The mechanism of nickel(0)-catalyzed reductive coupling of aldehydes and alkynes has been studied. Extensive double-labeling crossover studies have been conducted. While previous studies illustrated that phosphine- and N-heterocyclic carbene-derived catalysts exhibited differing behavior, the origin of these effects has now been evaluated in detail. Many variables, including ligand class, sterics of the ligand and alkyne, temperature, and ring size being formed in intramolecular versions, all influence the extent of crossover observed. A computational evaluation of these effects suggests that dimerization of a key metallacyclic intermediate provides the origin of crossover. Protocols that proceed with crossover are typically less efficient than those without crossover given the thermodynamic stability and low reactivity of the dimeric metallacycles involved in crossover pathways.

The control of regiochemistry is a considerable challenge in the development of a wide array of catalytic processes. Simple π-components such as alkenes, alkynes, 1,3-dienes, and allenes are among the many classes of substrates that present complexities in regioselective catalysis. Considering an internal alkyne as a representative example, when steric and electronic differences between the two substituents are minimal, differentiating among the two termini of the alkyne presents a great challenge. In cases where the differences between the alkyne substituents are substantial, overcoming those biases to access the regioisomer opposite that favored by substrate biases often presents an even greater challenge. Nickel-catalyzed reductive couplings of unsymmetrical π-components make up a group of reactions where control of regiochemistry presents a challenging but important objective. In the course of our studies of aldehyde-alkyne reductive couplings, complementary solutions to challenges in regiocontrol have been developed. Through careful selection of the ligand and reductant, as well as the more subtle reaction variables such as temperature and concentration, effective protocols have been established that allow highly selective access to either regiosiomer of the allylic alcohol products using a wide range of unsymmetrical alkynes. Computational studies and an evaluation of reaction kinetics have provided an understanding of the origin of the regioselectivity control. Throughout the various procedures described, the development of ligand-substrate interactions plays an essential role, and the overall kinetic descriptions were found to differ between protocols. Rational alteration of the rate-determining step plays a key role in the regiochemistry reversal strategy, and in one instance, the two possible regioisomeric outcomes in a single reaction were found to operate by different kinetic descriptions. With this mechanistic information in hand, the empirical factors that influence regiochemistry can be readily understood, and more importantly, the insights suggest simple and predictable experimental variables to achieving a desired reaction outcome. These studies thus present a detailed picture of the influences that control regioselectivity in a specific catalytic reaction, but they also delineate strategies for regiocontrol that may extend to numerous classes of reactions. The work provides an illustration of how insights into the kinetics and mechanism of a catalytic process can rationalize subtle empirical findings and suggest simple and rational modifications in procedure to access a desirable reaction outcome. Furthermore, these studies present an illustration of how important challenges in organic synthesis can be met by novel reactivity afforded by base metal catalysis. The use of nickel catalysis in this instance not only provides an inexpensive and sustainable method for catalysis but also enables unique reactivity patterns not accessible to other metals.

Highly regioselective remote hydroxylation of a natural product scaffold is demonstrated by exploiting the anchoring mechanism of the biosynthetic P450 monooxygenase PikCD50N-RhFRED. Previous studies have revealed structural and biochemical evidence for the role of a salt bridge between the desosamine N,N-dimethylamino functionality of the natural substrate YC-17 and carboxylate residues within the active site of the enzyme, and selectivity in subsequent C-H bond functionalization. In the present study, a substrate-engineering approach was conducted that involves replacing desosamine with varied synthetic N,N-dimethylamino anchoring groups. We then determined their ability to mediate enzymatic total turnover numbers approaching or exceeding that of the natural sugar, while enabling ready introduction and removal of these amino anchoring groups from the substrate. The data establish that the size, stereochemistry, and rigidity of the anchoring group influence the regioselectivity of enzymatic hydroxylation. The natural anchoring group desosamine affords a 1:1 mixture of regioisomers, while synthetic anchors shift YC-17 analogue C-10/C-12 hydroxylation from 20:1 to 1:4. The work demonstrates the utility of substrate engineering as an orthogonal approach to protein engineering for modulation of regioselective C-H functionalization in biocatalysis.

The diversification of late stage synthetic intermediates provides significant advantages in efficiency in comparison to conventional linear approaches. Despite these advantages, accessing varying ring scaffolds and functional group patterns from a common intermediate poses considerable challenges using existing methods. The combination of regiodivergent nickel-catalyzed C-C couplings and site-selective biocatalytic C-H oxidations using the cytochrome P450 enzyme PikC addresses this problem by enabling a single late-stage linear intermediate to be converted to macrolactones of differing ring size and with diverse patterns of oxidation. The approach is made possible by a novel strategy for site-selective biocatalytic oxidation using a single biocatalyst, with site selectivity being governed by a temporarily installed directing group. Site selectivities of C-H oxidation by this directed approach can overcome positional bias due to C-H bond strength, acidity, inductive influences, steric accessibility, or immediate proximity to the directing group, thus providing complementarity to existing approaches.

The hallmark of enzymes from secondary metabolic pathways is the pairing of powerful reactivity with exquisite site selectivity. The application of these biocatalytic tools in organic synthesis, however, remains under-utilized due to limitations in substrate scope and scalability. Here, we report how the reactivity of a monooxygenase (PikC) from the pikromycin pathway is modified through computationally guided protein and substrate engineering, and applied to the oxidation of unactivated methylene C-H bonds. Molecular dynamics and quantum mechanical calculations were used to develop a predictive model for substrate scope, site selectivity and stereoselectivity of PikC-mediated C-H oxidation. A suite of menthol derivatives was screened computationally and evaluated through in vitro reactions, where each substrate adhered to the predicted models for selectivity and conversion to product. This platform was also expanded beyond menthol-based substrates to the selective hydroxylation of a variety of substrate cores ranging from cyclic to fused bicyclic and bridged bicyclic compounds.

Iterative P450 enzymes are powerful biocatalysts for selective late-stage C-H oxidation of complex natural product scaffolds. These enzymes represent useful tools for selectivity and cascade reactions, facilitating direct access to core structure diversification. Recently, we reported the structure of the multifunctional bacterial P450 TamI and elucidated the molecular basis of its substrate binding and strict reaction sequence at distinct carbon atoms of the substrate. Here, we report the design and characterization of a toolbox of TamI biocatalysts, generated by mutations at Leu101, Leu244, and/or Leu295, that alter the native selectivity, step sequence, and number of reactions catalyzed, including the engineering of a variant capable of catalyzing a four-step oxidative cascade without the assistance of the flavoprotein and oxidative partner TamL. The tuned enzymes override inherent substrate reactivity, enabling catalyst-controlled C-H functionalization and alkene epoxidation of the tetramic acid-containing natural product tirandamycin. Five bioactive tirandamycin derivatives (6-10) were generated through TamI-mediated enzymatic synthesis. Quantum mechanics calculations and MD simulations provide important insights into the basis of altered selectivity and underlying biocatalytic mechanisms for enhanced continuous oxidation of the iterative P450 TamI.

Knowledge of the three-dimensional movement patterns of elasmobranchs is vital to understand their ecological roles and exposure to anthropogenic pressures. To date, comparative studies among species at global scales have mostly focused on horizontal movements. Our study addresses the knowledge gap of vertical movements by compiling the first global synthesis of vertical habitat use by elasmobranchs from data obtained by deployment of 989 biotelemetry tags on 38 elasmobranch species. Elasmobranchs displayed high intra- and interspecific variability in vertical movement patterns. Substantial vertical overlap was observed for many epipelagic elasmobranchs, indicating an increased likelihood to display spatial overlap, biologically interact, and share similar risk to anthropogenic threats that vary on a vertical gradient. We highlight the critical next steps toward incorporating vertical movement into global management and monitoring strategies for elasmobranchs, emphasizing the need to address geographic and taxonomic biases in deployments and to concurrently consider both horizontal and vertical movements.