Michael W. George

[FeFe]-hydrogenases are the fastest enzymes for hydrogen evolution, yet their irreversible inhibition by oxygen has thwarted their industrial use. CbA5H is an exception as its inhibition by oxygen is reversible. Protein scaffold rearrangement near the active site, allowing a ligand to coordinate the metal center in addition to the formation of a highly oxidized state of the metal center named H_inact, is the current hypothesis for CbA5H oxygen stability. However, the ligand identity has been disputed and there is no evidence to suggest that protein scaffold rearrangement is the sole reason for oxygen stability. Here, we investigate CbA5H oxygen stability by providing a high-resolution (1.96 Å) X-ray structure that shows that the protective ligand is a conserved cysteine thiol group, which directly coordinates the metal center. The local rearrangement also encompasses structural water molecules and the side chain of E341, associated with proton transfer. In addition, we illustrate that C236 and H245, located close to accessory iron sulfur clusters in the Fd domain, influence oxygen stability. We show that mutating these residues significantly decreases oxygen stability but not the ability to form H_inact. Variant C236A displays a slower inactivation rate, which we suggest is due to tuning the redox properties of one of the accessory iron sulfur clusters. We also show that the soluble ligand-binding β-grasp domain (SLBB) may not be required for oxygen stability by comparing CbA5H to a novel homolog lacking this domain. Collectively, these findings expand our understanding of oxygen stability in [FeFe]-hydrogenases.

A potential reference sample was developed for facile quantification of near-field Raman and photoluminescence contrast factors in gap and non-gap modes.

We report the development and optimization of a scalable flow process for metallaphotoredox (Ir/Ni) C-O coupling, a mild and efficient approach for forming alkyl-aryl ethers, a common motif in medicinal and process chemistry settings. Time-resolved infrared spectroscopy (TRIR) highlighted the amine as the major quencher of the photocatalyst triplet excited state, along with the formation of an Ir(II) species that, in the presence of the Ni cocatalyst, has its lifetime shortened, suggesting reductive quenching of Ir(III)*, followed by reoxidation facilitated by the Ni cocatalyst. TRIR and batch reaction screening was used to develop conditions transferrable to flow, and many processing benefits of performing the reaction in flow were then demonstrated using a simple to construct/operate, small-footprint FEP coil flow reactor, including short (10 kg day^-1 in a large PhotoVortex (185 mL irradiated volume) with good product yields (>90%) and low catalyst loadings (0.1 to 0.5 mol % of [Ir{dF(CF₃)ppy}₂dtbbpy]PF₆), enabled by excellent mixing ensuring sufficient mass transfer between short-lived photoexcited and other transient species.

The counter-electrode process of an organic electrochemical reaction is integral for the success and sustainability of the process. Unlike for oxidation reactions, counter-electrode processes for reduction reactions remain limited, especially for deep reductions that apply very negative potentials. Herein, we report the development of a bromide-mediated silane oxidation counter-electrode process for nonaqueous electrochemical reduction reactions in undivided cells. The system is found to be suitable for replacing either sacrificial anodes or a divided cell in several reported reactions. The conditions are metal-free, use inexpensive reagents and a graphite anode, are scalable, and the byproducts are reductively stable and readily removed. We showcase the translation of a previously reported divided cell reaction to a >100 g scale in continuous flow.

We describe the optimization and scale-up of two consecutive reaction steps in the synthesis of bio-derived alkoxybutenolide monomers that have been reported as potential replacements for acrylate-based coatings (Sci. Adv.2020, 6, eabe0026). These monomers are synthesized by (i) oxidation of furfural with photogenerated singlet oxygen followed by (ii) thermal condensation of the desired 5-hydroxyfuranone intermediate product with an alcohol, a step which until now has involved a lengthy batch reaction. The two steps have been successfully telescoped into a single kilogram-scale process without any need to isolate the 5-hydroxyfuranone between the steps. Our process development involved FTIR reaction monitoring, FTIR data analysis via 2D visualization, and two different photoreactors: (i) a semicontinuous photoreactor based on a modified rotary evaporator, where FTIR and 2D correlation spectroscopy (2D-COS) revealed the loss of the methyl formate coproduct, and (ii) our fully continuous Taylor Vortex photoreactor, which enhanced the mass transfer and permitted the use of near-stoichiometric equivalents of O₂. The use of in-line FTIR monitoring and modeling greatly accelerated process optimization in the Vortex reactor. This led to scale-up of the photo-oxidation in 85% yield with a projected productivity of 1.3 kg day^-1 and a space-time yield of 0.06 mol day^-1 mL^-1. Higher productivities could be achieved while sacrificing yield (e.g., 4 kg day^-1 at 40% yield). The use of superheated methanol at 200 °C in a pressurized thermal flow reactor accelerated the second step, the thermal condensation of 5-hydroxyfuranone, from a 20 h batch reflux reaction (0.5 L, 85 g) to a space time of 700 g day^-1. Proof of concept for telescoping the two steps was established with an overall two-step yield of 67%, producing a process with a projected productivity of 1.1 kg day^-1 for the methoxybutenolide monomer without any purification of the 5-hydroxyfuranone intermediate.

Amide bonds are ubiquitous and found in a myriad of functional molecules. Although formed in a reliable and robust fashion, alternative amide bond disconnections provide flexibility and synthetic control. Herein we describe an electrochemical method to form the non-amide C-N bond from direct benzylic C(sp³)-H amidation. Our approach is applied toward the synthesis of secondary amides by coupling secondary benzylic substrates with substituted primary benzamides. The reaction has been scaled up to a multigram scale in flow.

We describe the optimization and scale-up of two consecutive reaction steps in the synthesis of bio-derived alkoxybutenolide monomers that have been reported as potential replacements for acrylate-based coatings (Sci. Adv., 2020, 6, eabe0026). These monomers are synthesized by (i) oxidation of furfural with photo-generated singlet oxygen followed by (ii) thermal condensation of the desired 5-hydroxyfuranone intermediate product with an alcohol, a step which until now has involved a lengthy batch reaction. The two steps have been successfully telescoped into a single kilogram-scale process without any need to isolate the 5-hydroxyfuranone between the steps. Our process development involved FTIR reaction monitoring, FTIR data analysis via 2D-visualization and two different photo-reactors (i) a semi-continuous photo-reactor based on a modified rotary evaporator where FTIR and 2D-correlation spectroscopy (2D-COS) revealed the loss of the methyl formate co-product and (ii) our fully continuous Taylor Vortex photo-reactor which enhanced the mass transfer and permitted the use of near-stoichiometric equivalents of O2. The use of inline FTIR monitoring and modelling greatly accelerated process optimization in the Vortex reactor. This led to scale up of the photo-oxidation with an 85% yield and a projected productivity of 1.3 kg day-1, with a space time yield of 0.06 mol day-1 mL-1. Higher productivities could be achieved whilst sacrificing yield; e.g. 4 kg day-1 at 40% yield. The second step, the thermal condensation of 5-hydroxyfuranone, was transformed from a 20 h batch reflux reaction to a 700 g day-1. Proof of concept for telescoping the two steps was established with an overall two-step yield of 67%, producing a process with projected productivity of 1.1 kg day-1 of the methoxybutenolide monomer without any purification of the 5-hydroxyfuranone intermediate.

Functionally similar to the tight junctions present in animal guts, plant roots have evolved a lignified Casparian strip as an extracellular diffusion barrier in the endodermis to seal the root apoplast and maintain nutrient homeostasis. How this diffusion barrier is structured has been partially defined, but its lignin polymerization and assembly steps remain elusive. Here, we characterize a family of dirigent proteins (DPs) essential for both the localized polymerization of lignin required for Casparian strip biogenesis in the cell wall and for attachment of the strip to the plasma membrane to seal the apoplast. We reveal a Casparian strip lignification mechanism that requires cooperation between DPs and the Schengen pathway. Furthermore, we demonstrate that DPs directly mediate lignin polymerization as part of this mechanism.