Protein-ECE MEtallopincer Hybrids

Abstract

Modification of proteins with metal complexes is a promising and a relatively new field which conceals many challenges and potential applications. The field is a balance of contributions from the biological (protein engineering, bioconjugation) and chemical sciences (organic, inorganic and organometallic chemistry) and requires a multidisciplinary approach. Amongst the many prominent reasons to develop protein-metal complex hybrid materials are their potential use as artificial metalloenzymes with high performance as stereoselective non-enzymatic catalysts, as a tool for the isomorphous replacement method in X-ray structure determination, as diagnostic biosensors, as well as for novel pharmaceutical and medical treatments. The introduction of organometallic complexes into proteins has been challenged in numerous cases and has shown to be a promising extension to the existing hybrid systems. A crucial difference of organometallic complexes, as compared with other inorganic complexes, is the presence of a covalent metal-carbon bond which prevents facile metal dissociation from the protein-metal complex hybrid materials. One of the challenges in this field lies in the generation of organometallic complexes that are 'biocompatible', i.e. complexes that are stable under physiological conditions. The main objective of the work described in this thesis has been the development of a versatile and straightforward method to covalently and irreversibly incorporate organometallic complexes into a protein structure in a single site-directed manner. In this work, the versatile organometallic ECE-pincer metal complex system was chosen as the metal containing component of the protein-metal complex hybrid. As metal complex anchoring functionality the p-nitrophenyl phosphonate ester group have been selected as these have shown to be highly potent and irreversible inhibitors of the large family of serine hydrolases. Cutinase from Fusarium solani pisi was selected as model enzyme to demonstrate the versatility of the developed approach. The general concept of our molecular design is a pincer metal complex covalently connected through a carbon-phosphorous bond to the phosphonate active ester. The first part of this thesis is focused on the design and synthesis of these complexes as well as on the subsequent reactivity of such compounds with cutinase. In the second part several aspects of ECE-pincer metal complexes are described that are of importance when such complexes are applied in protein modification strategies. This entails a study of the conformational behavior of SCS-pincer metal complexes in solution and solid state, as well as interesting structural features and behavior in solution of highly water-soluble pincer metal complexes having a sulfate moiety as ligand or counterion.