The NiCEST approach: Nickel(II) ParaCEST MRI contrast agents

Article abstract of DOI:10.1021/ja307909x

Paramagnetic Ni(II) complexes are shown here to form paraCEST MRI contrast agents (paraCEST = paramagnetic chemical exchange saturation transfer; NiCEST = Ni(II) based CEST agents). Three azamacrocycles with amide pendent groups bind Ni(II) to form stable NiCEST contrast agents including 1,4,7- tris(carbamoylmethyl)-1,4,7-triazacyclononane (L1), 1,4,8,11- tetrakis(carbamoylmethyl)-1,4,8,11-tetraazacyclotetradecane (L2), and 7,13-bis(carbamoylmethyl)-1,4,10-trioxa-7,13-diazacyclopentadecane (L3). [Ni(L3)]²⁺, [Ni(L1)]²⁺, and [Ni(L2)]²⁺ have CEST peaks attributed to amide protons that are shifted 72, 76, and 76 ppm from the bulk water resonance, respectively. Both CEST MR images and CEST spectroscopy show that [Ni(L3)]²⁺ has the largest CEST effect in 100 mM NaCl, 20 mM HEPES pH 7.4 at 37 'C. This larger CEST effect is attributed to the sharper proton resonances of the complex which arise from a rigid structure and low relaxivity.

Full text of DOI:10.1021/ja307909x

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The NiCEST Approach: Nickel(II) ParaCEST MRI Contrast Agents
Abiola O. Olatunde,† Sarina J. Dorazio,† Joseph A. Spernyak,§ and Janet R. Morrow*,†
†Department of Chemistry, University at Buffalo, State University of New York, Amherst, New York 14260, United States
§Department of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, New York 14263, United States
S
* Supporting Information
complexes tend to have shorter T1e and correspondingly
sharper proton resonances than do octahedral complexes.5−9
ABSTRACT: Paramagnetic Ni(II) complexes are shown
here to form paraCEST MRI contrast agents (paraCEST =
paramagnetic chemical exchange saturation transfer;
NiCEST = Ni(II) based CEST agents). Three azamacro-
cycles with amide pendent groups bind Ni(II) to form
stable NiCEST contrast agents including 1,4,7-tris-
(carbamoylmethyl)-1,4,7-triazacyclononane (L1),
1,4,8,11-tetrakis(carbamoylmethyl)-1,4,8,11-tetraazacyclo-
tetradecane (L2), and 7,13-bis(carbamoylmethyl)-1,4,10-
trioxa-7,13-diazacyclopentadecane (L3). [Ni(L3)]2+, [Ni-
(L1)]2+, and [Ni(L2)]2+ have CEST peaks attributed to
amide protons that are shifted 72, 76, and 76 ppm from
the bulk water resonance, respectively. Both CEST MR
images and CEST spectroscopy show that [Ni(L3)]2+ has
the largest CEST effect in 100 mM NaCl, 20 mM HEPES
pH 7.4 at 37 °C. This larger CEST effect is attributed to
the sharper proton resonances of the complex which arise
from a rigid structure and low relaxivity.
To probe different coordination geometries, three Ni(II)
complexes were studied (Chart 1). [Ni(L1)]2+ forms a six-
Chart 1. Structures of Ni(II) Complexes
coordinate complex with a twisted trigonal antiprismatic
geometry.10 [Ni(L2)]2+ also is most likely a six-coordinate
complex, as the tetracarboxylate analogue of L2 exhibits four
bound amino groups and two bound pendent amide donor
groups.11 By contrast, [Ni(L3)]2+ is likely to be a seven-
coordinate pentagonal bipyramidal complex with the oxygen
and nitrogen donors of the macrocycle forming the pentagonal
base and the two pendent groups capping the axial sites, similar
to that observed for a Ni(II) complex of an analogous
macrocycle containing two benzimidazole pendents.12,13 In
aqueous solution, the effective magnetic moments of [Ni-
(L1)]2+, [Ni(L2)]2+, and [Ni(L3)]2+ at 25 °C are 3.2, 3.1, and
3.4 μB, consistent with high spin Ni(II) (eq S2 in Supporting
Information).
ne of the greatest challenges in the design of effective
O
paraCEST MRI contrast agents is the selection of a
paramagnetic metal ion. The paramagnetic center must
produce highly shifted yet relatively narrow proton resonances
and at least one of these highly shifted protons must be in
chemical exchange with water. Irradiation at the resonant
frequency of the exchangeable proton saturates the magnet-
ization and, upon exchange, reduces the intensity of the bulk
water proton resonance.1 To date, Ln(III) ions, especially
Eu(III), Yb(III), and Tm(III), have been the paramagnetic
metal ions of choice because complexes of these ions provide
the best compromise between large chemical shift dispersion
and narrow line widths.1 As an alternative to Ln(III) contrast
agents, we recently reported the first example of a macrocyclic
Fe(II) paraCEST contrast agent.2−4 Transition metal ions have
the dual advantage of very versatile coordination chemistry and
a more substantial contact (through-bond) contribution to
paramagnetic shifts compared to Ln(III) ions.5 Here we report
the first Ni(II) macrocyclic complexes that are paraCEST MRI
contrast agents. Similar to Fe(II), the key to forming successful
paraCEST agents for Ni(II) is control of spin state, metal ion
dissociation kinetics and fluxionality of the macrocyclic complex
on the NMR time scale. However, stabilizing the divalent
oxidation state under biologically relevant conditions is more
straightforward for Ni(II) than for Fe(II).
The appearance of the paramagnetic 1H NMR spectra of the
three complexes is quite different (Figures S1−S3). [Ni(L3)]2+
shows 12 distinct nonexchangeable proton resonances of equal
intensity and two distinct exchangeable NH protons, consistent
with a complex of apparent C2 symmetry in CD3CN. The
amide NH protons are identified by their disappearance upon
addition of a few drops of D2O. Similar to Fe(II) amide
complexes,3,14 the two chemically inequivalent amide NH
proton resonances have a large chemical shift difference (82
and 16 ppm). All proton resonances are quite sharp, with
FWHH ranging from 150 to 400 Hz. This proton NMR
spectrum is consistent with a static structure on the NMR time
scale. In contrast, [Ni(L1)]2+ has two distinct NH amide
protons in CD3CN (83 and 14 ppm), but the remaining proton
resonances are quite broad. This proton spectrum is
reminiscent of that of [Fe(L1)]2+ which also contains relatively
The Ni(II) ion has relatively short electronic relaxation time
constants (T1e) that produce highly shifted and, in some cases,
narrow proton resonances. Tetrahedral and bipyramidal Ni(II)
Received: August 13, 2012
Published: October 27, 2012
© 2012 American Chemical Society
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dx.doi.org/10.1021/ja307909x | J. Am. Chem. Soc. 2012, 134, 18503−18505

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Figure 1. CEST spectra recorded at 11.7 T of a solution containing 10 mM [Ni(L1)]2+ (left), 100 mM NaCl, 20 mM HEPES, pH 7.3; 10 mM
[Ni(L2]2+ (middle), 100 mM NaCl, 20 mM HEPES, pH 7.4; 10 mM [Ni(L3)]2+ (right), 100 mM NaCl, 20 mM HEPES, pH 7.4. RF presaturation
applied for 2 s, B1 = 24 μT at 37 °C. The large peak arises from direct irradiation of water protons, set to 0 ppm.
sharp amide NH proton resonances and extremely broad
macrocycle proton resonances.3 The broad resonances are
consistent with the fluxional nature of the transition metal ion
macrocyclic complexes on the NMR time scale, presumably in a
process that interconverts between diastereomeric forms that
differ in pendent group and macrocycle backbone arrange-
ment.4 The [Ni(L2)]2+ complex also has amide NH proton
resonances that are more narrow than those of the macrocycle
backbone. [Ni(L2)]2+ is most likely six coordinate with two
bound amide pendents and two unbound pendents.11 The
proton NMR of this complex in CD3CN shows three amide
proton resonances that are slightly shifted downfield from the
diamagnetic region of the proton NMR spectrum at 6, 14, and
19 ppm. In addition, there is at least one highly shifted amide
proton resonance at 84 ppm and possibly a second one at 80
ppm in CD3CN that decrease in intensity upon addition of
D2O. The number of amide proton resonances is consistent
with the low symmetry of the [Ni(L2)]2+ complex, but the
broad nature of the proton resonances in the NMR spectrum
makes it difficult to obtain further information about
coordination geometry.
CEST spectra were obtained by applying a presaturation
pulse in 1 ppm increments and were plotted as normalized
water signal intensity (Mz/Mo %) against frequency offset
(ppm). Spectra were collected at 37 °C in the presence of 20
mM buffer and 100 mM NaCl, at pH 7.3−7.4 (Figure 1). The
CEST peaks of all three Ni(II) complexes clearly arise from the
amide NH protons that are identified by their signature
appearance as two highly separated resonances for the two
chemically inequivalent protons. For [Ni(L1)]2+ and [Ni-
(L3)]2+, the two CEST peaks for the distinct amide NH can be
identified as one highly shifted at 76 and 72 ppm, respectively,
and one close to solvent (11 ppm). [Ni(L2)]2+ shows only one
broadened CEST peak at 76 ppm from bulk water. The pH
dependence of the CEST peak is also consistent with amide
pendent groups. The CEST effect for [Ni(L1)]2+ increases
from pH 6.6 to 7.5 and slightly decreases at more basic pH,
consistent with base-catalyzed exchange (Figure S4). Super-
position of the three CEST spectra show that [Ni(L3)]2+ has
the most pronounced CEST peaks by 2−3 fold (Figure S5).
To validate the observed CEST spectra of the Ni(II)
complexes, CEST imaging was done on a 4.7 T scanner using a
phantom array containing solutions of 2, 4, or 8 mM Ni(II)
complex, 100 mM NaCl, 20 mM buffer, pH 7.4 at 37 °C. A pair
of gradient echo images was acquired with a presaturation pulse
either on-resonance or off-resonance of the exchangeable
protons ( 76 ppm for [Ni(L1)]2+ and [Ni(L2)]2+ or 73
ppm for [Ni(L3)]2+). The ratio between these two images is
subtracted from 100% to generate a CEST image. The
phantoms show that CEST increases with concentration of
the Ni(II) complex over the range 2−8 mM (Figure S6). At all
concentrations, [Ni(L3)]2+ showed more substantial CEST
images by 2−4 fold (Figures 2 and S7). The T1 and T2
Figure 2. CEST images of phantoms on a MRI 4.7 T scanner with a
pulse train comprised of five Gauss pulses at 12 μT for 1 s each,
interpulse delay of 200 μs applied symmetrically about the bulk water
resonance ( 73 ppm) Arrow: 20 mM HEPES buffer pH 7.4 and 100
mM NaCl only. Other samples contain [Ni(L3)]2+: D1 (2 mM), D2
(4 mM), D3 (8 mM) with 20 mM HEPES pH 7.4 and 100 mM NaCl
at 37 °C. Scale represents the percent loss of signal due to CEST
saturation pulse.
relaxivities were low for [Ni(L2)]2+ (0.097 and 0.15 mM−1 s−1)
and [Ni(L3)]2+ (0.012 and 0.092 mM−1 s−1). [Ni(L1)]2+ had
larger relaxivities of 0.21 and 0.39 mM−1 s−1 for T1 and T2,
respectively. The T1 values for the Ni(II) complexes are
comparable to or lower than those of Fe(II)3 and Eu(III)
complexes.15
It is important to consider why [Ni(L3)]2+ has a larger
CEST effect than do the other two complexes. For paraCEST
agents with protons that are in slow exchange on the NMR
time scale, the magnitude of the CEST effect is anticipated to
increase with the number of chemically equivalent exchangeable
protons and with increasing rate constants for proton exchange,
but to decrease with greater T1 relaxivity.1 [Ni(L3)]2+ has fewer
chemically equivalent exchangeable amide protons than does
[Ni(L1)]2+, and rate constants for the amide proton exchange
are similar for all three complexes (240−360 s−1, Figure S8)
suggesting that neither of these properties are responsible for
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dx.doi.org/10.1021/ja307909x | J. Am. Chem. Soc. 2012, 134, 18503−18505

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Notes
the larger CEST effect. However, the low T1 relaxivity value for
[Ni(L3)]2+ may contribute to a greater CEST effect as it is 5−
10-fold smaller than those of the other two complexes. In
addition, the narrow exchangeable proton resonances of
[Ni(L3)]2+ are beneficial for CEST because the magnetization
of these protons are more easily saturated with low pulse
power. The broader NH resonances of [Ni(L1)]2+ and
[Ni(L2)]2+ may result from longer T1e and correspondingly
greater proton relaxivity of these six-coordinate complexes, or
alternately from the fluxional character of these complexes on
the NMR time scale.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the Mark Diamond Research Fund of the Graduate
Student Association at the University at Buffalo, the State
University of New York and the John R. Oishei Foundation for
support.
REFERENCES
■
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The Ni(II) complexes were examined for their resistance to
dissociation in the presence of acid, metal cations and
biologically relevant anions, as preliminary studies to determine
suitability for in vivo applications. [Ni(L1)]2+ and [Ni(L2)]2+
were remarkably resistant to dissociation at acidic pH. No
detectable dissociation was observed for [Ni(L1)]2+ after
incubation for 4 h at pD 1.8, 37 °C (Figures S9−11 and
Table S2). [Ni(L2)]2+ showed intermediate tendency to resist
dissociation with 20% dissociation after 4 h. [Ni(L3)]2+ was the
most labile of the complexes and dissociated completely at
acidic pH within 1 h. Cu(II) displacement assays showed
similar results. There was no detectable dissociation of
[Ni(L1)]2+ or [Ni(L2)]2+ upon incubation of the complexes
with excess Cu(II) at 37 °C for 5 h (Figure S12). Neither did
biologically relevant concentrations of phosphate (0.40 mM)
and carbonate (25 mM) affect [Ni(L1)]2+ or [Ni(L2)]2+ as
shown by a similar CEST spectrum of these complexes
following incubation at 37 °C for five days (Figures S13 and
S14). In contrast, [Ni(L3)]2+ showed the appearance of new
proton resonances upon addition of 25 mM carbonate and 0.40
mM phosphate, signifying a change in the coordination sphere
by anion binding (Figure S15). Surprisingly, the CEST
spectrum of the complex was not markedly changed under
similar conditions (Figure S16). This suggests that the two
amide pendent groups remain coordinated and relatively
unaffected by the change in the coordination sphere of
[Ni(L3)]2+.
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In conclusion, we show here that Ni(II) complexes with
amide pendent groups are effective paraCEST agents with
paramagnetic induced proton shifts that are similar to those of
Fe(II) analogs. Ni(II) complexes of L1 and L2 are highly
resistant to dissociation in the presence of acid, metal cations or
anions which bodes well for their use in vivo. The Ni(II)
complex of L3, which is likely seven-coordinate, has very
narrow proton resonances that give rise to a pronounced CEST
peak. The other complexes are six-coordinate and show
dynamic behavior on the NMR time scale that, while producing
paraCEST, may lead to broadening of the peak. This shows that
both the dynamic nature of the Ni(II) complexes and their
coordination geometry are important in the development of the
NiCEST approach.
ASSOCIATED CONTENT
■
S
* Supporting Information
Materials and methods, synthesis, additional CEST and NMR
spectra. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
jmorrow@buffalo.edu
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dx.doi.org/10.1021/ja307909x | J. Am. Chem. Soc. 2012, 134, 18503−18505