Loreto, Domenico (2023) Functionalization of protein crystals with metal complexes. [Tesi di dottorato]

Anteprima

Testo Loreto_Domenico_35.pdf Download (34MB) | Anteprima

Abstract

Dirhodium paddlewheel complexes are useful in several application fields, like catalysis, material or medicinal chemistry. Although artificial metalloenzymes based on the dirhodium tetracarboxylate reactivity with proteins have been developed, little is known on their molecular structures. The aim of this thesis is to provide new insights about the dirhodium tetracarboxylates/proteins recognition process and to explore the catalytic properties of dirhodium-conjugated cross-linked protein crystals. The interaction between dirhodium tetraacetate ([Rh2(OAc)4]) and two of its derivatives, cis-[Rh2(OAc)2(tfa)2] and [Rh2(OAc)(tfa)3] (tfa = trifluoroacetate ion), with the two model proteins hen egg white lysozyme (HEWL) and bovine pancreatic ribonuclease (RNase A) was investigated both in solution and in the solid state. X-ray crystallography studies revealed that the three complexes degrade upon reaction with HEWL, while their dimetallic core is retained upon reaction with RNase A. In the dirhodium tetraacetate/RNase A adduct, the dimetallic centre axially binds His side chains. However, the bridging ligands surrounding the dirhodium core have a significant influence in directing the interaction between these complexes and proteins, since upon reaction of [Rh2(OAc)(tfa)3] with RNase A equatorial binding of dirhodium core to His side chains can also be observed. The reactivity of the [Rh2(OAc)4]/RNase A adduct in the solid state toward imidazole and glycine was also studied. Surprisingly, imidazole binds the dirhodium tetraacetate at equatorial rather than axial position. Three acetate ligands are replaced by six water molecules. DFT studies clarified that this unexpected reactivity is triggered upon hydrolysis of the dirhodium core, once that the metal compound is axially bound to a first molecule of imidazole (provided by His residues). Electronic structure analysis reveals that the Rh atom which is coordinated to the protein acts as a better Lewis acid than the other Rh atom. Glycine also binds the dirhodium core replacing equatorial acetate ligands. Once verified that dirhodium tetraacetate retains its ability to react with small molecules when bound to RNase A in the solid state, catalysis experiments were carried out. Unfortunately, crystals of RNase A used to verify the binding of [Rh2(OAc)4], cis-[Rh2(OAc)2(tfa)2] and [Rh2(OAc)(tfa)3] to the protein (C2 space group with two molecules in the asymmetric unit) are not the best choice to perform catalysis experiments since they need long preparation times (3 weeks). For this reason, crystals of the dirhodium/RNase A adduct were prepared in a different space group (P3221), that allows the protein crystal growth and functionalization with [Rh2(OAc)4] in shorter times (26 h). X-ray structures of the [Rh2(OAc)4]/RNase A adducts from P3221 crystals exposed to the dirhodium compound for different times reveal that the protein is extensively metalated by [Rh2(OAc)4] just after 2 h of soaking. Also in this adduct, the metal compound structure is unperturbed upon 2 h protein binding. The dirhodium core is axially coordinated to His105 and His119 side chains, as observed for the metal/protein adduct crystals obtained into C2 space group. Cross-linking crystals of the adduct were then prepared using glutaraldehyde as cross-linking agent and the gentle diffusion technique. The structure of the adduct was solved by X-ray crystallography at two different temperatures (-173 and 0 °C). The results reveal no significant differences when the two structures are compared to each other and with the structure of the adduct from crystals in the C2 space group. The use of the cross-linked crystals of the [Rh2(OAc)4]/RNase A adduct as catalysts towards olefine cyclopropanation and self-coupling of diazo compounds reactions was investigated using styrene and ethyl diazoacetate as model reagents. The results revealed that they catalyse both the reactions. While several by-products are observed upon ethyl diazoacetate homocoupling reaction, the styrene cyclopropanation occurs producing the ethyl-phenylcyclopropane-1-carboxylate with high selectivity. Overall, these data provide the proof-of-concept for the catalytic use of cross-linked crystals of proteins in adduct with dirhodium compounds.

Downloads

Actions (login required)

Modifica documento