Synthesis and spectral studies of 2-[(N-ethyl carbazole)-3-sulfonyl ethylenediamine]-1-N,N-2-(2-methypyridy) as a fluorescence probe for Zn 2+

Article abstract of DOI:10.1016/j.bmcl.2011.11.004

A novel Zn²⁺ fluorescence probe, 2-[(N-ethyl carbazole)-3-sulfonyl ethylenediamine]-1-N,N-bis(2-methypyrbidy), was designed and synthesized via simple steps, and its structure was confirmed by IR and ¹H NMR. The probe gives significant fluorescence enhancement immediately following Zn²⁺ addition at neutral pH and exhibits improved selectivity for Zn²⁺ compared to the other metal ions in aqueous solution. The spectra and fluorescence quantum yield of the synthesized compound were carefully investigated by UV-vis absorption and fluorescence spectra in various solvents.

Full text of DOI:10.1016/j.bmcl.2011.11.004

Bioorganic & Medicinal Chemistry Letters 22 (2012) 343–346
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and spectral studies of 2-[(N-ethyl carbazole)-3-sulfonyl
ethylenediamine]-1-N,N-2-(2-methypyridy) as a fluorescence probe for Zn2+
Jun Zhang a, Huiling Cui a, Masashi Hojo b, Shaomin Shuang a, Chuan Dong a,
⇑
a Research Center of Environmental Science and Engineering, School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006, PR China
b Department of Chemistry, Faculty of Science, Kochi University, Kochi 780-8520, Japan
a r t i c l e i n f o
a b s t r a c t
A novel Zn2+ fluorescence probe, 2-[(N-ethyl carbazole)-3-sulfonyl ethylenediamine]-1-N,N-bis(2-methy-
pyrbidy), was designed and synthesized via simple steps, and its structure was confirmed by IR and 1H
NMR. The probe gives significant fluorescence enhancement immediately following Zn2+ addition at neu-
tral pH and exhibits improved selectivity for Zn2+ compared to the other metal ions in aqueous solution.
The spectra and fluorescence quantum yield of the synthesized compound were carefully investigated by
UV–vis absorption and fluorescence spectra in various solvents.
Article history:
Received 7 June 2011
Revised 18 October 2011
Accepted 2 November 2011
Available online 9 November 2011
Keywords:
Carbazole
Ó 2011 Elsevier Ltd. All rights reserved.
Absorption spectrum
Fluorescence probe
Solvent effect
Selectivity
Because the availability of better Zn2+-specific probes would
provide additional insight into the cell biology of Zn2+, interest in
the field remains high, and many fluorescent sensors for Zn2+ have
been described in the past decade.1–3 Many factors determine the
performance of fluorescence probe in vivo, including their photo-
physical properties, sensitivity, and selectivity for the analyte of
interest, affinity for the analyte and other species in the biological
milieu.4 In the current study, there is still scope for improvement
in the design of such sensors as they often suffer from disadvan-
tages such as sensitivity to H+, Ca2+ and Mg2+, short excitation
and emission wavelengths, small stokes shifts and cumbersome
synthesis.5
Some fluorophore or its modificated derivatives exhibit a weak
emission in the free state but they fluoresce intensely accompanied
by a spectral shift when bound to Zn2+ or other metal ions.6,7 This
property is desirable for fluorescence ratiometric determination of
the metal ions. Carbazole derivatives are fluorescent material pos-
sessing excellent fluorescent characteristics, which have been
widely used in construction of light-emitting device and fluorescent
detection.8–10 With perfect planarity and conjugation in molecular
structure, carbazole may be desirable for fluorescence ratiometric
determination of Zn2+, although there have been very few studies re-
lated to carbazole derivatives as fluorescent probes for metal ions. It
is also known that one of the most outstanding properties of Zn2+ ion
is a strong affinity for aromatic sulfonamides or N,N,N0,N0-tetrakis
(2-pyridylmethyl)-ethylenediamine (TPEN). Therefore, if aromatic
sulfonamides and TPEN were assembled together with carbazole,
the target compound should be a good fluorescence probe for Zn2+
.
In previous papers, we reported synthesis and spectrum charac-
teristic of new organic fluorescent dyes of pyrazoline compounds
and determination of human serum albumin using an intramolec-
ular charge transfer fluorescence probe: 40-Dimethylamino- 2,5-
dihydroxychalcone.11,12 The direct interaction of alkali metal or
alkaline earth metal ions not only with Rhodamine B base or a
fluoran-based color former but also with 1-(2-pyridylazo)-2-naph-
thol and its derivative has been demonstrated.13,14
The present work aims to describe the synthesis, chemical char-
acterization and spectral properties of a new compound containing
TPEN derivative, N,N-bis(2-pyridylmethyl)ethylenediamine linked
to aromatic sulfonamides units which are connected carbazole
(fluorescent signaling probe) units (see compound e). As we know,
nitrogen-rich tri- and tetra-dentate ligands coordinate with Zn2+
with high affinity. We have synthesized the new derivative of car-
bazole (compound e), exploiting methodology for coupling TPEN
with carbazole. This approach provides a straightforward and con-
venient means to access a compound that otherwise would require
a difficult, multistep route using conventional organic techniques.
The structures of the target compound have been fully character-
ized by IR, 1H NMR and fluorescence techniques. The synthesized
compound was used as a fluorescent probe for Zn2+ in aqueous
media under physiological buffer conditions and showed its out-
standing characteristics including nice sensitivity and selectivity
for zinc over other metal ions.
⇑
Corresponding author. Tel.: +86 0351 7018613; fax: +86 0351 7011322.
E-mail address: dc104@sxu.edu.cn (C. Dong).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.004

344
J. Zhang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 343–346
The synthesis route of compound 2-[(N-ethyl carbazol)-3-
sulfonyl ethylenediamine]-1-N,N-2-(2-methypyridy) is shown in
Scheme 1.
200
150
100
50
b
Figure 1 shows the drastic changes in the fluorescence spectrum
from the parent compound, carbazole, to the product (e). From car-
bazole to N-ethyl carbazole, a shift towards longer wavelengths in
the fluorescence-emission spectra and an increase in fluorescence
intensity are observed. It is known that the molecular structure
of the carbazole possesses a good conjugated system and rigid
plane. According to the intramolecular charge transfer (ICT) theory,
a conjugate connection between the fluorophore and electron do-
nor is formed by the introduction of electron-donating ethyl group,
results in a ICT from donor to acceptor, and then reduces the en-
ergy levels of electronic transitions in the molecule, which causes
the changes in fluorescence spectra.
a
e
d
c
0
350
400
450
500
Wavelength (nm)
From N-ethyl carbazole to N-ethyl carbazole-3-sulfonyl chlo-
ride, significantly reduced fluorescence intensity and red shift in
fluorescence spectra are detected. These phenomenon can be ex-
plained by the electron withdrawing effect caused by the directly
connection of strong electron-accepter chlorosulfonyl group with
carbazole at the benzene ring.
Figure 1. The fluorescent emission spectra of (a) carbazole (1.2 ꢀ 10ꢁ7 M), (b) N-
ethyl carbazole (3.2 ꢀ 10ꢁ7 M), (c) N-ethyl carbazole-3-sulfonyl chloride
(4.1 ꢀ 10ꢁ6 M),
(d)
2-[(N-ethy
carbazole)-3-sulfonyl
ethylenediamine
(2.4 ꢀ 10ꢁ7 M), (e) 2-[(N-ethy carbazole)-3-sulfonyl ethylenediamine]-1-N,N-2-(2-
methypyridy) (5.6 ꢀ 10ꢁ7 M) in CH2Cl2.
From N-ethyl carbazole-3-sulfonyl chloride to 2-[(N-ethyl car-
bazole)-3-sulfonyl ethylenediamine, the introduction of two elec-
tron-donating amidocyanogen weakens the electron withdrawing
effect of chlorosulfonyl group, which are responsible for the slight
recovery of fluorescence intensity and the red shift in fluorescence
spectra.
As for the spectrum change from 2-[(N-ethyl carbazole)-3-sulfo-
nyl ethylenediamine to 2-[(N-ethy carbazole)-3-sulfonyl ethylene-
diamine]-1-N,N-2-(2-methypyridy), a large conjugated structure is
resulted in when two methypyridy groups are joined. The strong
ICT occurs in the first excited singlet state can induce large molec-
ular polarizability, making a red shift of the fluorescence emission
wavelength and a concomitant increase of half-band-width value.
As seen in Figure 1 for compound (e), the fluorescence intensity is
moderately weaker comparing with other compounds which sug-
gests that the formed compound system is so large that the rigid
planar structure of molecular may have been distorted to some
extent.
chloride. As shown in Figure 2, the absorption spectra of target
compound (e) exhibits a redshift in chloroform but a blueshift in
ethanol and tetrahydrofuran respectively compared to carbon tet-
rachloride (nonpolar solvent). The blueshift value is 62 nm in eth-
anol and 44 nm in tetrahydrofuran. The redshift value is 24 nm in
chloroform. The spectral behavior suggests that the compound (e)
is relatively sensitive to variations in the solvent polarity. Such
sensitivity to microenvironment may be an advantage toward
achieving a sensor with good photophysical properties and affinity
or Zn2+ selectivity.
The fluorescence spectrum of the probe compound (e) in vari-
ous solvents is shown in Figure 3. By a detailed analysis of the
absorption and fluorescence spectra, it is clear that the fluores-
cence spectra follow the same trend as the absorption spectra.
Judging from the blueshift in both fluorescence and absorption
spectra, it can be concluded that a hydrogen-bonding complex is
formed between the solvent and the fluorophore in ground state
and the slight blue shift in the fluorescence spectra can be rational-
ized by the hydrogen bonding interactions.
The spectra of product were studied in four solvents of different
polarity: ethanol, tetrahydrofuran, chloroform and carbon tetra-
The hydrogen bond donor-acceptor ability and the polarity of a
solvent will generally influence the actual fluorescence spectral shift
of fluorophores.15 In addition, the hydrogen-bonding interaction be-
tween the solvents and the compound molecules partly inhibits the
excited-state charge transfer between the lone-pair electrons on the
electron donating substituents and aromatic ring in the compounds
SO2Cl
ClSO3H
CH3CH2Br
N
N
N
H
C2H5
(b)
C2H5
(c)
(a)
1
4
1.0
0.8
0.6
0.4
0.2
0.0
SO2NHCH2CH2NH2
NH2CH2CH2NH2
N
C2H5
(d)
N
SO2NHCH2CH2 N
N
Cl·HCl
N
N
200
250
300
Wavelength (nm)
350
400
C2H5
(e)
Scheme 1. Synthesis of 2-[(N-ethyl carbazole)-3-sulfonylethylenediamine]-1-N,N-
Figure 2. Normalized absorption spectrum of the probe compound (e) in various
2-(2-methypyridy).
solvents: (1) ethanol, (2) tetrahydrofuran, (3) carbon tetrachloride, (4) chloroform.

J. Zhang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 343–346
345
a much weaker hydrogen bonding or protonation effects. This
behavior of compound (e) is also in accord with many known flu-
oroionophores carrying nitrogen heterocyclic, which also display
highly quenched fluorescence.17,18
1
4
800
600
400
200
0
Figure 4 shows the effect of Zn2+ ion on the fluorescence emis-
sion spectra of the synthesized compound (e) in solution of phys-
iological pH. The excitation, emission wavelength of compound (e)
is 303, 422 nm, respectively. The fluorescence quantum yield of
compound (e) is found to be 0.037. When ZnSO4 solution was
added, a significant linear increase in fluorescence intensity was
observed, and the quantum yield increased to 0.10 with only minor
changes in the maximum absorption and emission, indicating that
a complex may be formed between compound (e) and Zn2+
.
When concentration ratio of Zn2+/compound (e) reaches 1, the
fluorescence intensity of the solution does not change basically
with increasing concentration of Zn2+. The dissociation constant
Kd of the Zn2+ complex of compound (e) were determined as
1.2 nM by fitting the fluorescence data with a 1:1 association equa-
tion. That is to say, the probe can be used to quantitatively deter-
mine the concentration of Zn2+ around the sub-nanomolar range,
which affords sufficient sensitivity for application in mammalian
cells. The fluorescent response to Zn2+ by compound (e) can be
attributed to a inhibition of photoinduced electron transfer pro-
cess. It is possible that incorporation of Zn2+ ion enhances the rigid-
ity of molecular thus inhibiting some of non-radiative transitions
between electronic states or shuts down the photoinduced elec-
tron-transfer pathway of the excited free ligand.
350
400
450
500
Wavelength (nm)
Figure 3. Fluorescence emission spectra of the probe compound (e) (1 ꢀ 10ꢁ5 M) in
various solvents. (1) ethanol (kex = 224 nm, kem = 352/368 nm), (2) tetrahydrofuran
kem = 360/375 nm),
kem = 380 nm), (4) chloroform(kex = 310 nm, kem = 417 nm).
(kex = 242 nm,
(3)
carbon
tetrachloride(kex = 286 nm,
molecules, causing thus the blue shift of fluorescence spectra in the
solvents. On the other hand, the presence of hydrogen bonds may in-
crease the molecular coplanarity in excited state, which promote ICT
and thus exhibit a red shift in the fluorescence spectra. Therefore,
the actual measured shift of fluorescence spectral is the net result
of two operations, that is, the combination of solvent effect and
hydrogen bonding interactions on solute molecules. As shown in
Figure 3, the high fluorescence intensities are exhibited in strong po-
lar solvents accompanied by the appearance of vibrational fine
structure (the typical two sub-peaks) in fluorescence spectrum,
while the low fluorescence intensities are displayed in weakly polar
solvents with the disappearance of vibrational fine structure.
The ability of the molecules to emit the absorbed light energy is
characterized quantitatively by the fluorescence quantum yield.
The photoluminescent quantum yields of different compounds
were measured by relative method using Quinine sulfate as the
standard. The quantum yield was calculated from the following
Eq. 1:
The interference from other metal ions with the detection of the
Zn2+ ion was evaluated, which shows that other ions do not hinder
the Zn2+ detection. As can be seen from Table 2, the results clearly
demonstrate the selectivity for Zn2+ over the other metal ions. The
fluorescence of compound (e) is not influenced by cations such as
Na+, K+, Ca2+, Mg2+, which exist at high concentration under phys-
iological conditions, even at 9 mM. Thus, this molecule can be used
even under biological conditions involving an increase of Ca2+ con-
centration. The alkaline metals or alkaline earth metals ions have
no effect on the detection of Zn2+ ion, which can be attributed to
the poor complexation of these metal ions with the synthesized
fluorescent substance. What’s more, the fluorescence of this mole-
cule can be quenched to some extent by Fe3+, Cu2+, Co2+, probably
because there is electron or energy transfer between metal cation
In the above expression, Yu and Ys are the fluorescent quantum
yield of test and reference materials, Fu and Fs are the integration of
the emission intensities of test and reference materials, Au and As
are the absorbance of the test and reference material solutions
Fu As
Fs Au
Yu ¼ Ys ꢂ
ꢂ
ð1Þ
400
1.0
at the exciting wavelength, respectively.
0.8
350
Under the same conditions, the fluorescence quantum yields of
the goal product and the other compounds were measured with
the standard value of 0.55 at the excitation wavelength of
313 nm for quinine sulfate.16 Table 1 details the quantum yields
of the different compounds in various solvents.
When the planar configuration is distorted by the increased size
of the molecules, the fluorescence of products are gradually weak-
ened, due to an increase of the non-radioactive decay rate induced
by hampered ICT. Relatively poor fluorescence quantum yields for
dichloromethane compared to ethanol is understandable in view of
0.6
300
0.4
250
0.2
[Zn2+]
0.0
200
0
10
20
30
40
50
[Zn2+](µm)
150
100
50
Table 1
0
The fluorescence quantum yields of the compounds in various solvents
400
450
500
550
Compound
Solvent
Fluorescence quantum yield
Wavelength (nm)
Carbazole (a)
Ethanol
Ethanol
Ethanol
Dichloromethane
0.57
0.64
0.19
0.11
Figure 4. Fluorescence spectra of the compound (e) (1 ꢀ 10ꢁ5 M) in H2O (HEPES:
100 mM, pH7.4, I = 0.1 (KNO3), 10 mM NTA) with incremental addition of Zn2+ (0–
5 ꢀ 10ꢁ5 M). Inset: Determination of Kd by the increase in If as a function of Zn2+
concentration.
Ethyl carbazole (b)
Compound (e)
Compound (e)

346
J. Zhang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 343–346
Table 2
of probe can be prepare to improve the lipophility so that it could
be permeated into the cell where it will be transformed into probe
by esterase in the cytosol.21
Relative fluorescent intensities of the compound (e) with various biologically
important metal cations
Entry
Metal cations
Relative intensity (If/I0)a
In conclusion, a novel fluorescence probe 2-[(N-ethyl carba-
zole)-3-sulfonyl ethylenediamine]-1-N,N-2-(2-methypyridy) has
been synthesized. It should be emphasized that the probe can be
easily synthesized in four steps from readily available starting
materials. The reactivity of the fluorescence probe with Zn2+ has
been examined in solution of physiological pH by fluorescence
spectroscopy. The UV/vis spectra of the compound in various sol-
vents were measured and the interactions between the probe
and solvents were also discussed. Finally, it should be noted that
the target fluorescent probe, due to its unique fluorescence proper-
ties and high selectivity, could be very promising for further re-
search on the Zn2+ analysis under physiological conditions. We
are currently in the process of modifying carbazole compound (e)
to shift its excitation and emission spectra in the visible to protect
biological sample from photodamage upon UV irradiation region as
well as to circumvent the spectral window with pronounced cellu-
lar autofluorescence and we will report the findings subsequently.
1
2
3
4
5
6
7
8
None
1.0
1.0
1.0
1.0
1.0
3.6
0.5
0.3
1.0
0.2
1.0
1.2
3.6
3.6
3.6
3.6
9 ꢀ 10ꢁ3 M Na+
9 ꢀ 10ꢁ3 M K+
9 ꢀ 10ꢁ3 M Ca2+
9 ꢀ 10ꢁ3 M Mg2+
1 ꢀ 10ꢁ5 M Zn2+
1 ꢀ 10ꢁ5 M Fe3+
1 ꢀ 10ꢁ5 M Cu2+
9
1 ꢀ 10ꢁ5 M Ni2+
10
11
12
13
14
15
16
1 ꢀ 10ꢁ5 M Co2+
1 ꢀ 10ꢁ5 M Mn2+
1 ꢀ 10ꢁ5 M Cd2+
1 ꢀ 10ꢁ5 M Zn2+ + 9 ꢀ 10ꢁ3 M Na+
1 ꢀ 10ꢁ5 M Zn2+ + 9 ꢀ 10ꢁ3 M K+
1 ꢀ 10ꢁ5 M Zn2+ + 9 ꢀ 10ꢁ3 M Ca2+
1 ꢀ 10ꢁ5 M Zn2+ + 9 ꢀ 10ꢁ3 M Mg2+
a
I0 and If indicate the average fluorescence intensities of fluorescence probe
compound (e) (1 ꢀ 10ꢁ5 M) in the absence and presence of various metal cations,
respectively.
Acknowledgments
350
300
250
200
150
100
50
This work was supported by the National Natural Science Foun-
dation of China(No. 21175086) and the Research Project Supported
by Shanxi Scholarship Council of China. All the authors express
their deep thanks.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.11.004.
References and notes
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Figure 5. Effect of pH on the fluorescence intensity of compound (e) (1.0 ꢀ 10ꢁ5 M)
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