Gd3+ complexes as potential spin labels for high field pulsed EPR distance measurements

Article abstract of DOI:10.1021/ja075544g

Pulse electron-electron double resonance distance measurements between two high spin Gd³⁺ ions in a novel bis-Gd³⁺ complex involving two pyridine-based gadolinium tetracarboxylate systems linked by a rigid aryl-alkyne unit were carri

Full text of DOI:10.1021/ja075544g

Published on Web 10/27/2007
Gd3+ Complexes as Potential Spin Labels for High Field Pulsed EPR Distance
Measurements
Arnold M. Raitsimring,† Chidambaram Gunanathan,‡ Alexey Potapov,§ Irena Efremenko,‡
Jan M. L. Martin,‡ David Milstein,*,‡ and Daniella Goldfarb*,§
Department of Chemistry, UniVersity of Arizona, Tucson, Arizona 85721, and Departments of Organic Chemistry
and Chemical Physics, Weizmann Institute of Science, RehoVot 76100, Israel
Received August 4, 2007; E-mail: daniella.goldfarb@weizmann.ac.il; david.milstein@weizmann.ac.il
Scheme 1. Synthesis of the Bis-Gd3+ Complexa
Distance measurements between spin labels by means of pulse
EPR have attracted considerable attention because of the applica-
tions to biological systems.1,2 These determine the dipolar interaction
1
between two electron spins, which for two S ) /2 spins is
g1g2â2µ0
4πhr3
ν
DD(θ,r) )
(3 cos2 θ - 1)
(1)
In eq 1, g1 and g2 are the g values, r is the interspin distance, and
θ is the angle between r and the external magnetic field, B0. The
accessible distance range, 1.5-6.0 nm, allows one to obtain
structural information on biomolecules such as proteins, DNA and
RNA, and their complexes.3,4 Two nitroxide spin labels (NSL) are
introduced in the biomolecule, and the measurements are usually
carried out at X-band frequencies (∼9.5 GHz) on samples of ∼50
µL of 0.1-0.2 mM solutions (∼12 h accumulation time). Distance
measurements, however, are not limited to organic radicals, and
measurements involving metal ions were reported as well.5-7
In principle, high field (HF)/high microwave frequency (HM-
WF)8,9 measurements can offer improvement in sensitivity over
X-band. However, due to the g anisotropy, the width of the NSL
EPR spectrum increases at HF, leading to a decrease in the spectral
density and of the flip probability of the pumped spins. Therefore,
excluding cases where the relative orientation of the g frames of
the NSLs is of prime interest, HF measurements provide only
moderate gain (if any).
i
a Conditions: (a) trans-PdCl2(PPh3)2, CuI, dry Pr2NH, rt, 40 h, 67%;
(b) CF3CO2H/CH2Cl2, rt, 12 h, 82%; (c) pH 5-7, GdCl3‚3H2O, rt, 5 h,
86%.
For HF distance measurements, a different class of spin labels,
the spectrum of which narrows with increasing B0, such as high
spin half integer systems (HSS) like Gd3+ (S ) 7/2) may be
attractive. While the width of the entire spectrum of these ions is
determined by the crystal field interaction (cfi), which is magnetic
field independent, the sub-spectrum of the central |-1/2 f | /2
1
Figure 1. (a) The geometry of the bis-Gd3+ complex with a total charge
of -2 and 14 unpaired electrons, optimized at the PBE0/sdd level of theory
with D2h symmetry constrain.11 (b) Two-pulse ED-EPR spectra of the bis-
Gd3+ complex recorded at Ka-band (∼1.5 mM, 13 K), W-band (0.2 mM,
10, 25 K). The positions of the pump and observed pulses are shown on
the figure.
transition (CT) narrows as B0 increases. Here we present first results
on the synthesis and pulse double electron-electron resonance
(DEER) measurements between two Gd3+ ions connected via a rigid
linker, shown in Scheme 1.
The bis-Gd3+ complex comprises two units of a pyridine-based
tetracarboxylate gadolinium complex, developed as an MRI contrast
agent.10 Double Sonogashira coupling of the pyridine tetracarboxy-
late ester 1 and 1,4-diethynylbenzene (2) provided the corresponding
octa-ester11 (Scheme 1). Deprotection of the octa-ester11 and
subsequent complexation of the octa-acid with excess of an aqueous
solution of GdCl3‚3H2O gave the desired bis-Gd3+ complex 5 in
86% yield. The paramagnetic complex 5 was characterized by IR,
MS (ESI), HRMS, and magnetic moment (p ) 15.63(2) µB)
analyses.11
The DEER measurements were carried out at Ka- (33.78 and
29.6 GHz) and W- (94.9 GHZ) bands.12,13 The corresponding echo-
detected (ED) EPR spectra are shown in Figure 1b. The width of
the W-band CT is about 0.4 of that at Ka-band, as expected.14
The broad background the CT is superimposed on is due to all
other transitions, and their relative intensities are a function of the
temperature and spectrometer frequency. The relative populations,
P(MS), of the MS ) (1/2 energy levels for the frequencies and
temperatures at which the experiments were carried out are listed
in Table 1. The four-pulse,15 time domain DEER traces, shown in
Figure 2a, exhibit a steep initial decay and shallow but clear
† University of Arizona.
‡ Department of Organic Chemistry, Weizmann Institute of Science.
§ Department of Chemical Physics, Weizmann Institute of Science.
9
14138
J. AM. CHEM. SOC. 2007, 129, 14138-14139
10.1021/ja075544g CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
Table 1. The Relative Populations of the MS ) (1/2 levels and the
DEER Modulation Depth, λef ) λ[P(1/2) + P(-1/2)]
1
) /2. This is a consequence of the selection of the CT which
excludes the detection of all pairs with MS ) (1/2 for one Gd3+
but |MS| > 1/2 for the other. Therefore, λef depends on λ, determined
from the sub-spectrum of the CT, and the populations of the
([1/2] states, P((1/2). The estimated λ11 and λef values are listed
in Table 1. The experimental values are nonetheless smaller by a
factor of about 2. This could be due to some distortion of the initial
part of the decay caused by the finite duration of the pumping pulse
and B1 inhomogeneity along the sample.
P(
−
1/2)
P(1/2)
λ
λef
33.78 GHz, 13 K
94.9 GHz, 10 K
94.9 GHz, 25 K
0.128
0.096
0.126
0.113
0.061
0.105
0.19
0.36
0.36
4.6%
5.6%
8.3%
To conclude, this work shows that Gd3+ complexes have a
potential of being used as spin labels for HF distance measurements
in biological systems, provided that suitable labeling techniques,
similar to those used for NSL, are developed. They feature high
sensitivity, requiring only 2 µL of 0.1 mM solutions, affording high
effective B1 values, allowing for very high repetition rates due to
short spin-lattice relaxation times (<300 µs), and have negligible
orientation selectivity. More work is certainly needed to optimize
the modulation depth and to understand the exact contribution of
the transitions other than the CT.
Acknowledgment. A.R. acknowledges the help of Dr. A.
Astashkin in Ka-band measurements and discussions. We thank
Y. Lipkin and Y. Gorodetsky for their work on the W-band bridge,
and B. Epel for his constant support of the SpecMan software. The
support of NIH (S10RR 020959) and NSF (DBI-9604939) for Ka-
band instrumentation development and of the Binational USA-
Israel Science Foundation (BSF 2002175 to A.R. and D.G.) are
appreciated. D.M. thanks the Israel Science Foundation for Financial
Support. D.G. holds the Erich Klieger Professorial chair in Chemical
Physics, D.M. is the Israel Matz Professor of Organic Chemistry.
and C.G. is the recipient of Deans of Faculties Postdoctoral
Fellowship.
Figure 2. (a) The four-pulse DEER traces of the bis-Gd3+ complex recorded
at (top) Ka-band, 13 K; data shown are after removal of the background
(observer pulse 15/25/25 ns, pump 12 ns, ∆ν ) 160 MHz; see Figure S1),
(middle) W-band, 10 K (observer pulse 20/40/40 ns, pump 16 ns, ∆ν ) 83
MHz, accumulation time 5 h), (bottom) W-band, 25 K (observer pulse 16/
32/32 ns, pump 16 ns, ∆ν ) 83 MHz, accumulation time 9 h). (b) Fourier
transform of the time domain traces after background removal. (c) The
corresponding distance distributions. Peak positions are indicated on the
figure.
modulations. This behavior is characteristic of a short distance with
some distribution. The corresponding magnitude real Fourier
transforms (FT) are shown in Figure 2b, and the distance distribu-
tions obtained using DeerAnalysis200616 are shown in Figure 2c.
All FT spectra show a peak at 6.0-6.4 MHz, which corresponds
to a distance of 2-2.05 nm. The distance was derived from eq 1,
neglecting exchange interaction and cfi effects on νDD. The latter
depends on ratio of the cfi parameter D and B0. For D ∼ 37 mT
(see simulations in Supporting Information (SI) and Figure S3),
the deviation of νDD from the nominal one was found minor even
at Ka-band.11,17 Density functional theory (DFT) calculations carried
out for bis-Gd3+ complex 5 (Figure 1a) gave r ) 2.213 nm.11
Calculations performed on the monocomplex (Figure S2) showed
that solvation and counterion effects should decrease r and increase
its distribution by relaxing the D2h symmetry constrain used, in
agreement with the experimental results.11
In addition to the clear peak at ∼6.2 MHz, all FT spectra show
low-frequency features, which we attribute primarily to artifacts
due to some uncertainty in the background removal. The shallow
modulation depth and the fast initial decay characteristic of short
distances make the shape of the FT spectrum highly susceptible to
small variations in background removal. The possibility that the
low-frequency features are also related to some intrinsic properties
of the HSS, however, cannot be excluded and needs further
investigation.
Supporting Information Available: Simulations of the Ka- and
W-band ED-EPR spectra and brief discussion of its effect of cfi on
ν
DD, description of the calculations of the modulation depth, detailed
synthesis and characterization of the bis-Gd3+ complex, and results of
DFT calculations. This material is available free of charge via the
Internet at http://pubs.acs.org.
References
(1) Jeschke, G.; Polyhach, Y. Phys. Chem. Chem. Phys. 2007, 9, 1895-1910.
(2) Borbat P. P.; Freed, J. H. In Biological Magnetic Resonance; Berliner, L.
J., Eaton, S. S., Eaton G. R., Eds.; Kluwer Academic/Plenum Publishers:
New York, 2000; Vol. 19, pp 383-456.
(3) Borbat, P.; Ramlall, T. F.; Freed, J. H.; Eliezer, D. J. Am. Chem. Soc.
2006, 128, 10004-10005.
(4) Schiemann, O.; Weber, A.; Edwards, T. E.; Prisner, T. F.; Sigurdsson, S.
T. J. Am. Chem. Soc. 2003, 125, 3434-3435.
(5) Codd, R.; Astashkin, A. V.; Pacheco, A.; Raitsimring, A. M.; Enemark,
J. H. J. Biol. Inorg. Chem. 2002, 7, 338-350.
(6) Elsasser, C.; Brecht, M.; Bittl, R. J. Am. Chem. Soc. 2002, 124, 12606-
12611.
(7) van Amsterdam, I. M. C.; Ubbink, M.; Canters, G. W.; Huber, M. Angew.
Chem. Int. Ed. 2003, 42, 62-64.
(8) Polyhach, Y.; Godt, A.; Bauer, C.; Jeschke, G. J. Magn. Reson. 2007,
185, 118-129.
(9) Denysenkov, V. P.; Prisner, T. F.; Stubbe, J.; Bennati, M. Proc. Natl.
Acad. Sci. U.S.A. 2006, 103, 13386-13390.
(10) Gunanathan, C.; Pais, A.; Furman-Haran, E.; Seger, D.; Eyal, E.;
Mukhopadhyay, S.; Ben-David, Y.; Leitus, G.; Cohen, H.; Vilan, A.;
Degani, H.; Milstein, D. Bioconjugate Chem. 2007, 18, 1361-1365.
(11) See Supporting Information.
(12) Astashkin, A. V.; Enemark, J.; Raitsimring, A. M. Concepts Magn. Reson.
B 2006, 29, 125-136.
(13) Gromov, I.; Krymov, V.; Manikandan, P.; Arieli, D.; Goldfarb, D. J. Magn.
Reson. 1999, 139, 8-17. A new W-band bridge was used; details are not
published yet.
Figure 2a shows that a deeper modulation and a narrower distance
distribution around 2 nm are observed at W-band, 25 K, where the
contributions of the background signal from the other transitions
are the smallest and the intensity of the central transition is the
highest. Hence, a higher frequency and a higher temperature are
desired while performing distance measurements on Gd3+ pairs.
Besides the modulation frequency, νDD, the DEER traces are
(14) Raitsimring, A. M.; Astashkin, A. V.; Poluektov, O.; Caravan, P. Appl.
Magn. Reson. 2005, 28, 281-295.
(15) Pannier, M.; Veit, S.; Godt, A.; Jeschke, G.; Spiess, H. W. J. Magn. Reson.
2000, 142, 331-340.
(16) Jeschke, G.; Chechik, V.; Ionita, P.; Godt, A.; Zimmermann, H.; Banham,
J.; Timmel, C. R.; Hilger, D. Appl. Magn. Reson. 2006, 3, 473-498.
(17) Astashkin, A. V.; Raitsimring, A. M. J. Chem. Phys, 2002, 117,
6121-6132.
1
characterized by modulation depth λ which for S ) /2 is the flip
probability of the “pumped” spins. The observed modulation depth,
λef, as seen from Figure 2 is smaller than would be expected for S
JA075544G
9
J. AM. CHEM. SOC. VOL. 129, NO. 46, 2007 14139