A responsive europium(III) chelate that provides a direct readout of pH by MRI

Article abstract of DOI:10.1021/ja106018n

A europium(III) DO3A-tris(amide) complex is reported for imaging pH by MRI using ratiometric chemical exchange saturation transfer (CEST) principles. Deprotonation of a single phenolic proton between pH 6 and 7.6 results in an ～5 ppm shift in the water ex

Full text of DOI:10.1021/ja106018n

Published on Web 09/20/2010
A Responsive Europium(III) Chelate That Provides a Direct Readout of pH by
MRI
†
‡
†,§
†
,†,‡
Yunkou Wu, Todd C. Soesbe, Garry E. Kiefer, Piyu Zhao, and A. Dean Sherry*
Department of Chemistry, UniVersity of Texas at Dallas, P.O. Box 830668, Richardson, Texas 75083, AdVanced
Imaging Research Center, The UniVersity of Texas Southwestern Medical Center, 5323 Harry Hines BouleVard,
Dallas, Texas 75390, and Macrocyclics, Inc., 1309 Record Crossing, Dallas, Texas 75235
Received July 8, 2010; E-mail: sherry@utdallas.edu; dean.sherry@utsouthwestern.edu
3+
Previously reported Eu -based PARACEST agents have a highly
shifted water exchange peak that is independent of pH between 5
Abstract: A europium(III) DO3A-tris(amide) complex is reported
for imaging pH by MRI using ratiometric chemical exchange
saturation transfer (CEST) principles. Deprotonation of a single
phenolic proton between pH 6 and 7.6 results in an ∼5 ppm shift
in the water exchange CEST peak that is easily detected by MRI.
Collection of two CEST images at two slightly different activation
frequencies provides a direct readout of solution pH without the
need of a concentration marker.
5
and 8. More recently, we discovered that the chemical shift of the
3
+
Eu -bound water exchange peak can be altered considerably by
varying the electron density on a single amide oxygen donor in
14
these DOTA-tetraamide systems. This observation was later
15
expanded to multifrequency PARACEST agents, but again none
of those were pH sensitive. Given these prior observations, we
envisioned a new type of complex, Eu-1, wherein one of the amide
side chains is replaced by a ketone oxygen donor that is directly
conjugated to an ionizable group, in this case a phenol unit (Figure 1).
Magnetic resonance imaging (MRI) is one of the most widely
1
used, noninvasive imaging modalities in clinical medicine. One
reason for its popularity is that image contrast arises from inherent
differences in water proton densities and relaxation rates between
various tissue components. Even with these natural contrast
differences between tissues, exogenous contrast agents that alter
1 2 2
proton relaxation times (T , T , or T *) are still widely used to
2
enhance contrast between various tissue compartments.
A new type of image contrast based on a chemical exchange
3
saturation transfer (CEST) mechanism was recently introduced.
Paramagnetic versions of CEST agents have the potential of offering
3+
4
two major advantages over Gd -based imaging agents. First,
image contrast produced by a PARACEST agent can be switched
“on” or “off” by application of a frequency-selective radio frequency
pulse. This feature allows potential multiplexing of agents in a single
study. Second, since contrast in these systems is based on chemical
exchange of either liable protons or water molecules, the agents
are extremely sensitive to exchange rates (kex). This feature makes
them attractive for developing biologically responsive sensors.
PARACEST sensors using a variety of design platforms have been
Figure 1. Structure of the pH-responsive PARACEST agent, Eu-1.
Deprotonation of the phenolic proton results in conjugation of the resulting
3+
quinone-like structure with the acetyl oxygen atom coordinated to the Eu
5
,6
7
2+
8
9
reported for measuring pH, temperature, Zn , glucose, nitric
ion (bottom).
1
0
11
12,13
oxide, phosphate esters, and enzyme activity.
Most PARACEST sensors reported to date have a CEST signal
that changes intensity in response to external stimuli. This of course
requires a separate measure of agent concentration to obtain quantitative
results. Some exceptions include agents that use either a cocktail of
5
agents or single agents having multiple weakly shifted -NH
6
exchangeable protons for ratiometric imaging. The latter design feature
has the disadvantage of relying on exchange sites that are relatively
close to the bulk water frequency (typically <15 ppm). Here, we address
this problem by presenting a novel europium(III) DOTA-monoketone-
tris(amide) complex that has a single highly shifted water exchange
peak whose frequency varies as a function of solution pH. We
demonstrate that this single agent is nearly ideal for measuring pH by
use of ratiometric CEST imaging without the need of a second
concentration marker.
Figure 2. UV-vis spectra of Eu-1 (20 µM) recorded as a function of
solution pH. The arrow indicates the direction of the absorbance changes
with increasing pH. Insert: titration curve showing the increase in absorbance
at 360 nm as a function of pH.
†
University of Texas at Dallas.
‡
The absorption spectrum of Eu-1 displays a bathochromic shift from
310 to 360 nm as the phenolic proton is removed (Figure 2), consistent
University of Texas Southwestern Medical Center.
§
Macrocyclics, Inc.
1
4002 9 J. AM. CHEM. SOC. 2010, 132, 14002–14003
10.1021/ja106018n  2010 American Chemical Society

C O M M U N I C A T I O N S
with extended delocalization of the phenolate anion through the π
that Eu-1 will also be effective for ratiometric CEST imaging of
system to form the quinone-like structure. This would place a more
negative charge on the carbonyl oxygen atom coordinated to the Eu
pH at physiological temperatures as well (Figure S8).
3+
ion (Figure 1). The pK derived from these optical data (6.7 ( 0.1) is
a
nearly ideal for pH measurements in biological systems. One would
predict a priori that such a resonance structure would also alter the
water exchange rate and potentially the frequency of the exchanging
15
water molecule. The CEST spectra of Eu-1 recorded at five different
pH values (Figure 3) showed a surprisingly large change in exchange
frequency over this same pH range. The pK of Eu-1 derived from
a
the CEST data was 6.5 ( 0.1 (Figure S2), nearly identical to the value
determined optically.
Figure 4. Images of a phantom containing either water (w) or 10 mM
Eu-1 adjusted to the indicated pH (9.4 T, 298 K). (a) Proton density images,
(
b) ratio water intensities after activation at 54 versus 47 ppm, and (c)
calculated pH values as determined by ratiometric CEST imaging.
3
+
In summary, a novel Eu -based PARACEST agent is reported
that provides a concentration independent measure of pH by
ratiometric CEST imaging. A unique aspect of this agent is that
the ratiometric image data can be collected at CEST activation
frequencies widely separated from the bulk water frequency and
Figure 3. pH dependence of CEST spectra for Eu-1 recorded at 9.4 T and
2
98 K. Insert: expanded view of the water exchange peak as a function of
3+
pH. [Eu ] ) 10 mM, B ) 14.1 µT, and saturation time ) 2 s.
1
a
the pK of the sensor is nearly ideal for imaging pH over a range
This unusual CEST feature suggests it may be possible to image
pH directly using Eu-1 and ratiometric CEST imaging. For example,
the ratio of CEST intensities after activation of Eu-1 at 55 versus
of interest for detecting abnormal physiology. The basic platform
technology reported here will likely prove useful in the design of
other types of responsive imaging agents as well.
4
9 ppm was found to be nearly linear between pH 6.0 and 7.6 and,
Acknowledgment. Financial support from the National Institutes
of Health (CA115531, RR02584, and EB004582) and the Robert
A. Welch Foundation (AT-584) is gratefully acknowledged.
more importantly, independent of Eu-1 concentration (Figure S5).
Other combinations of activation frequencies gave similar results.
Thus, Eu-1 offers several advantages over previously reported
ratiometric pH indicators. First, the pH measurement can be made
using a single reagent rather than a cocktail of agents, and, second,
the exchange peak in Eu-1 is shifted well away from the frequency
Supporting Information Available: Complete synthetic details,
CEST results, and ratiometric plots are provided. This material is
available free of charge via the Internet at http://pubs.acs.org.
5
of solvent protons so it can be activated without concern about
6
,16,17
partial off-resonance saturation of bulk water protons.
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To demonstrate the simplicity of using this agent for imaging pH
by MRI, CEST images of a phantom prepared from five Eu-1 samples
adjusted to different pH values (plus a control sample lacking Eu-1)
were collected at two presaturation frequencies, 54 and 47 ppm. The
CEST intensity ratio in these two images is shown in Figure 4b as a
color map. The sample containing water alone showed nearly perfect
cancellation while the CEST ratio in samples of Eu-1 varied from 0.43
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1
8
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(
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1
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JA106018N
J. AM. CHEM. SOC. 9 VOL. 132, NO. 40, 2010 14003