Abstract
Laboratory Automation and High-Throughput Chemistry
Solution-Phase Synthesis of a Tetrasubstituted Pyrrole-Amides Library
L. F. Raveglia et al. describe an efficient strategy for the solution-phase parallel synthesis of a library of pyrrole-amides. Key reactions include functional homologation of β-ketoesters with a set of aldehydes followed by oxidation to produce a series of differently substituted 1,4-dicarbonyl compounds. Rapid cyclization using a microwave-assisted Paal–Knorr reaction provides a set of 24 pyrrole esters that are further functionalized through a trimethylaluminum-mediated aminolysis to obtain a larger library of 288 diverse pyrrole-3-amides. The tetrasubstitution offers a good exploration of the chemical space around the central pyrrole core. The last step is entirely automated with a Bohdan Myriad personal synthesizer (J. Comb. Chem.
Further Development of a Robust Workup Process for Solution-Phase High-Throughput Library Synthesis to Address Environmental and Sample Tracking Issues
During further improvement of a high-throughput, solution-phase synthesis system, new workup tools and apparatus for parallel liquid–liquid extraction and evaporation have been developed by N. Kuroda et al. A parallel liquid–liquid extraction unit, containing miniature high-speed magnetic stirrers for efficient mixing of organic and aqueous phases, is used on a multichannel liquid handler. Separation of the phases is achieved by dispensing them into a newly patented filter tube containing a vertical hydrophobic porous membrane, which allows only the organic phase to pass into collection vials positioned below. The vertical positioning of the membrane overcomes the dependence on the use of heavier-than-water, bottom-phase, organic solvents such as dichloromethane, which present environmental compliance challenges. In addition, an apparatus for parallel solvent evaporation to remove solvent from the samples with highly efficient recovery and to avoid the movement of individual samples between their collection on a liquid handler prevents sample identification errors (J. Comb. Chem.
Evaluation of “Credit Card” Libraries for Inhibition of Human Immunodeficiency Virus (HIV)-1 gp41 Fusogenic Core Formation
Protein–protein interactions are of critical importance to biological systems, and small-molecule modulators of such protein recognition and intervention processes are of particular interest. K. D. Janda et al. have synthesized small-molecule libraries that can disrupt a number of biologically relevant protein–protein interactions. These library members are designed upon planar motif, and appended with a variety of chemical functions. The authors call them “credit card” structures. In the process, a series of molecules is revealed to act as inhibitors against the HIV-1 gp41 fusogenic six-helix bundle core formation, viral antigen p24 formation, and cell–cell fusion at low micromolar concentrations (J. Comb. Chem.
On the Reactivity of Some 2-Methyleneindolines with β-Nitroamines, α-Nitroalkenes, and 1,2-Diaza-1,3-Butadienes
A study of the behavior of some electron-rich 2-methyleneindolines with different electron-poor reagents (formation of new carbon–carbon and nitrogen–carbon bonds) furnishes interesting results from both synthetic and the mechanistic viewpoints (Orazio A. Attanasi et al., Tetrahedron
Enamines react with the β-nitroamines (reaction CeCl3.7H2O promoted), creating polymethine dyes. The same bases are nitroalkylated with nitroolefins to produce indolines, and the diastereoselectivity reaction is thoroughly investigated. The most unexpected results come from the first example of Fischer's bases with 1,2-diaza-1,3-butadienes where previously unknown indoline spirodihydropyrroles are formed.
The new reaction provides a convenient route to interesting, partially reduced benzocondensed pyrrole derivatives that are intermediates in natural product synthesis.
Dowex-Promoted General Synthesis of N,N′-Disubstituted-4-Aryl-3,4-Dihydropyrimidinones Using a Solvent-Free Biginelli Condensation Protocol
Dowex-50W ion exchange resin-promoted solvent-free heating of an intimate mixture of an aldehyde, an active methylene compound, and N,N′-dimethylurea generates title compounds in moderate to good yields (Kamaljit Singh et al., Tetrahedron Lett.
The highly efficient method is environmentally benign, amenable to parallel synthesis, and offers operational advantages, such as clean reaction profiles and simple experimental/product isolation procedures, which makes it a useful and attractive strategy for the preparation of 4-aryl-3,4-dihydropyrimidinones (DHPMs) of synthetic importance.
The library preparation of these compounds is useful for screening in drug discovery. In the area of calcium channel modulators, appropriately functionalized DHPMs occupy a place of prominence. More recently, these substances have emerged as potent antihypertensive agents, α1a-adrenergic antagonists, and mitotic kinesin inhibitors.
An Efficient One-Pot Synthesis of Tetrahydroquinoline Derivatives via an Aza Diels–Alder Reaction Mediated by Ceric Ammonium Nitrate (CAN) in an Aqueous Medium and Oxidation to Heteroaryl Quinolines
Tetrahydroquinoline derivatives are an important class of natural products that exhibit a broad spectrum of biological activity. For this reason, substituted tetrahydroquinolines are the core structures in many important pharmacological agents and drug molecules, such as anti-arrhythmic and cardiovascular agents, anticancer drugs, immunosuppressants, and as ligands for 5-hydroxytryptamin 1A (5-HTIA) and N-methyl-D-asparat (NMDA) receptors.
In Tetrahedron Lett.
Highly Substituted Indol-2-Ones, Quinoxalin-2-Ones and Benzodiazepin-2,5-Diones via a New Ugi(4CR)-Pd–Assisted N-Aryl Amidation Strategy
A new strategy using an Ugi four-component reaction and a palladium-assisted intramolecular N-aryl amidation reaction is reported by Cédric Kalinski et al. (Tetrahedron Lett.
With final products containing four points of potential diversity and a facile and rapid production protocol, access to thousands of compounds containing important pharmacophoric scaffolds is now feasible. Thus, this new reaction is suitable for the generation of different compound libraries.
Microwave-Assisted Parallel Synthesis of a 14-Helical β-Peptide Library
J. K. Murray and S. H. Gellam describe a microwave-based method for parallel synthesis of a peptide library (J. Comb. Chem.
The authors adapt reaction conditions for the solid-phase synthesis of 14-helical β-peptides for use in a multimode microwave reactor to facilitate the preparation of β-peptide libraries in parallel. They describe a blend of parallel and sequential microwave irradiation steps in a multimode reactor, which combines the best aspects of both methods, for rapid preparation of a β-peptide library in inexpensive 96-well polypropylene filter plates.
Although microwave irradiation is attractive for accelerating the discovery of bioactive molecules, harnessing this method of rapid heating for the preparation of combinatorial libraries can be challenging. The major problem with microwave-assisted parallel library synthesis was the inhomogeneous heating and poor mechanical stability at elevated temperatures and pressures associated with using polypropylene well plates for microwave-assisted synthesis. To solve this problem, reaction blocks composed of microwave-absorbent material have been developed for accurate temperature measurement and uniform heating.
The experimental conditions developed for the solid-phase synthesis of β-peptides with microwave irradiation uses relatively low temperatures and high boiling solvents in open vessels. Multiwell polypropylene filter plates are sufficiently heat-stable for these conditions, and these plates are inexpensive and allow bottom-filtration of the solid support.
In conclusion, the article shows that microwave irradiation is used to improve the initial purity of β-peptides and reduce synthesis time (10-fold reduction). Use of a multimode microwave reactor for solid-phase β-peptide synthesis facilitates the preparation of a hexa-β-peptide library using a combination of parallel and sequential irradiation techniques. This library is sufficiently pure for initial screening without HPLC purification, although further improvement in synthetic efficiency would be desirable. The use of inexpensive polypropylene multiwell filter plates for microwave-assisted parallel library solid-phase synthesis is a simple alternative to more complex and expensive equipment for the rapid generation of peptide libraries.
A Chemical Reaction Engineering Laboratory Experiment: Isothermal Laminar-Flow Reactor
Nabeel S. Aboghander and Abdullah A. Shaikh describe the results of an experiment by an undergraduate chemical reaction engineering (CRE) laboratory (Int. J. Chem. Reactor Eng.
A sodium hydroxide–ethyl acetate reaction is chosen as the reaction system. The reaction between sodium hydroxide and ethyl acetate is conducted isothermally in a tubular reactor under isothermal, laminar-flow conditions. Steady-state reactor performance is followed at different space times and Reynolds numbers. Analysis of reactor performance is followed easily by direct sampling and titration. In the comparisons of the theoretical and experimental results, the authors find reasonable agreement. The experiment demonstrates how concepts in transport phenomena are related to reactor engineering.
Microwave Acoustic Sensor: From Chemical to Electrical Signals
G. Fischerauer reports on a mass-sensitive chemical sensor on an acoustical basis, the functional principle, the verification, and the signal processing (Tech. Mess.
A thin section is applied on a microacoustic element (SAW—surface acoustic waves). The selective sorption of molecules in the thin section causes a mass change of the piezoelectric oscillator, and a measurable change of frequency. The system performance is analyzed and an analytic model is derived. It enables the prediction of the dynamic response to the different concentrations that can be measured. Two examples of the model and the numeric results are compared for the measurement of Diesel steam concentration. An algorithm is presented for the real time, model-based estimation of the concentration.
Microfluidic Chip Technology and Microreactor Technology
The Origins and the Future of Microfluidics
G. M. Whitesides summarizes in a perspective article how microfluidics has the potential to influence subject areas from chemical synthesis and biological analysis to optics and information technology. He also offers an outlook on future developments, problems, and trends (Nature
Scaling and the Design of Miniaturized Chemical–Analysis Systems
A. Manz et al. describe the various reasons why micrometer-scale analytical devices are more attractive than their macroscale counterparts. These systems use smaller volumes of reagents, and are therefore cheaper, quicker, less hazardous to use, and more environmentally appealing. Scaling laws compare the relative performance of a system as the dimensions of the system change, and can predict the operational success of miniaturized chemical separation, reaction, and detection devices before they are fabricated. Some devices using basic principles of scaling are now commercially available, and opportunities for miniaturizing new and challenging analytical systems continue to increase (Nature
Developing Optofluidic Technology Through the Fusion of Microfluidics and Optics
D. Psaltis et al. describe devices in which optics and fluidics are used synergistically to synthesize novel functionalities. Fluidic replacement or modification leads to reconfigurable optical systems, whereas the implementation of optics through the microfluidic toolkit creates highly compact and integrated devices. The authors categorize optofluidics according to three broad categories of interactions: fluid–solid interfaces, purely fluidic interfaces, and colloidal suspensions. Examples of optofluidic devices in each category are given (Nature
Future Lab-on-a-Chip Technologies for Interrogating Individual Molecules
Advances in technology have allowed chemical sampling with high spatial resolution, and the manipulation and measurement of individual molecules. H. Craighead describes how adaptation of these approaches to lab-on-a-chip formats provides a new class of research tools for the investigation of biochemistry and life processes (Nature
Control and Detection of Chemical Reactions in Microfluidic Systems
Considerable progress in the development of microfabricated systems for use in chemical and biological sciences has been achieved during the last few years. Much of this development has been driven by a need to perform rapid measurements on small sample volumes. Interest in miniaturized analytical systems has been stimulated by the fact that physical processes can be more easily controlled and harnessed when instrumental dimensions are reduced to the micrometer scale. A. J. de Mello summarizes how such systems can define new operational paradigms, and how molecular synthesis might be revolutionized in the fields of high-throughput synthesis and chemical production (Nature
Cells on Chips
Microsystems create new opportunities for the spatial and temporal control of cell growth and stimuli by combining surfaces that mimic complex biochemistries and geometries of the extracellular matrix with microfluidic channels that regulate transport of fluids and soluble factors. K. F. Jensen et al. review how further integration with bioanalytic microsystems results in multifunctional platforms for basic biological insights into cells and tissues, as well as cell-based sensors with biochemical, biomedical, and environmental functions. Highly integrated micro devices show great promise for basic biomedical and pharmaceutical research, and robust and portable devices could be used in clinical settings (Nature
Microfluidic Diagnostic Technologies for Global Public Health
The developing world does not have access to many of the best medical diagnostic technologies because they have been designed for air-conditioned laboratories, and require refrigerated storage of chemicals, a constant supply of calibrators and reagents, stable electrical power, highly trained personnel, and rapid transportation of samples. P. Yager et al. review how microfluidic systems allow miniaturization and integration of complex functions, which could move sophisticated diagnostic tools out of developed-world laboratories. These systems must be inexpensive, but also accurate, reliable, rugged, and well suited to the medical and social contexts of developing nations (Nature
High-Throughput Analytics
Gas Chromatography–Microchip Atmospheric Pressure Chemical Ionization–Mass Spectrometry
The improvement of sensitivity and speed of analysis using microchip technology are two research goals of analytical chemistry. R. Kostiainen, T. Kotiaho, S. Franssila, and coworkers present a modified atmospheric pressure chemical ionization (APCI) microchip with an external corona discharge needle for coupling gas chromatography to mass spectrometry (Anal. Chem.
Novel Ion Mobility Setup Combined with Collision Cell and Time-of-Flight Mass Spectrometer
Ion mobility spectrometers in combination with mass spectrometers and soft ionization techniques are well suited for the analysis of biomolecules. Alexander Loboda from PerkinElmer SCIEX (Concord, Ontario, Canada) describes a novel ion mobility cell integrated into a matrix-assisted laser desorption/ionization (MALDI) source, collision-induced dissociation (CID) cell, and an orthogonal injection time-of-flight (TOF) mass spectrometer (J. Am. Soc. Mass Spectrom.
A new, low-pressure ion mobility setup, based on a segmented radio frequency (RF) ion guide, provides an axial confinement via gas drag and additional radial confinement via RF field, and allows ions to be trapped in the ion guide for an extended period of time. This extension of the separation times offers improved performance in combination with a CID cell and a TOF mass spectrometer. Furthermore, strikingly different from other typical MS/MS operation modes, a sequential fragmentation of multiple precursor ions can be practiced, and facilitates the efficient use of ions when analyzing complex samples such as a bovine serum albumine (BSA) digest. For testing the mobility separation of peptide fragment ions, the peptides M28, GluF, and adrenocorticotropic hormone (ACTH) are fragmented in the ion source before entering the ion mobility region, and the experimental results are discussed in detail. The generation of pseudo-MS3 spectra of the former can be achieved by further fragmentation in the adjacent CID cell. Finally, the important mass range capabilities of the setup are presented, based on mass and mobility spectra of protein and peptide mixtures, and technical improvements for the future are pointed out.
Bioautomation and Screening
Reading Dynamic Kinase Activity in Living Cells for High-Throughput Screening
Protein kinases represent an emerging class of drug targets, and the ability to assay their activities in living cells with high-throughput screening should provide exciting opportunities for drug discovery and chemical and functional genomics. J. Zhang et al. describe a general method for high-throughput reading of dynamic kinase activities using ratiometric fluorescent sensors. The authors perform the first successful compound screen based on the ability of compounds to modulate dynamic kinase activities in living cells (ACS Chem. Biol.
Quantitative High-Throughput Screening: A Titration-Based Approach that Efficiently Identifies Biological Activities in Large Chemical Libraries
High-throughput screening (HTS) is being used to identify modulators and chemical probes of gene, pathway, and cell functions, with the ultimate goal of comprehensively delineating relationships between chemical structures and biological activities. Achieving this goal will require methodologies that efficiently generate pharmacological data from the primary screen, and reliably profile the range of biological activities associated with large chemical libraries. Traditional HTS tests compounds at a single concentration, and therefore is not fully suited to this task, because of many false positives and false negatives that require extensive follow-up testing. J. Inglese et al. have developed a paradigm, quantitative HTS (qHTS), tested with the enzyme pyruvate kinase, to generate concentration–response curves for >60,000 compounds in a single experiment. The authors claim this method is precise, refractory to variations in sample preparation, and identifies compounds with a wide range of activities. qHTS produces large data sets that can be mined for biological activities, thereby providing a platform for chemical genomics and accelerating the identification of leads for drug discovery (Proc. Natl. Acad. Sci. U.S.A.
Image Subtraction Approach to Screening One-Bead-One-Compound Combinatorial Libraries with Complex Protein Mixtures
To screen one-bead-one-compound (OBOC) combinatorial bead libraries, generally tagged purified protein is used as the screening probe. Compound beads that interact with the purified protein can then be identified via an enzyme-linked colorimetric assay, and isolated for structure determination. K. S. Lam et al. demonstrate a rapid and efficient method for screening OBOC combinatorial libraries using two protein mixtures as screening probes. By comparing optical images of the beads stained by one protein mixture but not the other, ligand beads unique to one of the two protein mixtures can be identified. The significance of this method is that it allows for rapid selection of ligands directed against proteins unique to one mixture while screening out positive beads resulting from proteins common to both mixtures as well as beads that are positive as a result of interactions with chemical and protein components found in the assay itself. The authors describe their method as fast and efficient, using off-the-shelf equipment (J. Comb. Chem.
A Method for Rapid Protease Substrate Evaluation and Optimization
M. Lebl et al. have developed a high-throughput assay for the measurement of protease activity in solution. This technology might accelerate research in functional proteomics, and enable biologists to streamline protease substrate evaluation and optimization. The peptide sequences that serve as protease substrates in this assay are labeled on the carboxy terminus with a biotin moiety, and a fluorescent tag is attached to the amino terminus. Protease cleavage causes the biotin-containing fragment to be detached from the labeled peptide fragment. After the protease treatment, all biotin-containing species (uncleaved substrates and the cleaved carboxy terminal fragment of the substrate) are removed by incubation with streptavidin beads. The cleaved and fluorescently labeled amino terminal part of the substrate remains in solution. The measured fluorescence intensity of the solution is directly proportional to the activity of the protease (Comb. Chem. High Throughput Screen.
Design and Characterization of a Functional Library for NMR Screening Against Novel Protein Targets
In the past few years, nuclear magnetic resonance (NMR) has been extensively used as a screening tool for drug discovery using various types of compound libraries. The designs of NMR-specific chemical libraries that use a fragment-based approach based on drug-like characteristics have been previously reported. R. Powers et al. now describe a new type of compound library that focuses on aiding the functional annotation of novel proteins that have been identified from various genomics efforts. The NMR functional chemical library comprises small molecules with known biological activity such as cofactors, inhibitors, metabolites, and substrates. This functional library is developed through an extensive effort of mining several databases based on known ligand interactions with protein systems. To increase the efficiency of screening the NMR functional library, the compounds are screened as mixtures of 3–4 compounds that avoid the need to deconvolute positive hits by maintaining a unique NMR resonance and function for each compound in the mixture (Comb. Chem. High Throughput Screen.
High Content Cellular Screening
High content screening has established itself as a high-throughput technology for the analysis of microscopy-based cellular assays over the last few years. It has opened new areas of cell biology to the large-scale analysis of cellular phenotypes, and has enabled the application of increasingly sophisticated assays for large-scale genetic and compound screening. O. Rausch reviews the various technologies and applications of high content screening (Curr. Opin. Chem. Biol.
Streamlining Lead Discovery by Aligning In Silico and High-Throughput Screening
Lead discovery in the pharmaceutical environment is largely an industrial-scale process in which it is typical to screen 1–5 million compounds in a matter of weeks using high-throughput screening. This process is a very costly endeavor. Therefore, a great deal of pressure is on the drug discovery units to maximize the return on investment by finding fast and more effective ways of screening. A technology that has emerged over the past few years to help address this issue is in silico screening. J. W. Davies et al. review how in silico screening is incorporated in all areas of lead discovery; from target identification and library design, to hit analysis and compound profiling (Curr. Opin. Chem. Biol.
How Good is Your Screening Library?
Efficient library design is an ongoing challenge for investigators seeking novel ligands for drug discovery or chemical biology projects. Strategies that add neglected chemistry or exclude unproductive compounds are two important recent themes, as is a growing awareness of molecular complexity and its implications. J. J. Irwin discusses the various aspects that are important to enhancement programs for screening libraries with special focus on complexity (Curr. Opin. Chem. Biol.
A Practical View of “Druggability”
The introduction of Lipinski's “Rule of Five” has initiated a profound shift in the thinking paradigm of medicinal chemists. Understanding the difference between biologically active small molecules and drugs became a priority in the drug discovery process, and the importance of addressing pharmacokinetic properties early during lead optimization is a clear result. These concepts of “drug-likeness” and “druggability” are extended to proteins and genes for target identification and selection. T. H. Keller et al. summarize how these concepts could be integrated practically into the drug discovery process, as well as recent advances in the field (Curr. Opin. Chem. Biol.
Novel Method for High-Throughput DNA Methylation Marker Evaluation Using Peptide Nucleic Acid–Probe Library Hybridization and MALDI-TOF Detection
In March 2006, Schatz et al. published a novel high-throughput screening method to evaluate DNA methylation markers (Nucleic Acids Res.
The current publication introduces a new assay based on peptide nucleic acid (PNA)-library hybridization and subsequent MALDI-TOF analysis. The authors highlight the high flexibility, stability, and multiplexing properties of the method, allowing the use of standard 384-well automated pipetting techniques. In addition, the application provides more stability than established methods, such as microarrays and MS-SNuPE (methylation-sensitive single-nucleotide primer extension). The approach is used to evaluate three-candidate colon cancer methylation markers previously identified in a microarray study. The methylation of the genes Adenomatous polyposis coli (APC), glycogen synthase kinase-b-3 (GSK3b), and eyes absent 4 (EYA4) was analyzed in 12 colon cancer and 12 normal tissues. Using this method, APC and EYA4 are confirmed as being differentially methylated in colon cancer patients, whereas GSK3b did not show differential methylation. The method describes a potential way to analyze thousands of methylation markers, elevating the level of understanding that the role of methylation has in cancer diseases.