A combined optical and EPR spectroscopy study: azobenzene-based biradicals as reversible molecular photoswitches.

Azobenzene compounds are known as versatile examples for photoswitchable systems because of their isomeric cis- and trans-configurations. The switching between these isomers can be reversibly controlled by light excitation. In this study we characterize two members of this class by joining the azobenzene moiety with each two paramagnetic nitroxide spin labels. Two different linkers were chosen to tune the molecular properties. The combined approach using optical and EPR spectroscopy proved the reversibility of photoexcitation and high fatigue resistance. Furthermore, depending on the nature of the linker, PELDOR distance measurements monitored clearly the photo-induced structural changes of the azobenzene unit. Thus, a powerful concept is presented resulting from the combination of these two complementary spectroscopic techniques.

Double quantum coherence ESR spectroscopy and quantum chemical calculations on a BDPA biradical.

Carbon-centered radicals are interesting alternatives to otherwise commonly used nitroxide spin labels for dipolar spectroscopy techniques because of their narrow ESR linewidth. Herein, we present a novel BDPA biradical, where two BDPA (α,α,γ,γ-bisdiphenylene-ß-phenylallyl) radicals are covalently tethered by a saturated biphenyl acetylene linker. The inter-spin distance between the two spin carrier fragments was measured using double quantum coherence (DQC) ESR methodology. The DQC experiment revealed a mean distance of only 1.8 nm between the two unpaired electron spins. This distance is shorter than the predictions based on a simple modelling of the biradical geometry with the electron spins located at the central carbon atoms. Therefore, DFT (density functional theory) calculations were performed to obtain a picture of the spin delocalization, which may give rise to a modified dipolar interaction tensor, and to find those conformations that correspond best to the experimentally observed inter-spin distance. Quantum chemical calculations showed that the attachment of the biphenyl acetylene linker at the second position of the fluorenyl ring of BDPA did not affect the spin population or geometry of the BDPA radical. Therefore, spin delocalization and geometry optimization of each BDPA moiety could be performed on the monomeric unit alone. The allylic dihedral angle θ1 between the fluorenyl rings in the monomer subunit was determined to be 30° or 150° using quantum chemical calculations. The proton hyperfine coupling constant calculated from both energy minima was in very good agreement with literature values. Based on the optimal monomer geometries and spin density distributions, the dipolar coupling interaction between both BDPA units could be calculated for several dimer geometries. It was shown that the rotation of the BDPA units around the linker axis (θ2) does not significantly influence the dipolar coupling strength when compared to the allylic dihedral angle θ1. A good agreement between the experimental and calculated dipolar coupling was found for θ1 = 30°.

Separation of intra- and intermolecular contributions to the PELDOR signal.

Pulsed Electron-electron Double Resonance (PELDOR) is commonly used to measure distances between native paramagnetic centers or spin labels attached to complex biological macromolecules. In PELDOR the energies of electron magnetic dipolar interactions are measured by analyzing the oscillation frequencies of the recorded time resolved signal. Since PELDOR is an ensemble method, the detected signal contains contributions from intramolecular, as well as intermolecular electron spin interactions. The intramolecular part of the signal contains the information about the structure of the studied molecules, thus it is very important to accurately separate intra- and intermolecular contributions to the total signal. This separation can become ambiguous, when the length of the PELDOR signal is not much longer than twice the oscillation period of the signal. In this work we suggest a modulation depth scaling method, which can use short PELDOR signals in order to extract the intermolecular contribution. Using synthetic data we demonstrate the advantages of the new approach and analyze its stability with regard to signal noise. The method was also successfully tested on experimental data of three systems measured at Q-Band frequencies, two model compounds in deuterated and protonated solvents and one biological sample, namely BetP. The application of the new method with an assigned value of the signal modulation depth enables us to determine the interspin distances in all cases. This is especially interesting for the model compound with an interspin distance of 5.2nm in the protonated solvent and the biological sample, since an accurate separation of the intra- and intermolecular PELDOR signal contributions would be difficult with the standard approach in those cases.

Carr-Purcell Pulsed Electron Double Resonance with Shaped Inversion Pulses.

Pulsed electron paramagnetic resonance (EPR) spectroscopy allows the determination of distances, in the range of 1.5-8 nm, between two spin-labels attached to macromolecules containing protons. Unfortunately, for hydrophobic lipid-bound or detergent-solubilized membrane proteins, the maximum distance accessible is much lower, because of a strongly reduced coherence time of the electron spins. Here we introduce a pulse sequence, based on a Carr-Purcell decoupling scheme on the observer spin, where each π-pulse is accompanied by a shaped sech/tanh inversion pulse applied to the second spin, to overcome this limitation. This pump/probe excitation scheme efficiently recouples the dipolar interaction, allowing a substantially longer observation time window to be achieved. This increases the upper limit and accuracy of distances that can be determined in membrane protein complexes. We validated the method on a bis-nitroxide model compound and applied this technique to the trimeric betaine transporter BetP. Interprotomer distances as long as 6 nm could be reliably determined, which is impossible with the existing methods.

Host-guest complexes as water-soluble high-performance DNP polarizing agents.

Dynamic nuclear polarization (DNP) enhances the sensitivity of solid-state NMR (SSNMR) spectroscopy by orders of magnitude and, therefore, opens possibilities for novel applications from biology to materials science. This multitude of opportunities implicates a need for high-performance polarizing agents, which integrate specific physical and chemical features tailored for various applications. Here, we demonstrate that for the biradical bTbK in complex with captisol (CAP), a ß-cyclodextrin derivative, host-guest assembling offers a new and easily accessible approach for the development of new polarizing agents. In contrast to bTbK, the CAP-bTbK complex is water-soluble and shows significantly improved DNP performance compared to the commonly used DNP agent TOTAPOL. Furthermore, NMR and EPR data reveal improved electron and nuclear spin relaxation properties for bTbK within the host molecule. The numerous possibilities to functionalize host molecules will permit designing novel radical complexes targeting diverse applications.

PELDOR measurements on a nitroxide-labeled Cu(II) porphyrin: orientation selection, spin-density distribution, and conformational flexibility.

Metal ions are functionally or structurally important centers in metalloproteins or RNAs, which makes them interesting targets for spectroscopic investigations. In combination with site-directed spin labeling, pulsed electron-electron double resonance (PELDOR or DEER) could be a well-suited method to characterize and localize them. Here, we report on the synthesis, full characterization, and PELDOR study of a copper(II) porphyrin/nitroxide model system. The X-band PELDOR time traces contain besides the distance information a convolution of orientational selectivity, conformational flexibility, exchange coupling, and spin density distribution, which can be deconvoluted by experiments with different frequency offsets and simulations. The simulations are based on the known experimental and spin Hamiltonian parameters and make use of a geometric model as employed for structurally similar bis-nitroxides and spin density parameters as obtained from density functional theory calculations. It is found that orientation selection with respect to dipolar angles is only weakly resolvable at X-band frequencies due to the large nitrogen hyperfine coupling of the copper porphyrin. On the other hand, the PELDOR time traces reveal a much faster oscillation damping than observed for structurally similar bis-nitroxides, which is mainly assigned to a small distribution in exchange couplings J. Taking the effects of orientation selectivity, distribution in J, and spin density distribution into account leads finally to a narrow distance distribution caused solely by the flexibility of the structure, which is in agreement with distributions from known bis-nitroxides of similar structure. Thus, X-band PELDOR measurements at different frequency offsets in combination with explicit time trace simulations allow for distinguishing between structural models and quantitative interpretation of copper-nitroxide PELDOR data gives access to localization of copper(II) ions.

Counting the monomers in nanometer-sized oligomers by pulsed electron-electron double resonance.

In a lot of cases active biomolecules are complexes of higher order, thus methods capable of counting the number of building blocks and elucidating their geometric arrangement are needed. Therefore, we experimentally validate here spin-counting via 4-pulse electron-electron double resonance (PELDOR) on well-defined test samples. Two biradicals, a symmetric and an asymmetric triradical, and a tetraradical were synthesized in a convergent reaction scheme via palladium-catalyzed cross-coupling reactions. PELDOR was then used to obtain geometric information and the number of spin centers per molecule in a single experiment. The measurement yielded the expected distances (2.2-3.8 nm) and showed that different spin-spin distances in one molecule can be resolved even if the difference amounts to only 5 A. The number of spins n has been determined to be 2.1 in both biradicals, to 3.1 and 3.0 in the symmetric and asymmetric triradicals, respectively, and to 3.9 in the tetraradical. The overall error of PELDOR spin-counting was found to be 5% for up to four spins. Thus, this method is a valuable tool to determine the number of constituting spin-bearing monomers in biologically relevant homo- and heterooligomers and how their oligomerization state and geometric arrangement changes during function.

Spin labeling of oligonucleotides with the nitroxide TPA and use of PELDOR, a pulse EPR method, to measure intramolecular distances.

In this protocol, we describe the facile synthesis of the nitroxide spin-label 2,2,5,5-tetramethyl-pyrrolin-1-oxyl-3-acetylene (TPA) and then its coupling to DNA/RNA through Sonogashira cross-coupling during automated solid-phase synthesis. Subsequently, we explain how to perform distance measurements between two such spin-labels on RNA/DNA using the pulsed electron paramagnetic resonance method pulsed electron double resonance (PELDOR). This combination of methods can be used to study global structure elements of oligonucleotides in frozen solution at RNA/DNA amounts of approximately 10 nmol. We especially focus on the Sonogashira cross-coupling step, the advantages of the ACE chemistry together with the appropriate parameters for the RNA synthesizer and on the PELDOR data analysis. This procedure is applicable to RNA/DNA strands of up to approximately 80 bases in length and PELDOR yields reliably spin-spin distances up to approximately 6.5 nm. The synthesis of TPA takes approximately 5 days and spin labeling together with purification approximately 4 days. The PELDOR measurements usually take approximately 16 h and data analysis from an hour up to several days depending on the extent of analysis.

Electro- and magnetocommunication in [5,5]ditrovacenyls, [(eta7-C7H7)V(eta5-C5H4-X-eta5-C5H4)V(eta7-C7H7)], mediated by the spacers X = (Z)-CH=CH-, (E)-CH=CH-, > C=CH2, -CH2CH2-, and -CH2-.

Five new paramagnetic dinuclear complexes containing [5]trovacenyl groups, (eta7-C7H7)V(eta5-C5H4-), have been prepared and characterized, including by single-crystal X-ray diffraction. As intervening spacers, ethenediyl units in the geminal and vicinal (Z)- and (E)-bridging modes as well as methanediyl and ethanediyl units have been included with the aim of studying their propensity to transmit electric and magnetic information. It is found that redox splitting of consecutive electron-transfer steps is resolved for reduction (0-->1- -->2-) only, unsaturation of the C2 bridge not being requisite, since the -CH2CH2- spacer also gives rise to a small redox splitting. Magnetic communication is quantified in terms of the exchange coupling constant J, accessible from the EPR hyperfine pattern in solution and from magnetic susceptometry in the solid state. The results obtained from these methods generally differ; this fact is not surprising in view of conformational differences in the respective states of aggregation. It is concluded that orientation-dependent mechanisms of spin-spin interactions (pi-orbital overlap, hyperconjugation) contribute extensively although, as implied by sizeable J values for -CH2- and -C2H4- linked di[5]trovacenyl groups, coupling mediated by the sigma-orbital chain must also be considered.

Di([5]trovacenyl)ethyne, di([5]trovacenyl)butadiyne, and di-1,4-([5]trovacenylethynyl)benzene: electrocommunication and magnetocommunication mediated by --C triple bond C--, --C triple bond C--C triple bond C-- and --C triple bond C--C6H4--C triple bond C-- spacers.

The synthesis of dinuclear derivatives of trovacene (eta7-C7H7)V(eta5-C5H5) (1.) is reported, in which ethynyl (6..), butadiynyl (7..), and 1,4-di(ethynyl)phenyl (8..) groups serve as spacers between paramagnetic (S = 1/2) [5]trovacenyl units. The mononuclear precursors [5]trovacenylcarbaldehyde (2.) and [5]trovacenylacetylene (4.) are also described. Structural characterization by X-ray diffraction has been performed for 4., 6.., 7.., and 8.. Electronic communication as gleaned from cyclic voltammetry only manifests itself in the reduction processes where redox splitting deltaE1/2(0/ 1-, 1-/2-) is resolved for 6.. (deltaE1/2 = 150 mV) and indicated for 7.. (deltaE1/2 < or approximately 80 mV). Magnetocommunication leads to exchange coupling of the two electron spins which reside in vanadium centered orbitals. The values JEPR(6..) = (-)0.92, JEPR(7..) = (-)0.56, and JEPR(8..) = (-)0.005 cm(-1) are derived from the 51V hyperfine patterns. Accordingly, attenuation of exchange interaction by oligoalkyne spacers is weak, corresponding to a factor of 0.6 only per added --C triple bond C-- unit. In the determination of very weak long distance exchange interactions, EPR excels because of the range 5 x 10(-4) < or approximately J < or approximately 1.5 cm(-1) accessible in the case of 51V as a reporting magnetic nucleus and because competing intermolecular exchange is quenched in dilute fluid solution. This is demonstrated by the value Jchi(7..) = -3.84 cm(-1) obtained from a magnetic susceptibility study, which exceeds JEPR(7..) by a factor of 7. The small magnitude of spin exchange interaction between trovacene units reflects the fact that the spin bearing V3dz2 orbital is virtually orthogonal to the pi-perimeter ligand orbitals and weakly overlapping only with the a1g(sigma) ring orbitals, creating two bottlenecks for spin-exchange in the spacer-containing ditrovacenes.