Final Activity Report Summary - MEDIUM EFFECT (Using chemical models to characterize potential specific medium effects in active sites of enzymes) Enzymes are outstanding catalysts for a very large range of difficult reactions, able to work in mild conditions in water. To do so, they use pockets, or active sites, in which the microenvironment is designed to catalyse one reaction or another. These pockets usually bear localised charges to stabilise transition states within a hydrophobic environment to enhance electrostatic interactions. However, even though these parameters are commonly accepted as crucial for the enzyme activity, the chemical models demonstrating the importance of a specific microenvironment for enzyme catalysis are very rare. In order to model enzyme active sites, we used a water soluble polymer (polyethylene Imine) which we chemically modified with positively charged guadinium group and aliphatic groups. Their activity was then tested for the biologically relevant, phopshate transfer reaction. However, it was not obvious in which proportions these modifications were optimal for the reaction, so we synthesised a wide range of modified polymer using a combinatorial approach and screened their activity using a chromogenic substrate (HPNP). This screening showed a synergetic effect of both modifications on the polymer, demonstrating that both localised charges and hydrophobic environment were crucial for catalysis. A detailed kinetic study of the best catalyst also demonstrated that this system was the best metal-free catalyst for phosphate transfer and that its activity was comparable to the best metal-based catalysts. Furthermore, this polymeric catalyst was reminiscent to an enzyme by many characteristics, such as solubility in water, hydrophobic pockets, low KM and specific inhibition by small organic molecules. Since metals are known to be important cofactors for phosphate hydrolysis, we also studied the activity of these polymers with metals coordinated to the amines of the backbone. The gain in activity for the transesterification reaction was not particularly high, but pH rate profiles and inhibition studies with different metals gave us interesting indications that could be relevant to the biological systems. Concerning other reactions, such as phosphate diester, sulfate monoester or phopsphonate hydrolysis, it seemed that changing the metal and the specific microenvironment could be important for the specificity of the catalyst generating a very specific or a promiscuous catalyst.