No abstract

Dysfunctional interactions of metal ions, especially Cu, Zn, and Fe, with the amyloid-beta (A beta) peptide are hypothesized to play an important role in the etiology of Alzheimer's disease (AD). In addition to direct effects on A beta aggregation, both Cu and Fe catalyze the generation of reactive oxygen species (ROS) in the brain further contributing to neurodegeneration. Disruption of these aberrant metal-peptide interactions via chelation therapy holds considerable promise as a therapeutic strategy to combat this presently incurable disease. To this end, we developed two multifunctional carbohydrate-containing compounds N,N'-bis[(5-beta-D-glucopyranosyloxy-2-hydroxy)benzyl]-N,N'-dimethyl-ethane-1,2-diamine (H2GL1) and N,N'-bis[(5-beta-D-glucopyranosyloxy-3-tert-butyl-2-hydroxy)benzyl]-N,N'-dimethyl-ethane-1,2-diamine (H2GL2) for brain-directed metal chelation and redistribution. Acidity constants were determined by potentiometry aided by UV-vis and 1H NMR measurements to identify the protonation sites of H2GL1,2. Intramolecular H bonding between the amine nitrogen atoms and the H atoms of the hydroxyl groups was determined to have an important stabilizing effect in solution for the H2GL1 and H2GL2 species. Both H2GL1 and H2GL2 were found to have significant antioxidant capacity on the basis of an in vitro antioxidant assay. The neutral metal complexes CuGL1, NiGL1, CuGL2, and NiGL2 were synthesized and fully characterized. A square-planar arrangement of the tetradentate ligand around CuGL2 and NiGL2 was determined by X-ray crystallography with the sugar moieties remaining pendant. The coordination properties of H2GL1,2 were also investigated by potentiometry, and as expected, both ligands displayed a higher affinity for Cu2+ over Zn2+ with H2GL1 displaying better coordinating ability at physiological pH. Both H2GL1 and H2GL2 were found to reduce Zn2+- and Cu2+- induced Abeta1-40 aggregation in vitro, further demonstrating the potential of these multifunctional agents as AD therapeutics.

X-ray spectral data and self-consistent field-Xa-scattered wave (SCF-Xa-SW) calculations are presented for D4h and D u CuC12in order to evaluate specific contributions to the small hyperfine in distorted tetrahedral copper sites and to compare the mechanism of hyperfine reduction in these complexes with that for the blue copper site in plastocyanin. Comparison of the 2p3,2 XPS data for the two geometries indicates that the extent of delocalization of the d,z+ ground state in the D u salt is slightly less than in the D4h complex. Multiplet splitting of satellite structure from Cu 3s photoemission shows no change going from D4h to D u CuCIz-, indicating that the indirect Fermi contact contribution to the hyperfine from the core Cu 3s level does not change between the two complexes. Analysis of X-ray edge data for Cs2CuC14 ( D u ) indicates that there is at most 5.8% 4p, mixed into the ground-state wave function, an amount that is insufficient to explain the reduced hyperfine in this distorted Td complex. SCF-Xa-SW calculations performed with sphere radii adjusted so that the ground-state wave function fits the experimental g values indicate that -70% of the reduction in All between D4h and D2d comes from increased orbital angular momentum in the ground state of the Du salt arising from decreased ligand field transition energies. In contrast, increased delocalization relative to D4h CUCI,~accounts for most of the reduction in the blue copper proteins. The remaining 30% of the reduction in All is associated with a -50 X lo4 cm-I reduction in Fermi contact between the two salts. Xa calculations of a number of Cu complexes with < D u symmetry indicate that this reduction is not associated with direct 4s mixing into the half-occupied ground state but is most likely due to increased polarization of the filled totally symmetric valence levels in DZd CuCI," resulting from increased 4s mixing as compared with D,,, CuCIt-. These studies are then extended to include copper sites exhibiting rhombically split g and A values. Single-crystal optical and EPR studies on copper-doped bis( 1,2-dimethylimidazole)zinc(II) dichloride (Zn[Cu] (dmi),C12) combined with ligand field and SCF-Xa-SW calculations indicate that the rhombic features in these complexes can be explained through admixture of -3% d,z character in the ground-state wave function. Finally, the structurally uncharacterized blue copper protein stellacyanin, which also shows a rhombic EPR spectrum similar to that in Zn[C~](dmi)~Cl~, is considered. In contrast to the C, effective symmetry found in the structurally defined site in plastocyanin, stellacyanin is predicted to have C, effective symmetry. A ligand field calculation using the plastocyanin site as a starting point indicates that stellacyanin requires a stronger field ligand along the Cu-methionine coordinate to produce the observed dzz mixing.

Electronic spectra over the 50 000-20 000-cm-1 region are reported for well-characterized chromophores having Cu(ll)-imidazole (ImH) and Cu(Il)-imidazolate (Im_) units. For tetragonal Cu(Il)-lmH chromophores, three ligand to metal charge-transfer (LMCT) absorptions originate from the -symmetry nitrogen donor lone pair and from two -symmetry ring orbitals, one having primarily carbon character ( ) and the other having primarily nitrogen character (7 2). These ff(lmH) 2( ) -and TTi(lmH) ->• Cu(Il) LMCT absorptions occur at ~220, ~260, and ~330 nm, respectively. Ligand rotation causes the -symmetry absorptions to be broadened for solutions containing geometrically unconstrained Cu(ll)-ImH complexes. The -symmetry absorptions generally are well-resolved spectral features of crystalline complexes, and may be split when the ImH groups have nonequivalent orientations. The cr(ImH) -Cu(II) absorption at 220 nm is insensitive to ligand rotation about the Cu-N axis, and is well resolved from the ligand-localized absorption at ~205 nm. The Cu(l l)-lm" complexes exhibit an additional and characteristic broad absorption at --375 nm for which a tentative assignment has been suggested. Tetragonal type 2 and type 3 copper protein chromophores are expected to exhibit corresponding ( ) -* Cu(ll) LMCT transitions in the near-UV region. Such absorptions are expected to be red shifted for the approximately tetrahedral type 1 copper chromophores. The reported spectra of the above types of proteins briefly are reconsidered from this point of view.

Two of the biochemical features of Alzheimers disease (AD) that contribute to neurodegeneration are intracellular oxidative stress and elevated levels of trace metal ions, especially Fe III , Cu II , and Zn II . [1] Both are factors involved in formation of the histological features in the brain used typically for postmortem diagnosis of AD, namely b-amyloid (Ab) plaques and neurofibrillary tangles. Therapeutic interventions under current investigation elsewhere include clioquinol [2] and desferrioxamine, [3] which are metal chelators that target elevated trace-metal ions in the brain, although neither are intended to affect oxidative stress directly and nor are they targeted to the brain. Antioxidant supplements have been studied separately as palliative-only measures for alleviation of the symptoms of AD.[4]Herein, we present for the first time a trifunctional approach to AD therapy. Modified and functionalized bidentate hydroxypyridinone pro-ligands (Scheme 1) address both the metal-ion and the oxidative imbalances inherent in AD while incorporating a glucose-receptor targeting feature.These prodrugs are designed to cross the blood-brain barrier (BBB), lose the pendant carbohydrate by enzymatic cleavage, passivate excess metal ions in the brain, and also protect neuronal cells against reactive oxygen species (ROS). Each of these functionalities has been demonstrated, thereby establishing the trifunctional principle as a valid goal in AD therapy. The prodrug strategy solves the potential problem of premature metal binding by using carbohydrates as both masking and directing substituents. In the context of increasing empirical support for re-establishing normal metal-ion homeostasis in neurodegenerative diseases, including AD, the trifunctional approach permits selective, tissue-dependent metal binding as a tailor-made, biologically compatible therapy.To demonstrate the utility of this approach, a series of assays on prototype compounds have been undertaken, including both in vitro and in vivo studies. This strategy is aimed at reducing neurodegeneration from oxidative stress; by passivating the pro-oxidant metal ions Fe III and Cu II , the production of ROS can be expected to be lower. By changing the R group on the pyridinone ring, the aqueous solubility, lipophilicity, and BBB permeability can be modified. Prodrug hydroxy (OH) groups have been elaborated by glycosylation (Scheme 1 b) such that, after enzymatic deprotection, the free ligands will have ring OH groups available that can either efficiently trap radicals or bind metal complexes (Scheme 1 a). Removing metal ions that promote Ab aggregation, such as Cu II and Zn II, also serves to prevent or reverse Scheme 1. Hydroxypyridinones: a) nonglycosylated pro-ligands and b) their glycosylated prodrug forms designed for metal passivation in the brain as a therapeutic intervention in Alzheimer's disease (AD).