Near-IR core-substituted naphthalenediimide fluorescent chemosensors for zinc ions: Ligand effects on PET and ICT channels

Article abstract of DOI:10.1002/chem.201000461

Near-IR (NIR) emission can offer distinct advantages for both in vitro and in vivo biological applications. Two NIR fluorescent turn-on sensors N,N′-di-n-butyl-2-(Ar-{2-[bis-(pyridin-2-ylmethyl)amino]ethyl}) -6-(N-piperidinyl)naphthalene-1,4,5,8-tetracarboxylic acid bisimide and N,N′-din-butyl-2-[N,N,N′-tri(pyridin-2-ylmethyl)amino] ethyl-6-(N-piperidinyl)naphthalene-1,4,5,8-tetracarboxylic acid bisimide (PND and PNT) for Zn²⁺ based on naphthalenediimide fluorophore are reported. Our strategy was to choose core-substituted naphthalenediimide (NDI) as a novel NIR fluorophore and N,N-di(pyridin-2-ylmethyl)ethane-1,2-diamine (DPEA) or N,N,N′-tri(pyridin2-ylmethyl)ethane-1,2-diamine (TPEA) as the receptor, respectively, so as to improve the selectivity to Zn²⁺. In the case of PND, the negligible shift in absorption and emission spectra is strongly suggestive that the secondary nitrogen atom (directly connected to the NDI moiety, N¹) is little disturbed with Zn²⁺. The fluorescence enhancement of PND with Zn²⁺ titration is dominated with a typical photoinduced electron-transfer (PET) process. In contrast, the N ¹ atom for PNT can participate in the coordination of Zn²⁺ ion, diminishing the electron derealization of the NDI moiety and resulting in intramolecular charge-transfer (ICT) disturbance. For PNT, the distinct blueshift in both absorbance and fluorescence is indicative of a combination of PET and ICT processes, which unexpectedly decreases the sensitivity to Zn ²⁺. Due to the differential binding mode caused by the ligand effect, PND shows excellent selectivity to Zn²⁺ over other metal ions, with a larger fluorescent enhancement centered at 650 nm. Also both PND and PNT were successfully used to image intracellular Zn²⁺ ions in the living KB cells.

Full text of DOI:10.1002/chem.201000461

FULL PAPER
DOI: 10.1002/chem.201000461
Near-IR Core-Substituted Naphthalenediimide Fluorescent Chemosensors
for Zinc Ions: Ligand Effects on PET and ICT Channels
Xinyu Lu,[a] Weihong Zhu,*[a] Yongshu Xie,[a] Xin Li,[a] Yuan Gao,[b] Fuyou Li,[b] and
He Tian*[a]
Abstract: Near-IR (NIR) emission can
offer distinct advantages for both in
vitro and in vivo biological applica-
tions. Two NIR fluorescent turn-on
2-ylmethyl)ethane-1,2-diamine (TPEA)
as the receptor, respectively, so as to
improve the selectivity to Zn2+. In the
case of PND, the negligible shift in ab-
sorption and emission spectra is strong-
ly suggestive that the secondary nitro-
gen atom (directly connected to the
NDI moiety, N1) is little disturbed with
Zn2+. The fluorescence enhancement
of PND with Zn2+ titration is dominat-
ed with a typical photoinduced elec-
tron-transfer (PET) process. In con-
trast, the N1 atom for PNT can partici-
pate in the coordination of Zn2+ ion,
diminishing the electron delocalization
of the NDI moiety and resulting in in-
tramolecular charge-transfer (ICT) dis-
turbance. For PNT, the distinct blue-
shift in both absorbance and fluores-
cence is indicative of a combination of
PET and ICT processes, which unex-
pectedly decreases the sensitivity to
Zn2+. Due to the differential binding
mode caused by the ligand effect, PND
shows excellent selectivity to Zn2+
over other metal ions, with a larger flu-
orescent enhancement centered at
650 nm. Also both PND and PNT were
successfully used to image intracellular
Zn2+ ions in the living KB cells.
sensors
N,N’-di-n-butyl-2-(N-{2-[bis-
(pyridin-2-ylmethyl)amino]ethyl})-6-
(N-piperidinyl)naphthalene-1,4,5,8-tetra-
carboxylic acid bisimide and N,N’-di-
n-butyl-2-[N,N,N’-tri(pyridin-2-ylmeth-
yl)amino]ethyl-6-(N-piperidinyl)naph-
thalene-1,4,5,8-tetracarboxylic acid bis-
imide (PND and PNT) for Zn2+ based
on naphthalenediimide fluorophore are
reported. Our strategy was to choose
core-substituted
naphthalenediimide
Keywords: fluorescent sensors · li-
gand effects · naphthalene diimides ·
NMR spectroscopy · zinc
(NDI) as a novel NIR fluorophore and
N,N-di(pyridin-2-ylmethyl)ethane-1,2-
diamine (DPEA) or N,N,N’-tri(pyridin-
Introduction
sion.[1] It is well-believed that the lack of zinc ions could
result in an increasing risk of several diseases.[2] Therefore, it
is necessary to get an insight into the vital roles of Zn2+ in
biological processes with the growing demand for sensing
Zn2+ in living systems. For this purpose, a variety of fluores-
cent Zn2+ sensors have been documented based on various
fluorophores, such as acridine, anthracene, benzoxazole, bor-
adiazaindacene, coumarin, dansyl, fluorescein, naphthali-
mide, nitrobenzoxadiazole, quinoline, or squaraines.[3] For
example, Lippard et al. have reported several series of zinc
probes by making alterations to a Zn2+-binding unit and flu-
orophore platform, and even successfully imaged the distri-
bution, uptake, and mobilization of Zn2+ in various types of
cells and neuronal cultures.[4] However, the cases on Zn2+
sensors in the near-IR (NIR) fluorescent emission between
650 and 900 nm are still less common.[5] As a matter of fact,
such longer wavelength emission can offer distinct advantag-
es for both in vitro and in vivo biological applications in
that they can avoid the strong interference from UV-in-
duced phototoxicity/autofluorescence and sensor-induced in-
As the second most abundant transition metal in the human
body, the zinc ion plays a great role in biological systems,
such as DNA binding, apoptosis, signal transduction, gene
expression, metalloenzyme catalysis, and neurotransmis-
[a] X. Lu, Prof. Dr. W. Zhu, Prof. Dr. Y. Xie, X. Li, Prof. Dr. H. Tian
Key Laboratory for Advanced Materials and
Institute of Fine Chemicals
East China University of Science and Technology
Shanghai 200237 (P. R. China)
Fax : (+86)21-6425-2758
E-mail: whzhu@ecust.edu.cn
tianhe@ecust.edu.cn
[b] Y. Gao, Prof. Dr. F. Li
Department of Chemistry & Laboratory of
Advanced Materials, Fudan University
Handan Road 220, Shanghai 200433 (P. R. China)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201000461.
Chem. Eur. J. 2010, 16, 8355 – 8364
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8355

terference encountered in the shorter wavelength region. In
this regard, it is highly desirable to develop Zn2+-selective
NIR fluorescent sensors to match harmless imaging and vis-
ualization of Zn2+ in living cells.
dination behavior between DPEA and TPEA. Unexpected-
ly, upon comparing with PNT, competition experiments
demonstrate that PND shows higher selectivity to Zn2+ with
less interference by Cd2+
.
With these considerations in mind, we designed and syn-
thesized two novel NIR fluorescent turn-on sensors N,N’-di-
n-butyl-2-(N-{2-[bis(pyridin-2-ylmethyl)amino]ethyl})-6-(N-
piperidinyl)naphthalene-1,4,5,8-tetracarboxylic acid bisimide
Results and Discussion
and
N,N’-di-n-butyl-2-[N,N,N’-tri(pyridin-2-ylmethyl)ami-
Design and synthesis of PND and PNT: NDI is an exquisite
building block for supramolecular self-assemblies,[10] excel-
lent n-type semiconductors for organic field-effect transis-
tors (OFETs),[11] and n-type functional materials (electron
acceptor) for solar energy conversion materials.[12] Consider-
ing their richness in spectroscopic and electrochemical prop-
erties, NDI derivatives are ideally suitable for sensor design.
Recently, two NDI-based fluorescent-ion sensors through
forming excimers have been reported.[13] Generally, most
functionalizations of NDI are through its dianhydride group
or core substitution at the 2-, 3-, 6- and/or, 7-position, pro-
viding a facial way to tune the emission wavelength from
387 to 623 nm,[14a] even longer than 650 nm[14b,c] (falling into
the NIR region). Meanwhile, the hydrophilic, lipophilic, or
biophilic unit can be easily incorporated into the specific
NDI chromophore for developing novel NIR fluorescent
systems to meet a variety of requirements in chemical and
biological analysis.
no]ethyl-6-(N-piperidinyl)naphtalene-1,4,5,8-tetracarboxylic
acid bisimide (PND and PNT) for Zn2+ ion based on naph-
In our synthesis, we first attempted to incorporate the
ligand unit at both the 2- and 6-positions of the NDI core
for preparing a symmetric chelate-appended sensor. Due to
the easy cleavage of the pyridylmethyl carbon–nitrogen
bond in DPEA or TPEA, we prefer to treat N,N’-dibutyl-
2,6-dibromonaphthalene bisimide (2) with the ligand at
moderate temperature (Scheme 1). Unfortunately, only one
bromo group can be substituted with the ligands of DPEA
or TPEA at 608C. It was very difficult to introduce a second
thalenediimide fluorophore (NDI). Also a piperidine unit
was incorporated as a strong electron-donor to the NDI
core by nucleophilic substitution for extending the push–pull
electronic system with an emission band centered at 650 nm,
thus successfully tuning the determining wavelength to fall
in the desirable NIR region. Furthermore, among the avail-
able ionophore groups, two water-compatible and mem-
brane-permeable chelators, N,N-di(pyridin-2-ylmethyl)-
ethane-1,2-diamine (DPEA)[6] and N,N,N’-tri(pyridin-2-yl-
methyl)ethane-1,2-diamine (TPEA),[7] were incorporated for
considering the selectivity of Zn2+ over other metal ions
under physiological conditions.[3i,8a,9] With respect to the di-
pyridine amine unit in the ligand of DPEA, the additional
pyridine in the ligand of TPEA might be expected to im-
prove the selectivity due to the effect of cooperative chelat-
ing. Interestingly, in the target sensors of PND and PNT,
two ionophores exhibit completely different ligand effects
on photoinduced electron-transfer (PET) and intramolecu-
lar charge-transfer (ICT) channels due to the different coor-
Scheme 1. Synthetic route of target sensors: i) n-butylamine, acetic acid,
1188C, 5 h, 58%; ii) DPEA or TPEA, NMP, 608C, 70 h; iii) piperidine,
NMP, 508C, 24 h.
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Naphthalenediimide Fluorescent Chemosensors
FULL PAPER
ligand to the NDI core, which might be due to the poor re-
activity of the aliphatic amines in the DPEA or TPEA
ligand. When using a more reactive and nucleophilic piperi-
dine group, the remaining bromo group in the core of NDI
can be successfully substituted (Scheme 1). As expected, the
second substituted piperidine group with strong electron-do-
nating capability plays an essential role in realizing a large
bathochromic shift, thus obtaining target sensors with the
emission located at the NIR region.
As illustrated in Scheme 1, the target molecules of PND
were conveniently synthesized by three steps from 2,6-dibro-
monaphthalene bisanhydride (1), which was prepared ac-
cording to the published procedure.[15] Firstly, compound 1
was treated with n-butylamine in hot acetic acid under
reflux to give N,N’-dibutyl-2,6-dibromonaphthalene bisimide
(2) in 58% yield. In the following steps, by using triethyla-
mine as an acid-trap agent in dry N-methyl-2-pyrrollidone
(NMP), the two bromo groups of intermediate 2 were nucle-
ophilically stepwise-substituted with DPEA and piperidine,
respectively, to give the target core-substituted Zn2+ sensor
PND with a yield of 31%. Similarly, 2 was treated with
TPEA instead of DPEA at the second step, which finally re-
sulted in PNT with a yield of 33%. The chemical structures
of PND and PNT were fully characterized by 1H and
13C NMR spectroscopy and HRMS as shown in the Experi-
mental Section.
PNT exhibit obviously bathochromic shifts of 72 and 77 nm,
respectively, which are accompanied by a distinct color
change from red to dark blue (see Figure S1 in the Support-
ing Information). A similar change in the emission can be
also observed, that is, there is a bathochromic shift from the
monosubstituted BND (lem 565 nm) to the disubstituted
PND (lem 644 nm) and PNT (lem 638 nm). Clearly, the incor-
poration of a piperidine group at the 6-position of the NDI-
core by nucleophilic displacement can extend the push–pull
electronic system with positive mesomeric effects (+M
effect, bathochromic shift)[14b] on both absorption and emis-
sion, thus successfully tuning the wavelength to fall in the
desirable long-wavelength region.
Absorption and fluorescence response of PND and PNT to
Zn2+: Spectroscopic response measurements with PND and
PNT were studied in a mixture of acetone/water (90:10, v/v)
with a buffer solution of 3-(N-morpholino)propanesulfonic
acid (MOPS, 10 mm, pH 7.0). Notably in buffered acetone/
water solution, PND shows a bathochromic shift of 6 nm in
emission to 650 nm due to the solvent effect, and a similar
change in the absorption and emission of PNT can be also
observed (see Table S1 in the Supporting Information).
Titrations of PND and PNT with Zn2+ were then per-
formed in MOPS buffer system to characterize Zn2+ coordi-
nation. As shown in Figure 2A, the absorption band of free
PND exhibits a maximum centered at 610 nm. Upon adding
one equivalent of Zn2+, the absorption band and intensity
Optical properties of BND, PND, and PNT: By using the
monosubstituted intermediate as a reference (N,N’-di-n-
butyl-2-(N-{2-[bis(pyridin-2-ylmethyl)amino]ethyl})-6-bro-
monaphthalene-1,4,5,8-tetracarboxylic
acid
bisimide
(BND)), the normalized absorption and fluorescence spectra
of sensors PND and PNT in chloroform were initially stud-
ied (Figure 1). All of them display a characteristic intense
absorption band corresponding to the core-substituted naph-
thalenediimide moiety in the visible region.[16] With respect
to BND in the UV region, both PND and PNT exhibit two
similar intense absorption bands located at about 356 and
374 nm, which have little effect on the core substituents.
Meanwhile, upon comparison with the absorption band of
BND (located at 528 nm) in the visible region, PND and
Figure 2. Changes in absorption spectra upon titration of Zn2+ from 0 to
1.0 equiv: A) PND (50 mm). B) PNT (50 mm). All data were obtained in
aqueous solution (acetone/100 mm MOPS buffer 90:10, pH 7.0).
Figure 1. Normalized absorption and fluorescence spectra of BND (g),
PND (a), and PNT (c) in chloroform (2.0ꢁ10À5 m).
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W. Zhu, H. Tian et al.
did not change. To understand the interaction between PND
and Zn2+, the fluorescence titration with Zn2+ was also
monitored. When excited at 610 nm, there is a very small
blueshift of 3 nm in the emission peak wavelength for PND
with a sequential addition of Zn2+, but showing a turn-on
fluorescent response with zinc addition (Figure 3A). When
lone electron pair of the tertiary nitrogen atom (dipicolyla-
mine, N2) in the NDI fluorophore (Scheme 2A). Further-
more in the case of PND, the neglect of a shift in absorption
and emission spectra strongly suggested that the secondary
nitrogen atom (directly connected to the NDI moiety, N1) is
little disturbed by Zn2+, that is, the coordination between
Zn2+ and N1 can be possibly ruled out.
On the other hand, PNT undergoes different phenomena,
that is, a large blueshift in both absorption and emission
peak is observed. Upon adding one equivalent of Zn2+ into
the PNT system, the absorption peak at 603 nm became de-
creased, and simultaneously a blueshifted absorption peak
centered at 577 nm was developed with a well-defined iso-
sbestic point at 566 nm (Figure 2B, Table 1). Upon choosing
the isosbestic point as the excitation wavelength, the emis-
sion intensity of PNT was also enhanced to some extent
with a distinct blueshift from 640 to 631 nm upon the se-
quential addition of Zn2+ (Figure 3B). Such a blueshift
means that the nitrogen N1 attached to the NDI moiety is
disturbed with Zn2+, thus resulting in a decrease in the elec-
tron-donating ability. Here, upon comparing with PND, the
nitrogen atom N1 for PNT shows a much stronger coordina-
tion tendency with Zn2+. This might be attributed to the ad-
ditional pyridinyl nitrogen N5 in TPEA, which helps N1–N4
to form a five-coordinating Zn2+ complex by a synergistic
chelating effect (Scheme 2B). Consequently, the moderate
“off–on” enhancement and the distinct blueshift in both ab-
sorption and emission peak for PNT is suggestive of a com-
bination of PET and ICT processes[7d], which unexpectedly
decreases the sensitivity to Zn2+
.
pH effects on the fluorescence of PND and PNT were
also examined (Figure 4). For PND, the weak emission (lem
at 650 nm) was observed with the excitation at 610 nm. Its
emission intensity does not change significantly in the range
of pH 6.0–9.0. However, a distinct emission enhancement of
three-fold at 650 nm can be observed when decreasing the
pH from 6.0 to 3.0 (Figure 4), which shows a typical PET
block caused by the protonation of the nitrogen atom N2[18]
and a negligible induced PET process caused by the proto-
nation of pyridine subunits[8b,c] (see Figure S3A in the Sup-
porting Information). On the other hand, the fluorescence
intensity of PNT (lem =635 nm) does not change as signifi-
cantly as PND in the range of pH 3.0–9.0 (lex =566 nm, see
Figure S3B in the Supporting Information). Again, the de-
creased sensitivity to low pH for PNT suggests a dominated
control of ICT[9] with some degree of PET process. Notably,
the stable fluorescence of PND and PNT at around pH 7.0 is
favorable for in vivo application.
Figure 3. Changes in fluorescence spectra upon titration of Zn2+ from 0
to 1.4 equiv: A) PND (50 mm, lex =610 nm). B) PNT (50 mm, lex
=
566 nm). All data were obtained in aqueous solution (acetone/100 mm
MOPS buffer=90:10, pH 7.0).
adding more than one equivalent of Zn2+, the maximum of
fluorescence intensity at 647 nm is reached (Table 1). It also
shows a fast fluorescent response within less than 20 s (see
Figure S2 in the Supporting Information). The fluorescence
enhancement of PND during the titration with Zn2+ is a
typical PET characteristic. It is blocking PET[8] from the
Table 1. Optical properties of PND, PNT, and their zinc complexes.[a]
Systems
Absorption Extinction co-
Emission
peak lem
[nm]
Fluorescence
yield Fem
[b]
peak lab
efficient
[nm]
[ꢁ103 mÀ1 cmÀ1
]
PND
610
610
603
577
9.74
9.72
9.12
7.81
650
647
640
631
0.17
0.55
0.23
0.42
1H NMR titration of PND and PNT with Zn2+: Interesting-
PND+Zn2+[c]
PNT
ly, the target sensors of PND and PNT exhibit different opti-
PNT+Zn2+[c]
cal properties due to different coordination modes of Zn2+
,
[a] All data were obtained in aqueous solution (acetone/100 mm MOPS
buffer 90:10, pH 7.0). [b] Fluorescent quantum yields were determined in
reference to N,N’-di(2,6-dibutyl)-1,6,7,12-tetrakis(4-tert-butylphenoxy)-
3,4:9,10-perylenetetracarboxylic diimide (F=0.66, in CH2Cl2).[17] [c] The
data were measured in the presence of Zn2+ (1.0 equiv with respect to
PND or PNT).
respectively. Actually, their binding behaviors are more pro-
nounced with NMR titration in [D6]DMSO. As shown in
Figure 5, upon adding an increasing amount of Zn2+, anoth-
er set of NMR spectroscopic signals for the Zn2+-coordina-
tion complex appears besides the set of signals for free
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Naphthalenediimide Fluorescent Chemosensors
FULL PAPER
Tables S2 and S3 in the Sup-
porting Information). For probe
PND, Zn2+ coordination trig-
gers a clear downfield shift of
Ha in the pyridine unit from d=
8.45 to 8.62 ppm. The signal for
Hb of pyridine groups show a
significant downfield shift up to
Dd=0.88 ppm, whereas the sig-
nals for Hc and Hd in pyridine
at about d7.6 ppm were almost
unchanged, which is consistent
with the previous observation,[3i]
indicative of the possible direc-
tion of charge transfer from the
pyridine to Zn2+. As for the
methylene hydrogen (He), the
singlet at d=3.86 ppm splits
into two doublets at d=4.13
and 4.50 ppm, respectively. It
might be attributed to two dif-
ferent proton environments,
one of them much more affect-
ed by the presence of Zn2+
probably by closer proximity.[19]
Meanwhile, a triplet for Hf at
d=2.88 ppm also shows an ob-
vious downfield shift by d=
0.15 ppm, which indicates that
the tertiary amino-N2 adjacent
to the methylene are strongly
Scheme 2. Proposed Zn2+ binding mode: A) PND and B) PNT (note: in the case of PND, the neglect of a
shift in absorption and emission spectra strongly suggested that the secondary nitrogen atom (directly connect-
ed to the NDI moiety, N1) is little disturbed by Zn2+, that is, the coordination between Zn2+ and N1 can be
possibly ruled out).
bonded with Zn2+. The singlet for Hh at d=9.75 ppm corre-
sponding to the N1H of PND disappeared upon titration of
Zn2+, mainly due to the active proton exchanged with trace
water. In addition, the slightly downfield shift for Hi and Hj
of the NDI moiety by d=0.11 and 0.07 ppm, respectively, is
indicative of little change in charge density on the core of
PND during its binding with Zn2+
.
Compared with PND, Ha–d in pyridines of PNT show a
similar downfield shift upon the titration of one equivalent
of Zn2+. Notably, the triplets for Hg of PNT shift downfield
with a Dd of 0.51 ppm, much larger than that of PND (Dd
of 0.26 ppm for Hg in the case of PND), also supporting a
stronger coordination occurring between Zn2+ and tertiary
amino N1 of PNT than that of PND. This difference is exact-
ly consistent with the phenomena in absorption and emis-
sion spectra.
&
Figure 4. Effect of pH on the fluorescence intensity of PND ( , 50 mm)
*
and PNT ( , 50 mm) in 0.1m sodium phosphate solution adjusted to vari-
ous pH values, which was determined at 650 nm with an excitation at
610 nm for PND, and at 635 nm with excitation at 577 nm for PNT.
Again, the DFT calculation by the Gaussian 03 software
package demonstrated two different coordination modes for
Zn2+–PND and Zn2+–PNT in support of the proposed bind-
ing process. As shown in Figure S4 and Table S4 in the Sup-
porting Information, the bonding lengths of N–Zn are close
to the reported ones[20] in the optimized structures. In the
case of the Zn2+–PND complex, the Zn2+ ion is chelated by
the DPEA unit with the participation of possible anions (for
example, nitrate anions), thus leaving the N1 atom alone,
not directly coordinated with Zn2+. This is why PET domi-
probes. When 0.5 equivalents of Zn2+ was added, two sets of
NMR spectroscopic signals display almost identical intensity,
whereas when one equivalents of Zn2+ was added, only the
signals corresponding to the complex can be observed. How-
ever, more equivalents of Zn2+ does not lead to any further
1
evident change in the H NMR spectrum, which is indicative
of the 1:1 binding ratio.
The chemical shifts in Zn2+ complexes are also compared
with those in free probes of PND and PNT (Figure 5, see
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W. Zhu, H. Tian et al.
Figure 5. Partial 1H NMR spectra (400 MHz, [D6]DMSO) in the absence (a) and in the presence of b) 0.5 and c) 1.0 equiv of Zn2+: A) PND (5 mm) and
B) PNT (5 mm). The signals marked with +, <, and ꢁ are for the protons from the free sensor, zinc-bound sensor, and H2O, respectively.
nates with the negligible shift in absorption and emission
spectra of PND. In contrast, in the Zn2+–PNT complex, the
N1 atom can participate in the coordination of the Zn2+ ion.
And the N1 amine group is found to be perpendicular to the
naphthalene plane, which diminishes the electron delocaliza-
tion of the NDI moiety for PNT, and results in the ICT dis-
turbance to tune the blueshift in both absorbance and fluo-
rescence.
The selectivity of the fluorescent response of PND and
PNT to zinc ions was examined. Competition experiments
were conducted to further check the Zn2+ selectivity of
PND and PNT in the presence of other metal ions, which
demonstrates that PND shows a higher selectivity to Zn2+
than that of PNT (Figure 6 and Figure S7 in the Supporting
Information). As shown in Figure 6, the emission profile of
Zn2+–PND complex is unperturbed in the presence of
10 equivalents of Na+, K+, Mg2+, and Ca2+, which are phys-
iologically important and abundant cations existing in living
cells. More importantly, most heavy and transition-metal
Zn2+ selectivity, competition experiments, and in vivo imag-
ing: Job plot analyses of Zn2+ with PND or PNT were also
performed with fluorescence. Both of them show breaks at
0.5, indicative of a same 1:1 binding behavior in solution
(see Figure S5 in the Supporting Information). Moreover,
the apparent association constants (Kass) of Zn2+ with PND
ions (HTM ions), including Co2+, Ni2+, Ag+, Hg2+, Pb2+
,
and Mn2+, also have a very negligible effect. Only Cu2+ and
Fe2+ quench fluorescence to some extent, which was often
met in other metal-ion sensors,[21,22] and Cu2+ could be sup-
pressed by the addition of acetylacetone (see Figure S8 in
the Supporting Information). Since there are rare examples
of PET-based fluorescent sensors that can discriminate Zn2+
from Cd2+ owing to their closely related properties,[23,24] we
are pleased to find that PND exhibits a much higher fluores-
cence enhancement to Zn2+, which suggests that PND is a
Zn2+-selective fluorescence probe that is less affected by
Cd2+. PND shows high selectivity toward Zn2+ over other
competing metal ions in aqueous solution. Unexpectedly, al-
and PNT were determined to be 2.3ACHTNUGTRENNUNG
A
ting of the fluorescence data (see Figure S6 in the Support-
ing Information). As expected, the Kass for PNT is larger by
almost one order of magnitude than that for PND since the
cooperative coordination induced by the additional pyridine
in the TPEA group increases the Zn2+ coordination ability
of PNT (Scheme 2).
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Naphthalenediimide Fluorescent Chemosensors
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Figure 7. Confocal fluorescence images in KB cells: Top, A–C) Cells in-
cubated with PND (20 mm) for 0.5 h. Bottom, D–F) Cells incubated with
ZnCl2 (20 mm) for 0.5 h then PND (20 mm) for 0.5 h. Emission was collect-
ed at 598–698 nm upon excitation at 543 nm. Bright field (A and D),
fluorescence (B and E), and overlap field (C and F).
the fluorescence signals are localized in the perinuclear area
of the cytosol, which indicates a subcellular distribution of
Zn2+ and good cell-membrane permeability of PND. A simi-
lar fluorescence image of PNT was shown in Figure S9 (Sup-
porting Information). These cell experiments demonstrated
the usefulness of both PND and PNT for the imaging of
Zn2+ in living cells.
Figure 6. The relative fluorescence intensity at pH 7.00 (10 mm MOPS) of
various metal ions: A) PND (lem =650 nm, 30 mm). B) PNT (lem
=
630 nm, 30 mm). Light gray bars represent the addition of the analytes:
1) Na+ (300 mm), 2) K+ (300 mm), 3) Mg2+ (300 mm), 4) Ca2+ (300 mm),
5) Cr2+ (30 mm), 6) Mn2+ (30 mm), 7) Co2+ (30 mm), 8) Ni2+ (30 mm),
9) Ag+ (30 mm), 10) Pb2+ (30 mm), 11) Hg2+ (30 mm), 12) Fe2+ (30 mm),
13) Cu2+ (30 mm), and 14) Cd2+ (30 mm). Black bars represent the subse-
quent addition of Zn2+ (30 mm) to the solution.
Conclusion
We have designed and synthesized PND and PNT, two
novel and highly sensitive fluorescent probes for Zn2+ by
using core-substituted naphthalenediimide fluorophore as an
NIR fluorophore, and DPEA or TPEA derivatives as Zn2+
binding moieties. Interestingly, in the target sensors of PND
and PNT, two ionophores exhibit completely different
ligand effects on photoinduced electron-transfer (PET) and
intramolecular charge transfer (ICT) channels due to the
different coordination behavior between DPEA and TPEA.
The fluorescence enhancement of PND during the titration
with Zn2+ is dominated with a typical PET characteristic. In
contrast, the N1 atom for PNT can participate in the coordi-
nation of Zn2+ ion, diminishing the electron delocalization
of the NDI moiety and resulting in the ICT disturbance. For
PNT, the distinct blueshift in both absorbance and fluores-
cence is suggestive of a combination of PET and ICT pro-
cesses. PND shows several advantages as a probe for sensing
Zn2+, such as the determining wavelength falling in the de-
sired NIR region beneficial to high light penetration depth
and weak autofluorescence in biological tissues, a PET-
based turn-on fluorescent type to get the maximum signal-
to-noise ratio, and discriminating Zn2+ from Cd2+ and other
cations apparently through the difference in fluorescence in-
tensity. PND and PNT are long wavelength, cell-permeable,
and can be applied to image Zn2+ in living cells and in vivo
though the additional pyridine promotes the stability of
Zn2+–PNT complexes, PNT showed less selectivity with less
fluorescent enhancement with respect to PND (see Figur-
e S7B in the Supporting Information), and was interfered
with by the presence of Co2+, Ni2+, and Cd2+, but with less
fluorescent quenching by Fe2+ or Cu2+ (Figure 6B). As
mentioned above, the different selectivity toward the same
metal ion (Zn2+) might be also caused by the different bind-
ing mode with other HTM ions.
Finally, we apply the NIR sensor of PND to KB cells
(human nasopharyngeal epidermal carcinoma cell) for ex-
amining whether it could work in living systems with the
help of a confocal laser scanning microscopy. Bright-field
measurements confirmed that the cells after treatment with
Zn2+ and PND were viable throughout the imaging experi-
ments (Figure 7A and D). Under selective excitation of
PND, staining KB cells with a 20 mm solution of PND for
30 min at 378C led to very weak intracellular fluorescence
(Figure 7B). After the cells were supplemented with ZnCl2
(20 mm) in the growth medium for 30 min at 378C and then
loaded with PND under the same conditions, a significant
increase in the fluorescence from the intracellular area was
observed (Figure 7E). As depicted in Figure 7C and F, the
overlay of fluorescence and bright-field images reveals that
Chem. Eur. J. 2010, 16, 8355 – 8364
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
8361

W. Zhu, H. Tian et al.
aqueous solution of 0.1m HCl (50 mL) and extracted with dichlorome-
thane (3ꢁ40 mL). The combined organic layer was washed with saturat-
ed brine, dried over anhydrous MgSO4, filtered, and rotary evaporated.
The crude product was purified by silica-gel chromatography by using
methanol/dichloromethane 1:120 as the elute to afford PND as an indigo
potentially and should, therefore, be useful for clarifying the
roles of Zn2+ in biological processes.
blue solid (43 mg, 31% yield). Rf =0.3 (methanol/dichloromethane 1:15);
Experimental Section
1
À
H NMR (400 MHz, CDCl3, TMS): d=9.88 (s, 1H; NH), 8.50 (d, J=
4.4 Hz, 2H; pyridyl-H), 8.43 (s, 1H; naphthalene-H), 8.06 (s, 1H; naph-
Materials: 1,4,5,8-Naphthalenetetracarboxylic dianhydride was purchased
from Tokyo Chemical Industry Co. All other reagents and solvents were
purchased from commercial sources and are of analytical grade. N,N-Di-
(pyridin-2-ylmethyl)ethane-1,2-diamine and N,N,N’-tri(pyridin-2-ylme-
thyl)ethane-1,2-diamine were prepared by the established literature pro-
thalene-H), 7.80 (d, J=7.6 Hz, 2H; pyridyl-H), 7.62 (t, J=7.6 Hz, 2H;
À
pyridyl-H), 7.12 (t, J=4.4 Hz, 2H; pyridyl-H), 4.26 (t, J=7.2 Hz, 2H;
À
À
À
NCH2 ), 4.16 (t, J=7.2 Hz, 2H; NCH2 ), 3.95 (s, 4H; CH2-pyridyl),
À
3.66 (t, J=5.6 Hz, 2H; NHCH2CH2 ), 3.32 (s, 4H; piperidinyl-H), 3.02
(t, J=5.6 Hz, 2H; NHCH2CH2 ), 1.86 (s, 4H; piperidinyl-H), 1.74–1.80
(m, J=7.2 Hz, 2H; NCH2CH2 ), 1.74 (s, 2H; piperidinyl-H), 1.69 (m, J=
7.2 Hz, 2H; NCH2CH2 ), 1.49–1.55 (q, J=5.6 Hz, 2H; CH2CH3), 1.41–
À
cedure[7c,8]
.
À
Apparatus: 1H and 13C NMR in CDCl3 or [D6]DMSO were recorded on
a Bruker Avance-400 spectrometer with tetramethylsilane (TMS) as the
internal standard. Data for 1H NMR spectra are reported as follows:
chemical shift (ppm) and multiplicity (s=singlet, d=doublet, t=triplet,
q=quartet, m=multiplet). Data for 13C NMR spectra are reported in
ppm. HRMS were measured on a Waters LCT Premier XE spectrometer.
UV/Vis spectra were obtained by using a Varian Cary 500 spectropho-
tometer (1 cm quartz cell) at 258C. Fluorescent spectra were recorded on
a Varian Cary Eclipse fluorescence spectrophotometer (1 cm quartz cell)
at 258C. The slit width was 5 nm for both excitation and emission. TLC
analyses were performed on silica-gel plates and flash chromatography
was conducted by using silica-gel column packages purchased from Qing-
dao Haiyang Chemical Co. (China). The preparation of 2,6-dibromo-
naphthalene-1,4,5,8-tetracarboxylic dianhydride (1) was conducted ac-
cording to literature methods.[15]
À
À
À
À
1.46 (q, J=5.6 Hz, 2H; CH2CH3), 1.02–1.06 (t, J=5.6 Hz, 3H;
CH2CH3), 0.95–0.99 ppm (t, J=5.6 Hz, 3H; CH2CH3); 13C NMR
(100 MHz, CDCl3): d=13.86, 13.94, 20.35, 20.48, 24.03, 26.18, 30.26,
30.28, 40.07, 40.52, 40.58, 52.53, 53.81, 60.33, 101.10, 111.15, 118.78,
121.96, 122.11, 123.35, 123.69, 124.20, 125.40, 127.04, 136.49, 148.91,
149.60, 151.71, 158.98, 161.88, 162.93, 163.20, 165.94 ppm; HRMS (TOF-
ESI+): m/z: calcd for C41H48N7O4+: 702.3768 [M+H+]; found: 702.3773;
calcd for C41H47N7O4Na+: 724.3587 [M+Na+]; found: 724.3589.
À
N,N’-Di-n-butyl-2-[N,N,N’-tri(pyridin-2-ylmethyl)amino]ethyl-6-(N-piper-
idinyl)naphtalene-1,4,5,8-tetracarboxylic acid bisimide (PNT): N,N,N’-
Tri(pyridin-2-ylmethyl)ethane-1,2-diamine[7c] (400 mg, 1.2 mmol) in dry
NMP (10 mL) was added to a solution of 2 (108 mg, 0.2 mmol) and the
resulting mixture was stirred at 608C for 70 h. After having been evapo-
rated in vacuo, the residue was dissolved in an aqueous solution of 0.1m
HCl (50 mL) and was extracted with dichloromethane (3ꢁ40 mL). The
combined organic layer was dried with magnesium sulfate, filtered, and
evaporated in vacuo. The crude product was purified by silica-gel chro-
matography by using methanol/dichloromethane 1:120 as the elute to
afford a dark-red solid (60 mg), which was redissolved in a solution of
dry NMP (5 mL) and piperidine (170 mg, 2.0 mmol). The resulting mix-
ture was stirred at 508C for 24 h under an argon atmosphere, and was
then evaporated in vacuo. The residue was dissolved in an aqueous solu-
tion of 0.1m HCl (30 mL) and extracted with dichloromethane (3ꢁ
20 mL). The combined organic layer was dried with magnesium sulfate,
filtered, and evaporated in vacuo. The crude product was purified by
silica-gel chromatography by using methanol/dichloromethane 1:100 as
the elute to afford PNT as an indigo/blue solid (30 mg, 23% yield). Rf =
N,N’-Di-n-butyl-2,6-dibromonaphthalene-1,4,5,8-tetracarboxylic acid bisi-
mide (2): The mixture of compound 1 (2.13 g, 5 mmol), n-butylamine
(1.6 g, 21 mmol), and acetic acid (150 mL) was refluxed for 5 h. After
having been cooled to room temperature, the precipitate was filtered and
washed with water (300 mL) to give 2 as an orange crystal (3.1 g, 58%
yield). 1H NMR (400 MHz, CDCl3, TMS): d=9.01 (s, 2H; naphthalene-
À
À
À
H), 4.22 (t, J=8.0 Hz, 4H; NCH2 ), 1.71–1.79 (m, 4H; NCH2CH2 ),
À
À
1.44–1.53 (m, 4H; CH2CH3), 1.01 ppm (t, J=7.2 Hz, 6H; CH2CH3);
MS (EI+): m/z (%): 538.1 (43), 536.1 (100) [M]+, 534.1 (47).
N,N’-Di-n-butyl-2-(N-{2-[bis(pyridin-2-ylmethyl)amino]ethyl})-6-bromo-
naphthalene-1,4,5,8-tetracarboxylic acid bisimide (3a, BND): Compound
2 (108 mg, 0.2 mmol) was added to a solution of N,N-di(2-pyridylmeth-
yl)ethylenediamine[8] (400 mg, 1.2 mmol) in dry NMP (10 mL), and the
mixture was stirred at 608C for 70 h. After having been evaporated in
vacuo, the residue was dissolved in an aqueous solution of 0.1m HCl
(50 mL) and extracted with dichloromethane (3ꢁ40 mL). The combined
organic layer was washed with saturated brine, dried over anhydrous
MgSO4, filtered, and rotary evaporated. The crude product was purified
by silica-gel chromatography by using methanol/dichloromethane 1:150
as the elute to afford a dark-red solid (73 mg, 52% yield). Rf =0.3 (meth-
1
0.3 (methanol/dichloromethane 1:12); H NMR (400 MHz, CDCl3, TMS):
d=8.46 (d, J=6.0 Hz, 1H; pyridyl-H), 8.45 (s, 1H; naphthalene-H), 8.39
(s, 1H; naphthalene-H), 8.38 (d, J=6.0 Hz, 2H; pyridyl-H), 7.54 (t, J=
7.6 Hz, 1H; pyridyl-H), 7.46 (t, J=7.6 Hz, 2H; pyridyl-H), 7.31 (d, J=
7.6 Hz, 1H; pyridyl-H), 7.23 (d, J=7.6 Hz, 2H; pyridyl-H), 7.09 (t, J=
6.0 Hz, 1H; pyridyl-H), 7.03 (t, J=6.0 Hz, 2H; pyridyl-H), 4.64 (s, 2H;
À
À
CH2-pyridyl), 4.12–4.18 (m, 4H; NCH2 ), 3.68 (s, 4H; CH2-pyridyl),
1
anol/dichloromethane 1:16); H NMR (400 MHz, CDCl3, TMS): d=10.31
À
3.59 (t, J=6.0 Hz, 2H; NCH2CH2N ), 3.38 (s, 4H; piperidinyl-H), 2.85
À
(s, 1H; NH), 8.54 (s, 1H; naphthalene-H), 8.44 (d, J=4.4 Hz, 2H; pyr-
À
(t, J=6.0 Hz, 2H; NCH2CH2N ), 1.83 (s, 4H; piperidinyl-H), 1.65–1.72
(m, 4H; NCH2CH2 ), 1.60 (s, 2H; piperidinyl-H), 1.35–1.44 (m, 4H;
idyl-H), 8.24 (s, 1H; naphthalene-H), 7.72 (d, J=8.0 Hz, 2H; pyridyl-H),
À
À
7.56 (t, J=8.0 Hz, 2H; pyridyl-H), 7.12 (t, J=4.4 Hz, 2H; pyridyl-H),
CH2CH3), 0.94–0.99 ppm (m, 6H; CH2CH3); 13C NMR (100 MHz,
CDCl3): d=13.85, 13.87, 20.38, 23.99, 29.67, 30.32, 40.59, 40.67, 51.99,
52.46, 53.66, 58.86, 60.56, 109.19, 110.38, 121.93, 122.26, 122.33, 122.88,
124.04, 124.56, 125.06, 125.13, 125.47, 126.58, 136.30, 136.76, 148.86,
149.35, 151.12, 152.28, 157.61, 159.02, 161.77, 161.93, 163.01, 163.17 ppm;
HRMS (TOF-ESI+): m/z: calcd for C47H53N8O4+: 793.4190 [M+ H+];
found: 793.4201; calcd for C47H52N8O4Na+: 815.4009 [M+Na+]; found:
815.4001.
À
À
À
À
À
4.17 (t, J=7.2 Hz, 2H; NCH2 ), 4.07 (t, J=7.2 Hz, 2H; NCH2 ), 3.89
À
(s, 4H; CH2-pyridyl), 3.62 (t, J=5.6 Hz, 2H; NHCH2CH2 ), 2.97 (t, J=
À
À
5.6 Hz, 2H; NHCH2CH2 ), 1.67–1.74 (m, 2H; NCH2CH2 ), 1.58–1.66
(m, 2H; NCH2CH2 ), 1.42–1.48 (q, J=5.6 Hz, 2H; CH2CH3), 1.33–1.39
À
À
À
À
(q, J=5.6 Hz, 2H; CH2CH3), 0.95–0.99 (t, J=5.6 Hz, 3H; CH2CH3),
0.91–0.94 ppm (t, J=5.6 Hz, 3H; CH2CH3); 13C NMR (100 MHz,
À
CDCl3): d=13.83, 13.98, 20.33, 20.52, 30.15, 30.24, 40.14, 40.63, 40.67,
52.38, 60.31, 101.02, 119.37, 120.04, 122.23, 123.46, 123.54, 124.41, 126.13,
127.79, 129.43, 131.25, 138.56, 148.97, 151.96, 158.77, 162.88, 163.09,
163.38, 165.88 ppm.
UV/Vis absorption spectrum measurements: The absorption spectra of
PND (50 mm) were measured at 258C in aqueous solution (acetone/
100 mm MOPS buffer 90:10, pH 7.0). Zn2+ was added as Zn
ACHTUNGTRENNUNG(NO3)2 (0,
N,N’-Di-n-butyl-2-(N-{2-[bis(pyridin-2-ylmethyl)amino]ethyl})-6-(N-pi-
peridinyl)naphthalene-1,4,5,8-tetracarboxylic acid bisimide (PND): Com-
pound 3 (70 mg, 0.1 mmol), piperidine (170 mg, 2.0 mmol), and TEA
(200 mL) was dissolved in a solution of dry NMP (5 mL), and the result-
ing mixture was stirred at 508C for 24 h under an argon atmosphere.
After having been evaporated in vacuo, the residue was dissolved in an
0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, and 2.0 equiv of Zn2+
with respect to PND). The absorption spectra of PNT (50 mm) were mea-
sured under the same conditions.
Fluorescence spectral measurements: The fluorescence spectra of PND
(50 mm) were measured at 258C in aqueous solution (acetone/100 mm
8362
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ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 8355 – 8364

Naphthalenediimide Fluorescent Chemosensors
FULL PAPER
MOPS buffer 90:10, pH 7.0) upon excitation at l=610 nm. The amounts
of Zn2+ added were 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4 equiv. The fluo-
rescence spectra of PNT (50 mm) were also measured in MOPS buffer at
258C upon excitation at the isosbesitic wavelength of l=566 nm.
Higher Education (SRFDP 200802510011), and the State Key Laboratory
of Precision Spectroscopy (ECNU).
Quantum yield measurements: The quantum yields of fluorescence were
determined by comparison of the integrated area of the corrected emis-
sion spectrum with a reference of N,N’-di(2,6-dibutyl)-1,6,7,12-tetrakis(4-
tert-butylphenoxy)-3,4:9,10-perylenetetracarboxylic diimide (F=0.66, in
CH2Cl2).[17] For the metal-free study, 5 mL of 5 mm PND or PNT in aque-
ous solution (acetone/100 mm MOPS buffer 90:10, pH 7.0) was prepared.
[1] a) J. M. Berg, Y. G. Shi, Science 1996, 271, 1081–1085; b) J. J. R.
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Soc. 2007, 129, 1378–1385; e) D. D. Mott, M. Benveniste, R. J. Din-
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For the metal-bound studies, ZnACHTNUGTRNEUNG(NO3)2 (15 mL, 5 mm) was added to
PND/PNT (5 mL, 5 mm) in aqueous solution (acetone/100 mm MOPS
buffer 90:10, pH 7.0). The reference concentration was adjusted to match
the absorbance of a test sample at excitation wavelength. Emission for
PND was integrated from 615 to 800 nm with excitation at 610 nm, and
PNT was integrated from 576 to 800 nm with excitation at 566 nm. The
quantum yields were calculated with the reported expression.[20]
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Effect of pH on the fluorescence intensity: The following buffers were
used: ClCH2COOH/ClCH2COONa buffer (100 mm, pH 3.0 and 3.6),
AcOH/AcONa buffer (100 mm, pH 4.2, 4.8, and 5.4), MES buffer
(100 mm, pH 5.7, 6.1, and 6.5), MOPS buffer (100 mm, pH 7.0, 7.4, and
8.0), and CHES buffer (100 mm, pH 8.5 and 9.0). The fluorescence inten-
sity (lex =610, 650 nm) of PND (50 mm) and (lex =566, 635 nm) PNT were
plotted, respectively.[25]
Metal-ion selectivity measurements: The fluorescence selectivity of PND
or PNT to various metals was measured in aqueous solution (acetone/
100 mm MOPS buffer 90:10, pH 7.0) at 258C (lex =610, 650 nm). Heavy
metal ions (30 mm) were added as AgNO3, Cd
(NO3)2, MnSO4, FeSO4, NiSO4, and CuSO4. Other cations were added as
Zn(NO3)2 (30 mm), NaNO3 (300 mm), KNO3 (300 mm), CaCl2 (300 mm), and
ACHTUNGTREN(NUG NO3)2, HgACHTUGNTRNE(NUGN NO3)2, Pb-
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
MgSO4 (300 mm).
Cell culture: A human nasopharyngeal epidermal carcinoma cell line
(KB cell) was provided by the Institute of Biochemistry and Cell Biology,
SIBS, CAS (China). Cells were grown at 378C and with 5% CO2 in Ros-
well Park Memorial Institute medium (RPMI) 1640 supplemented with
10% fetal bovine serum (FBS). Cells (5ꢁ108 LÀ1) were plated on 14 mm
glass coverslips under 100% humidity and allowed to adhere for 24 h.
The cells were washed three times with PBS buffer, and the medium was
replaced with PBS buffer before imaging.
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Microscopy and imaging methods: Confocal luminescence imaging: Con-
focal luminescence imaging of cells was performed with a modified
Olympus FV1000 laser-scanning microscope. A 60ꢁoil-immersion objec-
tive lens was used. Excitation was carried out with an Ar laser at l=
543 nm, and emissions were collected in the range l=598–698 nm, in-
cluding the maximum excitation wavelength of PND (650 nm) and PNT
(634 nm). KB cells were incubated with a PBS solution of ZnCl2 (20 mm)
for 0.5 h, then the cells were incubated with a PBS solution (note: ca.
1% acetonitrile was added for solubility reasons) of PND or PNT
(20 mm) for dye loading for 0.5 h at 378C. The stained cells were washed
three times with PBS buffer, and the medium was replaced with PBS
buffer before imaging. Then the treated cells were imaged by fluores-
cence microscopy.
Computational details (LANL2DZ-optimized geometries of PND–Zn2+
and PNT–Zn2+): The DFT calculations were carried out by using a Gaus-
sian 03 software package.[26] The ground-state geometries of PND–Zn2+
and PNT–Zn2+ were optimized at the DFT level with the B3LYP hybrid
functional in combination with the LANL2DZ basis set for the Zn atom
and the D95V basis set for C, H, O, and N atoms.
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Acknowledgements
This work was financially supported by the NSFC/China, National Basic
Research 973 Project (2006CB806200), Project for New Century Excel-
lent Talents in University (NCET-06-0418), Shanghai Shuguang Project
(07SG34), Specialized Research Fund for the Doctoral Program of
Chem. Eur. J. 2010, 16, 8355 – 8364
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Received: February 22, 2010
Published online: June 11, 2010
8364
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