Cong Yu et al.
culated using the Stern–Volmer equation: F0/F=1+Ksv[Q], in which F0
and F refer to the fluorescence intensity in the absence and presence of
the quencher, respectively, and [Q] refers to the concentration of the
quencher in mM.[18]
Experimental Section
General Experimental Details
Materials: Alkaline phosphatase (from calf intestine) and Exo I were
purchased from Takara Biotechnology Co., Ltd. (Dalian, China). Ester-
ase and trypsin were purchased from Sigma–Aldrich (St. Louis, MO,
USA). ATP, ADP, AMP, and adenosine were purchased from Sangon
Biotechnology Co., Ltd. (Shanghai, China). 4,4’-diaminodiphenyl and p-
nitrophenyl phosphate were purchased from TCI (Shanghai, China).
3,4,9,10-perylenetetracarboxylic dianhydride (PTCDA) was purchased
from Acros Organics. Tetraoctylammonium bromide (TOAB) and 1,4-di-
bromobutane were purchased from Aladdin Chemistry Co., Ltd. (Shang-
hai, China). All other chemicals were of analytical grade and used with-
out further purification. All stock and buffer solutions were prepared
using water purified with a Milli-Q A10 filtration system (Millipore, Bill-
erica, MA, USA).
Binding Constant Calculation
Probe 1 (5 mm) and different amounts of ATP were mixed in a Tris-HAc
buffer solution and the corresponding UV/Vis spectra were recorded.
The binding constant was calculated according to the following equation:
[ATP]/
obs A[probe], the extinction coefficient of probe 1 in the fully bound
form, and the extinction coefficient of free probe 1, respectively.[14] Next,
[ATP]/
A
E
N
A
/CHTUNGTRENNUNG
A
stant (Ka) was determined as the ratio of the slope to the intercept (Fig-
ure S2 in the Supporting Information).
Fluorescence Recovery
1
Measurements: H NMR spectra were obtained with a Bruker AVANCE
ALP (2.5 UmLꢀ1) was added to the solution of probe 1 (5 mm) and ATP
(200 mm) in Tris-HAc buffer (5 mm, pH 8.2). The resulting solution was
incubated at 258C for a certain period of time (0–30 min), and the emis-
sion spectrum was measured.
600 (600 MHz) Fourier transform NMR spectrometer with chemical
shifts reported in parts per million (ppm) relative to tetramethylsilane.
Splitting patterns are reported as s (singlet), d (doublet), t (triplet), and
multiplet (m). UV/Vis absorption spectra were obtained using a Cary 50
Bio Spectrophotometer (Varian Inc., CA, USA) equipped with a xenon
flash lamp. Emission spectra were recorded using a Fluoromax-4 spectro-
fluorometer (Horiba Jobin Yvon Inc., USA) with an excitation wave-
length of 442 nm and corrected against PMT response. Excitation and
emission slit widths were both 2 nm at 5 mm probe 1 concentration.
Quartz cuvettes with 10 mm path length and 2 mm window width were
used for UV/Vis and emission measurements. Unless otherwise specified,
all spectra were taken at 258C in 5 mm Tris-HAc buffer at pH 8.2.
Assay Selectivity
ALP, esterase, trypsin, or Exo I (2.5 UmLꢀ1 each) were added to a solu-
tion of probe 1 (5 mm) and ATP (200 mm) in Tris-HAc buffer (5 mm,
pH 8.2). The solutions were incubated at 258C for 30 min, and the emis-
sion spectra were recorded.
Kinetic Assays
Different amounts of ALP (0, 0.01, 0.02, 0.1, 0.2, 0.5, 1.0, 2.5, and
5.0 UmLꢀ1, respectively) were added to a solution of probe 1 (5 mm) and
ATP (200 mm) in Tris-HAc buffer (5 mm, pH 8.2), and emission intensity
changes at 488 nm were monitored in real time. The concentration of
ATP in the assay solution at a certain reaction time was estimated from
the Stern–Volmer curve. The initial reaction rate (V0) from the linear
portion of the kinetic curve was then calculated (Figure S9 in the Sup-
porting Information).
Probe Synthesis
Synthesis of 3,4,9,10-tetra-(4-bromobutyloxy-carbonyl)-perylene (2): Per-
ylene-3,4,9,10-tetracarboxylic dianhydride (PTCDA) (784 mg, 2.0 mmol),
KOH (1.0 g, 17.7 mmol), and 30 mL deionized water were added into
a 100 mL flask and stirred at 708C for 30 min. The solution was filtered
and its pH value was adjusted to 8–9 with 1m HCl. Subsequently, tetraoc-
tylammonium bromide (TOAB) (400 mg, 0.7 mmol) was added. The mix-
ture was stirred vigorously for 10 min, and 1,4-dibromobutane (4.3 g,
20.0 mmol) was added. The solution was refluxed with vigorous stirring
for 2 h. The aqueous solution became clear and colorless, and on top of it
a layer of red oil formed. Next, CHCl3 (30 mL) was added to the reaction
mixture. The organic phase was washed three times with 15% aqueous
NaCl (30 mL) and concentrated under reduced pressure. The crude prod-
uct was purified by column chromatography on silica gel using a mixture
of CH2Cl2/petroleum ether (1:1) as eluent. After drying under vacuum,
compound 2 (1.2 g, 63%) was obtained as an orange solid. 1H NMR
(600 MHz, CDCl3): d=8.37 (d, 4H), 8.07 (d, 4H), 4.36–4.40 (t, 8H),
3.48–3.52 (t, 8H), 2.03–2.10 (m, 8H), 1.95–2.02 ppm (m, 8H); 13C NMR
(150 MHz, CDCl3): d=167.9, 132.4, 129.8, 129.5, 128.3, 128.2, 120.9, 64.0,
32.7, 28.8, 26.6 ppm.
ALP Inhibition
Different amounts of the inhibitors (0, 25, 50, 75, and 100 mm for
Na3VO4; 0, 100, 200, 500, and 1000 mm for 4,4’-diaminodiphenyl) were
added to the solution of probe 1 (5 mm), ATP (200 mm), and ALP
(5.0 UmLꢀ) in Tris-HAc buffer (5 mm, pH 8.2), and real-time emission
spectral changes of probe 1 at 488 nm were monitored. The inhibition ef-
ficiency was defined by the following equation:
ðIEÞ ¼ ½Fðno inhibitorÞꢀFðinhibitorÞꢁ=½Fðno inhibitorÞꢀF0ꢁ
in which F(inhibitor) and F(no
refer to the emission intensity of probe
inhibitor)
1 in the presence or absence of inhibitors, and F0 refers to background
emission of probe 1.[7b] The IC50 value refers to the inhibitor concentra-
tion required for 50% inhibition of the enzymatic activity.
Synthesis of 3,4,9,10-tetra-(4-trimethylammoniobutyloxy-carbonyl)-pery-
lene (1): Compound 2 (484 mg, 0.5 mmol) was dissolved in THF (50 mL)
in a 100 mL flask. An excess amount of trimethylamine (5 mL, 30%
aqueous solution) was added. The solution was refluxed for 3 days.
During this period, water was added at several intervals (total of 15 mL).
The organic solvent was evaporated under reduced pressure, and the
aqueous solution was washed with CHCl3 (30 mL) three times. After sol-
vent evaporation and drying under vacuum, probe 1 (0.57 g, 95%) was
Acknowledgements
This work was supported by the “100 Talents” program of the Chinese
Academy of Sciences, the National Natural Science Foundation of China
(21075119, 91027036, 21275139), the National Basic Research Program of
China (973 Program, No. 2011CB911002), the Pillar Program of Chang-
chun Municipal Bureau of Science and Technology (No. 2011225), and
the Open Project of State Key Laboratory of Supramolecular Structure
and Materials (Grant No. SKLSSM201215).
1
obtained as a red solid. H NMR (600 MHz, D2O): d=7.62–7.69 (m, 8H),
4.39–4.43 (t, 8H), 3.38–3.42 (t, 8H), 3.12 (s, 36H), 1.87–1.93 (m, 8H),
1.93–1.99 ppm (m, 8H); 13C NMR (150 MHz, D2O): d=169.1, 130.9,
129.7, 127.7, 126.6, 126.5, 120.7, 65.6, 65.0, 52.6, 24.5, 19.1 ppm; ESI-MS:
m/z 221.34 [M4+/4] (see Figure S21 in the Supporting Information).
Fluorescence Quenching
ATP (0–200 mm) was added to the solution of probe 1 (5 mm) in Tris-HAc
buffer (5 mm, pH 8.2). The solutions were thoroughly mixed for subse-
quent fluorescence and absorption measurements. The Ksv value was cal-
[1] J. E. Coleman, Annu. Rev. Biophys. Biomol. Struct. 1992, 21, 441–
483.
Chem. Asian J. 2013, 8, 276 – 281
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