Abstract
Laboratory Automation and High-Throughput Chemistry
Diaryl Ether and Diaryl Thioether Syntheses on Solid Supports via Copper (I)-Mediated Coupling
An efficient method for synthesizing diaryl ethers and thioethers on solid supports is described by S. Bräse and N. Jung. Starting with immobilized phenols or arylhalides and coupling with aryliodides/arylbromides or phenolic/thiophenolic substrates in solution is successful with CuCl and Cs2CO3 as a base. Coupling conditions known from solution-phase syntheses of diaryl ethers have been effectively modified and adapted to solid-phase synthesis. Optimized conditions enable the coupling of sterically hindered and electronically deactivated aryl moieties. A newly developed diversity-generating linker based on cinnamic acid allows the diaryl ethers to be cleaved from the resin either via saponification/transesterification or via ozonolysis. The target substances are generally isolated in good to excellent yields and high purities (J. Comb. Chem.
Parallel Synthesis of 2-Aryl-4-aminobenzimidazoles and Their Evaluation as Gonadotropin-Releasing Hormone Antagonists
2-Trifluoromethyl-4-aminobenzimidazoles were previously identified by screening to be active antagonists of the gonadotropin-releasing hormone receptor (GnRH-R). D. M. Green et al. discuss structure activity relationships and diversity-oriented synthesis. 2-Substituted benzimidazoles are synthesized in parallel by the coupling of carboxylic acids with a latent intermediate diamine monomer to yield the desired benzimidazoles in fair yields. A catch and release strategy is used as a product isolation technique, followed by RP-HPLC to obtain products of desired purity for biological evaluation. Two libraries are prepared and screened to determine the optimal substitution for inhibitory activity against GnRH-R. The initial library focuses on substituted phenyl, pyridine, and thiophenes. The follow-up library focuses on substitution patterns observed in the initial library members, and generates compounds with IC50 values lower than 100 nM at the GnRH-R (J. Comb. Chem.
Microfluidic Chip Technology and Micro-Reactor Technology
Novel, Transient Response Technique for Heterogeneous Catalysis in Liquid Phase with Electro Spray Ionization (ESI)
Heterogeneous catalysis is important in areas of production, such as bulk chemicals, fine chemicals and pharmaceuticals. Due to multi-step synthesis procedures and the use of stoichiometric reduction and oxidation, less efficiency of production of fine chemicals and pharmaceuticals compared with bulk chemicals is a well-known circumstance. Efficient and selective catalysts to improve these processes are required. Aimed molecules are complex and have limited thermal stability; therefore, most reactions require solvents and run in liquid phase.
To model such a catalyst, a real-time analysis of the output of a reactor was developed by Radivojevic et al. (2008). By coupling an ESI-MS to the exit of a reactor, they achieve direct online monitoring of catalytic experiments in liquid phase. Using the ability of ESI-MS to distinguish between different compounds, they take advantage of the most significant difference between existing transient analyzing methods.
The setup is verified at two model reactions; nitrite reduction with Pt/SiO2 and glucose oxidation with Pt/carbon nitro-fibers/Ni. The catalytic reactor is fed with one pulse, and a free flow of reactant is placed in an oven and controlled to hold a temperature of 60 °C.
In a nutshell, Radivojevic et al. have developed a method for real-time, semi-quantitative monitoring of reactants, intermediates, and products, especially for heterogeneous catalysis (J. Catalysis
Microfluidic Crystallization
Microfluidics offers a wide range of new tools that permit one to revisit the formation of crystals in solution and yield insight into crystallization processes. J. Leng and J.B. Salmon review recent microfluidic devices with particular emphasis on lab-on-chips dedicated to the HTS of crystallization conditions of proteins with nanoliter consumption. They also thoroughly discuss the possibilities offered by microfluidic tools to acquire thermodynamic and kinetic data that may improve industrial processes and shed a new light on nucleation and growth mechanisms (Lab on a Chip
High-Throughput Analytics
Improving the Signal Intensity and Sensitivity of MALDI Mass Spectrometry by Using Nanoliter Spots Deposited by Induction-Based Fluidics
The applicability of matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) for biological studies is often limited by the small quantities of sample, and hence, the impossibility of increasing the signal intensity by increasing the sample amount. In their article, Tu et al. (Center for Biomedical and Bioorganic Mass Spectrometry, Washington University of St. Louis, Missouri, USA) describe a new contact-free, small droplet deposition method using an induction-based fluidics (IBF) technique for MALDI-MS to overcome this obstacle (J. Am. Soc. Mass Spectrom.
The approach using micro-dispensing to produce smaller and more concentrated spots is motivated by the concept of creating “hot spots” for enhanced sensitivity and productivity. IBF transports liquids by inductively charging them, and allows the manipulation of sub-microliter drops without using any moving parts. With ionic-liquid matrices, the authors report a signal enhancement that is nearly proportional to the concentration enhancement, comparing conventional (500 nL) and nanoliter (50 and 20 nL) spots that contain equal amounts (0.1–1.25 pmol) of the analyte bradykinin. Furthermore, multiple spotting of nanodroplets at the same position permits an “on-plate” concentration enhancement. As expected, the signal intensities of a single spot and a multiple spotted sample, both with the same amount of analyte, are very similar. In comparison with an adequate normal-sized drop, 25-fold greater signal intensity is achieved. The nanoliter spotting with conventional solid matrices, like α-cyano-4-hydroxycinnamic acid, shows homologues improvement. At this, the additional enhancement is attributed to a “depth effect” caused by the smaller surface area of the IBF spotted drops.
Development of a Palm Portable Mass Spectrometer
Portable chemical analyzers have been of great interest for the identification of a vast variety of chemical substances in the field. M. Yang and colleagues from Analytical Instrumentation Research Institute (Sam Yang Chemical Company, Seoul, Korea) have developed a palm portable mass spectrometer (PPMS) with a weight of 1.48 kg and a size of 1.58 liter that can be operated with an average battery power of 5 W (J. Am. Soc. Mass Spectrom.
The main PPMS hardware consists of an ion-trap assembly, a vacuum chamber, an ion-getter pump, a power supply for the ion-getter pump, an air inlet, a pulsed vacuum valve, and an initiation vacuum valve for pre-evacuation. All accessory technical components are discussed in detail, too. The ionization is obtained with a standard electron gun (rhenium filament), and the scan mass range is variable up to m/z 300 with a mass resolution of three mass units. After programming the PPMS with a PC and a pre-evacuation with a roughing pump, the instrument can be operated for 10 h using a battery of 3000 mAh. Furthermore, even after a shutdown of 3 days, the ion-getter pump alone is able to restart the system. Further developments of the PPMS for prospective applications in other areas, such as environmental sciences, homeland security, and space exploration, are discussed at large.
Bioautomation and Screening
New Connections Across Pathways and Cellular Processes: Industrialized Mutant Screening Reveals Novel Associations between Diverse Phenotypes in Arabidopsis
It is widely known that HT approaches are not limited to the fields of drug discovery and white biotechnology. These techniques enter into many other areas, among them different parts of plant biology. HT analyses led to the sequencing of the first genome of a higher eukaryotic organism, the mouse-ear cress Arabidopsis thaliana, and were also essential for setting up large repositories of clones, microarray data, and mutants from different model and crop plants. These partially publicly available resources are nowadays indispensable for progress in any field of plant research, but in the future, HT approaches will not be limited to particular fields.
These techniques in plant biology also will apply to other areas of research. A study by Y. Lu et al. is an interesting example of this development. In this report, the authors analyze 13 different Arabidopsis mutants biochemically and phenotypically. The biochemical analyses are performed with different LC, GC, and MS methods to determine the contents of free amino acids, fatty acids, starch, and chlorophyll in different organs. One of the results of these data is several new phenotypes that are discovered in particular mutants. In a further approach, statistical and bioinformatic calculations are performed with the data sets to reveal novel connections and associations between different metabolic and catabolic pathways. As one conclusion, Y. Lu et al. show that parallel analyses of plant germplasm-like mutants or ecotypes harbor enormous potential for plant biology, especially for plant breeding. The exploitation of this potential depends on the application of HT methods, especially of automated ones. Such methods have either to be improved or even to be invented in the field of plant research to cover the future needs for crop plants (Plant Physiol.
In Silico Activity Profiling Reveals the Mechanism of Action of Antimalarials Discovered in a High-Throughput Screen
Malaria is still one of the most widely distributed serious diseases worldwide. It threatens 40% of the world population, of which 500 million currently are ill and at least one million are dying as a consequence of infection, mostly in sub-Saharan Africa. There is only one drug therapy available to fight malaria, the artemisinin-based combination therapy. The lack of alternatives to this medication, together with growing parasite resistance, increases pressure for the discovery of next generation compounds of antimalarial drugs. Because demand for these drugs is centered in over-populated and poor regions of the world, new drugs must be efficient and inexpensive.
As one approach, D. Plouffe et al. initiated a HTS of 1.7 million compounds to measure their antimalarial activity. As an assay system, they use erythrocytes infected with the parasite Plasmodium falciparum, the causative organism of malaria, and stain them with the dye SYBR green after compound application. This dye fluoresces when bound to DNA, and due to the lack of nuclei in red blood cells, the fluorescence correlates to the number of parasites.
The performance of the described assay in 1536-well format at a speed of 25,000 compounds per week resulted in 17,000 primary hits. After verification, the substances are first clustered based on chemical similarity. Using this approach, about 530 different scaffold classes are identified. Only 5% of them reveal similarities to known drugs, whereas the vast majority of scaffolds are new. Furthermore, 8457 compound hits are chosen for which data from 131 cellular assays are available and clustered according to their behavior. This approach, called “guilt by association,” which works analogous to the interpretation of gene expression data, unravels 31 classes of compounds with common assay patterns. One of these classes with 26 substances attracts special interest because it includes several antifolates acting on dihydrofolate reductase, which might be a novel antimalarial drug target.
The reported results are more proof of the power of the combination of uHTS and creative data mining for the discovery of new drugs. The number of yet unknown substances and structures found in this screen give hope for the future development of novel drugs against malaria and other widespread diseases (Proc. Natl. Acad. Sci. U. S. A.
Port-a-Patch and Patchliner: High Fidelity Electrophysiology for Secondary Screening and Safety Pharmacology
During the past decade, ion channel screening platforms have emerged to enable HT drug screening from a more functional perspective. N. Fertig et al. describe an entry-level device for automated patch clamping–the Port-a-Patch, which is claimed to be the world's smallest patch-clamp workstation. Another fully automated system is the Patch-liner, Nanion's higher throughput patch-clamp workstation. The system uses a robotic liquid handling environment for fully automated application of solutions, cells and compounds (Combinat. Chem. High-Throughput Screen.
High-Throughput Electrophysiology with Xenopus Oocytes
Voltage-clamp techniques are typically used to study plasma membrane proteins, such as ion channels and transporters, that control bioelectrical signals. Many of these proteins have been cloned and can now be studied as potential targets for drug development. The two approaches most commonly used for heterologous expression of cloned ion channels and transporters involve either transfection of the genes into small cells grown in tissue culture, or injection of genetic material into larger cells. The standard large cells used for the expression of cloned cDNA or synthetic RNA are the egg progenitor cells (oocytes) of the African frog, Xenopus laevis. Until recently, cellular electrophysiology was performed manually by a single operator, one cell at a time. However, methods of HT electrophysiology have been developed and automated, permitting data acquisition and analysis from multiple cells in parallel. These methods are breaking bottlenecks in drug discovery, and in some cases, have become useful for primary screening as well as for thorough characterization of new drugs. Increasing throughput of high-quality functional data greatly augments the efficiency of academic research and pharmaceutical drug development. R. L. Papke and C. Smith-Maxwell describe the main features and potential applications of OpusXpress, an efficient commercially available system for automated recording from X. laevis oocytes (Comb. Chem. High Throughput Screen.
Automated Planar Electrode Electrophysiology in Drug Discovery: Examples of the Use of QPatch in Basic Characterization and High-Content Screening
Planar chip technology has greatly facilitated progress toward fully automated electrophysiological systems that, in contrast to traditional patch-clamp technology, have the capability of parallel compound testing. The throughput has been increased from testing below 10 compounds per day to a realized capacity approaching HT levels. Many pharmaceutical companies have implemented automated planar chip electrophysiology in their drug discovery processes, particularly at the levels of lead optimization, secondary screening, and safety testing, whereas primary screening is generally not performed. P. Christophersen et al. discuss this technology and give examples from selected ion channel drug discovery programs, where one of the systems, the QPatch, has been evaluated for use in lead optimization and primary screening campaigns when high information content is a requirement (Comb. Chem. High Throughput Screen.
The Chiptip: A Novel Tool for Automated Patch Clamp
To facilitate automated patch-clamp measurements of ion channels in cells, the development of an all-glass Chiptip pipette that may be combined with the previously described Flip-the-Tip technology is reported by A. Lepple-Wienhues et al. A single measurement requires less than 50 cells, and the addition of drugs for screening can be limited to very low volumes down to 1 μL. This apparatus is suitable for the study of small cells, subcellular organelles, and bacteria (Comb. Chem. High Throughput Screen.
QPatch: The Missing Link between HTS and Ion Channel Drug Discovery
The conventional patch clamp has long been considered the best approach for studying ion channel function and pharmacology. However, its low throughput has been a major hurdle to overcome for ion channel drug discovery. The recent emergence of higher throughput, automated patch-clamp technology begins to break this bottleneck by providing medicinal chemists with high-quality, information-rich data in a more timely fashion. Therefore, these technologies have the potential to bridge a critical missing link between HT primary screening and meaningful ion channel drug discovery programs. One of these technologies, the QPatch automated patch clamp system, records whole-cell ion channel currents from 16 or 48 individual cells in a parallel fashion. C. Mathes et al. review the general applicability of the QPatch by studying a wide variety of ion channel types (voltage-/ligand-gated cationic/anionic channels) in various expression systems (Comb. Chem. High Throughput Screen.
Use of Planar Array Electrophysiology for the Development of Robust Ion Channel Cell Lines
The tractability of ion channels as drug targets has been significantly improved by the advent of planar array electro-physiology platforms that have dramatically increased the capacity for electrophysiological profiling of lead series compounds. However, the data quality and throughput obtained with these platforms is critically dependent on the robustness of the expression reagent being used. The generation of high-quality, recombinant cell lines is, therefore, a key step in the early phase of ion channel drug discovery and this can present significant challenges due to the diversity and organizational complexity of many channel types. J. J. Clare et al. discuss several complex and difficult to express ion channels, and illustrate how improved stable cell lines can be obtained by the integration of planar array electrophysiology systems into the cell line generation process (Comb. Chem. High Throughput Screen.
Sodium Channel Inhibitor Drug Discovery Using Automated High-Throughput Electrophysiology Platforms
Voltage-dependent sodium channels are widely recognized as valuable targets for the development of therapeutic interventions for neuroexcitatory disorders, such as epilepsy, pain, and cardiac arrhythmias. An ongoing challenge for sodium channel drug discovery is the ability to readily evaluate state-dependent interactions, which are known to underlie inhibition by many clinically used local anesthetic, antiepileptic, and antiarrhythmic sodium channel blockers. Although patch-clamp electrophysiology is still considered the most effective way of measuring ion channel function and pharmacology, it does not have the throughput to be useful in early stages of drug discovery where there is often a need to evaluate many thousands of compounds. Over the past five years, there has been significant progress in developing much higher throughput electrophysiology platforms, including the PatchXpress and Ion-Works, which are now widely used in drug discovery.
N. Castle et al. review the strengths and weaknesses of these two HT devices for use in sodium channel inhibitor drug discovery programs. Overall, the PatchXpress and Ion-Works electrophysiology platforms have individual strengths that make them complementary to each other. Both platforms are capable of measuring state-dependent modulation of sodium channels. IonWorks has the throughput to allow for effective screening of libraries of tens of thousands of compounds, whereas the PatchXpress has more flexibility to provide quantitative voltage clamp, which is useful in structure activity evaluations for the hit-to-lead and lead optimization stages of sodium channel drug discovery (Comb. Chem. High Throughput Screen.
3D Cell Culture Opens New Dimensions in Cell-Based Assays
3D cell culture technologies have revolutionized the understanding of cellular behavior, both in culture and in vivo, but adoption by cell-based screening groups has been slow, owing to problems of consistency, scale, and cost. The evolving field of high-content screening technologies requires a rethinking of 3D cell culture adoption to ensure the next generation of cells provide relevant in vivo-like data. Three current technologies are presented in a review by B. A. Justice et al.—membranes, sponges/gels, and microcarriers. A short history of these technologies and unique research applications are discussed, and the technologies are evaluated for usefulness in modern automated cell-based screening equipment (Drug Discov. Today
Information Technology
Comparability of Data in Process and Laboratory
In process automation, there is standardization for equipment and data. This also is required for laboratory automation. Laboratory information systems (LIMS) collect and organize information, but they are mostly proprietary solutions that do not communicate with production departments.
Profibus is the world's most popular fieldbus, and is unique in offering a fully integrated solution for discrete and process applications. The basic functions, controls, and communication services are defined in standardized device profiles. The Profibus user organization was the first of the different major fieldbus organizations to specify a device profile that is especially dedicated to the requirements of laboratory equipment.
An overview of the device profile “LabDevices” is presented by Jecht et al. (CIT plus
Data Sharing in Distributed Databases and Peer-to-Peer Databases
Content-sharing systems on the Web have renewed interest in the design and development of decentralized database management systems. The need for large-scale data sharing between autonomous, and possibly heterogeneous, decentralized systems on the Web gives rise to the concept of peer-to-peer (P2P) database systems. An article by A. Bonifati et al. provides insight to this new P2P data management technology with a comparison of more established decentralized models, commonly referred to as distributed, federated, and multi-databases. The goal is to clarify some of the essential differences and similarities from a data management point of view.
A database system (DBS) is software that manages one or more databases. A distributed database system (DDBS) is software that manages one or more logically related databases, spanning a network. Both a federated data base system (FDBS) and a multi-database system (MDBS) are collections of pre-existing DBSs in which operations can be applied to multiple heterogeneous component DBSs in a coordinated manner. The authors distinguish between DB-centric and P2P-centric features. The various DBS are characterized along the three dimensions of distribution, autonomy, and heterogeneity.
The most important features of P2P paradigm are scalability in terms of the number of nodes and distribution; direct access to the data at the source to guarantee freshness in contrast to centralized repositories; robustness and resilience against attacks and churn by exploiting self-organization principles; and simplified deployment because resources from the “edge” of the Internet can be used and no special infrastructure is required to join the network.
A P2P database system (PDBS) is conceived as a collection of autonomous local repositories that interact in a peer-to-peer style to establish correspondences, or exchange query and update requests. The local repositories are autonomous peers with equal rights, and are linked to only a small number of neighbors. The main data integration and interoperability idea in peer data management is to avoid a global schema by providing mappings between pairs of information sources. Mappings between all pairs is not necessary. An export schema contains only the elements of the local schema that a peer wants to share with the outside world. Open and challenging issues in P2P data management are anonymity, security, and access control problems (SIGMOD Record
Integration of LIMS and SDMS
Using Starlims as an example, G. Koberg presents a combination of two tools and their advantages for laboratory workflows. All scientific and laboratory information are integrated in one application. The advantages are the assurance of data and intellectual property, increased availability, prevention of double searches, and targeted information management within a company. SDMS are solutions for the capture and management of data, such as instrument files, reports, standard operating procedures (SOPs) in compliance with regulations. The processes are divided into four steps—capture, analysis, workflow management, and evaluation. Direct data capture and reporting is possible in the LIMS workflow. The files are managed in the SDMS. Innovative algorithms of artificial intelligence for automated processing of unstructured documents are used to extract content from documents and save it in a structured format (unified XML). Keywords, tables, or figures are identified and extracted automatically. Using the workflow, manager allows the management of documents in compliance with regulations. Electronic signature and an audit trail can be integrated. Another important function is the efficient extraction and use of information using searches based on keywords and metadata. Data from any device type with a known format can be captured. This system is advantageous for R&D projects in interdisciplinary teams. All relevant documents and information are in a central solution and compose an interconnected image of a project. Comprehensive knowledge management can be realized (GIT
Take Leave of Time-Based Data Storage
For historians logging process data with a timestamp as a process value in files or databases, as the amount of data rises, the need for extended analyses and more complex information about the data rises, making time-based data storage very extensive and complicated. (Küppers et al, Automation & Drives
An alternative is the Multi-Dimensional Data Logging (MDDL). Data from several sources are collected in addition to the time stored. In this way, a relationship between any information and any process data can be established. For example, a correlation can be made between process data and order data. An important method for the estimation of a production process is the determination of key performance indicators. These factors present economic success in reference to guidelines depending on the company or a goal and the degree of convergence. Overall equipment effectiveness is the performance of an engine, the standstill, and the produced quantity. This information can be retrieved directly from an MDDL database. MDDL databases build the backbone of a complete analysis system for the simple and effective storage of production coherences. Analysis and reporting tools allow the simple generation of reports, evaluations, and analysis that lead to new knowledge about coherences of several parameters.