A glycosylation strategy to develop a low toxic naphthalimide fluorescent probe for the detection of Fe3+ in aqueous medium

Article abstract of DOI:10.1039/c7dt01099k

A glycosylation strategy based on click chemistry was employed to develop a naphthalimide-based Fe³⁺ fluorescent probe with low cytotoxicity and good water-solubility. The selectivity and sensitivity to Fe³⁺ of three synthesized naphthalimide-based fluorescent probes follows a Nap-PZ < Nap-OH < Nap-Glc trend, because Nap-PZ was modified with a good water-soluble group. The cytotoxicity follows a Nap-PZ > Nap-OH > Nap-Glc trend, because the exposed toxic group of Nap-PZ was shielded by a good biocompatible group. The detection limit toward Fe³⁺ ion follows a Nap-PZ (7.40 × 10^-6 M) > Nap-OH (2.73 × 10^-7 M) > Nap-Glc (4.27 × 10^-8 M) trend. Moreover, Nap-Glc could be used to detect Fe³⁺ in living cells. The fluorescent "off-on" response of Nap-Glc towards Fe³⁺ could be recognized by the naked eye, and the "off-on" fluorescent mechanism also was demonstrated by theoretical calculations. Therefore, Nap-Glc is a novel glucosyl naphthalimide fluorescent probe for environmental or biological detection of Fe³⁺ with low cytotoxicity and good water-solubility.

Full text of DOI:10.1039/c7dt01099k

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Joseph T. Hupp, Omar K. Farha et al.
Efficient extraction of sulfate from water using
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A glycosylation strategy to develop low toxic naphthalimide
fluorescent probe for the detection of Fe3+ in aqueous medium
Received 00th January 20xx,
Accepted 00th January 20xx
Feiyang Liu,a Piaoping Tang,a Ruihua Ding,a Lujuan Liao,a Lisheng Wang,a Mian Wang*b and Jianyi
Wang* a,c
DOI: 10.1039/x0xx00000x
A glycosylation strategy based on click chemistry was employed to develop naphthalimide-based Fe3+ fluorescent probe
with low cytotoxicity and good water-solubility. The selectivity and sensitivity to Fe3+ of three synthesized naphthalimide-
based fluorescent probes follows a Nap-PZ<Nap-OH<Nap-Glc trend, because Nap-PZ was modified by good water-soluble
group. The cytotoxicity follows a Nap-PZ>Nap-OH>Nap-Glc trend, because the exposed toxic group of Nap-PZ was
shielded by good biocompatible group. The detection limit toward Fe3+ ion follows a Nap-PZ(7.40×10-6M)>Nap-
OH(2.73×10-7M)>Nap-Glc(4.27×10-8M) trend. Moreover, Nap-Glc could be used to detect Fe3+ in living cells. The
fluorescent “off-on” response of Nap-Glc towards Fe3+ could be recognized by naked-eye, and the “off-on” fluorescent
mechanism was demonstrated by theoretical calculations. Therefore, Nap-Glc was a novel glucosyl naphthalimide
fluorescent probe for environmental or biological detection of Fe3+ with low cytotoxicity and good water-solubility.
www.rsc.org/
because of their high efficiency, economy, convenience
manipulation13-15. However, several shortcomings limited wide
Introduction
Iron (especially Fe3+) is the most abundant trace elements in
human body and plays an essential role in various biochemical
processes involving oxygen transport, exotic metabolism, DNA
synthesis and repair1. The deficiency of iron would cause
anemia, hemochromatosis, liver damage, diabetes and
cancer2,3, and the excess of iron could induce Alzheimer’s,
Huntington’s, neurodegenerative diseases and Parkinson’s
diseases4, 5 etc. Also, Fe3+ was important pollutant source of
the environment. Therefore, the monitoring and detection of
Fe3+ was very significant in both biosystem and environment.
Spectrophotometry, atomic absorption spectroscopy and
voltammetry inductively coupled plasma mass spectroscopy
were familiar method to detect Fe3+. But high cost, time-
consuming and sampling complexity limited wide application
of these methods. The fluorescence probes towards Fe3+,
application of these probes towards Fe3+: poor water-
solubility7,9-12, high toxicity16-18, slow-response towards Fe3+
ions6-8,11
.
The fluorescent quenching caused by the
paramagnetism of iron was another shortcoming9
Among
these probes, naphthalimide was widely used as
chemosensors monitoring metal ions19-25 pH26,27 and
.
,
endogenous substances28-34 because of its good photo-physical
and chemical properties. The water-solubility and cytotoxicity
of naphthalimide could was overcome by introducing a
glycosyl group to the naphthalimide skeleton35, 36, but this
probe was mainly used to detect zinc ion rather than Fe3+ ion.
To date, the glycosylation from click reaction37-42 or glycosidic
reaction43-45 has emerged as a promising strategy for the
modification of hydrophobic and toxic materials.
Therefore, in this work, our aim was to develop novel
naphthalimide-based fluorescent probes towards Fe3+ with
high efficient, good water-solubility and low cytotoxicity, and
including rhodamine6-8
imidazole11, naphthalimide12 derivatives, recently drawn
tremendous attention from academia and industrial circles
,
fluorescein9, thiazolo-pyrimidine10,
the obtained target compound (Nap-PZ, Nap-OH and Nap-Glc)
was correspondingly characterized and evaluated
.
a.School of Chemistry and Chemical Engineering,Guangxi University, Nanning
530004, China. * email: jianyiwang@gxu.edu.cn
Results and Discussion
Synthesis of Fe3+ fluorescent probes
b.College of Life Science and Technology, Guangxi University, Nanning530004,
China.* email: mianwang23@163.com
c. Guangxi Colleges and Universities Key Laboratory of Applied Chemistry Technology
and Resource Development, Nanning 530004, China
† Electronic Supplementary Information (ESI) available: [absorption spectra of Nap-
PZ, Nap-OH and Nap-Glc towards metal ions; Fluorescence concentration titration
.
As shown in Scheme 1, compound Nap-PZ was synthesized by
conjugating 1, 8-naphthalimid with piperazine which acts as
electron-donator and fluorescent quencher based on photo
induced electron transfer (PET) effect. Nap-PZ was modified by
a hydrophilic azole group to afford Nap-OH. A hydrophilic and
biocompatible glucose unit was introduced to Nap-PZ through
spectra and titration curves of Nap-PZ and Nap-OH
; Acid-base titration
experiments of Nap-Glc; ESI-MS spectrum of Nap-Glc-Fe3+;theoretical calculations
of Nap-Glc and Nap-Glc-Fe3+ complex; Experimental procedures of compound
3,5, C NMR of all compounds; HRMS spectra of three probes]. See
6;1H/13
DOI: 10.1039/x0xx00000x
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click reaction to afford Nap-Glc. The structures of the targeted Nap-OH and Nap-Glc probes with good soluble gVroiewupArtsichleoOwnleinde
compounds were confirmed by 1H NMR, 13C NMR, and HRMS.
better selectivity for Fe3+ than Nap-PZ
.
DOI: 10.1039/C7DT01099K
a
O
N
O
O
N
O
O
N
O
O
O
N
O
O
O
O
N
O
n-Propylaminwe
ethanol
piperazine
2-Methoxyethanol
N
N
N
N
N
N
Br
HO
OH
OH
Br
N
N
N
N
OH
H
N
H
N
H
N
N
H
N
N
O
1
2
OH
Hydrophilicity
Nap-PZ
O
O
Hydrophilicity
Biocompatibility
Nap-PZ
Nap-Glc
Nap-OH
O
N
O
N3
HO
5
.
2O
5H
N
CuSO4
sodiumascorbate
THF/
N
N
N
O
N
O
O(1:1)
H2
N
OH
H
N
O
Br
N
O
OH
H
3
Nap-OH
HO
HO
N
N
O
acetone
K
2CO3
N3
O
N
O
6
OH
H
N
CuSO
.
sodiumascorbate
4 5H
2O
O
4
THF/
H2
O(1:1)
N
N
OH
OH
HO
N
N
H
N
N
O
OH
O
Nap-Glc
Scheme 1 Synthetic routes of Nap-PZ, Nap-OH and Nap-OH
Selectivity of Nap-PZ, Nap-OH and Nap-Glc for Fe3+ ion
To evaluate selectivity of Nap-PZ Nap-OH and Nap-Glc (Fig. 1a)
,
towards Fe3+, the fluorescence spectra of Nap-OH and Nap-Glc
could be directly measured in phosphate buffer (pH=6.50)
medium, whereas Nap-PZ required a mixed medium with 10%
ethanol. As shown in Fig.1b, Nap-PZ presented a strong
background fluorescence, which could be attributed to
protonation of aqueous medium to the piperazine nitrogen of
Nap-PZ, inhibiting PET effect of naphthalimide. And Nap-PZ
almost responded to all these metal ions, being difficult to
distinguish Fe3+ from other metal ions. When a hydrophilic
azole group was introduced to Nap-PZ to afford Nap-OH, the
background fluorescence disappeared, because the exposed
piperazine nitrogen of Nap-PZ was shielded. Upon addition of
Fe3+, the Nap-OH solution exhibited a clear fluorescent
enhancement (Fig. 1c) at 528 nm. While the addition of other
metal ions(Ce3+, Eu3+, Ba2+, Ca2+, Cd2+, Co2+, Fe2+, Mg2+, Mn2+,
Fig.1(a) Structure of Nap-PZ, Nap-OH and Nap-Glc. The Fluorescence
spectra of Nap-PZ (b), Nap-OH (c) and Nap-Glc (d) (10 µM) upon addition of
5 equiv different metal cations. (e) The color changes of three probes in the
presence of different metal cations under UV light (365 nm).
Sensitivity of Nap-PZ, Nap-OH and Nap-Glc for Fe3+ ion
To assess the sensitivity of these probes toward Fe3+, titration
experiments were carried out in phosphate buffer (pH=6.50). The
fluorescence intensity of Nap-Glc solution (Fig. 2) at 528 nm was
substantially increased when Fe3+ ions was gradually added (0–50
mM), which was linear with the concentration of Fe3+ ions in the
range of 0 to 20 mM. The detection limit of Nap-Glc toward Fe3+ ion
was calculated to be 4.27× 10-8M, while the detection limit was 7.40
×10-6 M for Nap-PZ (Fig .S2) and 2.73 ×10-7 M for Nap-OH (Fig .S3),
respectively. The detection limit was calculated by the formula 3δ/k,
where k is the slope between fluorescent intensity versus sample
concentration, and δ represents the standard deviation obtained by
maximal fluorescence intensity of free probes. The outstanding
detection level of Nap-Glc could be ascribed to their good water-
solubility compared with Nap-PZ.
+
Ni2+, Pb2+, Zn2+,Ag+, K+, Li+, Na+, NH4 ), the Nap-OH solution
only showed small fluorescent enhancement, although Nap-
OH was slightly disturbed Pb2+. When a better hydrophilic
glucose unit was introduced through click reaction to form
Nap-Glc
, the Nap-Glc solution exhibited a remarkable
fluorescent enhancement (Fig. 1d) at 528 nm toward Fe3+ ion
compared with other metal ion. Similar phenomenon could be
observed in absorption spectra of these probes (Fig. S1). Under
the irradiation of ultraviolet lamp(365 nm), one could hardly
distinguish the Nap-PZ-Fe3+ solution from Nap-PZ-other ions
solution by naked-eye, and the Nap-PZ solution was “lighted
up” by all metal ions(the first row). Comparatively, Nap-OH
and Nap-Glc could easily recognized Fe3+ rather than other
metal ions (Fig.1e). The reason should be attributed to the
water-solubility of Nap-OH and Nap-Glc over Nap-PZ. In
addition, the fluorescence of Nap-OH and Nap-Glc could be
quenched upon titration with Cu2+, which was caused by the
well-known paramagnetic effect of Cu2+ ion23, 46, Therefore, the
Fig.2 (a) Fluorescence changes of Nap-Glc (10 µM) in phosphate buffer
(pH=6.50) with 0-5 equiv Fe3+.Inset: The color changes of Nap-Glc in the
presence of 5 equiv Fe3+ under UV light of 365 nm. (b) Plots of fluorescence
intensity as a function of Fe3+ concentrations from 0– 20 µM.
2 | J. Name., 2017, 00, 1-3
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Cytotoxicity assays
ARTICLE
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DOI: 10.1039/C7DT01099K
Low toxicity is another requirement for the application of
probes in biosystem or environment. As shown in Fig. 3, Nap-
PZ displayed an obvious concentration-dependent toxicity
towards HepG2 cell and nearly killed all cells at
concentration of 80 M. When a hydrophilic azole group was
introduced to Nap-PZ Nap-OH showed much less toxic than
a
µ
,
Nap-PZ. The main reason was that the exposed piperazine
nitrogen of Nap-PZ, which might be a toxic group, was
shielded by a biocompatible azole group. When this exposed
piperazine nitrogen of Nap-PZ was modified by a better
biocompatible glycosyl moiety (a nutrient group), Nap-Glc
hardly showed toxic towards HepG2 cell in a wide range of
concentrations, was even favorable for growth of HepG2 cell.
Therefore, non-toxic Nap-Glc could well satisfy the detection
of Fe3+ in vitro or vivo.
Fig.4 The fluorescence intensity (528 nm) of Nap-Glc (10 µM)toward Fe3+
with other ions: 0, no ion; 1, Ag+;2, Ba2+; 3, Ca2+; 4, NH4+; 5, Cd2+;6, Co2+; 7,
Cu2+, 8, K+; 9, Li+; 10, Mg2+;11, Mn2+; 12, Na+; 13, Ni2+; 14, Zn+; 15 Ce3+; 16,
Eu3+; 17, Fe2+; 18, Pb2+
.
Fig.5 (a) Fluorescence changes of Nap-Glc in the presence of different
anions. (b) The fluorescence intensity (528 nm) of Nap-Glc (10 µM) toward
2-
Fe3+ upon addition of 0, no anions; 1, Br-;2, CH3COO-; 3, Cl-; 4, F-; 5, H2PO4
,
-
-
2-
6, HSO3 ; 7, I-; 8, NO3 ;9, S2O32-; 10, SO4
.
Fig. 3 Cell viability of HepG2 treated with different concentrations of Nap-
PZ, Nap-OH and Nap-Glc.
As shown in Fig.6, when 5 equiv. Fe3+ was added, the
fluorescence of Nap-Glc in phosphate buffer (pH=6.50)
instantly enhanced and reached the maximum within one
minute. Furthermore, the fluorescence intensity of the Nap-
Glc solution with Fe3+ ions was kept steady in the following 30
minutes. So short-response time of Nap-Glc toward Fe3+ could
well satisfy the requirement of real-time and quick detection.
The acid-base titration experiments (Fig.S4) showed that the
working pH range for Nap-Glc detecting Fe3+ was about 4-8,
where the fluorescent sensing behaviour was dominated by
Practical application of Nap-Glc
Since probe Nap-Glc with low cytotoxicity possessed desired
selectivity and sensitivity toward Fe3+, good competition, short
response time and wide working pH rang were also necessary
for practical application of probes. When Ag+, Ba2+, Ca2+, NH4 ,
+
Cd2+, Co2+, Cu2+, K+, Li+, Mg2+, Mn2+,Na+, Ni2+, Zn2+, Ce3+, Eu3+,
Fe2+, Pb2+ were added into the Nap-Glc solution (10 μM), no
significant fluorescent enhancement were observed at 528 nm
(black bars), and the fluorescent intensity was dramatically
enhanced when Fe3+ was added into the Nap-Glc solution with
other competitive metal ions (red bars) (Fig. 4). Similarly, Nap-
Glc hardly responded to the anions Br-, CH3COO-, Cl-, F-,
Fe3+. No fluorescence could be observed in the range of pH
>8,
which should be ascribed to the hydrolysis of Fe3+.
-
-
H2PO42-, HSO3 , I-, NO3 , S2O32-, SO42- etc, and the addition of
Fe3+ into the Nap-Glc solution with these anions caused
dramatic enhancement of the fluorescence (Fig. 5). Therefore,
Nap-Glc had an excellent anti-disturbance capacity, could be
independently applied for the detection of Fe3+ in aqueous
medium, and not disturbed by the ions listed above.
Fig.6 Time response of Nap-Glc (10 µM) towads 5 equiv Fe3+ in phosphate
buffer (pH=6.50).
To explore the application of Nap-Glc in biosystem, the
living cell imaging was conducted in HepG2 cells. As shown in
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Fig. 7, no fluorescence was observed when the cells were only
treated with Nap-Glc (10 µM) (Fig. 7a). However, obvious
fluorescence enhancement could be observed (Fig. 7b, c) when
the cells were treated with different concentrations of Fe3+
and Nap-Glc (10 µM), and the fluorescence intensity in HepG2
cells increased with the incremental concentrations of Fe3+.
Clearly, Nap-Glc showed good membrane-permeable and
could be used to detect Fe3+ in biosystem.
No
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51
Cu2+
no
2.2
×
10-6
No discuss
< 1 min
H2O-CH3OH (v/v=9/1)
DOI: 10.1039/C7DT01099K
discuss
No toxic
phosphate buffer
160 μM
Nap-Glc
4.27×10-8
Stoichiometry and recognition mode of Nap-Glc toward Fe3+
To explore the binding mode of Nap-Glc toward Fe3+, Job’s plot
analysis was carried out with the total concentration of Nap-
Glc and Fe3+ solution being constant, and the [Nap-Glc]/[Nap-
Glc+Fe3+] value varying from 0.1 to 0.9 (Fig.8). When [Nap-
Glc]/[Nap-Glc+Fe3+] value was about 1/3, the solution emitted
the strongest fluorescence, suggesting that one equiv Nap-Glc
was most likely to bind 2 equiv Fe3+ ion (1:2 stoichiometry). 1:2
stoichiometry of Nap-Glc–Fe3+ complex was also confirmed by
ESI-MS spectrum data (Fig.S5). Among these two equiv Fe3+ ion,
one equiv. Fe3+ bound to the piperazine nitrogen of Nap-Glc
,
Fig.7 Confocal fluorescence microscopic images of HepG2 cells. (a) Cells
treated with Nap-Glc (10 µM) alone; (b) Cells treated with Nap-Glc (10 µM)
and Fe3+ (1 eq); (c) Cells treated with Nap-Glc (10 µM) and Fe3+ (10 eq).
inhibiting PET effect from electron-donator piperazine to
electron-receptor 1, 8-naphthalimide moiety, recovered the
strong fluorescence of 1, 8-naphthalimide, and another equiv.
Fe3+ ion might bind to the glycosyl moiety52, 53. The recognition
mechanism of Nap-Glc toward Fe3+ was thus proposed in
Scheme 2.
Comparison of Nap-Glc with other Fe3+ fluorescent sensors.
The comparison of Nap-Glc and other Fe3+ fluorescence
probe were summarized in Table1. We noticed that Nap-Glc
exhibited high selectivity toward Fe3+ and was not disturbed by
other metals ions, while the reported fluorescence sensors for
Fe3+ usually were disturbed by other metals cations such as
Cu2+ ion47,51 and Hg2+ ion48-50
. Nap-Glc showed excellent
detection limit and very short response time compared with
these sensors3, 8, 11, 47, 48, 50, 51. More important, Nap-Glc could
be performed in 100% aqueous medium and most of reported
Fe3+ sensors9, 11, 12, 47-49 required a mixed medium of organic
solvent and water. Also, Nap-Glc showed less toxic in higher
dose range compared with the reported Fe3+ probes11, 12, 50
.
Table 1．Comparison of Nap-Glc with other reported Fe3+ fluorescent sensor.
Fig. 8 Job's plot of Nap-Glc with Fe3+ (the total concentration of Nap-Glc
and Fe3+ solution was 50 µM).
Ref of Disturbed Detection
response time
media
toxic
sensor
ions
limit (M)
4.5
10-6
10-7
No
discuss
No
3
no
×
No discuss
40 min
Tris–HCl buffer
O
N
O
O
N
O
8
9
no
no
1
×
H2O-CH3CN (95:5=v/v)
2
Fe3+
Fe3+
Fe3+
HO
discuss
No
N
N
N
N
PET
OH
OH
OH
OH
PET
HO
N
N
N
N
N
N
No discuss No discuss
MOPS buffer
H
N
O
N
O
H
OH
discuss
OH
O
O
Potassium phosphate
No toxic
10-7
10-8
40 min
< 1 min
on
Turn
Strong fluorescence
Weak fluorescence
11
12
no
no
1.2
3.0
×
×
buffer acetone (1:4v/v) at 5 μM
Scheme 2 Proposed “off-on” fluorescent mechanism of Nap-Glc towards
Fe3+
Tris HCl–CH3CN
(1:1v/v 0.01M)
No toxic
.
at 25 μM
No
discuss
No
47
48
49
50
Cu2+
Hg2+
Hg2+
Hg2+
7.65
×
10-6
No discuss
30 mim
DMF
Fluorescence mechanism reflected by theoretical calculations
To demonstrate “off-on” fluorescent mechanism of Nap-Glc
toward Fe3+, theoretical calculations were performed. When
Fe3+ bound to the piperazine nitrogen of Nap-Glc and PET
effect from piperazine to 1, 8-naphthalimide was inhibited, the
LUMO-HOMO energy gap was calculated to reduce from 3.464
ev for Nap-Glc to 2.874 ev for Nap-Glc-Fe3+ complex(Fig.9),
3.9
×
10-9
H2O-C2H5OH (4:1,V/V)
CH3CN-H2O (v/v=3:7)
H2O−CH3CN (9:1=v/v)
discuss
No
2.72
×
10-8
No discuss
> 60 min
discuss
No toxic
6.25 μM
1.3×10−7
4 | J. Name., 2017, 00, 1-3
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resulting in “Fluorescence on” of Nap-Glc. The UV-spectrum
was calculated to be around 409 nm with f=0.3326 for Nap-
Glc(close to the experimental value 405 nm, Table S1), and
about 376 nm for Nap-Glc-Fe3+ (close to the experiment value
385 nm, Table S2), also supporting that the calculated
mechanism was credible. Further calculations on
“Fluorescence on” mechanism of Nap-Glc were given in Figure
S7.
Experimental
Materials and instruments
View Article Online
DOI: 10.1039/C7DT01099K
All regents were commercially available and used without
further purification. 1H NMR and 13C NMR spectra were
recorded on Bruker 600MHz at 25°C, TMS as the internal
standard in CDCl3, and solvent peak as the internal reference in
CD3OD. HRMS analysis was performed by Thermo Fisher
Scientific LTQ FT Ultra. The UV-vis absorption and the
fluorescence spectra were carried out by Shanghai JingHua
7600 UV visible spectrophotometer (dual beam) and Shimadzu
RF-5301PC at room temperature, respectively. The cytotoxicity
assays was conducted by ELX800 absorbance microplate
reader. Cell image assays was performed by Multi-photon laser
confocal scanning microscopy (LEICA-TCS-SP8MP).
Synthesis of the fluorescent probes
Synthesis of compound 2
As shown in Scheme 1, 4-Bromo-1, 8-naphthalic anhydride (0.8
g, 2.89 mmol) and n-Propylamine (0.262 ml, 3.18 mmol) were
dissolved in ethanol (15 mL). The reaction mixture was stirred
and refluxed for 4.5 h. After cooling to room temperature, the
mixture was poured into ice water to form the precipitate. The
precipitate was isolated, washed with water and cool ethanol,
and then dried to afford grey white solid 2 (0.79 g; yield:
85.6%).1H NMR (600 MHz, CDCl3) δ 8.64 (d, J = 7.2 Hz, 1H), 8.54
(d, J = 8.4 Hz, 1H), 8.40 (d, J = 7.8 Hz, 1H), 8.03 (d, J = 7.8 Hz,
1H), 7.84 (t, J = 7.9 Hz, 1H), 4.23 – 4.03 (m, 2H), 1.84 – 1.67 (m,
2H), 1.03 (t, J = 7.4 Hz, 3H). 13C NMR (151 MHz, CDCl3) δ 163.64,
163.61, 133.19, 132.01, 131.20, 131.08, 130.62, 130.17, 129.01,
128.07, 123.16, 122.30, 77.24, 77.03, 76.82, 42.08, 21.37,
11.51.
Fig. 9 “off-on” fluorescent mechanism of Nap-Glc given by theoretical
calculations.
Conclusions
In this work, a glycosylation strategy based on click chemistry
was employed to develop naphthalimide-based Fe3+
fluorescent probe with good water-solubility and low
cytotoxicity. The selectivity and sensitivity to Fe3+ of three
synthesized naphthalimide-based fluorescent probes follows a
Nap-PZ<Nap-OH<Nap-Glc trend, where Nap-PZ was modified
by good water-soluble group. The cytotoxicity to cell follows a
Nap-PZ>Nap-OH>Nap-Glc trend. The reason was that the
exposed piperazine nitrogen of Nap-PZ was shielded by good
biocompatible group. The detection limit toward Fe3+ ion
Synthesis of Nap-PZ
Piperazine (0.855 g, 9.93 mmol) was dissolved in 2-
methoxyethanol (15 mL), and compound 2 (0.79 g, 2.48 mmol)
was added. The reaction mixture was stirred and refluxed for 4
h
under nitrogen atmosphere. After cooling to room
toward Fe3+ ion follows a Nap-PZ (7.40
(2.73
10-7M)>Nap-Glc(4.27 10-8M) trend. The fluorescent
“off-on” response of Nap-Glc at 528 nm ( ex=405 nm) towards
×
10-6M)>Nap-OH
temperature, the solvent was evaporated under reduced
pressure, and the crude product was purified by silica gel
column chromatography using CH2Cl2/MeOH (25:1, v/v) as
eluent to give yellow solid (0.6 g ; yield 74.72%).1H NMR (600
MHz, MeOD) δ 8.40 (ddd, J = 6.6, 5.0, 0.9 Hz, 2H), 8.35 (d, J =
8.1 Hz, 1H), 7.68 (dd, J = 8.3, 7.3 Hz, 1H), 7.24 (d, J = 8.1 Hz, 1H),
4.06 – 4.00 (m, 2H), 3.24 (d, J = 4.8 Hz, 4H), 3.19 – 3.14 (m, 4H),
1.75 – 1.66 (m, 2H), 0.99 (t, J = 7.5 Hz, 3H). 13C NMR (151 MHz,
MeOD) δ 164.44, 164.05, 156.53, 132.29, 130.69, 130.45,
129.57, 125.99, 125.51, 122.70, 116.03, 114.81, 53.43, 48.03,
47.88, 47.74, 47.60, 47.46, 47.32, 47.17, 45.18, 41.31, 20.98,
×
×
λ
Fe3+ was easily recognized by naked-eye under UV light. Nap-
Glc rapidly responded to Fe3+ within one minute. Thus, Nap-Glc
was a novel glucosyl naphthalimide fluorescent probe with
excellent water-solubility and low cytotoxicity, which could be
independently applied for the detection of Fe3+ in aqueous
+
medium, and not disturbed by Ag+, Ba2+, Ca2+, NH4 , Cd2+, Co2+,
Cu2+, K+, Li+, Mg2+, Mn2+,Na+, Ni2+, Zn2+, Ce3+, Eu3+, Fe2+, Pb2+
-
-
2-
and Br-, CH3COO-, Cl-, F-, H2PO42-, HSO3 , I-, NO3 , S2O32-, SO4
.1:2 stoichiometry was the most likely recognition mode of
Nap-Glc toward Fe3+, and the corresponding “off-on”
fluorescent mechanism of Nap-Glc was demonstrated by
theoretical calculations. The cell imaging assay indicated that
Nap-Glc was membrane-permeable and could be used to
detect Fe3+ in living cells.
+
10.33. HRMS calcd for C19H22N3O2 ([M + H] +) 324.1706, found
324.1700.
Synthesis of compound 4
A mixture of compound
3 (0.261 g, 1.48 mmol), K2CO3 (0.256 g,
1.86 mmol) and Nap-PZ (0.4, 1.24 mmol) in 15 mL dry acetone
was refluxed for
6
h, then cooled to room temperature,
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filtered, and the filter-cake was washed with acetone for three solution of probes (1 mM) was prepared in ViDewMArStiOcle. OTnlhinee
times. The crude product from the concentration of the filtrate fluorescence was measured with an excDitOaIt:io10n.10w3a9/vCe7lDenT0g1t0h99aKt
in vacuum was purified by silica gel column chromatography 405 nm by scanning the spectra between 350 nm and 680 nm.
using CH2Cl2/MeOH (60:1, v/v) as eluent to afford yellow solid The bandwidth for both excitation and emission spectra was 3
(0.43 g ; yield 83.07%). 1H NMR (600 MHz, CDCl3) δ 8.59 (dt, J = nm.
5.7, 2.8 Hz, 1H), 8.53 (dd, J = 8.0, 5.7 Hz, 1H), 8.42 – 8.37 (m,
1H), 7.74 – 7.68 (m, 1H), 7.35 (s, 1H), 7.25 (dd, J = 8.0, 4.5 Hz, Cytotoxicity assays
1H), 4.18 – 4.10 (m, 4H), 3.34 (s, 4H), 3.23 (s, 2H), 2.91 (s, 4H),
HepG2 cells were incubated overnight on 96-well plates in
2.27 (t, J = 2.5 Hz, 1H), 1.79 – 1.74 (m, 2H), 1.02 (td, J = 7.4, 3.2
growth medium, and each well contained 5000 HepG2 cells.
Hz, 3H). 13C NMR (151 MHz, CDCl3) δ 169.58, 164.43, 164.00,
The cells were then treated with different concentrations of
probes for 24 h. The medium was removed and 10µL of MTT (5
mg/mL) was added to the wells. After incubation of 4 h at
37 °C, the absorbance was recorded by an ELX800 absorbance
microplate reader at 490 nm. The relative viabilities of cells
were calculated based on the data of five parallel tests.
155.42, 132.38, 131.17, 129.98, 129.83, 126.20, 125.89, 123.37,
117.26, 115.07, 79.52, 71.53, 61.39, 53.62, 53.05, 41.81, 28.74,
21.43, 11.54.
Synthesis of Nap-OH
To a solution of 4 (0.25 g, 0.60 mmol) and 5 (63 mg, 0.72 mmol)
in THF (4 mL) was added a solution of sodium ascorbate (48
mg, 0.24 mmol) and CuSO4·5H2O (48 mg, 0.19 mmol) in
distilled H2O (4 mL). The reaction mixture was stirred for 4 h at
r.t. under argon. The solvent was evaporated under reduced
pressure, and the crude product was purified by silica gel
column chromatography using CH2Cl2/MeOH (10:1, v/v) as
Computational details
The ground state geometries optimizations and exited state
calculations were respectively performed at density functional
theory (DFT) and time-dependent density functional theory
(TD-DFT)54 level with hybrid B3LYP55, 56 functional using the
polarized continuum (PCM)57 solvation model. 6-31g (d, p)
basis set was employed for the main group atoms (C, H, O, N),
whereas lanl2dz basis set, an effective core potential basis set,
was used for Fe with optimized secondary p function and
additional f-polarization function (ζf=0.246)58, 59. Frequency
calculation was carried out at the same level of theory to
ensure the optimized structure was a minimum. TD-DFT
calculations based on the corresponding optimized structure
of the ground state were used to calculate the vertical
excitation energies and interpret the absorption spectra (UV-
vis spectrum) in solvent (water). To reveal the reason for
“fluorescent on” phenomenon, further TD-DFT calculation on
the first singlet excited state was also conducted on Nap-Glc to
provide its excited state geometry as well as reproduce
emission energies. The electronic transitions character was
assigned based on the frontier molecular orbitals analysis. All
calculations were carried out using the Gaussian 09 package60.
1
eluent to afford yellow solid (0.253 g; yield 83.77%). H NMR
(600 MHz, CDCl3) δ 8.62 – 8.56 (m, 1H), 8.52 (d, J = 8.0 Hz, 1H),
8.38 (d, J = 8.4 Hz, 1H), 7.85 (t, J = 5.8 Hz, 1H), 7.72 (s, 1H), 7.72
– 7.68 (m, 1H), 7.23 (d, J = 8.0 Hz, 1H), 4.57 (d, J = 6.0 Hz, 2H),
4.52 – 4.47 (m, 2H), 4.17 – 4.11 (m, 2H), 4.10 – 4.05 (m, 2H),
3.32 (s, 4H), 3.20 (s, 2H), 2.87 (s, 4H), 1.81 – 1.72 (m, 2H), 1.02
(t, J = 7.4 Hz, 3H). 13C NMR (151 MHz, CDCl3) δ 170.36, 164.48,
164.03, 155.49, 144.40, 132.46, 131.14, 130.02, 129.83, 126.17,
125.82, 123.33, 123.31, 117.13, 115.12, 61.46, 61.07, 53.61,
52.99, 52.69, 41.82, 34.48, 21.42, 11.53. HRMS calcd for
C26H32N7O4+ ([M + H]+) 506.2510, found 506.2503.
Synthesis of Nap-Glc
Nap-Glc was synthesized with similar procedures to Nap-
OH.1H NMR (600 MHz, CD3OD) δ 8.37 (ddd, J = 31.1, 15.0, 7.7
Hz, 3H), 8.12 (s, 1H), 7.67 (dt, J = 14.2, 4.7 Hz, 1H), 7.23 (dd, J =
10.3, 5.2 Hz, 1H), 5.62 (d, J = 9.2 Hz, 1H), 4.57 (s, 2H), 4.03 (dd,
J = 11.6, 6.1 Hz, 2H), 3.90 (dt, J = 12.3, 5.5 Hz, 2H), 3.72 (dd, J =
12.2, 5.5 Hz, 1H), 3.61 – 3.56 (m, 2H), 3.53 – 3.49 (m, 1H), 3.37
(s, 1H), 3.30 (s, 3H), 3.24 (s, 2H), 2.87 (s, 4H), 1.70 (dd, J = 13.7,
7.3 Hz, 2H), 0.99 (t, J = 7.3 Hz, 3H). 13C NMR (151 MHz, CD3OD)
δ 171.49, 164.42, 164.02, 156.14, 144.83, 132.27, 130.68,
130.45, 129.50, 125.86, 125.47, 122.64, 122.04, 115.90, 114.81,
88.19, 79.73, 77.07, 72.63, 69.51, 60.99, 60.82, 53.41, 53.10,
Cell imaging assay
HepG2 cell lines were incubated for 24 h in DMEM medium (5%
CO2, 37℃) and then treated with probe Nap-Glc (10 μM) for 30
min, washed twice with PBS to remove the extracellular probe.
The cells were fixed by 4% paraformaldehyde solution at room
temperature and washed 4 times with deionized water, after
that, the cells were treated with different concentrations of
Fe3+ and incubated for 20 min. Finally, the cell images of
HepG2 treated with Nap-Glc alone, Nap-Glc and different
concentrations Fe3+ were taken with Multi-photon laser
confocal scanning microscopy.
+
52.58, 41.32, 33.97, 20.99, 10.36. HRMS calcd for C30H38N7O8
([M + H] +) 624.2776, found 624.2758.
Spectroscopic measurements
The stock solutions of 1 mM of Fe(NO3)3·9H2O , Cu(NO3)2·3H2O ,
Pb(NO3)2, Eu(NO3)3·6H2O, Co(NO3)2·6H2O, Ce(NO3)3·6H2O,
Cd(NO3)2·4H2O, AgNO3, Ba(NO3)2, Ni(NO3)2·6H2O, NH4Cl, ZnCl2,
CaCl2 , CaCl2, NaCl, KCl, MgSO4, FeSO4·7H2O, MnSO4·H2O,
CH3COONa, NaHSO3, Na2SO3, NaF, NaBr, NaI, and NaNO3,
Na2HPO4·2H2O were prepared in deionized water. The stock
Acknowledgements
W. J. Y. thanks the support of the National Natural Science
Foundation of China (No.21262004) and Guangxi Key Laboratory of
Traditional Chinese Medicine Quality Standards (No.201602), and
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W. M. thanks the support of Study Abroad Program for Excellent
Ph.D Students of Guangxi Zhuang Autonomous Region.
27. M. H. Lee, N. Park, C. Yi, J. H. Han, J. H. Hong, KV.iePw. AKritmicle, ODn.liHne.
DOI: 10.1039/C7DT01099K
Kang, J. L. Sessler, C. Kang and J. S. Kim, J Am Chem Soc, 2014,
136, 14136-14142.
28. D. T. Shi, D. Zhou, Y. Zang, J. Li, G. R. Chen, T. D. James, X. P. He
and H. Tian, Chem Commun (Camb), 2015, 51, 3653-3655.
29. M. H. Lee, J. H. Han, P. S. Kwon, S. Bhuniya, J. Y. Kim, J. L.
Sessler, C. Kang and J. S. Kim, J Am Chem Soc, 2012, 134, 1316-
1322.
30. M. H. Lee, H. M. Jeon, J. H. Han, N. Park, C. Kang, J. L. Sessler
and J. S. Kim, J Am Chem Soc, 2014, 136, 8430-8437.
31. P. Jin, C. Jiao, Z. Guo, Y. He, S. Zhu, H. Tian and W. Zhu,
ChemSci, 2014, 5, 4012.
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