Antibody binding of amyloid beta peptide epimers/isomers and ramifications for immunotherapies and drug development.

Extracellular deposition of amyloid beta (Aß) peptide is a contributing factor of Alzheimer's disease (AD). Considerable effort has been expended to create effective antibodies, or immunotherapies, targeting Aß peptides. A few immunotherapies are thought to provide some benefit. It is possible that a contributing factor to the responses of such therapies may be the presence of modified, or aberrant, Aß peptides found in AD patients. These aberrations include the isomerization and epimerization of L-Asp and L-Ser residues to form D-Asp, L/D-isoAsp, and D-Ser residues, respectively. An effective methodology is essential to isolate all Aß peptides and then to quantify and locate the aberrant amino acids. Modifications to Aß peptides may elevate the deposition of Aß plaques and/or contribute to the neurodegeneration in AD patients, and may alter the binding affinity to antibodies. Herein, we used immunoprecipitation to examine the binding affinity of four antibodies against 18 epimeric and/or isomeric Aß peptides compared to wild type (all L) Aß peptide. Tandem mass spectrometry was used as a detection method, which also was found to produce highly variable results for epimeric and/or isomeric Aß.

Total synthesis of haploscleridamine, villagorgin A and an approach towards lissoclin C.

An investigation of asymmetric total syntheses of three indole-imidazole alkaloids from histidine are described. A common advanced piperidinone was contructed via a ring-closing metathesis which was then subjected to a modified Fischer indole synthesis. Deprotection of an N-tosyl group via a dissolving metal reduction affords haploscleridamine which upon reaction with aqueous formaldehyde in trifluoroethanol provided villagorgin A. On closer examination, it was found that villagorgin A was produced as a byproduct during the reductive detosylation in the presence of magnesium and methanol. Attempts to obtain the brominated haploscleridamine congener, lissoclin C through use of bromophenyl hydrazone were thwarted by reductive debromination during deprotection efforts. Investigation of the enantiopurity of the synthetic natural products revealed production of almost racemic materials in some batches as the result of partial racemization of an early stage intermediate. A revised approach routinely provided scalemic haploscleridamine and villagorgin in 30% ee. Analysis of the enantiomer composition of all intermediates by HPLC using columns with chiral stationary phases; this analysis revealed several steps where erosion of enantiomer composition occurred.

Recent advances in the field of chiral crystallization.

Crystallization is one of the largest and most economical bulk purification techniques used in industry today. There has been an increase in demand for enantiomerically pure compound production for research, organic synthesis, pharmaceutical drug production, and other applications. Even after asymmetric synthesis, chiral purification will always be necessary. The focus of this review is on recent advances in chiral crystallization for the purification of enantiomers. A comprehensive discussion of three techniques and their mechanisms is provided, namely: attrition-enhanced deracemization, cocrystallization, and inorganic ionic cocrystallization. Several examples of attrition-enhanced deracemization are discussed. The key advantage of this technique is that it eliminates enantiomeric waste and can be used to produce enantiomeric excesses of greater than 99% from racemic mixtures. Chiral cocrystallization is examined, with over 60 cocrystallizing compounds, as an excellent means for enantiomeric enrichment. Selective chiral inclusion complexation was shown to be a novel approach for the formation of cocrystals. Chiral inorganic ionic cocrystallization is a new technique involving the formation of cocrystals between chiral ligands and certain metal salts in order to produce conglomerate crystal behavior in otherwise racemic compounds.

Liquid chromatography enantiomeric separation of chiral ethanolamine substituted compounds.

Eleven racemic ethanolamine derivatives were prepared, and their enantiomers were separated using liquid chromatography with various chiral columns. These derivatives included chiral vicinal amino alcohols, ß-hydroxy ureas, ß-hydroxy thioureas, and ß-hydroxy guanidines, all of which are present in many active pharmaceutical ingredients. The screening study was performed with six chiral stationary phase containing columns, including four recently introduced superficially porous particles bonded with two macrocyclic glycopeptides, a cyclodextrin derivative and a cyclofructan derivative. The two remaining columns contained chiral stationary phases, based on either a cellulose derivative or derivatized amylose, both bonded to fully porous particles. The cyclodextrin and cellulose-based chiral stationary phases proved to be the most broadly effective selectors and were able to separate 8 and 7 of the 11 tested compounds, respectively. With respect to analyte structural features, marked differences in enantiorecognition were observed between compounds containing phenyl and cyclohexyl groups adjacent to the stereogenic center. Additionally, replacing a small electronegative oxygen atom by a larger and less electronegative sulfur atom induced a significant difference in chiral recognition by the cellulose derivative as well as by the vancomycin-based chiral selectors.

Enhanced carboxypeptidase efficacies and differentiation of peptide epimers.

Carboxypeptidases enzymatically cleave the peptide bond of C-terminal amino acids. In humans, it is involved in enzymatic synthesis and maturation of proteins and peptides. Carboxypeptidases A and Y have difficulty hydrolyzing the peptide bond of dipeptides and some other amino acid sequences. Early investigations into different N-blocking groups concluded that larger moieties increased substrate susceptibility to peptide bond hydrolysis with carboxypeptidases. This study conclusively demonstrates that 6-aminoquinoline-N-hydroxysuccimidyl carbamate (AQC) as an N-blocking group greatly enhances substrate hydrolysis with carboxypeptidase. AQC addition to the N-terminus of amino acids and peptides also improves chromatographic peak shapes and sensitivities via mass spectrometry detection. These enzymes have been used for amino acid sequence determination prior to the advent of modern proteomics. However, most modern proteomic methods assume that all peptides are comprised of l-amino acids and therefore cannot distinguish L-from d-amino acids within the peptide sequence. The majority of existing methods that allow for chiral differentiation either require synthetic standards or incur racemization in the process. This study highlights the resistance of d-amino acids within peptides to enzymatic hydrolysis by Carboxypeptidase Y. This stereoselectivity may be advantageous when screening for low abundance peptide stereoisomers.

Enantiomeric Separation of New Chiral Azole Compounds.

Twelve new azole compounds were synthesized through an ene reaction involving methylidene heterocycles and phenylmaleimide, producing four oxazoles, five thiazoles, and one pyridine derivative, and ethyl glyoxal for an oxazole and a thiazole compound. The twelve azoles have a stereogenic center in their structure. Hence, a method to separate the enantiomeric pairs, must be provided if any further study of chemical and pharmacological importance of these compounds is to be accomplished. Six chiral stationary phases were assayed: four were based on macrocyclic glycopeptide selectors and two on linear carbohydrates, i.e., derivatized maltodextrin and amylose. The enantiomers of the entire set of new chiral azole compounds were separated using the three different mobile phase elution modes: normal phase, polar organic, and reversed phase. The most effective chiral stationary phase was the MaltoShell column, which was able to separate ten of the twelve compounds in one elution mode or another. Structural similarities in the newly synthesized oxazoles provided some insights into possible chiral recognition mechanisms.