Noncanonical Amino Acids in Biocatalysis.

In recent years, powerful genetic code reprogramming methods have emerged that allow new functional components to be embedded into proteins as noncanonical amino acid (ncAA) side chains. In this review, we will illustrate how the availability of an expanded set of amino acid building blocks has opened a wealth of new opportunities in enzymology and biocatalysis research. Genetic code reprogramming has provided new insights into enzyme mechanisms by allowing introduction of new spectroscopic probes and the targeted replacement of individual atoms or functional groups. NcAAs have also been used to develop engineered biocatalysts with improved activity, selectivity, and stability, as well as enzymes with artificial regulatory elements that are responsive to external stimuli. Perhaps most ambitiously, the combination of genetic code reprogramming and laboratory evolution has given rise to new classes of enzymes that use ncAAs as key catalytic elements. With the framework for developing ncAA-containing biocatalysts now firmly established, we are optimistic that genetic code reprogramming will become a progressively more powerful tool in the armory of enzyme designers and engineers in the coming years.

Transition-Metal-Catalyzed C-H Bond Activation for the Formation of C-C Bonds in Complex Molecules.

Site-predictable and chemoselective C-H bond functionalization reactions offer synthetically powerful strategies for the step-economic diversification of both feedstock and fine chemicals. Many transition-metal-catalyzed methods have emerged for the selective activation and functionalization of C-H bonds. However, challenges of regio- and chemoselectivity have emerged with application to highly complex molecules bearing significant functional group density and diversity. As molecular complexity increases within molecular structures the risks of catalyst intolerance and limited applicability grow with the number of functional groups and potentially Lewis basic heteroatoms. Given the abundance of C-H bonds within highly complex and already diversified molecules such as pharmaceuticals, natural products, and materials, design and selection of reaction conditions and tolerant catalysts has proved critical for successful direct functionalization. As such, innovations within transition-metal-catalyzed C-H bond functionalization for the direct formation of carbon-carbon bonds have been discovered and developed to overcome these challenges and limitations. This review highlights progress made for the direct metal-catalyzed C-C bond forming reactions including alkylation, methylation, arylation, and olefination of C-H bonds within complex targets.

A non-canonical nucleophile unlocks a new mechanistic pathway in a designed enzyme.

Directed evolution of computationally designed enzymes has provided new insights into the emergence of sophisticated catalytic sites in proteins. In this regard, we have recently shown that a histidine nucleophile and a flexible arginine can work in synergy to accelerate the Morita-Baylis-Hillman (MBH) reaction with unrivalled efficiency. Here, we show that replacing the catalytic histidine with a non-canonical Nδ-methylhistidine (MeHis23) nucleophile leads to a substantially altered evolutionary outcome in which the catalytic Arg124 has been abandoned. Instead, Glu26 has emerged, which mediates a rate-limiting proton transfer step to deliver an enzyme (BHMeHis1.8) that is more than an order of magnitude more active than our earlier MBHase. Interestingly, although MeHis23 to His substitution in BHMeHis1.8 reduces activity by 4-fold, the resulting His containing variant is still a potent MBH biocatalyst. However, analysis of the BHMeHis1.8 evolutionary trajectory reveals that the MeHis nucleophile was crucial in the early stages of engineering to unlock the new mechanistic pathway. This study demonstrates how even subtle perturbations to key catalytic elements of designed enzymes can lead to vastly different evolutionary outcomes, resulting in new mechanistic solutions to complex chemical transformations.