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Biomedical EPR Part-B Methodology Instrumentation and Dynamics - Sandra R. Eaton

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150

FABIAN GERSON AND GEORG GESCHEIDT

electrolytically generated inside the cavity, cannot be studied by the ENDOR technique, as the electrodes interfere with the RF coils.

The number of the nuclei giving rise to an ENDOR signal must be verified by close examination, preferably by simulation, of the corresponding EPR spectrum. Other procedures can likewise be used to this aim, e.g. isotopic substitution, which also serves for the assignment of coupling constants to sets of equivalent nuclei. ENDOR spectroscopy is particularly suited for such an assignment, because the signals of different isotopes appear in separate frequency regions. Relative numbers of nuclei responsible for the signals can be determined by techniques such as specialTRIPLE resonance, while the relative signs of the coupling constants are obtainable by general-TRIPLE resonance.

1.4Triple Resonance

This technique requires a second powerful RF source. In the special- TRIPLE-resonance or double-ENDOR experiment (Kurreck et al., 1984; Kurreck, et al., 1988), the sample is irradiated simultaneously with two RF fields, in addition to the saturating MW irradiation, so that both NMR transitions and (Figure 1) are excited at the same time. According to schemes (c) and (d) of this Figure, such procedure should double the ENDOR enhancement, because it leads simultaneously to in and in thus yielding for the levels involved in the relevant EPR

transition a difference instead of achieved with a single RF source. The main advantage of special-TRIPLE resonance is that the signal intensities reproduce better the relative number of nuclei giving rise to them than it does ENDOR spectroscopy. The special-TRIPLE-resonance signal, associated with the coupling constant appears separated from the origin (NMR frequency by in unit of As shown in Figure 3 for the phenalenyl radical (Kurreck et al., 1984), the intensities of the special- TRIPLE-resonance signals from the six protons in the 1,3,4,6,7,9-positions relative to those from the three protons in the 2,5,8-positions exhibit more exactly the expected ratio 2.

Whereas in the special-TRIPLE-resonance experiment the NMR transitions of the same set of protons are irradiated (homonuclear-TRIPLE resonance), in its general-TRIPLE counterpart, transitions of different sets of nuclei are saturated simultaneously (heteronuclear-TRIPLE resonance). One NMR transition is “pumped” with the first (unmodulated) RF frequency, while the second (modulated) RF field is scanned over the whole range of NMR resonances. The “pumping” causes characteristic intensity changes of the highand low-frequency signals relative to those observed by ENDOR. When the high- (low-) frequency signal is pumped, its intensity strongly reduced, while that of the low- (high-) frequency partner, associated with the

SOLUTION ENDOR OF RADICAL IONS

151

same coupling constant, is enhanced, because, for the latter signal, the pumping corresponds to a special-TRIPLE-resonance experiment.

Figure 3. EPR (left, center), (right, top), and the corresponding special-TRIPLE- resonance (left, bottom) and general-TRIPLE-resonance (right, center and bottom) spectra of the phenalenyl radical; solvent mineral oil, temperature 300 K. The arrow above the EPR spectrum indicates the line selected for saturation, while those in the general-TRIPLE- resonance spectra mark the ENDOR signal chosen for pumping. is the frequency of the free proton. Taken with a Bruker-ER-200-D spectrometer and a Bruker ENB-ENDOR cavity. Reproduced from (Kurreck, et al., 1988) by permission of VCH Publishers.

Figure 3 demonstrates the effect of pumping the signals separated by the smaller coupling constant, of the three protons in the 2,5,8-positions

of phenalenyl radical

(Kurreck, et al., 1984). When the high-frequency

signal at

is pumped, it nearly disappears, while its low-

frequency partner at

is substantially strengthened. An

opposite effect on the intensities is observed on pumping this low-frequency signal. Simultaneously, striking intensity changes are observed for the pair

of signals separated by the larger coupling constant,

of the

six

protons in the 1,3,4,6,7,9-positions, although neither of these

signals

is

subjected to pumping. As is evident from Figure 3, such changes follow patterns which are diametrically opposed to those induced on the signals

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FABIAN GERSON AND GEORG GESCHEIDT

separated by

The intensity ratio of the high-frequency signal at

 

to its low-frequency partner at

increases

relative to that in the ENDOR spectrum when the high-frequency signal at

 

is pumped. In contrast, this ratio decreases when the pumping is

carried out

on the low-frequency signal at

This behavior

points to opposite signs of the two coupling constants, i.e.

and

corresponding to –0.629 and +0.181 mT,

respectively (Gerson, 1966).

 

2.QUINONES

2.1Introduction

Quinones are the most common electron acceptors and electron-transfer mediators in biological processes, in which their radical anions (semiquinone anions) play an important role (Morton, 1965). An early ENDOR study of biologically interesting semiquinone anions in fluid solution was published (Das et al., 1970) a few years after ENDOR spectra of organic radicals in solution had been reported for the first time (Hyde and Maki, 1964). The

SOLUTION ENDOR OF RADICAL IONS

153

pertinent semiquinones were the radical anions of (vitamin E quinone; 1) and ubiquinone (2), which are both derivatives of p- benzoquinone, as well as those of 2-methyl-3-phytyl-1,4-naphthoquinone (vitamin quinone; 3) and menadione (vitamin quinone; 4) which is another derivative of 1,4-naphthoquinone. Recently, a solution-ENDOR study of the semiquinone anion from emodic acid (5), an oxidation product of emodin, was reported (Rahimipour et al., 2001b); emodin, a derivative of 9,10-anthraquinone, is used as a laxative and has other pharmacological applications (Hartmann and Goldstein, 1989). Here, we describe solutionENDOR spectra of the semiquinone anion and dianion from hypericin (6H) (Gerson et al., 1995), a derivative of biphenoquinone, which has been isolated from St. John’s wort and displays antiviral, antidepressive, and photodynamic activity (Muldner and Zoller, 1984; Suzuki et al., 1984). Solution-ENDOR spectra of a radical anion and two radical dianions from hypericin derivatives (Rahimipour et al., 2001a) are also presented.

2.2Hypericin

Hypericin is claimed to exist as 7,14and 1,6-dihydroxy tautomers, of which the former (6H) is more stable and prevails in non-concentrated solutions (Dax et al., 1999; Etzlstorfer and Falk, 2000; Freeman et al., 2001). In neutral and alkaline media, it readily deprotonates to yield its conjugate base Both 6H and readily accept an additional electron, and ENDOR spectroscopy serves as a straightforward tool to find out whether deprotonation also occurs at the stage of the one-electron-reduced species.

Figure 4 shows the spectrum of the radical dianion generated from the sodium salt of with potassium in tetrahydrofuran (THF); identical spectra were observed under the same conditions from other salts of and very similar ones were obtained upon reduction of the sodium salt with zinc in N,N-dimethylformamide (DMF) and

(10:1) (Gerson et al., 1995). Use of (10:1) led to the radical dianion in which all five OH protons were replaced by deuterons; the pertinent and spectra are also reproduced in Figure 4. Contrary to the EPR and ENDOR spectra of the persistent radical dianion those of the radical anion its conjugated acid, were observable only under strictly anhydrous conditions, because rapidly deprotonated

to in the presence of traces of water. A spectrum of recorded immediately upon reaction of 6H with potassium in a carefully dried THF, is likewise displayed in Figure 4.

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FABIAN GERSON AND GEORG GESCHEIDT

Figure 4. Radical

ions from hypericin.

 

spectrum of the

anion

(top, left);

solvent THF, counterion

 

 

spectrum of the dianion

(top,

right); solvent

THF, counterions

and

and

 

spectra of the dianion

(bottom);

solvent

counterions

and

Temperature 298 K throughout.

and

are the frequencies

of the free proton and deuteron, respectively. Taken

with a Bruker-ESP-300 spectrometer. Reproduced from (Gerson et al., 1995) by permission of J. Am. Chem. Soc.

The hyperfine data for and obtained under various conditions, are listed in Table 1. Relying on simulation of the EPR spectra of

SOLUTION ENDOR OF RADICAL IONS

155

its coupling constants, of one and six protons were straightforwardly assigned to the single proton bridging the 3,4-O atoms and to the of the two 10,11-methyl substituents, respectively. Moreover, deuteration allowed to distinguish the proton pairs in the 1,6- and 8,13-OH groups from the sets of two at the and 9,12. (According to the conventional nomenclature of EPR spectroscopy, protons directly linked to the are called while those separated from such centers by one C atom are denoted The remaining ambiguities were resolved by UB3LYP/6-31G* calculations (Rahimipour et al., 2001a). The coupling constants for are similar to those for with the exception of the value due to the two protons in the non-dissociated 3,4-OH groups of which strongly differs from that of the single proton bridging the pertinent two O atoms in The signs of the coupling constants were determined by the general-TRIPLE-resonance experiments with the reasonable assumption that the values of the are negative. All signs were confirmed by theoretical calculations. The hyperfine data for and are compatible with a twisted helical geometry and aneffective symmetry.

2.3Hypericin Derivatives

These derivatives are 2,5-dibromohypericin (7H), 10,11desmethylhypericin (8H), and 1,3,4,6,8,13-hexaacetylhypericin (9). In contrast to the parent hypericin (6H), the of 8H can adopt a planar geometry, while the 3,4-OAc groups of 9 are not amenable to deprotonation. The three compounds were reduced under various experimental conditions (Rahimipour et al., 2001a), and the spectra of the paramagnetic species thus obtained with zinc in DMF are shown in Figure 5. These spectra allowed to identify the species in question as the radical dianions and and the radical anion respectively. Their hyperfine data are given in Table 2.

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FABIAN GERSON AND GEORG GESCHEIDT

Figure 5. Radical ions from hypericin derivatives. spectra of the dianions (top) and (left, bottom) and of the anion (right, bottom); solvent DMF, counterion , temperature 273 K. is the frequency of the free proton. The unsplit signals at in the spectra of and arise from protons with a very small coupling constant; the pertinent (absolute) values are 0.003 mT for 1,6-OH protons in (Table 2) and <0.005 mT for the acetyl protons in (not given in Table 2). Taken with a Bruker-ESP-300 spectrometer. Reproduced from (Rahimipour et al., 2001a) by permission of Photochem. Photobiol.

Assignments and signs of the coupling constants which compare favorably with the corresponding values for and were corroborated by UB3LYP/6-31G* calculations. The spin distribution in these radical dianions and radical anion is not markedly altered by the structural modifications of hypericin, as the bulk of spin population remains accommodated by the central biphenoquinone moiety. With respect to

SOLUTION ENDOR OF RADICAL IONS

157

biological processes it is important to note that the structure of like that of remains unaltered when organic solvents are replaced by an aqueous buffer solution.

3.PORPHYRINOIDS

3.1Introduction

Porphyrins in general, and metalloporphyrins in particular, are involved in many biological processes and, owing to their deep colors, have been called the pigments of life (Battersby and McDonald, 1979; Battersby and Frobel, 1982). The prominent property of these macrocyclic is the easy acceptance and donation of electrons, which leads to many redox stages. Because of their effective symmetry, the radical ions of the unsubstituted porphyrin and metalloporphyrins (10M) (Seth and Bocian, 1994), as well as those of tetraoxaporphyrin (11) (Bachmann et al., 1992; Bachmann, 1996), have a degenerate ground state and are subject to the dynamic Jahn-Teller effect which enhances the electron-spin relaxation. As a consequence, the EPR lines of these ions are excessively broadened and difficult to saturate, so that their solution-ENDOR spectra could not be observed. On the other hand, the effective symmetry is lowered to in the isomeric porphycene (Vogel et al., 1986), metalloporphycenes (12M), and tetraoxaporphycene (15) (Vogel et al., 1988) which also have

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FABIAN GERSON AND GEORG GESCHEIDT

applications to photobiology (Toporowicz et al., 1989). The corresponding radical ions have thus a nondegenerate ground state, and they gave rise to highly resolved EPR spectra along with readily observable solution-ENDOR signals (Schlüpmann et al., 1990; Bachmann et al., 1993; Bachmann, 1996), as is described below.

3.2Porphycenes and Metalloporphycenes

The porphycenes, of which the radical anions were studied, include the free base and its 2,7,12,17and 9,10,19,20-tetra-n-propyl derivatives and respectively), as well the metalloporphycenes 12Zn, 13Ni, 13Pd, and 13Pt (Schlüpmann et al., 1990).

Figure 6. Radical ions of porphycenes and metalloporphycenes. and spectra of the anions (left, from top to bottom) and and (right, from top to bottom); solvent THF, counterion temperature 220–240 K. and are the frequencies of the free proton and nucleus, respectively. For the constant, the low-frequency signal at 0.04 MHz was not observed, because it is below the range of the spectrometer. Taken with a home-built instrument. Reproduced from (Schlüpmann et al., 1990) by permission of J. Am. Chem. Soc.

Figure 6 presents the and spectra of the corresponding radical anions (except those of which were generated from their neutral precursors with sodium in THF. The MW power required for saturation of the EPR line, a prerequisite for a successful ENDOR experiment, had to be raised on going from to and to ENDOR signals of were not detected even with the MW power as

SOLUTION ENDOR OF RADICAL IONS

159

high as 200 mW. The increased reluctance to saturation is a consequence of the more efficient spin-orbit coupling and the enhanced electron-spin relaxation with the growing atomic order of the metal. This phenomenon was also manifested by the broadening of the EPR lines (that of is a single unresolved signal) and the higher g factor, as indicated in Table 3 which gives the hyperfine data for the six radical anions with observable and constants, and

The and values are compatible with the symmetry being effective on the hyperfine time-scale, especially with the equivalency of the four nuclei and the fast tautomerization of the two N-protons in and Assignments of the coupling constants relied (i) on special-TRIPLE-resonance experiments and simulation of the EPR spectra, from which the numbers of protons giving rise to the ENDOR signals were derived, (ii) on deuteration in the 9,10,19,20-positions of (Renner et al., 1989), and (iii) on the internal consistency of the hyperfine data in the series, by which sets with equal numbers of protons were distinguished. The relative signs of all coupling constants were derived from the general- TRIPLE-resonance spectra with the reasonable assumption that the values of the are negative. Both assignments and signs of the coupling constants were confirmed by all-valence-electrons-self-consistent-field MO calculations (RHF/INDO-/SP) for and The changes in the distribution along the series are moderate, and metallation has only a minor effect. On going from to and from to or there is an increase in the populations at the centers and a corresponding decrease at and 9,10,19,20.