Long-range distance measurements based on paramagnetic relaxation enhancement (PRE) in NMR, quantification of surface water dynamics near biomacromolecules by Overhauser dynamic nuclear polarization ...(DNP) and sensitivity enhancement by solid-state DNP all depend on introducing paramagnetic species into an otherwise diamagnetic NMR sample. The species can be introduced by site-directed spin labeling, which offers precise control for positioning the label in the sequence of a biopolymer. However, internal flexibility of the spin label gives rise to dynamic processes that potentially influence PRE and DNP behavior and leads to a spatial distribution of the electron spin even in solid samples. Internal dynamics of spin labels and their static conformational distributions have been studied mainly by electron paramagnetic resonance spectroscopy and molecular dynamics simulations, with a large body of results for the most widely applied methanethiosulfonate spin label MTSL. These results are critically discussed in a unifying picture based on rotameric states of the group that carries the spin label. Deficiencies in our current understanding of dynamics and conformations of spin labeled groups and of their influence on NMR observables are highlighted and directions for further research suggested.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Labelling techniques such as electron paramagnetic resonance spectroscopy and single-molecule fluorescence resonance energy transfer, allow access to distances in the range of tens of angstroms, ...corresponding to the size of proteins and small to medium-sized protein complexes. Such measurements do not require long-range ordering and are therefore applicable to systems with partial disorder. Data from spin-label-based measurements can be processed into distance distributions that provide information about the extent of such disorder. Using such information in modelling presents several challenges, including a small number of restraints, the influence of the label itself on the measured distance and distribution width, and balancing the fitting quality of the long-range restraints with the fitting quality of other restraint subsets. Starting with general considerations about integrative and hybrid structural modelling, this review provides an overview of recent approaches to these problems and identifies where further progress is needed.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
A simple trifluoromethoxylation method enables non‐directed functionalization of C−H bonds on a range of substrates, providing access to aryl trifluoromethyl ethers. This light‐driven process is ...distinctly different from conventional procedures and occurs through an OCF3 radical mechanism mediated by a photoredox catalyst, which triggers an N−O bond fragmentation. The pyridinium‐based trifluoromethoxylation reagent is bench‐stable and provides access to synthetic diversity in lead compounds in an operationally simple manner.
N‐O problem: A bench‐stable pyridinium reagent transfers the elusive trifluoromethoxyl radical to a variety of arenes, including a selection of biorelevant compounds. By sidestepping limitations of traditional OCF3 sources, this approach enables late‐stage diversification by direct C−H functionalization in a mechanistically unique fashion.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
A series of 37 dinitroxide biradicals have been prepared and their performance studied as polarizing agents in cross-effect DNP NMR experiments at 9.4 T and 100 K in 1,1,2,2-tetrachloroethane (TCE). ...We observe that in this regime the DNP performance is strongly correlated with the substituents on the polarizing agents, and electron and nuclear spin relaxation times, with longer relaxation times leading to better enhancements. We also observe that deuteration of the radicals generally leads to better DNP enhancement but with longer build-up time. One of the new radicals introduced here provides the best performance obtained so far under these conditions.
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IJS, KILJ, NUK, UL, UM, UPUK
Structural characterization of proteins and their complexes may require integration of restraints from various experimental techniques. MMM (Multiscale Modeling of Macromolecules) is a Matlab‐based ...open‐source modeling toolbox for this purpose with a particular emphasis on distance distribution restraints obtained from electron paramagnetic resonance experiments on spin‐labelled proteins and nucleic acids and their combination with atomistic structures of domains or whole protomers, small‐angle scattering data, secondary structure information, homology information, and elastic network models. MMM does not only integrate various types of restraints, but also various existing modeling tools by providing a common graphical user interface to them. The types of restraints that can support such modeling and the available model types are illustrated by recent application examples.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Many RNA-binding proteins undergo liquid-liquid phase separation, which underlies the formation of membraneless organelles, such as stress granules and P-bodies. Studies of the molecular mechanism of ...phase separation in vitro are hampered by the coalescence and sedimentation of organelle-sized droplets interacting with glass surfaces. Here, we demonstrate that liquid droplets of fused in sarcoma (FUS)-a protein found in cytoplasmic aggregates of amyotrophic lateral sclerosis and frontotemporal dementia patients-can be stabilized in vitro using an agarose hydrogel that acts as a cytoskeleton mimic. This allows their spectroscopic characterization by liquid-phase NMR and electron paramagnetic resonance spectroscopy. Protein signals from both dispersed and condensed phases can be observed simultaneously, and their respective proportions can be quantified precisely. Furthermore, the agarose hydrogel acts as a cryoprotectant during shock-freezing, which facilitates pulsed electron paramagnetic resonance measurements at cryogenic temperatures. Surprisingly, double electron-electron resonance measurements revealed a compaction of FUS in the condensed phase.
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GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
While two-dimensional correlation spectra contain more information as compared to one-dimensional spectra, typical spectral widths encountered in electron paramagnetic resonance (EPR) spectroscopy ...largely restrict the applicability of correlation techniques. In essence, the monochromatic excitation pulses established in pulsed EPR often cannot uniformly excite the entire spectrum. Here, this restriction is alleviated for nitroxide spin labels at Q-band microwave frequencies around 35 GHz. This is achieved by substitution of monochromatic pulses by frequency-swept chirp pulses tailored for uniform excitation. Unwanted interference effects brought by this substitution are analyzed for a pair of electron spins with secular dipolar coupling. Experimentally, the dipole-dipole interaction can be separated from other interactions by a constant-time Zeeman-compensated solid echo sequence called SIFTER. Such SIFTER experiments usually yield a one-dimensional dipolar spectrum. EPR-correlated dipolar spectra can be obtained when the four pulses are replaced by chirp pulses. These two-dimensional spectra encode additional information on the geometrical arrangement of the two spin labels. With the excitation parameters achieved by a home-built Q-band spectrometer capable of frequency-swept excitation, unwanted interference effects can be largely neglected for the examined model compound with a spin-spin distance of 4 nm. The experimentally obtained correlation pattern conforms to the expectation based on the inter-spin geometry of the investigated rigid model compound.
While natural photosynthesis serves as the model system for efficient charge separation and decoupling of redox reactions, bio‐inspired artificial systems typically lack applicability owing to ...synthetic challenges and structural complexity. We present herein a simple and inexpensive system that, under solar irradiation, forms highly reductive radicals in the presence of an electron donor, with lifetimes exceeding the diurnal cycle. This radical species is formed within a cyanamide‐functionalized polymeric network of heptazine units and can give off its trapped electrons in the dark to yield H2, triggered by a co‐catalyst, thus enabling the temporal decoupling of the light and dark reactions of photocatalytic hydrogen production through the radical′s longevity. The system introduced here thus demonstrates a new approach for storing sunlight as long‐lived radicals, and provides the structural basis for designing photocatalysts with long‐lived photo‐induced states.
Live long and prosper: A cyanamide‐functionalized carbon nitride can form ultra‐long‐lived radicals under solar irradiation in the presence of an electron donor. This longevity enables the temporal decoupling of the two half reactions in the photo‐redox production of hydrogen.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
We report a mild and operationally simple trifluoroacylation strategy of olefines, that utilizes trifluoroacetic anhydride as a low‐cost and readily available reagent. This light‐mediated process is ...fundamentally different from conventional methodologies and occurs through a trifluoroacyl radical mechanism promoted by a photocatalyst, which triggers a C−O bond fragmentation. Mechanistic studies (kinetic isotope effects, spectroelectrochemistry, optical spectroscopy, theoretical investigations) highlight the evidence of a fleeting CF3CO radical under photoredox conditions. The trifluoroacyl radical can be stabilized under CO atmosphere, delivering the trifluoroacetylation product with higher chemical efficiency. Furthermore, the method can be turned into a trifluoromethylation protocol by simply changing the reaction parameters. Beyond simple alkenes, this method allows for chemo‐ and regioselective functionalization of small‐molecule drugs and common pharmacophores.
The trifluoroacyl radical is generated by C−O bond fragmentation of trifluoroacetic anhydride under photoredox conditions and undergoes olefinic C−H bond trifluoroacetylation in a mechanistically unique fashion. The protocol is operationally simple, scalable, and enables the late‐stage diversification of complex biorelevant molecules delivering the corresponding α,β‐unsaturated trifluoromethyl ketones in one step.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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