Cell-Permeable Esterase-Activated Ca(II)-Sensitive MRI Contrast Agent

Article abstract of DOI:10.1021/acs.bioconjchem.5b00561

Calcium [Ca(II)] is a fundamental transducer of electrical activity in the central nervous system (CNS). Influx of Ca(II) into the cytosol is responsible for action potential initiation and propagation, and initiates interneuronal communication via releas

Full text of DOI:10.1021/acs.bioconjchem.5b00561

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Article
A Cell Permeable Esterase-Activated Ca(II)-Sensitive MRI Contrast Agent
Keith W. MacRenaris, Zhidong Ma, Ruby L. Krueger, Christiane E. Carney, and Thomas J. Meade
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.5b00561 • Publication Date (Web): 21 Dec 2015
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Despite the importance of Ca(II) in physiology, visualizing Ca(II) flux in the central nervous system (CNS) in
vivo remains challenging. To address these challenges we have developed a new generation, cell-permeable
Ca(II)-activated MRI contrast probe that utilizes ethyl esters to increase cell permeability and prevent
extracellular divalent Ca(II) binding. Upon cellular internalization, the ethyl esters are cleaved allowing the
agent to bind intracellular Ca(II) increasing relaxivity and resulting in enhanced positive MR image contrast.
44x23mm (300 x 300 DPI)
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A Cell Permeable Esterase-Activated Ca(II)-Sensitive MRI Contrast Agent
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Keith W. MacRenaris, Zhidong Ma, Ruby L. Krueger, Christane E. Carney, and Thomas
J. Meade*
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Departments of Chemistry, Molecular Biosciences, Neurobiology, Biomedical Engineering, and
Radiology, Northwestern University, Evanston IL, USA
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* Corresponding author: tmeade@northwestern.edu
Abstract
Calcium [Ca(II)] is a fundamental transducer of electrical activity in the central nervous
system (CNS). Influx of Ca(II) into the cytosol is responsible for action potential initiation and
propagation, and initiates interꢀneuronal communication via release of neurotransmitters and
activation of gene expression. Despite the importance of Ca(II) in physiology, it remains a
challenge to visualize Ca(II) flux in the central nervous system (CNS) in vivo. To address these
challenges, we have developed a new generation, Ca(II)ꢀactivated MRI contrast agent that
utilizes ethyl esters to increase cell labeling and prevent extracellular divalent Ca(II) binding.
Following labelling, the ethyl esters can be cleaved thus allowing the agent to bind Ca(II),
increasing relaxivity and resulting in enhanced positive MR image contrast. The ability of this
probe to discriminate between extraꢀ and intracellular Ca(II) may allow for spatiotemporal in
vivo imaging of Ca(II) flux during seizures or ischemia where large Ca(II) fluxes (1 – 10 µM)
can result in cell death.
Introduction
Gradients and fluctuations in Ca(II) concentration play critical roles in virtually all cellular
events including membrane potential maintenance, action potential propagation, signal
transduction cascades, and apoptosis.1ꢀ4 In the central nervous system (CNS), Ca(II) flux is
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critical to neuronal function where aberrant fluctuations of intracellular Ca(II) cause a number of
pathologies affecting brain activity resulting in seizures, ischemia, and hypoglycemia.1, 5ꢀ10
The pivotal role of Ca(II) flux in physiology has led to the development of Ca(II) sensitive
imaging techniques to investigate the fundamental mechanisms of this important cation. The
most common technique for Ca(II) visualization has been the use of optical imaging which relies
on the use of Ca(II)ꢀsensitive fluorescent dyes.11ꢀ13 The major challenge for optical techniques is
limited penetration depth (~5ꢀ10 mm) due to light scattering and tissue absorption. As a result,
nonꢀinvasive in vivo longitudinal studies using optical techniques such as bioluminescence and
twoꢀphoton confocal microscopy of Ca(II) flux are challenging.11 More recent methods have
combined electrophysiology or fiber optic Ca(II) imaging with MRI to record neural activity with
Ca(II) flux simultaneously but are technically challenging and have limited clinical potential.14
Additionally, the acquired data is in the form of maps that are not in real time and are
fundamentally static representations of dynamic activity.
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An alternative Ca(II) imaging strategy has been the use of bloodꢀoxygenꢀlevelꢀdependent
(BOLD) contrast generated in functional magnetic resonance imaging (fMRI).15ꢀ19 Further,
while fMRI is not limited by tissue depth, the alterations in BOLD contrast are used to measure
brain activity and not necessarily Ca(II) flux and are indirect, as they arise from changes in
cerebral blood flow, blood volume, and oxygenation.17ꢀ18 Although optical imaging and BOLD
fMRI approaches have yielded insights regarding Ca(II) signaling, transport and trafficking,
these techniques are unable to provide high resolution, realꢀtime in vivo imaging of Ca(II) flux.
In order to overcome the limitations, an alternative in vivo imaging strategy is required to
visualize in vivo Ca(II) flux.
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Contrastꢀenhanced magnetic resonance imaging (MRI) is an attractive alternative to both
optical imaging and BOLD fMRI due to its unlimited penetration depth, softꢀtissue contrast, high
resolution tomographic (3D) information, and the ability to provide anatomical and physiological
information about surrounding tissues.20 In order to visualize Ca(II) via contrastꢀenhanced MR
imaging we developed bioactivated contrast agents that are conditionally activated by the
presence of Ca(II).21ꢀ22 Since these initial studies, several agents have been prepared that detect
Ca(II) by a similar mechanism.23ꢀ27 However, these agents are either confined to the extracellular
space or cannot discern between intraꢀ and extracellular Ca(II) limiting their effectiveness to
report on intracellular Ca(II) flux.
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Our work has focused on conjugating two paramagnetic Gd(III) complexes (1,4,7,10ꢀ
tetraazacycloddodecaneꢀ1,4,7,10ꢀtetraacetic acid, Gd(III)ꢀDOTA) to the wellꢀknown Ca(II)
chelator 1,2ꢀbisꢀ(Oꢀaminophenoxy) ethaneꢀN,N,N’,N’ꢀtetraacetic acid (BAPTA). Complex 2
[DOPTA-Gd(III)] relies on the four carboxylates of the BAPTA domain to block water
coordination to the Gd(III) ions (q) in the absence of Ca(II) (low relaxivity or ‘off’ state). In the
presence of 1ꢀ100 µM Ca(II), the four carboxylates of BAPTA preferentially bind Ca(II)
allowing water access to the Gd(III) ions and resulting in an 80% increase in T1 relaxivity
allowing Ca(II) fluctuations to be visualized via small molecule contrastꢀenhanced MR imaging.
However, while the relaxivity enhancement of complex 2 (Figure 1) is adequate to detect Ca(II)
binding at low concentrations (~10 µM), the overall negative charge of the complex could limit
cell permeability. Further, complex 2 is not capable of distinguishing intracellular versus
extracellular Ca(II) (1ꢀ2 mM) in vivo.
To overcome these limitations we have designed and synthesized complex 1 [DOPTA-
Ethyl-Gd(III)] where the negativelyꢀcharged imino acetates of BAPTA are protected via
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esterification (Figure 1).28ꢀ30 The addition of the esters to the BAPTA ligand address the two
issues cited above simultaneously: i. increases the lipophilicity and cell permeability of the agent
and more importantly ii. prevents extracellular Ca(II) binding maintaining the contrast agent in a
low relaxivity (‘off’) state prior to cell internalization. Upon cell uptake, endogenous cytosolic
esterases cleave the esters of complex 1 deprotecting the aromatic imino acetates (resulting in
complex 2) and allowing intracellular Ca(II) binding with a concomitant increase in relaxivity
(Figure 1).
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Figure 1. Schematic of complex 1 activation after ethyl ester hydrolysis and subsequent Ca(II)
binding. Esterases cleave the ethyl esters on the BAPTA moiety unmasking four carboxylates
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that coordinate to the metal center blocking water access to the macrocyclic chelates maintaining
the agent in a low relaxivity state. In the presence of Ca(II) a conformational change occurs
whereby the free carboxylates bind Ca(II) providing water access to the Gd(III) centers thereby
increasing contrast agent relaxivity. Complex 3 was used in a parallel experiment as a control.
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Results and Discussion
Synthesis
Complex 1 was synthesized in five steps according to modified literature procedures31 using
1,2ꢀbis(oꢀaminophenoxy)ꢀethaneꢀN,N,N',N'ꢀtetraacetic acid (BAPTA) as a Ca(II)ꢀbinding
domain due to its 105ꢀfold selectivity for Ca(II) over other divalent metal cations such as Mg(II)
and protons.14, 21ꢀ22, 32 The intermediate diol, 4 was brominated to the dibromide 5 using bromine
and triphenylphosphine. Attachment of the 2,2',2''ꢀ(1,4,7,10ꢀtetraazacyclododecaneꢀ1,4,7ꢀtriyl
triacetate (DO3A) was accomplished using excess trisꢀtꢀbutylꢀDO3A in order to minimize the
formation of the monoalkylated product. The cyclen imino trisꢀtꢀbutyl acetates as part of 6 were
o
selectively hydrolyzed with excess formic acid at 50 C (the aromatic imino ethyl diacetates on
the BAPTA ligand remain). Metalation using GdCl3 was performed under weakly acidic
conditions (pH = 5.5) and after purification by semiꢀpreparative HPLC the final product 1 was
isolated (Figure 2).
The design of the agent incorporates a propyl linker to couple the aromatic iminoacetates of
BAPTA to the macrocycle. We have designed the linker to maximize acetate backꢀbinding (after
ester cleavage) to Gd(III) when the Ca(II) concentration is low (< 1 µM). As a result, the
relaxivity of the complex is minimized. An internal rearrangement allows water to bind to
Gd(III) when the Ca(II) concentration is between 5 and 10 µM. Utilizing the stable ethyl
bromoacetates in place of acetoxymethyl esters reduced synthetic challenges while decreasing
the rate of ethyl ester hydrolysis and as a result minimized cleavage by extracellular esterases.
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Figure 2. Synthetic scheme of DOPTAꢀEthylꢀGd(III) . (a) Br2, PPh3, TEA, DCM, 60%. (b) trisꢀ
tꢀbutylꢀDO3A, K2CO3, MeCN, 65%. (c) formic acid, 55oC. (d) GdCl3, NaOH, pH 5ꢀ6, 44%.
Effect of Ester Hydrolysis and Ca(II) Binding on Relaxivity
Ethyl ester hydrolysis of 1 was investigated using HPLCꢀMS equipped with an evaporative
light scattering detector (ELSD) to monitor the reaction with porcine esterase.28, 30, 33ꢀ35 The
kinetics of ester hydrolysis were determined by following the disappearance of the HPLC peak
of 1 postꢀincubation with porcine esterase at 37 °C in HEPES buffer at pH 7.4 (Figure 3, and
Supporting Figure 6). After 50 hours, 92.4% of 1 was hydrolyzed resulting in a rate constant of
1.69 × 10ꢀ5 sꢀ1 (kobs, 37 °C). Employing pseudo firstꢀorder kinetics this result corresponds to an
11.4 hour halfꢀlife.
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o
Figure 3. Plot of ethyl ester hydrolysis of 1 by porcine esterase at 37 C (in pH 7.4 HEPES
buffer) monitored by HPLCꢀMS using an evaporative light scattering detector (ELSD).
Individual data points represent the integration of the HPLCꢀMS peak of 1 after exposure to
porcine esterase divided by the HPLCꢀMS peak of 1 prior to porcine esterase exposure
(multiplied by 100 to give percent complex hydrolysis). The line corresponds to a pseudo firstꢀ
order kinetic fit of the data with an 11.4 h halfꢀlife for 1.
Relaxivities (r1) of 1, 2, and 1,4,7,10ꢀtetraazacyclododecaneꢀN,N',N'',N'''–tetraacetic acid, 3
[DOTA-Gd(III)] were measured. The r1 of 1, 2, and 3 were 10.6 mMꢀ1sꢀ1, 7.4 mMꢀ1sꢀ1, and 3.1
mMꢀ1sꢀ1, respectively prior to activation and Ca(II) binding (Table 1). Following ester hydrolysis
(in pH 7.4 HEPES buffer and 37 °C for 24 hours), the r1 of 1 is 7.6 mMꢀ1 secꢀ1 in the absence of
Ca(II) and reaches a maximum of 12.6 mMꢀ1 sꢀ1 in the presence of 1 mM Ca(II) with a dynamic
relaxivity range between 1 and 100 µM Ca(II) (Table 1, Figure 4). To confirm that ester
hydrolysis of 1 produces the same relaxivity changes as (2), this complex was independently
synthesized and the r1 measured in the presence of increasing Ca(II) concentration. The increase
in r1 upon ester hydrolysis and subsequent Ca(II) binding to 1 is 66%, while the increase in r1 of
2 post Ca(II) binding is 64% suggesting the product of the ester hydrolysis of 1 is complex 2
(Figure 4). Additionally, these same relaxivity samples were then run on a 7 T imaging
spectrometer to determine high field relaxivity. As shown in supporting figure 10 the high field
relaxivity of 2 is 6.21 mMꢀ1sꢀ1 and 9.14 mMꢀ1sꢀ1 for 1. This is very promising since the relaxivity
decrease due to increased field strength is less than 20% for both molecules.
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Figure 4. Relaxivity (r1, mMꢀ1sꢀ1) changes of complex 1 (after ethyl ester hydrolysis via porcine
esterase) and 2 upon addition of Ca(II) at 1.41 T and 37° C (in pH 7.4 0.1 M HEPES buffer wih
0.1 M KCl and 1% DMSO). Both synthesized 2 and ester hydrolyzed 1 show a > 64% change in
r1 upon Ca(II) binding. Lines represent nonꢀlinear sigmoidal doseꢀresponse fits to the data. EC50
value for compound 1 is 16.6 µM Ca2+ and for compound 2 is 11.5 µM Ca2+.
To determine q of 1, fluorescence lifetime of the Tb(III) analog (1b) was measured in D2O
and H2O. Tb(III) was chosen over Eu(III) due to its increased luminescence and longer
luminescent lifetime in both H2O and D2O.36ꢀ38 The difference in luminescence lifetimes in H2O
versus D2O reflects the number of innerꢀsphere water molecules, q, and can be calculated using
Equation (1).38
ꢃ
ꢃ
ꢀ ꢁ 4.2 ꢂꢄ
ꢈ
ꢈ 0.06ꢊ
ꢄ
ꢉ ꢇ
ꢆ
(1)
ꢅ
ꢆ
ꢇ
The fluorescence lifetimes of the complexes were measured and fit to an exponential curve with
observed decay times of 1.41 ms and 2.42 ms in H2O and D2O, respectively where q is 0.5 for 1b
and 0.3 for 2b (per metal). This result indicates that the ethyl esters of 1b prevent water from
coordinating to Gd(III) as efficiently as the acetates of 2b. Furthermore, q was measured for 1b
and 2b after the addition of porcine esterase and Ca(II) to determine whether the changes in the
relaxivities of the complexes are due to changes in the innerꢀsphere water coordination of the
Gd(III) chelates. As shown in Table 1, q for 2b increases from 0.3 to 1 in the presence of 1 mM
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Ca(II) whereas q for 1c (prior to ester hydrolysis) remains constant at 0.5 even in the presence of
1 mM Ca(II). This data shows the ethyl ester protected iminoacetates likely do not participate in
Ca(II) binding prior to enzymatic hydrolysis. Following hydrolysis, the q value of 1 increases
from 0.5 to 0.9 confirming that the ester hydrolysis of 1 results in 2.
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To investigate whether changes in low field relaxivity can be visualized via high field MRI
(7 T, 300 MHz) phantoms of a 100 µM solution of 2 [200 µM Gd(III)] were prepared with
different stoichiometric concentrations of CaCl2 with concentrations of 100, 50, 25, 12.5, and
6.25, and 0 µM Ca(II) (Figure 5). There is a 32% decrease in T1 from 0 µM Ca(II) to 100 µM
Ca(II) which is visible via high field MRI. Additionally, Ca(II) concentrations as low as 6.25 µM
produce a T1 decrease of at least 10% which is required to visualize large Ca(II) fluxes of
approximately 10 µM.
Figure 5. MR images and T1 analysis of 100 µM solution phantoms of 2 binding with increasing
amounts of Ca(II) corresponding to 100, 50, 25, 12.5, and 6.25 µM Ca(II) at 7T and 25 °C. Scale
bar (white) represents 1 mm. T1 values are calculated as the average of 5, 1 mm slices ± 1
standard deviation. %T1 decrease is calculated versus the 0 µM Ca(II) sample.
Table 1 – Relaxivity, octanolꢀwater partition coefficients and hydration numbers of
complexes 1, 2, and 3
+ Esterase
+ 1 mM Ca(II)
- Ca(II)
+ 1 mM Ca(II)
r1
(mM-1 s-1)
r1
(mM-1 s-1)
r1
log P
q
q
q
(mM-1 s-1)
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ꢀ2.44
ꢀ3.69
ꢀ3.42
10.6 ± 0.3
7.4 ± 0.2
3.1 ± 0.3
0.5 ± 0.1
0.3 ± 0.1
1 ± 0.1
10.2 ± 0.2
12.5 ± 0.4
3.1 ± 0.2
0.5 ± 0.1
1.0 ± 0.1
ꢀ
12.6 ± 0.3
0.9 ± 0.1
1
2
3
ꢀ
ꢀ
ꢀ
ꢀ
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In vitro Cellular Uptake and Retention
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The previous data showed that Ca(II) can only bind to complex 1 following ester hydrolysis
resulting in its detectable form (complex 2) which can then bind Ca(II) (5ꢀ10 µM) leading to a
decrease in T1 providing positive MR image contrast. This provides a novel bioꢀactivated probe
for determining intracellular Ca(II) fluxes but requires cellular uptake of the contrast agent. One
factor that has been used to predict the ability of a molecule or drug to diffuse into
biomembranes is lipophilicity where a balance in amphilicity is desired for ease of aqueous
administration and cell uptake.39ꢀ40 In order to determine the lipophilicity of Ca(II)ꢀsensitive MRI
contrast agents, octanol–water partition coefficients (log P) were determined for each complex
where 1 was one order of magnitude more hydrophobic than 2 (ꢀ2.44 versus ꢀ3.69) which should
lead to an increase in cell uptake.41ꢀ42
To determine the cell labelling properties of 1, 2, and 3 a hippocampal neuronal cell line
(HTꢀ22 cells were chosen as they have frequently been used to investigate neuronal activity).43ꢀ44
Concentrationꢀdependent uptake of 1, 2 and 3 was determined by incubating HTꢀ22 cells with
31.25, 62.5, and 125 µM of the complexes for 48 hours. After incubation, cells were washed,
trypsinized, counted, and digested with nitric acid. Gd(III) content was measured using ICPꢀMS
of the acidꢀdigested samples (Figure 6A, Supporting Table 1). As shown, cellular labelling is
concentration dependent and directly correlated to the octanol–water partition coefficients of the
complexes where the more hydrophobic 1 exhibited higher uptake than 2 at incubation
concentrations of 125 µM. At lower incubation concentrations, uptake of 1 and 2 were the same.
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Figure 6. Concentrationꢀdependent (A), timeꢀdependent (B), and cellular retention studies (C) of
1, 2, and 3 in the hippocampal neuronal cell line, HTꢀ22. Cellular uptake of 1 was significantly
higher than that of 2 and 3. A small amount of contrast agent (<7%) leached out of the cells in 24
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hours. All samples were washed 3x with DPBS, trpsyinized, counted, and digested in nitric acid
to determine Gd(III) content via ICPꢀMS. Data are mean ± standard deviation of triplicate runs
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To determine the timeꢀdependent cell labelling, HTꢀ22 cells were incubated with 125 µM of
the complexes for 1, 4, 8, 24 hours. After incubation, cells were washed, trypsinized, counted,
and digested with nitric acid. Finally, Gd(III) content was measured using ICPꢀMS of the acidꢀ
digested samples. As shown in Figure 6B (and Supporting Table 2) maximum Gd(III) labelling
occurs after a 4 or 8 hour incubation followed by a significant decrease after 24 hours.
To elucidate the relationship between cellular labelling and cellular retention HTꢀ22 cells
were incubated with 125 µM of each complex for 2 hours. After incubation, cells were washed
with DPBS (2x) followed by the addition of fresh media. Cells were allowed to efflux (leach) for
various time points (0, 1, 2, 3, 4, and 24 hrs) in fresh media followed by media removal, cell
washing with DPBS (2x), cell harvesting and Gd(III) determination via ICPꢀMS. At each time
point cell samples were counted and analyzed by ICPꢀMS.
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Figure 6C shows that, despite multiple washes, greater than 90% of the Gd(III) is retained.
The cells treated with 1 lost ~ 5% more of the agent compared to cells treated with the other two
agents. However, because the cellular uptake of 1 is more than 200% higher than complex 2 (and
more than 500% higher than complex 3), the overall amount of cell associated 1 remains higher
than 2 and 3 prior to and after leaching. The absolute quantities were divided by their respective
initial concentrations (those collected at time = 0) in order to quantify the relative amount of
cellular retention for each agent.
To determine whether the cell labelling and molecular relaxivity of 1 and 2 leads to
detectable image contrast, HTꢀ22 cells were treated with 125 ꢁM of 1, 2, and 3 complexes for 4
hours and centrifuged into capillaries (~ 1.0 mm diameter) prior to MR imaging. The greyscale
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and color intensity MR images of the cell pellets show increases in image intensity for cells
treated with 1 or 2 compared with cells treated with 3 or untreated cells where the contrast to
noise ratio (CNR) between 1 and 3 is 11 (Figure 7A). Lastly, The amount of Gd(III) per cell was
determined by ICPꢀMS for each cell pellet and as expected, T1 was found to be inversely
proportional to Gd(III) content (Figure 7B).
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Figure 7. (A) T1ꢀweighted MR image with corresponding imageꢀintensity color map of HTꢀ22
cells incubated with 1, 2, 3 and media alone for 4 h (scale bar = 1 mm). 9.4 T (400 MHz) and
25°C. (B) Calculated values corresponding to the MR image in (A). Values represent the average
of 4, 1 mm slices and the error is plus or minus one standard deviation of the mean.
The findings from these in vitro studies show that 1 labels cells up to 2ꢀfold more efficiently
than 2 or 3, which is directly correlated to the octanolꢀwater partition coefficients. The results are
in agreement with our hypothesis that esterification enhances lipophilicity and consequently cell
labelling.
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We have synthesized a cellꢀpermeable version of a previously reported Ca(II)ꢀsensitive
MRI contrast agent, 1, for visualizing intracellular Ca(II) signaling. By incorporating ethyl esters
in the BAPTA binding ligand the charge of the complex is neutralized resulting in increased
lipophilicity and concurrent cellular labelling. In addition, the ethyl esters maintain the contrast
agent in a low relaxivity or ‘off’ state until ethyl ester hydrolysis resulting in the active form, 2,
which is capable of binding Ca(II) producing a high relaxivity or ‘on’ state. This probe design
will allow for the discrimination between extraꢀ and intracellular Ca(II) by MR imaging and
thereby providing the ability to monitor intracellular Ca(II) signaling.
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Materials and Methods
General Methods
Unless otherwise noted, materials and solvents were purchased from SigmaꢀAldrich
Chemical Co. (St. Louis, MO) and used without further purification. Common precursor 4 (2ꢀ{2ꢀ
[3ꢀ(3ꢀhydroxypropoxy)ꢀ2ꢀdi(ethoxycarbonylmethyl)aminophenoxy]ethoxy}
ꢀ6ꢀ(3ꢀ
hydroxypropoxy)ꢀN,Nꢀdi(ethoxycarbonylmethyl)benzenamine), trisꢀtꢀbutylꢀDO3A (Trisꢀtertꢀ
butyl 2,2',2''ꢀ(1,4,7,10ꢀtetraazacyclododecaneꢀ1,4,7ꢀtriyl) triacetate) were prepared according to
literature procedures.3e, 20 Unless noted, all reactions were performed under a nitrogen or argon
atmosphere. Acetonitrile was purified using a Glass Contour Solvent system (Pure Process
Technology, Nashua, NH, USA). Deionized water (18.2 MΩꢁcm) was obtained from a Millipore
QꢀGuard System (Billerica, MA). Thinꢀlayer chromatography (TLC) was performed on EMD 60
F254 silica gel plates. Standard grade 60 Å 230 – 400 mesh silica gel (Sorbent Technologies,
Norcross, GA, USA) was used for flash column chromatography. 1H and 13C NMR spectra were
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obtained on a Bruker 500 MHz Avance III NMR spectrometer (Bruker Biospin, Billerica, MA,
USA) or a Varian Inova 400 MHz NMR spectrometer (Agilent Technologies, Santa Clara, CA,
USA) with deuterated solvents as noted. Electrospray ionization mass spectrometry (ESIꢀMS)
was carried out using a Varian 1200L singleꢀquadrupole mass spectrometer (Agilent
Technologies, Santa Clara, CA, USA). Matrixꢀassisted laser desportion ionization timeꢀofꢀflight
mass spectra (MALDIꢀTOFꢀMS) were recorded on a Bruker AutFlex III (Bruker, Billerica, MA,
USA), using 2, 5ꢀdihydroxybenzoic acid as the matrix. Analytical reverseꢀphase HPLCꢀMS was
performed on a Varian Prostar 500 system (Agilent Technologies, Santa Clara, CA, USA) using
either a Waters (Milford, MA, USA) Atlantis C18 column (4.6 × 250, 5 ꢂm) or an XBridge C18
column (4.6 × 150, 5 ꢂm). This system is equipped with a Varian 380 LC ELSD system, a
Varian 363 fluorescence detector, a Varian 335 UVvis detector, and a Varian 1200L quadrupole
MS detector (Agilent Technologies, Santa Clara, CA, USA). Preparative runs were performed on
a Waters (Milford, MA, USA) Atlantis C18 column (19 × 250, 10 ꢂm). The mobile phase
consisted of water or water with NH3•H2O (pH = 10.4) (solvent A) and HPLCꢀgrade acetonitrile
(solvent B).
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2-{2-[3-(3-bromopropoxy)-2-di(ethoxycarbonylmethyl)aminophenoxy]ethoxy}-6-(3-
bromopropoxy)-N,N-di(ethoxycarbonylmethyl)benzenamine, 5. 0.37 mL of TEA (2.66
mmol) was added via syringe to 0.70 g of triphenylphosphine (2.66 mmol) dissolved in 10 mL of
dry dichloromethane at 0 °C. 0.14 mL of bromine (2.66 mmol), diluted in 15 mL of dry
dichloromethane, was added to the reaction mixture. Stirring was continued at 0 °C for 30 min.
0.89 g of compound 4 (1.21 mmol), in 10 mL of dichloromethane, was added via syringe and the
reaction mixture was stirred at room temperature. Absence of starting material was confirmed by
TLC after 24 h. The crude material was purified by flash column chromatography over silica gel
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with hexanes: ethyl acetate (3:1) to afford a yellow oil (0.63g, 60%, Rf = 0.45). H NMR (500
MHz, CDCl3): δ 6.93 (t, 2H, J = 8.3 Hz), 6.60 (d, 2H, J = 8.2 Hz), 6.56 (d, 2H, J = 8.2 Hz), 4.30
(s, 4H), 4.09 (t, 4H, J = 5.8 Hz), 4.08 (s, 8H), 4.05 (q, 8H, J = 7.15 Hz), 3.63 (t, 4H, J = 6.5 Hz),
2.30 (m, 4H), 1.16 (t, 12H, J = 7.15 Hz); 13Cꢀ{H} NMR (126 MHz, CDCl3): δ 172.2, 154.2,
128.3, 123.6, 106.9, 106.7, 67.5, 66.5, 60.2, 53.9, 32.5, 30.4, 14.2
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2-{2-[3-(3-(tris-t-butyl-DO3A)propoxy)-2-di(ethoxycarbonylmethyl)aminophenoxy]
ethoxy}-6-(3-(tris-t-butyl-DO3A)propoxy)-N,N-di(ethoxycarbonylmethyl)
benzenamine,
DOPTA-Ethyl, 6. 131 mg (0.26 mmol) of trisꢀtꢀbutylꢀDO3A in 50 mL of acetonitrile and 49.7
mg (0.36 mmol) of K2CO3 was added to a 100 mL roundꢀbottom flask. The mixture was sealed,
stirred and placed under nitrogen atmosphere for 30 min before 100 mg (0.12 mmol) of 5 in 10
mL of acetonitrile was added. The mixture was heated to 75 oC and stirred for 48 h. The reaction
was monitored by mass spectrometry to follow the disappearance of the starting materials. The
reaction was filtered and evaporated to yield a yellow oil. The crude material was purified by
flash column chromatography over silica gel with dichloromethane: methanol (50:1) to afford a
1
o
yellow oil (135 mg, 65% Rf = 0.90). H NMR (400 MHz, DMSOꢀd6, 70 C): δ 6.92 (2H, br),
6.64 (4H, br), 4.28 (s, 4H), 3.97 (m, 20H,), 3.67ꢀ1.79 (m, 52H), 1.43 (s, 54H), 1.11 (t, 12H, J =
6.92 Hz); MALDIꢀMS m/z observed: 1730.33, calculated: 1730.17 [M + H]+.
2-{2-[3-(3-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecyl)propoxy)-2-
di(ethoxycarbonylmethyl)aminophenoxy]
ethoxy}-6-(3-(4,7,10-tris(carboxymethyl)-
benzenamine-
1,4,7,10-tetraazacyclododecyl)propoxy)-N,N-di(ethoxycarbonylmethyl)
gadolinium(III), DOPTA-Ethyl-Gd(III) , 1. 200 mg (0.12 mmol) of compound 6 was
o
dissolved in 50 mL of formic acid and heated to 55 C for 18 hours. After formic acid was
evaporated on a rotary evaporator, the resulting oil was redissolved in methanol 3 × 10 mL and
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evaporated again to remove most of the formic acid. The resulting glassy solid 7 was dissolved
in 20 mL of water and the pH of the solution was adjusted to 5. 107 mg (0.28 mmol) of GdCl3ꢁ
6H2O was added and the pH of the reaction was adjusted back to 6 every 4ꢀ6 h until no further
change occurred. (Typically the reaction was completed in 1ꢀ2 days). The reaction was
evaporated and purified by reverse phase HPLC according to method 1, retention time of 44.5
min, and 99% purity, followed by lyophilization to yield 87 mg of 1 as a white powder in 44%.
MALDIꢀTOFꢀMS m/z observed: 1703.2 (M + H+) calcd for C64H94Gd2N10O24 1702.0.
The terbium(III) analog of 1 were prepared in a similar manner for luminescence lifetime
measurements. HPLC and MALDIꢀTOFꢀMS were used to determine the purity. DOPTAꢀEthylꢀ
Tb(III), retention time: 47.0 min, yield: 42%, m/z observed: 1705.1, calcd for C64H94Yb2N10O24
1705.3
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Relaxation Time Measurements. 1 was dissolved in 500 ꢁL of filtered, 0.1 M HEPES, 0.1 M
KCl containing 1% DMSO for T1 acquisition. Relaxation times were measured on a Bruker
mq60 NMR analyzer equipped with Minispec v2.51 Rev.00/NT software (Bruker Biospin,
Billerica, MA, USA) operating at 1.41 T (60 MHz) and 37 °C T1 relaxation times were measured
using an inversion recovery pulse sequence (T1_ir_mb) using the following parameters: 4 scans
per point, 10 data points for fitting, monoꢀexponential curve fitting, phase cycling, 10 ms first
pulse separation, and a recycle delay and final pulse separation ≥ 5 T1. The Gd(III)
concentrations of each solution were determined using ICPꢀMS. The relaxivity r1 was calculated
from Equation (2). The inverse of the longitudinal relaxation time (1/T1, sꢀ1) was plotted against
Gd(III) concentration (mM) and fitted to a straight line with an R2 > 0.99. The slope of the fitted
line was recorded as the relaxivity, r1.
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ꢃ
ꢃ
ꢒ
ꢕ
ꢁ
+ ꢑ ꢓꢔ
(2)
ꢃ
ꢋ
ꢋ
ꢌ,ꢐ
ꢌ,ꢍꢎꢏ
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Octanol–Water Partition Coefficients. 0.5 mg of 1, 2 and 3 were dissolved in 1 mL of a 1:1
mixture of water:1ꢀoctanol. After shaking for 30 s, tubes containing the mixtures were placed on
a rotator for gentle mixing to equilibrate for 48 h. After mixing, samples were allowed to settle at
room temperature for 24 hours. Gd(III) concentrations of each layer were determined by ICPꢀ
MS. Partition coefficients were calculated from the Equation log P = log(Co/Cw), where log P is
the logarithm of the partition coefficient, Co is the concentration of Gd in the 1ꢀoctanol layer, and
Cw is the concentration of Gd in the water layer.
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Luminescence Lifetime Measurements of 1C. The luminescence lifetime of a 1 mM solution
of 1c was measured in H2O and D2O on a Hitachi (San Francisco, CA) F4500 fluorometer, using
a λex = 232 nm and λem = 544 nm. Twentyꢀfive scans were acquired, averaged and fit to a monoꢀ
exponential decay function.
Kinetics of Ethyl Ester Hydrolysis. Porcine liver esterase was diluted 10 times with 0.1 M
HEPES, 0.1 M KCl and 1% DMSO buffer and used to hydrolyze 1. The solution of 1 was
prepared by dissolving the agent in in the aforementioned buffer to give a final concentration of
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1 mM. 10 U/ml of porcine esterase was added to the solution and was maintained at 37 C. 200
µL aliquots of the reaction was taken at different time points for 72 h and centrifuged at 4000
RPM for 5 mins and filtered through microporous (0.2 µm) membrane filter. The filtrate was
analyzed by HPLCꢀMS with an evaporative light scattering detector (ELSD). Pseudoꢀfirstꢀorder
rate constants for enzymatic hydrolysis were determined based on the Equation, y = A0 × eꢀx/t
(where A0 = 1 and tꢀ1= kobs), using Origin 7 (OriginLab, Northampton, MA).
General Cell Culture. Dulbecco’s modified phosphate buffered saline (DPBS), media, and
TrypLE were purchased from Invitrogen (Carlsbad, CA). Serum is from VWR (Corning,
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Mediatech). Cell culture consumables (flasks, plates, etc.) were purchased from VWR (Radnor,
PA). Charcoal dextran stripped FBS was purchased from Atlanta Biologicals (Lawrenceville,
GA). The HTꢀ22 hippocampal neuronal cell line obtained from Professor Schubert at Salt
Institute (La Jolla, CA) and were cultured using phenol red free DMEM supplemented with 10%
FBS. Experiments and incubations were done in a 5.0% CO2 incubator at 37 oC unless otherwise
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noted. Cells were harvested by incubation with 0.25% TrypLE for 10 min at 37 C in a 5.0%
CO2 incubator.
Cellular Uptake Studies. Concentration-dependent uptake. Cell studies were performed to
determine the ability of 1, 2 and 3 to label cells. HTꢀ22 hippocampal neuronal cells were grown
to 60% confluence in a 24 well cell culture plate at 350 µL per well. Media was then replaced
with media containing 1% DMSO and increasing concentrations (31.25 µM, 62.5 µM and 125
µM) of the three agents in duplicate. The plate was incubated for 48 h and washed three times
with sterile phosphate buffered saline. Each sample was then trypsinized and tested for cell count
and viability on a Guava PCA system using the Guava viacount protocol according to the
manufacturer’s instructions. The remaining volume in each sample was dissolved in concentrated
nitric acid and diluted for analysis by ICPꢀMS to determine the amount of Gd(III) associated
with the cells. Viability analysis revealed no change in viability of treated cells.
Time-dependent uptake. The cells were prepared in a similar fashion to previous assays. HTꢀ22
hippocampal neuronal cells were grown to 60% confluence and incubated with 125 µM of the
three agents for 1, 4, 8, and 24 h in duplicate. After incubation, the medium was removed, and
the cells were rinsed three times with DPBS. These cells were trypsinized, counted, and analyzed
for Gd(III) content using Guava EasyCyte Mini PCA System and ICPꢀMS.
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Cellular Retention Studies. The cells were prepared in a similar fashion to previous assays. HTꢀ
22 hippocampal neuronal cells were grown to 60% confluence and incubated with 125 µM of the
three agents for 2 h. After incubation, the cells were washed with DPBS (time = 0, initial
amount), placed in fresh media, and allowed leach for 0, 1, 2, 3, 4, and 24 hours. After each time
point, cells were harvested, counted, and analysed for Gd(III) content by ICPꢀMS.
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Cell Counting Using A Guava EasyCyte Mini Personal Cell Analyzer (PCA) System. After
cell harvesting, an aliquot of the cell suspensions was mixed with Guava ViaCount reagent and
allowed to stain at room temperature for at least 5 min (no longer than 20 min). Stained samples
were vortexed for 10 s and percent cell viability was determined via manual analysis using a
Guava EasyCyte Mini Personal Cell Analyzer (PCA) and ViaCount software module. For each
sample, 1000 events were acquired with dilution factors that were determined based upon
optimum machine performance (∼25ꢀ100 cells/ꢂL). Instrument reproducibility was assessed
daily using GuavaCheck Beads and following the manufacturer’s suggested protocol using the
Daily Check software module.
MR Imaging and T1 analysis of Cell Pellets at 9.4 T. 9.4 T MR imaging and T1 measurements
were performed on a Bruker Biospec 9.4 T imaging spectrometer fitted with shielded gradient
coils at 25 °C. For cell pellet images, ~ 2.0 × 106 HTꢀ22 cells were incubated in 25ꢀcm2 Tꢀflasks
with 125 µM of 1, 2 and 3 for 4 h. The cells were rinsed with DPBS (3 × 5 mL/flask), and
harvested with 500 µL of a 0.25ꢀvol% trypLE Express solution. After addition of 500 µL of the
appropriate fresh media (1.0 mL total volume/flask), cells were transferred to 1.5ꢀmL
microcentrifuge tubes and centrifuged at 200 g for 5 min. The supernatant was removed, and cell
pellets were reꢀsuspended in 1 mL of complete media, added to 5 3/4″flameꢀsealed Pasteur
pipets, and centrifuged at 200 g and 4.0 °C for 5 min. The bottom sections of the flameꢀsealed
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pipets were scored with a glass scribe, broken into small capillaries, and imaged using a RF RES
400 1H 089/038 quadrature transmit receive 23ꢀmm volume coil (Bruker BioSpin, Billerica,
MA, USA).
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Spinꢀlattice relaxation times (T1) were measured using a rapidꢀacquisition rapidꢀecho
(RAREꢀVTR) T1ꢀmap pulse sequence, with static TE (10 ms) and variable TR (100, 200, 400,
500, 750, 1000, 2500, 5000, 7500, and 10000 ms) values. Imaging parameters were as follows:
field of view (FOV) = 25 × 25 mm2, matrix size (MTX) = 256 × 256, number of axial slices = 3,
slice thickness (SI) = 1.0 mm, and averages (NEX) = 4 (total scan time = 2 hrs 58 min). T1
analysis was carried out using the image sequence analysis tool in Paravision 5.0 pl3 software
(Bruker, Billerica, MA, USA) with monoꢀexponential curveꢀfitting of image intensities of
selected regions of interest (ROIs) for each axial slice.
Quantification of Gd(III) with Inductively Coupled Plasma Mass Spectrometry.
Quantification of Gd(III) was accomplished using ICPꢀMS of acid digested samples.
Specifically, solution samples were digested in ACS reagent grade nitric acid (70%, Sigma, St.
Louis, MO, USA) and incubated in a water bath at 70 °C for at least 2 h to allow for complete
sample digestion. A portion of the digested sample was added to a 15 mL conical tube along with
5 ng/mL of multiꢀelement internal standard containing Bi, Ho, In, Li, Sc, Tb, and Y (Inorganic
Ventures, Christiansburg, VA, USA) in filtered, deꢀionized H2O (18.2 MΩꢁcm). Instrument
calibration was accomplished by preparing individualꢀelement Gd standard (Inorganic Ventures,
Christiansburg, VA, USA) using concentrations of 1.000, 5.000, 10.00, 20.00, 50.00, 100.0, and
200.0 ng/mL containing 3.0% nitric acid (v/v) and 5.0 ng/mL of multiꢀelement internal standard.
ICPꢀMS was performed on a computerꢀcontrolled (Plasmalab software) Thermo X series
II ICPꢀMS (Thermo Fisher Scientific, Waltham, MA, USA) operating in standard mode
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equipped with an ESI SCꢀ2 autosampler (Omaha, NE, USA). Each sample was acquired using 1
survey run (10 sweeps) and 3 main (peak jumping) runs (100 sweeps). The isotopes selected for
analysis were 157,158Gd with 115In and 165Ho isotopes selected as internal standards for data
interpolation. Instrument performance is optimized daily by an autotune followed by verification
via a performance report (passing manufacturer specifications). Addition of all reagents for all
samples and standards were weighed using a Mettler Toledo (Columbus, OH, USA) XS205
DeltaRange analytical micro balance (with 0.01 mg resolution for up to 81 g of sample).
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Acknowledgements
This work was supported by the National Institutes of Health grant R01EB005866 and
R01EB014806 as well as a CBC Spark Award Sꢀ003. A portion of this work was completed at
the Northwestern University Integrated Molecular Structure Education and Research Center.
Imaging was performed at the Northwestern University Center for Advanced Molecular Imaging
generously supported by NCI CCSG P30 CA060553 awarded to the Robert H Lurie
Comprehensive Cancer Center. MRI was performed on the 7 T Bruker Pharmscan system
purchased with the support of NCRR 1S10RR025624ꢀ01. Metal analysis was performed at the
Northwestern University Quantitative Bioꢀelement Imaging Center generously supported by
NASA Ames Research Center NNA06CB93G.
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For Table of Contents Only
TOC Figure Synopsis:
Despite the importance of Ca(II) in physiology, visualizing Ca(II) flux in the central
nervous system (CNS) in vivo remains challenging. To address these challenges we have
developed a new generation, cellꢀpermeable Ca(II)ꢀactivated MRI contrast probe that utilizes
ethyl esters to increase cell permeability and prevent extracellular divalent Ca(II) binding. Upon
cellular internalization, the ethyl esters are cleaved allowing the agent to bind intracellular Ca(II)
increasing relaxivity and resulting in enhanced positive MR image contrast.
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