Ward, T.R.

J. Am. Chem. Soc. 2016, 138, 5781-5784, 10.1021/jacs.6b02470

Enzymes typically depend on either NAD(P)H or FADH2 as hydride source for reduction purposes. In contrast, organometallic catalysts most often rely on isopropanol or formate to generate the reactive hydride moiety. Here we show that incorporation of a Cp*Ir cofactor possessing a biotin moiety and 4,7-dihydroxy-1,10-phenanthroline into streptavidin yields an NAD(P)H-dependent artificial transfer hydrogenase (ATHase). This ATHase (0.1 mol%) catalyzes imine reduction with 1 mM NADPH (2 mol%), which can be concurrently regenerated by a glucose dehydrogenase (GDH) using only 1.2 equiv of glucose. A four-enzyme cascade consisting of the ATHase, the GDH, a monoamine oxidase, and a catalase leads to the production of enantiopure amines.

Ward, T.R.

J. Am. Chem. Soc. 2019, 141, 15869-15878, 10.1021/jacs.9b06923

The biotin–streptavidin technology has been extensively exploited to engineer artificial metalloenzymes (ArMs) that catalyze a dozen different reactions. Despite its versatility, the homotetrameric nature of streptavidin (Sav) and the noncooperative binding of biotinylated cofactors impose two limitations on the genetic optimization of ArMs: (i) point mutations are reflected in all four subunits of Sav, and (ii) the noncooperative binding of biotinylated cofactors to Sav may lead to an erosion in the catalytic performance, depending on the cofactor:biotin-binding site ratio. To address these challenges, we report on our efforts to engineer a (monovalent) single-chain dimeric streptavidin (scdSav) as scaffold for Sav-based ArMs. The versatility of scdSav as host protein is highlighted for the asymmetric transfer hydrogenation of prochiral imines using [Cp*Ir(biot-p-L)Cl] as cofactor. By capitalizing on a more precise genetic fine-tuning of the biotin-binding vestibule, unrivaled levels of activity and selectivity were achieved for the reduction of challenging prochiral imines. Comparison of the saturation kinetic data and X-ray structures of [Cp*Ir(biot-p-L)Cl]·scdSav with a structurally related [Cp*Ir(biot-p-L)Cl]·monovalent scdSav highlights the advantages of the presence of a single biotinylated cofactor precisely localized within the biotin-binding vestibule of the monovalent scdSav. The practicality of scdSav-based ArMs was illustrated for the reduction of the salsolidine precursor (500 mM) to afford (R)-salsolidine in 90% ee and >17 000 TONs. Monovalent scdSav thus provides a versatile scaffold to evolve more efficient ArMs for in vivo catalysis and large-scale applications.

Ward, T.R.

Angew. Chem. Int. Ed. 2017, 56, 10156-10160, 10.1002/anie.201702181

Cross‐regulation of complex biochemical reaction networks is an essential feature of living systems. In a biomimetic spirit, we report on our efforts to program the temporal activation of an artificial metalloenzyme via cross‐regulation by a natural enzyme. In the presence of urea, urease slowly releases ammonia that reversibly inhibits an artificial transfer hydrogenase. Addition of an acid, which acts as fuel, allows to maintain the system out of equilibrium.

Mangiatordi, G.F.; Salmain, M.

J. Mol. Catal. B: Enzym. 2015, 122, 314-322, 10.1016/j.molcatb.2015.10.007

Two half-sandwich d6-rhodium(III) complexes of the general formula [(η5-Cp*)Rh(N^N)Cl]Cl where N^N is a phenanthroline or a bispyridine methane derivative carrying a thiol-targeting maleimide or chloroacetamide function were synthesized and characterized. Both complexes were able to catalyse the transfer hydrogenation of 2,2,2-trifluoroacetophenone in aqueous medium using formate or phosphite as hydrogen donor. Covalent anchoring of these complexes to the cysteine endoproteinase papain yielded hybrid metalloproteins with transfer hydrogenase properties. Under optimized conditions of pH, hydrogen donor concentration and catalyst load, conversion of substrate was nearly quantitative within 24 h at 40 °C and the (S)-enantiomer was obtained preferably albeit with a modest enantiomeric excess of 7–10%. Covalent docking simulations complemented the experimental findings suggesting a molecular rationale for the observed low enantioselectivity. The harmonious use of experimental and theoretical approaches represents an unprecedented starting point for driving the rational design of artificial metalloenzymes built up from papain with higher catalytic efficiency.