Mechanistic analysis of carbon–carbon bond formation by deoxypodophyllotoxin synthase

Deoxypodophyllotoxin contains a core of four fused rings (A to D) with three consecutive chiral centers, the last being created by the attachment of a peripheral trimethoxyphenyl ring (E) to ring C. Previous studies have suggested that the iron(II)- and 2-oxoglutarate–dependent (Fe/2OG) oxygenase, deoxypodophyllotoxin synthase (DPS), catalyzes the oxidative coupling of ring B and ring E to form ring C and complete the tetracyclic core. Despite recent efforts to deploy DPS in the preparation of deoxypodophyllotoxin analogs, the mechanism underlying the regio- and stereoselectivity of this cyclization event has not been elucidated. Herein, we report 1) two structures of DPS in complex with 2OG and (±)-yatein, 2) in vitro analysis of enzymatic reactivity with substrate analogs, and 3) model reactions addressing DPS’s catalytic mechanism. The results disfavor a prior proposal of on-pathway benzylic hydroxylation. Rather, the DPS-catalyzed cyclization likely proceeds by hydrogen atom abstraction from C7', oxidation of the benzylic radical to a carbocation, Friedel–Crafts-like ring closure, and rearomatization of ring B by C6 deprotonation. This mechanism adds to the known pathways for transformation of the carbon-centered radical in Fe/2OG enzymes and suggests what types of substrate modification are likely tolerable in DPS-catalyzed production of deoxypodophyllotoxin analogs.

Lignin as a Multifunctional Photocatalyst for Solar-Powered Biocatalytic Oxyfunctionalization of C-H Bonds

Lignin is a key structural material in all terrestrial plants that is responsible for cell wall formation, water transportation, seed protection, and stress adaptation. Each year, pulp and paper industry produces approximately 50 million metric tons of lignin as waste, 95% of which is combusted or abandoned. Here, we report a new multifunctionality of lignin as a photocatalyst (e.g., synergistic formation of H2O2 formation through O2 reduction and H2O oxidation, use of H2O as an electron donor, and OH• -scavenging activity). Our spectroscopic and photoelectrochemical analyses reveal the photophysical characteristics (e.g., light absorption, charge separation/transfer) of lignin models [e.g., lignosulfonate (LS) and kraft lignin (KL)] and their electronic properties [HOMO-LUMO gap: 2.67 eV (LS), 2.95 eV (KL), LUMO: -0.VRHE (LS) and -0.26 VRHE (KL), HOMO: 2.44 VRHE (LS) and 2.69 VRHE (KL)]. We demonstrate lignin-sensitized redox chemistry, such as (i) H2O2 formation through O2 reduction using H2O as an electron donor and (ii) O2 evolution through H2O oxidation, under visible light. Furthermore, the integration of lignin and H2O2-dependent unspecific peroxygenases (UPOs) enables enantiospecific oxyfunctionalization reactions (e.g., benzylic hydroxylation, alkane hydroxylation, styrene epoxidation). Lignin photocatalysts solve existing issues (e.g., requirement of artificial electron donors, H2O2- or OH• -driven inactivation of UPO) related to the sustainable activation of UPO. The lignin/UPO hybrid achieves a total turnover number of enzyme of 81070, the highest value ever recorded for solar-powered biocatalytic oxyfunctionalization in photochemical platforms. This work demonstrates the propriety of lignin in robust photocatalyst/biocatalyst hybrids for artificial photosynthesis.

Lignin as a Multifunctional Photocatalyst for Solar-Powered Biocatalytic Oxyfunctionalization of C-H Bonds

Lignin is a key structural material in all terrestrial plants that is responsible for cell wall formation, water transportation, seed protection, and stress adaptation. Each year, pulp and paper industry produces approximately 50 million metric tons of lignin as waste, 95% of which is combusted or abandoned. Here, we report a new multifunctionality of lignin as a photocatalyst (e.g., synergistic formation of H2O2 formation through O2 reduction and H2O oxidation, use of H2O as an electron donor, and OH• -scavenging activity). Our spectroscopic and photoelectrochemical analyses reveal the photophysical characteristics (e.g., light absorption, charge separation/transfer) of lignin models [e.g., lignosulfonate (LS) and kraft lignin (KL)] and their electronic properties [HOMO-LUMO gap: 2.67 eV (LS), 2.95 eV (KL), LUMO: -0.VRHE (LS) and -0.26 VRHE (KL), HOMO: 2.44 VRHE (LS) and 2.69 VRHE (KL)]. We demonstrate lignin-sensitized redox chemistry, such as (i) H2O2 formation through O2 reduction using H2O as an electron donor and (ii) O2 evolution through H2O oxidation, under visible light. Furthermore, the integration of lignin and H2O2-dependent unspecific peroxygenases (UPOs) enables enantiospecific oxyfunctionalization reactions (e.g., benzylic hydroxylation, alkane hydroxylation, styrene epoxidation). Lignin photocatalysts solve existing issues (e.g., requirement of artificial electron donors, H2O2- or OH• -driven inactivation of UPO) related to the sustainable activation of UPO. The lignin/UPO hybrid achieves a total turnover number of enzyme of 81070, the highest value ever recorded for solar-powered biocatalytic oxyfunctionalization in photochemical platforms. This work demonstrates the propriety of lignin in robust photocatalyst/biocatalyst hybrids for artificial photosynthesis.

Accessing Chemo- and Regioselective Benzylic and Aromatic Oxidations by Protein Engineering of an Unspecific Peroxygenase

Unspecific peroxygenases (UPOs) enable oxyfunctionalisations of a broad substrate range with unparalleled activities. Tailoring these enzymes for chemo- and regioselective transformations represents a grand challenge due to the difficulties in their heterologous productions. Herein, we performed a protein engineering in <i>S. cerevisiae</i> with the novel <i>Mth</i>UPO. Experimental approaches were combined with computational modelling resulting in the screening of more than 5,300 transformants. This protein engineering led to a significant reshaping of the active site as elucidated by molecular dynamics. The k_cat/K_m was improved by 16.5-fold. Variants were identified with high chemo- and regioselectivities in the oxyfunctionalisation of aromatic and benzylic carbons, respectively. The benzylic hydroxylation was demonstrated to perform with excellent enantioselectivities of 95 % <i>ee</i>. Additionally, the first reported effective exchange of the conserved catalytic Glu residue was observed.

Accessing Chemo- and Regioselective Benzylic and Aromatic Oxidations by Protein Engineering of an Unspecific Peroxygenase

Unspecific peroxygenases (UPOs) enable oxyfunctionalisations of a broad substrate range with unparalleled activities. Tailoring these enzymes for chemo- and regioselective transformations represents a grand challenge due to the difficulties in their heterologous productions. Herein, we performed a protein engineering in <i>S. cerevisiae</i> with the novel <i>Mth</i>UPO. Experimental approaches were combined with computational modelling resulting in the screening of more than 5,300 transformants. This protein engineering led to a significant reshaping of the active site as elucidated by molecular dynamics. The k_cat/K_m was improved by 16.5-fold. Variants were identified with high chemo- and regioselectivities in the oxyfunctionalisation of aromatic and benzylic carbons, respectively. The benzylic hydroxylation was demonstrated to perform with excellent enantioselectivities of 95 % <i>ee</i>. Additionally, the first reported effective exchange of the conserved catalytic Glu residue was observed.

An Enantioconvergent Benzylic Hydroxylation Using a Chiral Aryl Iodide in a Dual Activation Mode

In this article we describe an enantioselective hydroxylation of benzylic C-H bonds with a unique activation mechanism. A chiral aryl iodide catalyst initially acts as precursor for a brominating reagent which subsequently brominates the benzylic C-H bond in a non-stereoselective fashion through a radical bromination. In the second step of this transofrmation, the same catalyst acts as a chiral ligand in a Cu-catalyzed enantioconvergent substitution. We present a broad substrate scope and an intial mechanistic proposal based on a plethora of control experiments.<br>

An Enantioconvergent Benzylic Hydroxylation Using a Chiral Aryl Iodide in a Dual Activation Mode

In this article we describe an enantioselective hydroxylation of benzylic C-H bonds with a unique activation mechanism. A chiral aryl iodide catalyst initially acts as precursor for a brominating reagent which subsequently brominates the benzylic C-H bond in a non-stereoselective fashion through a radical bromination. In the second step of this transofrmation, the same catalyst acts as a chiral ligand in a Cu-catalyzed enantioconvergent substitution. We present a broad substrate scope and an intial mechanistic proposal based on a plethora of control experiments.<br>