Michela Pellizzoni

Artificial Metalloenzymes: Reaction Scope and Optimization Strategies

The incorporation of a synthetic, catalytically competent metallocofactor into a protein scaffold to generate an artificial metalloenzyme (ArM) has been explored since the late 1970s. Progress in the ensuing years was limited by the tools available for both organometallic synthesis and protein engineering. Advances in both of these areas, combined with increased appreciation of the potential benefits of combining attractive features of both homogeneous catalysis and enzymatic catalysis, led to a resurgence of interest in ArMs starting in the early 2000s. Perhaps the most intriguing of potential ArM properties is their ability to endow homogeneous catalysts with a genetic memory. Indeed, incorporating a homogeneous catalyst into a genetically encoded scaffold offers the opportunity to improve ArM performance by directed evolution. This capability could, in turn, lead to improvements in ArM efficiency similar to those obtained for natural enzymes, providing systems suitable for practical applications and greater insight into the role of second coordination sphere interactions in organometallic catalysis. Since its renaissance in the early 2000s, different aspects of artificial metalloenzymes have been extensively reviewed and highlighted. Our intent is to provide a comprehensive overview of all work in the field up to December 2016, organized according to reaction class. Because of the wide range of non-natural reactions catalyzed by ArMs, this was done using a functional-group transformation classification. The review begins with a summary of the proteins and the anchoring strategies used to date for the creation of ArMs, followed by a historical perspective. Then follows a summary of the reactions catalyzed by ArMs and a concluding critical outlook. This analysis allows for comparison of similar reactions catalyzed by ArMs constructed using different metallocofactor anchoring strategies, cofactors, protein scaffolds, and mutagenesis strategies. These data will be used to construct a searchable Web site on ArMs that will be updated regularly by the authors.

Improving the Catalytic Performance of an Artificial Metalloenzyme by Computational Design.

Artifical metalloenzymes combine the reactivity of small molecule catalysts with the selectivity of enzymes, and new methods are required to tune the catalytic properties of these systems for an application of interest. Structure-based computational design could help to identify amino acid mutations leading to improved catalytic activity and enantioselectivity. Here we describe the application of Rosetta Design for the genetic optimization of an artificial transfer hydrogenase (ATHase hereafter), [(η(5)-Cp*)Ir(pico)Cl] ⊂ WT hCA II (Cp* = Me5C5(-)), for the asymmetric reduction of a cyclic imine, the precursor of salsolsidine. Based on a crystal structure of the ATHase, computational design afforded four hCAII variants with protein backbone-stabilizing and hydrophobic cofactor-embedding mutations. In dansylamide-competition assays, these designs showed 46-64-fold improved affinity for the iridium pianostool complex [(η(5)-Cp*)Ir(pico)Cl]. Gratifyingly, the new designs yielded a significant improvement in both activity and enantioselectivity (from 70% ee (WT hCA II) to up to 92% ee and a 4-fold increase in total turnover number) for the production of (S)-salsolidine. Introducing additional hydrophobicity in the Cp*-moiety of the Ir-catalyst provided by adding a propyl substituent on the Cp* moiety yields the most (S)-selective (96% ee) ATHase reported to date. X-ray structural data indicate that the high enantioselectivity results from embedding the piano stool moiety within the protein, consistent with the computational model.

Cytotoxic effect of (1-methyl-1H-imidazol-2-yl)-methanamine and its derivatives in PtII complexes on human carcinoma cell lines: A comparative study with cisplatin

The synthesis and pharmacological characterisation of (1-methyl-1H-imidazol-2-yl)-methanamine and its derivatives in Pt(II) complexes are described. Six out of eleven new Pt(II) complexes showed a significant cytotoxic effect on NCI-H460 lung cancer cell line with EC50 values between 1.1 and 0.115 mM, determined by MTT assay. Compound Pt-4a showed a particularly more potent cytotoxic effect than the previously described Pt(II) complex with 2,2-bipyridine, [Pt(bpy)Cl2], with an EC50 value equal to 172.7 μM versus 726.5 μM respectively, and similar potency of cisplatin (EC50=78.3 μM) in NCI-H460 cell line. The determination of the intracellular and DNA-bound concentrations of (195)Pt, as marker of the presence of the complexes, showed that the cytotoxic compound Pt-4a readily diffused into the cells to a similar extent of cisplatin and directly interacted with the nuclear DNA. Pt-4a induced both p53 and p21(Waf) expression in NCI-H460 cells similar to cisplatin. A direct comparison of the cytotoxic effect between compound Pt-4a and cisplatin on 12 different cancer cell lines demonstrated that compound Pt-4a was in general less potent than cisplatin, but it had a comparable cytotoxic effect on non-small-cell lung cancer NCI-H460 cells, and the colorectal cancer cells HCT-15 and HCT-116. Altogether, these results suggested that the Pt(II) complex with 1-methyl-1H-imidazol-2-yl)-methanamine (compound Pt-4a), displayed a significant cytotoxic activity in cancer cells. Similarly to cisplatin this compound interacts with nuclear DNA and induces both p53 and p21(waf), and thus it represents an interesting starting point for future optimisation of new Pt(II) complexes forming DNA adducts.

Simple 1,3-diamines and their application as ligands in ruthenium(II) catalysts for asymmetric transfer hydrogenation of aryl ketones

In this research work simple unsymmetrical 1,3-diamines were studied. The synthesis of the diamines started from non-commercial available compounds. S-5a and S,S-5c were obtained by biocatalysis with non conventional yeast, Rhodotorula rubra MIM 147, with excellent 99% e.e. and d.e. up to 90%. Different approaches of synthesis were applied to the same backbone to study both the steric and electronic effects of the ligands. The reactivity of the corresponding ruthenium complexes was evaluated in the asymmetric hydrogen transfer reduction of acetophenone as a standard substrate and of other different aryl ketones, highlighting the flexibility of the six membered chelating ring. A screening of the reaction conditions indicated aqueous media in the presence of HCOONa as a hydrogen donor to be the best system for overcoming the lack of stereocontrol thus allowing us to obtain 56% e.e. in the reduction of acetophenone with the complex in which the ligand was diamine 1, revealed as the best in terms of reactivity and stereoselectivity also in the reduction of the other different aryl ketones, in particular for α-tetralone, i (63% e.e.).

Evaluation of Chemical Diversity of Biotinylated Chiral 1,3‐Diamines as a Catalytic Moiety in Artificial Imine Reductase

The possibility of obtaining an efficient artificial imine reductase was investigated by introducing a chiral cofactor into artificial metalloenzymes based on biotin–streptavidin technology. In particular, a chiral biotinylated 1,3‐diamine ligand in coordination with iridium(III) complex was developed. Optimized chemogenetic studies afforded positive results in the stereoselective reduction of a cyclic imine, the salsolidine precursor, as a standard substrate with access to both enantiomers. Various factors such as pH, temperature, number of binding sites, and steric hindrance of the catalytic moiety have been proved to affect both efficiency and enantioselectivity, underlining the great flexibility of this system in comparison with the achiral system. Computational studies were also performed to explain how the metal configuration, in the proposed system, might affect the observed stereochemical outcome.