Automation Highlights from Literature

Kerstin Thurow, Ph.D.

Hilmar Weinmann, Ph.D.

Laboratory Automation and High-Throughput Chemistry

Chemical Development on the Chiral Auxiliary (S)-4-(Phenylmethyl)-2-oxazolidinone Utilizing Automated Synthesis and DOE

Enantiopure 4-substituted oxazolidinones are well-known chiral auxiliaries for asymmetric synthesis of carboxylic acid derivatives. O. W. Gooding et al. (Argonaut Technologies) conducted chemical development studies toward an efficient and scalable “one-pot” process for production of (S)-4-(phenylmethyl)-2-oxazolidinone (Org. Proc. Res. Dev. 2003, 7, 514). The first step in the synthesis employed a sodium borohydride reduction of phenylalanine mediated by iodine. The second step utilized triphosgene as a phosgene source to effect cyclization of the intermediate amino alcohol (Figure 1). Both chemical steps and workup procedures were rapidly screened and optimized utilizing statistical design of experiments (DoE) and parallel synthesis in an Advantage Series 2050 manual chemistry synthesizer. This system could perform five reactions at five different temperatures simultaneously in reaction volumes of 5 to 80 mL. The procedure was further characterized in an automated reactor system (Advantage Series 3400 Process Chemistry Workstation). This workstation was capable of running four computer-controlled reactions simultaneously in glass reactors of 130 or 250 mL. All process data (time, temperature, pressure, pH, heat flow) were logged to a protected run file. It was demonstrated that the efficiency of the process compared favorably to those described in the literature in view of raw material costs, time requirements, reaction hazards, and waste generation.

Figure 1.

Synthesis of chiral auxiliary (S)-4-(Phenylmethyl)-2-oxazolidinone.

Recent Advances in Automated Solid-Phase Carbohydrate Synthesis: From Screening to Vaccines

Throughout the past year, significant advances in a new automated technology for the synthesis of complex carbohydrates have occurred. Solid-phase methods have been applied to the construction of previously inaccessible carbohydrates. Furthermore, the application of automated solid-phase carbohydrate synthesis became promising. New methods for the synthesis of carbohydrates and potential applications were described in a review of this field by O. J. Plante and P. H. Seeberger (Current Opinion in Drug Discovery & Development 2003, 6(4), 521).

Fluorescence Resonance Energy Transfer (FRET) as a High-Throughput Assay for Coupling Reactions

A solution-phase assay based on fluorescence resonance energy transfer (FRET) was developed by J. F. Hartwig and S. R. Stauffer (J. Am. Chem. Soc. 2003, 125, 6977) for high-throughput screening of palladium-catalyzed aminations of aryl halides. Dansylpiperazine was used as the fluorescent component and a chloroarene or bromoarene tagged with an azodye as the quenching partner. Fluorescence intensities or reaction aliquots correlated linearly with reaction yields. A library of 119 phosphine and heterocyclic carbene ligands was evaluated in duplicate reactions of two combinations. In general, the FRET assay displayed excellent reproducibility, with less than 5% of the duplicate experiments showing significant variability in yields. In summary, this assay is a new tool for rapid determination of reaction outcome that potentially could be used in an automated environment.

Recent Advances in the Discovery of Organometallic Catalysts Using High-Throughput Screening Assays

Over the past year, many groups in academia and industry developed and utilized new high-throughput screening assays to reduce the time required to identify catalysts for asymmetric processes, cross-coupling reactions, and other metal-catalyzed transformations. The continued success of combinatorial techniques in organometallic chemistry should propagate the development of new and improved methods to facilitate catalyst discovery. An interesting review of this is presented by J. F. Hartwig and J. P. Stambuli in Current Opinion in Chemical Biology 2003, 7, 420.

High-Throughput Approaches to Catalyst Discovery

High-throughput synthesis and screening approaches to catalyst discovery and optimization are systematically changing the way in which catalyst research is conducted. Increased rates of innovation, cost effectiveness, improved intellectual property, reduced time-to-market, and an improved probability of success are some of the attractive features that demand consideration. High-throughput approaches to catalyst discovery are now being implemented broadly in industrial and academic laboratories. An overview of recent developments, technologies, and systems is outlined by V. Murphy and colleagues from Symyx Technologies in Current Opinion in Chemical Biology 2003, 7, 427.

A Practical High-Throughput Screening System for Enantioselectivity Using FTIR Spectroscopy

FTIR spectroscopy was applied for the first time to the measurement of enantiomeric purity by M. T. Reetz and coworkers at Max-Planck-Institut für Kohlenforschung, Mülheim/Ruhr (Chem. Eur. J. 2003, 9, 3882). The underlying concept was based on the use of pseudo-enantiomers that were ¹³C-labeled at appropriate positions. Upon applying Lambert-Beer's law in the determination of the concentration of both enantiomers of chiral acetates or acetamides, the ee values were accessible, with accuracy within ±5 % of the true values being possible. The application of a commercially available high-throughput FTIR system (Tensor 27 spectrometer in connection with a HTS-XT system from Bruker Optik GmbH) resulted in a slightly decreased accuracy (±7 % for the ee values) but allowed a throughput of up to 10,000 samples per day. This new method is of high interest in the area of combinatorial asymmetric catalysis and directed evolution of enantioselective enzymes.

Microfluidic Combinatorial Chemistry

Microreactors are finding increasing applications in the field of combinatorial chemistry. In the past few years, micro-reactor chemistry has shown great promise as a novel method on which to build new chemical technology and processes. It has been conclusively demonstrated that reactions performed within microreactors invariably generate relatively pure products in high yield. One of the obvious applications is, therefore, in combinatorial chemistry and drug discovery. P. Watts and S. Haswell (University of Hull) reviewed some recent trends in this field (Current Opinion in Chemical Biology 2003, 7, 380).

High-Speed Parallel Synthesis of 1,2-diaryl-1-ethanones via a Clean-Chemistry C-C Bond Formation Reaction

The parallel, as well as conventional synthesis of 1,2-diaryl-1-ethanones via environmentally benign acylation of arenes with in situ generated arylacetyl trifluoroacetates has been reported. According to V. R. Veeramaneni, M. Pal, and K. R. Yeleswarapu (Tetrahedron 2003, 59(18), 3283), a wide variety of arylacetic acids participated in trifluoroacetic anhydride/phosphoric acid-mediated C-C bond formation reaction when reacted with arenes to give 1,2-diaryl-1-ethanones in good-to-excellent yield. Under solvent-free conditions, these chemical transformations, which normally required longer reaction times, could be performed within minutes in good yields. Parallel synthesis was carried out using eight reaction flasks simultaneously on a Buchi Syncore® Reactor/ Polyvap where reaction temperature and time could be monitored with accuracy.

Asymmetric Rhodium Carbene Insertion into the Si-H Bond: Identification of New Dirhodium(II) Carboxylate Catalysts Using Parallel Synthesis Techniques

According to Richard T. Buck et al. in Tetrahedron: Asymmetry 2003, 14(7), 791–816, decomposition of methyl 2-diazophenylacetate in the presence of silanes and chiral dirhodium(II) catalyst resulted in Si-H insertion of the intermediate carbenoid with varying degrees of enantioselectivity. New chiral dirhodium(II) carboxylate catalysts were identified using solution phase parallel synthesis techniques. For a systematic investigation, the investigators selected 2000 carboxylic acids from 30,000 commercially available compounds by filtering to “chiral flags,” monocarboxylic acids, interfering functional groups and clustering the resulting set to find the centroids in 80 clusters that were selected as the representative acids. From this, 70 chiral dirhodium(II) carboxylate catalysts were isolated; the remaining 10 acids were recovered as unreacted starting material. The catalysts were screened with three silanes with varying degrees of steric bulk in a catalytic reaction (effecting the insertion of the carbenoid into the Si-H bond). Forty-eight catalysts showed significant catalytic activity.

Synthesis and Preliminary Studies on a Novel Class of Soluble Amino Alcohol Reagents Based on Methacrylate Copolymers

A class of soluble polymers containing chiral b-amino alcohol ligands, based on methacrylate copolymers, was described by Martin Wills et al. (Tetrahedron 2003, 59, 5823–5830). The use of copolymers permitted the selective introduction of material to control the solubility and a functional group in a controllable ratio and polymer length (Figure 2). Using a cobalt-based chain transfer reagent, it was possible to form polymers with a molecular weight average of about 5.000 Da in large quantities (> 100 g) in a reproducible manner.

Figure 2.

Copolymers of methacrylates.

The application of supported polymers to diethylzinc additions is described. Although the actual supported catalyst prepared in this paper was not optimal for the subject application, the results demonstrated the principle of the approach and in particular the ease of solubility and analysis of the products.

High-Throughput Methods in Material Sciences

Method for Combinatorial Screening of Moisture Vapor Transmission Rate

Materials with varying levels of gas or moisture barriers are used in a wide range of packaging applications, from food and medical packaging to the protection of electronic materials. A high-throughput method for screening the moisture vapor transmission rate of barrier films was developed by J. C. Grunlan et al. at Avery Dennison Corporation (J. Comb. Chem. 2003, 5, 362). This screening technique was based on a Nafion-crystal violet sensor that changed color from yellow to green upon absorption of water. Using an appropriate set of known standards, the slope of absorbance (at 630 nm) as a function of time could be converted into moisture vapor transmission rate values. High-throughput screening was demonstrated by depositing 20 emulsion-based poly(vinylidene chloride) films, using a 48-well template, of varying thicknesses onto the sensor film and aging at 40 °C and 90% relative humidity for 72 h. The moisture vapor transmission rate was accurately determined to a level of 0.9 g/m² · day.

Development of Combinatorial Chemistry Methods for Coatings: High-Throughput Adhesion Evaluation and Scale-Up of Combinatorial Leads

Coupling of combinatorial chemistry methods with high-throughput performance testing and measurements of resulting properties has provided a powerful set of tools for the 10-fold accelerated discovery of new high-performance coating materials for automotive applications. The approach of R. A. Potyrailo et al. at General Electric Company (J. Comb. Chem. 2003, 5, 472) replaced labor-intensive steps with automated systems for evaluation of adhesion of 8 × 6 arrays of coating elements that were discretely deposited on a single 9 × 12-cm plastic substrate. The high-throughput adhesion evaluation provided previously unavailable capabilities of high speed and reproducibility of testing by using a robotic automation. It was possible to test an expanded range of coatings by using the coating tagging strategy, and to achieve an improved quantitation using the coating tagging strategy, and an improved quantitation by using high signal-to-noise automatic imaging. The robotics and automation systems used for these studies are described in the article. The high-throughput screening results for the best coating compositions were validated on the traditional scales of coating formulation and adhesion loss testing, and the superb performance of combinatorially developed coatings over conventional coatings was confirmed on the traditional scale.

Automated MALDI-TOF-MS Sample Preparation in Combinatorial Polymer Research

A new, automated MALDI-TOF-MS sample-spotting technique that allows the integration of MALDI sample preparation in the workflow of combinatorial polymer research is described by U. Schubert et al. at Eindhoven University of Technology (J. Comb. Chem. 2003, 5, 369). The technique was performed utilizing a commercially available synthesis robot (Chemspeed ASW2000) and was first evaluated with polymer standards of known composition and later used for the monitoring of the living cationic ring-opening polymerization of 2-ethyl-2-oxazoline. The described technique reduced the time required for sample preparation and offered the possibility of automated sample spotting during polymerization reactions performed in a synthesis robot. This allowed the integration of molecular weight screening and polymer end group determination utilizing MALDI-TOF-MS as a high-throughput tool in combinatorial polymer research.

High-Throughput Analytics

A “Hot” New Nanoelectrospray Probe

C. Robinson, J. Benesch, and F. Sobott from the University of Cambridge (U.K.) reported the first nanoESI probe that included a thermoelectric cooler (TEC) for heating and cooling an aluminium block between the TEC and the capillary (Anal. Chem. 2003, 75, 249). The researchers embedded the thermocouple into the aluminium block to indirectly measure the solution temperature. Even though the NanoESI was well suited for the study of complexes in aqueous solutions, the new thermoelectrically controlled nanoprobe allowed the investigation of temperature sensitive systems, e.g., “small heat-shock proteins” that were inactive under normal conditions.

Mini MS Shows Big Results

A new approach for shrinking mass spectrometers down to size came from R.G. Cooks and his team at Purdue University (Anal. Chem. 2003, 75, 250). These researchers built a mass spectrometer of 17 kg weight. The Mini-CIT 7.0 is more portable and offers a better mass range, limits of detection, and resolving power than the older version. The Mini-CIT has an m/z range of ∼500 and needs only 136 W. Further miniaturization will be achieved by substitution of the large, complex and fragile turbopumps with diaphragm pumps. Beside this Mini-CIT, Cooks and co-workers reported new miniaturized MALDI-TOF mass spectrometers with a mass range of 70 kDa and a resolving power of ∼1000. To enhance the resolving power, Cooks modified the electrical field.

Mass and Composition MALDI-TOF Analysis Enabling Inference of the Sequence of Most Peptides where the Protein Origin is Known

Protein sequencing with one MS is no longer a vision. Instead of using a tandem MS system to sequence proteins, R. Phelps and co-workers at the University of Edinburgh (U.K.) used a MALDI-TOF instrument and mass and composition analyses. They combined accurate mass data obtained by MALDI-TOF and partial composition data to infer the sequence of peptide ions where the identity of the starting protein was known (Anal. Chem. 2003, 75, 2653). The researchers found that the probability of mistaken sequence inference was between 0.0007 and 0.06, depending on the conditions.

Investigative Proteomics: Identification of an Unknown Plant Virus from Infected Plants Using Mass Spectrometry

D. Eckert, N. Andon, and co-workers presented a new approach for identification of a previously uncharacterized plant virus infecting different plants (Am. Soc. Mass Spec. 2003, 14, 732). In this procedure, protein extracts were prepared first and then determined with two-dimensional electrophoresis (2-DE). After visual comparison, the unidentified protein spots were analyzed by HPLC-MS/MS. The obtained mass spectrometry data of the individual peptides were searched with SEQUEST. With this approach, Eckert and co-workers demonstrated a successful proof-of-concept experiment by identifying the TMV proteins present in the total protein extract as well as a successful identification of an unknown tobacco plant virus.

Capillary High-Performance Liquid Chromatography/Mass Spectrometric Analysis of Proteins from Affinity-Purified Plasma Membrane

A key factor for successful analysis of plasma membrane and its proteins is the preparation of plasma membrane fractions with low contamination from subcellular organelles. Y. Zhao and co-workers at the University of Texas Southwestern Medical Center, Dallas used an affinity purification step to isolate plasma membrane fractions prior to MS analysis (Anal. Chem. 2003, 75, 3751). The cell surface proteins that bind to the streptavidin beads were biotinylated. They found 918 unique proteins after capillary HPLC and MS analysis of the affinity-purified samples. Zhao and co-workers demonstrated that their procedure reduced contamination of subcellular organelles and that biotinylation reaction did not activate or suppress any receptors. With this improved strategy for the analysis of plasma membrane, many new proteins were detected. This should help increase knowledge of cell structure and regulation of cellular behaviors.

Voltammetric Mass Spectrometry—a New Tool for High-Throughput Early ADME-Tox Screening

ADME-Tox assays in an early stage of drug discovery have become important to avoid costs for irrelevant compounds in more costly follow-up stages of drug development. Compound failure during drug development is commonly a result of adverse metabolism and toxicity. The metabolic stability of a compound, nature of metabolites, and resulting biological effects are very often based on redox reactions in biotransformation. Since compound bioactivity, metabolism, chemical stability, and toxicity frequently involve redox reactions, a rapid assessment of a library component's electrochemical activity and reaction products is highly relevant to its “druglike” properties. Gamache et al. (Drug Plus International 2003, 2, 6) described a study where coulometric cells were used as a simple and rapid redox reactor, the products of which were monitored by a serial coupled mass spectrometer (MS). Serial coupling of electrochemistry (EC) with MS is a very useful tool to monitor a wide range of biological and chemical redox processes such as N-dealkylation, sulfoxidation, desulfuration, purine metabolism, dopamine oxidation, and other redox reactions. Gamache et al. used substrate-product pairs to examine Phase 1-type oxidative reactions as a function of electrode potential (i.e., voltammetric mass spectrometry or VMS). The nature and reactivity of oxidation products was then further studied by co-injecting nucleophiles and examining the formation via MS of covalent adducts.

The serial coulometric EC-MS described was an easily automated high-throughput means of obtaining data for prediction of drug-like properties with primary emphasis on metabolism and toxicity. In addition, the EC-MS technique was more readily standardized, had higher throughput potential than biological assays, and could be readily integrated with LC-MS-based systems.

Bioautomation and Screening

Systematic Discovery of Multi-Component Therapeutics

Multi-component therapies have a successful history in a number of areas of medicine, including cancer, infectious diseases, and CNS disorders. Borisy et al. (PNAS 2003, 100, 7977) developed a combination high-throughput screening (cHTS) procedure for identifying effective combinations of therapeutic compounds. Through systematic screening of ≈120,000 different two-component combinations of reference-listed drugs, they identified potential multicomponent therapeutics, e.g., fungistatic and analgesic agents that together generated fungicidal activity in drug-resistant Candida albicans yet did not significantly affect human cells.

Compound solutions were transferred into 384-well master plates containing appropriate volumes of diluent by using a Perkin-Elmer Life Sciences Multiprobe. Compounds were then serially diluted by using a Perkin-Elmer MiniTrak. Dilution daughter plates were created. All dilution and assay plates were filled by using an Apogent PlateMate 384-well pipettor. Aliquots from two or more compound dilution plates were then dispensed to the final combination assay plate by using MiniTrak protocols. Addition of cells was carried out with a Thermo Labsystems Multidrop in a biosafety cabinet. Further instruments used for the different cell-based assays included an incubator, a centrifuge, and analytic devices such as NEPHELOstar (BMG Labtechnologies) for direct readout of cell number and a Wallac Victor V plate reader for fluorescence detection.

A practical application of this cHTS methodology is the creation of combination drugs through systematic screening of compounds in disease-relevant assays. Mining the combinatorics of multicomponent effects promises to result in rapid production of medicines and will define an approach to the systematic discovery of therapeutic agents.

Automated Screening of Neurite Outgrowth

Outgrowth of neurites in culture is used for assessing neurotrophic activity. Traditionally, assessment is done manually from images, using methods that are labor intensive and time consuming. Medium-throughput and noninteractive image analysis of neurite screens have not been well described. Most published neurite outgrowth measurements are still made with minimally automated image analysers. Although flexible, these research image analyzers are highly interactive and very low in throughput. The key challenge of automation is the discrimination of valid neurite material from other cellular components without human interaction.

Ramm et al. (J. Biomol. Screen. 2003, 8, 7) report the performance of a fully automated image acquisition and analysis system (IN Cell Analyzer 1000) applied to both fluorescent and brightfield specimens. Neuronal cells were seeded in 96-well plates and exposed to retinoic acid. Immunofluorescence labelling of the cytoskeleton was used to detect neurites and cell bodies. Image acquisition was done automatically by the IN Cell Analyzer 1000 system, which consisted of a computer-controlled, modified Nikon-inverted microscope. A 1392 × 1040 pixel digital camera was used for the readout of the signals. The image set was then analyzed by both manual tracing and automated algorithms. Automated scoring required initial training on the system and a series of computationally intensive steps. On five relevant parameters, such as number of neurites, total cell area, etc., the authors did not observe a difference between the automated analysis and the manual analysis. The throughput of the automated system was orders of magnitude higher compared to manual methods.

Improving Quality: High-Content Screening with Acumen Explorer

In HTS, one of the challenging goals is to improve, as early as possible, the quality and the relevance of selected compounds. That is why Aventis combined HTS robotic systems with the high-content screening (HSC) tool Acumen Explorer™. Acumen is a fluorescence plate reader, dedicated to cell- and bead-based assays.

The reader was integrated into an existing robotic system, the ROBOLAB™ from CRS ROBOCON. The system is equipped with CyBio liquid handling devices, an incubator with CO₂ and humidity control, a CRS robotic arm, and three different readers (FLIPR, VIEWLUX, Acumen). The assays could be carried out in different plate formats such as 24-, 96-, 384-, and 1536-wells. A 488-nm excitation laser scans the bottom of a plate containing the appropriate cells labeled with the specific fluorescent probes. Simultaneous reading of more than one well allows increased throughput. Acumen could record up to 15 result parameters for each object (bead or cell) in each well: size, perimeter, diameter, area, mean intensity, width, depth, etc. According to preset parameters, an algorithm could reconstitute the object in 3D and calculate the desired result value.

The reader can be used for a wide range of cellular assays. Grepin et al. (Current Drug Discovery 2003, Heft 1, 37) reported using the Acumen reader for the measurement of various biological phenomena in living cells such as quantification of a cell surface protein, respectively, cell surface sugar, measurement of receptor-ligand binding, and cytotoxicity test. The use of the Acumen technology in primary screening allows the speedup of the lead discovery process by identifying hits of higher quality at the primary screening level. Cytotoxic compounds could be eliminated at an early stage to reduce workload in secondary follow-up assays.

Going to the Well No More: Lawn Format Assays for Ultra-High-Throughput Screening

Screening in a “well-less” or lawn format provides a means to screen large compound collections against many targets in a fast, versatile, and cost-effective manner. The development of generic lawn format assays to screen various gene families against large compound collections should facilitate the identification of hits and tools to use in drug discovery and chemogenomic endeavors. Lawn format holds particular promise for screening GPCRs and selected enzyme families with potential use in other gene families. In addition, the application of this format to ADMET type assays, including cytochrome P450 drug–drug interactions and P-glycoprotein assays, would greatly expand the throughput of these assays and potentially impact their drug discovery efforts. Given the advantages in compound handling and flexibility in the format, it is surprising that more groups have not reported the advancement and development of well-less assays but instead focus on the miniaturization of wells. A review on recent developments in this field was published by B. E. Marron (Icagen) and C. K. Jayawickreme (GlaxoSmithKline) in Current Opinion in Chemical Biology 2003, 7, 395.