Langmuir
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radical (168 mg, 0.6 mmol), and degassed 1,4-dioxane (4 mL) were
placed in a three-necked flask and bubbled carefully with N2 gas for 30
min. To this reaction mixture, a 10% Na2CO3(aq) solution (10 mL)
was added and stirred at 100 °C for 6 h. Brine added to the reaction
mixture was extracted with CHCl3 three times, and the combined
organic layer was dried over MgSO4 and then evaporated under
reduced pressure. The crude residue was chromatographed on silica
gel using CHCl3/MeOH (100:1−50:1) as the eluent to obtain an
orange oil (197 mg, 0.30 mmol) with a 59% yield. IR (NaCl, cm−1)
3516, 3339, 2929, 2862, 1696, 1668, 1602, 1558, 1453, 1360, 1249,
1201, 1114, 1033, 850. 1H NMR (DMSO-d6 + ascorbic acid, 500
MHz) δ 8.38 (s, 1H), 7.33 (d, J = 8.5 Hz, 2H), 7.26 (d, J = 8.2 Hz,
2H), 6.07 (t, J = 5.8 Hz, 1H), 5.84 (s, 1H), 3.50−3.45 (m, 26H), 3.23
(s, 3H), 3.06 (q, J = 6.5 Hz, 2H), 2.37 (s, 2H), 1.49 (quin, J = 7.0 Hz,
4H), 1.42 (quin, J = 7.0 Hz, 4H), 1.34−1.26 (m, 4H), 1.21 (s, 6H),
1.13 (s, 6H). 13C NMR (DMSO-d6 + ascorbic acid, 126 MHz) δ
155.13, 132.64, 128.88, 128.66, 126.88, 126.66, 126.02, 125.07, 117.34,
71.32, 70.31, 69.83, 69.62, 69.53, 58.09, 29.78, 29.24, 26.28, 25.47. ESI-
MS m/z 689.42 [M + Na]+. HRMS (ESI) calculated for
C35H60N3O9Na [M + Na]+, 689.4222; found, 689.4245.
log P Calculation. log P values were obtained from the calculation
methods. The modeling studies were performed using MacroModel
ver. 11.2 as implemented in Maestro ver. 10.5. The structures of 1′ and
2′ (with the nitroxyl radical in TEMDO replaced by a carbonyl group)
were optimized in molecular mechanics with the OPLS3 force field
with a dielectric constant of 1.0. The convergence criterion was set to
an energy gradient of 0.05 kJ/mol. Conformational searches were then
performed using the Monte Carlo molecular modeling (MCMM)
method to generate 1000 structures, which were individually
minimized into local minima. The most stable conformations of 1′
and 2′ were subsequently optimized at the DFT (B97-D/3-21G*)
level using the D.01 revision of the Gaussian 09 program package.21,22
Their corresponding nitroxyl radicals 1 and 2 were also optimized at
the same level of theory (Figure S6). Frequencies were analytically
computed at the B97-D/6-31G* level of theory to give gas-phase
Gibbs free energies (298 K, 1 atm) and to confirm whether the
structures are minima (no imaginary frequencies) or transition states
(only one imaginary frequency).
log P was estimated from the computed free transfer energy
according to equation23
1,1′-(5-(1-Oxyl-2,2,6,6-tetramethyl-3,6-dihydro(2H)pyridin-4-yl)-
phenyl-1,3-diyl)bis(3-(2,5,8,11,14,17,20-heptaoxahexacosan-26-yl)-
urea) (TEMPO-UBD-DA (2)). Iodo-UBD-DA (539 mg, 0.5 mmol),
Pd(PPh3)4 (28.9 mg, 0.025 mmol), 2,2,6,6-tetramethyl-4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridin-1(2H)-yloxyl
radical (168 mg, 0.6 mmol), and degassed 1,4-dioxane (4 mL) were
placed in a three-necked flask and carefully bubbled with N2 gas for 30
min. To this reaction mixture, a 10% Na2CO3(aq) solution (10 mL)
was added and stirred at 100 °C for 6 h. Brine added to the reaction
mixture was extracted with CHCl3 three times, and the combined
organic layer was dried over MgSO4 and then evaporated under
reduced pressure. The crude residue was chromatographed on silica
gel using CHCl3/MeOH (100:1−50:1) as the eluent to obtain an
orange oil (211 mg, 0.20 mmol) with a 40% yield. IR (NaCl, cm−1)
3516, 3339, 2929, 2862, 1696, 1668, 1602, 1558, 1453, 1360, 1249,
1201, 1114, 1033, 850. 1H NMR (DMSO-d6 + ascorbic acid, 500
MHz) δ 8.35 (s, 2H), 7.28 (t, J = 1.7 Hz, 1H), 7.05 (d, J = 1.7 Hz, 2H),
5.99 (t, J = 5.5 Hz, 2H), 5.79 (s, 1H), 3.52−3.46 (m, 36H), 3.40−3.37
(m, 16H), 3.23 (s, 6H), 3.05 (q, J = 6.3 Hz, 4H), 2.33 (s, 2H), 1.49
(quin, J = 6.5 Hz, 4H), 1.41 (quin, J = 6.5 Hz, 4H), 1.33−1.28 (m, 8H),
1.22 (s, 6H), 1.14 (s, 6H). 13C NMR (DMSO-d6 + ascorbic acid, 126
MHz) δ 172.80, 116.02, 108.32, 105.43, 91.32, 87.91, 84.49, 83.48,
79.24, 78.98, 78.71, 74.81, 73.28, 71.28, 70.28, 69.79, 69.58, 69.49,
58.05, 29.77, 29.21, 26.23, 25.44. ESI-MS m/z 574.34 [M + 2Na]2+.
HRMS (ESI) calculated for C55H100N5O17Na2 [M + 2Na]2+, 574.3449;
found, 574.3459.
Standard Preparation Method of Radical Nanoparticles
(RNP). Radicals 1 and 2 were dissolved in saline solutions with
sonication in an ice bath for approximately 120 s. The dissolved
transparent solutions without further filtration were used as samples
for various measurements.
Electron Magnetic Resonance (ESR). ESR spectra were recorded
on a Bruker Biospin ESR300 EPR X-band (9.4 GHz) spectrometer
equipped with a microwave frequency counter. Sample solutions in
saline were placed in capillary tubes and were analyzed at 20−22 °C.
Dynamic Light Scattering (DLS) and Zeta Potential. DLS
measurements were performed on a Zetasizer Nano ZS (Malvern
Instruments Ltd.). Sample solutions in saline were placed in
polystyrol/polystyrene tubes. The zeta potential of the samples was
analyzed using the above-described apparatus at 20 °C for 1 and 25 °C
for 2, above and below a critical transition concentration.
(ΔGsol(n‐octanol) − ΔGsol(water)
)
log P =
2.30RT
where R is the gas constant and T is the temperature.
To estimate log P (n-octanol/water) values, gas-phase DFT-
optimized conformers (1′, 2′, 1, and 2) were reoptimized in n-
octanol and water, respectively ((U)B97-D/6-31G*//(U)B97-D/3-
21G*:SMD = n-octanol or water) . The results of each optimization
were used to evaluate the free-energy difference for the two solvents.
Calculated log P values were 5.03 for 1′, 2.85 for 2′, 5.05 for 1, and
T1- and T2-Weighted MRI and Relaxivity of the Samples. MRI
acquisitions of CAs were performed on a 1.0 T-MRI scanner
(BrukerBiospin, Ettlingen, Germany) with a solenoid coil (35 mm
inner diameter, transmission, and reception, Aspect Imaging, Shoham,
Israel). An aqueous solution of the contrast agents was initially put
into a polymerization chain reaction (PCR) tube cluster plate, and the
PCR tube cluster plate was set in the center of the volume coil. The
sample temperature was maintained at 23.0 0.5 °C throughout all of
the experiments by using an air condition. Using the MRI scanner,
horizontal single-slice T1-weighted MR images were acquired with the
following parameters: spin echo, TR/TE = 400/10 ms, slice thickness
= 2.0 mm, matrix = 256 × 256, field of view (FOV) = 38.4 × 38.4
mm2, number of averages (NA) = 1, and number of slices = 1. For
longitudinal relaxation time (T1) and longitudinal relaxivity (r1)
calculations, horizontal single-slice inversion−recovery MRI was
performed using RARE (rapid acquisition with relaxation enhance-
ment) acquisition with the following parameters: TR = 10 000 ms, TE
= 20 ms, inversion time = 52, 100, 200, 400, 800, 1600, 3200, 6400 ms,
matrix size = 128 × 128, FOV = 38.4 × 38.4 mm2, slice thickness = 2.0
mm, RARE factor = 4, and NA = 1. For transverse relaxation time (T2)
and longitudinal relaxivity (r2) calculations, spin-echo mulch slice
mulch echo sequence was used with the following parameters: TR =
20000 ms, TE = 20 ms (256 echoes, for contrast agent measurement)
or 40 ms (256 echoes, for saline measurement), matrix size = 64 × 64,
FOV = 38.4 × 38.4 mm2, slice thickness = 2.0 mm, and NA = 1.
In Vivo Examination Using Mice. BALB/c nude mice bearing
colon-26 tumors on the lower back were used to test the compatibility
of the MRI CAs. After intravenous administration of 2 (100 mM, 100
μL) and 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL)
(300 mM, 200 μL) saline solutions, the intensities of the T1-weighted
images in the tumor and the muscle tissue (regions of interest, ROIs)
were noted every 20 s during a period of 30 min using a 7.0 T-MRI
scanner (BrukerBiospin, Ettlingen, Germany) equipped with a volume
coil (35 mm inner diameter, transmission and reception, Rapid
Biomedical, Rimpar, Germany).
Transmission Electron Microscopy (TEM). TEM images were
taken on a FEI Tecnai20 machine. The samples (0.5, 1.0, and 5 mM)
for TEM measurement were prepared as follows: The corresponding
solutions (50 μL) were dropped onto a carbon grid. After
approximately 30 s, the residual solution was blotted using KimWipes.
To prepare negative stained images, 50 μL of a solution containing 5%
uranyl acetate solution was again dropped on the grid, and then the
residual solution was blotted using KimWipes. The resulting grids
were air dried for 15 min and used as samples.
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Langmuir 2017, 33, 7810−7817