Controlling ion-sensing specificity of N-amidothioureas: From anion-selective sensors to highly Zn2+-selective sensors by tuning electronic effects

Article abstract of DOI:10.1021/ol302313x

N-Amidothioureas bearing nitro groups showed different sensitivities to various anions, while those with methoxy moieties exhibited the sensitive and selective fluorescence turn-on property for Zn²⁺ in aqueous solution and in cells. N-Amidothio

Full text of DOI:10.1021/ol302313x

ORGANIC
LETTERS
2012
Vol. 14, No. 19
5070–5073
Controlling Ion-Sensing Specificity of
N‑Amidothioureas: From Anion-Selective
Sensors to Highly Zn2þ-Selective Sensors
by Tuning Electronic Effects
Hua-Long Chen, Zhi-Fo Guo, and Zhong-lin Lu*
College of Chemistry, Beijing Normal University, Xinjiekouwai Street 19, Beijing,
100875, China
luzl@bnu.edu.cn
Received August 19, 2012
ABSTRACT
N-Amidothioureas bearing nitro groups showed different sensitivities to various anions, while those with methoxy moieties exhibited the sensitive
and selective fluorescence turn-on property for Zn2þ in aqueous solution and in cells. N-Amidothioureas can therefore be adjusted from anion-
selective sensors to Zn2þ-selective sensors by varying the electronic effects of their substituents.
Due to their ability to form multiple inter- and intra-
molecular hydrogen bonds, especially pairs of NꢀH...S
hydrogen bonds,1 amidoureas and amidothioureas have
been successfully developed as colorimetric and fluorescent
anion sensors in recent years. Various mono- and disub-
stituted N-amidothiourea receptors have been designed
and synthesized for their selective and efficient binding of
different anions in both organic solvents and aqueous
media.2 The mechnisms for anion sensing by these com-
pounds are suggested to be either through hydrogen
bonding and/or via deprotonation. Based on the reported
examples, cleft-like bis-N-amidothiourea receptors show
enhanced affinities and colorimetric sensing for anions
as compared to monosubstituted receptors.3,4 It is also
known that electron-withdrawing substituents such as
nitro and trifluoromethyl groups attached to the N-phenyl
rings reinforce the anion binding and afford high sensiti-
vity.4 However, to the best our knowledge, there hae been
no reports of these compounds acting as sensors for the
detection of cations based on coordination mode.5 Different
from anion recognition, which relies on both the acidity
of the thioureido NH protons and the electron-deficient
(3) (a) Duke, R. M.; O’Brien, J. E.; McCabe, T.; Gunnlaugsson, T.
Org. Biomol. Chem. 2008, 6, 4089. (b) Duke, R. M.; Gunnlaugsson, T.
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W.; Gunnlaugsson, T. J. Org. Chem. 2012, 77, 3115. (d) Wei, W.; Zhang,
Y. M.; Wei, T. B. Chin. J. Chem. 2008, 26, 1935.
(4) (a) Evans, L. S.; Gale, P. A.; Light, M. E.; Quesada, R. New J.
Chem. 2006, 30, 1019. (b) Wu, J. L.; He, Y. B.; Wei, L. H.; Liu, S. Y.;
Meng, L. Z.; Hu, L. Supramol. Chem. 2004, 16, 353. (c) Wei, L. H.; He,
Y. B.; Wu, J. L.; Wu, X. J.; Meng, L. Z.; Yang, X. Supramol. Chem. 2004,
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(1) (a) Moragues, M. E.; Martınez-Manez, R.; Sancenon, F. Chem.
Soc. Rev. 2011, 40, 2593. (b) Jiang, Q. Q.; Darhkijav, B.; Liu, H.; Wang,
F.; Li, Z.; Jiang, Y. B. Chem.;Asian J. 2010, 5, 543. (c) Li, A. F.; Wang,
J. H.; Wang, F.; Jiang, Y. B. Chem. Soc. Rev. 2010, 39, 3729. (d) Evans,
L. S.; Gale, P. A.; Light, M. E.; Quesada, R. Chem. Commun. 2006, 42,
965. (e) Zeng, Z. Y.; He, Y. B.; Wu, J. L.; Wei, L. H.; Liu, X.; Meng,
L. Z.; Yang, X. Eur. J. Org. Chem. 2004, 13, 2888.
(5) Some Hg2þ sensors are due to the desulfurization reactions; see:
(a) Yang, Y. K.; Yook, K. J.; Tae, J. J. Am. Chem. Soc. 2005, 127, 16760.
(b) Mahapatra, A. K.; Roy, J.; Sahoo, P.; Mukhopadhyay, S. K.;
Chattopadhyay, A. Org. Biomol. Chem. 2012, 10, 2231. (c) Liu, J.; Yu,
M.; Wang, X. C.; Zhang, Z. Spectrochim. Acta. A 2012, 93, 245. (d)
Koteeswari, R.; Ashokkumar, P.; Malar, E. J. P.; Ramakrishnan, V. T.;
Ramamurthy, P. Chem. Commun. 2011, 47, 7695.
(2) (a) Nie, L.; Li, Z.; Han, J.; Zhang, X.; Yang, R.; Liu, W. X.; Wu,
F. Y.; Xie, J. W.; Zhao, Y. F.; Jiang, Y. B. J. Org. Chem. 2004, 69, 6449.
(b) Liu, W. X.; Yang, R.; Li, A. F.; Li, Z.; Gao, Y. F.; Luo, X. X.; Ruan,
Y. B.; Jiang, Y. B. Org. Biomol. Chem. 2009, 7, 4021.
r
10.1021/ol302313x
Published on Web 09/20/2012
2012 American Chemical Society

thiourea center for high sensitivity, an elelctron-rich thio-
urea center in the N-amidothiourea compounds may lead
to an increase in sensitivity for cations. Given the thio-
affinity of zinc(II) ions, N-amidothioureas with elelctron-
donating substituentes should serve as good sensors for
Zn2þ. On the other hand, Zn2þ, as the second most abun-
dant d-block metal ion in the human body, plays critical
roles in regulating enzyme activities as well as in structure
and function, neural signal transmission, gene expres-
sion, and apoptosis. Although various Zn2þ sensors have
been developed,6 highly Zn2þ-selective, water-soluble, cell
permeable, and easily accessible fluorescent sensors are still
in high demand.
found that their absorption spectra of these conpounds
showedclear butdifferent changesuponthe additionof the
above-mentioned anions (Figures 1 and S3a).
Herein, we report the synthesis of a series of N-amido-
thiourea compounds with different substituents and the
switchable anions/cations sensing behavior of these com-
pounds. While N-amidothioureas containing elelctron-
withdrawing nitro substituents are able to recognize
many biologically important anions and show almost no
response to metal ions, those with electron-donating meth-
oxyl moieties exhibited a high selectivity and sensitivity for
Zn2þ ions in aqueous solution as well as in cells. Ours are
the first series of Zn2þ sensors based on N-amidothioureas.
The syntheses of the new N-amidothioureas were
straightforward(Scheme 1). All newcompounds werefully
characterized with 1H, 13C NMR, mass spectroscopy and
IR spectra (see Supporting Information). A single crystal
of 2b was analyzed by X-raydiffraction. (Figure S1aꢀS1b).
The 3D supramolecular frameworks was established by
H-bondings and strong πꢀπ interaction between adjacent
Figure 1. (a) Changes in the UVꢀvis of 4a (25 μM) in 4:1
DMSO/H2O upon addition of various anions. (b) Changes in
the UVꢀvis of 2a upon addition of HPO4 (0f8.0 equiv) in
2ꢀ
DMSO.
Several points are noteworthy: (1) The sensitivity of the
five receptors to the anions follows essentially the same
direction, i.e., it varies in the order P2O74ꢀ > HPO42ꢀ g
Fꢀ >CH3CO2ꢀ >HPO42ꢀ >AMP. ADP ∼ATP. Their
recognition of pyrophosphate is very interesting. An in-
significant change in their absorption spectra upon the
addition of ADP and ATP was observed. (2) The absorp-
tion spectra of the nitro-containing amidothioureas 3, 4a,
and 4b displayed a new strong band at ca. 420 nm, which is
visible to naked eyes since the color of the solution changed
from colorless to yellow. For the methoxyl-containing 2a
and 2b, their sensitivity toward anions clearly decreased as
shown by the changes of the absorption spectra and the
apperance of new bands at short wavelengths. Among
the anions tested, only P2O74ꢀ, HPO42ꢀ, OAcꢀ, and Fꢀ
were obviously differentiated with new bands appearing
at λmax = 390 and 350 nm, respectively (Figure S3a).
Results from the anion titrations (Figure S3b) clearly
indicated that N-amidothioureas with electron-withdraw-
ing substituents showed strong affinity toward these ions.
1H NMR titrations (Figure S7aꢀ7c) demonstrated that
both hydrongen-bonding and/or deprotonation interac-
tions are responsible for anion sensing. These results are in
good agreement with those in the literatures.3 The distinct
selectivity for anions can be attributed to the presence
of the multihydrogen bonding interactions. The presence
of electron-withdawing substituents in the N-phenyl ring
favors the recognition of anions due to the complementary
electrostatic interactions, and the enhanced internal charge
transfer (ICT) which shifted the spectra to a longer
wavelength.3
˚
thiophene planes (3.725 A) located in the two dimers.
The sensing abilities of the five N-amidothiourea re-
ceptors toward anions such as H2PO4ꢀ, HPO42ꢀ, P2O7
4ꢀ
,
AMP, ADP, ATP, OAcꢀ, and Fꢀ were determined in 4:1
DMSO/H2O following the reported procedure.3a It was
Scheme 1. Synthesis of the N-Amidothiourea Compounds
(6) For recent reviews for Zn2þ sensors, see: (a) Nolan, E. M.;
Lippard, S. J. Acc. Chem. Res. 2009, 42, 193. (b) Zhang, J. F.; Zhou,
Y.; Yoon, J. Y.; Kim, Y. M.; Kim, S. J.; Kim, J. S. Org. Lett. 2010, 12,
3852. (c) Zhang, J. F.; Kim, S. Y.; Han, J. H.; Lee, S. J.; Pradhan, T.;
Cao, Q. Y.; Kang, C. H.; Kim, J. S. Org. Lett. 2011, 13, 5294.
(d) Yamaguchi, S.; Miuraa, C.; Kikuchi, K.; Celino, F. T.; Agusa, T.;
Tanabe, S.; Miura, T. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 10859.
(f) Que, E. L.; Domaille, D. W.; Chang, C. J. Chem. Rev. 2008, 108, 1517.
(g) Wu, J. S.; Liu, W. M.; Zhuang, X. Q.; Wang, P. F.; Wu, S. K.; Lee,
S. T. Org. Lett. 2007, 9, 33. (h) Xu, Z.; Yoon, J.; Spring, D. R. Chem. Soc.
Rev. 2010, 39, 1996.
In the sensing of cations using the above N-amidothioureas,
the perchlorate salts of Liþ, Agþ, Ca2þ, Cd2þ, Co2þ
,
Cu2þ, Fe3þ, Hg2þ, Kþ, Mg2þ, Naþ, Ni2þ, Pb2þ, and
Zn2þ ions were examined in aqueous buffer (DMSO/H2O,
1:99, v/v, tris-HCl, 10 mM, 0.1 M KNO3, pH 7.4). The three
nitro-containing ligands showed no sigiunificant fluorescence
changes upon the addition of the 14 cations (Figure S4).
However, for 2a (Φfree = 0.043) and 2b (Φfree = 0.030),
Org. Lett., Vol. 14, No. 19, 2012
5071

Figure 2. Fluorescence spectra of 2a (a) and 2b (b) at 10 μM in
the absence and presence of 1.0 equiv of different metal ions
Figure 3. Fluorescence titration spectra of 2a (a) and 2b (b) at 10
μM upon addition of Zn2þ at 0ꢀ20 μM (0ꢀ2.0 equiv) in DMSO/
H2O (1:99, v/v). Inset: ratiometric fluorescence intensity [F/F0]
as a function of [Zn2þ].
(Liþ, Agþ, Ca2þ, Cd2þ, Co2þ, Cu2þ, Fe3þ, Hg2þ, Kþ, Mg2þ
,
Naþ, Ni2þ, Pb2þ, Zn2þ). Inset: Fluorescent emission change
irradiated at 365 nm by fluorescent lamp.
730, and 693 cmꢀ1, respectively, indicating that the coordi-
nation involved the sulfur atom in the Zn2þ complexes.7c,d
1H NMR titration experiments of 2a and Zn2þ in DMSO-
d6 found that the three signals at 10.69, 9.78, and 9.65 ppm,
derived from the amide and the two thiourea protons
on each arm of the ligand, had no significant change
upon the addition of Zn2þ (0ꢀ10 equiv),3a,4a indicating
that there was no significant amide deprotonation observed;
i.e., no enolation occurred in the metal coordination sites
(Figure S7dꢀS7e).
upon the addition of Zn2þ, the probes immediately gave
a yellow-green (λmax = 541 nm, ca. 14-fold) and a bright-
blue emission enhancement (λmax = 479 nm, ca. 19-fold)
(Figure 2). For 2a, the addition of Cd2þ and Agþ showed
a slight enhancement in emission, while only Zn2þ triggered
a sharp fluorescence enhancement for 2b (Φ2a‑Zn(II)
0.270, and Φ2b‑Zn(II) = 0.352).
=
Competing experiments among metal ions (Figure S6)
proved that Liþ, Naþ, Kþ, Ca2þ, Mg2þ, Agþ, and Fe3þ
showed no interference in the recognition of Zn2þ. ForPb2þ
,
The emission intensities of 2a and 2b in the presence
of Zn2þ ion were stable in the pH range 6.0 ꢀ10.0, which
would be beneficial for biological applicability (Figure S2).
For the absorption spectra of 2a and 2b, the addition of
Zn2þ ions resulted in the appearance of new bands at 448
and 336 nm. Complexes with stoichiometries of 1:1 and 1:2
were clearly formed (Figure S3c).
Hg2þ, and Cd2þ ions, the fluorescence were not completely
restored by the addition of Zn2þ, but strong enhancements
were still observed. In the case of Cu2þ, Ni2þ, and Co2þ, the
quenched fluorescence was not recovered after the addition
of Zn2þ, which is common for most Zn2þ sensors. It needs to
be pointed out that the influence of these ions could be
neglected due to their low concentration in vivo.
Table 1 showed the results from molecular orbital
calculations for these ligands. In HOMOs, the charge
was mainly located on the center of all compounds. How-
ever, in LUMOs, the extent of π-electron delocalization of
the nitro-containing ligands was drastically increased,
which clearly weakens the coordination capability to metal
ions.
A fluorescence titration experiment (Figure 3) revealed
that a linear enhancement with increasing [Zn2þ] up to
10 μM (1:1) was observed. Higher Zn2þ concentrations
only caused insignificant emission enhancement for 2a.
The emission enhancement of 2b plateaued with an excess
of 5 μM of Zn2þ (2:1). The 1:1 and 2:1 stoichiometric ratios
between 2a and 2b to Zn2þ were also derived from Job’s
plot (Figure S5). The binding constant Ka of 2a for Zn2þ
was determined to be 7.87 ꢁ 104 Mꢀ1, which is higher
than that of 2b (2.16 ꢁ 102 Mꢀ2, Figure S9) because of the
cleft effect. The detection limits were found to be 1.05 and
2.30 μM for 2a and 2b. ESI-MS results also confirmed
the formation of 1:1 (2a:Zn) and 2:1 (2b:Zn) complexes
(Figure S8).
However, such delcocalization should enhance interac-
tions with anions. In contrast, the π-electrons of 2a and
2b were mainly found on the electron-deficient moieties
of these ligands (;(CdO);NH;NH;(CdS);) due to
the electron-donating effect of the methoxyl groups, which
greatly improved the ability of chelating metal ions by
these compounds (Tables S5ꢀS6).8
A likely mechanism for the sensing of anions and Zn2þ
is shown in Figure 4. The anionꢀrecepotor interaction
can be both a hydrongen-bonding and/or deprotona-
tion course as reported in the literatures.3,9 Zn2þ chelation
IR spectra indicated that the amide-I stretching vibra-
tions of free 2a and 2b were blue-shifted by 39 and 14 cmꢀ1
in those of the two complexes, illustrating the coordination
of the amido-carbonyl oxygen atom to Zn2þ 7a,b
The CdS
.
stretching at 1462, 740 cmꢀ1 in 2a and 1415, 732 cmꢀ1 in 2b
(8) (a) Yousef, T. A.; El-Gammal, O. A.; Ghazy, S. E.; Abu El-Reash,
G. M. J. Mol. Struct. 2011, 1004, 271. (b) Hudson, G. A.; Cheng, L.; Yu,
J.; Yan, Y.; Dyer, D. J.; McCarroll, M. E.; Wang, L. J. Phys. Chem. B
2010, 114, 870.
were split and shifted to 1447, 1382, 720 cmꢀ1 and 1413,
(7) (a) Desseyn, H. O.; Jacob, W. A.; Herman, M. A. Spectrochim.
Acta 1969, 25A, 1685. (b) Nishat, N.; Ahamad, T.; Zulfequar, M.;
Hasnain, S. J. Appl. Polym. Sci. 2008, 110, 3305. (c) Swaminathan, K.;
Irving, H. M. N. H. J. Inorg. Nucl. Chem. 1964, 26, 1291. (d) Pedrido, R.;
Romero, M. J.; Bermejo, M. R.; Gonzalez-Noya, A. M.; Garcıa-Lema,
I.; Zaragoza, G. Chem.;Eur. J. 2008, 14, 500.
(9) For the hydrogen bonding and deprotonation mechanism of the
N-Amidothioureas compounds with anions, see: (a) Perez-Casas, C.;
Yatsimirsky, A. K. J. Org. Chem. 2008, 73, 2275. (b) Gunnlaugsson, T.;
Kruger, P. E.; Lee, T. C.; Parkesh, R.; Pfeffer, F. M.; Hussey, G. M.
Tetrahedron Lett. 2003, 44, 6575. (c) Camiolo, S.; Gale, P. A.; Hurst-
house, M. B.; Light, M. E. Org. Biomol. Chem. 2003, 1, 741.
ꢀ
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Org. Lett., Vol. 14, No. 19, 2012

Table 1. Calculated HOMOs and LUMOs of the Derivatives Using DFT Method (6-31G* Basis Sets)
Figure 5. Intracellular Zn2þ was imaged in living cells at 37 °C.
(a) HepG2 cells incubated with 2b (20 μM) buffer solution for
30 min. (b) Bright field transmission image of living HepG2 cells
in parts a and c. (c) HepG2 cells further incubated with Zn2þ
(10 μM) for 30 min, then washed three times with PBS.
to those for cations. N-Amidothioureas containing elec-
tron-withdrawing nitro groups are responsive to a host of
biologically important anions, whereas those containing
electron-donating methoxy moieties can recoginize Zn2þ
with high selectivity and sensitivity. To the best of our
knowledge, ours is also the first example that “turns on”
fluorescent sensors for Zn2þ based on amidothioureas, a
class of compounds that are easily available, highly selec-
tive, water-soluble, and cell-permeable.
Figure 4. Proposed binding mode and sensing process of the
amidothioureas with anions and Zn2þ
.
resulted in better planarity and rigidity of the amidothioureas
and hence inhibited the distortion of the molecular struc-
tures. This promoted strong ICT from ꢀOCH3 moieties
to the thiourea center and fulfilled the CHEF course.
Furthermore, intracellular Zn2þ sensing and imaging
were investigated using 2b in live HepG2 cells as shown in
Figure 5. When the cells were stained with only 2b (20 μM),
a negligible intracellular fluorescence was detected. In
contrast, with the addition of Zn2þ (10 μM), a significant
enhancement of the intracellular blue fluorescence were
observed. In view of the good match between Figure 5b
and 5c, it can be concluded that the probe has excellent
membrane permeability. The binding of 2b with Zn2þ
happened within HepG2 cells.
Acknowledgment. We are thankful for funding from
the NSFC (20972019), NCET-08-0054, and 2009SC-1. The
authors thank Prof. B. Gong at NYSU, Buffalo, for his
helpful discussions.
Supporting Information Available. Experimental details,
compound characterizations, and the X-ray single crys-
tal diffraction data for compound 2b. This material
is available free of charge via the Internet at http://
pubs.acs.org.
In summary, this work clearly demonstrated that tuning
the electronic properties of the substituents in N-ami-
dothioureas results in the conversion of sensors for anions
The authors declare no competing financial interest.
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