Abstract
Laboratory Automation and High-Throughput Chemistry
Automated Liquid–Liquid Extraction Workstation for Library Synthesis and its Use in the Parallel and Chromatography-Free Synthesis of 2-Alkyl-3-alkyl-4-(3H)-quinazolinones
An automated liquid–liquid extraction workstation developed by M. A. Toledo et al. processes up to 96 samples in an automated and parallel mode, avoiding the time-consuming and intensive sample manipulation during the workup process. To validate the workstation, a highly automated and chromatography-free synthesis of differentially substituted quinazolin-4(3H)-ones with two diversity points is carried out successfully, and highly pure products are received (J. Comb. Chem.
96-Well Plate-to-Plate Gravity Fluorous Solid-Phase Extraction for Solution-Phase Library Purification
Large particle size (125–210 μm) fluorous silica gel bonded with a – SiCH2CH2C8F17 stationary phase is used for gravity-driven fluorous solid-phase extraction (F-SPE) on two types of 96-well plates by W. Zhang and Y. Lu. A 1 or 0.75 g portion of fluorous silica is packed into each well of the 3.5-mL Ex-Blok and the 2.2-mL deep-well filtration plates. Up to 50 mg of reaction mixture is loaded and then eluted with a fluorophobic solvent dimethyl sulfoxide (DMSO), dimethyl formamide (DMF), or 85:15 DMF-H2O). Products collected in 96-well receiving plates are directly concentrated on a GeneVac vacuum centrifuge. This simple and highly efficient plate-to-plate F-SPE technique has been demonstrated in the purification of four 96-compound libraries. Approximately, 80% of products in each library have greater than 85% purity after F-SPE without conducting chromatography (J. Comb. Chem.
Microwave-Assisted Solid-Supported Alkyne Cyclotrimerization Reactions for Combinatorial Chemistry
A. Deiters et al. demonstrate the reactivity enhancing effects of microwave irradiation combined with the effects of spatial diyne separation on a polymeric support on the ruthenium-catalyzed [2 + 2 + 2] cyclotrimerization reaction. The conducted transformations are highly efficient and a high level of chemoselectivity is observed. Microwave irradiation does not affect the regioselectivity of the cyclotrimerization reaction when differentially substituted diyne precursors are used. The developed methodology provides rapid access to a variety of carbo- and heterocyclic structures from simple starting materials. It can be directly used in the synthesis of small-molecule arrays of pharmacologically relevant structures (e.g., isoindolines and tetrahydroisoquinolines) (J. Comb. Chem.
Microfluidic Chip Technology and Micro Reactor Technology
High-Throughput Screening and Optimization Approaches for Chiral Compounds by Means of Microfluidic Devices
D. Mangelings and Y. V. Heyden review chiral separations facilitated by using microchip devices. The first research paper on this topic was published in 1999. It showed that analysis times are greatly reduced compared with more conventional techniques such as liquid chromatography and capillary electrophoresis, and that these devices enable the separation of chiral molecules. Method optimization can be conducted in a rather easy manner, reducing the total method development time. Finally, minute amounts of sample and buffer are used during analysis, which makes the systems highly economical. Although the number of applications in the chiral separation field on these miniaturized systems is still rather limited, they exhibit much potential for high-throughput screening. Some efforts are, however, still needed regarding detection modes, because derivatisation of the samples is often needed to enable their detection (Comb. Chem. High Throughput Screen.
Assessment of the Ecological Potential of Microreaction Technology
An article in the Chem. Eng. Sic.
The authors investigate the ecological potential of microreaction technology using life-cycle assessment. The focus is on the ecological advantages associated with the transfer of the chemical synthesis from a macroscale semicontinuous batch process to a continuous microscale setup. As model reaction, the authors chose the highly exothermic synthesis of m-anisaldehyde from m-bromoanisole. This reaction makes high demands on safety and cooling efficient.
Two scales of producing are analyzed. In laboratory scale, a synthesis of 10 kg product under lab conditions is carried out by use of two Cytos-Lab-Systems. In industrial scale, a synthesis of 1000 kg product under real production conditions is carried out by use of the Cytos-Pilot-Systems with 11 microreactors in parallel. This can be compared with a conventional macroscale batch production.
The authors can show significant ecological advantages in case by using microreaction technology in comparison to the macroscale batch technology. On the lab scale, the advantages exist in energy savings, the reduction of solvents, and the increase of the reaction yield. The most important feature in industrial scale is the avoidance of a cryogenic system by increasing the reaction temperature.
In summary, the article shows that by using microreaction, technology can be a promising way to increase ecological sustainability of production processes.
Controlled Hydrogen Oxidation in a Microstructured Mixer-Reactor Module
In various fields of applications, for example, space flights, fuel cells, and the generation of pure water, controlled oxidations of hydrogen are of interest. Those reactions are highly exothermic, and safety is very important. Microreactors have to be cooled by a heat transfer medium, and must be able to withstand great stress.
To avoid stress in the microstructured component, reaction, mixing, and heat transfer must be combined in one reactor consisting of three stainless steel foils arranged with a cooling passage. The stack is completed with bottom and cover plates and joined by diffusion welding.
The authors show that this new microstructured reactor design allows a controlled, very rapid, and high exothermal reaction. In this case, it is hydrogen oxidation. The advantages of this concept include the avoidance of hot spots and the reaching of the operation temperature in very short periods of time. Furthermore, a high heat transfer to the cooling air flow is obtained.
The device also can be used for other gas-phase reactions, and its outer dimensions may vary (Chem. Eng. Technol.
High-Throughput Analytics
A Pulsed Triple-Ionization Source for Sequential Ion/Ion Reactions in an Electrodynamic Ion Trap
Gas-phase ion/ion reactions are being explored and used for the analysis of peptides, proteins, and other biomolecules. Scott A. McLuckey and coworkers from Purdue University (West Lafayette, USA) have developed a pulsed triple-ionization source, using a common atmosphere/vacuum interface and ion path to generate different ions for sequential ion/ion reaction experiments (J. Am. Soc. Mass Spectrom.
Differentiation of Maturity and Quality of Fruit Using Noninvasive Extractive Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometry
Maturity is an essential factor in determining storage life and final quality of most fruits and vegetables. R. Zenobi and coworkers have developed a new extractive electrospray ionization (EESI) method in combination with a quadrupole time-of-flight mass spectrometer (QTOF-MS) for fruit maturity and quality monitoring (Anal. Chem. 2007, 79, 1447–1455). For this purpose, fruits are placed in a clean and dry gas container, and air with about 45% relative humidity is infused as carrier gas to introduce the compounds released from the fruits into the EESI source. Here, the sampling gas is used as desolvation gas for the ESI spray and micro droplet–droplet extraction/ionization occurs. This setup allows a fast, highly sensitive spectral fingerprinting without any chemical contamination of the fruit. The unequivocal classification of unripe, ripe, and overripe fruits like bananas, grapes, and strawberries is demonstrated by differentiation patterns in their mass spectra. Furthermore, the mass spectral fingerprints of the investigated fruits can become the basis for the differentiation of ripening stages by performing principal component analyses (PCAs) of the raw data obtained in EESI-QTOF-MS. 3-D graphs of three different PCA scores to facilitate a clear classification and verify stability and reproducibility of the measurements.
Calibrant Delivery for Mass Spectrometry
Accurate mass assignment provides improved identification confidence for mass spectrometry, and is dependent on a reliable internal or external calibration. Thomas R. Covey and Bradley B. Schneider from MDS SCIEX (Ontario, Canada) describe the principal use of a new independent calibrant introduction methodology for different ion sources (J. Am. Soc. Mass Spectrom.
Molecular Formula Analysis by an MS/MS/MS Technique to Expedite Dereplication of Natural Products
One of the major problems encountered in natural product research is the repeated discovery of known compounds after the tedious work of purification and structural determination. Yasuo Konishi and coworkers developed a facile and sensitive mass spectrometric method for the dereplication of natural products that provide information about the molecular formula and substructure of a precursor molecule and its fragments (Anal. Chem.
Bioautomation and Screening
Identification of New Small-Molecule Inhibitors of Cystic Fibrosis Transmembrane Conductance Regulator Protein: In Vitro and in Vivo Studies
Cholera is life-threatening secretory diarrhea that results from intestinal infection with gram-negative bacteria Vibrio cholera. Cholera toxin, an enterotoxin produced by Vibriocholera, acts on enterocytes to increase intracellular cAMP, which in turn leads to massive cystic fibrosis transmembrane conductance regulator (CFTR) dependent intestinal chloride, and secondarily, fluid secretion. The volume of secreted fluid far exceeds the intestinal absorptive capacity, resulting in severe intestinal fluid loss and associated morbidity and mortality in affected individuals. Therefore, there are considerable efforts focused on developing antisecretory agents for patients suffering from cholera. At present, two classes of potent CFTR inhibitors have been identified by high-throughput screening. In spite of the above mentioned current-lead compounds, efforts to develop them as antidiarrheal drugs may be hampered by such drawbacks as poor water solubility and potential off-target effects. Therefore, the authors’ study is aimed at identifying new classes of CFTR inhibitors with equal or better properties as additional compounds of choice. They perform high-throughput screening using the iodide influx assay in Fisher rat thyroid (FRT) cells, stably expressing human wild-type CFTR and halide-sensitive yellow fluorescence protein H148Q (YFP-H148Q). Coexpression of the YFP-H148Q provides a quantitative fluorescence readout of inhibition potency of test compounds. After screening of 50,000 chemically diverse drug-like small molecules, they can identify two new classes of CFTR inhibitors. To determine potency of CFTR inhibitory actions of those identified inhibitors, they determine dose–response relationship by iodide influx assays and apical chloride current measurements in the FRT cells. The reversibility of CFTR inhibition by each compound is tested in apical chloride current measurements. However, only one compound, INH 1, is effective in reducing cholera toxin-induced intestinal fluid secretion in vivo. This in vivo study demonstrates that INH 1 could be the first of a new class of blockers of secretory diarrhea and a CFTR inhibitor (Muanprasat, C.; Kaewmokul, S.; Chatsudthipong, V. Biol. Pharm. Bull. 30(3),
Antifungal Chemical Compounds Identified Using a C. elegans Pathogenicity Assay
Breger et al. (PLoS Pathog.
Design Considerations for High-Throughput Screening and In Vitro Diagnostic Assays
K. E. Achyuthan and D. G. Whitten review the various factors that must be considered during the development of assays for high-throughput screening (HTS) or in vitro diagnostic (IVD) applications. The reader is introduced to the terminology used in assay development and the statistical approaches for evaluating the data. The authors state that the review is intended to serve as a tutorial to biotechnology, pharmaceutical, and clinical professionals. Elementary mathematical and statistical tools for designing robust assays and data management are described. An overview of the regulatory requirements for IVDs is included in the context of the U.S. Food and Drug Administration. Quality concepts and high-content screening are also briefly described (Comb. Chem. High Throughput Screen.
Optical Chemical Biosensors for High-Throughput Screening of Drugs
Optical biosensors have been commercially available since the early 1990s, and have been used extensively in many areas of research in the life sciences. Optical biosensors developed for drug analysis generally exploit the high selectivity of the antigen–antibody and drug–protein interaction. Optical biosensors can be made based on optical diffraction or electrochemiluminescence. High-throughput screening, which includes the automated preparation of a large number of samples and then a screening of their properties in multiwell plates, improves the efficiency of research in many scientific areas, including catalyst screening, food processing, chemical synthesis, drug discovery, absorption, distribution, metabolism, and excretion and toxicological and cell-based screening. The three most common detection techniques used in HTS are UV–VIS absorbance, fluorescence, and luminescence. F. S. Rojas et al. summarize some recent trends and developments in the construction of optical chemical biosensors used in high-throughput screening of drugs (Comb. Chem. High Throughput Screen.
Optical Biosensors as a Tool for Early Determination of Absorption and Distribution Parameters of Lead Candidates and Drugs
Specific molecular interactions provide a fundamental mechanism for selectivity in every aspect of biological structure and function. The ability to measure quantitatively such interaction properties across a wide range of affinity, size, and purity is a growing need. C. Bertucci et al. review the use of the optical biosensor techniques, focusing on applications for determining the absorption and distribution parameters of drugs and lead compounds. Basic biosensor technology principles are described with some immobilization methods commonly used for the preparation of selective and specific biosensor surfaces for assays. Some relevant research topics in the field of small-molecule recognition phenomena are presented as examples, including binding to plasma proteins, and binding to lipid membranes, in the frame of ADME (absorption, distribution, metabolism, and excretion) parameter determinations. These applications demonstrate the applicability of such techniques to the study of low-mass compounds, and illustrate their potential for the screening of libraries of compounds with regard to their binding to target biomolecules as part of drug development (Comb. Chem. High Throughput Screen.
Application of Liposome-Based Sensors in High-Throughput Screening Systems
High-throughout screening is an approach based on a concept that assumes that when a sufficiently large library of compounds is tested, the chance of discovering a new active compound is increased. To meet this expectation, proper testing criteria need to be devised. These criteria should be related to the fate of a compound in the organism to have any predictive power. Until recently, the main criteria have been based exclusively on parameters defined by the highest activity. With this approach, the activity criteria are not included, therefore, a compound's ability to reach a target is not accounted for. Considering that, the construction of yet another set of parameters has been initiated. The parameters are, in fact, semiempirical numbers that need to be tested on real, physical models. Whereas, the activity tests are straightforward, the pharmacokinetic tests are difficult and controversial. One such parameter describes the critical property of an active compound to cross biological membranes. M. Langner et al. describe new concepts for the determination of the permeability coefficient with the help of methods that are based on liposome biosensors. Two methods using fluorescence probes incorporated in the lipid bilayer of liposome are described in detail and compared to other currently available techniques (Comb. Chem. High Throughput Screen.
High-Speed Screening and Quantitative SAR Analysis of Human ABC Transporter ABCG2 for Molecular Modeling of Anticancer Drugs to Circumvent Multidrug Resistance
The transport mechanism-based molecular design strategy can provide an effective tool for rationalized chemotherapy against tumors. To develop a platform for molecular modeling to circumvent multidrug resistance, T. Ishikawa et al. report new methods of high-speed screening for human ABCG2–drug interactions, quantitative structure-activity relationship analysis, and quantum chemical calculation for lead optimization (Mini Rev. Med. Chem.
High-Throughput Analysis of Signals Regulating Stem-Cell Fate and Function
Stem cells exhibit promise in many areas of regenerative medicine. Their fate and function are governed by a combination of intrinsic determinants and signals from within their local microenvironments. An understanding of the mechanisms underlying both embryonic and adult stem-cell functions has been greatly enhanced by the recent development of several high-throughput technologies: microfabricated platforms, including cellular microarrays, to investigate the combinatorial effects of microenvironmental stimuli and large-scale screens using small molecules and short-interfering RNAs to identify crucial genetic and signaling elements. G. H. Underhill and S. N. Bhatia give an overview on this research topic (Curr. Opin. Chem. Biol.
The Development of High-Throughput Screening Approaches for Stem-Cell Engineering
Experimental studies find that both soluble factors, such as growth factors, and insoluble factors, including the surfaces on which cells grow, can have controlling effects on stem-cell behavior and differentiation. Although much progress has been made in biomaterial design and application, the rational design of biomaterial cues to direct stem-cell behavior and differentiation remains challenging. Recent advances in automated, high-throughput methods for synthesizing and screening combinatorial biomaterial libraries and cellular microenvironments promise to accelerate the discovery of factors that control stem-cell behavior. Specific examples include miniaturized, automated, combinatorial material synthesis, and extracellular matrix screening methods as well as microarrayed methods for creating local microenvironments of soluble factors, such as small molecules, siRNA, and other signaling molecules. Y. Mei et al. review the current status of high-throughput screening approaches for stem-cell studies (Curr. Opin. Chem. Biol.