An esterase-activated magnetic resonance contrast agent

Article abstract of DOI:10.1039/b711989e

A Gd(III) complex bearing pendant acetoxymethyl esters is activated on exposure to porcine liver esterase; the 84% increase in relaxivity is a result of suppression of HCO₃^-/CO₃^2- binding by the resulting negative charge. The Royal Society of Chemistry.

Full text of DOI:10.1039/b711989e

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COMMUNICATION
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An esterase-activated magnetic resonance contrast agent{
a
Marco Giardiello, Mark P. Lowe* and Mauro Botta
a
b
Received (in Cambridge, UK) 6th August 2007, Accepted 7th September 2007
First published as an Advance Article on the web 14th September 2007
DOI: 10.1039/b711989e
A Gd(III) complex bearing pendant acetoxymethyl esters is
activated on exposure to porcine liver esterase; the 84% increase
into the negatively charged species, i.e. activated, resulting in a
2
change in hydration state (HCO
no longer binding to Gd(III) at
3
2
22
in relaxivity is a result of suppression of HCO3 /CO3 binding
by the resulting negative charge.
physiological pH). The outflow from the cell of the now negatively
charged complex is expected to be significantly reduced, hence the
7
agent is accumulated in the cell. This hypothesis is illustrated in
In recent years the focus of research into Gd(III)-based contrast
agents has shifted to the development of ‘smarter’, responsive or
Scheme 1. The switch from neutral to negatively charged species is
achieved by masking the negative charge with appended carboxylic
esters. Acetoxymethyl esters have been used for a number of years
to mask negative charge, e.g. to ‘smuggle’ charged complexes into
1
activated contrast agents, i.e. complexes whose relaxivities are
2
modulated by a particular in vivo stimulus such as pH, metal ion
3
4
7
concentration or enzyme-activity. In order to image at the
molecular level, smarter contrast agents are required that display
significant changes in relaxivity, i.e. signal intensity on activation.
This communication describes an activation and accumulation
approach to attempt to address the inherent insensitivity of the
magnetic resonance imaging technique. Cyclen-based complexes of
Gd(III) that are 7-coordinate with respect to ligand tend to possess
two inner-sphere water molecules, i.e. their hydration state q = 2. It
is well-established that such complexes generally make poor
contrast agents; the expected high relaxivity engendered by two
inner-sphere waters is not manifested in vivo. This is due to the
affinity of this type of often cationic or neutral complex for
endogenous serum anions such as hydrogencarbonate, phosphate,
cells. Such esters are excellent substrates for non-specific
intracellular esterases. A recent example of the use of acetoxy-
methyl esters is the trapping of fluorescent lipophilic ferrichrome
3
+
analogues inside cells (for Fe chelation) by Shanzer and co-
8
workers. Lippard and co-workers have recently utilised intracel-
2+ 9
lular esterase activity to activate fluorescent sensors for Zn . The
more hydrolytically robust ethyl ester-containing 2 was prepared
as a model for the acetoxymethyl-containing 3 used in this study.
The interaction of their Gd(III) complexes with carbonate in the
presence and absence of porcine liver esterase is reported.
The Eu(III) and Gd(III) complexes of 2 and 3 were synthesised as
32
32
shown in Scheme 2. [Eu.4] and [Gd.4] were prepared for
6
comparison as the post-enzyme-activity complexes. The ethyl
5
lactate and citrate, as well as carboxylate residues on proteins. To
ester-containing [Eu.2] and [Gd.2] were prepared as models for the
corresponding acetoxymethyl-containing complexes [Eu.3] and
[Gd.3]. The ethyl esters are more resistant to base-catalysed
hydrolysis, thus enabling the pH-dependency of carbonate binding
some extent, such anions displace the inner-sphere waters,
rendering the complex a poor contrast agent. Of these coordinat-
2
ing anions, HCO
3
22
(bound as carbonate, CO
3
) is the most
abundant in serum (20–30 mM), possesses a relatively high affinity
for this type of q = 2 complex and is the focus of this
communication.
2
It has been demonstrated that this affinity for HCO
3
can be
suppressed by introduction of negative charge into complexes of
6
this type. The work reported herein takes advantage of this
2
difference in affinity for HCO3 between neutral and negatively
charged complexes, by using enzyme-activation to switch from a
neutral contrast agent with an affinity for carbonate-binding to a
negatively charged one where the suppression of carbonate-
binding is exploited. This enzyme-activation is manifested as a
concomitant change in hydration state and hence relaxivity.
The rationale behind the design of these complexes is that the
neutral species (in equilibrium with the carbonate-bound species)
will be capable of crossing the cell membrane, e.g. via pinocytosis.
Once internalised, the neutral complex is designed to be converted
a
Department of Chemistry, University Road, University of Leicester,
Leicester, UK LE1 7RH. E-mail: mplowe@le.ac.uk
b
Dipartimento di Scienze dell’Ambiente e della Vita, Universit a` del
Piemonte Orientale ‘‘Amedeo Avogadro’’, Via Bellini 25/G, I-15100,
Alessandria, Italy
{ Electronic supplementary information (ESI) available: Experimental and
details of lifetime and relaxivity studies. See DOI: 10.1039/b711989e
Scheme 1 Proposed activation and accumulation strategy.
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4
044 | Chem. Commun., 2007, 4044–4046

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2
concentration of HCO
3
, it is lowered significantly, to give an
apparent q = 1.2.{ This indicates the presence of y40% carbonate-
bound species in solution. This carbonate-binding equilibrium has
been shown previously to be pH-dependent for related q = 2
5
complexes, irrespective of the charge on the complex. It is
2
noteworthy that the affinity for HCO
3
of the negatively charged
32
complex [Eu.4] is essentially negligible at pH 7.4. In the presence
2
of HCO3 , there is a y70% increase in q moving from
3
2
[
Eu.2]/[Eu.3] to [Eu.4]
The effect of carbonate-binding to the neutral and negatively
charged Gd(III) complexes can be seen in Fig. 1. The plot shows
.
32
the change in relaxivity of [Gd.2] and [Gd.4] vs. pH in the
presence of 30 mM NaHCO (the acetoxymethyl esters of [Gd.3]
3
are too sensitive to base hydrolysis to allow titration to basic pH).
The plot clearly shows that both neutral ([Gd.2]) and negatively
3
2
charged ([Gd.4] ) complexes will bind carbonate at high enough
pH. At pH 10, both complexes have a similar relaxivity of
21 21
y3.0 mM
s , characteristic of a q = 0 complex; however, the
major difference between these two species is the pH at the onset of
carbonate binding. At pH , 5.0 both complexes are q = 2 as no
carbonate is bound, but as the pH increases from 5.0 the neutral
Scheme 2
[Gd.2] starts to bind carbonate, indicated by a gradual decrease in
to be probed (acetoxymethyl esters are very susceptible to
hydrolysis under even mildly basic conditions). The mixed
relaxivity as inner-sphere water molecules are displaced. A similar
32
change is seen for negatively charged [Gd.4] , but the binding of
carbonate and the subsequent decrease in relaxivity do not occur
until the pH of the solution is . 7.0. The complicated form of the
plotted data is due to the presence of various interlinked
equilibrium processes: the pH-dependent speciation of carbonate,
the pH-dependent deprotonation of bound water molecules and
the carbonate-binding equilibrium. What Fig. 1 clearly demon-
t
ethyl/ butyl ester of racemic 2-bromoadipic acid was reacted with
2 3 3
cyclen in the presence of K CO in CH CN to give 1 as a statistical
mix of six stereoisomeric forms due to the absolute configuration
at the a-carbon (RRR/SSS, RRS/SSR, RSR/SRS). Acid hydro-
t
lysis of the butyl esters in TFA yielded the pro-ligand, which was
reacted with EuCl ?6H O or GdCl ?6H O in H O to give [Eu.2]
3
2
3
2
2
32
and [Gd.2] respectively. Synthesis of [Eu.3] and [Gd.3] was slightly
strates is the difference in relaxivity of [Gd.2] and [Gd.4] at
less straightforward: following base hydrolysis of the ethyl esters of
physiological pH. There is a pronounced difference between the
32
pre- and post-enzyme activated complexes ([Gd.2] and [Gd.4]
1
and Boc-protection, the acetoxymethyl esters were introduced by
reaction with bromomethyl acetate and DIPEA in CH Cl
Removal of the Boc and butyl groups in TFA yielded the pro-
ligand which was reacted with EuCl ?6H O or GdCl ?6H O in
MeOH to give [Eu.3] and [Gd.3] respectively.
Luminescent lifetime measurements on the Eu(III) complexes in
O and D O enable the determination of the hydration state (q)
of the complexes; the same values can be inferred for the
2
.
respectively). The negatively charged post-ester hydrolysis species
32
2
t
2
[Gd.4] has less affinity for HCO , suggesting a significant
3
potential increase in relaxivity can be obtained upon activation of
3
2
3
2
the contrast agent by esterase.
2
In the absence of HCO , all three of the Gd(III) complexes
3
H
2
2
have a high relaxivity (Table 2). These values are a result of their
10
2
corresponding Gd(III) complexes. In the absence of HCO3 at
pH 7.4, all three complexes possess two inner-sphere water
32
molecules. For [Eu.2], [Eu.3] and [Eu.4] calculated values of q =
.1 were obtained (Table 1). In the presence of 30 mM NaHCO a
2
3
2
significant change in excited state lifetime in H O (1/k) is noted for
the two neutral complexes [Eu.2] and [Eu.3] (displacement of
quenching inner-sphere waters giving longer lifetimes). Whilst the
hydration state does not fall to zero at physiological pH at this
Table 1 Radiative rate constants (k) and calculated hydration states
q) for Eu(III) complexes (1 mM, lex = 395 nm, pH 7.4, 298 K)
(
2
1
21
k
H O/ms
k
D O/ms
q (¡0.2)
2
2
[
[
[
[
[
[
Eu.2]
Eu.3]
Eu.4]
3.32
3.26
3.30
2.83
2.75
3.26
1.36
1.23
1.33
1.61
1.46
1.38
2.1
2.1
2.1
1.2
1.2
2.0
3
2
Eu.2] + 30 mM NaHCO
Eu.3] + 30 mM NaHCO
Eu.4] + 30 mM NaHCO
3
3
Fig. 1 Relaxivity (r1p) vs. pH for 1 mM [Gd.2] (closed circles) and
32
3
32
3
[Gd.4] (open circles) in 30 mM NaHCO (298 K, 20 MHz).
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2
1
21
Table 2 Relaxivities r1p (mM
complexes in the presence and absence of NaHCO
porcine liver esterase (100 units) (pH 7.4, 298 K, 20 MHz)
s
) ¡5% of Gd(III) (0.2 mM)
The authors thank Prof. Silvio Aime (Universit a` di Torino)
for the use of his relaxometers and the EPSRC National
Mass Spectrometry Service Centre, Swansea for high resolution
ESMS.
3
(10 mM) and
Complex +
Complex
only
Complex +
esterase
Complex +
NaHCO
3
NaHCO +
esterase
3
Notes and references
[Gd.2]
[Gd.3]
[Gd.4]
10.2
9.9
11.3
10.8
10.5
10.8
5.7
5.7
10.8
10.8
10.5
10.8
{ More details on the fitting of the decay curves are contained in the
supplementary information.
32
1
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R
t cf. [GdDOTA] ). These values are similar to those of
11
GdHOPO-based q = 2 complexes.
To demonstrate the ability of an enzyme to activate the contrast
agents, the relaxivities of the three Gd(III) complexes were
2
measured in the presence and absence of HCO3 and porcine
liver esterase. The results of these studies are shown in Table 2.
Solutions were prepared containing complex alone; complex
+
10 mM NaHCO
NaHCO + esterase (intracellular concentration of HCO
y10 mM). Relaxivities of the solutions were measured (at 298 K,
0 MHz) 2 h after incubation at 310 K. The results clearly
3
; complex + esterase; and complex + 10 mM
2
3
3
is
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2
demonstrate ester hydrolysis is occurring; an increased relaxivity is
noted for the neutral complexes [Gd.2] and [Gd.3] in the presence
3
2
of the enzyme as they are converted to [Gd.4] . This is entirely
3
3
2
expected as [Gd.4] possesses a slightly higher relaxivity than
[
Gd.2] or [Gd.3]. Both [Gd.2] and [Gd.3] exhibit a fall in relaxivity
2
1 21
s
in the presence of 10 mM NaHCO
3
(r1p = 5.7 mM
for both);
this correlates with the pH-dependency of carbonate-binding
32
depicted in Fig. 1. The slight lowering of relaxivity of [Gd.4]
21 21
from 11.3 to 10.8 mM
s
3
in the presence of 10 mM NaHCO is
4
again expected due to the low affinity for carbonate of this
complex at pH 7.4. The most important observation is the
relaxivity enhancement of both [Gd.2] or [Gd.3] in the presence of
1
0 mM NaHCO
3
when exposed to porcine liver esterase.
21
21
The increase in relaxivity from 5.7 mM
21 21
s
to 10.8 and
10.5 mM
s
respectively for [Gd.2] or [Gd.3] is due to their
32
conversion to the negatively charged complex [Gd.4] with its
much reduced affinity for carbonate at pH 7.4, i.e. complete
conversion occurs. The effect of enzyme-activation produces an
5
89% and 84% increase in relaxivity for complexes [Gd.2] and
[Gd.3] respectively. Such a large percentage increase is a significant
change with respect to magnetic resonance imaging.
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and their propensity to bind endogenous HCO
3
has been
exploited. On activation by esterase, the relaxivities of these
complexes increased by y85% at physiological pH and NaHCO3
concentration, as anion binding is inhibited by the unmasked
negative charge. This augurs well for developing the proposed
accumulation and activation strategy for cellular MR imaging.
Indeed, one of the few enzyme-activated agents to be used in
6
7
8 M. M. Meijler, R. Arad-Yellin, Z. I. Cabantchik and A. Shanzer, J. Am.
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1
2
enzyme activation; sufficient for in vivo imaging of gene
4
expression. Studies are underway to incorporate targeting
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046 | Chem. Commun., 2007, 4044–4046
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