4950
J. Phys. Chem. A 2007, 111, 4950-4957
Combined ESR and Thermodynamic Studies of the Superoxide Adduct of 5-(Diethoxyphosphoryl)-5-Methyl-1-Pyrroline N-Oxide (DEPMPO): Hindered Rotation around the O-O Bond Evidenced by Two-Dimensional Simulation of Temperature-Dependent Spectra Antal Rockenbauer,† Jean-Louis Cle´ ment,‡ Marcel Culcasi,*,‡ Anne Mercier,‡ Paul Tordo,‡ and Sylvia Pietri‡ Chemical Research Center, Institute of Structural Chemistry, H-1025 Budapest, Pusztaszeri u´ t 59, Hungary, and Laboratoire Structure et Re´ actiVite´ des Espe` ces Paramagne´ tiques-Sondes Mole´ culaires en Biologie, CNRS-UMR 6517, UniVersite´ s d’Aix-Marseille 1 et 3, Faculte´ des Sciences de Saint-Je´ roˆ me (case 522), 13397 Marseille Cedex 20, France ReceiVed: January 26, 2007; In Final Form: April 4, 2007
Experiments were performed to elucidate the origin of the superhyperfine structure and line width alternation (LWA) seen in the ESR spectrum of the major diastereoisomer (1) of DEPMPO-OOH, the remarkably persistent superoxide adduct of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO). Using selectively deuterated DEPMPO derivatives, we demonstrated that the superhyperfine pattern can be unambiguously attributed to long-range couplings. The recording in pyridine of highly resolved spectra in a wide temperature range, combined with two-dimensional simulation, allowed us to characterize an inverted LWA in 1 and revealed a uniform line broadening in the spectrum of the minor DEPMPO-OOH diastereoisomer (2), with both effects originating from a chemical exchange between conformers. When the individual spectra of 1 presenting LWA in the fast-exchange regime were simulated, four equally good fits were obtained and this ambiguity could be resolved by using a two-dimensional simulation technique. The thermodynamic and kinetic constants of this exchange were consistent with a rotation around the O-O bond. We propose that line broadening effects in 1 and 2 result from this O-O rotation concerted with the pseudo-rotation of the pyrrolidine ring.
1. Introduction The variation of line width in the hyperfine pattern of ESR spectra bears crucial information about molecular dynamics. This phenomenon has been extensively studied in the case of nitroxides probes or spin labels, having outstanding applications in the evaluation of motional parameters of biological systems.1 The origin of line broadening occurs along with certain dynamic phenomena such as molecular reorientational tumbling partially averaging the anisotropy of the g and A tensors, or the intramolecular conversion between conformations. This latter situation, termed as chemical exchange, results in line width alternation (LWA) in the hyperfine pattern, provided at least two nuclei have rather different hyperfine coupling constants (hfcs) in the interconverting species. Such unusual shape of the ESR spectra is generally an unwanted feature in spin trapping, where nitroxide spin adducts are intended to unequivocally provide structural and quantitative information on the primary radical species formed. Very typical of this situation are the hydroperoxyl radical adducts of five-membered ring nitrones such as 5,5-dimethyl-1-pyrroline N-oxide (DMPO, Scheme 1A), in particular, 2,2-dimethyl-5-hydroperoxy-1-pyrrolidinyloxyl (DMPO-OOH, Scheme 1A), the protonated DMPO adduct of superoxide (O2•-). The X-band ESR signal of DMPO-OOH was first reported more than 30 years ago,2 being composed of 12 * Corresponding author. Tel: (33) 4 91 28 90 25. Fax: (33) 4 91 28 87 58. E-mail:
[email protected]. † Institute of Structural Chemistry. ‡ Universite ´ s d’Aix-Marseille 1 et 3.
lines with hfcs consistent with nitrogen, β-hydrogen, and one long-range hydrogen. However, the DMPO-OOH spectrum cannot be simulated as a unique species because of its asymmetry, the origin of which has been a matter of debate.3-7 The main difficulty in correctly modeling the ESR line shapes of DMPO-OOH results from its intramolecular decay pattern forming the more persistent 2,2-dimethyl-5-hydroxy-1-pyrrolidinyloxyl (DMPO-OH), the DMPO adduct of hydroxyl radical (HO•), whose quartet spectrum superimposes to that of DMPOOOH within seconds at room temperature.8 Despite a technique to produce DMPO-OOH virtually devoid of any contamination with DMPO-OH having been reported,9 the resulting DMPOOOH signals nevertheless retain asymmetry, which therefore appears to be intrinsic to the nitroxide. At room temperature, this line asymmetry may be imitated considering either the existence of two superimposed signals from different species3,4 or a slow intramolecular chemical exchange,5,10 but only in the latter case was a hfcs of about 0.12 mT from one hydrogen atom of C-3 introduced in the calculation. Using DMPO derivatives either fully deuterated or 2H-substituted at C-2 and C-3 (DMPO-d3, Scheme 1A), Pou et al.11 first obtained direct evidence for the contribution of one C-3 γ-hydrogen coupling to the spectrum pattern of DMPO-OOH. This was definitively confirmed in our laboratory, because DMPO selectively deuterated at C-3 (DMPO-d2, Scheme 1A) yields a symmetrical ESR sextet upon trapping of O2•- and we therefore assumed that it is the significant difference (calculated as 0.14 mT) in γ-couplings between two exchanging conformers of DMPO-
10.1021/jp070679u CCC: $37.00 © 2007 American Chemical Society Published on Web 05/23/2007
Superoxide Adduct of DEPMPO
J. Phys. Chem. A, Vol. 111, No. 23, 2007 4951
SCHEME 1: Structures of Nitrones and Related Spin Adducts
OOH that causes spectral asymmetry5 with no LWA. As additional support for the hypothesis of interconverting conformers, a temperature-dependence of the DMPO-OOH spectral asymmetry was recently reported.6 In our search for spin traps yielding O2•- adducts more persistent than DMPO-OOH, we,10,12 followed by others,13 have developed a new family of pyrroline N-oxides bearing at least one 31P-containing substituent at C-5, the most popular compound in biological studies still being 5-(diethoxyphosphoryl)5-methyl-1-pyrroline N-oxide (DEPMPO, Scheme 1B). Many of the DEPMPO-type nitrones (DEPMPOs) reported to date meet the stability criterion stated above, the half-life of the O2•adducts being 8-20 times larger than that of DMPO-OOH.10,12,13 Meanwhile, two main difficulties are encountered in the spectral simulation of DEPMPOs-OOR adducts (R ) H or alkyl). First, regardless of their mechanism of formation, they are often obtained as mixtures of cis/trans diastereoisomers as a consequence of nonstereoselective radical addition on both diastereotopic faces of the parent nitrones. Scheme 1B depicts the situation for 2-diethoxyphosphoryl-2-methyl-5-hydroperoxy1-pyrrolidinyloxyl (DEPMPO-OOH), the O2•- adduct of DEPMPO. We assigned the major diastereoisomer to trans-DEPMPOOOH (1), because the enantiomeric excess found in DEPMPOsOOR (R ) H or alkyl) seems to depend upon steric hindrance around the nitrone function,10,12,13 e.g., using a standard O2•generator at room temperature, cis-DEPMPO-OOH (2) typically accounts for only 7% in the mixture.14 According to computations by Villamena et al., however, a hydrogen bond specific to 2 can invert the formation probabilities, making this diastereoisomer the preferential adduct.15 Second, in addition to spectral asymmetry similar to that of DMPO-OOH, the major trans-DEPMPOs-OOR signals exhibit a spectacular LWA and, if adequate acquisition settings are used, superhyperfine resolution of the sharpest lines.6,10,12c-g,13a Because it could not be explained by any proton exchange mechanism10 and it was not observed in DEPMPOs-OR adducts12d,13a,b,16, an explanation10 for LWA in the major transDEPMPOs-OOH and DEPMPOs-OOR ESR signals involved intramolecular conformational changes within the pyrrolidine ring associated with restricted rotation of the peroxyl bond.17 In the case of 1 in aqueous medium, the best simulations of highly resolved spectra were consistent with two individual conformers having significant differences in their β-H and 31P hfcs of 0.36 mT and 0.63 mT, respectively, thus being at the origin of LWA.14 In this study14 the observed superhyperfine
structure of DEPMPO-OOH was best simulated assuming two conformers for 1 having γ-couplings in the range 0.09-0.05 mT. An alternative model was recently proposed in which this superhyperfine structure was attributed to the β-epoxy ringcontaining nitroxide 3, considered as a decomposition product of 1 (Scheme 1B).6 In this paper, we used selective deuterium labeling of DEPMPO and O2•- spin trapping as a model of DEPMPOsOOR adducts to assess the participation in the ESR signal of a nitroxide bearing only one γ-hydrogen at C-3, such as in 3. We then carried out experimental conditions allowing to record high-resolution spectra of mixtures of 1 and 2 in a wide temperature range, and a two-dimensional simulation procedure18 to investigate the existence of chemical exchange for both diastereoisomers and extract their thermodynamic parameters. 2. Experimental Section Pyridine from Acros Organics was freshly distilled and stored over potassium hydroxide. Other solvents, starting materials, and reagents were of laboratory reagent grade from Acros Organics or Sigma-Aldrich. Water was produced with an Elga Purelab Option deionizer. DEPMPO and its pentadeutero (4) or selectively C-3 deuterated (5) analogues (Scheme 1) were prepared and purified by published procedures.10,19 Isotopic purity was estimated >95% by 1H NMR. ESR measurements were carried out in either a cylindrical tube or a standard aqueous flat cell using a Bruker ESP 300 X-band spectrometer equipped with a ER 4111 VT, calibrated ((2 °C) variable temperature unit, a ER 053M gaussmeter, a Hewlett-Packard 5350B frequency counter, a TM110 cavity, and a 100-kHz field modulation. Samples set at a given temperature are allowed to stabilize for at least 2 min prior to ESR acquisition. Sample solutions in aqueous KH2PO4 buffer (0.16 M; pH 7.0) contained 0.9 mM diethylenetriaminepentaacetic acid (DTPA), 10% v/v dimethyl sulfoxide (DMSO), 0.01 M 18-crown-6 ether (1,4,7,10,13,16-hexaoxacyclooctadecane), 0.05 M nitrone, and 0.01 M potassium superoxide (KO2) as the O2•- generator and were bubbled with nitrogen gas for 45 s prior to spectral acquisition. Sample solutions in anhydrous pyridine contained DEPMPO (0.1 M) and 0.32 M of hydrogen peroxide (H2O2) from a 30% aqueous solution and were bubbled with air for 3 min and then deoxygenated by freeze/thaw cycles before heating. ESR spectra were sequentially simulated using a previously described program.17
4952 J. Phys. Chem. A, Vol. 111, No. 23, 2007
Rockenbauer et al.
Figure 1. ESR spectra of O2•- adducts of DEPMPO and its deutero analogues 4 and 5 at 295 K in phosphate buffer. (Left) Experimental spectra showing contributions from the minor cis (b), HO• (O), and alkyl (0) adducts. (Right) Experimental and calculated spectra of the selected region.
3. Results and Discussion (a) Definitive Assignment of the Superhyperfine Structure of DEPMPO-OOH Using Selective Deuteration. A phosphate buffer reaction of DEPMPO with KO2 at room temperature
produced DEPMPO-OOH that, under low modulation amplitude conditions (i.e., 0.01 mT), yielded the well-known highly resolved ESR spectrum shown in Figure 1a. In this spectrum, the two inner lines in the superhyperfine structure pattern of the major nitroxide clearly consisted of nine lines with a maximal hfcs
