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G. Zhao et al. / Inorganica Chimica Acta 406 (2013) 146–152
or 2b. After the mixture was kept under reflux with stirring over-
night, it was cooled to room temperature and filtered to remove
the inorganic salts. The filtrate was concentrated under reduced
pressure to afford the resultant crude product, which was then
purified by silica gel column chromatography with CH2Cl2/CH3OH
to give the desired compound 3a or 3b.
2.2.5. Synthesis of general procedure for the synthesis of the lanthanide
complexes
Two or three equivalent of the corresponding lanthanide chlo-
ride (GdCl3, EuCl3) was added into the solution (5–7 mL) of
(DO3A)2–DAB or (DO3A)3–TAB at pH 7.0. The reaction was kept
at 70 °C for 24 h while the pH was maintained at 7.0 with 1 M
NaOH. The completion of the Gd3+ complexation reaction was
monitored by using xylenol orange as indicator.
2.2.3.1. (tBu-DO3A)2–DAB (3a). Prepared according to general pro-
cedure A: tBu-DO3AꢁHBr (1.5 g, 2.52 mmol), NaHCO3 (1.06 g,
12.6 mmol), dry acetonitrile (50 mL), compound 2a (0.5 g,
1.56 mmol). Chromatography (CH2Cl2/CH3OH 70:6). Yield: 64%
(0.96 g). dH (600 MHz, CDCl3): 8.09 (s, 4H, Ph), 4.20 (s, 4H,
PhCOCH2), 2.0–3.6 (m, 44H, NCH2, CH2COO), 1.47 (s, 54H, CCH3).
2.2.5.1. (Gd-DO3A)2–DAB. The MALDI-MS (DHB-H2O): m/z 1162.5
[M+H]+. UV–Vis (H2O) kmax = 273 nm (
e
= 12527 Mꢀ1 cmꢀ1). IR
(FT)
m .
= 3419, 1607, 1402, 1132, 1086 cmꢀ1
2.2.5.2. (Gd-DO3A)3–TAB). The MALDI-MS (DHB-H2O): m/z 1701.3
[M+H]+. UV–Vis (H2O) kmax = 245 nm ( = 9275 Mꢀ1 cmꢀ1). IR (FT)
= 3417, 1610, 1402, 1323, 1131, 1086 cmꢀ1
@
dC (151 MHz, CDCl3): 199.65 (PhC O), 172.92, 172.59 (COO),
@
e
138.99, 128.06 (PhC), 81.97 (CCH3), 60.84 (PhCOCH2), 55.68,
55.45 (CH2COO), 52.56, 48.46 (NCH2), 28.03, 27.89, 27.76 (CCH3).
MS (MALDI-DCTB): m/z 1209.8 [M+Na]+.
m
.
2.3. The proton relaxation time measurement (T1)
The longitudinal relaxation times (T1) of (Gd-DO3A)2–DAB and
(Gd-DO3A)3–TAB in 100 mM HEPES–NaOH at pH 7.0 were mea-
sured using an inversion-recovery pulse sequence at 20 MHz and
37 °C on a MQ-20 Minispec (Bruker). The relaxivity was derived
from the plots of 1/T1 versus the concentration of Gd3+ using Eq.
(1):
2.2.3.2. (tBu-DO3A)3–TAB (3b). Prepared according to general pro-
cedure A: tBu-DO3AꢁHBr (2.68 g, 4.5 mmol), NaHCO3 (1.1 g,
12 mmol), dry acetonitrile (16 mL), compound 2b (0.7 g,
1.6 mmol). Chromatography (CH2Cl2/CH3OH 10:1). Yield: 55%
(1.44 g). dH (300 MHz, CDCl3): 8.75 (s, 3H, Ph), 4.43 (s, 6H,
PhCOCH2), 2.17–3.47 (m, 66H, NCH2, CH2COO), 1.46 (m, 81H,
@
CCH3). dC (151 MHz, CDCl3): 199.37 (PhC O), 173.14, 172.58,
@
ð1=T1Þobsd ¼ ð1=T1Þd þ ½Mꢃ ꢂ r1
ð1Þ
170.42(COO), 136.93, 130.93 (PhC), 81.86 (CCH3), 61.82 (PhCOCH2),
57.99, 55.78 (CH2COO), 52.69, 48.42 (NCH2), 28.06 (CCH3). MS
(MALDI-DCTB): m/z 1764.1 [M+Na]+.
where (1/T1)obsd and (1/T1)d are the measured solvent relaxation
rate values in the presence and absence of the paramagnetic species
respectively (sꢀ1), [M] is the concentration of Gd3+ (mM) and r1 is
the relaxivity of the agent in a unit of mMꢀ1 sꢀ1. To investigate
the effects of BSA and dipole–dipole interactions on relaxivity, sim-
ilar measurements were carried out on Gd-complex samples with
the addition of BSA (0.72 mM) and 10-fold of La3+ (0.1 M LaCl3 solu-
tion), respectively. The pH dependence of the relaxivity at 20 MHz
and 37 °C of (Gd-DO3A)3–TAB was measured at the following buffer
systems: chloroacetic acid/NaOH (pH 2 and 3), acetic acid/NaOH
(pH 4 and 5), Mes/NaOH (pH 6), HEPES/NaOH (pH 7 and 8), ammo-
nia/HCl (pH 9 and 10), 0.1 M NaOH (pH 11 and 12). The 100 mM
buffers were used to maintain the solution pH.
2.2.4. Synthesis of general procedure B for the tert-butyl ester
deprotection (4a = (DO3A)2–DAB, 4b = (DO3A)3–TAB)
The compound 3a or 3b was dissolved in trifluoroacetic acid
(TFA) at 0 °C and stirred for 1 h. Then the reaction mixture was al-
lowed to stir at room temperature for 24 h. After all volatiles were
removed under reduced pressure, the residue was repeatedly dis-
solved in methanol (30 mL ꢂ 2) and CH2Cl2 (30 mL ꢂ 2), which
were removed in vacuo. The remained residue was dissolved in
minimum methanol and then precipitated by adding diethyl ether
at 0 °C. The obtained solid product was filtered and washed with
diethyl ether to afford the target product as trifluoroacetate salt
adduct.
2.4. T1-weighted MRI imaging in vitro
The in vitro T1-weighted MR imaging of (Gd-DO3A)3–TAB and
(Gd-DO3A)2–DAB was obtained on a HT-MRSI60-25 MRI Imaging
system (Shanghai Shinning Globe Science and Education Equip-
ment Co., Ltd.) at 25 °C in 100 mM HEPES buffer solution (pH 7).
The instrumental parameters were set as follows: a 1.5T magnet,
2.2.4.1. (DO3A)2–DAB (4a). Prepared according to general proce-
dure B: The compound 3a (0.96 g, 0.783 mmol), TFA (20 mL,
@
270 mmol). Yield: 66% (0.7 g). dC (151 MHz, D2O): 198.81 (PhC O),
@
173.82, 169.37 (COO), 163.13, 162.90, 162.66, 162.43 (CF3COOH),
138.61, 128.42 (PhC), 119.24, 117.31, 115.37, 113.44 (CF3COOH),
58.86 (PhCOCH2), 55.29, 53.39 (CH2COO), 51.73, 48.26 (NCH2).
ESꢀ MS (CH3OH): m/z 849.8 [MꢀH]ꢀ, 871.8 [Mꢀ2H + Na]ꢀ. Anal.
Calc. for C38H58N8O14ꢁ4CF3COOHꢁ3H2O: N, 8.23; C, 40.59; H, 5.04.
Found: N, 8.23; C, 41.88; H, 5.03%. UV–Vis (H2O) kmax = 261 nm
matrix = 256 ꢂ 256,
slice
thickness = 1 mm,
TE = 13 ms,
TR = 100 ms, number of acquisitions = 4.
2.5. Luminescence measurements
Samples for luminescence measurements were 0.3 mM of the
Eu-chelates in 50 mM HEPES–NaOH or HEPES–NaOD buffer at pH
7.0. The steady state emission spectra of the (Eu-DO3A)2–DAB
and (Eu-DO3A)3–TAB were measured on a Perkin-Elmer LS 55
luminescence spectrometer. The luminescence delay curves were
obtained from a Lecroy Wave Runner 6100 digital oscilloscope
(1 GHz) using a tunable laser (pulse width = 4 ns, gate = 50 ns) as
the excitation source [Continuum Sunlite optical parametric oscil-
lator (OPO)]. The luminescence lifetimes were obtained by fitting
the signal decays with a single-exponential function. The hydration
number (q) of the (Eu-DO3A)2–DAB and (Eu-DO3A)3–TAB centers
within the complexes was calculated from the luminescence life-
times in H2O and D2O using Eq. (2) [38]:
(e m .
= 12457 Mꢀ1 cmꢀ1). IR (FT) = 3425, 1694, 1406, 1089 cmꢀ1
2.2.4.2. (DO3A)3–TAB (4b). Prepared according to general procedure
B: The compound 3a (1.24 g, 0.71 mmol), TFA (40 mL, 540 mmol).
@
Yield: 67% (0.96 g). dC (151 MHz, D2O): 198.21 (PhC O), 174.29,
@
169.71 (COO), 163.14, 162.90, 162.67, 162.43 (CF3COOH), 136.05,
131.83 (PhC), 119.21, 117.27, 115.34, 113.40 (CF3COOH), 58.34
(PhCOCH2), 55.57, 53.06 (CH2COO), 51.73, 48.23 (NCH2). ESꢀ MS
(CH3OH): m/z 1236.1 [MꢀH]ꢀ, 1258.2 [Mꢀ2H + Na]ꢀ. Anal. Calc.
for C54H84N12O21ꢁ6CF3COOHꢁ5H2O: C, 39.41; H, 5.01; N, 8.36.
Found: C, 39.86; H, 5.38; N, 8.31%. UV–Vis (H2O) kmax = 229 nm
(e m .
= 16421 Mꢀ1 cmꢀ1). IR (FT) = 3427, 1692, 1388, 1089 cmꢀ1