Automation Highlights from the Literature

Laboratory Automation and High-Throughput Chemistry

Applications of the Combination of Microwave and Parallel Synthesis in Medicinal Chemistry

Kerstin Thurow, Ph.D.

Hilmar Weinmann, Ph.D.

The combination of microwave heating and parallel chemistry is a logical consequence of the significant rate enhancement and higher product yield afforded by microwave technology and the increase in productivity afforded by combinatorial chemistry. Therefore, this combination has become increasingly popular in the organic chemistry community. J. Alcazar et al. discuss the various aspects of this approach as a way to increase the rate of analog synthesis in medicinal chemistry (Comb. Chem. High Throughput Screen. 2007, 10, 918–932).

Microwave Reactions under Continuous Flow Conditions

The adoption and integration of microwave technology has permitted a resurrection of many synthetic transformations that previously were considered too extreme in their conditions (temperatures, pressures, reaction times) to be synthetically useful. S. V. Ley et al. describe the continuous flow processing of chemical intermediates taking advantage of a unique heating mechanism, and how characteristics of microwave irradiation will be the next evolutionary step forward in this arena. The synergistic combination afforded by the simultaneous application of these two core-processing tools further enhances synthetic capabilities (Comb. Chem. High Throughput Screen. 2007, 10, 802–836).

Microwave-Assisted Reactions in Heterocyclic Compounds with Applications in Medicinal and Supramolecular Chemistry

Recent advances in microwave-assisted combinatorial chemistry include high-speed solid-phase and polymer-supported organic synthesis, rapid parallel synthesis of compound libraries, and library generation by automated sequential microwave irradiation. New instrumentation for high-throughput microwave-assisted synthesis continues to be developed at a steady pace. Impressive speed combined with unmatched control over reaction parameters are the main reasons for the growing interest in this application of microwave heating. A. de la Hoz et al. review some recent advances in this area, with particular emphasis on cycloaddition reactions of heterocyclic compounds with and without supports, applications in supramolecular chemistry, and the reproducibility and scalability of organic reactions using microwave irradiation techniques (Comb. Chem. High Throughput Screen. 2007, 10, 877–902).

Diversity-Oriented Synthesis and Solid-Phase Organic Synthesis under Controlled Microwave Heating

Diversity-oriented organic synthesis (DOS) and solid-phase organic synthesis (SPOS) are proven technologies for generating small molecule libraries for chemical genetics studies. W. -M. Dai and J. Shi discuss how integration of controlled microwave heating with DOS and SPOS not only speeds up the library preparation process, but also offers unique opportunities to tackle issues rarely addressed by thermal heating. Microwave-assisted synthesis is illustrated with examples of selective reactions within a broad variety of chemicals (Comb. Chem. High Throughput Screen. 2007, 10, 837–856).

Comprehensive Survey of Chemical Libraries for Drug Discovery and Chemical Biology: 2006

R. E. Dolle et al. present the tenth installment of their comprehensive survey series in combinatorial chemistry. The title of this latest review is updated to reflect the trend in high-throughput chemistry toward synthesis of smaller numbered arrays (< 1000 members), and their applications in drug discovery and chemical biology. The 469 entries published in 2006 are captured in various tables (J. Comb. Chem. 2007, 9, 855–902).

Skeletal Diversity in Small Molecule Synthesis Using Ligand-Controlled Catalysis

S. L. Schreiber and B. L. Gray describe two Pd-catalyzed reductive transformations of diynes tethered through a silyl ether linkage, where the reaction outcomes are controlled solely by selection of phosphine ligand. The authors screen Pd precatalysts, ligands, and additives to optimize conditions selective either for reductive cyclization or hydrogenation of this substrate class. These studies illustrate how small modifications to a transition metal catalyst can be used to access a diverse set of small molecules, in a fashion analogous to biosynthetic pathways such as terpene biosynthesis, where minor changes to enzyme structure direct skeletal differentiation (J. Comb. Chem. 2007, 9, 1028–1035).

Fully Automated Open Access Platform for Rapid, Combined Serial Evaporation and Sample Reformatting

O. Benali et al. describe a novel evaporator and its integration with an automated sample handling system to create a high-throughput evaporation platform. The Vaportec V-10 evaporator uses a high-speed rotation motor to spin the vial containing a sample, creating a thin film of solvent that can be readily evaporated by the application of heat to the vial. An intelligent algorithm controls pressure and temperature for optimum solvent removal conditions and end of run detection. The system allows the option of evaporation directly from a sample source vial, or alternatively, integrated liquid-handling facilities provide the capability to transfer samples portion wise from a larger source vial. Their open access system is a significant improvement over current vacuum centrifugal evaporators in terms of evaporation rate and ease of automation (J. Comb. Chem. 2008, 10, 52–55).

Screening for Polymorphs on Polymer Microarrays

M. Bradley et al. report a high-throughput method for studying polymorphism in small molecules. The approach uses arrays of polymers to generate or trigger different polymorphic forms, and Raman is used to characterize the different polymorphic forms. Results show that crystal habit forms of the small molecule solids are poor indicators of polymorphic forms, and powder X-ray diffraction is not suitable because of the small scale of the high-throughput method. Although the hydrophilic glass surface yields just amorphic forms of the studied compounds, many of the polymers are selectively triggering specific polymorphic forms and demonstrating the role of polymers in the crystallization process. This method is used to screen three different small molecule compounds with 128 polymers, and requires just milligram quantities of compound and 27 μg of each polymer per array, while generating large numbers of polymorphic forms (J. Comb. Chem. 2008, 10, 24–27).

Construction and Validation of an Automated Flow Hydrogenation Instrument for Application in High-Throughput Organic Chemistry

B. Clapham et al. describe the construction of a fully automated flow hydrogenation apparatus for use in high-throughput organic synthesis. The instrument comprises a Bohdan robot platform coupled with a ThalesNano H-cube hydrogenator and a series of solvent valves and pumping mechanisms. Using this instrument, the authors are able to fully automate a number of transformations that could not otherwise be conveniently undertaken in a high-throughput manner (J. Comb. Chem. 2008, 10, 88–93).

Automated Oligosaccharide Synthesis

Peptides and oligonucleotides are prepared by automated synthesizers that can be operated by nonspecialists. Carbohydrates have been hard to assemble, but the increasing awareness of the biological importance of this class of complex repeating biopolymers has prompted efforts to accelerate their synthesis. In a tutorial review by P. H. Seeberger, the state of the art of automated solid-phase oligosaccharide synthesis is defined and areas in need of further innovation are identified. Application of the automated synthesis method to prepare a malaria vaccine candidate is briefly highlighted (Chem. Soc. Rev. 2008, 37, 19–28).

An Integrated High-Throughput Screening Approach for Purification of Solid Organic Compounds by Trituration and Crystallization in Solvents

Trituration and crystallization are essential techniques for purification of intermediates and drug substances in pharmaceutical process development. Traditionally, identifying the optimal solvent conditions for impurity rejection demands an empirical approach that can be a time and material consuming process. H. Tan et al. describe the development and implementation of a high-throughput screening workflow in a 96-well array format for identifying optimal purification conditions. The impure samples are first triturated at room temperature, and subsequently subjected to a thermal cycle in 96 unique solvents or solvent mixtures at a volume of 0.6 mL per well. The compound solubility and the impurity profiles of both the supernatant and recovered solid are analyzed by HPLC. The authors claim that this high-throughput screening approach is valuable as an integrated part of process development to identify the thermodynamically favored solvent conditions for purification of pharmaceutical compounds (Org. Process Res. Dev. 2008, 12, 58–65).

Microfluidic Chip Technology and Micro Reactor Technology

Microfluidics for Drug Discovery and Development: From Target Selection to Product Lifecycle Management

Because microfluidic technologies have the ability to miniaturize assays and increase experimental throughput, they have generated significant interest in the drug discovery and development domain. These characteristics make microfluidic systems a potentially valuable tool for many applications in this area. L. Kang et al. review recent advances of microfluidic devices for drug discovery and development, and highlight their applications in different stages of the process, including target selection, lead identification, preclinical tests, clinical trials, chemical synthesis, formulations studies, and product management (Drug Discov. Today 2008, 13, 1–13).

Adventures in Inner Space: Microflow Systems for Practical Organic Synthesis

I. Ryu et al. present an overview of their recent efforts to achieve practical organic syntheses using a variety of microreaction devices with special focus on ionic liquid-based catalytic reactions, photochemical transformation, and heterogenous hydrogenation reactions (Synlett 2008, 151–163).

An Integrated High-Throughput Screening Approach for Purification of Solid Microbioreactors for High-Throughput Cytotoxicity Assays

In drug discovery, cell-based assays are being used more and more for drug target validation and ADMET (absorption, distribution, metabolism, elimination, and toxicity) studies, because cells can provide more representative responses to drugs than simple biochemical assays, and they are easier to use in a high-throughput format than animals. S.T. Yang et al. describe how microfluidic bioreactors with miniaturized culturing vessels and high controllability for operation and on-line monitoring have gained popularity in bioprocess development and cell-based assays. The advancement in this field has been enabled by the development of novel cell lines and reporter gene techniques, as well as new microfabrication, microfluidics, and optical and electrochemical sensor technologies. Microbioreactors with continuous perfusion allow for long-term culturing to study chronic toxicity effects. Systemic toxicity and interactions between different cell types also can be studied on a biochip. High-density microfluidic arrays provide a platform for future high-throughput and high-content screening that will contribute to drug discovery and bioprocess development (Curr. Opin. Drug Discov. Dev. 2008, 11, 111–127).

Analysis of a Membrane Reactor: Influence of Membrane Characteristics and Operating Conditions

P. Nikunj et al. report on investigations of the influence of membrane characteristics and operating conditions in a membrane reactor (Int. J. Chem. Reactor Eng. 2007, 5, Article A5). The authors model a batch pervaporation membrane reactor based on the underlying reaction and separation phenomenon using the esterification of ethanol with acetic acid as the model reaction. Two intrinsic characteristics that decide the performance of the membrane are the flux and the selectivity. The membrane flux dictates the rate of removal of the byproduct (water) in the model reaction, whereas the selectivity decides the amount of the reactant (alcohol) carried away along with water. The analysis of the authors shows that while selectivity (up to a certain minimum value) contributes to the membrane reactor performance, higher values do not have any profound influence on membrane reactor performance. The membrane reactor performance is more sensitive to the product of flux and membrane area. The performance of a membrane reactor can be enhanced by proper selection of the membrane area in relation to its flux such that the byproduct removal rate matches its generation by reaction. The disadvantages of using a membrane with low selectivity can be overcome by slightly tuning the feed ratio to compensate for the reactant loss.

The reaction time in a batch membrane reactor can be divided into three regimes, the kinetic regime, the equilibrium regime, and the intermediate regime. The batch reactor and membrane reactor exhibit similar performance when the reaction is in the kinetic regime. As the reaction nears equilibrium, the difference between the performance of the batch and membrane reactors becomes prominent. If tubular membrane reactors are operated in the kinetic regime, there may not be any difference in the conversion and product yield as compared to a simple plug-flow reactor under similar reaction conditions. When the residence time in a tubular membrane reactor is selected, such that the reaction is in the intermediate regime, in addition to product/byproduct separation, considerable enhancement in conversion and yield compared to the conventional flow reactor can be obtained.

Development of a New Swirling Micro Mixer for Continuous Hydrothermal Synthesis of Nano-Size Particles

A new swirling micro mixer for the synthesis of nano-size particles is described by Yuichiro Wakashima et al. (J. Chem. Eng. Japan. 2007, 40(8), 622–629). The continuous production of various nano-size particles is normally based on supercritical water hydrothermal synthesis. It involves the direct mixing of supercritical water with aqueous metal salt solutions at a normal temperature. This mixing process of the two fluids is extremely crucial for obtaining a highly supersaturated crystallization field, and for growth process control of fine metal oxides.

The authors explain how a swirling micro mixer can replace the conventional mixing tee-union for improved mixing of the two fluids. In addition, they use flow motion energy for mixing enhancement. By using the mixer, preliminarily boehmite nanoparticle synthesis from an Al(NO₃)₃ solution is experimentally performed. The synthesis results show some improvements in the average particle size (down from 100–200 to 60 nm), size distributions, and dispersibility in comparison to the existing data by using a conventional mixing tee. The experimental results also show how well the swirling micro mixer works for the synthesis of desired particles, and propose advantages for the reduced possibility of channel blockage by particle aggregated solids.

“Signal Processing Algorithms for an Atomic Force Microscope Operating in TREC Mode”

Atomic force microscopes (AFMs) belong to the raster probe microscope group, and they are used for mechanical scanning of surfaces and to measure atomic forces in nanometer scale. They can be used for conductive and nonconductive materials in different media. S. Adamsmair et al. describe a special operation mode of an AFM—the TREC mode (Topography and RECognition) (tm—Technisches Messen 2007, 74(4), 196–203). This TREC mode is particularly well suited for molecular biology, because it permits the simultaneous scanning of surface roughness and biological interaction places (Epitope).

After an introduction to the theoretical bases of an AFM, the function and realization of the TREC mode are described. Within this special mode, a ligand is attached to the probe tip. AFMs are still mostly built of analog parts, but this article describes how a new digital signal processing algorithm, based on a DSP (Digital Signal Processor) solution can be used. A block diagram illustrates the software modules for the TREC mode, including their links to the hardware. The algorithm realizes complete control and measurement in real time, and is written in assembler language for the DSP ADSP-2185 from Analog Devices. The application of the algorithms presented is demonstrated in biological applications by the biomolecules biotin and avidin under physiological conditions.

High-Throughput Analytics

Remote Mass Spectrometric Sampling of Electrospray- and Desorption Electrospray-Generated Ions Using an Air Ejector

The introduction, development, and refinement of novel ionization sources, especially ambient direct analysis methods, continue to expand the analytical utility of mass spectrometry. R. B. Dixon et al. from W. M. Keck FT-ICR Mass Spectrometry Laboratory (North Carolina State University, Raleigh, USA) propose a generally applicable remote sampling method that uses a commercial air ejector (AE) to transport ions from the point of ion formation to the MS inlet by a flexible polyethylene tube (J. Am. Soc. Mass Spectrom. 2007, 18, 1844–1847).

The AE consists of an unmodified Series EIX Air Ejector from Bosch Rexroth AG interfaced with an LTQ-MS from Thermo Electron. Electrospray ionization and desorption electrospray ionization ion generation are achieved 3 ft away from the inlet of the mass spectrometer. An applied flow of high-pressure gas within the AE creates a vacuum by the Venturi effect, and entrains the ions generated at the sample intake. The experimental configuration is depicted in detail, and crucial parameters like inlet pressure, sample intake, and total ion abundance are examined and optimized.

For both sources, the analytes rhodamine 6G, melittin, and polypropylene glycol (average molecular weight = 1000 Da [PPG-1000]) are used as subjects of investigation. Limits of detection for these analytes are calculated to be in the lower ng range. The authors convincingly demonstrate the potential using the AE as a remote sampling device for large or geometrically complex samples. Furthermore, the source they present does not require extensive modifications to the MS, and could be readily adaptable to other direct ionization techniques such as ASAP, DART, LD-APCI, ELDI, and MALDESI.

Mass Spectrometric Techniques for Label-free High-Throughput Screening in Drug Discovery

High-throughput screening (HTS) is an important tool for finding active compounds to initiate medical chemistry programs in pharmaceutical discovery research. In an article by T. P. Roddy et al. from Novartis Institute for Biomedical Research (MA, USA), three techniques that have been adapted for large-scale (∼175,000 sample) compound library screening are described. They use mass spectrometry-based high-throughput screening (MS-HTS) to avoid the inherent limitations of methodologies based on labeling and coupling enzymes (Anal. Chem. 2007, 79, 8207–8213).

The techniques presented, namely four-way parallel multiplexed electrospray liquid chromatography tandem mass spectrometry (MUX-LC/MS/MS), four-way parallel staggered gradient liquid chromatography tandem mass spectrometry, and eight-way staggered flow injection MS/MS following 384-well plate solid-phase extraction (SPE), are described by means of instrument setups, injection procedures, and analysis parameters. The total cycle time for analyzing four samples is in the range of 0.3–1.5min, respectively, for a 384-well plate between 37 min and 2.4 h. Two different primary screenings are presented and show high-quality screening data, regarding an average plate Z' value greater than 0.7. Overall, with further development, MS-HTS is expected to become a powerful mainstream tool that can be used for most screens in which an enzymatic conversion of substrate to product is required.

LIMS and Software Technology

Development of an Integrated Informatics Toolbox: HT Kinetic and Virtual Screening

A paper by D. Farrusseng et al. (Comb. Chem. High Throughput Screen. 2007, 10, 85–97) discusses aspects and issues for the development of a bespoke, generic infrastructure designed for high-throughput experimentation and knowledge management. It is composed of a collection of modules dealing with laboratory analytics, robotics, data handling and analytics, optimization, in-database processing, and visualization using the workflow paradigm. Scientific workflows promise to become an important area of research within workflow and process automation, and will lead to the development of the next generation of problem-solving and decision-support environment.

The widely used analytical workstation from Agilent and its corresponding software Chemstation for hardware monitoring, data acquisition, and data processing is integrated. A Visual Basic application is developed to manage the data collection from the Chemstation software. The advantages of this system are illustrated via two different case studies: HT screening of catalyst for glycerol selective oxidation in batch reactors, and virtual screening of catalysts.

Data Reduction and Representation in Drug Discovery

Drug discovery produces huge volumes of interrelated multivariate data. To make sense of these data and enable quality decision making, they must be presented in an interpretable form. The human eye and mind cannot easily interpret more than three dimensions, and so some form of data reduction is required. Reducing the dimensionality of the data often results in a data set that is too complex to interpret readily, so intuitive visualization methods are needed. Data in high-dimensional space, which has to be reduced and reinterpreted in as few meaningful dimensions as possible, have been the province of the mathematical and statistical community for many hundreds of years. These techniques are modified frequently and applied to biosciences. This report shows that as statistical literature is used increasingly by biosciences to help reduce data size and dimensionality, the methodology of the math becomes linked inextricably to its graphical representation. A drug discovery team often can be blind to obvious data trends when interpreting large compound data sets that contain a variety of in vivo, in vitro, and in silico tests. A section of this report highlights the most common tool in the pharmaceutical industry for data visualization, Spotfire (Spotfire AB. Göteborg, Sweden), and its special features for interactive data analysis (Howe, T. J.; Mahieu, G. et al. Drug Disc. Today 2007, 12(1/2), 45–53).

Value and Relation Display: Interactive Visual Exploration of Large Data Sets with Hundreds of Dimensions

Large data sets with hundreds of dimensions are common in applications such as image analysis or bioinformatics. For example, to detect the contents of large image collections, it is common to analyze hundreds of low-level visual attributes of the images. A paper by J. Yang et al. (IEEE Trans. Vis. Comp. Graph. 2007, 13(3), 494–507) presents a significantly improved multidimensional visualization approach named Value and Relation (VaR) display that allows users to effectively and efficiently explore large data sets with several hundred dimensions.

The essential idea of the VaR display is to represent each dimension in a high-dimensional data set using an information-rich glyph, and arrange the glyphs to reveal the relationships among the dimensions. In particular, pixel-oriented techniques and density-based scatterplots are used to create dimension glyphs to convey values. Multidimensional scaling, Jigsaw map hierarchy visualization techniques, and an animation metaphor named Rainfall are used to convey relationships among dimensions. The case studies presented in this paper show how the prototype supports interactive exploration of data sets of several hundred dimensions.

Bioautomation and Screening

Integrating High-Content Screening and Ligand-Target Prediction to Identify Mechanism of Action

High-content screening studies typically generate immense data sets of image-based phenotypic information, and how best to mine relevant phenotypic data are an unsolved challenge. Y. Feng et al. introduce factor analysis as a data-driven tool for defining cell phenotypes and profiling compound activities. This method allows large data reduction while retaining relevant information, and the data-derived factors used to quantify phenotype have discernable biological meaning. The authors use factor analysis of cells stained with fluorescent markers of cell cycle states to profile a compound library and cluster the hits into seven phenotypic categories. In addition, they compare phenotypic profiles, chemical similarity, and predicted protein binding activities of active compounds. By integrating these different descriptors of measured and potential biological activity, they effectively draw mechanism-of-action inferences (Nat. Chem. Biol. 2008, 4, 59–68).

Case Study: Impact of Technology Investments in Lead Discovery at Bristol–Myers Squibb

J. G. Houston et al. review strategies used over an 8-year period at Bristol-Myer Squibb (BMS) to implement new high-throughput approaches to lead discovery. Investments in compound management infrastructure and chemistry library production capabilities allow significant growth in the size, diversity, and quality of the BMS compound collection. Screening platforms are upgraded with robust automated technology to support miniaturized assay formats, while workflows and information handling technologies are streamlined for improved performance. These technology changes drive the need for a supporting organization in which critical engineering, informatics, and scientific skills are more strongly represented. The authors claim that these investments lead to significant improvements in speed and productivity as well a greater impact of screening campaigns on the initiation of new drug discovery programs (Drug Disc. Today 2008, 13, 44–51).