Automation Highlights from the Literature

Laboratory Automation and High-Throughput Chemistry

High-Throughput Screening of Low-Temperature CO Oxidation Catalysts Using IR Thermography

The catalytic oxidation of carbon monoxide to carbon dioxide is an important process used in several areas such as respiratory protection, industrial air purification, automotive emissions control, and CO cleanup of flue gases and fuel cells. Research in this area has mainly focused on the improvement of catalytic activity at low temperatures, and numerous catalyst systems have been proposed. Important commercial catalyst formulations for room temperature CO oxidation are based on CuMn₂O₄ and CuCoAgMnO _x mixed oxides. A. Hagemeyer et al. apply high-throughput and combinatorial methodologies to the discovery of more efficient catalysts for low-temperature CO oxidation. The screening approach is based on a hierarchy of qualitative and semiquantitative primary screens for the discovery of hits and quantitative secondary screens for hit confirmation, lead optimization, and scale-up. Parallel IR thermography is used as the primary screen, allowing one wafer-formatted library of 256 catalysts to be screened in approximately 1 h. Multichannel fixed bed reactors equipped with imaging reflection FTIR spectroscopy or GC are used for secondary screening. Novel RuCoCe compositions are discovered and optimized for CO oxidation, and the effect of doping was investigated for supported and bulk mixed oxide catalysts (Comb. Chem. High Throughput Screen. 2007, 10, 25–35).

Retrospective Hit-Deconvolution of Mixed Metal Oxides: Spotting Structure–Property Relationships in Gas-Phase Oxidation Catalysis Through High-Throughput Experimentation

Complex multi-element lead structures of mixed metal oxides that may be identified as hits during high-throughput experimentation campaigns can be deconvoluted retrospectively on the basis of simple binary and ternary oxides as illustrated by S. A. Schunk et al. Simple binary and ternary mixed metal oxide structure–property relationships can be established, providing insight into the roles of the complex mixed metal oxides’ different components, helping to establish a reaction mechanism, and converting the hit into a development candidate (Comb. Chem. High Throughput Screen. 2007, 10, 51–57).

Implementation of the Multichannel Monolith Reactor in an Optimization Procedure for Heterogeneous Oxidation Catalysts Based on Genetic Algorithms

A multicriteria optimization procedure based on genetic algorithms was carried out by P. Claus et al. in search of advanced heterogeneous catalysts for total oxidation. Simple but flexible software routines are created for use within a search space of more then 150,000 individuals. The general catalyst design includes mono-, bi-, and trimetallic compositions assembled out of 49 different metals and depleted on an Al₂O₃ support in up to nine amount levels. As an efficient tool for high-throughput screening and perfectly matched to the requirements of heterogeneous gas-phase catalysis, the multichannel monolith reactor is implemented to evaluate the catalyst performances (Comb. Chem. High Throughput Screen. 2007, 10, 59–70).

High-Throughput Microwave-Assisted Organic Synthesis: Moving from Automated Sequential to Parallel Library-Generation Formats in Silicon Carbide Microtiter Plates

A 48-deep-well microtiter plate system for sealed vessel parallel microwave synthesis is described by C. O. Kappe et al. The plate consists of a standard matrix of 48 wells with a maximum working volume of 300 μL and is made out of strongly microwave-absorbing sintered silicon carbide. In combination with an alumina sealing plate equipped with adequate conical bore holes for sample withdrawal, the setup can be used for microwave processing at temperatures up to 200 °C and 20 bar of pressure. The microtiter plate setup displays excellent temperature and reaction homogeneity, and is used for the generation of a 30-member library of 2-aminopyrimidines (J. Comb. Chem. 2007, 9, 285–291).

Nanoporous Magnesium Aluminometasilicate Tablets for Precise, Controlled, and Continuous Dosing of Chemical Reagents and Catalysts: Applications in Parallel Solution-Phase Synthesis

Mechanically robust tablets of nanoporous magnesium aluminometasilicate with high surface area and porosity can be loaded with a variety of organic and inorganic reagents and catalysts. The scope of this novel dosing methodology is demonstrated by T. Ruhland et al. through the evaluation of 14 diverse organic reactions, including Mitsunobu, Suzuki, and bromination reactions (J. Comb. Chem. 2007, 9, 301–305).

Microfluidic Chip Technology and Microreactor Technology

Microreactors: A New Tool for the Synthetic Chemist

P. Watts and C. Wiles review the use of microreactors as tools in synthetic organic chemistry. Special attention is given to the many advantages associated with their use, in particular, their ability to synthesize products with high yield, purity, and selectivity (Org. Biomol. Chem. 2007, 5, 727–732).

Recent Advances in Synthetic Microreaction Technology

The field of microreaction technology is growing rapidly, with many research groups investigating the practical advantages associated with reaction miniaturization. In a feature article, P. Watts and C. Wiles provide an overview of the progress made in the past decade, with particular attention to the field of synthetic organic chemistry (Chem. Commun. 2007, 443–467).

Accelerating Reactions with Microreactors at Elevated Temperatures and Pressures: Profiling Aminocarbonylation Reactions

The use of a pressurized microreactor system greatly expands the range of reaction conditions available to the bench chemist. S. L. Buchwald and K.F. Jenssen et al. use pressures from 4.5 to 14.8 bar and temperatures from 98 to 160 °C with greater flexibility, in terms of loading and sampling, than would be possible with traditional high-pressure chemical equipment. In addition, the use of injection valves on the inlet lines offers the possibility of using a wide range of substrates to efficiently produce a library of products. Furthermore, with the microreactor system, the reaction conditions themselves are improved. The carbonylation case study demonstrates the considerable potential of continuous-flow, microreactor-based experiments under conditions not easily achieved in conventional benchtop experiments. In particular, the technique provides a useful tool for rapidly and safely scanning reaction conditions and reagents (Angew. Chem. 2007, 119, 1764–1767).

Integrated Continuous Microfluidic Liquid–Liquid Extraction

F. Jensen et al. describe continuous-flow liquid–liquid phase separation in microfluidic devices based upon capillary forces and selective wetting surfaces. Effective liquid–liquid phase separation is achieved by using a thin porous fluoropolymer membrane that selectively wets nonaqueous solvents, has average pore sizes in the 0.1–1-mm range, and has a high pore density for high separation throughput. Pressure drops throughout the microfluidic network are modeled, and operating regimes for the membrane phase separator are determined based on hydrodynamic pressure drops and capillary forces. A microfluidic extraction device integrating mixing and phase separation is realized by using silicon micromachining. The device is capable of completely separating several organic–aqueous and fluorous–aqueous liquid–liquid systems, even with high fractions of partially miscible compounds (Lab Chip 2007, 7, 256–263).

The Use of Solid-Supported Reagents for the Multistep Synthesis of Analytically Pure α,β-Unsaturated Compounds in Miniaturized Flow Reactors

Microreaction technology offers a safe, controllable, and information-rich technique suitable for the long-term production of pharmaceutical agents and fine chemicals. To date, however, few of the syntheses performed using this technology have addressed the problems associated with product purification. P. Watts et al. report the incorporation of multiple supported reagents into electro osmotic flow (EOF)-based miniaturized flow reactors for the two-step synthesis of analytically pure compounds. Using this approach, the successful synthesis of 20 α,β-unsaturated compounds in excellent yields and purities has been achieved (Lab Chip 2007, 7, 322–330).

Integrated Microreaction System for Optical Resolution of Racemic Amino Acids

H. Maeda et al. present a novel microreaction system for optical resolution of racemic amino acids. This device, which is based on a continuous microfluidic system, consists of an enzyme-immobilized microreactor and a microextractor. Use of the enzyme microreactor, which is prepared by membrane formation on the microchannel surface, enables a highly enantioselective reaction for a racemic amino acid derivative. The microextractor provides a laminar flow of two immiscible solutions, which enables selective extraction of the product. Using this integrated device, the authors perform efficient continuous production of optically pure unnatural amino acids (Lab Chip 2007, 7, 366–372).

CFD Study of the Effect of the Fluid Flow in a Channel on the Generation of Oil-in-Water Emulsion Droplets in Straight-Through Microchannel Emulsification

I. Kobayashi et al. describe investigations on the effect of fluid flow in a microchannel (MC) (J. Chem. Eng. Jpn. 2006, 39 (8), 855–863).

Emulsions, thermodynamically metastable dispersions of two immiscible liquids that are stabilized by surface-active components, are the basis of many products, for example pharmaceuticals, cosmetics, and chemicals. The investigated straight-through MC emulsification enables formulating monodisperse emulsions using a channel array vertically microfabricated on a plate surface.

This study investigates the effect of the fluid flow in a channel on the generation of soybean oil-in-water emulsion droplets from an elliptic channel using computational fluid dynamics (CFD). The visualized CFD results demonstrate that droplets with diameters of 32–38 μm are stably generated from the channel below the critical flow velocity of the to-be-dispersed phase (U_d). Above the critical U_d, a drastic increase of the resultant droplet diameter is observed. The calculated droplet generation rate achieves a maximum value of about 15 droplets per second at the critical U_d. The numerical results obtained in this study suggest that monodisperse droplets can be stably generated from the elliptic channel at the highest generation rate at the critical U_d. The shrinkage trends of the neck explain the droplet generation behavior in the channel.

This study demonstrates that the CFD approach can be useful for optimal device and process design for droplet generation in straight-through MC emulsification.

Faster Selective Chemistry by Microflow and Continuous Microwave Synthesis

The advantages of microflow and continuous microwave synthesis are reported by T. Schwalbe and K. Simons (Chimica Oggi—Chemistry Today 2006, 24 (2), 56–61). This article gives an overview of recent applications of microflow and continuous microwave with improved performance. It shows a rationale for these findings, and discusses applications and the state of technology in accelerating information gathering and library synthesis with continuous synthesis technology. Synthetic methodology has evolved over the past 50 years, and emphasis on the selectivity and yield enhancement of chemical reactions has resulted in the development of microflow and microwave tools. What chemists need now are tools for faster cycle times—more new compounds, and then faster development times.

Microflow provides a means to accelerate mixing, improve temperature control, largely reduce back-mixing, and provide a scalable system. It is an alternative to using traditional stirrers or in-line mixers, which achieve agitation via turbulent mixing. When microflow reagent streams are brought together in narrow channels (0.5 mm or less), mixing is achieved through axial diffusion of the (multi)laminar streams. In addition to superior mixing, the high surface area to volume also assists thermal transfer.

Microwave in a continuous fashion additionally provides an alternative means of heating reactions. Microwave and continuous microwave can accelerate chemical reactions. In many instances, microflow can substitute rapid microwave heating in a controlled fashion. Reaction temperature would, if anything, rather safely be overstated than underestimated. Additionally, in microflow (whether thermal or microwave), reaction control is enhanced by lesser back-mixing and a defined reaction time. Further to the emergence of microflow and continuous microwave in single experimentation, adaptation of the sequential organic synthesis technology for faster access to libraries and faster information gathering is gaining momentum. For the foreseeable future, microflow synthesis is seen as more scalable and supports the retrieval of kinetic information for cost-efficient manufacturing plant design, though microwave has won a well-deserved place in the medicinal chemistry laboratory.

High-Throughput Analytics

Approaches to High-Throughput Physical Organic Chemistry

High-throughput (HT) techniques are extensively used for the synthesis of libraries of thousands of compounds. More recently, HT methods are being applied to other areas, such as physical organic chemistry. This has allowed the development of tools for HT reaction assessment, HT kinetic and thermodynamic measurements, and physicochemical property profiling, using a broad set of analytical tools ranging from mass spectrometry to image-analysis-based techniques.

M. Bradley and C. Portal provide an overview of recent HT physical organic chemistry techniques. Special attention is given to the application of quantitative analytical constructs for HT monomer reactivity profiling and HT evaluation of Hammett parameters (Org. Biomol. Chem. 2007, 5, 587–592).

Imaging of Peptides in the Rat Brain using MALDI-FTICR Mass Spectrometry

Recently, imaging mass spectrometry (IMS) has been recognized as a proteomic tool for in situ spatial analysis of tissue. Ron M. A. Heeren and coworkers from the FOM Institute for Atomic and Molecular Physics (Amsterdam, The Netherlands) study imaging FTICR-MS for the spatially resolved mass analysis of rat brain tissue with the aim to optimize protein identification by the high mass accuracy and online MS/MS capabilities of the technique. For a direct comparison, mass microscope MALDI-TOF imaging was performed parallel thereto (J. Am. Soc. Mass Spectrom. 2007, 18, 145–151). The authors demonstrate that the high resolving power of FTICR-MS enables the differentiation of biomolecules differing in mass by less than 0.1 Da. For instance, for FTICR-selected ion images of three different compounds with Δm/z <0.1 Th, it is possible to distinguish between their spatial distributions. Furthermore, mass accuracies up to 6 ppm are obtained for phosphatidylcholine (m/z 760.5856) and vasopressin (m/z 1084.4346) with external calibration in the direct MALDI analysis of the tissue together with a spatial resolution of 200 μm. In addition, online FTICR-MS fragmentation techniques are used to identify neuropeptides found during the tissue analysis. Due to the time-consuming realization of high-resolution FTICR-MS imaging experiments, the real benefit of the technique is in profiling small tissue areas to elucidate the peptide sequences of unknowns.

Use of a Variably Sensitive Selected Reaction Monitoring Method to Extend the Linear Dynamic Range for Quantitative High-Performance Liquid Chromatography/Tandem Mass Spectrometry

To avoid the time-consuming procedures of pre- and post-analysis dilution schemes of biosamples where large concentration ranges are expected, H. E. Fries and coworkers illustrate a method to extend the linear dynamic range of a triple quadrupole mass spectrometer operating in the selected reaction monitoring (SRM) mode of analysis (Rapid Commun. Mass Spectrom. 2007, 21, 369–374). For this purpose, an SRM method for the determination of the analyte GSK-A is developed, and a detailed collision energy (CE) versus analyte response profile for the assigned transition is collected. With this, a variably sensitive SRM method is created by adding multiple acquisition lines, due to different CE values, within the same method with only one precursor-to-product ion mass transition. Because of software limitations, it is necessary to collect data for transitions with 0.1-m/z unit differences for the first and/or third quadrupole to produce unique data sets. With both quadrupoles set to low resolution (~1.5 m/z units at half peak-height), the 0.1-m/z unit differences do not substantially affect the signal response, and the linear analytical range for GSK-A can be extended from 2–10,000 ng/mL to 0.5–10,0000 ng/mL.

Bioautomation and Screening

Microarray Platforms for Enzymatic and Cell-Based Assays

In a tutorial review, M. Bradley et al. introduce the basic concepts of microarrays and cover a variety of aspects such as substrates and surfaces, printing, and analysis. Newer applications of microarray technology, which include enzyme analysis (notably kinases and proteases), as well as the growing enchantment with cell-based microarrays that offer a unique approach to high-throughput cellular analysis are discussed. In addition, a look at possible future trends and directions in the microarray arena is shared (Chem. Soc. Rev. 2007, 36, 449–457).

Droplets as Microreactors for High-Throughput Biology

Inspired by the principles of biological evolution, biologists and other scientists have in recent decades harnessed the power of “natural” selection to sift through huge libraries of genes to find those with desirable properties. At the same time, the demand for high-throughput biochemical and genetic assays and screens has driven the development of increasingly miniaturized assay systems. An exciting synergy is now emerging between these two fields, whereby the tools of ultrahigh-throughput screening promise to open up new directions in molecular engineering. Recent trends in this area are summarized by A. D. Griffith et al. (ChemBioChem 2007, 8, 262–273).

A Pipeline for Ligand Discovery Using Small-Molecule Microarrays

Uncovering the functions of thousands of gene products, in various states of post-translational modification, is a key challenge in the postgenome era. To identify small-molecule probes for each protein function, high-throughput methods for ligand discovery are needed. In recent years, small-molecule microarrays (SMMs) have emerged as high-throughput and miniaturized screening tools for discovering protein–small-molecule interactions. Microarrays of small molecules from a variety of sources, including FDA-approved drugs, natural products, and products of combinatorial chemistry and diversity-oriented synthesis, are prepared and screened by several laboratories, leading to several newly discovered protein–ligand pairs. A. Koehler et al. offer an introduction to microarray technology and its application at the Broad Institute (Curr. Opin. Chem. Biol. 2007, 11, 74–82).

Chemical Genetic Approaches to Probing Cell Death

Chemical genetics has become a tool for the discovery of pathways and proteins in mammalian systems. This approach, comprising small-molecule screening combined with biochemical and genomic target identification methods, enables one to assess which proteins are involved in regulating a particular phenotype. When applied to cell death, this strategy can reveal novel targets and pathways regulating the demise of mammalian cells. Numerous diseases have been linked to the loss of regulation of cell death. Recent advances described by B. R. Stockwell et al. include the discovery of novel small molecules regulating cell death pathways—necrostatin and erastin—as well as the elucidation of the mechanism of death induced in cancer cells by the cytotoxic agent Apratoxin A (Curr. Opin. Chem. Biol. 2007, 11, 83–87).

Fluorogenic Substrates for High-Throughput Measurements of Endothelial Lipase Activity

In November 2006, Mitnaul et al. published work on new flurogenic substrates for high-throughput measurements of endothelial lipase (EL) activity in a 3456-well-format (J. Lipid Res. 2006, 21, 369–374). Lipases are known to function on the surface of vascular endothelial cells in contact with the plasma compartment, but it was previously reported that endothelial phospholipase and triglyceride lipase activity could not be measured in vitro in the presence of animal serum at concentrations greater than 5%. EL, however, is a critical determinant for high-density lipoprotein cholesterol (HDL-C) levels. It is known that Triton X-100 detergent micelles and synthetic HDL particles containing bodipy-labeled phospholipid or bodipy-labeled triglyceride substrates are catalytic substrates for EL, lipoprotein lipase (LPL), and hepatic lipase (HL), but, according to the authors, only synthetic HDL particles containing bodipy-labeled triglyceride are ideal substrates for EL, LPL, and HL in the presence of high concentrations of different sera types. This paper describes new fluorescent bodipy-labeled substrates that can be used in vitro in homogeneous, ultrahigh-throughput kinetic assays that measure EL phospholipase or triglyceride lipase activities. It is well known that lipase enzyme activity is sensitive to substrate–enzyme interfaces. The authors assume that substrate presentation is a critical factor when determining EL activity in the presence of serum.

Converting a Breast Cancer Microarray Signature into a High-Throughput Diagnostic Test

Recently, a 70-gene prognosis profile was identified by using complex microarrays, and the process revealed a powerful predictor for the outcome of disease in young breast cancer patients. This profile is generated using 78 tumor samples of patients having lymph node negative disease by hybridization of fluorescent-dye-labeled RNA to microarrays containing 25,000 60-mer oligonucleotide probes. This profile, however, is generated on microarrays containing 25,000 60-mer oligonucleotides that are not designed for processing many samples on a routine basis. To facilitate its use for diagnostic approaches in a high-throughput setting, the authors translate this profile in a custom-made eight-pack mini-array (Agilent Technologies). This mini-array is a single slide containing eight identically printed regions or subar-rays, each containing 1900 60-mer oligonucleotide probes, including the 70 prognostic classifier genes. This allows eight individual hybridizations to be carried out simultaneously on a single microarray slide. By using the same material used for the 70-gene prognosis profile, Glas et al. demonstrate the reproducibility of the current test and the similarity of its results to those obtained from the original data. In conclusion, they could demonstrate that microarrays are an excellent tool to predict the outcome of disease in breast cancer patients and are highly suitable for an upscalable clinical diagnostic setting. As a direct result, many patients could be potentially spared the side effects and risks of breast cancer treatment, improving their quality of life and reducing their health care costs (BMC Genomics 2006, 7, 278, doi:10.1186/1471-2164-7-278).

Src Homology 2 Domain-Based High-Throughput Assays for Profiling Downstream Molecules in Receptor Tyrosine Kinase Pathways

Tyrosine phosphorylation has a central role in signal transduction, controlling many important biological processes such as proliferation, differentiation, apoptosis, and cell motility. In addition to the regulatory importance for healthy cells, the uncontrolled activation of tyrosine phosphorylation can result in disease. Therefore, a number of techniques are used to study interactions with phosphoproteins, for example co-immunoprecipitation of tyrosine-phosphorylated proteins complexed with their interacting partners, specific antibodies that are used in conjunction with mass spectrometry detection systems or gene chip technology for profiling gene expression. These techniques, however, each have shortcomings, which may explain the increasing demand for new technologies. These authors describe a SH2 domain-based assay developed to profile the interaction of tyrosine kinases or other phosphotyrosine proteins with downstream molecules in their signaling pathways. This assay is a microsphere-based method in which various SH2 domains are coupled to different fluorescent microspheres to capture any binding proteins in a sample. The SH2 domain-bound proteins are then detected with specific GST antibodies against either phosphotyrosine residues or specific phosphotyrosine proteins. In a primary screen, Takuro Yaoi et al. demonstrate that both synthetic phosphotyrosine peptides and cellular phosphotyrosine proteins can be profiled. The GST–SH2 fusion proteins conjugated to the microspheres retained their functional binding activity to the target phosphotyrosine peptide, and do not cross-react with target peptides belonging to other domain families. The authors state that the main advantage of the fluorescent microsphere-based technology is its multiplexed high-throughput format with its ability to analyze 96 samples in 3.5 h. To target all the SH2 domains found in the human genome, they are planning to construct five sets of microspheres in the 25-plex format. With these assays, phosphotyrosine proteins that bind to genome-wide SH2 domains can be profiled. This will greatly facilitate the elucidation of underlying cellular signaling pathways with particular significance for use in drug screening (Mol. Cell. Proteomics 2006, 5, 959–968).

LIMS and Software Technologies

Information Science Integrates Systems in Laboratories

Cross-system processing of orders, samples, and results requires a continuous cross-linking of information, making identification by barcode or radio frequency identification (RF-ID) a key factor in the process to ensure that all relevant information stored in the system can be retrieved. Samples are often examined on several different analyzers, and information from all equipment used needs to be linked. Unfortunately, available equipment does not support the exchange of information well. Thus, concepts are necessary to enable the assumption and processing of data by the devices. A complete information chain (including raw data and processed data) could be provided with a digital signature to contribute to the quality assurance after 21 CFR part 11. A new approach is described that permits complete traceability of all procedures in the database (GIT 2006, 10, 930–932). Complete documentation and reproducibility of all procedures are secured.

Another important problem with information transfer between systems is syntactical and semantical compatibility. The author notes the XML standard in development AnIML. This standard permits uniform results representation, experiment information capture, representation of assigned methods, and a digital signature. All information from analytical measurements can thus be described and transferred in a well-defined format.