Analytical Chemistry
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with NTR. Unlike the previously reported HyCL-2, this system in
7H), 1.32 (t, J = 7.1 Hz, 2H). 13C NMR (150 MHz, CDCl3) δ
166.5, 153.2, 147.8, 143.3, 139.2, 138.3, 137.9, 132.9, 129.5,
129.4, 128.7, 128.2, 125.2, 123.8, 120.9, 74.4, 67.9, 60.7, 57.3,
39.2, 39.0, 38.6, 38.5, 37.0, 32.9, 29.7, 28.3, 28.1, 25.6, 14.2. MS
(ESI) m/z 536.18 [C30H32ClNO6-H].
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current work does not require the addition of enhancer reagent to
enhance the chemiluminescence output signal under aqueous
conditions, and surprisingly the response property is much higher
than HyCL-2 system. The general design strategy and decompose
process of this system are presented in Scheme 1. The probe CL-
NTR was prepared by introducing 4-nitrobenzyl as a masking
group and recognition unit to 1,2-dioxetane scaffold via an ether
bond. Under hypoxic conditions, reaction of CL-NTR with NTR
in the presence of NADH causes the reduction of 4-nitrobenzyl
moiety to corresponding 4- hydroxylaminebenzyl, followed by the
1,6-rearrangement elimination reaction and initiating an electron
transfer cascade that leads to dioxetane ring decompose, and it's
accompanied by the release of chemiluminescence. CL-NTR
exhibited a high selectivity to NTR activity with 6000-fold total
light emission photons, and the detection limit of 0.947 ng/mL. It
was then successfully applied to imaging exogenous and
endogenous NTR in tumor mice.
Syntheses of 3. Compound 2 (0.43 mmol, 230 mg) and methylene
blue were dissolved in 10 mL DCM, oxygen was bubbled through
the solution while irradiating with yellow light for 2 h. The
reaction was monitored by TLC, and then the mixture was diluted
in 20 mL water, extracted with ethyl acetate (30 mL x 3) for three
times and dried over MgSO4. The solvent was removed under
vacuum, and the product was purified by silica gel column
chromatography (petroleum ether/ethyl acetate = 10:1-3:1) to
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1
afford the desired product 3 in 41% yield. H NMR (600 MHz,
CDCl3) δ 8.57-8.09 (m, 1H), 7.87 (dd, J = 23.1, 16.2 Hz, 1H),
7.75-7.65 (m, 1H), 7.65-7.48 (m, 1H), 6.50 (dd, J = 22.5, 16.2 Hz,
1H), 5.06 (d, J = 30.1 Hz, 1H), 4.33-4.17 (m, 1H), 3.97 (s, 1H),
3.24 (s, 1H), 2.35-1.45 (m, 5H), 1.32 (td, J = 7.2, 5.1 Hz, 2H).13C
NMR (150 MHz, CDCl3) δ166.2, 153.7, 147.9, 142.9, 137.4,137.1,
133.1, 131.4, 129.4, 128.7, 127.0, 125.4, 125.3, 123.8, 122.7,
122,1, 111.6, 96.3, 74.5, 60.8, 52.7, 49.7, 46.9, 39.2, 36.5, 36.3,
33.6, 32,2, 31.5, 27.4, 25.8, 14.2.
Syntheses of CL-NTR. Compound 3 (0.14 mmol, 80 mg) and
lithium hydroxide (1 mmol, 42 mg) were dissolved in 10 mL of
MeOH/H2O=1:1, then the mixture was stirred overnight at room
temperature and was monitored by TLC. After completion, 1 M
hydrochloric acid was added to adjust the pH to 2-3, stand for 30
1
min and filter to get the white solid CL-NTR in yield 39%. H
NMR (600 MHz, CDCl3) δ 8.30 (d, J = 8.7 Hz, 1H), 8.00 (t, J =
11.0 Hz, 1H), 7.69 (dd, J = 13.5, 8.6 Hz, 1H), 6.55 (d, J = 16.1 Hz,
1H), 5.07 (s, 1H), 3.27 (s, 1H), 2.43-1.16 (m, 7H). 13C NMR (150
MHz, DMSO-D6) δ166.9, 164.9, 153.3, 153.1, 147.3, 143.5, 143.4,
136.2, 135.9, 134.1, 132.7, 131.3, 128.9, 126.5, 126.3, 123.6,
111.1, 95.4, 74.3, 52.7, 49.4, 46.3, 38.4, 35.8, 35.5, 33.3, 33.0,
31.8, 31.6, 31.0, 30.8, 26.8, 25.5, 25.1. HRMS (ESI) m/z
calculates for C28H28ClNO8, 541.1503, found 540.1440
[C28H28ClNO8 -H].
Scheme
1.
1,2-dioxetane-based
probe
for
selective
chemiluminescent detection of nitroreductase.
EXPERIMENTAL SECTION
Reagents and Apparatus. All chemicals reagents are
commercially available and used without further purification.
Nitroreductase (≥90 units/mg) from Escherichia coli and NADH
were purchased from Sigma-Aldrich. The powder of NTR was
dissolved into ultrapure water. All these enzyme solutions were
frozen immediately at -20 °C for storage and allowed to thaw
before use, which results in no change of the enzyme activity. A
stock solution (10 mM) of CL-NTR was prepared by dissolving
an appropriate amount of CL-NTR in DMSO. Ultrapure water
Spectroscopic Measurements. Chemiluminescent spectra were
acquired using an Ultra-Weak Luminescence Analyzer for
Chemiluminescence and Bioluminescence (BPCL-GP-TGC).
2970 μL of Tris-HCl (50 mM, pH=7.4) buffered, 10 μL of a 0.03
mM CL-NTR in DMSO, 10 μL of a 50 mM NADH in 0.01 mM
NaOH solution, 10 μL of nitroreductase from Escherichia coli
(NTR, Sigma-Aldrich #N9284-1mg) in DI-H2O (1 mg of NTR
dissolved in 100 μL of DI-H2O, 10 mg/mL) were added to glass
dish. Time scans were acquired using the time scan module after
adding probes. For the dose-dependent response to nitroreductase,
0.1 μM CL-NTR was treated with 8 μL of a 50 mM NADH and 1
μL different concentrations of NTR solution (finally concentration,
0, 2.5, 5, 15, 25, 55, 110, 220, 550 ng/mL) were measured.
Selectivity for CL-NTR was measured by monitoring the time-
dependent chemiluminescent emission at 540 nm. All assays were
performed in Tris-HCl (50 mM, pH 7.4) buffer.
was purified by ULTRAPURE WATER system. 1H NMR and 13
C
NMR spectra were measured on a Bruker DMX-600 spectrometer.
Electrospray ionization mass spectra (ESI-MS) were recorded in
LTQ mass spectrometry. Probe CL-NTR was synthesized
following the synthetic routes shown in Scheme 2.
Syntheses of 2. Compound 1 (0.75 mmol, 300 mg) and K2CO3
(1.12 mmol, 155 mg) dissolved in DMF (10 mL) at room
temperature for 30 minutes, 4-nitrobenzyl bromide (0.97 mmol,
210 mg) was added, and then stirred at room temperature 18 h, the
reaction mixture was diluted in 20 mL water, then extracted with
ethyl acetate (30 mL x 3) for three times and dried over MgSO4.
The solvent was removed under vacuum, and the product was
purified by silica gel column chromatography (petroleum
ether/ethyl acetate = 10:1-4:1) to afford the desired product 2 in
63% yield. 1H NMR (600 MHz, CDCl3) δ 8.44-8.07 (m, 1H), 7.90
(d, J = 16.2 Hz, 1H), 7.70 (d, J = 8.7 Hz, 1H), 7.48 (d, J = 8.0 Hz,
1H), 7.13 (d, J = 8.0 Hz, 1H), 6.47 (d, J = 16.1 Hz, 1H), 5.11 (d, J
= 9.9 Hz, 1H), 4.25 (q, J = 7.1 Hz, 1H), 3.34 (s, 2H), 2.26-1.65 (m,
ESI-MS analysis of the reaction of CL-NTR with NTR. 50 µL
of 10 mM CL-NTR in DMSO was dispersed in 850 µL Tris-HCl
(50 mM, pH=7.4) buffer solution, 50 µL of 100 mM NADH in
0.01 mM NaOH, 50 µL of 1 mg/mL NTR were added into a vial
and mixed well. After 1 h of incubation at room temperature, the
mixture was poured into 30 mL DI-H2O, 1 M HCl was added to
2
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