Hydrogen radical-shuttle (HRS)-enabled photoredox synthesis of indanones via decarboxylative annulation

Hydrogen atom transfer (HAT) process is a powerful and effective strategy for activating C-H bonds followed by further functionalization. Intramolecular 1,n (n = 5 or 6)-HATs are common and frequently encountered in organic synthesis. However, intramolecular 1,n (n = 2 or 3)-HAT is very challenging due to slow kinetics. Compared to proton-shuttle process, which is well established for organic synthesis, hydrogen radical-shuttle (HRS) is unexplored. In this work, a HRS-enabled decarboxylative annulation of carbonyl compounds via photoredox catalysis for the synthesis of indanones is developed. This protocol features broad substrate scope, excellent functional group tolerance, internal hydrogen radical transfer, atom- and step-economy. Critical to the success of this process is the introduction of water, acting as both HRS and hydrogen source, which was demonstrated by mechanistic experiments and density functional theory (DFT) calculations. Importantly, this mechanistically distinctive HAT provides a complement to that of typical proton-shuttle-promoted, representing a breakthrough in hydrogen radical transfer, especially in the inherently challenging 1,2- or 1,3-HAT.

How ATP and dATP act as molecular switches to regulate enzymatic activity in the prototypic bacterial class Ia ribonucleotide reductase

Class Ia ribonucleotide reductases (RNRs) are subject to allosteric regulation to maintain the appropriate deoxyribonucleotide levels for accurate DNA biosynthesis and repair. RNR activity requires a precise alignment of its α2 and β2 subunits such that a catalytically-essential radical species is transferred from β2 to α2. In E. coli, when too many deoxyribonucleotides are produced, dATP binding to RNR generates an inactive α4β4 state in which α2 and β2 are separated, preventing radical transfer. ATP binding breaks the α−β interface, freeing β2 and restoring activity. Here we investigate the molecular basis for allosteric activity regulation in the prototypic E. coli class Ia RNR. Through the determination of six crystal structures we are able to establish how dATP binding creates a binding pocket for β on α that traps β2 in the inactive α4β4 state. These structural snapshots also reveal the numerous ATP-induced conformational rearrangements that are responsible for freeing β2. We further discover, and validate through binding and mutagenesis studies, a previously unknown nucleotide binding site on the α subunit that is crucial for the ability of ATP to dismantle the inactive α4β4 state. These findings have implications for the design of allosteric inhibitors for bacterial RNRs.

Does Tyrosine Protect S. Coelicolor Laccase from Oxidative Degradation?

We have investigated the roles of tyrosine (Tyr) and tryptophan (Trp) residues in the four-electron reduction of oxygen catalyzed by <i>Streptomyces coelicolor</i> laccase (SLAC). During normal enzymatic turnover in laccases, reducing equivalents are delivered to a type 1 Cu center (Cu_T1) and then are transferred over 13 Å to a trinuclear Cu site (TNC: (Cu_T3)₂Cu_T2) where O₂ reduction occurs. The TNC in SLAC is surrounded by a large cluster of Tyr and Trp residues that can provide reducing equivalents when the normal flow of electrons is disrupted. Canters and coworkers have shown that when O₂ reacts with a reduced SLAC variant lacking the Cu_T1 center, a Tyr108^· radical near the TNC forms rapidly. We have found that ascorbate reduces the Tyr108^·radical in wild-type SLAC about 10 times faster than it reacts with the Cu_T1²⁺ center, possibly owing to radical transfer along a Tyr/Trp chain. Aerobic oxidation of two reduced SLAC mutants (Y108F and W132F) leads to the formation of a long-lived (~15 min) Tyr^·radical with distinct absorption at 408 nm. The diffusion of redox equivalents away from the primary enzymatic pathway in SLAC may indicate a poorly optimized enzyme or a mechanism to protect against protein damage.

Does Tyrosine Protect S. Coelicolor Laccase from Oxidative Degradation?

We have investigated the roles of tyrosine (Tyr) and tryptophan (Trp) residues in the four-electron reduction of oxygen catalyzed by <i>Streptomyces coelicolor</i> laccase (SLAC). During normal enzymatic turnover in laccases, reducing equivalents are delivered to a type 1 Cu center (Cu_T1) and then are transferred over 13 Å to a trinuclear Cu site (TNC: (Cu_T3)₂Cu_T2) where O₂ reduction occurs. The TNC in SLAC is surrounded by a large cluster of Tyr and Trp residues that can provide reducing equivalents when the normal flow of electrons is disrupted. Canters and coworkers have shown that when O₂ reacts with a reduced SLAC variant lacking the Cu_T1 center, a Tyr108^· radical near the TNC forms rapidly. We have found that ascorbate reduces the Tyr108^·radical in wild-type SLAC about 10 times faster than it reacts with the Cu_T1²⁺ center, possibly owing to radical transfer along a Tyr/Trp chain. Aerobic oxidation of two reduced SLAC mutants (Y108F and W132F) leads to the formation of a long-lived (~15 min) Tyr^·radical with distinct absorption at 408 nm. The diffusion of redox equivalents away from the primary enzymatic pathway in SLAC may indicate a poorly optimized enzyme or a mechanism to protect against protein damage.

Structure of a trapped radical transfer pathway within a ribonucleotide reductase holocomplex

Ribonucleotide reductases (RNRs) are a diverse family of enzymes that are alone capable of generating 2′-deoxynucleotides de novo and are thus critical in DNA biosynthesis and repair. The nucleotide reduction reaction in all RNRs requires the generation of a transient active site thiyl radical, and in class I RNRs, this process involves a long-range radical transfer between two subunits, α and β. Because of the transient subunit association, an atomic resolution structure of an active α2β2 RNR complex has been elusive. We used a doubly substituted β2, E52Q/(2,3,5)-trifluorotyrosine122-β2, to trap wild-type α2 in a long-lived α2β2 complex. We report the structure of this complex by means of cryo–electron microscopy to 3.6-angstrom resolution, allowing for structural visualization of a 32-angstrom-long radical transfer pathway that affords RNR activity.