Glycosamino Acids: New Building Blocks for Combinatorial Synthesis

In order to produce inexpensive, chemically diverse carbohydrate building blocks more amenable for use in combinatorial organic synthesis, amine and carboxylic acid functional groups were incorporated into several monosaccharides. A series of 12 new glycosamino acids was prepared from commercially available starting materials. Conventional peptide synthesis solution coupling techniques were used to ligate glycosamino acids, producing oligomeric “glycotides”. Finally, a library of glycotides was produced by coupling of a glycosamino acid mixture to a rigid template

Synthesis of Two Bicyclic Surfactants Which Form Reversed Micelles Capable of Selective Protein Extraction

Synthesis of Two Bicyclic Surfactants Which Form Reversed Micelles Capable of Selective Protein Extractio

Glycosamino Acids: New Building Blocks for Combinatorial Synthesis

In order to produce inexpensive, chemically diverse carbohydrate building blocks more amenable for use in combinatorial organic synthesis, amine and carboxylic acid functional groups were incorporated into several monosaccharides. A series of 12 new glycosamino acids was prepared from commercially available starting materials. Conventional peptide synthesis solution coupling techniques were used to ligate glycosamino acids, producing oligomeric “glycotides”. Finally, a library of glycotides was produced by coupling of a glycosamino acid mixture to a rigid template

Technetium Complexes for the Quantitation of Brain Amyloid

Technetium Complexes for the Quantitation of Brain Amyloi

Technetium Complexes for the Quantitation of Brain Amyloid

Technetium Complexes for the Quantitation of Brain Amyloi

Fibrils Formed in Vitro from α-Synuclein and Two Mutant Forms Linked to Parkinson's Disease are Typical Amyloid^†

Two missense mutations in the gene encoding α-synuclein have been linked to rare, early-onset forms of Parkinson's disease (PD). These forms of PD, as well as the common idiopathic form, are characterized by the presence of cytoplasmic neuronal deposits, called Lewy bodies, in the affected region of the brain. Lewy bodies contain α-synuclein in a form that resembles fibrillar Aβ derived from Alzheimer's disease (AD) amyloid plaques. One of the mutant forms of α-synuclein (A53T) fibrillizes more rapidly in vitro than does the wild-type protein, suggesting that a correlation may exist between the rate of in vitro fibrillization and/or oligomerization and the progression of PD, analogous to the relationship between Aβ fibrillization in vitro and familial AD. In this paper, fibrils generated in vitro from α-synuclein, wild-type and both mutant forms, are shown to possess very similar features that are characteristic of amyloid fibrils, including a wound and predominantly unbranched morphology (demonstrated by atomic force and electron microscopies), distinctive dye-binding properties (Congo red and thioflavin T), and antiparallel β-sheet structure (Fourier transform infrared spectroscopy and circular dichroism spectroscopy). α-Synuclein fibrils are relatively resistant to proteolysis, a property shared by fibrillar Aβ and the disease-associated fibrillar form of the prion protein. These data suggest that PD, like AD, is a brain amyloid disease that, unlike AD, is characterized by cytoplasmic amyloid (Lewy bodies). In addition to amyloid fibrils, a small oligomeric form of α-synuclein, which may be analogous to the Aβ protofibril, was observed prior to the appearance of fibrils. This species or a related one, rather than the fibril itself, may be responsible for neuronal death

The N-Terminal Repeat Domain of α-Synuclein Inhibits β-Sheet and Amyloid Fibril Formation^†

The conversion of α-synuclein into amyloid fibrils in the substantia nigra is linked to Parkinson's disease. α-Synuclein is natively unfolded in solution, but can be induced to form either α-helical or β-sheet structure depending on its concentration and the solution conditions. The N-terminus of α-synuclein comprises seven 11-amino acid repeats (XKTKEGVXXXX) which can form an amphipathic α-helix. Why seven repeats, rather than six or eight, survived the evolutionary process is not clear. To probe this question, two sequence variants of α-synuclein, one with two fewer (del2) and one with two additional (plus2) repeats, were studied. As compared to wild-type α-synuclein, the plus2 variant disfavors the formation of β-sheet-rich oligomers, including amyloid fibrils. In contrast, the truncated variant, del2, favors β-sheet and fibril formation. We propose that the repeat number in WT α-synuclein represents an evolutionary balance between the functional conformer of α-synuclein (α-helix and/or random coil) and its pathogenic β-sheet conformation. N-Terminal truncation of α-synuclein may promote pathogenesis

Improving Binding Specificity of Pharmacological Chaperones That Target Mutant Superoxide Dismutase-1 Linked to Familial Amyotrophic Lateral Sclerosis Using Computational Methods

We recently described a set of drug-like molecules obtained from an in silico screen that stabilize mutant superoxide dismutase-1 (SOD-1) linked to familial amyotrophic lateral sclerosis (ALS) against unfolding and aggregation but exhibited poor binding specificity toward SOD-1 in presence of blood plasma. A reasonable but not a conclusive model for the binding of these molecules was proposed on the basis of restricted docking calculations and site-directed mutagenesis of key residues at the dimer interface. A set of hydrogen bonding constraints obtained from these experiments were used to guide docking calculations with compound library around the dimer interface. A series of chemically unrelated hits were predicted, which were experimentally tested for their ability to block aggregation. At least six of the new molecules exhibited high specificity of binding toward SOD-1 in the presence of blood plasma. These molecules represent a new class of molecules for further development into clinical candidates

The Impact of the E46K Mutation on the Properties of α-Synuclein in Its Monomeric and Oligomeric States^†

The third and most recently identified Parkinson's disease-linked variant of the neuronal protein α-synuclein to be identified (E46K) results in widespread brain pathology and early onset Parkinson symptoms (Zarranz et al. (2004) Ann. Neurol. 55, 164−173). Herein, we present biochemical and biophysical characterization of E46K α-synuclein in various states of aggregation. Circular dichroism and nuclear magnetic resonance spectroscopy illustrate that the E46K mutation results in subtle changes in the conformation of the monomeric protein both free in solution and in the presence of SDS micelles. However, it does not alter the overall helical propensity of the protein in the presence of phospholipids. E46K α-synuclein formed insoluble fibrils in vitro more rapidly than the wild type protein, and electron microscopy revealed that E46K α-synuclein fibrils possess a typical amyloid ultrastructure. E46K α-synuclein protofibrils, soluble aggregates that form during the transition from the monomeric form to the fibrillar form of α-synuclein, were characterized by electron microscopy and gel filtration and were found to include annular species. The unique ability of a subfraction of E46K and wild type α-synuclein protofibrils containing porelike species to permeabilize lipid vesicles was demonstrated in vitro using a real-time chromatographic method. In contrast to simplistic expectations, the total amount of protofibrils and the amount of permeabilizing activity per mole protein in the protofibril fraction were reduced by the E46K mutation. These results suggest that if the porelike activity of α-synuclein is important for neurotoxicity, there must be factors in the neuronal cytoplasm that reverse the trends in the intrinsic properties of E46K versus WT α-synuclein that are observed in vitro