DNA complexes with human apurinic/apyrimidinic endonuclease 1: structural insights revealed by pulsed dipolar EPR with orthogonal spin labeling.

A DNA molecule is under continuous influence of endogenous and exogenous damaging factors, which produce a variety of DNA lesions. Apurinic/apyrimidinic sites (abasic or AP sites) are among the most common DNA lesions. In this work, we applied pulse dipolar electron paramagnetic resonance (EPR) spectroscopy in combination with molecular dynamics (MD) simulations to investigate in-depth conformational changes in DNA containing an AP site and in a complex of this DNA with AP endonuclease 1 (APE1). For this purpose, triarylmethyl (TAM)-based spin labels were attached to the 5' ends of an oligonucleotide duplex, and nitroxide spin labels were introduced into APE1. In this way, we created a system that enabled monitoring the conformational changes of the main APE1 substrate by EPR. In addition, we were able to trace substrate-to-product transformation in this system. The use of different (orthogonal) spin labels in the enzyme and in the DNA substrate has a crucial advantage allowing for detailed investigation of local damage and conformational changes in AP-DNA alone and in its complex with APE1.

Spin State Switching in Heptauthrene Nanostructure by Electric Field: Computational Study.

Recent experimental studies proved the presence of the triplet spin state in atomically precise heptauthrene nanostructure of nanographene type (composed of two interconnected triangles with zigzag edge). In the paper, we report the computational study predicting the possibility of controlling this spin state with an external in-plane electric field by causing the spin switching. We construct and discuss the ground state magnetic phase diagram involving S=1 (triplet) state, S=0 antiferromagnetic state and non-magnetic state and predict the switching possibility with the critical electric field of the order of 0.1 V/Å. We discuss the spin distribution across the nanostructure, finding its concentration along the longest zigzag edge. To model our system of interest, we use the mean-field Hubbard Hamiltonian, taking into account the in-plane external electric field as well as the in-plane magnetic field (in a form of the exchange field from the substrate). We also assess the effect of uniaxial strain on the magnetic phase diagram.

Synthesis of Spin-Labelled Bergamottin: A Potent CYP3A4 Inhibitor with Antiproliferative Activity.

Bergamottin (BM, 1), a component of grapefruit juice, acts as an inhibitor of some isoforms of the cytochrome P450 (CYP) enzyme, particularly CYP3A4. Herein, a new bergamottin containing a nitroxide moiety (SL-bergamottin, SL-BM, 10) was synthesized; chemically characterized, evaluated as a potential inhibitor of the CYP2C19, CYP3A4, and CYP2C9 enzymes; and compared to BM and known inhibitors such as ketoconazole (KET) (3A4), warfarin (WAR) (2C9), and ticlopidine (TIC) (2C19). The antitumor activity of the new SL-bergamottin was also investigated. Among the compounds studied, BM showed the strongest inhibition of the CYP2C9 and 2C19 enzymes. SL-BM is a more potent inhibitor of CYP3A4 than the parent compound; this finding was also supported by docking studies, suggesting that the binding positions of BM and SL-BM to the active site of CYP3A4 are very similar, but that SL-BM had a better ∆Gbind value than that of BM. The nitroxide moiety markedly increased the antitumor activity of BM toward HeLa cells and marginally increased its toxicity toward a normal cell line. In conclusion, modification of the geranyl sidechain of BM can result in new CYP3A4 enzyme inhibitors with strong antitumor effects.

Doxyl Nitroxide Spin Probes Can Modify Toxicity of Doxorubicin towards Fibroblast Cells.

The biological properties of doxyl stearate nitroxides (DSs): 5-DS, Met-12-DS, and 16-DS, commonly used as spin probes, have not been explored in much detail so far. Furthermore, the influence of DSs on the cellular changes induced by the anticancer drug doxorubicin (DOX) has not yet been investigated. Therefore, we examined the cytotoxicity of DSs and their ability to induce cell death and to influence on fluidity and lipid peroxidation (LPO) in the plasma membrane of immortalised B14 fibroblasts, used as a model neoplastic cells, susceptible to DOX-induced changes. The influence of DSs on DOX toxicity was also investigated and compared with that of a natural reference antioxidant α-Tocopherol. By employing the trypan blue exclusion test and double fluorescent staining, we found a significant level of cytotoxicity for DSs and showed that their ability to induce apoptosis and modify plasma membrane fluidity (measured fluorimetrically) is more potent than for α-Tocopherol. The most cytotoxic nitroxide was 5-DS. The electron paramagnetic resonance (EPR) measurements revealed that 5-DS was reduced in B14 cells at the fastest and Met-12-DS at the slowest rate. In the presence of DOX, DSs were reduced slower than alone. The investigated compounds, administered with DOX, enhanced DOX-induced cell death and demonstrated concentration-dependent biphasic influence on membrane fluidity. A-Tocopherol showed weaker effects than DSs, regardless the mode of its application-alone or with DOX. High concentrations of α-Tocopherol and DSs decreased DOX-induced LPO. Substantial cytotoxicity of the DSs suggests that they should be used more carefully in the investigations performed on sensitive cells. Enhancement of DOX toxicity by DSs showed their potential to act as chemosensitizers of cancer cells to anthracycline chemotherapy.

Measurement of Angstrom to Nanometer Molecular Distances with 19 F 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 19 F 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 19 F 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.

Semi-Rigid Nitroxide Spin Label for Long-Range EPR Distance Measurements of Lipid Bilayer Embedded ß-Peptides.

ß-Peptides are an interesting new class of transmembrane model peptides based on their conformationally stable and well-defined secondary structures. Herein, we present the synthesis of the paramagnetic ß-amino acid ß3 -hTOPP (4-(3,3,5,5-tetramethyl-2,6-dioxo-4-oxylpiperazin-1-yl)-d-ß3 -homophenylglycine) that enables investigations of ß-peptides by EPR spectroscopy. This amino acid adds to the, to date, sparse number of ß-peptide spin labels. Its performance was evaluated by investigating the helical turn of a 314 -helical transmembrane model ß-peptide. Nanometer distances between two incorporated ß3 -hTOPP labels in different environments were measured by using pulsed electron/electron double resonance (PELDOR/DEER) spectroscopy. Due to the semi-rigid conformational design, the label delivers reliable distances and sharp (one-peak) distance distributions even in the lipid bilayer. The results indicate that the investigated ß-peptide folds into a 3.2514 helix and maintains this conformation in the lipid bilayer.

Efficient Building Blocks for Solid-Phase Peptide Synthesis of Spin Labeled Peptides for Electron Paramagnetic Resonance and Dynamic Nuclear Polarization Applications.

Specific spin labeling allows the site-selective investigation of biomolecules by EPR and DNP enhanced NMR spectroscopy. A novel spin labeling strategy for commercially available Fmoc-amino acids is developed. In this approach, the PROXYL spin label is covalently attached to the hydroxyl side chain of three amino acids hydroxyproline (Hyp), serine (Ser) and tyrosine (Tyr) by a simple three-step synthesis route. The obtained PROXYL containing building-blocks are N-terminally protected by the Fmoc-protection group, which makes them applicable for the use in solid-phase peptide synthesis (SPPS). This approach allows the insertion of the spin label at any desired position during SPPS, which makes it more versatile than the widely used post synthetic spin labeling strategies. For the final building-blocks, the radical activity is proven by EPR. DNP enhanced solid-state NMR experiments employing these building-blocks in a TCE solution show enhancement factors of up to 26 for 1 H and 13 C (1 H→13 C cross-polarization). To proof the viability of the presented building-blocks for insertion of the spin label during SPPS the penta-peptide Acetyl-Gly-Ser(PROXYL)-Gly-Gly-Gly was synthesized employing the spin labeled Ser building-block. This peptide could successfully be isolated and the spin label activity proved by EPR and DNP NMR measurements, showing enhancement factors of 12.1±0.1 for 1 H and 13.9±0.5 for 13 C (direct polarization).

A semi-rigid isoindoline-derived nitroxide spin label for RNA.

A new isoindoline-derived benzimidazole nitroxide spin label, ImUm, was synthesized and incorporated into RNA oligoribonucleotides. ImUm is the first example of a conformationally unambiguous spin label for RNA, in which the nitroxide N-O bond lies on the same axis as the single bond used to attach the rigid isoindoline-based spin label to a uridine base. This results in minimal displacement of the nitroxide upon rotation of this single bond, which is a useful property for a label to be used for distance measurements. Continuous-wave (CW) EPR measurements of RNA duplexes containing ImUm indicate a restricted rotation around this single bond, presumably due to an intramolecular hydrogen bond between the benzimidazole N-H and O4 of the uracil. Orientation-selective pulsed electron-electron double resonance (PELDOR, also called double electron-electron resonance, or DEER) distance measurements between two spin labels in two RNA duplexes showed in one case a strong orientation dependence, further confirming the restricted motion of the spin labels in RNA duplexes.

Synthesis of Nanometer Sized Bis- and Tris-trityl Model Compounds with Different Extent of Spin-Spin Coupling.

Tris(2,3,5,6-tetrathiaaryl)methyl radicals, so-called trityl radicals, are emerging as spin labels for distance measurements in biological systems based on Electron Paramagnetic Resonance (EPR). Here, the synthesis and characterization of rigid model systems carrying either two or three trityl moieties is reported. The monofunctionalized trityl radicals are connected to the molecular bridging scaffold via an esterification reaction employing the Mukaiyama reagent 2-chloro-methylpyridinium iodide. The bis- and tris-trityl compounds exhibit different inter-spin distances, strength of electron-electron exchange and dipolar coupling and can give rise to multi-spin effects. They are to serve as benchmark systems in comparing EPR distance measurement methods.

Nucleotide-Independent Copper(II)-Based Distance Measurements in DNA by Pulsed ESR Spectroscopy.

A site-specific Cu2+ binding motif within a DNA duplex for distance measurements by ESR spectroscopy is reported. This motif utilizes a commercially available 2,2'-dipicolylamine (DPA) phosphormadite easily incorporated into any DNA oligonucleotide during initial DNA synthesis. The method only requires the simple post-synthetic addition of Cu2+ without the need for further chemical modification. Notably, the label is positioned within the DNA duplex, as opposed to outside the helical perimeter, for an accurate measurement of duplex distance. A distance of 2.7 nm was measured on a doubly Cu2+ -labeled DNA sequence, which is in exact agreement with the expected distance from both DNA modeling and molecular dynamic simulations. This result suggests that with this labeling strategy the ESR measured distance directly reports on backbone DNA distance, without the need for further modeling. Furthermore, the labeling strategy is structure- and nucleotide-independent.

Versatile Trityl Spin Labels for Nanometer Distance Measurements on Biomolecules In†Vitro and within Cells.

Structure determination of biomacromolecules under in-cell conditions is a relevant yet challenging task. Electron paramagnetic resonance (EPR) distance measurements in combination with site-directed spin labeling (SDSL) are a valuable tool in this endeavor but the usually used nitroxide spin labels are not well-suited for in-cell measurements. In contrast, triarylmethyl (trityl) radicals are highly persistent, exhibit a long relaxation time and a narrow spectral width. Here, the synthesis of a versatile collection of trityl spin labels and their application in in vitro and in-cell trityl-iron distance measurements on a cytochrome P450 protein are described. The trityl labels show similar labeling efficiencies and better signal-to-noise ratios (SNR) as compared to the popular methanethiosulfonate spin label (MTSSL) and enabled a successful in-cell measurement.

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 dU. 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 dU and Çm on a self-complementary RNA duplex. Our results demonstrate that the conformational diversity of dU is significantly reduced near the freezing temperature of a phosphate buffer, resulting in strongly orientation-selective PELDOR time traces of the dU-labeled RNA duplex.

Poly-arginine conjugated triarylmethyl radical as intracellular spin label.

Stable triarylmethyl radicals are ideal spin labels used for biomedical electron paramagnetic resonance applications. Previously reported structures exhibit polar charged functions for water solubilization preventing them from crossing the cell membrane. We report the synthesis of a triarylmethyl radical conjugated to poly-arginine peptide allowing intracellular delivery of the paramagnetic label.

Synthesis of fluorophosphate nucleotide analogues and their characterization as tools for ¹9F NMR studies.

To broaden the scope of existing methods based on (19)F nucleotide labeling, we developed a new method for the synthesis of fluorophosphate (oligo)nucleotide analogues containing an O to F substitution at the terminal position of the (oligo)phosphate moiety and evaluated them as tools for (19)F NMR studies. Using three efficient and comprehensive synthetic approaches based on phosphorimidazolide chemistry and tetra-n-butylammonium fluoride, fluoromonophosphate, or fluorophosphate imidazolide as fluorine sources, we prepared over 30 fluorophosphate-containing nucleotides, varying in nucleobase type (A, G, C, U, m(7)G), phosphate chain length (from mono to tetra), and presence of additional phosphate modifications (thio, borano, imido, methylene). Using fluorophosphate imidazolide as fluorophosphorylating reagent for 5'-phosphorylated oligos we also synthesized oligonucleotide 5'-(2-fluorodiphosphates), which are potentially useful as (19)F NMR hybridization probes. The compounds were characterized by (19)F NMR and evaluated as (19)F NMR molecular probes. We found that fluorophosphate nucleotide analogues can be used to monitor activity of enzymes with various specificities and metal ion requirements, including human DcpS enzyme, a therapeutic target for spinal muscular atrophy. The compounds can also serve as reporter ligands for protein binding studies, as exemplified by studying interaction of fluorophosphate mRNA cap analogues with eukaryotic translation initiation factor (eIF4E).

Combined molecular MRI and immuno-spin-trapping for in vivo detection of free radicals in orthotopic mouse GL261 gliomas.

Free radicals play a major role in gliomas. By combining immuno-spin-trapping (IST) and molecular magnetic resonance imaging (mMRI), in vivo levels of free radicals were detected within mice bearing orthotopic GL261 gliomas. The nitrone spin trap DMPO (5,5-dimethyl pyrroline N-oxide) was administered prior to injection of an anti-DMPO probe (anti-DMPO antibody covalently bound to a bovine serum albumin (BSA)-Gd (gadolinium)-DTPA (diethylene triamine penta acetic acid)-biotin MRI contrast agent) to trap tumor-associated free radicals. mMRI detected the presence of anti-DMPO adducts by either a significant sustained increase (p<0.001) in MR signal intensity or a significant decrease (p<0.001) in T1 relaxation, measured as %T1 change. In vitro assessment of the anti-DMPO probe indicated a significant decrease (p<0.0001) in T1 relaxation in GL261 cells that were oxidatively stressed with hydrogen peroxide, compared to controls. The biotin moiety of the anti-DMPO probe was targeted with fluorescently-labeled streptavidin to locate the anti-DMPO probe in excised brain tissues. As a negative control a non-specific IgG antibody covalently bound to the albumin-Gd-DTPA-biotin construct was used. DMPO adducts were also confirmed in tumor tissue from animals administered DMPO, compared to non-tumor brain tissue. GL261 gliomas were found to have significantly increased malondialdehyde (MDA) protein adducts (p<0.001) and 3-nitrotyrosine (3-NT) (p<0.05) compared to normal mouse brain tissue, indicating increased oxidized lipids and proteins, respectively. Co-localization of the anti-DMPO probe with either 3-NT or 4-hydroxynonenal was also observed. This is the first report regarding the detection of in vivo levels of free radicals from a glioma model.

Gd(III)-PyMTA label is suitable for in-cell EPR.

Distance measurement in the nanometer range by electron paramagnetic resonance spectroscopy (EPR) in combination with site-directed spin labeling is a very powerful tool to monitor the structure and dynamics of biomacromolecules in their natural environment. However, in-cell application is hampered by the short lifetime of the commonly used nitroxide spin labels in the reducing milieu inside a cell. Here, we demonstrate that the Gd(III) based spin label Gd-PyMTA is suitable for in-cell EPR. Gd-PyMTA turned out to be cell compatible and was proven to be inert in in-cell extracts of Xenopus laevis oocytes at 18 °C for more than 24 h. The proline rich peptide H-AP10CP10CP10-NH2 was site-directedly spin labeled with Gd-PyMTA at both cysteine moieties. The resulting peptide, H-AP10C(Gd-PyMTA)P10C(Gd-PyMTA)P10-NH2, as well as the model compound Gd-spacer-Gd, which consists of a spacer of well-known stiffness, were microinjected into Xenopus laevis oocytes, and the Gd(III)-Gd(III) distances were determined by double electron-electron resonance (DEER) spectroscopy. To analyze the intracellular peptide conformation, a rotamer library was set up to take the conformational flexibility of the tether between the Gd(III) ion and the Cα of the cysteine moiety into account. The results suggest that the spin labeled peptide H-AP10C(Gd-PyMTA)P10C(Gd-PyMTA)P10-NH2 is inserted into cell membranes, coinciding with a conformational change of the oligoproline from a PPII into a PPI helix.

In vivo proton-electron double-resonance imaging of extracellular tumor pH using an advanced nitroxide probe.

A variable radio frequency proton-electron double-resonance imaging (VRF PEDRI) approach for pH mapping of aqueous samples has been recently developed (Efimova et al. J. Magn. Reson. 2011, 209, 227-232). A pH map is extracted from two PEDRI acquisitions performed at electron paramagnetic resonance (EPR) frequencies of protonated and unprotonated forms of a pH-sensitive probe. To translate VRF PEDRI to an in vivo setting, an advanced pH probe was synthesized. Probe deuteration resulted in a narrow spectral line of 1.2 G compared to a nondeuterated analogue line width of 2.1 G allowing for an increase of Overhauser enhancements and reduction in rf power deposition. Binding of the probe to the cell-impermeable tripeptide, glutathione (GSH), allows for targeting to extracellular tissue space for monitoring extracellular tumor acidosis, a prognostic factor in tumor pathophysiology. The probe demonstrated pH sensitivity in the 5.8-7.8 range, optimum for measurement of acidic extracellular tumor pH (pH(e)). In vivo VRF PEDRI was performed on Met-1 tumor-bearing mice. Compared to normal mammary glands with a neutral mean pH(e) (7.1 ± 0.1), we observed broader pH distribution with acidic mean pH(e) (6.8 ± 0.1) in tumor tissue. In summary, VRF PEDRI in combination with a newly developed pH probe provides an analytical approach for spatially resolved noninvasive pHe monitoring, in vivo.

Conversion rate of para-hydrogen to ortho-hydrogen by oxygen: implications for PHIP gas storage and utilization.

OBJECT: To determine the storability of para-hydrogen before reestablishment of the room temperature thermal equilibrium mixture. MATERIALS AND METHODS: Para-hydrogen was produced at near 100% purity and mixed with different oxygen quantities to determine the rate of conversion to the thermal equilibrium mixture of 75: 25% (ortho: para) by detecting the ortho-hydrogen (1)H nuclear magnetic resonance using a 9.4 T imager. RESULTS: The para-hydrogen to ortho-hydrogen velocity constant, k, near room temperature (292 K) was determined to be 8.27 ± 1.30 L/mol · min(-1). This value was calculated utilizing four different oxygen fractions. CONCLUSIONS: Para-hydrogen conversion to ortho-hydrogen by oxygen can be minimized for long term storage with judicious removal of oxygen contamination. Prior calculated velocity rates were confirmed demonstrating a dependence on only the oxygen concentration.

Synthesis of spin-labeled riboswitch RNAs using convertible nucleosides and DNA-catalyzed RNA ligation.

Chemically stable nitroxide radicals that can be monitored by electron paramagnetic resonance (EPR) spectroscopy can provide information on structural and dynamic properties of functional RNA such as riboswitches. The convertible nucleoside approach is used to install 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO) and 2,2,5,5-tetramethylpyrrolidin-1-oxyl (proxyl) labels at the exocyclic N(4)-amino group of cytidine and 2'-O-methylcytidine nucleotides in RNA. To obtain site-specifically labeled long riboswitch RNAs beyond the limit of solid-phase synthesis, we report the ligation of spin-labeled RNA using an in vitro selected deoxyribozyme as catalyst, and demonstrate the synthesis of TEMPO-labeled 53 nt SAM-III and 118 nt SAM-I riboswitch domains (SAM=S-adenosylmethionine).

Pulsed ESR dipolar spectroscopy for distance measurements in immobilized spin labeled proteins in liquid solution.

Pulsed electron spin resonance (ESR) dipolar spectroscopy (PDS) in combination with site-directed spin labeling is unique in providing nanometer-range distances and distributions in biological systems. To date, most of the pulsed ESR techniques require frozen solutions at cryogenic temperatures to reduce the rapid electron spin relaxation rate and to prevent averaging of electron-electron dipolar interaction due to the rapid molecular tumbling. To enable measurements in liquid solution, we are exploring a triarylmethyl (TAM)-based spin label with a relatively long relaxation time where the protein is immobilized by attachment to a solid support. In this preliminary study, TAM radicals were attached via disulfide linkages to substituted cysteine residues at positions 65 and 80 or 65 and 76 in T4 lysozyme immobilized on Sepharose. Interspin distances determined using double quantum coherence (DQC) in solution are close to those expected from models, and the narrow distance distribution in each case indicates that the TAM-based spin label is relatively localized.