Inorganic Chemistry
Article
70.58, 71.63, 72.46, 100.54, 108.78, 111.13, 112.09, 112.13, 112.17, 112.20,
113.48, 118.07, 118.18, 142.84, 142.83, 166.75, 169.36, 170.02. ESI-MS:
calcd m/z 475.4, found 476.8 [M + H]+. Anal. Calcd for C20H23F2NO10: C
50.53, H 4.88, N 2.95. Found: C 50.59, H 5.16, N 2.84.
2.2. Methods. 2.2.1. Reversed Phase High-Performance Liquid
Chromatography (HPLC) Method. The HPLC experiments were per-
formed on an Amersham AKTAbasic 10 equipped with an Amersham
UV-900 detector and Amersham Frac-920 fraction collector. A Supelcosil
RP-C18 column (5 μm, 4.6 mm × 250 mm) was used.
̈
2.1.1.4. 1-(2-Difluoromethyl-4-(1-bromoacetamido)phenyl)-2,3,4-
triacetyl-β-D-glucopyronuronate (6). To a solution of 1-(2-difluor-
omethyl-4-(1-bromoacetamido) phenyl)-2,3,4-triacetyl-β-D-glucopyro-
nuronate (2.50 g, 5.26 mmol) in anhydrous dichloromethane (50 mL)
was added potassium carbonate (0.87 g, 6.31 mmol), and the solution
was cooled in ice bath with stirring. Bromoacetyl bromide (1.59 g,
7.89 mmol) was added dropwise into the stirred reaction mixture on ice
bath overnight. The crude reaction mixture was filtered and washed with
saturated sodium bicarbonate (3 × 200 mL), water, and brine and dried
over anhydrous magnesium sulfate. After filtration, the solvent was
removed under reduced pressure, and the residue thus obtained was
purified by silica gel column chromatography (17% ethyl acetate in
dichloromethane) to give the product as yellow powder (2.84 g, 90.5%).
1H NMR (400 MHz, CDCl3): δ 2.06 (s, 9H, OAc), 3.76 (s, 3H,
2.2.2. Relaxation Time Measurement. The longitudinal relaxation
times (T1) of Gd(III) complex were measured to determine relaxivity
(r1). The measurements were made using a relaxometer operating at
20 MHz and 37.0 0.1 °C (NMR-120 minispec, Bruker). Before each
measurement the relaxometer was tuned and calibrated. The values of r1
were determined from 5 data points generated by an inversion−recovery
pulse sequence. To study the effect of β-glucuronidase cleavage of β-D-
glucopyranuronic acid on the T1 value of [Gd(DOTA-FPβGu)] solu-
tions, β-glucuronidase isolated from Escherichia coli was used. The
Escherichia coli enzyme was reconstituted with 0.1 M sodium phosphate
buffer solution (PBS), pH = 7.4 at 25.0 0.1 °C. For the longitudinal
relaxation time (T1) measurements of [Gd(DOTA-FPβGu)] in the pre-
sence or absence of β-glucuronidase and HSA, the follow reactions were
set up: (a) 0.5 mM [Gd(DOTA-FPβGu))] in 100 mM PBS; (b) 0.5 mM
[Gd(DOTA-FPβGu)] and 0.1 mg/mL β-glucuronidase in 100 mM
PBS; (c) 0.5 mM [Gd(DOTA-FPβGu)] and 0.5 mM HSA in 100 mM
PBS; (d) 0.5 mM [Gd(DOTA-FPβGu)], 0.5 mM HSA, and 0.01 mg/mL
β-glucuronidase in 100 mM PBS; (e) 0.5 mM [Gd(DOTA-FPβGu)],
0.5 mM HSA, and 0.1 mg/mL β-glucuronidase in 100 mM PBS. The
percentage change of T1 value in these solutions was plotted against the
incubation time. These measurements were made in triplicate to reduce
systematic error in the relaxation time (T1) measurements.
2.2.3. Enzyme Kinetics. All reactions were performed in a quartz cell
at 37.0 0.1 °C. The final reaction mixture had a volume of 3.0 mL
containing [Eu(DOTA-FPβGu)] (0.25, 0.5, 1.0, 2.0, 4.0, and 6.0 mM)
in 100 mM PBS (pH 7.4 0.1). Luminescence intensity at 616 nm
(excitation λmax = 318 nm) was measured after addition of the β-glucuronidase
(0.1 mg/mL) to initiate the enzymatic reaction and was recorded every
30 s continuously by a Varian Cary Eclipse fluorescence spectropho-
tometer. The initial velocity for each enzymatic reaction in the presence
of a specific [Eu(DOTA-FPβGu)] concentration was obtained by
determining the slope of the change of luminescence within the first
3 min of the reaction. Acquired initial velocity for each concentration of
[Eu(DOTA-FPβGu)] was plotted to fit the data into the Michaelis−
Menten equation [v = (Vmax[Eu(DOTA-FPβGu)])/(KM + [Eu(DOTA-
FPβGu)])] and obtain KM, kcat, and kcat/KM.
COOCH3), 4.02 (s, 2H, CH2Br), 4.22 (dd, 1H, sugar, CH), 5.10 (d, 1H,
sugar, CH), 5.34 (m, 3H, sugar, CH), 6.81 (t, 1H, CHF2), 7.11 (d, 1H,
ArH), 7.58 (d, 1H, ArH), 7.77 (dd, 1H, ArH), 8.25 (s, 1H, NH). 13C
NMR (100 MHz, CDCl3): δ 20.41, 20.47, 20.54, 53.08, 68.96, 70.33,
71.35, 72.49, 99.51, 108.33, 110.68, 113.04, 116.52, 118.24, 118.28,
118.32, 118.36, 124.07, 132.79, 132.81, 163.63, 166.62, 169.35, 169.97.
ESI-MS: calcd m/z 596.3, found 597.1 [M + H]+. Anal. Calcd for
C22H24BrF2NO11: C 44.31, H 4.06, N 2.35. Found: C 44.42, H 4.14, N 2.49.
2.1.1.5. 1-(2-Difluoromethyl-4-(1-(4,7,10-triscarboxymethyl-
(1,4,7,10-tetraazacyclodecyl)) acetamido)phenyl)-β-D-glucopyro-
nuronate (DOTA-FPβGu, 8). To a solution of DO3A-tris-tbutyl ester
(1.87 g, 2.04 mmol) in acetonitrile (50 mL) was added triethylamine
(0.55 mL, 3.93 mmol) and 1-(2-difluoromethyl-4-(1-bromoacetamido)-
phenyl)-2,3,4-triacetyl-β-D-glucopyronuronate (1.18 g, 1.93 mmol).
The mixture was heated to 70 °C and stirred for 24 h. After filtration
solvent was evaporated under reduced pressure. The acetyl group was
removed by reaction with sodium methoxide and dry methanol (50 mL)
for 2 h. Deprotections of tert-butyl ester groups and methyl groups
were achieved by stirring at ice bath in 1 N sodium hydroxide solution
(20 mL) for 8 h. This compound was purified by AG 1 × 8 anion
−
exchange resin column (200−400 mesh, HCO2 form, eluted first
with H2O and then with a gradient of formic acid) and then collected
and concentrated by 0.12−0.15 N formic acid solution. The trace of
formic acid was removed by coevaporation with water (3 × 100 mL),
2.2.4. Butanol Buffer Partition Coefficient. [Gd(DOTA-FPβGu)]
(0.07 mM) in 10 mL of PBS was equilibrated at room temperature for
1−2 h with PBS-saturated butanol (10 mL). The vials were centrifuged
at 2000 g for 5 min to ensure that the layers were separated. Aliquots (5 mL)
from each phase were removed and analyzed via inductively coupled
plasma−atomic emission spectroscopy (ICP-AES, PerkinElmer OPTIMA
2000 PV). The partition coefficient (P) was calculated by eq 125−27
1
and freeze-dried to give a white powder (0.37 g, 13.0%). H NMR
(400 MHz, CDCl3): δ 3.06 (m, 8H, DOTA, N−CH2CH2−N), 3.30 (m,
8H, DOTA, N−CH2CH2−N), 3.55 (m, 6H, DOTA, N−CH2COOH),
3.75 (m, 5H, sugar and DOTA, CH and N−CH2-CO), 4.00 (d, 1H,
sugar, CH), 5.07 (d, 1H, sugar, −CH−), 7.00 (t, 1H, −CHF2), 7.15 (d,
1H, ArH), 7.43 (dd, 1H, ArH), 7.61 (s, 1H, ArH). 13C NMR (100 MHz,
CDCl3): δ 48.85, 51.06, 55.35, 55.74, 59.94, 71.42, 72.66, 75.19, 75.28,
100.78, 104.72, 114.45, 116.80, 119.11, 123.97, 125.37, 132.59, 162.61,
172.96. ESI-MS: calcd m/z 721.7, found 722.2 [M + H]+. Anal. Calcd for
C29H41F2N5O14: C 48.27, H 5.73, N 9.70. Found: C 48.35, H 5.84,
N 9.75. The purity of DOTA-FPβGu was determined by HPLC and
mass. The HPLC chromatogram has been deposited as Supporting
Information (Figure S1)
2.1.2. Preparation of Lanthanide Complexes. The Gd(III) and
Eu(III) complexes were prepared by dissolving the DOTA-FPβGu
(0.42 g, 0.58 mmol) in H2O (10 mL) and adjusting the pH of the solu-
tion to 6.5 with dilute sodium hydroxide. LnCl3 (0.57 mmol, dissolved in
5 mL H2O and brought to pH = 6.5 with sodium hydroxide) was added
dropwise, maintaining pH at 5.5−6.5 with dilute sodium hydroxide
periodically. The mixture solution was stirred over an ice bath for 1 day,
the pH was maintained, and the formation of Ln(III) chelate was con-
sidered complete. The pH was brought to 8.0, and the solution was
centrifuged to remove excess lanthanide ions as Ln(OH)3 and verified
by the xylenol orange test. The trace Ln(OH)3 was removed by filtrating
with 200 nm nylon filter, and the solution was freeze-dried under
reduced pressure. The purity of [Gd(DOTA-FPβGu)] was determined
by HPLC and mass. The HPLC chromatogram has been deposited as
Supporting Information (Figure S2)
avg conc of Gd(III) complex in butanol
avg conc of Gd(III) complex in PBS
P =
(1)
2.2.5. Cells and Animals. CT26 and CT26/mβG-eB7 (Supporting
Information S1)28 murine colon carcinoma cells were grown in Dulbecco’s
minimal essential medium (DMEM) supplemented with 5% bovine calf
serum and 100 U/mL penicillin, 100 μg/mL streptomycin (Sigma) in a
humidified 37.0 0.1 °C, 5% CO2 atmosphere.
Eight-week-old Balb/c mice were purchased from the National Labo-
ratory Animal Center of Taiwan (Taipei, Taiwan). All animal experi-
ments were performed in accordance with institute guidelines. 2 ×106
CT26/mβG-eB7 and 2 × 106 CT26 tumor cells were injected sub-
cutaneous to right and left hind limb, respectively. Whole body image
was performed a MR imaging scanner (Sigma; GE Medical Systems)
two to three weeks after the injection when tumors grew to a diameter of
5−10 mm.
2.2.6. MR Imaging Studies of Enzymatic Activation of [Gd(DOTA-
FPβGu)]. Four reactions were set up to study the MR images of
[Gd(DOTA-FPβGu)] and its β-glucuronidase-cleavaged product: (a)
0.5 mM [Gd(DOTA-FPβGu)] in 100 mM PBS (pH = 7.4), (b) 0.5 mM
[Gd(DOTA-FPβGu)] and 0.5 mM HSA in 100 mM PBS (pH = 7.4),
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dx.doi.org/10.1021/ic301827p | Inorg. Chem. 2012, 51, 12426−12435