Semisynthetic Pincer Metalloenzymes : A Match between Organometallic and Protein Building Blocks

Abstract

The modification of biomolecules by transition metal complexes has become an increasingly important area of research in recent years. As transition metal complexes possess a high electron density around the metal centre, they display a wide range of reactivities rendering them extremely useful target molecules in e.g. catalysis and coordination studies. When transition metal complexes are used to modify biological molecules such as carbohydrates, peptides and proteins, these special properties can be used to alter the structure and function of the respective biomolecule. This combination of transition metal and biochemistry opens up exciting new possibilities in the structural study of biomolecules and drug targeting, in which the special properties of the metal centre can be exploited to elucidate the structure and the mechanism of action. Furthermore, by synthetic modification with metal complexes, the intrinsic properties of the biomolecule can be altered and novel functionalities can be added, e.g. novel spectroscopic or non-natural catalytic features. A group of transition metal complexes, which have been studied intensively, are the so-called pincer complexes. Due to their specific structural features and remarkable stability, ECE-pincer metal complexes have found numerous applications, ranging from their uses such as catalysts, organometallic switches, and heavy atom probes to sensoring applications. Recently, several ECE-pincer metal complexes substituted by protein-reactive phosphonate groups have been developed in our group. As phosphonates have been known to be site-selective inhibitors for serine hydrolases binding covalently to the reactive nucleophile serine in the active site of serine hydrolases, the ECE-pincer metal substituted phosphonates could be used for the single-site covalent labelling of the lipase cutinase (lipases belong to the superfamily of serine hydrolases), as shown recently. In this thesis various phosphonate functionalized organometallic complexes were immobilized onto the active site of lipases and used in structural, coordination, catalytic and protein labeling studies. First, a comprehensive overview of bioorganometallic pincer complexes known from literature is given (Chapter 1). In Chapter 2, five crystal structures of cutinase covalently modified with two different ECE-pincer metal phosphonate inhibitors were resolved. A study investigating the binding of the two opposite enantiomers of two different phosphonate pincer substrates to cutinase is described in Chapter 3. In Chapter 4 the coordination between a cationic NCN-pincer platinum cutinase hybrid and several phosphines is studied. Based on the successful coordination studies, a catalytic study with an SCS-pincer palladium cutinase hybrid was performed in Chapter 5. In Chapter 6 a novel luminescent organometallic biolabel based on a luminescent NCN-platinum complex is described. The coordination of a fluorescent stilbazole to a cationic NCN pincer platinum complex is investigated in Chapter 7. The synthesis and site-specific immobilization of a ruthenium racemization catalyst onto lipase beads is described in Chapter 8. This catalyst was successfully applied in a Dynamic Kinetic Resolution study of a secondary alcohol. The various applications of these transition metal complex lipase hybrids show the versatility of these systems and open up new possibilities for further studies in proteomics, novel catalytic reactions and supramolecular metal-biohybrid systems. By exploiting the unique structural and electronic properties of protein-embedded transition metal complexes, novel protein modification and studying tools are at hand, which can tremendously advance the field of protein science.