A water-soluble 1,8-naphthyridine-based imidazolium molecular gripper for fluorescence sensing and discriminating of GMP

Article abstract of DOI:10.1016/j.dyepig.2019.108103

A selective receptor for sensing and discriminating of GMP is useful not only in the energy metabolism but also in the processes of DNA replication and transcription-related to GMP; such a receptor is presently rare especially in a pure water environment. Herein, a novel 1,8-naphthyridine-based tripodal imidazolium gripper-like molecular served as a “turn-on” fluorescent receptor (TINP) was designed and synthesized for selective sensing and discriminating GMP from structurally similar GNPs (N = D and T) and XMPs (X = U, T, A, and C) in 100% aqueous solution. TINP consists of 1,8-naphthyridines and imidazolium cations. 2-acetylamino-1,8-naphthyridine was chosen as fluorophore and tri-hydrogen bonds interactions sites for the nucleobase guanine. Imidazolium cations were identified as the phosphate part receiving moieties and communicators, while the three imidazolium cations also served as indispensable water-soluble parts. GMP caused a remarkable fluorescence enhancement (ca. 6.5-fold) with a quantum yield (Φ_f) of 0.26 at 399 nm, displaying an efficient “turn on” behavior. The sensing mechanisms and fluorescence response were explained by Job's plot, NMR spectroscopic analysis, and theoretical calculations. The “turn-on” fluorescent property for GMP can be attributed to photoinduced electron transfer (PeT) transitions with some mixed intraligand charge-transfer (ILCT) transitions. Finally, the preliminary results of cell experiments show that the receptor can be applied for the imaging of GMP in living mammalian cells.

Full text of DOI:10.1016/j.dyepig.2019.108103

Dyes and Pigments xxx (xxxx) xxx
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: http://www.elsevier.com/locate/dyepig
A water-soluble 1,8-naphthyridine-based imidazolium molecular gripper
for fluorescence sensing and discriminating of GMP
a,
b
a
a
a,
d,
*
c,**
Yuan Zhang , Ran Sun , Xiaoxue Kang , De-Hui Wang
, Yong Chen
a
Liaoning Shihua University, Fushun, 113001, China
b
Liaoning Institute for Food Control, Shenyang, 110015, China
c
Key Laboratory of Photochemical Conversion and Optoelectronic Materials and HKU-CAS Joint Laboratory on New Materials, Technical Institute of Physics and
Chemistry, Chinese Academy of Sciences, Beijing, 100190, China
d
Department of Chemistry, University of Utah, 315 South 1400 East, Room 2020, Salt Lake City, UT, 84112, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
A selective receptor for sensing and discriminating of GMP is useful not only in the energy metabolism but also in
the processes of DNA replication and transcription-related to GMP; such a receptor is presently rare especially in
a pure water environment. Herein, a novel 1,8-naphthyridine-based tripodal imidazolium gripper-like molecular
served as a “turn-on” fluorescent receptor (TINP) was designed and synthesized for selective sensing and
discriminating GMP from structurally similar GNPs (N ¼ D and T) and XMPs (X ¼ U, T, A, and C) in 100%
aqueous solution. TINP consists of 1,8-naphthyridines and imidazolium cations. 2-acetylamino-1,8-naphthyri-
dine was chosen as fluorophore and tri-hydrogen bonds interactions sites for the nucleobase guanine. Imida-
zolium cations were identified as the phosphate part receiving moieties and communicators, while the three
imidazolium cations also served as indispensable water-soluble parts. GMP caused a remarkable fluorescence
1
,8-Naphthyridine
GMP
Turn on
Fluorescent discriminating
Bioimaging
enhancement (ca. 6.5-fold) with a quantum yield (Φ ) of 0.26 at 399 nm, displaying an efficient “turn on”
f
behavior. The sensing mechanisms and fluorescence response were explained by Job’s plot, NMR spectroscopic
analysis, and theoretical calculations. The “turn-on” fluorescent property for GMP can be attributed to photo-
induced electron transfer (PeT) transitions with some mixed intraligand charge-transfer (ILCT) transitions.
Finally, the preliminary results of cell experiments show that the receptor can be applied for the imaging of GMP
in living mammalian cells.
1
. Introduction
[9–12]. Of all the nucleotides, the sensing of GMP is a field of intense
research activity, because it is potentially attractive for use in such areas
0
0
Nucleotides, such as adenosine 5 -triphosphate (ATP), guanosine 5 -
as an intermediate in the synthesis of nucleic acids and plays important
roles in several metabolic processes [13–15]. Apart from its roles in
energy metabolism, GMP is also incorporated into nucleic acids by
polymerases in the processes of DNA replication and transcription.
Recently, considerable efforts have been devoted to developing fluo-
rescent chemosensors for GMP [16–22]. However, only a limited num-
ber of “fluorescence-on” sensors for GMP with high selectivity have been
reported because of the structures of various types of nucleotides are
similar, rich in a number of hydrogen bonds, such a recognition in the
aqueous medium would be limited due to the interference from hydroxyl
groups of the sugar moiety and competitive hydrogen bonding of the
solvent [23–25]. The poor binding selectivity for GMP over other
0
triphosphate (GTP), and guanosine 5 -monophosphate (GMP), play
central roles in understanding and evaluating several key biological
processes [1–3]. Thus, specific detection of nucleotides is very impor-
tant. Fluorescence detection techniques for sensing and imaging of trace
amounts of various nucleotides and nucleosides have attracted immense
attention due to their conveniently and easily monitor target molecule in
the visible range with fast response, high selectivity and sensitivity, and
instantaneous response [4–8]. More importantly, a great deal of fluo-
rescent nucleotide receptors are ready for in vivo and/or in vitro cellular
imaging to make the fluorescence approach superior to other analytical
methods, such as NMR, X-ray crystallography, and electrophoresis
*
Corresponding author. Liaoning Shihua University, Fushun, 113001, China.
* Corresponding author.
E-mail addresses: dhuiwang@aliyun.com (D.-H. Wang), chenyong@mail.ipc.ac.cn (Y. Chen).
*
https://doi.org/10.1016/j.dyepig.2019.108103
Received 6 May 2019; Received in revised form 29 November 2019; Accepted 3 December 2019
Available online 9 December 2019
0
143-7208/© 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Yuan Zhang, Dyes and Pigments, https://doi.org/10.1016/j.dyepig.2019.108103

Y. Zhang et al.
Dyes and Pigments xxx (xxxx) xxx
0
0
disodium hydrate (GTP), Uridine-5 -monophosphate disodium (UMP),
structurally similar guanosine 5 -phosphate (GNPs, N ¼ di (D) or tri (T))
0
0
0
and nucleotide 5 -monophosphate (XMPs, X ¼ uridine (U), thymidine
Adenosine-5 -monophosphate acid monohydrate (AMP), Cytidine-5 -
0
0
(
T), adenosine (A), and cytidine (C)), decrease the accuracy of GMP
diphosphate acid (CMP), 2 -Deoxythymidine-5 -monophosphate diso-
dium (dTMP), Guanosine, HeLa cells, phosphate-buffered saline (PBS)
and sodium cacodylate buffer solution (containing NaCl, pH ¼ 7.0) were
purchased from Bio Basic Inc. (BBI) (Shanghai, China). Milli-Q (Milli-
pore, Milford, MA) ultrapure water was used in all of the experiments.
Caution! Sodium perchlorate is potentially explosive and should be handled
with care and in small amounts.
detection in the analysis media. Ramaiah and co-workers have reported
the first selectively GMP probe relied on a metallocyclophane by a
fluorescence quenching output manner in DMSO-water solution [17].
However, development of bright chemosensors for GMP with high
selectivity and sensitivity, which can clearly discriminate GMP from
structurally similar XMPs and GNPs still remains rare up to now, espe-
cially in 100% aqueous solution.
1H NMR and 13C NMR spectra were recorded using a Varian Inova-
The imidazolium podand-shaped fluorescent chemosensors for
sensing nucleotides were pioneered by Yoon and Kim [26] and devel-
oped by the groups of Ramaiah [27], Duan [28], and others [29,30]. Our
group has reported an aminonaphthalimide imidazolium podand which
worked as luminescence chemosensor for selectively sensing ADP
through a “turn-on” manner [28]. Accordingly, the successful applica-
tions of the podands with arms comprising imidazolium bonding moi-
eties encouraged us finding a new fluorescent substituent with special
structure to further extend this excellent research. Extending this
approach [28,31–33], herein, we report the synthesis, binding proper-
ties, and biological activities of a new tripodal imidazolium receptor
400 spectrometer with chemical shifts reported as ppm (in DMSO‑d
or CDCl , TMS as internal standard). ESI mass spectra were obtained
6
3
using an LC/Q-TOF MS spectrometer. Melting points (Mp) were deter-
mined using an MP100. Elemental analyses (EA) were performed using a
Vario EL cube analyzer. Fluorescence emission spectra were recorded
using a SPEX Fluorolog-2 Model F111 fluorescence spectrophotometer.
For all fluorescent measurements, both excitation and emission slit
widths were 1 nm. Optical absorption spectra were measured using a
Hitachi U-0080D spectrometer at room temperature. Cell images were
measured on
microscopy.
a Nikon Eclipse TE2000-5 inverted fluorescence
(
TINP) for fluorescent discriminating GMP from structurally similar
GNPs and XMPs in 100% aqueous solution. Indeed, 2-acetylamino-1,
-naphthyridine which possesses hydrogen bonding site fully comple-
2
.2. General procedures for spectroscopy
8
mentary to nucleobase guanine was chosen as fluorophore and
tri-hydrogen bonds interactions site for the nucleobase part [34–36].
Imidazolium cations were identified as the phosphate parts receiving
moieties and communicators, while the three imidazolium cations also
served as indispensable water-soluble parts (Scheme 1). The three
necessary units were introduced as trigger sites to achieve efficient in-
teractions and a consequently good fluorescence signal response in
Stock solutions (10 mM) of the sodium salts of nucleotides of GMP,
2À
GDP, GTP, UMP, dTMP, AMP, CMP, Guanosine, HPO
4
in 1 : 1 DMF/
2
H O (v: v) solvents were prepared and diluted to the appropriate con-
centration. Stock solutions of TINP (4 mM) were also prepared in ul-
trapure water containing 1% DMSO. Test solutions were prepared by
placing 20 L of host stock solution into a quartz cell of 1 cm optical path
μ
length, including 2 mL sodium cacodylate buffer solution (containing
NaCl, pH ¼ 7.0) solution, and then adding an appropriate aliquot of each
nucleotides stock by using a micro-syringe. Luminescent quantum yields
1
00% neutral aqueous solution. The “turn-on” fluorescence can be
attributed to a mixture of PeT and ILCT transitions. What is more, the
blue-purple region (λ ~ 350–450 nm) of TINP provide an opportunity to
luminescent image GMP in living cells.
were referenced to quinine sulfate in 0.1 N H
2
SO
4
(Ф
f
¼ 0.546) and
extrapolated to infinite dilution (estimated error � 3%). Excitation and
emission slit widths were modified to adjust the luminescent intensity in
a suitable range. All the spectroscopic measurements were performed at
2
. Experimental
least in triplicate and averaged.
2
.1. Materials and apparatuses
1H NMR titration experiments were measured on a Varian Inova-400
spectrometer at 298 K. A solution (1 mM) of receptor TINP in DMSO‑d
6
All materials were used as received. The solvents used for synthesis
was titrated with 4 equiv of GMP by using a micro-syringe. The chemical
shift changes of the proton of TINP and GMP were monitored.
were of analytical grade. All the solvents and reagents unless otherwise
stated, were purchased from Sinopharm Chemical Reagent Co.
(
Shanghai, China) and Sigma–Aldrich (USA), and were used without
2
.3. Association constants calculation
0
further purification. Guanosine-5 -monophosphate disodium (GMP),
0
0
Guanosine-5 -diphosphate disodium (GDP), Guanosine-5 -triphosphate
The binding constant was calculated from the fluorescent titration
3
þ
Scheme 1. The chemical structures of TINP and TIM. Insert: X-ray crystal structure of [TIM] with cis, trans, trans conformation. Thermal displacement ellipsoids
À
are drawn at the 30% probability level. The anions of ClO
4
and H atoms are omitted for clarity.
2

Y. Zhang et al.
Dyes and Pigments xxx (xxxx) xxx
curve according to the equation.
to give the desired tribromo anions product as a yellow powder. A so-
À
Log ((F-Fmin)/(Fmax-F)) ¼ log k þ n log [c].Where the F is fluorescent
of TINP at 399 nm upon addition of the different amount of the GMP. [c]
Stands for the concentration of GMP.
lution of the mixture of 3Br product (0.39 g, 0.35 mmol) and NaClO
4
3
(0.44 g, 1.3 mmol) was stirred at room temperature in CH OH (30 mL)
for 1 h. The yellow precipitated formed was filtered, washed with
methanol and diethyl ether, and dried in vacuo. Yield 0.32 g (73%). M.p.
ꢀ
2
.4. Cell incubation and imaging
146–148 C; Anal calc. for C54
H54Cl
3
N
15
O
15: C 51.50, H 4.32, N 16.68%.
1
Found: C 51.47, H 4.35, N 16.61%; H NMR (400 MHz, DMSO‑d
6
) δ:
HeLa cells were cultured in 1640 supplemented with 10% FCS
10.94 (s, 3H), 9.01 (s, 3H), 8.47 (m, 6H), 8.34 (d, J ¼ 8, 3H), 7.85 (d, J ¼
12, 3H), 7.70 (d, J ¼ 8, 3H), 7.50 (d, J ¼ 8, 3H), 5.65 (s, 6H), 5.64 (s,
(
Invitrogen). Cells were seeded on 18 mm glass coverslips for confocal
6H), 2.50 (s, 9H), 2.16 (s, 9H); 13C NMR (101 MHz, DMSO‑d
) δ 166.2,
fluorescence imaging and in 24-well flat-bottomed plates for Nikon
Eclipse TE2000-5 inverted fluorescence microscopy. After 12 h, HeLa
6
165.9, 164.2, 164.0, 158.2, 142.1, 137.2, 132.1, 131.5, 129.5, 126.8,
cells were incubated with 10
μ
M compound TINP (in the culture me-
124.1, 122.2, 115.1, 53.8, 49.1, 23.6, 18.2 ppm; LCQ-Tof MS: 320.2190
ꢀ
3þ
dium containing 0.5% DMSO) for 15 min at 37 C under 5% CO
2
and
[M]
.
then washed with phosphate-buffered saline (PBS) three times before
incubating with 20 equiv of GMP for another 30 min, and cells were
rinsed with PBS three times again. The fluorescence imaging of intra-
cellular GMP in HeLa cells was observed under Nikon Eclipse TE2000-5
inverted fluorescence microscopy with a 20 ꢁ objective lens (excited by
blue light). For all images, the microscope settings, such as brightness,
contrast, and exposure time were held constant to compare the relative
intensity of intracellular GMP fluorescence.
TIM: TIM was synthesized with 80% yield using the same method as
TINP.
3. Results and discussion
3.1. Design, synthesis, and structure
In our previous work, we have reported chemosensors based on
aminonaphthalimide derivatives for turn-on fluorescence sensing of
nucleoside polyphosphates [28]. Planar aminonaphthalimide groups
2
.5. Computational details
can react with ADP/ATP/GTP via the possible
π
…
π
stacking interaction
All theoretical calculations were performed in the Virtual Laboratory
between the nucleobase and 1,8-naphthalimide group. Amino-
naphthalimide groups could not increase the selectivity because of the
lack of nucleobase-targeting multiple hydrogen bonds [35]. Therefore,
we speculated that incorporating multiple hydrogen bonds onto planar
chromophore might generate a novel fluorescent probe candidate for
nucleotides. The planar 2-acetylamino-1,8-naphthyridine which pos-
sesses hydrogen bonding groups fully complementary to nucleobase
guanine was chosen as fluorophore and tri-hydrogen bonds interactions
site for the nucleobase part. Moreover, again, imidazolium cationic
for Computational Chemistry, CNIC, CAS. All calculations were carried
out with the Gaussian 09 programs [37]. The B3LYP calculations were
carried out by the 6-311G**(d, p) basis set. We employed the density
functional theory (DFT) with no symmetry constraints to investigate the
þ
optimized ground state (S
0
) geometries of the ligand TINP3 and
1/3TINP þ GMP] . UV–Vis energies (the first excited-state (S
geometrical optimization) were computed from Time-dependent DFT
TDDFT, nstates ¼ 30) method based on the optimized geometry of the
lowest excited state in water.
-
[
1
)
(
groups display water solubility like that of ionic liquids and support the
À
chances of anions exchange (the anions of ClO
4
are replaced with bigger
size of GMP2 ) [42,43]. In addition, the tripod and gripper-like structure
can enhance the chelated functional effectiveness, which may benefit
the selectively of sensing of GMP [44].
À
2
.6. X-ray structure determinations
Data collection: Intensity data of compound TIM was collected on a
BRUKER SMART APEXCCD diffractometer. A crystal of dimensions 0.26
The syntheses of TINP are shown in Scheme 2 in a high yield, which
can be prepared by the reaction of three-arm 1,3,5-tris-(imidazol-1-
ylmethyl) À 2,4,6-trimethylbenzene intermediate 2 with 2-bromo-
x 0.24 ꢁ 0.23 mm mounted in a glass capillary was used for data
ꢀ
collection at 28 C on a MAR diffractometer with a 300 mm image plate
detector using graphite monochromatized Mo-K
α
radiation (λ
¼
methyl-7-acetylamino-1,8-naphthyridine intermediate 1 in CHCl
3
, fol-
ꢀ
0
.71073 Å). Data collection was made with 1.5 oscillation step of ϕ, 5
lowed by an anion exchange reaction with NaClO . Notably, the
4
min exposure time and scanner distance at 120 mm. 130 images were
collected.
intermediate 2 has a total cis, trans, trans conformation after recrystal-
lization with ethanol. TINP was characterized by EA, NMR, and MS
(Fig. S8). The crystal of TINP was polycrystal and easy to efflorescence,
it was unsuitable for single crystal analysis. To better understand the
structure of TINP and the effect of 1,8-naphthyridine substituent, the
comparison of ligand TIM was designed and synthesized under the same
experimental conditions. Fortunately, crystals of TIM suitable for single
crystal analysis were easily obtained by slowing volatilizing the solution
at room temperature for about two days. From the crystal analysis, TIM
adopts a partial cone conformation with a molecular cavity defined by
two essentially coplanar imidazolium groups and two methyl sub-
stituents. The third imidazolium group is on the opposite face of the
benzene ring (Scheme 1 and Fig. S1). At room temperature, all spectra
display symmetry in contrast to the X-ray crystal structure of TIM. Thus,
we judged that the substituted 1,8-naphthyridine host TINP showed a
structural framework similar to that of TIM, which increases the char-
acter of the coordination site for G only, as reported by a few groups
[45–48].
Data reduction: The images were interpreted and intensities inte-
grated using program DENZO [38].
Structure solution: The structure was solved by direct methods
employing SHELXS-97 program [ [39]] on PC. Many non-H atoms were
located according to the direct methods. The positions of the other
non-hydrogen atoms were found after successful refinement by
full-matrix least-squares using program SHELXL-97 on PC. CCDC num-
ber 1543420 contains the supplementary crystallographic data for this
paper. These data can be obtained free of charge from the Cambridge
Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/cgi-bin/c
atreq.cgi.
2
.7. Synthesis of TINP
Compound 1 was synthesized with a low 30% yield according to the
published procedure [40].
Compound 2 was synthesized with 80% yield using published pro-
cedure [41].
3.2. Fluorescent detection of GMP
TINP: 2 (0.18 g, 0.5 mmol) and 1 (0.45 g, 1.6 mmol) were dissolved
in CHCl
3
(20 mL) and stirred at reflux for 15 h. During this time, a white
TINP exhibited two characteristic absorption bands centered at 262
nm and 321 nm in sodium cacodylate buffer solution (containing NaCl,
precipitate formed. The product was filtered off and washed with CHCl
3
3

Y. Zhang et al.
Dyes and Pigments xxx (xxxx) xxx
Scheme 2. The synthetic pathway of TINP and TIM.
pH ¼ 7.0) (Fig. S3). The first band located at 200–295 nm can be
assigned to the moderate energy ( *) transition of the
* and n →
aromatic rings, while the second band at 295–350 nm is due to the low
energy ( *) transition involving the -electrons of the 1,8-naphthyr-
idine group. TINP displayed a weak fluorescence band with a corre-
sponding emission maximum at 389 nm (Φ ¼ 0.08) in sodium
cacodylate buffer solution (containing NaCl, pH ¼ 7.0) when excited at
21 nm (Fig. 1). Upon addition of structurally similar GNPs (GMP, GDP,
and GTP) and XMPs (UMP, dTMP, AMP, and CMP), only GMP caused a
remarkable fluorescence enhancement with a quantum yield (Φ ) of
.26, displaying an efficient “turn on” behavior. The insert photograph
of the turn-on fluorescence of sensor TINP upon binding to GMP clearly
demonstrated the naked eye detection of GMP using this chemosensor.
While after the addition of various other GNPs and XMPs, almost
negligible changes in fluorescence intensity were induced. The addition
of GMP resulted in a significant fluorescence enhancement until a
plateau was reached. Upon adding GMP, the fluorescence emission
responsible for the observed fluorescence enhancement, and the asso-
π
→
π
π
ciation constant for GMP binding to TINP was calculated as 9.17 ꢂ 0.10
4
À 1
ꢁ 10 M [49]. To gain further information for the proposed binding
1
π
→
π
π
pattern, H NMR titration experiments of TINP with GMP are investi-
gated (Fig. 3). The addition of 4 equiv of GMP to the solution of TINP in
f
DMSO‑d
which indicates the strong ionic hydrogen bond interaction between
imidazolium and phosphate groups. The aromatic protons and –CH
protons of TINP split into irregular peaks. This result means that a strong
- protons of TINP
6
causes downfield shifts of the protons of imidazolium moieties
3
2
-
f
binding participation of the imidazolium ring and –CH
2
0
occur with GMP unequally [50]. More importantly, the peak at 9.01
þ
ppm, which corresponds to the characteristic (C–H) proton H
1
on the
imidazolium (N-heterocyclic carbene), was downfield shifted to 9.45
ppm which was accompanied by an irregular split. Meanwhile, the peak
2
of –NH– proton H at 10.94 ppm, was downfield shifted to 11.56 ppm,
which was accompanied by an appearance of a broad peak. The signif-
icant upfield shifts of the signals corresponding to the aromatic protons
within the 1,8-naphthyridine and downfield shift of the signal of -NH-
demonstrated the possible three-point hydrogen bonds interactions be-
tween the guanine and 1,8-naphthyridine group [51]. On the other
hand, upon addition of GMP, almost all the peaks of TINP split into
multiple-peaks with different up- or downfield chemical shifts. All the
above NMR results support the asymmetrical binding mode of TINP with
GMP.
maximum at 399 nm of TINP increased by ca. 6.5-fold (Fig. 2). The
À 8
detection limit of TINP toward GMP was obtained as 2.4 ꢁ 10
M
(
Fig. S9). Moreover, the competition experiments revealed that TINP
retained the excellent GMP specificity in the presence of a variety of
other GNPs and XMPs, this means that the luminescence enhancement
induced by GMP was little affected by these structurally similar nucle-
otides (Fig. S4). These results suggest that TINP can respond to GMP
with high selectivity by a fluorescence output manner.
3
.4. Binding pattern and luminescence mechanism between TINP and
3
.3. Nature of binding
GMP
Binding analysis using the method of the linear fitting of the fluo-
Based on the results mentioned above, the following mechanism was
rescence titration curve (Fig. S5) and the continues variations (Job’s
plot) (Fig. S6) established that a 1 : 1 TINP-GMP complex was
proposed to explain this good fluorescence signal response and
discriminating of GMP (Fig. 4). In view of the rich in a number of
4

Y. Zhang et al.
Dyes and Pigments xxx (xxxx) xxx
phosphate group from GMP, as well as water solubility and the chances
of anions exchange. In addition, the tripod and gripper-like structure can
enhance the chelated functional effectiveness, which may benefit the
selectively of sensing of GMP. These necessary units were introduced as
trigger sites to achieve efficient weak interactions and a consequently
good fluorescence signal response.
Since the 7-acetylamino-1,8-naphthyridines absorbance band did
not change upon the addition of increasing amounts of GMP (Fig. S3),
the enhancement of the fluorescence was attributed to the PeT “on”
motif [52]. In the presence of GMP, fully matched tri-hydrogen bonds of
the nucleobase guanine with 7-acetylamino-1,8-naphthyridine favors
electron transfer (ET) from the 7-acetylamino-1,8-naphthyridine to the
À 2À
guanine part. Meanwhile, anion exchange among ClO and GMP is
4
achieved via weak electrostatic interactions between the þ3 charged
imidazolium groups and the negatively charged guest anions. The more
negative the charge of the GMP2 , the higher its affinity for the posi-
tively charged TINP. This process of anion exchange will also effectively
reduce the charge density of the imidazolium part and favors ET from 1,
À
8
-naphthyridine to the imidazolium parts. Accordingly, this two ET
processes increase the chance of electronic transitions from LUMO to
HOMO of the 1,8-naphthyridine part and consequently enhance lumi-
nescence [53]. It is worth noting that there is a slight shoulder at 460 nm
for TINP þ GMP. This phenomena may be ascribed to
π
–
π
stacking of two
1
,8- naphthyridine in the sensing process. Although these interactions
are weak in water, we postulated that they would be sufficient to
distinguish the structurally similar nucleotides at their millimolar
intracellular concentrations.
In order to further rationalizing the binding pattern, we optimized
the ground state of TINP3 based on the crystal data of TIM and S
þ
state
0
þ
optimum structures of one arm of TINP ([1/3TINP] ) and [1/3TINP þ
Fig. 1. Fluorescence changes of TINP (4.0 ꢁ 10À 5 M) upon titration with 5.0
-
GMP] complex in water (Fig. S7 and Fig. 5) following the previous es-
2
4
À
in 10 mM so-
equiv of each of the guest XMPs, GNPs, guanosine, and HPO
timates. The optimum geometry for the ground electronic state of TINP
is an open structure (Fig. S7) which closes when the complex is formed
dium cacodylate buffer solution (containing 50 mM NaCl, pH ¼ 7.0). Excitation
wavelength: 321 nm.
(
Fig. 4). The open structure of TINP exhibited a similar structure to that
þ
in the crystal state of TIM. We selected one arm of TINP ([1/3TINP] ) as
ligand and created the theoretical model to reduce the computational
pressure of macromolecules. Although this reduced computation will
affect the strength of the spectra, it will not affect the results of the
positions of the absorption peaks. The calculated model demonstrated
that the 2-amino-1,8-naphthyridine possessed hydrogen bonding groups
fully complementary to G base and a planar bicyclic ring structure for
þ
binding. Meanwhile, there is an obvious strong effect of (C–H) … O
charged hydrogen bonds between the imidazolium of TINP and phos-
phate group in GMP. The time-dependent density functional theory
(
TDDFT) calculations on the UV–vis absorption (vertical excitation) was
summarized in Table S3. Indeed, the peaks of calculated absorbance of
-
[
1/3TINP þ GMP] in water are in good agreement with the experi-
mental results (~318–328 nm, TINP þ GMP in sodium cacodylate buffer
solution) (Fig. S1).
3
.5. Fluorescent detection of GMP in living organisms
Furthermore, preliminary inverted microscopy fluorescent images of
TINP-loaded HeLa cells in the absence and presence of GMP were
examined. HeLa cells were incubated with 10 M TINP for 30 min at
Fig. 2. Fluorescence titrations of TINP (4.0 ꢁ 10À 5 M) with GMP in 10 mM
μ
room temperature to allow the probe to permeate into the cells. When
exciting at 320 nm, HeLa cells showed no obvious intracellular fluo-
rescence (Fig. 6a). A significant fluorescence emission was observed
when cells stained were further incubated with GMP (0.2 mM) for
another 30 min (Fig. 6b), indicating the possible usage in fluorescence
images involving GMP within living cells. Bright-field measurement
confirmed that the cells remained viable and no apparent toxicity and
side effects were observed throughout the imaging experiments
(Fig. 6c).
sodium cacodylate buffer solution (containing 50 mM NaCl, pH ¼ 7.0). Exci-
tation wavelength: 321 nm [GMP]: 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0
equiv. Inset: Detection of GMP based on the changes in fluorescence of TINP.
The samples are excited with a UV lamp at 302 nm.
hydrogen bonds for GMP, TINP could function as a multiple functional
chelator to ligate with GMP in a 1 : 1 stoichiometric ratio. The 7-acety-
lamino-1,8-naphthyridine from the ligand is chosen as fluorophore and
are the most likely tri-hydrogen binding sites for the G part of GMP.
Imidazoline cationic groups display electrostatic interactions with
5

Y. Zhang et al.
Dyes and Pigments xxx (xxxx) xxx
1
Fig. 3. Partial H NMR spectra for (a) pure GMP, (b) TINP þ GMP (4 eq) and (c) TINP in DMSO‑d
6
, respectively.
Fig. 4. Proposed sensing mechanism of the receptor TINP towards GMP.
4
. Conclusions
In summary, we have successfully developed a water-soluble 1,8-
naphthyridine-based tripodal imidazolium receptor for fluorescent
discriminating GMP from structurally similar GNPs and XMPs in 100%
aqueous solution. The key feature of this sensor is a gripper-like guest
biding site, the 1,8-naphthyridine moiety incorporated into the imida-
zolium arms of TINP. Further binding with GMP increases the fluores-
cence of the 1,8-naphthyridine fluorophore enabling the use of TINP as a
fluorescence turn-on sensor. We also demonstrated that the selectivity of
TINP for GMP is a result of combining charge-charge electrostatic and H-
bonding interactions, steric and size effects. The utility of TINP as a
bioanalytical molecular tool has also been demonstrated by fluorescence
imaging of GMP in living cells. Actually, the accurate mechanism of high
selectivity for GMP is still unclear. Future studies on the recognition
mechanisms, recognition of Guanine-Guanine mismatches by fluores-
cent and footprinting analysis, is in progress.
Author contribution statement
þ
Fig. 5. DFT calculated interactions between [1/3TINP] with GMP in water.
Yuan Zhang: Conceptualization, Methodology, Software, Data
curation, Writing- Original draft preparation; Ran Sun: Methodology,
Data curation; Xiaoxue Kang: Software (DFT); De-Hui Wang:
Conceptualization, Methodology, Resources, Writing- Reviewing and
6

Y. Zhang et al.
Dyes and Pigments xxx (xxxx) xxx
Fig. 6. Fluorescence images of HeLa cells (λex ¼ 320 nm). (a) Cells supplemented with TINP for 15 min. (b) Cells supplemented with TINP for 15 min and then
incubated with GMP for 30 min. (c) A bright-field image of the cells in the panel.
Editing; Yong Chen: Supervision, Project administration.
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Declaration of competing interests
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sensing of nucleoside monophosphates by a dicopper(II) cryptate. J Am Chem Soc
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The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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(
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