High-throughput synthesis and analysis of acylated cyanohydrins

The yields and optical purities of products obtained from chiral Lewis acid/Lewis base-catalysed additions of alpha-ketonitriles to prochiral aldehydes could be accurately determined by an enzymatic method. The amount of remaining aldehyde was determined after its reduction to an alcohol, whilst the two product enantiomers were analysed after subsequent hydrolysis first by the (S)-selective Candida antarctica lipase B and then by the unselective pig liver esterase. The method could be used for analysis of products obtained from a number of aromatic aldehydes and aliphatic ketonitriles. Microreactor technology was successfully combined with high-throughput analysis for efficient catalyst optimization.     

Analytical pitfalls in hair testing

This review focuses on possible pitfalls in hair testing procedures. Knowledge of such pitfalls is useful when developing and validating methods, since it can be used to avoid wrong results as well as wrong interpretations of correct results. In recent years, remarkable advances in sensitive and specific analytical techniques have enabled the analysis of drugs in alternative biological specimens such as hair. Modern analytical procedures for the determination of drugs in hair specimens—mainly by gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–mass spectrometry (LC–MS)—are reviewed and critically discussed. Many tables containing information related to this topic are provided.     

Novel approaches to the analysis of steroid estrogens in river sediments

A wide range of estrogenic contaminants has been detected in the aquatic environment. Among these, natural and synthetic steroid estrogens, typically present in municipal sewage-treatment plant (STP) effluents, are the most potent. In this study a new GC–MS method has been developed for direct analysis of five major steroid estrogens (estrone, 17β-estradiol, 17α-ethinylestradiol, dienestrol, and diethylstilbestrol) in river sediments. Four GC–MS systems used for analysis of underivatized analytes in purified extracts were compared. Relatively low detection limits (1.5–5 ng g⁻¹ dried sediment) and good repeatability of GC splitless injection (RSD 1–2%) were achieved by use of a system combining low-pressure gas chromatography with a single-quadrupole mass analyzer (LP-GC–MS). Use of orthogonal gas chromatography (GC×GC) hyphenated with high-speed time-of-flight mass spectrometry (HSTOF-MS) enabled not only significantly better resolution of target analytes, and their unequivocal identification, but also further improvement (decrease) of their detection limits. In addition to these outcomes, use of this unique GC×GC–TOF-MS system enabled identification of several other non-target chemicals, including pharmaceutical steroids, present in purified sediment extracts.     

Rapid detection of cyanobacterial toxins in precursor ion mode by liquid chromatography tandem mass spectrometry

We established an analytical method based on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in the precursor ion mode for simultaneous qualitative monitoring of various groups of cyanobacterial toxins. The toxin groups investigated were paralytic shellfish poisoning (PSP) toxins, anatoxins (ANAs), cylindrospermopsins (CYNs), microcystins (MCs), and nodularins (NODs), including rare and uncharacterized derivatives found in plankton and water matrices. Alternative analytical methods based on tandem mass spectrometry commonly operate in multiple reaction monitoring (MRM) mode and depend on prior knowledge of putative toxigenicity of the cyanobacterium species and strain, and the expected toxin variants. In contrast, the precursor ion mode yields diagnostic mass fragments for the detection of characteristic compounds of the different toxin classes and thus allows monitoring of a large set of unspecified cyanotoxins of various groups, even when the species composition is undetermined or uncertain. This rapid method enables screening for a wide spectrum of toxic cyanobacterial metabolites and degradation products in a single chromatographic separation with detection limits at nanogram levels. The precursor ion technique is a valuable adjunct to existing mass spectrometric methods for cyanotoxins, although it is not a complete replacement for detailed quantitative analysis requiring comprehensive sample cleanup.     

Effective virtual screening protocol for CYP2C9 ligands using a screening site constructed from flurbiprofen and S-warfarin pockets

An effective virtual screening protocol was developed against an extended active site of CYP2C9, which was derived from X-ray structures complexed with flubiprofen and S-warfarin. Virtual screening has been effectively supported by our structure-based pharmacophore model. Importance of hot residues identified by mutation data and structural analysis was first estimated in an enrichment study. Key role of Arg108 and Phe114 in ligand binding was also underlined. Our screening protocol successfully identified 76% of known CYP2C9 ligands in the top 1% of the ranked database resulting 76-fold enrichment relative to random situation. Relevance of the protocol was further confirmed in selectivity studies, when 89% of CYP2C9 ligands were retrieved from a mixture of CYP2C9 and CYP2C8 ligands, while only 22% of CYP2C8 ligands were found applying the structure-based pharmacophore constraints. Moderate discrimination of CYP2C9 ligands from CYP2C18 and CYP2C19 ligands could also be achieved extending the application domain of our virtual screening protocol for the entire CYP2C family. Our findings further demonstrate the existence of an active site comprising of at least two binding pockets and strengthens the need of involvement of protein flexibility in virtual screening.     

Sensitivity enhancement in saturation transfer difference (STD) experiments through optimized excitation schemes

Investigation of ligand-protein interactions by the saturation transfer difference (STD) experiment has been well established in the drug discovery process through numerous examples. Thus, binding epitopes may be mapped by comparing signals of the ligand with and without saturation of the protein. Herein, it is shown that a less selective process allows more protons to assist in the saturation of the protein, thereby considerably enhancing the sensitivity of the STD experiment. Increasing the saturation power entails a greater risk of perturbing the ligand; however, an amplitude modulation of the waveform assists this procedure by distributing the applied energy in sidebands.     

Virtual Screening and Molecular Dynamics Simulation Study of Influenza Polymerase PB2 Inhibitors

Influenza A virus is the main cause of worldwide epidemics and annual influenza outbreaks in humans. In this study, a virtual screen was performed to identify compounds that interact with the PB2 cap-binding domain (CBD) of influenza A polymerase. A virtual screening workflow based on Glide docking was used to screen an internal database containing 8417 molecules, and then the output compounds were selected based on solubility, absorbance, and structural fingerprints. Of the 16 compounds selected for biological evaluation, six compounds were identified that rescued cells from H1N1 virus-mediated death at non-cytotoxic concentrations, with EC50 values ranging from 2.5–55.43 μM, and that could bind to the PB2 CBD of H1N1, with Kd values ranging from 0.081–1.53 μM. Molecular dynamics (MD) simulations of the docking complexes of our active compounds revealed that each compound had its own binding characteristics that differed from those of VX-787. Our active compounds have novel structures and unique binding modes with PB2 proteins, and are suitable to serve as lead compounds for the development of PB2 inhibitors. An analysis of the MD simulation also helped us to identify the dominant amino acid residues that play a key role in binding the ligand to PB2, suggesting that we should focus on increasing and enhancing the interaction between inhibitors and these major amino acids during lead compound optimization to obtain more active PB2 inhibitors.     

A Method for High-Throughput Screening of Enantioselective Catalysts

About 1000 catalytic or stoichiometric asymmetric reactions of racemic compounds or prochiral substrates bearing enantiotopic groups can be analyzed per day. In this highly efficient method the enantioselectivity is determined by electrospray ionization mass spectrometry using isotopically labeled substrates. The picture shows the mass spectrum of the mixture obtained upon hydrolysis of 1 to afford the pseudo-enantiomeric products 2 and 3 .     

“Scaffold-Hopping” by Topological Pharmacophore Search: A Contribution to Virtual Screening

A chemically advanced template search (CATS) based on topological pharmacophore models has been developed as a technique for virtual screening. This technique has successfully identified novel potent Ca²⁺ antagonists (such as 2 ) that have a similar activity to 1 (a known T-channel blocking agent) in a library of several hundred thousand compounds on the basis of a correlation vector representation.