDS), followed by geminate recombination on the picosecond time scale. Interestingly, while recombination in water (k(rec) = 1.7 x 10(9) s(-)(1)) occurs preferentially at the carboxylate group, at the SDS micelle surface, recombination (k(rec) = 9.2 x 10(9) s(-)(1)) occurs at the hydroxyl group. The important conclusion is that proton mobility at the SDS micelle surface is substantially reduced with respect to the mobility in water, which implies that geminate recombination should be a general phenomenon in SDS micelles.

Novel ground- and excited-state prototropic reactivity of a hydroxycarboxyflavylium salt.

Synthetic and natural hydroxyflavylium salts are super-photoacids, exhibiting values of the rate constant for proton transfer to water in the excited state as high as 1.5 x 10(11) s(-1). The synthetic flavylium salt 4-carboxy-7-hydroxy-4'-methoxyflavylium chloride (CHMF) has an additional carboxyl group at the 4-position of the flavylium cation that deprotonates in the ground state at a lower pH (pK(a1) = 0.73; AH2+ --> Z) than the 7-hydroxy group (pK(a2) = 4.84; Z --> A-). Ground-state deprotonation of the carboxyl group of the acid (AH2+) to form the zwitterion (Z) is too fast to be detected by nanosecond laser flash perturbation of the ground-state equilibrium, while deprotonation of the hydroxyl group of Z to form the anionic base (A-) occurs in the microsecond time range (k(d2) = 0.6 x 10(6) s(-1) and k(p2) = 4.2 x 10(10) M(-1) x s(-1)). In the excited state, the cationic form (AH2+) deprotonates in approximately 9 ps, resulting in the excited neutral base form (AH), which is unstable in the ground state. Deprotonation of Z occurs in 30 ps (k(d2) = 2.9 x 10(10) s(-1)), to form excited A-, which either reprotonates (k(p3)* = 3.7 x 10(10) M(-1) x s(-1)) or decays in 149 ps, and shows an important contribution from geminate recombination to give the excited neutral base (AH). Predominant reprotonation of A- at the carboxylate group reflects both the presence of the negative charge on the carboxylate and the increase in the excited-state pK(a) of the carboxyl group. Thus, while the hydroxyl pK(a) decreases by approximately 5 units upon going from the ground state (pK(a) = 4.84) to the excited state (pK(a) = -0.2), that of the carboxyl group increases by at least this much. Consequently, the excited state of the Z form of CHMF acts as a molecular proton transporter in the picosecond time range.

Charge-transfer complexation as a general phenomenon in the copigmentation of anthocyanins.

Color intensification of anthocyanin solutions in the presence of natural polyphenols (copigmentation) is re-interpreted in terms of charge transfer from the copigment to the anthocyanin. Flavylium cations are shown to be excellent electron acceptors (E(red) approximately -0.3 V vs SCE). It is also demonstrated, for a large series of anthocyanin-copigment pairs, that the standard Gibbs free energy of complex formation decreases linearly with EA(Anthoc) - IP(Cop), the difference between the electron affinity of the anthocyanin, EA(Anthoc), and the ionization potential of the copigment, IP(Cop). Based on this correlation, copigmentation strengths of potential candidates for copigments can be predicted.