Insights into the conformational passage of a polypeptide chain across its free energy landscape have come from the judicious combination of experimental studies and computer simulations 1,2 . Even though some unfolded and partially folded proteins are now known to possess biological function 3 or to be involved in aggregation phenomena associated with disease states 1,4 , experimentally derived atomic-level information on these structures remains sparse as a result of conformational heterogeneity and dynamics. Here we present a technique that can provide such information. Using a 'Trp-cage' miniprotein known as TC5b (ref. 5), we report photochemically induced dynamic nuclear polarization NMR 6 pulse-labelling experiments that involve rapid in situ protein refolding 7,8 . These experiments allow dipolar cross-relaxation with hyperpolarized aromatic side chain nuclei in the unfolded state to be identified and quantified in the resulting folded-state spectrum. We find that there is residual structure due to hydrophobic collapse in the unfolded state of this small protein, with strong inter-residue contacts between side chains that are relatively distant from one another in the native state. Prior structuring, even with the formation of non-native rather than native contacts, may be a feature associated with fast folding events in proteins.Experimental advances in nuclear magnetic resonance (NMR) spectroscopy have led to the characterization of a diverse range of unfolded states of proteins 9 . In many cases the presence of residual structure has been shown 10-13 , but with some significant exceptions 14 the poorly resolved spectra of the unfolded state, arising from conformational exchange and dynamic averaging, have generally hampered structural analysis by NMR. We report here the use of an NMR technique that circumvents some of these problems by transferring Three methodologies are combined in this 'pulse-labelling' experiment ( Fig. 1). (1) Photo-CIDNP (chemically induced dynamic nuclear polarization) 6,22 , a technique for enhancing the NMR signals ('hyperpolarization') of solvent-accessible tryptophan, tyrosine and histidine side chains by means of a laser-induced reaction of the protein with a flavin photosensitizer. (2) Rapid homogeneous mixing of solutions in the NMR sample tube to trigger the folding of a denatured protein on a timescale faster than nuclear spin-lattice relaxation ( Supplementary Fig. 1) 7,8 . To these two techniques we add here, for the first time, (3) transfer of nuclear magnetization via nuclear Overhauser effects (NOEs) from the hyperpolarized side chain protons to neighbouring atoms before the refolding step. As a result, inter-residue contacts in unfolded conformations can be detected in the well-resolved NMR spectrum of the refolded native state.1 H photo-CIDNP measurements were initially performed on the native and denatured states of TC5b. The photo-CIDNP spectrum of native TC5b (Fig. 2b) is considerably simpler than the conventional NMR spectrum (Fig. 2a), because only t...

A combined photokinetical approach helped develop and optimize a green-light driven photoredox catalytic system that generates a “super-reductant” with simple instrumentation, consumes only a bioavailable donor, and provides very high turnover numbers.

We present the first working system for accessing and utilizing laboratory-scale concentrations of hydrated electrons by photoredox catalysis with a green light-emitting diode (LED). Decisive are micellar compartmentalization and photon pooling in an intermediate that decays with second-order kinetics. The only consumable is the nontoxic and bioavailable vitamin C. A turnover number of 1380 shows the LED method to be on par with electron generation by high-power pulsed lasers, but at a fraction of the cost. The extreme reducing power of the electron and its long unquenched life as a ground-state species are synergistic. We demonstrate the applicability to the dechlorination, defluorination, and hydrogenation of compounds that are inert towards all other visible-light photoredox catalysts known to date. A comprehensive mechanistic investigation from microseconds to hours yields results of general validity for photoredox catalysis with photon pooling, allowing optimization and upscaling.

The interaction of an anionic micelle with the molecular charges and dipole moments ensures the desired sequence of processes and suppresses cross reactions in an “all-green” photoredox catalytic access to hydrated electrons e˙–aq, which reductively decompose nonactivated chloro-organics.

Hydrated electrons are highly aggressive species that can force chemical transformations of otherwise unreactive molecules such as the reductive detoxification of halogenated organic compounds. We present the first example of the sustainable production of hydrated electrons through a homogeneous catalytic cycle driven entirely by green light (532 nm, coinciding with the maximum of the terrestrial solar spectrum). The catalyst is a metal complex serving as a "container" for a radical anion. This active center is generated from a ligand through quenching by a sacrificial electron donor, is shielded by the complex such that it stores the energy of the photon for much longer than a free radical anion could, and is finally ionized by another photon to regenerate the ligand and recover the starting complex quantitatively. The sacrificial donor can be a bioavailable reagent such as ascorbic acid.

No abstract

A sustainable generation of hydrated electrons with green light would allow solar-driven applications of this potent reductant, such as the detoxification of halogenated organic waste. Using two-color laser flash photolysis, we have studied the photoionizations of the 1,5-naphthalene disulfonate radical anion and triplet with 532 nm as well as 355 nm. The radical anion is prepared by reducing the triplet with the bioavailable ascorbate monoanion under physiological conditions; its photoionization recovers the starting substrate, so turns the reaction sequence into a catalytic cycle. A comparison of the four ionizations suggests that their efficiency is strongly influenced by the electronic configuration of the state ejecting the electron. The quantum yield for ionizing the radical anion with 532 nm (0.27) is at least four times higher than for the very few known examples of such green-light ionizations and comparable to the most efficient UV ionizations known to date, so this system might represent a breakthrough towards the "green" production of hydrated electrons.

The hydrated electron represents a "super-reductant" in water, providing 2.9 eV of reductive power, which suffices to decompose nonactivated aliphatic halides. We show that 3-amino-perylene in SDS micelles, when combined with the bioavailable ascorbate as an extramicellar sacrificial donor, sustainably produces hydrated electrons through photoredox catalysis with green light, from a metal-free system, and at near-physiological pH. Photoionization of the amine with a 532 nm laser yields an extremely long-lived radical cation as the by-product, and a subsequent reaction of the latter with the sacrificial donor across the micelle/water interface regenerates the catalyst. The regeneration step involves parallel reactions between differently protonated forms, causing a bell-shaped pH dependence in basic medium. We have separated these processes kinetically. Employing this catalytic cycle for the laboratory-scale decomposition of chloroacetate, an accepted model compound for toxic and persistent halo-organic waste, gave turnover numbers of about 170. Even though both the substrate and the sacrificial donor compete for the hydrated electron, their consumption ratio is practically independent of the initial concentration ratio because the formal radical anion of the ascorbate undergoes secondary scavenging by the chloroacetate. In the course of the reaction, the initial hydrophobic catalyst is converted into a secondary species that is hydrophilic and still exhibits catalytic activity.