A Gd3+-based magnetic resonance imaging contrast agent sensitive to β-Galactosidase activity utilizing a receptor-induced magnetization enhancement (RIME) phenomenon

Article abstract of DOI:10.1002/chem.200700785

Magnetic resonance imaging (MRI) permits noninvasive three-dimensional imaging of opaque organisms. Gadolinium (Gd³⁺) complexes have become important imaging tools as MRI contrast agents for MRI studies, though most of them are nonspecific and report solely on anatomy. Recently, MRI contrast agents have been reported whose ability to relax water protons is triggered or greatly enhanced by recognition of a particular biomolecule. This new class of MRI contrast agents could open up the possibility of reporting on the physiological state or metabolic activity deep within living specimens. One possible strategy for this purpose is to utilize the increase in the longitudinal water proton r₁ relaxivity that occurs upon slowing the molecular rotation of a small paramagnetic complex, a phenomenon which is known as receptor-induced magnetization enhancement (RIME), by either binding to a macro-molecule or polymerization of the agent itself. Here we describe the design and synthesis of a novel β-galactosidase-activated MRI contrast agent, the Gd³⁺ complex [Gd-5], by using the RIME approach. β-Galactosidase is commonly used as a marker gene to monitor gene expression. This newly synthesized compound exhibited a 57% increase in the r₁ relaxivity in phosphate-buffered saline (PBS) with 4.5% w/v human serum albumin (HSA) in the presence of β-galactosidase. Detailed investigations revealed that RIME is the dominant factor in this increase of the observed r₁ relaxivity, based on analysis of Gd³⁺ complexes [Gd-5] and [Gd-8], which is generated from [Gd-5] by the activity of β-galactosidase, and spectroscopic analysis of their corresponding Tb³⁺ complexes, [Tb-5] and [Tb-8].

Full text of DOI:10.1002/chem.200700785

FULL PAPER
DOI: 10.1002/chem.200700785
A Gd3+-Based Magnetic Resonance Imaging Contrast Agent Sensitive to
ACHTREUNGb-Galactosidase Activity Utilizing a Receptor-Induced Magnetization
Enhancement (RIME) Phenomenon
Kenjiro Hanaoka,[a] Kazuya Kikuchi,[b] Takuya Terai,[a] Toru Komatsu,[a] and
Tetsuo Nagano*[a]
Abstract: Magnetic resonance imaging
(MRI) permits noninvasive three-di-
mensional imaging of opaque organ-
the increase in the longitudinal water
proton r1 relaxivity that occurs upon
slowing the molecular rotation of a
small paramagnetic complex, a phe-
nomenon which is known as receptor-
induced magnetization enhancement
(RIME), by either binding to a macro-
molecule or polymerization of the
agent itself. Here we describe the
design and synthesis of a novel b-galac-
tosidase-activated MRI contrast agent,
commonly used as a marker gene to
monitor gene expression. This newly
synthesized compound exhibited
a
isms. Gadolinium (Gd3+
)
complexes
57% increase in the r1 relaxivity in
phosphate-buffered saline (PBS) with
4.5% w/v human serum albumin
(HSA) in the presence of b-galactosi-
dase. Detailed investigations revealed
that RIME is the dominant factor in
this increase of the observed r1 relaxivi-
ty, based on analysis of Gd3+ com-
have become important imaging tools
as MRI contrast agents for MRI stud-
ies, though most of them are nonspecif-
ic and report solely on anatomy. Re-
cently, MRI contrast agents have been
reported whose ability to relax water
protons is triggered or greatly en-
hanced by recognition of a particular
biomolecule. This new class of MRI
contrast agents could open up the pos-
sibility of reporting on the physiologi-
cal state or metabolic activity deep
within living specimens. One possible
strategy for this purpose is to utilize
the Gd3+ complex
[Gd-5], by using the
RIME approach. b-Galactosidase is
plexes
A
ACHTRE[UNG Gd-8], which is
generated from ACHTREUNG
of b-galactosidase, and spectroscopic
analysis of their corresponding Tb3+
Keywords: biosensors · gadolinium
complexes · lanthanides · lumines-
cence · magnetic resonance imaging
complexes, ACTHERNGU[Tb-5] and ACHRTE[UGN Tb-8].
Introduction
observed in light-based microscopy imaging experiments.[1]
Therefore, MRI is useful not only in clinical medicine, but
also in experimental research.[1,2] Nowadays, there is a con-
siderable interest in MRI contrast agents, which can im-
prove the resolution of MR images.[1,2] The MR images are
based upon the NMR signal from water protons, and the
signal intensity depends upon the water concentration and
relaxation time (T1 and T2).[2] Paramagnetic ions like the ga-
dolinium ion (Gd3+), primarily shorten the T1 (spin–lattice)
relaxation time with high efficacy by rapid exchange of
inner-sphere water molecules with bulk solvent.[3] Thus,
Gd3+-based MRI contrast agents increase tissue contrast by
increasing water proton relaxation, and are widely used in
clinical diagnostics.[4] In Gd3+-based MRI contrast agents,
chelation of Gd3+ is required for safety reasons: Dissocia-
tion of Gd3+ from a MRI contrast agent is undesirable, as
both the free metal and unchelated ligands are generally
more toxic than the complex itself.[2,4] Commonly used MRI
Magnetic resonance imaging (MRI) is a noninvasive imag-
ing technique that can provide images of intact, opaque or-
ganisms in three dimensions, even deep within a specimen,
without photobleaching or light scattering which are often
[a] Dr. K. Hanaoka, T. Terai, T. Komatsu, Prof. Dr. T. Nagano
Graduate School of Pharmaceutical Sciences
The University of Tokyo
7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan)
Fax : (+81)3-5841-4855
E-mail: tlong@mol.f.u-tokyo.ac.jp
[b] Prof. Dr. K. Kikuchi
Department of Materials and Life Sciences
Graduate School of Engineering, Osaka University
2-1 Yamada-oka, Suita City, Osaka 565-0871 (Japan)
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
Chem. Eur. J. 2008, 14, 987 – 995
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
987

contrast agents are mainly extracellular agents with nonspe-
cific biodistribution.[4,5] In contrast, it is also possible to de-
of the agents to human serum albumin (HSA).[12,13] Another
agent permitted the detection of yeast transcription repress-
or protein (Gal80) as MR images by utilizing a specific pep-
tide-protein binding event,[14] and the enzyme carbonic an-
hydrase was selectively targeted with a sulfonamide substitu-
ent.[15] Moreover, oligonucleotide sequences have also been
detected with iron oxide nanoparticles derivatized with oli-
gonucleotide.[16] Hybridization with oligonucleotide-derived
particles resulted in changes mainly in the spin–spin relaxa-
tion time (T2) of adjacent water protons. Efficient polymeri-
zation of Gd3+ complexes can also be used to directly image
the activity of enzymes such as myeloperoxidase (MPO) and
matrix metalloproteinase 2 (MMP-2).[17,18]
velop Gd3+ complexes with various chemical properties by
[6]
means of appropriate ligand design for Gd3+
,
and, indeed,
some bioactivated MRI contrast agents have been reported
for monitoring enzyme activity, Ca2+, pH, p(O2), Zn2+, and
so on.[7] These MRI contrast agents show a change in the
water proton relaxation time (T1 or T2) in response to the
presence of specific biomolecules. Recently, attempts have
been made to utilize MR imaging techniques to detect gene
expression, and the development of these methods would
allow MR imaging of the expression of specific genes.[8] For
example, b-galactosidase is a commonly used gene expres-
sion marker, that is, gene expression is monitored by intro-
ducing a marker gene, lacZ, to follow the regulation of a
gene of interest because it can easily be assayed and is not
normally expressed in most mammalian tissues or cells.[9]
Meade and co-workers developed the bioactivated MRI
contrast agent, EgadMe, which reports on b-galactosidase
activity to image the expression of a transgene.[10] The mech-
anism of the T1 relaxation time change between two distinct
relaxation states, long and short, is as follows. The enzyme
substrate, galactopyranose, which is linked to the ligand,
blocks the one remaining open coordination site of the che-
lated Gd3+, inhibiting access of water to the chelated Gd3+
ion. The contrast agent is switched on when b-galactosidase
cleaves the galactopyranose from the Gd3+ complex and the
chelated Gd3+ ion becomes accessible to water. This agent
has been successfully used in vivo to monitor gene expres-
sion in Xenopus laevis.[10a] Thus, this MRI contrast agent
showed a change in the longitudinal relaxation time (T1) in
the presence of b-galactosidase by modulating the access of
water molecules to the chelated Gd3+ ion. In addition to
above results, b-galactosidase-activated MRI contrast agents
with a range of chemical properties are also needed for fur-
ther biological studies, so the development of novel b-galac-
tosidase-activated MRI contrast agents with a different
design approach would be helpful for studies of biological
phenomena by monitoring gene expression. One possible
approach for the development of biomolecule-activated
MRI contrast agents is the RIME (receptor-induced mag-
netization enhancement) approach.[11] The binding of a MRI
contrast agent to a macromolecule substantially slows mo-
lecular rotation of the Gd3+ complex, resulting in an addi-
tional increase in the r1 relaxivity through the rotational cor-
relation time tR.[2] When the Gd3+ complex binds to a mac-
romolecule, the tR increases from that of a small molecule
to that of the protein, and the r1 relaxivity increases. The
slower the Gd3+ complex tumbles, the longer the tR, leading
to faster relaxation rates and, hence, higher r1 relaxivity. The
tR for small Gd3+ complexes is usually in the picosecond
range (typically 50–200 ps), whereas the tR for a macromole-
cule such as albumin is in the nanosecond range (about
50 ns).[6a] This phenomenon is known as RIME. RIME
agents have been reported for alkaline phosphatase and for
carboxypeptidase B (part of the thrombin-activatable fibri-
nolysis inhibitor family), which regulate noncovalent binding
Here, we report the design and synthesis of a novel b-gal-
actosidase-activated MRI contrast agent based on the
RIME approach (Figure 1). Reaction with b-galactosidase
Figure 1. The RIME mechanism for the b-galactosidase-activated MRI
contrast agent
min binding moiety that is masked by the galactopyranose residue. The
galactopyranose residue of [Gd-5] is designed to be cleaved by b-galacto-
[Gd-8], and this cleavage promotes albu-
[Gd-5], a Gd3+ complex is coupled to an albu-
ACHRTUNEG[Gd-5]. In ACHTREUGN
AHCTREUNG
sidase, transforming ACHTER[UNG Gd-5] to ACHTREUNG
min binding of the Gd3+ complex.
yielded a 57% increase in the r1 relaxivity in phosphate-buf-
fered saline (PBS) with 4.5% w/v HSA. Cleavage of the gal-
actopyranose moiety from the aryl group of the Gd3+ com-
plex increases the hydrophobicity of the aryl group, thereby
increasing the HSA binding affinity. The greater binding of
the Gd3+ complex to the macromolecule, HSA, increases
988
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 987 – 995

A Gd3+-Based Magnetic Resonance Imaging Contrast Agent
FULL PAPER
the r1 relaxivity. We also confirmed the mechanism of this
increase in the r1 relaxivity.
from 3.5 to 5.5 sꢀ1 between 0 and 250min in the presence of
b-galactosidase (113 nm), whereas the value of 1/T1 changed
only slightly from 3.8 to 4.0 sꢀ1 between 0 and 250 min on
exposure to heat-inactivated b-galactosidase (113 nm). We
also assessed the ability of b-galactosidase to remove the
Results and Discussion
galactopyranose masking group from
ACHTRE[UNG Gd-5] by high-pres-
Design and synthesis of [Gd-5] and [Gd-8]: The Gd3+ com-
sure liquid chromatography (HPLC) analysis (Figure 3).
plex,
through conversion of the MRI-silent agent into an activat-
ed MRI agent, [Gd-8] (Figure 1). The [Gd-5] is composed
ACHTREUNG[Gd-5], was designed to detect b-galactosidase activity
A
ACHTREUNG
of three moieties: 1) a masking group consisting of galacto-
pyranose; 2) an albumin-binding moiety, the biphenyl
group; 3) an MRI signal-generating moiety, which is a Gd3+
complex. This design relies upon enzymatic transformation
of a Gd3+ complex with poor albumin affinity and concomi-
tant low relaxivity into one with high albumin affinity and
high relaxivity. The biphenyl group was selected as the albu-
min binding group, because the biphenyl residue is known
to possess high albumin binding affinity.[4,13,19] A substrate
for b-galactosidase, galactopyranose, was used as a masking
group, affording extremely high hydrophilicity compared
with the hydrophobicity of the biphenyl group. Hydrolysis
of the galactopyranose moiety unblocks the hydrophobicity
of the biphenyl group, thereby increasing the albumin bind-
ing affinity. Thus, in ACHTRE[UNG Gd-5], a masking group that inhibits
albumin binding was expected to be removed by the enzy-
matic activity, to expose an albumin-binding group with high
affinity. The strong interaction of the Gd3+ complex with a
macromolecule such as albumin increases the r1 relaxivity
owing to the RIME phenomenon. The synthetic schemes for
the lanthanide complexes, ACHTRENGU[Gd-5] and ACHTRE[UGN Gd-8], and details of
the chemical characterization of compounds are provided in
the Supporting Information.
Longitudinal relaxation time T1 measurements of [Gd-5]
with b-galactosidase: The longitudinal relaxation times T1 of
ACHTREUNG[Gd-5] were measured in the presence of b-galactosidase or
heat-inactivated b-galactosidase with 4.5% w/v HSA in
phosphate-buffered saline (PBS; pH 7.4), at 20 MHz
(0.47 T), at 378C (Figure 2). The value of 1/T1 increased
Figure 3. Time course of the conversion of
tion of reaction species was quantified by HPLC analysis on the basis of
the absorbance at 300 nm. The Gd3+ complex,
[Gd-5] (0.5 mm) was incu-
bated with: a) b-Galactosidase (113 nm) [Gd-5]:
~
b) heat-inactivated b-galactosidase (113 nm) ([Gd-5]: , AHCTRE[UNG Gd-8]: ) at
ACHRTUNEG[Gd-5] to ACHTRE[UGN Gd-8]; the distribu-
AHCTREUNG
~
,
&
(
N
[Gd-8]:
) or;
&
A
pH 7.4, and 378C, in phosphate-buffered saline (PBS) in the presence of
4.5% w/v human serum albumin (HSA).
This HPLC analysis showed that the Gd3+ complexes,
5] and [Gd-8], had distinct retention times, and [Gd-5] was
converted into [Gd-8] in the presence of b-galactosidase
ACHTREUNG[Gd-
A
ACHTREUNG
AHCTREUNG
(113 nm), whereas essentially no change was observed upon
the addition of heat-inactivated b-galactosidase (113 nm).
The HPLC experiments confirmed the enzymatic processing
of
ACHRTUNEG[Gd-5] by the b-galactosidase. Thus, CAHTRE[UNG Gd-5] exhibited a
b-galactosidase-induced RIME effect, accompanying the re-
~
Figure 2. Time course of the b-galactosidase-induced (113 nm; ) and
moval of the galactopyranose residue of ACTHRE[UNG Gd-5].
&
heat-inactivated b-galactosidase-induced (113 nm; ) changes in the value
The r1 relaxivity of Gd3+ complexes, [Gd-5] and [Gd-8]:
The paramagnetic species, Gd3+, acts as a catalyst to relax
bulk water protons by fast exchange of the coordinated
of 1/T1 [sꢀ1], of 0.5 mm
ACHTREUNG[Gd-5] solution at 20 MHz, 378C in phosphate-
buffered saline (PBS; pH 7.4) with 4.5% w/v human serum albumin
(HSA).
Chem. Eur. J. 2008, 14, 987 – 995
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
989

T. Nagano et al.
water with bulk water (inner-sphere contribution). There is
also a relaxation increase provided by the Gd3+ ion to water
molecules which are diffusing close to the Gd3+ ion
(second-sphere and outer-sphere contributions). The ob-
served longitudinal relaxation rate (1/T1)obs of the solvent
water protons is known to depend on the concentration of
Gd3+ ions according to Equation (1) :
ð1=T1Þobs ¼ ð1=T1Þd þ r1½GdŠ
ð1Þ
where (1/T1)obs is the observed relaxation rate of water pro-
tons in the presence of Gd3+, and (1/T1)d is the diamagnetic
relaxation rate of water protons in the absence of Gd3+. The
longitudinal relaxivity value, r1, refers to the amount of in-
crease in 1/T1 sꢀ1 per millimolar concentration of agent
(given as mmꢀ1 Gd), and [Gd] is the millimolar concentra-
tion of Gd3+ ions. Therefore, a plot of (1/T1)obs versus Gd3+
concentration would give the r1 relaxivity as the slope, and
the r1 relaxivity, normally expressed in units of mmꢀ1 secꢀ1,
reflects the ability of a Gd3+ complex to increase relaxation.
Figure 4. E-titration data for ACTHRENU[G Gd-5] and ACHTRE[UGN Gd-8]. e* versus [human serum
albumin (HSA)] in mm, at 378C, in phosphate-buffered saline (PBS,
pH 7.4) at 20 MHz, 0.47 T. Each solution contains various concentrations
~
of HSA (0–22.5% w/v (=0–3.35 mm) with 0.1 mm
[Gd-5] ( ) or
G
&
( ).
rates ((1/T1)para [sꢀ1]) in the presence and the absence of
HSA were plotted versus increasing HSA concentration at a
The water proton relaxivities, r1, of ACHTRENGU[Gd-5] and AHCTRE[UGN Gd-8] were
constant concentration of
[Eq. (2)] :
ACHRTEUG[N Gd-5] or AHCTRE[UGN Gd-8] (0.1 mm)
determined at 20 MHz (0.47 T), at 25 or 378C, and are
shown in Table 1. In the absence of HSA, the r1 relaxivities
Alb
Alb
Alb
Table 1. The r1 relaxivity [mmꢀ1 sꢀ1] (20 MHz) in PBS with 4.5% HSA or
PBS.
ð1=T1Þpara
ð1=T1Þobsꢀð1=T1Þdia
*
e
¼
¼
ð2Þ
PBS
PBS
PBS
ð1=T1Þobs ꢀð1=T1Þdia
ð1=T1Þpara
Compound
HSA[a]
PBS[b]
258C
6.34
8.76
378C
6.06
9.51
258C
5.80
4.11
378C
5.35
3.87
where the obs, para, and dia subscripts refer to the ob-
served, paramagnetic, and diamagnetic species, respectively,
and the Alb and PBS superscripts refer to “in PBS contain-
ing human serum albumin” and “in PBS”, respectively. The
longitudinal water proton relaxation times T1 of aqueous
solutions without Gd3+ complexes were measured as the di-
amagnetic contribution. The longitudinal relaxation rate (1/
ACHTREUNG
ACHTREUNG
[a] Human serum albumin (HSA) (4.5% w/v) in phosphate-buffered
saline (PBS; 137 mm NaCl, 8.10 mm Na2HPO4, 2.68 mm KCl, 1.47 mm
KH2PO4, pH 7.4). [b] PBS only.
of
378C. However, in PBS with 4.5% w/v HSA at 258C, the r1
relaxivity of [Gd-8] is higher than that of [Gd-5] by 38% as
[Gd-5] are higher than those of
[Gd-8] at both 25 and
T1 [sꢀ1]) increase of
HSA was much larger than that of AHCTREUNG
ACHTREUNG
not increase linearly with the concentration of HSA, sug-
gesting protein binding. There was a 1.9- or 4.5-fold increase
G
ACHTREUNG
a consequence of a higher albumin binding affinity; at 378C,
where the exchange of Gd3+-bound water molecules is more
in the enhancement factor e* upon binding to HSA for
ACHTREUNG[Gd-
5] or [Gd-8], respectively. Small parts of these increases in
AHCTREUNG
facile, the r1 relaxivity of
5] by 57%. From these results, it was considered that the in-
crease of the 1/T1 value of [Gd-5] in Figure 2 was a conse-
quence of enzymatic cleavage of the galactopyranose resi-
due of [Gd-5] by the b-galactosidase activity. Moreover, the
r1 relaxivity of [Gd-5] at 20 MHz showed similar values in
ACHRTUNEG[Gd-8] is higher than that of HCATRE[UGN Gd-
e* can be ascribed to the misleading apparent amount of
water molecules (ref. [4], p. 2342). For example, ꢁ3 mm
HSA solution contains more than 20% protein and hence
less than 80% water, because of the high molecular weight
of HSA. The molar concentration of 1 mmol Gd3+ in a liter
of 20% w/v HSA is written as 1 mm, but the actual molar
concentration would be 1.25 mm. However, the increase of
AHCTREUNG
ACHTREUNG
ACHTREUNG
the absence and the presence of HSA, indicating that ACHTRE[UNG Gd-
5] hardly interacts with HSA.
e* for
taken into consideration. Therefore, the enhancement factor
e* for [Gd-8] suggests a high affinity of [Gd-8] for the albu-
min, whereas the slight increase of e* for [Gd-5] can be in-
terpreted as indicating a weak interaction with the albumin.
Further, the binding interaction strengths of [Gd-5] and
ACHTRE[UNG Gd-8] is sufficiently large even when this problem is
Albumin binding study: We investigated the noncovalent
interaction between [Gd-5] or [Gd-8], and HSA. To demon-
G
ACHTREUNG
strate the extent of relaxation enhancement, the E-titration
E
ACHTREUNG
is shown in Figure 4.[2,11a] The longitudinal water proton re-
AHCTREUNG
laxation times T1 of a 0.1 mm solution of ACTHERNGU[Gd-5] or ACHTRE[UGN Gd-8]
in PBS with various concentrations of HSA (0–3.35 mm (0–
22.5% w/v)) were measured at 20 MHz (0.47 T), 378C. The
results were expressed in terms of the enhancement factor
e*, that is, the ratio of paramagnetic longitudinal relaxation
ACHTREUNG
AHCTRE[UNG Gd-8] to HSA were calculated by using the above E-titra-
tion experimental data.[2,11b,c,20] The observed longitudinal
water proton relaxation rate, r1obs [sꢀ1] is given by the sum of
990
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Chem. Eur. J. 2008, 14, 987 – 995

A Gd3+-Based Magnetic Resonance Imaging Contrast Agent
FULL PAPER
F
B
three contributions, r1p [mmꢀ1 sꢀ1], r1p [mmꢀ1 sꢀ1], and r1dia
tageous spectroscopic characteristics, such as, long lumines-
cence lifetimes of the order of milliseconds, narrow emission
peaks, a large Stokeꢁs shift of >150 nm, and excellent water
solubility.[21] This extremely long luminescence lifetime of
lanthanide ions allows a time-resolved detection procedure
to be employed, because typical fluorescence lifetimes are
in the nanosecond region; that is, a delay time is set be-
tween the excitation pulse and the measurement of the lan-
thanide luminescence, during which the background fluores-
cence and scattered light decay to negligible levels.[22] There-
fore, time-resolved luminescence measurements offer a
better signal-to-noise ratio, and the lanthanide luminescence
has been exploited in a number of useful detection systems
for time-resolved assays in the fields of medicine, biotech-
[sꢀ1] :
F
B
r1obsꢀr1dia ¼ ðr1P ½GdŠ þ r1p ½Gd-HSAŠÞ  1000
ð3Þ
where r1p and r1p are the r1 relaxivity [mmꢀ1 sꢀ1] of the
Gd3+ complex and of the paramagnetic macromolecular,
Gd3+ complex-HSA adduct, respectively, and r1dia [sꢀ1], is
the diamagnetic contribution of the observed longitudinal
water proton relaxation rate r1obs [sꢀ1], at 20 MHz, 378C.
[Gd], in m, is the concentration of the Gd3+ complex, and
[Gd-HSA], in m, is the concentration of the Gd3+ complex-
HSA adduct. The determination of the binding parameter
nKA [mꢀ1] (KA: association constant; n: number of inde-
pendent binding sites on the protein) for the equilibrium :
F
B
nology, and biological science.[23] We prepared
ACHTREUNG
AHCTRE[UNG Tb-8] as homologues of ACHERT[UNG Gd-5] and ACHTREUNG
KA
Tb3+ ion possesses the same charge as Gd3+, as well as simi-
lar ionic radius and coordination chemistry to the Gd3+
Gd þ HSA
GdꢀHSA
ð4Þ
G
H
is possible through the following equations :
ion.[24] The synthetic schemes for
ACTHERU[NG Tb-5] and HCATRE[UGN Tb-8] and de-
tails of the chemical characterization of compounds are pro-
vided in the Supporting Information. As regards the metal-
based luminescence properties, 25 mm aqueous solutions of
½GdꢀHSAŠ
ð5Þ
KA
¼
½GdŠ Á ½n Á HSAŠ
A
ACHTRE[UNG Tb-8] (in 100 mm HEPES buffer; pH 7.4) were
By combining Equations (3) and (5) we obtain Equa-
tion (6), which allows the nonlinear fitting of the experimen-
tal data :
ACHTREUNG
AHCTREUNG
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ꢀ
ꢁ
the naked eye (see Supporting
Information).
First, the UV/Vis absorption
KAA þ KAB þ 1ꢀ ðKAA þ KAB þ 1Þ ꢀ4KA2AB
2
r1obsꢀrid
¼
rF Á B þ ðrBꢀrFÞ Â
 1000
2KA
ð6Þ
spectrum of
[Tb-8] (50 mm) was measured in
100 mm HEPES buffer at pH 7.4, 258C. The absorption
spectra of [Tb-5] and [Tb-8] were similar, that is, [Tb-5]
showed a lmax at 279 nm tailing out to 330 nm, and [Tb-8]
showed a lmax at 281 nm tailing to 330 nm (Figure 6a). These
absorption spectra can be mainly ascribed to the biphenyl
substituent, because the bands observed in Tb3+ absorption
spectra are usually very weak, that is, molar absorption coef-
ACHTRE[UNG Tb-5] (50 mm) and
AHCTREUNG
where A and B [both in m], are the total molar concentra-
tions of HSA and the Gd3+ complex, respectively. The fit-
ting of the experimental data into Equation (6) provided an
assessment of the binding strength, nKA [mꢀ1], and the r1 re-
G
A
ACHTREUNG
ACHTREUNG
B
laxivity [mmꢀ1 sꢀ1], of the macromolecular adduct (r1
[mmꢀ1 sꢀ1]) (see the Supporting Information). The interac-
tion strength of ACHTREUNG[Gd-8] with HSA was fairly strong (nKA =
7.0”102 mꢀ1) and the r1 relaxivity of the paramagnetic mac-
ficients (e) of lanthanide
<1 dm3 molꢀ1 cmꢀ1.[21] Second, the time-resolved lumines-
cence spectrum of [Tb-5] (50 mm) or [Tb-8] (50 mm) was
ACHTRE(UNG III) ions are usually
B
romolecular
[Gd-8]-HSA adduct (r1 ) was 20 mmꢀ1 sꢀ1, at
C
ACHTREUNG
20 MHz and 378C, whereas the interaction of
ACHTRE[UGN Gd-5] with
HSA was rather weak (nKA <1.0”102 mꢀ1).
measured in 100 mm HEPES buffer (pH 7.4) upon excita-
Further, we examined whether various other species of al-
bumins, such as, rat, bovine, and rabbit serum albumins,
tion of the biphenyl substituent (excitation at 280 nm) for
both
A
ACHTREUNG
could serve as host macromolecules for
G
spectra of
G
ACHTREUNG
When [Gd-5] (0.1 mm) was incubated with b-galactosidase
N
played four bands (490, 545, 586, and 622 nm), arising from
5
(1.13 mm), at 378C, for 30 min, in PBS, with 4.5% w/v
human, rat, bovine, or rabbit serum albumin, all solutions
showed similar decreases of the longitudinal relaxation time
(T1) of water protons (see Supporting Information).
transitions from the emissive D4 state to the ground-state
7
7
7
7
manifolds, F6, F5, F4, and F3, respectively (Figure 6b).[21a,b]
On the basis of the above spectroscopic results, the biphenyl
substituent, which is the albumin-binding group, serves as a
sensitizing chromophore for Tb3+ in the luminescent Tb3+
Time-resolved luminescence and UV/Vis absorption spec-
tra of [Tb-5] and [Tb-8]: The chemical properties of
and [Gd-8] were further assessed by analyzing the lumines-
cence and chemical properties of the terbium trivalent ion
(Tb3+) complexes of chelator 5 and 8,
[Tb-5] and [Tb-8]
N
complexes,
The time-resolved luminescence spectra, with a delay
time of 50 msec, of [Tb-5] (50 mm) were measured in 100 mm
HEPES buffer at pH 7.4, 258C excited at the absorbance
maximum (lmax) wavelength of the biphenyl group (280 nm)
during the enzyme (b-galactosidase) reaction. Addition of b-
ACHRTUNEG[Tb-5] and ACHTREU[GN Tb-8].
ACHTREUNG
AHCTREUNG
U
(Figure 5). Lanthanide complexes, in particular complexes
of Tb3+ and Eu3+ (the europium trivalent ion), have advan-
Chem. Eur. J. 2008, 14, 987 – 995
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991

T. Nagano et al.
Figure 5. a) Structures of Tb3+ complexes,
ACTHERU[NG Tb-5] and AHCTRE[UGN Tb-8], and sche-
matic view of a chromophore incorporated into a terbium emitter. The
emission from Tb3+ after excitation of the sensitizing chromophore on
Tb3+ complexes,
ACHTREUNG[Tb-5] and ACHTRE[UGN Tb-8], is shown. b) The general chromo-
phore-to-terbium-ion sensitization process. Light absorption and lowest-
lying singlet excited state (S1) formation at the sensitizing chromophore
are followed by intersystem crossing (ISC), resulting in population of the
triplet excited state (T1) of the sensitizing chromophore. Subsequent
chromophore-to-Tb3+ energy transfer leads to a metal-centered emission,
which is derived from transitions from Tb3+-emitting states to the rele-
vant ground states.
Figure 6. Spectroscopic characteristics of solutions of
upon addition of b-galactosidase. a) Absorbance spectra of 50 mm aque-
ous solution (100 mm HEPES buffer, pH 7.4) of [Tb-5] and [Tb-8] at
258C ([Tb-5]: b, [Tb-8]: c). b) Time-resolved emission spectra (ex-
citation at 280 nm) of [Tb-5] and [Tb-8] (50 mm) ([Tb-5]: b, [Tb-8]:
ACHRTUNEG[Tb-5] and ACHTREUNG[Tb-8]
G
ACHTREUNG
U
ACHTREUNG
U
G
G
ACHTREUNG
c). These spectra were measured in 100 mm HEPES buffer at pH 7.4
and 258C by using a delay time of 50 ms and a gate time of 1.00 ms. The
galactosidase (113 nm) to an aqueous solution of ACTHRE[UNG Tb-5] re-
sulted in a decrease in the luminescence of Tb3+ as shown
in Figure 6c. The luminescence intensity at 545 nm of the
7
bands arise from 5D4! FJ transitions; the J values of the bands are la-
beled. c) Time-resolved emission spectra (excitation at 280 nm) of ACHTREUNG[Tb-5]
(50 mm) after the addition of b-galactosidase (113 nm) in 100 mm HEPES
buffer (pH 7.4) at 258C. Time-resolved emission spectra of [Tb-5] were
ACHTREUNG
AHCTREUNG
measured every 10 min (0ꢁ100 min) after the addition of b-galactosi-
A
ACHTREUNG
dase.
AHCTREUNG
a
rates, 1/T1, of the water protons is valid only if the concen-
tration of the paramagnetic species, Gd3+, is at the level of
mmol or submmol per kilogram of solvent (millimolality or
submillimolality),[2] whereas for the determination of the ki-
ACHTREUNG
AHCTREUNG
992
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ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 987 – 995

A Gd3+-Based Magnetic Resonance Imaging Contrast Agent
FULL PAPER
netic parameters, the b-galactosidase substrate concentration
should be in the range of several mmꢁseveral hundred mm.
Kinetic parameters Km and kcat were determined by direct
fitting of the initial velocity versus substrate concentration
data to the Michaelis–Menten equation (see Supporting In-
numbers of coordinated water molecules (q values) at the
metal center, Tb3+, were 1.35 and 9.8 for
ACTHERNGU[Tb-5] and ACHTREUNG[Tb-8],
respectively (Table 2), according to Equation (7).[29a,b]
Number of water molecules :
qTb ¼ 5ð1=tH Oꢀ1=tD Oꢀ0:06Þ
formation). The values of Km, kcat, and kcat/Km of
C
ð7Þ
2
2
The q value of ACTHRE[UNG Tb-5] is reasonable, considering the ex-
perimental data of longitudinal relaxation time (T1) and
long-lived luminescence measurements in this paper, and in-
k
cat =600 and 35 sꢀ1, kcat/Km =6000 and 389 mmꢀ1 sꢀ1, respec-
dicates that
coordinated to the chelated Tb3+. Further, in general, the
lanthanide trivalent ion (Ln3+) (such as, Eu3+, Gd3+, Tb3+
complexes of DTPA-monoamide or DTPA-bisamide deriva-
ACHTRE[UNG Tb-5] has approximately one water molecule
tively.[25] The reactivity of
ACHTRE[UGN Tb-5] for b-galactosidase is likely
to be sufficient for the detection of enzyme activity in bio-
logical systems, on the basis of the kinetic parameters of
ONPG and PG, and the experimental data from refs. [10a,b]
)
tives, which have a coordination number of eight for Ln3+
,
have approximately one metal-bound water molecule.[4,21
and [26]. The kcat/Km value of ACHTER[UNG Tb-5] is probably determined
a,23f,30]
by the structure of the biphenyl group which is the albumin
However, the q value of ACHTREU[NG Tb-8] was extremely large,
binding moiety,[27] so it should be possible to modulate the
9.8, showing inconsistency with other experimental data
herein, and therefore this calculated value seems not to re-
flect the number of water molecules coordinated to the cen-
tral metal ion, Tb3+. Other factors could account for this ex-
kcat/Km value of
structure of the albumin binding moiety. Moreover, the re-
activity of [Tb-5] with b-galactosidase appears to be much
ACHTREUNG[Tb-5] for b-galactosidase by modifying the
ACHTREUNG
higher than that of the b-galactosidase-activated MRI probe
tremely large q value of ACHTREUNG
which was reported by Meade and co-workers.[10b]
abnormal feature of
ACHTREUNG
Luminescence and chemical properties of [Tb-5] and [Tb-
8]: We further investigated in the luminescence and chemi-
cal properties of
summarized in Table 2. The luminescence quantum yields
(f) of [Tb-5] and [Tb-8] were 0.4 and 0.2%, respectively,
under air-equilibrated conditions (Table 2). This result cor-
responds to the phenomenon of the luminescence intensity
ACHTREUNG[Tb-5] and ACHTRE[UNG Tb-8], and these properties are
Conclusion
The Gd3+ complex
AHCTRE[UNG Gd-5] is the first RIME-based b-galac-
A
ACHTREUNG
tosidase-activated MRI contrast agent, and our design strat-
egy should be applicable to a range of new types of b-galac-
tosidase-activated MRI contrast agents, which may possess
novel chemical characteristics, such as, various reactivity
with b-galactosidase, specific biodistribution in living speci-
mens and cells, differing extent of r1 relaxivity change, and
decrease of ACHTREUNG[Tb-5] in the presence of b-galactosidase. These
luminescence quantum yields are relatively low, as com-
pared with those reported for highly luminescent lanthanide
complexes,[23f,g,k] but they are sufficiently large for lumines-
cence detection, as described above. Measurement of the
so on. This bioactive MRI contrast agent ACHTRE[UGN Gd-5] should be
useful for studies on the gene expression of lacZ in biologi-
decay rate constants of the Tb3+ excited state for
ACHTREUNG
ACHTREUNG
cal systems.[1a,8,10]
A
ACHTREUNG
and 0.23 ms in H2O (tH O), and 1.03 and 0.43 ms in D2O
2
(tD O), respectively (Table 2). The luminescence lifetime of
2
Experimental Section
G
ACHTREUNG
AHCTREUNG
All reagents were purchased from Tokyo Kasei Kogyo Co. Ltd. (Japan),
Wako Pure Chemical Industries Ltd. (Japan), or Aldrich Chemical Co.
Inc. (St. Louis, MO), and were used directly without further purification.
All solvents were used after distillation. b-Galactosidase [EC 3.2.1.23]
Sigma cat G 6008 (Grade VI: From Escherichia coli), HSA (human
serum albumin, 97–99%) Sigma cat A 9511 (1”crystallized and lyophi-
lized), albumin from rat serum Sigma cat A 6272, BSA (bovine serum al-
bumin) Sigma cat A 7906 (minimum 98% electrophoresis), and albumin
from rabbit serum Sigma cat A 0764 (ca. 99% agarose gel electrophore-
sis) were purchased from Sigma. Dulbeccoꢁs phosphate-buffered saline
(D-PBS(ꢀ)) Sigma cat 14190–136 was purchased from GIBCO, and was
used as phosphate-buffered saline (PBS). Silica gel column chromatogra-
phy was performed by using BW-300, and Chromatorex-ODS (both from
Fuji Silysia Chemical Ltd., Japan). Amberlite IR-120 Plus(H) was pur-
chased from ICN Biomedicals, Inc. (USA). Chelex 100 resin (100–200
mesh, sodium form) was purchased from Bio-Rad Laboratories (USA).
ACHTREUNG
Table 2. Luminescence and chemical properties.
Compound
[Tb-5]
[Tb-8]
f [%][a]
tH O [ms][b]
tD O [ms][c]
q[d]
2
2
R
0.4
0.2
0.77
0.23
1.03
0.43
1.35
[e]
E
–
[a] Quantum yields were calculated by using quinine sulfate (f=0.546 in
1n H2SO4)[28] as a standard, and measured in 100 mm HEPES buffer;
pH 7.4. [b] In H2O-based buffer (100 mm HEPES buffer, pH 7.4). [c] In
D2O-based buffer (100 mm HEPES buffer, pD 7.4). [d] The q values were
estimated by using the equation qTb =5(1/tH Oꢀ1/tD Oꢀ0.06), which
2
2
allows for the contribution of unbound water molecules.[29a,b] [e] The che-
lated q value of ACHTREUNG[Tb-8] was extremely large (9.8), and this value seems
not to reflect the number of coordinated water molecules to the centered
metal ion, Tb3+. Other factors may account for this extremely large q
value.
Instruments: 1H and 13C NMR spectra were recorded by using a JEOL
JNM-LA300 spectrometer. Mass spectra were measured by using
a
JEOL-T100LC AccuTOF mass spectrometer (ESI+ and ESIꢀ). HPLC
Chem. Eur. J. 2008, 14, 987 – 995
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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993

T. Nagano et al.
purification was performed on a reversed-phase column, Inertsil Prep-
ODS 30 mm”250 mm (GL Sciences, Inc. (Tokyo, Japan)) fitted on a
Jasco PU-1587 system. Measurements of longitudinal water proton relax-
ation times (T1) were made by using an NMR analyzer operating at
20 MHz, 0.47 T (Minispec mq20, Bruker). Time-resolved luminescence
spectra were recorded by using a Perkin–Elmer LS-55 (Beaconsfield,
Buckinghamshire, England). UV/Vis spectra were obtained by using a
Shimadzu UV-1650PC (Tokyo, Japan). Normal fluorescence spectra were
measured by using a Hitachi F4500 spectrofluorometer (Tokyo, Japan).
Aqueous solutions of Tb3+ complexes illuminated at 254 nm were photo-
graphed by using a Handy UV lamp (Handy UV Lamp, SLUV-4, AS O-
NE Co., Japan) (see Supporting Information).
crease of the Tb3+ luminescence of
ACHTRE[GNU Tb-5] solutions at 378C in PBS
(pH 7.4) (excitation 280 nm, emission 545 nm) with a Hitachi F4500 spec-
trofluorometer, in the presence of b-galactosidase (151 nm) and various
concentrations of CAHTRE[UNG Tb-5] (5, 10, 20, 40, 80, and 160 mm). The slit width
was 5 nm for both excitation and emission. The photomultiplier voltage
was 700 V.
Quantum yield measurements: The luminescence spectra were measured
with a Hitachi F4500 spectrofluorometer. The slit width was 2.5 nm for
both excitation and emission. The photomultiplier voltage was 700 V. The
luminescence spectra of ACTHER[GNU Tb-5] or ACHTREU[GN Tb-8] were measured in 100 mm
HEPES buffer at pH 7.4, 258C, with irradiation at 280 nm. The quantum
yields of Tb3+ complexes were evaluated by using a relative method with
reference to a luminescence standard, quinine sulfate (f=0.546 in 1 n
H2SO4).[28] The quantum yields of Tb3+ complexes can be expressed by
Relaxation-time measurements: The longitudinal water proton relaxation
times, T1, of aqueous solutions of the Gd3+ complex
ACTHERNGU[Gd-5] or ACHTRE[UGN Gd-8]
Equation (9)[32]
:
were measured in phosphate-buffered saline (PBS, Dulbeccoꢁs phos-
phate-buffered saline, pH 7.4) or PBS with albumin at 20 MHz, 0.47 T
(Minispec mq20, Bruker). The values of T1 were measured from 10
points generated by using the standard inversion-recovery procedure.
2
Fx=Fst ¼ ½Ast=AxŠ½nx2=nst Š½Dx=DstŠ
ð8Þ
The r1 relaxivity [mMꢀ1 sꢀ1] of
the slope of the plot of 1/T1 versus [
and 0.475 mM) in PBS or PBS with 4.5% w/v HSA at 258C or 378C.
HPLCanalysis : The transformation of [Gd-5] or [Tb-5] to [Gd-8] or
[Tb-8] was monitored by using HPLC analysis. The HPLC analysis for
the transformation of [Gd-5] to [Gd-8] was performed on a reversed-
A
ACHTREUNG
G
ACHTREUNG
where F is the quantum yield (subscript “st” stands for the reference and
“x” for the sample), A is the absorbance at the excitation wavelength, n
is the refractive index, and D is the peak area (on an energy scale) of the
luminescence spectra. The samples and the reference were excited at the
same wavelength (280 nm). The sample and the reference absorbance at
the excitation wavelength were kept as low as possible to avoid fluores-
cence errors (Aexc <0.05).
G
E
ACHTREUNG
ACHTREUNG
G
ACHTREUNG
phase column (Inertsil ODS-3 4.6”250 mm (GL Sciences); eluent, a 20-
min linear gradient, from 0 to 80% solvent B (solvent A, 0.1 m triethy-
lammonium acetate (pH 6.5); solvent B, acetonitrile/H2O 4:1); flow rate,
Luminescence lifetime measurements: The luminescence lifetimes of the
Tb3+ complexes were recorded on a Perkin–Elmer LS-55 instrument.
The data were collected with a 10-msec resolution in H2O (100 mm
HEPES buffer at pH 7.4) and D2O (100 mm HEPES buffer at pD 7.4,
based on the equation pD=pH+0.40[33]) at 258C, and fitted to a single-
exponential curve obeying Equation (9) :
1.0 mLminꢀ1; UV 300 nm). The retention times of
A
ACHTRE[UNG Gd-8]
A
ACHTREUNG
formed by using a reversed-phase column (Inertsil ODS-3 4.6”250 mm
(GL Sciences); eluent, a 70-min linear gradient, from 10 to 80% solvent
B (solvent A, 0.1 m triethylammonium acetate (pH 6.5); solvent B, aceto-
nitrile/H2O 4:1); flow rate, 1.0 mLminꢀ1; UV 280 nm). The retention
I ¼ I0 exp ðꢀt=tÞ
ð9Þ
times of
respectively.
T1 relaxation time measurements of [Gd-5] with b-galactosidase: The
longitudinal relaxation T1 times were measured for [Gd-5] in the pres-
ence of b-galactosidase (113 nm) or heat-inactivated b-galactosidase
(10 min at 808C) at 113 nm, with 4.5% w/v HSA in PBS (pH 7.4), at
20 MHz, 0.47 T, at 378C. The concentration of b-galactosidase was calcu-
lated based on a monomer of MW =116.3 kDa.[31] On HPLC analysis of
ACHTREUNG[Tb-5] and ACHTREUNG[Tb-8] under these conditions were 7.7 and 14.9 min,
AHCTREUNG
where I0 and I are the luminescence intensities at the time t=0 and time
t, respectively, and t is the luminescence emission lifetime. Lifetimes
were obtained by monitoring the emission intensity at 545 nm (excitation
at 280 nm).
the reaction mixture, only two peaks of
at 300 nm.
ACHRTUNEG[Gd-5] and ACHRTE[UGN Gd-8] were detected
Acknowledgement
Albumin binding study: The T1 relaxation times of
ACHTREUNG
ACHTRE[UNG Gd-5] (0.1 mm) or
tions of HSA (0, 0.335, 0.67, 1.005, 1.34, 1.675, 2.01, 2.345, 2.68, 3.015,
and 3.35 mm). The concentrations of HSA were determined on the basis
of 4.5% w/v= ꢁ0.67 mm.[4,11a]
This work was supported by the Ministry of Education, Culture, Sports,
Science and Technology of Japan (Grants for The Advanced and Innova-
tional Research Program in Life Sciences, 16370071 and 16659003 to
T.N., 15681012, 17035019, 17036012, 017048006, and 17651119 to K.K.).
T.N. was also supported by the Hoh-ansha Foundation. K.K. was also
supported by the Sankyo Foundation, by the Kanagawa Academy of Sci-
ence, and by the Shimadzu Foundation. K.H. was the recipient of Re-
search Fellowships of the Japan Society for the Promotion of Science for
Young Scientists. We thank Professor Haruhiko Bito for valuable sugges-
tions.
Comparison of various species of serum albumins: The longitudinal
water proton relaxation times T1 of ACTHER[UGN Gd-5] or ACHTRE[UNG Gd-8] were measured at
378C in PBS (pH 7.4) in the presence of 4.5% w/v serum albumin from
four different species (human, rat, bovine, and rabbit) in the presence or
absence of b-galactosidase (1.13 mm).
UV/Vis absorption spectral measurements: The absorption spectra of
ACHTREUNG[Tb-5] (50 mm) or ACHTREUNG[Tb-8] (50 mm) were measured at 258C in aqueous solu-
tion buffered to pH 7.4 (100 mm HEPES buffer).
Time-resolved luminescence spectral measurements: The time-resolved
[1] a) G. Genove, U. DeMarco, H. Xu, W. F. Goins, E. T. Ahrens, Nat.
Med. 2005, 11, 450–454; b) K. H. Thompson, C. Orvig, Science 2003,
300, 936–939; c) K. Nakahara, T. Hayashi, S. Konishi, Y. Miyashita,
Science 2002, 295, 1532–1536; d) H. Degani, V. Gusis, D. Weinstein,
S. Fields, S. Strano, Nat. Med. 1997, 3, 780–782; e) R. E. Jacobs,
S. E. Fraser, Science 1994, 263, 681–684; f) J. C. Frias, K. J. Williams,
E. A. Fisher, Z. A. Fayad, J. Am. Chem. Soc. 2004, 126, 16316–
16317.
luminescence spectra of
sured in 100 mm HEPES buffer at pH 7.4, 258C (excitation at 280 nm for
[Tb-5] and [Tb-8], respectively). The slit width was 10 nm for both exci-
ACHTREUNG[Tb-5] or ACHTRE[UGN Tb-8] (50 mm, respectively) were mea-
A
ACHTREUNG
tation and emission. A delay time of 50 ms and a gate time of 1.00 ms
were used.
Kinetic studies: Kinetic parameters Km and kcat were determined by
direct fitting of the initial velocity versus substrate (ACHTRE[UGN Tb-5]) concentration
data to the Michaelis–Menten equation as shown in the Supporting Infor-
mation. The initial velocities were determined by monitoring the de-
[2] A. E. Merbach, É. Tóth, The Chemistry of Contrast Agents in Medi-
cal Magnetic Resonance Imaging, Wiley, New York, 2001.
994
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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A Gd3+-Based Magnetic Resonance Imaging Contrast Agent
FULL PAPER
[3] a) S. Aime, M. Botta, M. Fasano, E. Terreno, Acc. Chem. Res. 1999,
32, 941–949; b) S. Aime, M. Botta, M. Fasano, E. Terreno, Chem.
Soc. Rev. 1998, 27, 19–29.
[13] A. L. Nivorozhkin, A. F. Kolodziej, P. Caravan, M. T. Greenfield,
R. B. Lauffer, T. J. McMurry, Angew. Chem. 2001, 113, 2987–2990;
Angew. Chem. Int. Ed. 2001, 40, 2903–2906.
[14] L. M. De León-Rodrꢃguez, A. Ortiz, A. L. Weiner, S. Zhang, Z.
Kovacs, T. Kodadek, A. D. Sherry, J. Am. Chem. Soc. 2002, 124,
3514–3515.
[15] P. L. Anelli, I. Bertini, M. Fragai, L. Lattuada, C. Luchinat, G.
Parigi, Eur. J. Inorg. Chem. 2000, 625–630.
[16] L. Josephson, J. M. Perez, R. Weissleder, Angew. Chem. 2001, 113,
3304–3306; Angew. Chem. Int. Ed. 2001, 40, 3204–3206.
[17] J. W. Chen, W. Pham, R. Weissleder, A. Bogdanov, Jr., Magn.
Reson. Med. 2004, 52, 1021–1028.
[18] M. Zhao, L. Josephson, Y. Tang, R. Weissleder, Angew. Chem. 2003,
115, 1413–1416; Angew. Chem. Int. Ed. 2003, 42, 1375–1378.
[19] S. Aime, M. Chiaussa, G. Digilio, E. Gianolio, E. Terreno, J. Biol.
Inorg. Chem. 1999, 4, 766–774.
[20] S. Aime, M. Botta, M. Fasano, S. G. Crich, E. Terreno, J. Biol. Inorg.
Chem. 1996, 1, 312–319.
[21] a) D. Parker, J. A. G. Williams, J. Chem. Soc. Dalton Trans. 1996,
3613–3628; b) D. Parker, Coord. Chem. Rev. 2000, 205, 109–130;
c) G. R. Choppin, D. R. Peterman, Coord. Chem. Rev. 1998, 174,
283–299.
[4] P. Caravan, J. J. Ellison, T. J. McMurry, R. B. Lauffer, Chem. Rev.
1999, 99, 2293–2352.
[5] V. M. Runge, D. Y. Gelblum, M. L. Pacetti, F. Carolan, G. Heard,
Radiology 1990, 177, 393–400.
[6] a) M. P. Lowe, Aust. J. Chem. 2002, 55, 551–556; b) L. Thunus, R.
Lejeune, Coord. Chem. Rev. 1999, 184, 125–155; c) V. Comblin, D.
Gilsoul, M. Hermann, V. Humblet, V. Jacques, M. Mesbahi, C. Sauv-
age, J. F. Desreux, Coord. Chem. Rev. 1999, 185–186, 451–470;
d) M. K. Thompson, B. Misselwitz, L. S. Tso, D. M. J. Doble, H.
Schmitt-Willich, K. N. Raymond, J. Med. Chem. 2005, 48, 3874–
3877; e) V. C. Pierre, M. Botta, K. N. Raymond, J. Am. Chem. Soc.
2005, 127, 504–505; f) H. Kato, Y. Kanazawa, M. Okumura, A. Ta-
ninaka, T. Yokawa, H. Shinohara, J. Am. Chem. Soc. 2003, 125,
4391–4397; g) M. Mikawa, H. Kato, M. Okumura, M. Narazaki, Y.
Kanazawa, N. Miwa, H. Shinohara, Bioconjugate Chem. 2001, 12,
510–514; h) A. Accardo, D. Tesauro, P. Roscigno, E. Gianolio, L.
Paduano, G. D’Errico, C. Pedone, G. Morelli, J. Am. Chem. Soc.
2004, 126, 3097–3107; i) J. Lee, M. J. Zylka, D. J. Anderson, J. E.
Burdette, T. K. Woodruff, T. J. Meade, J. Am. Chem. Soc. 2005, 127,
13164–13166.
[22] P. R. Selvin, T. M. Rana, J. E. Hearst, J. Am. Chem. Soc. 1994, 116,
6029–6030.
[7] a) T. J. Meade, A. K. Taylor, S. R. Bull, Curr. Opin. Neurobiol. 2003,
13, 597–602; b) J. A. Duimstra, F. J. Femia, T. J. Meade, J. Am.
Chem. Soc. 2005, 127, 12847–12855; c) W. Li, S. E. Fraser, T. J.
Meade, J. Am. Chem. Soc. 1999, 121, 1413–1414; d) W. Li, G.
Parigi, M. Fragai, C. Luchinat, T. J. Meade, Inorg. Chem. 2002, 41,
4018–4024; e) S. Zhang, K. Wu, A. D. Sherry, Angew. Chem. 1999,
111, 3382–3384; Angew. Chem. Int. Ed. 1999, 38, 3192–3194; f) N.
Raghunand, C. Howison, A. D. Sherry, S. Zhang, R. J. Gillies, Magn.
Reson. Med. 2003, 49, 249–257; g) É. Tóth, R. D. Bolskar, A. Borel,
G. Gonzµlez, L. Helm, A. E. Merbach, B. Sitharaman, L. J. Wilson,
J. Am. Chem. Soc. 2005, 127, 799–805; h) M. Mikawa, N. Miwa, M.
Bräutigam, T. Akaike, A. Maruyama, J. Biomed. Mater. Res. 2000,
49, 390–395; i) M. Mikawa, T. Yokawa, N. Miwa, M. Brautigam, T.
Akaike, A. Maruyama, Acad. Radiol. 2002, 9, S109–S111; j) K. E.
Løkling, R. Skurtveit, A. Bjørnerud, S. L. Fossheim, Magn. Reson.
Med. 2004, 51, 688–696; k) S. Aime, M. Botta, E. Gianolio, E. Ter-
reno, Angew. Chem. 2000, 112, 763–766; Angew. Chem. Int. Ed.
2000, 39, 747–750; l) K. Hanaoka, K. Kikuchi, Y. Urano, M. Naraza-
ki, T. Yokawa, S. Sakamoto, K. Yamaguchi, T. Nagano, Chem. Biol.
2002, 9, 1027–1032; m) K. Hanaoka, K. Kikuchi, Y. Urano, T.
Nagano, J. Chem. Soc. Perkin Trans. 2 2001, 1840–1843; n) R. Tro-
kowski, S. Zhang, A. D. Sherry, Bioconjugate Chem. 2004, 15, 1431–
1440.
[23] a) I. Hemmilä, S. Webb, Drug Discovery Today 1997, 2, 373–381;
b) A. J. Kolb, P. V. Kaplita, D. J. Hayes, Y. W. Park, C. Pernell, J. S.
Major, G. Mathis, Drug Discovery Today 1998, 3, 333–342; c) V.
Laitala, I. Hemmilä, Anal. Chem. 2005, 77, 1483–1487; d) J. Karvi-
nen, V. Laitala, M. L. Mäkinen, O. Mulari, J. Tamminen, J. Hermo-
nen, P. Hurskainen, I. Hemmilä, Anal. Chem. 2004, 76, 1429–1436;
e) Y. Koshi, E. Nakata, I. Hamachi, ChemBioChem 2005, 6, 1349–
1352; f) K. Hanaoka, K. Kikuchi, H. Kojima, Y. Urano, T. Nagano,
J. Am. Chem. Soc. 2004, 126, 12470–12476; g) N. Weibel, L. J. Char-
bonniꢄre, M. Guardigli, A. Roda, R. Ziessel, J. Am. Chem. Soc.
2004, 126, 4888–4896; h) M. K. Johansson, R. M. Cook, J. Xu, K. N.
Raymond, J. Am. Chem. Soc. 2004, 126, 16451–16455; i) Z. Lin, M.
Wu, M. Schäferling, O. S. Wolfbeis, Angew. Chem. 2004, 116, 1767–
1770; Angew. Chem. Int. Ed. 2004, 43, 1735–1738; j) K. Lee, V. Dzu-
beck, L. Latshaw, J. P. Schneider, J. Am. Chem. Soc. 2004, 126,
13616–13617; k) S. Mameri, L. J. Charbonniꢄre, R. F. Ziessel, Inorg.
Chem. 2004, 43, 1819–1821; l) C. Li, G. L. Law, W. T. Wong, Org.
Lett. 2004, 6, 4841–4844; m) J. C. Frias, G. Bobba, M. J. Cann, C. J.
Hutchison, D. Parker, Org. Biomol. Chem. 2003, 1, 905–907; n) K. J.
Franz, M. Nitz, B. Imperiali, ChemBioChem 2003, 4, 265–271;
o) J. P. Cross, M. Lauz, P. D. Badger, S. Petoud, J. Am. Chem. Soc.
2004, 126, 16278–16279.
[24] D. Parker, R. S. Dickins, H. Puschmann, C. Crossland, J. A. K.
Howard, Chem. Rev. 2002, 102, 1977–2010.
[25] O. Viratelle, J. P. Tenu, J. Garnier, J. Yon, Biochem. Biophys. Res.
Commun. 1969, 37, 1036–1041.
[26] a) Y. Urano, M. Kamiya, K. Kanda, T. Ueno, K. Hirose, T. Nagano,
J. Am. Chem. Soc. 2005, 127, 4888–4894; b) J. Hofmann, M. Sernetz,
Anal. Biochem. 1983, 131, 180–186.
[8] a) D. Hçgemann, J. P. Basilion, Eur. J. Nucl. Med. 2002, 29, 400–
408; b) R. Weissleder, A. Moore, R. Mahmood, R. Bhorade, H.
Benveniste, E. A. Chiocca, J. P. Basilion, Nat. Med. 2000, 6, 351–
354.
[9] D. J. Spergel, U. Krꢂth, D. R. Shimshek, R. Sprengel, P. H. Seeburg,
Prog. Neurobiol. 2001, 63, 673–686.
[10] a) A. Y. Louie, M. M. Hꢂber, E. T. Ahrens, U. Rothbächer, R.
Moats, R. E. Jacobs, S. E. Fraser, T. J. Meade, Nat. Biotechnol. 2000,
18, 321–325; b) R. A. Moats, S. E. Fraser, T. J. Meade, Angew.
Chem. 1997, 109, 749–752; Angew. Chem. Int. Ed. Engl. 1997, 36,
726–728; c) M. M. Alauddin, A. Y. Louie, A. Shahinian, T. J.
Meade, P. S. Conti, Nucl. Med. Biol. 2003, 30, 261–265.
[11] a) P. Cravan, N. J. Cloutier, M. T. Greenfield, S. A. McDermid, S. U.
Dunham, J. W. M. Bulte, J. C. Amedio, Jr., R. J. Looby, R. M. Sup-
kowski, W. Dew. Horrocks, Jr., T. J. McMurry, R. B. Lauffer, J. Am.
Chem. Soc. 2002, 124, 3152–3162; b) S. Amie, E. Gianolio, E. Terre-
no, G. B. Giovenzana, R. Pagliarin, M. Sisti, G. Palmisano, M. Botta,
M. P. Lowe, D. Parker, J. Biol. Inorg. Chem. 2000, 5, 488–497; c) S.
Aime, M. Botta, S. G. Crich, G. B. Giovenzana, R. Pagliarin, M. Pic-
cinini, M. Sisti, E. Terreno, J. Biol. Inorg. Chem. 1997, 2, 470–479.
[12] R. B. Lauffer, T. J. McMurry, S. O. Dunham, D. M. Scott, D. J. Par-
melee, S. Dumas, PCT Int. Appl. WO 9736619, 1997.
[27] J. P. Tenu, O. M. Viratelle, J. Garnier, J. Yon, Eur. J. Biochem. 1971,
20, 363–370.
[28] W. H. Helhuish, J. Phys. Chem. 1961, 65, 229–235.
[29] a) A. Beeby, I. M. Clarkson, R. S. Dickins, S. Faulkner, D. Parker, L.
Royle, A. S. de Sousa, J. A. G. Williams, M. Woods, J. Chem. Soc.
Perkin Trans. 2 1999, 493–503; b) S. Quici, G. Marzanni, M. Cavaz-
zini, P. L. Anelli, M. Botta, E. Gianolio, G. Accorsi, N. Armaroli, F.
Barigelletti, Inorg. Chem. 2002, 41, 2777–2784.
[30] M. Li, P. R. Selvin, J. Am. Chem. Soc. 1995, 117, 8132–8138.
[31] A. V. Fowler, I. Zabin, J. Biol. Chem. 1978, 253, 5521–5525.
[32] Q. Y. Chen, C. J. Feng, Q. H. Luo, C. Y. Duan, X. S. Yu, D. J. Liu,
Eur. J. Inorg. Chem. 2001, 1063–1069.
[33] Y. M. Wang, Y. J. Wang, Y. L. Wu, Polyhedron 1999, 18, 109–117.
Received: May 24, 2007
Published online: November 8, 2007
Chem. Eur. J. 2008, 14, 987 – 995
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