2-: A new MRI contrast agent for copper ion sensing

Article abstract of DOI:10.1039/c1dt10033e

In this study, we have developed two new l-tryptophan based contrast agents [Gd(Try-TTDA)(H₂O)]^2- and [Gd(Try-ac-DOTA)(H ₂O)]^-. Upon addition of Cu(ii) to [Gd(Try-TTDA)(H ₂O)]^2-, significant increases in the relaxivity (r ₁) and hydration number of [Gd(Try-TTDA)(H₂O)] ^2- were observed. However, it only induced a minute increase in the relaxivity (r₁) in the case of [Gd(Try-ac-DOTA)(H₂O)] ^-. Furthermore, the interaction of Cu(ii) with the indole ring of Gd(iii) complexes was explored by measuring the intrinsic fluorescence of the tryptophan of the Gd(iii) complex. With the addition of one equivalent of Cu(ii) to [Gd(Try-TTDA)(H₂O)]^2- the indole fluorescence was completely quenched. Moreover, the [Gd(Try-TTDA)(H₂O)]^2- complex shows excellent selectivity towards Cu(ii) over other metal ions (Cu(ii) > La(iii) > Mg(ii)). Importantly, the significant signal intensity (2073 ± 67) for in vitro MR imaging using [Gd(Try-TTDA)(H₂O)] ^2- in the presence of Cu(ii) implicates that this new smart contrast agent ([Gd(Try-TTDA)(H₂O)]^2-) can serve as a Cu(ii) sensor for MR imaging.

Full text of DOI:10.1039/c1dt10033e

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PAPER
2
-
[
Gd(Try-TTDA)(H O)] : A new MRI contrast agent for copper ion sensing†
2
Dayananda Kasala, Tsung-Sheng Lin, Chiao-Yun Chen, Gin-Chung Liu,c,d Chai-Lin Kao,e
a,b
a
c,d
f
a,b
Tian-Lu Cheng and Yun-Ming Wang*
Received 9th January 2011, Accepted 4th March 2011
DOI: 10.1039/c1dt10033e
2
-
In this study, we have developed two new L-tryptophan based contrast agents [Gd(Try-TTDA)(H O)]
2
-
2-
and [Gd(Try-ac-DOTA)(H
increases in the relaxivity (r
2
O)] . Upon addition of Cu(II) to [Gd(Try-TTDA)(H O)] , significant
2
2
-
1
) and hydration number of [Gd(Try-TTDA)(H
2
O)] were observed.
However, it only induced a minute increase in the relaxivity (r
1
) in the case of
-
[
Gd(Try-ac-DOTA)(H
2
O)] . Furthermore, the interaction of Cu(II) with the indole ring of Gd(III)
complexes was explored by measuring the intrinsic fluorescence of the tryptophan of the Gd(III)
2
-
complex. With the addition of one equivalent of Cu(II) to [Gd(Try-TTDA)(H O)] the indole
2
2
-
fluorescence was completely quenched. Moreover, the [Gd(Try-TTDA)(H
2
O)] complex shows
excellent selectivity towards Cu(II) over other metal ions (Cu(II) > La(III) > Mg(II)). Importantly, the
2
-
significant signal intensity (2073 ± 67) for in vitro MR imaging using [Gd(Try-TTDA)(H O)] in the
2
2
-
presence of Cu(II) implicates that this new smart contrast agent ([Gd(Try-TTDA)(H
2
O)] ) can serve as
a Cu(II) sensor for MR imaging.
Introduction
a large magnetic moment with a long electron spin relaxation
time (~10 s at the magnetic field strengths of interest for MRI
-
9
Magnetic resonance imaging (MRI) offers several advantages
over other clinical diagnostic techniques in molecular imaging,
including high spatial resolution, noninvasiveness, high anatom-
ical contrast and lack of harmful radiation. On the other
hand, the sensitivity of MRI in depicting a small molecule is
constrained by the ubiquitous protons in the body, resulting in a
high background and low signal to noise ratio (SNR). To enhance
the MR image signal intensity, gadolinium ion based contrast
agents have been developed. This metal ion, with the high electron
applications), these properties ensure an optimum efficiency for the
4
nuclear spin relaxation of the interacting nuclei. Currently, most
of the MRI contrast agents approved in clinical use are Gd(III)-
1
–3
1,2
poly(aminocarboxylate) complexes. However, these contrast
agents are non-specific and provide only anatomical information.
The MR signal enhancement depends on the relaxation rate of
the protons in water molecules interacting with the metal centre.
The relaxation rate is influenced by a variety of factors such as
the number of inner-sphere water molecules (q), the rotational
8
spin and a symmetric electronic ground state ( S7/2), couples to
correlation time (t
Gd(III) coordinated/bound water molecule (t
smart contrast agents, which have abilities to respond to a micro-
R
), and the mean residence lifetime of the
M
). Recently, several
a
Department of Biological Science and Technology, National Chiao Tung
5
6
7
8
environment such as pH, partial oxygen pressure, Ca(II), Zn(II),
University, 75 Bo-Ai Street, Hsinchu 300, Taiwan. E-mail: ymwang@
mail.nctu.edu.tw; Fax: 886-3-5729288; Tel: 886-3-5721212 ext 56972
9
10
11
12
K(I), Cu(I)/Cu(II), and Fe(II) metal ions, enzyme activity
b
13
Institute of Molecular Medicine and Bioengineering, National Chiao Tung
and sugars have been reported. These smart MR contrast
agents furnish structural changes upon target interaction, which
significantly change their signal properties.
University, 75 Bo-Ai Street, Hsinchu 300, Taiwan. E-mail: ymwang@
mail.nctu.edu.tw; Fax: 886-3-5729288; Tel: 886-3-5721212 ext 56972
c
Department of Medical Imaging, Kaohsiung Medical University Hospital.
Among all the bio-relevant metal ions, copper plays an im-
Kaohsiung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung 807,
Taiwan
14,15
portant role in various biological processes.
An imbalance
d
Department of Radiology, Kaohsiung Medical University, 100 Shih-Chuan
of Cu(II) ions within the human body can cause many diseases
1
st Road, Kaohsiung 807, Taiwan
16,17
18
including Menkes disease,
Wilson’s disease.
occipital horn syndrome and
e
Department of Medicinal and Applied Chemistry, Kaohsiung Medical
University, 100 Shih-Chuan 1st Road, Kaohsiung 807, Taiwan
19,20
The normal concentration of copper in
17
f
normal blood is 100–150 mg/dL (15.7–23.6 mM). The deci-
phering of the relationship between copper regulation and its
physiological consequences is interesting, however, understanding
remains elusive at the molecular level.
Department of Biomedical Science and Environmental Biology, Kaohsiung
Medical University, 100 Shih-Chuan 1st Road, Kaohsiung 807, Taiwan
Electronic supplementary information (ESI) available: Procedures for
†
1
7
d
O NMR and dissociation constant (K ), ESI mass spectra (S1, S2, S4,
S6, S8), HPLC chromatograms (S3, S5), Job’s plot (S7), determination
of dissociation constant (S9), emission spectra (S10), and quenching plot
Over the past few decades, several fluorescence-based sensors
21,22
(
S11). See DOI: 10.1039/c1dt10033e
for the detection of Cu(II) ions have been reported.
In contrast,
5
018 | Dalton Trans., 2011, 40, 5018–5025
This journal is © The Royal Society of Chemistry 2011

View Article Online
the reported MR-based copper ion sensors are limited. Given
that noncovalent binding forces, such as interactions between
a cation and an aromatic ring, play important functional and
23,24
structural roles in biological molecules,
the bio-related indole
side chain bearing a poly(aminocarboxylate) such as EDTA-
bis(L-tryptophan methyl ester) has been demonstrated to interact
significantly with the metal ion (Cu(II)), exhibiting the strongest
25
interaction. Therefore, the development of an optical and
MRI dual mode Cu(II) sensing molecule with an indole group
incorporated in the Gd(III) complex is fascinating.
In this report, we designed and synthesized (4S)-4-indolyl-
3
,6,10-tri(carboxymethyl)-3,6,10-triazadodecanedioic acid (Try-
TTDA) and N-L-tryptophan-(4,7,10-tris(carboxymethyl)-4,7,10-
tetraaza-cyclododec-1-yl)-acetamide (Try-ac-DOTA) and their
Gd(III) complexes. The synthetic strategy of [Gd(Try-
2
-
-
TTDA)(H
2
O)] and [Gd(Try-ac-DOTA)(H O)] was depicted in
2
Schemes 1 and 2. Subsequently, the T
1
relaxivity was determined
◦
-
in the presence and absence of Cu(II) at 37.0 ± 0.1 C and 20 MHz.
Furthermore, the quenching of the intrinsic fluorescence of the
indole with various biologically relevant metal ions was studied.
Scheme 2 Synthetic scheme of [Gd(Try-ac-DOTA)(H O)] .
2
29
by using our previously reported procedure. All commercial
grade chemicals were used without further purification. Fluores-
cence and luminescence were measured by using a Varian Cary
Eclipse fluorescence spectrophotometer and H (400 MHz),
100 MHz), and O (54.2 MHz) NMR spectra were recorded
In addition, in vitro T
1
weighted MR imaging studies were also
investigated to demonstrate a smart Cu(II) sensing contrast agent.
1
13
C
1
7
(
on a Varian Gemini-400 spectrometer. The concentration of the
Gd(III) complex was determined by ICP-MS with a Perkin–Elmer
OPTIMA 2000. The relaxivity measurements were performed
◦
using a relaxometer operating at 20 MHz and 37.0 ± 0.1
C
(NMR-120 Minispec, Bruker). MR imaging was performed with
a 3.0 T MR scanner (Sigma; GE Medical Systems, Milwaukee,
WI) using a knee coil. The HPLC experiments were performed
¨
on an Amersham AKTAbasic 10 instrument equipped with an
Amersham UV-900 detector and Amersham Frac-920 fraction
collector. LC- mass spectral analyses were performed with a
Waters Micromass-ZQ mass spectrometer.
Preparations
Synthesis of methyl ester-L-tryptophan hydrochloride (1). L-
Tryptophan (10.0 g, 49.0 mmol) was dissolved in methanol
(
200 mL) and then thionyl chloride (4.29 mL, 58.8 mmol) was
added slowly. Upon completion of the addition, the reaction
mixture was stirred at room temperature for 9 h. The reaction
mixture was concentrated under reduced pressure and then diethyl
ether was added, which resulted in a white precipitate. The
resulting product was filtered and dried under vacuum for 48 h.
2-
2
Scheme 1 Synthetic scheme of [Gd(Try-TTDA)(H O)] .
Experimental
1
The white powder 1 was obtained (8.0 g, 67.0%). H NMR
(
DMSO-d
protons), 4.2 (m, CH), 3.64 (s,OCH
(DMSO-d , 100 MHz): d (ppm) = 169.77, 136.21, 126.90, 125.01,
121.16, 118.6, 118.00, 115.88, 106.32, 52.65, 26.09.
Synthesis of 2-amino-N-(3-aminopropyl)-3-(1H-indol-2-
6
, 400 MHz): d (ppm) = 6.96 –7.60 (m, 5H, indolyl
Materials and instrumentation
13
3
) 3.15 (m, CH ). C NMR
2
L-Tryptophan and bromoacetylbromide were purchased from
Alfa Aesar. Thionyl chloride, BH
hexahydrate, europium chloride, copper chloride, magnesium
chloride, N-chloroacetyl-L-tryptophan and phosphate buffered
saline were purchased from Aldrich. Cyclen was purchased
from Strem chemicals. 4,7,10-(Tris-tert-butyl carboxymethyl)-
6
3
·THF, gadolinium(III) chloride
yl)propanamide trihydrochloride (2). L-Tryptophan methyl ester
hydrochloride (8.23 g, 32.4 mmol) was dissolved in 100 mL of
methanol and then 1,3-diaminopropane (4.29 mL, 162.0 mmol)
was added. The resulting reaction mixture was stirred at room
temperature for 24 h. The reaction mixture was concentrated
under reduced pressure to obtain a yellow viscous oil. Then, 7 M
(
1,4,7,10-tetraazacyclodecane) (DO3A-tris-tert-butyl ester) was
26,27
synthesized using the previously reported procedure.
tert-
Butylbromoacetate was synthesized according to the reported
28
2-
procedure. The [Gd(TTDA)(H
2
O)] complex was prepared
NH
4
OH (100 mL) solution was added to this oil and the pH
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was adjusted to 10–11.5. The aqueous phase was extracted with
chloroform (five portions of 500 mL). The organic layer was dried
terminal, NCH
(m, NH CH CH
C NMR (DMSO-d
2
COOH), 3.4 (s, central, NCH
CH NH ), 2.5 (quint, NH
, 100 MHz): d (ppm) = 178.00, 176.00,
2
COOH), 3.08–2.94
2
2
2
2
2
2
CH
2
CH
2
CH
2
NH ).
2
1
3
over anhydrous MgSO
4
and filtered. The filtrate was concentrated
6
under reduced pressure and the crude amide was purified using
cationic exchange (AG 50 W ¥ 8 column (200–400 mesh, H form,
174.60, 171.55, 140.25, 130.96, 126.73, 125.16, 122.52, 122.33,
115.61, 113.34, 49.11, 45.48, 36.52, 27.48, 23.73.
+
1
00 mL resin), 4.2 cm column diameter). The product was eluted
Synthesis of N-L-tryptophan-(4,7,10-tris(carboxymethyl)-4,7,
10-tetraaza-cyclododec-1-yl)-acetamide(Try-ac-DOTA)(6). 4,7,
with 4.0–4.5 M HCl. The solution was evaporated to dryness to
1
obtain 2 as a hygroscopic powder (2.4 g, 20.2%). H NMR (D
00 MHz): d (ppm) = 7.1–7.60 (m, 5H, indolyl protons), 4.1 (t,
indolyl-CH –CH-), 3.31–2.93 (m, NHCH CH CH NH ), 2.4
m, indolyl-CH -CH), 1.42 (quint, NHCH CH CH NH
O, 100 MHz): d (ppm) = 169.69, 136.25, 126.73, 125.23,
2
O,
1
0-(Tris-tert-butylcarboxymethyl)-(1,4,7,10 tetraazacyclodecane)
DO3A-tris-tert-butyl ester) (1.22 g, 2.38 mmol), KI (0.078 g,
0.48 mmol) and N,N-diisopropylamine (DIPEA) (1.36 mL,
.8 mmol) were dissolved in acetonitrile (150 mL) and then the
4
(
2
2
2
2
2
1
3
(
2
2
2
2
2
).
C
7
NMR (D
1
2
2
mixture was stirred for 2 h. Subsequently, the N-chloroacetyl-
22.29, 119.70, 118.35, 112.12, 106.78, 54.11, 48.99, 36.79, 36.44,
6.92, 26.17.
L-tryptophan (0.7 g, 2.5 mmol) was added to the solution. The
◦
reaction mixture was heated to 60–70 C and stirred for 48 h.
Synthesis of N-(2-amino-3-(1H-indol-2-yl)propyl)propane-1,3-
The solvent was removed by rotary evaporation. Then, 6 M HCl
diamine trihydrochloride (3). Amide (2) (2.4 g, 6.55 mmol) was
(
100 mL) was added to the residue and the mixture was stirred
placed in a 250 mL two necked round bottom flask, fitted with
at room temperature for 4 h. The solution was evaporated by
rotary evaporation. The acid treatment was repeated three times.
The residue was dissolved in water and adjusted to pH 11 with
a reflux condenser. After purging with nitrogen, the flask was
◦
cooled to 0–10 C. The 1 M BH
3
·THF (30 mL) was cannulated
into the reaction flask. The reaction mixture was warmed up to
room temperature and brought to reflux for 24 h. The solution
was cooled to 0–10 C and the excess borane was quenched by
ammonium hydroxide, and subjected to anion exchange (AG1 ¥
-
8
column (200–400 mesh, HCO
2
form, 100 mL resin); 4.2-cm
◦
column diameter). The product was eluted with 2.0 L of water
and a formic acid gradient. The trace amount of formic acid was
removed by co-evaporation with water (3¥ 100 mL), yielding a
a slow and dropwise addition of water until the evolution of
gas ceased. The reaction mixture was evaporated and the residue
was dissolved in 6 M HCl (100 mL). The resulting solution was
refluxed for 3 h, followed by stirring at room temperature for 24 h.
The acidic reaction mixture was loaded into a cationic exchange
+
yellow viscous oil of Try-ac-DOTA (6) (0.70 g, 50%) MS ESI
+
for [C27
H
38
N
6
O
9
] : calculated 590.27, found 591.10, (see Fig. S2†)
H NMR (D O, 400 MHz): d (ppm) = 7.0–7.60 (m, 5H, indolyl
1
2
+
13
column (AG 50 W ¥ 8 column (200–400 mesh, H form, 100 mL
protons), 2.6–3.81(m, 27H, cycle CH
NMR (D O, 100 MHz): d (ppm) = 176.40, 175.0, 170.55, 169.89,
35.82, 127.06, 124.36, 121.73, 119.34, 118.57, 111.70, 110.13,
7.5–56.14 (12C, cycle CH ), 42.25, 27.15.
2
, indolyl-CH, CH
2
),
C
resin); 4.2 cm column diameter). The product was eluted with
2
5
.5–6.0 M HCl. The solution was evaporated to dryness. A pale
1
4
1
yellow compound was obtained (3) (2.02 g, 86.6%). H NMR
O, 400 MHz): d (ppm) = 7.1–7.60 (m, 5H, indolyl protons),
.95 (m, indolyl-CH –CH), 3.4 (m, indolyl-CH -CH), 3.28–2.94
m,NH CH CH CH NH ), 2.03 (quint, NH CH CH CH NH ).
C NMR (D O, 100 MHz): d (ppm) = 136.25, 126.73, 125.63,
2
(
D
2
Complexation. The Gd(III) and Eu(III) complexes were pre-
pared by dissolving the ligand (Try-TTDA or Try-ac-DOTA)
3
2
2
(
2
2
2
2
2
2
2
2
2
2
1
3
(0.05 mmol) in H O (8 mL) and adjusting the pH of the
2
2
solution to 6.5 with 1 N NaOH. To these solutions, 2.0 mL of
122.52, 119.88, 118.20, 112.34, 106.40, 49.01, 45.48, 36.52, 26.78,
an aqueous LnCl solution (0.05 mmol) was added dropwise,
3
23.73.
while maintaining the pH at 5.5–6.0 with 1 N NaOH (aq). The
mixture solution was stirred at room temperature. The absence
of free lanthanide ions in the solutions was verified by the
xylenol orange test. The solutions were evaporated under reduced
pressure. The purity of the Gd(III) complexes was determined
Synthesis of (4S)-4-indolyl-3,6,10-tri(carboxymethyl)-3,6,10-
triazadodecanedioic acid (Try-TTDA) (4). To a solution of
amine (3) (2.02 g, 5.74 mmol) and anhydrous K CO (7.12 g,
1.6 mmol) in CH CN (200 mL), tert-butyl bromoacetate
4.64 mL, 31.35 mmol) was added. The resulting reaction mixture
was refluxed for 24 h. The K CO was removed by filtration
2
3
5
(
3
+
by HPLC and identified by a mass spectrometer. MS ESI for
2
3
2-
+
[
Gd(Try-TTDA)(H
2
O)] [C24
H
29GdN
4
O
10] : calculated 689.10,
through a Buchner funnel and the solvent was removed under
reduced pressure. Then, 6 M HCl (100 mL) was added to the
residue, and the mixture was stirred at room temperature for 4 h.
The solution was evaporated by rotary evaporation. The acid
treatment was repeated three times. The residue was dissolved
in distilled water and adjusted to pH 11.0 by adding aqueous
+
found 689.69, (see Fig. S3 and S4†). MS ESI for [Gd(Try-ac-
3+
DOTA)(H
2
O)] [C27
H
35GdN
6
O ] : calculated 745.15, found 748.10
9
(see Figs. S5 and S6†)
Relaxation time measurement (r )
1
ammonia and loaded to the anion exchange column (AG1 ¥
Relaxation times (T
were measured to determine relaxivity (r
1
) of aqueous solutions of Gd(III) complexes
). All measurements
-
8
column (200–400 mesh, HCO
2
form, 100 mL resin); 4.2 cm
1
column diameter). The product was eluted with 500 mL of water
and a formic acid gradient. The trace of formic acid was removed
by co-evaporation with water (3 ¥ 100 mL), and the product was
dried under vacuum to give a pale yellow powder of Try-TTDA
were made using a relaxometer operating at 20 MHz and 37.0
± 0.1 C (NMS-120 Minispec, Bruker). Before each measurement,
◦
the relaxometer was tuned and calibrated. The values of r
were determined from six data points generated by an inversion-
recovery pulse sequence. The proton spin–lattice relaxation (T
was measured by using various concentrations of [Gd(Try-
1
+
+
(
4) (1.0 g, 31.0%). MS ESI for [C24
H
32
N
4
O
10] calculated 536.21,
1
)
1
found 536.77, (see Fig. S1†). H NMR (DMSO-d
6
, 400 MHz):
2
-
-
◦
d (ppm) = 7.1–7.60 (m, 5H, indolyl protons), 3.81 (s, 8H,
TTDA)(H
2
O)] or [Gd(Try-ac-DOTA)(H O)] at 37.0 ± 0.1 C
2
5
020 | Dalton Trans., 2011, 40, 5018–5025
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in pH 7.4 PBS buffer. The interaction with metal ions was
confirmed by titration method. The different concentrations of
metal ions were added with a fixed concentration of the [Gd(Try-
complex was shown in Scheme 1. Synthesis of Try-TTDA was
achieved by the following steps: the reaction of L-tryptophan
with thionyl chloride in methanol at room temperature led to
the formation of compound 1, followed by the addition of 1,3-
2
-
-
TTDA)(H
2
O)] or [Gd(Try-ac-DOTA)(H
2
O)] complex in acetate
◦
buffer at pH 5.5, and 37.0 ± 0.1 C and their relaxivity values were
diaminopropane in methanol, which yielded compound 2. The
◦
measured.
mixed solution of 2 and BH
3
·THF was stirred for 15 h at 60 C,
followed by the addition of 6 M HCl (aq) solution, which results
Luminescence method for establishing solution hydration state
in the formation of compound 3. The compound 3 was treated
with tert-butylbromoacetate in the presence of anhydrous K
2
CO
3
Luminescence lifetime data has been obtained for the Eu(III)
complex to determine the number of inner-sphere water molecules
◦
in CH
3
CN at 80 C to provide the tert-butyl ester of Try-TTDA
and, subsequently, the hydrolysis of the tert-butyl ester using 6 M
30
in the aqueous solution. The luminescence lifetime (t) has been
determined in both H O and D O. The number of inner-sphere
2
-
HCl gave Try-TTDA (4). Finally, the [Gd(Try-TTDA)(H O)] (5)
2
2
2
complex was achieved by the complexation of the Gd(III) ion with
the Try-TTDA ligand in water at room temperature. The resulting
complex was identified by MS (ESI†).
water molecules contained in the complex was calculated by
eqn (1):
q = ([1/tH2O - 1/tD2O] - 0.25) ¥ 1.2
where q is the number of water molecules bound to metal ions,
is the luminescence half-life in water solution and t is the
luminescence half-life in deuterium oxide solution, respectively.
(1)
The synthesis of the L-tryptophan-based DOTA ligand and its
Gd(III) complex was shown in Scheme 2. Preparation of Try-ac-
DOTA was accomplished by the following steps: the reaction
of N-chloroacetyl-L-tryptophan with DO3A-tris-tert-butyl ester
t
H
O
D O
2
2
in the presence of a catalytic amount of KI in acetonitrile, at
◦
8
0 C, led to the formation of the tert-butyl ester of Try-ac-DOTA
2
-
Fluorescence study of [Gd(Try-TTDA)(H
2
O)] and
and, subsequently, the hydrolysis of the tert-butyl ester using 3 N
HCl(aq) at room temperature was completed to obtain Try-ac-
-
[
Gd(Try-ac-DOTA)(H
2
O)] for metal ion interaction
To examine the metal ion-indole ring interaction, the fluorescence
DOTA (6). Furthermore, the ligand was treated with GdCl
3
·6H
2
O
2
-
quenching studies of [Gd(Try-TTDA)(H
2
O)] and [Gd(Try-ac-
in water at room temperature to afford the complex [Gd(Try-ac-
O)]- with various metal ions were performed. The
-
DOTA)(H
2
DOTA)(H
(ESI†).
2
O)] (7). The resulting complex was identified by MS
metal ions were titrated into a solution of 0.1 mM Gd(III) complex
in acetate buffer at pH 5.5. The excitation wavelength was fixed at
2
81 nm, and the emission intensity at 360 nm was recorded.
In vitro MR imaging study
The in vitro T -weighted MR imaging of [Gd(Try-TTDA)(H
Luminescence study
To determine the number of inner-sphere water molecules before
and after Cu(II) interaction with [Gd(Try-TTDA)(H
2
-
2
O)] or
2
-
1
2
O)]
-
[
Gd(Try-ac-DOTA)(H
2
O)] , the luminescence method was used.
was performed by the concentration-dependent imaging. The
different concentrations (0.01, 0.05, 0.10 and 0.20 mM) of
We conducted the luminescence lifetime (t) measurements of the
equivalent Eu(III) complexes in both H
in Table S1†. The number of inner sphere molecules (q) was
calculated by using eqn (1). The estimated number of inner sphere
molecules for [Eu(Try-TTDA)(H
Cu(II) and 1.94 in the presence of Cu(II). These results indicate that
the interaction of Cu(II) with the indole ring changes the chelating
mode for [Eu(Try-TTDA)(H
30
2
O and D
2
O, as shown
2
-
[
Gd(Try-TTDA)(H O)] and metal ions in the molar ratio of 1 : 1
2
were mixed at room temperature. The MR imaging was performed
with a 3.0 T MR imaging scanner (Sigma, GE Medical Systems,
2-
2
O)] is 1.23 in the absence of
Milwaukee, WI) using a knee coil. The T
performed under the following condition: fast gradient echo, TR =
00 ms, TE = 1.5 ms, coronal view, and section thickness = mm,
1
-weighted scanning was
5
2-
2
O)] . Presumably, a carboxylate arm
FOV = 10 cm.
switches to bind with Cu(II), leaving space for the coordination
of a second water molecule to the lanthanide, which leads to
an increase in the hydration number (Dq = 0.7). On the other
Results and discussion
hand, in the presence or absence of Cu(II), the hydration number
Synthesis
-
remains the same for [Eu(Try-ac-DOTA)(H
2
O)] , since no change
in the chelation is necessary to accommodate the Cu(II). These
results support the proposed structures of the addition of Cu(II) to
In this study, we reported two L-tryptophan moiety-bearing
ligands (Try-TTDA and Try-ac-DOTA), as shown in Fig. 1.
Synthesis of L-tryptophan-based TTDA ligand and its Gd(III)
2
-
-
[
Gd(Try-TTDA)(H
2
O)] or [Gd(Try-ac-DOTA)(H O)] , as shown
2
in Scheme 3.
Stoichiometry analysis
In order to investigate the binding between Cu(II) and [Gd(Try-
2
-
TTDA)(H O)] , the fluorescence based Job’s method was
2
31
performed. A plot of the fluorescence intensity as a function
2
-
of [Cu(II)]/([Cu(II) + [Gd(Try-TTDA)(H
2
O)] ) is shown in Fig.
S7.† The inflection point at 0.5 indicates that the [Gd(Try-
2
-
Fig. 1 Structural formula of L- tryptophan based ligands.
This journal is © The Royal Society of Chemistry 2011
TTDA)(H
2
O)] –Cu(II) complex exists with 1 : 1 stoichiometry.
Dalton Trans., 2011, 40, 5018–5025 | 5021

View Article Online
Fig.
2
1
Dependence of the proton spin-lattice relaxivity (r ) on
2-
metal ion concentration for [Gd(Try-TTDA) (H
2
O)] {Cu(II) (ꢀ),
O)] {Cu(II) (ꢁ)} at 37.0 ± 0.1 C and 20 MHz in pH 5.5
Mg(II) (ꢀ), La(III) (᭹)}, [Gd(Try-ac-DOTA)(H
2
O)]- {Cu(II) (᭢)}, and
◦
2-
[
Gd(TTDA)(H
2
acetate buffer solution.
Scheme
3
Proposed structures of Cu(II) ion interaction with
confirmed by mass spectroscopic analysis. The expected mass
O)] - and [Gd(Try-ac-DOTA)(H
2
O)]-.
2-
[
Gd(Try-TTDA)(H
2
2
was observed at m/z 752, corresponding to ([Gd(Try-TTDA)]
+
[
(K
Cu(II)]), as shown in Fig. S8.† Moreover, the dissociation constant
2
Table 1 The relaxivity of [Gd(Try-TTDA)(H
2
O)] - and [Gd(Try-
O)]-
d
) is estimated to be 2.52 mM (Fig. S9†). On the other
ac-DOTA)(H
2
in the presence and absence of Cu(II),
-
2
-
2
-
O)]-
[
Gd(TTDA)(H
2
O)] , [Gd(DTPA)(H
2
O)] , and [Gd(DOTA)(H
2
hand, after addition of Cu(II) to [Gd(Try-ac-DOTA)(H
[Gd(TTDA)(H O)] , only a minor increase in the relaxivity was
2
O)] and
◦
2-
at 37.0 ± 0.1 C and 20 MHz
2
observed because there is no increase in the number of inner sphere
r
1
/mM- s-1
1
Complexes
water molecules, as shown in Table 1. In addition, we measured
2
-
2
-
-
the relaxivity of [Gd(Try-TTDA)(H O)] titrated by a variable
[
[
[
[
[
[
[
Gd(Try-TTDA)(H
Gd(Try-TTDA)(H
Gd(Try-DOTA)(H
Gd(Try-DOTA)(H
2
O)]
4.22 ± 0.03
7.42 ± 0.03
4.02 ± 0.05
4.65 ± 0.04
3.85 ± 0.03
3.89 ± 0.03
3.56
2
2
◦
2
O)] +Cu(II)
concentration of different metal ions at 37.0 ± 0.1 C and 20 MHz
2
-
-
2
O)]
in pH 5.5 acetate buffer solution, as shown in Fig. 2. The result
showed that there was no remarkable increase in the relaxivity for
Mg(II) and La(III). These relaxivity studies also indicate that the
interaction of the indole ring with Cu(II) is significantly stronger
than those of the indole ring with La(III) and Mg(II).
2
2
-
O)] +Cu(II)
2
a
2
Gd(TTDA)(H O)]
2-
b
Gd(DTPA)(H
2
O)]
-
c
Gd(DOTA)(H
2
O)]
a
b
c
Data was obtained from ref. 29 ref. 32 ref. 33
1
7
O NMR Study
These results also support the proposed structure of [Gd(Try-
2
-
The kinetic parameters were determined for [Gd(Try-
TTDA)(H
2
O)] in the presence of Cu(II), as shown in Scheme 3.
2
-
17
TTDA)(H
chemical shifts (Dw
1/T2r) relaxation rates were simultaneously analyzed (see ESI†).
2
O)] by variable-temperature O NMR studies. The
r
), the longitudinal (1/T1r) and transverse
Relaxivity measurements
(
2
-
2-
The relaxivity (r
1
) values of [Gd(Try-TTDA)(H
2
O)] and
The data for [Gd(Try-TTDA)(H O)] are plotted in Fig. 3,
2
-
[
Gd(Try-ac-DOTA)(H
2
O)] in the presence and absence
with the corresponding curves representing the results of the
best fitting of the data according to equations (1S–9S). The
kinetic parameters obtained were illustrated in Table 2, the
large number of parameters influence the data obtained by
2
-
2-
of Cu(II), [Gd(TTDA)(H
2
O)] , [Gd(DTPA)(H
2
O)] , and
-
◦
[
Gd(DOTA)(H
2
O)] at 37.0 ± 0.1 C and 20 MHz are shown
2
-
in Table 1. The relaxivity values of [Gd(Try-TTDA)(H
2
O)] and
-
17
[
(
Gd(Try-ac-DOTA)(H
2
O)] are higher than those of [Gd(TTDA)-
the different techniques. Nevertheless, the O NMR technique
2
-
29
2- 32
- 33
H
2
O)] ,
[Gd(DTPA)(H
2
O)] ,
and [Gd(DOTA)(H
2
O)] .
has an advantage that the outer-sphere contributions to the
relaxation rates are negligibly small, which is a result of the
oxygen nucleus being closer to the paramagnetic centre when it
2
-
Fig. 2 represents the relaxivity of [Gd(Try-TTDA)(H
2
O)] titrated
◦
by a variable concentration of metal ions at 37.0 ± 0.1 C and
34
36
2
0 MHz in pH 5.5 acetate buffer solution.
is bound in the inner sphere. The scalar coupling constant (A/©)
2
-
Upon the addition of one equivalent of Cu(II) to [Gd(Try-
of [Gd(Try-TTDA)(H
2
O)] is very similar to those obtained for
2
-
6
-1
TTDA)(H O)] , the relaxivity significantly increases, owing to
2
other Gd(III) complexes (-3.2 ± 0.3 and -3.8 ± 0.2 ¥ 10 rad s
2
-
2-
an increase in the number of inner sphere water molecules.
for [Gd(TTDA)(H
2
O)] and [Gd(DTPA)(H O)] , respectively)
2
Moreover, Cu(II) itself is paramagnetic and has a relaxivity (r
1
)
with one inner-sphere water molecule. In the overall temperature
range, the transverse O relaxation rates (1/T2r) decrease with
value of 1.0 mM- s , measured at 10 MHz in water. This
1
-1
35
17
relaxivity increase compares favourably with other reported Cu(II)
an increasing temperature, indicating that this system is in the
fast exchange regime. Consequently, 1/T2r is determined by
the relaxation rate of the coordinated water molecule (1/T2m),
10
37
sensors, such as the Copper-Gad (CG) family. The addition
of more Cu(II) has no effect on the relaxivity. This result is in
good agreement with the fluorescence based Job’s method for the
which is influenced by the water residence lifetime (t
M
= 1/kex),
2
-
1
: 1 molar ratio binding of [Gd (Try-TTDA)(H
2
O)] to Cu(II).
the longitudinal electronic relaxation rate (1/T1e), and the scalar
2
-
298
The Cu(II) interaction with [Gd(Try-TTDA)(H
2
O)] was also
coupling constant (A/©). The water-exchange rate (kex ) for
5
022 | Dalton Trans., 2011, 40, 5018–5025
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2 2 2 2
Table 2 Kinetic and NMR parameters of [Gd(Try-TTDA)(H O)] , [Gd(TTDA)(H O)] , [Gd(DTPA)(H O)] , and [Gd(ETPTA)(H
O)]2- as obtained
2
-
2-
2-
17
a
from the simultaneous fit of O-NMR data
Parameter
[Gd(TryTTDA)(H
2
O] -
2
[Gd(TTDA)(H
2
O]2-a
[Gd(DTPA)(H
2
O]2-b
[Gd(ETPTA)(H
2
O]2-c
2
98
6
-1
k
ex
(10 s )
40.9 ± 2.7
28.7 ± 0.2
-2.7 ± 0.1
-3.4 ± 0.1
562 ± 2
146 ± 17
23.1 ± 0.5
-11.1 ± 3.1
-3.2 ± 0.3
104 ± 12
0
4.1 ± 0.3
52.0 ± 1.4
56.2 ± 5.0
-3.8 ± 0.2
103 ± 10
0.13 ± 0.06
18 ± 2
330 ± 40
27.9 ± 11
11.0 ± 3.0
-3.9 ± 0.2
75 ± 6
π
-1
DH (kJ mol )
π
-1
-1
DS (Jmol k )
6
-1
A/©(10 rad s )
298
t
R
(ps)
C
E
os
0
0.1
-1
R
(kJ mol )
16.9 ± 1.6
24.8 ± 1.5
17.7 ± 1.0
17
17
17
17
Method
O
O
O
O, EPR, NMRD
a
b
c
Data was obtained from ref. 29 ref. 32. ref. 38.
2
-
2-
higher than those of [Gd(TTDA)(H
2
O)] , [Gd(DTPA)(H O)] ,
2
-
and [Gd(DOTA)(H
2
O)] as shown in Table 1.
Fluorescence quenching study
The interaction between the metal ions and the side chain in-
2
-
-
dole of [Gd(Try-TTDA)(H
2
O)] and [Gd(Try-ac-DOTA)(H O)]
2
was investigated by the quenching of the intrinsic trypto-
phan fluorescence using fluorescence spectroscopy. The [Gd(Try-
2
-
TTDA)(H O)] was titrated with various biologically relevant
2
metal ions, such as Na(I), Ca(II), Mg(II), Zn(II), Cu(II), and
La(III) in acetate buffer at pH = 5.5, and the quenching of the
intrinsic tryptophan fluorescence was measured with excitation
wavelength at 281 nm and emission wavelength at 360 nm. As
2
-
shown in Fig. 4a, the fluorescence of [Gd(Try-TTDA)(H O)]
2
is not quenched by the addition of Na(I), Ca(II), Mg(II), Zn(II)
or La(III) (in molar ratio 1 : 1). However, under the same mol
ratio conditions, the fluorescence emission intensity dramatically
decreases after adding Cu(II). We further studied the titration
2
-
of [Gd(Try-TTDA)(H
2
O)] with various amounts of Cu(II). The
2
-
fluorescence intensity of [Gd(Try-TTDA)(H
2
O)] is completely
quenched when one equivalent of Cu(II) is added, as shown in
Fig. 4b. The consequence is consistent with stoichiometry analysis
and mass spectroscopic studies. These results indicate that the
2
-
interaction between the indole ring of [Gd(Try-TTDA)(H O)]
2
and Cu(II) is stable in solution state and quenches more strongly
than those of other metal ions. Furthermore, we studied the
-
titration of [Gd(Try-ac-DOTA)(H
2
O)] with various amounts of
Cu(II), as shown in Fig. S10.† However, the fluorescence quenching
plot is nonlinear with a downward curve (Fig. S11†). Therefore,
-
the fluorescence quenching of [Gd(Try-ac-DOTA)(H
2
O)] after
Fig. 3 Temperature dependence of (a) transverse and (b) longitudinal
binding with Cu(II) is significantly lower than that of [Gd(Try-
17
17
2-
O relaxation rates and (c) O chemical shifts at B = 9.4 T for
TTDA)(H
2
O)] .
2-
[
2
Gd(Try-TTDA)(H O)] . The lines represent simultaneous least-squares
fits to all data points displayed.
In vitro MR imaging study
To demonstrate the potential utility of this new MRI con-
2
-
6
2-
[
Gd(Try-TTDA)(H
those of other [Gd(TTDA)(H
higher than that of [Gd(DTPA)(H
Additionally, the value of correlation time (t
2
O)] (40.9 ± 2.7 ¥ 10 s-1) is lower than
trast agent ([Gd(Try-TTDA)(H
2
O)] ), MR imaging studies were
2
-
2
O)] derivatives, but is significantly
performed using a 3.0 T MR imaging scanner. The different
2
-
6
-1
2-
2
O)] (4.1 ± 0.3 ¥ 10 s ).
) for the Gd(III)
concentrations of [Gd(Try-TTDA)(H
2
O)] (0.01, 0.05, 0.10 and
R
0.20 mM) and metal ions (Cu(II), La(III) and Mg(II)) in the molar
ratio of 1 : 1 were mixed at room temperature and T weighted MR
complex with Try-TTDA was obtained at 298 K from the fitting
of the O NMR. The t
562 ps) is higher than those of [Gd(TTDA)(H
O)] (103 ps), and [Gd(EPTPA)(H
ps). Therefore, the relaxivity of [Gd(Try-TTDA)(H
O)]2- is
1
1
7
298
2-
R
value of [Gd(Try-TTDA)(H
2
O)]
images were performed. As shown in Fig. 5, the signal intensity
values for the 0.2 mM concentration of Cu(II), La(III) and Mg(II)
are 2073 ± 67, 1101 ± 16 and 1106 ± 24, respectively. The signal
intensity of in vitro MR imaging increases as the concentration
2
-
29
(
[
2
O)] (104 ps),
2
-
32
2-
Gd(DTPA)(H
2
2
O)] (75
38
2
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Dalton Trans., 2011, 40, 5018–5025 | 5023

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Conclusion
In summary, two new L-tryptophan based contrast agents,
2
-
-
[
Gd(Try-TTDA)(H
2
O)] and [Gd(Try-ac-DOTA)(H O)] , were
2
-
2
successfully synthesized. The [Gd(Try-TTDA)(H
2
O)] effectively
recognizes Cu(II) among the metal ions examined at pH 5.5 acetate
buffer solution using fluorescence measurements. This Gd(III)
complex displays high selectivity for Cu(II) via quenching of in-
trinsic tryptophan fluorescence. In particular, the relaxivity studies
show a significant increase in relaxivity upon Cu(II) detection. In
2
-
addition, the sensing of Cu(II) by [Gd(Try-TTDA)(H
proven by in vitro T -weighted MR imaging. Hence, [Gd(Try-
O)] may serve as a smart contrast agent for the
2
O)] was
1
2
-
TTDA)(H
2
detection of Cu(II) ions.
Acknowledgements
Funding from National Science Council of Taiwan (Grant no.
NSC 98-2627-M-009-009 and NSC 97-2113-M-009-016-MY3) is
gratefully acknowledged.
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