Evolution of Fluorescein
A R T I C L E S
purchased from Sigma-Aldrich Japan K.K. (Tokyo, Japan). NMR
spectra were recorded on a JNM-LA300 (JEOL) instrument at 300 MHz
for 1H NMR and 75 MHz for 13C NMR. Mass spectra were measured
with a JMS-DX300 (JEOL) for EI and a JMS-T100LC (JEOL) for ESI.
All experiments were carried out at 298 K, unless otherwise specified.
General Procedure for the Syntheses of TokyoGreens. Xanthone
diTBDMS ether was prepared according to the literature.3 Magnesium
turnings (4.50 mmol) were dried under Ar at 250 °C for 3 h. After the
magnesium turnings were cooled to room temperature, 2 mL of
bromobenzene derivatives (0.45 mmol) in distilled THF was added,
and the mixture was stirred at 60 °C to yield phenylmagnesium bromide
derivatives. The solution was cooled to 0 °C, then 2 mL of xanthone
diTBDMS ether (0.30 mmol) in distilled THF was added, and the
mixture was stirred for 10 min. The reaction was quenched by adding
10 mL of 2 N HCl(aq), and the resulting yellow precipitate was
collected by filtration, washed with a small quantity of distilled THF,
and dried in vacuo to yield almost pure product. Further purification
by silica gel column chromatography was done if required.
3H), 6.86 (dd, 1H, J ) 2.2, 8.4 Hz), 6.89 (d, 1H, J ) 2.0 Hz), 7.23
(dd, 2H, J ) 2.2, 9.2 Hz), 7.29 (d, 1H, J ) 8.8 Hz), 7.31 (d, 2H, J )
2.2 Hz), 7.77 (d, 2H, J ) 9.2 Hz). 13C NMR (75 MHz, CD3OD): δ
56.35, 56.36, 100.16, 103.26, 107.11, 113.62, 118.48, 121.03, 133.47,
135.22, 159.58, 161.02, 165.72, 166.39, 172.57. MS (EI+): m/z 348
(M+). HRMS (ESI+): m/z calcd for (M + H)+, 349.10760; found,
349.10457.
9-[1-(2,5-Dimethoxyphenyl)]-6-hydroxy-3H-xanthen-3-one (8).
1
Yield: 85%. H NMR (300 MHz, CD3OD): δ 3.68 (s, 3H), 3.82 (s,
3H), 6.97 (d, 1H, J ) 1.8 Hz), 7.26 (dd, 2H, J ) 2.4, 9.2 Hz), 7.29 (m,
2H), 7.37 (d, 2H, J ) 2.4 Hz), 7.74 (d, 2H, J ) 9.2 Hz). 13C NMR (75
MHz, CD3OD): δ 56.45, 56.70, 103.29, 114.35, 117.44, 118.35, 118.87,
121.31, 121.75, 135.02, 151.87, 155.22, 161.20, 166.20, 172.79. MS
(EI+): m/z 348 (M+). HRMS (ESI+): m/z calcd for (M + H)+,
349.10760; found, 349.10382.
Preparation of 2-Me-4-MeO TokyoGreen Mono(2′,3′,4′,6′-tetra-
O-acetyl-â-D-galactopyranoside) (TG-âGal Tetraacetate). A mixture
of 2-Me-4-OMe TokyoGreen (5) (10 mg, 30 µmol), Cs2CO3 (100 mg,
300 µmol), and 2,3,4,6-tetra-O-acetyl-R-D-galactopyranosyl bromide
(100 mg, 250 µmol) in dry dimethylformamide (0.5 mL) was stirred
overnight at room temperature under Ar. The inorganic precipitate was
filtered off, and the filtrate was concentrated under reduced pressure.
The residue was diluted with water and extracted with CH2Cl2 three
times. The combined organic solution was washed with water and
saturated NaCl(aq), dried over Na2SO4, and evaporated. The residue
was chromatographed on silica gel with CH2Cl2-MeOH (100:3) as
the eluent to give TG-âGal tetraacetate (11.3 mg, 57%) as an orange
NMR and Mass Spectral Data of TokyoGreens. 9-[1-(2-Meth-
1
ylphenyl)]-6-hydroxy-3H-xanthen-3-one (1). Yield: 96%. H NMR
(300 MHz, CD3OD): δ 2.05 (s, 3H), 7.18 (dd, 2H, J ) 2.2, 9.2 Hz),
7.29-7.32 (m, 3H), 7.47-7.64 (m, 5H). 13C NMR (75 MHz, CD3-
OD): δ 19.74, 103.62, 118.05, 121.72, 127.31, 130.11, 131.93, 132.06,
132.65, 134.31, 137.21, 161.11, 166.84, 173.47. MS (EI+): m/z 302
(M+). HRMS (ESI+): m/z calcd for (M + H)+, 303.10212; found,
303.10060.
9-[1-(2,4-Dimethylphenyl)]-6-hydroxy-3H-xanthen-3-one (2).
1
Yield: 86%. H NMR (300 MHz, CD3OD): δ 2.01 (s, 3H), 2.49 (s,
1
powder. H NMR (300 MHz, CDCl3): δ 2.03 (s, 3H), 2.05 (s, 3H),
3H), 7.19-7.26 (m, 3H), 7.32-7.39 (m, 4H), 7.60 (d, 2H, J ) 9.2
Hz). 13C NMR (75 MHz, CD3OD): δ 19.74, 21.40, 103.52, 118.36,
121.53, 127.96, 129.62, 130.17, 132.72, 134.64, 137.10, 142.56, 161.23,
168.48, 173.04. MS (EI+): m/z 316 (M+). HRMS (ESI+): m/z calcd
for (M + H)+, 317.11777; found, 317.11538.
2.07 (s, 3H), 2.14 (s, 3H), 2.20 (s, 3H), 3.90 (s, 3H), 4.10-4.25 (m,
3H), 5.12-5.19 (m,2H), 5.48-5.58 (m, 2H), 6.40 (d, 1H, J ) 1.4 Hz),
6.57 (dd, 1H, J ) 1.4, 9.3 Hz), 6.80-7.11 (m, 7H). HRMS (ESI+):
m/z calcd for (M + Na)+, 685.18971; found, 685.18920.
Preparation of 2-Me-4-MeO TokyoGreen Mono-â-D-galactopy-
ranoside (TG-âGal). To a solution containing 34 mg (51.3 µmol) of
2-Me-4-OMe TokyoGreen mono(2′,3′,4′,6′-tetra-O-acetyl-â-D-galac-
topyranoside) in 2 mL of methanol was added 10.5 µL of 4.9 M NaOMe
(51.3 µmol). After being stirred at 0 °C for 30 min, the reaction mixture
was neutralized with Amberlite IR-120 plus (H+). The Amberlite was
filtered off, and the filtrate was evaporated. The obtained residue was
chromatographed on reversed-phase preparative TLC (RP18W) with
CH3CN-H2O (1:1) as the eluent to give 2-Me-4-OMe TokyoGreen
mono-â-D-galactopyranoside (21.7 mg, 85.4%) as an orange pow-
9-[1-(2,5-Dimethylphenyl)]-6-hydroxy-3H-xanthen-3-one (3).
Yield: 89%. H NMR (300 MHz, CD3OD): δ 1.99 (s, 3H), 2.42 (s,
1
3H), 7.13 (s, 1H), 7.19 (dd, 2H, J ) 2.4, 9.2 Hz), 7.30 (d, 2H, J ) 2.2
Hz), 7.43 (s, 2H), 7.54 (d, 2H, J ) 9.4 Hz). 13C NMR (75 MHz, CD3-
OD): δ 19.21, 20.90, 103.62, 118.01, 121.72, 130.40, 131.97, 132.54,
132.60, 134.06, 134.37, 137.37, 161.05, 166.90, 173.53. MS (EI+): m/z
316 (M+). HRMS (ESI+): m/z calcd for (M + H)+, 317.11777; found,
317.11472.
9-[1-(2-Methoxyphenyl)]-6-hydroxy-3H-xanthen-3-one (4). Yield:
1
91%. H NMR (300 MHz, CD3OD): δ 3.74 (s, 3H), 7.20-7.38 (m,
1
der. H NMR(300 MHz, CD3OD): 2.03 (s, 3H), 3.61 (dd, 1H, J )
7H), 7.67-7.77 (m, 3H). 13C NMR (75 MHz, CD3OD): δ 56.36,
103.33, 113.18, 118.37, 121.28, 122.07, 131.87, 134.24, 134.95, 158.00,
161.15, 166.44, 172.81. MS (EI+): m/z 318 (M+). HRMS (ESI+): m/z
calcd for (M + H)+, 319.09703; found, 319.09433.
3.4, 9.5 Hz), 3.74-3.88 (m, 4H), 3.89 (s, 3H), 3.92 (d, 1H, J ) 3.9
Hz), 5.10 (dd, 1H, J ) 2.8, 7.9 Hz), 6.47 (d, 1H, J ) 2.0 Hz), 6.62
(dd, 1H, J ) 2.0, 9.5 Hz), 7.00-7.21 (m, 6H), 7.37 (d, 1H, J ) 2.4
Hz). HRMS (ESI+): m/z calcd for (M + Na)+, 517.14745; found,
517.15056.
9-[1-(2-Methyl-4-methoxyphenyl)]-6-hydroxy-3H-xanthen-3-
1
one (5). Yield: 89%. H NMR (300 MHz, CD3OD): δ 2.03 (s, 3H),
Fluorescence Properties and Quantum Efficiency of Fluores-
cence. Steady-state fluorescence spectroscopic studies were performed
on an F4500 (Hitachi). UV-vis spectra were obtained on a UV-1600
(Shimadzu), with 0.1 mol L-1 sodium phosphate buffer (pH 3.4) or
NaOH(aq) (pH 13) as the solvent. Each solution contained up to 0.1%
(v/v) DMSO as a cosolvent. For determination of the quantum efficiency
of fluorescence (Φfl), fluorescein in 0.1 mol L-1 NaOH(aq) (Φfl ) 0.85)
was used as a fluorescence standard.6 Curve fittings in Figure 2b,c were
done by the Marcus17 and Rehm-Weller18 equation with the constant
values of λ ) 0.81 eV, kf ) 2.01 × 108 s-1, Φfl ) 0.85 for the anion
form of fluorescein, and Φfl ) 0.32 for the neutral form of fluorescein
and with wp and V values as variables.13
Cyclic Voltammetry. Cyclic voltammetry was performed on a 600A
electrochemical analyzer (ALS). A three-electrode arrangement in a
single cell was used for the measurements: a Pt wire as the auxiliary
electrode, a glassy carbon electrode as the working electrode, and a
Ag/Ag+ electrode as the reference electrode. The sample solutions
contained 1.0 × 10-3 M sample and 0.1 M tetrabutylammo-
3.93 (s, 3H), 7.06 (dd, 1H, J ) 2.2, 8.4 Hz), 7.11 (d, 1H, J ) 2.0 Hz),
7.21 (dd, 2H, J ) 2.2, 9.2 Hz), 7.24 (d, 1H, J ) 7.5 Hz), 7.32 (d, 2H,
J ) 2.2 Hz), 7.61 (d, 2H, J ) 9.3 Hz). 13C NMR (75 MHz, CD3OD):
δ 20.16, 56.02, 103.56, 112.97, 117.38, 118.49, 121.54, 124.60, 131.90,
134.61, 139.24, 161.07, 163.19, 167.58, 173.20. MS (EI+): m/z 332
(M+). HRMS (ESI+): m/z calcd for (M + H)+, 333.11268; found,
333.10856.
9-[1-(2-Methoxy-5-methylphenyl)]-6-hydroxy-3H-xanthen-3-
1
one (6). Yield: 87%. H NMR (300 MHz, CD3OD): δ 2.40 (s, 3H),
3.70 (s, 3H), 7.17 (d, 1H, J ) 1.8 Hz), 7.24 (dd, 2H, J ) 2.2, 9.2 Hz),
7.25 (d, 1H, J ) 8.6 Hz), 7.35 (d, 2H, J ) 2.2 Hz), 7.54 (dd, 1H, J )
2.2, 8.6 Hz), 7.72 (d, 2H, J ) 9.2 Hz). 13C NMR (75 MHz, CD3OD):
δ 20.39, 56.36, 103.29, 113.07, 118.36, 121.03, 121.23, 131.86, 132.07,
134.57, 135.05, 155.93, 161.11, 166.65, 172.77. MS (EI+): m/z 332
(M+). HRMS (ESI+): m/z calcd for (M + H)+, 333.11268; found,
333.10945.
9-[1-(2,4-Dimethoxyphenyl)]-6-hydroxy-3H-xanthen-3-one (7).
1
Yield: 89%. H NMR (300 MHz, CD3OD): δ 3.74 (s, 3H), 3.97 (s,
9
J. AM. CHEM. SOC. VOL. 127, NO. 13, 2005 4893