A novel fluorescent molecule based on 1,8-naphthalimide: Synthesis, spectral properties, and application in cell imaging

Article abstract of DOI:10.1007/s11164-010-0214-6

A novel naphthalimide molecule, 4-(2-methoxyethoxy)-N-butyl-1,8- naphthalimide (MEBN), has been synthesized and characterized. The UV-visible absorption and fluorescence emission spectral properties in n-hexane, toluene, CHCl₃, and CH₃OH were studied. Its optical properties depend on solvent polarity. The application of MEBN in cell imaging was investigated.

Full text of DOI:10.1007/s11164-010-0214-6

Res Chem Intermed (2010) 36:1021–1026
DOI 10.1007/s11164-010-0214-6
A novel fluorescent molecule based on 1,8-
naphthalimide: synthesis, spectral properties,
and application in cell imaging
Haibo Xiao
Lei Wang
•
Minjuan Chen
•
Guanghao Shi
•
•
Hongyao Yin Chong Mei
•
Received: 28 June 2010 / Accepted: 14 October 2010 / Published online: 23 November 2010
Ó The Author(s) 2010. This article is published with open access at Springerlink.com
Abstract A novel naphthalimide molecule, 4-(2-methoxyethoxy)-N-butyl-1,8-
naphthalimide (MEBN), has been synthesized and characterized. The UV–visible
absorption and fluorescence emission spectral properties in n-hexane, toluene,
CHCl , and CH OH were studied. Its optical properties depend on solvent polarity.
3
3
The application of MEBN in cell imaging was investigated.
Keywords 1,8-Naphthalimide Á Fluorescence Á Synthesis Á Cell imaging
Introduction
Derivatives of 1,8-naphthalimide usually exhibit strong fluorescence. They have
been widely used as colorants in the polymer industry, fluorescent probes for
medical and biological purposes, and as n-type materials in organic light-emitting
diodes [1]; they are potential anti-HIV drugs, fluorescent cell markers, DNA-
cleaving agents, liquid-crystal additives, and laser dyes [1].
The excellent fluorescent properties of commercial 1,8-naphthalimide com-
pounds depend largely on the presence of strong electron-donating groups, typically
such groups as alkoxy and alkylamine, at the C-4 position [2]. It is known that
4
-alkoxy-1,8-naphthalimides have been widely used as fluorescent brightening agents.
In this paper, a novel 4-alkoxy-1,8-naphthalimide compound, 4-(2-methoxyethoxy)-
N-butyl-1,8-naphthalimide (MEBN) was prepared by a simple method. The
UV–visible absorption and fluorescence emission spectral properties were studied.
Application in cell imaging was investigated.
H. Xiao (&) Á M. Chen Á G. Shi Á L. Wang Á H. Yin Á C. Mei
Department of Chemistry, Shanghai Normal University, 200234 Shanghai,
People’s Republic of China
e-mail: xiaohb@shnu.edu.cn
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H. Xiao et al.
Experimental
Solvents and reagents were obtained from commercial sources. 1,8-Naphthalic
anhydride was recrystallized from chlorobenzene before use. Other chemicals were
analytical regents and used as received. Further purification and drying were
employed by standard methods, and they were distilled before use when necessary.
Melting points were determined with an XT4A apparatus and are uncorrected. Mass
1
spectral studies were carried out using a VG12-250 mass spectrometer. H NMR
1
and C NMR spectra were obtained on a Bruker DRX 400 MHz spectrometer.
3
Chemical shifts are reported in ppm relative to Me Si as standard. Carbon, nitrogen,
4
and hydrogen were determined on an Elementar Vario EL. Steady-state emission
and excitation spectra were recorded on a Perkin–Elmer LS55 instrument. Visible
absorption spectra were determined on a Perkin–Elmer Lambda 35 spectrophotom-
eter. Cell images were acquired by use of a luminescence microscope.
Preparation of 4-bromo-N-butyl-1,8-naphthalimide
A mixture of 4-bromo-N-butyl-1,8-naphthalic anhydride (5.0 g, 18 mmol), butyl-
amine (1.7 g, 23.3 mmol), and 50 mL acetic acid was heated under reflux for 3 h
under nitrogen. After cooling, the mixture was dissolved in 100 mL water. The
crude solid was further purified by recrystallization from alcohol and 5.0 g white
1
solid was obtained, Yield 83.5%. mp 109–110 °C. H NMR (500 MHz, CDCl ):
3
d 8.68 (d, J = 7.2 Hz, 1H), 8.59 (d, J = 8.4 Hz, 1H), 8.43 (d, J = 7.6 Hz, 1H),
8
.06 (d, J = 8.0 Hz, 1H), 7.87 (t, J = 7.2, 7.2 Hz, 1H), 4.19 (t, J = 7.6, 7.6 Hz,
H), 1.73 (m, 2H), 1.46 (m, 2H), 1.00 (t, J = 7.2, 7.2 Hz, 3H).
2
Preparation of MEBN
A 50-mL round-bottomed, one-necked flask was charged with 1.2 g (3.01 mmol)
4-bromo-N-butyl-1,8-naphthalimide, 4 mL 2-methoxyethanol, 0.096 g (0.503
mmol) CuI, and 1.96 g (20 mmol) K CO . The reaction mixture was heated under
2
3
reflux for 3 h. After cooling, the mixture was extracted with ethyl acetate and
dried over MgSO . The crude solid was further purified by silica gel column
4
chromatography using petroleum ether and ethyl acetate (5:1, v/v) as eluent to
1
afford 1.1 g yellow solid, Yield 93%. mp 103–105 °C. H NMR (500 MHz,
CDCl ): d 8.61 (dd, J = 7.6, 2, 1.2 Hz, 2H), 8.54 (d, J = 8.4 Hz, 1H), 7.72
3
(
t, J = 7.2, 8.4 Hz, 1H), 7.05 (d, J = 8.4 Hz, 1H), 4.44 (t, J = 4.4, 4.8 Hz, 2H),
4
2
1
1
.18 (t, J = 7.2, 7.6 Hz, 2H), 3.95 (t, J = 4.4, 4.8 Hz, 2H), 3.54 (s, 3H), 1.72 (m,
1
3
H), 1.46 (m, 2H), 0.98 (t, J = 7.6, 7.2 Hz, 3H). C NMR (CDCl , 400 MHz)
3
4.08, 20.63, 29.90, 40.31, 59.59, 68.60, 70.90, 106.15, 115.56, 122.68, 123.74,
26.12, 128.94, 128.96, 129.61, 131.75, 133.47, 160.16, 164.15, 164.73. Ms:
?
m/z = 327 (M , 95.47%). Elemental analysis for: C H NO Calc.: C, 69.71; H,
1
9
21
4
6
.47; N, 4.28. Found: C, 69.50; H, 6.49; N, 4.26.
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A novel fluorescent molecule based on 1,8-naphthalimide
1023
Results and discussion
Synthesis
4
-Alkoxy-1,8-naphthalimides have been obtained from naphthalimides substituted
at the 4-position by electron-withdrawing groups by many procedures [3]. Most of
the synthetic methods for preparation of 4-alkoxy-1,8-naphthalimides are based on
nucleophilic aromatic substitution (S Ar) of halogens at the C-4 position by
N
alkoxylating agents in dipolar aprotic solvents such as N-methylpyrrolidinone,
DMF, and quinoline [3, 4]. Catalysts such as potassium or copper acetates and
phase-transfer catalysts are commonly used [3]. Some of these reactions are carried
out under reflux at high pressures up to 10 atm. The route to the synthesis of
MEBN is presented in Scheme 1. MEBN was obtained by nucleophilic substitution
of the bromide atom at the 4-position in 4-bromo-N-butyl-1,8-naphthalimide with a
methoxyethoxy group under copper-catalyzed conditions and use the reactant
2
-methoxyethanol itself as solvent. No phase-transfer catalyst is needed. In
comparison with other literature procedures for synthesis of 4-alkoxy-N-substituted-
,8-naphthalimides, our method has the advantages of employing simple and mild
1
conditions, having an easy work-up, and giving excellent yields.
Photophysical properties
The UV–visible absorption of MEBN in n-hexane, toluene, CHCl , and CH OH was
3
3
recorded to study its spectral properties in various solvents of different polarity
Fig. 1). The wavelengths corresponding to the spectral peaks are shown in Table 1.
(
As indicated in the absorption spectra, MEBN has a common low-energy broad
band at 350–360 nm. MEBN has structured absorption (there appear to be two
shoulder peaks) in nonpolar solvents such as hexane, which could be ascribed to the
lowest p–p* transitions [4, 5]. On the other hand, bathochromic shifts in absorption
due to solvent polarity were observed. For example, in hexane, MEBN had a kmax
value at 355 nm and a red-shift in absorbance for the polar solvent methanol at
3
65 nm. It is interesting to compare the effect of the substituents at C-4 on the
absorption spectrum. The absorption peak is observed at 355 nm for MEBN and
50 nm for 4-methoxy-N-butyl-1,8-naphthalimide (MBN) [4]. It can be seen that
3
methoxyethoxy has stronger electron-donating ability than methoxy [4, 6–8].
O
O
O
O
N
O
O
N
O
HOCH CH OCH
3
CH (CH ) NH
2
2
2
3
2 3
CH COOH
CuI/K CO
2 3
3
reflux, 3 h
reflux, 3 h
Br
Br
OCH CH OCH
3
2
2
Scheme 1 Synthesis of MEBN
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H. Xiao et al.
1.0
0.8
0.6
0.4
0.2
0.0
Hexane
Toluene
CHCl3
CH OH
3
320
340
360
380
400
420
440
Wavelength (nm)
Fig. 1 Absorption spectra of MEBN in solvents of different polarity
Table 1 Absorption and
fluorescence data for MEBN in
solvents of different polarity
Solvent
Absorption
maxima (nm)
Emission
maxima (nm)
n-Hexane
342(sh), 355, 370(sh)
406
421
426
448
Toluene
360
364
365
CHCl
3
CH OH
3
sh, shoulder
The fluorescence spectra of MEBN are displayed in Fig. 2. It can be observed
from Fig. 2 that the fluorescence emission spectra of MEBN were also sensitive to
solvent polarity. They move gradually to lower energies with increasing solvent
polarity (Table 1). The effect of the polarity of the medium on the photoluminescent
maximum was more pronounced than for the absorption maximum. The long
wavelength of fluorescence emission observed in more polar solvents is undoubt-
edly due to an intramolecular charge-transfer transition. This observation demon-
strates the highly polar nature of the S excited state of MEBN [9, 10]. Comparing
1
MEBN and MBN, by changing the methoxy in MBN with 2-methoxyethoxy, the
fluorescence emission maximum (in hexane) is increased by approximately 16 nm.
This behavior can be understood by taking into consideration the different electron-
donating ability of the two substituents.
Cell imaging
To examine the potential of MEBN for imaging in cells, Hela cells were treated with
0.9% saline solution of MEBN (500 lg/mL, the cell culture medium was added
also). The water solubility of MEBN is good. It was accordingly possible to directly
1
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A novel fluorescent molecule based on 1,8-naphthalimide
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1.0
0.8
0.6
0.4
0.2
0.0
Hexane
Tolulene
CHCl3
CH OH
3
350
400
450
500
550
600
Wavelength (nm)
Fig. 2 Normalized fluorescence emission spectra of MEBN in solvents of different polarity
Fig. 3 Luminescence (left, kex = 365 nm) and phase contrast (right) images of Hela cells
employ a 0.9% saline solution of the dye without use of any solubilizing agent such
as CrEL (polyoxyethylene castor oil) [11]. Cells were incubated at room
temperature for 2 h and were then washed with aqueous PBS solution to remove
excess unbound dye. The slides were mounted and imaged by epifluorescence
microscopy. The one-photon epifluorescence microscopy images are presented in
Fig. 3. From Fig. 3, it is clear the dye had good permeability. After 48 h of
incubation, 73.9% of cells are alive, which indicates that the toxicity of MEBN is
low (In contrast, 100% survival is observed for Hela cells not treated with MEBN
[
12].)
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H. Xiao et al.
Conclusion
A novel naphthalimide molecule, MEBN, was synthesized by use of a simple
method. The absorption and photoluminescent spectra of MEBN in various solvents
were investigated. Its optical properties depend on solvent polarity. By fluorescence
cell imaging with MEBN, it was found that MEBN has good cell permeability, low
toxicity, and has potential in cell imaging techniques.
Acknowledgments This work was financially supported by the Innovation Program of Shanghai
Municipal Education Commission (no. 09YZ160).
Open Access This article is distributed under the terms of the Creative Commons Attribution
Noncommercial License which permits any noncommercial use, distribution, and reproduction in any
medium, provided the original author(s) and source are credited.
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