Development of isoindoline nitroxides for EPR oximetry in viable systems

Nitroxides are widely used as biophysical probes to study molecular motion, intracellular oxygen, pH, transmembrane potential, and cellular redox metabolism, etc. They may be rapidly metabolized to hydroxylamines by cells, which limits their use in viable systems. In this study, we have characterized relevant properties in cells of several isoindoline nitroxides that have been prepared to have different physicochemical properties: 1,1,3,3-tetramethylisoindolin-2-yloxyl (TMIO) and its analogs 5-carboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl (CTMIO), 5-(N,N,N-trimethylammonio)-1,1,3,3-tetramethyl isoindolin-2-yloxyl iodide (QATMIO) and 2-hydroxy-1,1,3,3-tetramethylisoindoline hydrochloride (TMIOH.HCI). The oxygen sensitivity and metabolic kinetics of these were compared in CHO cells under different oxygen tensions with 1-oxyl-2,2,6,6-tetramethyl-4-piperidione (Tempone) and 3-carboxyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl (PCA). Cytotoxicity was evaluated by the measurement of oxygen consumption rates, trypan blue exclusion, and clone formation. TMIO and its analogues have a higher relative oxygen sensitivity than Tempone and PCA with the oxygen sensitivity in electron paramagnetic resonance (EPR) spectrometry in the order of: TMIO=TMIOH=CTMIO>QATMIO=Tempone<PCA. The rates of metabolism of these nitroxides are moderate and depend on oxygen concentration, ring type, ring substituent, and membrane permeation. These nitroxides have low cytotoxicity. The results indicate that TMIO and its analogues are potentially useful for EPR studies of viable systems, especially for oximetry.     

Interactions between Diamagnetic Metal Ions and pH-Sensitive Nitroxides

Specific interactions between pH-sensitive nitroxide radicals and selected diamagnetic metal ions were investigated. To this end, the influence of different metal salts at varying concentrations on the continuous-wave electron paramagnetic resonance spectra of two imidazoline nitroxides was studied. Among the screened metal ions, Zn(II) most significantly affected the spectral profile, analogous to the effect attributed to protonation of the nitroxide imino nitrogen known from pH studies. Simulations showed the acquired spectra to result from the superposition of the signals of the coordinated and the uncoordinated species. The complex formation between Zn(II) and (4-amino-2,5-dihydro-2,2,5,5-tetramethyl-3-imidazoline-1-yloxyl) was modelled by theoretical methods revealing the rather specific selectivity of the nitroxide toward Zn(II). The results suggest imidazoline nitroxides as promising candidates for the development of new specific metal ion probes.     

EPR studies of15N- and2H-substituted pH-sensitive spin probes of imidazoline and imidazolidine types

The isotopically substituted analogs of pH-sensitive imidazoline and imidazolidine radicals have been synthesized and investigated with electron paramagnetic resonance (EPR) spectroscopy. The introduction of²H and¹⁵N into the structure of the radical is a useful approach to enhance the information obtained from spin-labeling experiments. The spectra of the radicals have been analyzed with 9.8 (X-band) and 130 GHz (D-band) EPR spectroscopy. The substitution of¹H for²H leads to significant narrowing of Gaussian line width, while the substitution of¹⁴N for¹⁵N in the nitroxyl fragment decreases both the number of spectral lines and Lorentzian line width. These effects result in a significant increase in the peak intensities up to 5–7 times for X-band EPR spectra of one of the imidazoline radicals (R4). The increase in spectral resolution allowed us to reveal the hyperfine interaction splitting with the attached proton (0.36 G) in the protonated form of the radical R4. The influence of proton exchange of the radicals with phosphate and acetate buffers on their EPR spectra has been studied in H₂O and D₂O. The corresponding rate constants of the proton exchange have been calculated from fitting of the simulated EPR spectra line shapes to experimental spectra. The data obtained demonstrated the advantages of the isotopically substituted spin pH probes in spectral resolution and sensitivity which can be an important factor particularly for applications in vivo where the fundamental sensitivity is much lower. The sensitivity of EPR spectra of these spin probes to the buffer capacity could be of practical importance taking into account the biological relevance of monitoring this parameter in some pathological states.     

Fast and accurate characterization of biological membranes by EPR spectral simulations of nitroxides

A method by which it is possible to characterize the membranes of biological samples on the basis of the EPR spectral lineshape simulation of membrane-dissolved nitroxide spin probes is described. The presented simulation procedure allows the determination of the heterogeneous structure of biological membranes and fluidity characteristics of individual membrane domains. The method can deal with isotropic and anisotropic orientations of nitroxides introduced into the biological samples described by restricted fast motion with a correlation time between 0.01 and 10 ns. The linewidths of the Lorentzian lineshapes are calculated in a restricted fast-motion approximation. In the special case of samples with high concentrations of nitroxides or in the presence of paramagnetic ions, the lineshapes are calculated directly from the exchange-coupled Bloch equations. The parameters describing ordering, relaxation, polarity, and the portions of the individual spectral components are extracted by optimizing the simulated spectra to the experimental spectrum with either a Simplex or a Monte Carlo algorithm. To improve the algorithm's efficiency, a new way of characterizing the goodness of fits is introduced. The new criterion is based on the standard least-squares function, but with special weighting of the partial sums. Its benefits are confirmed with membrane spectral simulation. Two classes of examples-simulation and optimizations of synthetic spectra to evaluate the accuracy of the optimization algorithms and simulation and optimization of EPR spectra of nitroxides in liposome suspensions in the presence of a broadening agent and in human leukocytes are shown.     

EPR and Quantum Chemical Studies of the pH-sensitive Imidazoline and Imidazolidine Nitroxides with Bulky Substituents

The X- and W-band electron paramagnetic resonance (EPR) spectroscopies were employed to investigate a series of imidazolidine nitroxide radicals with different number of ethyl and methyl substituents at positions 2 and 5 of a heterocycle in liquid and frozen solutions. The influence of the substituents on the line shape and width was studied experimentally and analyzed using quantum chemical calculations. Each pair of the geminal ethyl groups in the positions 2 or 5 of the imidazolidine ring was found to produce an additional hyperfine splitting (hfs) of about 0.2 mT in the EPR spectra of the nitroxides. The effect was attributed to the hfs constant of only one of four methylene hydrogen atoms of two geminal ethyl substituents not fully averaged by ethyl group rotation and ring puckering. In accordance with this assumption, the substitution of hydrogen atoms of CH₂ groups in 2,2,5,5-tetraethyl-substituted imidazolidine nitroxides by deuterium leads to the substantial narrowing of EPR lines which could be useful for many biochemical and biomedical applications, including pH-monitoring. W-band EPR spectra of 2,2,5,5-tetraethyl-substituted imidazolidine nitroxide and its 2,2,5,5-tetraethyl–d₈ deuterium-substituted analog measured at low temperatures demonstrated high sensitivity of their g-factors to pH, which indicates their applicability as spin labels possessing high stability.     

An EPR study of lipid vesicles as paramagnetic agent vectors

Paramagnetic nitroxides have been proposed as probes in electron paramagnetic resonance (EPR) imaging and in clinical diagnosis. However, nitroxides are rapidly reducedin vivo to hydroxylamines, diamagnetic EPR-inactive species. Reduction occurs in blood via soluble agents such as ascorbic acid, as well as in the cells via enzymatic and non-enzymatic endocellular systems. To prevent the reduction, a water soluble nitroxide, i.e., potassium peroxylamine disulfonate, is entrapped in reverse phase evaporation vesicles. The loaded liposomes have a high entrapment capacity, and vesicles with the encapsulated agent are stable for days, even at room temperature. The vesiclesin vitro can almost completely prevent the reduction of the entrapped nitroxide by ascorbic acid. In blood of a rat, enriched with a homogenate of rat liver proteins, the vesicles are able to greatly prolong the life time of the nitroxide. In particular, the encapsulated nitroxide has a half-life of more than one hour, compared to two minutes for free nitroxide under the same conditions. Due to these protective effects, the lipid vesicles might be useful as a delivery system for paramagnetic agents.     

CW EPR, echo-detected EPR, and field-step ELDOR study of molecular motions of nitroxides ino-terphenyl glass: Dynamical transition, dynamical heterogeneity and β-relaxation

Continuous-wave electron paramagnetic resonance (CW EPR), echo-detected (ED) EPR, and field-step electron-electron double resonance (FS ELDOR) were simultaneously applied to study molecular motions of nitroxide spin probes of two different types in glassyo-terphenyl. A strong linear temperature dependence of the overall splitting of the CW EPR lineshape was found for nitroxide Tempone and only a weak one for a phenyl-ring-containing imidasoline nitroxide. The linear temperature dependence of the splitting is explained within the model of harmonic librations. The assessed libration frequency for Tempone is of the order of 3·10¹² rad/s. The observed remarkable difference between the two nitroxides is explained by the different strength of interactions between guest and host molecules and by dynamical heterogeneity of the glass. The nonlinear temperature dependence above 250 K is attributed to the onset of anharmonic motion that is postulated in a number of neutron scattering and Mössbauer spectroscopy studies for molecular glasses and proteins (the so-called dynamical transition). Above 245 K also ED EPR spectra change drastically, which may be explained on the same ground. Magnetization transfer was observed in FS ELDOR for nitroxide Tempone, with a time constant around 10^?5 s. It was found to be almost temperature-independent between 160 K and 265 K and was attributed to the Johari-Goldstein β-relaxation process. For the phenyl-ring-containing imidasoline nitroxide this transfer was not observed, which may be explained again by the dynamical heterogeneity of the glass and by small effectivity of the β-relaxation process in this case.     

Assessment of Nitrones as In Vivo Redox Sensors

Spin traps such as 5,5-dimethyl-1-pyrroline N-oxide, α-phenyl-tert-butyl nitrone, α-(4-pyridyl-1-oxide)-N-tert-butyl nitrone and newer generation 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide analogs have been known for years. What are the desired properties of good probes for measuring in vivo redox reactions in biological systems? These are specificity, sensitivity, rapid, high yield kinetics, low toxicity, high stability and easy to administer and target. Unfortunately, the nitrones perform poorly in almost all of these categories. Typical in vivo concentrations of spin trap approach 100 mM (assuming solubility and toxicity are not an issue), frequently yield 1% nitroxide or less stoichiometry, are typically unstable with time and frequently lack specificity. In vivo electron paramagnetic resonance (EPR) experiments need to have strong signals that correlate with redox chemistry. The resultant signal should be stable and not rapidly interconvert to other diamagnetic species. Fortunately, some newer probes of in vivo redox reactions in biological systems have come upon the horizon. In fact some have been around for a long time, but their virtues are becoming increasingly appreciated. This paper summarizes the disadvantages of nitrones versus the clear advantages of other probes of free radicals, redox state and the like by EPR. It also expands on the properties of nitroxides and nitrones as therapeutics.     

Polarity contributions to hyperfine splittings of hydrogen-bonded nitroxides--the microenvironment of spin labels

A self-consistent treatment of reaction field effects on isotropic (14)N hyperfine coupling constants of nitroxide spin labels in mixtures of polar and apolar solvents is given based on the Onsager approach. It is shown that this works reasonably well for mixtures of water or methanol with dioxane, far better than do conventional approaches using the Clausius-Mossotti relation. Association constants, K(A,h), for hydrogen bonding of protic solvents to nitroxides are derived in this way from published EPR data. A value of K(A,h) approximately 1.0 M x (-1) is argued to be reasonable for water in a hydrophobic environment. Data from spin-labelled lipids can then be used to estimate effective water concentrations in biological membranes.     

Kinetics of nitroxide spin label removal in biological systems: An in vitro and in vivo ESR study

A systematic study on the disappearance of the electron spin resonance (ESR) signal of nitroxides based on six- or five-membered ring and bearing various charges was carried out in vitro and in vivo. The second-order kinetic rate constants of the reaction of spin probes with ascorbate were measured in vitro at various temperatures in phosphate buffered saline, and the relative activation energies were calculated. Clearance rates of the nitroxide radicals in rat brain homogenates and in blood indicate that the ascorbate contribution to nitroxide removal is about 50–70% in brain and 50–90% in blood. These rates can be easily calculated on the basis of the ascorbate concentration and of the second-order kinetic rate constants measured in phosphate buffered saline. ESR spectra acquired in vivo in rat head and tail, by an L-band resonator, indicated that the nitroxide decay rate is a first-order kinetic process in both domains and that the positively charged nitroxides are not retained in the brain, whereas the anionic and uncharged nitroxides are. Once nitroxides with piperidine ring enter the brain, their decay appears controlled mainly by ascorbate, while the ascorbate has a negligible influence on disappearance in brain of five-membered ring proxyl nitroxides.     

Inherent Microporosity and Photostability of Fluoroacrylic Polymer Films Studied by Electron Paramagnetic Resonance of Nitroxide Spin Probes

Efficient and straightforward methods for characterization of polymers with inherent microporosity are demanded in their targeted design for particular applications. Among critical parameters to be obtained are the size of the pores and polymer stability against photoirradiation. Herewith, we demonstrate the efficiency of electron paramagnetic resonance (EPR) spectroscopy applied to this task. We use stable nitroxide radicals (2,2,6,6-tetramethylpyperidine-1-oxyl) (TEMPO) as reporter spin probes for EPR and investigate a series of perspective polymers with inherent microporosity developed for pressure sensitive paints (PSP), namely, poly(1,1,1,3,3,3-hexafluoroisopropylmethacrylate-co-2,2,3,3,4,4,4-heptafluorobutylmethacrylate) (FIB), its two modifications poly(1,1,1,3,3,3-hexafluoroisopropylmethacrylate-co-2,2,3,3,4,4,4-heptafluorobutylmethacrylate-co-1-(4-(4-chloro-2,3,5,6-tetrafluorophenyl)piperazin-1-yl)prop-2-en-1-one) (NS4) and poly(1,1,1,3,3,3-hexafluoroisopropylmethacrylate-co-2,2,3,3,4,4,4-heptafluorobutylmethacrylate-co-1-(4-(4-tert-butylphenylsulfonyl)piperazin-1-yl)prop-2-en-1-one) (NS5), as well as poly(1-trimethylsilyl-1-propyne) (PTMSP) and poly(1,1,2,2-perfluorooctylmethacrylate) (PFOMA). Nitroxides were incorporated into the pores of the polymers post-synthetically via a gas-phase sorption, and the mobility of nitroxides tracked by EPR yielded information on the pore sizes and polymer degradation under ultraviolet light. The conclusions obtained by EPR have been supported by a variety of other techniques, thus demonstrating EPR to be a very convenient tool for express analysis of porous polymers.     

Comparing continuous wave progressive saturation EPR and time domain saturation recovery EPR over the entire motional range of nitroxide spin labels

The measurement of spin-lattice relaxation rates from spin labels, such as nitroxides, in the presence and absence of spin relaxants provides information that is useful for determining biomolecular properties such as nucleic acid dynamics and the interaction of proteins with membranes. We compare X-band continuous wave (CW) and pulsed or time domain (TD) EPR methods for obtaining spin-lattice relaxation rates of spin labels across the entire range of rotational motion to which relaxation rates are sensitive. Model nitroxides and spin-labeled biological species are used to illustrate the potential complications that arise in extracting relaxation data under conditions typical to biological experiments. The effect of super hyperfine (SHF) structure is investigated for both CW and TD spectra. First and second harmonic absorption and dispersion CW spectra of the nitroxide spin label, TEMPOL, are all fit simultaneously to a model of SHF structure over a range of microwave amplitudes. The CW spectra are novel because all harmonics and microwave phases were acquired simultaneously using our homebuilt CW/TD spectrometer. The effect of the SHF structure on the pulsed free induction decay (FID) and pulsed saturation recovery spectrum is shown for both protonated and deuterated TEMPOL. We present novel pulsed saturation recovery measurements on biological molecules, including spin-lattice relaxation rates of spin-labeled proteins and spin-labeled double-stranded DNA. The impact of structure and dynamics on relaxation rates are discussed in the context of each of these examples. Collisional relaxation rates with oxygen and transition metal paramagnetic relaxants are extracted using both continuous wave and time domain methods. The extent of the errors inherent in the CW method and the advantages of pulsed methods for unambiguously measuring collisional relaxation rates are discussed. Spin-lattice relaxation rates, determined by both CW and pulsed methods, are used to determine the electrostatic potential on the surface of a protein.     

Real-time monitoring of drug-induced changes in the stomach acidity of living rats using improved pH-sensitive nitroxides and low-field EPR techniques

New improved pH-sensitive nitroxides were applied for in vivo studies. An increased stability of the probes towards reduction was achieved by the introduction of the bulky ethyl groups in the vicinity of the paramagnetic NO fragment. In addition, the range of pH sensitivity of the approach was extended by the synthesis of probes with two ionizable groups, and, therefore, with two pKa values. Stability towards reduction and spectral characteristics of the three new probes were determined in vitro using 290 MHz radiofrequency (RF)- and X-band electron paramagnetic resonance (EPR), longitudinally detected EPR (LODEPR), and field-cycled dynamic nuclear polarization (FC-DNP) techniques. The newly synthesized probe, 4-[bis(2-hydroxyethyl)amino]-2-pyridine-4-yl-2,5,5-triethyl-2,5-dihydro-1H-imidazol-oxyl, was found to be the most appropriate for the application in the stomach due to both higher stability and convenient pH sensitivity range from pH 1.8 to 6. LODEPR, FC-DNP and proton-electron double resonance imaging (PEDRI) techniques were used to detect the nitroxide localization and acidity in the rat stomach. Improved probe characteristics allowed us to follow in vivo the drug-induced perturbation in the stomach acidity and its normalization afterwards during 1 h or longer period of time. The results show the applicability of the techniques for monitoring drug pharmacology and disease in the living animals.     

Nitroxide Side-Chain Dynamics in a Spin-Labeled Helix-Forming Peptide Revealed by High-Frequency (139.5-GHz) EPR Spectroscopy

High-frequency electron paramagnetic resonance (EPR) spectroscopy has been performed on a nitroxide spin-labeled peptide in fluid aqueous solution. The peptide, which follows the single letter sequence, was reacted with the methanethiosulfonate spin label at the cysteine sulfur. The spin sensitivity of high-frequency EPR is excellent with less than 20 pmol of sample required to obtain spectra with good signal-to-noise ratios. Simulation of the temperature-dependent spectral lineshapes reveals the existence of local anisotropic motion about the nitroxide N-O bond with a motional anisotropy tau( perpendicular)/tau( parallel) ( identical with N) approaching 2.6 at 306 K. Comparison with previous work on rigidly labeled peptides suggests that the spin label is reorienting about its side-chain tether. This study demonstrates the feasibility of performing 140-GHz EPR on biological samples in fluid aqueous solution.     

PEG化氘代单磷酸三苯甲基自由基的高效合成及性能研究

电子顺磁共振（EPR）波谱联用外源性自旋探针技术是测量体内外pH的有效手段.有研究表明,单磷酸取代三苯甲基（p₁TAM）自由基是目前用于检测pH的最理想的EPR探针.然而,这类探针的合成产率低、易受蛋白影响,极大限制了其生物应用.针对上述问题,本文提出了高效合成p₁TAM自由基的方法,使其总产率从文献报道的1.6%提高到了25%;同时采用PEG修饰的方法避免了白蛋白（BSA）对其产生的干扰.进一步氘代实验结果证实：非氘代PEG化（p₁TAM-H）衍生物（POP）虽与氘代PEG化（p₁TAM-D）衍生物（dPOP）具有相似的pH敏感性（其pKa分别为6.80和6.79）,但POP的EPR谱图较为复杂,而dPOP无论在低pH还是在高pH条件下均具有简单的EPR双峰信号;而且,dPOP具有比POP和p₁TAM-H更好的检测灵敏性;此外,dPOP还具有较好的生物稳定性和氧气敏感性.因此,dPOP能够用来同时检测pH和氧气,有望在生物医学方面得到更好的应用.     

ESR Studies of Nitroxides Produced from Secondary Amines Through Oxidation by Photoexcited N-Oxides

The photolysis of chloroform solutions containing 2-methyl-3-acetylquinoxalin N, N-dioxide (MAQO) and dialkyl amines leads to the observation of ESR spectra of the corresponding dialkyl nitroxides. Oxygen-transfer exciplex 5 which derived from the triplet state of MAQO is suggested as intermediate to give nitroxide through N-H bond cleavage in the secondary amine within 5.     

Spin-Label EPR for Determining Polarity and Proticity in Biomolecular Assemblies: Transmembrane Profiles

Hyperfine couplings and g-values of nitroxyl spin labels are sensitive to polarity and hydrogen bonding in the environment probed. The dependences of these electronic paramagnetic resonance (EPR) properties on environmental dielectric permittivity and proticity are reviewed. Calibrations are given, in terms of the Block–Walker reaction field and local proton donor concentration, for the nitroxides that are commonly used in spin labeling of lipids and proteins. Applications to studies of the transverse polarity profiles in lipid bilayers, which constitute the permeability barrier of biological membranes, are reviewed. Emphasis is given to parallels with the permeation profiles of oxygen and nitric oxide that are determined from spin-label relaxation enhancements by using nonlinear continuous-wave EPR and saturation recovery EPR, and with permeation profiles of D₂O that are determined by using ²H electron spin echo envelope modulation spectroscopy.     

Isotope selection in distance measurements between nitroxides

Self-assembly of spin-labeled synthetic macromolecules or biomacromolecules can lead to structures that contain more than two nitroxide radicals. Label-to-label distance distributions are then poorly resolved since established electron paramagnetic resonance techniques for distance measurements cannot select between the different pairs of nitroxides. A separation into different contributions can be achieved by partially labeling the nitroxide radicals by (15)N or by deuterium and applying pulse electron electron double resonance techniques. With (15)N labeling, strong suppression of either the (14)N or the (15)N contribution can be achieved by suitable choices of the excitation bandwidths and frequencies of the observer subsequence and pump pulse and linear combination of data sets. With deuterium labeling, interactions between only the isotope-labeled nitroxides can be selected by a two-dimensional version of the four-pulse double electron electron resonance experiment. This selection is based on the deep electron spin echo envelope modulation of deuterated nitroxides.     

Saturation recovery EPR and ELDOR at W-band for spin labels

A reference arm W-band (94 GHz) microwave bridge with two sample-irradiation arms for saturation recovery (SR) EPR and ELDOR experiments is described. Frequencies in each arm are derived from 2 GHz synthesizers that have a common time-base and are translated to 94 GHz in steps of 33 and 59 GHz. Intended applications are to nitroxide radical spin labels and spin probes in the liquid phase. An enabling technology is the use of a W-band loop-gap resonator (LGR) [J.W. Sidabras, R.R. Mett, W. Froncisz, T.G. Camenisch, J.R. Anderson, J.S. Hyde, Multipurpose EPR loop-gap resonator and cylindrical TE₀₁₁ cavity for aqueous samples at 94 GHz, Rev. Sci. Instrum. 78 (2007) 034701]. The high efficiency parameter (8.2 GW^−1/2 with sample) permits the saturating pump pulse level to be just 5 mW or less. Applications of SR EPR and ELDOR to the hydrophilic spin labels 3-carbamoyl-2,2,5,5-tetra-methyl-3-pyrroline-1-yloxyl (CTPO) and 2,2,6,6,-tetramethyl-4-piperidone-1-oxyl (TEMPONE) are described in detail. In the SR ELDOR experiment, nitrogen nuclear relaxation as well as Heisenberg exchange transfer saturation from pumped to observed hyperfine transitions. SR ELDOR was found to be an essential method for measurements of saturation transfer rates for small molecules such as TEMPONE. Free induction decay (FID) signals for small nitroxides at W-band are also reported. Results are compared with multifrequency measurements of T_1e previously reported for these molecules in the range of 2–35 GHz [J.S. Hyde, J.-J. Yin, W.K. Subczynski, T.G. Camenisch, J.J. Ratke, W. Froncisz, Spin label EPR T₁ values using saturation recovery from 2 to 35 GHz. J. Phys. Chem. B 108 (2004) 9524–9529]. The values of T_1e decrease at 94 GHz relative to values at 35 GHz.