PELDOR at S- and X-band frequencies and the separation of exchange coupling from dipolar coupling

Article abstract of DOI:10.1006/jmre.2002.2596

A pulsed electron double resonance (PELDOR) setup working at S-band frequencies is introduced and its performance compared with an X-band setup. Furthermore, to verify experimentally that it is possible to disentangle the dipolar coupling v_Dip from the exchange coupling J by PELDOR we synthesized and investigated four bisnitroxide radicals. They exhibit in pairs the same distances r_AB between the nitroxide moieties but only one of each pair possesses a non-zero J. The experimental values for r_AB match the ones from molecular modeling very well for the molecules without exchange coupling. For one bisnitroxide it was possible to separate v_Dip from J and to ascertain the magnitude and sign of J to +11 MHz (antiferromagnetic spin-spin coupling).

Full text of DOI:10.1006/jmre.2002.2596

Journal of Magnetic Resonance 157, 277–285 (2002)
doi:10.1006/jmre.2002.2596
PELDOR at S- and X-Band Frequencies and the Separation
of Exchange Coupling from Dipolar Coupling
Axel Weber, Olav Schiemann,1 Bela Bode, and Thomas F. Prisner
Institut fu¨r Physikalische und Theoretische Chemie, Johann Wolfgang Goethe-Universita¨t, Marie-Curie-Strasse 11, 60439 Frankfurt/Main, Germany
Received March 22, 2002; revised June 24, 2002; published online August 28, 2002
A pulsed electron double resonance (PELDOR) setup working
at S-band frequencies is introduced and its performance compared
with an X-band setup. Furthermore, to verify experimentally that
it is possible to disentangle the dipolar coupling νDip from the ex-
change coupling J by PELDOR we synthesized and investigated
four bisnitroxide radicals. They exhibit in pairs the same distances
rAB between the nitroxide moieties but only one of each pair pos-
sesses a non-zero J. The experimental values for rAB match the ones
from molecular modeling very well for the molecules without ex-
changecoupling. ForonebisnitroxideitwaspossibletoseparateνDip
from J and to ascertain the magnitude and sign of J to +11 MHz
Hamiltonian HAB [1], which is defined as the sum of the
Hamiltonian HD [2] for the magnetic dipole–dipole coupling
and the Hamiltonian HJ [3] for the exchange coupling.
HAB = HD + HJ
HD = SA DSB
HJ = J SASB.
[1]
[2]
[3]
with
and
(antiferromagnetic spin–spin coupling). ꢀ 2002 Elsevier Science (USA)
C
Key Words: dipolar coupling; PELDOR; S-band EPR; pulse EPR;
exchange coupling.
Here D is the magnetic dipole–dipole coupling tensor and J is
the electron–electron exchange coupling constant. In first-order
high-field approximation the electron–electron coupling νAB can
be expressed by Eqs. [4] and [5] as the sum of the orientation-
dependent magnetic dipole–dipole coupling constant νDip and
the isotropic exchange coupling constant J.
INTRODUCTION
There is a strong interest in structures and dynamics of or-
ganic polymers (1) and biomolecules like proteins (2) or RNA
(3) driven by the paradigm that the structure determines its prop-
erties or function. Commonly used methods to gain insight into
the structure of these molecules are X-ray crystallography (4),
fluorescence energy transfer (FRET) measurements (5), as well
as nuclear magnetic resonance (NMR) (6), and electron para-
magnetic resonance (EPR) spectroscopy (7). X-ray crystallogra-
phy can provide precise global and local structure information.
But it is restricted to studies of molecules in crystals, where,
for example, an enzyme may not be in the catalytically active
form. Complementary to that, NMR, FRET, and EPR are meth-
ods capable of collecting structural data from these molecules
in solution. However, to be able to apply EPR spectroscopic
tools the molecule needs to possess paramagnetic centers. If it
does not site-directed spin-labeling by nitroxides is a commonly
used way to overcome this problem (8). Twofold spin-labeling
makes it then possible to gather structural information about the
labeled molecule by measuring the coupling energy EAB = hνAB
between the two unpaired electrons with spins SA and SB
(9). EAB is the eigenvalue of the electron–electron coupling
νAB = νDip(3 cos2 θ − 1) + J
[4]
with
µ2BgAgBµ0
4πh
1
rA3B
νDip
=
.
[5]
θ is the angle between the applied magnetic field B0 and the
interspin distance vector rAB, µB is the Bohr magneton, µ0 is
the field constant and gA, gB are the g values of the unpaired
electrons A and B, respectively. The orientation dependence of
νAB is depicted in Fig. 1. From νDip the spin–spin distance rAB
can be calculated and it may, for example, be rationalized if a
molecule is stretched or bent or if its structure changes upon
binding to another molecule.
With continuous wave (cw) EPR spectroscopy distance mea-
surements are limited due to the spectral linewidth of the nitrox-
˚
ides to a maximum distance of about 20 A (10). However, since
1 To whom correspondence should be addressed. Fax: 069-798-29404.
E-mail: olav@prisner.de.
the linewidth of the nitroxides is determined by unresolved hy-
perfine splittings and inhomogeneous line broadening, pulsed
277
1090-7807/02 $35.00
2002 Elsevier Science (USA)
All rights reserved.
ꢀC

278
WEBER ET AL.
sequence at microwave frequency ν1 excites spin A in a birad-
rAB ⊥ B0
ical and creates a Hahn echo. Spin B in the same molecule is
excited by an additional inversion pulse at microwave frequency
ν2 whereupon the amplitude of the Hahn echo shows an os-
cillation dependent on the position T of the inversion pulse. The
frequency of this oscillation is the coupling νAB. Jeschke and
co-workers extended PELDOR to a dead time free four-pulse
sequence (12) and derived a single frequency technique for re-
focusing dipolar couplings (SIFTER) (13) from the solid-echo
well known in NMR (14). Two other pulse experiments capa-
ble of measuring νAB are “2 + 1” from Kurshev et al. (15) and
“DQC” from Saxena and Freed (16).
In the paper presented here the technical setup for PELDOR
measurements at S-band frequencies, its successful testing, and
its performance with respect to an X-band PELDOR setup are
described. The advantage of carrying out PELDOR experiments
at two different frequencies is to be able to separate the wanted
νAB coupling from residual hyperfine coupling contributions
(17). In all previous publications PELDOR measurements were
performed at X-band frequencies. However, Raitsimring and co-
workers announced S- and C-band PELDOR measurements in
two conference contributions (18). Furthermore we show that in
the case of exchange-coupled biradicals it is possible to deter-
mine experimentally not only νDip but also the magnitude and
sign of J. This is achieved by detecting with PELDOR both
tensor singularities of νAB (Fig. 1) and using (19)
rAB  B0
θ = 90˚
(3cos2θ - 1) = -1
54.7˚
0˚
+2
0
J
ν
AB = - ν Dip + J
2ν Dip + J
FIG. 1. The powder pattern for νAB with the singularities and the related θ
angles.
EPR spectroscopy is a way to raise this limit by designing pulse
sequences which recover the spin–spin coupling and suppress
other spectral contributions. In 1981 Milov et al. established the
pulsed electron double resonance (PELDOR) method (11) with
the well-known three-pulse pattern. Here a π/2–τ–π detection
O
N
2•
1•
H
N
H
O
N
O
νDip = (ν(θ ) − ν(θ ))/3
[6]
[7]
ꢁ
⊥
O
O
J = (2ν(θ ) + ν(θ ))/3.
⊥
ꢁ
O
O
O
3••
In this way it is possible to calculate the spin–spin distancerAB
even in the case of an exchange-coupled biradical. As examples
the bisnitroxides 5••–8•• were synthesized (Fig. 2), where the
conjugated bridges in 7•• and 8•• facilitate an exchange coupling
through bonds whereas the ester linkages in 5•• and 6•• disrupt
this conjugation and therefore diminish the exchange coupling
while leaving the rAB almost unchanged.
O
O
O
N
O
O
Hex
O
4••
N
O
O
N
O
SYNTHESIS AND SAMPLE PREPARATION
O
Hex
Radical 2• (2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-3-acety-
lene) was prepared from 2,2,5,5-tetramethyl-3-pyrroline-1-
oxyl-3-carboxylic acid 1• with slight modifications to the pro-
cedure described by Hideg et al. (20). Biradicals 3•• and 4••
were donated to us by G. Jeschke and used as received. The new
biradicals 5••–6•• were synthesized in analogy to (21).
Biradical 5••. To a solution of 46 mg (0.42 mmol) 1,4-
dihydroxybenzene, 230 mg (1.25 mmol) 2,2,5,5-tetramethyl-3-
pyrroline-1-oxyl-3-carboxylic acid 1•, and 171 mg (1.4 mmol)
dimethylaminopyridine in 5 ml dry THF were added 1.25 ml
of a 1 M solution of N,N-dicyclohexylcarbodiimide in methyl-
enechloride (1.25 mmol) at 0◦C. The yellow reaction mixture
was stirred at room temperature for 20 h. The precipitate was
O
O
N
n = 1 => 5••
n = 2 => 6••
O
O
N
O
n
n
O
O
n = 1 => 7••
n = 2 => 8••
N
N
O
FIG. 2. Structures of the inquired radicals 1• to 8•• in the stretched confor-
mation as used for molecular modeling (Hex, n-hexyl groups).

PELDOR AT S- AND X-BAND FREQUENCIES
279
Biradical 8••. Biradical 8•• was synthesized and purified
according to the procedure described for 7•• but with the use
of 4,4ꢂ-diiodbiphenyl instead of 1,4-diiodbenzene (yield 20 mg,
7%). EI-MS:m/z = 478.6(20,3%M+), 463.6(6%–CH3), 448.5
(22% –CH3), 433.6 (8% –CH3), 418.5 (100% –CH3). FT-IR
(KBr): 3050(m), 2980(s), 2930(m), 2215(w), 825(m) cm−1. Ele-
mental analysis calculated for C32H34N2O2 (478.64): C 80.30,
H 7.16, N 5.85; found C 80.40, H 7.10, N 5.92.
For the EPR measurements appropriate amounts of biradicals
3••–8•• were dissolved in h8- or d8-toluene to yield 160 µl of
1 mM solutions. The pulsed S-band measurements were carried
out at 10 K and the respective X-band measurements at 20 K.
filtered off and washed with THF until the solid was color-
less. The yellow filtrate was washed with 2 N HCl and brine.
The organic phase was dried with MgSO4 and concentrated in
vacuo. The remaining yellow solid was redissolved in warm
dichloromethylene and applied onto a silica gel column. Using
diethyl ether : pentane (1 : 1) as the eluent yielded 5•• in the
second band (Rf = 0.35). An additional HPLC purification
(CH2Cl2 : hexane : ethyl acetate = 7 : 5 : 2) provided pure 5•• as
yellow powder with a yield of 25 mg (11%). MALDI-MS (ATT-
Matrix): m/z = 444.2 (100% M+ + 2H), 442.2 (100% M+),
428.6 (60% –CH3). FT-IR (KBr): 3080(m), 2980(m), 2930(m),
1730(s), 1625(m), 1505(s), 1175(s), 790(m) cm−1. Elemental
analysis calculated for C24H30N2O6 (442.5): C 65.14, H 6.83,
N 6.33; found C 64.92, H 7.00, N 5.91.
EPR INSTRUMENTATION AND DEVELOPMENTS
Biradical 6••. To a solution of 78 mg (0.42 mmol) 4,4ꢂ-
dihydroxybiphenyl, 230 mg (1.25 mmol) 2,2,5,5-tetramethyl-3-
pyrroline-1-oxyl-3-carboxylic acid 1•, and 171 mg (1.4 mmol)
dimethylaminopyridine in 5 ml dry THF were added 1.25 ml
of a 1 M solution of N,N-dicyclohexylcarbodiimide in methyl-
enechloride(1.25mmol)at0◦C. Thereactionmixturewasstirred
at room temperature for 20 h, and the precipitate filtered off and
washed with THF until the solid was colorless. The yellow fil-
trate was washed with 0.5 N HCl and brine. The organic phase
was dried with MgSO4 and concentrated in vacuo. The solid
was redissolved in warm dichloromethylene and applied onto a
silica gel column. Using diethyl ether : pentane (1 : 1) as the elu-
ent yielded 6•• in the second band (Rf = 0.3). An additional
HPLC purification (CH2Cl2 : hexane : ethyl acetate = 7 : 5 : 2)
gave pure 6•• as a yellow powder with a yield of 30 mg (16%).
MALDI-MS (ATT-Matrix): m/z = 520.4 (95% M+ + 2H),
518.5 (100% M+), 504.3 (50% -CH3). FT-IR (KBr): 3065(m),
2980(m), 2935(m), 1735(s), 1625(m), 1490(s), 1195(s),
795(m) cm−1. Elemental analysis calculated for C30H34N2O6
(518.61):C69.48, H6.61, N5.40;foundC68.95, H6.89, N4.78.
X-band. The cw X-band EPR spectra were recorded on a
Bruker ESP300e EPR spectrometer. The pulsed X-band exper-
iments were carried out on an ELEXSYS E580 pulsed X-band
EPR spectrometer from Bruker with the technical specifications
as described in (22). For the PELDOR experiments this spec-
trometer was extended in such a way that a second microwave
frequency could be fed into the microwave pulse forming unit
(MPFU) within the bridge. The second microwave frequency
was generated by a HP 8672A synthesizer and amplified by a
Miteq AMF-5B-080120-30-25P amplifier to achieve the desired
powerof+17dBm. Followingtheamplifieradirectionalcoupler
Narda 3004-10 fed −10 dB for power measurements into a spec-
trum analyzer 492 from Tektronix. The signal from the other exit
was coupled into one of the two MPFUs in the ELEXSYS E580
microwave bridge. A phase-insensitive detection of the power
level of both pulse frequencies was accomplished by placing a
Narda 3004-10 directional coupler before the TWT and feeding
−10 dB into a MDC 1118-S diode from Midisco. The signal
from this diode was viewed by a LeCroy 9350AM oscilloscope.
In all X-band experiments a commercial flex line probehead
with a dielectric ring resonator from Bruker was used which
exhibits in overcoupled conditions a resonance frequency ν0 of
Biradical 7••. Biradical 7•• was synthesized from 2•
as follows. A solution of 100 mg (0.6 mmol) of 2• and
99 mg (0.3 mmol) of 1,4-diiodbenzene in 5 ml of N,N-
dimethylformamide was deoxygenated by repeated exposure to
vacuum, followed by the addition of nitrogen. The amounts of
132 mg (0.114 mmol) tetrakis(triphenylphosphine)palladium(0)
and 290 mg (1.53 mmol) copper(I)iodide were added, and the
9.7 GHz, a quality factor Q of about 100, a conversion factor κ
√
of 4 µT/ W and a bandwidth of 97 MHz. Accordingly, even
with a frequency offset of 80 MHz between ν0 and ν2 both pulses
are still within the bandwidth of the resonator.
S-band. The S-band EPR experiments were performed on
solution was deoxygenated once more. The amount of 73 mg a home-built cw/pulsed S-band EPR spectrometer (23). As the
(0.72 mmol) triethylamine was added via a syringe, and the re- second source a synthesizer 4002 from Schlumberger was used.
sulting mixture was stirred at 25◦C for 12 h. The volatiles were Since it generates only microwave frequencies up to 2.16 GHz
removed in vacuo and the residue was dissolved in toluene. a frequency doubler MX2J020040 from Miteq was necessary
Chromatography on silica gel (Toluol : THF = 17 : 1) followed to convert the frequency up to S-band. A variable microwave
byHPLC(hexane : CH2Cl2 : ethylacetate = 5 : 2 : 1)purification power attenuator Narda 791FM following the frequency doubler
oftheproductfractionyielded7•• asapaleyellowpowder(yield: makes it possible to adjust the power of the microwave signal.
30 mg, 10%). EI-MS: m/z = 403.1 (79,5% M+), 388.2 (13% From there the microwave propagates through an isolator FG1-1
–CH3), 373.5 (15% –CH3), 358.2 (100% –CH3), 342.0 (12% L1FFL1 and reaches a PIN switch HP 33144A. If the PIN switch
–CH3). FT-IR (KBr): 3050(m), 2980(s), 2930(m), 2220(w), is closed the microwave is reflected and absorbed by the isolator,
835(m) cm−1. Elemental analysis calculated for C26H30N2O2 iftheswitchisopenthemicrowaveiscombinedwiththesignalof
(402.54):C77.58, H7.51, N6.96;foundC77.38, H7.61, N6.74. the first source at a two-way power combiner ANAREN 41130.

280
WEBER ET AL.
FIG. 3. Computed orientation selection in the hyperfine axis system for the pump (points) and the detection pulses (open circles) at (a) ꢀν = 80 MHz and (b)
ꢀν = 40 MHz. The calculations were done with rectangular pulses of 25 MHz width and for only one hemisphere.
Both microwaves are then guided through an isolator M312040 selected depends on the position of the pulses with respect to the
to a fast PIN switch S136ADU1 from Miteq (switching time field swept spectrum. In all experiments the inversion pulse was
<2 ns). A GaAs-amplifier AMA2040B (gain 45 dB) amplifies exciting the 14N mI = 0 hyperfine line resulting in the excitation
the pulses to +30 dBm in order to provide an optimal power of only a fraction of the nitroxide moieties but of all orientations
level for the subsequent Litton 624 pulsed 1 kW traveling wave with respect to B0. From these molecules those are contribut-
tube (TWT) amplifier. A 10 dB directional coupler Narda 3003- ing to the PELDOR modulation for which the second nitroxide
10 placed before the TWT amplifier serves in conjunction with group is selected by the detection sequence. If the detection se-
a detector diode MDC1118-S from Midisco as a transmitter quence has a frequency offset ꢀν of 80 MHz from the inversion
monitor, which is necessary to compare the amplitude of the pulse the field swept spectra of the nitroxides under study con-
pulses of the second frequency with the amplitude of the pulses sist mostly of the mI = +1 component of the AZZ(14N) hyperfine
of the first one.
coupling leading to a selection of 1/3 of those molecules with
All S-band experiments were performed with a home-built the second nitroxide oriented with AZZ parallel to B0 (Fig. 3a).
bridged loop gap resonator exhibiting in overcoupled condi- If ꢀν equals 40 MHz the selection profile of the detection pulses
tions a resonance frequency ν0 of 3.60 GHz, a quality factor is shifted closer to equatorial orientations of the hyperfine tensor
Q of 150 and a bandwidth of 24 MHz. Therefore even with the (Fig. 3b). This hyperfine orientation selectivity results for both
smallest frequency offset ꢀν = ν1 − ν2 of 40 MHz used in our ꢀν’s in a θ selection depending on the orientation of rAB with
experiments it is not possible to have both frequencies ν1 and respect to the hyperfine tensor. However, additional orientations
ν2 within the bandwidth of the resonator. Nevertheless, due to to those displayed in Fig. 3 are excited by the whole profile of the
√
the large conversion factor κ of 15 µT/ W it is possible to pulsesbuttoalesserextent. Thismayleadtothedetectionofboth
achieve a flip angle of 180◦ for typical pulses of 40 ns duration, dipolar tensor singularities with one measurement especially if
up to a frequency offset of 100 MHz from the center ν0 of the the weak θ = 0◦ dipolar tensor singularity is excited within the
ꢁ
resonator. To ensure a flip angle of 180◦ for the inversion pulse bandwidth of the detection pulses whereas the intense θ = 90◦
⊥
at the off-resonant frequency ν2 a Hahn echo was created at this is excited by the side loops only. The θ values can then be as-
frequency and the power optimized.
The sensitivity of the pulsed S-band spectrometer was mea- and νDip and J can be calculated according to Eqs. [6] and [7].
signed by comparing the measurements at ꢀν = 80 and 40 MHz
sured to be three times smaller then at X-band and in agreement
with theory (24, 25).
MEASUREMENTS AND DISCUSSION
The S- and X-band PELDOR setups described above were
ORIENTATION SELECTIVITY
tested on the bisnitroxides 3•• (26) and 4•• (12a) known from
The PELDOR measurements were performed with pulse literature. Withaꢀν of80 MHz3•• and4•• displayedinbothfre-
lengths of 40 ns at S-band and of 32 ns at X-band for all three quency bands an oscillation frequency νAB of 6.8 and 2.1 MHz,
pulses. Since the corresponding spectral widths of the pulses respectively. These data match the X-band PELDOR data from
of 25 and 35 MHz for S- and X-band, respectively, are smaller the literature nicely (Table 1) and confirm that both PELDOR
than the nitroxide field swept spectra (∼200 MHz) the pulses are units are working properly. To calculate the spin–spin distance
selecting certain molecular orientations. Which orientations are rAB according to Eqs. [4] and [5] the θ value belonging to the

PELDOR AT S- AND X-BAND FREQUENCIES
281
TABLE 1
ESEEM depth from 100 to 80% and shortened its period but at
the same time also the T2-relaxation time from 0.8 to 0.4 µs so
that in the end no improvement could be achieved. The PELDOR
modulation depth is comparable in S- and X-band.
Data for 5••–8•• Gathered from S- and X-Band
PELDOR Measurements
b
νAB(θ ) νAB(θ )
νDip
JPELDOR/JCW
rAB
rAB
⊥
ꢁ
❛
❛
Molecule (MHz)a (MHz)a (MHz)
(MHz)
(A)exp. (A)theor.
3••
4••
5••
6••
7••
8••
6.8/6.8 13.4/13.8 6.8(2)
2.1/2.1 4.3/4.3 2.1(2)
14.1/14.2 —/— 14.2(2)
6.9/6.9 —/13.7 6.9(2)
—
—
—
19.7(3) 19.7(10)c
29.2(4) 29.2(10)d
15.4(3) 15.5(10)
19.7(3) 19.7(10)
—
—
—
—
—/73(3)
—
16.4(3)
—/7.2
—/18.3 3.7(2) +11.0(3)/12(3) 24.0(4) 21.0(3)
a The first number is from the S-band the second one from the X-band mea-
surement.
b For the molecular modeling the N–O groups were replaced by N–OH groups
andthetheoreticalrAB measuredfromthemidpointofoneN–Obondtotheother.
❛
c Larsen and Singel find the same experimental rAB of 19.7 A(26).
❛
d Jeschke and co-workers measured an rAB of 28.3(5) A(12a).
measured νAB as well as the magnitude and sign of J need to
be known. Thus the measurements were repeated with a ꢀν
of 40 MHz where the simulations showed that different orienta-
tions are selected (Fig. 3). The spectra of 3•• with ꢀν = 40 MHz
are shown as an example in Fig. 4. The Fourier-transformed
PELDOR spectra display at S-/X-band one peak at 6.8/6.8 MHz
assigned to θ and another weaker one at 13.4/13.8 MHz cor-
⊥
responding to θ . Since the frequency at θ is two times the
ꢁ
ꢁ
frequency at θ it can be concluded that both singularities of
the dipolar tensor were excited (28). It also confirms that the θ
⊥
⊥
singularity was detected with ꢀν = 80 MHz. Using Eqs. [6] and
[7] a negligible J and a νDip of 6.8 MHz were found. With this
˚
an rAB of 19.7 A was calculated from Eq. [4]. That the weak
peak assigned to θ is not an artifact caused by hyperfine in-
ꢁ
teraction is proven by the occurrence of this peak at the same
frequency in the S- and X-band. In principle hyperfine interac-
tions can contribute to PELDOR spectra as the occurrence of
the free deuterium oscillation in the spectrum of monoradical 2•
showed. 4•• and 6•• showed in principle the same PELDOR be-
havior; their data are collected in Table 1. The experimental rAB
match for all three molecules those rAB obtained by molecular
modeling where the molecules adopted a stretched conforma-
tion (Table 1). The stretched conformation is also in agreement
with the finding that AZZ is perpendicular to rAB
.
The shorter time window τ for the S-band compared to the
X-band PELDOR measurements is caused by several properties
of the electron spin echo (ESE) spectroscopy at low frequen-
cies/low magnetic fields: (a) the deeper S-band electron spin-
echo envelope modulation (ESEEM) (Fig. 5) due to weak hy-
perfine interactions (25, 29), (b) the longer period of the Lamor
frequencies, and (c) the reduced sensitivity as noted above. Ad-
ditionally a shorter T2-relaxation time was found for this sample
even at the lower temperature compared to the X-band. There-
fore, it was only possible to choose a few τ values where the ESE
amplitude exhibits a maximum and a reasonable signal-to-noise
FIG. 4. Three-pulse PELDOR spectra of 3•• in d8-toluene (a) at X-band
with ꢀν = 40 MHz, the Fourier-transformed spectrum is shown below, and (b) at
S-band with ꢀν = 40 MHz, the Fourier-transformed spectrum is shown below.
Both time spectra were Fourier-transformed after subtracting the background
ratio. The use of h8-toluene as the solvent reduced the S-band caused by instantaneous diffusion (27).

282
WEBER ET AL.
the importance of being able to perform PELDOR at two differ-
ent principal frequencies.
Biradical 7•• differs from 5•• by the extended π-conjugation
in the bridge, due to the replacement of the ester linkages by
alkin groups. Therefore an exchange coupling J through bond
FIG. 5. Two-pulse ESEEM spectra of 3•• in d8-toluene at (a) S-band and
(b) X-band.
For biradical 5•• one peak was detectable at 14.2 MHz in
X-band with ꢀν = 80 MHz (Fig. 6a), the second component of
the tensor was not resolvable. However, since the nitroxide moi-
ety and the field swept spectrum are the same as for 3••, 4••,
and 6•• it is reasonable to attribute this peak to θ . Assuming
⊥
secondly that J is negligible, because the two ester linkages pre-
vent as in the case of 6•• an exchange coupling, the measured
νAB of 14.2 MHz can be assigned to νDip. A spin–spin distance
˚
rAB of 15.4 A is calculated from that, matching nicely the dis-
tance obtained by molecular modeling and supporting thereby
the assumptions. To ensure that the observed peak is not solely
due to the free proton frequency we also performed S-band mea-
surements (Fig. 6b), which revealed one peak at 14.1 MHz and
one at 5.3 MHz. Since the peak at 14.2 MHz did not change
its frequency upon changing the magnetic field from 3435 to
1249 G, this proves that it is due to dipolar electron–electron
coupling. Nevertheless, the second peak in the S-band spectrum
FIG. 6. Three-pulse PELDOR spectra of 5•• in d8-toluene (a) at X-band
with ꢀν = 80 MHz, the Fourier-transformed spectrum is shown below, and (b) at
at 5.3 MHz corresponds to the free proton frequency, showing S-band with ꢀν = 80 MHz, the Fourier-transformed spectrum is shown below.

PELDOR AT S- AND X-BAND FREQUENCIES
283
not the reason for the failure of PELDOR since the•e•ven shorter
a
˚
distance of 15.5 A can be measured in the case of 5 . This may
point to the possibility of measuring even shorter distances with
PELDOR as long as the exchange-coupling J is small enough.
The X-band PELDOR spectra of 8•• and their Fourier trans-
formations are shown in Fig. 8. The tensor singularities are
3400
3300
3325
3375
3425
3350
B0 [G]
b
3400
3360
3380
3320
3340
B0 [G]
FIG. 7. Continuous wave X-band EPR spectra of (a) 7•• and (b) 8•• in
d8-toluene at room temperature.
is possible for 7••, but suppressed for 5•• by the σ-bonds of the
ester groups. On the other hand molecular modeling revealed
••
˚
˚
that their rAB are similar with 15.5 A for 5 and 16.4 A for
7••. The same holds for the couple 6•• and 8••, here the rAB are
19.7 and 21.0 A, respectively. The existence of J in 7 and 8••
was proven by room temperature cw X-band EPR spectroscopy
(Fig. 7). Simulating the spectra yielded values for |J| of 73 and
12 MHz for 7•• and 8••, respectively. Contrarily, no splitting
or line broadening was observable in the room temperature cw
spectra of molecules 3••–6••, which confirms independently and
once more that J is negligible for them. The sign of J cannot be
determined by this method.
••
˚
Biradical 7•• exhibits in the PELDOR spectrum no νAB os-
cillation or echo decay at S- as well as at X-bands, which is
ascribed to the relatively strong exchange coupling of 73 MHz
leading to a spin state of the molecule which is rather described
by a triplet and a singlet state than by two weakly interacting
doublet states needed for PELDOR to be applied successfully.
FIG. 8. X-band 3-pulse PELDOR spectra of 8•• with (a) ꢀν = 80 MHz
(in d8-toluene) and (b) ꢀν = 40 MHz (in h8-toluene). The Fourier-transformed
˚
The small distance of 16.4 A between the two N–O groups is spectra are shown below the time spectra.

284
WEBER ET AL.
(SFB 579 and 472 and a habilitation fellowship for O.S.). O.S. wishes to dedicate
this work to Ch. Elschenbroich.
visible at 7.2 MHz for ꢀν = 80 MHz (Fig. 8a) and at 18.3 MHz
for ꢀν = 40 MHz (Fig. 8b), corresponding to θ and θ , respec-
⊥
ꢁ
tively. As expected in the event of a non-zero J the frequency νAB
REFERENCES
at θ is not twice the one at θ . Since both singularities are de-
ꢁ
⊥
tected νDip and J can be disentangled from νAB using Eqs. [6] and
[7]. The calculation yields J = +11 MHz (antiferromagnetic)
and νDip = 3.7 MHz. The absolute value of J gathered from the
PELDOR measurements fits perfectly the one from the cw EPR
measurements. Additionally it reveals the sign and therefore
the antiferromagnetic nature of J in 8•• in agreement with the
knowledge that a para-substituted benzene facilitates usually an
antiferromagnetic and not a ferromagnetic spin–spin coupling
1. S. Schlick (Ed.), “Ionomers: Characterization, Theory, and Applications,”
CRC Press, New York (1996).
2. A. Messerschmidt, R. Huber, T. Poulos, and K. Wieghardt (Eds.), “Hand-
book of Metalloproteins,” Wiley, New York (2001).
3. R. F. Gesteland, T. R. Cech, and J. F. Atkins (Eds.), “The RNA World,” Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1999).
4. N. Ban, P. Nissen, J. Hansen, P. B. Moore, and T. A. Steitz, The complete
˚
atomic structure of the large ribosomal subunit at 2.4 A resolution, Science
˚
289, 905 (2000).
(30). From νDip an rAB of 24 A is calculated which is longer
5. N. G. Walter, K. J. Hampel, K. M. Brown, and J. M. Burke, Tertiary structure
formation in the hairpin ribozyme monitored by fluorescence resonance
energy transfer, EMBO 17, 2378 (1998).
˚
than the computed one of 21 A. The reason for that may be the
failure of the point dipole approximation used for the derivation
of Eqs. [4] and [5] owing to the influence of the exchange cou-
pling J. It is interesting that J also seems to have an influence on
6. C. Richter, B. Reif, C. Griesinger, and H. Schwalbe, NMR spectroscopic
determination of angles α and ζ in RNA from CH-dipolar coupling, P-CSA
cross-correlated relaxation, J. Am. Chem. Soc. 122, 12728 (2000).
the orientation selection, since the peak for θ is stronger than
ꢁ
7. T. F. Prisner, M. Rohrer, and F. MacMillan, Pulsed EPR spectroscopy:
Biological applications, Annu. Rev. Phys. Chem. 52, 279 (2001).
the one for θ at ꢀν = 40 MHz, in contrast to the orientation
⊥
∼
selection for the molecules with J 0.
=
8. L. J. Berliner (Ed.), “Biological Magnetic Resonance,” Vol. 14, Plenum,
New York (1998).
CONCLUSIONS
9. E. J. HustedtandA. H. Beth, StructuralinformationfromCW-EPRspectraof
dipolar coupled nitroxide spin labels, in “Biological Magnetic Resonance”
(L. J. Berliner, S. S. Eaton, and G. R. Eaton, Eds.), Vol. 19, pp. 155–184,
Kluwer Academic/Plenum, New York (2000).
We introduced a technical setup for PELDOR working at S-
band frequencies and tested it successfully on two bisnitroxides
known in the literature. Its performance is compared with a setup
at X-band frequencies. Being able to perform PELDOR in two
frequency bands makes it possible to identify hyperfine cou-
pling contributions. A disadvantage for PELDOR at S-band is
the ESEEM behavior at this low field, which allows only the use
of a few τ values for the detection sequence. The orientation
selectivity is not significantly different at S-band compared to
X-band, attributed to the small g anisotropy and the dominat-
ing AZZ(14N) hyperfine-coupling contribution of the nitroxides
used here. This may be different for PELDOR measurements on
systems containing metal ions, e.g., molybdenum or copper.
Furthermore we synthesized the biradicals 5••–8••. For
molecules 5•• and 6•• the experimental rAB match the theo-
10. S. S. Eaton and G. R. Eaton, Distance measurements by CW and pulsed
EPR, in “Biological Magnetic Resonance” (L. J. Berliner, S. S. Eaton, and
G. R. Eaton, Eds.), Vol. 19, p. 8, Kluwer Academic/Plenum, New York
(2000).
11. (a) A. D. Milov, K. M. Salikhov, and M. D. Shirov, Application of ELDOR
in electron-spin echo for paramagnetic center space distributions in solids,
Fiz. Tverd. Tela 23, 975 (1981); (b) A. D. Milov, A. B. Ponomarev, and
Yu. D. Tsvetkov, Electron electron-double resonance in electron-spin echo
model biradical systems and the sensitized photolysis of decalin, Chem.
Phys. Lett. 110, 67 (1984).
12. (a) M. Pannier, S. Veit, A. Godt, G. Jeschke, and H. W. Spiess, Dead-
time free measurement of dipole-dipole interactions between electron spins,
J. Magn. Reson. 142, 331 (2000); (b) R. E. Martin, M. Pannier, F. Diederich,
V. Gramlich, M. Hubrich, and H. W. Spiess, Determination of end-to-end
distances in a series of TEMPO diradicals of up to 2.8 nm length with a new
four-pulse double electron electron resonance experiment, Angew. Chem.
Int. Ed. 37, 2834 (1998).
retical ones very well. For biradical 7•• with an rAB of 16.4 A
˚
and a J of 73 MHz no PELDOR oscillation was observable,
13. G. Jeschke, M. Pannier, A. Godt, and H. W. Spiess, Dipolar spectroscopy
and spin alignment in electron paramagnetic resonance, Chem. Phys. Lett.
331, 243 (2000).
whereas the PELDOR oscillation was recovered for 5•• with an
∼
˚
even shorter rAB of 15.4 A but with J 0 MHz. For bisnitroxide
=
8•• it was possible to disentangle νDip from J and to ascertain the
magnitude and sign of J to +11 MHz (antiferromagnetic spin–
14. J. G. Powles and P. Mansfield, Phys. Lett. 2, 58 (1962).
15. V. V. Kurshev, A. M. Raitsimring, and Y. D. Tsvetkov, Selection of the
dipolarinteractionbythe“2 + 1”pulsetrain, J. Magn. Reson. 81, 441(1989).
˚
spin coupling). However, the experimental rAB of 24 A came out
longer than the theoretically predicted one of 21 A. This may
˚
16. S. Saxena and J. H. Freed, Double quantum two-dimensional Fourier trans-
form spin resonance: Distance measurements, Chem. Phys. Lett. 251, 102
(1996).
indicate that the lower distance limit for PELDOR is dictated
by the magnitude of J. Currently we are using PELDOR for
structure determination of doubly spin-labeled RNA.
17. A. Raitsimring, R. H. Crepeau, and J. H. Freed, Nuclear modulation effects
in “2 + 1” electron spin-echo correlation spectroscopy, J. Chem. Phys. 102,
8746 (1995).
ACKNOWLEDGMENTS
18. (a) A. V. Astashkin, J. H. Enemark, V. V. Kozlyuk, J. MacMaster, and A. M.
Raitsimring, Pulsed electron-electron double resonance of paramagnetic
centers with large g-value separation, in “41st Rocky Mountain Conference
on Analytical Chemistry,” p. 69 (1999); (b) J. H. Enemark, A. Astashkin,
The authors gratefully acknowledge the donation of biradicals 3•• and 4••
by G. Jeschke, MPI Mainz. The work was financially supported by the DFG

PELDOR AT S- AND X-BAND FREQUENCIES
285
R. Kedia, V. Kozliouk, J. McMaster, A. Pacheco, A. M. Raitsimring, and
M. Valek, Pulsed electron–electron double resonance (ELDOR) studies of
Mo(V)/Fe(III) centers, J. Inorg. Biochem. 74, 123 (1999).
25. M. Willer, J. Forrer, J. Keller, S. Van Doorslaer, and A. Schweiger, S-band
(2–4 GHz) pulse electron paramagnetic resonance spectrometer: Construc-
tion, probe head design, and performance, Rev. Sci. Instrum. 71, 2807
(2000).
19. A. D. Milov, A. G. Maryasov, and Y. D. Tsvetkov, Pulsed electron double
resonance (PELDOR) and its applications in free-radicals research, Appl.
Magn. Reson. 15, 107 (1998).
26. R. G. Larsen and D. J. Singel, Double electron–electron resonance spin-echo
modulation: Spectroscopic measurement of electron spin pair separations
in orientationally disordered solids, J. Chem. Phys. 98, 5134 (1993).
20. T. Ka´lai, M. Balog, J. Jeko¨, and K. Hideg, Synthesis and reactions of a
symmetric paramagnetic pyrrolidine diene, Synthesis 6, 973 (1999).
27. M. Pannier, V. Scha¨dler, M. Scho¨ps, U. Wiesner, G. Jeschke, and H. W.
Spiess, Determination of ion cluster sizes and cluster-to-cluster distances
in ionomers by four-pulse double electron electron resonance spectroscopy,
Macromolecules 33, 7812 (2000).
21. A. Godt, C. Franzen, S. Veit, V. Enkelmann, M. Pannier, and G. Jeschke,
EPR probes with well-defined, long distances between two or three unpaired
electrons, J. Org. Chem. 65, 7575 (2000).
28. The broad components of the tensor vanish in the dead time of the
3-pulse experiment. However, they were recovered by applying the 4-pulse
PELDOR sequence. Nevertheless, it turned out that it is difficult to obtain
22. S. Grimaldi, F. MacMillan, T. Ostermann, B. Ludwig, H. Michel, and T. F.
Prisner, QH ubisemiquinone radical in the bo3-Type ubiquinol oxidase stud-
ied by pulsed electron paramagnetic resonance and hyperfine sublevel cor-
relation spectroscopy, Biochemistry 40, 1037 (2001).
a precise frequency for the θ singularity from these spectra.
ꢁ
23. A. Weber, M. Rohrer, J. T. To¨rring, and T. F. Prisner, A CW and pulsed
S-band spectrometer, in “Proceedings of the 29th Congress AMPERE/13th
ISMAR” (D. Ziessow, W. Lubitz, and F. Lendzian, Eds.), Vol. 2, p. 1138
(1998).
29. (a) R. B. Clarkson, M. D. Timken, D. R. Brown, H. C. Crookham, and
R. L. Belford, Enhancement of nuclear modulation in electron spin echoes
at low magnetic fields: S-band ESE spectrometer, Chem. Phys. Lett. 163,
277 (1989). (b) J. H. Hankiewicz, C. Stenland, and L. Kevan, Pulsed S-band
electron spin resonance spectrometer, Rev. Sci. Instrum. 64, 2850 (1993).
24. G. Feher, Sensitivity considerations in microwave paramagnetic resonance
absorption techniques, Bell Syst. Tech. J. 36, 449 (1957).
30. O. Kahn, “Molecular Magnetism,” VCH, New York (1993).