Using chiral peptide substitutions to probe the structure function relationship of a key residue of Aß42.

Amyloid beta-protein 42 plays an important role in the onset and progression of Alzheimer's disease. Familial mutations have identified the glutamate residue 22 as a hotspot with regard to peptide neurotoxicity. We introduce an approach to study the influence of systematic sidechain modification at this residue, employing chirality as a structural probe. Circular dichroism experiments reveal that charge-preserving alterations of the amino acid sidechain attenuate the characteristic random coil to ß-sheet transition associated with the wildtype peptide. Removal of the negative charge from residue 22, a trait observed with all known familial mutations at this residue, gives rise to a peptide with limited random coil propensity and high ß-sheet characteristics. Our approach can be extended to other residues of Aß, as well as further amyloidogenic peptides.

Suppression of Oligomer Formation and Formation of Non-Toxic Fibrils upon Addition of Mirror-Image Aß42 to the Natural l-Enantiomer.

Racemates often have lower solubility than enantiopure compounds, and the mixing of enantiomers can enhance the aggregation propensity of peptides. Amyloid beta (Aß) 42 is an aggregation-prone peptide that is believed to play a key role in Alzheimer's disease. Soluble Aß42 aggregation intermediates (oligomers) have emerged as being particularly neurotoxic. We hypothesized that the addition of mirror-image d-Aß42 should reduce the concentration of toxic oligomers formed from natural l-Aß42. We synthesized l- and D-Aß42 and found their equimolar mixing to lead to accelerated fibril formation. Confocal microscopy with fluorescently labeled analogues of the enantiomers showed their colocalization in racemic fibrils. Owing to the enhanced fibril formation propensity, racemic Aß42 was less prone to form soluble oligomers. This resulted in the protection of cells from the toxicity of l-Aß42 at concentrations up to 50 µm. The mixing of Aß42 enantiomers thus accelerates the formation of non-toxic fibrils.

Introduction of d-Glutamate at a Critical Residue of Aß42 Stabilizes a Prefibrillary Aggregate with Enhanced Toxicity.

The amyloid beta peptide 42 (Aß42) is an aggregation-prone peptide that plays a pivotal role in Alzheimer's disease. We report that a subtle perturbation to the peptide through a single chirality change at glutamate 22 leads to a pronounced delay in the ß-sheet adoption of the peptide. This was accompanied by an attenuated propensity of the peptide to form fibrils, which was correlated with changes at the level of the fibrillary architecture. Strikingly, the incorporation of d-glutamate was found to stabilize a soluble, ordered macromolecular assembly with enhanced cytotoxicity to PC12 cells, highlighting the importance of advanced prefibrillary Aß aggregates in neurotoxicity.

Trichlorosilane mediated asymmetric reductions of the C=N bond.

Chiral amines are key components in numerous bioactive molecules. The development of efficient and economical ways to access molecules containing this functional group still remains a challenge at the forefront of synthetic chemistry. Of the methods that do exist, the trichlorosilane mediated organocatalytic reduction of ketimines offers significant potential as an alternative strategy. In this perspective, we wish to highlight the progress made in the past decade in this field and offer a direct quantitative comparison to transition-metal mediated process.

The Bioactive Lipid (S)-Sebastenoic Acid Impacts Motility and Dispersion in Vibrio cholerae.

Although Gram-negative bacterial pathogens continue to impart a substantial burden on global healthcare systems, much remains to be understood about aspects of basic physiology in these organisms. In recent years, cyclic-diguanylate (c-di-GMP) has emerged as a key regulator of a number of important processes related to pathogenicity, including biofilm formation, motility and virulence. In an effort to discover chemical genetic probes for studying V. cholerae we have developed a new motility-based high-throughput screen to identify compounds that modulate c-di-GMP levels. Using this new screening platform, we tested a library of microbially-derived marine natural products extracts, leading to the discovery of the bioactive lipid (S)-sebastenoic acid. Evaluation of the effect of this new compound on bacterial motility, vpsL expression and biofilm formation implied that (S)-sebastenoic acid may alter phenotypes associated to c-di-GMP signaling in V. cholerae.

A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation.

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (Aß), can present a significant challenge when trying to obtain high purity material. Here we present a tailored HPLC purification protocol to produce high-purity amyloid beta 42 (Aß42) and amyloid beta 40 (Aß40) peptides. We have found that the combination of commercially available hydrophobic poly(styrene/divinylbenzene) stationary phase, polymer laboratory reverse phase - styrenedivinylbenzene (PLRP-S) under high pH conditions, enables the attainment of high purity (>95%) Aß42 in a single chromatographic run. The purification is highly reproducible and can be amended to both semi-preparative and analytical conditions depending upon the amount of material wished to be purified. The protocol can also be applied to the Aß40 peptide with identical success and without the need to alter the method.

Biofilm Formation and Detachment in Gram-Negative Pathogens Is Modulated by Select Bile Acids.

Biofilms are a ubiquitous feature of microbial community structure in both natural and host environments; they enhance transmission and infectivity of pathogens and provide protection from human defense mechanisms and antibiotics. However, few natural products are known that impact biofilm formation or persistence for either environmental or pathogenic bacteria. Using the combination of a novel natural products library from the fish microbiome and an image-based screen for biofilm inhibition, we describe the identification of taurine-conjugated bile acids as inhibitors of biofilm formation against both Vibrio cholerae and Pseudomonas aeruginosa. Taurocholic acid (1) was isolated from the fermentation broth of the fish microbiome-derived strain of Rhodococcus erythropolis and identified using standard NMR and MS methods. Screening of the twelve predominant human steroidal bile acid components revealed that a subset of these compounds can inhibit biofilm formation, induce detachment of preformed biofilms under static conditions, and that these compounds display distinct structure-activity relationships against V. cholerae and P. aeruginosa. Our findings highlight the significance of distinct bile acid components in the regulation of biofilm formation and dispersion in two different clinically relevant bacterial pathogens, and suggest that the bile acids, which are endogenous mammalian metabolites used to solubilize dietary fats, may also play a role in maintaining host health against bacterial infection.

Development of benzo[1,4]oxazines as biofilm inhibitors and dispersal agents against Vibrio cholerae.

Bacterial biofilms are estimated to be associated with over 65 percent of all nosocomial infections. However, no therapeutics have been approved by the FDA which directly mediate biofilm formation or persistence. Herein we report oxazine as a highly potent inhibitor, disperser and in the presence of the appropriate antibiotic eradicator of V. cholerae biofilms.

Living in the matrix: assembly and control of Vibrio cholerae biofilms.

Nearly all bacteria form biofilms as a strategy for survival and persistence. Biofilms are associated with biotic and abiotic surfaces and are composed of aggregates of cells that are encased by a self-produced or acquired extracellular matrix. Vibrio cholerae has been studied as a model organism for understanding biofilm formation in environmental pathogens, as it spends much of its life cycle outside of the human host in the aquatic environment. Given the important role of biofilm formation in the V. cholerae life cycle, the molecular mechanisms underlying this process and the signals that trigger biofilm assembly or dispersal have been areas of intense investigation over the past 20 years. In this Review, we discuss V. cholerae surface attachment, various matrix components and the regulatory networks controlling biofilm formation.

Enantioselective preparation of P-chiral phosphine oxides.

A highly efficient chiral auxiliary-based strategy for the asymmetric synthesis of P-chiral phosphine oxides in >98:2 er has been developed. The methodology involves the highly stereoselective formation of P-chiral oxazolidinones that then undergo displacement with a variety of Grignard reagents to prepare the desired phosphine oxides.