Modulating the Fibrillization of Parathyroid-Hormone (PTH) Peptides: Azo-Switches as Reversible and Catalytic Entities.

We here report a novel strategy to control the bioavailability of the fibrillizing parathyroid hormone (PTH)-derived peptides, where the concentration of the bioactive form is controlled by an reversible, photoswitchable peptide. PTH1-84, a human hormone secreted by the parathyroid glands, is important for the maintenance of extracellular fluid calcium and phosphorus homeostasis. Controlling fibrillization of PTH1-84 represents an important approach for in vivo applications, in view of the pharmaceutical applications for this protein. We embed the azobenzene derivate 3-{[(4-aminomethyl)phenyl]diazenyl}benzoic acid (3,4'-AMPB) into the PTH-derived peptide PTH25-37 to generate the artificial peptide AzoPTH25-37 via solid-phase synthesis. AzoPTH25-37 shows excellent photostability (more than 20 h in the dark) and can be reversibly photoswitched between its cis/trans forms. As investigated by ThT-monitored fibrillization assays, the trans-form of AzoPTH25-37 fibrillizes similar to PTH25-37, while the cis-form of AzoPTH25-37 generates only amorphous aggregates. Additionally, cis-AzoPTH25-37 catalytically inhibits the fibrillization of PTH25-37 in ratios of up to one-fifth. The approach reported here is designed to control the concentration of PTH-peptides, where the bioactive form can be catalytically controlled by an added photoswitchable peptide.

The radicals of quercetin-derived antioxidants in Triton X-100 micelles.

We have employed photoionization with a pulsed laser (5 ns, 355 nm) as a direct access to the radicals of quercetin, five of its monoethers and three of its diethers in nonionic micelles. On a submicrosecond timescale, the first detectable intermediates are neutral radicals NRx, which can then be deprotonated to give radical anions RANxy, where x and y denote the phenoxyl positions bearing spin and/or charge. Alkylation at oxygen x blocks the formation of NRx and RANxy but barely changes the spectra of all other structurally possible radical isomers. Through systematic comparison, this allowed unambiguous radical identification and spectral assignment by experiment in all cases: NR3 is preferred over NR4', all other NRx are negligible; NR3 and NR4' are deprotonated at oxygens 4' and 3', respectively, unless barred by substitution, or at oxygen 7. As a caveat, B3LYP calculations on the radicals with a 6-311++g(2d,2p) basis set and a PCM solvation model gave only partially correct energy orderings and spectra, the former most likely due to an inability fully to describe the intramolecular hydrogen bonds and the latter possibly due to spin contamination. The favored deprotonation of NR3 is associated with a typical pKa of 4.8 and first-order kinetics, that of NR4' with a pKa of 3.9 and complex kinetics, suggesting NR3' as a fleeting intermediate. Both reverse reactions are diffusion controlled.

Do equilibrium and rate constants of intramicellar reactions depend on micelle size?

We have studied the combined static and dynamic quenching of pyrene by methyl viologen in sodium alkyl sulfate micelles varying in volume by a factor of more than 4. Size controls were the temperature T (283 K-333 K) and the alkyl chain length n (9-14) as well as, with n = 12 only, added NaCl (up to 9 times the surfactant weight-in concentration). At high [NaCl], up to 40% of the viologen resides in the aqueous bulk and quenches dynamically across the micelle-water interface with a rate limited by its diffusion-controlled attachment to the micelle. The micellar aggregation numbers depend linearly both on n and on the difference between T and the Krafft temperature; we have derived interpolation formulas for them as well as for the associated molar volumes of the micelles; the aggregation numbers at the critical micelle concentration are also linear functions of T, and the exponent relating them to the aggregation numbers at other concentrations is temperature independent. At given T, the volume-based quenching rate constants for different n or [NaCl] are very similar, and the same holds true for the equilibrium constants of the static quenching. Arrhenius plots identify the microviscosity inside the micelles as octanol-like; van't Hoff plots give virtually the same reaction enthalpies and entropies as in homogeneous methanolic solution; and the underlying kinetic and thermodynamic parameters are not modified by the micelle size.

Laser Access to Quercetin Radicals and Their Repair by Co-antioxidants.

We have demonstrated the feasibility and ease of producing quercetin radicals by photoionization with a pulsed 355 nm laser. A conversion efficiency into radicals of 0.4 is routinely achieved throughout the pH range investigated (pH 2-9), and the radical generation is completed within a few ns. No precursor other than the parent compound is needed, and the ionization by-products do not interfere with the further fate of the radicals. With this generation method, we have characterized the quercetin radicals and studied the kinetics of their repairs by co-antioxidants such as ascorbate and 4-aminophenol. Bell-shaped pH dependences of the observed rate constants reflect opposite trends in the availability of the reacting protonation forms of radical and co-antioxidant and even at their maxima mask the much higher true rate constants. Kinetic isotope effects identify the repairs as proton-coupled electron transfers. An examination of which co-antioxidants are capable of repairing the quercetin radicals and which are not confines the bond dissociation energies of quercetin and its monoanion experimentally to 75-77 kcal mol-1 and 72-75 kcal mol-1 , a much narrower interval in the case of the former than previously estimated by theoretical calculations.

Pyrene-viologen complexes in SDS micelles: quenching parameters and use as probes of aggregation numbers.

Through a formal-kinetic treatment, we rigorously derive the micellar occupations in the presence of fluorophore-quencher ground-state complexes and the resulting expressions for the time-dependent and stationary observables in the case of combined static and dynamic quenching. We present a protocol for data analysis that effectively isolates the processes, thereby ensuring rapid fit convergence and unique parameter sets. By this approach, we interpret time-resolved fluorescence measurements on pyrene quenched by a homologous series of viologens in SDS micelles. The dynamic intramicellar quenching is diffusion limited; and the formation of the ground-state complexes is entropy driven, with a constant increment per methylene group in the viologen sidechains. The micellar aggregation numbers are obtained with a precision comparable to neutron scattering, including their temperature dependence.

Laser-Induced Wurtz-Type Syntheses with a Metal-Free Photoredox Catalytic Source of Hydrated Electrons.

Upon irradiation with ns laser pulses at 355 nm, 2-aminoanthracene in SDS micelles readily produces hydrated electrons. These "super-reductants" rapidly attack substrates such as chloro-organics and convert them into carbon-centred radicals through dissociative electron transfer. For a catalytic cycle, the aminoanthracene needs to be restored from its photoionization by-product, the radical cation, by a sacrificial donor. The ascorbate monoanion can only achieve this across the micelle-water interface, but the monoanion of ascorbyl palmitate results in a fully micelle-contained regenerative electron source. The shielding by the micelle in the latter case not only increases the life of the catalyst but also strongly suppresses the interception of the carbon-centred radicals by the hydrogen-donating ascorbate moiety; and in conjunction with the high local concentrations effected by the pulsed laser, termination by radical dimerization thus dominates. We have obtained a complete and consistent picture through monitoring the individual steps and the assembled system by flash photolysis on fast and slow timescales, from microseconds to minutes; and in preparative studies on a variety of substrates, we have achieved up to quantitative dimerization with a turnover on the order of 1 mmol per hour.

Combined static and dynamic intramicellar fluorescence quenching: effects on stationary and time-resolved Stern-Volmer experiments.

We have conducted a theoretical and experimental study of Stern-Volmer experiments in micellar systems for the important case that fluorophore and quencher remain confined to their micelle during the luminescence decay (the "immobile probe/immobile quencher" scenario) and exhibit static quenching followed by dynamic quenching. By a comparative mathematical analysis, we have exposed inherent physical and mathematical contradictions of earlier theories. We present a general framework that allows a very simple derivation of consistent solutions. Even with the correct model, strong parameter correlations severely compromise fit uniqueness when the stationary luminescence is the only observable, but these correlations can be removed by parallel absorption measurements and do not occur in time-resolved luminescence experiments. The application of our protocol to pyrene quenching by substituted viologens in SDS micelles revealed a linear dependence of the apparent aggregation number of the surfactant on the equilibrium constant of formation of ground-state complexes, which can be quantitatively explained by a preference of the quencher for micelles containing the fluorophore. The complex formation is entropy controlled, as evidenced by a driving force that decreases linearly with the number of free rotors in the viologen sidechains.

3-Aminoperylene and ascorbate in aqueous SDS, one green laser flash and action! Sustainably detoxifying a recalcitrant chloro-organic.

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.

Combined static and dynamic quenching in micellar systems-closed-form integrated rate laws verified using a versatile probe.

We demonstrate that the 3-aminoperylene radical cation is a near-ideal probe for investigating kinetic and transport processes in SDS micellar systems. Its isolated generation by two-photon ionization at a wavelength where most quenchers are transparent (532 nm) is free from side reactions; no exit from the micelles is detectable on a millisecond timescale; and its unquenched lifetime is as long as 350 ms, thus allowing the study of quenching processes over a time frame spanning at least 7 orders of magnitude. The lipophilic antioxidant ascorbyl palmitate reconverts it to its parent compound through the interplay of static and fast dynamic intramicellar quenching as well as through subsequent slow intermicellar migration. Using this radical-cation probe, we have successfully validated closed-form expressions which we derived for the probe decay in all these situations. From these functions, we also obtained an exact and closed-form analytical result for Stern-Volmer experiments with combined static and dynamic intramicellar quenching.

Green-light ionization of 3-aminoperylene in SDS micelles-a promising access to hydrated electrons despite a myth debunked.

Using an improved methodology, we have carefully reinvestigated the title reaction by laser flash photolysis and disproved an earlier study (J. K. Thomas and P. Piciulo, J. Am. Chem. Soc., 1978, 100, 3239), which claimed this green-light ionization to be monophotonic, the only instance of such a scenario ever reported for a stable compound. We show it to be biphotonic instead, in accordance with thermodynamic considerations, and present a photokinetic model that accurately represents the intensity dependences throughout the whole excitation range in the green (532 nm) and the near UV (355 nm), up to near-quantitative electron release in the latter case. A major artifact deceptively similar to a chemical decay arises from an SDS-related laser-induced turbidity but can be eliminated by difference experiments or careful selection of excitation intensities and temporal windows. The ionization step is not accompanied by side processes, and affords an extremely long-lived (0.35 s) radical cation remaining solubilized. The micelles completely block attacks of hydrated electrons or hydroxyl radicals on the starting material and its radical cation but allow a post-ionization regeneration by high concentrations of the hydrophilic ascorbate monoanion.

CASP10-BCL::Fold efficiently samples topologies of large proteins.

During CASP10 in summer 2012, we tested BCL::Fold for prediction of free modeling (FM) and template-based modeling (TBM) targets. BCL::Fold assembles the tertiary structure of a protein from predicted secondary structure elements (SSEs) omitting more flexible loop regions early on. This approach enables the sampling of conformational space for larger proteins with more complex topologies. In preparation of CASP11, we analyzed the quality of CASP10 models throughout the prediction pipeline to understand BCL::Fold's ability to sample the native topology, identify native-like models by scoring and/or clustering approaches, and our ability to add loop regions and side chains to initial SSE-only models. The standout observation is that BCL::Fold sampled topologies with a GDT_TS score > 33% for 12 of 18 and with a topology score > 0.8 for 11 of 18 test cases de novo. Despite the sampling success of BCL::Fold, significant challenges still exist in clustering and loop generation stages of the pipeline. The clustering approach employed for model selection often failed to identify the most native-like assembly of SSEs for further refinement and submission. It was also observed that for some ß-strand proteins model refinement failed as ß-strands were not properly aligned to form hydrogen bonds removing otherwise accurate models from the pool. Further, BCL::Fold samples frequently non-natural topologies that require loop regions to pass through the center of the protein.