Intermolecular contributions, filtration effects and signal composition of SIFTER (single-frequency technique for refocusing).

To characterize structure and molecular order in the nanometre range, distances between electron spins and their distributions can be measured via dipolar spin-spin interactions by different pulsed electron paramagnetic resonance experiments. Here, for the single-frequency technique for refocusing dipolar couplings (SIFTER), the buildup of dipolar modulation signal and intermolecular contributions is analysed for a uniform random distribution of monoradicals and biradicals in frozen glassy solvent by using the product operator formalism for electron spin S=1/2. A dipolar oscillation artefact appearing at both ends of the SIFTER time trace is predicted, which originates from the weak coherence transfer between biradicals. The relative intensity of this artefact is predicted to be temperature independent but to increase with the spin concentration in the sample. Different compositions of the intermolecular background are predicted in the case of biradicals and in the case of monoradicals. Our theoretical account suggests that the appropriate procedure of extracting the intramolecular dipolar contribution (form factor) requires fitting and subtracting the unmodulated part, followed by division by an intermolecular background function that is different in shape. This scheme differs from the previously used heuristic background division approach. We compare our theoretical derivations to experimental SIFTER traces for nitroxide and trityl monoradicals and biradicals. Our analysis demonstrates a good qualitative match with the proposed theoretical description. The resulting perspectives for a quantitative analysis of SIFTER data are discussed.

Strategies to identify and suppress crosstalk signals in double electron-electron resonance (DEER) experiments with gadoliniumIII and nitroxide spin-labeled compounds.

Double electron-electron resonance (DEER) spectroscopy applied to orthogonally spin-labeled biomolecular complexes simplifies the assignment of intra- and intermolecular distances, thereby increasing the information content per sample. In fact, various spin labels can be addressed independently in DEER experiments due to spectroscopically nonoverlapping central transitions, distinct relaxation times, and/or transition moments; hence, they are referred to as spectroscopically orthogonal. Molecular complexes which are, for example, orthogonally spin-labeled with nitroxide (NO) and gadolinium (Gd) labels give access to three distinct DEER channels that are optimized to selectively probe NO-NO, NO-Gd, and Gd-Gd distances. Nevertheless, it has been previously recognized that crosstalk signals between individual DEER channels can occur, for example, when a Gd-Gd distance appears in a DEER channel optimized to detect NO-Gd distances. This is caused by residual spectral overlap between NO and Gd spins which, therefore, cannot be considered as perfectly orthogonal. Here, we present a systematic study on how to identify and suppress crosstalk signals that can appear in DEER experiments using mixtures of NO-NO, NO-Gd, and Gd-Gd molecular rulers characterized by distinct, nonoverlapping distance distributions. This study will help to correctly assign the distance peaks in homo- and heterocomplexes of biomolecules carrying not perfectly orthogonal spin labels.

Distance measurement between trityl radicals by pulse dressed electron paramagnetic resonance with phase modulation.

Distance measurement in the nanometre range is among the most important applications of pulse electron paramagnetic resonance today, especially in biological applications. The longest distance that can be measured by all presently used pulse sequences is determined by the phase memory time Tm of the observed spins. Here we show that one can measure the dipolar coupling during strong microwave irradiation by using an appropriate frequency- or phase-modulation scheme, i.e. by applying pulse sequences in the nutating frame. This decouples the electron spins from the surrounding nuclear spins and thus leads to significantly longer relaxation times of the microwave-dressed spins (i.e. the rotating frame relaxation times T1ρ and T2ρ) compared to Tm. The electron-electron dipolar coupling is not decoupled as long as both spins are excited, which can be implemented for trityl radicals at Q-band frequencies (35 GHz, 1.2 T). We show results for two bis-trityl rulers with inter-electron distances of about 4.1 and 5.3 nm and discuss technical challenges and possible next steps.

Pulsed EPR Methods to Study Biomolecular Interactions.

Orthogonal site-directed spin labelling in combination with pulsed EPR spectroscopy is a powerful approach to study biomolecular interactions on a molecular level. Following a surge in pulse EPR method development, it is now possible to access distance distributions in the nanometre range in systems of complex composition. In this article we briefly outline the necessary considerations for measurements of distance distributions in macromolecular systems labelled with two or more different types of paramagnetic centres. We illustrate the approach with two examples: an application of the Double Electron-Electron Resonance (DEER) method on a triple spin-labelled protein dimer labelled with nitroxide and Gd(III), and an optimisation study of the Relaxation Induced Dipolar Modulation Enhancement (RIDME) experiment for the orthogonal spin pair Cu(II)-nitroxide.

Improving the accuracy of Cu(ii)-nitroxide RIDME in the presence of orientation correlation in water-soluble Cu(ii)-nitroxide rulers.

Orientation selection is a challenge in distance determination with double electron electron resonance (DEER) spectroscopy of rigid molecules. The problem is reduced when applying the Relaxation-Induced Dipolar Modulation Enhancement (RIDME) experiment. Here we present an in-depth study on nitroxide-detected RIDME in Cu(ii)-nitroxide spin pairs using two Cu(ii)-nitroxide rulers that are both water soluble and have comparable spin-spin distances. They differ in the type of the ligand (TAHA and PyMTA) for the Cu(ii) ion which results in different contributions of exchange coupling. Both rulers feature substantial orientation correlation between the molecular frames of the Cu(ii) complex and the nitroxide. We discuss how the spin-spin couplings can be accurately measured and how they can be correlated to the nitroxide resonance frequencies. In that, we pay particular attention to the suppression of nuclear modulation and of echo crossing artefacts, to background correction, and to orientation averaging. With a nitroxide observer sequence based on chirp pulses, we achieve wideband detection of all nitroxide orientations. Two-dimensional Fourier transformation of data obtained in this manner affords observer-EPR correlated RIDME spectra that enable visual understanding of the orientation correlation. The syntheses of the Cu(ii)-nitroxide rulers are presented. The synthetic route is considered to be of general use for the preparation of [metal ion complex]-nitroxide rulers, including water soluble ones.

Trityl Radicals with a Combination of the Orthogonal Functional Groups Ethyne and Carboxyl: Synthesis without a Statistical Step and EPR Characterization.

Finland trityl radical (FTR) shows very attractive EPR spectroscopic properties for a manifold of applications. For most of its applications only one chemically reactive functional group is needed. The presence of three equally reactive carboxyl groups leads to FTR modifications through reactions which give statistical mixtures of 1-fold-, 2-fold-, and 3-fold-modified and unmodified FTR. To avoid the side effects of such a statistical reaction-limited yields and separation challenges-we took a route to FTR-type trityl radicals with scaffold assembly by addition of an aryllithium with one type of substituent to a diarylketone with another type of substituent. This gave the two FTR-type trityl radicals 1 and 2 which carry a combination of the chemically orthogonal groups, carboxyl and triisopropylsilylethynyl. Standard column chromatography was sufficient for product isolation on all stages, whereby polar tagging helped. The EPR spectroscopic properties of the trityl radicals 1 and 2 in ethanol were determined in X and W bands. Their g anisotropy and T1 and T2 relaxation times make them spin labels as good as the benchmark FTR. This paper discloses also details on the synthesis of building blocks used for FTR preparation and improved access to the bare FTR scaffold.

Quantitative analysis of zero-field splitting parameter distributions in Gd(iii) complexes.

The magnetic properties of paramagnetic species with spin S > 1/2 are parameterized by the familiar g tensor as well as "zero-field splitting" (ZFS) terms that break the degeneracy between spin states even in the absence of a magnetic field. In this work, we determine the mean values and distributions of the ZFS parameters D and E for six Gd(iii) complexes (S = 7/2) and critically discuss the accuracy of such determination. EPR spectra of the Gd(iii) complexes were recorded in glassy frozen solutions at 10 K or below at Q-band (∼34 GHz), W-band (∼94 GHz) and G-band (240 GHz) frequencies, and simulated with two widely used models for the form of the distributions of the ZFS parameters D and E. We find that the form of the distribution of the ZFS parameter D is bimodal, consisting roughly of two Gaussians centered at D and -D with unequal amplitudes. The extracted values of D (σD) for the six complexes are, in MHz: Gd-NO3Pic, 485 ± 20 (155 ± 37); Gd-DOTA/Gd-maleimide-DOTA, -714 ± 43 (328 ± 99); iodo-(Gd-PyMTA)/MOMethynyl-(Gd-PyMTA), 1213 ± 60 (418 ± 141); Gd-TAHA, 1361 ± 69 (457 ± 178); iodo-Gd-PCTA-[12], 1861 ± 135 (467 ± 292); and Gd-PyDTTA, 1830 ± 105 (390 ± 242). The sign of D was adjusted based on the Gaussian component with larger amplitude. We relate the extracted P(D) distributions to the structure of the individual Gd(iii) complexes by fitting them to a model that superposes the contribution to the D tensor from each coordinating atom of the ligand. Using this model, we predict D, σD, and E values for several additional Gd(iii) complexes that were not measured in this work. The results of this paper may be useful as benchmarks for the verification of quantum chemical calculations of ZFS parameters, and point the way to designing Gd(iii) complexes for particular applications and estimating their magnetic properties a priori.

EPR characterization of Mn(ii) complexes for distance determination with pulsed dipolar spectroscopy.

The four Mn(ii) complexes Mn-DOTA, Mn-TAHA, Mn-PyMTA, and Mn-NO3Py were characterized by electron paramagnetic resonance (EPR), electron-nuclear double resonance (ENDOR), and relaxation measurements, to predict their relative performance in the EPR pulse dipolar spectroscopy (PDS) experiments. High spin density localization on the metal ions was proven by ENDOR on 1H, D, 14N, and 55Mn nuclei. The transverse relaxation of the Mn(ii) complexes appears to be slow enough for PDS-based spin-spin distance determination. Rather advantageous ratios of T1/Tm were measured allowing for good relaxation induced dipolar modulation enhancement (RIDME) performance and, in general, fast shot repetitions in any PDS experiment. Relaxation properties of the Mn(ii) complexes correlate with the strengths of their zero field splitting (ZFS). Further, a comparison of Mn(ii)-DOTA and Gd(iii)-DOTA based spin labels is presented. The RIDME technique to measure nanometer-range Mn(ii)-Mn(ii) distances in biomolecules is discussed as an alternative to the well-known DEER technique that often appears challenging in cases of metal-metal distance measurements. The use of a modified kernel function that includes dipolar harmonic overtones allows model-free computation of the Mn(ii)-Mn(ii) distance distributions. Mn(ii)-Mn(ii) distances are computed from RIDME data of Mn-rulers consisting of two Mn-PyMTA complexes connected by a rodlike spacer of defined length. Level crossing effects seem to have only a weak influence on the distance distributions computed from this set of Mn(ii)-Mn(ii) RIDME data.

Switch-On Fluorescence of a Perylene-Dye-Functionalized Metal-Organic Framework through Postsynthetic Modification.

A perylene dye was introduced directly as a linker into a metal-organic framework (MOF) during synthesis. Depending on the dye concentration in the MOF synthesis mixture, different fluorescent materials were generated. The successful incorporation of the dye was proven by using (13) C and (27) Al MAS NMR spectroscopy, by solution NMR spectroscopy after digestion of the MOF sample, and by synthesizing a reference dye without connecting groups, which could coordinate on the metal-oxo cluster inside the MOF. Fluorescence quenching effects of the MOF linker, 2-aminoterephthalate, were observed and overcome by postsynthetic modification with acetic anhydride. We show here for the first time that amino groups, which can be used as anchoring points for covalent attachment of other molecules, are responsible for fluorescence quenching. Thus, a very promising strategy to implement switchable fluorescence into MOFs is shown here.

Postsynthetic modification of an amino-tagged MOF using peptide coupling reagents: a comparative study.

The suitability of four peptide coupling reagents for postsynthetic modification (PSM) of amino-tagged metal-organic frameworks (MOFs) with carboxylic acids was investigated. Mild reaction conditions at room temperature allow effective covalent attachment of drugs and biomolecules inside the pores of MOFs with moderate chemical stability.

Turn-on fluorescence triggered by selective internal dye replacement in MOFs.

Coordinatively unsaturated metal sites (CUS) are used to create dye-functionalized metal-organic frameworks (MOFs). The quenching of dye fluorescence through interactions with the CUS can be utilised for chemical sensing of Lewis bases that displace the dye from the CUS, resulting in a triggered turn-on fluorescence signal.

Adsorption and orientation of the physiological extracellular peptide glutathione disulfide on surface functionalized colloidal alumina particles.

Understanding the interrelation between surface chemistry of colloidal particles and surface adsorption of biomolecules is a crucial prerequisite for the design of materials for biotechnological and nanomedical applications. Here, we elucidate how tailoring the surface chemistry of colloidal alumina particles (d50 = 180 nm) with amino (-NH2), carboxylate (-COOH), phosphate (-PO3H2) or sulfonate (-SO3H) groups affects adsorption and orientation of the model peptide glutathione disulfide (GSSG). GSSG adsorbed on native, -NH2-functionalized, and -SO3H-functionalized alumina but not on -COOH- and -PO3H2-functionalized particles. When adsorption occurred, the process was rapid (≤5 min), reversible by application of salts, and followed a Langmuir adsorption isotherm dependent on the particle surface functionalization and ζ potential. The orientation of particle bound GSSG was assessed by the release of glutathione after reducing the GSSG disulfide bond and by ζ potential measurements. GSSG is likely to bind via the carboxylate groups of one of its two glutathionyl (GS) moieties onto native and -NH2-modified alumina, whereas GSSG is suggested to bind to -SO3H-modified alumina via the primary amino groups of both GS moieties. Thus, GSSG adsorption and orientation can be tailored by varying the molecular composition of the particle surface, demonstrating a step toward guiding interactions of biomolecules with colloidal particles.