An OFF-ON fluorescent probe for Zn2+ based on a GFP-inspired imidazolone derivative attached to a 1,10-phenanthroline moiety

Article abstract of DOI:10.1039/c1cc10210a

A green fluorescent protein chromophore inspired chemosenor for Zn ²⁺ was designed and synthesized. A Zn²⁺ specific fluorescence enhancement was observed due to restricted rotation between the 1,10-phenanthroline and imidazolone moie

Full text of DOI:10.1039/c1cc10210a

Chemical Communications
www.rsc.org/chemcomm
Volume 47 | Number 15 | 21 April 2011 | Pages 4305–4556
ISSN 1359-7345
COMMUNICATION
Yi-Tao Long et al.
An OFF-ON fluorescent probe for Zn2+ based on a GFP-inspired imidazolone
derivative attached to a 1,10-phenanthroline moiety

Dynamic Article Links
ChemComm
Cite this: Chem. Commun., 2011, 47, 4361–4363
www.rsc.org/chemcomm
COMMUNICATION
An OFF–ON fluorescent probe for Zn2+ based on a GFP-inspired
imidazolone derivative attached to a 1,10-phenanthroline moietyw
Yang Li,a Lei Shi,a Li-Xia Qin,a Lu-Lu Qu,a Chao Jing,a Minbo Lan,a Tony D. Jamesb and
Yi-Tao Long*a
Received 12th January 2011, Accepted 16th February 2011
DOI: 10.1039/c1cc10210a
A green fluorescent protein chromophore inspired chemosenor
for Zn2+ was designed and synthesized. A Zn2+ specific
fluorescence enhancement was observed due to restricted rota-
tion between the 1,10-phenanthroline and imidazolone moieties.
Green fluorescent protein (GFP) is an indispensable tool for live
cell labeling.1 The GFP chromophore is formed via autocatalytic
Scheme 1 Chemical structure and synthesis of probe 1.
cyclization and dehydration of a Ser-Tyr-Gly tripeptide motif
followed by air-oxidization to give an imidazolinone, which
produces high fluorescent emission efficiency and a respectable
fluorescence lifetime. However, the imidazolinone unit does
not fluoresce in solution unless it is anchored covalently and
embedded deeply within the hydrogen-bond network of GFP.
Seven-membered-ring hydrogen-bonding2 and complexation by
a BF2 entity3 have previously been used to restrict rotation
about the aryl–alkene bond of the GFP chromophore. With our
present work, we set out to design a new sensory system based
on restricted rotation about the aryl–alkene bond controlled by
metal complexation within the GFP chromophore.4 Recently,
Tolbert et al.4b have reported the GFP analog 1,2-dimethyl-
4-(pyridin-2-ylmethylene)-1H-imidiazol-5(4H)-one and produced
a chromophore which turns on its fluorescence in the presence
of Zn2+ or Cd2+ ions.
The biological significance of Zn2+ has led to the development
of numerous fluorescent chemosensors.5 A number of Zn2+
chemosensors have been developed using photoinduced electron
transfer (PET) and intermolecular charge transfer (ICT) signaling
mechanisms.5b,6 Diverse receptors including di-2-picolylamine,7
quinoline,8 and bipyridine9 etc. have exhibited excellent selectivity
and sensitivity toward Zn2+. Zn2+ induced conformational
changes have also been employed to convert the emission of
pyrene from monomer to excimer.10 Other different methodologies
for the inhibition of the free rotation have also been reported.11
Herein, a GFP analogue 1 was designed and synthesized,
where the formation of a complex between 1 and Zn2+ results
in a remarkable fluorescence enhancement of imidazolinone
units, attached to a 1,10-phenanthroline moiety. Our design
combines elements of the Mg2+ selective porphyrin-like
macrocycle12 and the enhanced metal ion selectivity of the
2,9-di-(pyrid-2-yl)-1,10-phenanthroline unit.13 We anticipated
that the complexation of Zn2+ would restrict free rotation of
the aryl–alkene bond and result in enhanced fluorescence. The
fluorescent spectra changes observed upon addition of various
metal ions indicate that 1 is highly selective for Zn2+ over other
metal ions. We believe this opens the way for the development
of many other applications using the GFP chromophore.
Compound 1 was prepared according to Scheme 1. Facile
condensation of 1,10-phenanthroline-2,9-dicarboxaldehyde14 with
1-methyl-2-phenyl-1H-imidazol-5(4H)-one3 produces probe 1 in a
yield of 35% as confirmed by NMR and HRMS analyses.
The spectroscopic properties of 1 were recorded in aqueous
buffer (CH3CN–HEPES, 4 : 1, v/v; HEPES, 50 mM, pH 7.4). An
absorption around 400 nm was observed in the absorption
spectrum (see Fig. S1, ESIw) and is ascribed to the neutral (A)
form15 similar to that observed for natural GFP. Several metal
cations (Na+, Mg2+, Ca2+, Ni2+, Ag+, Pb2+, Co2+, Hg2+
,
Cu2+, Cd2+ and Zn2+) as their perchlorate salts were titrated
with probe 1 and as expected a weak fluorescence emission at
506 nm was observed, due to the free rotation of aryl–alkene
bonds between the 1,10-phenanthroline and imidazolone units.
However, upon the addition of 1 equivalent of Zn2+, the fluores-
cence emission of 1 shifts from 506 nm to 485 nm indicating a
decrease in the electron-donating ability of the nitrogen of the
imidazolone moieties, and the intensity increases by more than
10-fold. While with the addition of the other metal ions, no
enhancement of emission can be observed in the fluorescence
spectra, there is no change with Ca2+ and Na+, and quenching
a Shanghai Key Laboratory of Functional Materials Chemistry &
Department of Chemistry, East China University of Science and
Technology, 130 Meilong Road, Shanghai 200237, P. R. China.
E-mail: ytlong@ecust.edu.cn; Fax: +86-21-64250032;
Tel: +86-21-64250032
b Department of Chemistry, University of Bath, Bath, BA2 7AY, UK
w Electronic supplementary information (ESI) available: Synthesis,
characterization, absorption, mass spectra, NMR and DFT data of 1.
See DOI: 10.1039/c1cc10210a
effects by other heavy metal ions (Mg2+, Cd2+, Ag+, Hg2+
Pb2+, Cu2+, Co2+, and Ni2+) are observed. The excited state of
,
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 4361–4363 4361

Fig. 1 Top: fluorescence emission of 1 (15 mM) in the presence of
1 equiv. Co2+, Hg2+, Cd2+, Ni2+, Ag+, Cu2+, Mg2+, Pb2+, Na+
,
Ca2+, Zn2+ (from bottom to top) in CH3CN–HEPES (4 : 1, v/v;
HEPES, 50 mM, pH 7.4). Inset: proposed mechanism for fluorescence
enhancement of 1 with Zn2+ ion. Bottom: visible fluorescence emission
responses of 1.5 Â 10À5 M of 1 with 1 equiv. of various metal ions
excited using a UV lamp (l = 365 nm).
1
Fig. 3 (A) H NMR spectra of 1 in the absence and in the presence of
excessive Zn2+ in CDCl3/CD3CN; (B) SERS spectra of 1 Â 10À5 M of 1 in
the absence and in the presence of excessive Zn2+ in CDCl3/CD3CN; (C)
density functional theory (DFT)-optimized geometry of 1; (D) DFT-
optimized geometry of the complexation of 1 with Zn2+. Bond length is
in angstrom and unimportant hydrogen atoms are deleted for clarity.
chromophore model systems (e.g. HBDI) has significant dihedral
freedom, which may lead to fluorescence quenching through
intersystem crossing.16 As proposed in the inset of Fig. 1, the
bonding of Zn2+ with compound 1 may lead to a restriction in the
rotational freedom of the chromophore, and result in an increase
in fluorescence intensity.
Raman spectroscopy has been used to study the cis–trans
isomerization of GFP chromophores.18 Therefore, we decided to
probe the effect of Zn2+ on a single molecule of 1 using surface
enhanced resonance Raman spectroscopy (SERS). On mixing 1
with Zn2+ and AgNPs colloid stabilized by citrate, a remarkable
difference was observed in SERS spectra (see Fig. 3B). The peaks
attributable to modes which are not related to the chromophore
do not change, while most of the signals weaken after the addition
of Zn2+, which may be due to the enhanced rigidity. Fig. 3B
shows the SERS spectra of 1 in the absence and in the presence of
Zn2+. Compared to cis–trans isomerization of Y66F GFP
chromophore (BFPF),18a similar changes of the peaks, especially
a group of bands around 1100–1300 cmÀ1, were observed due to
the weakening and red-shift of the stretching mode of the CQC
bond. This observation gives clear evidence for the conformational
change of 1 upon the addition of Zn2+ ions.
Fig. S2 (ESIw) depicts the pH dependent fluorescence changes
obtained for 1. The plot of the integrated emission intensity
reveals that at lower pH values (o6) the fluorescence of 1 is
quenched, which is in agreement with Tolbert’s work.4b The
Benesi–Hildebrand analysis17 of the emission data gives a 1 : 1
stoichiometry for the 1–Zn2+ complex, and the association
equilibrium constant (Kass) is 2.44 Â 106 MÀ1 (Fig. 2).
The interaction of receptor 1 with zinc ion was further
investigated by 1H NMR (see Fig. 3A). After addition of
excessive zinc ions, all the hydrogens shift upfield due to
complexation with the zinc ions and paramagnetic effect.
Density functional theory (DFT) calculations were performed
on the proposed Zn2+ complexes without (Fig. S3a, ESIw) and
with one additional water molecule ligation (Fig. S3b, ESIw), at
the B3LYP/lanl2dz level. Without the additional water ligation,
the relative energies of the three species in Fig. S4a (ESIw) are
0.0, 9.9, and 13.8 kcal molÀ1 for the non-oxygen, one-oxygen
and two-oxygen ligated conformations, respectively, which
means that the non-oxygen-ligated conformation is the most
stable structure. With one additional water molecule ligation,
the relative sequence is not changed, but, with a smaller energy
gap, that is, 0, 3.3 and 7.7 kcal molÀ1. The optimized geometries
by DFT calculations are presented in Fig. 3C and D, which
demonstrate that Zn2+ complexation promotes a p–p stacking
interaction between the ligands two phenyl groups, and coordi-
nation shortens the distance of the four nitrogen atoms. The
complex of Zn2+ was also analyzed further by high-resolution
mass spectroscopy (Fig. S7, ESIw), which shows a peak at
629.1285 assigned to [1 + Zn + H2O–H]+.
Fig. 2 Fluorescence emission spectra of 1 (1.5 Â 10À5 M) upon
addition of various amounts of Zn2+ in CH3CN–HEPES (4 : 1, v/v;
HEPES, 50 mM, pH 7.4). Excitation at 400 nm. Inset: plot of
fluorescence intensity at 485 nm against the concentration of Zn2+
.
c
4362 Chem. Commun., 2011, 47, 4361–4363
This journal is The Royal Society of Chemistry 2011

4534–4535; (b) J. Kang, G. Zhao, J. Xu and W. Yang, Chem.
Commun., 2010, 46, 2868–2870.
3 L. Wu and K. Burgess, J. Am. Chem. Soc., 2008, 130, 4089–4096.
4 (a) D. P. Barondeau, C. J. Kassmann, J. A. Tainer and E. D. Getzoff,
J. Am. Chem. Soc., 2002, 124, 3522–3524; (b) A. Baldridge,
K. M. Solntsev, C. Song, T. Tanioka, J. Kowalik, K. Hardcastle
and L. M. Tolbert, Chem. Commun., 2010, 46, 5686–5688.
5 (a) E. L. Que, D. W. Domaille and C. J. Chang, Chem. Rev., 2008,
108, 1517–1549; (b) Z. Xu, J. Yoon and D. R. Spring, Chem. Soc.
Rev., 2010, 39, 1996–2006; (c) C. Lodeiro, J. L. Capelo,
J. C. Mejuto, E. Oliveira, H. M. Santos, B. Pedras and
C. Nunez, Chem. Soc. Rev., 2010, 39, 2948–2976; (d) P. Jiang
and Z. Guo, Coord. Chem. Rev., 2004, 248, 205–229;
(e) K. Kikuchi, K. Komatsu and T. Nagano, Curr. Opin. Chem.
Biol., 2004, 8, 182–191; (f) E. Tomata and S. J. Lippard, Curr.
Opin. Chem. Biol., 2010, 14, 225–230.
6 (a) J. F. Callan, A. P. De Silva and D. C. Magri, Tetrahedron, 2005,
61, 8551–8588; (b) Z. Xu, K.-H. Baek, H. N. Kim, J. Cui, X. Qian,
D. R. Spring, I. Shin and J. Yoon, J. Am. Chem. Soc., 2010, 132,
601–610.
7 (a) G. K. Walkup, S. C. Burdette, S. J. Lippard and R. Y. Tsien,
J. Am. Chem. Soc., 2000, 122, 5644–5645; (b) T. Hirano, K. Kikuchi,
Y. Urano, T. Higuchi and T. Nagano, J. Am. Chem. Soc., 2000, 122,
12399–12400; (c) F. Qian, C. Zhang, Y. Zhang, W. He, X. Gao,
P. Hu and Z. Guo, J. Am. Chem. Soc., 2009, 131, 1460–1468;
(d) L. Zhang, C. S. Murphy, G.-C. Kuang, K. L. Hazelwood,
M. H. Constantino, M. W. Davidson and L. Zhu, Chem. Commun.,
2009, 7408–7410; (e) X. Lu, W. Zhu, Y. Xie, X. Li, Y. Gao, F. Li
and H. Tian, Chem.–Eur. J., 2010, 16, 8355–8364.
8 (a) M. Royzen, A. Durandin, V. G. Young, N. E. Geacintov and
J. W. Canary, J. Am. Chem. Soc., 2006, 128, 3854–3855;
(b) Y. Zhang, X. Guo, W. Si, L. Jia and X. Qian, Org. Lett.,
2008, 10, 473–476; (c) L. Xue, C. Liu and H. Jiang, Chem. Commun.,
2009, 1061–1063; (d) X. Zhou, B. Yu, Y. Guo, X. Tang, H. Zhang
and W. Liu, Inorg. Chem., 2010, 49, 4002–4007; (e) Y. Liu,
N. Zhang, Y. Chen and L.-H. Wang, Org. Lett., 2007, 9, 315–318;
(f) M. A. Palacios, Z. Wang, V. A. Montes, G. V. Zyryanov and
P. Anzenbacher, J. Am. Chem. Soc., 2008, 130, 10307–10314.
9 (a) A. Ajayaghosh, P. Carol and S. Sreejith, J. Am. Chem. Soc.,
2005, 127, 14962–14963; (b) P. N. W. Baxter, J. Org. Chem., 2001,
66, 4170–4179; (c) A. E. Dennis and R. C. Smith, Chem. Commun.,
2007, 4641–4643; (d) H. Huang and D. G. Drueckhammer, Chem.
Commun., 2005, 5196–5198.
Fig. 4 Phase contrast (left) and fluorescence (right) microscopy
images of HeLa cells incubated for 30 min with 1 (40 mM), without
(top) and with (bottom) the addition of Zn2+ (1 equiv.).
To further explore the selectivity of 1 for Zn2+, we measured
the fluorescence intensity of 1 in the presence of Zn2+ and other
metal ions in aqueous buffer (CH3CN–HEPES, 4 : 1, v/v;
HEPES, 50 mM, pH 7.4) (Fig. S8, ESIw). The emission intensity
of Zn2+-bound 1 is unperturbed in the presence of 1 equiv.
Na+, Ca2+, Mg2+, Cd2+, Ag+, Hg2+, Pb2+ and Ni2+
,
indicating excellent selectivity for Zn2+ over these competing
cations, whereas the same amounts of Cu2+ and Co2+ quench
the fluorescence, which is due to the displacement of Zn2+ by
Cu2+ or Co2+ from the Zn2+–1 complex.8d,19 It is important to
note that Cd2+ does not interfere with the detection of Zn2+ by
1, in stark contrast to the previously reported GFP analogue,4b
this is due to the porphyrin-like structure of 1 which results in
selective complexation of Zn2+
.
In vitro Zn2+ sensing and imaging was investigated using
probe 1 in live HeLa cells (Fig. 4). The cells exhibited strong
blue-green fluorescence with the addition of Zn2+. These
results indicate that 1 may be used as a possible sensor to
detect Zn2+ released from stimulated cells.
10 (a) H. Yuasa, N. Miyagawa, T. Izumi, M. Nakatani, M. Izumi and
H. Hashimoto, Org. Lett., 2004, 6, 1489–1492; (b) S. Y. Park,
J. H. Yoon, C. S. Hong, R. Souane, J. S. Kim, S. E. Matthews and
J. Vicens, J. Org. Chem., 2008, 73, 8212–8218; (c) Z. Zhong and
Y. Zhao, Org. Lett., 2007, 9, 2891–2894.
In conclusion, a GFP-inspired imidazolone derivative con-
taining a 1,10-phenanthroline moiety was designed and synthesized
as a Zn2+ specific turn-on fluorescent chemosensor. We are
currently exploring the use of this design strategy in the construc-
tion of other fluorescent chemosensors.
11 (a) W. R. Browne and B. L. Feringa, Annu. Rev. Phys. Chem.,
2009, 60, 407–428; (b) P. Molina, A. Tarraga and A. Caballero,
´
Eur. J. Inorg. Chem., 2008, 3401–3417; (c) D. Zhang, Q. Zhang,
J. Su and H. Tian, Chem. Commun., 2009, 1700–1702.
12 M. Ishida, Y. Naruta and F. Tani, Angew. Chem., Int. Ed., 2010,
49, 91–94.
13 (a) A. Bencin and V. Lippolis, Coord. Chem. Rev., 2010, 254,
2096–2180; (b) G. M. Cockrell, G. Zhang, D. G. VanDerveer,
R. P. Thummel and R. D. Hancock, J. Am. Chem. Soc., 2008, 130,
1420–1430.
14 C. J. Chandler, L. W. Deady and J. A. Reiss, J. Heterocycl. Chem.,
1981, 18, 599–601.
15 J. Dong, K. M. Solntsev and L. M. Tolbert, J. Am. Chem. Soc.,
2006, 128, 12038–12039.
16 C. M. Megley, L. A. Dickson, S. L. Maddalo, G. J. Chandler and
M. Zimmer, J. Phys. Chem. B, 2009, 113, 302–308.
17 H. A. Benesi and J. H. Hildebrand, J. Am. Chem. Soc., 1949, 71,
2703–2707.
18 (a) S. Luin, V. Voliani, G. Lanza, R. Bizzarri, R. Nifosı, P. Amat,
V. Tozzini, M. Serresi and F. Beltram, J. Am. Chem. Soc., 2009,
131, 96–103; (b) V. Tozzini, A. R. Bizzarrib, V. Pellegrinia,
R. Nifosıa, P. Giannozzia, A. Iulianoc, S. Cannistrarob and
This work is supported by Major Research Plan of National
Natural Science Foundation of China (Grant No. 91027035)
and the Fundamental Research Funds for the Central Univer-
sities (Grant No. WK1013002). Y.-T.L. is supported by the
Program for Professor of Special Appointment (Eastern Scholar)
at Shanghai Institutions of Higher Learning. We also thank the
Catalysis And Sensing for our Environment (CASE) consortium
for networking opportunities and Dr Yong Wang at Dalian
Institute of Chemical Physics (Chinese Academy of Sciences) for
the help in DFT calculation.
Notes and references
1 (a) Green Fluorescent Protein: Properties, Applications and Protocols,
ed. M. Chale and S. R. Kain, John Wiley & Sons, Hoboken,
New Jersey, 2006; (b) M. Zimmer, Chem. Rev., 2002, 102, 759–782.
2 (a) K.-Y. Chen, Y.-M. Cheng, C.-H. Lai, C.-C. Hsu, M.-L. Ho,
G.-H. Lee and P.-T. Chou, J. Am. Chem. Soc., 2007, 129,
F. Beltram, Chem. Phys., 2003, 287, 33–42; (c) P. Altoe,
´
F. Bernardi, M. Garavelli, G. Orlandi and F. Negri, J. Am. Chem.
Soc., 2005, 127, 3952–3963.
19 S. C. Burdette, C. J. Frederickson, W. Bu and S. J. Lippard, J. Am.
Chem. Soc., 2003, 125, 1778–1787.
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 4361–4363 4363