Abstract
Laboratory Automation and High-Throughput Chemistry
Novel Enzymatic Synthesis of Levulinyl Protected Nucleosides Useful for Solution Phase Synthesis of Oligonucleotides
An efficient synthesis of 3′- and 5′-O-levulinyl-2′-deoxy- and 2′-O-alkylribonucleosides has been developed from appropriate nucleosides by enzyme-catalyzed regioselective acylation in organic solvents (Javier García et al., Tetrahedron: Asymmetry 2003, 22(14), 3533). Therefore several lipases were screened in combination with acetonoxime levulinate as an acylating agent. Immobilized Pseudomonas cepacia lipase (PSL-C) was selected for acylation of the 3′-hydroxyl group in nucleosides, furnishing 3′-O-levulinylated products in excellent yields. Similarly, Candida antarctica lipase B (CAL-B) provided 5′-O-levulinyl nucleosides in high yields. Furthermore base-protected cytidine and adenosine analogs were found to be good substrates for lipase-mediated acylations.
For demonstrating the industrial utility of this method, 3′-O-levulinyl thymidine and N2-Ibu-5′-O-levulinyl-2′deoxyguanosine were synthesized on a 25 g scale. In addition, PSL-C was reused, making the processes even more economical.
Catalytic Transfer Reduction of Conjugated Alkenes and an Imine Using Polymer-Supported Formates
An efficient and mild method for catalytic transfer hydrogenation of C = C and C = N double bonds with the aid of polymer-supported formate (PSF) as the hydrogen donor and palladium acetate as the catalyst is reported by Basudeb Basu et al. (Tetrahedron Letters 2003, 50(44), 8931).
The PSF was prepared by washing Amberlite resin (IRA 420, Cl−) packed in a column with 10 % aqueous formic acid solution, then with water several times and after that dried under vacuum. For the reaction a mixture of unsaturated compound, palladium acetate (2 mol%), and resin formate in DMF was stirred at 70–75 °C for 10–16 h. After the mixture was cooled and diluted with water, it was filtered and extracted with ether, which on evaporation afforded the desired product.
The generality of this methodology has been investigated with different types of electron-deficient alkenes and an imine. Cyano and ester groups remained unaffected under the reaction conditions. Surprisingly, a simple alkyl cinnamate and a nitro olefin failed to undergo hydrogenation.
Self-Indicating Amine Scavenger Resins
A self-indicating methylisocyanate resin, which functions as both a scavenger and an indicator for amines, was used by M. Bradley et al. (University of Southampton) for in-situ reaction monitoring and purification of a urea-based library (Chem. Commun. 2004, 502). The dye used was bromophenol blue, which is blue in the presence of amines. As the excess amine in solution was removed by the resin-bound isocyanate, the resin's color gradually changed to yellow.
Discovery and Evaluation of Highly Active Imidotitanium Ethylene Polymerization Catalysts Using High-Throughput Catalyst Screening
A family of ∼50 imidotitanium precatalysts was prepared by P. Mountford et al. (University of Oxford) in good yields using semi-automated procedures (Chem. Commun. 2004, 434). By using high-throughput screening techniques, seven highly active ethylene polymerization pre-catalysts with activities in the range of ∼3,400 to 10,000 kg (PE) mol−1h−1bar−1 were identified.
Combinatorial Electrochemical Screening of Fuel Cell Electrocatalysts
Combinatorial methods have been applied to the preparation and screening of fuel cell electrocatalysts by B. E. Hayden and colleagues from the University of Southampton (J. Comb. Chem. 2004, 6, 149). Hardware and software have been developed for fast sequential measurement of cyclic voltammetric and steady-state currents in 64-element half-cell arrays. The arrays were designed for the screening of high-surface-area supported electrocatalysts. Analysis software developed allowed the semi-automated processing of the large quantities of data, applying filters that defined figures of merit relevant to fuel cell catalyst activity and tolerance. Results are reported on the screening of carbon-supported platinum catalysts of varying platinum metal loading on carbon in order to demonstrate the speed and sensitivity of the screening methodology.
Combinatorial Synthesis of Oxide Powders with an Autopipetting System
P. J. McGinn et al. (University of Notre Dame, Indiana) described the development of a relatively simple, rapid route to produce combinatorial compositional oxide powder libraries by autopipetting of liquid precursors (J. Comb. Chem. 2004, 6, 86). This partitioning approach should apply equally well to any low viscosity, liquid precursors for the synthesis of oxide powders. A commercially available TomTec 96 well pipettor that can aspirate or dispense to 96 wells simultaneously was modified by fitting a plastic “mask” assembly beneath the pipette array in order to partition and direct liquids into crucibles. A series of 10 mixtures from two precursor solutions can be produced in 5–6 h, as compared to solid state serial techniques taking 24–48 h. After they are dispensed, the liquid mixtures are dried in the crucibles and reacted at elevated temperatures to produce oxide powders.
Two-Dimensional and Three-Dimensional Spatially Addressed Arrays for High-Throughput Automated Synthesis of Combinatorial Libraries
One of the key elements in the drug discovery process is the use of automation to synthesize libraries of compounds for biological screening. The “split-and mix” approaches in combinatorial chemistry have been recognized as extremely powerful techniques to access large numbers of compounds, although requiring only few reaction steps. However, the need for effective encoding and deconvolution strategies and demands for larger amounts of compounds have limited the use of these techniques in the pharmaceutical industry. M. Patek et al. (Selectide/Aventis) now described a concept of directed sort and combine synthesis with spatially arranged arrays of macroscopic supports (J. Comb. Chem. 2004, 6, 43). This concept attempts to balance the number of reaction steps, the confidence in compound identity, and the quantity of synthesized compounds. Using three-dimensional arrays of frames, each containing a two-dimensional array of macroscopic solid support, a modular semi-automated system with a capacity of up to 100,000 compounds per batch was developed. Modularity of this system enables flexibility either to produce large diverse combinatorial libraries or to synthesize more focused smaller libraries, both as single compounds in 12–15 μmol quantities. This method using sortable and spatially addressed arrays is exemplified by the synthesis of a 15,360 compound library.
A Simple, Inexpensive Reactor with Condenser for Use in Combinatorial Chemistry
S. M. Firestine and W. Xie from the Duquesne University, Pittsburgh, published the construction of a simple, inexpensive reaction vessel that contains a built-in condenser. The vessel (total volume 30 mL) can be constructed from easily available materials found in the common organic laboratory and each reaction vessel can be prepared for about one dollar. This inexpensive system might therefore find some interest in combinatorial chemistry for the synthesis of libraries of compounds (J. Comb. Chem. 2004, 6, 24).
Automated Solid-Phase Synthesis of Protected Tumor-Associated Antigen and Blood Group Determinant Oligosaccharides
Automation of oligosaccharide assembly has the potential to revolutionize the biological understanding of complex oligosaccharides as non-specialists gain access to a variety of structures. Automated assembly of the Ley-Lex tumor marker and the Lewis X and Lewis Y blood group antigens by P. Seeberger and K. Routenberg Love (ETH Zuerich) serves as a first example of how to create a host of structures of biological significance with a stepwise monosaccharide approach (Angew. Chem. 2004, 116, 612). The adaptation of coupling cycles to include Fmoc protection and the introduction of linker strategies to enable base cleavage brings automated methods of oligosaccharide synthesis a step closer to existing protocols for peptide synthesis.
The Application of Microreactor Technology for the Synthesis of 1,2-Azoles
The successful syntheses of pyrazole and isoxazole derivatives within a borosilicate glass microreactor have been demonstrated by S. J. Haswell and co-workers from the University of Hull (Org. Proc. Res. Dev. 2004, 8, 28). By applying the technique of electro-osmotic flow (EOF) conversions in the range of 98–100% were obtained. In terms of large-scale production, this corresponds to 0.339 g day−1 per microreactor when employing reagent concentrations of 1.0 M. By numbering parallel microreactors it would be possible to rapidly produce larger amounts of these compounds with potential interest as building blocks in the life sciences industries.
Chemistry in Microreactors
A comprehensive review of a recent development of chemistry in microreactors was published by K. Jähnisch, V. Hessel, H. Löwe and M. Baerns (Institut für Angewandte Chemie Berlin and Institut für Mikrotechnik Mainz, Germany) (Angew. Chem., 2004, 116, 410). A wide variety of catalyzed and stoichiometric chemical reactions in liquid, as well as in gas phase, are described. The authors report on various microreactor systems and first industrial applications in process development and production.
High-Throughput Analytics
High-Throughput Miniature Cylindrical Ion Trap Array Mass Spectrometer
A fully multiplexed cylindrical ion trap (CIT) array mass spectrometer with four parallel ion source/mass analyzer/ detector channels has been built to allow simultaneous high-throughput analysis of multiple samples (Anal. Chem. 2003, 75, 5656). A multi-element external chemical ionization/electron ionization source was coupled to a parallel array of CITs and the signal was recorded using an array of four miniature electron multipliers. The instrument provides a mass/charge range of m/z 50–500, and its modular design allows scale-up to many more channels of analysis for future applications in the areas of industrial process monitoring, combinatorial analysis, proteomics, and meta-bolomics.
High Sequence Coverage of Proteins Isolated from Liquid Separations of Breast Cancer Cells Using Capillary Electrophoresis-Time-of-Flight MS and MALDI-TOF MS Mapping
A method has been developed for high sequence coverage analysis of proteins isolated from breast cancer cell lines (Anal. Chem. 2003, 75, 6209). Intact proteins are isolated using multidimensional LC-separations that permit the collection of individual protein fractions. Protein digests are then analyzed by MALDI-TOF MS peptide mass fingerprinting and by CE-ESI-TOF MS peptide mapping. Using combined sequence information provide by both mapping methods, 100% sequence coverage is often obtained for smaller proteins, while for larger proteins up to 75 kDa, over 90% coverage can be obtained. Further, an accurate intact protein MW value (within 150 ppm) can be obtained by ESI-TOF MS. The intact MW, together with high coverage sequence information, provides accurate identification. This capability is critical in the analysis of human cancer cells where a large number of expressed proteins are modified, and these modifications may play an important role in the cancer process.
Use of Design of Experiments to Optimize High-Throughput Semi-Preparative LC and LC/MS Methods
D. C. Cole et al. (Wyeth Research) described an investigation into decreasing the run time for high-throughput semi-preparative RP-HPLC methods without compromising the resolution (J. Comb. Chem. 2004, 6, 78). Experimental design software (Modde 6.0) was used to devise a small set of experiments in which factors, including solvent flow rate, solvent/column temperature, at column-dilution, and run time were varied systematically. The results were analyzed by means of multiple regression and partial least squares to generate a model relating the factors to the results, and identifying which factors are important. The model was then used to determine the optimal conditions. Optimized conditions resulted in a 50% reduction in run time and a 25% reduction in solvent usage and at the same time increasing the resolution. Increased resolution also leads to sharper peaks and therefore fewer fractions to combine and less solvent to evaporate.
A Fully Automated Kinetic Solubility Screen in 384-Well Plate Format Using Nephelometry
Within the drug discovery industry there is a growing trend toward measuring ADME and physical chemical properties for larger numbers of compounds at an earlier stage, and at a higher throughput, in an attempt to highlight potential ADME issues and to reduce attrition. Solubility is one of the most important physicochemical properties of a discovery compound and recognizing solubility issues at an early stage is invaluable. Not only are low solubility compounds more difficult to develop, obtaining reproducible data for ADME screens such as Caco-2 and lipophilicity is also more time-consuming and costly. Therefore, a rapid, low cost method for determining solubility prior to running the more costly ADME screens is a useful tool. Laser nephelometry has been shown to be an important technique for the measurement of kinetic solubility in 96-well plate format. Laser nephelometry is the measurement of forward scattered light when a laser beam is directed through a solution. The more particulate there is in the solution, the greater the amount of forward scattered light. An article published by BMG Labtechnologies, Inc.—which is available for free downloading at http://www.bmg-labtechnologies.com/db_assets/applications/downloads/applications/AN%20Nephelometry%205101.pdf—demonstrates how this technique has been advanced into a fully automated and rapid kinetic solubility screen in 384-well plate format.
Bioautomation and Screening
β Galactosidase Enzyme Fragment Complementation as a Novel Technology for High-Throughput Screening
β Galactosidase is one of several enzymes to undergo complementation, i.e., biological activity is restored by non-covalent interaction of different polypeptides. This phenomenon can be used to monitor protein-protein interactions by coupling enzyme fragments to proteins which have a certain affinity to each other (weakly and strongly interacting fragments are distinguished). Eglen et al. (Comb. Chem. High Throughput Screen. 2003, 6, 381) discuss in their review aspects of p galactosidase complementation as a strategy for high throughput assay development and compound screening and they describe applications of this technology. Two companies have developed β galactosidase complementation as HTS assay systems with different approaches.
Eglen et al. point out that enzyme fragment complementation is a technology that possesses several advantages for HTS. A major advantage of using β galactosidase is that signal amplification occurs due to enzymatic amplification that provides the basis for highly sensitive bioassays and thus very low levels of interacting proteins or analytes (e.g., cAMP, IP3) can be measured. Several substrates are available, allowing for colorimetric, chemiluminescent or fluorescent signals, all of which can be measured in a homogeneous fashion. Further, the inhibitors can be detected at the target, resulting in a low false positive hit rate in contrast to other cell-based functional approaches including reporter gene assays. The use of β galactosidase complementation provides a novel and flexible technology for highly sensitive HTS assay development and is adaptable to many cell-based or cell free screens.
Microplate-Based Biosensing with a Computer Screen-aided Technique
The use of intact biochemical-signalling pathways coupled with easily detected physical phenomena is an advantageous condition for biosensing (Biosens. Bioelect. 2003, 19, 35). Melanophores, dark pigment cells from the frog Xenopus laevis, provide this possibility. They have the ability to change light absorbance upon stimulation by different biological agents. Filippini et al. used hormone exposure (e.g., melatonin) as a reversible stimulus to test a new compact microplate reading platform. As an application, they detected the asthma drug formoterol in blood plasma samples. The cells can be grown on conventional microplates, providing a highly specific and flexible biochemical platform because of spatial codification of different target reactions. The instrument setup consists of a computer screen as a programmable large area light source, and a standard Web camera to record multiple kinetic responses. These components are broadly available, making it advantageous and versatile for the evaluation of numerous bioassays (especially point-of-care or self test applications) compared with highly dedicated, comparatively expensive commercial systems.
High-Resolution Screening in an Expanded Chemical Space
H. Irth, S. Long, and T. Schenk (Kiadis, The Netherlands) reported a high-resolution screening method (HRS) for the identification of bioactive molecules in complex mixtures (Curr. Drug Disc. 2004, 19). The HRS technology integrates, in parallel in a single instrument, the three key technologies required for the efficient screening of impure compounds and mixtures: high-performance separation via HPLC, chemical analysis by mass spectrometry, and biochemical screening. With a single HRS measurement, information about the total number of active compounds in the mixture and their retention times can be provided as well as their correlated UV-VIS and MS spectra. Therefore, this might be an interesting addition to existing discovery tools.