A Bis-Manganese(II)–DOTA Complex for Pulsed Dipolar Spectroscopy

Article abstract of DOI:10.1002/cphc.201600234

High-spin gadolinium(III) and manganese(II) complexes have emerged as alternatives to standard nitroxide radical spin labels for measuring nanometric distances by using pulsed electron–electron double resonance (PELDOR or DEER) at high fields/frequencies.

Full text of DOI:10.1002/cphc.201600234

DOI: 10.1002/cphc.201600234
Articles
A Bis-Manganese(II)–DOTA Complex for Pulsed Dipolar
Spectroscopy**
Paul Demay-Drouhard,[a] H. Y. Vincent Ching,[b] Dmitry Akhmetzyanov,[c] Rꢀgis Guillot,[d]
Leandro C. Tabares,[b] Hꢀlꢁne C. Bertrand,*[a] and Clotilde Policar*[a]
High-spin gadolinium(III) and manganese(II) complexes have
emerged as alternatives to standard nitroxide radical spin
labels for measuring nanometric distances by using pulsed
electron–electron double resonance (PELDOR or DEER) at high
fields/frequencies. For certain complexes, particularly those
with relatively small zero-field splitting (ZFS) and short distan-
ces between the two metal centers, the pseudosecular term of
the dipolar coupling Hamiltonian is non-negligible. However,
in general, the contribution from this term during conventional
data analysis is masked by the flexibility of the molecule of in-
terest and/or the long tethers connecting them to the spin
labels. The efficient synthesis of a model system consisting of
two [Mn(dota)]2ꢀ (MnDOTA; DOTA4ꢀ =1,4,7,10-tetraazacyclodo-
decane-1,4,7,10-tetraacetate) directly connected to the ends of
a central rodlike oligo(phenylene–ethynylene) (OPE) spacer is
reported. The rigidity of the OPE is confirmed by Q-band
PELDOR measurements on a bis-nitroxide analogue. The MnIIꢀ
MnII distance distribution profile determined by W-band
PELDOR is in reasonable agreement with one simulated by
using a simple rotamer analysis. The small degree of flexibility
arising from the linking MnDOTA arm appears to outweigh the
contribution from the pseudosecular term at this interspin dis-
tance. This study illustrates the potential of MnDOTA-based
spin labels for measuring fairly short nanometer distances, and
also presents an interesting candidate for in-depth studies of
pulsed dipolar spectroscopy methods on MnIIꢀMnII systems.
1. Introduction
Measuring nanometer-scale distances in biomacromolecules,
such as proteins or nucleic acids, is an efficient way to unravel
useful information about their structure and dynamics.[1–6] One
of the ways this can be achieved is by using pulsed electron
paramagnetic resonance (EPR) techniques, through which dis-
tances can be measured by determining the magnitude of
magnetic dipolar coupling between pairs of paramagnetic cen-
ters.[7–9] The most commonly used method is pulsed electron–
electron double resonance (PELDOR, also known as DEER).[10–13]
PELDOR measurements with nitroxide radicals (S=1/2), which
can be incorporated into biomacromolecules through site-di-
rected mutagenesis and spin labeling,[14,15] have been exten-
sively studied;[7–9] most measurements are performed at X
(9.5 GHz) and Q-band (34 GHz) frequencies.[16,17] Theoretically,
the sensitivity of the measurement can be further improved by
using higher fields/frequencies. However, the spectral width
for organic radicals increases with field as their g anisotropy
becomes resolved, which potentially provides information on
orientations,[18–20] but can also reduce the expected sensitivity
gain and complicate data analysis.[21] Furthermore, commonly
used nitroxide radicals, such as (S)-(1-oxyl-2,2,5,5-tetramethyl-
[a] Dr. P. Demay-Drouhard, Dr. H. C. Bertrand, Prof. C. Policar
Ecole Normale Supꢀrieure—PSL Research University
Dꢀpartement de Chimie, Sorbonne Universitꢀs—UPMC Univ Paris 06
CNRS UMR 7203 LBM, 24 rue Lhomond
75005 Paris (France)
E-mail: helene.bertrand@ens.fr
2,5-dihydro-1H-pyrrol-3-yl)methyl
methanesulfonothioate
clotilde.policar@ens.fr
(MTSL) and (2,2,6,6-tetramethyl piperidin-1-yl)oxyl (TEMPO) de-
rivatives, can be readily converted into EPR-silent N-hydroxyla-
mines in the reducing environment of a cell;[22] this compli-
cates PELDOR measurements in complex cellular environ-
ments.
[b] Dr. H. Y. V. Ching, Dr. L. C. Tabares
Institute for Integrative Biology of the Cell (I2BC)
Department of Biochemistry, Biophysics and Structural Biology
Universitꢀ Paris-Saclay, CEA, CNRS UMR 9198
Gif-sur-Yvette, F-91198 (France)
[c] D. Akhmetzyanov
In the last decade, high-spin gadolinium(III) (S=7/2) and
manganese(II) (S=5/2) complexes have emerged as attractive
alternatives.[23–28] At higher fields, the main feature in their EPR
spectra is the jꢀ1/2>!j+1/2> “central transition”. The spec-
tral width of this transition is proportional to D2/n0, in which D
is the size of the zero-field splitting (ZFS), assuming the asym-
metry parameter E is equal to zero, and n0 is the spectrometer
frequency. Consequently, unlike radicals, the spectral width of
the GdIII or MnII central transition becomes narrower and better
resolved as the observation frequency increases. Furthermore,
Goethe-University Frankfurt am Main
Institute of Physical and Theoretical Chemistry and
Center for Biomolecular Magnetic Resonance
Max von Laue Str. 7, 60438 Frankfurt am Main (Germany)
[d] Dr. R. Guillot
Institut de Chimie Molꢀculaire et des Matꢀriaux O’Orsay
Universitꢀ Paris-Sud, UMR CNRS 8182, Universitꢀ Paris-Saclay
91405 Orsay (France)
[**] DOTA4ꢀ =1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate
Supporting Information for this article can be found under: http://
dx.doi.org/10.1002/cphc.201600234.
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these metal centers are generally stable within biological envi-
ronments. GdIIIꢀGdIII PELDOR distance measurements at Q
(34 GHz) and W-band (95 GHz) frequencies have been per-
formed in vitro on synthetic model compounds,[29–31] pro-
teins,[32–35] peptides,[36,37] and DNA,[38] and also on proteins,[39]
peptides,[40] and DNA[41] inside cells. MnII centers, which have
received much less attention, are very promising in a biological
context because MnII is endogenous in biological environ-
ments and present at the active site of numerous enzymes.[42]
MnII can also replace MgII in other biomacromolecules due to
similarities in size and charge.[43] Unlike GdIII, the central transi-
tion of MnII centers is split into a sextet by the hyperfine inter-
action to the 55Mn nucleus (I=5/2), which reduces the sensitiv-
ity of MnIIꢀMnII PELDOR measurements by a factor of six if D
values are comparable.[26] However, this is partially compensat-
ed for by the lower spin multiplicity of MnII compared with
that of GdIII.[26] To date, only three MnIIꢀMnII PELDOR studies
have been reported and all were performed at the W-band fre-
quency.[26–28] In the first instance, the distance between two
[Mn(edta)]2ꢀ (MnEDTA; EDTA4ꢀ =ethylenediaminetetraacetate)
derivatives, grafted through a disulfide tether onto cysteine
residues of a protein fragment, was measured.[26] The dipolar
modulation depth (l), an important sensitivity parameter, was
found to be low (0.4%), and this was attributed to the large D
value (D=3000 MHz) of MnEDTA. Recently, Martorana et al. re-
ever, for MnDOTA, these effects were mostly obscured by the
intrinsic flexibility of the macromolecules and/or the tether to
the metal spin label.[27] In contrast, the pseudosecular term
contributions were expected to be much smaller for MnEDTA
and MnPEDTA due to their larger and more distributed D
values.[28]
Taken together, it appears that a compromise between ZFS
parameters, their distribution, and flexibility needs to be
reached for MnII spin labels. However, despite the shortcom-
ings of MnDOTA in specific cases, spin labels based on this
motif are still very promising for biological MnII PELDOR and
other pulsed dipolar EPR spectroscopic distance measurements
because of sensitivity and stability[46] considerations. To assess
the limitations of MnDOTA, in particular, at shorter and less
flexible distances, we present herein the preparation and char-
acterization of a water-soluble MnIIꢀMnII complex with an ex-
pected MnꢀMn distance of around 2.5 nm, in which the chelat-
ing DOTA ligands have been directly attached to the backbone
of a rigid oligo(phenylene–ethynylene) (OPE)[30,45,47–54] spacer
through amide bonds. A bis-nitroxide analogue was also syn-
thesized to evaluate the size and flexibility of the OPE spacer
in the bis-MnII complex. PELDOR measurements performed on
both systems are discussed.
ported the attachment of [Mn(pedta)]ꢀ derivatives (MnPEDTA; 2. Results
PEDTA3ꢀ =N-(pyrid-2-ylmethyl)ethylenediamine-N,N’,N’-triace-
2.1. Synthesis of the Target Compounds
tate) to cysteine residues of ubiquitin mutants through CꢀS
conjugation.[28] For two of the derivatives, the D values were
1860(900) and 3060(600) MHz; the values in parentheses
denote their distributions. As expected, in the corresponding
MnIIꢀMnII PELDOR measurements, l values (ꢁ0.7–1%), which
were comparable to or somewhat greater than that of
MnEDTA, were observed. However, in contrast to MnEDTA,
narrow distance distribution profiles, down to 0.6 nm full-
width at half-height (fwhh), were observed in the MnPEDTA
measurements; this was attributed to the short length and ri-
gidity of the tether connecting the spin label to the protein.[28]
Previously, we reported the grafting of two [Mn(dota)]2ꢀ
(MnDOTA; DOTA4ꢀ =1,4,7,10-tetraazacyclododecane-1,4,7,10-
tetraacetate; D=280(150) MHz) derivatives through a flexible
succinimidyl thioether tether onto a series on polyproline
spacers.[27] The PELDOR measurements gave higher l values
(1.2 to 2%) and the MnꢀMn distance distributions were in
good agreement with those obtained from molecular dynam-
ics simulations. However, under certain acquisition conditions,
additional features in the frequency-domain spectra became
apparent and they were attributed to contributions from the
pseudosecular term of the dipolar coupling Hamiltonian,
which was found to be non-negligible for MnDOTA.[27] Similar
observations have also been reported for GdIIIꢀGdIII PELDOR
measurements,[30] for which they were found to be more ap-
parent for shorter interspin distances (<3.4 nm). Thus, conven-
tional application of Tikhonov regularization, as implemented
in the DeerAnalysis toolbox,[44] which disregards the contribu-
tion from the pseudosecular term,[45] resulted in extra peaks in
and/or broadening of the distance distribution profiles.[30] How-
A convergent synthesis of symmetric OPE spacers containing
alternate phenyl rings with and without small polyethylene-
glycol (PEG) chains (diethyleneglycol methyl ether), which
were introduced to improve water solubility, was devised by
a double Sonogashira coupling between a para-substituted
ethynylbenzene and a central diiodinated building block
equipped with the PEG chains, which was synthesized in three
steps. Compound 2, prepared by tosylation of diethylene
glycol monomethyl ether 1,[55] was reacted with hydroquinone
in a double Williamson reaction to afford the intermediate 3 in
good yield.[56] Subsequent diiodination of 3 by using iodine in
conjunction with KIO3 in acetic acid afforded the diiodo build-
ing block 4 in satisfactory yield after recrystallization.[57] This
protocol has been scaled up and provides an easy access to
grams (30 g) of intermediate 4 (Scheme 1).
A double Sonogashira coupling between 4 and commercial-
ly available p-ethynylaniline (5), by using [Pd(PPh3)2Cl2]
(0.1 equiv) and CuI (0.2 equiv) in a mixture of triethylamine/
THF (1:1), afforded the dicoupled product 6 (OPE-diNH2) in
modest yield (52%; Scheme 2). To functionalize both extremi-
ties of this spacer with DOTA macrocycles, OPE-diNH2 6 was re-
acted with bromoacetyl bromide in the presence of K2CO3 to
afford bromide 7, which was subjected to double nucleophilic
substitution with tri-tBu-DO3A under classical conditions,[58–63]
providing the tBu-protected bis-DOTA compound 8 in good
yield after column chromatography (Scheme 2).
Removal of the tBu ester protecting groups of compound 8
was attempted under several conditions [1:1 trifluoroacetic
acid (TFA)/CH2Cl2 with or without scavengers,[59] HCO2H at
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the standard tBu-protected DOTA.[66] Tri-Pp-DO3A 11 was syn-
thesized in two steps from 2-phenyl-2-propanol 9 by treatment
with bromoacetic acid in the presence of N,N’-dicyclohexylcar-
bodiimide (DCC) and 4-dimethylaminopyridine (DMAP) to give
intermediate 10, followed by reaction with 1,4,7,10-tetraazacy-
clododecane (cyclen; 0.33 equiv). Despite the low yield due to
the concomitant formation of diversely alkylated cyclen deriva-
tives as side products, tri-Pp-DO3A 11 was easily purified and
obtained on a gram scale (Scheme 3).
The reaction between tri-Pp-DO3A 11 and bromide 7 under
the conditions described above provided the Pp-protected bis-
DOTA module 12, which was cleanly deprotected in 4 h with
a 2:2:96 TFA/TIS/CH2Cl2 cleavage cocktail, as monitored by re-
versed-phase HPLC (Figure S2 in the Supporting Information).
The bis-DOTA-OPE model system 13 was obtained in high
purity after purification by reversed-phase HPLC (Scheme 3).
Due to the presence of the small PEG chains and the DOTA
moieties, bis-DOTA-OPE 13 was very soluble in H2O. The corre-
sponding MnII complex (labeled MnDOTA2OPE) was generated
in situ by the addition of 1.8 equivalents of Mn(ClO4)2 to a buf-
fered solution [4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid (HEPES) 100 mm, pH 8] of 13 with 20% v/v glycerol.
The bis-TEMPO derivative 19 was also prepared by adopting
a similar overall strategy. Commercially available p-bromoben-
zaldehyde (14) was reacted with trimethylsilylacetylene (TMSA)
under Sonogashira coupling conditions to give intermediate
15 in good yield. The TMS group was then removed with
Scheme 1. Synthesis of the diiodo building block 4. TsCl=4-toluenesulfonyl
chloride.
608C,[64] 6m aqueous solution of HCl]; however, complex mix-
tures of unidentified products were obtained in each attempt
(see HPLC profiles in Figure S1 in the Supporting Information).
This deprotection step has been described to proceed smooth-
ly, even on related compounds,[60] although low yields have
sometimes been reported.[59] We surmised that the strong
acidic conditions needed for the removal of the tBu esters
from the DOTA moieties, which is known to be sluggish,[65,66]
led to the decomposition of compound 8.
We turned our attention to the use of the more acid-labile
phenylisopropyl (Pp) protecting group described by Mier
et al.,[67] which could be cleaved in the presence of 2% TFA in
CH2Cl2.[68] The Pp group has been successfully employed to
generate peptides that incorporate a DOTA core with a much
cleaner HPLC profile after cleavage from the resin than with
K2CO3 in MeOH to afford p-ethynylbenzaldehyde 16.[69]
A
double Sonogashira coupling between this compound and
building block 4 gave OPE-diCOH 17 in 80% yield; compound
17 was oxidized with Oxone to generate the corresponding di-
Scheme 2. Synthesis of the tBu-protected bis-DOTA 8.
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Scheme 3. Synthesis of the bis-DOTA-OPE 13. TIS=triisopropylsilane.
carboxylic acid 18. Amide-bond formation between OPE-
diCO2H 18 and commercially available 4-NH2-TEMPO in the
presence of DCC and 1-hydroxybenzotriazole (HOBt) gave bis-
TEMPO-OPE 19 in excellent yield (Scheme 4). Interestingly, the
yield obtained with this method was higher than those of stan-
dard coupling methods used to build systems incorporating
two nitroxide radicals connected to a central rigid rod through
ester bonds (usually in the region of 20%).[70]
Scheme 4. Synthesis of the bis-TEMPO-OPE 19.
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¯
2.2. X-ray Crystallography
ethyl acetate. Compound 17 crystallized in the P1 space group
(triclinic system). The aromatic rings were twisted relative to
each other with dihedral angles of 18 and 318 relative to the
central ring. The molecules were slightly bent (approximately
78), which reflected the nonideal unidirectional arrangement of
OPE spacers. No p stacking is observed, presumably because
of the steric hindrance imposed by the PEG chains (Figure 1A).
The length of the OPE spacer (the CꢀC distance between the
carbon atoms of the two aldehydes) was 1.93 nm.
Orange crystals of OPE-diCOH 17 suitable for XRD were ob-
tained by slow evaporation from a 1:1 mixture of chloroform/
Orange prisms of bis-TEMPO-OPE 19 were obtained by slow
evaporation from a solution of the biradical in toluene. Com-
pound 19 crystallized in the P21/n space group (monoclinic
system), with the three aromatic rings twisted by 398 from
each other. The distance between the two NꢀO bonds (point-
dipole approximation) was 3.02 nm (Figure 1B) and the length
of the OPE spacer (the distance between the two carbons of
the amide bonds) was 1.93 nm, which was identical to that in
OPE-diCOH 17 (Figure 1B).
2.3. EPR Measurements
The W-band Hahn echo-detected field-swept EPR spectrum of
MnDOTA2OPE in frozen glassy solution is depicted in Fig-
ure 2A. A narrow sextet corresponding to the central transition
superposing a broad component arising from the other transi-
tions was observed, and was essentially the same as that of
MnDOTA and MnDOTA-grafted polyprolines.[27] PELDOR meas-
urements were performed with the pump pulse frequency
(npump) resonant with the highest field hyperfine line and the
detection pulses frequency (ndetect) set 70 MHz lower (Fig-
Figure 1. CSD Mercury[71] ellipsoid views of the structure of OPEs A) com-
pound 17 and B) bis-TEMPO-OPE 19. Ellipsoids are drawn at the 30% proba-
bility level.
Figure 2. W-band PELDOR measurements on MnDOTA2OPE (100 mm) in HEPES (100 mm, pH 8) with 20% v/v glycerol at 10 K (black). A) Hahn echo-detected
field-swept EPR spectrum with the pump and detection positions indicated schematically by the down and up arrows, respectively. B) Raw PELDOR time trace
and background (dashed red). C) Background-corrected PELDOR time trace and its fit based on Tikhonov regularization (dashed red). D) Frequency-domain
spectra corresponding to the experimental background-corrected time trace and its fit based on Tikhonov regularization (dashed red). E) Distance distribution
profiles obtained by Tikhonov regularization and predicted by rotamer analysis (blue).
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ure 2B–E). These frequency positions were chosen because
previous studies suggested that the contribution from the
pseudosecular term of the dipolar coupling Hamiltonian was
least apparent when pumping at the hyperfine line and detect-
ing off to the side.[27] The first modulation was clearly visible in
the raw time trace and after background division the l value
was 1.9%, which was comparable to previous PELDOR meas-
urements with MnDOTA.[27] The corresponding frequency-
domain spectra showed a doublet with a splitting of 2.0 MHz.
Tikhonov regularization, as conventionally implemented in
DeerAnalysis,[44] gave a most probable distance of 2.6 nm. The
fwhh of the distribution was 0.6 nm, which was comparable to
sponding frequency domain spectra (Figure 3C), a large
3.6 MHz frequency component was observed. In contrast, for
the other time traces, the main frequency component was at
about 1.85 MHz. The orientation selection effects can be quali-
tatively rationalized in terms of the geometry of the biradical
from its X-ray crystal structure. The g tensor of the nitroxide
moieties is oriented with the gxx component collinear to the
mean axis of the molecule. For the orange and green time
traces, the detection pulses sampled significant amounts of
the gxx component, such that the parallel orientation of the di-
polar coupling tensor was excited to a high extent, which re-
sulted in oscillations that were doubled in frequency (3.6 MHz)
the narrowest distance distribution profile observed in MnIIꢀ relative to the major component of the other time traces. In
MnII PELDOR measurements.[28]
the green time trace, increased contributions from the gyy and
gzz components meant that a single frequency (1.85 MHz) com-
ponent was also observed. For all other time traces, the gxx
component only had a minor contribution to the signal, which
resulted in time traces that lacked the parallel orientation of
the dipolar coupling tensor. As expected, orientation selection
was also observed at W-band frequencies (see Figure S4 in the
Supporting Information).
The Q-band Hahn echo-detected field-swept EPR spectrum
of bis-TEMPO-OPE 19 is depicted in Figure 3A. PELDOR time
traces (Figure 3B) were recorded across the whole EPR spec-
trum by using a constant ndetectꢀnpump offset of 50 MHz, with
the exception of the magenta time trace for which an offset of
ꢀ50 MHz was used. In the time traces, up to 7 full periods of
a dampened dipolar oscillation were observed with modula-
tion depths of up to 24%. Differences between the traces
clearly indicate orientation selection.[19–21,72–76] For the orange
and green time traces, the detection pulses were resonant
with the lower field edge of the EPR spectrum, in which the gxx
component of the g tensor had a relatively large contribution
to the excited orientations. In these two traces and their corre-
The orientation selection effects were minimized by sum-
ming the Q-band PELDOR time traces (Figure 3D–F), which, in
effect, provided a more uniform excitation of the dipolar cou-
pling tensor orientations. The corresponding frequency
domain spectrum resembled a complete Pake pattern. The
summed time trace was analyzed by means of Tikhonov regu-
Figure 3. Q-band PELDOR measurements on bis-TEMPO-OPE 19 (100 mm) in 1:1 v/v [D8]toluene/CDCl3 at 50 K. A) Hahn echo-detected field-swept EPR spec-
trum with different pump and detection PELDOR positions indicated schematically in different colors by the down and up arrows, respectively. B) Raw
PELDOR time traces recorded at different positions of the EPR spectrum, with colors corresponding to the arrows depicted in A). C) Fourier transform of the
background-corrected time traces. D) The summed PELDOR time trace (black) after background correction and its fit based on Tikhonov regularization
(dashed red), as implemented in DeerAnalysis.[40] The black arrow indicates a deviation due to imperfections of averaging of the orientation selection (dis-
cussed in detail in the text). E) Frequency-domain spectra corresponding to the background-corrected summed time trace (black) and its fit based on Tikho-
nov regularization (dashed red). F) Distance distribution profile obtained by Tikhonov regularization analysis of the summed PELDOR time trace and predicted
by rotamer analysis (blue). The black asterisk indicates an artefact due to imperfection of averaging of orientation selection (details described in the text).
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larization (Figure 3D–F). Small differences can be seen be-
tween the summed time trace (black) and the fit based on Ti-
khonov regularization (red), for example, at the first minimum
of the dipolar oscillations, indicated with a black arrow in Fig-
ure 3D. This indicates that orientation selection is still present
in the averaged time trace that gives rise to the small peak
seen at 2.4 nm in Figure 3F. However, such imperfections are
not expected to cause a significant error in the position of the
main peak and the distance distribution. The most probable
distance observed from the Tikhonov fit of the summed trace
was 2.98 nm with a fwhh of 0.1 nm and width at 10% height
of 0.2 nm.
tributed to bending of the OPE spacer and rotations of the
single bonds of the molecule. This profile was in agreement
with the prediction that had a sampling inaccuracy of 0.1 nm,
but did not take into account bending of the molecule.
Compared with the bis-TEMPO-OPE results, the MnꢀMn dis-
tance obtained in W-band PELDOR measurements on Mn-DO-
TA2OPE were around six times more distributed. The bulk of
this broadening was unlikely to have arisen from the bending
of the OPE, but rather was most likely to be due to the inher-
ent flexibility of the MnDOTA spin label. Indeed, the MnꢀMn
distance distribution predicted by using a rotamer analysis was
remarkably in reasonable agreement with the experimental
data. Compared with PELDOR results, the predicted distance
distribution profile was slightly shifted to longer distances; this
was possibly due to sampling inaccuracy and/or inaccuracy of
the method of modeling, such as 1) rotamer analysis did not
take into account the small bending flexibility of the OPE
spacer, which would give slightly shorter MnꢀMn distances;
and 2) a dynamic coordination process in which the amide O
atom coordinates to the MnII center,[80] which would favor
shorter MnꢀMn distances. Alternatively, the differences be-
tween the experimental and predicted distance distribution
profiles might also have originated from experimental data
analysis, which did not take into account the pseudosecular
term of the dipolar coupling Hamiltonian. For specific cases, in
previous MnDOTA polyproline studies, this approach resulted
in extra features at shorter distances.[27] The small feature at
around 3.3 nm in the distance distribution profile, obtained in
the PELDOR experiment, is possibly due to uncertainty in de-
termining the background. Nevertheless, overall, the MnDO-
TA2OPE PELDOR results closely matched expectations, which
suggested that MnDOTA spin labels could be used for measur-
ing nanometric distances with relatively narrow distributions
predictably, even at distances as short as 2.6 nm. Compared
with the OPE spacer alone, each MnDOTA spin label only con-
tributed around 0.3 nm to the distance and 0.3 nm to the dis-
tribution at fwhh. Although relatively small, the size and flexi-
bility of the tethering DOTA arm appeared to outweigh the
contribution from the pseudosecular term of the dipolar cou-
pling Hamiltonian during Tikhonov regularization analysis.
These advantages can potentially be further exploited by affix-
ing MnDOTA to the biomolecule of interest in a more con-
strained manner, for example, by using 2,2’,2’’-(10-{2-[(2,5-diox-
opyrrolidin-1-yl)oxy]-2-oxoethyl}-1,4,7,10-tetraazacyclodode-
cane-1,4,7-triyl)triacetic acid (DOTA-NHS ester), the protein
backbone can be directly labeled at the N terminus,[81] or by
using a derivative with short thiol-reactive tethers, similar to
the GdIII complexes very recently reported by Abdelkader
et al.[79]
2.4. Simulations of Distance Distribution Profiles
The MnꢀMn distance was simulated by using a rotamer analy-
sis that accounted for the inherent flexibility of spin labels.
This approach has been used to adequately predict distance
distributions obtained from PELDOR measurements.[27,40,77–79]
Starting from known X-ray crystal structures, we constructed
a model of MnDOTA2OPE (Figure 4). Subject to certain con-
Figure 4. Schematic structure of MnDOTA2OPE. The relevant dihedral angles,
c, of rotatable bonds of each MnDOTA are labeled. Hydrogen atoms have
been omitted for clarity.
straints (see the Experimental Section), all possible rotamers
that avoided clashes were generated and each was given the
same probability. The predicted most probable MnꢀMn dis-
tance was 2.7 nm with a distribution of 0.5 nm at fwhh (Fig-
ure 2E). Similarly, starting from the X-ray structure of bis-
TEMPO-OPE, its rotamers were generated and each was given
the same probability. The predicted most probable spin–spin
distance was 3.0 nm with a distribution of 0.1 nm at fwhh (Fig-
ure 3F).
3. Discussion
The distance determined in the Q-band PELDOR measure-
ments on bis-TEMPO-OPE 19 was in good agreement with that
obtained from X-ray crystallography by using the point-dipole
approximation and rotamer analysis. The strong orientation se-
lection effect observed in the PELDOR time traces at these fre-
quencies highlighted the rigidity of the molecule (and corre-
spondingly of the OPE spacer). The determined width of the
distance distribution profile was very narrow and could be at-
4. Conclusions
A model system incorporating two DOTA ligands on a short
OPE spacer was designed, synthesized, and fully characterized.
PELDOR measurements were performed on the corresponding
MnII complex and on a bis-nitroxide analogue. The experimen-
tal MnꢀMn distance distribution obtained by Tikhonov regula-
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1
a colorless oil. H NMR (CDCl3, 300 MHz): d=7.80 (d, 2H, J=8.0 Hz),
7.34 (d, 2H, J=8.0 Hz), 4.17 (t, 2H, J=4.8 Hz), 3.69 (t, 2H, J=
4.8 Hz), 3.61–3.54 (m, 2H), 3.51–3.44 (m, 2H), 3.35 (s, 3H), 2.44 ppm
(s, 3H); 13C NMR (CDCl3, 75 MHz): d=144.9 (Cq), 133.0 (Cq), 129.9
(CH), 128.1 (CH), 71.9 (CH2), 70.8 (CH2), 69.3 (CH2), 68.8 (CH2), 59.2
(CH3), 21.8 ppm (CH3); HRMS (ESI): m/z calcd for C12H19O5S+ [M+
H]+, C12H18NaO5S+ [M+Na]+: 275.0948, 297.0766; found: 275.0950,
297.0767.
rization was in good agreement with that simulated by using
a simple rotamer analysis. However, there were small devia-
tions, which might have arisen from a combination of short-
comings in the modeling and data analysis. Overall, our results
suggested that PELDOR measurements with MnDOTA spin
labels directly grafted on an object of interest could be used
to determine nanometer distances with relatively narrow distri-
butions predictably. Currently, we are studying the potential
use of other MnDOTA derivatives as spin labels for PELDOR, as
well as other dipolar EPR spectroscopic methods for MnIIꢀMnII
nanometer distance measurements, and these results will be
reported in due course.
1,4-Bis[2-(2-methoxyethoxy)ethoxy]benzene (3)
Hydroquinone (14.64 g, 133.1 mmol, 1.0 equiv) and K2CO3 (73.47 g,
532.4 mmol, 4.0 equiv) were suspended in MeCN (1.1 L). The result-
ing suspension was heated at reflux for 30 min and cooled to RT.
Compound 2 (72.95 g, 266.2 mmol, 2.0 equiv) dissolved in MeCN
(220 mL) was added dropwise. The resulting mixture was heated at
reflux for 72 h, cooled to RT, filtered, and concentrated. Column
chromatography (SiO2, cyclohexane/EtOAc 90:10 to 50:50) afforded
3 (32.81 g, 104.5 mmol, 79%) as an orange oil. 1H NMR (CDCl3,
300 MHz): d=6.83 (s, 4H), 4.11–4.05 (m, 4H), 3.86–3.80 (m, 4H),
3.74–3.68 (m, 4H), 3.60–3.54 (m, 4H), 3.39 ppm (s, 6H); 13C NMR
(CDCl3, 75 MHz): d=153.2 (Cq), 115.6 (CH), 72.1 (CH2), 70.9 (CH2),
70.0 (CH2), 68.1 (CH2), 59.2 ppm (CH3); HRMS (ESI): m/z calcd for
Experimental Section
Material and methods
1H and 13C NMR spectra were recorded on Bruker Avance 300 or
600 (300 or 400 MHz) spectrometers at the Ecole Normale Supꢀr-
ieure (ENS) in the Laboratoire des Biomolꢀcules (LBM, UMR 7203)
or at UPMC (Universitꢀ Pierre et Marie Curie) in IPCM (Institut Pari-
sien de Chimie Molꢀculaire, UMR 7201), with residual solvent sig-
nals as internal references. The following abbreviations were used:
singlet (s), doublet (d), triplet (t), multiplet (m) and broad (br); d in-
dicates chemical shifts in ppm, J are coupling constants in Hz, and
Cq are quaternary carbons. HRMS by using electrospray ionization
(ESI) or atmospheric pressure chemical ionization (APCI) methods
was performed at the Universitꢀ Paris Sud in the Service de Spec-
tromꢀtrie de masse of the ICMMO (Institut de Chimie Molꢀculaire
et des Matꢀriaux d’Orsay). ESI-HRMS was also performed at UPMC
(IPCM). MALDI-TOF MS was performed at UPMC in the Plate-forme
de Spectromꢀtrie de masse et Protꢀomique. The matrix was a satu-
rated solution of a-cyano-4-hydroxycinnamic acid (HCCA) in
MeCN/H2O 1:1 with 0.1% TFA. Analytical TLC analysis was per-
formed on silica gel plates (Merck 60F-254) with UV visualization at
l=254 and 366 nm. Preparative column chromatography was per-
formed with Merck silica gel (Si 60, 40–63 mm). Analytical HPLC
measurements were performed on a Dionex Ultimate 3000 instru-
ment by using C18A ACE columns. Preparative HPLC was per-
formed on a Waters 600 instrument by using an XBridgeTM Prep
C18 OBDTM column. Gradients of MeCN in H2O, both containing
0.1% TFA, were employed. Products were monitored with UV de-
tection. Unless otherwise stated, all syntheses were performed
under an inert atmosphere (argon or nitrogen). Reagents and
chemicals were purchased from Sigma–Aldrich, Alfa Aesar, Strem
Chemicals, or Chematech. Dry solvents (CH2Cl2, MeCN, toluene,
THF, dioxane, DMF, DMSO) were purchased from Sigma and used
without further purification. EtOAc and triethylamine were dried
with CaH2, distilled under argon, and stored over 4 ꢃ molecular
sieves under argon.
C16H27O6 [M+H]+ , C16H26NaO6 [M+Na]+; 315.1802, 337.1622;
found: 315.1796, 337.1617.
+
+
1,4-Diiodo-2,5-bis[2-(2-methoxyethoxy)ethoxy]benzene (4)
Compound
3 (25.0 g, 79.6 mmol, 1.0 equiv), iodine (22.3 g,
87.6 mmol, 1.1 equiv), and KIO3 (6.8 g, 31.8 mmol, 0.4 equiv) were
dissolved in glacial AcOH (250 mL) and H2O (25 mL). Concentrated
H2SO4 (3.3 mL) was added, the resulting mixture was heated at
reflux for 48 h and cooled to RT. CH2Cl2 and a 10% aqueous solu-
tion of Na2S2O3 were added, the organic layer was recovered and
washed with a saturated aqueous solution of NaHCO3 (1ꢄ), H2O
(1ꢄ), and a saturated aqueous solution of NaCl (1ꢄ); dried over
Na2SO4; filtered; and concentrated. Recrystallization from EtOH af-
forded 4 (27.27 g, 48.1 mmol, 60%) as a white solid. 1H NMR
(CDCl3, 300 MHz): d=7.23 (s, 2H), 4.14–4.07 (m, 4H), 3.92–3.86 (m,
4H), 3.80–3.74 (m, 4H), 3.61–3.55 (m, 4H), 3.40 ppm (s, 6H);
13C NMR (CDCl3, 75 MHz): d=153.2 (Cq), 123.6 (CH), 86.5 (Cq), 72.2
(CH2), 71.2 (CH2), 70.5 (CH2), 69.8 (CH2), 59.3 ppm (CH3); HRMS (ESI):
+
+
m/z calcd for C16H25I2O6
566.9735, 588.9554; found: 566.9707, 588.9544.
,
[M+H]+, C16H24I2NaO6 [M+Na]+:
4,4’-({2,5-Bis[2-(2-methoxyethoxy)ethoxy]-1,4-phenylene}-
bis(ethyne-2,1-diyl))dianiline (OPE-NH2 6)
Compounds
4 (2.78 g, 5.0 mmol, 1.0 equiv) and 5 (1.17 g,
10.0 mmol, 2.0 equiv) were dissolved in dry THF (20 mL) and dry
triethylamine (20 mL). Argon was bubbled for 15 min and
[Pd(PPh3)2Cl2] (351.0 mg, 0.5 mmol, 0.1 equiv) and CuI (191.0 mg,
1.0 mmol, 0.2 equiv) were added. The resulting suspension was
stirred for 24 h at RT; a saturated aqueous solution of NH4Cl was
added and the mixture was extracted with CH2Cl2 (3ꢄ). The com-
bined organic layers were dried over Na2SO4, filtered, and concen-
trated. Column chromatography (SiO2, 50:50 cyclohexane/EtOAc to
100% EtOAc) afforded 6 (1.41 g, 2.59 mmol, 52%) as an orange
2-(2-Methoxyethoxy)ethyl 4-methylbenzenesulfonate (2)
Compound 1 (39.1 mL, 332.7 mmol, 1.0 equiv) was dissolved in
THF (110 mL). The resulting solution was cooled to 08C and NaOH
(26.44 g, 661.0 mmol, 2.0 equiv) dissolved in H2O (110 mL) was
added dropwise, followed by TsCl (95.16 g, 499.0 mmol, 1.5 equiv)
dissolved in THF (110 mL) dropwise. The resulting mixture was
stirred at RT for 2 h and H2O (400 mL) was added. The organic
layer was recovered and washed without shaking with a 1m aque-
ous solution of NaOH (2ꢄ) and H2O (1ꢄ), dried over Na2SO4, fil-
tered, and concentrated to afford 2 (72.95 g, 266.2 mmol, 80%) as
1
solid. H NMR (CDCl3, 300 MHz): d=7.32 (d, J=8.4 Hz, 4H), 6.99 (s,
2H), 6.63 (d, J=8.4 Hz, 4H), 4.20 (t, J=5.0 Hz, 4H), 3.92 (t, J=
5.0 Hz, 4H), 3.86–3.77 (m, 8H), 3.57–3.50 (m, 4H), 3.36 ppm (s, 6H);
13C NMR (CDCl3, 75 MHz): d=153.5 (Cq), 146.9 (Cq), 133.1 (CH), 117.5
&
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(CH), 114.9 (CH), 114.5 (Cq), 112.9 (Cq), 95.1 (Cq), 84.0 (Cq), 72.2 (CH2),
was cooled to 08C. DCC (15.2 g, 74 mmol, 1.03 equiv) was added
portionwise and the resulting suspension was allowed to warm to
RT for 3 h, filtered, and washed with CH2Cl2. The filtrate was
washed with a 0.5m aqueous solution of HCl (2ꢄ) and a saturated
aqueous solution of NaHCO3 (1ꢄ). The organic layer was dried over
Na2SO4, filtered, and concentrated. Column chromatography (SiO2,
95:5 cyclohexane/EtOAc) afforded 10 (10.82 g, 42.1 mmol, 58%) as
71.2 (CH2), 69.9 (CH2), 69.9 (CH2), 59.2 ppm (CH3); HRMS (ESI): m/z
+
+
calcd for C32H37N2O6
545.2646, 567.2466; found: 545.2609, 567.2443.
[M+H]+, C32H36N2NaO6
[M+Na]+:
N,N’-[({2,5-Bis[2-(2-methoxyethoxy)ethoxy]-1,4-phenylene}-
bis(ethyne-2,1-diyl))bis(4,1-phenylene)]bis(2-bromoacet-
amide) (7)
1
a yellow oil. H NMR (CDCl3, 300 MHz): d=7.47–7.24 (m, 5H), 3.80
(s, 2H), 1.84 ppm (s, 6H); 13C NMR (CDCl3, 100 MHz): d=165.1 (Cq),
144.6 (Cq), 128.1 (CH), 127.0 (CH), 123.9 (CH), 83.4 (Cq), 28.0 (CH3),
OPE-NH2 6 (798 mg, 1.465 mmol, 1.0 equiv) was dissolved in dry
CH2Cl2 (20 mL). The resulting solution was cooled to 08C, and trie-
thylamine (496 mL, 3.66 mmol, 2.5 equiv) was added, followed by
the dropwise addition of bromoacetyl bromide (319 mL, 3.66 mmol,
2.5 equiv) dissolved in dry CH2Cl2 (3 mL). The resulting mixture was
allowed to warm to RT for 3 h and washed with a saturated aque-
ous solution of NaHCO3 (1ꢄ) and a saturated aqueous solution of
NaCl (1ꢄ). The organic layer was dried over Na2SO4, filtered, and
concentrated. Compound 7 (459 mg, 0.584 mmol, 40%) was ob-
tained after column chromatography (SiO2, 40:60 to 20:80 cyclo-
hexane/EtOAc) as a pale yellow solid. 1H NMR (CDCl3, 300 MHz):
d=8.17 (s, 2H), 7.59–7.48 (m, 8H), 7.03 (s, 2H), 4.22 (t, J=4.8 Hz,
4H), 4.04 (s, 4H), 3.93 (t, J=4.8 Hz, 4H), 3.83–3.78 (m, 4H), 3.57–
3.52 (m, 4H), 3.37 ppm (s, 6H); 13C NMR (CDCl3, 75 MHz): d=163.4
(Cq), 153.7 (Cq), 137.1 (Cq), 132.6 (CH), 120.1 (Cq), 119.7 (CH), 117.6
(CH), 114.3 (Cq), 94.7 (Cq), 86.0 (Cq), 72.2 (CH2), 71.2 (CH2), 69.9 (CH2),
69.8 (CH2), 59.2 (CH3), 29.6 ppm (CH2); HRMS (ESI): m/z calcd for
+
27.0 ppm (CH2); HRMS (ESI): m/z calcd for C11H13BrNaO2 [M+Na]+:
278.9991; found: 278.9992.
Tris(2-phenylpropan-2-yl) 2,2’,2’’-(1,4,7,10-Tetraazacyclodo-
decane-1,4,7-triyl)triacetate (tri-Pp-DO3A 11)
Cyclen (2.41 g, 14 mmol, 1.0 equiv) was dissolved in MeCN
(150 mL). The resulting solution was cooled to 08C and NaHCO3
(3.53 g, 42 mmol, 3.0 equiv) was added. Compound 10 (10.8 g,
42 mmol, 3.0 equiv) dissolved in MeCN (40 mL) was added drop-
wise (ꢁ30 min). The resulting suspension was allowed to warm to
RT for 15 h and concentrated. CH2Cl2 was added and the mixture
was washed with H2O (1ꢄ) and a saturated aqueous solution of
NaCl (1ꢄ). The organic layer was dried over Na2SO4, filtered, and
concentrated. Column chromatography (SiO2, 98:2 to 95:5 CH2Cl2/
MeOH) afforded 11 (2.21 g, 3.15 mmol, 24%) as a light brown
foam. 1H NMR (CDCl3, 300 MHz): d=9.82 (s, 1H), 7.37–7.20 (m,
15H), 3.40–3.35 (m, 4H), 3.31 (s, 2H), 3.06–2.97 (m, 4H), 2.85–2.72
(m, 12H), 1.78 (s, 12H), 1.77 ppm (s, 6H); 13C NMR (CDCl3,
100 MHz): d=170.3 (Cq), 169.3 (Cq), 145.4 (Cq), 145.2 (Cq), 128.5
(CH), 127.5 (CH), 127.4 (CH), 124.4 (CH), 124.4 (CH), 82.9 (Cq), 82.8
(Cq), 57.9 (CH2), 52.1 (CH2), 51.8 (CH2), 49.7 (CH2), 49.3 (CH2), 47.3
(CH2), 28.8 (CH3), 28.8 ppm (CH3); HRMS (ESI): m/z calcd for
C36H38Br2N2NaO8 [M+Na]+: 809.0867; found: 809.0859.
+
General Procedure A: Synthesis of Protected Bis-DOTA Com-
pounds
Bromide 7 (1.0 equiv) and a DO3A derivative (tri-tBu-DO3A or tri-
Pp-DO3A 11) (2.5 equiv) were mixed in dry MeCN. K2CO3
(10.0 equiv) was added and the resulting mixture was heated at
608C for 15 h and concentrated. CH2Cl2 was added and the mixture
was washed with H2O (1ꢄ) and a saturated aqueous solution of
NaCl (1ꢄ). The organic layer was dried over Na2SO4, filtered, and
concentrated. Column chromatography afforded the correspond-
ing protected bis-DOTA-OPE compound as a yellow solid.
+
+
C41H57N4O6
[M+H]+, C41H56N4NaO6
[M+Na]+: 701.4273,
723.4092; found: 701.4315, 723.4096.
Pp-Protected Bis-DOTA-OPE (12)
By using general procedure A with 7 (71 mg, 0.09 mmol), 11
(161 mg, 0.23 mmol), and K2CO3 (124 mg, 0.9 mmol) in dry MeCN
(8 mL), Pp-protected bis-DOTA-OPE 12 (99 mg, 0.049 mmol, 54%)
was obtained after column chromatography (SiO2, 95:5 to 85:15
tBu-Protected Bis-DOTA-OPE (8)
1
CH2Cl2/MeOH). H NMR (CDCl3, 300 MHz): d=10.95 (s, 2H), 7.91 (d,
By using general procedure A with 7 (55 mg, 0.07 mmol), tri-tBu-
DO3A (100 mg, 0.174 mmol), and K2CO3 (96 mg, 0.7 mmol) in dry
MeCN (10 mL), tBu-protected bis-DOTA-OPE 8 (96 mg, 0.058 mmol,
83%) was obtained after column chromatography (SiO2, 90:10 to
80:20 CH2Cl2/MeOH). 1H NMR (CDCl3, 300 MHz): d=11.49 (s, 2H),
7.97 (d, J=8.5 Hz, 4H), 7.34 (d, J=8.5 Hz, 4H), 7.01 (s, 2H), 4.19 (t,
J=5.0 Hz, 4H), 3.91 (t, J=5.0 Hz, 4H), 3.83–3.78 (m, 4H), 3.75 (s,
4H), 3.55–3.50 (m, 4H), 3.34 (s, 6H), 3.30–1.85 (m, 44H), 1.48 (s,
18H), 1.43 ppm (s, 36H); 13C NMR (CDCl3, 75 MHz): d=172.34 (Cq),
171.6 (Cq), 153.6 (Cq), 140.1 (Cq), 131.8 (CH), 120.2 (CH), 117.6 (Cq),
114.5 (Cq), 95.9 (Cq), 84.7 (Cq), 82.4 (Cq), 82.2 (Cq), 72.2 (CH2), 71.2
(CH2), 69.9 (CH2), 69.9 (CH2), 59.1 (CH3), 57.0 (CH2), 55.9 (CH2), 55.8
(CH2), 51.3 (CH2), 28.2 (CH3), 28.1 ppm (CH3); HRMS (ESI): m/z calcd
J=8.4 Hz, 4H), 7.39 (d, J=8.4 Hz, 4H), 7.34–7.08 (m, 30H), 7.04 (s,
2H), 4.21 (t, J=4.9 Hz, 4H), 3.92 (t, J=4.9 Hz, 4H), 3.84–3.76 (m,
4H), 3.56–3.49 (m, 4H), 3.34 (s, 6H), 3.25–1.95 (m, 44H), 1.62 ppm
(s, 36H); 13C NMR (CDCl3, 100 MHz): d=171.6 (Cq), 171.0 (Cq), 153.6
(Cq), 145.7 (Cq), 140.1 (Cq), 131.8 (CH), 128.4 (CH), 127.2 (CH), 124.2
(CH), 124.1 (CH), 120.1 (CH), 117.6 (CH), 117.4 (Cq), 114.5 (Cq), 95.9
(Cq), 84.8 (Cq), 83.4 (Cq), 83.3 (Cq), 72.2 (CH2), 71.2 (CH2), 69.9 (CH2),
59.1 (CH3), 56.6 (CH2), 55.8 (CH2), 55.7 (CH2), 54–50.5 (CH2, broad
cluster), 50–47.5 (CH2, broad cluster), 33–24 ppm (CH3, broad clus-
ter); HRMS (ESI): m/z calcd for C118H148N10Na2O202+/2 [M+2Na]2+
,
C118H149N10NaO202+/2 [M+H+Na]2+: 1035.5328, 1025.0435; found:
1035.5329, 1025.0453.
for C88H136N10NaO20 [M+Na]+: 1675.9825; found: 1675.9832.
+
Bis-DOTA-OPE (13)
2-Phenylpropan-2-yl 2-Bromoacetate (10)
Pp-protected bis-DOTA-OPE 12 was dissolved in a mixture of TFA/
TIS/CH2Cl2 (2:2:96, 10 mgmLꢀ1). The resulting solution was stirred
at RT for 4 h and concentrated. MeOH was added and the product
was precipitated by slow addition of Et2O, filtered, and dried. The
Bromoacetic acid (10.0 g, 72 mmol, 1.0 equiv) and
109 mmol, 1.5 equiv) were dissolved in CH2Cl2 (100 mL). DMAP
(880 mg, 7.2 mmol, 0.1 equiv) was added and the resulting mixture
9 (14.8 g,
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crude product was purified by preparative HPLC (30 to 50%
MeCN) to afford bis-DOTA-OPE 13 as a yellow solid. HPLC: 5.02 min
(5 to 100% MeCN in 10 min, >98%); MALDI-TOF MS (HCCA): m/z
(m, 4H), 7.72–7.64 (m, 4H), 7.08 (s, 2H), 4.27- 4.21 (m, 4H), 3.98–
3.92 (m, 4H), 3.83–3.77 (m, 4H), 3.58–3.53 (m, 4H), 3.37 ppm (s,
6H); 13C NMR (CDCl3, 75 MHz): d=191.4 (CH), 153.9 (Cq), 135.6 (Cq),
132.2 (CH), 129.7 (CH), 129.6 (Cq), 117.5 (CH), 114.3 (Cq), 94.5 (Cq),
89.9 (Cq), 72.1 (CH2), 71.2 (CH2), 69.9 (CH2), 69.7 (CH2), 59.2 ppm
calcd for C64H89N10O20 [M+H]+: 1317.62; found: 1317.43.
+
(CH3); HRMS (ESI): m/z calcd for C34H35O8 [M+H]+, C34H34NaO8
+
+
[M+Na]+: 571.2326, 593.2146; found: 571.2315, 593.2134.
MnDOTA2OPE
The MnII complex of bis-DOTA-OPE (13) was generated in situ for
mass spectrometry analysis by the addition of Mn(ClO4)2 (2.0 equiv)
to a buffered solution (HEPES 100 mm, pH 7.5) of 13. MALDI-TOF
MS (HCCA): m/z (%): 1423.46 (100) [Bis-DOTA-OPEꢀ3H+2Mn]+.
4,4’-({2,5-Bis[2-(2-methoxyethoxy)ethoxy]-1,4-phenylene}-
bis(ethyne-2,1-diyl))dibenzoic acid (OPE-diCO2H 18)
OPE-diCHO 17 (1.5 g, 2.63 mmol, 1.0 equiv) was suspended in dry
DMF (65 mL). KHSO5·KHSO4·K2SO4 (Oxoneꢅ; 3.23 g, 5.26 mmol,
2.0 equiv) was added and the resulting suspension was stirred at
RT for 24 h. H2O was added and the resulting precipitate was fil-
tered and dried to afford OPE-diCO2H 18 (1.294 g, 2.15 mmol,
4-[(Trimethylsilyl)ethynyl]benzaldehyde (15)
Compound 14 (10.0 g, 54.05 mmol, 1.0 equiv) was dissolved in dry
THF (40 mL) and dry triethylamine (20 mL). [Pd(PPh)3Cl2] (380 mg,
0.541 mmol, 0.01 equiv) and CuI (103.3 mg, 0.541 mmol,
0.01 equiv) were added, and argon was bubbled for 15 min. TMSA
(9.2 mL, 64.86 mmol, 1.2 equiv) was added dropwise and the re-
sulting mixture was heated at reflux for 24 h and then cooled to
RT. CH2Cl2 (200 mL) was added, the organic layer was recovered
and washed with H2O (1ꢄ), a 10% aqueous solution of HCl (1ꢄ),
and H2O (1ꢄ); dried over Na2SO4; filtered; and concentrated.
Column chromatography (SiO2, 100:0 to 90:10 cyclohexane/EtOAc)
afforded 15 (9.31 g, 46.02 mmol, 85%) as a yellow solid. 1H NMR
(CDCl3, 300 MHz): d=9.99 (s, 1H), 7.81 (d, 2H, J=8.3 Hz), 7.60 (d,
2H, J=8.3 Hz), 0.27 ppm (s, 9H); 13C NMR (CDCl3, 75 MHz): d=
191.5 (CH), 135.7 (Cq), 132.6 (CH), 129.6 (CH), 129.5 (Cq), 104.0 (Cq),
99.2 (Cq), 0.1 ppm (CH3); HRMS (ESI): m/z calcd for C12H15OSi+ [M+
H]+, C12H14NaOSi+ [M+Na]+: 203.0887, 225.0706; found: 203.0886,
225.0668.
1
82%) as a yellow solid. H NMR ([D6]DMSO, 300 MHz): d=13.13 (s,
2H), 8.03–7.94 (m, 4H), 7.68–7.60 (m, 4H), 7.27 (s, 2H), 4.20 (t, J=
4.5 Hz, 4H), 3.80 (t, J=4.5 Hz, 4H), 3.71–3.64 (m, 4H), 3.48–3.41 (m,
4H), 3.21 ppm (s, 6H); 13C NMR ([D6]DMSO, 75 MHz): d=166.7 (Cq),
153.2 (Cq), 131.4 (CH), 130.6 (Cq), 129.6 (CH), 126.8 (Cq), 116.9 (CH),
113.1 (Cq), 94.3 (Cq), 88.8 (Cq), 71.4 (CH2), 70.1 (CH2), 69.1 (CH2), 69.0
ꢀ
(CH2), 58.1 ppm (CH3); HRMS (ESI): m/z calcd for C34H33O10
[MꢀH]ꢀ, C34H32O102ꢀ/2 [Mꢀ2H]2ꢀ
: 601.2079, 300.1003; found:
601.2076, 300.1022.
Bis-TEMPO-OPE (19)
OPE-diCO2H 18 (57 mg, 0.094 mmol, 1.0 equiv) and 4-NH2-TEMPO
(40 mg, 0.234 mmol, 2.5 equiv) were dissolved in dry DMF (2 mL).
The resulting solution was cooled to 08C, HOBt·H2O (43 mg,
0.282 mmol, 3.0 equiv) and DCC (58 mg, 0.282 mmol, 3 equiv) were
added, and the resulting mixture was stirred at RT for 24 h. H2O
was added and the mixture was extracted with CH2Cl2 (3ꢄ). The
combined organic layers were dried over Na2SO4, filtered, and con-
centrated. Column chromatography (SiO2, 30:70 to 20:80 cyclohex-
ane/EtOAc) afforded bis-TEMPO-OPE 19 (79 mg, 0.087 mmol, 93%)
as an orange solid. Rf (SiO2): 0.39 (100% EtOAc); 1H NMR (CDCl3,
300 MHz; all signals broadened and some obscured due to para-
magnetism): d=8.05–7.53 (m, 8H), 7.09 (s, 2H), 4.25 (brs, 4H), 3.96
(brs, 4H), 3.82 (brs, 4H), 3.57 (brs, 4H), 3.39 (s, 6H), 2.12–0.79 ppm
(m, 32H); HPLC: 6.92 min (40 to 100% MeCN in 10 min, 95%);
4-Ethynylbenzaldehyde (16)
Compound 15 (9.31 g, 46.02 mmol, 1.0 equiv) was dissolved in
MeOH (450 mL). K2CO3 (4.77 g, 34.5 mmol, 0.75 equiv) was added
and the resulting suspension was stirred at RT for 30 min. A satu-
rated aqueous solution of NH4Cl was added and the mixture was
extracted with CH2Cl2 (3ꢄ). The combined organic layers were
washed with H2O (1ꢄ), dried over Na2SO4, filtered, and concentrat-
ed. Column chromatography (SiO2, 100:0 to 90:10 cyclohexane/
EtOAc) afforded 16 (5.87 g, 45.12 mmol, 98%) as a brown solid.
1H NMR (CDCl3, 300 MHz): d=10.02 (s, 1H), 7.87–7.81 (m, 2H),
7.67–7.60 (m, 2H), 3.29 ppm (s, 1H); 13C NMR (CDCl3, 75 MHz): d=
191.5 (CH), 136.1 (Cq), 132.9 (CH), 129.6 (CH), 128.5 (Cq), 82.8 (Cq),
HRMS (ESI): m/z calcd for C52H69N4O10 [M+H]2+, C52H68N4NaO10
2+
2+
[M+Na]2+: 909.5008, 931.4828; found: 909.4968, 931.4793.
81.2 ppm (CH); HRMS (APCI): m/z calcd for C6H7O9 [M+H]+:
+
131.0491; found: 131.0493.
X-ray Crystallography
XRD data for compound 17 was collected by using a Kappa X8
APEX II Bruker diffractometer with graphite-monochromated MoKa
radiation (l=0.71073 ꢃ). XRD data for compound 19 was collected
by using a Kappa VENTURE PHOTON 100 Bruker diffractometer
with ImS microfocus graphite-monochromated MoKa radiation (l=
0.71073 ꢃ). Crystals were mounted on a CryoLoop (Hampton Re-
search) with Paratone-N (Hampton Research) as a cryoprotectant
and then flash frozen in a nitrogen gas stream at 100 K. The tem-
perature of the crystal was maintained at the selected value
(100 K) by means of a 700 series Cryostream cooling device to
within an accuracy of ꢂ1 K. The data were corrected for Lorentz
polarization and absorption effects. The structures were solved by
direct methods by using SIR-97[82] and refined against F2 by full-
matrix least-squares techniques by using SHELXL-2013[83] with ani-
sotropic displacement parameters for all non-hydrogen atoms. All
4,4’-({2,5-Bis[2-(2-methoxyethoxy)ethoxy]-1,4-phenylene}bi-
s(ethyne-2,1-diyl))dibenzaldehyde (OPE-diCHO 17)
Compounds 4 (639 mg, 1.15 mmol, 1.0 equiv) and 16 (300 mg,
2.31 mmol, 2.0 equiv) were dissolved in dry THF (5 mL) and dry
triethylamine (5 mL). Argon was bubbled for 15 min and then
[Pd(PPh3)2Cl2] (81 mg, 0.115 mmol, 0.1 equiv) and CuI (44 mg,
0.23 mmol, 0.2 equiv) were added. The resulting suspension was
stirred for 24 h at RT; a saturated aqueous solution of NH4Cl was
added and the mixture was extracted with CH2Cl2 (3ꢄ). The com-
bined organic layers were dried over Na2SO4, filtered, and concen-
trated. Column chromatography (SiO2, 80:20 to 0:100 cyclohexane/
EtOAc) afforded OPE-diCHO 17 (528 mg, 0.925 mmol, 80%) as an
1
orange solid. H NMR (CDCl3, 300 MHz): d=10.03 (s, 2H), 7.91–7.84
&
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Articles
calculations were performed by using the crystal structure crystal-
lographic software package WINGX.[84]
from 8 to 19, depending on the spectral position at which the
PELDOR time traces were recorded. The accumulation time of the
full set of PELDOR time traces was about 5 h.
The crystal data collection and refinement parameters are given in
Table S1 in the Supporting Information.
Rotamer Analysis
The experimental data indicated positional static disorder of tolu-
ene of compound 19 (on inversion center). In the figures, the dis-
ordered toluene molecule is omitted for clarity.
Starting from the X-ray crystal structures of MnH2DOTA[80] and the
OPE spacer of bis-TEMPO-OPE 19, a model of MnDOTA2OPE was
constructed in Spartan 14 (Wavefunction Inc.) For the MnDOTA
arm that linked each spin label to the OPE, dihedral angles for the
CꢀN bond (c1 and c1’) and CꢀC bond (c2 and c2’) were restricted to
staggered conformations. The values of c3 and c3’ were fixed be-
cause previous studies showed that the acetanilide unit was over-
whelmingly in the trans configuration.[86] Relative to each other,
the two spin-labeled ends were allowed to fully rotate around the
molecular axis in 308 intervals. From these parameters, all 972 pos-
sible rotamers were systematically generated and molecular me-
chanic force field calculations were performed on each to identify
and eliminate those with clashes. For the remainder, 406 rotamers,
each was given the same probability and the MnꢀMn distances
were compiled in 0.1 nm boxes. Similarly, starting from the X-ray
structure of bis-TEMPO-OPE, the dihedral angles of the rotatable
CꢀN bonds were restricted to staggered conformations and, rela-
tive to each other, the two spin-labeled ends were allowed to fully
rotate around the molecular axis in 308 intervals. From these pa-
rameters, all 108 possible clash-free rotamers were systematically
generated. Each was given the same probability and the distances
between the middle points of the NꢀO bonds were compiled in
0.1 nm boxes.
CCDC 1431929-1431930 contains the supplementary crystallo-
graphic data for this paper. These data can be obtained free of
charge from The Cambridge Crystallographic Data Centre.
EPR Measurements
W-band PELDOR measurements were performed on a Bruker Elex-
sys II 680 EPR spectrometer equipped with a Bruker “power up-
grade 2” and an Oxford Instruments CF935 flow cryostat. For
MnDOTA2OPE all experiments were performed at a sample temper-
ature of 10 K. Spin-echo-detected EPR spectra were taken by using
a Hahn-echo sequence with p/2 and p pulse durations of 12 and
24 ns, an interpulse delay time of 2500 ns, a shot repetition time of
800 ms, and a sweep width of 1000 G. For PELDOR measurements,
the four-pulse, dead-time-free sequence[11,12] was used and the dif-
ference between the pump and detection frequencies was
+70 MHz. The pump pulse duration was 24 ns and the duration of
the p/2 and p detection pulses was 12 and 24 ns. The initial inter-
pulse delay (between detection p/2 and p pulses) was 544 ns and
the dipolar evolution window was 2226 ns. A four-step phase cycle
procedure was used. The shot repetition time was 800 ms with 100
shots per point and the number of scans was 156, which equated
to an accumulation time of 1.5 h. For bis-TEMPO-OPE 19, all experi-
ments were performed at a sample temperature of 40 K. Spin-
echo-detected EPR spectra were recorded by using a Hahn-echo
sequence with p/2 and p pulse durations of 40 and 80 ns, an inter-
pulse delay time of 1500 ns, a shot repetition time of 5 ms, and
a sweep width of 400 G. For PELDOR measurements, the difference
between the pump and detection frequencies was +70 MHz. The
pump pulse duration was 80 ns and the duration of the p/2 and p
detection pulses was 40 and 80 ns. The initial interpulse delay (be-
tween detection p/2 and p pulses) was 400 ns and the dipolar evo-
lution window was 1500 ms. A four-step phase cycle procedure was
used. The shot repetition time was 5 ms with 100 shots per point
and the number of scans was 25. The total accumulation time of
the 3 PELDOR time traces was about 4.5 h.
Acknowledgements
This work was financially supported by ANR MnHFPELDOR, ANR-
DFG Chemistry 2011-INTB-1010-01 (P.D.-D. PhD studentship and
H.Y.V.C. post-doctoral fellowship) and DFG (GZ: PR 294/14-1, D.A.
PhD studentship). We thank the French Infrastructure for Inte-
grated Structural Biology (FRISBI) for financial support. The W-
band spectrometer was funded by the Rꢀgion Ile-de-France
“Sesame” program, the CEA, and CNRS. We thank UPS, IUF,
Rꢀgion Ile de France SESAME program 2012 no. 12018501, LabEx
CHARM3AT, and CNRS for X-ray crystallography. We are very
grateful to Dr. Sun Un (I2BC-CEA Saclay), Prof. Thomas Prisner
(Goethe Universitꢁt Frankfurt), Dr. Burkhard Endeward (Goethe
Universitꢁt Frankfurt), and Dr. Alice Bowen (Goethe Universitꢁt
Frankfurt) for spectrometer access and fruitful and constructive
discussions.
Q-band PELDOR measurements were performed on a Bruker Elex-
sys E580 spectrometer, equipped with a 10 W AMP-Q-Band solid-
state amplifier. A more detailed description of the Q-band setup is
given elsewhere.[85] All experiments were performed with the
ER5107D2 probe at a sample temperature of 50 K. Spin-echo-de-
tected EPR spectra were recorded by using a Hahn-echo sequence
with p/2 and p pulse durations of 16 and 32 ns, an interpulse
delay time of 130 ns, a shot repetition time of 3 ms, and a sweep
width of 140 G. For PELDOR measurements, the difference be-
tween the pump and detection frequencies was +50 or ꢀ50 MHz.
The pump pulse duration was 22 ns and the duration of all detec-
tion pulses were 32 ns. The initial interpulse delay (between detec-
tion p/2 and p pulses) was 332 ns and the dipolar evolution
window was 5 ms. To suppress deuterium modulations in the
PELDOR time traces, the initial interpulse delay time was incre-
mented by 16 ns over 8 steps (tau averaging procedure). A two-
step phase cycle procedure was used. The shot repetition time was
3 ms with 20 shots per point and the number of scans was varied
Keywords: EPR spectroscopy · gadolinium · manganese ·
pulsed electron–electron double resonance · spin labels
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Manuscript received: March 7, 2016
Accepted Article published: March 27, 2016
Final Article published: && &&, 2016
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P. Demay-Drouhard, H. Y. V. Ching,
D. Akhmetzyanov, R. Guillot, L. C. Tabares,
H. C. Bertrand,* C. Policar*
&& – &&
A Bis-Manganese(II)–DOTA Complex
for Pulsed Dipolar Spectroscopy
New labels for spin: The efficient syn-
thesis of a model system consisting of
two [Mn(dota)]2ꢀ (DOTA4ꢀ =1,4,7,10-tet-
raazacyclododecane-1,4,7,10-tetraace-
tate; see figure) molecules directly con-
nected to a central rodlike oligo(pheny-
lene–ethynylene) spacer is described.
This study illustrates the potential of
these spin labels for measuring short
nanometer distances.
&
ChemPhysChem 2016, 17, 1 – 14
www.chemphyschem.org
14
ꢂ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!