Automation Highlights from the Literature

Automated Synthesis of Heterocycles on Solid Supports

The creation of novel diverse heterocycle libraries is an indispensable requirement of modern drug discovery processes. S. Braese et al. discuss in a review the recent advances in the automated solid-phase syntheses of heterocycles to generate libraries of bioactive products and illustrate library sizes that are obtained, robots used for production of libraries, points of diversity, and number of steps on the resin (Curr. Opin. Drug Discov. Devel. 2006, 9(6), 713–728).

Lab Automation in Support of Safer Process Design for HWE Reaction

A scaleable process technology by D. Levin et al. for a Horner–Wadsworth–Emmons reaction uses the basic calorimetry capability of the MultiMax automated reactor system. This system affords some interesting insights regarding scale-up safety of the original process and helps improve alternative chemistry more suitable for safe scale-up (Organic Process Res. Dev. 2006, 10, 1296–1298).

Microwave-Assisted, Solid-Phase Synthesis of a Chiral 1,2,3,4-Tetrahydroquinoline Library

A. Silvani et al. explain an efficient route to a small library of chiral, highly functionalized tetrahydroisoquinolines. Both loading on a Merrifield resin and the key acid-catalyzed Pictet–Spengler condensation are efficiently promoted by microwave irradiation. The library is designed so that up to five points of diversity can be introduced, making the strategy in principle suitable for the generation of a large number of compounds (Comb. Chem. High Throughput Screen. 2006, 9, 691–701).

Microwave-Assisted Solid-Phase Synthesis of 2,5-Diketopiperazines: Solvent and Resin Dependence

M. Grotli et al. describe the solid-phase synthesis of diketopiperazines (DKPs) by using various combinations of resins (polystyrene, TentaGel, ArgoGel, and PEGA) and solvents (toluene, tert-butyl alcohol, water, and toluene/2-butanol). The DKPs are synthesized from solid-phase bound dipeptides via intramolecular aminolysis. Both thermal and microwave-assisted solid-phase synthesis of DKPs generate high yields of products independently of resin and organic solvent used; however, only the PEGA resin results in high yields of DKPs in water independent of heating method. The short reaction times, high yields, and the possibility to run reactions in water when an appropriate resin is used makes the microwave-assisted solid-phase synthesis the method of choice. The method should be suitable for solid-phase synthesis of diketopiperazine-based libraries (J. Comb. Chem. 2006, 8, 915–922).

Automation of Fluorous Solid-Phase Extraction for Parallel Synthesis

W. Zhang and Y. Lu share an automatic fluorous solid-phase extraction (F-SPE) technique by using FluoroFlash SPE cartridges on the RapidTrace workstation. A 10-module workstation has the capability to complete a maximum of 100 SPEs in 1–2 h. Another important feature of the Rapid-Trace system is that it has the capability to load slurry samples onto the F-SPE cartridges. The F-SPE cartridge charged with 2 g of fluorous silica gel can be used to purify up to 200 mg of crude sample. Sample loading, elution solvent, cartridge reuse, and SPE reproducibility are evaluated. The automatic SPE system is used for purification of a small urea library generated from amine-scavenging reactions using fluorous dichlorotriazine, a 96-membered amide library is generated using 2-chloro-4,6-bis[(perfluorohexyl)propyloxy]-1,3,5-triazine as the coupling agent and another 96-membered library is generated from fluorous Mitsunobu reactions. Approximately 90% of the products have >90% purity after F-SPE (J. Comb. Chem. 2006, 8, 890–896).

Synthetic Applications of Fluorous Solid-Phase Extraction (F-SPE)

W. Zhang and D. Curran review the basic concept and synthetic applications of fluorous solid-phase extraction as well as automation equipment used for these methods of reaction work-up and extractions (Tetrahedron 2006, 62, 11890–11896).

Minimization of Palladium Content in Suzuki Cross-Coupling Reactions

A protocol for conducting Suzuki reactions in a plate format amenable to use in library synthesis of biaryl compounds is illustrated by H. Tye et al. A key objective is to determine reaction conditions that result in biaryl products containing <10 ppm of Pd for a wide variety of building blocks. A Design of Experiments approach for the identification of an optimized set of reaction conditions is successfully applied. The utility of the protocol is demonstrated by preparation of an array of 96 biaryl compounds in a parallel manner (Comb. Chem. High Throughput Screen., 2006, 9, 703–710).

Further Exploration of Antimicrobial Ketodihydronicotinic Acid Derivatives by Multiple Parallel Syntheses

A synthetic re-examination of a series of ketodihydronicotinic acid class antibacterial agents is undertaken by L. A. Mitscher et al. in an attempt to improve therapeutic potential. A convenient new synthesis involving hetero Diels-Alder chemistry produces 74 new analogs in a multiple parallel synthetic manner, and these are examined in vitro for antimicrobial potential. Several compounds demonstrate significant broad-spectrum activity against clinically derived bacterial strains, but previously known 1-(2,4-difluorophenyl)-6-(4-dimethylaminophenyl)-4-pyridone-3-carboxylic acid remains the most potent compound in this class (Comb. Chem. High Throughput Screen. 2006, 9, 663–681).

Efficient Route to Benzo[4,5]imidazo [2,1-a]phthalazines

Benzo[4,5]imidazo[2,1-a]phthalazines (BIPH) are obtained from various o-nitrophenylhydrazines through different 2-(2-nitrophenyl)-1,2-dihydro-1-phthalazinones as intermediates using an elaborated advanced procedure (Viktor A. Kuznetsov et al., Tetrahedron 2006, 42(62), 10018–10030).

An activated chlorine atom in 2-nitrophenyl moiety of the latter is able to undergo nucleophilic substitution for secondary alicyclic amines yielding novel substituted phthalazinones. Their one-pot reduction and cyclodehydration yield a series of novel substituted benzo[4,5]imidazo[2,1-a]phthalazines. Substituents in five and nine positions may be varied in a wide range, and an amino group may be introduced in 10 positions. All the steps of the BIPH synthesis are reliable, result in high yields, and require only simple laboratory implementation.

Phenylsilane as an Active Amidation Reagent for the Preparation of Carboxamides and Peptides

The use of phenylsilane as a mild coupling reagent for amidation reactions is reported by Zheming Ruan et al. (Tetrahedron Lett. 2006, 43(47), 7649–7651). Applicability to both solution- and solid-phase chemistry is demonstrated for a variety of amines and carboxylic acids.

The scope of this reaction is first examined with a series of diverse carboxylic acids and amines. A 77-compound array is constructed from a matrix of seven carboxylic acids crossed with 11 amines and anilines in solution. An excess of PhSiH₃ (3 equiv) is necessary to drive the coupling reaction to completion. To explore the extension of these coupling conditions to solid-phase, two representative acid resins are prepared from 4-formyl-3-methoxyphenoxy methyl resin and amino methyl esters, (NH₂R₁COOMe) by reductive amidation, acylation, and deprotection. The acid resins are then tested with representative amines under conditions of 10 equiv of amines and 20 equiv of PhSiH₃. Further investigations to explore the scope and limitations of this transformation are ongoing.

Pharmaceutical Industrial Experiments on Continuous Cryogenic Reactions Using Mini-Sized Multistage Reactors

Masahiro Takasuga et al. describe investigations in the field of mini-sized multistage reactors (J. Chem. Eng. Jpn. 2006, 39(7), 722–776). Microreactor technology is becoming important in the fine-chemical and pharmaceutical industries. Therefore, research on microreactors has become well established, and it was reported that 50% of all reactions conducted in the fine-chemical and pharmaceutical industries could benefit from continuous processes based on microreactor technology. The frequent presence of a solid phase in these reactions, however, hinders the widespread application of microreactors, which tend to be easily blocked with solid deposits. However, a minireactor has been developed that has several attributes of microreactors, such as fast mixing and good heat transfer. Cryogenic conditions will be used in the pharmaceutical industry for organometallic reactions and for the stereoselective synthesis of active pharmaceutical ingredients. They are required for these reactions because of the thermal instability of the organometallic compound. A further point for using these conditions is to improve the selectivity of the reaction.

This paper focuses on continuous cryogenic organic reactions using mini-sized multistage reactors. The reactors have a volume in the range from 10⁻⁶ m³ to 10⁻³ m³. They have many advantages, such as high heat exchange and efficient and fast mixing comparable to microscale reactors. Mini-sized reactors, however, are easier to operate than microscale reactors. In this paper, two successive organometallic reactions are used to synthesize the spirolactone fragment of neuropeptide Y using a mini-sized reactor. Successive cryogenic organometallic reactions are studied using a milli-sized reactor. Improved heat removal resulted in a 10% product yield increase compared with the pilot-scale batch process. In addition, forced mixing and heat transfer affect both product yield and selectivity, decreasing by-product and undesirable cis-product generation.

Stabilization of a CSTR in an Oscillatory State by Varying the Thermal Characteristics of the Reactor Vessel

Investigations on the stabilization of a continuous stirred tank reactor in an oscillatory state are reported by Luis F. Razon (Int. J. Chem. Reactor Eng. 2006, 4, Note S1).

The problem of a first-order exothermic reaction in a diabatic continuous stirred tank reactor is often investigated in chemical engineering. In this article, the author considers an extension of this classic problem in which the reactor vessel temperature varies independently of its contents. Through a bifurcation analysis, the author shows that a continuous stirred tank reactor in an oscillatory state may be stabilized. This is made possible by using a reactor with the appropriate combination of thermal characteristics.

Stabilization of an oscillatory state is demonstrated for a variety of scenarios that had been previously shown by Uppal, Ray, and Poore to be representative cases of phase diagrams that exhibit limit-cycle behavior.

This article demonstrates that, under a varied set of conditions, it is possible to stabilize a reactor in an oscillatory state by simply changing the thermal characteristics of the reactor. Reactor designers would thus be well-advised to consider the dynamics of the reactor vessel when designing their reactors.

Synthesis of SiO₂ Nanoparticles in RF Plasma Reactors: Effect of Feed Rate and Quench Gas Injection

Effects of the synthesis of SiO₂ nanoparticles in a RF plasma reactor are reported by Behnam Mostajeran Goortani et al. (Int. J. Chem. Reactor Eng. 2006, 4, Article A33). Radio frequency thermal plasma processes are effective techniques for the production of high quality metallic and ceramic materials in the nanometric size range. These processes involve a complex sequence of transport phenomena, thermodynamics, and chemical kinetics.

Nanoparticles of SiO₂, which have been produced in an inductively coupled thermal plasma reactor, are characterized based on their morphology and size distribution.

The authors offer experimental and numerical simulation results on the synthesis of SiO₂ nanoparticles, created in a pilot-scale RF induction plasma reactor. The overall process of silica nanoparticle synthesis in a radio frequency reactor is studied experimentally and numerically. Good agreement between the experimental and the simulation results are observed. The exact control of the feed rate in the plasma reactor plays an important role in generating aggregates of the desired size and morphology. The equivalent particle diameters varied from 37 nm to 65 nm (without quench). Moreover, by injection of quench, this equivalent diameter suddenly decreased to 25 nm. The powder consisted mostly of aggregates in the size range of 1–4 μm with a peak at 2.5 μm. In the absence of quench, there are no significant differences in size of the aggregates prepared under different feeding conditions in the range of the powder feeding rates used.

With the quench used, as a result of higher temperature gradients, the equivalent diameters of the primary particles decrease to 25 nm, while, as a result of the higher fluid recirculation, higher levels of aggregation are observed.

The simulation results are further used to provide more explanation for the phenomena taking place in the reactor (e.g., temperature contours and velocity profiles). Reducing the temperature to the boiling point of silica in the zone around the plasma flame results in a minimum value of the diameter of particles deposited on the reactor wall.

Multiphase Microfluidics: From Flow Characteristics to Chemical and Materials Synthesis

K. F. Jensen and A. Guenther review transport characteristics of pressure-driven, multiphase flows through micro-channel networks tens of nanometers to several hundred micrometers wide with emphasis on conditions resulting in enhanced mixing and reduced axial dispersion. Dimensionless scaling parameters for characterizing multiphase flows are summarized along with experimental flow visualization techniques. Static and dynamic stability considerations also are included along with methods for stabilizing multiphase flows through surface modifications. Observed gas–liquid and immiscible liquid–liquid flows are summarized in terms of flow regime diagrams and the different flows are related to applications in chemistry and materials synthesis (Lab Chip 2006, 6, 1487–1503).

Fully Automated Continuous-Flow Synthesis of 4,5-Disubstituted Oxazoles

A multipurpose mesofluidic flow reactor capable of producing gram quantities of material is presented by S. V. Ley et al. as an automated synthesis platform for rapid, on-demand synthesis of key building blocks and small exploratory libraries. The reactor is configured to provide the maximum flexibility for screening of reaction parameters that incorporate on-chip mixing and columns of solid supported reagents to expedite chemical syntheses (Org. Lett. 2006, 8, 5231–5234).

A Flow Reactor Process for the Synthesis of Peptides Utilizing Immobilized Reagents, Scavengers and Catch and Release Protocols

A general flow process for the multistep assembly of peptides is shared by S. V. Ley et al., and this procedure is used to successfully construct a series of N-protected dipeptides in excellent yields and purities, including an extension of the method to enable the preparation of a tripeptide derivative (Chem. Commun. 2006, 4835–4837).

Addition of Secondary Amines to α,β-Unsaturated Carbonyl Compounds and Nitriles by Using Microstructured Reactors

Several additions of amines to α,β-unsaturated carbonyl compounds (Michael additions) are performed by H. Loewe et al. in a continuous flow microstructured reactor rig and compared to the respective batch reaction. Dimethylamine/diethylamine/piperidine and acrylic acid ethyl ester/acrylonitrile are used as two sets of reactants, resulting in six reactions. Some of these reactions are highly exothermal. Using the traditional batch procedure, the olefin must be added slowly to the diluted amine to ensure temperature control and safe operation. Good yields (>85%) can be achieved by this method. Processing time, however, is very long (17–25 h). To reveal the intrinsic kinetic potential and thus to accelerate these reactions, the reactants are mixed in a continuous-flow microstructured reactor rig that allows rapid mixing and efficient removal of the reaction heat. Reaction time is decreased to a few seconds up to about half an hour, which is a change by 2 orders of magnitude. While the yields achieved with the continuous-flow microstructured reactor rig match those for the batch procedure, the throughput for the microflow processing is much higher; in the best case by a factor of about 650 (Organic Process Res. Dev. 2006, 10, 1144–1152).

Synthesis of β-Peptides in a Microreactor

P. H. Seeberger et al. describe the first application of a silicon microreactor for the synthesis of peptides. They demonstrate that reaction conditions can be rapidly optimized and larger amounts of the peptide can be produced. In addition, this paper describes the first peptide coupling reactions of Boc- and Fmoc-protected amino acids within 1–5 min at an unusually high temperature of 120 °C, the use of β²- and β³-homo amino acid fluorides for the peptide coupling reaction, and the successful application of a C₁₀H₄F₁₇-substituted benzylic ester protecting group for peptide synthesis in solution (Angew. Chem. 2006, 118, 7157–7160).

Applications of Microfluidics in Chemical Biology

D. B. Weibel and G. M. Whitesides discuss in a review the application of microfluidics in chemical biology. An introduction to microfluidics is given and characteristics of microfluidic systems that are useful in studying chemical biology are described. The review concludes with an assessment of future directions and opportunities of microfluidics in chemical biology (Curr. Opin. Chem. Biol. 2006, 10, 584–591).

Addition of Secondary Amines to α,β-Unsaturated Carbonyl Compounds and Radical Polymerization Using Microflow System: Numbering-up of Microreactors and Continuous Operation

J. Yoshida et al. share a microchemical pilot plant for radical polymerization of methyl methacrylate by numbering up eight microtube reactors. Continuous operation is accomplished for 6 days without any problems. These results indicate that microflow systems can be applied to relatively large-scale production of polymers (Organic Process Res. Dev. 2006, 10, 1126–1131).

Enhanced Performance Test Mix for High-Throughput LC/MS Analysis of Pharmaceutical Compounds

LC/MS is being used for the routine analysis of small molecules in both the discovery and development stages within the pharmaceutical industry. In drug discovery, LC/MS is relied upon to confirm the identity and assess the purity of chemical entities. To ensure the quality of LC/MS analysis, it is important that the LC/MS system is operating within defined performance criteria. Performance monitoring of a system with a standard compound mix offers many advantages over other alternatives, because it monitors the LC/MS system as an integrated unit under the same working conditions as those used for the analysis of samples. It is also a convenient approach, because the test mix can be injected as part of the automated sequence. W. Goetzinger et al. select a set of eight compounds from a collection of 137 commercially available “druglike” compounds. This mixture has a balanced selection of pKa values and covers the typical range of hydrophobicity and molecular masses of pharmaceutical compounds. Furthermore, the selected compounds can generally be ionized using ESI and APCI modes with positive and negative polarity. Performance monitoring with the enhanced mix is demonstrated with respect to ionization and mass measurement, as well as changes in gradient profile, flow rate, buffer pH, and ionic strength (J. Comb. Chem. 2006, 8, 820–828).

Nanoliter High-Throughput Quantitative PCR

The understanding of biological complexity arising from patterns of gene expression requires an accurate and precise measurement of RNA levels across large numbers of genes simultaneously. Nowadays, real time PCR (RT-PCR) in a microtiter plate is the preferred method for quantitative transcriptional analysis. Nevertheless, even in 384 format, this process is limited by cost and logistic considerations. In a recent publication, Morrison et al. (Nucleic Acids Res. 2006, Vol. 00, No. 00 e1–9 doi:10.1093/nar/gkl639) refer to a nanofluidic system for performing solution-phase RT-PCR, sufficient for scaling RT-PCR to higher throughputs. Here, the authors describe a hybrid approach that combines accuracy, precision, and dynamic range of RT-PCR with the parallelism of a microarray in an array of 3,072 real time, 33 nl PCRs in the size of a microscope slide. To assess the performance of the system with a biologically relevant example, they construct an array with primer pairs targeting expression of 508 human kinases, 13 endogenous (housekeeping) controls in quadruplicate, and 208 negative controls for analyzing four samples per array. The results demonstrate that this novel procedure is equivalent with an accuracy and high replicate precision to the same assay in a 384-well microplate, but in a 64-fold smaller reaction volume, a 24-fold higher analytical throughput, and a workflow compatible with standard microplate.

A High-Throughput Mammalian Protein Expression, Purification, Aliquoting, and Storage Pipeline to Assemble a Library of the Human Secretome

In the post-human genome-sequencing era, the availability of recombinant proteins has become crucial for the identification of proteins with therapeutic potential. Based upon bioinformatic coding predictions of the genes for putative secreted proteins, T. Battle et al. establish a high-throughput protein pipeline (HTPP) for the production of a subset of the human secretome. The HTPP is based on a transient expression system in HEK293-EBNA cells at 100–500 mL culture scale, combined with an automated affinity purification procedure targeting >75% purity. This is followed by semi-automated protein sample logistics to provide biologists with quality-controlled and 96-well formatted protein aliquots amenable to cell-based assays. Over a 4-year period, more than 7,500 transfections are performed representing more than 2,200 registered proteins, at an average production of 280 proteins/month with a peak production of 320 proteins/month. All these proteins are tested in more than 50 different cell-based assays. The major process steps and the optimization required to maintain novel protein production while supporting both stock replenishment and scale-up productions are described in this article (Comb. Chem. High Throughput Screen., 2006, 9, 639–649).

Orphan Nuclear Receptors Excellent Targets of Drug Discovery

One of the most challenging areas of drug discovery is the search for novel receptor–ligand pairs. Nuclear receptors comprise a large superfamily of ligand-dependent transcription factors that regulate the expression of genes critical for a variety of biological processes. Orphan nuclear receptors, for which the ligands are not yet identified, represent the most ancient component of the nuclear receptor superfamily. Orphan nuclear receptors offer a unique system to uncover novel signaling pathways that affect human health, and they also provide excellent targets of drug discoveries for a variety of human diseases. Y. Shi highlights in a review, advances made in ligand identification for orphan nuclear receptors using transgenic mouse models, cell-based screening, direct binding, structure-based assays, and computer-aided virtual screening (Comb. Chem. High Throughput Screen., 2006, 9, 683–689).

A Chemical Genetic Screen for Cell-Cycle Inhibitors in Zebrafish Embryos

Chemical genetic screening is an effective strategy to identify compounds that alter a specific biological phenotype. As a complement to cell line screens, multicellular organism screens may reveal additional compounds. The zebrafish embryo is ideal for small molecule studies because of its small size and its ease of waterborne treatment. L. I. Zon et al. examine a broad range of known cell-cycle compounds in embryos using the mitotic marker phospho-histone H3. The majority of the known compounds exhibit the predicted cell-cycle effect in embryos. A 16 320-compound library for alterations of pH3 is screened. This screen reveals 14 compounds that had not been previously identified as having cell-cycle activity despite numerous mitotic screens of the same library with mammalian cell lines. With six of the novel compounds, sensitivity is greater in embryos than cell lines, but activity is still detected in cell lines at higher doses. These findings demonstrate that small molecule screens in zebrafish can identify compounds with novel activity, and thus may be useful tools for chemical genetics and drug discovery (Chem. Biol. Drug Des. 2006, 68, 213–219).

Safety in IT-Systems Close to Production

The risks in IT-systems close to production, like process control systems (PLS), lab systems (CDS, LIMS), advanced control or manufacturing execution systems (MES), clearly differ from those in office environments. Office safety requirements and preventive measures are not directly transferable to production environments. Many production processes are supported by IT systems, and critically depend on whether these systems work without interruption, making availability of substantially greater importance than in an office where five minutes of down time can be tolerated.

Confidentiality is a primary safety goal for a company's office network. Likewise, in production environments, protecting information such as recipes, production results, and system safety status are important. In special areas like the pharmaceutical industry, nonrepudiation and audit ability are important.

In areas closest to processes, less is more. Every addition of hardware and software must be critically evaluated to ensure that it will not compromise the availability of the system. Firewalls, virus scans, and other typical technological safety measures can increase safety in both office and production/process environments. Choosing the most effective measures requires an analysis of the risks, definition of protective goals, and the support of standardized assessment tools. (Chemietechnik 2006, 8, 34–35).

Visualization of Microarray and Contextual Bioinformatic Data

The interpretation of microarray and other high-throughput data is highly dependent on the biological context of experiments. Standard analysis software packages often are poor at simultaneously presenting both the array and related bioinformatics data. This article presents a system that enables several related data sources to be dynamically combined while highlighting one particular feature. The goal is to visualize the high dimensional data in 2-D while combining data from several sources. Each data source is represented as a network of nodes connected by weighted edges. The power of this combined display lies in its interactive feedback and exploitation of human visual pattern recognition. The high dimensionality and high noise level of the data can obscure patterns that would be recognized easily in smaller data sets. For these reasons, visualization of the results of such experiments is difficult and requires sophisticated mathematical tools. It is often through a combination of different experimental data that scientific insight is generated, and this is made easier if all relevant information sources are synthesized in a single view. The representation of the data as a network allows the simultaneous combination of many different sources of information. The solution described is flexible in that the influence of each information source can be varied by the user. (Bioinformatics 2006, 14, 99–107).