Anuj Kumar Sharma

Bifunctional compounds for controlling metal-mediated aggregation of the aβ42 peptide.

Abnormal interactions of Cu and Zn ions with the amyloid β (Aβ) peptide are proposed to play an important role in the pathogenesis of Alzheimers disease (AD). Disruption of these metal-peptide interactions using chemical agents holds considerable promise as a therapeutic strategy to combat this incurable disease. Reported herein are two bifunctional compounds (BFCs) L1 and L2 that contain both amyloid-binding and metal-chelating molecular motifs. Both L1 and L2 exhibit high stability constants for Cu(2+) and Zn(2+) and thus are good chelators for these metal ions. In addition, L1 and L2 show strong affinity toward Aβ species. Both compounds are efficient inhibitors of the metal-mediated aggregation of the Aβ(42) peptide and promote disaggregation of amyloid fibrils, as observed by ThT fluorescence, native gel electrophoresis/Western blotting, and transmission electron microscopy (TEM). Interestingly, the formation of soluble Aβ(42) oligomers in the presence of metal ions and BFCs leads to an increased cellular toxicity. These results suggest that for the Aβ(42) peptide-in contrast to the Aβ(40) peptide-the previously employed strategy of inhibiting Aβ aggregation and promoting amyloid fibril dissagregation may not be optimal for the development of potential AD therapeutics, due to formation of neurotoxic soluble Aβ(42) oligomers.

Pulsed hydrogen–deuterium exchange mass spectrometry probes conformational changes in amyloid beta (Aβ) peptide aggregation

Probing the conformational changes of amyloid beta (Aβ) peptide aggregation is challenging owing to the vast heterogeneity of the resulting soluble aggregates. To investigate the formation of these aggregates in solution, we designed an MS-based biophysical approach and applied it to the formation of soluble aggregates of the Aβ42 peptide, the proposed causative agent in Alzheimer’s disease. The approach incorporates pulsed hydrogen–deuterium exchange coupled with MS analysis. The combined approach provides evidence for a self-catalyzed aggregation with a lag phase, as observed previously by fluorescence methods. Unlike those approaches, pulsed hydrogen–deuterium exchange does not require modified Aβ42 (e.g., labeling with a fluorophore). Furthermore, the approach reveals that the center region of Aβ42 is first to aggregate, followed by the C and N termini. We also found that the lag phase in the aggregation of soluble species is affected by temperature and Cu2+ ions. This MS approach has sufficient structural resolution to allow interrogation of Aβ aggregation in physiologically relevant environments. This platform should be generally useful for investigating the aggregation of other amyloid-forming proteins and neurotoxic soluble peptide aggregates.

Neutral, cationic, and anionic low-spin iron(III) complexes stabilized by amidophenolate and iminobenzosemiquinonate radical in N,N,O ligands.

A brownish-black complex [Fe(III)(L)2] (1) (S = 0), supported by two tridentate redox-active azo-appended o-amidophenolates [H2L = 2-(2-phenylazo)-anilino-4,6-di-tert-butylphenol], has been synthesized and structurally characterized. In CH2Cl2 1 displays two oxidative and two reductive 1e(-) redox processes at E1/2 values of 0.48 and 1.06 V and -0.42 and -1.48 V vs SCE, respectively. The one-electron oxidized form [1](+) isolated as a green solid [Fe(III)(L)2][BF4] (2) (S = 1/2) has been structurally characterized. Isolation of a dark ink-blue one-electron reduced form [1](-) has also been achieved [Co(III)(η(5)-C10H15)2][Fe(III)(L)2] (3) (S = 1/2). Mössbauer spectral parameters unequivocally establish that 1 is a low-spin (LS) Fe(III) complex. Careful analysis of Mössbauer spectral data of 2 and 3 at 200 and 80 K reveal that each complex has a major LS Fe(III) and a minor LS Fe(II) component (redox isomers): [Fe(III){(L(ISQ))(-•)}2](+) and [Fe(II){(L(IBQ))(0)}{(L(ISQ))(-•)}](+) (2) and [Fe(III){(L(AP))(2-)}2](-) and [Fe(II){(L(ISQ))(-•)}{(L(AP))(2-)}](-) (3). Notably, for both at 8 K mainly the major component exists. Broken-Symmetry (BS) Density Functional Theory (DFT) calculations at the B3LYP level reveals that in 1 the unpaired electron of LS Fe(III) is strongly antiferromagnetically coupled with a π-radical of o-iminobenzosemiquinonate(1-) (L(ISQ))(-•) form of the ligand, delocalized over two ligands providing 3- charge (X-ray structure). DFT calculations reveal that the unpaired electron in 2 is due to (L(ISQ))(-•) [LS Fe(III) (SFe = 1/2) is strongly antiferromagnetically coupled to one of the (L(ISQ))(-•) radicals (Srad = 1/2)] and 3 is primarily a LS Fe(III) complex, supported by two o-amidophenolate(2-) ligands. Time-Dependent-DFT calculations shed light on the origin of UV-vis-NIR spectral absorptions for 1-3. The collective consideration of Mössbauer, variable-temperature (77-298 K) electron paramagnetic resonance (EPR), and absorption spectral behavior at 298 K, and DFT results reveals that in 2 and 3 the valence-tautomerism is operative in the temperature range 80-300 K.

A crude distillation unit model suitable for online applications

Abstract A steady state, multicomponent distillation model particularly suited for fractionation of crude oil has been developed based on equilibrium stage relations. For a mixture of C components, the present formulation uses C +3 iteration variables namely the mole fractions of the components, temperature, total liquid and total vapor flow rates on each stage. This choice of variables makes the present model numerically stable and robust rendering a separate initial guess computation unnecessary. An improved scheme of numbering the equilibrium stages when side strippers are present, was found to be advantageous with respect to computation time. Selected example problems have been included from literature as well as industry to demonstrate the efficacy and usefulness of the method. The accuracy of predictions and speed of solution of the model equations are particularly suited for online applications such as online optimization.

Small bifunctional chelators that do not disaggregate amyloid β fibrils exhibit reduced cellular toxicity.

Multifunctional metal chelators that can modulate the amyloid β (Aβ) peptide aggregation and its interaction with metal ions such as copper and zinc hold considerable promise as therapeutic agents for Alzheimer’s disease (AD). However, specific rather than systemic metal chelation by these compounds is needed in order to limit any side effects. Reported herein are two novel small bifunctional chelators, 2-[2-hydroxy-4-(diethylamino)phenyl]benzothiazole (L1) and 2-(2-hydroxy-3-methoxyphenyl)benzothiazole (L2), in which the metal-binding donor atoms are integrated within a molecular framework derived from the amyloid-binding fluorescent dye thioflavin T (ThT). The metal-binding properties of L1 and L2 were probed by pH spectrophotometric titrations to determine their pKa values and the corresponding metal complex stability constants, and the isolated metal complexes were structurally characterized. The amyloid-fibril-binding properties of L1 and L2 were investigated by fluorescence titrations and ThT competition assays. Interestingly, L1 and L2 do not lead to the formation of neurotoxic Aβ42 oligomers in the presence or absence of metal ions, as observed by native gel electrophoresis, Western blotting, and transmission electron microscopy. In addition, L1 and L2 were able to reduce the cell toxicity of preformed Aβ42 oligomers and of the copper-stabilized Aβ42 oligomers. Given their ability to reduce the toxicity of soluble Aβ42 and Cu-Aβ42 species, L1 and L2 are promising lead compounds for the development of chemical agents that can control the neurotoxicity of soluble Aβ42 species in AD.

Evaluation of 64Cu-Based Radiopharmaceuticals that Target Aβ Peptide Aggregates as Diagnostic Tools for Alzheimer’s Disease

Positron emission tomography (PET) imaging agents that detect amyloid plaques containing amyloid beta (Aβ) peptide aggregates in the brain of Alzheimer’s disease (AD) patients have been successfully developed and recently approved by the FDA for clinical use. However, the short half-lives of the currently used radionuclides 11C (20.4 min) and 18F (109.8 min) may limit the widespread use of these imaging agents. Therefore, we have begun to evaluate novel AD diagnostic agents that can be radiolabeled with 64Cu, a radionuclide with a half-life of 12.7 h, ideal for PET imaging. Described herein are a series of bifunctional chelators (BFCs), L1–L5, that were designed to tightly bind 64Cu and shown to interact with Aβ aggregates both in vitro and in transgenic AD mouse brain sections. Importantly, biodistribution studies show that these compounds exhibit promising brain uptake and rapid clearance in wild-type mice, and initial microPET imaging studies of transgenic AD mice suggest that these compounds could serve as lead compounds for the development of improved diagnostic agents for AD.

Coordination Chemistry of Bifunctional Chemical Agents Designed for Applications in 64Cu PET Imaging for Alzheimer’s Disease

Positron emission tomography (PET) is emerging as one of the most important diagnostic tools for brain imaging, yet the most commonly used radioisotopes in PET imaging, 11C and 18F, have short half-lives, and their usage is thus somewhat limited. By comparison, the 64Cu radionuclide has a half-life of 12.7 h, which is ideal for administering and imaging purposes. In spite of appreciable research efforts, high-affinity copper chelators suitable for brain imaging applications are still lacking. Herein, we present the synthesis and characterization of a series of bifunctional compounds (BFCs) based on macrocyclic 1,4,7-triazacyclononane and 2,11-diaza[3.3](2,6)pyridinophane ligand frameworks that exhibit a high affinity for Cu2+ ions. In addition, these BFCs contain a 2-phenylbenzothiazole fragment that is known to interact tightly with amyloid β fibrillar aggregates. Determination of the protonation constants (pKa values) and stability constants (log β values) of these BFCs, as well as characterization of the isolated copper complexes using X-ray crystallography, electron paramagnetic resonance spectroscopy, and electrochemical studies, suggests that these BFCs exhibit desirable properties for the development of novel 64Cu PET imaging agents for Alzheimer’s disease.

Azo-dyes based small bifunctional molecules for metal chelation and controlling amyloid formation

Chemical tools are needed to discover new effective drugs for tackling multifaceted complex neurodegenerative diseases like Alzheimers disease (AD). Multifunctional nature of two compounds, 5-((4-nitro-phenyl)diazenyl)quinolin-8-ol (HL1) and 4-((4-nitrophenyl)diazenyl)benzene-1,3-diol (HL2) is reported w.r.t. their ability to bind Cu2+ ions and amyloid aggregates related to AD. HL1 and HL2 have half congo-red type azo-stilbene structural framework incorporated with metal chelating groups, designed to chelate metal ions from metal-amyloid species. Metal binding studies of HL1 and HL2 are established by the methods of Jobs Plot, UV-vis spectra with metal ions and stability constant determination. In addition, their metal complexes are isolated, purity checked by elemental analysis, spectroscopically characterized and their structural analyses were obtained from DFT based calculations including binding energy determination. Chicken egg white Lysozyme (CEWL) was used as a model peptide for fibrillation studies. HL1 is found as an excellent colorimetric sensor for amyloid fibrils. Inhibitory effect of HL1 and HL2 and their isolated metal complexes L1-Cu and L2-Cu on CEWL fibrillation was studied using ThT and ANS fluorescence assay along with TEM imaging. In addition, the cell toxicity studies on these compounds suggest that although azo dyes may be non-toxic but having a nitro-substitution lead to significant cell toxicity. Overall, these results suggest that this new class of multifunctional small molecules can interact with amyloids as well as metal ions and could be potential anti-aggregation metal chelating agents.