Selective Hg(II) detection in aqueous solution with thiol derivatized fluoresceins

Article abstract of DOI:10.1021/ic052083w

The syntheses and photophysical properties of mercury sensors 2 and 3 (MS2 and MS3), two asymmetrically derivatized fluorescein-based dyes designed for Hg(II) detection, are described. These sensors each contain a single pyridyl-amine-thiol metal-binding moiety, form 1:1 complexes with Hg(II), and exhibit selectivity for Hg(II) over other Group 12 metals, alkali and alkaline earth metals, and most divalent first-row transition metals. Both dyes display superior brightness (Φ X ∈) and fluorescence enhancement following Hg(II) coordination in aqueous solution. At neutral pH, the fluorescence turn-on derives from greater brightness due to increased molar absorptivity. At higher pH, photoinduced electron transfer quenching of the free dye is enhanced, and the Hg(II)-induced turn-on also benefits from alleviation of this pathway. MS2 can detect ppb levels of Hg(II) in aqueous solution, demonstrating its ability to identify environmentally relevant concentrations of Hg(II).

Full text of DOI:10.1021/ic052083w

Inorg. Chem. 2006, 45, 2742−2749
Selective Hg(II) Detection in Aqueous Solution with Thiol Derivatized
Fluoresceins
Elizabeth M. Nolan, Maryann E. Racine, and Stephen J. Lippard*
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Received December 5, 2005
The syntheses and photophysical properties of mercury sensors 2 and 3 (MS2 and MS3), two asymmetrically
derivatized fluorescein-based dyes designed for Hg(II) detection, are described. These sensors each contain a
single pyridyl−amine−thiol metal-binding moiety, form 1:1 complexes with Hg(II), and exhibit selectivity for Hg(II)
over other Group 12 metals, alkali and alkaline earth metals, and most divalent first-row transition metals. Both
dyes display superior brightness (Φ × ꢀ) and fluorescence enhancement following Hg(II) coordination in aqueous
solution. At neutral pH, the fluorescence turn-on derives from greater brightness due to increased molar absorptivity.
At higher pH, photoinduced electron transfer quenching of the free dye is enhanced, and the Hg(II)-induced turn-
on also benefits from alleviation of this pathway. MS2 can detect ppb levels of Hg(II) in aqueous solution,
demonstrating its ability to identify environmentally relevant concentrations of Hg(II).
Introduction
The design of sensors that give fluorescence enhancement
upon Hg(II) binding is an intriguing challenge since Hg(II),
like many heavy elements, often causes fluorescence quench-
Facile, sensitive, and reliable methods for detecting heavy
metal ions are important for research in environmental
science and toxicology. Concern about human exposure to
mercury through a variety of routes that include fish
1
7
ing. Restricting such turn-on Hg(II) sensors to aqueous
environments introduces additional complexity to this prob-
lem. A variety of small-molecule Hg(II) detection strategies
1
-5
6,7
8
consumption, vaccination, dental amalgams, and both
18-34
the household8 and workplace environments suggests the
,9
10
that use fluorescence have been documented.
Neverthe-
less, many of these Hg(II) probes have one or more
need for new tools capable of detecting inorganic mercury
in aqueous solution and in biological samples. Small-
molecule fluorescent chemosensors that selectively bind to
and report on the target metal ion constitute one practical
route toward this goal.1
(
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*
To whom the correspondence should be addressed. E-mail:
8551-8588.
lippard@mit.edu.
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2742 Inorganic Chemistry, Vol. 45, No. 6, 2006
10.1021/ic052083w CCC: $33.50
© 2006 American Chemical Society
Published on Web 02/11/2006

SelectiWe Hg(II) Detection in Aqueous Solution
limitations, which include irreversibility, slow response,
incompatibility with aqueous solution, low selectivity, and
fluorescence quenching upon Hg(II) coordination. Recently,
several Hg(II) sensors that exhibit fluorescence turn-on and
compatibility with the aqueous milieu have been described
and include a biaryl pyridine species linked to an argogel
resin35 and xanthene-based MF1. Other reported Hg(II)
detection strategies relying on fluorescence output include
32
nanoparticles,3 polymers, and biomolecules.
6,37
38
39,40
A num-
ber of colorimetric4
documented.
1-48
Hg(II) indicators have also been
Figure 1. Mercury Sensors 1 (MS1) and 4 (MS4). MS1 gives a
fluorescence enhancement upon addition of Hg(II) at neutral pH (ref 49).
MS4 provides a ratiometric response to Hg(II) at pH 8 (ref 50).
As part of an ongoing investigation focused on the
preparation and utilization of small-molecule metal ion
4
9
sensors, we reported mercury sensors 1 and 4 (MS1 and
Hg(II) binding properties of chemosensors containing py-
ridyl-amine-thiol ligands. Sensors MS2 and MS3, depicted
in Scheme 1, are selective for Hg(II) over its Group 12
congeners, exhibit fluorescence turn-on immediately follow-
ing Hg(II) coordination in aqueous solution, and are among
the brightest fluorescent Hg(II) sensors to date.
50
MS4 ) (Figure 1), both of which give a positive fluorescence
response to Hg(II) in aqueous solution. In the design of MS1,
we employed fluorescein as the reporting group because of
its water solubility and excellent photophysical properties
(
Φ ≈ 1, high ꢀ). Sensor MS4 uses the seminaphthofluores-
cein chromophore, which affords single-excitation dual-
emission ratiometric Hg(II) detection. MS1 and MS4 contain
one or more thioether moieties in their metal-binding unit,
which confers selectivity for Hg(II) over Zn(II) and Cd(II).
Given the high affinity of Hg(II) for soft sulfur donors,
including thiols, we have expanded our MS sensor family
to include thiol-derivatized fluoresceins. In this work, we
describe the syntheses, photophysical characterization, and
Experimental Section
Reagents. Toluene and acetonitrile were purged with Ar and
dried by being passed through columns of Al
dried over 3 Å molecular sieves, and anhydrous 1,2-dichloroethane
DCE) was purchased from Aldrich. The compounds isobutylene
2 3
O . Ethyl acetate was
(
51
52
53
sulfide, 2, MEPAH, 3, MEPAHTr, 5, and 7′-chloro-4′-
5
4
bromomethylfluorescein di-tert-butyldimethylsilyl ether, 7, were
synthesized as previously described. Isobutylene oxide, used in the
synthesis of 2, was purchased from TCI America. All other reagents
were obtained from Aldrich and used as received, with the exception
of 2-(aminomethyl)pyridine, which was vacuum-distilled im-
mediately before use.
(
28) Liu, B.; Tian, H. Chem. Commun. 2005, 3156-3158.
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1
6760-16761.
(
(
(
(
(
(
(
(
(
30) Sakamoto, H.; Ishikawa, J.; Nakao, S.; Wada, H. Chem. Commun.
2001, 2395-2396.
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J. J. Am. Chem. Soc. 2003, 125, 3418-3419.
32) Yoon, S.; Albers, A. E.; Wong, A. P.; Chang, C. J. J. Am. Chem. Soc.
Methods. Merck F254 silica gel-60 plates, Merck F254 alumi-
num oxide-60 plates, and octadecyl-functionalized (reverse phase
2005, 127, 16030-16031.
33) Rurack, K.; Kollmannsberger, M.; Resch-Genger, U.; Daub, J. J. Am.
Chem. Soc. 2000, 122, 968-969.
(RP), RP18) silica gel plates were used for analytical thin-layer
34) Rurack, K.; Resch-Genger, U.; Bricks, J. L.; Spieles, M. Chem.
Commun. 2000, 2103-2104.
chromatography. TLC plates were viewed with UV light or after
being developed with ninhydrin stain. Either Whatman silica gel-
35) Mello, J. V.; Finney, N. S. J. Am. Chem. Soc. 2005, 127, 10124-
60 plates or EM Science RP18 F254 plates of 1 mm thickness were
10125.
used as the solid phase for preparative TLC. A Varian 300 MHz
or a Varian 500 MHz spectrophometer operating at 283 K was used
36) Chen, B.; Yu, Y.; Zhou, Z.; Zhong, P. Chem. Lett. 2004, 33, 1608-
609.
37) Zhu, C.; Li, L.; Fang, F.; Chen, J.; Wu, Y. Chem. Lett. 2005, 34,
98-899.
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849.
1
1
13
to obtain H and C NMR spectra, which were referenced to the
solvent peaks. IR spectra were obtained by using an Avatar FTIR
instrument. An Agilent 1100 Series LCMS was used to obtain low-
resolution electrospray ionization mass (ESI) spectrometry. High-
resolution ESI spectrometry was performed by staff in the MIT
Department of Chemistry Instrumentation Facility.
8
2
(
(
(
39) Ono, A.; Togashi, H. Angew. Chem., Int. Ed 2004, 43, 4300-4302.
40) Chen, P.; He, C. J. Am. Chem. Soc. 2004, 126, 728-729.
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418.
(
(
(
(
(
42) Sancen o´ n, F.; M a´ rtinez-M a´ n˜ ez, R.; Soto, J. Chem. Commun. 2001,
262-2263.
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2, 4321-4323.
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665.
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63.
2
-Methyl-1-[(pyridin-2-ylmethyl)amino]propane-2-thiol (4). A
2
portion (12.0 g, 111 mmol) of freshly distilled 2-aminomethylpy-
ridine was dissolved in 40 mL of toluene and heated to reflux.
Isobutylene sulfide (2, 5.0 g, 57 mmol) was dissolved in 60 mL of
toluene and added dropwise to the refluxing solution over 4 h. The
reaction was refluxed for an additional 24 h, cooled, and the solvent
was removed in vacuo. Vacuum distillation yielded the purified
4
1
3
46) Coronado, E.; Gal a´ n-Mascar o´ s, J. R.; Mart ´ı -Gastaldo, C.; Palomares,
E.; Durrant, J. R.; Vilar, R.; Gratzel, M.; Nazeeruddin, M. K. J. Am.
Chem. Soc. 2005, 127, 12351-12356.
(
(
(
(
47) Ros-Lis, J. V.; M a´ rtinez-M a´ n˜ ez, R.; Rurack, K.; Sancen o´ n, F.; Soto,
(51) Snyder, H. R.; Stewart, J. M.; Ziegler, J. B. J. Am. Chem. Soc. 1947,
69, 2672-2674.
J.; Spieles, M. Inorg. Chem. 2004, 43, 5183-5185.
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2002, 41, 6181-6183.
1167.
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4271.
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1
(54) Burdette, S. C.; Frederickson, C. J.; Bu, W.; Lippard, S. J. J. Am.
Chem. Soc. 2003, 125, 1778-1787.
Inorganic Chemistry, Vol. 45, No. 6, 2006 2743

Nolan et al.
Scheme 1
1
product as a colorless oil (4.9 g, 21%). H NMR (CDCl
δ 1.35 (6H, s, CH ), 2.00 (2H, s, br, NH and SH), 2.60 (2H, s,
CH CS), 3.93 (2H, s, NCH ), 7.10 (1H, m, py-H), 7.30 (1H, d,
py-H), 7.59 (1H, td, py-H), 8.49 (1H, dq, py-H). C NMR (CDCl
3
, 300 MHz)
mg, 2.17 mmol), and the mixture was stirred overnight at room
temperature. An orange precipitate formed, and the reaction was
filtered. TLC analysis showed the product to be present in both
the precipitate and the filtrate. Preparative TLC of the combined
3
2
2
13
3
,
3
1
00 MHz) δ 30.73, 45.46, 55.78, 63.16, 121.85, 122.03, 136.35,
49.13, 159.97. An alternative preparation, which requires excess
material on RP silica gel (9:1 CHCl
product as an orange solid (142 mg, 82%). H NMR (CD
MHz) δ 2.48 (4H, m, CH CH S), 3.68 (2H, s, fl-CH N), 3.90 (2H,
s, py-CH N), 6.50-6.58 (2H, m, fl-H), 6.78 (1H, s, fl-H), 6.88
3
/MeOH) yielded the pure
1
2 2
Cl , 300
isobutylene sulfide, and further characterization are available
elsewhere.55
2
2
2
2
(
2-Methyl-2-triphenylsulfanylpropyl)pyridin-2-ylmethyl-
(1H, s, fl-H), 7.15-7.37 (18H, m, Tr-H, py-H and fl-H), 7.63-
7.72 (3H, m, fl-H and py-H), 8.02 (1H, d, fl-H), 8.51 (1H, d, py-
amine (6). A portion (1.44 g, 5.54 mmol) of triphenylmethanol
and 4 (1.08 g, 5.51 mmol) were combined in a dry round-bottom
flask, and 9 mL of TFA was added. The reaction became brown-
red, and the triphenylmethanol slowly dissolved. The reaction was
stirred at room temperature for 2 h. The TFA was then removed
under reduced pressure to yield an orange-red viscous oil. The oil
was dissolved in 100 mL of ether and partitioned with 100 mL of
M NaOH, and the layers were separated. The ether layer was
concentrated under reduced pressure to afford the crude product
as an off-white oily solid. Chromatography on Al with a solvent
gradient (3:1 to 2:1 hexanes/EtOAc with 1% PrNH ) gave the
purified product as an off-white solid (1.52 g, 63%). TLC R
.43 (Al
CDCl , 300 MHz) δ 1.17 (6H, s, CH
1H, s, br, NH), 3.67 (2H, s, NCH ), 7.06-7.33 (12H, m, Tr-H
and py-H), 7.56 (1H, td, py-H), 7.66 (5H, m, Tr-H), 8.49 (1H, dd,
py-H). 13C NMR (CDCl
, 125 MHz) δ 28.75, 52.58, 55.35, 59.23,
7.28, 121.58, 121.66, 126.33, 127.51, 129.81, 136.20, 145.20,
-
1
H). FTIR (KBr, cm ) 3431 (s), 2922 (vs), 2852 (s), 1763 (m),
1581 (m), 1514 (w), 1489 (m), 1465 (m), 1444 (m), 1370 (w), 1259
(m), 1210 (m), 1150 (m), 1090 (m), 1012 (m), 873 (w), 743 (m),
+
700 (s), 617(m), 473 (w), 444 (w). HRMS (ESI) Calcd [M + H] ,
789.2184; Found, 789.2200.
2-(2-Chloro-6-hydroxy-5-{[(2-mercaptoethyl)pyridin-2-yl-
methylamino]methyl}-3-oxo-3H-xanthen-9-yl)benzoic acid (10,
MS2). A portion (142 mg, 0.180 mmol) of 8 was dissolved in 5
1
2
i
O
3
mL of TFA, and a few drops of Et SiH were added. A white
3
2
precipitate formed. The mixture was washed with hexanes (4 × 5
f
)
mL), and the TFA was removed under reduced pressure. Preparative
TLC of the dark residue on RP silica gel (6:1 MeOH/0.1 N HCl)
afforded the purified product as an orange-red solid (55 mg, 56%).
f
1
0
(
(
2
O
3
, 3:1 hexanes/EtOAc); mp ) 82-84 °C. H NMR
3
3
), 1.75 (2H, s, CH CS), 1.92
2
2
TLC R ) 0.25 (RP silica, 6:1 MeOH/0.1 N HCl); mp ) 194-196
1
°C, dec. H NMR (CD OD, 300 MHz) δ 1.28 (1H, s, SH), 2.92
3
3
(2H, m, CH CS), 3.27 (2H, m, NCH ), 4.27-4.46 (4H, m, NCH ),
2
2
2
6
1
3
(
1
(
1
(
4
6.56 (2H, m, fl-H), 6.64 (1H, d, fl-H), 6.93 (1H, d, fl-H), 7.21
(1H, d, fl-H), 7.34 (1H, t, py-H), 7.43 (1H, d, py-H), 7.70-7.83
(3H, m, fl-H and py-H), 8.03 (1H, d, fl-H), 8.50 (1H, d, py-H).
-
1
48.88, 160.46. FTIR (KBr, cm ) 3435 (s), 3348 (m), 3049 (w),
026 (w), 2993 (w), 2972 (w), 2959 (w), 2950 (w), 2905 (w), 2851
w), 1642 (w), 1625 (w), 1589 (m), 1567 (m), 1486 (m), 1473 (m),
444 (s), 1424 (m), 1380 (w), 1359 (m), 1319 (w), 1284 (w), 1242
w), 1209 (w), 1178 (w), 1157 (w), 1130 (m), 1083 (w), 1031 (m),
002 (w), 994 (w), 984 (w), 761 (m), 745 (s), 737 (s), 705 (s), 673
w), 665 (w), 629 (m), 621 (m). HRMS (ESI) Calcd [M + H] ,
-
1
FTIR (KBr, cm ) 3434 (vs), 3055 (w), 2958 (w), 2918 (w), 2851
(w), 1762 (s), 1627 (s), 1600 (s), 1581 (s), 1514 (w), 1489 (s),
1464 (s), 1452 (s), 1430 (s), 1370 (m), 1283 (s), 1260 (s), 1215
(s), 1149 (m), 1090 (m), 1106 (m), 1065 (w), 1012 (w), 874 (w),
822 (w), 801 (w), 760 (w), 702 (w), 621 (w), 596 (w). HRMS (ESI)
+
-
39.2202; Found, 439.2209.
-(2-Chloro-6-hydroxy-3-oxo-5-{[pyridin-2-ylmethyl-(2-triph-
enylsulfanyl-ethyl)amino]methyl}-3H-xanthen-9-yl)benzoic acid
8, MS2Tr). To 8 mL of MeCN were combined 7′-chloro-4′-
bromomethylfluorescein di-tert-butyldimethylsilyl ether (7, 150 mg,
.22 mmol), MEPAHTr (5, 91 mg, 0.22 mmol), and K CO (300
Calcd [M - H] , 545.0938; Found, 545.0963.
2
2-(2-Chloro-6-hydroxy-5-{[(2-mercapto-2-methylpropyl)py-
ridin-2-ylmethyl-amino]methyl}-3-oxo-3H-xanthen-9-yl)benzo-
ic acid (11, MS3). To 10 mL of MeCN and 1 mL of CH Cl were
(
2
2
added 7′-chloro-4′-bromomethylfluorescein di-tert-butyldi-methyl-
silyl ether (7, 201 mg, 0.293 mmol), 6 (133 mg, 0.303 mmol), and
0
2
3
2 3
K CO (300 mg, 2.17 mmol). The reaction was stirred at room
(55) Brand, U.; Vahrenkamp, H. Inorg. Chim. Acta 2000, 308, 97-102.
temperature for 24 h and filtered. The filtrate was evaporated to
2744 Inorganic Chemistry, Vol. 45, No. 6, 2006

SelectiWe Hg(II) Detection in Aqueous Solution
yield a dark orange residue, 9, which was carried on without
purification. Crude 9 was dissolved in 5 mL of TFA to yield a
deep red solution, and several drops of Et SiH were added. A white
3
precipitate formed, and the solution turned bright orange. The
mixture was washed with hexanes (5 × 6 mL), and the TFA was
removed in vacuo. Preparative TLC on silica gel (1:1:1:1 n-butanol/
acetic acid/water/ethanol) followed by preparative TLC on RP silica
gel (3:1 MeOH/0.1 N HCl) afforded MS3 as an orange solid (30
Fluorescence Spectroscopy. Fluorescence spectra were collected
by using a Hitachi F-3010 spectrofluorimeter maintained at 25 (
1 °C by a circulating water bath. Quartz cuvettes (Starna) with a
1-cm path length and 3.5-mL volume were used for all fluorescence
measurements. Excitation and emission slit widths of 3 nm were
used for data acquisition. Quantum yields were measured relative
to fluorescein in 0.1 N NaOH (Φ ) 0.95).56 The pH-dependent
pK
0.1 N HCl to a 1 µM solution of the sensor in 10 mM KOH, 100
mM KCl. The volume change for each pK experiment did not
a
values were determined by adding aliquots of 6, 2, 1, 0.5, and
mg, 17%). TLC R
194-196 °C, dec. H NMR (CD
CH and SH), 3.30 (2H, m, CH CS) 4.31-4.45 (4H, m, NCH
.51 (2H, m, fl-H), 6.64 (1H, d, fl-H), 6.99 (1H, s, fl-H), 7.17 (1H,
d, fl-H), 7.32-7.36 (2H, m, py-H), 7.70-7.79 (3H, m, fl-H and
f
) 0.23 (RP silica, 3:1 MeOH/0.1 N HCl); mp
1
)
3
OD, 300 MHz) δ 1.28 (7H, m,
),
a
3
2
2
exceed 3%. The emission spectra were recorded at pH intervals of
∼0.25, and the spectra were integrated, normalized, and plotted
against pH. The data were fit to the nonlinear equation previously
6
-
1
57
py-H), 8.02 (1H, d, fl-H), 8.40 (1H, d, py-H). FTIR (KBr, cm
430 (vs), 2957 (w), 2918 (m), 2850 (w), 1764 (m), 1628 (s), 1611
s), 1462 (m), 1452 (m), 1428 (m), 1370 (w), 1284 (m), 1255 (m),
)
described. Metal ion selectivity experiments were conducted as
3
(
described for MS1.49
1
219 (w), 1151 (w), 1107 (w), 1090 (w), 1014 (w), 875 (w), 762
Results and Discussion
+
(w), 698 (w). HRMS (ESI) Calcd [M + H] , 575.1402; Found,
Syntheses of Sensors MS2 and MS3. Several small-
molecule Hg(II) detectors that employ sulfur donors have
been reported by our laboratory
575.1394.
General Spectroscopic Methods. All aqueous solutions were
4
9,50
22,30-34
and others.
These
thio-
prepared with Millipore water (18.2 MΩ‚cm at 25 °C) obtained
from a Mili-Q Biocel purifier outfitted with a Quantum VX
cartridge. PIPES, piperazine-N,N′-bis(2-ethanesulfonic acid), CHES,
3
1-34
probes have sulfur-containing macrocycles,
3
0,49,50
35
ether,
or thiourea groups in the metal-binding units
and generally exhibit good selectivity for Hg(II). Given the
propensity of Hg(II) to bind thiols, we anticipated that
incorporation of thiol donors into the metal-binding unit of
a sensor would elicit both high selectivity and sensitivity
for Hg(II).
2
-(N-cyclohexylamino)ethanesulfonic acid, and CABS, 4-cyclo-
hexylamino-1-butanesulfonic acid, buffers were purchased from
Sigma and used as received. Puratonic grade KCl was purchased
from Calbiochem. Anhydrous HgCl (99.998%) was purchased from
2
Aldrich and used to prepare 10 mM Hg(II) stock solutions in water.
Aqueous solutions of Li(I), Na(I), Rb(I), Mg(II), Ca(II), Sr(II),
Ba(II), Cr(III), Mn(II), Co(II), Ni(II), Zn(II), and Cd(II) were
prepared from the chloride salts, solutions of Fe(II) were prepared
from ferrous ammonium sulfate and argon-purged water im-
mediately before use, and solutions of Cu(II) were prepared from
The syntheses for MS2 and MS3 are illustrated in Scheme
1
. MEPAHTr, 5, was prepared according to a literature
5
3
procedure. Combination of 5 with the silyl-protected
5
4
bromomethyl fluorescein, 7, in the presence of excess
CO and subsequent preparative TLC on RP silica gel (9:1
CHCl /MeOH) afforded MS2Tr, 8, in high yield (82%) as
an orange solid. As observed in the reported synthesis of
K
2
3
a
copper(II) sulfate. With the exception of the pK titrations,
measurements were made in buffered aqueous solution with 50 mM
buffer and 100 mM KCl at pH 7 (PIPES buffer), 9 (CHES buffer),
or 11 (CABS buffer). A starting solution consisting of 10 mM KOH,
3
5
8
sensor ZS3, the TBS protecting groups of 7 were unstable
to these conditions, and deprotection occurred during the
course of the reaction. The trityl protecting group was
1
00 mM KCl (pH ∼12) was used for the pK
in the Fluorescence Spectroscopy section below. Stock solutions
1 mM) of MS2 and MS3 in DMSO were prepared, stored at -25
C, and thawed in the dark before use. These stock solutions are
a
titrations as described
(
°
3
subsequently removed by dropwise addition of Et SiH to a
solution of 8 in TFA followed by washing with hexanes.
Preparative TLC of the crude product on RP silica gel (4.5:1
MeOH/0.1 N HCl) yielded pure MS2, 10, as an orange solid
in moderate yield (56%).
stable for >12 months under these conditions, judging by UV-vis
and fluorescence spectroscopy. After dilution of this stock in the
appropriate buffer, the resulting solutions contained 0.1% or 1%
DMSO for fluorescence or absorption measurements, respectively.
MS3 differs from MS2 by having gem-dimethyl substit-
uents incorporated into the pyridyl-amine-thiol ligand.
Compound 4 was prepared from isobutylene sulfide and
2-(aminomethyl)pyridine by analogy to the published pro-
cedure for 3 and was obtained in moderate yield as a
colorless oil following vacuum distillation. The thiol was
protected with a trityl group, and 6 was isolated as a white
solid in 63% yield following workup and chromatography
a
With the exception of the pK titrations, which were performed in
duplicate, all manipulations were performed at least in triplicate,
and the averages are reported. All data were manipulated by using
the KaleidaGraph software package.
5
2
Optical Absorption Spectroscopy. UV-visible spectra were
collected on a Hewlett-Packard 8453 diode array spectrophotometer
maintained at 25 ( 1 °C with a circulating water bath. Quartz
cuvettes (Starna) with a 1-cm path length and 3.5-mL volume were
used for all measurements. Job analyses and metal-binding titrations
were conducted to determine the stoichiometry of the MS2 and
MS3 mercury-bound complexes in solution. In a typical Job
2 3
on Al O using a hexanes/EtOAc solvent gradient. The
assembly of MS3 from 6 and 7 is analogous to that described
above for MS2. During this procedure, however, intermediate
2
analysis, starting solutions of 10 µM dye and 10 µM HgCl were
9
was not purified before deprotection of the thiol moiety.
used and the data were analyzed according to the standard equation
A* ) Aobs - ꢀlig[lig], where ꢀlig is the molar absorptivity at the
wavelength of interest and lig refers to the metal-free probe. For a
typical metal-binding titration, 3 µL aliquots of a 1 mM Hg(II)
solution were added to a 10 µM solution of dye. The absorbance
changes were plotted against equivalents of Hg(II) in solution.
Pure MS3, 11, was isolated as an orange-red solid in
(
(
56) Brannon, J. H.; Magde, D. J. Phys. Chem. 1978, 82, 705-709.
57) Burdette, S. C.; Walkup, G. K.; Spingler, B.; Tsien, R. Y.; Lippard,
S. J. J. Am. Chem. Soc. 2001, 123, 7831-7841.
(58) Nolan, E. M.; Lippard, S. J. Inorg. Chem. 2004, 43, 8310-8317.
Inorganic Chemistry, Vol. 45, No. 6, 2006 2745

Nolan et al.
Table 1. Spectroscopic and Thermodynamic Data for
Fluorescein-Based MS Sensors
absorption λ (nm),
× 10 (M cm
4
-1
-1
emission λ (nm), Φa
unbound Hg(II)
ꢀ
)
EC50c
pH unbound
Hg(II)
pKab (µM)
MS1d
MS2
7
7
9
1
7
1
505, 6.1 501, 7.3 524, 0.04 528, 0.11 7.1 0.410
500, 6.2 505, 6.9 526, 0.28 531, 0.27 8.0
502, 6.3 505, 8.2 526, 0.10 531, 0.28
1
1
504, 6.1 507, 7.8 526, 0.07 531, 0.24
501, 5.1 507, 5.9 526, 0.27 531, 0.28 8.2
504, 5.5 507, 6.4 525, 0.10 531, 0.26
0.384
MS3
1.6
a
Fluorescein (Φ ) 0.95 in 0.1 N NaOH, ref 56) was used as the standard
b
for the quantum yield measurements. The pKa value of the aniline (MS1)
or tertiary (MS2, MS3) nitrogen atom. This pKa corresponds to a
c
fluorescence increase when moving from high to low pH. The EC50 is
Figure 2. Fluorescence dependence on pH for free MS2 (circles) and MS3
(squares). A 1 µM solution of each dye was prepared and the emission
spectrum was taken. Aliquots of 6 2, 1, 0.5, and 0.1 N HCl were added to
achieve pH changes of ∼0.25 units and the emission spectrum was recorded
at each interval. The emission was integrated from 500 to 650 nm (MS2)
or from 450 to 650 nm (MS3), normalized, and plotted against pH.
Excitation was provided at 498 nm. Two protonation events affect the
fluorescence of MS2 and MS3 with pKa1 ) 8.0 (MS2) or 8.2 (MS3) and
pKa2 ) 4.6.
the concentration of Hg(II) required to achieve 50% of the maximum
d
fluorescence increase with 1 µM sensor. Data for MS1 were taken from
ref 49.
relatively low yield (17%) after purification by preparative
TLC. Both MS2 and MS3 have a faint sulfurous odor, and
the purified sensors are stable for >12 months when stored
in the dark at 4 °C as judged by absorption and fluorescence
spectroscopy.
Spectroscopic Properties of MS2 and MS3. MS2 and
MS3, shown in Scheme 1, were designed as turn-on
photoinduced electron transfer (PET) sensors that utilize
tertiary amines in the quenching unit. The generally accepted
mechanism for fluorescence turn-on in amine-based PET
sensors involves fluorescence quenching of the free dye by
PET through back-electron transfer from a nitrogen lone pair
electron in the photoexcited state, followed by diminution
of PET via coordination to a closed shell metal ion, which
results in fluorescence enhancement.12
Table 1 summarizes the results from spectroscopic char-
acterization of MS2 and MS3. Given the structural similarity
between these two compounds, we anticipated that they
would exhibit comparable photophysical properties. In all
instances, the results met our expectations.
Unbound MS2 and MS3 show essentially identical pH-
emission profiles, which are illustrated in Figure 2. The
fluorescence of each probe is ∼75% quenched at pH 12,
and maximum fluorescence is reached at pH ∼7. Two
protonation events affect the fluorescence of MS2 (pK )
a
8
.2, 4.6) and MS3 (pK
a
) 8.0, 4.6). The pK
a
∼8 transition
is assigned to the tertiary nitrogen atom, protonation of which
interferes with the PET quenching pathway and yields
enhanced fluorescence at neutral pH. This assignment is
a
based on comparison of the pK profiles of tertiary amine-
Figure 3. The fluorescence response of 1 µM MS2 (top) and MS3 (bottom)
to 50 equiv of Hg(II) at pH 7 (50 mM PIPES, 100 mM KCl). Dotted line:
MS2 or MS3; solid line: +Hg(II). Excitation was provided at 500 nm for
MS2 and at 504 nm for MS3.
5
7
58
based ZP dyes and related compounds, although we note
that pK values for thiols could also fall in this range.
Fluorescein itself displays pH-dependent fluorescence with
quenched emission at low pH, and the pK of 4.6 observed
a
a
which suggests that the thiol moiety also plays a role in
quenching the fluorescein excited state. Addition of Hg(II)
causes an ∼1.5- (MS2) or ∼2-fold (MS3) fluorescence
enhancement, and the emission spectra for both species red-
shift to 531 nm (Figure 3). The absorption spectra also
undergo red-shifts upon Hg(II) complexation. For MS2, a 5
in Figure 2 corresponds to fluorescein protonation and
formation of a nonfluorescent species at acidic pH.
MS2 and MS3 have quantum yields of 0.28 and 0.27 and
emission maxima at 526 and 525 nm, respectively, at neutral
pH (50 mM PIPES, 100 mM KCl) and in the presence of
the metal ion scavenger of EDTA (Table 1). These values
are ∼50% lower than that observed for the corresponding
asymmetrical di(2-picolyl)amine-based sensor (Φ ≈ 0.6),
-
1
-1
nm shift is observed from 500 (ꢀ ) 62 000 M cm ) to
-
1
-1
505 nm (ꢀ ) 69 000 M cm ). The optical absorption
spectrum for MS3 exhibits a similar shift from 501 (ꢀ )
2746 Inorganic Chemistry, Vol. 45, No. 6, 2006

SelectiWe Hg(II) Detection in Aqueous Solution
1 000 M- cm ) to 507 nm (ꢀ ) 59 000 M cm )
1
-1
-1
-1
5
following Hg(II) binding. Since introduction of Hg(II) to a
solution of MS2 or MS3 at pH 7 results in essentially no
change in quantum yield for either species (MS2, ΦHg
.27; MS3, ΦHg ) 0.28) while causing an increase in molar
)
0
absorptivity, the resulting fluorescence enhancement is due
exclusively to the absorption increase since brightness is
(
Φ × ꢀ).
Although modest, the fluorescence enhancement observed
for these sensors upon addition of Hg(II) was intriguing. The
pK
a
of ∼8 for MS2 and MS3 suggests that proton-induced
background fluorescence compromises Hg(II)-induced emis-
sion turn-on at neutral pH. Raising the solution pH to
deprotonate the tertiary amine nitrogen atom should enhance
PET quenching, lower the quantum efficiency of each free
dye, and result in greater fluorescence enhancement upon
Hg(II) complexation. As anticipated, the quantum yield of
free MS2 decreases substantially when the pH is raised to 9
Figure 4. Difference spectra obtained from the addition of Hg(II) to a
solution of MS2 at pH 11 (50 mM CABS, 100 mM KCl). The concentration
of MS2 was 10 µM. Aliquots of 1 mM HgCl in water were added to yield
2
Hg(II) concentrations of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
and 20 µM. Absorption increases occur at ca. 300 and 510 nm, and a
decrease occurs at 480 nm upon Hg(II) coordination. Inset: Absorption
change versus equiv of Hg(II) in solution for MS2, which indicates formation
of a 1:1 MS2/Hg(II) complex.
(
50 mM CHES, 100 mM KCl) or 11 (50 mM CABS, 100
mM KCl) with values of 0.10 and 0.07, respectively. An
5-fold increase in integrated emission immediately results
∼
after addition of Hg(II) to MS2 in this pH regime (Figure
S1, Supporting Information), and the quantum yield of the
Hg(II) complex is 0.28 (pH 9) or 0.24 (pH 11). MS3 exhibits
analogous behavior, as summarized in Table 1. Despite the
relatively modest degree of turn-on at pH 7, the choice of
tertiary amine as a PET switch and fluorescein as the
reporting group achieves greater brightness (Φ × ꢀ) upon
Hg(II) coordination than that observed for the aniline-
derivatized members of the MS family (Figure 1) and Hg(II)
sensors reported by others. To the best of our knowledge,
MS2 and MS3 are the brightest water-soluble Hg(II) sensors
to date.
Sensors MS2 and MS3 both show diminished fluorescence
turn-on following Hg(II) binding at neutral pH in the absence
49
50
of chloride ion, as previously observed for MS1 and MS4.
For instance, at neutral pH (50 mM PIPES, 1000 mM KX,
-
-
-
X ) OAc , F , NO
3
), only negligible (KOAc, KF) or no
(
KNO ) fluorescence enhancement occurs following Hg(II)
3
binding to MS2 (Figure S3). The presence of chloride ion
has no effect on the emission profile of the free dyes,
suggesting that a Hg-Cl bond or ion pairing causes the
chloride-dependent fluorescence enhancement. Results ob-
tained from low-resolution ESI mass spectrometric analyses
of solutions of MS2 and Hg(II) with added chloride ion
support this notion. When a methanolic solution of equimolar
MS2 and Hg(II) is treated with aqueous KCl, two new intense
peaks with m/z of 780.6 and 816.6 are observed in the
negative ion mode mass spectrum (Figure S4). The former
m/z ratio corresponds to the monochloro adduct, [MS2 +
Hg + Cl - 2H] (calcd 781.0) and the latter to the dichloro
species [MS2 + Hg + 2Cl - H] (calcd 817.0). Although
the precise nature of these chloride-containing complexes
cannot be ascertained from this experiment, the data clearly
reveal that the MS2:Hg(II) complex associates with one or
more chloride ions in solution. This association, in turn,
modulates the fluorescence emission of the Hg(II) complex.
Metal-Binding Studies of MS2 and MS3. Asymmetrical
MS2 and MS3 were designed for 1:1 metal ion complexation,
and the metal-binding properties of these probes were
investigated by UV-visible and fluorescence spectroscopies.
Job plots for the addition of Hg(II) to solutions of MS2 and
MS3 are given as Supporting Information (Figure S2). The
inflection points at 0.5 indicate the formation of 1:1 MS2:
Hg(II) and MS3:Hg(II) complexes in aqueous solution.
Metal-binding titrations reveal a break at 1 equiv of Hg(II),
providing further evidence for 1:1 binding stiochiometry
(
Figure 4). At pH 7 (50 mM PIPES, 100 mM KCl), the
absorbance difference spectrum for the addition of Hg(II)
to MS2 shows a decrease at 483 nm and increases at ca.
3
Fluorescence spectroscopy was used to determine an EC50
value, the concentration of Hg(II) required for 50% of the
total fluorescence enhancement at [MS] ) 1 µM, for Hg(II)
binding to MS2 and MS3 (Figure S1). Essentially complete
turn-on (∼96%) results upon addition of 1 equiv of Hg(II)
to a solution of MS2, and the EC50 of MS2 for Hg(II) is 384
nM. Addition of 1 equiv of Hg(II) to MS3 results in ∼34%
turn-on and maximum fluorescence is reached with ∼6 equiv
of Hg(II) in solution. The EC50 for MS3 for Hg(II) is 1.6
µM, indicating that gem-dimethyl susbtituents adjacent to
00 and 512 nm. The difference spectrum for MS3 is similar
decrease at 482 nm; increases at ca. 300 and 510 nm).
(
Comparable absorption changes are observed for Hg(II)
binding at high pH. For instance, at pH 11 (50 mM CABS,
1
00 mM KCl), Hg(II) binding to MS2 results in an
absorbance increase at 510 nm and a decrease at 490 nm
Figure 4). The absorbance changes at ca. 500 nm are
(
attributed to perturbation of the fluorescein π-π* transition
upon Hg(II) coordination and the increase at ca. 300 nm to
RS f Hg(II) charge-transfer bands.
-
59,60
(
59) Watton, S. P.; Wright, J. G.; MacDonnell, F. M.; Bryson, J. W.; Sabat,
(60) Fleissner, G.; Kozlowski, P. M.; Vargek, M.; Bryson, J. W.;
M.; O’Halloran, T. V. J. Am. Chem. Soc. 1990, 112, 2824-2826.
O’Halloran, T. V.; Spiro, T. G. Inorg. Chem. 1999, 38, 3523-3528.
Inorganic Chemistry, Vol. 45, No. 6, 2006 2747

Nolan et al.
affinity for metals in the “copper triangle” of the periodic
6
2
table. Like MS1 and MS4, sensors MS2 and MS3 both
display greater affinity for Hg(II) than its Group 12 congeners
Zn(II) and Cd(II). The positive fluorescence responses of
MS2 and MS3 are Hg(II)-specific (Figure 5).
Reversibility is a criterion in sensor development since
the ability to regenerate the free sensor is important for
practical applications. During our investigation of anion
effects, we observed that MS2 and MS3 do not coordinate
Hg(II) in buffer containing 100 mM KI (50 mM PIPES, 100
mM KI, pH 7). Optical absorption spectroscopy showed that
addition of Hg(II) to solutions of the sensors in this buffer
resulted in no change in the chromophore absorption band
centered at 500 nm and the formation of intense absorption
2
-
4
bands centered at 265 and 321 nm attributed to [HgI ]
6
3,64
(Figure S6).
This finding and a recent report that Hg(II)
complexation to a solid-state colorimetric Hg(II) sensor was
4
6
reversed by washing with KI motivated us to use KI to
achieve reversible Hg(II) binding with MS2. Optical absorp-
tion spectroscopy shows that addition of excess KI to
solutions of the MS2:Hg(II) complex causes an immediate
blue shift in the fluorescein π-π* transition from 505 to
Figure 5. Metal-ion selectivity of MS3 at pH 7 (50 mM PIPES, 100 mM
KCl) and 11 (50 mM CABS, 100 mM KCl). The light bars represent the
fluorescence emission of a solution of MS3 and 67 equiv of the cation of
interest: 1, Li(I); 2, Na(I); 3, Rb(I); 4, Mg(II); 5, Ca(II); 6, Sr(II); 7, Ba(II);
500 nm with a concomitant intensity decrease, consistent with
8
1
, Cr(III); 9, Mn(II); 10, Fe(II); 11, Co(II); 12, Ni(II); 13, Cu(II); 14, Zn(II);
5, Cd(II); 16, Hg(II); 17, Pb(II). The dark bars show the fluorescence
Hg(II) release. As anticipated, formation of new bands in
change that occurs upon addition of 67 equiv of Hg(II) to the solution
containing MS3 and the cation. The concentration of MS3 was 1 µM, and
excitation was provided at 500 nm. The response (F) is normalized with
respect to the emission of the free dye (Fo). See Figure S5 of Supporting
Information for MS2 data. The positive fluorescence response of MS2 and
MS3 to Hg(II) also occur in the presence of millimolar concentrations of
Li(I), Na(I), Rb(I), Mg(II), Ca(II), Sr(II), and Ba(II) (data not shown).
2-
4
the 260-320 nm region signaled formation of [HgI ]
(Figure S6).
The EPA-mandated upper limit of allowable inorganic
3
mercury in drinking water is 2 ppb. A fluorescence change
of 15.3% ( 5% in MS2 (500 nM) occurs in the presence of
2
ppb Hg(II) (50 mM CABS, 100 mM KCl, pH 11),
the thiol donor decrease the binding affinity. These values
may be compared to the EC50 value of 410 nM for sensor
MS1 (1 µM, pH 7).49
demonstrating that MS2 can detect environmentally relevant
concentrations of Hg(II).
Summary. MS2 and MS3, two water-soluble, fluorescein-
based Hg(II) sensors that each contain a pyridyl-amine-
thiol ligand were prepared and characterized. These probes
afford Hg(II)-selective fluorescence enhancement in aqueous
solution at neutral pH and EC50 values for Hg(II) in the mid-
nanomolar to low micromolar range. MS2 and MS3 bind
Hg(II) over Zn(II), Cd(II), Mn(II), Fe(II), Co(II), Cr(III),
Pb(II), and millimolar concentrations of various alkali and
alkaline earth metals. The choice of tertiary amine nitrogen
atoms as the PET switch yields fluorescein-based sensors
and Hg(II) complexes with superior brightness (Φ × ꢀ)
compared to aniline-containing MS1 and other reported
water-soluble Hg(II) sensors. The MS2 and MS3 sensors may
be useful for applications in toxicology and environmental
sciences.
Achieving high selectivity for the analyte of interest over
a complex background of potentially competing species is a
challenge in sensor development. Figure 5 illustrates the
fluorescence response of MS3 to Hg(II) in the presence of
6
1
alkali and alkaline earth metals found in natural waters,
select divalent first-row transition metals, and Zn(II) and
Cd(II). A background of Group 1 and 2 metals does not
interfere with Hg(II) coordination and subsequent fluores-
cence turn-on. Neither Cr(III) nor Pb(II) prevent the Hg(II)-
induced fluorescence enhancement. MS3 is selective for
Hg(II) over the open-shell divalent first-row transition metals
Mn(II), Fe(II), and Co(II). MS3 preferentially binds Ni(II)
and Cu(II) over Hg(II) at neutral pH, as observed for sensor
MS4,50 which also employs one sulfur and one pyridyl
nitrogen donor atom. The selectivity of MS2 for Hg(II) is
generally similar, although slightly less fluorescence en-
hancement occurs upon addition of Hg(II) to solutions of
MS2 and Co(II) (Figure S5). The preference of these sensors
Acknowledgment. This work was supported by Grant
No. GM65519 from the National Institute of General Med-
ical Sciences. Spectroscopic instrumentation at the MIT
2
for Cu(II) is not surprising given the N SO donor set and
the observation that sulfur-containing ligands exhibit high
(62) Cooper, T. H.; Mayer, M. J.; Leung, K.-H.; Ochrymowycz, L. A.;
Rorabacher, D. B. Inorg. Chem. 1992, 31, 3796-3804.
(
61) Drever, J. I. The Geochemistry of Natural Waters: Surface and
Groundwater EnVironments, 3rd ed.; Prentice Hall: Upper Saddle
River, NH, 1997.
(63) Griffiths, T. R.; Symons, M. C. R. J. Chem. Soc. Trans. Farady Soc.
1960, 56, 1752-1760.
(64) Griffiths, T. R.; Anderson, R. A. Can. J. Chem. 1991, 69, 451-457.
2748 Inorganic Chemistry, Vol. 45, No. 6, 2006

SelectiWe Hg(II) Detection in Aqueous Solution
DCIF is maintained with funding from NIH Grant No.
by the Undergraduate Research Opportunities Program at
MIT.
1
S10RR13886-01 and NSF Grants No. CH3-9808063,
DBI9729592, and CHE-9808061. E.M.N. thanks NDSEG for
a graduate fellowship, Dr. Daniel J. Kramer for helpful
discussions, and Dr. Xiao-an Zhang for assistance with low-
resolution mass spectrometry. M.E.R. was partially supported
Supporting Information Available: Figures S1-S6. This mat-
erial is available free of charge via the Internet at http://pubs.acs.org.
IC052083W
Inorganic Chemistry, Vol. 45, No. 6, 2006 2749