Metallocarbene Artificial Enzymes : Extending Transition Metal Selectivity and Protein Activity

Abstract

A series of new semi-synthetic metalloprotein hybrids were created via the covalent binding of organometallic species in the active site of lipases, accordingly resulting in the first active site-directed (ASD) homogeneous artificial metalloenzymes. The use of this method promises the generation of a 2nd coordination sphere by the protein scaffold over an incorporated transition metal complex, thereby promoting stereodirecting properties, i.e. catalytic selectivity, and water solubility to the organometallic fragment, and diversifying the hosting enzyme’s activity.

A review of the different metalloenzyme hybridization strategies is given in Chapter 1, with comparisons between their corresponding advantages. The ASD method in combination with N-heterocyclic carbene (NHC) ligands is proposed as a versatile strategy due to the good catalytic activity and water-tolerance of a myriad of known metal(NHC) species.

In Chapter 2, two covalent ASD Rh(NHC)-lipase hybrids were created. These and other hybrids of the thesis were characterized by ESI-MS and by the resulting catalytic activity. A Rh-cutinase hybrid showed catalytic activity in the hydrogenation of the ketone acetophenone and the olefin methyl 2-acetamidoacrylate with an enhanced chemoselectivity towards the olefin promoted by the protein scaffold compared to unsupported Rh catalyst. A rhodium-CalB hybrid showed pronounced chemoselective behavior due to the deeper location of the lipase’s active pocket.

In Chapter 3, Grubbs-Hoveyda II complexes were reacted with cutinase to form the first covalent ASD artificial enzymes for olefin metathesis. Important metal-protein interactions were observed, like hindered hybridization when bulky N-substituents in the NHC ligands were used and protein-metal distance influence over the catalytic activity with successfully formed hybrids. The ring-closing metathesis (RCM) of N,N-diallyl p-toluenesulfonamide and the cross metathesis (CM) of allylbenzene were achieved, the latter representing the first example of formal CM with metalloenzymes.

In Chapter 4, the catalytic asymmetric allylic alkylation of allyl aryls was explored with the first covalent artificial metallo-enzyme based on palladium. PPh3 was needed as an additive to achieve catalytic activity. Its role was studied, discarding Pd(NHC)-protein cleavage and showing activation of the Pd center. Inactive in the alkylation of 1,3-diphenyl allyl acetate with malonate due to sterical hindrance, the hybrids were however active in the alkylation of the smaller 1-phenylallyl acetate. In this case, the stereodirecting influence of the protein scaffold drastically inverted the linear/branched ratio of the products in comparison with unsupported Pd catalysts.

In Chapter 5, it was pursued to complement the promotion of a second coordination sphere over a Rh(NHC) hydrogenation catalyst by its compartmentalization between two enzyme hosting units, forming the first monometallic ditopic artificial enzyme. Catalytic studies and computational modeling of the hybrid showed that the catalytic center retained its racemic and chemoselective character as if it were not supported, due to large metal-protein tail allowing for flexibility in the protein-metal-protein conformation.

In conclusion, the first covalent homogeneous ASD metalloprotein catalysts have been created. This hybridization method shows its effectiveness for the anchoring of a single catalytic fragment in a family of enzymes and the formation of strong protein-metal interactions, generating catalytic chemoselectivity and regioselectivity.