Amplifying the Sensitivity of Zinc(II) Responsive MRI Contrast Agents by Altering Water Exchange Rates

Article abstract of DOI:10.1021/jacs.5b09158

Given the known water exchange rate limitations of a previously reported Zn(II)-sensitive MRI contrast agent, GdDOTA-diBPEN, new structural targets were rationally designed to increase the rate of water exchange to improve MRI detection sensitivity. These new sensors exhibit fine-tuned water exchange properties and, depending on the individual structure, demonstrate significantly improved longitudinal relaxivities (r₁). Two sensors in particular demonstrate optimized parameters and, therefore, show exceptionally high longitudinal relaxivities of about 50 mM^-1 s^-1 upon binding to Zn(II) and human serum albumin (HSA). This value demonstrates a 3-fold increase in r₁ compared to that displayed by the original sensor, GdDOTA-diBPEN. In addition, this study provides important insights into the interplay between structural modifications, water exchange rate, and kinetic stability properties of the sensors. The new high relaxivity agents were used to successfully image Zn(II) release from the mouse pancreas in vivo during glucose stimulated insulin secretion.

Full text of DOI:10.1021/jacs.5b09158

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Article
Amplifying the Sensitivity of Zinc(II) Responsive MRI
Contrast Agents by Altering Water Exchange Rates
Jing Yu, André F. Martins, Christian Preihs, Veronica Clavijo Jordan, Sara
Chirayil, Piyu Zhao, Yunkou Wu, Khaled Nasr, Garry E Kiefer, and A. Dean Sherry
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b09158 • Publication Date (Web): 14 Oct 2015
Downloaded from http://pubs.acs.org on October 20, 2015
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Amplifying the Sensitivity of Zinc(II) Responsive MRI Contrast
Agents by Altering Water Exchange Rates
Jing Yu,† André F. Martins,† Christian Preihs,‡ Veronica Clavijo Jordan,‡ Sara Chirayil,‡ Piyu
Zhao,† Yunkou Wu,‡ Khaled Nasr,‡ Garry E. Kiefer,†,& 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, Dal-
las, Texas 75390, and &Macrocyclics, Inc., 1309 Record Crossing, Dallas, TX 75235
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ABSTRACT: Given the known water exchange rate limitations of a previously reported Zn(II)-sensitive MRI contrast
agent, GdDOTA-diBPEN, new structural targets were rationally designed to increase the rate of water exchange to im-
prove MRI detection sensitivity. These new sensors exhibit fine-tuned water exchange properties and, depending on the
individual structure, demonstrate significantly improved longitudinal relaxivities (r1). Two sensors in particular demon-
strate optimized parameters and therefore show exceptionally high longitudinal relaxivities of about 50 mM-1s-1 upon
binding to Zn(II) and human serum albumin (HSA). This value demonstrates a 3-fold increase in r1 compared to that dis-
played by the original sensor, GdDOTA-diBPEN. In addition, this study provides important insights into the interplay
between structural modifications, water exchange rate, and kinetic stability properties of the sensors. The new high re-
laxivity agents were used to successfully image Zn(II) release from in the mouse pancreas in vivo during glucose stimulat-
ed insulin secretion.
1. INTRODUCTION
water protons. The first Zn(II) responsive MRI contrast
agent reported in 200112 was designed to detect a change
in water access to the inner-sphere of a Gd(III) ion upon
Zn(II) binding. Since then, several other designs based
on changes in q in response to Zn(II) have been report-
ed13–16 but none show particularly large changes in r1
relaxivity in response to Zn(II). Even so, a Mn(II) por-
phyrin derivative did show signal enhancement in brain
regions known to contain the highest Zn(II) levels but
this required direct injection of the agent.14 This obser-
vation demonstrated the feasibility of detecting Zn(II) in
tissues by T1-weighted imaging. In 2009, a new type of
Zn(II) sensor design based on a change in molecular
rotation, τR, was reported.17 Upon binding of two Zn(II)
ions to the high affinity N,N-bis(2-pyridyl-methyl) eth-
ylene diamine (BPEN) sites on GdDOTA-diBPEN 1 (cf.
Figure 1), the resulting ternary GdL-(Zn)2 complex binds
to site 2 of subdomain IIIa in human serum albumin
(HSA). This results in slowing of molecular rotation and
a change in r1 from 5.0 mM-1s-1 to 17.5 mM-1s-1 at 0.47 T.
Although the increase in r1 is not nearly this large at 9.4
T, where subsequent mouse imaging experiments were
performed, even a 2-fold change in r1 was sufficient to
detect Zn(II) ions co-released with insulin from pancre-
atic β-cells.18 The approach taken in that imaging study
was to administer a dose of GdDOTA-diBPEN 1 such
that its extracellular concentration is near the detection
limit of MRI (i.e. 0.025 mmol/kg), then assign any in-
crease in image intensity in the pancreas after a bolus of
glucose, to an increase in local Zn(II) released from β-
cells. Although the method proved useful for detecting
As the second most abundant transition metal in mam-
malian tissue, divalent zinc (Zn(II)) plays a critical role
in many cellular processes including structural, catalyt-
ic, and signal transduction processes.1 The total concen-
tration of Zn(II) in blood is 12-16 µM, mostly in chelated,
protein-bound forms.2 Zn(II) concentrations are particu-
larly high in the pancreas,3 prostate4 and brain,5 all tis-
sues that require Zn(II) for signal transduction. Pancre-
atic beta cells store insulin with two equivalents of
Zn(II) in granules and release Zn(II) with insulin in re-
sponse to an increase in plasma glucose. Upon release of
insulin, the local concentration of Zn(II) in the vicinity
of the beta cells rises transiently to ~450 ꢀM6 and may
signal other cells in the same islet.7 Zn(II) is tightly
regulated by multiple different transporters and imbal-
ances in Zn(II) content in these various tissues is associ-
ated with diabetes, Alzheimer’s disease, and prostate
cancer.8 For more than two decades, extensive efforts
have been devoted to the development of optical sen-
sors for detection of free Zn(II) ions.9,10 Optical Zn(II)
sensors offer an appropriate detection sensitivity but
show limited applicability for monitoring Zn(II) levels in
vivo.11 Thus, optical-based Zn(II) sensors have been
largely restricted to cell-based imaging. Magnetic reso-
nance imaging (MRI) is an attractive modality for imag-
ing physiology in vivo because tissue penetration is not a
limiting factor. Unfortunately, MRI is inherently much
less sensitive than optical imaging which is why MR
reporter molecules cannot be detected directly but ra-
ther must be detected indirectly through the abundant
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Figure 1. Chemical structures of GdDOTA-diBPEN 1 and the modified Zn(II) sensors Gd-2-6 reported in this work.
the expansion of pancreatic tissue in mice fed with a
high fat diet over 12 weeks, it would be highly desirable
to modify the structure of GdDOTA-diBPEN 1 to amplify
the sensitivity of the agent for detecting Zn(II) release
from secretory tissues.
2. RESULTS AND DISCUSSION
2.1. Synthesis of Sensors
GdDOTA-diBPEN 1 was prepared as reported previous-
ly.17 The macrocyclic Gd complexes, Gd-2 to Gd-6, in
Figure 1 were prepared using synthetic procedures fully
described in Supporting Information. Each Gd(III) com-
plex was purified and characterized using standard
It is well-known that the water exchange rate (kex)
in bis-amide derivatives like GdDOTA-diBPEN 1 (cf.
Figure 1) is typically 20-50 fold slower than that consid-
ered optimal for achieving a maximal increase in r1
when the agent binds to a macromolecule.19 This sug-
gested to us that the r1 relaxivity of GdDOTA-diBPEN-
(Zn(II))2 when bound to albumin is likely limited by
slow kex. Based on other known τM (τM = kex-1) values of
modified derivatives of GdDOTA, a series of new com-
plexes were designed with a goal of increasing the rate
of water exchange from the inner coordination sphere of
the Gd(III) ion while preserving the Zn(II) binding sites
and, hopefully, albumin-binding characteristics of
GdDOTA-diBPEN 1. In compound Gd-2, two phos-
phinate groups were introduced as oxygen donors to
introduce greater steric hindrance around the Gd(III)-
water binding site.20 This should in principle increase
the rate of water exchange. The two piperazine units in
compound Gd-3 were introduced to increase the popu-
lation of the twisted square antiprism (TSAP) isomer, a
coordination isomer known to display much faster water
exchange.21,22 In compounds Gd-4, Gd-5, and Gd-6, an
extra methylene carbon was included in either an ace-
tate (Gd-4) or acetamide (Gd-5 and Gd-6) side-chain, a
modification also known to increase steric hindrance
around the Gd(III)-water coordination site.23 The impact
of expanding the chelate ring size on τM can be rather
dramatic. For example, a structural analogue of GdDO-
TA bearing an extra methylene carbon on one acetate
arm exhibits a 15-fold faster water exchange rate com-
pared to GdDOTA.23,24 Given this prior information,
compounds Gd-4, Gd-5, and Gd-6 were predicted to
display considerably faster water exchange rates.
1
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methods (preparative HPLC, H NMR, C NMR and LC-
MS). Those details can also be found in the Supporting
Information section.
2.2. 17O NMR Measurements
To evaluate the principal physical parameters that gov-
ern r1 relaxivity, 25 mM samples of each Gd(III) complex
were prepared in 5%-enriched 17O water for 17O T1 and T2
measurements over the temperature range 277-333 K.
Figure S2 summarizes the temperature dependence of
the reduced 17O chemical shifts (ꢁωr), transverse (1/T2r)
and longitudinal (1/T1r) relaxation rates for all six com-
plexes. τR and kex were determined by fitting the longi-
tudinal (T1r) and transverse (T2r) data simultaneously to
paramagnetic relaxation theory.25,26 The 17O transverse
relaxation rates for GdDOTA-diBPEN 1 increase with
temperature above 333 K, indicating that water ex-
change lies in the slow-to-intermediate exchange regime
where 1/T2r provides a direct measure of kex. The τM =
1/kex value obtained for GdDOTA-diBPEN 1 at 298 K
(1362 ns) is consistent with previous observations that
replacement of carboxylate by an amide typically de-
creases the rate of water exchange by 3-4 fold. The τM
values found for Gd-4 and Gd-5 (190± 7 and 130± 2 ns,
respectively) were ~7-fold and ~11-fold shorter than the
τM found for GdDOTA-diBPEN 1, and even modestly
shorter than the τM of GdDOTA (243 ns).26 This nicely
demonstrates that introducing one extra carbon spacer
into either the acetate or amide coordinating side-chain
had the anticipated impact of increasing the rate of wa-
ter exchange. Clearly, introducing an extra methylene
carbon in the acetamide side-chain had a larger impact
on water exchange than expansion of the acetate side-
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10-fold faster than GdDOTA-diBPEN 1. This series illus-
chain. This effect was further amplified in Gd-6, where
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extending both amide side-chains by one carbon de-
trates that one can use a variety of different coordina-
tion chemistry principles to modify water exchange
creased τM by another ~35-fold to 3.7 ± 0.1 ns.
298
rates in Gd(III) complexes. The τRO values for these
The τM values summarized in Table 1 show that all of the
new Zn(II) sensors display faster water exchange than
GdDOTA-diBPEN 1. The largest change was observed
for the phosphinate derivative (Gd-2) and the bis-amide
complex with extra methylene carbons on both append-
ed amide side-chain ligating groups (Gd-6). According
to paramagnetic relaxation theory, complexes with τM
values in this range (<5 ns) are too short to achieve an
optimal r1 when bound to a protein. The τM value meas-
ured for Gd-3 (8.7 ± 0.1 ns) was closest to the value con-
sidered optimal for achieving maximal r1 relaxivity.19
This finding suggests that insertion of the bulky cyclo-
hexane groups likely increased the population of TSAP
isomer. To verify that this is indeed the origin of this
increase in τM, a sample of Eu-3 was prepared for high
resolution 1H NMR. The spectrum (cf. Figure S4.1) veri-
fied that the fraction of TSAP isomer in this complex
was ~80%, much larger than the TSAP fractions in Eu-2
(~66%), Eu-4 (~5%), or Eu-5 (<5%) or EuDOTA-diBPEN
(~40%) (cf. Figure S4.2-S4.4).17 Finally, sensors Gd-4 and
Gd-5 with one extra methylene carbon inserted into one
ligating side-chain displayed water exchange rates about
complexes calculated from the 17O T1 data were all in the
range 0.3-0.4 ns, indicating that these complexes rotate
more slowly than GdDOTA, as one would expect on the
basis of molecular weight.27,28 The inner-sphere q value
of each complex was estimated from the 17O chemical
shift of the fully-bound water molecule (δꢀ).29 All q val-
ues obtained were close to 1, except for sensor Gd-2,
where q was found equal to be 0.41 (Table 1). This likely
reflects the presence of multiple coordination isomers in
solution, perhaps one with q = 0 (59%) and one with q =
1 (41%). Complexes with q = 0 have been observed previ-
ously in a variety of phosphonate and phosphinate com-
plexes.30 To confirm the q values measured from 17O
NMR, q was also evaluated by luminescence lifetime
measurements on the corresponding Eu(III) complexes.
Those measurements gave q values of 0.44 and 0.92 for
Eu-2 and Eu-6, respectively, indicating the q values ob-
tained by 17O NMR on the Gd complexes are in good
agreement with those measured by luminescence life-
time methods.
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2.3. Relaxivity Measurements
Table 1. Fitted physical parameters, HSA binding constants, and r1 relaxivities (mM-1s-1) measured at 0.47 T at 310 K.
GdDOTA-diBPEN
1.02
Gd-2
0.41
Gd-3
0.96
Gd-4
0.98
Gd-5
1.01
Gd-6
0.92
q
τRO298 (ns)
kex298 (106 s-1) /
kex310 (106 s-1)
0.4 ± 0.1
0.3 ± 0.1
0.4 ± 0.1
0.3 ± 0. 1
0.3 ± 0. 1
0.3 ± 0.1
0.72±0.1 /
1.1±0.1
220±2 /
350±3
110±2 /
250±1
5.3±0.2 /
6.3±0.2
7.8±0.1 /
9.2±0.1
270±3 /
490±4
298τM (ns) / 310τM (ns)
4.5±0.1 /
2.8±0.1
8.7±0.1 /
4.0±0.1
3.7±0.1 /
2.0±0.1
1400±100 / 910±60
190±7 / 160±5
130 ±2 / 110±1
r1 (mM-1s-1)
5.0±0.1
6.6±0.1
3.7±0.1
4.9±0.2
20.8 ±0.5
7.6±0.3
6.5±0.1
6.4±0.2
7.0±0.2
47.6 ±1.2
6.2±0.2
7.0±0.3
50.1 ±1.2
4.1±0.1
3.5±0.1
r1 sensor + 2Zn(II) (mM-1s-1)
r1 sensor + 2Zn(II) + albumin
(mM-1s-1)
17.4 ±0.5
27.9 ±0.8
15.6 ±0.6
KD with HSA (µM)a
42b
-
383 ± 60
23.8 ± 2
227 ± 52
29.7 ± 1
48 ± 15
42 ± 15
130 ± 25
14.2 ± 1
r1c (mM-1s-1) a
48.4 ± 10
54.8 ± 7
a Obtained by fitting proton relaxation enhancement data to equation (1). bfrom Ref. 17.
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The r1 relaxivity of each new sensor in the absence and each complex on this curve, additional r1 measurements
1
presence of Zn(II) and HSA are compared with the cor-
responding values for GdDOTA-diBPEN 1 in Table 1. In
the absence of Zn(II) and HSA, the r1 values of complex-
es roughly parallel the molecular weights of the com-
plexes in solution, with the possible exception of Gd-6.
Given that q for Gd-6 as measured by two different
methods seems to be near 1 and the complex was pure
by all analytical measurements, the origin of the unusu-
ally low r1 relaxivity of this complex remains unknown.
Like GdDOTA-diBPEN 1, the r1 relaxivities of the new
sensors did not change significantly upon addition of
two equivalents of Zn(II) (as ZnCl2), but did increase
dramatically upon addition of both Zn(II) and a physio-
logical amount of HSA (600 µM). This indicates that the
five new Zn(II) sensors reported here retain their ability
to bind to HSA in the presence of Zn(II) ions and bind-
ing significantly slows molecular rotation and increases
r1. Relaxivity theory predicts that the r1 of each Gd(III)
sensor-Zn(II)-HSA adduct will depend upon the water
exchange rates with the complexes bound to HSA.19 Un-
were performed in the presence of Zn(II) and HSA at 333
K (Table S2). As expected, the r1 relaxivities of GdDOTA-
diBPEN 1, Gd-4 and Gd-5 were higher at 333 K while the
r1 relaxivities of Gd-2, Gd-3 and Gd-6 were lower. Given
that the rate of water exchange should increase with
temperature, this validates the positioning of the
GdDOTA-diBPEN, Gd-4 and Gd-5 date on the “slow
side” of the peak maximum of Figure 2 and Gd-2, Gd-3
and Gd-6 on the “fast side” of this peak maximum. The
observation that the data for GdDOTA-diBPEN, Gd-4
and Gd-5 fall at least near this theoretical curve suggests
but does not prove that kex in these three complexes is
not altered upon binding to HSA while kex may differ
when Gd-2, Gd-3 or Gd-6 bind to the protein. It is also
known that the r1 relaxivity of Gd(III) complexes such as
these are magnetic field dependent and the data in Ta-
ble S3 (Supporting Information) show that the r1 relaxiv-
ities of GdDOTA-diBPEN, Gd-4 and Gd-5 all decrease
significantly between 0.5 T and 9.4 T. The change in r1
relaxivity is not large for the complexes in aqueous solu-
tion but is quite dramatic for the Gd-4 and Gd-5 when
bound to HSA. These data suggest that there should be
a sensitivity advantage in detecting Zn(II) release from
the pancreas at lower magnetic fields (1.5T or lower) but
this advantage could be partially offset by the inherent
lower 1H sensitivity at the lower magnetic field.
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fortunately, we were unable to use O NMR techniques
to measure τM for the HSA-bound sensors because of
limitations in concentration imposed by the protein.
Despite this limitation, it is useful to compare the exper-
imental r1 values measured in the presence of HSA with
the τM values measured in the absence of Zn(II) and pro-
2.4. Albumin Binding Measurements
It is well known that HSA can bind many different types
of substrates via site 1 or 2 of subdomain IIA or IIIA.
Both are characterized by hydrophobic pockets sur-
rounded by a positively charged external surface.26 Stud-
ies indicate that, in the case of amphiphilic molecules,
like MS-325 and MP-2269, the hydrophobic side-chains
in these molecules bind in these hydrophobic pockets
on the protein, while the Gd(III) chelate has minimal
interactions with the protein surface.31–34 Nevertheless, it
has been found that the rate of water exchange between
a Gd(III) complex can be reduced quite substantially
upon binding of the agent to HSA.31 Interestingly, it has
Figure 2. A plot of r1 for each Zn(II) sensor when bound
to HSA versus τM measured for each unbound complex-
es in aqueous buffer at 310 K. The solid line shows the
relationship predicted by paramagnetic relaxation theo-
ry at 0.47 T for a molecule with τR = 10 ns. The data
point labeled Gd-2’ is the relaxivity value for Gd-2 (q =
0.41) after normalization to q = 1. Other parameters
used in calculating the theoretical curve include rGd-O
3.1 Å, q = 1 and T1e = 5 ns.
=
tein (Figure 2). If the rate of water exchange is unal-
tered upon binding of these agents to HSA, then the r1
values measured in aqueous solution in the absence of
HSA should reasonably fit the theoretical plots shown in
Figure 2. Three of the six complexes (GdDOTA-diBPEN
1, Gd-4 and Gd-5) agree reasonably well with theory
while three other complexes (Gd-2, Gd-3 and Gd-6) do
not. To validate the positioning of the data point for
Figure 3. Proton relaxation enhancement of Gd-3-(Zn(II))2
and Gd-4-(Zn(II))2 as a function of increasing concentration of
HSA at 0.47 T and 310 K in 100 mM Tris buffer at pH 7.5. The
solid line is the best fit of the data to equation 1 while the
dashed and dotted lines are simulated curves for a fixed fully-
bound r1 relaxivity (29.7 mM-1s-1 for Gd-3 and 48.4 mM-1s-1 for
Gd-4) with different dissociation constants (KD). The simulat-
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Gd(III) and the Zn(II)-DPA units have a substantial in-
also been shown that the rate of water exchange in vari-
1
2
3
4
5
6
7
8
ous Ln(III) derivatives of MS-325 is quite sensitive to
relatively minor structural differences between albumin
from different mammalian sources35 and when various
Mn(II) complexes bind to albumin.36 For the Zn(II) sen-
fluence on the binding interactions on HSA. Interesting-
ly, 1, Gd-4 and Gd-5, the three complexes with the high-
est affinity for HSA, fall onto the theoretical r1 curve of
Figure 2 while Gd-2, Gd-3 and Gd-6, those with the low-
est affinity for HSA, do not. The abnormally low r1 of
Gd-2 can be partly explained by this complex having a q
value less than 1 (Table 1 and Figure 2, mark Gd-2’) while
Gd-3 and Gd-6 must fall off this relationship for other
reasons. It is also interesting to observe that these same
three complexes have a weaker binding affinity for HSA.
Given that the propensity of Gd-3 to favor the TSAP
form in solution, it is reasonable to assume that this
complex may favor a different coordination isomer
when bound to the protein and thereby may have quite
a different water exchange rate. Similarly, the side-chain
groups on Gd-6 are highly flexible so this complex may
also adopt a different structure when bound to HSA.
(1)
9
sors presented here, HSA binding takes place mostly by
the electrostatic interaction between the Zn(II)-DPA
subunits and the tyrosine residue Y411 in the pocket site
II.37 The binding affinity of each Zn(II) sensor to HSA
was estimated by proton relaxation enhancement (PRE)
titrations, a relaxometric technique commonly used to
determine the dissociation constants (KD = 1/KA) for
binding of Gd(III) complexes to HSA.17 This experiment
consists of measuring the proton relaxation rates R1obs at
increasing concentrations of the protein at a fixed con-
centration of complex (Figure 3). Data such as these
were fitted to equation (1), where r1f and r1c are the pro-
ton relaxivities of the free and the bound state, cHSA and
c1 are the concentration of HSA and of the complex, re-
spectively, and n determines the number of binding
sites on the protein. Assuming that the complexes only
bind to HSA-binding site 2 of subdomain IIIA (n = 1), a
fit of the data to eqn. 1 revealed relatively strong binding
for 1, Gd-4 and Gd-5 with HSA (KD ~ 42-48 ꢀM), in good
agreement with literature values17 while Gd-2, Gd-3 and
Gd-6 exhibit lower affinities with HSA (383 ± 60 ꢀM, 227
± 52 ꢀM, 130 ± 25 ꢀM, respectively). This suggests that
the length and flexibility of the side-chains between the
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With the exception of Gd-6, the other four new com-
plexes displayed higher r1 values when bound to HSA
than GdDOTA-diBPEN 1, consistent with faster water
exchange. Gd-4 and Gd-5 in particular exhibit remarka-
bly high relaxivities of 47.6 ± 1.2 and 50.1 ± 1.2 mM-1s-1,
respectively, when bound to Zn(II) and HSA (Table 1).
These values, about 3-fold higher than the correspond-
ing r1 value of GdDOTA-diBPEN 1-Zn(II)-HSA, suggest
that detection of Zn(II) released from the pancreas in
response to glucose stimulation should be about 3-fold
more sensitive when using these newer agents at 0.47 T.
This difference however would not be nearly as large at
9.4 T.
2.5. In Vivo Imaging of Glucose-Stimulated Insulin
Figure 4. A comparison of normalized signal enhancement in pancreatic tissues of mice after infusion of two different Zn(II)
sensors followed by a bolus of glucose to stimulate insulin secretion. The images were collected at 11 minutes post glucose in-
jection. The top panel shows the time-dependent infusion protocol. Gd-HPDO3A (ProHance®) and no agent were used as con-
trols. The portion of the pancreas that could be identified in these slices is outlined in yellow. The bar graph shows the % sig-
nal enhancement of normalized pancreatic tissue calculated from ROIs of images before and after glucose stimulation (n = 5).
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and Zn(II) Release from the Mouse Pancreas.
plexes for clinical translation, was assessed by use of a
published trans-metalation method.39 Here, the Gd(III)
complexes were incubated with four equivalents of
Zn(II) in phosphate buffer (pH = 7), two equivalents of
which are expected to coordinate with the DPA subunits
present in each sensor and the remaining two equiva-
lents of Zn(II) are intended to gradually replace the
Gd(III) from its macrocyclic binding site if the complex
is kinetically labile on the time-scale of a few days. Any
Gd(III) released by the macrocyclic ligands will in turn
precipitate from solution as insoluble gadolinium phos-
phate, and, as a consequence, the T1 of the water protons
will increase. The kinetic inertness of each new com-
pound was compared with that of the FDA-approved
contrast agent Magnevist® (GdDTPA, cf. Figure 5). As
anticipated, all six of the macrocyclic complexes were
kinetically more inert than the non-cyclic Gd(III) che-
late Magnevist. In particular, Gd-2 was the most inert
complex in this series, with essentially no trans-
metalation occurring over 7 days. Gd-3 and GdDOTA-
diBPEN 1 exhibited similar kinetic inertness with their
relaxation rates dropping only slightly in the beginning
of the experiment. Gd-4 and Gd-5 were somewhat more
labile but were still noticeably more inert than Magne-
vist. Gd-6 was the most labile complex among this se-
ries. The combined data show that the coordinating
phosphinate groups in Gd-2 can dramatically stabilize
the complex while an extra methylene spacer in the car-
boxylate ligand results in reduced kinetic stability.
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We previously reported that GdDOTA-diBPEN 1 detects
release of Zn(II) ions from pancreatic β-cells in response
to an increase in blood glucose by MRI.18 Given that two
of the new Zn(II) sensors, Gd-4 and Gd-5, display about
a 3-fold improvement in r1 relaxivity upon binding to
Zn(II) and HSA in vitro in comparison to GdDOTA-
diBPEN, additional in vivo imaging experiments were
performed in mice to evaluate whether this enhanced in
vitro relaxivity translates to improved signal enhance-
ment of the pancreas in vivo. In separate experiments,
three different agents were infused into C57Bl/6 mice
using an identical infusion protocol to compare their
effectiveness. Figure 4 summarizes the imaging protocol
(top panel) and shows typical T1-weighted MR images of
mice in axial view at 10-15 min after stimulation of Zn(II)
release by glucose. Initially, each agent was infused at 5
ꢀl/min for ~30 minutes while monitoring the image in-
tensity of the kidneys. Once constant enhancement of
the kidneys was observed, 50 µl of 20% w/v D-glucose
was injected into the intraperitoneal (IP) space while
continuous monitoring of the pancreas by sequential T1-
weighted MRI. Maximal enhancement was observed at
10-15 minutes after glucose injection correlating with our
previous reports of zinc/insulin co-release after stimula-
tion.18 Figure 4 summarizes the % signal enhancement
of selected ROIs of pancreas at 11 minute time point af-
ter glucose stimulation. While significantly higher signal
enhancement of the pancreas was detected during infu-
sion of GdDOTA-diBPEN (as reported previously) and
Gd-5 when compared to Gd-HPDO3A, the % signal en-
hancement differences between GdDOTA-diBPEN and
Gd-5 did not reach statistical significance (p < 0.125).
There may be several reasons why the higher in vitro r1
of Gd-5 did not translate to greater tissue enhancement
in vivo including, 1) given that the pancreas is not a solid
organ but rather a thin tissue, there are likely significant
variations in selection of identical ROI’s in every mouse
and, 2) the r1 difference between GdDOTA-diBPEN ver-
sus Gd-5 when bound to Zn(II) and HSA is only 1.3-fold
greater at 9.4T compared the much larger difference
observed at 0.47T. The fact that we do detect a trend
toward higher signal enhancement using Gd-5 com-
pared to GdDOTA-diBPEN (Figure 4) is encouraging
because it suggests that the new higher relaxivity agents,
Gd-4 and Gd-5, will show significantly improved signal
enhancement upon Zn(II) release from the pancreas at
clinical imaging fields.
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Figure 5. Normalized T1 relaxivity rates for GdDOTA-
diBPEN 1 = ꢀ, Gd-2 = ꢀ, Gd-3 = ꢁ, Gd-4 = ꢁ, Gd-5 = ꢂ,
Gd-6 = ꢃ, and GdDTPA = ꢀ as a function of time. Each
complex was initially 1.5 mM in 30 mM sodium phosphate
buffer (pH = 7) in the presence of 6 mM of Zn(II). The
samples were maintained at 310 K. The symbols corre-
spond to experimental data points while the solid lines
represent a biexponential fit of the experimental data.
2.6. Kinetic Inertness
Thermodynamic stability and kinetic inertness are two
critical parameters for the successful translation of new
agents into clinical medicine. The new agents presented
here based on the DO2A scaffold typically exhibit stabil-
ity constants in the range of log KML = 20 or higher.38
Therefore, thermodynamic stability should not be a lim-
iting factor. Kinetic inertness, arguably the more im-
portant factor in determining the viability of these com-
3. CONCLUSIONS
A series of Zn(II)-sensitive MRI sensors was designed to
fine-tune the rate of water exchange from the inner-
sphere of Gd(III) in order to maximize the r1 of the
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Journal of the American Chemical Society
SUPPORTING INFORMATION
complexes bound to Zn(II) and HSA. Two complexes in
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particular, Gd-4 and Gd-5, displayed r1 relaxivity values
close to 50 mM-1s-1. This illustrates that optimizing the
water exchange rates can be a successful molecular de-
sign strategy to construct more sensitive MRI contrast
agents for Zn(II) detection. The 3-fold increase in r1
measured for two of the new agents in vitro at 0.47T did
not result in significantly improved signal enhancement
of the mouse pancreas in vivo at 9.4T. Nevertheless, we
anticipate that the higher r1 relaxivity of these new
agents will be much more evident when imaging larger
animals at clinical imaging fields.
Supporting information includes general methods, supple-
mentary schemes, synthetic protocols, and analytical data
for the new compounds.
AUTHOR INFORMATION
Corresponding Author
A Dean Sherry, PhD
Director, Advanced Imaging Research Center
Professor of Radiology
UT Southwestern Medical Center, Dallas TX 75390-8568
214-645-2730
E-mail: dean.sherry@utsouthwestern.edu;
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It should be noted that MR-responsive agents such as
those described herein offer the possibility of detecting
only qualitative changes in the amount of Zn(II) re-
leased from secretory tissues, not quantitative total
Zn(II) concentrations in tissue. Nonetheless, MRI-based
Zn(II) sensors such as these can provide added insights
into physiological events occurring in vivo that are simp-
ly not available with other molecular imaging modalities.
Given that binding affinity of HSA for Zn(II) is ~30 nM40
and the affinity of these BPEN-based Gd(III) complexes
for Zn(II) is also around 33 nM (add ref 2 here), one
should consider the relative concentrations of the agent
(~50 ꢀM) and HSA (~0.6 mM) in the extracellular space
around β-cells to gain some insight into the various
Zn(II) species that can potentially be formed upon re-
lease of Zn(II) ions from β-cells. The concentration dif-
ferences between the Gd(III) sensor and HSA suggests
that most of the Zn(II) released from β-cells should bind
directly with HSA and not the Gd(III) sensor, assuming
of course that the Zn(II) binding sites on HSA are not
already occupied. Given that HSA is considered to be a
Professor of Chemistry
UT Dallas, Richardson, TX 75083-3021
972-883-2907
E-mail: sherry@utdallas.edu
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENTS
Financial support from the National Institutes of Health
(P41-EB015908, R01-DK095416 and, in part, by the Harold
C. Simmons Cancer Center through an NCI Cancer Center
Support Grant, 1P30-CA142543), the American Diabetes
Association (7-12-MN-76 and 7-12-IN-42), and the Robert A.
Welch Foundation (AT-584) is gratefully acknowledged.
ABBREVIATIONS
q, hydration number; τR, rotational correlation time; τM,
water residence lifetime; kex, water exchange rate; r1, T1
relaxivity; rGd-O, gadolinium–water distance; T1e, electronic
relaxation time; TSAP, Twisted Square Antiprism; DPA, di-
(2-picolyl)amine; HSA, human serum albumin; KD, dissoci-
ation constant; KA, association constant; BPEN, N,N-bis(2-
pyridyl-methyl) ethylene diamine; DTPA (2-[Bis[2-
[bis(carboxymethyl) amino]ethyl]amino]acetic acid).
41
Zn(II) buffer and involved in delivery of Zn(II) to cells
and the fact that we observe image enhancement of the
pancreas in response to glucose indicates that the Zn(II)
binding sites on HSA must be largely occupied with
Zn(II) before more ions are released from β-cells. This
then allows the excess Zn(II) ions released from cells to
bind to the Gd(III) sensor and subsequently enhance the
MRI signal.
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It is also important to point out that 4 of 5 of the new
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the concentrations used here. Given that positively
charged Gd(III) complexes are generally consider toxic,
our observations suggest that either the charge on these
complexes is masked by associated counter anions in
vivo or perhaps chemical toxicity will be revealed when
these agents are infused at concentrations higher than
those used here.
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