Alex I. Smirnov

Darobactins are ribosomally synthesized and post-translationally modified peptides (RiPPs) with potent activity against Gram-negative pathogens. Their antibiotic function depends on a distinctive β-strand architecture stabilized by an ether cross-link between two tryptophan residues. Unlike most ether linkages in natural products, which arise from hydroxylated precursors, the darobactin ether bond is formed between two unmodified Trp residues by the radical S-adenosyl-l-methionine (SAM) enzyme DarE. DarE catalyzes both O-insertion and ether bond formation using molecular oxygen as a cosubstrate, but its catalytic mechanism remains largely unexplored. Here, we report detection and characterization of a key radical intermediate at the C_β position of W3 (W3-C_β•) using isotopically labeled DarA, kinetic analysis, and spectroscopy methods including EPR, ENDOR, and HYSCORE. Similar radical species were also observed in reactions with W3 variants of DarA. These results provide direct evidence that DarE catalyzes ether cross-link formation through reaction of the W3-C_β• intermediate with O₂, revealing an unprecedented radical-mediated strategy for O-atom incorporation in RiPP biosynthesis.

This perspective article highlights an interesting and useful discovery: gallium‐based liquid metals can initiate free radical polymerization to form hydrogels. Normally, hydrogels are polymerized using molecular initiators that generate radicals in response to heat or light. Liquid metal particles can replace the role of molecular initiators and are interesting for several reasons: 1) Liquid metal particles are easy to make by stirring or sonicating liquid metal in a non‐solvent such as water. 2) Conventional initiators that remain in hydrogels can be problematic (e.g., toxic); in contrast, some gel formulations can slowly dissolve the liquid metal particles as salts. 3) The polymers formed using liquid metals are, in some cases, softer, more extensible, and tougher than the same polymer formed using conventional molecular initiators and crosslinkers. 4) In addition to initiating polymerization, in some cases, the liquid metal particles appear to serve as crosslinkers through interactions with the polymers or by releasing Ga ³⁺ ions that can ionically crosslink negatively charged polymers. The purpose of this perspective is to summarize what is and what is not understood about liquid metal initiators for forming hydrogels (and several other polymers) while highlighting opportunities for further research.

Pulsed dynamic nuclear polarization (DNP) can enhance NMR signals by more than 2 orders of magnitude and is applicable to spin systems with short-lived electronic polarization. Recently, several pulsed DNP sequences were demonstrated at low magnetic fields. However, pulsed DNP at magnetic fields of modern NMR spectrometers (7 T and above) necessitates addressing major technical challenges for generating sufficiently high mm-wave B_1e amplitudes to match the nuclear Larmor frequencies. Here we describe a first-of-its-kind ESR/NMR spectrometer for pulsed DNP and phase-sensitive electronic detection operating at 7 T magnetic field. Generation of B_1e fields with amplitudes of ca. 75 MHz was made possible by employing a pulsed extended interaction klystron and piezo-tunable photonic-band gap resonators with unloaded quality factors Q ≈ 1500. Room-temperature NOVEL DNP yielded natural-abundance ¹³C NMR signal gains of up to 800 for a 30 μm thick crystal of a synthetic diamond. Simultaneous coherent manipulation of the electronic and nuclear spins was demonstrated by ¹³C-detected electron Rabi nutations.

Chlamydia protein associating with death domains (CtCADD) is involved in the biosynthesis of p-aminobenzoic acid (pABA) for integration into folate, a critical cofactor that is required for pathogenic survival. CADD activates dioxygen and utilizes its own tyrosine and lysine as synthons to furnish the carboxylate, carbon backbone, and amine group of pABA in a complex multistep mechanism. Unlike other members of the heme oxygenase-like dimetal oxidase (HDO) superfamily that typically house an Fe₂ cofactor, previous activity studies have shown that CtCADD likely uses a heterobimetallic Fe/Mn center. The structure of the Fe²⁺/Mn²⁺ cofactor and how the conserved HDO scaffold mediates metal selectivity have remained enigmatic. Adopting an in crystallo metalation approach, CtCADD was solved in the apo, Fe²⁺₂, Mn²⁺₂, and catalytically active Fe²⁺/Mn²⁺ forms to identify the probable site for Mn binding. The analysis of CtCADD active-site variants further reinforces the importance of the secondary coordination sphere on cofactor preference for competent pABA formation. Rapid kinetic optical and electron paramagnetic resonance (EPR) studies show that the heterobimetallic cofactor selectively reacts with dioxygen and likely initiates pABA assembly through the formation of a transient tyrosine radical intermediate and a resultant heterobimetallic Mn³⁺/Fe³⁺ cluster.

The natural aromatic polymer lignin and its lignin-like oligomeric fragments have attracted attention for their antioxidant capacity and free radical scavenging activities. In this study, a 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay was employed to assess the antioxidant capacity of fractionated and partially depolymerized organosolv lignin by electron paramagnetic resonance (EPR) and UV-Vis spectroscopy. The results show significant antioxidant activity for both the lignin and oligomeric fragments, with the EPR measurements demonstrating their efficiency in quenching the free radicals. The EPR data were analyzed to derive the kinetic rate constants. The radical scavenging activity (RSA) of lignins was then determined by UV-Vis spectroscopy and the results were compared with the EPR method. This two-method approach improves the reliability and understanding of the antioxidant potential of lignin and its derivatives and provides valuable insights for their potential applications in various industries, including pharmaceuticals, food preservation, and cosmetics.