Analytical Chemistry
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(A) Design of HA-specific site based on aza-Michael addition
= 7.2 Hz), 7.99 (s, 1H), 7.86 (t, 1H), 7.78 (m, 4H), 7.58 (m, 2H),
1
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3
4
5
6
7
8
7.36 (s, 1H), 7.25 (m, 1H), 7.16 (m, 7H), 6.95 (s, 2H), 3.64 (m,
20 H), 1.24 (t, 6H), 1.19 (t, 12H). 13C NMR (100 MHz, DMSO-
d6) δ 167.17, 166.98, 158.51, 158.20, 157.54, 156.16, 155.57, 154.67,
142.49, 139.51, 135.61, 134.79, 133.03, 132.72, 132.17, 131.36, 131.26,
131.03, 130.96, 130.85, 130.75, 130.37, 130.28, 130.21, 129.56,
128.04, 125.65, 124.49, 121.96, 118.92, 118.05, 117.72, 117.58, 117.02,
115.99, 115.96, 114.79, 114.27, 113.53, 107.03, 96.74, 96.39, 46.57,
NH2OH
C
C
H
EWG
EWG = electron withdrawing group
C
C
EWG
NH
HO
EWG =
C
N
COOH
Potential HA-specific site
C
C C
N
O
N
N
Michael accepor
HN
Chr
2+
45.84, 45.58, 12.89. HRMS m/z calcd. for C58H61N5O5 [M]+
(B) Highly specific ratiometric fluorescent probe for imaging cellular HA
COOH
HO
453.7331, found 453.7366.
O
H
9
N
OH
N
10
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Imaging of HA in living cells. (A) For the imaging of
exogenous HA, HepG2 cells were first treated with 5 μM
RhChr for 30 min at 37 °C, and washed with PBS (pH 7.4) to
remove excess RhChr. Then cells were incubated with 100
μM HA for 30 min. (B) For the imaging of endogenous HA in
living cells: RAW 264.7 macrophages were incubated with 20
μg/mL LPS, 50 μg/mL L-Arg, 0.01 μg/mL IFN-γ and 5 μM
RhChr for 12 h at 37 °C, and washed three times with PBS
(pH 7.4). Images were acquired with Nikon A1R confocal
microscope with a 100× objective lens.
O
O
N
O
O
NH2OH
N
N
N
N
T
T
E
E
R
R
F
F
N
O
N
N
O
N
RhChr-HA
RhChr
Scheme 1. (A) Design of a novel HA-specific site based on aza-
Michael addition of HA to unsaturated system. (B) Design of HA-
specific ratiometric fluorescent probe RhChr.
EXPERIMENTAL SECTION
Imaging of HA in tissues. Tissues were obtained from the
mice liver, and treated with 5 μM RhChr in an incubator for
30 min and then washed with PBS (pH 7.4) three times.
Subsequently, the tissues were treated with 100 μM HA for 30
min. Images were acquired with Nikon A1R confocal
microscope at Z-scan mode with a 10× objective lens.
Synthesis of compound Chr. Piperazine (4.30 g, 50 mmol)
in 20 mL mixed solvent (H2O: 2-methoxyethanol, v:v= 1:1)
was heated at 120 °C for 0.5 h. Then, 1-(4-
fluorophenyl)ethanone (1.38 g, 10 mmol) in 5 mL 2-
methoxyethanol was added to the above solution within 1 h
at 120 °C. After 4 h, the mixture was poured into 100 mL ice
water slowly, and the white solid was precipitated and
purified by column chromatography to afford 1-(4-
(piperazin-1-yl)phenyl)ethanone (1.53 g, 75%). Subequently,
1-(4-(piperazin-1-yl)phenyl)ethanone (612 mg, 3 mmol) and
2-(4-(diethylamino)benzoyl)benzoic acid (891 mg, 3 mmol)
were dissolved in 5 mL concentrated sulfuric acid, and this
mixture was heated at 90 °C for 4 h. After cooled to room
temperature, the mixture was poured into 20 mL ice water
slowly, and 5 mL perchloric acid was added dropwise. The
mixture was filtrated to afford red solid, which was further
purified by column chromatography using DCM and MeOH
(V/V, 3:1) as eluent to afford compound Chr perchlorate (1.20
Detection of xanthine oxidase in living organs. After the
euthanasia of mice by cervical dislocation, we got different
organs (liver, heart, lung, etc) from mice carefully. The organ
tissues were dried out by filter papers and weighted. Then,
saline solution (0.9% NaCl) was added in accordance with
the proportion of 10% (weight/volume). The mixture was
ground for 20 min below 0 °C and centrifuged at for 10 min at
4 °C to obtain the supernatants. Subsequently, the saline
solution was added into the super natants to prepare 10%
organ homogenates for the measurement.
For the detection of XOD relative level in different living
organs. (1) Control group: A mixture of 0.15 mL HA (1 mM),
1
0.50 mL RhChr (10 μM in MeOH) and 1.25 mL PBS (pH = 7.4)
were mixed, and then its fluorescence was determined at
different times. (2) XOD group: 0.50 mL HX (1 mM), 0.50 mL
XOD (0.04 U/mL) and 0.25 mL NaCl (0.9%) were mixed and
kept at 37 °C for 30 min. Subsequently, 0.15 mL HA (1 mM)
was added and kept at room temperature for 10 min. After
that, 0.50 mL RhChr (10 μM in MeOH) was added, and the
fluorescence of the mixture was determined at different
times. (3) Heart, liver, lung, kidney and spleen groups: 0.50
mL HX (1 mM), 0.50 mL PBS (pH = 7.4), 0.10 mL NaCl (0.9%)
and 0.15 mL 10% tissue sample from different living organs
were mixed and kept at 37 °C for 30 min. Subsequently, 0.15
mL HA (1 mM) was added. After 10 min, 0.50 mL RhChr (10
μM in MeOH) was added, and the fluorescence spectra of the
mixture was determined at different times.
For the detection of XOD activity in living liver organs.
Allopurinol (I) group: 0.50 mL HX (1 mM), 0.40 mL PBS (pH
= 7.4), 0.10 mL allopurinol (0.1 mM), 0.10 mL NaCl (0.9%)
and 0.15 mL 10% liver tissue sample were mixed and kept at
37 °C for 30 min. Subsequently, 0.15 mL HA (1 mM) was
added and kept at room temperature for 10 min. After that,
0.50 mL RhChr (10 μM in MeOH) was added, and the
g, 69%). H NMR (400 MHz, DMSO-d6) δ 12.23 (s, 1H), 8.82
(s, 1H), 8.34 (d, 2H, J = 8.4 Hz), 8.17 (d, 1H, J = 7.6 Hz), 7.87 (t,
1H), 7.79 (t, 1H), 7.53 (d, 1H, J = 7.2 Hz), 7.34 (s, 1H), 7.21 (d,
3H, J = 9.2 Hz), 7.13 7.21 (d, 1H, J = 8.8 Hz), 3.74 (t, 4H), 3.64
(q, 4H), 3.27 (t, 4H), 3.17 (s, 1H), 1.24 (t, 6H). 13C NMR (100
MHz, DMSO-d6) δ 167.34, 165.93, 164.84, 158.15, 157.98, 154.71,
154.10, 133.25, 130.75, 130.64, 130.46, 129.83, 129.56, 118.78,
114.79, 96.84, 49.07, 45.53, 43.98, 42.98, 12.90. HRMS m/z
calcd. for C30H32N3O3+ [M]+ 482.2438, found 482.2450.
Synthesis of the probe RhChr. A mixture of rhodamine B
(479 mg, 1 mmol) and 1 mL POCl3 in 15 mL C2H4Cl2 was
stirred at 60 °C for 4 h. Then, the mixture was evaporated
and dissolved in 5 mL CH2Cl2 to form red solution.
Subsequently, the red solution was added dropwise to the
solution of compound Chr (581 mg, 1 mmol) and 0.2 mL EtN3
in 5 mL CH2Cl2 at 0 °C within 0.5 h, and the mixture was
then stirred for 3 h. 0.5 mL perchloric acid was added
dropwise to the mixture, and then stirred for 0.5 h. The
resulting mixture was evaporated and purified by pre-HPLC
1
to afford product RhChr perchlorate (55.2 mg, 5%). H NMR
(400 MHz, DMSO-d6) δ 8.34 (d, 2H, J = 10.4 Hz), 8.18 (d, 1H, J
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