Models for Galactose Oxidase (GOase) Based on Combinatorial Peptoid Libraries

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Low molecular weight models for metalloenzymes are usually designed based on information available for the spatial arrangement of functional groups, metal ions, co-enzymes etc. in active sites (ideally such information is obtained via X-ray crystal analyses). This approach largely neglects the influence of the protein matrices, in particular their dynamics, local dipole moments, the inhibition of metal complex aggregation etc. As a consequence, many biomimetic low molecular weight transition metal complexes are able to reproduce structural and spectroscopical aspects of the corresponding enzymes, but most of them fail to show significant catalytic activity. In our approach we also deduce, from a given enzyme structure, which ligands are bound to the catalytically active metal center. However, in the next step, combinatorial ligand libraries are synthesized which place the metal-coordinating groups at different positions on an oligopeptide. By suitable assays, catalytically active members of the library are identified. In other words, the system is given a significant degree of freedom both in terms of constitution and dynamics, and only those entities that are catalytically active are examined further. The current project aims at the identification of functional models for galactose oxidase, i.e. biomimetic copper complexes that are able to oxidize alcohols with oxygen to the corresponding carbonyl compounds. The ligand libraries will be prepared on solid phase, encoding will be done by the IRORI radiofrequency labelling technique. The design of the libraries is based on the X-ray crystal structure of the fungal enzyme from Dactylium dendroides: histidine, tyrosine and non-natural amino acids mimicking the Cys228-Tyr272 conjugate of the wild-type enzyme will be used as metal binding entities. The non-natural Cys-Tyr mimics will be prepared in enantiomerically pure form from appropriate chirons. Assays for GOase-activity will be developed based on existing enzymatic methods for H2O2-detection, or on dye-forming reactions of the product carbonyl compounds. These reactivity assays, which are amenable to high-throughput screening, will be complemented by "classical" (chiral) GC- and HPLC-analysis (e.g. for the detection of enantioselectivity).