Azalysine analogues as probes for protein lysine deacetylation and demethylation

Reversible lysine acetylation and methylation regulate the function of a wide variety of proteins, including histones. Here, we have synthesized azalysine-containing peptides in acetylated and unacetylated forms as chemical probes of the histone deacetylases (HDAC8, Sir2Tm, and SIRT1) and the histone demethylase, LSD1. We have shown that the acetyl-azalysine modification is a fairly efficient substrate for the sirtuins, but a weaker substrate for HDAC8, a classical HDAC. In addition to deacetylation by sirtuins, the acetyl-azalysine analogue generates a novel ADP-ribose adduct that was characterized by mass spectrometry, Western blot analysis, and nuclear magnetic resonance spectroscopy. This peptide-ADP-ribose adduct is proposed to correspond to a derailed reaction intermediate, providing unique evidence for the direct 2'-hydroxyl attack on the O-alkylimidate intermediate that is formed in the course of sirtuin catalyzed deacetylation. An unacetylated azalysine-containing H3 peptide proved to be a potent inhibitor of the LSD1 demethylase, forming an FAD adduct characteristic of previously reported related structures, providing a new chemical probe for mechanistic analysis.     

High-field NMR studies of molecular recognition and structure-function relationships in antimicrobial piscidins at the water-lipid bilayer interface

High magnetic field solid-state NMR was performed on amphipathic cationic antimicrobial peptides from fish to characterize their secondary structure and orientation in hydrated phospholipid bilayers. High-resolution distance and orientational restraints on 13C- and 15N-labeled amidated piscidins 1 and 3 provided site-specific information establishing alpha-helicity and an orientation parallel to the membrane surface. Few membrane-bound natural peptides with this topology have been structurally studied at high resolution in the presence of hydrated lipid bilayers. This orientation was foreseen since the partitioning of amphipathic cationic antimicrobial peptides at the water-bilayer interface allows for favorable peptide-lipid interactions, and it may be related to the mechanism of action. The enhanced resolution obtained at 900 MHz evidences a determinant advantage of ultra-high-field NMR for the structural determination of multiple-labeled peptides and proteins.     

Chromatographic behaviour of peptides on a mixed-mode stationary phase with an embedded charged group by capillary electrochromatography and high-performance liquid chromatography

Retention behaviour of biological peptides was investigated on a stationary phase bearing an embedded quaternary ammonium group in a C21 alkyl chain by both high-performance liquid chromatography (HPLC) and capillary electrochromatography (CEC). In HPLC experiments, variation of acetonitrile (ACN) content in the mobile phase showed that peptides are mainly separated by RP mechanism. The weak or negative retention factors observed as compared to C18 silica stationary phase suggested the involvement of an electrostatic repulsion phenomenon in acidic conditions. Comparison of HPLC and CEC studies indicated that (i) ion-exclusion phenomenon is more pronounced in HPLC and (ii) higher ACN percentage in mobile phase induce for some peptides an increase of retention in CEC, pointing out the existence of mechanisms of retention other than partitioning mainly involved in chromatographic process. This comparative study demonstrated the critical role of electric field on peptide retention in CEC and supports the solvatation model of hydrolytic pillow proposed by Szumski and Buszewski for CEC using mixed mode stationary phase in CEC.     

Development and validation of a rapid and sensitive LC-ESI-MS/MS method for ondansetron quantification in human plasma and its application in comparative bioavailability study

The validation of a high throughput and specific method using a high‐performance liquid chromatography coupled to electrospray (ES+) ionization tandem triple quadrupole mass spectrometric (LC‐ESI‐MS/MS) method for ondansetron quantification in human plasma is described. Human plasma samples were extracted by liquid–liquid extraction (LLE) using methyl tert‐butyl ether and analyzed by LC‐ESI‐MS/MS. The limit of quantification was 0.2 ng/mL and the method was linear in the range 0.2–60 ng/mL. The intra‐assay precisions ranged from 1.6 to 7.7%, while inter‐assay precisions ranged from 2.1 to 5.1%. The intra‐assay accuracies ranged from 97.5 to 108.2%, and the inter‐assay accuracies ranged from 97.3 to 107.0%. The analytical method was applied to evaluate the relative bioavailability of two pharmaceutical formulations containing 8 mg of ondansetron each in 25 healthy volunteers using a randomized, two‐period crossover design. The geometric mean and respective 90% confidence interval (CI) of ondansetron test/reference percent ratios were 90.15% (81.74–99.44%) for C_max and 93.11% (83.01–104.43%) for AUC_0–t. Based on the 90% confidence interval of the individual ratios (test formulation/reference formulation) for C_max and AUC_0‐inf, it was concluded that the test formulation is bioequivalent to the reference one with respect to the rate and extent of absorption of ondansetron. Copyright © 2010 John Wiley & Sons, Ltd.     

Characterizing path graphs by forbidden induced subgraphs

A path graph is the intersection graph of subpaths of a tree. In 1970, Renz asked for a characterization of path graphs by forbidden induced subgraphs. We answer this question by determining the complete list of graphs that are not path graphs and are minimal with this property. © 2009 Wiley Periodicals, Inc. J Graph Theory 62: 369–384, 2009     

Practical and General Alcohol Deoxygenation Protocol

Herein, we describe a practical protocol for the removal of alcohol functional groups through reductive cleavage of their benzoate ester analogs. This transformation requires a strong single electron transfer (SET) reductant and a means to accelerate slow fragmentation following substrate reduction. To accomplish this, we developed a photocatalytic system that generates a potent reductant from formate salts alongside Brønsted or Lewis acids that promote fragmentation of the reduced intermediate. This deoxygenation procedure is effective across structurally and electronically diverse alcohols and enables a variety of difficult net transformations. This protocol requires no precautions to exclude air or moisture and remains efficient on multigram scale. Finally, the system can be adapted to a one-pot benzoylation-deoxygenation sequence to enable direct alcohol deletion. Mechanistic studies validate that the role of acidic additives is to promote the key C(sp³)−O bond fragmentation step.