Automation Highlights from Literature

Unusual Coupling Reactions of Aldehydes and Alkynes: A Novel Preparation of Substituted Phthalic Acid Derivatives by Automated Synthesis

Based upon a highly versatile multicomponent methodology, a new one-pot synthesis of substituted phthalic acid derivatives from α,β-unsaturated aldehydes was developed by M. Beller, K. Thurow et al. (University of Rostock) (Chem. Eur. J., 2003, 9, 2273). The reaction involves the intermediacy of an acetamidodiene species that undergoes Diels-Alder addition to diethyl acetylenedicarboxylate. The resultant acetamidocyclohexadiene is subject to elimination of acetamide under the reaction conditions to give rise to substituted diethyl phthalates in good yields. This domino condensation-cyclo-addition-elimination sequence has been applied to a variety of α,β-unsaturated aldehydes. Detailed studies reveal the catalytic role of the employed acetamide and the occurrence of a stereoselective 1,4-syn elimination pathway under standard conditions. Parallel-automated synthesis technology was used successfully for the rapid screening of reaction conditions and compositions. Sample preparation, reaction processing and work-up steps were performed by a Zymate XP Robot mounted on a linear track that uses a variety of interchangeable hands to move consumables, liquids, vials, tubes, and containers of various sizes to the different stations and modules. The reactions were run in 4-mL screw cap vials in a 60-well Büchi Syncore reactor station.

OPEN IN VIEWER Kerstin Thurow Ph.D.

OPEN IN VIEWER Hilmar Weinmann Ph.D.

Thorough Examination of a Wittig-Horner Reaction Using Reaction Calorimetry (RC-I), LabMax, and ReactIR

The kinetics and thermodynamics of a Wittig-Horner reaction for the production of a stilbene derivative was investigated by S. Zamir and M. Grabarnick (Organic Process Research & Dev., 2003, 7, 237) by using automated laboratory reactors RC-1 and LabMax in combination with ReactIR and statistical Design of Experiments. It was found that the water content in the reaction mass has a crucial effect on the rates both of the unwanted hydrolysis of the phosphonate and of the main reaction. The reaction was found to be fourth-order. This means that the reaction is very fast at the start but trails off toward the end. Based on the kinetic and thermodynamic parameters, a simulation of the whole process was made, including the expected concentrations of the product under the required conditions. The experimental values of the concentrations were obtained from spectra collected via a rapid reaction mode in the ReactIR (a spectrum was collected app. every 3 s). Exact agreement between experimental and theoretical values confirms the correctness of the mathematical model and enables right planning of the industrial process.

Traceless Liquid-Phase Synthesis of Piperazinediones

Construction of heterocyclic ring systems through combinatorial synthesis on polymer support promises to generate potential lead molecules rapidly. Recently, solid-phase synthesis has gained acceptance throughout the pharmaceutical industry as a powerful tool to assist in the identification and development of therapeutic agents. Now, liquid-phase combinatorial synthesis (LPCS) of piperazinedions using soluble polymer support to generate libraries has been reported by Chung-Ming Sun et al. (Com. Chem. & HTS, 2003, 6, 133).

Proline anchored polyethylene glycol monomethyl ether underwent dipeptide formation with different Fmoc-amino acids in the presence of dicyclohexyl carbodiimide. Deprotection of Fmoc, accompanied with the cleavage from polymer support with cyclization of dipetide to piperazinediones, offers a facile and effective way to prepare diverse combinatorial libraries. Excellent yields and purities were achieved by a simple wash and precipitation method.

Development of a Custom High-Throughput Preparative Liquid Chromatography/Mass Spectrometer Platform for the Preparative Purification and Analytical Analysis of Compound Libraries

Solution-phase parallel synthesis has had a profound impact on the speed of compound synthesis, delivering relatively pure compounds (>80%) in short order. However, to develop structure activity relationships (SAR) for a compound series, each library member should preferably be >95% pure. Historically, achieving and quantifying such high-purity criteria for each library member proved to be the rate-limiting step for most lead discovery groups. To address this issue, W. Leister and colleagues from Merck Research Laboratories made significant modifications to a commercial Agilent 1100 preparative LC/MS system to allow for the general mass-guided purification of diverse compound libraries (J. Comb. Chem., 2003, 5, 322). With their modified system, they achieve excellent mass recoveries (70–95%) and purities (>95%) in a single chromatographic run with a capacity to purify app. 6 samples/hour.

High-Performance Thin-Layer Chromatography Method for Assessment of the Quality of Combinatorial Libraries, and Comparison with Liquid Chromatography-Ultraviolet-Mass Spectrometry

A high-performance thin-layer chromatography (HPTLC) method was developed by R. K. Kostiainen et al. (University of Helsinki) for fast evaluation of the purity of solid-phase synthesis products (J. Comb. Chem., 2003, 5, 223). The results obtained were in agreement with results obtained by the LC-MS or the LC-UV-method, confirming the suitability of HPTLC for purity analysis of combinatorial syntheses. The synthesis products can be quantified and identified by measuring UV densitograms or in situ UV spectra or by ESI-MS after isolation of the zone of interest. A new, simple, and fast method for transferring the zone of the analyte from the plate to the ESI-MS equipment is described. The new HPTLC method enables rapid and efficient analysis of app. 40 samples per hour in parallel, compared to app. 3–10 samples per hour by using LC-MS. As such, HPTLC offers a less expensive and easier way to analyze the purity of synthesis products than the commonly used LC-UV-MS.

High-Throughput Analysis in Catalysis Research Using Novel Approaches to Transmission Infrared Spectroscopy

Anne Leugers et al. from The Dow Chemical Company demonstrated recently that high-throughput FTIR transmission measurements using a newly designed array-based support of silicon wells and a silicon wafer is a very useful and robust tool for the characterization of polymer composition for combinatorial materials research (J. Comb. Chem., 2003, 5, 238). The comonomer content in copolymers can be measured accurately with a fully automated throughput of more than 300 samples/day (8 h). The transmission measurement is more robust, reliable, and easier to automate than other spectroscopic methods. The support itself provides excellent resistance to aggressive organic solvents at elevated temperatures and allows the unattended deposition and preparation of polymer films for infrared analysis. This high-throughput approach to infrared transmission spectroscopy can be used for measuring a wide array of polymer characteristics like vinyl content, geometrical isomers, crystallinity, and tacticity.

Micro-X-ray Fluorescence as a General High-Throughput Screening Method for Catalyst Discovery and Small Molecule Recognition

Combinatorial chemistry has revolutionized the synthesis of compounds on solid supports, but the major bottleneck of this process is the reliable and rapid screening of desired properties of resin-bound components. Whether it is for the combinatorial development of new drugs, catalysts, or molecular receptors, the activity and selectivity of library components have to be determined quickly and reliably. A powerful high-throughput technique for the rapid on-bead screening of libraries for catalyst discovery and molecular recognition was developed by George J. Havrilla and his colleagues from the Chemistry Division of Los Alamos National Laboratory (J. Comb. Chem., 2003, 5, 245). They used micro-X-ray fluorescence (MXRF) to screen materials for elemental composition with mesoscale analysis. This method is nondestructive and requires minimal sample preparation and no special tags for analysis. The speed, sensitivity, and simplicity of MXRF as a high-throughput screening technique was demonstrated in the screening of bead-based libraries of oligopeptides for phosphate hydrolysis catalysts and molecular recognition of selective receptors for the degradation products and analogues of chemical warfare agents. Depending on the desired sensitivity, the current system has the capability of screening up to 3200 library components in one hour.

Two-photon Microscopy to Spatially Resolve and Quantify Fluorophores in Single-Bead Chemistry

For a better understanding of solid-phase synthesis in automated chemistry, it is highly desirable to describe the distribution of chemical groups within the solid-phase supports. R. V. Ulijn et al. (Universities of Edinburgh and Strathclyde, Polymer Laboratories Ltd.) have now reported the use of two-photon microscopy (TPM) for the direct quantification of fluorophore distributions within single PEGA₁₉₀₀ (poly(ethylene glycol)acrylamide) beads (J. Comb. Chem., 2003, 5, 215). Within the common size and loading range of beads used in combinatorial chemistry, the two-photon method gives reliable quantification of fluorophores present. Attenuation effects can be corrected for by taking into account an exponential drop in fluorescence with depth. Individual PEGA₁₉₀₀ beads were found to have a homogeneous distribution of chemical groups within each bead.

An Improved Method for Rapid Sequencing of Support-Bound Peptides by Partial Edman Degradation and Mass Spectrometry

Peptide libraries have found widespread applications and are usually synthesized and screened on the solid phase in the one-bead-one-peptide format. This results in a large number of positive beads that need to be individually characterized, requiring a rapid and inexpensive method for high-throughput sequencing of library-derived peptides. Several methods have been used for this purpose, including conventional Edman sequencing and others, but all of these methods have some disadvantages that compromise their utility in the routine high-throughput sequencing of library-derived peptides. D. Pei and M. C. Sweeney (Ohio State University, Columbus) developed an improved method for the partial Edman degradation by using N-hydroxysuccinimidyl esters as capping agents (J. Comb. Chem., 2003, 5, 218). This provides a highly reliable, sensitive, inexpensive, and rapid method for sequencing support-bound peptides derived from combinatorial libraries. This new sequencing strategy might have the potential to become the method of choice in the sequence determination of combinatorial peptide libraries in the future.

A High Repetition Rate (I KHz) Microcrystal Laser for High-Throughput Atmospheric Pressure MALDI-Quadrupole-Time-of-Flight Mass Spectrometry

Time-of-Flight mass analyzers are now considered standard hardware for bioanalytical mass spectrometry. In conventional MALDI-TOF MS, the low repetition rate of the nitrogen laser (337 nm) limits the duty cycle and overall throughput. John A. McLean and his co-workers developed an application of a 1-kHz repetition rate Nd:YAG laser (355 nm, <500-ps pulse widths) for atmospheric pressure MALDI-QqTOFMS that leads to a signal enhancement up to factor 80 and high-quality data in seconds (Anal. Chem., 2003, 75, 648).

In comparison with low-rate repetition lasers (1–30 Hz), the new one provides a significant improvement in sample throughput while retaining a high level of data quality in terms of sensitivity and mass accuracy. Furthermore, the high repetition rate leads to a nearly continuous ion beam, making it ideally suited for MALDI sample introduction for orthogonal TOF hybrid instruments.

Structural Biology and Drug Discovery: Astex, Structural Genomix, and Syrrx

The structure elucidation of proteins plays a crucial role in modern structure-based drug discovery and for the rational design of new small molecule inhibitors. Because crystallization and structure determination of proteins is, in many cases, still a bottleneck in the drug discovery process, a lot of effort is put into achieving a higher throughput. In a very interesting article, V. Mountain (Chemistry & Biology, 2003, 10, 95) presents an overview of the three pioneers in the field of high-throughput applications and structural biology: Astex, Structural Genomix, and Syrrx. All three of these companies have adapted X-ray crystallography for the fast-paced biotech world in similar ways but with some unique differences. Syrrx has highly automated high-throughput crystallography. Using a parallel fermentation system and the Sonic Hedgehog robot, Syrrx can make and purify 100 proteins in 5 h. The purified protein is then moved to the reengineered Agincourt crystallization robot, which can accurately generate 100 nL crystallization droplets, enabling the structural information to be obtained from very small amounts of protein. Structural Genomix has adopted a modular approach to their highly automated protein crystallization technology to enable expression, purification, and crystallization conditions to be precisely controlled for each particular protein family of interest. Protein crystals are then analyzed with the company's proprietary third-generation beamline synchrotron at the Advanced Photon Source in Chicago, which can use very small crystals due to its extremely narrow beam. Astex's core focus is on fragment-based drug discovery, using 24/7 high-throughput X-ray cocrystallography to identify critical fragments with trade-marked technologies known as Autosolve and Pyramid.

Despite the differences in their scientific strategies and technologies, all three companies share a common vision: that structure based drug design will have a strong impact on the future of drug discovery.

Absolute Quantification of the G Protein-Coupled Receptor Rhodopsin by LC/MS/MS Using Proteolytic Product Peptides and Synthetic Peptide Standards

Methods for the absolute quantification of a membrane protein are described using isotopically labeled or unlabeled synthetic peptides as standard. The new method presented by David R. Barnidge and his co-workers (Anal. Chem., 2003, 75, 445) has a much shorter analytical cycle time (minutes vs. hours for the global proteome experiments), making it well suited for high-throughput environments. The present approach uses synthetic peptides as standards in conjunction with proteolytic or chemical cleavage of target proteins for which no standard is available. The quantification of the important G protein-coupled receptors (GPCRs) was demonstrated with rhodopsin as a model membrane protein that shows this method is simple and reliable using LC/MS/MS triple quadrupole mass spectrometer.

Measuring Intracellular Calcium Fluxes in High-Throughput Mode

The measurement of intracellular calcium fluxes in real time is widely applied within the pharmaceutical industry. The development of new calcium-sensitive dyes and assays provides sensitive, homogeneous assays that can be readily applied to high-throughput screening (HTS). Chambers et al. (Comb. Chem. & HTS, 2003, 6, 355) describe the full automation of this assay type using a fluorometric imaging plate reader (FLIPR™) integrated into a Beckman/Sagian system. A carousel and CO₂ incubator have been incorporated for compound plate and cell plate storage, and a Biomek FX and a Labsystems Multidrop dispenser have been used as liquid handling systems for plate reagent addition and calcium indicator dye addition. Plate washer, lidding station, barcode reader, and tip storage hotels complete the setup of the system.

The system was used to screen 891787 compounds for identifying novel agonists of a GPRC. Identified were 7711 activities; 3617 compounds showed reproducible activity. The described FLIPR assay meets quality and robustness criteria for screening. A significant ongoing cost for FLIPR-based screenings is cell culture with its expensive consumables and its high demands on staff. Automated cell culturing reduces the necessary manual input but is only cost effective in larger companies because of the high costs of equipment.

For cost reduction, assay volumes can be further reduced in full-size, 384-well plates with standard liquid handling equipment. Further throughput increases are possible in 1536-well plates but require adaptation of the FLIPR software.

Long-Term Stimulation of Mouse Hippocampal Slice Culture on Microelectrode Array

To understand the central nervous system, simultaneous multisite recording and stimulation have become extremely helpful. Van Bergen et al. (Brain Research Protocols, 2003, 11, 123) have further developed an innovative approach, recording from intact neural networks using planar micro-electrode arrays (MEAs) with 60 nano-columnar electrodes. To allow for long-term stimulation, mouse hippocampal tissue slices were immobilized onto MEAs and permanently moved in a specifically designed tilting incubator that made it possible to electrically contact up to 90 MEAs with 5400 electrodes simultaneously. The system enables stimulation over extended periods of hours and days without the need to interrupt the culture. Stimulation was done over periods of about 20 min with 20 sweeps (bipolar, biphasic rectangular pulses of about 1 V, 300 or 700 μs duration).

The time required to run the total protocol is from 9 days up to several weeks, depending on the desired long-term culture period and the length of long-term stimulation.

Electronic Laboratory Notebooks: Product or Process?

Currently, researchers spend many hours per week capturing various data types in paper format and combining them for summary documents and project reports. This data diversity is driven by the fact that vendors and other suppliers create unique (and competitive) hardware and software solutions to the scientific marketplace. Electronic laboratory notebooks are one way to minimize the administrative burdens of everyday laboratory work. John P. Helfrich reports about recent developments on the properties and requirements of e-laboratory notebooks and explains the enhancements of this process (New Drugs, 2003, 3, 28). Key components to a process-based e-laboratory notebook include the capturing and cataloging of all data directly from source. These sources can be analytical devices (HPLC or mass spec) or spreadsheets and word-processing programs performed by the researchers themselves. The data have to be time and date stamped with full audit trail documentation. In addition, access to all stored data must be controlled and limited to authorized personnel. The development of software platforms is necessary to provide a secure method for automated archiving and retrieving raw data within short timeframes. Another capability request is the possibility of browsing the database by sorting and filtering report data.