Automation Highlights from Literature

Laboratory Automation and High-Throughput Chemistry

The Manual and Automated Solid-Phase Synthesis of α-Substituted Prolines and Homologues

A seven-step, fully automated synthesis of α-substituted prolines and homologues has been developed by W. L. Scott (Eli Lilly), J. Alsina, and M. J. O'Donnell (Purdue University, Indianapolis) (J. Comb. Chem. 2003, 5, 684). Activation of N-terminal resin-bound amino acids as aldimines, followed by alkylation with α,ω-dihaloalkanes, provides key intermediates for the solid-phase preparation of racemic α-substituted proline ring homologues bearing amino acid side chains. Two intramolecular displacement strategies, one involving an amine nucleophile, the other an amide nucleophile, are used to convert the intermediate ω-chloro or ω-bromo derivatives to the desired cyclic products. Using one of these routes and a fully automated Argonaut Technologies Trident Library Synthesizer, a 48-member combinatorial library (50-μmol scale) of α-substituted prolines can be prepared. The Trident Library Synthesizer is capable of running up to 192 reactions in parallel. The main part of the system is the Trident Reaction Cassette that holds 48 5-mL glass vessels. Purification of the crude samples is carried out using automated reversed-phase chromatography.

Using a Lipase as a High-Throughput Screening Method for Measuring the Enantiomeric Excess of Allylic Acetates

C. T. Seto and M. B. Onaran (Brown University, Providence) describe a high-throughput method for measuring the enantiomeric excess of allylic acetates (J. Org. Chem. 2003, 68, 8136). Such methods are useful tools for screening libraries of potential catalysts for enantioselective reactions. This technique, which is called EMDee for an enzymatic method for determining enantiomeric excess, uses the lipase from Pseudomonas cepacia to hydrolyze the (R)-enantiomer of an allylic acetate, while the (S)-enantiomer does not react. The rate of the reaction is monitored by measuring the acetic acid that is produced during the hydrolysis reaction with a pH indicator. Using the Michaelis-Menten equation, the rate of the reaction can be correlated with the concentration of the (R)-enantiomer. This method can process 88 samples in less than 30 min. Due to the very broad substrate specificity of this lipase, this assay procedure should be useful for screening potential enantioselective catalysts for a variety of synthetic transformations.

Self-Indicating Resins: Sensor Beads and In Situ Reaction Monitoring

Solid-phase organic synthesis and solution-phase parallel synthesis assisted by polymer-supported reagents, or scavenger resins, enable painstaking purifications to be avoided during synthesis. This concept is applied by M. Bradley et al. (University of Southampton) to reaction monitoring through the development of resin-bound indicators for the efficient analysis of various chemistries (J. Comb. Chem. 2003, 5, 632). Bromophenol blue was chosen as the indicator and attached to the resin after derivatization. The resin is successfully used as a “sensor” for monitoring solid-phase peptide synthesis and is applied successfully for in situ reaction monitoring during the synthesis of a library of ureas.

The Many Faces of Combinatorial Chemistry

S. Borman gives a broad and interesting progress report on key areas of combinatorial chemistry such as natural-product-like libraries, dynamic combichem, catalyst optimization, and multicomponent reactions (Chem. Eng. News 2003, 81(43), 45). Currently, the industry trend is shifting away from very large libraries toward synthesis of smaller, more-focused compound libraries.

Combinatorial Synthesis of SAPO-34 via Vapor-Phase Transport

Combinatorial synthesis is a very efficient way for the systematic evaluation and production of both inorganic and organic materials, but its application to the synthesis of zeolites and their aluminophosphate counterparts is still limited. In a new paper, L. Zang et al. present the first application of the combinatorial method to the synthesis of SAPO-34, a silicon-substituted aluminophosphate molecular sieve, using a vapor-phase transport (VPT) method (Chem. Commun. 2003, 17, 2232). A special multiwell reactor suitable for the combinatorial VPT synthesis was designed and installed into a Teflon liner of a conventional autoclave.

For the analysis, X-ray diffraction and scanning electron microscopy are used. With this new methodology, the synthesis parameters for SAPO-34 molecular sieve by VPT technique can be quickly and effectively examined.

Combinatorial Chemistry in Food Research

Combinatorial chemistry is not only a tool for drug discovery or material screening; it is also applicable in the food industry. Recent advances in the use of combinatorial methods have enabled the identification and analysis of flavors. “Food chemical” libraries as a potential source of enzyme inhibitors for the food industry and the utility of biocatalysis for the generation of molecular diversity are discussed by J. A. Khan and E. N. Vulfson in Combinatorial Chemistry & High Throughput Screening, 2003, 6(6), 569.

High-throughput analytics

Optimizing Preparative LC/MS Configurations and Methods for Parallel Synthesis Purification

Preparative LC/MS is widely employed to address the high-throughput purification demands of parallel synthesis. However, conflicting chromatography requirements and the complexities of applying MS-directed fractionation can limit its effectiveness in high-throughput parallel synthesis schemes. K. F. Blom and colleagues from Bristol-Myers Squibb report an at-column dilution, small-scale preparative LC configuration that satisfies the mass loading (>20 mg) and small fraction volume (<1.5 mL) requirements of discovery parallel synthesis schemes employing plate mapping (J. Comb. Chem. 2003, 5, 670). They also present a protocol for compound-specific optimization of the preparative gradient LC method and MS threshold for fractionation from a “prepreparative” LC/MS analysis of the crude material. The authors claim significantly improved preparative separations relative to universal preparative LC methods and the selection of reliable and effective fraction threshold methods for diverse libraries. The methods and configurations are simple to implement and are equally suited for open access and expert applications of preparative LC/MS purification.

Proteome Analyses Using Accurate Mass and Elution Time Peptide Tags with Capillary LC Time-Of-Flight Mass Spectrometry

Eric F. Strittmatter and co-workers describe the application of capillary LC-TOF mass spectrometric instrumentation for rapid characterization of microbial proteomes (J. Am. Soc. Mass Spectrom. 2003, 14, 980). Using an accurate mass and time (AMT) tag database, detected peptides can be assigned using measurements obtained on a TOF-MS due to the additional use of retention time data as a constraint. The peptide identification approach developed in this work enables high-throughput identification of peptides with the help of time-of-flight mass spectrometry and HPLC. This method can provide proteomic information for approximately 1.000 proteins in a single run of <3 h. In contrast, in MS/MS experiments, the number of peptide identifications is severely restricted because of the time required to dissociate the peptides individually.

Development of High-Sensitivity Ion Trap Ion Mobility Spectrometry Time-of-Flight Techniques: A High-Throughput Nano-LC-IMS-TOF Separation of Peptides Arising from a Drosophila Protein Extract

A linear octopole trap interface for an ion mobility time-of-flight MS has been developed for focusing and accumulating continuous beams of ions produced by electrospray ionization (Anal. Chem. 2003, 75, 5137). This interface improves experimental efficiencies by factors of 50 to 200 compared with an analogous configuration that utilizes a conventional ion trap. S. Myung and co-workers demonstrate the utility of the approach in the field of proteomics by an online, three-dimensional nano-LC-ion mobility-TOF separation of tryptic peptides from the Drosophila proteome. With these improvements, it is possible to record datasets on short time scales. In addition, detection limits in the 10 to 100 amol range are accessible.

Total Protein Determination by Particle Beam/Hollow Cathode Optical Emission Spectroscopy

A novel method for quantitative total protein determinations is presented by F. Jin and associates (Anal. Chem. 2003, 75, 4801). Total protein content is determined by particle beam/hollow cathode optical emission spectroscopy (PB/HC-OES) through monitoring of carbon atomic emission. The PB/HC-OES offers advantages such as ease of operation, exclusion of labor-intensive sample pretreatment processes, rapid analysis, high sensitivity, and low detection limit. The method could also be adapted to be integrated to microfluidics devices. The analytical performance observed here suggests that this technique could be effectively used in a number of bioanalytical applications, including the monitoring of proteins in MEMS devices. Furthermore, it is reasonable to postulate that the entire apparatus (including microfluidics, nebulizer, momentum separator, and plasma source) could be integrated into a lab-on-a-chip platform.

Microfluidic chip technology

Integrated Nanoliter Systems

Microfluidic chip platforms for manipulating liquid volumes in the nanoliter range are slowly inching their way into mainstream genomic and proteomic research. The principal challenge faced by these technologies is the need for high-throughput processing of increasingly smaller volumes with higher degrees of parallelization. Significant advances have been made over the past few years in addressing these needs through electrokinetic manipulation, vesicle encapsulation, and mechanical valve approaches. These strategies allow levels of integration density and platform complexity that promise to make them into serious alternatives to current robotic systems. An overview on this exciting technology is given by J. W. Hong and S. R. Quake (California Institute of Technology) in Nature Biotechnology 2003, 2(10), 1179.

Integrated Microfluidic Systems

In another short review by R. F. Ismagilov (University of Chicago), potential applications of integrated microfluidic systems are discussed (Angew. Chem. 2003, 115, 4262). These “lab on a chip technologies” have great potential for synthesis, purification, analytics, and diagnostic applications in chemical or medicinal laboratories.

Bioautomation and screening

The Lab of the Future: A Drug Discovery Factory?

Drug discovery is beginning to move in the same direction as more-traditional manufacturing industries. Increasing the level of automation promises increased capacity, shorter lead times, and reduced attrition rates. In an interesting special report, L. Evans describes the major trends in automation and screening technology with special focus on the new drug discovery factory of GlaxoSmithKline (Current Drug Discovery September 2003, 23). This new ultra high-throughput screening facility in Tres Cantos, Spain is capable of storing 2.8 million individual samples and performing 300,000 assays per day.

HTS and Cellular Biology at Serono

The mapping of the human genome has placed renewed emphasis on the identification of novel proteins. D. Besson, K. Yeow, P. Lang, and A. Scheer report on how the expertise acquired through its small molecule programs has enabled Serono to develop more than 60 robust cell assays using disease-relevant cellular systems to discover new protein therapeutics (Current Drug Discovery September 2003, 29). The Serono HTS environment, which was originally designed solely to support the small molecule discovery program, had to undergo significant changes to adapt to the specific needs of cellular screening, e.g., each of the robotic stations now has 37 °C incubators as well as 96-well automatic pipettors integrated into the system. In order to speed up the turnaround time of complex assays, an automated cell cytometer was co-developed with Beckman Coulter.

Gearing Up for Speedy Relief

In a feature article on automation, M. S. Lesnick presents an overview of the use of automated equipment in the different phases of the drug discovery process (Modern Drug Discovery August 2003, 39). Beginning with automated gene sequencing and microarrays for automated gene expression in the target selection phase, and continuing with automated synthesis and high-throughput screening of potential lead candidates, automation accelerates the overall processes. Finally, automated ADMET studies help to characterize the compounds further.

High-Throughput, Real-Time Monitoring of G_s-Coupled Receptor Activation in Intact Cells Using Cyclic Nucleotide-Gated Channels

Cyclic adenosine-monophosphate (cAMP) belongs to the second messengers transmitting extracellular signals from neurotransmitters or hormones into the intracellular environment. Measurements of cellular cAMP levels are suitable for studying signaling by G protein-coupled receptors. Current assays for cAMP determination are usually end-point assays involving cell lysis. Reinscheid et al. (European Journal of Pharmacology 2003, 478, 27) have developed a new technology for monitoring real-time changes of cAMP levels in living cells by linking intracellular cAMP levels to Ca²⁺ influx. The method uses a modified cyclic nucleotide-gated (CNG) Ca²⁺ channel that is opened by intracellular cAMP. Changes in cAMP levels lead to changes in free Ca²⁺, which can easily be measured using fluorimetric technologies suitable for high-throughput screening. Reinscheid et al. used the new assay method to characterize various endogenously and heterologously expressed G protein-coupled receptors involving the fluorimetric imaging plate reader FLIPR (Molecular Devices) for the addition of drugs and measurement of changes of intracellular calcium. With the new method, it is possible to study G_s-, G_r-, and G_q-linked receptors in the same cell population.

Enzyme Screening with Synthetic Multifunctional Pores: Focus on Biopolymers

Sordé et al. (PNAS 2003, 100, 11964) developed a “universal enzyme sensor” using a single set of identical synthetic multifunctional pores (SMP) that are able to detect the activity of many different enzymes in a non-invasive manner. The principle relies on the fact that anionic, cationic, and neutral substrates can gain access to the interior of complementary functionalized pores. That is why such pores can be the basis for a straightforward screening of a broad range of enzymes. If substrate binds and blocks the pores better than products, enzyme activity gradually removes the blocking agent and the pores can open. On the other hand, if products bind and block the pores better than the substrates, enzyme activity gradually produces blocking agents that can block the pores. Enzyme-gated pore opening and closing is detected by measuring the emission of 5(6)-carboxyfluorescein (CF) using fluorometric real-time technique. The method is based on vesicles containing CF that can dilute into the external medium when the pore is open. This results in an increase of emission intensity that correlates with the enzyme activity. Because of the broad adaptability of the described enzyme sensors, they are ideal for practical applications and might be interesting for automation.