Enzyme-catalyzed cationic epoxide rearrangements in quinolone alkaloid biosynthesis.

Epoxides are highly useful synthons and biosynthons for the construction of complex natural products during total synthesis and biosynthesis, respectively. Among enzyme-catalyzed epoxide transformations, a reaction that is notably missing, in regard to the synthetic toolbox, is cationic rearrangement that takes place under strong acid. This is a challenging transformation for enzyme catalysis, as stabilization of the carbocation intermediate upon epoxide cleavage is required. Here, we discovered two Brønsted acid enzymes that can catalyze two unprecedented epoxide transformations in biology. PenF from the penigequinolone pathway catalyzes a cationic epoxide rearrangement under physiological conditions to generate a quaternary carbon center, while AsqO from the aspoquinolone pathway catalyzes a 3-exo-tet cyclization to forge a cyclopropane-tetrahydrofuran ring system. The discovery of these new epoxide-modifying enzymes further highlights the versatility of epoxides in complexity generation during natural product biosynthesis

Computational Protocol to Understand P450 Mechanisms and Design of Efficient and Selective Biocatalysts

Cytochrome P450 enzymes have gained significant interest as selective oxidants in late-stage chemical synthesis. Their broad substrate scope enables them to be good candidates for their use in non-natural reactivity. Directed evolution evolves new enzyme biocatalysts that promote alternative reactivity for chemical synthesis. While directed evolution has proven useful in developing biocatalysts for specific purposes, this process is very time and labor intensive, and therefore not easily repurposed. Computational analysis of these P450 enzymes provides great insights into the broad substrate scope, the variety of reactions catalyzed, the binding specificity and the study of novel biosynthetic reaction mechanisms. By discovering new P450s and studying their reactivities, we uncover new insights into how this reactivity can be harnessed. We discuss a standard protocol using both DFT calculations and MD simulations to study a variety of cytochrome P450 enzymes. The approach entails theozyme models to study the mechanism and transition states via DFT calculations and subsequent MD simulations to understand the conformational poses and binding mechanisms within the enzyme. We discuss a few examples done in collaboration with the Tang and Sherman/Montgomery groups toward elucidating enzyme mechanisms and rationally designing new enzyme mutants as tools for selective C–H functionalization methods

Selective Enzymatic Oxidation of Silanes to Silanols

Compared to the biological world's rich chemistry for functionalizing carbon, enzymatic transformations of the heavier homologue silicon are rare. We report that a wild‐type cytochrome P450 monooxygenase (P450_(BM3) from Bacillus megaterium, CYP102A1) has promiscuous activity for oxidation of hydrosilanes to give silanols. Directed evolution was applied to enhance this non‐native activity and create a highly efficient catalyst for selective silane oxidation under mild conditions with oxygen as the terminal oxidant. The evolved enzyme leaves C−H bonds present in the silane substrates untouched, and this biotransformation does not lead to disiloxane formation, a common problem in silanol syntheses. Computational studies reveal that catalysis proceeds through hydrogen atom abstraction followed by radical rebound, as observed in the native C−H hydroxylation mechanism of the P450 enzyme. This enzymatic silane oxidation extends nature's impressive catalytic repertoire

A Biocatalytic Platform for Synthesis of Chiral α-Trifluoromethylated Organoborons

There are few biocatalytic transformations that produce fluorine-containing molecules prevalent in modern pharmaceuticals. To expand the scope of biocatalysis for organofluorine synthesis, we have developed an enzymatic platform for highly enantioselective carbene B–H bond insertion to yield versatile α-trifluoromethylated (α-CF_3) organoborons, an important class of organofluorine molecules that contain stereogenic centers bearing both CF_3 and boron groups. In contrast to current “carbene transferase” enzymes that use a limited set of simple diazo compounds as carbene precursors, this system based on Rhodothermus marinus cytochrome c (Rma cyt c) can accept a broad range of trifluorodiazo alkanes and deliver versatile chiral α-CF_3 organoborons with total turnovers up to 2870 and enantiomeric ratios up to 98.5:1.5. Computational modeling reveals that this broad diazo scope is enabled by an active-site environment that directs the alkyl substituent on the heme CF_3-carbene intermediate toward the solvent-exposed face, thereby allowing the protein to accommodate diazo compounds with diverse structural features

Selective Enzymatic Oxidation of Silanes to Silanols

Compared to the biological world's rich chemistry for functionalizing carbon, enzymatic transformations of the heavier homologue silicon are rare. We report that a wild‐type cytochrome P450 monooxygenase (P450_(BM3) from Bacillus megaterium, CYP102A1) has promiscuous activity for oxidation of hydrosilanes to give silanols. Directed evolution was applied to enhance this non‐native activity and create a highly efficient catalyst for selective silane oxidation under mild conditions with oxygen as the terminal oxidant. The evolved enzyme leaves C−H bonds present in the silane substrates untouched, and this biotransformation does not lead to disiloxane formation, a common problem in silanol syntheses. Computational studies reveal that catalysis proceeds through hydrogen atom abstraction followed by radical rebound, as observed in the native C−H hydroxylation mechanism of the P450 enzyme. This enzymatic silane oxidation extends nature's impressive catalytic repertoire

Origin and Control of Chemoselectivity in Cytochrome c Catalyzed Carbene Transfer into Si–H and N–H bonds

A cytochrome c heme protein was recently engineered to catalyze the formation of carbon–silicon bonds via carbene insertion into Si–H bonds, a reaction that was not previously known to be catalyzed by a protein. High chemoselectivity toward C–Si bond formation over competing C–N bond formation was achieved, although this trait was not screened for during directed evolution. Using computational and experimental tools, we now establish that activity and chemoselectivity are modulated by conformational dynamics of a protein loop that covers the substrate access to the iron–carbene active species. Mutagenesis of residues computationally predicted to control the loop conformation altered the protein’s chemoselectivity from preferred silylation to preferred amination of a substrate containing both N–H and Si–H functionalities. We demonstrate that information on protein structure and conformational dynamics, combined with knowledge of mechanism, leads to understanding of how non-natural and selective chemical transformations can be introduced into the biological world

Sponge-like molecular cage for purification of fullerenes

Since fullerenes are available in macroscopic quantities from fullerene soot, large efforts have been geared toward designing efficient strategies to obtain highly pure fullerenes, which can be subsequently applied in multiple research fields. Here we present a supramolecular nanocage synthesized by metal-directed self-assembly, which encapsulates fullerenes of different sizes. Direct experimental evidence is provided for the 1:1 encapsulation of C 60, C 70, C 76, C 78 and C 84, and solid state structures for the host-guest adducts with C 60 and C 70 have been obtained using X-ray synchrotron radiation. Furthermore, we design a washing-based strategy to exclusively extract pure C 60 from a solid sample of cage charged with a mixture of fullerenes. These results showcase an attractive methodology to selectively extract C 60 from fullerene mixtures, providing a platform to design tuned cages for selective extraction of higher fullerenes. The solid-phase fullerene encapsulation and liberation represent a twist in host-guest chemistry for molecular nanocage structures

Supramolecular fullerene sponges as catalytic masks for regioselective functionalization of C60

Isomer-pure poly-functionalized fullerenes are required to boost the development of fullerene chemistry in all fields. On a general basis, multi-adduct mixtures with uncontrolled regioselectivity are obtained, and the use of chromatographic purification is prohibitively costly and time consuming, especially in the production of solar cells. Single-isomer poly-functionalized fullerenes are only accessible via stoichiometric, multistep paths entailing protecting-unprotecting sequences. Herein, a nanocapsule is used as a supramolecular tetragonal prismatic mask to exert full control on the reactivity and the equatorial regioselectivity of Bingel-Hirsch cyclopropanation reactions of a confined C guest. Thus, equatorial bis-, tris-, and tetrakis-C homo-adducts are exclusively obtained in a stepwise manner. Furthermore, isomer-pure equatorial hetero-tetrakis-adducts or hetero-Th-hexakis-adducts are synthesized at will in one-pot synthesis for the first time. This work provides a synthetically valuable path to produce a plethora of new pure-isomer poly-functionalized C-based compounds as candidates for testing in solar cell devices and biomedical applications