Automation Highlights from Literature

Laboratory Automation and High-Throughput Chemistry

Adsorbent Screening for Metal Impurity Removal in Pharmaceutical Process Research

Impurity removal is a critically important task in pharmaceutical process research, where final products must meet stringent purity requirements. A microtube screening approach described by C. J. Welch and colleagues affords a simple and convenient assessment of the selective adsorption of metal impurities by a variety of different process adsorbents. This approach is helpful in identifying rapid solutions to metal impurity problems in pharmaceutical process research. Several examples illustrating the utility of the approach are presented (Org. Process Res. Dev. 2005, 9, 198–205).

Automated Electrochemical Synthesis and Photoelectrochemical Characterization of Zn_1-xCo_xO Thin Films for Solar Hydrogen Production

High-throughput electrochemical methods have been developed by E. W. McFarland et al. for the investigation of Zn_1-xCo_xO films for photoelectrochemical hydrogen production from water. A library of 120 samples containing 27 different compositions was synthesized by automated serial electrochemical deposition. High-throughput photo-electrochemical screening reveals improved solar hydrogen μproduction for the cobalt-doped films. Flat-band potential, bias-dependent photocurrent, and action spectra are also measured automatically with the high-throughput screening system. The 200-nm-thick films are subsequently characterized by numerous techniques, including scanning electron microscope (SEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and UV-vis, which show that the depositions are well-controlled. Zn/Co stoichiometry in the films is controlled by the ratio of the Zn and Co precursors in each deposition bath (J. Comb. Chem. 2005, 7, 264–271).

Combinatorial Approach toward High-Throughput Analysis of Direct Methanol Fuel Cells

A 40-member array of direct methanol fuel cells is generated by R. Jiang et al. by electrically connecting the fuel cells in series. High-throughput analysis of these fuel cells is realized by fast screening of voltages between the two terminals of a fuel cell at constant current discharge. A large number of voltage-current curves are obtained by screening the voltages through multiple small-current steps. Gaussian distribution is used to statistically analyze the large number of experimental data (J. Comb. Chem. 2005, 7, 272–278).

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

Combinatorial screening of material formulations followed by the scale-up of combinatorial leads is applied by R. A. Potyrailo and colleagues in the development of high-performance coating materials for automotive applications. They replace labor-intensive coating formulation, testing, and measurement with a “combinatorial factory” that includes robotic formulation of coatings, their deposition as 48 coatings on a plastic substrate, accelerated performance testing, and automated spectroscopic and image analysis of resulting performance. This high-throughput (HT) performance testing and measurement of the resulting properties provides a powerful set of tools for the tenfold-accelerated discovery of these coating materials. Performance of coatings is evaluated with respect to their weathering, because this parameter is one of the primary considerations in end-use automotive applications. Using their high-throughput methodology, they have developed several cost-competitive coating leads that match the performance of more costly coatings. These HT screening results for the best coating compositions are validated on the traditional scales of coating formulation and weathering testing. These validation results confirm the improved weathering performance of combinatorially developed coatings over conventional coatings on the traditional scale. (J. Comb. Chem. 2005, 7, 190–196).

Combinatorial Chemistry in Glycobiology

The application of combinatorial chemistry to glycobiology historically has proven challenging due to numerous synthetic hurdles. The advent of novel methodologies has enabled the production of natural as well as mimetic analogues for proof-of-concept experiments and structure-activity relationship (SAR). O. J. Plante reviews some recent synthetic advances in combinatorial carbohydrate synthesis. The application of carbohydrate libraries in glycobiology is also discussed (Comb. Chem. High Throughput Screen. 2005, 8, 153–159).

Purifying the Masses: Integrating Prepurification Quality Control, High-Throughput LC/MS Purification, and Compound Plating to Feed High-Throughput Screening

J. J. Isbell et al. report the use of a parallel, four-channel HPLC/MUX/MS purification system, the Purification Factory, to purify thousands of compounds destined for high-throughput screening in a single month. The maximum sample throughput during this 20-workday month was 704 samples/day. Because this purification throughput exceeds the postpurification sample and data handling capabilities provided by commercial solutions, a custom-integrated solution was designed to address these shortcomings. The key improvements in automation, solvent handling, and sample handling logistics implemented to sustain a mean throughput of 528 samples/day over a multimonth period are described (J. Comb. Chem. 2005, 7, 210–217).

Reproducibility across Microwave Instruments: First Example of Genuine Parallel Scale-Up of Compounds under Microwave Irradiation

One of the major issues related to microwave-assisted organic chemistry is reproducibility. J. Alcazar et al. demonstrate that reproducibility of results can be obtained from commercially available single-mode and multimode microwave reactors. In order to make an appropriate comparison of results obtained from both systems, alkylation reactions are used as model reactions. Based upon the reproducibility observed in these experiments, parallel scale-up of compounds in one irradiation experiment is reported for the first time (QSAR Comb. Sci., 2004, 23, 906).

SynCar: An Approach to Automated Synthesis

The automation of all aspects of manual solution-phase synthesis into one integrated, efficient, and reliable system could be regarded as something of an unmet challenge in organic chemistry. The requirements for modern solution-phase libraries in mainstream drug discovery is typically 50–250 high-purity compounds on a 10–100 mg scale, whether for target class libraries or lead optimization, and short cycle time in combination with high capacity is critical. To achieve these goals, in a codevelopment between Aventis Pharma Deutschland GmbH and Accelab GmbH (Kusterdingen, Germany), E. von Roedern and colleagues designed a completely novel system of independent workstations connected by a shuttle transfer system. Seven modular workstations process four reactions on each shuttle in parallel, with the ability to perform synthesis (temperature control and liquid reagent handling), filtration, liquid-liquid extraction, evaporation, weighing, solid-phase extraction, and HPLC/MS analysis. The modular design enables the continuous loading of shuttles at any time, and each shuttle can have its own workflow. The design also allows easy expansion for future needs. The result is a combination of high-flexibility and high-throughput. (J. Comb. Chem. 2005, 7, 178–184).

Design and Synthesis of a New Polymer-Supported Evans-Type Oxazolidinone: An Efficient Chiral Auxiliary in the Solid-Phase Asymmetric Alkylation Reactions

Wang resin-supported Evans chiral auxiliary was designed by Tomoya Kotake et al. (Tetrahedron 2005, 15, 3819–3833). It is based on a novel polymer-anchoring strategy that uses the 5-position of the oxazolidinone ring. The new synthetic route, which is applicable to multigram preparation, was developed in just a day. Solid-phase Evans asymmetric alkylation on N-acylimide resin and following lithium hydroperoxide-mediated chemoselective hydrolysis affords the corresponding a-branched carboxylic acids desired high stereoselectivities (up to 97% ee) and moderate-to-good overall yield (up to 70%, for 3 steps), which are comparable to those of the conventional solution-phase methods. In addition, recovery and recycling of the polymer-supported chiral auxiliary are successfully achieved without decreasing the stereoselectivity of the product. This is the first successful example of the solid-phase Evans asymmetric enolatealkylation being efficiently performed on the solid-support, and the conclusion is that the connection to the solid-support via the 5-position of the oxazolidinone ring is an ideal strategy in the solid-phase Evans chiral auxiliary.

One-Pot Synthesis of Substituted Catechols From the Corresponding Phenols

The catechol structural entity exists in a number of important, naturally occurring compounds and in other molecules with interesting biological activity. Therefore, synthetic methods leading to substituted catechols are of considerable interest. A new synthesis strategy for getting substituted catechols from the corresponding phenols is shared by Trond Vidar Hansen and Lars Skattebøl in Tetrahedron Lett. 2005, 19, 3357–3358. At first, the phenols are converted to salicylaldehydes with paraformaldehyde and MgCl₂-Et₃N in THF. Subsequent treatment with aqueous NaOH and H₂O₂ affords the corresponding catechols. The sequence is conveniently carried out as a one-pot procedure, and leads after 4–8 h at room temperature yields between 55 and 70%. The yields are determined for recrystallized materials and are not optimized. Substituted phenols are well available, and the present method appears to be a convenient way of transforming them into the corresponding catechols.

Microfluidic Chip Technology and Microreactor Technology

Simulation, Optimation, and Parametric Studies of a Solid Catalyzed Gas Phase Ethylene Polymerization Fluidized Bed Reactor

An article in the J. Chem. Eng. Jpn 2005, 38 (5), 171–175 by Nayef Mohamed Ghasem et al. describes an optimization program for a fluidized bed reactor. For the production of polyolefins, the industry normally uses the gas phase technology with the fluidized bed reactor (10–30 bar). For diminishing the concentration and temperature gradients in the reactor, the conversion per pass is kept low at 1–3%. The bed is maintained in the bubbling regime by recycling the reaction gases. The heat of reaction is removed by cooling the circulating gas, and sometimes by partial condensation to use the heat generated by evaporation of the condensates. Industrial gas phase polyethylene reactors are prone to exhibit unstable steady states, limit cycles, and temperature oscillations. Investigations reveal that the maximum polyethylene production rate occurs when the reactor operates on unstable steady states. For this reason, a suitable controller is required to stabilize the unstable steady states. The authors describe a developed optimization program for identifying the optimum reactor operating conditions for maximizing polyethylene production rates. Using the optimum parameter values found, the parametric investigations for the effect of the catalyst deactivation rate constant and process operating parameters, such the catalyst feed rate and the gas superficial to minimum fluidization velocity on the system behavior, are also conducted. The investigations show that the optimum polyethylene production rate is achieved when it operates close to its melting point under a suitable controller to avoid polymer melting.

An Improved Sonochemical Reactor

An article in the Ultrasonics Sonochem. 2005, 12, 213–217 by Giancarlo Cravotto et al. describes a novel sonochemical reactor. Commercial ultrasonic emitters are used for cleaning surfaces and preparing biological homogenates; cleaner baths are the simplest models. In this case, transducers are fixed to the underside of a tank that is usually to be filled with water, and the object to be sonicated is placed in the area of maximal irradiation. Higher irradiation intensities can be obtained using an immersion horn. The basic parameter in ultrasonic engineering is power density, which is defined as the electrical power fed into the transducer divided by the radiating surface area. For organic chemistry, instruments capable of continuous operation at high intensities under stringent reaction conditions (modified atmosphere, controlled temperature, etc.) are required. The authors present a novel cooling system for the ultrasonic transducer and a more efficiently thermostated reactor. Their ultrasonic transducer consists of high-efficiency piezoelectric (PZT) rings compressed between two erga discs, and is cooled by oil that is refrigerated by a chiller. The bottom of the housing is cemented to the horn. The frequency is tunable (between 17 and 45 kHz), but in this case, 18.2 kHz is used as the standard operational frequency. Power can be varied up to a maximum of 1000 W. Reactions are carried out in a PTFE (Teflon) tube (0.8 mm thick, 80 mm high, 35 mm in diameter). The reactor can rotate at 30 rpm on a vertical axis, and the distance between reactor and horn is variable (max 10 mm). With the help of a patented electronic device, the ultrasonic frequency is continuously adjusted to the actual resonance value of the reaction system. Good results are achieved within 2–3 mm of the radiating surface, but the best results are achieved by combining two movements: the reactor rotates eccentrically around the horn axis while the horn moves alternatively up-and-down via a predetermined excursion at a chosen speed. A more efficient refrigeration system cartridge provided for transducer and probe make the instrument suitable for work at high intensities for virtually unlimited time periods. In all cases, reaction times are considerably reduced and yields for heterogeneous-phase reactions improved.

The Application of Microreactors for Organic Synthesis

P. Watts and S. J. Haswell describe in a tutorial review how microreactors are being applied to synthetic chemistry to cover a wide range of applications, from the preparation of nanograms of material for drug discovery and screening to the multi-ton production of fine chemicals. This article explores how miniaturization may revolutionize chemical synthesis and demonstrates that products are generated in higher yield and purity compared to the equivalent bulk reactions, and in much shorter periods of time (Chem. Soc. Rev., 2005, 34, 235–246).

On-Microchip Multiphase Chemistry

A. van den Berg et al. review microreactor design principles and reagent contacting modes for multiphase systems with special focus on immiscible aqueous-organic liquid systems, gas-liquid systems, gas-liquid-solid systems, and gas-gas-solid systems (Tetrahedron, 2005, 61, 2733–2742).

Room-Temperature Swern Oxidations by Using a Microscale Flow System

Microreactors offer many practical advantages, including safety, easy modulation, and easy scale-up for industrial production when compared with conventional macroscale batch reactors. It is advantageous that highly exothermic reactions can be conducted on the basis of efficient mass and heat transfer. Microreactors also enable the precise control of reactive intermediates, and thereby facilitate highly selective reactions that are difficult to achieve in conventional reactors. J.-I. Yoshida reports that the Swern oxidation of alcohols to ketones can be accomplished by using a micro-reactor at temperatures between −20 and 20°C, much higher temperatures than those required for conventional macro-scale batch reactors (-50°C or below) (Angew. Chem., 2005, 117, 2–4).

Laboratory Information Management Systems

Standardize LIMS and Reduce Total Cost of Ownership

Pharmaceutical companies that are planning or deploying LIMS solutions face significant challenges. The new and recent needs of enterprise knowledge management, knowledge sharing, compatible reporting formats, and business process harmonization from lab-to-lab are discussed in eLAB - European Laboratory Scientist, 2004, 21–26. New concepts for e-business—in or between companies—require that businesses contend with increased LIMS solutions, from internal lab processes to administrative or business workflow. The challenge is to standardize as much of the business workflow as possible to realize global interfaces for business-to-business solutions. Another point of interest is the improvement of integration to other existing IT systems. The impact of standardization of total cost of ownership (TCO) or return on investment (ROI) is shown. The conclusion is a general trend to LIM systems for different user groups and dispersing labs by respecting high-level process standardizations

High-Throughput Analytics

Investigation of the Rapid Scan on an Electrospray Ion Trap Mass Spectrometer

Mark E. Bier and C. G. Yang describe how important characteristics like mass accuracy, ion signal, peak shape, and resolution are affected by an increased scan rate on a quadrupole ion trap mass spectrometer. (Anal. Chem. 2005, 77, 1663.) The consequential advantages and disadvantages are discussed in detail and are illustrated on the basis of the determination of peptide mixtures (e.g., angiotensin I, brandykinin) and polymers, for instance, the molecular weight determination of apomyoglobin (MW = 16951) from a 40 fmol/μL sample solution. This can be achieved with only one 3.2-second rapid scan, which consists of 3 microsecond scans at 12 times the normal scan rate using the same ion injection time, whereas the deconvolution of the data of a homologous normal scan does not result in a molecular weight determination. Despite drawbacks such as decreased resolution and current mass accuracy limitations, the rapid scan can significantly improve important parameters like signal-to-noise ratio, ion statistics, and scan time, making protein molecular weight determinations at low amounts possible.

Instrumentation and Method for Ultrahigh Resolution Field Desorption Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry of Nonpolar Species

The capability to generate molecular ions, the low tendency to cause fragmentation, and the suitability for ionizing nonpolar species are only a few of the particular features that depict the field ionization/field desorption mass spectrometry in association with fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) as a powerful tool for complex mixture analysis. Alan G. Marshall and coworkers illustrate how the FD FT-ICR MS can be utilized for the challenging applications of petroleum analysis (Anal. Chem. 2005, 77, 1317). A detailed introduction to the specific application and construction capabilities of the new FD FT-ICR MS system are provided. As model compounds n-alkanes, alkylbenzenes, cholestane, and squalene are chosen to obtain good test results; there is a representative selection of nonpolar compounds in mass range of several hundred Daltons. Special attention also is given to the detrimental influence of sample overload and the integrity of the nanoscale structure of carbon microneedle dendrites on commercial emitters after repeated petroleum use. As an example, a broadband FD FT-ICR mass spectrum of a sample of crude oil from the U.S. Gulf of Mexico demonstrates the complexity of a “real” mixture of nonpolar compounds and the efficiency of the FD FT-ICR MS at hand. An internal calibration from 19 members of the hydrocarbon DBE = 4 homologous series allows an assignment of 588 elemental compositions from 250 < m/z < 620 at an average mass resolving power, m/Δm(50%) = 442000.

Fourier Transform Infrared Imaging for High-Throughput Analysis of Pharmaceutical Formulations

Fourier transform infrared (FTIR) spectroscopic imaging with infrared array detectors has emerged as a powerful materials characterization tool. S. G. Kazarian and K. L. A. Chan report a novel application of FTIR imaging for high-throughput analysis of materials in a controlled environment. This approach combines spectroscopic imaging with an attenuated total reflection (ATR)-IR cell, microdroplet sample deposition system, and a device that controls humidity inside the cell. With this approach, it is possible to obtain “chemical snapshots” from a spatially defined array of many different polymer and drug formulations (more than 100) under identical conditions. This method provides direct measurement of material properties for high-throughput formulation design and optimization. Simultaneous response (water sorption, crystallization, etc.) of the array from formulations to environmental parameters are studied. Implications range from studies of smart polymeric materials and sensors to screening of pharmaceuticals and biomaterials (J. Comb. Chem. 2005, 7, 185–189).

Bioautomation and Screening

A Method for Screening Enzyme Inhibitors Using Size Exclusion Chromatography and ESI-LC-MS/MS

Most binding assays are commonly carried out under conditions in which salts may be present at concentrations high enough to interfere with MS detection. Consequently, the removal of salts from samples before MS analysis is desirable. The available methods, such as desalting using solid-phase extraction (SPE), liquid-liquid extraction (LLE), or ion-exchange chromatography, are time-consuming and thus not suitable for high-throughput analyses. Mathur et al. (J. Biomol. Screen., 2005, 10, 30) have developed a method to screen compound libraries, using an electrospray mass spectrometer interfaced with liquid chromatography. Carboxypeptidase A (CPA), a zinc-containing proteolytic enzyme, is incubated with a mixture of low-molecular weight compounds, and subsequently, the enzyme-bound compounds are separated by size exclusion chromatography (SEC) from unbound compounds. These compounds are quantitated using an automatic switching valve to avoid buffer salt interference with the detection of analytes. This methodology allows the detection and quantitation of protein bound compounds in samples containing salts in high concentrations. In addition, it does not require denaturation of the protein and desalting prior to LC-MS analyses, thereby reducing steps for library screening. The described method has a higher throughput than previously described methods and might be used for the screening of large compound libraries, and the quantitation of bound ligands for other enzyme inhibitor complexes.

Volume Cytometry: Microfluidic Sensor for High-Throughput Screening in Real Time

Cell volume and its physiological function are related to each other, so that a real-time monitor of cell volume can serve as a screen for drugs or other environmental influences in the same manner as cell-based calcium assays. The perturbations that affect cell volume are multifarious and include, for example, excitability, metabolism, and apoptosis. Ateya et al. (Anal. Chem., 2005, 77, 1290) have developed a microfluidic/electrical sensor chip (made of silicon) that is noninvasive and provides real-time measurement of changes in cell volume for both adherent and suspended cells. The small volume of the sensor enables rapid screening for test compounds that are only available in small quantities. The principle of measuring cell volume in the sensor is based on the fact that cells are electrical insulators at low frequencies. With cells in a chamber of fixed cross section, a change of cell volume displaces the extracellular fluid, thereby changing the chamber conductance. The chip is actually sensitive to any object that does not conduct ions, and therefore there is great potential for its use.

Automated Microscope System for Determining Factors that Predict Neuronal Fate

Unraveling cause-and-effect relationships in the nervous system is challenging because some biological processes begin stochastically, require a significant amount of time to unfold, and affect small neuronal subpopulations that can be difficult to isolate and measure. Single-cell approaches are slow, subject to user bias, and sometimes too laborious to achieve sample sizes large enough to detect important effects. M. Arrasate and S. Finkbeiner describe an automated imaging and analysis system that enables them to follow the fates of individual cells and intracellular proteins over time. Observations can be quantified in a high-throughput manner with minimal user bias. They adapt survival analysis methods to determine whether and how factors measured during longitudinal analysis predict a particular biological outcome. The ability to monitor complex processes at single-cell resolution quickly, quantitatively, and over long intervals could have wide applications for biology (PNAS, 2005, 102, 3840–3845).

Kinetic Assay for High-Throughput Screening of in Vitro Transthyretin Amyloid Fibrillogenesis Inhibitors

Stabilization of tetrameric transthyretin (TTR) by binding of small ligands is a current strategy aimed at inhibiting amyloid fibrillogenesis in transthyretin-associated pathologies such as senile systemic amyloidosis (SSA) and familial amyloidotic polyneuropathy (FAP). A kinetic assay was developed by A. Planas et al. for rapid evaluation of compounds as potential in vitro inhibitors in a high-throughput screening format. It is based on monitoring the time-dependent increase of absorbance due to turbidity occurring by acid-induced protein aggregation. The method uses the highly amyloidogenic Y78F mutant of human transthyretin (heterogously expressed in Escherichia coli cells). Initial rates of protein aggregation at different inhibitor concentrations follow a monoexponential dose-response curve from which inhibition parameters are calculated. For assay development, thyroid hormones and nonsteroidal antiinflamatory drugs are chosen among other reference compounds. Some of them are already known to be in vitro inhibitors of TTR amyloidogenesis. Analysis time is optimized to last 1.5 h, and the method is implemented in microtiter plates for screening of libraries of potential fibrillogenesis inhibitors (J. Comb. Chem. 2005, 7, 246–252).

Screening for Cell Migration Inhibitors via Automated Microscopy Reveals a Rho-Kinase Inhibitor

Small-molecule kinase inhibitors are predominantly discovered in pure protein assays. J. C. Yarrow et al. have discovered an inhibitor of Rho-kinase (ROCK) through an image-based, high-throughput screen of cell monomigrationlayer wound healing. Using automated microscopy, they screened a library of 16,000 compounds, finding many that affect cell migration or cell morphology, as well as compounds that block mitotic progression. The authors test 200 compounds in a series of subassays and choose one, 3-(4-pyridyl)indole (Rockout), for more detailed characterization. Rockout inhibits blebbing and causes dissolution of actin stress fibers, phenocopying Rho-kinase inhibitors. Testing Rho-kinase activity in vitro, Rockout inhibits with an IC50 of 25 μM (fivefold less potent than Y-27632), but has a similar specificity profile (Chem. Biol. 2005, 12, 385–395).

High-Throughput Screening for Protein Kinase Inhibitors

The pivotal role of kinases in signal transduction and cellular regulation has lent them considerable appeal as pharmacological targets across a broad spectrum of pathologies. Since the discovery that the v-Src oncogene encoded a protein kinase in 1978, kinases have remained a focus of research for pharmaceutical laboratories and academic groups alike. Many have sought to develop orally available low molecular weight synthetic kinase modulators (predominantly inhibitors) to capitalize on the links between aberrant regulation and disease. This interest in kinases as drug targets has been fueled in recent years by the success of several kinase inhibitors in the clinic—primarily Gleevec for the treatment of chronic myelogenous leukemia, and Iressa for the treatment of advanced nonsmall cell lung cancer. A review by H. Wesche et al. focuses on the development of small molecule drugs, most of them binding in or close to the ATP binding pocket. The majority of this review is dedicated to a detailed description and discussion of the various assay formats currently being employed for high-throughput screening (Comb. Chem. High Throughput Screen. 2005, 8, 181–195).