Incorporation of a Cp*Rh(III)-dithiophosphate Cofactor with Latent Activity into a Protein Scaffold Generates a Biohybrid Catalyst Promoting C(sp²)–H Bond Functionalization

Abstract

A Cp*Rh(III)-dithiophosphate cofactor with "latent" catalytic activity was developed to construct an artificial metalloenzyme representing a new type of biohybrid catalyst which is capable of promoting C(sp 2 )−H bond functionalization within the β-barrel structure of nitrobindin (NB). To covalently conjugate the Cp*Rh(III) cofactor into a specific position of the hydrophobic cavity of NB via a maleimide−Cys linkage, strong chelation of the dithiophosphate ligand is employed to protect the rhodium metal center against attack by nucleophilic amino acid residues in the protein. It is found that subsequent addition of the Ag + ion induces dissociation of the dithiophosphate ligands, thereby activating the catalytic activity of the Cp*Rh(III) cofactor. The resulting "active" biohybrid catalyst promotes cycloaddition of acetophenone oxime with diphenylacetylene via C(sp 2 )−H bond activation. This catalytic activity is enhanced 2.3-fold with the introduction of two glutamate residues (A100E/L125E) adjacent to the Cp*Rh(III) cofactor. The Cp*Rh(III) cofactor with switchable activity from a "latent" form to an "active" form provides a new strategy for generating biohybrid catalysts incorporating a variety of highly reactive transition metal complexes specifically within its protein scaffolds.

Directed Evolution of a Cp*RhIII‐Linked Biohybrid Catalyst Based on a Screening Platform with Affinity Purification

“…The prepared biohybrid catalysts were then investigated for the cycloaddition reaction of 2 a with 3 a . The reactions were performed under the optimized conditions according to our previous study (20 μM catalyst in AcOH buffer at pH 4.0), [6] and it was found that all three biohybrid catalyst variants had enhanced catalytic activities for the cycloaddition reaction (Figure 5). In particular, NB(T98H/L100K/K127E)‐ 1 was found to provide product 4 aa with a 4.9‐fold increase in activity compared to the original NB‐ 1 .…”

“…Biohybrid catalysts incorporating a synthetic Cp*Rh III complex as a cofactor have significant potential to enable a broad range of abiotic C−H bond functionalizations [4,5] . Our group has recently prepared a biohybrid catalyst incorporating a Cp*Rh III cofactor 1 within a hydrophobic cavity of nitrobindin (NB) protein (Figure 1a and b) [6] . Protection of a reactive rhodium center using dithiophosphate ligands enables us to incorporate a highly electrophilic Cp*Rh III complex at a defined position within NB (Figure 1c).…”

Designing Artificial Metalloenzymes by Tuning of the Environment beyond the Primary Coordination Sphere

“…C96 nitrobindin was employed to generate an ArM capable of C­(sp 2 )–H bond functionalization via concerted metalation deprotonation (CMD) with 15% yield and 92:8 regioselectivity through the covalent binding of a maleimide-tagged Rh complex. Carboxylates are known to facilitate the CMD-type C­(sp 2 )–H bond functionalization, and therefore, glutamate residues were introduced near the active site in a L100E/A125E mutant, which increased the yields to 40% with similar regioselectivity (90/10) . Directed evolution was later applied to this system, and the T98H/L100K/K127E mutant was identified to increase the activity 2.2-fold.…”

“…Carboxylates are known to facilitate the CMD-type C(sp 2 )−H bond functionalization, and therefore, glutamate residues were introduced near the active site in a L100E/A125E mutant, which increased the yields to 40% with similar regioselectivity (90/10). 593 Directed evolution was later applied to this system, and the T98H/L100K/K127E mutant was identified to increase the activity 2.2-fold. Interestingly, MD simulations suggested that all three of these residues are located opposite to the Rh metal center.…”

Chemogenetic Evolution of a Peroxidase-like Artificial Metalloenzyme

“…Nitrobindin (Arabidopsis thaliana) is a 10-stranded β-barrel heme protein, which has widely been used as a scaffold protein for the construction of ArMs. − In particular, the hydrophobic cavity of the heme-free nitrobindin variant NB4 (amino acid substitutions compared to the wildtype protein: M75L/H76L/Q96C/M148L/H158L) has proven to be a very versatile active site. After covalent anchoring of a wide variety of artificial cofactors, transformations were accomplished, including carbon–carbon bond forming reactions, − epoxidation, and hydrogenase activity …”