Well-plates

The rapid synthesis of heteroaromatic Hantzsch pyridines can be achieved by aromatization of the corresponding 1,4-DHP derivative under microwave-assisted conditions [51]. However, the domino synthesis of these derivatives has been reported in a domestic microwave oven [58,59] using bentonite clay and ammoniiun nitrate, the latter serving as both the source of ammonia and the oxidant, hi spite of some contradictory findings [51,58,59], this approach has been employed in the automated high-throughput parallel synthesis of pyridine libraries in a 96-well plate [59]. In each well, a mixture of an aldehyde, ethyl acetoacetate and a second 1,3-dicarbonyl compound was irradiated for 5 min in the presence of bentonite/ammonium nitrate. For some reactions, depending upon the specific 1,3-dicarbonyl compound used. 

Some of the compUcations Usted above could be flagged and, sometimes, remedied in a manual shake-flask experiment, but that is unlikely to be the case in automated shake-vial procedures, especially if performed in a 96-well plate setting. Nevertheless, the demands of modern pharmaceutical discovery operations emphasize high-throughput measurements, low compound consumphon... 

An automated log P workstation using a shake-flask method and robotic liquid handling in 96-well plate format is commercially available [30]. The system is equipped with a diode-array spectrophotometer and equimolar nitrogen detector. 

In addition, further automation will be needed in what is still very much a hands-on art. Autoinjectors coupled to complete analytical data systems and readers for 96-well plates are the beginning of what will continue to be a necessary trend of residue chemistry. The application of the techniques of combinatorial chemistry/biochemistry, which has produced screening methodology for handling many variables, might be appropriate to residue chemistry. 

The gold-standard assay used for all chemokine receptor inhibitors that reach clinical-phase trials is the chemotaxis functional assay. This assay relies on the ability of chemokines to recruit cells expressing their respective receptor to areas of inflammation. In vitro, this assay was first described in detail by Taub et al. (16) for 24/48-well plates currently, this can be achieved by using 96-well plates. Cells are incubated in the upper chamber with an antagonist for a particular receptor (at different concentrations or with buffer) and challenged to migrate to the lower chamber, which has the relevant chemokine. After 2 to 4 hours of incubation at 37°C, the upper chamber inlet is removed and the cells in the lower chamber quantified by fluorescence with, for example, Calcein AM (Invitrogen, Carlsbad, CA). 

Chiron provides a microwell plate heater, a luminometer, and data management software. The plate heater is specially designed to provide precise control of the hybridization temperature (0 0.5°C) and to distribute heat evenly throughout the microwell plate. The luminometer maintains a temperature of 37°C and accommodates the 96-well plates. The data management software runs on an IBM PC or compatible computer with a minimum of 80386,16-Mhz microprocessor, 2 Mb of RAM, monitor, mouse, compatible printer, MS DOS (version 5.0 or greater), and Windows (version 3.1 or greater). 

Well Plate Format for Inhibitor Modality Studies... 

Figure 7.6. Purification of protein from pooled yeast strains. Each yeast ORF was cloned as a fusion to glutathione-S-transferase in a protein expression vector to create 6144 yeast strains. The individual strains were pooled in groups of 96 to create a set of 64 pools. Each pool was grown and the 96 fusion proteins are purified in batch. Each pool was then assayed for a biochemical function (Martzen et al., 1999). Pools positive for function were then deconvoluted using smaller pools consisting of strains from rows and columns of a 96-well plate.

Samples are taken directly from 10 mM DMSO-solvated stocks in 96-well plates. Typically, 2 pL of sample is aspirated, and injected on to the column - this represents about 8 pg of sample with MW = 400. The retention time (R,) of peaks eluting from the column is related to the capacity factor log k by Eq. (8) ... 

HTS 96-well conical plates Translucent 96-well plates with conical bottoms (Costar 3897). 

We have found that many compounds identified in our screen are nonspecific inhibitors of luciferase enzyme activity. To eliminate these, we test the hits in a luciferase enzyme-based counterscreen. Firefly and renilla luciferase are produced in vitro by programming Krebs-2 extracts with FF/HCV/Ren mRNA and allowing the translations to proceed at 30° for 1 h. Ten microliters are then pipetted into a 96-well plate and compound is added to a final concentration of 20 /iM (1% DMSO). Luciferase activity is then determined as described previously in step 3. Since compound is added only after the translation reaction is complete, inhibitors of translation should not score positively in this assay. Typically, a 1-ml in vitro translation reaction is sufficient to screen 45 candidate hits in duplicate for nonspecific luciferase inhibitory activity. Compounds that inhibit in this counterscreen are eliminated from future analysis. 

About 5 ng of modified DNA is coated onto the surface of a 96 well plate (Figure 2, Step 1). In a competitive version of this assay, antibody with specificity against the PAH-DNA adduct of interest is incubated with samples if DNA to be tested (Step 2). 

Turntable Vessel volume Operation volume Vessel material Max. temperature Temp, measurement 3 x 96-well plates 1 mL min. 0.1 mL glass 150 °C fiber optic 52 x 50 mL max. 30 mL glass or PFA 150 °C fiber optic 120 x 15 mL max. 10 mL glass or PFA 150 °C fiber optic... 

Scheme 4.23 Generation of Hantzsch-type pyridines in a 96-well plate.

Several articles in the area of microwave-assisted parallel synthesis have described irradiation of 96-well filter-bottom polypropylene plates in conventional household microwave ovens for high-throughput synthesis. While some authors have not reported any difficulties in relation to the use of such equipment (see Scheme 4.24) [77], others have experienced problems in connection with the thermal instability of the polypropylene material itself [89], and with respect to the creation of temperature gradients between individual wells upon microwave heating [89, 90]. Figure 4.5 shows the temperature gradients after irradiation of a conventional 96-well plate for 1 min in a domestic microwave oven. For the particular chemistry involved (Scheme 7.45), the 20 °C difference between the inner and outer wells was, however, not critical. 

A recent study concerned the microwave-assisted parallel synthesis of di- and tri-substituted ureas utilizing dedicated 96-well plates in the CombiCHEM system [60], In a typical procedure, modification of the Marshall resin utilized was achieved by treatment with p-nitrophenyl chloroformate and N-methylmorpholine (NMM) in dichloromethane at low temperatures. The resulting resin was further modified by attaching various amines to obtain a set of polymer-bound carbamates (Scheme 7.48). 

Khmelnitsky and coworkers have also examined microwave-assisted parallel Hantzsch pyridine synthesis [28], They have demonstrated the benefits of microwave irradiation in a 96-well plate reactor for high throughput, automated production of a pyridine combinatorial library (Scheme 8.20). 

However, luminescence-based detection techniques often require a high number of steps. Consider ELISA as an example. As a first step, the sample is introduced into a 96-well plate an antibody targeting the antigen of interest has been immobilized to the wells of the plate. After a rinse, the wells contain the antibody and any bound antigen. However, although the antigen has been isolated, the protocol is nowhere near completion. The remaining steps include another antibody (different from the first) to form a sandwich assay, a secondary antibody with an enzymatic label, and a substrate that is luminescent when activated by the enzyme. Finally, the sample is analyzed by relatively expensive detection optics to determine the amount of analyte that was captured in the assay. The steps are illustrated in Fig. 14.1a. 

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