The intermediacy of alkoxy radicals in ceriumcatalyzed C−H functionalization via H-atom abstraction has been unambiguously confirmed. Catalytically relevant Ce(IV)−alkoxide complexes have been synthesized and characterized by X-ray diffraction. Operando electron paramagnetic resonance and transient absorption spectroscopy experiments on isolated pentachloro Ce(IV) alkoxides identified alkoxy radicals as the sole heteroatom-centered radical species generated via ligand-tometal charge transfer (LMCT) excitation. Alkoxy-radical-mediated hydrogen atom transfer (HAT) has been verified via kinetic analysis, density functional theory (DFT) calculations, and reactions under strictly chloride-free conditions. These experimental findings unambiguously establish the critical role of alkoxy radicals in Ce-LMCT catalysis and definitively preclude the involvement of chlorine radical. This study has also reinforced the necessity of a high relative ratio of alcohol vs Ce for the selective alkoxy-radical-mediated HAT, as seemingly trivial changes in the relative ratio of alcohol vs Ce can lead to drastically different mechanistic pathways. Importantly, the previously proposed chlorine radical−alcohol complex, postulated to explain alkoxy-radicalenabled selectivities in this system, has been examined under scrutiny and ruled out by regioselectivity studies, transient absorption experiments, and high-level calculations. Moreover, the peculiar selectivity of alkoxy radical generation in the LMCT homolysis of Ce(IV) heteroleptic complexes has been analyzed and back-electron transfer (BET) may have regulated the efficiency and selectivity for the formation of ligand-centered radicals.

Time-resolved fluorescence properties of quenched fluorescein sodium, including self-quenching and collisional quenching by iodide, have been studied by using a picosecond time-correlated single-photon counting (TCSPC) apparatus, together with an upconversion spectrophotofluorometer with a time resolution better than 300 fs. The steady-state fluorescence intensity of fluorescein sodium reached the maximum when its concentration was 510 μM with pH > 9. Both the fluorescence intensity and lifetime decreased with increasing concentrations of NaI quencher. When the NaI concentration was 12.2 M, a monoexponential decay with a lifetime as short as 17 ps was exactly determined for the first time using the femtosecond-resolved upconversion system. Picosecond time-resolved fluorescence measurements of circular permuted green and yellow fluorescent proteins (cpGFP and cpYFP) were reported, demonstrating that the fluorescence decay of quenched fluorescein sodium is a better approximation of the instrument response function (IRF) needed for the accurate deconvolution of fluorescence lifetime data, particularly for detectors used in the visible spectral region. We believe that this picosecond lifetime standard will find wide applications in fluorescence lifetime imaging microscopy (FLIM).

Protein surface hydration is critical to its structural stability, flexibility, dynamics, and function. Recent observations of surface solvation on picosecond time scales have evoked debate on the origin of such relatively slow motions, from hydration water or protein charged side chains, especially with molecular dynamics simulations. Here we used a unique nuclease with a single tryptophan as a local probe and systematically mutated three neighboring charged residues to differentiate the contributions from hydration water and charged side chains. By various mutations of one, two, and all three charged residues, we observed slight increases in the total tryptophan Stokes shifts with fewer neighboring charged residue(s) and found insensitivity of charged side chains to the relaxation patterns. The dynamics is correlated with hydration water relaxation with the slowest time in a dense charged environment and the fastest time at a hydrophobic site. On such picosecond time scales, the protein surface motion is restricted. The total Stokes shifts are dominantly from hydration water relaxation and the slow dynamics is from water-driven relaxation, coupled to local protein fluctuations.

The fluctuations of hydration water and the protein are coupled together at the protein surface and often such water-protein dynamic interactions are controlled presumably by hydration water motions. However, direct evidence is scarce and it requires measuring the dynamics of hydration water and protein sidechain simultaneously. Here, we use a unique protein with a single tryptophan to directly probe interfacial water and related sidechain relaxations with temperature dependence. With systematic mutations to change local chemical identity and structural flexibility, we found that the sidechain relaxations are always slower than hydration water motions and the two dynamic processes are linearly correlated with the same energy barriers, indicating the same origin of both relaxations. The charge mutations change the rates of hydration water relaxations but not the relaxation barriers. These results convincingly show that the water-protein relaxations are strongly coupled and the hydration water molecules govern such fluctuations on the picosecond timescales.

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