The impact of the second coordination sphere in phosphatase model complexes

The enzyme Purple Acid Phosphatase (PAP) is an important target for the development of new anti-osteoporotic drug leads. The major goal of this thesis is to better emulate the synergy that occurs between the primary and secondary coordination sphere within the active site of phosphatase enzymes, such as PAP. This was perceived by development of synthetic methods for new biomimetics, i.e. low-molecular weight metal complexes. Therefore, asymmetric dinucleating ligands which mimic the peptide backbone of the active site of PAP including the ability to form hydrogen bonds with a phosphoester substrate or a nucleophile were designed and synthesized. Using these ligands, more accurate model systems for the enzyme active site were achievable as they combine the two essential structural features known to influence the catalytic activity towards the hydrolysis of phosphoesters, i.e. the asymmetry of the dinuclear active site and the capacity for hydrogen bond formation. The latter was verified by the observation of hydrogen bonds in the X-ray structures of dizinc(II) and diiron(II) complexes. Moreover, two asymmetric dizinc(II) complexes were formed with two different Zn(II) sites and an unusual hydroxido co-ligand, representing two important features in the active site of PAP, the difference of the metal sites and the nucleophile needed for the phosphoester hydrolysis. In addition, hydrogen bond formation detected in these X-ray structures was accounted for the stabilization of the hydroxido co-ligand. In this work, a new synthetic approach towards more sophisticated model systems for the active form of mammalian PAP was developed. Chemical oxidation of the diiron(II) complex of an asymmetric ligand results in the generation of the Fe(III)Fe(II) complex that has been specifically designed to both satisfy the requirement of a heterovalent diiron core and to mimic the second coordination sphere of the active site of PAP. Similarly, more accurate model systems for the active site of plant PAPs have been generated in the form of heterovalent heterodinuclear complexes of asymmetric ligands bearing, adjacent to the Ga(III)Zn(II) core, functionalities capable of forming hydrogen bonds. Analysis of the complexation behavior of the respective ligands revealed the selective formation of the Ga(III)Zn(II) complexes in solution. The model complexes described above imitate successfully the extensive hydrogen bond network that is formed by the second coordination sphere within the active site of PAP as well as in a structural and functional similar phosphatase, Alkaline Phosphatase. Thus, those complexes allow to study the impact of hydrogen bonds on the reaction mechanism. The main impact of the secondary interactions in the dizinc(II) complexes was found to be the increased substrate affinity. This catalytic parameter was shown to be dependent on both the hydrogen bonding sites and the type of the hydrogen bonding groups. Although the substrate affinity of the Ga(III)Zn(II) complex was revealed to be lower compared to the dizinc(II) complex, a 50-fold faster hydrolysis rate and a 6-fold increased efficiency was detected for the heterodinuclear complex. Moreover, the mechanism previously proposed, in which the phosphoester is activated by the Zn(II) center and the Ga(III) being accountable for providing the hydroxide nucleophile at near physiological condition, was supported in this study and an accelerating effect by the interplay of the two metal ions was detected. However, the inhibition was found to be favored in the Ga(III)Zn(II) complex compared to the respective monogallium(III) complex, most likely due to bridging coordination of the hydrolysis product, additionally stabilized by coordination of the adjacent pivaloyl-amide residue. The proposed arrangement in the catalyst-hydrolysis product adduct derives from a structure of a stable phosphoester-bridged dizinc(II) complex bearing the same ligand backbone. However, the Ga(III)Zn(II) complex is the first heterodinuclear model complex that mimics the essential function of PAPs, the ability to cleave phosphomonoesters and therefore supports the crucial impact of the second coordination sphere in the active site of PAP.