Oh Oh

HFpy/THF; then TMSOMe

Scheme 6.2.1. Activation of glass slides and the covalent attachment of alcohols.

Cl Cl

Scheme 6.2.1. Activation of glass slides and the covalent attachment of alcohols.

Lam et al. have introduced two new ligation methods for glyoxylic acid-modified glass slides with which they could immobilize biotin or peptide ligands derivatized either via an amino-oxy group by oxime formation or a 1,2-aminothiol group by thiazolidine ring formation [19, 28]. The microrarray of immobilized ligands was analyzed in three different biological assays.

1. In a protein-binding assay with fluorescence detection a microarray of biotin, HPYPP-peptide and WSHHPQFEK-peptide was screened against streptavidin-Cy3 and avidin-Cy5. By following the same principle an anti-human insulin monoclonal antibody was also screened against a set of different peptides.

2. In a functional phosphorylation assay [g33P]-ATP and a specific protein kinase were used to label peptide substrate spots.

3. In an adhesion assay different areas of a slide with a certain peptide sequence were covered separately with different cell lines. After washing and staining only the WEHI 2312 cells were found to bind to the spotted area of the glass slide.

Schultz et al. devised an ingenious approach to convert a spatially separable, but not addressable split-pool (OBOC) library into a spatially addressable microarray (Scheme 6.2.2) [29]. Conventional solid-phase techniques were used for the synthesis of small molecules tethered to peptidonucleic acid (PNA) tags. The chemically robust and, by iterative amide formation, easily constructable PNA tags serve two purposes - first to encode the synthetic history of the small molecule and second to positionally encode the identity of the small molecule, by its location, on hybridization to a commercial oligonucleotide microarray. Overcoming the limitation of other screening technologies, which require fluorescence-labeled proteins or samples, they described an approach in which the fluorophore is conjugated to the PNA-small molecule entity. The library is then incubated with the target protein. Size-exclusion filtration enables one to separate unbound ligands from the protein-ligand mixture. After hybridization with the oligonucleotide array fluorescent spots encode molecules which have bound to the target protein. The feasibility of this approach has been shown by the screening of six mechanism-based cysteine protease inhibitors against cathepsin L.

Surpassing the mere proof-of-concept character of the work highlighted above, Schreiber et al. have prepared a high-density microarray of 3780 structurally complex 1,3-dioxane small molecules which have been synthesized by one-bead-one-stock-solution technology with the use of macrobeads [30]. By probing the array with fluorescence-labeled Ure2p, a protein which represses the transcription factors Gln3p and Nillp, several compounds which bind Ure2p were identified. One of these compounds, named uretupamine, specifically activates a glucose-sensitive transcriptional pathway downstream of Ure2p.

So far undisclosed in the peer-reviewed literature are contributions by the company Graffinty (www.graffinity.de) [31]. It has built up a technology platform in which combinatorial libraries are generated by solid-phase methodology using an acid-labile S-trityl linker. After cleavage the free thiols of the small molecules react

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