Automation Highlights from the Literature

Kerstin Thurow, Ph.D.

Hilmar Weinmann, Ph.D.

Laboratory Automation and High-Throughput Chemistry

Advances in Solution- and Solid-Phase Synthesis Toward the Generation of Natural Product-Like Libraries

There is a growing interest in the use of small molecules as chemical probes in parallel to classical genomic tools (gene expression, gene profiling, gene knockouts, and siRNA-based gene silencing, etc.) to understand biological functions. Small-molecule probes have the ability to modulate macromolecules, such as proteins, DNA, RNA, or carbohydrates, in a controlled, selective, and nondestructive manner. In addition to using them as chemical probes to understand biological function, these small molecules offer an excellent starting point for launching drug discovery programs and could be further developed as therapeutic candidates. In this regard, the development of natural product-inspired, high-throughput organic synthesis programs could be useful. Several examples of generating natural product-like compound libraries that use the diversity-oriented synthesis and the biology-oriented synthesis are discussed in a review by P. Arya et al. Both of these programs are aimed at populating the unexplored natural products-based chemical space that is currently unoccupied by conventional combinatorial chemistry (Chem. Rev. 2009, 109, 1999–2060).

Visualization of High-Dimensional Combinatorial Catalysis Data

The role of various techniques for visualization of high-dimensional data is demonstrated by K. Rajan et al. in the context of combinatorial high-throughput experimentation. Applying visualization tools, the authors identify that constituents of catalysts are associated with final products in a huge combinatorially generated data set of heterogeneous catalysts, and catalytic activity regions are identified with respect to pentanary composition spreads of catalysts. A radial visualization scheme directly visualizes pentanary composition spreads in two-dimensional space and catalytic activity of a final product by combining high-throughput results from five libraries. For catalyst discovery and lead optimization, this work demonstrates how large multidimensional catalysis data sets are visualized in terms of quantitative composition activity relationships to effectively identify the relevant key role of compositions (i.e., lead compositions) of catalysts (J. Comb. Chem. 2009, 11, 385–392).

High-Throughput Experimentation Platform: Parallel Microwave Chemistry in HPLC/GC Vials

A high-throughput reaction platform for performing parallel microwave chemistry in sealed HPLC/GC vials is described by M. Damm and C. O. Kappe. The system consists of a strongly microwave-absorbing silicon carbide plate with 20 cylindrical wells of appropriate dimensions fitted with standard HPLC/GC autosampler vials serving as reaction vessels. In combination with an aluminum sealing plate, the setup can be used for microwave processing reaction volumes from 0.5 to 1.5 mL at a maximum temperature/pressure limit of 250 °C/20 bar. The parallel reaction platform displays excellent temperature and reaction homogeneity and has been used for high-throughput reaction optimization studies involving the parallel screening of catalyst, solvent, and substrate reactivity for esterification reactions and metalcatalyzed dehydrative C–C couplings (J. Comb. Chem. 2009, 11, 460–468).

Parallel Solid-Phase Synthesis and High-Throughput ¹H-NMR Evaluation of a 96-Member 1,2,4-Trisubstituted-Pyrimidin-6-One-5-Carboxylic Acid Library

A solid-phase organic synthesis method has been developed by B. C. Hamper et al. for the preparation of trisubstituted-pyrimidin-6-one carboxylic acids, which allows elaboration to a three-dimensional combinatorial library. The scope of the method for library production is determined by investigation of a 3 × 4 pilot library of 12 compounds. A library is prepared using 8 aldehydes, 3 nitriles, and 4 amines to give a full combinatorial set of 96 pyrimidinones (J. Comb. Chem. 2009, 11, 469–480).

One-Pot Multicomponent Synthesis of 1-Aryl-5-Methyl-N-R2-1H-1,2,3-Triazole-4-Carboxamides: An Easy Procedure for Combinatorial Chemistry

A convenient synthetic protocol is described by N. T. Pokhodylo et al. for creation of combinatorial libraries of 1-(R1-phenyl)-5-methyl-N-R2-1H-1,2,3-triazole-4-carboxamides. As starting materials, commercially available or readily prepared azides, amines, and diketene are selected for a one-pot system reaction with high yields in a short time (J. Comb. Chem. 2009, 11, 481–485).

Controlled Microwave Heating in Modern Organic Synthesis: Highlights from the 2004–2008 Literature

Direct and rapid heating by microwave irradiation in combination with sealed vessel processing in many cases enables reactions to be carried out in a fraction of the time generally required by conventional conditions. This makes microwave chemistry an ideal tool for rapid reaction scouting and optimization of conditions, allowing very rapid progress through hypotheses–experiment–results iterations. C. O. Kappe and D. Dallinger discuss in a review more than 220 published examples of microwave-assisted synthetic organic transformations from the 2004 to 2008 literature. In addition ca. 500 reaction schemes are presented in the electronic supplementary material, providing an overall number of ca. 930 references in this fast-moving and exciting field (Mol. Div. 2009, 13, 71–193).

Microfluidic Chip Technology and Microreactor Technology

Microreactors for Radiopharmaceutical Synthesis

Multiple advantages of microfluidics have been demonstrated in the area of organic synthesis, but only a limited number of them have found applications in radiopharmaceutical synthesis. The need in this area is to create an environment where all reactions involving short-lived radioisotopes such as 18F (110 min half-life) or 11C (20 min half-life) are rapid and high yielding, while the devices are controlled remotely. Several groups have identified the potential of microfluidics in this area and have demonstrated that various steps of conventional radiosynthesis can be replaced by microfluidic devices. A. M. Elizarov reviews the technologies reported to date and analyzes the unmet needs that will have to be addressed before microfluidic technology has a chance of becoming a viable and truly advantageous method of preparation of commercial radiopharmaceuticals (Lab Chip. 2009, 9, 1326–1333).

Sandwich Mixer-Reactor: Influence of the Diffusion Coefficient and Flow Rate Ratio

Mixing in microfluidic devices poses some difficulties, as flows in such a small scale are mostly of laminar nature. Turbulence disappears and only diffusion between two solutions remains. Passive micromixing relies on geometrical or surface effects.

Bending the flow to enhance convective effects by inducing transverse components of flow, achieved by applying a t-junction, twisting the channel, or placing flow resistance in the channel, increases the mixing effect. A technique where only pure molecular diffusion remains as the only mixing mechanism is a flow splitting technology where fluids are divided into several streams and rejoined together after mixing is complete.

In a sandwich mixer fed with two solutions, a central laminar flow is surrounded by two outer flow solutions. Abonnenc et al. report that the reactor extent at the end of the sandwich mixer increases by 60% if the species with the lowest diffusion coefficient is located in the outer solution, where the flow velocity is small compared with that of the central part (Lab Chip. 2009, 9, 440–448). An even higher increase of 300% is achieved by decreasing the outer flow rate and simultaneously increasing the local concentration to keep the flux ratio.

Effective Mixing in a Microfluidic Chip Using Magnetic Particles

Rapid mixing in microfluidic channels is desirable, but difficult to realize due to laminar flows. S.W. Lee et al. present a novel active mixing method in a microfluidic chip where the controlled stirring of magnetic particles is used to achieve effective mixing in fluids (Lab Chip. 2009, 9, 479–482). The developed chip is fabricated using standard multilayer soft lithography and consists of two layers. The bottom layer, with a Y-shaped channel system, contains the mixing chamber. The top layer has pneumatic channels and valves to control the fluid. The incorporated barriers are located in the three channels around the mixing chamber to keep the beads in place, except for the particle solution inlet channel. The used ferromagnetic particles retain their magnetism and stay in aggregated form even in the absence of the magnetic field. Because of the coating with carboxyl groups, subsequent surface modifications or attachment of biological materials are possible. The used particles are biocompatible and do not contaminate the solutions. Therefore, this mixing method can be applied to various biological fields including glucose sensor, PCR, and continuous flow fast reaction kinetic study.

A Microfluidic Droplet Generator Based on a Piezoelectric Actuator

For biological and chemical applications, droplet-based microfluidic systems are a field of research that is gaining interest. S. Levenberg et al. from Biomedical Engineering (Technion, Haifa, Israel) describe a simple and robust droplet generator based on a piezoelectric actuator that is integrated into a microfluidic device (Lab Chip. 2009, 9, 516–520). After a brief introduction to the field of dropletbased microfluidics, detailed descriptions of device fabrication and test settings are presented. Practical investigations focus on a highly uniform single drop generation and are examined on the basis of different inlet to main channel ratios, piezoelectric pulse length experiments, flow rate variations, and pressure scenarios of the microfluids. The method turns out to be the most suitable for biological applications because no electric field or heating is used, which normally damages cell membranes or causes protein denaturation. Moreover, a piezo-driven generator can easily be integrated into any droplet-based microfluidic device using inexpensive commercially available actuators.

Modular Reconfigurable Robot Gripper for Limp Material Handling in Garment Industries

The design of special purpose grippers for handling limp materials in garment and textile industries is an interesting and emerging research area in robotics. An article depicts increasing competitiveness along with better quality standardization and process visibility as the main reasons for this trend.

The most challenging problem to applying robotic automation in cloth manufacturing industries is the proper grasping and transportation of fabric materials. After sketching the history in garment industry automation, the authors describe present technologies as admittedly effective for solving handling problems, but frequently inflexible as they use specific disposals and techniques. Because garment industries especially have to comply with continuously changing requests from different customers to possess high production flexibility, a new reconfigurable robotic gripper based on pneumatic technology has been developed.

This end effector, mounted on an industrial robot with 6 degrees of freedom, provides several pickup points, whose locations are selected based on the shape of the part to be grasped. A vision detection system analyzes the object, and gathered pose information is used to control the movement of the suction cups (pickup points). When a contact force appears between the gripper and the object surface, the system actuates the vacuum cups and grasps the fabric.

As the basic step in designing the gripper, the article outlines the dynamic simulation and analysis of the reconfigurable gripper system by means of the simulation package ADAMS. The developers put value in keeping the ADAMS Model physically realistic, dynamically stable, and modular to preserve the possibility of changing factors, such as the lengths of different parts. Finally, the article describes the dynamic Mock-Up Testing of the new approach and depicts the results of a simulation of grasping a shirtfront.

In conclusion, a reconfigurable mechatronic gripper is presented, described, and conceived to grant robust and smooth grasping, firm hold and reliable transfer capability of limp and soft materials. The structural modularity and the easy frame reshaping of the arm position allow this prehensor concept's application to manufacturing fields other than garment industry (Int. J. Robot. Autom. 2008, 23, 213–219).

High-Throughput Analytics

Bioanalysis Without Sample Cleanup or Chromatography: The Evaluation and Initial Implementation of Direct Analysis in Real-Time Ionization Mass Spectrometry for the Quantification of Drugs in Biological Matrices

Sample cleanup and chromatography in pharmaceutical bioanalysis, especially in high-throughput analysis, are often bottlenecks in a process. Yu et al. present the development, evaluation, and initial implementation of direct analysis in real-time (DART) ionization mass spectrometry for the direct and quantitative determination of pharmaceutical compounds in biological matrices (Anal. Chem. 2009, 81 (1), 193–202). A DART ion source is coupled with a triple-quadrupol mass spectrometer over an interface involving a novel gas ion separator (Vapur interface) that is developed. The Vapur interface works with helium as ionization gas and enhances the sensitivity of the system. An automated-sample introduction device is developed to fulfill high-throughput requirements. The DART/MS/MS system is evaluated for analytical aspects, such as reproducibility, sensitivity, linearity, matrix effects, precision, and accuracy. Different applications in pharmaceutical bioanalysis are investigated, for example, in vivo and in vitro ADME (absorption, distribution, metabolism, and excretion) studies. The comparison with classical analytical measurements, such as LC/MS/MS, shows similar results. The DART/MS/MS system enables a direct analysis of samples in biological matrices without sample cleanup or chromatography. Therefore, it is an efficient instrument for high-throughput and real-time bioanalysis.

Matrix Assisted Laser Desorption Ionization (MALDI) Mass Spectrometry Imaging, from its Origins Upto Today: The State of the Art

MALDI mass spectrometry imaging (MALDI MSI) is a MS technique with great potential in high impact fields, such as clinical proteomics, preclinical studies, and medical diagnosis. Francese et al. deliver insight into general principles of this exciting technique (Comb. Chem. High Throughput Screen. 2009, 12 (2), 156–174). Instrumental, technical, and experimental details, such as image resolution, spatial resolution, and analysis time, are described. Different applications in the fields of proteomics, drug discovery, lipids, and single cell analysis are explicated in detail. In addition to sample preparation, a further important aspect for profiling and imaging mass spectrometry concerns the matrix. In this article, the nature of the molecules, types of tissues, and matrix deposition methods subject to experiment type (profiling or imaging) are discussed. MALDI MSI is a MS technique with a number of very interesting features. On tissue, MALDI MSI enables acquisition of molecular distributions of several different analyte classes, such as protein, peptides, lipids, carbohydrates, and organic molecules, such as drugs. Some parameters will require optimization in the future. For example, better sample preparation as well as faster data acquisition and analysis are greatly needed. MALDI MSI has serious potential as a discovery tool, and in the future this technique could become a routine method in clinical diagnostics and drug discovery.

Detection of Honeybee Venom in Envenomed Tissues by Direct MALDI MSI

Hymenoptera venom can trigger fatal allergic reactions in humans. The investigation of the venom of European honeybees (Apis mellifera) is an interesting research field. Honeybee venom (HBV) consists of a wide spectrum of biomolecules containing biogenic amines, peptides, and protein. Francese et al. describe a new analytical approach to provide molecular maps of HBV in envenomed tissues using direct MALDI mass spectrometry imaging (MALDI MSI) over time (J. Am. Soc. Mass Spectrom. 2009, 20 (1), 112–123). The composition of the HBV is additionally analyzed with MALDI time of flight (TOF) MS and LC–ESI high-resolution MS. The diffusion and the distribution of three venom allergens (Api m 1, Api m 4, and Api m 6) and two venom toxins (apamine and mast cell degranulating peptide) in envenomed tissues are investigated with MALDI MSI. Therefore, in vitro analysis using honeybees and porcine ears as well as in vivo analysis using honeybees and rats are performed. Molecular mass images of the venom distribution are obtained for all biomolecules. This analytical approach shows the potential of MALDI MSI for further studies of venom action and organism response with a view to developing and testing new venom immunotherapy.

Bioautomation and Screening

A Multiplexed Approach to Hit Finding

Assay formats and screening technologies can deliver more than one read-out per measurement and therefore can be considered to be information rich. This holds true for biophysical and selected biochemical assays, and in particular for cellular assay formats where the term “high-content assay” describes the most complex and advanced paradigm of small-molecule screening available to date. Given the multifactorial nature of the lead generation and optimization process in drug discovery, the enhanced information density offered by multiplex screening technologies is considered to be beneficial to an informed hit selection for optimization. T. Hesterkamp et al. claim that although hard evidence for an enhancement in hit and lead selection is only now emerging, multiplexed screening technologies will fulfill their promise in drug discovery if the understanding of target pharmacology and the complexity of biological systems can be advanced in parallel (Curr. Opin. Drug Disc. Develop. 2009, 12, 351–357).

Diel Growth Cycle of Isolated Leaf Discs Analyzed with a Novel, High-Throughput Three-Dimensional Imaging Method is Identical to That of Intact Leaves

It is a long known fact that cuttings from plant leaves, so-called leaf discs, can grow as rapidly as intact leaves. This observation has been exploited in leaf disc assays for different investigations such as the analysis of effects of hormones or agrochemicals on plant growth. Yet, the use of leaf disc assays to decipher the control of leaf growth dynamics in HTS has been limited, because precise and automatic detection of leaf disc area is difficult. The most important shortcoming of two-dimensional projection and analysis depends on the inclination of the leaf discs. Their growth rates can be estimated only with substantial errors due to different problems. For example, when the leaf discs are tilted or strongly curved, aberrations of the surface area measurements appear. This also happens when the liquid level decreases in the course of an experiment. In this case, the measurement values decrease solely because the object distance increases. To overcome these problems, the authors implement a stereoscopic system to analyze the growth of the three-dimensional (3D) surface area of leaf discs. This system, called GROWSCREEN 3D, consists of a camera mounted to a scaffold that allows movements in three axes. For each leaf disc grown in 24-well plates, two pictures are taken at different positions. These images are segmented, and the leaf surface area is computed after stereo matching and 3D reconstruction. With this system, the authors are able to measure single leaf discs in less than 12 s with high precision. Furthermore, they show that this system is not limited to the measurement of particular species, but is applicable to a wide range of plants because they demonstrate with the use of tobacco and Arabidopsis. In the discussion, the authors make several suggestions for improvement of their system concerning the hardware and the software. Nevertheless, it can be concluded that the invention of GROWSCREEN 3D provides feasible solutions for the high-throughput analysis of growth measurements of leaf discs (Plant Physiol. 2009, 149, 1452–1461).

A Small Molecule That Directs Differentiation of Human ESCs into the Pancreatic Lineage

Human embryonic stem cells (hESCs) have a virtually endless replicative capacity and have the potential to differentiate into most cell types. One possible application is the stepwise differentiation from hESCs to functional insulin-secreting beta cells for cell replacement therapy for diabetics. Shuibing Chen et al. of Harvard University in Cambridge, MA, generate a cell population that is a key milepost on the path to making beta cells. As an alternative to using coculture with other cell types and/or growth factors as inducers, they develop a high-content screen using a chemical library of 5000 compounds to find cell-permeable small molecules that can increase the number of Pdx1-expressing cells derived from hESCs. In this way, they identify (–)-indolactam V as a molecule which, when combined with growth factors, can direct the differentiation of hESCs such that >45% of the cells become Pdx1-expressing pancreatic progenitor cells. Some of these Pdx1-expressing cells form insulin-expressing cells after transplantation into the kidney capsule of nude mice. In addition, Chen et al. find that (–)-indolactam V might differentiate hESCs through Ca²⁺-dependent protein kinase C activation.

The study shows that high-content chemical screening is a powerful tool as a means to control in vitro differentiation of hESCs. Remaining challenges include finding small molecules for the efficient induction of cells at each step of the differentiation pathway and studies to determine which cell stage is optimal for transplantation (Nat. Chem. Biol. 2009, 5 (4), 258–265).