Nitroxide Moiety Research Articles

“Snorkeling” of the Charged Sidechain of a Transmembrane Peptide as Directly Observed by Double Electron-Electron Resonance Experiment

While hydrophobic amino acids constitute the bulk of transmembrane protein domains, polar and even charged amino acids are not uncommon and often play significant roles in membrane protein function. Positioning a polar residue within the bilayer core is highly unfavorable thermodynamically; however, the free energy penalty could be minimized by “stretching” the side chain of the amino acid to bring the charged moiety closer to the bilayer surface while keeping the rest of the side chain inside the hydrophobic core. This biophysical phenomenon is known as “snorkeling”. Here we report experimental observations of “snorkeling” for nitroxide-modified side-chains upon protonation, its dependence upon the location along the transmembrane peptide helix, and how this snorkeling is affected by the membrane electrostatic surface potential. pH sensitive spin labels, either IMTSL or IKMTSL (JPCB 2009, 113, 3453) were attached to two cysteine residues positioned equidistant from the center of the WALP peptide so that the primary sequence of each peptide is palindromic, thus, ensuring symmetric location of the labels with respect to the bilayer. The change in protonation states of the nitroxide was directly observed from EPR spectra. The distance between two nitroxide moieties was measured by Q-band double electron-electron resonance (DEER) experiment. Upon protonation, the distance between the two IMTSL probes increased compared to that of the neutral forms, by approximately 3 A indicating displacements of the charged nitroxide sidechain towards the polar head region. The “snorkeling” of the label was observed to be depth dependent - no changes in the positioning of the sidechain upon protonation was observed for labels located within 10-8 A from the center of the bilayer. Supported by NSF-0843632 to TIS.

Advanced EPR Methods for Studying Conformational Dynamics of Nucleic Acids.

Pulsed electron paramagnetic resonance (EPR) spectroscopy has become an important tool for structural characterization of biomolecules allowing measurement of the distances between two paramagnetic spin labels attached to a biomolecule in the 2-8 nm range. In this chapter, we will focus on applications of this approach to investigate tertiary structure elements as well as conformational dynamics of nucleic acid molecules. Both aspects take advantage of using specific spin labels that are rigidly attached to the nucleobases, as they allow obtaining not only the distance but also the relative orientation between both nitroxide moieties with high accuracy. Thus, not only the distance but additionally the three Euler angles between both the nitroxide axis systems and the two polar angles of the interconnecting vector with respect to the nitroxide axis systems can be extracted from a single pair of spin labels. To extract all these parameters independently and unambiguously, a set of multifrequency/multifield pulsed EPR experiments have to be performed. We will describe the experimental procedure as well as newly developed spin labels, which are helpful to disentangle all these parameters, and tools which we have developed to analyze such data sets. The procedures and analyses will be illustrated by examples from our laboratory.

Spin exchange monitoring of the strong positive homotropic allosteric binding of a tetraradical by a synthetic receptor in water.

The flexible tetranitroxide 4T has been prepared and was shown to exhibit a nine line EPR spectrum in water, characteristic of significant through space spin exchange (J(ij)) between four electron spins interacting with four nitrogen nuclei (J(ij) ≫ a(N)). Addition of CB[8] to 4T decreases dramatically all the Jij couplings, and the nine line spectrum is replaced by the characteristic three line spectrum of a mononitroxide. The supramolecular association between 4T and CB[8] involves a highly cooperative asymmetric complexation by two CB[8] (K1 = 4027 M(-1); K2 = 202,800 M(-1); α = 201) leading to a rigid complex with remote nitroxide moieties. The remarkable enhancement for the affinity of the second CB[8] corresponds to an allosteric interaction energy of ≈13 kJ mol(-1), which is comparable to that of the binding of oxygen by hemoglobin. These results are confirmed by competition and reduction experiments, DFT and molecular dynamics calculations, mass spectrometry, and liquid state NMR of the corresponding reduced complex bearing hydroxylamine moieties. This study shows that suitably designed molecules can generate allosteric complexation with CB[8]. The molecule must (i) carry several recognizable groups for CB[8] and (ii) be folded so that the first binding event reorganizes the molecule (unfold) for a better subsequent recognition. The presence of accessible protonable amines and H-bond donors to fit with the second point are also further stabilizing groups of CB[8] complexation. In these conditions, the spin exchange coupling between four radicals has been efficiently and finely tuned and the resulting allosteric complexation induced a dramatic stabilization enhancement of the included paramagnetic moieties in highly reducing conditions through the formation of the supramolecular 4T@CB[8]2 complex.

Dynamic, in vivo, real-time detection of retinal oxidative status in a model of elevated intraocular pressure using a novel, reversibly responsive, profluorescent nitroxide probe

Changes to the redox status of biological systems have been implicated in the pathogenesis of a wide variety of disorders including cancer, Ischemia-reperfusion (I/R) injury and neurodegeneration. In times of metabolic stress e.g. ischaemia/reperfusion, reactive oxygen species (ROS) production overwhelms the intrinsic antioxidant capacity of the cell, damaging vital cellular components. The ability to quantify ROS changes in vivo, is therefore essential to understanding their biological role. Here we evaluate the suitability of a novel reversible profluorescent probe containing a redox-sensitive nitroxide moiety (methyl ester tetraethylrhodamine nitroxide, ME-TRN), as an in vivo, real-time reporter of retinal oxidative status. The reversible nature of the probe's response offers the unique advantage of being able to monitor redox changes in both oxidizing and reducing directions in real time. After intravitreal administration of the ME-TRN probe, we induced ROS production in rat retina using an established model of complete, acute retinal ischaemia followed by reperfusion. After restoration of blood flow, retinas were imaged using a Micron III rodent fundus fluorescence imaging system, to quantify the redox-response of the probe. Fluorescent intensity declined during the first 60 min of reperfusion. The ROS-induced change in probe fluorescence was ameliorated with the retinal antioxidant, lutein. Fluorescence intensity in non-Ischemia eyes did not change significantly. This new probe and imaging technology provide a reversible and real-time response to oxidative changes and may allow the in vivo testing of antioxidant therapies of potential benefit to a range of diseases linked to oxidative stress.

Abstract 498: Blunting the radical scavenging property of mitochondria-targeted, triphenylphosphonium-linked antioxidants failed to mitigate their anti-proliferative and antitumor effects

Abstract Reactive oxygen species (ROS), including superoxide and hydrogen peroxide, are implicated in cancer cell proliferation. Therefore, strategies to mitigate ROS levels were proposed as a viable mechanism to inhibit cancer cell proliferation. As mitochondria are believed to be a major contributor to intracellular ROS production, mitochondria-targeted antioxidants (MTAs) (e.g., Mito-Q, Mito-CP, Mito-Vitamin-E or Mito-chromanol) were successfully used to stop proliferation and kill cancer cells in vitro and attenuate tumor growth in vivo. MTAs also inhibited mitochondrial oxygen consumption and ATP production. In this study, we tested whether the antioxidant moiety and related antioxidant function of MTAs or other mitochondrial mechanism is responsible for inhibiting tumor cell proliferation and resulting cytotoxicity. To this end, we compared the anti-proliferative and antitumor effects of Mito-CP, a mitochondrially-targeted nitroxide exhibiting superoxide dismutase (SOD)-like activity, and a synthetic analog of Mito-CP (Mito-CP acetamide), lacking the nitroxide moiety and SOD-like activity. Both Mito-CP and Mito-CP acetamide were equally potent in different cancer cell types (breast, pancreatic, colon, skin, bladder and lung). Additionally, both compounds inhibited mitochondrial function and decreased intracellular ATP levels to the same extent. We conclude that MTAs such as Mito-CP, Mito-Vitamin-E exert their antiproliferative effects in cancer cells due to mitochondrial localization and targeting cellular bioenergetics and not due to their antioxidative activity. Results from this study strongly question the present dogma on mitochondrial ROS involvement in cancer cell proliferation. Citation Format: Gang Cheng, Jacek Zielonka, Joy Joseph, Balaraman Kalyanaraman. Blunting the radical scavenging property of mitochondria-targeted, triphenylphosphonium-linked antioxidants failed to mitigate their anti-proliferative and antitumor effects. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 498. doi:10.1158/1538-7445.AM2014-498

A sensitive and selective chemosensor for ascorbic acid based on a fluorescent nitroxide switch

Ascorbic acid (AsA), also known as vitamin C, is a vital small-molecule antioxidant with multiple functions in vivo. It’s the major natural antioxidant found in plants and is also an essential component of human nutrition. AsA plays a key role in many diseases-related biological metabolism. Therefore, sensitive and selective detection of AsA is greatly important in pharmaceutical, clinical and food industry. Here a sensitive and selective sensor for ascorbic acid detection based on the recovered fluorescence of NAPS-NO (N-propyl-triethoxysilane-4-(4-ylamino-1-oxy-2,2,6,6-tetramethylpiperdine)- naphthalimide) probe is described. The fluorescence of the naphthalimide moiety of NAPS-NO is inhibited by the nitroxide group, which is covalently linked to the fluorophore. Then, ascorbic acid reacts rapidly with the nitroxide moiety of NAPS-NO to form hydroxylamine, and the fluorescence properties of the naphthalimide moiety are recovered and the ESR signal decayed. Over a wide range from 80nM to 50μM, a good linear relationship between the fluorescence intensity and the concentration of ascorbic acid was found and the detection limit was estimated to be as low as 20nM. To confirm the practical usefulness of the fluorophore–nitroxide probe, we demonstrated the use of NAPS-NO for the measurement of AsA in human blood serum and also successfully determined the concentration of AsA in HEK 293 cell lysate. Results from confocal laser scanning microscopy experiments demonstrated that this chemosensor is cell permeable and can be used as a fluorescent probe for monitoring ascorbic acid in living cells.

Radical-triplet pair interactions as probes of long-range polymer motion in solution.

Radical-triplet pair interactions are used to investigate the dynamics of acrylic polymers in dilute solution. Methyl methacrylate was randomly copolymerized with a small amount of an amine-containing monomer to create the polymers. The amine subunits were then oxidized to nitroxide moieties (stable free radicals). Using time-resolved electron paramagnetic resonance (TREPR) spectroscopy on the sub-microsecond time scale, competition is observed between two deactivation processes of the ester side chain photoexcited triplet state: (1) Norrish I α-cleavage, leading to a 21-line main chain polymeric free radical TREPR spectrum with emissive triplet mechanism spin polarization, and (2) spin polarization transfer or quenching by a nearby stable nitroxide radical, which gives a 3-line spectrum exhibiting emissive radical-triplet pair spin polarization. The main chain polymer radical signal dominates the TREPR spectrum if the probability of radical-triplet pair encounters is low. These competing reactions show a strong dependence on nitroxide incorporation (mol %), temperature, solvent, and acrylic polymer ester side chain structure. A comparison of steady state EPR and TREPR signals from inter- vs intramolecular processes clearly demonstrates the influence of polymer chain dynamics on the observed phenomena.

Mitochondria-targeted spin traps: synthesis, superoxide spin trapping, and mitochondrial uptake.

Development of reliable methods and site-specific detection of free radicals is an active area of research. Here, we describe the synthesis and radical-trapping properties of new derivatives of DEPMPO and DIPPMPO, bearing a mitochondria-targeting triphenylphosphonium cationic moiety or guanidinium cationic group. All of the spin traps prepared have been observed to efficiently trap superoxide radical anions in a cell-free system. The superoxide spin adducts exhibited similar spectral properties, indicating no significant differences in the geometry of the cyclic nitroxide moieties of the spin adducts. The superoxide adduct stability was measured and observed to be highest (t1/2 = 73 min) for DIPPMPO nitrone linked to triphenylphosphonium moiety via a short carbon chain (Mito-DIPPMPO). The experimental results and DFT quantum chemical calculations indicate that the cationic property of the triphenylphosphonium group may be responsible for increased superoxide trapping efficiency and adduct stability of Mito-DIPPMPO, as compared to the DIPPMPO spin trap. The studies of uptake of the synthesized traps into isolated mitochondria indicated the importance of both cationic and lipophilic properties, with the DEPMPO nitrone linked to the triphenylphosphonium moiety via a long carbon chain (Mito10-DEPMPO) exhibiting the highest mitochondrial uptake. We conclude that, of the synthesized traps, Mito-DIPPMPO and Mito10-DEPMPO are the best candidates for potential mitochondria-specific spin traps for use in biologically relevant systems.

In vivo Overhauser-enhanced MRI of proteolytic activity.

There is an increasing interest in developing novel imaging strategies for sensing proteolytic activities in intact organisms in vivo. Overhauser-enhanced MRI (OMRI) offers the possibility to reveal the proteolysis of nitroxide-labeled macromolecules thanks to a sharp decrease of the rotational correlation time of the nitroxide moiety upon cleavage. In this paper, this concept is illustrated in vivo at 0.2 T using nitroxide-labeled elastin orally administered in mice. In vitro, this elastin derivative was OMRI-visible and gave rise to high Overhauser enhancements (19-fold at 18 mm nitroxide) upon proteolysis by pancreatic porcine elastase. In vivo three-dimensional OMRI detection of proteolysis was carried out. A keyhole fully balanced steady-state free precession sequence was used, which allowed 3D OMRI acquisition within 20 s at 0.125 mm(3) resolution. About 30 min after mouse gavage, proteolysis was detected in the duodenum, where Overhauser enhancements were 7.2 ± 2.4 (n = 7) and was not observed in the stomach. Conversely, orally administered free nitroxides or pre-digested nitroxide-labeled elastin were detected in the mouse's stomach by OMRI. Combined with specific molecular probes, this Overhauser-enhanced MRI technique can be used to evaluate unregulated proteolytic activities in various models of experimental diseases and for drug testing.

Synthesis of new paramagnetic retinal analogues

We synthesized new paramagnetic retinal analogues using Horner-Wadsworth-Emmons and Wittig reactions. In these new analogues, the pyrroline nitroxide moiety is situated in the place of the β-ionone ring or at the end of the polyene chain.

Hepatoprotective activity of betulonic acid amides containing a piperidine or pyrrolidine nitroxide moiety

Betulonic acid amides containing a nitroxyl radical moiety possess antiholestatic effects in mice. Introduction ofpiperidine nitroxide moiety into lupan core increases its hepatoprotective activity. Oral administration of piperidine nitroxide derivative in dose 50 mg/kg doesn't stimulate transplanted tumor growth and raises a lifespan of mice.

Interaction of Antidepressant Drug, Clomipramine, with Model and Biological Stratum Corneum Membrane as Studied by Electron Paramagnetic Resonance

The interactions of tricyclic antidepressant drug, clomipramine (CLO), with pig ear stratum corneum (SC) and model membranes were investigated by electron paramagnetic resonance (EPR) spin labeling to get some insight into the possible application of this drug in transdermal delivery. The changes in membrane characteristics caused by CLO in the regions that are close to the water-lipid interfaces and the central parts of the membranes were searched. The experimental results were supported by computer simulation of EPR spectra, which showed heterogeneity of the membranes composed of regions with different fluidity characteristics. CLO was effective in both parts of the layers, indicating intercalation of the drug into model membranes as well as into the pig ear SC. Introduction of various molar ratios of CLO caused a decrease in the order parameter and an increase in the rotational diffusion of nitroxide moiety in different membrane regions as well as an increase in the polarity of spin probe environment. It also changed the number of resolved spectral components, which reflects the heterogeneity of the membrane. The fluidizing effect of CLO on pig ear SC throughout the whole membrane layers indicates that CLO penetrates into the SC, which is important for its transdermal delivery.

Modulation of CEST Images in Vivo by T1 Relaxation: A New Approach in the Design of Responsive PARACEST Agents

A novel approach for the design of responsive paramagnetic chemical exchange saturation transfer (PARACEST) magnetic resonance imaging (MRI) agents has been developed where the signal is "turned on" by altering the longitudinal relaxation time (T1) of bulk water protons. To demonstrate this approach, a model Eu(DOTA-tetraamide) complex (DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) containing two nitroxide free radical units was synthesized. The nitroxide groups substantially shortened the T1 of the bulk water protons which, in turn, resulted in quenching of the CEST signal. Reduction of paramagnetic nitroxide moieties to a diamagnetic species resulted in the appearance of CEST. The modulation of CEST by T1 relaxation provides a new platform for designing biologically responsive MRI agents.

Molecular Conformations and Magnetic Parameters of the Compact Trimethylenemethane-Type Triplet Diradical

The ESR spectrum of compact nitroxide (NO)-substituted nitronyl nitroxide (NN) triplet diradical N-tert-butyl-N-oxidanyl-2-amino-4,4,5,5-tetramethyl-4,5-dihydro-1H-imidazole-3-oxide-1-oxyl (1) was recorded in solid argon matrix at 15 K. The zero-field splitting (ZFS) parameters of 1 were derived from the recorded ESR spectrum: |D| = 0.0248 cm(-1) and E = 0.0025 cm(-1). Quantum chemical calculations have been performed using DFT and multiconfigurational ab initio (CAS) methods in order to establish equilibrium geometries of the conformational isomers resulting from twisted conformations of NO and NN moieties. The ZFS parameters of 1 were calculated at these levels of theory to test validity of the calculated structures. The calculation results were analyzed using the measured ZFS parameters and magnetic and structural data from the previous studies (Suzuki, S.; et al. J. Am. Chem. Soc. 2010, 132, 15908; Tretyakov, E. V.; et al. Russ. Chem. Bull. 2011, 60, 2608). It was found that the ab initio method is most successful for accurate predictions of molecular and magnetic parameters. Diradical 1 has only one stable enantiomeric pair in pseudoeclipsed conformations. The two chiral isomers exist in racemic crystals 1 and in solid matrices with molecular parameters close to those attributed to a free molecule. The analysis of the spin density distribution suggests that one unpaired electron occupies NO group at the equilibrium geometry, whereas the torsion of NO group governs the spin density distribution of the second unpaired electron on a conjugated fragment in NN group. The increase in planarity by torsion of NO group enhances the trimethylenemethane-type properties and, therefore, gives rise to larger ferromagnetic exchange interaction. More planar equilibrium geometry and greater (three times) exchange interaction constant J were predicted for hypothetical diradical 1a, where bulky tert-butyl group is replaced by a methyl group in the nitroxide fragment.

Perylene-based profluorescent nitroxides for the rapid monitoring of polyester degradation upon weathering: An assessment

A profluorescent nitroxide possessing an isoindoline nitroxide moiety linked to a perylene fluorophore was developed to monitor radical mediated degradation of melamine-formaldehyde crosslinked polyester coil coatings in an industry standard accelerated weathering tester. Trapping of polyester-derived radicals (most likely C-radicals) that are generated during polymer degradation leads to fluorescent closed-shell alkoxy amines, which was used to obtain time-dependent degradation profiles to assess the relative stability of different polyesters towards weathering. The nitroxide probe couples excellent thermal stability and satisfactory photostability with high sensitivity and enables detection of free radical damage in polyesters under conditions that mimic exposure to the environment on a time scale of hours rather than months or years required by other testing methods. There are indications that the profluorescent nitroxide undergoes partial photo-degradation in the absence of polymer-derived radicals. Unexpectedly, it was also found that UV-induced fragmentation of the NO–C bond in closed-shell alkoxy amines leads to regeneration of the profluorescent nitroxide and the respective C-radical. The maximum fluorescence intensity that could be achieved with a given probe concentration is therefore not only determined by the amount of polyester radicals formed during accelerated weathering, but also by the light-driven side reactions of the profluorescent nitroxide and the corresponding alkoxy amine radical trapping products. Studies to determine the optimum probe concentration in the polymer matrix revealed that aggregation and re-absorption effects lowered the fluorescence intensity at higher concentrations of the profluorescent nitroxide, but too low probe concentrations, where these effects would be avoided, were not sufficient to trap the amount of polyester radicals formed upon weathering. The optimized experimental conditions were used to assess the impact of temperature and UV irradiance on polymer degradation during accelerated weathering.

Alamethicin in bicelles: Orientation, aggregation, and bilayer modification as a function of peptide concentration

Alamethicin is a 19-amino-acid residue hydrophobic peptide of the peptaibol family that has been the object of numerous studies for its ability to produce voltage-dependent ion channels in membranes. In this work, for the first time electron paramagnetic resonance spectroscopy was applied to study the interaction of alamethicin with oriented bicelles. We highlighted the effects of increasing peptide concentrations on both the peptide and the membrane in identical conditions, by adopting a twofold spin labeling approach, placing a nitroxide moiety either on the peptide or on the phospholipids. The employment of bicelles affords additional spectral resolution, thanks to the formation of a macroscopically oriented phase that allows to gain information on alamethicin orientation and dynamics. Moreover, the high viscosity of the bicellar solution permits the investigation of the peptide aggregation properties at physiological temperature. We observed that, at 35°C, alamethicin adopts a transmembrane orientation with the peptide axis forming an average angle of 25° with respect to the bilayer normal. Moreover, alamethicin maintains its dynamics and helical tilt constant at all concentrations studied. On the other hand, by increasing the peptide concentration, the bilayer experiences an exponential decrease of the order parameter, but does not undergo micellization, even at the highest peptide to lipid ratio studied (1:20). Finally, the aggregation of the peptide at physiological temperature shows that the peptide is monomeric at peptide to lipid ratios lower than 1:50, then it aggregates with a rather broad distribution in the number of peptides (from 6 to 8) per oligomer.

Pulsed electron spin nutation spectroscopy for weakly exchange-coupled multi-spin molecular systems with nuclear hyperfine couplings: a general approach to bi- and triradicals and determination of their spin dipolar and exchange interactions

Weakly exchange-coupled biradicals have attracted much attention in terms of their dynamic nuclear polarisation application in NMR spectroscopy for biological systems or the use of synthetic electron-spin qubits in quantum information processing/quantum-computing technology. Analogues multi-partite molecular systems are important in entering a new phase of the relevant fields. Many stable organic biradicals known so far have nitrogen nuclei at their electron spin sites, where singly occupied molecular orbitals are dominating and large hyperfine couplings occur. A salient feature of such weakly exchange-coupled molecular systems in terms of electronic spin structures is underlain by small zero-field splitting (ZFS) parameters comparable with nuclear hyperfine and/or exchange interactions. Pulse-based electron spin nutation (ESN) spectroscopy of weakly exchange-coupled biradicals, applicable to oriented or non-oriented media, has proven to be a useful and facile approach to the determination of ZFS parameters, which reflect relatively short distances between unpaired electron spins. In the present study, we first treat two-dimensional single-crystal ESN spectroscopy (Q-band) of a 15N-labelled weakly exchange-coupled biradical, showing the nuclear hyperfine effects on the ESN phenomena from both the experimental and theoretical side. ESN spectroscopy is transition moment spectroscopy, in which the nutation frequency as a function of the microwave irradiation strength ω1 (angular frequency) for any cases of weakly exchange-coupled systems can be treated. The results provide a testing ground for the simplified but general approach to the ESN analysis. In this study, we have invoked single-crystal electron-electron double resonance measurements on a typical biradical well incorporated in a diamagnetic host lattice and checked the accuracy of our ESN analysis for the spin dipolar tensor and exchange interaction. Next, we extend the general approach to analogues multi-partite molecular systems such as stable organic triradical, in which the exchange interaction can be governed by a significant amount of the delocalisation of three unpaired spins over the molecular frame of a triangular structure. The triangular structure maintains π-conjugation in which each spin-bearing nitroxide at the vertex participates and the exchange interaction is greatly controlled by the dihedral angle between the π-conjugation plane and nitroxide moiety at the vertex. In this context, the ZFS parameters do not correspond to spin distances (1.0 nm) in a straightforward manner, but reflect a salient electronic structure associated with both the π-electron network and the symmetry property of the triradical under study. Thus, both the D-value and exchange interaction J have been controlled in this study. In order to interpret the experimental ZFS parameters and exchange interaction, which is three-order of magnitude reduced in the present poly(methyl methacrylate) polymer matrix compared with that in the crystal, sophisticated quantum chemical calculations of the ZFS tensor and exchange interaction were carried out and reproduced the experimental values, concluding that the present triradical of the triangular structure undergoes significant twisting at the nitroxide sites in the polymer matrix. In this study, we observed electron spin resonance forbidden transitions between the Ms-manifolds belonging to the spin-doublet ground state and spin-quartet excited state. The observation enables us to derive the magnitude of the exchange coupling.

Polynitroxylated-Pegylated Hemoglobin Attenuates Fluid Requirements and Brain Edema in Combined Traumatic Brain Injury Plus Hemorrhagic Shock in Mice

Polynitroxylated-pegylated hemoglobin (PNPH), a bovine hemoglobin decorated with nitroxide and polyethylene glycol moieties, showed neuroprotection vs. lactated Ringer's (LR) in experimental traumatic brain injury plus hemorrhagic shock (TBI+HS). Resuscitation with PNPH will reduce intracranial pressure (ICP) and brain edema and improve cerebral perfusion pressure (CPP) vs. LR in experimental TBI+HS. C57/BL6 mice (n=20) underwent controlled cortical impact followed by severe HS to mean arterial pressure (MAP) of 25 to 27 mm Hg for 35 minutes. Mice (n=10/group) were then resuscitated with a 20 mL/kg bolus of 4% PNPH or LR followed by 10 mL/kg boluses targeting MAP>70 mm Hg for 90 minutes. Shed blood was then reinfused. Intracranial pressure was monitored. Mice were killed and %brain water (%BW) was measured (wet/dry weight). Mice resuscitated with PNPH vs. LR required less fluid (26.0±0.0 vs. 167.0±10.7 mL/kg, P<0.001) and had a higher MAP (79.4±0.40 vs. 59.7±0.83 mm Hg, P<0.001). The PNPH-treated mice required only 20 mL/kg while LR-resuscitated mice required multiple boluses. The PNPH-treated mice had a lower peak ICP (14.5±0.97 vs. 19.7±1.12 mm Hg, P=0.002), higher CPP during resuscitation (69.2±0.46 vs. 45.5±0.68 mm Hg, P<0.001), and lower %BW vs. LR (80.3±0.12 vs. 80.9±0.12%, P=0.003). After TBI+HS, resuscitation with PNPH lowers fluid requirements, improves ICP and CPP, and reduces brain edema vs. LR, supporting its development.

Orientational order of guest molecules in aligned liquid crystal as measured by EPR and UV–vis techniques

Orientational order of guest molecules in aligned liquid crystal 4-cyano-4′-pentylbiphenyl (5CB) is studied via optical dichroism and electron paramagnetic resonance (EPR) spectra measurements. The guest molecules used are bifunctional molecules bearing paramagnetic nitroxide group and photochromic azobenzene moiety. The bifunctional probe with rigidly bonded nitroxide and azobenzene moieties was found to align as a whole, while flexible long spacer between the moieties provides independent alignment for the nitroxide and azobenzene parts. Intermolecular interactions responsible for the alignment of azobenzene and nitroxide moieties of the probe molecules are discussed. The molecules with cis-configuration of azobenzene moiety are able to align in the liquid-crystalline medium, but to a lesser extent than the molecules with trans-configuration. Directions of orientational axes and characteristics of rotational mobility of spin probes are determined. Second, fourth and, in some cases, sixth rank order parameter values are found.

Synthesis of analogs of the radiation mitigator JP4-039 and visualization of BODIPY derivatives in mitochondria

JP4-039 is a lead structure in a series of nitroxide conjugates that are capable of accumulating in mitochondria and scavenging reactive oxygen species (ROS). To explore structure-activity relationships (SAR), new analogs with variable nitroxide moieties were prepared. Furthermore, fluorophore-tagged analogs were synthesized and provided the opportunity for visualization in mitochondria. All analogs were tested for radioprotective and radiomitigative effects in 32Dcl3 cells.