Dynamic nuclear polarization with a water-soluble rigid biradical

Article abstract of DOI:10.1021/ja212054e

A new biradical polarizing agent, bTbtk-py, for dynamic nuclear polarization (DNP) experiments in aqueous media is reported. The synthesis is discussed in light of the requirements of the optimum, theoretical, biradical system. To date, the DNP NMR signal enhancement resulting from bTbtk-py is the largest of any biradical in the ideal glycerol/water solvent matrix, ε = 230. EPR and X-ray crystallography are used to characterize the molecule and suggest approaches for further optimizing the biradical distance and relative orientation.

Full text of DOI:10.1021/ja212054e

Communication
pubs.acs.org/JACS
Dynamic Nuclear Polarization with a Water-Soluble Rigid Biradical
Matthew K. Kiesewetter,† Bjorn Corzilius,†,‡ Albert A. Smith,†,‡ Robert G. Griffin,*,†,‡
̈
and Timothy M. Swager*,†
†Department of Chemistry and ‡Francis Bitter Magnet Laboratory, Massachusetts Institute of Technology, Cambridge, Massachusetts
02139, United States
S
* Supporting Information
tration.20 In particular, because the low-temperature EPR
ABSTRACT: A new biradical polarizing agent, bTbtk-py,
for dynamic nuclear polarization (DNP) experiments in
aqueous media is reported. The synthesis is discussed in
light of the requirements of the optimum, theoretical,
biradical system. To date, the DNP NMR signal
enhancement resulting from bTbtk-py is the largest of
any biradical in the ideal glycerol/water solvent matrix, ε =
230. EPR and X-ray crystallography are used to character-
ize the molecule and suggest approaches for further
optimizing the biradical distance and relative orientation.
spectra of nitroxide radicals are anisotropic, the construction of
discrete biradical systems permits control of the relative
orientation of the g tensors of the radicals and their separation,
hence further augmenting ε.
Simulation of field-dependent EPR spectra of the biradical
TOTAPOL suggests that its nitroxide moieties are, somewhat
serendipitously, of nearly optimal orientation for DNP.23
Indeed, no other biradical that is soluble in glycerol/water
(60/40) has been reported to yield a higher ε. DNP from the
highly crystalline bTbk in DMSO/water mixtures did yield a
larger ε than TOTAPOL.24 However, this spiro-cyclic biradical
is insoluble in glycerol/water mixtures and only sparingly
soluble in DMSO/water. This limits the applicability of bTbk as
a polarizing agent because glycerol/water serves as a
cryoprotectant and is the solvent of choice for DNP of
proteins. The near-ideal geometry and experimental results with
bTbk and the reported synthesis of 125 stimulated us to
investigate water-soluble analogues of bTbk.
ynamic nuclear polarization (DNP)1−3 allows for the
D
intrinsically large spin polarization of electrons to be
transferred to nuclei for detection in an NMR experiment with
a theoretical signal enhancement of 2−3 orders of magnitude
over the Boltzmann population of the nuclear spins. In a typical
DNP experiment, the electron paramagnetic resonance (EPR)
spectrum of a paramagnetic molecule is irradiated with
microwaves resulting in the transfer of spin polarization to
surrounding nuclei. The particular appeal of DNP lies in its
ability to facilitate structural measurements (internuclear
distances and torsion angles) that are limited by signal-to-
noise in multidimensional biomolecular magic angle spinning
(MAS) experiments.4,5 In these situations, optimal NMR signal
enhancements are usually observed from experiments con-
ducted at cryogenic temperatures.6,7 Thus, glassy solvents,
particularly those that form a glass regardless of cooling rate,
are required to ensure a homogeneous dispersion of polarizing
agent and to prevent cold denaturation of proteins.8,9
Starting from the modified TEMPone structure, 1, and
drawing upon previous research,26 we made the bis-thioketal in
moderate yield, Scheme 1. The fully oxidized biradical 3 was
generated in a two-stage oxidation using potassium perox-
ymonosulfate as the oxidant. The low pH of the first oxidation
step acts as a protecting group for the amines,27 which are
subsequently oxidized under basic conditions using dimethyl
dioxirane generated in situ from the oxidation of acetone.28,29
Biradical 3, however, is effectively insoluble in glycerol/water
mixtures (solubility <0.1 mM). Accordingly, we decided to
pursue a mixed sulfur oxidation state biradical with the
knowledge that a mixture of species should enhance solubility.
The partial oxidation of 2 was performed by the reaction with
m-chloroperoxybenzoic acid (m-CPBA). The resulting mixture,
bTbtk-py, an extension of the established naming system short
for bis-TEMPO-bis-thioketal-tetra-tetrahydropyran,24 was char-
acterized by NMR (the reduced form having N−OH groups),
CHN analysis, and electrospray ionization high-resolution mass
spectrometry (HRMS-ESI), and it was found to be a biradical
possessing a mixture of sulfur oxidation states, 0−5 SO
The proper instrumentation (microwave sources, low-
temperature MAS probes, etc.)7,10−12 is essential to the
successful implementation of DNP. However, an equally
important aspect of the methodology is the development of
polarizing agents from which large signal enhancements, ε, are
observed. Currently, the most efficient high field in situ DNP
mechanism, the cross effect (CE), arises from the interaction of
three spins, two electrons and one nucleus.13−21 Irradiation of
the EPR spectrum results in a spin “flip-flop” process between
the electrons that leads to transfer of polarization to the
nucleus. The process is most efficient when the difference in
the Larmor frequencies of the electrons matches that of the
nucleus, ω0S1 − ω0S2 ≈ ω0I, where ω0S and ω0I are the Larmor
frequencies of the electron spin S or nuclear spin I, respectively.
Although monomeric nitroxide radicals were among the early
cross-effect DNP agents,18,19,22 tethering the radicals resulted in
larger signal enhancements with lower paramagnet concen-
Received: December 24, 2011
Published: February 28, 2012
© 2012 American Chemical Society
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Scheme 1. Synthesis of bTbtk-py
bonds. The X-band EPR spectrum is typical for a nitroxide
radical; the broad 1:1:1 triplet due to hyperfine coupling to a
nitrogen, aiso(14N) = 41.8 MHz, is consistent with a biradical
featuring a short, rigid tether with a weak electron−electron J-
coupling (exchange integral), J < 10·a(14N).23,30 The biradical
exhibited excellent solubility in the desired 60/40 glycerol/
water mixture (10.9 mM in glycerol/water (60/40); 3.0 mM in
D2O).
Figure 1. DNP-enhanced polarization was measured under microwave
irradiation using a gyrotron beam current of 34 mA, which
corresponds to ∼12 W microwave power. (Top) 13C NMR
(CPMAS) of 1 M urea-13C in 60/30/10 glycerol-d8/D2O/H2O (v/
v/v) with 10 mM of the appropriate biradical acquired with and
without microwave irradiation. (Bottom) Buildup curves were
recorded at the respective field, yielding the maximum enhancement
for each radical (i.e., 4.982 T for bTbtk-py, 4.980 T for TOTAPOL;
see Figure 3) and subsequently scaled in order to normalize the off
signal intensities at t = ∞. Proton longitudinal relaxation time
constants were determined as T1I = 3.9 s for bTbtk-py and T1I = 4.5 s
for TOTAPOL. Note that aside from the radical used and the
magnetic field strength, the experimental conditions are the same for
each trace.
The DNP-enhanced 13C−CPMAS NMR of 13C-urea (1 M in
60/40 glycerol/water) showed ε = 230 versus thermal
polarization. Experimentally, 13C-urea and bTbtk-py in the
glassy glycerol/water matrix is continuously irradiated with
microwaves under cryogenic (T ≈ 82 K) MAS conditions to
1
polarize the H’s, and this polarization is transferred to 13C for
detection via a cross-polarization step. In the absence of
microwaves, the thermal equilibrium polarization develops. In
either event, the signal intensity as measured after a
presaturation sequence versus recovery time shows a
monoexponential polarization buildup, Figure 1. The enhance-
ment, ε, is given by the ratio of signal intensity with the
microwaves on versus microwaves off. Interrogated under
identical conditions, TOTAPOL shows a lower enhancement
factor, ε = 191. The direct comparison with an external
standard, TOTAPOL in this case, is crucial in order to
determine the relative performance of different polarizing
agents. Absolute enhancements are greatly influenced by
instrumental (e.g., rotor size, microwave coupling to the
sample) and experimental (e.g., MAS frequency, sample
temperature, optimum magnetic field) parameters, thus
impeding comparison between studies performed under varying
conditions. The DNP enhancement derived from bTbtk-py
constitutes the largest signal enhancement observed so far for
any biradical in a biologically relevant glycerol/water mixture
under given conditions.31−34
Figure 2. Power dependence of the steady-state DNP enhancement
ε∞ for bTbtk-py and TOTAPOL given by: 1/ε = 1/ε∞(1 + 1/aP).
The experiments with the two polarizing agents were carried out at the
field position giving the optimal enhancement for each. Otherwise, the
experimental conditions were identical (see Supporting Information).
The enhancement field profile of a polarizing agent shows
the NMR signal intensity as a function of magnetic field and,
hence, the electron Larmor frequency and provides the field for
optimum enhancement. The breadth and intensity of the
enhancement profile of bTbtk-py versus TOTAPOL (Figure 3)
suggest that the nitroxide moieties have slightly different
relative orientation and electron−electron coupling in the two
biradicals. Nevertheless, the shape of the enhancement profile
Another metric of the efficiency of a polarizing agent is given
by measuring ε as a function of microwave power and
extrapolating the enhancement to infinite power, ε∞ in Figure
2. This factor, ε∞, is independent of microwave power, Pmw
,
and the saturation parameter, a, which depend on microwave
transmission efficiency and EPR relaxation properties.23,24
However, care must be taken to control sample temperature
and rotor size.
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Figure 4. Two views of the X-ray crystal structure of 3.
nitroxide moieties may be acting synergistically with the
increased (vis-a-vis bTbk) ketal ring size to produce the larger
̀
NO−NO distance and smaller NO dihedral angle. Insofar as
structure determines DNP enhancement, the nonideal NO−
NO relationships might be improved by removing the THP
moieties or sulfone bridges. Certainly, the lower DNP
enhancement of bTbtk-py relative to bTbk suggests avenues
for the further exploration of the bTxk structure space.
In summary, the reported biradical, bTbtk-py, yields the
largest enhancement, ε = 230, to date of any biradical in the
ideal glycerol/water solvent matrix. The spiro-cycle structure is
highly crystalline in its fully oxidized form (3), but a partially
oxidized mixture of species is soluble in the desired medium.
The 13C NMR signal enhancement has been shown to depend
with extreme sensitivity upon the modulation of the electron−
electron distance and relative orientation of the NO moieties of
the biradical. This extreme sensitivity suggests that additional
synthetic fine-tuning of the linkers separating nitroxide radicals
can produce superior DNP polarizing agents.
Figure 3. (Top) EPR spectrum of bTbtk-py measured by echo
detection at 140 GHz and T = 80 K and (bottom) field-dependent
enhancement profiles of bTbtk-py and TOTAPOL (see Supporting
Information). The enhancement profile was recorded by directly
observing the 1H polarization under ∼8 W of microwave power using
a Bloch decay and scaling the obtained data with respect to the
maximum enhancement determined by cross-polarization to 13C.
TOTAPOL field profile adapted from Hu, et al.23
for bTbtk-py is qualitatively similar to that of all nitroxide
biradicals whose electron−electron dipolar coupling (∼20
MHz) is small relative to the g-anisotropy and a(14N),20,23,24
̀
yet the different ε, vis-a-vis that of TOTAPOL, corroborates
ASSOCIATED CONTENT
* Supporting Information
Experimental details, DEER spectra, crystallographic details and
a CIF file. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
the observation that enhancement is extremely sensitive to
electron−electron distance and relative orientation. Further, the
inhomogeneous line shape of the EPR spectrum and the field
profile, shown in Figure 3, is consistent with bTbtk-py
supporting the cross-effect DNP mechanism.
S
This study indicates that bTbtk-py is superior to TOTAPOL
AUTHOR INFORMATION
Corresponding Author
tswager@mit.edu; rgg@mit.edu
■
as a DNP biradical polarizing agent by a factor of (εbTbtk‑py
εTOTAPOL=) 1.20 0.02. However, previous studies in nonideal
solvents have determined this ratio is larger for bTbk, εbTbk
/
/
εTOTAPOL= 1.41 0.09. The difference in these numbers belies
the similar architecture of the two spiro-biradicals. Therefore,
suggesting why bTbtk-py ‘underperforms’ in ε may guide the
development of the next generation of biradicals for DNP.
Differences in NO−NO distance change the dipolar coupling
between the two electrons, with larger distances leading to
smaller couplings. The dipolar coupling, in conjunction with
the J-coupling, leads to the state mixing that is critical for
efficient cross effect.21 Note that in the case of TOTAPOL,
bTbk, and bTbtk-py the dipolar coupling is the dominant
contributor to state mixing. Therefore, the structural differences
in bTbk and bTbtk-py, including the NO−NO distance, were
examined by measuring the electron−electron dipole coupling
with double electron−electron resonance (DEER). The DEER-
measured interelectron distances (r) were determined to be
Author Contributions
The manuscript was written through contributions of all
authors.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by the National Institutes of
Health (GM095843 to T.M.S. and EB002804 and EB002026 to
R.G.G.). B.C. was partially supported by the Deutsche
Forschungsgemeinschaft (Research Fellowship CO802/1-1).
M.K. thanks the National Institute of Biomedical Imaging and
Bioengineering for a NRSA Fellowship (F32EB012937).
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