70
S. Sato et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 169 (2016) 66–71
H, 5.71; N, 5.12. Found: C, 65.74; H, 5.68; N, 5.08. FABMS 530 (M + H)+.
g = 2.0059. aN = 1.70 mT.
residue was purified by silica-gel column chromatography (CHCl
afford 4 (100 mg, Y: 39.0%).
3
) to
Data for 2b: yellow powder. Mp = 274–277 °C (dec.). IRν (KBr)
Pale-yellow powder. Mp = 174–176 °C. IRν (KBr) 3527, 3176, 2975,
3
1
264, 2977, 2938, 1747, 1637, 1610, 1542, 1508, 1457, 1380, 1367,
2937, 1766, 1731, 1612, 1498, 1465, 1427, 1315, 1288, 1245,
322, 1268, 1241, 1214, 1180, 1112 cm−1
+ hydrazobenzene, 500 MHz) δ 1.01 (s, 6H, CH
.40 (t, J = 12.2, 2H, CH ), 1.65 (m, 2H, CH ), 4.13 (m, 1H, NCH), 6.55
.
1
H NMR (DMSO-
), 1.04 (s, 6H, CH ),
1166 cm . H NMR (DMSO-d
(s, 6H, CH × 2), 1.08 (s, 6H, CH
(m, 2H, CH ), 2.92 (m, 1H, NCH), 6.58 (dd, J = 1.9, 8.7, 1H, ArH), 6.60
−1
1
+ hydrazobenzene, 500 MHz) δ 1.10
6
d
6
3
3
3
3
× 2), 1.56 (t, J = 12.8, 2H, CH ), 1.89
2
1
2
2
2
(
2
1
dd, J = 2.1, 8.8, 2H, ArH), 6.58 (d, J = 8.8, 2H, ArH), 6.69 (d, J = 2.1,
H, ArH), 7.96 (s, 1H, ArH), 8.06 (d, J = 8.1, 1H, ArH), 8.16 (dd, J =
(dd, J = 0.9, 8.7, 1H, ArH), 6.71 (dd, J = 0.9, 1.9, 1H, ArH), 6.81 (d,
J = 8.7, 1H, ArH), 6.89 (dd, J = 2.3, 8.7, 1H, ArH), 7.23 (d, J = 2.3, 1H,
ArH), 7.35 (dt, J = 1.0, 7.6, 1H, ArH), 7.73 (dt, J = 7.6, 7.5, 1H, ArH),
7.80 (dt, J = 7.6, 7.5, 1H, ArH), 8.02 (dt, J = 1.0, 7.5, 1H, ArH), 10.24
.5, 8.1, 1H, ArH), 8.36 (brd, J = 7.9, 1H, NHCO), 10.20 (brs, 2H,
1
3
OH × 2). C NMR (DMSO-d
6
+ hydrazobenzene, 125 MHz) δ 19.8,
2.7, 41.4, 44.6, 58.1, 83.5, 102.4, 109.4, 112.9, 122.3, 124.9, 128.4,
29.4, 129.8, 140.9, 152.0, 152.8, 159.8, 163.9, 168.2. Anal. Calcd for
·H O: C, 65.80; H, 5.71; N, 5.12. Found: C, 65.66; H, 5.79;
3
1
C
(brs, 1H, OH). 13C NMR (DMSO-d
6
+ hydrazobenzene, 500 MHz) δ
19.5, 32.7, 34.6, 41.3, 57.6, 82.3, 102.5, 109.3, 110.6, 113.3, 116.8,
118.3, 124.3, 125.1, 126.1, 129.3, 129.4, 130.6, 136.0, 151.4, 151.8,
30
H
29
N
2
O
7
2
+
N, 5.09. FABMS 530 (M + H) . g = 2.0058. aN = 1.70 mT.
7 2
152.1, 152.5, 160.0, 168.8, 173.6. Anal. Calcd for C30H28NO ·1.5H O: C,
6
6.53; H, 5.77; N, 2.57. Found: C, 66.20; H, 5.87; N, 2.57. FABMS (m/z)
+
4
.1.2. 5- and 6-(TEMPO-4-carbonylamino)fluorescein (3a and 3b)
To a stirred solution of 4-carboxy-TEMPO (139 mg) and 13a
515 (M + H) .
(
2 2
300 mg) in dry CH Cl (5 mL), DCC (151 mg) and DMAP (9 mg)
4
.1.4. 3′-O-(3.4-dehydroPROXYL-3-methyl)fluorescein (5)
To a suspension of fluorescein (332 mg) in dry EtOH (5 mL), NaOEt
were added in an ice bath under argon atmosphere. After stirring at
room temperature for 14 h, the reaction mixture was filtered and
(
136 mg) was added under argon atmosphere and the suspension solu-
washed with CHCl
was purified by silica-gel column chromatography (CHCl
.10-di-O-acetyl 3a (329 mg, 77.0%). To a stirred solution of acetate in
3
. After the removal of organic solvents, the residue
tion was cleared. To a stirred solution, methanesulfonate (496 mg) was
added by portions at room temperature. After stirring at room temper-
ature for 1 h, the reaction mixture was stirred at 42 °C for 1.7 h. After
standing at room temperature, the reaction mixture was poured into
an ice-1 M HCl solution and extracted with AcOEt for three times. The
extract was separated and purified by silica-gel column chromatogra-
phy (toluene-AcOEt-AcOH = 6:1:0.2) to afford 5 (300 mg, Y: 62%).
Yellow powder. Mp = 130–132 °C (dec.). IRν (KBr) 3448, 2975,
3
) to afford
3
MeOH (3 mL), 1 mL of 2 N NaOH aqueous solution was added and the
mixture was stirred at room temperature for 5 h. After being acidified
by the addition of Dowex® 50Wx8 (H ) resin, the reaction mixture
was filtered and washed with MeOH. After the removal of MeOH, the
+
3
residue was purified by silica-gel column chromatography (CHCl -
MeOH = 10:1) to afford 3a (237 mg, 83.6%, overall yield: 64.4%). By
the similar way as 3a, 3b (208 mg, overall yield: 56.6%) was afforded
from 13b (300 mg).
−1 1
2
929, 1734, 1541, 1508, 1458, 1249, 1184, 1111 cm
DMSO-d + hydrazobenzene, 500 MHz) δ (PROXYL moiety): 1.09
and 1.15 (each s, 6H, CH × 2), 4.61 (s, 2H, CH ), 5.66 (s, 1H, olefinic
. H NMR
(
6
3
2
Data for 3a: yellow prism (from EtOH). Mp = 262–264 °C (dec.). IRν
H), (fluorescein moiety): 6.63 (d, J = 9.1, 1H, H8′), 6.69 (s, 1H, H4′ or
H5′), 6.81 (d, J = 7.5, 1H, H1′), 6.71 (d, J = 9 0.1, 1H, H7′), 7.26 (s, 1H,
H4′ or H5′), 7.26 (d, J = 7.4, 1H, H7), 7.28 (d, J = 7.5, 1H, H2′), 7.78
(
KBr) 3448, 2929, 1734, 1541, 1508, 1458, 1338, 1240, 1176,
−
1 1
1
(
CH
111 cm . H NMR (DMSO-d
× 4), 1.59 (t, 2H, J = 12.4, CH
), 2.77 (m, 1H, NCH), 6.54 (dd, 2H, J = 2.3, 8.3, H2′,7′), 6.58 (d, 2H,
J = 8.3, H1′,8′), 6.67 (d, 2H, J = 2.3, H4′,5′), 7.18 (d, 1H, J = 8.3, H7),
6
+ hydrazobenzene, 500 MHz) δ 1.08
s, 12H, CH
3
2
), 1.66 (d, 2H, J = 12.4,
13
(
t, J = 7.4, 1H, H6), 8.00 (d, J = 7.4, 1H, H4′), 10.19 (s, 1H, OH).
C
2
NMR (DMSO-d
2
1
6
+ hydrazobenzene, 125 MHz) δ (PROXYL moiety):
4.493, 24.512, 25.546, 25.575 (CH × 4), 66.44 and 68.50 (C1″, 4″),
39.79 (C3″), 149.86 (C2″), 64.76 (C5″), (fluorescein moiety): 82.72
3
7
2
(
1
1
.82 (dd, 1H, J = 8.3, 2.5, H6), 8.33 (d, 1H, J = 2.3, H4), 10.70 (br. s,
1
3
6
H, OH × 2). C NMR (DMSO-d + hydrazobenzene, 125 MHz) δ 19.9
(C1), 101.36 (C5′), 102.16 (C4′), 111.04 (C9′), 111.60 (C12′), 112.36
(C7′), 112.74 (C2′), 123.92 (C7), 124.12 (C4), 126.03 (C3), 128.75
(C8′), 128.83 (C1′), 130.08 (C5), 135.56 (C6), 151.74 (C10′), 151.79
(C11′), 152.35 (C8), 159.54 (C6′), 160.01 (C3′), 168.60 (C2). Anal.
×2), 32.8 (×2), 36.7, 42.2 (×2), 57.6 (×2), 83.7, 102.5 (×2),
10.1(×2), 112.0 (×2), 118.1(×2), 120.1, 124.7, 126.6, 127.3, 146.9,
50.1(×2), 150.2, 159.7 (×2), 169.0 (C = O), 174.6 (C = O). Anal.
Calcd for C30
7.56; H, 5.47; N, 5.21. FABMS (m/z) 530 (M + H) . g = 2.0058.
aN = 1.70 mT.
29 2 7 2
H N O ·0.1H O: C, 67.81; H, 5.54; N, 5.27. Found: C,
6
Calcd for C29H26NO : C, 71.89; H, 5.41; N, 2.89. Found: C, 71.55;
H, 5.69; N, 2.71. FABMS 485 (M + H) . g = 2.0056. aN = 1.62 mT.
+
6
+
Data for 3b: Yellow powder. Mp = 235–239 °C. IRν (KBr) 3431,
978, 2937, 1734, 1610, 1541, 1508, 1466, 1338, 1178, 1115 cm−1. 1H
4.2. Analysis
2
NMR (DMSO-d
s, 6H, CH × 4), 1.44 (t, 2H, J = 12.1, CH
.67 (m, 1H, NCH), 6.55 (dd, 2H, J = 2.3, 8.3, H2′,7′), 6.59 (d, 2H, J =
.3, H1′,8′), 6.66 (d, 2H, J = 2.3, H4′,5′), 7.53 (d, 1H, J = 1.5, H7), 7.78
6
+ hydrazobenzene, 500 MHz) δ 1.01 and 1.02 (each
4
.2.1. Apparatus
3
2
), 1.56 (d, 2H, J = 11.4, CH ),
2
Fluorescence spectra and relative fluorescence intensity were mea-
2
8
sured using a Hitachi F7000 fluorescence spectrophotometer. Both the
excitation and emission wavelength band passes were set at 5 nm and
the range was 1. Each sample was measured at a concentration of
(
dd, 1H, J = 8.4, 1.5, H5), 7.91 (d, 1H, J = 8.3, H4), 10.24 (br. s, 2H,
1
3
OH × 2). C NMR (DMSO-d
6
+ hydrazobenzene, 125 MHz) δ 19.7
0
.1 μM. Absorption spectra were obtained using a Hitachi U-3000 UV–
(
(
(
×2), 32.7 (×2), 36.7, 41.9 (×2), 57.5 (×2), 82.4, 102.5 (×2), 109.9
×2), 113.0 (×2), 120.1, 120.2, 120.7, 125.9, 129.1, 129.3, 145.9, 151.9
×2), 154.9, 159.7 (×2), 168.7 (C = O), 174.8 (C = O). Anal. Calcd for
VIS spectrometer. Electron spin resonance (ESR) spectra were obtained
on a JEOL JES-FR30 ESR spectrometer. Samples were drawn into quartz
capillaries, the bottoms of the capillaries were sealed and the capillaries
were placed in standard 2-mm-i.d. quartz ESR tubes. The ESR spectrom-
eter settings were as follows: microwave power, 4.0 mW; frequency,
C
30
H
29
N
2
O
7
·1.5H
2
O: C, 64.74; H, 5.80; N, 5.03. Found: C, 65.05; H,
+
5
.80; N, 5.03. FABMS (m/z) 530 (M + H) . g = 2.0059. aN = 1.70 mT.
9
.5 GHz; and, modulation amplitude, 1.25 G. All pH measurements
4
.1.3. 3′–O–(TEMPO-4-carbonyl)fluorescein (4)
To a stirred solution of 4-carboxy-TEMPO (100 mg) and fluorescein
were performed using a Fisher AB15 pH meter.
(
166 mg) in dry DMF (0.7 mL) and dry pyridine (0.7 mL), DCC (309 mg)
and DMAP (33 mg) were added in an ice bath under argon atmosphere.
4.2.2. ESR measurement
After stirring at room temperature for 17 h, the reaction mixture was fil-
tered and washed with CHCl . After the removal of halogen solvents, the
3
A 20 μM sample in 0.1 M PBS (pH 7.0 or 6.0) and a 20 mM AsA in
0.1 M PBS (pH 7.0 or 6.0) were mixed in a ratio of 1:1 (v/v). The