A Bis‐Manganese(II)–DOTA Complex for Pulsed Dipolar Spectroscopy

High‐spin gadolinium(III) and manganese(II) complexes have emerged as alternatives to standard nitroxide radical spin labels for measuring nanometric distances by using pulsed electron–electron double resonance (PELDOR or DEER) at high fields/frequencies. For certain complexes, particularly those with relatively small zero‐field splitting (ZFS) and short distances between the two metal centers, the pseudosecular term of the dipolar coupling Hamiltonian is non‐negligible. However, in general, the contribution from this term during conventional data analysis is masked by the flexibility of the molecule of interest and/or the long tethers connecting them to the spin labels. The efficient synthesis of a model system consisting of two [Mn(dota)]^2? (MnDOTA; DOTA^4?=1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetate) directly connected to the ends of a central rodlike oligo(phenylene–ethynylene) (OPE) spacer is reported. The rigidity of the OPE is confirmed by Q‐band PELDOR measurements on a bis‐nitroxide analogue. The Mn^II?Mn^II distance distribution profile determined by W‐band PELDOR is in reasonable agreement with one simulated by using a simple rotamer analysis. The small degree of flexibility arising from the linking MnDOTA arm appears to outweigh the contribution from the pseudosecular term at this interspin distance. This study illustrates the potential of MnDOTA‐based spin labels for measuring fairly short nanometer distances, and also presents an interesting candidate for in‐depth studies of pulsed dipolar spectroscopy methods on Mn^II?Mn^II systems.     

Selective High‐Resolution Detection of Membrane Protein–Ligand Interaction in Native Membranes Using Trityl–Nitroxide PELDOR

The orchestrated interaction of transmembrane proteins with other molecules mediates several crucial biological processes. Detergent solubilization may significantly alter or even abolish such hetero‐oligomeric interactions, which makes observing them at high resolution in their native environment technically challenging. Dipolar electron paramagnetic resonance (EPR) techniques such as pulsed electro–electron double resonance (PELDOR) can provide very precise distances within biomolecules. To concurrently determine the inter‐subunit interaction and the intra‐subunit conformational changes in hetero‐oligomeric complexes, a combination of different spin labels is required. Orthogonal spin labeling using a triarylmethyl (TAM) label in combination with a nitroxide label is used to detect protein–ligand interactions in native lipid bilayers. This approach provides a higher sensitivity and total selectivity and will greatly facilitate the investigation of multimeric transmembrane complexes employing different spin labels in the native lipid environment.     

Post‐synthetic Spin‐Labeling of RNA through Click Chemistry for PELDOR Measurements

Site‐directed spin labeling of RNA based on click chemistry is used in combination with pulsed electron‐electron double resonance (PELDOR) to benchmark a nitroxide spin label, called here d? . We compare this approach with another established method that employs the rigid spin label Çm for RNA labeling. By using CD spectroscopy, thermal denaturation measurements, CW‐EPR as well as PELDOR we analyzed and compared the influence of d? and Çm on a self‐complementary RNA duplex. Our results demonstrate that the conformational diversity of d? is significantly reduced near the freezing temperature of a phosphate buffer, resulting in strongly orientation‐selective PELDOR time traces of the d? ‐labeled RNA duplex.     

Accurate Extraction of Nanometer Distances in Multimers by Pulse EPR

Pulse electron paramagnetic resonance (EPR) is gaining increasing importance in structural biology. The PELDOR (pulsed electron–electron double resonance) method allows extracting distance information on the nanometer scale. Here, we demonstrate the efficient extraction of distances from multimeric systems such as membrane‐embedded ion channels where data analysis is commonly hindered by multi‐spin effects.     

Light‐Driven Coordination‐Induced Spin‐State Switching: Rational Design of Photodissociable Ligands

The bistability of spin states (e.g., spin crossover) in bulk materials is well investigated and understood. We recently extended spin‐state switching to isolated molecules at room temperature (light‐driven coordination‐induced spin‐state switching, or LD‐CISSS). Whereas bistability and hysteresis in conventional spin‐crossover materials are caused by cooperative effects in the crystal lattice, spin switching in LD‐CISSS is achieved by reversibly changing the coordination number of a metal complex by means of a photochromic ligand that binds in one configuration but dissociates in the other form. We present mathematical proof that the maximum efficiency in property switching by such a photodissociable ligand (PDL) is only dependent on the ratio of the association constants of both configurations. Rational design by using DFT calculations was applied to develop a photoswitchable ligand with a high switching efficiency. The starting point was a nickel–porphyrin as the transition‐metal complex and 3‐phenylazopyridine as the photodissociable ligand. Calculations and experiments were performed in two iterative steps to find a substitution pattern at the phenylazopyridine ligand that provided optimum performance. Following this strategy, we synthesized an improved photodissociable ligand that binds to the Ni–porphyrin with an association constant that is 5.36 times higher in its trans form than in the cis form. The switching efficiency between the diamagnetic and paramagnetic state is efficient as well (72 % paramagnetic Ni–porphyrin after irradiation at 365 nm, 32 % paramagnetic species after irradiation at 440 nm). Potential applications arise from the fact that the LD‐CISSS approach for the first time allows reversible switching of the magnetic susceptibility of a homogeneous solution. Photoswitchable contrast agents for magnetic resonance imaging and light‐controlled magnetic levitation are conceivable applications.     

Pd–Porphyrin Oligomers Sensitized for Green‐to‐Blue Photon Upconversion: The More the Better?

A series of directly meso‐meso‐linked Pd–porphyrin oligomers (PdDTP‐M, PdDTP‐D, and PdDTP‐T) have been prepared. The absorption region and the light‐harvesting ability of the Pd–porphyrin oligomers are broadened and enhanced by increasing the number of Pd–porphyrin units. Triplet–triplet annihilation upconversion (TTA‐UC) systems were constructed by utilizing the Pd–porphyrin oligomers as the sensitizer and 9,10‐diphenylanthracene (DPA) as the acceptor in deaerated toluene and green‐to‐blue photon upconversion was observed upon excitation with a 532 nm laser. The triplet–triplet annihilation upconversion quantum efficiencies were found to be 6.2 %, 10.5 %, and 1.6 % for the [PdDTP‐M]/DPA, [PdDTP‐D]/DPA, and [PdDTP‐T]/DPA systems, respectively, under an excitation power density of 500 mW cm^?2. The photophysical processes of the TTA‐UC systems have been investigated in detail. The higher triplet–triplet annihilation upconversion quantum efficiency observed in the [PdDTP‐D]/DPA system can be rationalized by the enhanced light‐harvesting ability of PdDTP‐D at 532 nm. Under the same experimental conditions, the [PdDTP‐D]/DPA system produces more ³DPA* than the other two TTA‐UC systems, benefiting the triplet–triplet annihilation process. This work provides a useful way to develop efficient TTA‐UC systems with broad spectral response by using Pd–porphyrin oligomers as sensitizers.     

Bis‐Gadolinium Complexes for Solid Effect and Cross Effect Dynamic Nuclear Polarization

High‐spin complexes act as polarizing agents (PAs) for dynamic nuclear polarization (DNP) in solid‐state NMR spectroscopy and feature promising aspects towards biomolecular DNP. We present a study on bis(Gd‐chelate)s which enable cross effect (CE) DNP owing to spatial confinement of two dipolar‐coupled electron spins. Their well‐defined Gd⋅⋅⋅Gd distances in the range of 1.2–3.4 nm allowed us to elucidate the Gd⋅⋅⋅Gd distance dependence of the DNP mechanism and NMR signal enhancement. We found that Gd⋅⋅⋅Gd distances above 2.1 nm result in solid effect DNP while distances between 1.2 and 2.1 nm enable CE for ¹H, ¹³C, and ¹⁵N nuclear spins. We compare 263 GHz electron paramagnetic resonance (EPR) spectra with the obtained DNP field profiles and discuss possible CE matching conditions within the high‐spin system and the influence of dipolar broadening of the EPR signal. Our findings foster the understanding of the CE mechanism and the design of high‐spin PAs for specific applications of DNP.     

Site‐Directed Spin‐Labeling of DNA by the Azide–Alkyne ‘Click’ Reaction: Nanometer Distance Measurements on 7‐Deaza‐2′‐deoxyadenosine and 2′‐Deoxyuridine Nitroxide Conjugates Spatially Separated or Linked to a ‘dA‐dT’ Base Pair

Nucleobase‐directed spin‐labeling by the azide‐alkyne ‘click’ (CuAAC) reaction has been performed for the first time with oligonucleotides. 7‐Deaza‐7‐ethynyl‐2′‐deoxyadenosine ( 1 ) and 5‐ethynyl‐2′‐deoxyuridine ( 2 ) were chosen to incorporate terminal triple bonds into DNA. Oligonucleotides containing 1 or 2 were synthesized on a solid phase and spin labeling with 4‐azido‐2,2,6,6‐tetramethylpiperidine 1‐oxyl (4‐azido‐TEMPO, 3 ) was performed by post‐modification in solution. Two spin labels ( 3 ) were incorporated with high efficiency into the DNA duplex at spatially separated positions or into a ‘dA‐dT’ base pair. Modification at the 5‐position of the pyrimidine base or at the 7‐position of the 7‐deazapurine residue gave steric freedom to the spin label in the major groove of duplex DNA. By applying cw and pulse EPR spectroscopy, very accurate distances between spin labels, within the range of 1–2 nm, were measured. The spin–spin distance was 1.8±0.2 nm for DNA duplex 17 ( dA*^7,10 ) ?11 containing two spin labels that are separated by two nucleotides within one individual strand. A distance of 1.4±0.2 nm was found for the spin‐labeled ‘dA‐dT’ base pair 15 ( dA*⁷ ) ?16 ( dT*⁶ ). The ‘click’ approach has the potential to be applied to all four constituents of DNA, which indicates the universal applicability of the method. New insights into the structural changes of canonical or modified DNA are expected to provide additional information on novel DNA structures, protein interaction, DNA architecture, and synthetic biology.     

Conformationally Restricted Isoindoline‐Derived Spin Labels in Duplex DNA: Distances and Rotational Flexibility by Pulsed Electron–Electron Double Resonance Spectroscopy

Three structurally related isoindoline‐derived spin labels that have different mobilities were incorporated into duplex DNA to systematically study the effect of motion on orientation‐dependent pulsed electron–electron double resonance (PELDOR) measurements. To that end, a new nitroxide spin label, ^ExIm U , was synthesized and incorporated into DNA oligonucleotides. ^ExIm U is the first example of a conformationally unambiguous spin label for nucleic acids, in which the nitroxide N?O bond lies on the same axis as the three single bonds used to attach the otherwise rigid isoindoline‐based spin label to a uridine base. Continuous‐wave (CW) EPR measurements of ^ExIm U confirm a very high rotational mobility of the spin label in duplex DNA relative to the structurally related spin label ^Im U , which has restricted mobility due to an intramolecular hydrogen bond. The X‐band CW‐EPR spectra of ^ExIm U can be used to identify mismatches in duplex DNA. PELDOR distance measurements between pairs of the spin labels ^Im U , ^Ox U , and ^ExIm U in duplex DNA showed a strong angular dependence for ^Im U , a medium dependence for ^Ox U , and no orientation effect for ^ExIm U . Thus, precise distances can be extracted from ^ExIm U without having to take orientational effects into account.     

Long Distance Measurements up to 160 Å in the GroEL Tetradecamer Using Q‐Band DEER EPR Spectroscopy

Current distance measurements between spin‐labels on multimeric protonated proteins using double electron–electron resonance (DEER) EPR spectroscopy are generally limited to the 15–60 Å range. Here we show how DEER experiments can be extended to dipolar evolution times of ca. 80 μs, permitting distances up to 170 Å to be accessed in multimeric proteins. The method relies on sparse spin‐labeling, supplemented by deuteration of protein and solvent, to minimize the deleterious impact of multispin effects and substantially increase the apparent spin‐label phase memory relaxation time, complemented by high sensitivity afforded by measurements at Q‐band. We demonstrate the approach using the tetradecameric molecular machine GroEL as an example. Two engineered surface‐exposed mutants, R268C and E315C, are used to measure pairwise distance distributions with mean values ranging from 20 to 100 Å and from 30 to 160 Å, respectively, both within and between the two heptameric rings of GroEL. The measured distance distributions are consistent with the known crystal structure of apo GroEL. The methodology presented here should significantly expand the use of DEER for the structural characterization of conformational changes in higher order oligomers.     

Long‐Lived Charge‐Transfer State Induced by Spin‐Orbit Charge Transfer Intersystem Crossing (SOCT‐ISC) in a Compact Spiro Electron Donor/Acceptor Dyad

We prepared conceptually novel, fully rigid, spiro compact electron donor (Rhodamine B, lactam form, RB)/acceptor (naphthalimide; NI) orthogonal dyad to attain the long‐lived triplet charge‐transfer (³CT) state, based on the electron spin control using spin‐orbit charge transfer intersystem crossing (SOCT‐ISC). Transient absorption (TA) spectra indicate the first charge separation (CS) takes place within 2.5 ps, subsequent SOCT‐ISC takes 8 ns to produce the ³NI* state. Then the slow secondary CS (125 ns) gives the long‐lived ³CT state (0.94 μs in deaerated n‐hexane) with high energy level (ca. 2.12 eV). The cascade photophysical processes of the dyad upon photoexcitation are summarized as ¹NI*→¹CT→³NI*→³CT. With time‐resolved electron paramagnetic resonance (TREPR) spectra, an EEEAAA electron‐spin polarization pattern was observed for the naphthalimide‐localized triplet state. Our spiro compact dyad structure and the electron spin‐control approach is different to previous methods for which invoking transition‐metal coordination or chromophores with intrinsic ISC ability is mandatory.     

Measurement of Angstrom to Nanometer Molecular Distances with 19F Nuclear Spins by EPR/ENDOR Spectroscopy

Spectroscopic and biophysical methods for structural determination at atomic resolution are fundamental in studies of biological function. Here we introduce an approach to measure molecular distances in bio‐macromolecules using ¹⁹F nuclear spins and nitroxide radicals in combination with high‐frequency (94 GHz/3.4 T) electron–nuclear double resonance (ENDOR). The small size and large gyromagnetic ratio of the ¹⁹F label enables to access distances up to about 1.5 nm with an accuracy of 0.1–1 Å. The experiment is not limited by the size of the bio‐macromolecule. Performance is illustrated on synthesized fluorinated model compounds as well as spin‐labelled RNA duplexes. The results demonstrate that our simple but strategic spin‐labelling procedure combined with state‐of‐the‐art spectroscopy accesses a distance range crucial to elucidate active sites of nucleic acids or proteins in the solution state.     

One‐component thioxanthone‐based polymeric photoinitiators

The photochemical and photophysical properties of new polymeric photoinitiators (PPIs) containing pendant thioxanthone (TX) and amine moieties are studied. The PPIs are synthesized by copolymerization of tert‐butyl 2‐((9‐oxo‐9H‐thioxanthen‐2‐yloxy)methyl)acrylate (TX1) with N,N‐dimethylaminoethyl methacrylate (DMAEM) at two different ratios using free radical polymerization. UV–vis spectra indicate that PPIs possess similar absorption characteristics to TX1 in the violet range (～400 nm; absorption red‐shift 20 nm). The photochemical mechanisms are studied by electron spin resonance (ESR), steady state photolysis, laser flash photolysis, and cyclic voltammetry. ESR studies indicate formation of two different aminoalkyl radicals on the hydrogen donor amine. The triplet state of the PPIs is short‐lived compared to isopropyl thioxanthone and TX1, due to the built‐in amine functionality. Photopolymerization of trimethylolpropane triacrylate (TMPTA) initiated by these photoinitiators under LED exposure at 385 and 405 nm using real‐time FTIR spectroscopy shows that they exhibit higher efficiency than TX/N‐methyldiethanolamine (MDEA) and TX1/MDEA systems with the advantage of a much higher molecular weight that can be very helpful to overcome migration issues. © 2016 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016 , 54, 3370–3378     

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.     

Structural Changes of a Doubly Spin‐Labeled Chemically Driven Molecular Shuttle Probed by PELDOR Spectroscopy

Gaining detailed information on the structural rearrangements associated with stimuli‐induced molecular movements is of utmost importance for understanding the operation of molecular machines. Pulsed electron–electron double resonance (PELDOR) was employed to monitor the geometrical changes arising upon chemical switching of a [2]rotaxane that behaves as an acid–base‐controlled molecular shuttle. To this aim, the rotaxane was endowed with stable nitroxide radical units in both the ring and axle components. The combination of PELDOR data and molecular dynamic calculations indicates that in the investigated rotaxane, the ring displacement along the axle, caused by the addition of a base, does not alter significantly the distance between the nitroxide labels, but it is accompanied by a profound change in the geometry adopted by the macrocycle.     

A Charge‐Stabilizing,Multimodular, Ferrocene–Bis(triphenylamine)–Zinc‐porphyrin–Fullerene Polyad

A novel multimodular donor–acceptor polyad featuring zinc porphyrin, fullerene, ferrocene, and triphenylamine entities was designed, synthesized, and studied as a charge‐stabilizing, photosynthetic‐antenna/reaction‐center mimic. The ferrocene and fullerene entities, covalently linked to the porphyrin ring, were distantly separated to accomplish the charge‐separation/hole‐migration events leading to the creation of a long‐lived charge‐separated state. The geometry and electronic structures of the newly synthesized compound was deduced by B3LYP/3‐21G(*) optimization, while the energy levels for different photochemical events was established using data from the optical absorption and emission, and electrochemical studies. Excitation of the triphenylamine entities revealed singlet‐singlet energy transfer to the appended zinc porphyrin. As predicted from the energy levels, photoinduced electron transfer from both the singlet and triplet excited states of the zinc porphyrin to fullerene followed by subsequent hole migration involving ferrocene was witnessed from the transient absorption studies. The charge‐separated state persisted for about 8.5 μs and was governed by the distance between the final charge‐transfer product, that is, a species involving a ferrocenium cation and a fullerene radical anion, with additional influence from the charge‐stabilizing triphenylamine entities located on the zinc‐porphyrin macrocycle.     

Long‐Lived Charge Separation in Novel Axial Donor–Porphyrin–Acceptor Triads Based on Tetrathiafulvalene,Aluminum(III) Porphyrin and Naphthalenediimide

Two self‐assembled supramolecular donor–acceptor triads consisting of Al^III porphyrin (AlPor) with axially bound naphthalenediimide (NDI) as an acceptor and tetrathiafulvalene (TTF) as a secondary donor are reported. In the triads, the NDI and TTF units are attached to Al^III on opposite faces of the porphyrin, through covalent and coordination bonds, respectively. Fluorescence studies show that the lowest excited singlet state of the porphyrin is quenched through electron transfer to NDI and hole transfer to TTF. In dichloromethane hole transfer to TTF dominates, whereas in benzonitrile (BN) electron transfer to NDI is the main quenching pathway. In the nematic phase of the liquid crystalline solvent 4‐(n‐pentyl)‐4′‐cyanobiphenyl (5CB), a spin‐polarized transient EPR spectrum that is readily assigned to the weakly coupled radical pair TTF^.+NDI^.? is obtained. The initial polarization pattern indicates that the charge separation occurs through the singlet channel and that singlet–triplet mixing occurs in the primary radical pair. At later time the polarization pattern inverts as a result of depopulation of the states with singlet character by recombination to the ground state. The singlet lifetime of TTF^.+NDI^.? is estimated to be 200–300 ns, whereas the triplet lifetime in the approximately 350 mT magnetic field of the X‐band EPR spectrometer is about 10 μs. In contrast, in dichloromethane and BN the lifetime of the charge separation is <10 ns.     

Novel Pulsed Electron Paramagnetic Resonance Techniques for the Studies of Structure and Dynamics of Photo‐excited Triplet State of Organic Molecules: A Professional Journey

We present a few novel pulsed electron paramagnetic resonance techniques developed in our laboratory for the studies of structure and dynamics of the photo‐excited triplet state of organic molecules. We discuss many aspects of these new techniques and the significances of these measurements: (1) enhancing NMR signal intensity by dynamic nuclear polarization ‐ integrated solid effect, (2) performing magnetic resonance in zero‐field and low‐field by pulsed microwave, (3) mapping molecular motion of organic crystals by pulsed zero‐field and low‐field experiments, (4) probing spin dynamics at level anti‐crossing by fast field switching, (5) measuring hyperfine interaction by electron spin echo envelop modulation and spin‐echo electron nuclear double resonance and (6) detecting spin dynamics, nuclear quantum oscillation, entanglements and new avenues for quantum computer. We have employed the highly electron spin polarized pentacene triplet state as the model system in all of our pulsed EPR experiments. We performed most of our experiments at room temperature. The goals of our studies are aiming to improve spin detectability, to probe molecular dynamics, to determine electronic structures, to measure molecular interaction and motion, and to examine quantum coherence and oscillation which may yield new avenues in the applications of pulsed EPR techniques to quantum computer.     

Broadband Visible‐Light‐Harvesting trans‐Bis(alkylphosphine) Platinum(II)‐Alkynyl Complexes with Singlet Energy Transfer between BODIPY and Naphthalene Diimide Ligands

A heteroleptic bis(tributylphosphine) platinum(II)‐alkynyl complex ( Pt‐1 ) showing broadband visible‐light absorption was prepared. Two different visible‐light‐absorbing ligands, that is, ethynylated boron‐dipyrromethene (BODIPY) and a functionalized naphthalene diimide (NDI) were used in the molecule. Two reference complexes, Pt‐2 and Pt‐3 , which contain only the NDI or BODIPY ligand, respectively, were also prepared. The coordinated BODIPY ligand shows absorption at 503 nm and fluorescence at 516 nm, whereas the coordinated NDI ligand absorbs at 594 nm; the spectral overlap between the two ligands ensures intramolecular resonance energy transfer in Pt‐1 , with BODIPY as the singlet energy donor and NDI as the energy acceptor. The complex shows strong absorption in the region 450 nm–640 nm, with molar absorption coefficient up to 88 000 M ^?1 cm^?1. Long‐lived triplet excited states lifetimes were observed for Pt‐1 – Pt‐3 (36.9 μs, 28.3 μs, and 818.6 μs, respectively). Singlet and triplet energy transfer processes were studied by the fluorescence/phosphorescence excitation spectra, steady‐state and time‐resolved UV/Vis absorption and luminescence spectra, as well as nanosecond time‐resolved transient difference absorption spectra. A triplet‐state equilibrium was observed for Pt‐1 . The complexes were used as triplet photosensitizers for triplet–triplet annihilation upconversion, with upconversion quantum yields up to 18.4 % being observed for Pt‐1 .