Abstract
Laboratory Automation and High-throughput Chemistry
High-Throughput Heterogeneous Catalytic Science
High-throughput experimentation in heterogeneous catalysis recently has experienced nearly exponential growth. Initial qualitative screening has evolved into quantitative high-throughput experimentation, characterization, and analysis. This now allows high-throughput catalysis to rise above simple screening to the level of fundamental understanding of reaction mechanisms. R. J. Hendershot, C. M. Snively, and J. Lauterbach have published an interesting concept article on high-throughput heterogeneous catalytic science (Chem.—Eur. J., 2005, 11, 806).
Accelerating the Living Polymerization of 2-Nonyl-2-oxazoline by Implementing a Microwave Synthesizer into a High-Throughput Experimentation Workflow
U. S. Schubert et al. report the successful implementation of a microwave synthesizer (Emrys Liberator, Biotage, Uppsala, Sweden) into a high-throughput workflow with a Chemspeed ASW 2000 synthesizer robot. The resulting workflow is utilized to screen different solvent mixtures for the microwave-assisted cationic ring-opening polymerization of 2-nonyl-2-oxazoline at 140 °C to prevent precipitation of the polymer during the polymerization process. Polymerizations in solvent mixtures containing more than 20% dichloromethane are found to proceed to full monomer conversion, whereas a lower dichloromethane content leads to a maximum conversion of only 85%. Kinetic investigations of the microwave-assisted polymerizations of 2-nonyl-2-oxazoline in dichloromethane reveal a living polymerization mechanism for all investigated temperatures (100-180 °C). The polymerizations under microwave irradiation proceed significantly faster than conventionally heated polymerizations (at ambient pressure). The authors demonstrate the successful living polymerization of 2-oxazolines in dichloromethane under microwave irradiation for the first time (J. Comb. Chem., 2005, 7, 10).
Simple Tools for Resin Distribution
M. Lebl et al. describe two simple and efficient instruments that allow uniform distribution of resin beads for solid-phase synthesis (J. Comb. Chem., 2005, 7, 42). The first tool simplifies distribution of resin into microtiterplates. The second tool is designed for distribution of a variable amount of resin beads into any number of reaction vessels, and the technique is applicable to resin beads with a wide range of physical properties (density).
Orthogonality and Compatibility Between Tsc and Fmoc Amino-Protecting Groups
New deprotection conditions that provide a complete orthogonality between Tsc and Fmoc aminoprotecting groups are described by Jin Seok Choi et al. (Tetrahedron, 2005, 9, 2493-2503). The potential of these orthogonal deprotection conditions is demonstrated by the efficient solid-phase synthesis of branched peptides using doubly protected amino acids such as Tsc-Lys(Fmoc)-OH and Fmoc-Lys(Tsc)-OH. They showed that 1-methylpyrrolidine and LiOH are the reagents of choice for mild and orthogonal deprotection of Fmoc and Tsc. The new Tsc/Fmoc strategy should greatly extend the scope of the Tsc group to dual protections under mild basic conditions. Tsc/ Fmoc will be a potential alternative to standard Dde/Fmoc, particularly where use of the Dde amino-protecting group is not possible.
Transglutaminase Surface Recognition by Peptidocalix[4]arene Diversomers
A series of N-linked tetrakis(tetrapeptido)calix[4]arene diversomers has been synthesized by coupling a cone calix[4]arene tetracarboxylic acid chloride with tetrapeptides obtained in a parallel fashion (Simona Francese et al., Tetrahedron Lett., 2005, 10, 1611-1615). The inhibition activity of the diversomers towards tissue and microbial transglutaminase is evaluated by in vitro assays with a labeled substrate. Kinetic analysis using one of the most active derivatives shows a noncompetitive inhibition with respect to the amino acceptor substrate, and an uncompetitive inhibition with respect to amino donor substrate. The experimental results are in accordance with an inhibition due to protein-specific surface recognition on a region noncomprising the enzyme active site.
Alkene Epoxidation with Urea-Hydrogen Peroxide Complex and PS-DVB Supported Phthalic Anhydride
Chiara Ghiron et al. report the use of a new PS-DVB supported phthalic anhydride and urea-hydrogen peroxide complex (UHP) for metal-free alkene epoxidation reactions (Tetrahedron Lett., 2005, 10, 1643-1645). The resin is prepared by microwave mediated “PEGylation” of Merrifield resin followed by esterification with trimellitic anhydride chloride. Successful epoxidation of several alkenes is carried out with this resin and UHP.
Preparation of Kinase-Biased Compounds in the Search for Lead Inhibitors of Kinase Targets
Justine Y. Q. Lai, Steven Langston, and colleagues report the preparation of approximately 13,000 compounds for rapid identification of hits in high-throughput screening (HTS) of kinase targets. These compounds are designed as potential serine/threonine or tyrosine kinase inhibitors. The library consists of various scaffolds, for example, purines, oxindoles, and imidazoles, whereby each core scaffold generally includes the hydrogen bond acceptor/donor properties known to be important for kinase binding. The routes to their preparation are outlined. A variety of automation techniques are used to prepare >500 compounds per scaffold. Where applicable, scavenger resins are employed to remove excess reagents, and when necessary, preparative high-performance liquid chromatography (HPLC) is used for purification. These compounds are screened against an in-house kinase panel, and the success rate in HTS is significantly higher than the corporate compound collection (Med. Res. Rev., 2005, 25, 310).
Microfluidic Chip Technology and Micro Reactor Technology
Microreactor-Based Reaction Optimization in Organic Chemistry: Glycosylation as a Challenge
K. F. Jensen, P. H. Seeberger, and coworkers use a continuous flow silicon microreactor to study glycosylation reactions in a rapid manner over a wide range of conditions. These experiments are a very interesting example of microreactor-based method optimization and process development in organic chemistry. Unlike batch methods, which are challenged by the difficulty of handling microliter quantities of volatile solvents and the possibility of external contamination, the enclosed microreactor system serves to rapidly obtain comprehensive information about a given transformation. With a single preparation of reagents, 44 reactions are completed at varying temperatures and reaction times requiring just over 2 mg of glycosylating agent for each reaction (Chem. Commun., 2005, 578).
Continuous Hydrogenation Reactions in a Tube Reactor Packed with Pd/C
Nungruethai Yoswathananont et al. report the functionality of a tube reactor for a hydrogenation reaction (Chem. Commun., 2005, 40-42). The authors introduce a continuous tube reactor from stainless steel. The dimensions of the reactor are 6.3 mm outer diameter, 1.0 mm inner diameter, and 25 cm length. At the reactor inlet is a T-shaped mixer with a transparent Teflon tube. The gas-liquid-solid reactor works as follows. The catalyst fills in the hole of the tube. The substrate solution and the gas are mixed in the T-shaped mixer. The most efficient relationship between the substrate solution and the gas can be controlled by the flow rate of the solution and the gas pressure. The gas-liquid relationship can be observed in the transparent Teflon tube. The efficiency of the reactor is evaluated with the hydrogenation of 4-cyanobenzaldehyde in methanol. The catalyst is palladium on carbon. The results of the experiments show efficiency at different temperatures and higher yields than batch systems.
Versatile Thin-Film Gas-Liquid Multi-Channel Microreactors for Effective Scale-Out
The development and construction of a versatile gasliquid multi-channel microreactor is described by Richard D. Chambers et al. (Lab Chip, 2005, 5, 191-198). In today's laboratory, small conti reactors typically are used with batch reactors because they make strong exothermic reactions safe. An example of such a reaction is direct fluorination. The safe handling and control of such thermodynamically exothermic processes limit the viable potential for direct fluorination on an industrial scale. For these reactions, microreactors offer considerable benefits to laboratory and manufacturing processes. They are highly safe and can facilitate efficient gas/ liquid mixing and effective heat exchanges for very large gas/ liquid surface: volume ratios. Furthermore, what happens in the laboratory also can occur on an industrial scale because a manufacturing process can be constructed with many of the same laboratory reactors in parallel. The reactor is developed in multiple steps. At first it consists of a single groove cut into a nickel block. The cover of the groove is made from polychlorotriofluoroethylene (PCTFE), and the flow regime in the reactor is the pipe flow. Pipe flow is the best possible scenario to obtain excellent reaction surface. The next step is to develop a multi-channel plate. Here, each channel requires a separate feed system for each reactant and multiple outlet port. This is achieved with the use of reservoirs for the inlet and the outlet. With integration of these reservoirs in the reactor base block, the authors construct a modular microreactor device for laboratory and large-scale synthesis. The materials of the reactor are stainless steel, nickel, and PTFCE. The reactor and channel plates can be manufactured simply and inexpensively with mechanical workshop techniques. In addition, the modular device can be assembled and disassembled quickly, permitting simple replacement of the different channel plates.
High-throughput Analytics
ALARM NMR: A Rapid and Robust Experimental Method to Detect Reactive False Positives in Biochemical Screens
The escalating cost of drug discovery is due in large part to financing the discovery and development of compounds that fail to become drugs. High-throughput screening (HTS) of large compound collections typically results in numerous small molecule hits that must be carefully evaluated to identify valid drug leads. Although several filtering mechanisms and other tools exist to assist the chemist in this process, it is often the case that costly synthetic resources are expended in pursuing false positives. Philip J. Hajduk and colleagues report a rapid and reliable nuclear magnetic resonance (NMR) based method for identifying reactive false positives, including those that oxidize or alkylate a protein target. The reactive species does not need to be the parent compound, because both reactive impurities and breakdown products can be detected. The assay is called ALARM NMR (a La assay to detect reactive molecules by nuclear magnetic resonance) and is based on monitoring DTT-dependent 13C chemical shift changes of the human La antigen in the presence of a test compound or mixture. Extensive validation is performed to demonstrate the reliability and utility of using ALARM NMR to assess thiol reactivity. This includes comparing ALARM NMR to a glutathione-based fluorescence assay, as well as testing a collection of more than 3500 compounds containing HTS hits from 23 drug targets. The data show that current in silico filtering tools fail to identify more than half of the compounds that can act via reactive mechanisms. ALARM NMR data is critical in identifying reactive compounds that would otherwise have been prioritized for lead optimization. In addition, a new filtering tool has been developed on the basis of the ALARM NMR data, and it can augment current in silico programs for identifying nuisance compounds and improving the process of hit selection and optimization (J. Am. Chem. Soc., 2005, 127, 217).
High-Throughput Microcoil NMR of Compound Libraries Using Zero-Dispersion Segmented Flow Analysis
NMR analysis is regarded as one of the best means to confirm the identity of synthetic products and to establish purity quantitatively. However, performing routine NMR analyses on a library of 10,000 compounds can require months. In addition, conventional high-throughput NMR methods using liquid chromatography (LC) NMR probes typically require 100 μg of analyte, and several milliliters of expensive deuterated solvent. Roger A. Kautz et al. has developed an automated system for loading samples into a microcoil NMR probe by using segmented flow analysis. This approach enhances twofold the throughput of the known direct injection and flow injection methods, improves sample utilization threefold, and is applicable to high field NMR facilities with long transfer lines between the sample handler and NMR magnet. Sample volumes of 2 μL are drawn from a 96-well microtiter plate by a sample handler, then pumped to a 0.5-μL microcoil NMR probe as a queue of closely spaced plugs separated by an immiscible fluorocarbon fluid. Individual sample plugs are detected by their NMR signal and automatically positioned for stopped-flow data acquisition. The sample in the NMR coil can be changed within 35 s by advancing the queue. The fluorocarbon liquid wets the wall of the Teflon transfer line, preventing the dimethyl sulfoxide (DMSO) samples from contacting the capillary wall and thus reducing sample losses to below 5% after passage through the 3-m transfer line. With a wash plug of solvent between samples, sample-to-sample carryover is < 1%. The samples do not disperse into the carrier liquid during loading or during acquisitions. Several days are required for trace analysis. For automated high-throughput analysis using a 16-s acquisition time, spectra were recorded at a rate of 1.5 min/sample and total deuterated solvent consumption was <0.5 mL ($1) per 96-well plate (J. Comb. Chem., 2005, 7, 14).
UV-Triggered Main-Component Fraction Collection Method and Its Application for High-Throughput Chromatographic Purification of Combinatorial Libraries
A maximum-seeking, algorithm-driven fraction collection method is shared by T. Karancsi et al. to support high-throughput chromatographic purification that provides new possibilities for off-line high-performance liquid chromatography mass spectroscopy (HPLC/MS) quality control experiments. The method is based on manipulation of a six-port valve that is installed downstream from the UV detector and equipped with a fraction collector loop. The detector signal is monitored by a programmable microcontroller that controls the state of the fraction collector valve. After detecting a chromatographic peak, the appropriate fraction is stored in the collector loop. The height of the next peak is compared to the previous one (using a maximum seeking algorithm) and, depending on the result, the collected fraction is or is not exchanged with the new one. At the end of the run, the stored UV main component is pumped into the external fraction vial. This configuration is used for chromatographic purification of large compound libraries (up to 5324 compounds), as well as for high-throughput off-line HPLC quality control experiments where the collected main component fractions of an analytical-scale separation are subject to further mass spectrometric molecular weight verification (J. Comb. Chem., 2005, 7, 58).
Microchip Atmospheric Pressure Chemical Ionization Source for Mass Spectrometry
For life science, biopharmaceutical chemistry, and environmental research, the new ionization techniques electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photo ionization (APPI) play key roles and therefore are involved in state-of-the-art developments. R. Konstantinen, S. Franssila, and coworkers (University of Helsinki) combine the well established APCI technology with the latest microchip technology for improved sensitivity and faster analysis via miniaturization (Anal. Chem. 2004, 76, 6659). This new two-dimensional APCI source is based on a silicon wafer with etched flow channels, inlet, nozzle, and a glass wafer with integrated aluminium heater jointed by anodic bonding. Moreover, the implementation of an external corona-discharge needle provides a multiplex connection of the microchip to any MS equipped with an atmospheric pressure ionization source. The explored configuration provides long-term stable flow rates down to 50 nL/min and an excellent sensitivity, even below 1 fmol/s, predestined for microfluidic applications of nonpolar or neutral targets below a molecular weight of 1000 amu. Further advantages of the microchip APCI are the fast stabilization of the operating temperature and the fact that cooling takes only 10-20 s. Therefore, during each LC run, most compounds can be measured under optimal conditions. Several compounds with different physical and chemical properties are tested and the results are compared with other standard macro APCI devices to show the inherent advantages.
Preparative Linear Ion Trap Mass Spectrometer for Separation and Collection of Purified Proteins and Peptides in Arrays Using Ion Soft Landing
When reducing the amount of material needed for many chemical and biochemical tests, the drawbacks of collecting highly purified molecular species by means of mass spectrometric separation are not as severe as they once were. R. G. Cooks and coworkers from Purdue University (West Lafayette, IN) demonstrate the utilization of an enhanced linear ion trap mass spectrometer instrumentation for the production of peptide and protein arrays, separated by mass/ charge ratio and subsequently collected by soft landing technique (Anal. Chem. 2004, 76, 6293). All important technical requirements of the utilized instrument are described in detail including how each component of the system influences the final soft-landing experiments. In the second part, the instrument is characterized by means of its analytical mass spectrometer performance, the soft landing and array production, and the efficiency of the overall process. For this purpose compounds like apomyoglobin, insulin, trypsin, brandykinin, or neurotensin are used. The ability to retain bioactivity in the mass-selected and collected biomolecules is demonstrated, as well as the usability of the integrated soft-landing instrument to create arrays of chemically different spots from a mixture of proteins without cross-contamination.
Stereoselectivity in the Collision-Activated Reactions of Gas-Phase Salt Complexes
During the past decade, major efforts have been made to gain stereochemical information from mass spectrometric data thanks to the high sensitivity and contemporary analysis capabilities of mass spectrometry. S. Gronert and coworkers present two new gas-phase reaction systems to study and understand molecular interactions that lead to stereoselectivity in selected chemical transformations (J. Am. Soc. Mass Spectrom. 2004, 15, 1499). The two describe in detail collision-activated dissociation (CAD) reactions of gas-phase salt complexes based on the formation of diastereromeric complexes, which consist of two chiral counterparts. The CAD of the first kind of complexes is composed of a deprotonated binaphthol, and a bistetraalkylammonium dication leads to a stereoseletive distribution of mono-charged tetraalkylammonium cation products, arising from a proton or an alkyl cation transfer to the binaphtholate. Secondly, the product distribution of a peptide dianion and a tetraalkylammonium cation is investigated. In general, reactive sites with an asymmetric center nearby showed a more effective and reliable cause to achieve stereo-selectivity. Representative transition states are modeled computationally, and energies of the structures are determined by single-point calculations at the B3LYP level to get an insight into factors that lead to the observed stereo-selectivity.
Bioautomation and Screening
High-Throughput Mutation Detection Underlying Adaptive Evolution of Escherichia coli-K12
MALDI-TOF mass spectrometry is an efficient and highly accurate tool for identifying DNA sequence changes by analyzing base-specific cleavage products. Honisch et al. (Genome Research, 2004, 14, 2495) use the MassArray system from Sequenom Inc, San Diego, CA, to conduct fully automated high-throughput mutation detection in microbial genomes, specifically the bacterium Escherichia coli-K12. In contrast to Sanger-Sequencing that delivers direct DNA sequence information, the MALDI-TOF-based analysis focuses on the detection of sequence deviations. The result is an automated output of mutations and their localization. In addition, the system allows parallel screening of multiple samples and offers a solution for rapid large-scale resequencing studies.
Microfluidic Sorting of Mammalian Cells by Optical Force Switching: Microfluidic Devices Allow Miniaturization and a High Parallel Screening for Many Functions in Biological Assays
Wang et al. (Nature Biotechnology, 2005, 23, 83) share a practical technology for microfluidic-based fluorescence-activated cell sorting. They show that optical forces can be used for the active control of cell routing on a microfluidic chip. The performance of the device is evaluated on live, stably transfected HeLa cells expressing a fused histonegreen fluorescent protein. Recovered populations are verified to be both viable and unstressed by evaluation of the expression of two genes (HSP A6 and FOS) known as indicators of cellular stress.