7412
T. Hirayama et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7410–7413
Because the emission intensity of lanthanide ions is directly depen-
dent on the q value, some lanthanide-based luminescent sensors
have utilized the displacement of metal-bound water by an ana-
lyte-binding as a luminescent switch of the sensor.16 In case of
TbOTZ, however, the luminescence OFF–ON mechanism involving
water displacement can be excluded. We then compared the phos-
phorescence spectra of TbOTZ before and after binding with Zn2+
.
In the absence of Zn2+, the phosphorescence emission (22,500 cm
À1) from the triplet state of the cs124 chromophore17 and intense
sharp peaks of Tb3+ were observed in MeOH/EtOH (4/1, v/v) glasses
at 77 K (Fig. S7). On the other hand, the triplet state completely dis-
appeared upon the addition of Zn2+. These indicate that Zn2+ bind-
ing triggers enhancement of the energy transfer rate from the
triplet excited-state of chromophore to Tb3+, which strongly sup-
port the conclusion that the terminals of TbOTZ become spatially
close as illustrated in Scheme 1.
Finally, we tested the application of TbOTZ to TRL detection of
Zn2+ in a biological buffer containing 10% fetal bovine serum
(FBS). TRL measurement provided distinct turn-on response to
Zn2+ without being affected by biological contaminants in the
serum (Fig. S8).
Figure 2. Time-resolved emission spectra (excitation at 330 nm) of TbOTZ (10 lM)
upon addition of Zn2+ (0–1.0 mM, 0.1 mM each). These spectra were measured in
50 mM HEPES buffer (pH 7.4, 0.1 M KNO3) using a delay time of 0.05 ms and a gate
time of 1.00 ms.
In conclusion, we have successfully developed a new peptidic
dual functional sensor TbOTZ, which enables both the ratiometric
and TRL measurements for the detection of Zn2+ with a single mol-
ecule. Furthermore, TbOTz is able to correctly detect Zn2+ in the
presence of FBS by TRL measurement. The main advantage of pep-
tidic sensors is the high versatility of the sensor design. For in-
stance, the binding affinity for Zn2+ would be increased by tuning
the b-turn sequence of the peptide in a way that enables peptide
to adopt a more stable b-hairpin conformation.10d Moreover, the
metal-binding sequence in TbOTZ can be altered with other chelat-
able amino acids such as methionine and aspartic acid in order to
change the metal ion selectivity. Such the peptide-based fluores-
cence probes would be useful chemical sensors for monitoring
extracellular and environmental metal ion concentrations.
Acknowledgments
This work was financially supported by a Grant-in-Aid for
Young Scientists (B) (No. 21750168 to M.T.) and Grant-in-Aid for
JSPS Fellows (T.H.).
Supplementary data
Supplementary data associated with this article can be found, in
Figure 3. Metal ion selectivity profiles of TbOTZ (10
0.1 M KNO3). Bars represent (a) emission intensity ratio (F545/F365
luminescence intensity at 545 nm to various metal cations (10 mM for Na+, K+,
l
M) in 50 mM HEPES (pH 7.4,
)
and (b)
Ca2+, and Mg2+, 500
5: Ni2+; 6: Cu2+; 7: Zn2+; 8: Cd2+; 9: Na+; 10: K+; 11: Mg2+; 12: Ca2+; 13: Na+ + Zn2+
14: K+ + Zn2+; 15: Mg2+ + Zn2+; 16: Ca2+ + Zn2+
l
M for all other metals); 1: no metal; 2: Mn2+; 3: Fe2+; 4: Co2+
;
;
.
References and notes
1. de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley, A. J. M.; McCoy, C.
P.; Rademacher, J. T.; Rice, T. E. Chem. Rev. 1997, 97, 1515.
2. Outten, C. E.; O’Halloran, T. V. Science 2001, 292, 2488.
very low concentrations as compared with Zn2+ 15
.
It should be
noted that the fluorescence of TbOTZ was not affected by Cd2+
(Fig. S5), which often exhibits the same or similar fluorescence re-
sponses to Zn2+ ions as well as competitive inhibition of the re-
sponse because of their chemical similarities. This may suggest
the metal-binding site consisting of six histidines in TbOTZ creates
a highly restricted binding pocket for first-row transition metals
rather than for Cd2+ with large ionic radius.
3. Joseph, E. C. Curr. Opin. Chem. Biol. 1998, 2, 222.
4. (a) Lee, J. Y.; Son, H. J.; Choi, J. H.; Cho, E.; Kim, J.; Chung, S. J.; Hwang, O.; Koh, J.
Y. Brain Res. 2009, 1286, 208; (b) Sensi, S. L.; Paoletti, P.; Bush, A. I.; Sekler, I. Nat.
Rev. Neurosci. 2009, 10, 780; (c) Assaf, S. Y.; Chung, S. H. Nature 1984, 308, 734.
5. Gyulkhandanyan, A. V.; Lu, H.; Lee, S. C.; Bhattacharjee, A.; Wijesekara, N.; Fox,
J. E. M.; MacDonald, P. E.; Chimienti, F.; Dai, F. F.; Wheeler, M. B. J. Biol. Chem.
2008, 283, 10184.
6. Zalewski, P. D.; Jian, X.; Soon, L. L.; Breed, W. G.; Seamark, R. F.; Lincoln, S. F.;
Ward, A. D.; Sun, F. Z. Reprod. Fertil. Dev. 1996, 8, 1097.
7. (a) Nolan, E. M.; Lippard, S. J. Acc. Chem. Res. 2009, 42, 193; (b) Que, E. L.;
Domaille, D. W.; Chang, C. J. Chem. Rev. 2008, 108, 1517; (c) Domaille, D. W.;
Que, E. L.; Chang, C. J. Nat. Chem. Biol. 2008, 4, 168; (d) Kikuchi, K.; Komatsu, K.;
Nagano, T. Curr. Opin. Chem. Biol. 2004, 8, 182.
In order to confirm whether the Zn2+-induced luminescence
enhancement occurred with conformational change of the peptide
platform, lifetimes of Tb3+ luminescence (
s) were measured in the
absence and presence of Zn2+ (Table 1 and Fig. S6). Both before and
after the Zn2+ binding, the numbers of coordinated water mole-
cules (q values) on the Tb3+ center were unchanged to be ꢀ1.
8. (a) van Dongen, E. M. W. M.; Dekkers, L. M.; Spijker, K.; Meijer, E. W.; Klomp, L.
W. J.; Merkx, M. J. Am. Chem. Soc. 2006, 128, 10754; (b) Vinkenborg, J. L.;
Nicolson, T. J.; Bellomo, E. A.; Koay, M. S.; Rutter, G. A.; Merkx, M. Nat. Methods