Bifunctional fluorescent probes for hydrogen peroxide and diols based on a 1,8-naphthalimide fluorophore

Article abstract of DOI:10.1007/s11426-013-4870-4

This report discloses a series of naphthalimide-based bifunctional fluorescent probes for hydrogen peroxide and diols. As a result, these molecules not only demonstrated high turn-on fluorescent response and good selectivity towards hydrogen peroxide over other relevant reactive oxygen species, but also displayed different responses to diols. Therefore, these fluorescent probes could be served as sensitive, selective and practical chemosensors for both hydrogen peroxide and diols under physiological-like conditions.

Full text of DOI:10.1007/s11426-013-4870-4

SCIENCE CHINA
Chemistry
January 2013 Vol.5 No.1: 1–6
doi: 10.1007/s11426-013-4870-4
• ARTICLES •
doi: 10.1007/s11426-013-4870-4
Bifunctional fluorescent probes for hydrogen peroxide and diols
based on a 1,8-naphthalimide fluorophore
SUN Wei, MA Zhao, LI Jing, LI WenHua, DU LuPei* & LI MinYong*
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (MOE), School of Pharmacy, Shandong University,
Jinan 250012, China
Received January 30, 2013; accepted February 28, 2013
This report discloses a series of naphthalimide-based bifunctional fluorescent probes for hydrogen peroxide and diols. As a re-
sult, these molecules not only demonstrated high turn-on fluorescent response and good selectivity towards hydrogen peroxide
over other relevant reactive oxygen species, but also displayed different responses to diols. Therefore, these fluorescent probes
could be served as sensitive, selective and practical chemosensors for both hydrogen peroxide and diols under physiological-
like conditions.
1,8-naphthalimide, bifunctional fluorescent probe, hydrogen peroxide, diols
strategy [19]. In general, the boronate groups were attached
1 Introduction
to the chromophores to adopt a non-fluorescent form, which
would generate the fluorescent phenol product upon the
cleavage of carbon-boron bond with H2O2. Herein, we
chose 1,8-naphthalimide as the fluorescent chromophore to
design boronate-probes, and further investigated their
hydrolyzed products, i.e., the corresponding boronic acids.
As a Lewis acid, boronic acid can bind with diols to form
five- or six-membered cyclic esters, which can result in fluo-
rescence change as well [20]. Therefore, we also examined
the binding behavior of our synthetic probes with diols.
As an increasingly recognized small molecule, hydrogen
peroxide (H2O2) has experienced much attention in the
fields of chemical, biological, and medical sciences [1, 2]. It
can be generated by responding to various stimuli, such as
growth factors [3, 4] and cytokines [5–8], and can act as a
second messenger in the cell signaling of proliferation, dif-
ferentiation and migration [9, 10]. Consequently, the detec-
tion of H2O2 is becoming the indispensable footstep to in-
quire its biological and physiological actions. As providing
necessary and critical information about target biomolecules
in in vivo cellular systems, fluorescent probe emerged as
one of the encouraging tools for the detection of hydrogen
peroxide. So far, numerous research papers have been pub-
lished in discovering novel fluorescent probes for hydrogen
peroxide, in which several reviews have encapsulated the
prevalent state of this field very well [11–14]. One interest-
ing case is the application of boronate as fluorogenic switch,
which relies on the selective H2O2-mediated transformation
of arylboronates to phenols [15–18]. Recently, several
fluorogenic probes were accordingly developed using this
2 Experimental
2.1 Materials and instruments
All reagents were purchased from Acros and Aldrich.
Twice-distilled water was used throughout all experiments.
Melting points were determined on an electrothermal melt-
1
ing point apparatus and were uncorrected. H NMR, 13C
NMR were recorded on a Bruker 300 NMR spectrometer.
Mass spectra were performed by the analytical and the mass
spectrometry facilities at Shandong University. Infrared
spectrum was recorded with a Nicolet-470 spectrophoto-
*Corresponding authors (email: dulupei@sdu.edu.cn, mli@sdu.edu.cn)
© Science China Press and Springer-Verlag Berlin Heidelberg 2013
chem.scichina.com www.springerlink.com

2
Sun W, et al. Sci China Chem January (2013) Vol.56 No.1
meter using KBr pellets in the 4000–400 cm1 region.
135.57, 134.48, 134.12, 130.33, 129.25, 127.47, 127.14,
124.41, 122.38, 84.46, 55.98, 55.86, 34.81, 24.68, 18.51,
13.05; IR (KBr, cm1): 2990, 2926, 1697, 1659, 1592, 1443,
1364, 1339, 1320, 1245, 1151, 1064, 858, 790, 758; HRMS
m/z calcd. for C20H23BNO4 [(M+H)+] 352.1715; found
352.1702.
2.2 Synthesis
As shown in Scheme 1, two boronate-probes 2a and 2b
were synthesized in two steps. Bromo-substituted naph-
thalimides 1a and 1b, which were prepared according to the
previous method [21], were converted to probes 2a and 2b
by palladium-mediated borylation in the total yields of 71.3
and 60.2%, respectively. The further hydrolysis of bono-
rates was achieved by reacting 2a and 2b with NaIO4 and
NH4OAc to afford the boronic acids 3a and 3b.
N-ethanol-4-(4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1,
8-naphthalimide (2b)
The synthesis is analogous to 2a with compound 1b as the
starting material. The product was obtained as a white solid,
yield 69.2% [22].
2-Ethyl-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-
N-ethyl-4-amino-1,8-naphthalimide (1a)
ylboronic acid (3a)
A mixture of 4-bromo-1,8-naphthalic anhydride (2.00 g, 7.2
mmol) and ethylamine (70% soln in water) (0.69 mL, 8.66
mmol) in 70 mL 1,4-dioxane were refluxed for 7 h. The
mixture was then poured into water to precipitate out a solid,
which was collected by filtration, washed with water, and dried
to yield 1 as a creamcolored solid (1.98 g, yield 90%) [21].
The compound 2a (52 mg, 0.15 mmol), NaIO4 (95 mg, 0.44
mmol), NH4OAc (24 mg, 0.44 mmol) were dissolved in 6
mL acetone/water (2/1) mixture. After stirred at room tem-
perature for 8 h, the mixture was filtrated, and the filtrate
was added with 6 mL cold water. The resulting white nee-
1
dles of 1a were obtained in 69.6% yield. H NMR (300
2-Ethanol-6-hydrazinobenzo[de]isoquinoline-1,3-dione (1b)
MHz, CDCl3):  1.227 (t, J = 7.2 Hz, 3 H), 4.097 (m, 2H),
7.866 (t, J = 7.8 Hz, 1 H), 8.057 (d, J = 7.2 Hz, 1 H), 8.488
(m, 2 H), 8.794 (m, 3 H); 13C NMR (DMSO, 75 MHz):
 163.29, 135.22, 133.81, 133.47, 132.55, 130.18, 129.39,
127.12, 126.70, 122.51, 122.17, 34.71, 13.10; IR (KBr, cm1):
3373, 2980, 1697, 1643, 1606, 1589, 1445, 1371, 1336,
1246, 1229, 1150, 1062, 870, 782, 759; HRMS m/z calcd.
for C14H13BNO4 [(M+H)+] 270.0932; found 270.0921.
The synthesis is analogous to 1a with ethanolamine as the
starting material. The product was obtained as a light yellow
solid. Yield 87%.
N-ethyl-4-(4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1,8-
naphthalimide (2a)
A mixture of compound 1a (230 mg, 0.76 mmol),
bis(pinacolato)diboron (270 mg, 1.13 mmol), Pd(dppf)Cl2
(15 mg, 0.04 mmol) and potassium acetate (250 mg, 2.27
mmol) in dioxane (5 mL) was stirred at 90 °C overnight
under nitrogen atmosphere. After cooling to room tempera-
ture, the reaction mixture was diluted with CH2Cl2, washed
with H2O and brine, and dried over Na2SO4. After removal
of solvent, the reaction mixture was purified by flash chro-
matography on silica gel (petroleum ether/methylene chlo-
ride = 2/1) to afford 2a as a white powder, 0.21 g, yield
79.2%. 1H NMR (300 MHz, CDCl3):  1.225 (t, J = 7.2 Hz,
3 H), 1.418 (s, 12 H), 4.087 (m, 2H), 7.926 (t, J = 7.8 Hz, 1
H), 8.243 (d, J = 7.2 Hz, 1 H), 8.502 (m, 2 H), 9.020 (d, J =
8.4 Hz, 1 H); 13C NMR (DMSO-d6, 75 MHz):  163.11,
2-Ethanol-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-
ylboronic acid (3b)
The synthesis is analogous to 3a with compound 2b as the
starting material. The product was obtained as a white solid,
1
yield 63.5%. H NMR (300 MHz, CDCl3):  3.635 (t, J =
6.6 Hz, 2 H), 4.166 (t, J = 6.6 Hz, 2 H), 4.798 (s, 1H), 7.863
(t, J = 7.8 Hz, 1 H), 8.056 (d, J = 7.2 Hz, 1 H), 8.478 (m, 2 H),
8.789 (m, 3 H); 13C NMR (DMSO-d6, 75 MHz):  163.66,
142.82, 135.14, 133.82, 132.56, 130.17, 129.38, 127.22,
126.69, 122.62, 122.27, 57.79, 41.81; IR (KBr, cm1): 3420,
2963, 1694, 1648, 1608, 1588, 1442, 1370, 1301, 1276, 1234,
1110, 1058, 784, 759; HRMS m/z calcd. for C14H13BNO5
Scheme 1 Synthesis of probes 2a, 2b, 3a and 3b.

Sun W, et al. Sci China Chem January (2013) Vol.56 No.1
3
[(M+H)+] 286.0918; found 286.0902.
displaying one major absorption band centered at ca. 352
nm ( = 11600–12850 M1 cm1) with the corresponding
blue-colored fluorescence maximum at 400 nm ( =
0.120–0.129) (Figure S1).
2.3 Fluorometeric analysis
Buffer reagents were purchased from Aldrich and Acros and
were used without purification. Water used for the fluores-
cence studies was doubly distilled and further purified with
a Mill-Q filtration system. Quartz cuvettes were used in all
studies. A Shimadzu UV-1700 UV-visible spectrometer was
used for all absorbance studies. Nanodrop ND3300 Fluoro-
spectrometer was used for all fluorescent studies. Solutions
of compound were prepared in 0.1 M phosphate buffer at
pH 7.4 (0.2% DMSO). Then the solutions were transferred
into a 1 cm quartz cell and UV absorbance data were rec-
orded immediately. The fluorescence excitation wave-
lengths used were the UV max.
3.2 Fluorescent responses to hydrogen peroxide
Afterwards, the fluorescent responses of these probes to-
ward H2O2 were evaluated. Upon treatment with H2O2, the
absorbance at 350 nm diminished and one major absorbance
(450 nm) appeared. The addition of H2O2 (400 M) trig-
gered very significant fluorescence increase (109-fold for
2a; 128-fold for 2b; 64-fold for 3a; 100-fold for 3b) at 560
nm as indicated in Figure 1 (excited at 450 nm). The fluo-
rescence intensities of compounds increased perceptibly as
a concentration-dependent function at more than 6.25 μM
level. The specificity of these probes was also investigated.
Figure 2 compares the relative reactivities of all four com-
pounds toward various reactive oxygen species (ROS). The
selectivity data are displayed at several time points. As de-
scribed in Figure 2, the four probes showed no significant
difference. All probes exhibited 80–110-fold higher re-
sponse to hydrogen peroxide than tert-butoxy radicals
(t-BuO·) and hydroxyl radical (·OH), about 2.0–3.6-fold
3 Results and discussion
3.1 Spectroscopic properties
We assessed the spectroscopic properties of the probes un-
der physiological-like conditions (100 mM PBS, pH 7.4).
These four probes have similar spectroscopic properties,
Figure 1 Concentration-dependent emission intensity changes of probes 2a (a), 2b (b), 3a (c) and 3b (d) at room temperature. Experiments were conducted
in 0.1 M phosphate buffer (0.2% DMSO), pH 7.4 with excitation at 450 nm. The emission spectra were obtained at 120 min after the addition of hydrogen
peroxide to a 10 M solution of probes. The concentrations of hydrogen peroxide are 0, 6.25, 12.5, 25, 50, 100, 200 and 400 M from bottom to top.

4
Sun W, et al. Sci China Chem January (2013) Vol.56 No.1
1

IH O /IKO
I
H O /I O
I
H O /ITBHP
IH O /It-BuO
IH O /I·OH
2 2
Probes
2a
IH O /IClO
2 2
2
2
2
2
2
2
2
2
2
2
3.6
2.0
2.1
2.1
2.1
11.0
9.1
20.6
74.1
81.7
94
2b
2.0
2.8
2.2
22.4
19.8
14.0
81
3a
10.4
7.3
94.5
101
3b
107.0
110.0
Figure 2 Fluorescence response of probes 2a (a), 2b (b), 3a (c) and 3b (d) to various reactive oxygen species (ROS). Data shown are for 100 M of H2O2,
1O2 and KO2, and 400 M of all other ROS. Hydrogen peroxide (H2O2), tert-butyl hydroperoxide (TBHP), and hypochlorite (OCl) were diluted from 30%,

70% and 5% aqueous solutions, respectively. Singlet oxygen (1O2) was generated by reaction of 1 mM OCl with 100 M H2O2. Superoxide (O2 ) was added
as solid KO2. Hydroxyl radical (·OH) and tert-butoxy radical (t-BuO·) were generated by reaction of 4 mM Fe2+ with 400 M H2O2 or 400 M TBHP, re-
spectively. Spectra were acquired in 0.1M phosphate buffer, pH 7.4, and all data were obtained after incubation (10 M solution of probes) with the appro-
priate ROS at room temperature. Emission intensity was collected at 560 nm (ex = 450 nm). Insert table: the intensity ratio of different ROS at 120 min.
more sensitive to hydrogen peroxide than singlet oxygen
and the fluorescence intensity increased significantly for
both boronic acids (Figure S5, S6 and S7). The probes had
no response to mannose, fucose, sucrose and lactose.
Observed 1:1 stability constants (Kobs) between the
probes and the diols were determined in 0.1 M phosphate
buffer (0.2% DMSO, pH = 7.4, Table 1), with the binding
ability of boronic acids being a little better than that of
bonorates. The affinity trend of diols with probes followed
the order: pyrogallol > catechol > D-sorbitol > D-fructose >
D-arabinose > D-galactose > D-glucose, which is consistent
with previous reports for other boronic acids [23–25]. This
phenomenon about fluorescence change could be explained
by electron-induced effect and the photoinduced electron
transfer (PET) quenching. With the formation of the cyclic
boronic esters in the reaction of boronic acid and diols, the
electron-induced effect resulted in fluorescence enhancement
while PET quenching [20] from cyclic esters reduced the
fluorescence.

(1O2) and superoxide (O2 , approximately 7.311.1-fold
more reactive than hypochlorite (ClO), and about 14.0–
22.4-fold more reactive than tert-butyl hydroperoxide
(TBHP). In other words, the four probes exhibit high
turn-on fluorescent response and high-pitched selectivity
toward H2O2 over other ROS, thus could be applied as
on/off fluorescent switches to detect hydrogen peroxide.
3.3 Fluorescent responses to diols
In view that the boric acid can bind with diols, we also tested
the fluorescent response of four probes toward different diols.
As depicted in Figure S2, S3, S4 and S8, the emission at 400
nm (ex = 350 nm) showed a remarkable decreasing trend
with increasing concentration of pyrogallol, catechol, sorbi-
tol and glucose. Whereas, upon treatment with fructose,
arabinose and galactose, the emission red shifted to 420 nm

Sun W, et al. Sci China Chem January (2013) Vol.56 No.1
5
Table 1 The Kobs values between the probes and the diols
2a
2b
3a
3b
Keq
Fluo.
response
Keq
Fluo.
response
Keq
Fluo.
response
Keq
Fluo.
response
(M1 × 103)
12.3 ± 0.8
(M1 × 103)
13.4 ± 0.7
(M1 × 103)
17.0 ± 0.8
(M1 × 103)
18.9 ± 1.0
pyrogallol
catechol
↓
↓
↓
↑+
↑+
↑+
↓

↓
↓
↓
↑+
↑+
↑+
↓

↓
↓
↓
↑+
↑+
↑+
↓

↓
↓
↓
↑+
↑+
↑+
↓

8.7 ± 0.5
10.2 ± 0.6
13.1 ± 1.1
15.4 ± 1.0
sorbitolum
D-fructose
D-arabinose
D-galactose
D-glucose
D-mannose
L-fucose
4.0 ± 0.2
4.1 ± 0.2
4.1 ± 0.2
4.1 ± 0.2
3.1 ± 0.1
3.0 ± 0.1
3.4 ± 0.1
3.4 ± 0.1
0.26 ± 0.03
0.22 ± 0.02
0.29 ± 0.04
0.37 ± 0.05
0.22 ± 0.01
0.20 ± 0.02
0.22 ± 0.04
0.22 ± 0.04
0.052 ± 0.008
0.072 ± 0.01
0.060 ± 0.004
0.099 ± 0.012















sucrose








lactose








Notes: “↓” indicates the decrease of fluorescent intensity; “↑+”indicates the increase of fluorescent intensity and the red shift of emission peak; “” in-
dicates no significant changes.
5
6
Ohba M, Shibanuma M, Kuroki T, Nose K. Production of hydrogen
peroxide by transforming growth factor-beta 1 and its involvement in
induction of egr-1 in mouse osteoblastic cells. J Cell Biol, 1994, 126:
1079–1088
Strassheim D, Asehnoune K, Park JS, Kim JY, He QB, Richter D,
Mitra S, Arcaroli J, Kuhn K, Abraham E. Modulation of bone
marrow-derived neutrophil signaling by H2O2: Disparate effects on
kinases, NF-kappa B, and cytokine expression. Am J Physiol Cell
Physiol, 2004, 286: C683–C692
Geiszt M, Leto TL. The Nox family of NAD(P)H oxidases: Host
defense and beyond. J Biol Chem, 2004, 279: 51715–51718
Park HS, Lee SH, Park D, Lee JS, Ryu SH, Lee WJ, Rhee SG, Bae
YS. Sequential activation of phosphatidylinositol 3-kinase, beta Pix,
Rac1, and Nox1 in growth factor-induced production of H2O2. Mol
Cell Biol, 2004, 24: 4384–4394
Sundaresan M, Yu ZX, Ferrans VJ, Irani K, Finkel T. Requirement
for generation of H2O2 for platelet-derived growth factor signal
transduction. Science, 1995, 270: 296–299
4 Conclusions
In summary, four probes were designed and facilely synthe-
sized from readily available cheap starting materials. They
were examined under various conditions and demonstrated
to be bifunctional fluorescent probes for H2O2 and diols.
The probes themselves displayed no fluorescence when
being excited at 450 nm, however, the addition of H2O2
triggered an obvious fluorescence increase at 560 nm. And
these probes exemplified high-pitched selectivity toward
H2O2 over other ROS. Therefore, compared with the
previous probes, our naphthalimide-based boronates and
boronic acids are accessible, switchable, sensitive, selective
and practical. In addition, our four probes were able to re-
spond to different diols. Their selective binding to pyrogal-
lol, catechol, sorbitol and glucose caused immediate and
remarkable fluorescence decrease, while to fructose, arabi-
nose and galactose caused immediate and obvious fluores-
cence enhancement at longer emission wavelength. Hence,
these probes could be served as sensitive and selective bi-
functional chemosensors for H2O2 and diols.
7
8
9
10 Rhee SG, Bae YS, Lee SR, Kwon J. Hydrogen peroxide: A key
messenger that modulates protein phosphorylation through cysteine
oxidation. Sci STKE, 2000, 2000: 1–6
11 Sun W, Wu J, Li J, Fang H, Du L, Li M. Boronate can be the
fluorogenic switch for the detection of hydrogen peroxide. Curr Med
Chem, 2012, 19: 3622–3634
12 Kalyanaraman B, Darley-Usmar V, Davies KJ, Dennery PA, Forman
HJ, Grisham MB, Mann GE, Moore K, Roberts LJ, 2nd, Ischiropoulos
H. Measuring reactive oxygen and nitrogen species with fluorescent
probes: Challenges and limitations. Free Radic Biol Med, 2012, 52: 1–6
13 Lippert AR, Van de Bittner GC, Chang CJ. Boronate oxidation as a
bioorthogonal reaction approach for studying the chemistry of
hydrogen peroxide in living systems. Acc Chem Res, 2011, 44:
793–804
The present research was supported by the Shandong Natural Science
Foundation (JQ201019) and the Independent Innovation Foundation of
Shandong University, IIFSDU (2010JQ005).
14 Rhee SG, Chang TS, Jeong W, Kang D. Methods for detection and
measurement of hydrogen peroxide inside and outside of cells. Mol
Cells, 2010, 29: 539–549
15 Du LP, Ni NT, Li MY, Wang BH. A fluorescent hydrogen peroxide
probe based on a ‘click’ modified coumarin fluorophore. Tetrahedron
Lett, 2010, 51: 1152–1154
16 Du LP, Li MY, Zheng SL, Wang BH. Rational design of a
fluorescent hydrogen peroxide probe based on the umbelliferone
fluorophore. Tetrahedron Lett, 2008, 49: 3045–3048
17 Kuivila HG, Armour AG. Electrophilic displacement reactions. IX.
Effects of substituents on rates of reactions between hydrogen
peroxide and benzeneboronic Acid1-3. J Am Chem Soc, 1957, 79:
5659–5662
1
2
3
Rhee SG. H2O2, a necessary evil for cell signaling. Science, 2006,
312: 1882–1883
Veal EA, Day AM, Morgan BA. Hydrogen peroxide sensing and
signaling. Molecular Cell, 2007, 26: 1–14
Bae YS, Kang SW, Seo MS, Baines IC, Tekle E, Chock PB, Rhee SG.
Epidermal growth factor (EGF)-induced generation of hydrogen
peroxide. Role in EGF receptor-mediated tyrosine phosphorylation. J
Biol Chem, 1997, 272: 217–221
Azar ZM, Mehdi MZ, Srivastava AK. Activation of insulin-like
growth factor type-1 receptor is required for H2O2-induced PKB
phosphorylation in vascular smooth muscle cells. Can J Physiol
Pharmacol, 2006, 84: 777–786
4
18 Kuivila HG, Wiles RA. Electrophilic displacement reactions. VII.

6
Sun W, et al. Sci China Chem January (2013) Vol.56 No.1
Catalysis by chelating agents in the reaction between hydrogen
22 Sun W, Li W, Li J, Zhang J, Du L, Li M. Naphthalimide-based fluo-
rescent off/on probes for the detection of thiols. Tetrahedron, 2012,
68: 5363–5367
peroxide and benzeneboronic acid1,2,3. J Am Chem Soc, 1955, 77:
4830–4834
19 Zielonka J, Sikora A, Hardy M, Joseph J, Dranka BP, Kalyanaraman
B. Boronate probes as diagnostic tools for real time monitoring of
peroxynitrite and hydroperoxides. Chem Res Toxicol, 2012, 25:
1793–1799
23 Shen QJ, Jin WJ. Chemical and photophysical mechanism of
fluorescence enhancement of 3-quinolineboronic acid upon change of
pH and binding with carbohydrates. Luminescence, 2010, 26: 494–
499
20 Wu Z, Li M, Fang H, Wang B. A new boronic acid based fluorescent
reporter for catechol. Bioorg Med Chem Lett, 2012, 22: 7179–7182
21 Gunnlaugsson T, Kruger PE, Jensen P, Tierney J, Ali HD, Hussey
GM. Colorimetric “naked eye” sensing of anions in aqueous solution.
J Org Chem, 2005, 70: 10875–10878
24 Jin S, Wang J, Li M, Wang B. Synthesis, evaluation, and computational
studies of naphthalimide-based long-wavelength fluorescent boronic
Acid reporters. Chemistry, 2008, 14: 2795–2804
25 Springsteen G, Wang BH. A detailed examination of boronic
acid-diol complexation. Tetrahedron, 2002, 58: 5291–5300