Abstract
Laboratory Automation and High-Throughput Chemistry
Solid-Phase Synthesis of Biologically Active Benzoannelated Nitrogen Heterocycles: An Update
C. Gil and S. Bräse update their previous review in the field and summarize the literature published from 2002 describing methods for solid-phase synthesis of benzoannelated nitrogen heterocycles, motivated by the fact that these heterocycles are a pivotal element in modern drug discovery programs. The chapters on the cyclization methods for the preparation of five-, six-, and seven-membered benzoannelated rings are subdivided depending on the number of nitrogen atoms present in the ring system (J. Comb. Chem.
Simple and Economical High-Throughput Equilibrium Dialysis System
As more areas of research embrace the advantages of high-throughput strategies, simple yet reliable technology is critical to fully realize the benefits that such strategies can offer. D. Putnam et al. report a dialysis system that offers an improved high-throughput approach to purifying high molecular weight products from solutions. Constructed from materials that are commercially available, the apparatus can be affordably and easily fabricated in a standard machine shop. The system can be readily integrated with in-line automated high-throughput technology (e.g., robotic liquid and solid handling systems, 96-well plate readers), is resistant to acidic environments, creates minimal waste, and is easy to assemble. The dialysis apparatus and the validation approach described for a 96-well assay platform provides a readily transferable template upon which other multiwell assay platforms can be produced (J. Comb. Chem.
Enhancing the Reagent Selection Workflow via Real-Time Vendor Inventory
In an effort to increase the use and effectiveness of parallel array synthesis in the drug discovery process, D. S. Nirschl et al. evaluate and optimize the array synthesis workflow using lean techniques as a guiding principle. An analysis of the workflow, from reagent selection through synthesis, purification, and compound registration, identified reagent sourcing as a major bottleneck where reagent sourcing is defined as the process of acquiring appropriate array building blocks within a given budget and time frame. The authors describe their partnership with a key reagent vendor to provide access to real-time reagent inventory. The real-time data integrate seamlessly into a reagent selection application alongside non—real-time availability information. They find that access to this real-time data significantly simplifies the presynthesis portion of the array synthesis workflow (J. Comb. Chem.
Screening the Synthesis of 2-Substituted-2-Oxazolines
2-Oxazolines are well-known structural moieties that are included in a variety of complex biologically active structures and play a role as catalyst ligands and intermediates for functional compounds. In addition, 2-oxazolines serve as monomers for the synthesis of substituted poly(imine)s by cationic ring-opening polymerization. For the latter application, the feasibility of preparing new 2-substituted-2-oxazolines is investigated by U. S. Schubert et al. using an automated synthesizer. The reaction of various nitriles with 2-aminoethanol under Lewis acid catalysis is used for this purpose. Twenty-nine different substituted nitriles are selected out of more than 2000 commercial available nitriles to form the corresponding 2-oxazolines. At first, the reaction conditions are optimized for seven nitriles with regard to solvent and catalyst, including reproducibility tests in an automated parallel robot system. In the next step, the synthesis of all 29 2-oxazolines is screened in an automated parallel manner, whereby the reactions are monitored by GC—MS measurements providing novel insights into the scope of this synthesis route (J. Comb. Chem.
Practical Synthesis of 5-Aryl-3-Alkylsulfonyl-Phenol and 5-Aryl-3-Arylsulfonyl-Phenol Libraries
A straightforward and cost-efficient synthesis of 5-aryl-3-alkylsulfonyl-phenols by a sequential scaffold derivatization strategy has been developed by G. Jeges et al. The procedure is suitable for parallel synthesis of small libraries around the biphenyl privileged core having an unusual 1,3,5-substitution pattern. The synthesis is exemplified by a pilot library of 30 compounds (J. Comb. Chem.
Design and Combinatorial Synthesis of a Library of Methylenesulfonamides and Related Compounds as Potential Kinase Inhibitors
An effective parallel solid-phase route to methylenesulfonamides and amides bearing a wide variety of substituents is described by E. McDonald et al. The three key reaction steps are reductive amination of a haloheteroaromatic aldehyde onto a benzhydrylamine type polystyrene resin, sulfonamide, or amide formation, and palladium catalyzed transformation of the remaining heteroaromatic halogen. A process of virtual library design and filtering, together with solution and solid-phase optimizations, aids the preparation of several novel drug-like product classes in high purities and enables access to a variety of further useful analogs (Comb. Chem. High Throughput Screen.
Microfluidic Chip Technology and Micro Reactor Technologyz
Effective Mixing of Laminar Flows by an Ultrasonic Transducer
L. Johnsson et al. (Lab Chip.
The system evaluated by L. Johnsson et al. produces an acoustic standing wave, perpendicular to fluid flow, generated by a miniaturized piezoelectric transducer. Mixing occurs at a fluid—fluid density interface using the acoustic radiation force on the interface between two liquids with different density. Only a small density difference of 2–5% is required. The operation frequency of 10 MHz provokes only small viscous absorption losses in the fluid. The absorption coefficient increases quadratically with the driving frequency. At this low frequency resonance in the fluid occurs, generating high acoustic amplitudes and resulting in acoustic forces. Due to little energy losses, temperature in the fluid will not increase. So this system can be suitable for temperature sensitive reactions.
A Novel Pressure-Driven Piezodispenser for Nanoliter Volumes
A successful dispensing device for use in biotechnology applications requiring nanoliter volume liquid transfer is reported by S. Mcguire et al. A big drawback of current piezoelectric dispensing systems is their sensitivity against air entrainment during loading and use of the reagents. If the entrapped air bubble is of a critical size, the piezodispenser cannot provide enough acoustic energy to dispense droplets of fluid, and the system must be manually evacuated and reloaded with reagent. The authors present a dispensing architecture using air pressure as the primary driving force, controlled via a high-speed miniature solenoid valve. Unlike many existing systems that use a valve in line with constantly pressurized fluid to start and stop the dispensing action, this automated pressure-driven system is used to improve a typical piezodriven microdispenser. The resulting system is much less prone to failures resulting from air entrainment, and can dispense much higher viscosity fluids than the microdispenser alone.
The air pressure is applied to a supply reservoir via a microfluidic valve. The primary design requirement is to pressurize the reagent in the supply reservoir very quickly to overcome the surface tension and viscous forces, and then vent to ambient to end the dispensing. This requires keeping the air volume above the fluid as small as possible to reduce the capacitive effect, as well as constant. To satisfy these requirements, a custom-made piston/vial assembly is fabricated. A stepper motor linear actuator moves a piston inside the reservoir. A fiber optic sensor indicates when the reagent level is at the required height. This monitoring of the height and movement of the piston occurs between dispenses to keep the air volume constant and maintain consistent dispensing. As with more standard solenoid valve dispensers where the fluid passes through the valve, the volume that is dispensed by the presented test bed prototype has a linear relation with the valve open time duration (Rev. Sci. Instrum.
Simple, Robust Storage of Drops and Fluids in a Microfluidic Device
Drops in microfluidic can be considered as “micro-reactors.” Each drop can be seen as the site of an independent experiment. Besides processing steps like formulation, drop creation, and mixing of drop contents, weight is put on on-chip drop storage and extraction.
Boukellal et al. describe a technology that passively stores aqueous drops in a continuous stream of oil only by hydrodynamic flow. The drops are aqueous phase, whereas the continuous phase is immiscible oil. The device is based on capillary valves and changes in hydrodynamic resistance by the presence of drops; considering the fact that hydrodynamic resistance is the dominant factor influencing the choice of which channel a drop enters. It uses the fact that the presence of a drop itself increases the resistance in a channel. Advantages of these methods are minimization of the external intervention from the user and the avoidance of drop-to-drop contact. Moreover, the need of a surfactant to stabilize the drops is eliminated.
One method is to store drops that were created upstream from the storage zone. When single drops in a stream of oil flow through the device, each one is stored in small well. A second application of the same device creates and stores drops simultaneously. The only difference is the way the stream is built. Single drops in oil do not flow through the device, but a continuous fluid stream of aqueous phase surrounded by a phase of oil. Due to the hydrodynamic design, drops are created and stored the same way as in the first method. Drop extraction is as simple as reversing the direction of the flow.
Designing the hydrodynamic resistance of a channel network is a passive way to control droplet operations in micro-fluidic devices, and offers the advantages of being easy to manufacture and being robust in operation (Lab Chip.
High-Throughput Analytics
Simultaneous Determination of LogD, LogP, and pKa of Drugs by Using a Reverse Phase HPLC Coupled with a 96-Well Plate Auto Injector
For years, the physicochemical properties of drug candidates have been used to predict their in vivo pharmacokinetic behaviors. Several theories and empirical correlations have been established with the overall goal of expediting the drug candidate selection process with greater confidence and faster turnaround. P.C. Chiang and Y. Hu describe a 96-well reverse phase HPLC method that simultaneously determines LogD, LogP, and pKa values of drugs in a high-throughput mode. Determination of the LogD and LogP values of each compound are based on the octanol-aqueous partitioning behavior of the charged and noncharged species under different pH values. The advantages of this method are low sample consumption, suitability for low solubility compounds, less restriction on compound purity, potential for higher throughput, precise data, and multiple determinations in one assay (Comb. Chem. High Throughput Screen.
High-Throughput Sequencing of Peptoids and Peptide—Peptoid Hybrids by Partial Edman Degradation and Mass Spectrometry
A method for the rapid sequence determination of peptoids [oligo(N-substituted glycines)] and peptide—peptoid hybrids selected from one-bead-one-compound combinatorial libraries has been developed by D. Pei et al. In this method, beads carrying unique peptoid (or peptide—peptoid) sequences are subjected to multiple cycles of partial Edman degradation (PED) by treatment with a 1:3 (mol/mol) mixture of phenyl isothiocyanate and 9-fluorenylmethyl chloroformate (Fmoc-Cl) to generate a series of N-terminal truncation products for each resin-bound peptoid. After PED, the Fmoc group is removed from the N-terminus and any reacted side chains via piperidine treatment. The resulting mixture of the full-length peptoid and its truncation products is analyzed by matrix-assisted laser desorption ionization mass spectrometry to reveal the sequence of the full-length peptoid. This rapid, high-throughput, sensitive, and inexpensive sequencing method should greatly expand the utility of combinatorial peptoid libraries in biomedical and materials research (J. Comb. Chem.
Recent Developments in Ambient Ionization Techniques for Analytical Mass Spectrometry
A multitude of samples are investigated in their native state by mass spectrometry using ambient ionization techniques. These new ionization approaches, reported since 2004, are applied for analytics in the field of broadband, respectively, high-throughput screenings. G.A. Harris et al. illustrate the latest ambient ionization techniques and classify them in three main groups (Analyst.
Microwave-Induced Combustion Coupled to Flame Furnace Atomic Absorption Spectrometry for Determination of Cadmium and Lead in Botanical Samples
The determination of metals in biological samples is the emerging field of trace analysis. Barin et al. present a method for the determination of cadmium and lead in solid biological samples, such as aquatic plants, olive leaves, oriental tobacco leaves, pine needles, and tea (Anal. Chem.
Revisitation of the Phenyl Isothiocyanate Derivatives Procedure for Amino Acid Determination by HPLC—UV
Paintings, easels, polychrome sculptures, and parchments contain organic binders, which are used for pigment fixative and dispersing properties. Information about these organic binders and protein binders, respectively, is very important for reconstructing the working techniques and for defining a program for restoration and conservation of the works of art. Checa-Moreno et al. revisits a well-known and long used HPLC method for the determination of phenyl isothiocyanate amino acid derivates to check and adjust its characteristics to the analysis of protein binders in artistic samples (J. Sep. Sci.
Bioautomation and Screening
The Four Human γ-Aminobutyric Acid (GABA) Transporters: Pharmacological Characterization and Validation of a Highly Efficient Screening Assay
The neurotransmission mediated by γ-aminobutyric acid (GABA) in the mammalian brain is terminated by a family of four GABA transporters (GATs). Inhibition of GATs is currently used in the treatment of epilepsy, and these proteins are generally considered to be important drug targets. H. Bräuner-Osborne et al. report the first elaborate pharmacological characterization of all four human GAT subtypes. They conduct experiments in parallel in a [3H]GABA uptake assay using 14 standard GAT substrates and inhibitors. This setup enables direct comparison of the absolute values of inhibitory activities of the compounds between the different GAT subtypes. Given the potential therapeutic relevance of the individual GAT subtypes, a search for novel structures displaying selectivities for specific GAT subtypes is important (Comb. Chem. High Throughput Screen.
Development and Validation of a High-Density Fluorescence Polarization-Based Assay for the Trypanosoma RNA Triphosphatase TbCet1
RNA triphosphatases are attractive and mostly unexplored therapeutic targets for the development of broad spectrum antiprotozoal, antiviral, and antifungal agents. The use of malachite green asa readout for phosphatases is well characterized and widely used. However, the reaction depends on high quantities of inorganic phosphate to be generated, which makes this assay not easily amenable to screening in 1536-well format. The overly long reading times required also prohibit its use for screening large chemical libraries. To overcome these limitations, H. Djaballah et al. report the development of a fluorescence polarization-based assay for triphosphatases, compatible with miniaturization and fast readouts. For this purpose, they take advantage of the nucleoside triphosphatase activity of this class of enzyme to successfully adapt the Transcreener Adenosindiphosphate (ADP) assay based on the detection of generated ADP by immunocompetition fluorescence polarization to the RNA triphosphatase TbCet1 in 1536-well format. The authors also test the performance of this newly developed assay in a pilot screen of 3000 compounds and confirm the activity of the obtained hits (Comb. Chem. High Throughput Screen.
A Multistep NMR Screen for the Identification and Evaluation of Chemical Leads for Drug Discovery
A multistep nuclear magnetic resonance (NMR)-based screening assay is described by R. Powers et al. for identifying and evaluating chemical leads for their ability to bind a target protein. The multistep NMR assay provides structure-related information while being an integral part of a structure-based drug discovery and design program. The fundamental principle of the multistep NMR assay is to combine distinct 1D and 2D NMR techniques so that the inherent strengths and weakness associated with each technique are complementary to each other in the screen. By taking advantage of the combined strengths of 1D and 2D NMR experiments, it is possible to minimize protein requirements and experiment time and differentiate between nonspecific and stoichiometric binders while being able to verify ligand binding, determine a semiquantitative dissociation constant, identify the ligand binding site, and rapidly determine a protein—ligand costructure. The quality and physical behavior of the ligand is readily evaluated to determine its appropriateness as a chemical lead (Comb. Chem. High Throughput Screen.
Docking-Based Virtual Screening: Recent Developments
Virtual (database) screening (VS) of molecules promises to accelerate the discovery of new drugs and reduce costs by identifying molecules with high probabilities of binding to a target receptor. The large amount of available protein X-ray crystal structures, together with the development of more effective homology modeling techniques, has led recently to a steep increase in docking-based VS studies. This approach needs computational fitting of molecules into a receptor active site using advanced algorithms followed by the scoring and ranking of these molecules to identify potential leads. T. Tuccinardi reviews the main published docking-based VS studies developed over the last 8 years (Comb. Chem. High Throughput Screen.
High-Throughput Screening of Physicochemical Properties and in Vitro ADME Profiling in Drug Discovery
Current advances of new technologies with robotic automated assays combined with highly selective and sensitive LC—MS enable high-speed screening of lead series libraries in many in vitro assays. H. Wan and A. G. Holmen summarize state-of-the-art high-throughput assays for screening of key physicochemical properties such as solubility, lipophilicity, pKa, drug—plasma protein binding, and brain tissue binding, as well as in vitro absorption, distribution, metabolism, excretion (ADME) profiling. Two primary approaches for high-throughput screening of solubility using an automated 96-well plate assay integrated with LC—MS and a rapid multiwavelength UV plate reader are discussed. The advantages of newly developed miniaturized techniques for high-throughput pKa screening by capillary electrophoresis combined with mass spectrometry with automated data analysis flow are also addressed. Several new lipophilicity approaches other than octanol—water partitioning are critically reviewed, including a rapid liquid chromatographic retention-based approach, immobilized artificial membrane partitioning and liposome, and potential microemulsion electrokinetic chromatography for accurate screening of LogP. The sample pooling (cassette dosing) as an efficient approach for high-throughput screening of physicochemical properties and in vitro ADME profiling with emphasis on the benefit of on-line quality control is described. This cassette dosing approach has been widely adapted in drug discovery for rapid screening of in vivo pharmacokinetic parameters with significantly increased capacity and dramatically reduced animal usage (Comb. Chem. High Throughput Screen.
Minitags for Small Molecules: Detecting Targets of Reactive Small Molecules in Living Plant Tissues Using Click Chemistry
The detection and analysis of small molecules in cells is mostly a substantial problem. Despite their central function in many cellular processes, the tools for their analysis are still limited. A paper by Kaschani et al. proposes and validates a procedure for the detection of small molecules covalently bound to proteins in plants, which seems to have the potential for the analysis of such interactions in general. Proteins as targets of small molecules are often detected with the help of reporter tags like biotin or rhodamine. But large reporter tags can disturb the specificity of small molecules and their membrane permeability. As an alternative, the authors suggest the use of minitags. These are small chemical moieties (azide or alkyne) coupled to a small molecule that are inert under biological conditions. In a two-step process, the mini-tagged small molecule labels the targeted proteins in vivo, which has little influence on membrane permeability and binding specificity. Then, this complex is coupled to an alkyne- or azide-modified reporter tag through a so-called click chemistry reaction, which is a Cu1+ catalyzed Huisgen's 1,3-dipolar cycloaddition. With this highly specific reaction, the authors can label and detect proteases in vivo with mini-tagged E-64 and MG132, both low molecular cysteine protease inhibitors. This method, which was first introduced in mice, can be applied in various tissues and also is suited to identify targets of compounds like hormones, drugs, and other low molecular substances. Due to its applicability in different organisms, its high reaction specificity, and its advantages compared with big labels, the use of minitags can evolve into an attractive alternative to classical identification of protein targets (Plant J.
Selective Molecular Imaging of Viable Cancer Cells with pH-Activatable Fluorescence Probes
In cancer diagnosis, a long-term goal is the development of a tool for cancer detection and real-time monitoring of the anti-cancer therapy. Yasuteru Urano et al. from the University of Tokyo now move a step toward this goal by designing a cancer-targeting macromolecule with a small-molecule fluorescent moiety that is only activated within cancer cells. This enables them to visualize cancer cells and to discriminate between live and dead cells. Urano et al. target human epidermal growth factor receptor type 2 (HER2) with the monoclonal antibody trastuzumab, which, after binding to HER2, is internalized via the endosomal—lysosomal degradation pathway. The pH in the lysosomes is low (5–6) relative to the cytoplasm (pH ∼ 7.4), which led to the idea to develop a probe that becomes activated in an acidic environment. They show that tumor cells expressing the HER2 emit fluorescence, whereas control cells without HER2 do not. In addition, they prove that pH-activatable probes produce fluorescence only in living cells. Exchanging the antibody Urano et al. also targets ovarian cancer in a mouse model. They perform in vivo fluorescence microendoscopic experiments in live mice bearing peritoneal ovarian cancer micrometastases, and show that the method is amenable to an endoscopic or laparoscopic approach, making it potentially useful as a surgical aid to improve the completeness of resection.
In addition to its main potential application as a clinical tool for cancer detection, the small-molecule, pH-activatable fluorescent probe conjugates can also potentially be used as in vitro tools for evaluating intracellular receptor kinetics, cell viability, and real-time monitoring of cell death (Nat. Med.