Zn2+ responsive bimodal magnetic resonance imaging and fluorescent imaging probe based on a gadolinium(III) complex

Article abstract of DOI:10.1021/ic301308z

A Zn²⁺-responsive bimodal magnetic resonance imaging (MRI) and luminescence imaging probe GdL was synthesized. The relaxivity and luminescence properties were examined. In the presence of 0.5 equiv of Zn²⁺, the longitudinal relaxivity is increased from 3.8 mM^-1 s^-1 to 5.9 mM^-1 s^-1 at 23 MHz and 25 °C with 55% enhancement, whereas the fluorescence exhibits a 7-fold increase. The Zn²⁺ responsive imaging probe shows favorable selectivity and tolerance over a variety of biologically relevant anions and metal ions in physiological pH range for both relaxivity and luminescence. In vitro phantom images and confocal fluorescence images in living cells show that the bimodal Zn²⁺ probe can effectively enhance T₁-weighted imaging contrast and luminescence imaging effect through Zn²⁺ coordination with excellent cellmembrane permeability and biocompatibility. Spectral and electrospray ionization mass spectrometry (ESI-MS) studies indicate that two different Zn²⁺-bound species, (GdL)₂Zn and GdLZn, are formed when 0.5 and 1 equiv of Zn²⁺ are bound to GdL complex, respectively. Crystal structural determination and dysprosium-induced ¹⁷O NMR shift (DIS) experiment demonstrate that the increased molecular weight and the improved molecular rigidity upon complexation of Zn²⁺ with GdL is the primary factor for relaxivity enhancement. Significant enhancement of the luminescence is due to a heavy atom effect and much increased molecular rigidity upon Zn²⁺ binding to 8-sulfonamidoquinoline chromophore.

Full text of DOI:10.1021/ic301308z

Article
pubs.acs.org/IC
Zn2+ Responsive Bimodal Magnetic Resonance Imaging and
Fluorescent Imaging Probe Based on a Gadolinium(III) Complex
Jian Luo, Wei-Sheng Li, Peng Xu, Li-Yi Zhang, and Zhong-Ning Chen*
State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences,
155 Yangqiao Road West, Fuzhou 350002, China
S
* Supporting Information
ABSTRACT: A Zn2+-responsive bimodal magnetic resonance
imaging (MRI) and luminescence imaging probe GdL was
synthesized. The relaxivity and luminescence properties were
examined. In the presence of 0.5 equiv of Zn2+, the
longitudinal relaxivity is increased from 3.8 mM−1 s−1 to 5.9
mM−1 s−1 at 23 MHz and 25 °C with 55% enhancement,
whereas the fluorescence exhibits a 7-fold increase. The Zn2+
responsive imaging probe shows favorable selectivity and
tolerance over a variety of biologically relevant anions and
metal ions in physiological pH range for both relaxivity and
luminescence. In vitro phantom images and confocal fluorescence images in living cells show that the bimodal Zn2+ probe can
effectively enhance T1-weighted imaging contrast and luminescence imaging effect through Zn2+ coordination with excellent
cellmembrane permeability and biocompatibility. Spectral and electrospray ionization mass spectrometry (ESI-MS) studies
indicate that two different Zn2+-bound species, (GdL)2Zn and GdLZn, are formed when 0.5 and 1 equiv of Zn2+ are bound to
GdL complex, respectively. Crystal structural determination and dysprosium-induced 17O NMR shift (DIS) experiment
demonstrate that the increased molecular weight and the improved molecular rigidity upon complexation of Zn2+ with GdL is the
primary factor for relaxivity enhancement. Significant enhancement of the luminescence is due to a heavy atom effect and much
increased molecular rigidity upon Zn2+ binding to 8-sulfonamidoquinoline chromophore.
imaging agents that respond to specific metal ions or chemical
species in living biological systems have been less explored
except for one Cu2+-selective GdDO3A-based MRI contrast
agent that exhibits luminescence turn-off response.6 It
represents a significant challenge for synthetic chemists to
achieve responsive bimodal MRI/luminescence imaging probes
that exhibit simultaneous turn-on effect in both luminescence
and relaxivity in response to a specific metal ion or a
biologically relevant substance.
INTRODUCTION
■
Multiple imaging techniques have been widely applied in
clinical diagnostics although none of them could provide
comprehensive information of the human body each because of
their own limitations.1 Recently, the development of bimodal or
multimodal imaging agents with the combination of two or
more molecular imaging techniques has been receiving more
and more attention.1b Luminescence imaging and magnetic
resonance imaging (MRI) are both powerful approaches in
disease diagnostic, and in fact they are complementary
analytical techniques. As a sensitive, specific, and fast response
analytical method, luminescence imaging is suitable for
generating high-resolution images but could not penetrate in
thick tissues owing to lack of optical transparency.2 In contrast,
MRI is a safe, noninvasive, and nonradiative technique that is
perfect for providing whole body images, but its spatial
resolution is only in the 0.1 mm range.2b,3 When luminescence
imaging and MRI are synchronously used, much more
complete information regarding tissue and organs in the
human body can thus be obtained.
According to the Solomon−Bloembergen−Morgan equa-
tion,7 the factors that contribute to the relaxivity for a MRI
contrast agent include the number of inner-sphere water
molecules (q), the rotational tumbling time (τR), and the
residence lifetime of inner-sphere water molecules (τm).3,5a,8 To
design a responsive dual-mode contrast agent with turn-on
effect in both luminescence and relaxivity, it is necessary to
increase the number of inner-sphere water molecules (q),
increase molecular weight, or improve molecular rigidity of the
contrast agents upon metal ion binding to chromophores with
switch-on luminescence.
Since quinoline-based compounds are ideal probes for Zn2+
because of their high selectivity, low toxicity, cell permeability,
and relatively convenient synthetic procedure and functional-
Several bimodal MRI and luminescence imaging agents based
on gadolinium(III) systems or magnetic nanoparticles (NPs)
have been reported in recent years.1b,2b,4 Some GdDO3A-based
smart MRI contrast agents with selective response to specific
metal ions or anions in the human body have been also
developed.5 Nevertheless, bimodal MRI and luminescence
Received: June 20, 2012
Published: August 10, 2012
© 2012 American Chemical Society
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a
Scheme 1. Synthetic Routes to GdL
a
(i) Chloroacetyl chloride, triethylamine, rt, 4 h; (ii) tris-tert-butyl-DO3A, K2CO3, CH3CN, 70°C, 1 d; (iii) TFA/CH2Cl2 (1: 1), rt, 1 d; (iv)
GdCl3·6H2O, water, pH = 7.0, rt, 1 d.
ization,9 8-sulfonamidoquinoline chromophore is elaborately
introduced to a Gd-DO3A moiety so that Zn2+-responsive MRI
and luminescence imaging agent GdL (Scheme 1) was thus
accessed with significant enhancement in both relaxivity and
luminescence. This Zn2+-responsive bimodal imaging probe
shows favorable selectivity and tolerance over a variety of
biologically relevant anions and metal ions in physiological pH
range. Effectively improved T1-weighted imaging contrast and
strikingly enhanced luminescence imaging effect together with
good cell membrane permeability and biocompatibility were
demonstrated through in vitro phantom images and confocal
fluorescence images in living cells.
Figure 1. Relaxivity titration of GdL with Zn2+ in 100 mM HEPES
buffer at pH = 7.2. All the measurements were performed at 23 MHz
RESULTS AND DISCUSSION
and 25 °C. The inner picture of T1-weighted phantom MR images of
0.5 mM GdL in the absence (a) or presence of (b) 0.5 equiv of Zn2+
and (c) 1 equiv of Zn2+.
■
Synthetic routes to complex GdL are depicted in Scheme 1.
Reaction of 1 with 1.2 equiv of chloroacetyl chloride in the
presence of triethylamine gave 2 in 70% yield. Compound 3
was prepared by the reaction of 2 and tris-tert-butyl-DO3A in
the presence of K2CO3 in 53% yield. Ligand H3L in 92% yield
was then accessed through removal of protected tris-tert-
butylester in trifluoroacetic acid (TFA) and CH2Cl2 (v/v =
1:1). Addition of equimolar GdCl3·6H2O to an aqueous
solution of H3L by maintaining pH = 7.0 using 1 M NaOH
solution thus afforded complex GdL as a white powder.
The longitudinal relaxivity r1 of GdL was investigated by a 23
MHz NMR Analyzing & Imaging system at 25 °C in 100 mM
HEPES buffer solution at pH = 7.2. The r1 of GdL was
estimated as 3.8 mM−1 s−1, which was gradually increased with
the addition of Zn2+. Upon addition of 0.5 equiv of Zn2+, the r1
was increased to 5.9 mM−1 s−1 with 55% enhancement. When 1
equiv of Zn2+ was added, the r1 was measured as 5.2 mM−1 s−1.
Nevertheless, further change of r1 value was not observed by
addition of more than 1 equiv of Zn2+ (Figure 1). It is known
that the parent gadolinium(III) complex of 1,4,7,10-tetraaza-
dodecanetetraacetic acid (Gd-DOTA) exhibited a relaxivity of
3.9 mM−1 s−1 when it was recorded on a 400 MHz NMR
spectrometer at 25 °C.10 T1 measurements of GdL were also
performed at 400 MHz and 25 °C, and the r1 value of GdL was
estimated as 4.0 mM−1 s−1 in this condition. Although GdL
affords a comparable relaxivity with parent Gd-DOTA at high
field (9.4 T), it exhibits significantly Zn2+-responsive relaxivity
change with 55% relaxivity enhancement (5.9 mM−1 s−1) at low
field (0.5 T). The overall variation in relaxivity upon addition of
Zn2+ is sufficiently sensitive for specific detection of Zn2+ in
physiological conditions. As shown in Figure 1, T1-weighted
imaging contrast of GdL was strikingly enhanced upon addition
of 0.5 or 1 equiv of Zn2+.
To detect selectivity and tolerance of GdL in response to
Zn2+, the relaxivity measurement was carried out in the
presence of other biological metal ions (Figure 2). The r1 of
GdL was almost unchanged by the addition of 0.5 equiv of Ca2+
(3.9 mM−1 s−1), Mg2+ (3.9 mM−1 s−1), Fe2+ (3.8 mM−1 s−1), or
Fe3+ (3.7 mM−1 s−1). The interference experiments were
carried out in the same conditions by the addition of 0.5 equiv
of Zn2+ and 0.5 equiv of an interfering metal ion. As depicted in
Figure 2, Zn2+-enhanced relaxivity of GdL was little influenced
by other biological metal ions such as Ca2+ (5.9 mM−1 s−1),
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0.5 mM Fe3+, and 0.1 mM Cu2+ as well as a series of biological
anions (vide infra).
There are various anions that exist in human body, and some
of them display high concentration, which may affect the
relaxivity of Gd-based contrast agents.12 To determine the
tolerance of this Zn2+-responsive MRI system to various
biological anions, Zn2+-responsive relaxivity changes of GdL
were investigated in the presence of a variety of biological
anions at physiologically relevant concentrations. As shown in
Figure 3, the influence by most of the anions is negligible
except for citrate and lactate which induce a little less relaxivity
enhancement of GdL in response to 0.5 equiv of Zn2+.
Nevertheless, when a simulated extracellular anion solution
(EAS) containing 30 mM NaHCO3, 100 mM NaCl, 0.9 mM
KH2PO4, 2.3 mM sodium lactate, and 0.13 mM sodium citrate
(pH = 7.0)13 was used, GdL displayed 45% enhancement of the
relaxivity in response to 0.5 equiv of Zn2+. Consequently, GdL
displays a satisfactory tolerance to various biological anions and
metal ions.
Figure 2. Relaxivity of GdL at 23 MHz in the presence of various
metal ions at 25 °C. Blank bars represent the relaxivity upon addition
of 0.5 equiv of appropriate metal ion to the solutions of GdL. Shadow
bars represent the relaxivity upon addition of 0.5 equiv of Zn2+
together with 0.5 equiv of another interfering metal ion to the
solutions of GdL. All solutions were prepared in 100 mM HEPES
buffer at pH = 7.2.
Mg2+ (5.8 mM−1 s−1), Cu2+ (5.7 mM−1 s−1), Fe2+ (5.8 mM−1
s−1), and Fe3+ (5.7 mM−1 s−1). Thus, complex GdL exhibits
highly selectivity in response to Zn2+ with remarkable relaxivity
enhancement which is almost undisturbed by other biological
metal ions. Although GdL displays a little relaxivity enhance-
ment in response to 0.5 equiv of Cu2+ (4.6 mM−1 s−1), the
interference of Cu2+ is still limited considering that the content
of Cu2+ is only about 1/20 of Zn2+ in the human body.11
Furthermore, the calculated stability constant of GdLCu (4.2 ×
104 M−1) is much smaller than that of (GdL)2Zn (4.2 × 1011
M−2) or GdLZn (2.1 × 106 M−2) species (vide infra),
suggesting that the binding capability of GdL to Zn2+ is
much larger than that to Cu2+. As indicated in Figure 3, the
relaxivity of GdL was increased from 3.6 mM−1 s−1 to 5.0 mM−1
s−1 in response to 0.5 equiv of Zn2+ even if the measurement
was carried out in a simulated biological solution system that
contains 5 mM K+, 2 mM Ca2+, 0.7 mM Mg2+, 0.5 mM Fe2+,
Complex GdL is also a selective luminescence sensor for
Zn2+. Luminescence studies were carried out at 25 °C in a 100
mM HEPES buffer solution at pH = 7.2. Figure 4 depicts the
Figure 4. Fluorescence spectra of GdL (20 μM) in the presence of 0.5
or 1 equiv of Zn2+, and 1 equiv of K+, Na+, Ca2+, Mg2+, Fe2+, Fe3+, and
Cu2+ in 100 mM HEPES buffer solutions at 25 °C, pH = 7.2 (λex = 360
nm). Inset luminescence photographs were taken from 50 μM GdL
solutions in the absence and presence of 0.5 or 1 equiv of of Zn2+
under UV light irradiation at 365 nm.
emission spectral changes of GdL in the presence of various
metal ions. Although the 8-sulfonamidoquinoline chromophore
of GdL exhibited weak emission centered at 496 nm, addition
of 0.5 equiv of Zn2+ resulted in 7-fold luminescence
enhancement and a distinct red-shift to 503 nm because of
an internal charge transfer process from 8-sulfonamidoquino-
line chromophore to Zn2+.9d When 1 equiv of Zn2+ was added,
the emission spectrum was red-shifted to 530 nm with 5.5-fold
luminescence enhancement relative to GdL. As shown in Figure
4 (inset), bright green and yellow-green emitting was observed
when 0.5 and 1.0 equiv of Zn2+ was added to a 50 μM solution
of GdL with the emission centered at 503 and 530 nm,
respectively. Interference (Figure 5) experiments revealed that
distinct luminescence changes were not observed upon addition
of 0.5 equiv of biological metal ion to the solution of GdL
except that Cu2+ significantly quenched the emission. Although
both Fe3+ and Cu2+ are paramagnetic and redox-active, it is
likely that complexation of Cu2+ to GdL induces quenching of
the emission whereas Fe3+ is existent in free ion without
binding to GdL.
Figure 3. Relaxivity response of GdL to 0.5 equiv of Zn2+ at 23 MHz
in the presence of biologically relevant anions. Blank bars represent the
relaxivity of GdL in the presence of corresponding anions. Shadow
bars represent the relaxivity upon addition of 0.5 equiv of Zn2+ to GdL
in presence of corresponding anions. All measurements were
performed in 100 mM HEPES buffer, pH = 7.2 except for PBS,
EAS, and the complicated system. The relaxivity measurement with
PBS was acquired under similar conditions using PBS, pH = 7.4. EAS
contains 30 mM NaHCO3, 100 mM NaCl, 0.9 mM KH2PO4, 2.3 mM
sodium lactate, and 0.13 mM sodium citrate, pH = 7.0. A complicated
solution system contains all the anions in ESA as well as 5 mM KCl, 2
mM CaCl2, 0.7 mM MgCl2, 0.5 mM FeCl2, 0.5 mM FeCl3, and 0.1
mM CuCl2, pH = 7.0.
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The emission spectral changes were monitored by titration of
GdL with Zn2+ in 100 mM HEPES buffer solutions at pH = 7.2
(Figure 7). The luminescence was first progressively enhanced
Figure 5. Fluorescence (λex = 360 nm) intensity of GdL (20 μM) in
the presence of 0.5 equiv of biological metal ions (blank bars), or 0.5
equiv of Zn2+ together with 0.5 equiv of an interfering metal ion
(shadow bars). The last two bars represent the fluorescence intensity
of GdL (20 μM) in the absence (blank bar) and presence (shadow
bar) of 1 equiv of Zn2+.
Figure 7. Emission spectral changes of GdL (20 μM) upon addition of
Zn2+ ([Zn2+] = 0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5, and
Zn2+-responsive luminescence imaging of GdL in breast
cancer cell line MDA-MB-231 was investigated through
confocal luminescence microscopy. Incubation of MDA-MB-
231 cells with 20 μM GdL in PBS buffer for 30 min at 37 °C
gave a very weak intracellular luminescence (Figure 6a). When
the cells were subsequently incubated with Zn2+ (30 μM) at 37
°C for 20 min, the yellow-green luminescence became much
brighter which was clearly observable to the naked-eye (Figure
6d). Bright-field measurements (Figure 6b and 6e) indicated
that the cells were viable throughout the imaging experiments
upon treatment with GdL and Zn2+, respectively. It appears that
complex GdL can be ingested harmlessly by living cells. As
depicted in Figure 6f, the luminescence signals of GdL-Zn
species were mainly localized at the cytoplasm, implying good
cell membrane permeability and an intracellular distribution of
GdL-Zn species.
30 μM) in 100 mM HEPES buffer solutions at 25 °C, pH = 7.2 (λex
=
360 nm).
to reach a maximum at 503 nm when 0.5 equiv of Zn2+ was
added. Further addition of more than 0.5 equiv of Zn2+ induced
a red-shift of the emission at 503 to 530 nm with a little
reduced luminescence intensity. The two-step emission spectral
changes thus suggest the formation of two types of Zn2+-bound
species depending on the amount of Zn2+ added. As revealed by
electrospray ionization mass spectrometry (ESI-MS) studies,
when 0.5 equiv of Zn2+ was added, the base peak at 1741.5
corresponded to the molecular ion peak of (GdL)2Zn
(Supporting Information, Figure S6) through complexation of
one Zn2+ with two GdL moieties. When 1 equiv of Zn2+ was
used, the observed base peak at 938.0 ([M+2H2O−H+]−)
implied the formation of species GdLZn (Supporting
Figure 6. Confocal fluorescence images in MDA-MB-231cells. (a) Cells incubated with 20 μM GdL in PBS buffer for 30 min; (b) Bright-field image
of (a); (c) Overlay image of (a) and (b); (d) Cells incubated first with 20 μM GdL in PBS buffer for 30 min, and then incubated with 30 μM Zn2+
for 20 min; (e) Bright-field image of (d); (f) Overlay image of (d) and (e).
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Information, Figure S7) with a 1:1 ratio between Zn2+ and
GdL. The stability constants of (GdL)2Zn (ka) and GdLZn (kb)
species defined in eqs 1−3 were determined as 4.2 × 1011 M−2
and 2.1 × 106 M−1, respectively.
ka
2GdL + Zn2+ ⇌ (GdL)2Zn
[(GDL)2Zn]
ka =
[GdL]2[Zn2+
]
(1)
(2)
(3)
kb
GdL + Zn2+ ⇌ GdLZn
[GdLZn]
kb =
[GdL][Zn2+
]
kCu
GdL + Cu2+ HooI GdLCu
[GdLCu]
kCu
=
[GdL][Cu2+]
The UV−vis spectral changes by titration of GdL with Zn2+
revealed that a new absorption band at 360 nm (Supporting
Information, Figure S2) which reached the maximum upon
addition of 1 equiv of Zn2+. Similarly, titration of GdL with
Cu2+ resulted in a new absorption band centered at 370 nm
(Supporting Information, Figure S3), which was gradually
increased to the maximum upon addition of 1 equiv of Cu2+.
The calculated stability constant of GdLCu (kCu) as defined in
eq 3 is 4.2 × 104 M−1. The pH-dependent Zn2+-responsive
fluorescence changes (Supporting Information, Figure S4) of
GdL suggested that the fluorescence of (GdL)2Zn and GdLZn
complexes reached their maxima at pH = 6 and 5.5,
respectively. At pH = 7.2 in physiological condition, the
fluorescence exhibits 7- and 5.5-fold enhancement upon
formation of (GdL)2Zn and GdLZn species, respectively.
The structure of neutral complex GdLZn(H2O)3·5H2O was
successfully determined by X-ray crystallography. Because of
the difficulty in crystallization, only a limited number of Gd-
DO3A complexes have been characterized by single crystal X-
ray diffraction.2b,3b,14 As depicted in Figure 8, the Gd3+ is nine-
coordinated to adopt a monocapped square antiprismatic
geometry with eight apexes occupied by four amine N, three
carboxylate O, and one amide O donors whereas the capped
site is occupied by a H2O molecule. The Gd−OH2 length
(2.500(3) Å) is much longer than other four Gd−O bonds
(2.359(2)−2.382(3) Å), and also a little longer than that found
in Na[Gd(DOTA)(H2O)] (2.458 Å).15 The Zn2+ is five-
coordinated to give a distorted trigonal bipyramid, in which the
trigonal plane is composed of amide N (N5), quinoline N
(N7), and one coordinated H2O (O12) whereas two pyramidal
apexes are occupied by sulfamide N (N6) and another
coordination H2O (O11).
Figure 8. ORTEP drawing of neutral complex GdLZn with atom-
labeling scheme showing 30% thermal ellipsoid.
Figure 9. Plots of DIS of the 17O NMR signals of H2O (−Δ, ppm) vs
[Dy] for DyCl3 ( ), DyL ( ), and (DyL)2Zn ( ). The 17O NMR
spectra were recorded at room temperature in 100 mM HEPES buffer
solutions containing 20% D2O, pH = 7.2.
■
●
▲
water molecule. The measured 17O NMR shift for DyL was 52
ppm/M, corresponding to q = 1.4. When 0.5 equiv of Zn2+ was
added to the solution of DyL, the corresponding DIS was
measured as 48 ppm/M, corresponding to q = 1.3 in complex
(DyL)2Zn. The 17O NMR experimental studies thus suggest
that one coordination water remains unchanged upon
complexation of two DyL with one Zn2+ to form (DyL)2Zn.
This coincides is well with that in the heteronuclear complex
GdLZn, in which one H2O molecule (q = 1) bound to Gd3+
was unambiguously determined by X-ray crystallography.
Consequently, Zn2+-triggered relaxivity enhancement in this
system is not because of the changes in inner-sphere
coordination water molecules (q) upon complexation of GdL
with Zn2+.
Dysprosium-induced 17O NMR shift (DIS) experimental
studies on DyL and its Zn2+-bound complex (DyL)2Zn were
performed so that the number of coordination hydration (q) in
aqueous solution was determined, respectively. The corre-
sponding DIS data for Zn2+-bound DyLZn species, however,
was not obtained because of its solubility limitation in a high
concentration. Calibrations with standard DIS plots for DyCl3
(q = 9) to determine q values were performed by Peters’s
method.15 As depicted in Figure 9, a plot of DIS versus DyCl3
concentration in HEPES buffer solution revealed a slope of 332
ppm/M, corresponding to 37 ppm/M shift per coordination
To make a mechanistic elucidation of Zn2+-enhanced
relaxivity and luminescence, a proposed schematic diagram is
depicted in Scheme 2, showing formation of heterotrinuclear
array (GdL)2Zn through loss of a sulfamide proton as well as
heterobinuclear array GdLZn through further loss of an amide
proton upon GdL binding to 0.5 and 1 equiv of Zn2+,
respectively. The Gd3+ in GdL is nine-coordinated, in which
eight apexes are occupied by N and O donors from L, whereas
another site is bonded to one H2O molecule with q = 1. When
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Scheme 2. Schematic Binding Mode of GdL Bound to Zn2+
one Zn2+ is bound to two GdL moieties through monoanionic
N,N-bidentate 8-sulfonamidoquinoline moieties, heterotrinu-
clear complex (GdL)2Zn is formed in a 1:2 binding
stoichiometry between GdL and Zn2+, in which each Gd3+
remains in coordination to one H2O. The relaxivity enhance-
ment from 3.8 to 5.9 mM−1 s−1 is ascribed to an increased
molecular weight of (GdL)2Zn relative to the precursor GdL,
thus inducing a longer rotational tumbling time. The
luminescence is significantly enhanced because of a heavy
atom effect upon Zn2+ binding to 8-sulfonamidoquinoline
chromophore. There exist intermolecular hydrogen bonds
between pyridyl N atom and sulfanilamide of 8-sulfonamido-
quinoline chromophore in free precursor GdL, which results in
photoinduced electron transfer and nonradiative deactivation.9d
When 8-sulfonamidoquinoline is coordinated to Zn2+, the
electron-transfer deactivation is largely prohibited upon
formation of Zn2+-coordinated system with more extended π-
conjugation so that the luminescence exhibits significant
enhancement with a distinct red-shift from 496 to 503 nm.
When Zn2+ is bound to GdL with formation of GdLZn in a
1:1 ratio between GdL and Zn2+, the dianionic N-(quinolin-8-
yl)-2-acetamino-benzenesulfonamide moiety acts as a terden-
tate ligand to chelate Zn2+ through sulfamide N, quinoline N,
and amide N donors (Scheme 2) so that the luminescence is
red-shifted to 530 nm because of better π-conjugation in
GdLZn than that in (GdL)2Zn. Even if the molecular weight of
GdLZn (903) is not remarkably increased compared with GdL
(840), free rotation and wobble at amide C−N, C(phenyl)−
N(amide), sulfamide N−S, and S−C(phenyl) bonds of N-
(quinolin-8-yl)-2-acetamino-benzenesulfonamide moiety are
totally prohibited so that the molecular structure of GdLZn is
much more constrained to afford a significantly improved
rigidity compared with GdL, which induce a longer rotational
tumbling time. This makes GdLZn have a higher relaxivity
value than GdL. Nevertheless, the molecular weight of GdLZn
is much smaller than that of GdL)2Zn (1742) so that the
longitudinal relaxivity of GdLZn (5.2 mM−1 s−1) is a little lower
than that of (GdL)2Zn (5.9 mM−1 s−1), but still much higher
than that of GdL (3.8 mM−1 s−1). Therefore, Zn2+-responsive
relaxivity enhancement for GdL results mostly from the
increased molecular weight for (GdL)2Zn and improved
molecular rigidity3c,4d,16 for GdLZn when Zn2+ is chelated to
8-sulfonamidoquinoline chromophore. Overall, the increased
molecular weight is a more important factor than other factors
for Zn2+-coordination triggered relaxivity enhancement in this
system.
CONCLUSION
■
A Zn2+-responsive bimodal MRI and fluorescent imaging probe
was elaborately designed. It exhibits obvious Zn2+-responsive
relaxivity enhancement with improved signal contrast as well as
significant turn-on luminescence. The relaxivity enhancement
originates mostly from the increase in molecular weight as well
as the improvement in molecular rigidity through Zn2+
coordination. Significantly enhanced luminescence is ascribed
to a heavy atom effect and much increased molecular rigidity
upon Zn2+ binding to 8-sulfonamidoquinoline chromophore.
The sensitivity and tolerance of this Zn2+-responsive
luminescence and MRI bimodal sensor toward other common
biological metal ions and anions is satisfactory. Furthermore,
this Zn2+-responsive bimodal sensor displays good biocompat-
ibility and cell membrane permeability.
EXPERIMENTAL SECTION
■
General Procedures and Materials. All chemicals were reagent
grade and purchased from commercial sources. All reactions were
carried out under a dry argon atmosphere by using Schlenk techniques
and vacuum line systems unless otherwise specified. The solvents were
dried, distilled, and degassed prior to use. The N-(quinolin-8-yl)-2-
amino-benzenesulfonamide (1) was synthesized by Landry’s meth-
od.17 The tris-tert-butyl-DO3A (3) was modified by Finn’s method.18
N-(Quinolin-8-yl)-2-(2-chloroacetyl)-amino-benzenesulfona-
mide (2). To a CH2Cl2 (25 mL) solution of 1 (0.5 g, 1.7 mmol) and
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1
triethylamine (0.202 g, 2.0 mmol) cooled in an ice bath was added
dropwise a CH2Cl2 (5 mL) solution of chloroacetyl chloride (0.226 g,
2.0 mmol) with stirring. After 4 h stirring at room temperature, the
reaction mixture was washed with 1 M NaHCO3 aqueous (30 mL)
solution and brine (30 mL). The organic layer was dried over Na2SO4
and concentrated. The product was purified by chromatography on a
silica gel column using CH2Cl2 as eluent to give a yellow solid. Yield:
70% (0.44 g). ESI-MS (CH2Cl2): m/z 376.0 ([M+H]+). IR (KBr):
3342, 3248, 1687, 1159, 769 cm−1. 1H NMR (CDCl3, 400 MHz, TMS,
ppm): δ 4.33 (S, 2H), 7.12 (t, J = 7.72 Hz, 1H), 7.43 (m, 3H), 7.51
(m, 1H), 7.83 (d, J = 7.44 Hz, 1H), 7.95 (d, J = 8.00 Hz, 1H), 8.10 (d,
J = 8.23 Hz, 2H), 8.72 (m, 1H), 9.36 (s, 1H), 10.15 (s, 1H). 13C NMR
(CDCl3, 400 MHz, ppm): δ 43.09 (chloroacetyl), 116.74 (quinoline),
122.14 (quinoline), 123.35 (quinoline), 123.63 (phenyl), 124.48
(phenyl), 126.69 (phenyl), 127.75 (quinoline), 128.24 (quinoline),
129.91 (phenyl), 132.75 (phenyl), 134.20 (quinoline), 134.73
(phenyl), 136.53 (quinoline), 138.78 (quinoline), 148.91 (quinoline),
164.70 (chloroacetyl).
1-(2-N-quinolin-8-yl-benzenesulfonamino-acetamide)-
4,7,10-tris(tert-butoxy-carbonyl-methyl)-1,4,7,10-tetraaza-cy-
clododecane (3). A mixture of tris-tert-butyl-DO3A (0.26 g, 0.5
mmol), 2 (0.21 g, 0.56 mmol), and K2CO3 (0.5 g) in CH3CN (40
mL) was stirred at 70 °C for 1 d. After cooling, the mixture was
filtered, and the filtrate was evaporated to generate the crude residue,
which was purified by chromatography on a silica gel column using
CH2Cl2/CH3OH (v/v = 20: 1) as eluent to give a light olive brown
oil. Yield: 53% (0.23 g). ESI-MS (CH2Cl2): m/z 854.7 ([M+H]+).1H
NMR (CDCl3, 400 MHz, TMS, ppm): δ 1.42 (S, 18H), 1.47 (S, 9H),
2.80−3.50 (m, 24H), 7.06 (t, J = 7.68 Hz, 1H), 7.43 (m, 4H), 7.80 (d,
J = 7.24 Hz, 1H), 7.90 (s, 1H), 8.07 (d, J = 8.28 Hz, 1H), 8.13 (s, 1H),
8.73 (s, 1H). 13C NMR (CDCl3, 400 MHz, ppm): δ 28.14 (methyl),
52.01, 52.57 (cyclene), 55.46 (acetamide), 56.66 (carbonyl-methyl),
77.25 (tert-butyl), 122.04 (quinoline), 122.98 (quinoline), 126.64
(phenyl and quinoline), 128.23 (phenyl), 129.84 (quinoline and
phenyl), 133.21 (quinoline), 133.90 (phenyl), 135.47 (quinoline),
136.30 (quinoline and phenyl), 139.01 (quinoline), 148.94 (quino-
line), 170.42 (carbonyl).
1-(2-N-quinolin-8-yl-benzenesulfonamino-acetamide)-
4,7,10-tris(aceticacid)-1,4,7,10-tetraaza-cyclododecane (H3L).
To a CH2Cl2 (5 mL) solution of 3 (0.15 g, 0.14 mmol) was added
trifluoroacetic acid (5 mL) with stirring. After stirring at ambient
temperature for 1 d, the solution was evaporated, and the residue
redissolved in CH3OH. To the solution was added dropwise cold
diethyl ether to precipitate the product as a white solid, which was
filtered and dried in vacuo. Yield: 92% (0.11 g). ESI-MS (CH3OH):
m/z 686.7 ([M+H]+). 1H NMR (D2O, 400 MHz, ppm): δ 2.80−3.90
(m, 24H), 6.96 (t, J = 7.64 Hz, 1H), 7.22 (d, J = 7.72 Hz, 1H), 7.44 (t,
J = 8.26 Hz, 1H), 7.53 (t, J = 9.00 Hz, 2H), 7.60 (t, J = 15.69 Hz, 1H),
7.80 (m, 1H), 7.91 (d, J = 8.26 Hz, 1H), 8.77(d, J = 8.26, 1H), 8.92 (d,
J = 4.16 Hz, 1H). 13C NMR (D2O, 400 MHz, ppm): δ 48.22, 51.49
(tetraazacyclododecane), 54.67 (acetamide), 56.49 (acetic acid),
122.08 (quinoline), 126.23 (quinoline), 127.54 (quinoline), 127.86
(phenyl), 128.39 (phenyl), 129.41 (phenyl), 129.48 (quinoline),
129.79 (quinoline), 129.99 (phenyl), 131.79 (phenyl), 134.02
(quinoline), 134.75 (phenyl), 136.09 (quinoline), 145.43 (quinoline),
146.38 (quinoline), 170.32 (acetamide), 173.64 (carboxyl).
Physical Measurements. The H and 13C NMR spectra and 17O
NMR dysprosium induced shift (DIS) experiments were recorded at
400 MHz on a Bruker Avance III NMR spectrometer. Gadolinium
content was determined on an Ultima 2 inductively coupled plasma
OES (ICP-OES) spectrophotometer. Elemental analyses (C, H, and
N) were carried out on a Perkin-Elemer model 240C elemental
analyzer. ESI-MS were performed on a Finnigan LCQ mass
spectrometer. Fourier transform infrared spectra were recorded on a
Magna 750 FT-IR spectrophotometer with KBr pellet. Emission and
excitation spectra were recorded on a Perkin-Elmer LS55 lumines-
cence spectrometer with a red-sensitive photomultiplier type R928.
UV−vis absorption spectra were measured on a Perkin−Elmer
Lambda 25 UV−vis spectrometer. All of the spectra were recorded
at 25 °C in a 100 mM HEPES buffer solutions at pH = 7.2.
Crystal Structural Determination. Crystals of GdLZn-
(H2O)·5H2O suitable for X-ray diffraction studies were grown using
a concentrated aqueous solution of GdL with equal amount of ZnCl2
at pH = 7.0 through slow evaporation at ambient temperature. Data
collection was performed on a SATURN70 CCD diffractometer by the
ω scan technique at room temperature using graphite-monochromated
Mo−Kα (λ = 0.71073 Å) radiation. The Crystal Clear software
package was used for data reduction and empirical absorption
correction. The structures were solved by direct methods. The heavy
atoms were located from the E-map, and the rest of the non-hydrogen
atoms were found in subsequent Fourier maps. All non-hydrogen
atoms were refined anisotropically, while the hydrogen atoms were
generated geometrically and refined with isotropic thermal parameters.
The structures were refined on F2 by full-matrix least-squares methods
using the SHELXTL-97 program package.19 The crystallographic data
are summarized in Table 1.
Table 1. Crystallographic Data of GdLZn(H2O)3·5H2O
empirical formula
formula weight
crystal System
space group
a, Å
C31H50GdN7O17SZn
1047.46
triclinic
P1
̅
11.156(0)
12.17940(10)
17.03590(10)
70.941(6)
76.769(6)
64.777(5)
1968.32(8)
2
b, Å
c, Å
α, deg
β, deg
γ, deg
V, Å3
Z
ρcalcd g/cm−3
μ, mm−1
radiation (λ,Å)
temp, (K)
1.767
2.415
0.71073
293(2)
a
R1(Fo)
0.0336
b
2
wR2(Fo )
0.0827
GOF
1.066
2
a
b
R1 = ∑||Fo| − |Fc||/∑|Fo|. wR2 = ∑w(Fo − Fc2)2/∑w(Fo )]1/2
.
2
GdL Complex. To an aqueous solution of H3L (0.082 g, 0.12
mmol) was added dropwise 1 M NaOH solution until pH = 7.0. An
equimolar amount of an aqueous solution of GdCl3·6H2O was slowly
added. To maintain the pH at 7.0, 1 M NaOH solution was further
added. Upon stirring at room temperature for 1 d, the solution was
evaporated. The crude product was dissolved in a minimal volume of
water, and acetone was added carefully to precipitate the product as a
white solid. Yield: 87% (0.085 g). ESI-MS (CH3OH): m/z 839.3
([M−H]−). Anal. Calcd for GdL(H2O)·6H2O: C, 38.54, H, 5.22, N,
10.15, Gd 16.28%. Found: C, 38.34, H, 4.91, N, 10.03, Gd 16.77%.
DyL Complex. This complex was prepared by the same synthetic
procedure as that of the GdL complex. ESI-MS (CH3OH): m/z 845.2
([M−H]−).
T1 Measurements. The longitudinal relaxation times (T1) of
aqueous buffer solutions of GdL were measured by a standard
inversion−recovery pulse sequence on a PQ-001 NMR Analyzing &
Imaging system (Shanghai Niumag Corporation) at 0.5 T. In each
case, five samples were prepared separately whose concentrations were
0, 0.5, 1.0, 1.5, 2.0 mM Gd3+ at 25 °C in a 100 mM HEPES buffer
solutions at pH = 7.2. The relaxivity r1 is commonly used to express
the ability of proton relaxation enhancement of a paramagnetic
compound. It is defined as the slope of eq 1 with mM−1 s−1 as the unit.
(1/T) = (1/T) + r [M]
(1)
1 obs
1 d
1
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Inorganic Chemistry
Article
where (1/T1)obs and (1/T1)d are the observed values in the presence
and absence of the paramagnetic species, and [M] is the concentration
of the paramagnetic species.
3017−3021. (d) Ke, H. T.; Wang, J. R.; Dai, Z. F.; Jin, Y. S.; Qu, E. Z.;
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5561−5564.
T1-Weighted MRI Phantom Images. Phantom images were
obtained using a MiniMR-60 NMR Analyzing & Imaging system
(Shanghai Niumag Corporation). The instrumental parameters were
set as 0.5 T magnet, section thickness = 60 mm, TE = 1.4 s, and TR =
5 ms.
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17O Dysprosium Induced Shift (DIS) Experiments. DIS
experiments were performed on a 400 MHz Bruker Avance III
NMR spectrometer. Dy3+ was used to be the analogue of DyL and
(DyL)2Zn. DIS measurements were performed in 100 mM HEPES
buffer solutions containing 20% D2O at pH = 7.2. DIS values were
determined by plotting [Dy] versus −Δppm and calculating the slope
of the resulting line. The calculated slopes were referenced to the slope
obtained for DyCl3 (q = 9).
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Cell Culture. A human breast cancer cell line MDA-MB-231 was
purchased from the Shanghai Institute of Cell Biology, Chinese
Academy of Sciences, China. Cells were maintained in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 10% FBS
(Fetal Bovine Serum) and antibiotics at 37 °C in a humidified
incubator with 5% CO2 atmosphere. The viability of cells was
determined by Trypan blue dye exclusion. Cells were maintained in
logarithmic phase with viability >95%.
Microscopy and Imaging Methods. The cells were mounted for
direct microscopic observation at 37 °C. The confocal fluorescence
images of cells were performed with an Olympus Fluo View FV1000
laser-scanning microscope. A 60× oil-immersion objective lens was
used. Excitation was carried out with a semiconductor laser at λ = 360
nm, and the emission was collected in the range λ = 520 50 nm,
including the maximum emission wavelength of GdL (496 nm),
(GdL)2Zn (503 nm), and GdLZn (530 nm). A total of 30 × 104
MDA-MB-231 cells were planted in confocal chamber slides (NEST)
in the presence of medium for 1 d at 37 °C. After being washed three
times with PBS buffer solution, the cells were treated with a PBS buffer
solution of GdL (20 μM) for dye loading for 30 min at 37 °C; then the
cells were incubated with a PBS solution of ZnCl2 (30 μM) for 20 min
at 37 °C. The stained cells were washed three times with PBS buffer.
Then the treated cells were imaged by fluorescence microscopy. For
the control experiment, cells were incubated with a PBS solution of
GdL (20 μM) for dye loading for 30 min at 37 °C and washed three
times with PBS buffer, then imaged by fluorescence microscopy.
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ASSOCIATED CONTENT
* Supporting Information
■
S
(6) Zhang, X. L.; Jing, X.; Liu, T.; Han, G.; Li, H. Q.; Duan, C. Y.
Inorg. Chem. 2012, 51, 2325−2331.
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Figures giving additional UV−vis and emission spectra and X-
ray crystallographic file in CIF format for the structure
determination of compoundGdLZn(H2O)·5H2O. This material
is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: czn@fjirsm.ac.cn.
Notes
■
The authors declare no competing financial interest.
(10) Livramento, J. B.; Weidensteiner, C.; Prata, M. I. M.; Allegrini,
P. R.; Geraldes, C. F. G. C.; Helm, L.; Kneuer, R.; Merbach, A. E.;
ACKNOWLEDGMENTS
■
́
́
Santos, A. C.; Schmidt, P.; Toth, E. Contrast Media Mol. Imaging 2006,
1, 30−39.
We thankfully acknowledge the financial support from the
NSFC (20931006, U0934003, and 91122006), the 973 project
(2007CB815304) from MSTC, and the NSF of Fujian Province
(2011J01065).
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