Water-soluble narrow-line radicals for dynamic nuclear polarization

Article abstract of DOI:10.1021/ja304918g

The synthesis of air-stable, highly water-soluble organic radicals containing a 1,3-bis(diphenylene)-2-phenylallyl (BDPA) core is reported. A sulfonated derivative, SA-BDPA, retains the narrow electron paramagnetic resonance linewidth (<30 MHz at 5 T) of the parent BDPA in highly concentrated glycerol/water solutions (40 mM), which enables its use as polarizing agent for solid effect dynamic nuclear polarization (SE DNP). A sensitivity enhancement of 110 was obtained in high-field magic-angle-spinning (MAS) NMR experiments. The ease of synthesis and high maximum enhancements obtained with the BDPA-based radicals constitute a major advance over the trityl-type narrow-line polarization agents.

Full text of DOI:10.1021/ja304918g

Communication
pubs.acs.org/JACS
Water-Soluble Narrow-Line Radicals for Dynamic Nuclear
Polarization
Olesya Haze,† Bjorn Corzilius,†,‡ Albert A. Smith,†,‡ Robert G. Griffin,*,†,‡ and Timothy M. Swager*,†
̈
†Department of Chemistry and ‡Francis Bitter Magnet Laboratory, Massachusetts Institute of Technology, 77 Massachusetts Avenue,
Cambridge, Massachusetts 02139, United States
S
* Supporting Information
ABSTRACT: The synthesis of air-stable, highly water-
soluble organic radicals containing a 1,3-bis(diphenylene)-
2-phenylallyl (BDPA) core is reported. A sulfonated
derivative, SA-BDPA, retains the narrow electron para-
magnetic resonance linewidth (<30 MHz at 5 T) of the
parent BDPA in highly concentrated glycerol/water
solutions (40 mM), which enables its use as polarizing
agent for solid effect dynamic nuclear polarization (SE
DNP). A sensitivity enhancement of 110 was obtained in
high-field magic-angle-spinning (MAS) NMR experiments.
The ease of synthesis and high maximum enhancements
obtained with the BDPA-based radicals constitute a major
advance over the trityl-type narrow-line polarization
agents.
Figure 1. Trityl-type radicals and BDPA.
BDPA is an air-stable persistent radical that shows no
dimerization in solid or in solution and has a narrow EPR
linewidth9 (Δ ≈ 25 MHz at 140 GHz). The ability of BDPA to
transfer polarization was investigated via SE DNP in a
polystyrene matrix10 and recently via dissolution DNP in
sulfolane.11 While BDPA was proven to be an excellent
polarization agent, its utility in biological applications is limited
by its lack of solubility in aqueous media. Only one water-
soluble BDPA derivative has been described.12 But again, its
water solubility is limited, and DNP experiments with this
compound were not pursued. We report an efficient synthesis
ynamic nuclear polarization (DNP) is used to signifi-
Dcantly enhance the limited sensitivity of NMR studies of
small molecules and complex biological systems.1 The
increased sensitivity and decreased measurement time afforded
by DNP allow observation of transient (e.g., photochemical
intermediates) and metabolic processes. DNP coupled with
multidimensional magic-angle-spinning (MAS) NMR spectros-
copy is useful in structural studies of proteins that cannot be
crystallized, such as membrane and amyloid proteins.2
Scheme 1. Synthesis of Water-Soluble SA-BDPA Radicals
DNP relies on the transfer of polarization from relatively
highly polarized unpaired electron spins to nuclear spins (e.g.,
1H or 13C) via microwave irradiation of the electron
paramagnetic resonance (EPR) spectrum. Unpaired electrons
are supplied by the polarizing agents, which in most cases are
persistent radical species added to a glass-forming solvent. The
solid effect (SE) DNP3 is a two-spin process that relies on the
polarization transfer between a single electron spin and a
nuclear spin. This method requires a polarization agent with
both the homogeneous EPR linewidth (δ) and the inhomoge-
neous spectral breadth (Δ) smaller than the nuclear Larmor
frequency (ω0I). Trityl-type4 and bis(diphenylene)-2-phenyl-
allyl (BDPA)-based5 radicals satisfy these requirements at high
1
magnetic fields for SE DNP of H.
The trityl-type radicals CT-03 and OX0636 (Figure 1)
exhibit narrow EPR linewidths (Δ = 50 MHz at 140 GHz) and
have been used successfully in DNP NMR experiments and in
vivo EPR imaging of tissue oxygenation and pH.7 However, a
lengthy synthesis8 contributes to the high cost of these radicals
and limits their use as routine polarizing agents.
Received: May 25, 2012
© XXXX American Chemical Society
A
dx.doi.org/10.1021/ja304918g | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
of highly water-soluble BDPA derivatives that preserve the
desirable DNP properties of BDPA and expand its application
to aqueous systems.
The BDPA precursor BDPAH13 (1) can be prepared in four
steps from fluorene and benzaldehyde. Whereas BDPA radical
decomposes in strong acid, treatment of 1 with fuming sulfuric
acid followed by oxidation with silver nitrate yielded a mixture
of the extremely water-soluble sulfonated radicals SA-BDPA
(3). (Scheme 1)
The shape of the UV−vis spectrum, the redox behavior, and
the EPR spectrum of 3 closely resemble those of BDPA. The
sulfonate groups cause a red shift of the absorption maxima of
the carbanion 2 and SA-BDPA radical 3 by ∼20 nm relative to
those of BDPA anion and BDPA. As shown in Figure 2, SA-
BDPA in water has a strong absorption in the visible region
(λmax = 508 nm, D0 → D2) and a weak absorption in the near-
IR region (λmax = 881 nm, D0 → D1). BDPA in dichloro-
methane has similar absorption maxima (485 and 859 nm,
respectively).12,14 Similar to BDPA, SA-BDPA is reduced under
basic conditions [e.g., aqueous NaOH in the presence of
catalytic amounts of acetone, tetrahydrofuran (THF), dimethyl
sulfoxide, or ascorbate]. The resulting carbanion 2 can be
reoxidized to 3 chemically (e.g., by AgNO3) or electrochemi-
cally.
Figure 3. EPR spectra of SA-BDPA: (A) 1 mM solution in water
(9.856 GHz, RT, cw EPR); (B) 1 mM frozen solution in 60/40 (v/v)
glycerol-d8/D2O (9.745 GHz, 80 K, echo-detected); (C) 1 mM frozen
solution in 60/40 (v/v) glycerol-d8/D2O (140.0 GHz, 80 K, echo-
detected).
Figure 4 shows the field-dependent DNP enhancement at 5 T
(140 GHz microwave frequency) obtained with a 40 mM
frozen solution of SA-BDPA in 60/30/10 (v/v) glycerol-d8/
D2O/H2O compared with that of the commonly used trityl
polarizing agent OX063. The glass-forming glycerol/water
mixture is crucial because it prevents phase separation of the
solutes and allows for efficient nuclear spin diffusion.
Furthermore, it acts as a cryoprotectant to prevent potential
1
cold denaturation of proteins. H signal enhancement by DNP
was directly observed via a Bloch decay as function of the
external magnetic field. The two radicals gave frequency profiles
typical of the well-resolved SE, with a positive peak and a
negative peak separated by twice the Larmor frequency of the
polarized nucleus and centered around the EPR resonance. As
expected, the smaller linewidth of SA-BDPA was retained in the
DNP field profile. The enhancement factors were determined
with a Hartmann−Hahn cross-polarization (CP) step to 13C at
the respective fields of maximum enhancement (1 M [13C]urea
was added to provide sufficient 13C for detection of thermal
equilibrium polarization). SA-BDPA yielded an NMR signal
enhancement (ε) of 61 by comparison of the on and off signals
after a time of 1.3TB (where TB is the time constant of
polarization buildup) at a microwave power of 6 W; this is
∼30% higher than the enhancement obtained with OX063
under the same conditions.16
Figure 2. Absorption spectra and (inset) photographs of aqueous
In addition to the signals attributed to the SE, another
feature centered at the EPR resonance field of 4983 mT was
observed for SA-BDPA. Experiments have shown a 1H
enhancement of ∼8 independent of the applied microwave
power over the range of 2.4−10 W. This flat power dependence
together with the symmetric shape and increased width of this
feature led us to conclude that the underlying mechanism is
solely based on direct saturation of the EPR resonance and that
nuclear polarization is induced via cross-relaxation, similar to
the Overhauser effect. It is unlikely that the cross effect (CE) or
thermal mixing (TM) caused this peak because both
mechanisms require a much larger EPR linewidth and typically
yield a DNP field profile with regions of positive and negative
enhancements and an overall width comparable to the EPR
linewidth. For direct comparison, an overlay of the DNP field
profile and the EPR spectrum of SA-BDPA is shown in Figure
S3 in the Supporting Information. In fact, we attributed the
feature in the center of the trityl profile to CE/TM.16 However,
the SA-BDPA profile does not show any sign of CE/TM, in
accordance with the significantly reduced linewidth of SA-
BDPA compared with trityl (28 vs 50 MHz).
solutions of SA-BDPA and its anion.
Also, SA-BDPA is air-stable both in solution and as a solid.
Unlike BDPA, however, SA-BDPA does not partition into
organic solvents and is soluble in water in all proportions. The
EPR spectrum of SA-BDPA (Figure 3) shows no evidence of
aggregate formation or radical dimerization in liquid or frozen
solution. The solution EPR spectrum features a hyperfine
pattern typical of BDPA-type radicals, with nine lines resulting
from coupling to eight protons with similar coupling constants
of ∼5.1 MHz (Figure 3A). In frozen solution, the linewidth [Δ
= 26 MHz at 9 GHz, full width at half-maximum (fwhm)] is
dominated by unresolved hyperfine couplings to protons and
perfectly matches the envelope of the solution spectrum
(Figure 3B). At 140 GHz, the line broadens insignificantly to Δ
= 28 MHz as a result of a very small g anisotropy (Figure 3C).
This small g anisotropy qualifies SA-BDPA as an interesting
new polarizing agent for SE DNP at even higher fields (e.g., 9.4
T). Trityl’s linewidth, on the other hand, increases linearly with
the external field because g anisotropy is the dominant
broadening mechanism in that case.15
Previous studies of the solid effect have shown that the
polarization enhancement is accompanied by a decrease in the
time constant at which nuclear longitudinal polarization builds
Encouraged by SA-BDPA’s desirable properties, we
proceeded to test it as a polarization agent for SE DNP.
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Journal of the American Chemical Society
Communication
This explanation is further supported by the significantly
slower spin−lattice relaxation of the respective electron spins
(T1 = 56 ms in SA-BDPA vs 1.3 ms in trityl; Figure S2). This
longer spin−lattice relaxation time constant might also lead to a
“saturation effect” observed in microwave-power-dependent
measurements of the 1H DNP enhancement: while trityl
showed a near-linear power dependence, the enhancements
obtained with SA-BDPA were more than 50% larger at the
lowest applied power but approached those obtained with trityl
at the highest gyrotron output power available (Figure 4 inset).
Figure 4. Field-dependent 1H DNP enhancement by 40 mM SA-
BDPA (red) in 60/30/10 (v/v) glycerol-d8/D2O/H2O compared with
that of trityl OX063 (green) recorded at a microwave power of ∼6 W.
1
Inset: power dependence of the H enhancements measured at the
respective field maxima by 1H−13C CP. The trityl OX063 comparison
Figure 6. (left) DNP-enhanced 2D 13C−13C correlation spectrum
(spin diffusion) using mixing times of 2 ms (green) and 20 ms (red)
and (right) 13C CPMAS spectra of 0.1 M [U-13C5]proline polarized by
40 mM SA-BDPA in 60/30/10 (v/v) glycerol-d8/D2O/H2O under 8.9
W microwave irradiation.
data (recorded under similar conditions) were taken from ref 16.
up.16,17 Therefore, careful analysis of the buildup dynamics and
extrapolation of the signal intensity at infinite polarization time
are crucial to prevent misinterpretation of data (Figure 5). We
report enhancement values extrapolated to infinite polarization
time.
The buildup time constants TB were found to be between 43
and 31 s depending on the incident microwave power and
obtained enhancements (Figure 5). The reduction of TB by SE
In Figure 6 we demonstrate the application of SA-BDPA in
1D DNP-enhanced CPMAS and 2D 13C−13C correlation
spectra of a 0.1 M solution of uniformly 13C-labeled proline.
13C−13C mixing was achieved using spin diffusion assisted by a
dipolar-assisted rotational resonance (DARR) field applied to
1H.18 Mixing was either limited to a one-bond distance (Figure
6, green) or allowed to occur among all of the nuclei in the
small molecule (Figure 6, red) by allowing spin diffusion for a
mixing period of 2 or 20 ms, respectively. In each case all of the
expected cross-peaks were present and clearly resolved. The 1H
signal enhancement was determined to be ε = 50 by
comparison of the 1D signal amplitudes with and without
microwave irradiation (Figure 6, right). The slight decrease in
the DNP enhancement compared with the experiments using
urea could be caused by less efficient irradiation of the entire
sample volume (a fully packed NMR rotor was used for proline,
while the urea experiments were conducted in a center-packed
1
rotor). Additionally, different H relaxation properties induced
by alicyclic side chain dynamics could lead to lower equilibrium
polarization.
Figure 5. Polarization buildup curves obtained with 40 mM SA-BDPA
in 60/30/10 (v/v) glycerol-d8/D2O/H2O at different microwave
power levels and without microwave irradiation (multiplied by a factor
of 10 to enhance visibility).
SA-BDPA is an effective narrow-line DNP agent with
outstanding water solubility. However, access to a family of
peripherally substituted BDPA derivatives would be advanta-
geous because experience has indicated that a single polar-
ization agent may not suffice for all analytes.19 For example,
DNP NMR experiments may be compromised if the polarizing
agent binds to the analyte, which would cause significant
paramagnetic broadening of the NMR signals of interest.
Therefore, we set out to develop a modular route to access
differentially functionalized water-soluble BDPA derivatives.
The reaction of HOOC−BDPAH (4)20 with chlorosulfonic
acid produced chlorosulfonate 5, which could be hydrolyzed or
reacted with nucleophiles prior to oxidation to give both ionic
and neutral polarization agents.21 For example, treatment of 5
DNP compared with T1 leads to a further increase in sensitivity
due to more rapid recycling of NMR experiments.17 At the
highest microwave power, the enhancement factor was ε = 94,
while the gain in sensitivity was ε(T1/TB)1/2 = 110. However,
the overall TB values (including T1) were ∼50% larger in
comparison with those of trityl OX063 under similar
conditions,16 which is attributed to less efficient longitudinal
relaxation enhancement of protons by the paramagnetic
species.
C
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Journal of the American Chemical Society
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Scheme 2. Modular Approach to Water-Soluble BDPA-
Based Radicals
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BDPA derivatives can find application in this field. We
anticipate that biradicals and multiradicals incorporating the
water-soluble BDPA cores will be useful for cross effect DNP.
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ASSOCIATED CONTENT
* Supporting Information
Detailed experimental procedures and characterization of 3, 6a,
and 6b. This material is available free of charge via the Internet
at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
rgg@mit.edu; tswager@mit.edu
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This study was funded through NIH Grant GM095843 to
T.M.S. and Grants EB002804 and EB002026 to R.G.G. B.C.
was supported by the Deutsche Forschungsgemeinschaft (DFG
Research Fellowship CO802/1-1).
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