Automation Highlights from the Literature

Laboratory Automation and High-Throughput Chemistry

Direct Asymmetric Aldol Reaction in Aqueous Media using Polymer-Supported Peptide

A polyethyleneglycol grafted onto a cross-linked polystyrene resin-supported tripeptide/zinc chloride catalyst system is shared for use in the direct asymmetric aldol reaction of acetone with aldehydes in aqueous media (Kengo Akagawa et al., Tetrahedron Lett. 2005, 47(46), 8185-8187).

A major drawback of the proline(-derivative)-catalyzed aldol reaction is its low efficiency, and as much as 10-40mol%, typically 20mol%, of catalyst is needed. To indirectly overcome this problem, the authors investigate multiple use of the catalyst. The peptide catalyst can be separated easily from the reaction mixture by simple filtration and washing, and can be reused at least five times without significant change in its activity and selectivity. Reactions using recycled catalysts proceed even in the absence of ZnCl₂, but efficiency and selectivity are slightlylower. Basically, adding ZnCl₂ to each cycle results in a better outcome.

“Green” Synthesis of Important Pharmaceutical Building Blocks: Enzymatic Access to Enantiomerically Pure α-Chloroalcohols

Chiral halohydrins are important synthetic intermediates for the preparation of a wide range of biologically interesting compounds. Thirty-one recombinant ketoreductase enzymes are screened for the reduction of six α-chloroketones, the precursors of these α-chloroalcohols (Dunming et al., Tetrahedron: Asymmetry 2005, 19(16), 3275-3278).

The enantioselectivities of 12 selected ketoreductases with high activities are also studied. Several highly active and enantioselective ketoreductases that produce an enantiomeric excess greater than 99% in good to excellent isolated yields are found. Their applications to the synthesis of both enantiomers of these α-chloroalcohols are demonstrated on a preparative scale. This method offers a convenient, “green” access to these types of valuable pharmaceutical building blocks.

The authors use this method for genome mining with a protein engineering developed toolbox of 31 recombinant ketoreductase enzymes. The isolated enzymes can be stored and used as normal chemical reagents, and no microbiological knowledge is required.

Sandwich Immunoassay as a High-Throughput Screening Method for Cross-Coupling Reactions

Combinatorial and parallel methods have become an important focus of research in catalysis. In particular, techniques that allow the simultaneous screening of numerous catalyst candidates have gained attention because they may speed up the identification and optimization of active catalysts. Frederic Taran et al report a new versatile enzyme immunoassay format that is suitable for the fast screening of coupling reaction catalysts. As an example, they study the Sonogashira reaction. Although the assay presented is restricted to conditions and catalysts that are at least partially compatible with tag structures, its versatility and efficiency can be very useful in the discovery of catalysts and reactions. Improvement of the presented method would require the production of antibodies directed against chemically inert tags. Furthermore, the high sensitivity of this type of immunoassay allows the exploration of reactions that occur under very dilute conditions, an advantage that is of great importance in the search for new reactions compatible with bioconjugation (Angew. Chem. 2005, 117, 7023).

The Removal of the t-BOC Group by Electrochemically Generated Acid and Use of an Addressable Electrode Array for Peptide Synthesis

Karl Maurer et al report the use of an electrochemically generated acid for the removal of the t-BOC protecting group at selected positions on an electronically addressable array. This deprotection technique allows the construction of different peptide chains at different places on the array using commercially available t-BOC protected substrates. The device used for this work is a CombiMatrix CustomArray. This unit consists of a semiconductor silicon chip with an array of 1024 individually addressable platinum electrodes, and a porous overlaying layer on which is synthesized a DNA oligomer. The electrodes can be set to a specified voltage via connection to a PC, allowing different patterns of on and off electrodes to be used at each step in the construction of the array. This is used successfully to synthesize an array of peptides containing the N-terminus of the endorphin sequence (YGGFL) (J. Comb. Chem. 2005, 7, 637).

London University Search Instrument: A Combinatorial Robot for High-Throughput Methods in Ceramic Science

J. Wang and J. R. G. Evans describe the design, construction, and operation of the London University Search Instrument that was recently commissioned to create and test combinatorial libraries of ceramic compositions. This instrument uses commercially available powders, milled as necessary to create thick-film libraries by ink-jet printing. Multicomponent mixtures are prepared by well plate reformatting of ceramic inks. The library tiles are robotically loaded into a flatbed furnace and, when fired, transferred to a two-axis high resolution measurement table fitted with a hot plate where measurements of optical or electrical properties can be performed (J. Comb. Chem. 2005, 7, 665).

Microfluidic Chip Technology and Micro Reactor Technology

Heterogeneous Hydrogenation Reactions Using a Continuous Flow High Pressure Device

Bimbisar Desai and C. Oliver Kappe report on efficient continuous flow hydrogenation reactions using either Pd/C or Raney-Ni as heterogeneous catalysts in the novel H-cube device. The H-cube (produced by Thales Nanotechnology) is a compact, high-pressure heterogeneous hydrogenator with a HPLC-like platform for substrate delivery, enabling heterogeneous hydrogenations at temperatures up to 100°C and 100 bar of hydrogen pressure in a continuous flow mode. The electrolytic decomposition of water generates in situ hydrogen of up to 100 bar. A prepacked, replaceable cartridge of the heterogeneous catalyst contained in the system allows a uniform substrate flow (mixed with hydrogen) without catalyst leaching, circumventing the need to filter the catalyst from the substrate after the desired hydrogenation. The instrument allows a continuous monitoring of the reaction progress and modification of the reaction parameters (temperature and hydrogen pressure) during an experiment for rapid optimization of hydrogen retained in the Raney-Ni catalyst cartridge. The continuous flow reduction of an aromatic nitro group, benzyl deprotections, and desulfurization reactions of selected dihydropyrimidine analogues are performed successfully (J. Comb. Chem. 2005, 7, 641).

High-Throughput Analytics

Interpretation of High-Throughput Liquid Chromatography Mass Spectrometry Data for Quality Control Analysis and Analytical Method Development

Bernard K. Choi et al report an approach to rapidly process and interpret high-throughput liquid chromatography mass spectrometry data. This approach applies an in-house developed computer application to process LC/MS report files containing spectral and chromatographic data from four different detectors (i.e., electrospray positive ionization, electrospray negative ionization mass spectrometry, UV absorption, and evaporative light scattering detection (ELSD)). Properties characteristic of detection and chromatographic retention are extracted and populated into a database. Approaches to applying this analytical information database for quality control analysis of about 400,000 samples are presented. Structural similarity searches are applied with the analytical information database to identify compounds that may be undetectable by electrospray mass spectrometry. The analytical information database may also be applied to aid rapid analytical method development by providing a reference for the selection of detection methods and chromatographic conditions (Comb. Chem. High Throughput Screen. 2005, 8, 467).

Maximizing Automation in LC/MS High-Throughput Analysis and Purification

Marcus Koppitz et al. describe a system for LC/MS-based analysis and purification of compounds aimed at the minimization of manual interference in the overall process. Key elements of the concept are automated identification of target compounds, automated assignment of optimized preparative gradients for purification of target compounds, and automated purity assessment of fractions with subsequent pooling of validated product fractions. Additional support is provided by an automated solvent and waste management system. It is possible to process 100-200 compounds on a 150-mg scale per day on this system (J. Comb. Chem. 2005, 7, 714).

Integrity Profiling of High-Throughput Screening Hits Using LC–MS and Related Techniques

Integrity profiling of HTS hits is important for verification of hit identity and purity. Methodology for integrity profiling of HTS hits must be run in a high-throughput manner, consume little material, and selectively provide structure-based data. Analytical techniques that can be used for integrity profiling methods are reviewed by E. H. Kerns et al. for their appropriateness in sample preparation, component separation, detection, purity quantitation, and identity confirmation (Comb. Chem. High Throughput Screen. 2005, 8, 459).

Analytical and Biological Evaluation of High Throughput Screen Actives Using Evaporative Light Scattering, Chemiluminescent Nitrogen Detection, and Accurate Mass LC–MS–MS

M. A. Strege et al investigate the utility of ELSD combined with HPLC–MS as a key component of a bioassay-guided fractionation technique for the evaluation of high-throughput screen activities. ELSD provides online analytic mass information that is critical to the classification of samples. Chemiluminescent nitrogen detection is also evaluated for sample concentration estimation for nitrogen-containing compounds, and accurate mass LC/MS/MS analysis is used for rapid structural confirmation and elucidation of components previously identified as active via biofractionation (Comb. Chem. High Throughput Screen. 2005, 8, 477).

The Design and Characteristic Features of a New Time-of-Flight Mass Spectrometer with a Spiral Ion Trajectory

The characteristic features of a time-of-flight (TOF) mass spectrometer are high sensitivity, theoretically infinite mass range, and rapid measurement. T. Satoh et al from JEOL (Tokyo, Japan) present a newly designed and constructed spiral TOF mass spectrometer, and detail the characteristic features of the system and its basic performance (J. Am. Soc. Mass Spectrom. 2005, 16, 1969).

The new spiral TOF mass spectrometer consists of an electronic ionization (EI) source coupled with an orthogonal acceleration block, directing the ion packets toward the torodial electrostatic sectors (TESs) of the spiral ion optics. Each TES has 15 stories and the flight path length of one cycle is 1.308m, so that the maximum flight path length of the generated ions becomes approximately 20m before reaching the movable detector system. Because of problems with the limited area of the usual multichannel plate (MCP) detector surface, a secondary electron multiplier is used as an ion detector. Mass resolution measurements with isotope-tagged pyridine and benzene mixtures and perfluorokerosine reach 35,000 and 35.000 full width at half maximum (FWHM) at m/z>300 for the latter after 15 cycles. The determined mass accuracy of the TOF MS is better than 1ppm using internal standard peaks, and the usual temporal drift can be corrected likewise. The ion transmission, one important consideration for high sensitivity, is determined to be near 100% for the peak areas of the analyzed CF₃+ (m/z=69) in 1-15 cycles.

A Combination of Atmospheric Pressure LC/MS: GC/MS Ion Source: Advantages of Dual AP-LC/MS:GC/MS Instrumentation

C. N. McEwen and R. G. McKay from DuPont Corporate Center for Analytical Science (Wilmington, DE) describe the modification of a commercial LC/MS instrumentation to allow both atmospheric pressure LC/MS and GC/MS (J. Am. Soc. Mass Spectrom. 2005, 16, 1730).

After a short historical and technical overview, the authors elucidate in detail, how different mountings of LC and GC interface probes inside the ion source affect the method's inherent qualities—especially the combination and preservation of high chromatographic resolution, great peak capacity for complex mixture analysis, high sensitivity for volatile and low polarity compounds for GC/MS, and rapid switching between positive and negative ion mode, cone voltage fragmentation, MS ⁿ , high mass resolution, and accurate mass measurement for LC/MS instrumentations. Manifold measurements with a Restec 8270 mixture for the evaluation of advantages and drawbacks of the combined ion sources show that in addition to the ease of switching between GC/MS and LC/MS, less volatile compounds can be analyzed and also the speed of GC/MS can be increased by simple manipulations. In addition, all of the capabilities that these LC/MS instruments have in common (as mentioned in the preceding) can be applied to atmospheric pressure GC/MS.

Bioautomation and Screening

Chemical and Biological Integrity in Natural Products Screening

Because of pressure from combinatorial chemistry and the streamlining of the drug discovery process through automated high-throughput screening technologies, pharmaceutically based natural product programs are under increasing scrutiny. However, by taking advantage of technologies originally developed for high-throughput screening and combinatorial chemistry and applying them to processes considered to be bottlenecks in classical natural products chemistry (e.g., purification, structure elucidation, sample availability), George G. Harrigan and Gilles H. Goetz demonstrate that natural products still can contribute to the effective discovery of novel bioactive and pharmaceutically relevant metabolites (Comb. Chem. High Throughput Screen. 2005, 8, 529).

High Throughput Sonication: Evaluation for Compound Solubilization

Precipitation of compounds from DMSO screening stocks is a recognized problem in the HTS materials management process. The adverse effect of freeze–thaw cycles on DMSO stock solutions stored in plate format as a result of cherry picking operations has led to the gradual replacement of plate-based storage with tube-based storage to minimize the number of freeze–thaw cycles. Compound solubility in DMSO is markedly decreased with the uptake of small quantities of water. M. Kelly et al. report that efficient compound dissolution is possible even in 384-well format by using in-well plate-based sonication. They find that solids precipitated from DMSO stocks either by water uptake or repeated freeze–thaw cycles can be redissolved through low energy sonication (Comb. Chem. High Throughput Screen. 2005, 8, 499).

HTS Quality Control and Data Analysis: A Process to Maximize Information from a High-Throughput Screen

Changes in all aspects of HTS, from compound management through to evaluation of hits and leads, strengthened by improvements in automation and informatics, have made possible increased analysis and implementation of process and quality control throughout HTS. Ramesh Padmanabha et al describe their process of HTS with an emphasis on quality control, reducing the variability of all the processes that affect the final result (Comb. Chem. High Throughput Screen. 2005, 8, 521).

The Novartis Compound Archive: From Concept to Reality

Because of the importance of HTS technologies for modern drug discovery, pharmaceutical companies are focusing increasingly on the quality of their screening collections. Storage conditions and their influence on compound stability and solubility are checked intensely. The Novartis strategy for this is described by U. Schopfer et al and consists of the following elements:

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Compounds unsuitable for storage in solution are excluded by computational methods.

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Compounds are stored at 4°C/20% relative humidity in a DMSO/water mixture to avoid freeze-thaw cycles and water uptake and to allow rapid plate replication.

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Compounds are resolubilized at regular intervals.

To achieve these goals, automated systems for compound storage, weighing, and plate replication are used (Comb. Chem. High Throughput Screen. 2005, 8, 513).

High-Throughput P450 Inhibition Screens in Early Drug Discovery

G. Zlokarnik et al. reviewed high-throughput P450 inhibition technologies and their impact on early drug discovery. The relationship between P450 inhibition and drug-drug interactions is well understood. A wide variety of P450 inhibition detection technologies are readily available off-the-shelf, but what seems still to be missing is a general agreement on how much weight one should give to the various types of early discovery high-throughput P450 inhibition data. Method-dependent potency differences are a cause of concern, and to resolve this issue, the authors strongly encourage calibration of high-throughput methods with a large set of marketed drugs (Drug Discov. Today 2005, 10, 1443).

High-Throughput Generation of Small Antibacterial Peptides with Improved Activity

The treatment of bacterial infections has become limited by an increase of bacterial antibiotic resistance. In the field of antibiotic development, it is difficult to find new structures with the same characteristics as conventional antibiotics (i.e., low toxicity for the host and high efficiency against bacterial pathogens).

Cationic antimicrobial peptides show a high activity against a broad variety of Gram-negative and Gram-positive bacteria, and thus are good candidates for lead structures to develop a new class of antimicrobial drugs to treat multidrug-resistant bacteria.

Hilpert et al. (Nat. Biotechnol. 2005, 8, 1008-1012) describe a high-throughput method to screen large numbers of peptides for improved antimicrobial activity. The technique relies on peptide synthesis on a cellulose support (with the help of pipetting robots), and a Pseudomonas aeruginosa strain that constitutively expresses bacterial luciferase. A complete substitution library of 12 amino acid peptides of the bovine peptide bactenecin is screened and used to determine which substitutions at each position of the peptide chain improved activity. By combining the most favorable substitutions, the authors designed two optimized peptides showing broad-spectrum activities with inhibitory concentrations (MIC) as low as 0.5μg/ml against Escherichia coli, and with an MIC of 2μg/ml against E. coli and Staphylococcus aureus, respectively.

A Novel Approach to Determine Water Content in DMSO for a Compound Collection Repository

The integrity of a corporate compound collection is crucial to the follow-up quality of screening data, and can be affected by a complex combination of storage and processing factors. Water content in DMSO compound stock solution is a critical issue that can significantly affect solubility, degradation, and freeze–thaw cycle parameters, and therefore the value of the corporate compound collection.

Semin et al. (J. Biomol. Screen. 2005, 10, 568) report a novel approach to determine water content in DMSO compound stock solutions within the common storage vessel format of polypropylene microtubes using near-infrared (NIR) spectroscopy. The NIR instrument is designed and developed by The Automation Partnership (TAP, Royston, UK). Measurements can be performed only on a single tube at one time. The obtained spectra are stored and processed digitally using proprietary algorithms developed by TAP. The precision and accuracy of the NIR technique were compared to the standard Karl Fisher titration method, which involves the generation of iodine in direct proportion to the amount of water present in the reaction, which is known to be sample and time consuming.

The authors demonstrate that the advantages of the NIR technique include accuracy, precision, and speed to non-destructively assess water content in DMSO solutions. They present a case study to demonstrate the utility of the technique to aid an organization in optimizing processing parameters to minimize the potential deleterious effect of water content in DMSO compound stock solutions.

LIMS and Information Technology

From Island to Integrated Solutions

The global availability of an LIMS as an integrated solution sets the trend in the development of LIMS. This is the result of a current “Chemische Rundschau” survey by W. Suske (Chem. Rundsch. 2005, 7(58), 28-35).

An LIMS is no longer used only for managing the data and documents of a lab, and meeting quality demands. Today, it supports the entire workflow in R&D labs, and is used by lab assistants, scientists, members of the sales department, and others. The keyword is “integration”—the integration of an LIMS in a business as an enterprise solution. Therefore, an LIMS has to be standardized as well as adaptable, and adaptability is the greatest challenge in enterprise-wide LIMS developments. LIMS vendors like Thermo Electron, Creon Lab Control, and NuGenesis are working in this field.

CDS and LIMS Integration

In September 2004, the final Food and Drug Administration (FDA) report of the Pharmaceutical current good manufacturing processes (cGMPs) of the 21st century was published. It addresses pharmaceutical quality and manufacturing process improvement, and criticizes the hesitation of pharmaceutical companies to adopt new technology, even when it improves the quality of the products. The cGMP initiative focuses on updating the manufacturing process by using modern science and technology. In Europe, the counterpart to the U.S. FDA is the Office of New Drug Chemistry (ONDC), which intends to focus on critical pharmaceutical attributes. Less variability in manufacturing processes increases the FDA's confidence, and the article discusses the influence that the ONDC has with the FDA (LCGC Eur. 2005, 4(18), 226-227).

LIMS Security

Choosing the right LIMS for supporting lab processes is an extensive operation, and security is a critical factor in decisions being made today (Wallrafen, GIT Labor-Fachz. 2005, 8(49), 635-638).

LIMS security issues are becoming more and more important. An LIMS has to be secure, and a company's entire environment must fit its special requirements, for example, firewalls, virus checks, etc. Next to the hardware and software, system users also present special risks making it crucial to train users and deploy only skilled operators. Regulations are another concern that affects systems as a whole, and validations can help with requirements. In addition to security issues, the author discusses the key elements of an LIMS. Usually, an LIMS is adapted to the special requirements of a lab, and the result is a specialized LIMS.

The Challenges of Changing a LIMS System

Fish et al. discuss the challenges of transitioning from one LIMS to another (LaborPraxis 2005, 6(29), 28-30). A well-defined process is an important condition prerequisite to such a transition. This includes the analysis of source and destination systems to data migration and uses for the new system. LIMS migration is a complex process that requires considerable time, costs, analysis, process definition, configuration, validation, and training. Each of these factors has its own risks and challenges, making the total cost of an LIMS migration difficult to estimate. A clear concept and an experienced LIMS vendor are helpful.

Very often, the existing LIMS is multifariously integrated into existing business processes, which must not be disturbed during the migration process. This article presents the migration process in 10 reasonable steps.

Workflow Management in LIMS

By using add-on modules, the enterprise solution Win-LIMS by Quality Systems international (QSI, Midland Park, NJ) realizes the Integration of neighboring work areas, such as document and workflow management, into an LIMS (Strunk, LITUS 2005, 2(2), 8-9). This offers enormous synergy and many advantages. Information that is already stored in an LIMS does not need to be entered in the other systems, and all information can be stored in a closed area that can be secured by electronically signatures. By the exemplary creation of a new test specification, the article testifies to this approach.

Adaptive Online Control of Laboratory Systems

The lack of an online system coupling is a critical bottleneck in many HTS applications, because the direct integration of HTS systems to an LIMS offers many advantages. Göde et al. offer a new approach using extensible markup language (XML) to flexibly connect devices to an LIMS (LITUS 2005, 2(2), 12-14). In Process Control Systems (PCS), proprietary methods can be imported to an LIMS, including defined limits for method parameters. On the other hand, the modified data can be exported to a PCS. In addition, results data can be managed with respect to the entire workflow. The solution shared by the authors represents an open integration concept for a wide range of lab equipment in LIMS. A converter module for system administrators offers the adaptation of the process values and attributes of both sides, the PCS and the LIMS.