Development and applications of fluorogenic probes for mercury(II) based on vinyl ether oxymercuration

Article abstract of DOI:10.1021/ja108028m

Mercury is a major threat to the environment and to human health. It is highly desirable to develop a user-friendly kit for on-site mercury detection. Such a method must be able to detect mercury below the threshold levels for drinking water, 1-2 ppb. We developed a fluorescence method based on the oxymercuration of vinyl ethers to detect mercury in dental and environmental samples. Chloride ions interfered with the oxymercuration reaction, but the addition of AgNO₃ solved this problem. Fine electronic and structural tuning led to the development of a more responsive probe that was less sensitive to chloride ion interference. This second-generation probe could detect 1 ppb mercury ions in water.

Full text of DOI:10.1021/ja108028m

ARTICLE
pubs.acs.org/JACS
Development and Applications of Fluorogenic Probes for
Mercury(II) Based on Vinyl Ether Oxymercuration
Shin Ando and Kazunori Koide*
Department of Chemistry, University of Pittsburgh, 219 Parkman Avenue, Pittsburgh, Pennsylvania 15260, United States
S Supporting Information
b
ABSTRACT: Mercury is a major threat to the environment and to human health. It is highly desirable to develop a user-friendly kit
for on-site mercury detection. Such a method must be able to detect mercury below the threshold levels for drinking water, 1-2 ppb.
We developed a fluorescence method based on the oxymercuration of vinyl ethers to detect mercury in dental and environmental
samples. Chloride ions interfered with the oxymercuration reaction, but the addition of AgNO3 solved this problem. Fine electronic
and structural tuning led to the development of a more responsive probe that was less sensitive to chloride ion interference. This
second-generation probe could detect 1 ppb mercury ions in water.
’ INTRODUCTION
employed in developing countries in which mercury pollution is
severe and can be found even in tap water.18
Mercury is one of the most toxic metals, as seen in such un-
fortunate incidents as Minamata disease1 and mercury poisoning
in Iraq.2 As these cases showed, methylmercury is extremely toxic
and damages the central nervous system.3 The tragedy of
Professor Karen Wetterhahn, who was studying organic mercury
compounds,4 led to a moratorium on the use of these highly toxic
and skin-permeable organic mercury compounds at many American
institutions, including ours.5 Inorganic mercury compounds exhibit
severe effects on the human heart, kidney, stomach, and genes.6
Despite the indisputable toxicity of mercury, it remains unclear
whether there is a direct link between mercury and autism.7
Nonetheless, pregnant women are advised to avoid mercury-
rich diets such as shark and tuna as a precaution.8
Mercury metal is volatile and can travel far in the air. It is then
deposited on land or in water and oxidized to mercury(II).9
Oxidized inorganic mercury(II) may be converted to organic
mercury by microorganisms.10 Mercury in the environment ori-
ginates not only from geological events (e.g., volcano emis-
sions11) but also from modern human activities. For example, the
wastewater from dental offices is a major source of mercury pollu-
tion, partially because dentists are exempt from federal regula-
tions on waste.12 Coal-fired power plants generate substantial
amounts of mercury, which is monitored by the United States
Environmental Protection Agency (US EPA).13 In addition, the
combination of human activities and geological samples such as
the recent oil spill in the Gulf of Mexico has raised great concern
among the general public.14
Mercury is generally quantified by cold vapor atomic absorp-
tion spectroscopy15 or inductively coupled plasma mass spectros-
copy.16 Although these methods are quantitative and powerful,
the analyses require large and expensive instruments, highly trai-
ned personnel, and tedious maintenance. Mostly because of the
size of the instruments, mercury-contaminated samples are gene-
rally analyzed off-site. The off-site analysis format retards the
remediation of wastewater17 in coal-fired power plants and
makes it essentially impractical to monitor dental offices and
their surroundings.12 It is also important to note that these
expensive and sophisticated analytical techniques cannot be
Optical methods are more amenable to the on-site analysis of
mercury with fewer resources. Therefore, numerous fluorescent
chemosensors and chemodosimeters have been reported in the
literature.19-22 These methods may facilitate mercury analyses,
even if they are only semiquantitative. Because mercury is a soft
Lewis acid, a vast majority of the chemosensors and chemodosi-
meters for mercury contain sulfur atom(s) that can tightly
coordinate the metal as part of off-on fluorescence switches.19,21
Some of the chemosensors and chemodosimeters were success-
fully applied to real world “dirty” samples and in biological
settings with low concentrations of mercury ions.19,22
As the protocol of the US EPA indicates,23 mercury-contain-
ing environmental samples are pretreated with harsh oxidants
24
such as Cl-Br23 and H2O2 to transform various forms of
organic and sulfur-bound (e.g., cysteine-bound) mercury spe-
cies25 to sulfur-free inorganic mercury(II). Therefore, we felt that
chemodosimeters and chemosensors for mercury in environ-
mental and biological fluid samples might need to be compatible
with oxidants. Although others have successfully demonstrated
the utility of their sulfur-based indicators, we turned our atten-
tion to the π electrophilicity of mercury ions and developed a
chemodosimeter based on the oxymercuration of an alkyne.26
This chemodosimeter was found to be resistant to strong
oxidants such as H2O2 and N-chlorosuccinimide (NCS). This
method enabled us to detect mercury from fish and dental
samples. However, the oxymercuration reaction needed to be
heated to 90 °C. Moreover, we were not able to detect mercury
ions below 4 ppb. The ability to detect mercury ions below this
level is critically important, because the limit of mercury
concentration in drinking water is 2 ppb in the United States.27
We hypothesized that a more electron rich π bond might be
more reactive toward mercury ions, allowing for mer-
cury detection at a lower temperature and at lower mercury
concentrations. Herein, we report user-friendly and more sensitive
Received: September 6, 2010
Published: February 4, 2011
r
2011 American Chemical Society
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Scheme 1. Two-Step Sequence To Cleave an Allyl Ether (a),
Platform for a Fluorescence Off-On Switch (b), and Pre-
paration of 3 and Its Reaction with HgCl2 To Form 1 (c)
conditions in a refluxingacetone-water mixture,28 such hydrolysis
did not occur when vinyl ether 3 was incubated in a pH 4 buffer at
80 °C, ensuring its stability during storage.
Next, we optimized conditions for a Hg(II)-promoted hydrolysis
of vinyl ether 3 to form 133 in various buffers (for details, see Table
S1 in the Supporting Information) at 25 °C. The ratio of Hg(II)-
promoted and Hg(II)-free hydrolysis—the latter was negligible—
was optimal at pH 3 (Figure 1a). However, the pH 3 reaction media
was more difficult to neutralize than the pH 4 media for fluores-
cence measurement in a high-throughput manner. Therefore, pH 4
was chosen for the remaining part of this study.
In a pH 4 buffer, we subjected vinyl ether 3 to various metal
ions at 5 μM, demonstrating that the conversion of 3 to 1 was
most efficiently promoted by Hg(II) (Figure 1b and Table S2 in
the Supporting Information). A slight signal increase was ob-
served in the presence of Pt(II).
The conversion of 3 to 1 in the presence of Hg(II) (0.30 μM)
in a pH 4 buffer showed ∼40% completion after 1 h, and the reac-
tion continued to proceed (Figure 1c). The 1 h duration was
considered an optimal balance between sensitivity and conve-
nience. The fluorescence signals generated by the conversion of 3
to 1 was [Hg(II)] dependent with a distinct linear signal begin-
ning as low as 4 ppb (=20 nM) Hg(II) (b in Figure 1d; Figure
S3a and Table S3 in the Supporting Information).
Oxidative pretreatment of environmental samples with Br-Cl
is a standard procedure by the US EPA. We found that NCS
could also oxidatively disrupt the Hg-S bond.26 Because NCS
can react with olefins, we were concerned about the stability of
vinyl ether 3 toward NCS. However, this probe was stable against
NCS while remaining responsive to Hg(II) (O in Figure 1d and
Table S3 in the Supporting Information). These data indicate
that probe 3 could be used to detect Hg(II) in sulfur-containing
samples in the presence of NCS.
new fluorescent chemodosimeters that react with mercury ions at
ambient temperature. The second-generation chemodosimeter
in this study was found to be particularly powerful, detecting
mercury ions at a 1 ppb level.
Failed Application of 3 to Detect Hg and Revision of
Reaction Conditions. We proceeded to apply the above method
for the detection of Hg(II) in actual “dirty” samples in order to
evaluate the utility of the method. Known amounts of Hg(II) were
spiked into river water that contained <1 ppb total mercury.36 The
resulting solutions, after the pH was adjusted to 4, were treated
with 3 in an attempt to convert 3 to 1, but to no avail (Figure 3a,
left). This failure prompted us to revisit the generality of the
method. Rather than testing each metal separately, we examined
mixtures of HgCl2 (2.5 μM) with various inorganic reagents (25
μM). Figure 2a (white bars) shows that most reagents interfered
with the conversion of 3 to 1. It appeared that this interference was
not caused by metals but by chloride ions. We compared the effect
of NaCl and NaNO3 to verify the effect of chloride ions.
Treatment of compound 3 (1.0 μM) with HgCl2 (2.5 μM) and
excess NaCl (1.0 mM) in a pH 4 buffer showed no fluorescence
increase (Figure 2b). Under similar reaction conditions, we only
observed the starting material 3 by 1H NMR spectroscopy (Figure
S18, Supporting Information). In contrast, the presence of NaNO3
did not impact the conversion of 3 to 1. We concluded that chloride
ions interfered with our fluorescence method. This may also
account for the failure of our earlier river water study and limit
the applications of 3 in biological systems.
’ RESULTS AND DISCUSSION
Design and Synthesis of Probe 3. An allyl ether is used as a
protecting group and can be removed via a base-catalyzed olefin
migration followed by either an acid-catalyzed hydrolysis at elevated
temperature28 or mercury-promoted hydrolysis (Scheme 1a).29
Because the latter can be employed at ambient temperature, we
hypothesized that this transformation might be used as a means to
selectively convert a nonfluorescent molecule to a fluorescent mole-
cule with mercury ions.
We previously demonstrated the conversion shown in
Scheme 1b as a general platform for the development of fluores-
cence methods.26,30,31 In order to couple this platform with the
chemistry shown in Scheme 1a, allyl ether 230,32,33 was treated
with KOtBu in DMSO34 to form vinyl ether 3 in 86% yield as an
inseparable mixture of cis and trans isomers (Scheme 1c). The
purity of 3 was ensured by HPLC analysis (Figure S2, Supporting
Information). The absorption and emission spectra of 3 are
shown in Figure S1 (Supporting Information).
Reactivity of Probe 3 with Metal Ions. Treatment of 3 with
HgCl2 (1.0 equiv) at 25 °C afforded 1 in 82% isolated yield, indi-
cating that this transformation could be used to develop a fluo-
rescence method for Hg(II) (possibly HgClþ, because HgCl2 is
mostly dissociated into HgClþ and Cl-)35 at a more convenient
temperature. Although vinyl ethers can be hydrolyzed under acidic
We asked how chloride ions interfered with the Hg(II)-
promoted conversion of 3 to 1. The formation of 7 from the
electrophilic species 5 (Scheme 1c) could be ruled out by the
aforementioned NMR analysis. It is possible that the equilibrium
shifted from more reactive HgXþ (X = Cl, phosphate, etc.) to less
reactive HgXCl. This working hypothesis could account for the
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Figure 1. Conversion of 3 to 1 at 25 °C. [3] = 1.0 μM for all of the experiments. Except for (c), all of the reactions were performed for 1 h. Fluorescence
intensities were measured after addition of 1.23 M phosphate pH 7 buffer. (a) pH dependence. (b) Metal selectivity in a 50 mM phthalate pH 4 buffer.
Metal reagents: AgNO3, AuCl3, BaCl2, CaCl2, CdCl2 2.5H2O, CoCl2, CrCl3, CuCl2 2H2O, FeCl3, HgCl2, KCl, LiCl, MgCl2, MnCl2 4H2O, NaCl,
3
3
3
NiCl2, Pb(NO3)2, Pd(NO3)2, PtCl2, Rh(PPh3)3, RuCl3, and ZnCl2. (c) Time course of the oxymercuration reaction: [Hg(II)] = 0.30 μM, 50 mM
phthalate pH 4 buffer. (d) Correlation between fluorescence intensities and [Hg(II)] in the presence and absence of N-chlorosuccinimide (NCS). The
experiments were performed in a 50 mM phthalate pH 4 buffer in triplicate. The graph shows the mean values and standard deviations.
Figure 2. Compatibility of our method to detect Hg(II) in the presence of inorganic ions: (a) interference by inorganic materials ([HgCl2] = 2.5 μM,
[reagent shown] = 25 μM); (b) interference by NaCl but not by NaNO3. These experiments were performed in pH 4 buffer.
noninterference of the mixture of HgCl2 and AgNO3 (not
shown). These results prompted us to hypothesize that the
addition of excess AgNO3 to the mixtures of HgCl2 and MCln
(M = Li, Na, etc.) might facilitate the conversion of HgCl2
(XHg-Cl bond 24 kcal/mol37) to HgClþ by virtue of the
formation of poorly water soluble AgCl38 (Ag-Cl bond 71.7
kcal/mol37). In effect, the combination of HgCl2 (2.5 μM),
AgNO3 (100 μM), and MCln (25 μM) generated a nearly
uniform fluorescence signal (Figure 2a, gray bars). Thus, the
addition of AgNO3 was found to afford a more general fluoro-
metric detection method for Hg(II) in the presence of various
inorganic molecules.
Detection of Hg(II) in River Water. With this improvement,
we attempted to detect mercury species in environmental
samples again. In wastewater, the permitted discharge limits
for total mercury may be 5 ppb39 or 10 ppb.40 Thus, as a proof-
of-concept experiment,41 we proceeded to apply our method,
which involved the use of AgNO3 for the detection of spiked
Hg(II) (0-256 ppb) in river water (Figure 3a,b). Figure 3a
shows that, unlike the previously failed case (left), the addition
of AgNO3 improves the signal recovery to nearly 100%.
The standard curve in Figure 3b (also Table S4, Supporting
Information) indicates that compound 3 could detect Hg(II)
at ∼8 ppb in river water.
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Figure 3. Applications of probe 3 for the detection of mercury species in environmental and dental samples. All detection was performed after
adjustment of pH of samples to pH 4. (a) Comparison of river water and commercial pH 4 buffer ([HgCl2] = 2.2 μM (440 ppb)). In the absence of
AgNO3, Hg(II) cannot be detected by the method (left). In the presence of AgNO3, Hg(II) can be detected (middle). (b) Mercury detection in river
water ([HgCl2] = 0-256 ppb, [AgNO3] = 2.0 mM). (c) Mercury detection in river water in the presence of organic compound ([AgNO3] = 2.0 mM,
[compound] = 100 ppb). (d) Dental samples ([NCS] = 500 μM).
We proceeded to determine whether the method based on the
oxymercuration of vinyl ether 3 could be compatible with organic
contaminants in environmental samples. We applied our method
in the presence of various typical organic contaminants in waste-
water42 to assess the robustness of 3 with other functional groups
(Figure 3c and Table S5, Supporting Information). It was found
that this probe could detect Hg(II) even if the reaction solution
was contaminated with organic compounds, including an alkene.43
This may not be surprising, because the olefin of compound 3 is
more electron rich and thus more reactive toward Hg(II) than
most alkenes.
Scheme 2. Preparation of 12 and Its Reaction with HgCl2 To
Form 13
Detection of Hg(II) in Dental Samples. In addition to envir-
onmental samples, dental samples were examined to broaden the
applications of the fluorescence method. We hypothesized that
cysteine from food might facilitate the dissolution of Hg from
amalgam-filled teeth. Thus, the previously used two teeth26 were
stirred in a cysteine solution. After the teeth were removed, the
resulting solution was treated with NCS to oxidize the thiol and
Hg-bound sulfur atoms before the addition of 3. Figure 3d and
Table S6 (Supporting Information) show that we were able to
detect, although unable to quantify,44 leached mercury from
dental samples. The difference between bars 2 and 3 (3.63 ꢀ
104 in the raw data; see page S17 in the Supporting In-
formation) was greater than 3 times the standard deviation
of bar 2 (6.85 ꢀ 103), enabling us to detect Hg(II) with a
confidence level of over 90%.44 Additional studies of dental
samples are needed to validate the utility of the method in
dentistry. Nonetheless, this result may warrant further studies
on how sulfur-rich food may dissolve mercury amalgams.
It should be noted that, during this work, Ahn et al. reported a
very similar compound for Hg detection.45 There are notable
differences: 3 cannot be contaminated with heavy metals, it reacts
with Hg(II) in a 1:1 stoichiometry, and we discovered anion
interference and a solution to this interference.
Design, Preparation, and Characterization of 12 and
13. While developing a protocol to circumvent interference by
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Figure 4. (a) pH titration of compound 13. The fluorescence intensity at 515 nm was monitored at ∼0.3 pH intervals. (b) UV-vis absorption spectra of
12 and 13 in 1% DMSO/pH 8 buffer. (c) Emission spectra of 12 and 13 in 0.1% DMSO/pH 8 buffer.
chloride ions, we realized that the chlorine atoms that were built
into 3 might interfere, although organic and inorganic chlorides are
not the same. Further consideration on removing the chlorides from
3 suggested that the vinyl ether of 12 (Scheme 2) might be more
reactive toward Hg(II), due to the lack of electron-withdrawing
chloride groups. Our minor concern was related to the inferiority of
fluorescein over 20,70-dichlorofluorescein due to the higher pKa
(6.4) of fluorescein compared to that of 20,70-dichlorofluorescein
(4.3; the pKa of compound 1 is 4.3).33 Nonetheless, we proceeded
to synthesize compound 12 as shown in Scheme 2. This time, the
olefin migration of the allyl ether 11 was highly stereoselective, only
giving the cis product 12. Compound 12 also reacted with HgCl2
smoothly at 25 °C and gave the new fluorescent compound 13 in
72% isolated yield. The linear correlation was confirmed between
the concentration of 13 and the fluorescence signals (Figure S4,
Supporting Information).
The pKa of the phenolichydroxygroupof compound13 was 6.0
(Figure 4a and Figure S5, Supporting Information), noticeably
lower than that of fluorescein (6.4). This was unexpected, because
theconversionofthe strongerelectron-withdrawingcarboxygroup
of fluorescein to the weaker hydroxymethyl group should decrease
the acidity of the phenol hydroxy group. We postulate that the
carboxylate anion of fluorescein might destabilize the phenoxy
anion intramolecularly. Although the reason is not yet clear, the
fine tuning of the acidity of phenol in fluorescein derivatives is a
fruitful endeavor to develop new assay methods.46
Reactivity of 12 with Metal Ions. Solutions of the vinyl ether
12 were treated with Hg(II) (0.3 μM = 60 ppb) in pH 4, 5, 6, and
7 buffer (Figure 5a). Similarly to compound 3, compound 12 was
more reactive at a lower pH. The reactivity of 12 toward Hg(II)
was very high (Figure 5b). The only other metal that reacted with
12 was Pd(II) (cf. Figure 1b). In the presence of AgNO3, none of
the coexisting metal reagents interfered with the Hg(II)-pro-
moted conversion of 12 to 13 (Figure 5c). It was found that the
fluorescence method using 12 allowed for the detection of 1 ppb
of Hg(II) (Figure 5d and Table S7, Supporting Information).
Figure 5e indicates that chloride, bromide, and iodide ions
interfered with the Hg(II)-promoted conversion of 12 to 13
but nitrate and sulfate ions did not. The interference by chloride
ions was less severe for compound 12 than for compound 3
(Figure S6, Supporting Information). The halide-ion-mediated
interference could be overcome by the addition of AgNO3
(Figure 5c).
Next, we compared the new chemodosimeter 12 with 3 and
14.26 As Figure 6a shows, compound 12 was far more reactive
than 14 toward Hg(II) in a pH 4 buffer at 25 °C. For example,
after 10 min the fluorescence increase with 12 was 242 times
greater than that with 14. The reaction with 12 was >60% com-
plete after 1 h. The initial rate was about 12 times greater for 12
relative to 3 (Figure 6b). Additionally, compound 12 was more
reactive than compound 3 at pH 7 (Figure 6c), implying
potential biological applications.
Application with River Water. We applied chemodosimeter
12 for detection of Hg(II) spiked in river water again. Chemo-
dosimeter 12 was able to detect 1 ppb of Hg in river water
(Figure 7 and Table S8, Supporting Information). The height-
ened sensitivity of 12 in determining [Hg(II)] in such a complex
media as natural water indicates its robustness and potential
for facile on-site monitoring of water safety. When our method
The UV-vis absorption spectra of 12 and 13 are shown in
Figure4b. Compound 12 showed noabsorptionpeak at∼490 nm,
but compound 13 did. The fluorescence emission spectra of 12
and 13indicatedthat the signalof12was 140 timeslower thanthat
of 13 in a pH 8 buffer at 515 nm (λmax) at the same concentration
(Figure4c; see alsoFigureS1). Thisisconsistentwith theplatform
depicted in Scheme 1b.
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Figure 5. Comparison of reaction conditions for the conversion of 12 to 13. (a) pH dependence. Fluorescence intensities were measured after addition of 1.3 M
pH 7 buffer and 500 mM pH 10 buffer. (b) Metal selectivity. All the metals were tested at 5 μM in pH 4 buffer. (c) Hg detection in the presence of various metal
ions. All of the reactions were performed in pH 4 buffer. Metal reagents: AgNO3, AuCl3, BaCl2, CaCl2, CdCl2 2.5H2O, CoCl2, CrCl3, CuCl2 2H2O, FeCl3,
3
3
HgCl2, KCl, LiCl, MgCl2, MnCl2 4H2O, NaCl, NiCl2, Pb(NO3)2, Pd(NO3)2, PtCl2, Rh(PPh3)3, RuCl3, and ZnCl2. (d) Correlation between fluorescence
3
intensities at 515 nm and [Hg(II)]. The experiments were performed at pH 4 in triplicate. (e) Effect of anion. All detection was performed in pH 4 buffer.
was applied to the analysis of two wastewater samples from a
coal-fired power plant, we found compound 12 to be a quali-
tative indicator of Hg(II) (Supporting Information). Although
we were unable to quantitatively assess Hg(II) concentrations
in these more complex samples, we were able to qualitatively
discriminate between concentration values as low as 2 ppb and
<500 ppt. Further studies are needed to determine the scope
and limitations of the use of 12 with additional complex
samples.
contaminants. The method with 3 was effective in the detection
of Hg(II) in river water and dental samples. Further structural
fine tuning led to the development of the vinyl ether
12 (Scheme 3). Compound 12 could react with Hg(II) after
the removal of chloride ions with AgNO3. This compound
was 242 times more reactive than the previously reported
alkyne 14 toward Hg(II) at ambient temperature and could be
used to detect 1 ppb Hg(II). Further evaluations of this method
with additional real-world samples are underway in our labora-
tory.
’ CONCLUSIONS
’ EXPERIMENTAL SECTION
In summary, we have developed a sensitive and selective fluo-
rometric method to detect mercury species at ambient tempera-
ture in the presence of various organic, inorganic, and anionic
Synthetic Materials and Methods. All of the reactions in
Schemes 1c and 2 were carried out with commercial-grade reagents
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Figure 6. Comparisons of reactivity between probes developed by our group ([probe] = 1.0 μM, [Hg(II)] = 0.3 μM). (a) Time course of the
oxymercuration reaction at 25 °C in 50 mM phthalate pH 4.0 buffer. The comparison between probes 12 and 14 is shown. (b) Time course of the
oxymercuration reaction at 25 °C in 50 mM phthalate pH 4.0 buffer. The comparison between probes 3 and 12 is shown. To focus on their initial rate,
fluorescence measurements were carried out every 1 min for 18 min. (c) Time course of the oxymercuration reaction in 50 mM phosphate pH 7 buffer.
The comparison between probes 3 and 12 is shown.
Scheme 3. Summary of This Work
Figure 7. Application of 12 for the detection of Hg(II) in river water
([12] = 1.0 μM; [Hg(II)] = 0, 0.25, 0.5, 1, 2, and 4 ppb; [AgNO3] = 2.0
mM; 0.5% DMSO in 50 mM phthalate pH 4 water; 1 h; 25 °C). Titration
of Hg(II) in river water was performed.
7.34-7.27 (m, 1H, Ar), 7.05 (s, 0.67H, Ar (cis isomer)), 7.03 (s, 0.33H,
Ar (trans isomer)), 6.99-6.97 (m, 2H, Ar), 6.91-6.88 (m, 1H, Ar), 6.87
(s, 1H, Ar), 6.68 (dq, J = 12.0, 1.8 Hz, 0.33H, CH3CHdCHOAr (trans
isomer)), 6.65 (dq, J = 6.6 (a similar coupling constant was observed in a
related compound47), 1.8 Hz, 0.67H, CH3CHdCHOAr (cis isomer)),
5.51 (dq, J = 12.0, 6.6 Hz, 0.33H, CH3CHdCHOAr (trans isomer)),
5.42 (s, 2H, ArCH2OH), 5.13 (dq, J = 6.6,47 6.6 Hz, 0.67H, CH3CHd
CHOAr (cis isomer)), 1.76-1.66 (m, 3H, CH3CHdCHOAr) ppm;
13C NMR (75 MHz, acetone-d6, 293 K, Figure S8) δ 154.6, 154.2, 154.0,
150.44, 150.38, 145.8, 142.1, 140.9, 139.7, 130.7, 130.4, 129.5, 124.1,
122.3, 121.0, 118.8, 118.4, 118.3, 117.2, 111.7, 110.7, 104.7, 104.5, 104.2,
83.6, 73.4, 12.3, 9.6 ppm; HRMS (EIþ) m/z calcd for C23H16O4Cl2
[M]þ 426.0426, found 426.0425.
Conversion of Compound 3 to Compound 1. HgCl2 (14 mg,
50 μmol) was added to a solution of compound 3 (21 mg, 50 μmol) in a
1/9 DMSO/50 mM phthalate pH 4 buffer (20 mL) mixture. The
resulting mixture was stirred at 25 °C for 1 h and extracted with EtOAc
(2 ꢀ 20 mL). The combined organic layers were washed with brine
(2 ꢀ 30 mL), dried over Na2SO4, filtered, and evaporated under reduced
pressure. The residue was purified by flash column chromatography (10
f 70% EtOAc in hexanes) on silica gel (10 mL) to afford compound 1
(16 mg, 82%) as an orange solid. The spectroscopic data of compound 1
were consistent with the literature.30 Compound 8 was isolated from
other fractions. Rf = 0.66 (50% EtOAc in hexanes). 1H NMR (300 MHz,
acetone-d6, 293 K, Figure S9): δ 9.63 (d, J = 1.2 Hz, 1H, CHO), 3.66
(qd, J = 6.9, 1.2 Hz, 1H, OHC-CHHgX), 1.53 (d, J = 6.9 Hz, 3H, CH3)
ppm. Due to the extreme toxicity of organomercury species, we discarded
this material in collaboration with the Environmental Health and Safety office
at the University of Pittsburgh (http://www.ehs.pitt.edu) without obtaining
further spectroscopic data.
without further purification. DMF was used after distillation from silica
gel. CH2Cl2 was used after distillation from CaH2. Yields refer to chro-
matographically and spectroscopically (1H NMR) homogeneous mate-
rials. All reactions were monitored by thin-layer chromatography (TLC)
carried out on 0.25 mm E. Merck silica gel plates (60F-254) using UV
light (254 nm) for visualization or phosphomolybdic acid in ethanol as
developing agents and heat for visualization. TSI silica gel (230-400
mesh) was used for flash chromatography. NMR spectra were recorded
on AM300 or AM400 (Bruker) instruments and calibrated using a
solvent peak as an internal reference. The following abbreviations are
used to indicate the multiplicities: s = singlet, d = doublet, t = triplet, q =
quartet, m = multiplet, br = broad, app = apparent. High-resolution mass
spectra were obtained using EBE geometry.
Preparation of Compound 3. KOtBu (11 mg, 0.10 mmol) was
added to a solution of compound 230 (21 mg, 50 μmol) in DMSO (1.0
mL) at 25 °C under a nitrogen atmosphere, and the resulting solution
was heated in a 90 °C oil bath for 12 h. The reaction mixture was then
poured onto ice-cold H2O (5.0 mL), and the resulting mixture was
extracted with EtOAc (2 ꢀ 5 mL). The combined organic layers were
washed with brine (10 mL), dried over Na2SO4, filtered, and evaporated
under reduced pressure. The residue was purified by flash column
chromatography (5 f 20% EtOAc in hexanes) on silica gel (10 mL) to
afford compound 3 (18 mg, 86%, mixture of cis/trans (2:1)) as an
orange solid. Data for 3: mp 146-155 °C; Rf = 0.31 (30% EtOAc in
hexanes); IR (KBr pellet) νmax 3368 (broad, O-H), 2921, 2859, 1606
(CdO), 1482, 1435, 1411, 1269, 1175, 1034, 874, 725 cm-1; 1H NMR
(300 MHz, acetone-d6, 293 K, Figure S7) δ 7.52-7.39 (m, 2H, Ar),
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Preparation of Compound 10. K2CO3 (4.15 g, 30.0 mmol) was
added to a solution of fluorescein (3.32 g, 10.0 mmol) in DMF (20 mL)
at 25 °C under a nitrogen atmosphere, followed by allyl bromide (2.60
mL, 30 mmol). After it was stirred for 48 h at 25 °C, the reaction mixture
was poured onto H2O (500 mL). The resulting mixture was then
extracted with EtOAc (3 ꢀ 200 mL), and the combined extracts were
washed with H2O (3 ꢀ 500 mL) and brine (500 mL), dried over
Na2SO4, filtered, and evaporated under reduced pressure. The residue
was recrystallized from hexanes/EtOAc to afford compound 10 (3.17 g,
77%) as an orange solid. Data for 10: mp 153-155 °C; Rf = 0.34 (60%
EtOAc in hexanes); IR (KBr pellet) νmax 3054, 2986, 2932, 1727
(CdO), 1643 (CdO), 1595, 1517, 1481, 1256, 1211, 1106, 855, 759
cm-1; 1H NMR (300 MHz, CDCl3, 293 K, Figure S10) δ 8.27 (dd, J =
7.5, 1.5 Hz, 1H, Ar), 7.75 (ddd, J = 7.5, 7.5, 1.5 Hz, 1H, Ar), 7.68 (ddd, J =
7.5, 7.5, 1.5 Hz, 1H, Ar), 7.32 (dd, J = 7.5, 1.5 Hz, 1H, Ar), 6.96 (d, J = 2.4
Hz, 1H, Ar), 6.90 (d, J = 9.0 Hz, 1H, Ar), 6.87 (d, J = 9.6 Hz, 1H, Ar),
6.77 (dd, J = 9.0, 2.4 Hz, 1H, Ar), 6.56 (dd, J = 9.6, 1.8 Hz, 1H, Ar), 6.47
(d, J = 1.8 Hz, 1H, Ar), 6.07 (dddd, J = 17.4, 10.5, 5.4, 5.4 Hz, 1H,
CH2dCHCH2OAr), 5.60 (dddd, J = 17.4, 10.2, 6.0, 6.0 Hz, 1H,
ArCO2CH2CHdCH2), 5.46 (dd, J = 17.4, 1.2 Hz, 1H, HtransCHd
CHCH2OAr), 5.37 (dd, J = 10.5, 1.2 Hz, 1H, HCHcisdCHCH2OAr),
5.14-5.08 (m, 2H, ArCO2CH2CHdCH2), 4.66 (ddd, J = 5.4, 1.2, 1.2
Hz, 2H, CH2dCHCH2OAr), 4.48 (dddd, J = 12.9, 6.0, 1.2, 1.2 Hz, 1H,
ArCO2CHaHCHdCH2), 4.46 (dddd, J = 12.9, 6.0, 1.2, 1.2 Hz, 1H,
ArCO2CHHbCHdCH2) ppm; 13C NMR (75 MHz, CDCl3, 293 K,
Figure S11) δ 185.8, 165.2, 163.1, 159.1, 154.3, 150.2, 134.5, 132.9,
132.0, 131.4, 131.1, 130.67, 130.65, 130.4, 130.1, 129.9, 129.1, 119.3,
118.9, 117.9, 115.1, 113.9, 105.9, 101.3, 69.6, 66.2 ppm; HRMS (ESIþ)
m/z calcd for C26H21O5 [M]þ 413.1389, found 413.1373.
was heated in a 50 °C oil bath for 1 h. The reaction mixture was then
poured onto ice-cold H2O (10 mL), and the resulting mixture was
extracted with EtOAc (2 ꢀ 10 mL). The combined organic layers were
washed with brine (30 mL), dried over Na2SO4, filtered, and evaporated
under reduced pressure. The residue was purified by flash column
chromatography (10 f 30% EtOAc in hexanes) on silica gel (10 mL) to
afford compound 12 (70 mg, 97%) as an orange solid. Data for 12: mp
96-98 °C; Rf = 0.30 (40% EtOAc in hexanes); IR (KBr pellet) νmax
3304 (broad, O-H), 3045, 2921, 2858, 1610 (CdO), 1497, 1459, 1426,
1
1270, 1177, 1109, 1023, 846, 804, 725 cm-1; H NMR (400 MHz,
acetone-d6, 293 K, Figure S14) δ 7.47 (d, J = 7.6 Hz, 1H, Ar), 7.39 (dd, J
= 7.6, 7.6 Hz, 1H, Ar), 7.28 (dd, J = 7.6, 7.6 Hz, 1H, Ar), 6.96 (d, J = 8.8
Hz, 1H, Ar), 6.84 (d, J = 8.8 Hz, 1H, Ar), 6.83 (d, 6.82 J = 7.6 Hz, 1H,
Ar), (d, J = 2.4 Hz, 1H, Ar), 6.75 (dd, J = 8.8 2.4 Hz, 1H, Ar), 6.67 (d, J =
2.4 Hz, 1H, Ar), 6.60-6.55 (m, 2H, Ar and CH3CHdCHOAr), 5.30 (s,
2H, ArCH2OH), 4.96 (dq, J = 6.8, 6.8 Hz, 1H, CH3CHdCHOAr), 1.68
(dd, J = 6.8, 1.6 Hz, 3H, CH3CHdCHOAr) ppm; 13C NMR (100 MHz,
acetone-d6, 293 K, Figure S15) δ 159.2, 158.7, 152.12, 152.07, 146.7,
141.4, 140.0, 131.2, 131.0, 129.1, 128.9, 124.3, 121.9, 120.7, 117.6, 112.9,
112.7, 108.7, 103.5, 102.8, 84.0, 72.9, 9.6 ppm; HRMS (ESIþ) m/z calcd
for C23H18O4Na [M þ Na]þ 381.1103, found 381.1083.
Conversion of Compound 12 to Compound 13. HgCl2 (18
mg, 67 μmol) was added to a solution of compound 12 (24 mg, 67
μmol) in a 1/9 DMSO/pH 4 buffer (26 mL) mixture. The resulting
mixture was stirred at 25 °C for 1 h and extracted with EtOAc (2 ꢀ 20
mL). The combined organic layers were washed with brine (2 ꢀ 50 mL),
dried over Na2SO4, filtered, and evaporated under reduced pressure.
The residue was purified by flash column chromatography (10 f 70%
EtOAc in hexanes) on silica gel (10 mL) to afford compound 13 (15 mg,
72%) as an orange solid. Data for 13: mp >200 °C; Rf = 0.32 (50%
EtOAc in hexanes); IR (KBr pellet) νmax 3430 (broad, O-H), 2869,
2600, 1602 (CdO), 1455, 1379, 1309, 1241, 1207, 1117, 860, 757
cm-1; 1H NMR (300 MHz, acetone-d6, 293 K, Figure S16) δ 7.45 (d, J =
7.5 Hz, 1H, Ar), 7.34 (dd, J = 7.5, 7.5 Hz, 1H, Ar), 7.27 (dd, J = 7.5, 7.5
Hz, 1H, Ar), 6.83 (d, J = 7.5 Hz, 1H, Ar) 6.81 (d, J = 8.4 Hz, 2H, Ar), 6.64
(d, J = 2.4 Hz, 2H, Ar), 6.56 (dd, J = 8.4, 2.4 Hz, 2H, Ar), 5.26 (s, 2H,
ArCH2OH) ppm; 13C NMR (75 MHz, acetone-d6, 293 K, Figure S17) δ
159.0, 152.3, 146.9, 140.2, 131.0, 129.1, 128.8, 124.4, 121.8, 117.9, 112.6,
102.8, 84.2, 72.6 ppm; HRMS (EIþ) m/z calcd for C20H15O4 [M]þ
319.0970, found 319.0999.
Sample Preparations. Mercury standard solution (20 ppm in 5%
HNO3) was purchased from the RICCA Chemical Co. (Arlington, TX)
and used as received. The other mercury standard solution (10 000 ppm
in 2% HNO3) was purchased from Ultra Scientific (North Kingstown,
Rhode Island) and used as received. OmniTrace Ultra High Purity Acid
HNO3 (Hg < 10 ppt) was purchased from EMD (item number NX0408,
lot number 48157) and used as received. ARISTAR ULTRA water was
purchased from VWR (catalog number 7732-18-5) and used as received.
AgNO3 was purchased from EMD and used as received. River water was
collected from the Allegheny River on Jan 29, 2008. The pH 3 buffer
solution was made from a commercial pH 4 buffer (50 mM) and a pure
HNO3 solution.
Preparation of Compound 11. A solution of DIBALH (9.6 mL,
1.0 M in CH2Cl2) was added dropwise to a solution of compound 10
(0.83 g, 2.0 mmol) in CH2Cl2 (7.0 mL) over 15 min at -78 °C under a
nitrogen atmosphere. The resulting solution was stirred at the same
temperature for 10 min and then warmed to 25 °C. After stirring at the
same temperature for 2 h, Et2O (5.0 mL) was added to the resulting
solution at 0 °C with stirring, and then saturated aqueous NH4Cl
(3.5 mL) was added dropwise to the mixture at the same temperature.
This mixture was warmed to 25 °C again and stirred for 1 h. The reaction
mixture was then diluted with Et2O (5.0 mL), and DDQ (0.45 g,
2.2 mmol) was added slowly to this mixture at 0 °C. After being stirred
for 1 h at 25 °C, the mixture was filtered through a pad of Celite, and the
pad was rinsed with EtOAc. The filtrate was dried over Na2SO4, filtered,
and evaporated under reduced pressure. The residue was purified by
flash column chromatography (10 f 30% EtOAc in hexanes) on silica
gel (180 mL) to afford compound 11 (701 mg, 98%) as a pale yellow
solid. Data for 11: mp 144-146 °C; Rf = 0.32 (30% EtOAc in hexanes);
IR (KBr pellet) νmax 3228 (broad, O-H), 2921, 2852, 1616 (CdO),
1
1501, 1517, 1456, 1430, 1335, 1224, 1180, 1003, 841, 759 cm-1; H
NMR (300 MHz, acetone-d6, 293 K, Figure S12) δ 7.44 (d, J = 7.5 Hz,
1H, Ar), 7.37 (dd, J = 7.5, 7.5 Hz, 1H, Ar), 7.26 (dd, J = 7.5, 7.5 Hz, 1H,
Ar), 6.89 (d, J = 8.7 Hz, 1H, Ar), 6.84 (d, J = 7.5 Hz, 1H, Ar), 6.83 (d, J =
8.7 Hz, 1H, Ar), 6.76 (d, J = 2.4 Hz, 1H, Ar), 6.67 (d, J = 2.4 Hz, 1H, Ar),
6.66 (dd, J = 8.7, 2.4 Hz, 1H, Ar), 6.58 (dd, J = 8.7, 2.4 Hz, 1H, Ar), 6.07
(dddd,, J = 17.4, 10.5, 5.4, 5.4 Hz, 1H, CH2dCHCH2OAr), 5.42 (dd, J =
17.4, 1.2 Hz, 1H, HtransCHdCHCH2OAr), 5.28 (s, 2H, ArCH2OH),
5.24 (dd, J = 10.5, 1.2 Hz, 1H, HCHcisdCHCH2OAr), 4.61 (ddd, J = 5.4,
1.2, 1.2 Hz, 2H, CH2dCHCH2OAr) ppm; 13C NMR (75 MHz,
acetone-d6, 293 K, Figure S13) δ 160.2, 159.0, 152.2, 152.1, 146.7,
140.1, 134.5, 131.0, 130.9, 129.1, 128.8, 124.3, 121.9, 118.9, 117.74,
117.67, 112.7, 112.3, 107.8, 101.9, 84.1, 72.7, 69.5 ppm; HRMS (ESIþ)
m/z calcd for C23H18O4Na [M þ Na]þ 381.1103, found 381.1072.
Preparation of Compound 12. KOtBu (56 mg, 0.44 mmol) was
added to a solution of compound 11 (72 mg, 0.20 mmol) in DMSO (1.7
mL) at 25 °C under a nitrogen atmosphere, and the resulting solution
Metal Solutions (1.0 mM). AuCl3, BaCl2, NiCl2, CrCl3, Pb-
(NO3)2, NaCl, MnCl2 4H2O, MgCl2, CoCl2, HgCl2, AgNO3, ZnCl2,
3
LiCl, CuCl2 2H2O, and CaCl2 were dissolved in H2O. FeCl3, CdCl2
3
3
2.5H2O, KCl, Rh(PPh3)3, and RuCl3 were dissolved in MeOH. PtCl2
was dissolved in MeOH/acetone (1/1). Pd standard solution (High-
Purity Standards, Cat. No. 100038-1) was diluted with 1% HNO3. The
resulting solution was used as the Pd solution.
Fluorescence Measurement. All samples were incubated at 25
°C, and the pH values of the solutions were adjusted to an appropriate
pH range (pH >5 for 1, pH >8 for 13) by the addition of 1.23 M phos-
phate pH 7 buffer (for 1, 4.0% of the total volume of a reaction solution)
or a 1:5 mixture of 1.23 M phosphate pH 7 buffer and 500 mM borate
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pH 10 buffer (for 13, 24% of the total volume of a reaction solution).
The resulting samples were vortexed for 5 s prior to fluorescence mea-
surement. Fluorescence spectra were recorded in a 1 ꢀ 1 cm disposable
cuvette (VWR; catalog number 58017-880) on a Jobin Yvon Fluoro-
Max-3 spectrometer under the control of a Windows-based PC running
FluorEssence software. The samples were excited at 497 nm, and the
emission intensities were collected at 523 nm (for 1) or 515 nm (for 13).
All spectra were corrected for emission intensity using the manufacturer-
supplied photomultiplier curves.
pH Dependence of Hg Detection (Figures 1a and 5a).
Reaction conditions: [Hg(II)] = 0.30 μM, [3 or 12] = 1.0 μM, 0.05%
DMSO in buffer (5.0 mL), 25 °C, 1 h. Protocol: a 100 μM Hg standard
solution (15 μL each) was added to a pH 3, 4, 5, 6, or 7 buffer (4.98 mL
each). A 1.0 mM solution of 3 or 12 in 1/1 DMSO/50 mM phosphate
pH 8 buffer (5.0 μL) was added to each of these solutions, and the
resulting samples were shaken for 3 s and incubated at 25 °C for 1 h
before fluorescence measurement. See Fluorescence Measurement for
sample neutralization. Note: for the pH 3 sample, an additional 1.0 mL
of 500 mM borate pH 10 buffer was required to obtain a pH >5 solution.
Metal Selectivity (Figures 1b and 5b). Reaction conditions:
[metal] = 5.0 μM, [3 or 12] = 1.0 μM, 0.05% DMSO in a 50 mM
phthalate pH 4 buffer (5.0 mL), 25 °C, 1 h. Protocol: each of the 1.0 mM
solutions of metal reagents in appropriate solvents (25 μL, see the
Supporting Information for details) was added to a 50 mM phthalate pH
4 buffer (4.97 mL). A 1.0 mM solution of 3 or 12 in 1/1 DMSO/50 mM
phosphate pH 8 buffer (5.0 μL) was added to these solutions, and the
resulting samples were shaken for 3 s and incubated at 25 °C for 1 h
before fluorescence measurement. See Fluorescence Measurement for
sample neutralization.
Time Course of the Oxymercuration Reaction (Figures 1c
and 6). Reaction conditions: [Hg(II)] = 0.30 μM, [3, 12, or 14] = 1.0
μM, 0.05% DMSO in a 50 mM phthalate pH 4 buffer or a 50 mM pH 7
phosphate buffer (20 mL), 25 °C. Protocol: a 100 μM Hg standard
solution (60 μL) was added to a 50 mM phthalate pH 4 buffer
(Figures 1c and 6a,b) or a 50 mM phosphate pH 7 buffer (Figure 6c)
(20 mL). A 1.0 mM solution of 3, 12, or 14 in 1/1 DMSO/50 mM
phosphate pH 8 buffer (20 μL) was added to each of the solutions, and
the resulting reaction solutions were shaken for 3 s and incubated at
25 °C before fluorescence measurement. A fraction (1.5 mL) of each of
these reaction solutions was taken for the measurement at 10, 20, 30, 40,
50, 60, 90, 120, 240, 480, and 1140 min. For Figure 6b, this measurement
was carried out every 1 min for 18 min with 1.0 mL of the reaction
solution. See Fluorescence Measurement for sample neutralization.
Titration Curve (Figures 1d and 5d). Reaction conditions:
[Hg(II)] = 0, 1, 2, 4, 8, 16, and 32 ppb, [NCS] = 10 μM for NCS(þ)
samples and 0 μM for NCS(-) samples, [3 or 12] = 1.0 μM, 0.05%
DMSO in a 50 mM phthalate pH 4 buffer (5.0 mL), 25 °C, 1 h. Protocol:
a 0, 200, 400, 800, 1600, 3200, or 6400 ppb solution of HgCl2 in 5%
HNO3 (25 μL) and a 2.0 mM solution of NCS in water (25 μL)
were added to each of 50 mM phthalate pH 4 buffers (4.95 mL). A 1.0
mM solution of 3 or 12 in 1/1 DMSO/50 mM phosphate pH 8
buffer (5.0 μL) was added to each of these solutions, and the resulting
solutions were shaken for 3 s and incubated at 25 °C for 1 h before
fluorescence measurement. See Fluorescence Measurement for sample
neutralization.
incubated at 25 °C for 1 h before fluorescence measurement. See Fluore
scence Measurement for sample neutralization.
Effect of Chloride or Nitrate Ion (Figures 2b and S6
(Supporting Information)). Reaction conditions: [Hg(II)] = 2.5
μM, [Cl- or NO3-] = 1.0 mM, [3 or 12] = 1.0 μM, 0.05% DMSO in
a 50 mM phthalate pH 4 buffer (5.0 mL), 25 °C, 1 h. Protocol: a 1.0 mM
HgCl2 solution in 5% HNO3 (12.5 μL) and a 100 mM NaCl or NaNO3
solution in water (50 μL) were added to a 50 mM phthalate pH 4 buffer
(4.93 mL). Each of these solutions was treated with a 1.0 mM solution of
3 or 12 in 1/1 DMSO/50 mM phosphate pH 8 buffer (5.0 μL). The
resulting samples were shaken for 3 s and incubated at 25 °C for 15 min
(Figure 2b) or 1 h (Figure 5c) before fluorescence measurement. See
Fluorescence Measurement for sample neutralization.
Interference of Inorganic Materials with AgNO3 (Figures 2a
(Gray Bar) and 5c). Reaction conditions for Figure 2a:[Hg(II)] = 2.5 μM,
[other metal] = 25 μM, [AgNO3] = 100 μM, [3] = 1.0 μM, 0.05% DMSO
in a 50 mM phthalate pH 4 buffer (5.0 mL), 25 °C, 1 h. Protocol: a 1.0 mM
HgCl2 solution in 5% HNO3 (12.5 μL) and a 1.0 mM solution of another
metal (125 μL) were added to a 50 mM phthalate pH 4 buffer (4.86 mL).
These solutions were vortexed for 5 s, and then a 10 mM solution of AgNO3
in ultrapure water (50 μL) was added to the resulting solutions. After
vortexing for 5 s, a 1.0 mM solution of 3 in 1/1 DMSO/50 mM phosphate
pH 8 buffer (5.0 μL) was added to these solutions, and the resulting samples
were shaken for 3 s and incubated at 25 °C for 1 h before fluorescence
measurement. See Fluorescence Measurement for sample neutralization.
Reaction conditions for Figure 5c: [Hg(II)] = 1.0 μM, [other metal] =
10 μM, [AgNO3] = 2.0 mM, [12] = 1.0 μM, 0.05% DMSO in a 50 mM
phthalate pH 4 buffer (5.0 mL), 25 °C, 1 h. Protocol: a 1.0 mM HgCl2
solution in 5% HNO3 (5.0 μL) and a 1.0 mM solution of another metal
(50 μL) were added to a 50 mM phthalate pH 4 buffer (4.84 mL). These
solutions were vortexed for 5 s, and then a 100 mM solution of AgNO3 in
ultrapure water (100 μL) was added to the resulting solutions. After
vortexing for 5 s, a 1.0 mM solution of 12 in 1/1 DMSO/50 mM
phosphate pH 8 buffer (5.0 μL) was added to these solutions, and the
resulting samples were shaken for 3 s and incubated at 25 °C for 1 h
before fluorescence measurement. See Fluorescence Measurement for
sample neutralization.
Detection of Hg in River Water by Probe 3 (Figure 3a,
b). Reaction conditions for Figure 3a: [Hg(II)] = 440 ppb (550 ppb =
2.75 μM in river before dilution), [AgNO3] = 2.0 mM, [3] = 1.0 μM,
0.05% DMSO in a 50 mM phthalate pH 4 buffer (5.09 mL), 25 °C, 1 h.
Protocol: river water (3.98 mL) was spiked with a 440 μM solution of
HgCl2 in 5% HNO3 (25 μL). This solution was treated with a 250 mM
phthalate pH 4 buffer (1.0 mL) and then a 100 mM solution of AgNO3
in ultrapure water (100 μL). After vortexing for 5 s, a 1.0 mM solution of
3 in 1/1 DMSO/50 mM phosphate pH 8 buffer (5.0 μL) was added to
this solution, and the resulting sample was shaken for 3 s and incubated
at 25 °C for 1 h before fluorescence measurement. See Fluorescence
Measurement for sample neutralization.
Reaction conditions for Figure 3b: [Hg(II)] = 0, 1, 2, 4, 8, 16, 32, 64,
128, and 256 ppb, [AgNO3] = 2.0 mM, [3] = 1.0 μM, 0.05% DMSO in a
50 mM phthalate pH 4 buffer (5.1 mL), 25 °C, 1 h. Protocol: a 0, 0.2, 0.4,
0.8, 1.6, 3.2, 6.4, 12.8, 25.6, or 51.2 ppm solution of HgCl2 in 5% HNO3
(25 μL) was spiked into river water samples (4.0 mL each). Each of these
solutions was treated with a 250 mM phthalate pH 4 buffer (1.0 mL
each) and then a 100 mM solution of AgNO3 in ultrapure water (100 μL
each). After being shaken for 3 s, a 1.0 mM solution of 3 in 1/1 DMSO/
50 mM phosphate pH 8 buffer (5.0 μL) was added to these solutions,
and the resulting samples were shaken for 3 s and incubated at 25 °C for
1 h before fluorescence measurement. See Fluorescence Measurement
for sample neutralization.
Hg Detection in the Presence of Other Metal (Figure 2a
(White Bar)). Reaction conditions: [Hg(II)] = 2.5 μM, [other metal] =
25 μM, [3] = 1.0 μM, 0.05% DMSO in a 50 mM phthalate pH 4 buffer
(5.0 mL), 25 °C, 1 h. Protocol: a 100 μM Hg standard solution in 5%
HNO3 (125 μL) and each of the 1.0 mM metal solutions (125 μL; see
the Supporting Information for details) were added to each of 50 mM
phthalate pH 4 buffer (4.75 mL). Each of these solutions was treated
with a 1.0 mM solution of 3 in 1/1 DMSO/50 mM phosphate pH 8
buffer (5.0 μL). The resulting solutions were shaken for 3 s and
Hg Detection in the Presence of Organic Compound
(Figure 3c). Reaction conditions: ([Hg(II)] = 16 ppb, [organic con-
taminant] = 80 ppb, [AgNO3] = 2 mM, [3] = 1 μM, 1.3% DMSO in a 50
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mM phthalate pH 4 buffer (1.30 mL), 25 °C, 1 h. Protocol: river water
(1.0 mL) was spiked with a 10 μM HgCl2 solution in 5% HNO3 (10 μL)
and a 10 ppm solution of an organic contaminant in DMSO (phenol,
acetophenone, caffeine, or cholesterol, 10 μL). Each of these solutions
contained 0.1 μM (20 ppb) Hg(II) and 100 ppb of the compound. Each
of the resulting solutions was treated with a 250 mM phthalate pH 4
buffer (250 μL each) and then a 100 mM solution of AgNO3 in ultrapure
water (25 μL). After vortexing for 5 s, a 0.1 mM solution of 3 in 1/1
DMSO/50 mM phosphate pH 8 buffer (12.5 μL) was added to this
solution, and the resulting sample was shaken for 3 s and incubated at 25
°C for 1 h before fluorescence measurement. See Fluorescence Mea-
surement for sample neutralization.
and the resulting samples were shaken for 3 s and incubated at 25 °C
for 1 h before fluorescence measurement. See Fluorescence Mea-
surement for sample neutralization.
’ ASSOCIATED CONTENT
S
Supporting Information. Text, tables, and figures giving
b
experimental procedures for all fluorescence analyses and chemo-
dosimeter preparation. This material is available free of charge via
the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Detection of Hg from Dental Amalgam (Figure 3d). Reaction
conditions: two teeth filled with mercury amalgam, [cysteine] = 50 μM,
[NCS] = 500 μM, [3] = 1.0 μM, 0.05% DMSO in a 50 mM phthalate pH 4
buffer (4.06 mL), 25 °C, 1 h. Protocol: two teeth filled with an amalgam
were added to a 20 mM solution of cysteine in water (2.0 mL), and the
resulting mixture was shaken (200 rpm) for 1 h at 37 °C.26 A portion (5.0
μL) of the resulting solution was added to a 50 mM phthalate pH 4 buffer
(2.0 mL). After being shaken for 3 s, a 20 mM solution of NCS in water (50
μL) and a 1.0 mM solution of 3 in 1/1 DMSO/50 mM phosphate pH 8
buffer (2.0 μL) were added to the resulting solution. A negative control
sample was prepared by adding two teeth filled with an amalgam into water
(2.0 mL) and shaken (200 rpm) for 1 h at 37 °C. A portion (5.0 μL) of the
resulting mixture was added to a 50 mM phthalate pH 4 buffer (2.0 mL).
This mixture was then treated with a 20 mM solution of NCS in water (50
μL) and a 1.0 mM solution of 3 in 1/1 DMSO/50 mM phosphate pH 8
buffer (2.0 μL). The resulting samples were shaken for 3 s and incubated at
25 °C for 1 h before fluorescence measurement. See Fluorescence
Measurement for sample neutralization.
pH Titration of Compound 13 (Figure 4a). An aqueous
solution (100 mL) containing 13 (5 μM) and NaCl (1.0 M) was
prepared. To half of this mixture solution (50 mL) was added a small
magnetic stir bar in a vial with a pH electrode. The pH of the solution
was changed by adding 0.1 or 1 N HCl solution dropwise with stirring,
and the fluorescence spectrum was recorded at ∼0.3 pH intervals. The
other half of this mixture (50 mL) was titrated with 0.1 or 1 N NaOH
solution. In order to calculate the pKa, the pH dependence of fluores-
cence spectra were analyzed using the following equation: pH = pKa -
log[(Fmax - F)/(F - Fmin)].48
Corresponding Author
*koide@pitt.edu
’ ACKNOWLEDGMENT
We thank Nalco Co. for the wastewater samples. This work
was supported by the U.S. National Science Foundation (Nos.
CHE-0616577 and CHE-0911092) and the National Institutes
of Health (No. R01CA120792).
’ REFERENCES
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(2) (a) Amin-Zaki, L.; Elhassani, S.; Majeed, M. A.; Clarkson, T. W.;
Doherty, R. A.; Greenwood, M. Pediatrics 1974, 54, 587–595. (b) Bakir,
F.; Damluji, S. F.; Amin-Zaki, L.; Murtadha, M.; Khalidi, A.; Al-Rawi,
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