Combinatorial Chemistry & High Throughput Screening - Volume 10, Issue 3, 2007

The heating of different parallel arrays in domestic ovens offers the possibility to perform multiple reactions in one irradiation experiment, blending the advantages of microwave heating technology and parallel chemistry. However, they are usually performed without an appropriate temperature control; thus, reproducibility becomes a major issue limiting the application of such reactions. This is exemplified when working at a different scales or using different instruments. For the first time a typical solvent-free reaction described in a domestic oven has been reproduced in monomode reactor, scaled up in a controlled multimode oven and reproduced in parallel, 24 reactions were carried out in a well plate. Parallel reactions were performed in a Weflon® multiwell plate to assure identical conditions for each individual reaction. As many reactions under microwave irradiation have been performed in solvent-free conditions, this result opens new possibilities in reproducibility, scalability and combinatorial chemistry and permits to take advantage of many synthetic procedures described in domestic ovens.

Chemical synthesis, physicochemical characterization and kinetic investigations of a tetrapeptide library of chromogenic substrates containing the amide of 5-amino-2nitrobenzoic acid (Anb5,2-NH2) at their C-termini are reported. Anb5,2-NH2 served as a chromophore released upon enzymatic action. The library consisting of 9567 peptides was synthesized using the portioning-mixing method and was screened against bovine α-chymotrypsin and human leukocyte elastase in solution applying an iterative approach. The selected chromogenic substrates were resynthesized and further modified at their N- and C-termini. Finally, two sequences, Z-Phe-Ala-Thr-Tyr-Anb5,2-NH2 and Z-Phe-Phe-Pro-Val-Anb5,2-NH2, were obtained as highly specific substrates for bovine α-chymotrypsin and human leukocyte elastase, respectively. The method of synthesis and selection of chromogenic substrates of serine proteinases described herein is straightforward and can be applied to design substrates for other proteases.

Eukaryotic initiation factor (eIF) 4F plays a key role in recruiting 40S ribosomes and associated factors to mRNA templates during translation initiation. The function of this heterotrimeric complex is to deliver an RNA helicase to the 5' cap proximal region of mRNAs in preparation for ribosome binding. To study the interaction between subunits of this complex, as well as identify small molecules that could interfere with their association, we developed a time resolved fluorescence assay that allows monitoring of interactions between two subunits of eIF4F. We have performed a small molecule chemical screen of >73,000 compounds using this assay.

In many cases at the beginning of an HTS-campaign, some information about active molecules is already available. Often known active compounds (such as substrate analogues, natural products, inhibitors of a related protein or ligands published by a pharmaceutical company) are identified in low-throughput validation studies of the biochemical target. In this study we evaluate the effectiveness of a support vector machine applied for those compounds and used to classify a collection with unknown activity. This approach was aimed at reducing the number of compounds to be tested against the given target. Our method predicts the biological activity of chemical compounds based on only the atom pairs (AP) two dimensional topological descriptors. The supervised support vector machine (SVM) method herein is trained on compounds from the MDL drug data report (MDDR) known to be active for specific protein target. For detailed analysis, five different biological targets were selected including cyclooxygenase-2, dihydrofolate reductase, thrombin, HIVreverse transcriptase and antagonists of the estrogen receptor. The accuracy of compound identification was estimated using the recall and precision values. The sensitivities for all protein targets exceeded 80% and the classification performance reached 100% for selected targets. In another application of the method, we addressed the absence of an initial set of active compounds for a selected protein target at the beginning of an HTS-campaign. In such a case, virtual highthroughput screening (vHTS) is usually applied by using a flexible docking procedure. However, the vHTS experiment typically contains a large percentage of false positives that should be verified by costly and time-consuming experimental follow-up assays. The subsequent use of our machine learning method was found to improve the speed (since the docking procedure was not required for all compounds from the database) and also the accuracy of the HTS hit lists (the enrichment factor).

The field of directed evolution of oxygenases (mono-, di- and epoxygenases) is rapidly advancing as an increasing number of success stories indicate. A significant number of screening systems have been developed to specifically improve oxygenase properties. Oxygenases will become very valuable biocatalysts for synthetic applications in industry when stability, cofactor and activity properties match industrial demands. This review summarizes screening systems and principles of screening systems that have been used for directed evolution of oxygenases. Sections on mutagenic conditions, mutant library size and property improvements provide a comprehensive picture on performance and limitations of current directed evolution methodologies for oxygenases. A discussion of challenges in the directed evolution of oxygenases for industrial exploitation concludes this review.

Multicomponent reactions (MCRs) generate multiple bonds in a single reaction process, which is highly efficient to construct relatively complex molecules. Conducting post-MCR modification reactions further increases the molecular complexity and diversity. MCR has become a powerful approach to make drug-like molecules in lead generation chemistry. In fluorous MCR (F-MCR), one of the starting materials is attached to a fluorous tag and used as the limiting agent. After the MCR, the fluorous component is fished out from the reaction mixture and used for post-MCR modifications. The fluorous tag can be finally removed in traceless fashion by displacement or cyclization reactions. Unique fluorous technology such as fluorous solid-phase extraction (F-SPE) facilitates the separation process. Other techniques such as microwave irradiation and plate-to-plate SPE can also be used to make the F-MCR even more efficient. Syntheses of unique heterocyclic and natural product-like library scaffolds using Ugi/de-Boc/cyclization, MCR/Suzuki coupling, and [3+2] cycloaddition/de-tag/cyclization protocols are described in this paper.

Some 2D graphical representations of DNA sequences have been reported by several authors, which give visual characterizations of DNA sequences. In this paper, we present a new 2D graphical representation of DNA sequences without degeneracy. Furthermore, we propose two methods for the visualization and analysis of long DNA sequences.