126
M. Afri et al. / Chemistry and Physics of Lipids 131 (2004) 123–133
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It should be noted that NMR and UV spectral anal-
J = 8, 1.5 Hz, H4), 6.93 (1H, s, H1 /8 ), 6.92 (2H, d,
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ysis unambiguously verified that the labile DCFH did
not undergo autoxidation during the course of the
sometimes lengthy NMR measurements.
J = 8 Hz, H6), 6.72 (1H, s, H4 /5 ), 6.10 (1H, s, H9);
13C NMR (DMSO) δ 162.26 (C2a), 152.47 (C3 /6 ),
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149.07 (C4 a/10 a), 146.82 (C1), 132.15 (C5), 130.56
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(C6), 129.68 (C1 /8 ), 129.23 (C3), 126.39 (C4), 115.85
2.2. Substrates—preparation and spectral data
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(C8 a/9 a), 114.61 (C2 /7 ), 103.74 (C4 /5 ), 36.35 (C9 ).
HRMS calcd (C20H12O5Cl2, M+) 402.0062, obsd
402.0059.
DCF (3) and DCFH-DA (1) are known in the lit-
erature, but some of their spectral data is lacking;
DCFH-DA analog enriched with 13C at the ester car-
bonyls was prepared using acetyl-1-13C chloride, fol-
lowing the general procedure for the esterification as
previously described (Wheeler, 1963), and was diluted
with the 12C compound in a 1:1 ratio. The latter was
incorporated within DMPC liposomes in a substrate
to lipid ratio of 1:20 (4.75 mol%) and a substrate con-
centration of 0.01 M.
2.2.4. 2ꢀ,7ꢀ-Dichlorofluorescein (DCF, 3a)
1H NMR (DMSO) δ 11.09 (2H, bs, OH), 8.02 (1H,
d, J = 7.5 Hz, H3), 7.82 (1H, dt, J = 7.5, 1 Hz,
H5), 7.75 (1H, dt, J = 7.5, 1 Hz, H4), 7.35 (1H, d,
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J = 7.5 Hz, H6), 6.93 (2H, s, H4 /5 ), 6.67 (2H, s,
H1 /8 ); 13C NMR (DMSO) δ 168.17 (C2a), 155.06
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(C4 a/10 a), 151.41 (C1), 150.02 (C3 /6 ), 135.80 (C5),
130.42 (C4), 128.08 (C1 /8 ), 125.82 (C2), 124.99 (C3),
123.86 (C6), 116.17 (C8 a/9 a), 110.38 (C3 /7 ), 103.64
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1
2.2.1. 2ꢀ,7ꢀ-Dichlorodihydrofluorescein diacetate
(DCFH-DA, 1)
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(C4 /5 ), 81.45 (C9 ). H NMR (MeOH) δ 8.05 (1H,
d, J = 7.5 Hz, H3), 7.82 (1H, dt, J = 7.5, 1 Hz,
H5), 7.75 (1H, dt, J = 7.5, 1 Hz, H4), 7.25 (1H, d,
1H NMR (DMSO) δ 7.89 (1H, dd, J = 8, 1.5 Hz,
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J = 7.5 Hz, H6), 6.84 (2H, s, H4 /5 ), 6.62 (2H, s,
H3), 7.49 (1H, dt, J = 7.5, 1 Hz, H5), 7.38 (1H, dt,
H1 /8 ); 13C NMR (MeOH) δ 170.82 (C2a), 156.76
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J = 7.5, 1 Hz, H4), 7.27 (2H, s, H4 /5 ), 7.20 (2H,
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s, H1 /8 ), 7.07 (1H, d, J = 7.5 Hz, H6), 6.40 (1H, s,
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(C4 a/10 a), 153.48 (C1), 152.33 (C3 /6 ), 136.94 (C5),
131.65 (C4), 129.55 (C1 /8 ), 127.90 (C2), 126.27 (C3),
H9), 2.33 (6H, s, Me); 13C NMR (DMSO) δ 168.89
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=
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125.28 (C6), 118.37 (C8 a/9 a), 112.32 (C2 /7 ), 104.89
(C2a), 168.02 (C O), 148.80 (C4 a/10 a), 145.83 (C3 ),
145.26 (C1), 132.69 (C5), 131.13 (C6), 130.07 (C3),
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(C4 /5 ), 84.10 (C9 ).
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127.16 (C4), 129.95 (C2), 129.76 (C1 ), 123.09 (C9 a),
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2.2.5. DCF anion (DCF + KOH, 3b)
120.42 (C2 ), 112.37 (C4 ), 20.25 (Me).
1H NMR (0.5% KOH in MeOH) δ 8.11 (1H, dd,
J = 7, 1.5 Hz, H3), 7.65 (1H, dt, J = 7, 1 Hz, H4),
7.62 (1H, dt, J = 7, 1.5 Hz, H5), 7.21 (1H, dd, J = 7,
2.2.2. Synthesis of 2ꢀ,7ꢀ-dichlorodihydrofluorescein
(DCFH, 2) (Brandt and Keston, 1965)
1 Hz, H6), 7.12 (2H, s, H1 /8 ), 6.58 (2H, s, H4 /5 ); 13C
DCF (3; 0.53 g; 1.3 mol) was dissolved in hot
methanol (40 mL), and acetic acid (20 mL) was intro-
duced with continued heating and stirring. Zinc dust
(1.5 g; 0.023 mol) was added in three 0.5 g portions
with continued heating and stirring over a period of
5 min, during which time the solution turned colorless.
The zinc was removed by filtration, and the filtrate was
added to 200 mL of water. The resulting white solid
was vacuum filtered to give DCFH (0.42 g, 1.04 mol,
80% yield). The product oxidizes readily to DCF but
is relatively stable when stored in the dark and at low
temperature (−18 ◦C).
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NMR (0.5% KOH in MeOH) δ 176.10 (C9 ), 173.71
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(C2a), 160.10 (C4 a/10 a), 158.79 (C3 /6 ), 140.99 (C2),
134.13 (C1), 131.05 (C3), 130.61 (C6), 130.51 (C4),
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130.43 (C5), 129.77 (C1 /8 ), 129.01 (C8 a/9 a), 112.80
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(C2 /7 ), 104.74 (C4 /5 ).
2.3. Substrates—partition coefficients
Following literature procedures (Pooler and
Valenzeno, 1979; Leo et al., 1971), we carried out
partition coefficients studies on DCFH and DCF 3b
using equal volumes of octanol and deionized water
(pH 6). The results indicated that DCFH was to-
tally partitioned into the octanol, while DCF 3b was
partitioned in an octanol:water ratio of 2:1.
2.2.3. 2ꢀ,7ꢀ-Dichlorodihydrofluorescein (DCFH, 2)
1H NMR (DMSO) δ 7.78 (1H, dd, J = 8, 1.5 Hz,
H3), 7.39 (1H, dt, J = 8, 1.5 Hz, H5), 7.27 (1H, dt,