Liu et al.
JOCArticle
corresponding biradicals show high oxygen sensitivity, the
N-labeled TNN15-H with its doublet EPR signal exhibits
DIPEA (2.49 mL, 14.3 mmol), and N-hydroxysuccinimide
(0.66 g, 5.73 mmol) in DMF (20 mL) was added a solution of
EDCI (1.10 g, 5.73 mmol) in DMF (5 mL) at 0 °C. After
addition, the reaction mixture was stirred for 18 h at room
temperature. Solvents were removed under vacuum, and the
residue was purified by flash column chromatography using the
solvents ethyl acetate/petroleum ether (1:3, 1:2, and then 1:1 v/v) as
eluents. A light yellowish solid (0.71 g) was obtained. Yield: 88%.
15
improved EPR signal resolution compared with that of
TNN14-H with its EPR triplet. Therefore the new probes
also exhibit enhanced sensitivity for simultaneous measure-
ment of redox status and oxygenation compared to either
nitroxide or trityl radicals alone.
-1
IR (cm , neat): 2978, 2937, 1810, 1783, 1735, 1464, 1427, 1366,
1304, 1245, 1200, 1092, 1065, 1047, 994, 958, 920, 899, 813, 735,
Experimental Section
þ
EPR Measurements and Simulations. EPR measurements
were carried out on a Bruker EMX-X band with an HS
resonator at room temperature. General instrument settings
were as follows: modulation frequency, 100 kHz; microwave
frequency, 9.87 GHz; microwave power, 10 mW for biradicals
and nitroxide radicals and 0.5-2 mW for the trityl-hydroxyla-
mines; microwave frequency, 9.87 GHz; modulation amplitude,
647. GC-MS: retention time, 5.90 min; [M - C
4
H
4
NO
3
þ H] ,
þ
186.11(calculated), 186.00 (measured); [M - C
9
H15NO
3
þ H] ,
115.03 (calculated), 114.94 (measured).
1
5
3-Carbamoyl ( N)-2,2,5,5-Tetramethylpyrrolidin-1-yloxy (3-
CaP). To a solution of 3-CP-OSu (0.5 g, 1.77 mmol) and K CO
(0.3 g) in dioxane (5 mL) and water (1 mL) was added solid
2
3
1
5
NH4Cl (99% isotopic purity, 0.106 g, 1.94 mmol). The reac-
1
.0 G for biradicals and nitroxide radicals and 0.03-0.08 G for
tion mixture was stirred for 22 h at room temperature, filtered,
and evaporated to dryness under vacuum. The residue was
redissolved in 10 mL of ethyl acetate. After filtration, the filtrate
was concentrated and separated by flash column chromatogra-
phy using 2% methanol in dichlomethane as an eluent. The
yellow solid was obtained and recrystallized from hexane and
4
the trityl-hydroxylamines; receiver gain, (1-10.00) ꢀ 10 ; time
constant, 10.24-40.96 ms; sweep time, 10.49-41.94 s. Measure-
ments were performed using 50 μL capillary tubes.
Simulations were carried out using a well-developed EPR
simulation program (ROKI\EPR). The fitting routine to deter-
mine the J values of the trityl-nitroxide biradicals was similar to the
method described in our previous study. Since we could not find
any improvement about the quadratic error between experimental
and calculated spectra when J was larger than 400 G, the J values
for TNN14 and TNN15 were suggested to be >400 G.
Oxygen Sensitivity. Oxygen sensitivities of the trityl-hydro-
xylamine TNN14-H and TNN15-H were evaluated according to
our previous method. In brief, ascorbic acid (4 mM) was added
to a solution of the biradicals (50 μM) in PBS buffer. After 60
min, the solution was transferred into a gas-permeable Teflon
tube (i.d. = 0.8 mm) and was sealed at both ends. The sealed
sample was placed inside a quartz EPR tube with open ends.
Nitrogen or N /O gas mixture with varying concentrations of
28
-
1
ethyl acetate to give the pure 3-Cap (0.31 g, 94%). IR (cm ,
neat): 3346, 3195, 2977, 2934, 1668, 1462, 1426, 1365, 1321,
26
1283, 1244, 1171, 1154, 1102, 830, 739. GC-MS: retention time,
7.22 min; [M] , 186.13 (calculated), 185.99 (measured).
þ
15
15
3-Amino ( N)-2,2,5,5-Tetramethyl-1-pyrrolidin-1-yloxy ( NN).
To a solution of sodium hypobromite prepared from 0.36 g of
NaOH (9 mmol), 4 mL of water, and 0.34 g of bromide (2.13
mmol) was added 0.28 g of 3-CaP (1.50 mmol) at 0 °C. The
reaction mixture was stirred for 2 h at 0 °C, heated to 70 °C, and
kept this temperature for 1 h. The reaction mixture was cooled with
the ice bath, treated with 6 g KOH, and extracted with ether. The
extract was dried on anhydrous Na
rated by flash column chromatography using CH
NH OH = 10:1:0.05 as an eluent. A yellow solid (0.15 g) was
2 4
SO , concentrated, and sepa-
Cl /CH OH/
2
2
2
2
3
O
2
was allowed to bleed into the EPR tube and after about 4 min
4
-
1
was changed into another gas mixture. EPR spectra were
recorded using a model of incremental sweep. According to
the resulting spectra, the spectral ratio (I /Iout) was calculated.
Cyclic Voltammetry. Cyclic voltammetry was performed on a
potentiostat and computer-controlled electroanalytical system.
Electrochemical measurements were carried out in a 10 mL cell
obtained. Yield: 63%. IR (cm , neat): 3483, 3363, 3291, 3198,
2972, 2933, 1610, 1463, 1363, 1315, 1254, 1228, 1195, 1164, 1102,
1059, 1024, 895, 834, 763, 673. GC-MS: retention time, 5.13 min;
in
þ
[M] , 158.13 (calculated), 157.99 (measured).
TNN14. To a solution of CT-03 (100 mg, 0.1 mmol), HOBt
(40.5 mg, 0.3 mmol), and BOP (46.4 mg, 0.105 mmol) in dry
2
equipped with a glassy carbon working electrode (7.07 mm ), a
2
DMF (10 mL) was added DIPEA (90 μL) under N . The reaction
platinum-wire auxiliary electrode, and a Ag/AgCl reference elec-
trode. Solutions of biradicals (1 mM) were degassed by bubbling
with the nitrogen gas before the detection. The redox potentials
mixture was stirred at room temperature for 20 min, and then
NN (16.5 mg, 0.105 mmol) in 5 mL of DMF was added dropwise.
14
The resulting mixture was continuously stirred for 18 h at room
temperature. Solvent was removed under vacuum, and the residue
was dissolved in phosphate buffer (0.1 M, pH 7.4) and purified by
column chromatography on reverse phase C-18 using water
followed by 0-15% acetonitrile in water as eluants to give the
biradical TNN14 as a green solid (75 mg, 66%). Purity: >98% by
HPLC (see Supporting Information) and 97 ( 1% versus TEM-
a
c
were calculated according to the relation E = (E
p
þ E
p
)/2.
Reaction Kinetics of TNN14 with Ascorbate. Various concen-
trations of ascorbic acid (0.5, 1, 2, and 4 mM) were added to the
solution of TNN14 (50 μM) in PBS (50 mM, pH 7.4). Incre-
mental EPR spectra were recorded 45 s after mixing. The
concentration of the trityl-hydroxylamine TNN14-H at each
time point was obtained by comparing their double integrated
signal intensities relative to CT03 as standard. Since the ascorbic
acid concentration (0.5, 1, 2, and 4 mM) used was in greater
excess than the biradical concentration (50 μM), the reaction
kinetics of the biradical with ascorbic acid is a pseudo-first-order
reaction. The resulting curves in Figure 2B were fitted with the
17
-1
POL determined as previously reported. IR (cm , neat): 3425.1,
2971, 2933, 1646, 1580, 1454, 1367, 1312, 1237, 1168, 1150, 1113,
þ
þ
886, 820, 725, 697. MS ([MþH] , m/z): 1139.038 (measured),
1139.071 (calculated); ([M þ Na] , m/z): 1161.000 (measured),
1161.052 (calculated).
TNN15. To the solution of CT-03 (63.3 mg, 63.3 μmol), HOBt
(25.6 mg, 189.9 μmol) and BOP (29.4 mg, 66.5 μmol) in dry
equation ln[(C
concentration of TNN14, C
hydroxylamine TNN14-H at each time point, and kobs the
observed pseudo-first-order rate constant. Considering kobs
k [Asc], the approximated second-order rate constant k was
0
- C
t
)/C
0
] = -kobst, where C
0
is the initial
the concentration of the trityl-
t
DMF (8 mL) was added DIPEA (60 μL) under N . The reaction
2
mixture was stirred at room temperature for 20 min, and then
NN (10 mg, 63.3 μmol) in 5 mL of DMF was added dropwise.
1
5
=
The resulting mixture was continuously stirred for 18 h at room
temperature. Solvent was removed under vacuum, and the
residue was dissolved in phosphate buffer (0.1 M, pH 7.4) and
purified by column chromatography on reverse phase C-18
using water followed by 0-15% acetonitrile in water as eluants
2
2
finally calculated from the slope of the plot of kobs versus [Asc].
Synthesis. 1-Oxyl-2,2,5,5-tetramethylpyrrolidinyl-3-carboxy-
lic Acid N-Hydroxysuccinimide Ester (3-CP-OSu). To a solution
of 3-carboxy-proxyl (0.53 g, 2.86 mmol), HOBT (1.61 g, 8.60 mmol),
J. Org. Chem. Vol. 75, No. 22, 2010 7801