their application toward biological samples are hampered by
poor solubility in water and modest sensitivities.
With such problems at the forefront, we have focused on
the synthesis of a new derivative of Nile blue (1) and
investigated its chemodosimetric properties that can provide
sensitive measurement of the Hg2+ ion in a 100% aqueous
environment. Compound 1 absorbs and emits lights at 630
and 652 nm, respectively, with a high quantum yield. In
addition, and more importantly, its chloride salt shows an
excellent water-solubility and intense fluorescence, even in
aqueous media.5 Its reaction with the Hg2+ ion induces a
desulfurization-based cyclization followed by an absorption
and emission shift of 1. As such, the Nile blue derivative
(1) is a good candidate for the Hg(II) chemodosimeter. For
demonstration of its application in biological samples, various
experiments using BSA (bovine serum albumin) and human
serum were implemented and discussed.
Figure 1. (a) Absorption and (b) fluorescence spectra of 1 (10.0
As depicted in Scheme 1, Nile blue derivative (1) was
prepared in ∼90% yield from the reaction of 5-(ethylamino)-
and 5.0 µM, respectively) upon addition of Cl- salts of K+, Na+,
Hg2+, Co2+, Ni2+, Ba2+, Ca2+, Cd2+, Mg2+, Zn2+, Pb2+, and Fe2+
(5.0 equiv, respectively) in HEPES (pH ) 7.4) buffer at room
temperature, λex ) 540 nm. (c) Metal ion selectivity of 1 in the
presence of various metal cations: (A) 1; (B) 1 + Hg2+ (25 µM);
(C) 1 + Hg2+ (25.0 µM) + other metal ions (Na+ + K+ + Ca2+
+
Scheme 1. Synthetic Routes to 1 and 5
Mg2+, 1.0 mM, respectively); (D) 1 + Hg2+ (25.0 µM) + other
metal ions (Zn2+ + Cd2+ + Co2+ + Fe2+ + Ba2+ + Ni2+, 25.0
µM, respectively). Inset: color changes of A-D. (d) Fluorescence
response of 1 at 624 nm in the presence of thiophilic metal cations.
) 1.6 × 104 M-1 cm-1), respectively (Figure 1a). In the
fluorescence spectra of 1, an obvious blue shift from 652
nm (ΦF ) 0.20) to 626 nm (ΦF ) 0.35), with an intensity
increase, was observed only in the presence of Hg2+ ions
(Figure 1b).6 Figure S1 (Supporting Information) gives
detailed UV/vis and fluorescence changes of 1 as a function
of [Hg2+]. Upon increasing [Hg2+], the absorption bands of
1 decreased gradually, and new absorption bands attributable
to the Hg2+-induced cyclization concomitantly increased
(Figure S1a, Supporting Information). Similar results are also
observed in the fluorescence titration spectra (Figure S1b,
Supporting Information). For further photophysical studies,
fluorescence experiments of 1 were performed with an
excitation at 610 nm (Figure S2, Supporting Information).
In addition, the reaction responsible for these changes reaches
completion within the time frame (<1 min) of these
measurements.
The results of cation-competitive experiments are depicted
in Figure 1c. We found that the selectivity and sensitivity
of 1 toward Hg2+ are not influenced by biologically active
metal ions such as highly concentrated Na+, K+, Ca2+, and
Mg2+ (1.0 mM, 200 equiv). Compound 1 retains a high Hg2+-
selective chemodosimetric response, even under polluted
conditions containing heavy- and transition-metal cations
such as Zn2+, Cd2+, Co2+, Fe2+, Ba2+, and Ni2+ (25.0 µM
of each). Moreover, 1 can detect Hg2+ in the “naked-eye”
4-methyl-2-nitrosophenol (2) with naphthalene derivative (3)
in ethanol/H+ solution. To gain insight into the role of the
N-phenylthiourea unit in the Hg2+ chemodosimetric detec-
tion, a reference compound (5) lacking the dosimetric portion
was synthesized in high yield (90%) from the reaction of 2
with 4. The identities of all synthetic compounds were fullly
confirmed by 1H NMR, 13C NMR, and FAB-MS in Figures
S10-S18 (Supporting Information).
A stock solution of 1 was prepared in 100% distilled water,
and all photochemical experiments were carried out in 10
mM HEPES buffer at pH 7.4 without any organic solvent.
Parts a and b of Figure 1 show absorption and fluorescence
spectra of 1 upon addition of 5.0 equiv of various metal ions.
Compound 1 revealed two absorption maxima at 630 nm (ε
) 1.9 × 104 M-1 cm-1) and 592 nm (ε ) 1.6 × 104 M-1
cm-1). Upon addition of the Hg2+ ion, the maxima were
shifted to 583 nm (ε ) 1.2 × 104 M-1 cm-1) and 546 nm (ε
(6) (a) Briks, J. B. Photophysics of Aromatic Molecules; Wiley-
Interscience: London, 1970. (b) Magde, D.; Rojas, G. E.; Seybold, P. G.
Photochem. Photobiol. 1999, 70, 737. (c) Rhodamine B (ΦF ) 0.31 in
water)6b was used as the actinomer for the ΦF of Nile blue derivative
(1).
(5) Frade, V. H. J.; Gonc¸alves, M. S. T.; Moura, J. C. V. P. Tetrahedron
Lett. 2006, 47, 8567.
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Org. Lett., Vol. 11, No. 10, 2009