Aminoisoquinolines as colorimetric Hg2+ sensors: the importance of molecular structure and sacrificial base

Article abstract of DOI:10.1016/j.tet.2009.03.063

Here it is shown that 3-phenyl-2-amino isoquinoline acts as a simple mercury sensor. It is simple to synthesize. The molecule requires base/buffer to bind in a 1:1 stoichiometry with mercury ion, however. Otherwise, it acts as a sacrificial base, presumab

Full text of DOI:10.1016/j.tet.2009.03.063

Tetrahedron 65 (2009) 4293–4297
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Tetrahedron
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Aminoisoquinolines as colorimetric Hg2þ sensors: the importance
of molecular structure and sacrificial base
y
*
Yanjun Wan, Weijun Niu, William J. Behof, Yifei Wang , Paul Boyle, Christopher B. Gorman
Department of Chemistry, North Carolina State University, Box 8204, Raleigh, NC 27695-8204, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Here it is shown that 3-phenyl-2-amino isoquinoline acts as a simple mercury sensor. It is simple to
synthesize. The molecule requires base/buffer to bind in a 1:1 stoichiometry with mercury ion, however.
Otherwise, it acts as a sacrificial base, presumably to pick up a proton liberated during binding. This
binding is characterized quantitatively.
Received 6 January 2009
Received in revised form 9 March 2009
Accepted 13 March 2009
Available online 27 March 2009
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
categories usually have the advantage of lower detection limit,
their applications are highly constrained by the susceptibility to
There is an increasing public concern over the severe risk of
heavy metal pollution and poisoning in environment, food, and
products.1 Mercury ion is of particular interest because of its
high toxicity and wide variety of damage to kidneys and di-
gestive and neurological systems.2 Thus, a simple yet highly se-
lective routine of detecting mercury ions in both organic and
aqueous media has become an urgent call and has drawn a lot of
research attention.3 Most mercury sensors that have been re-
cently developed fall into one of the four categories: nano-particle
based sensors, DNA or other bio-molecule based sensors, polymer
based sensors, or organic dye based sensors. While the first three
a series of factors, including but not limited to, temperature, pH,
presence of other salts, etc. On the other hand, organic dye based
sensors are usually synthesized organic molecules with high
fluorescence and/or remarkable optical changes upon binding to
target metal ions. These dyes ideally have the advantages of low
cost, easy synthesis and storage, and more tolerance toward different
experimental conditions. However, organic dye based mercury
sensors often suffer from one or several disadvantagesdparticularly
the requirement for a multi-step synthesis with a low overall
yield, a low binding constant and/or poor sensitivity and/or
selectivity.
N
NH2
N
NH2
Br
CN
CN
CN CN
Cl
Cl
iv
i
iii
+
1
2
3a (97%)
3b (72%)
NH2
3c (79%)
Br
N
N
NH2
N
N
CN
CN CN
CN CN CN
iv
ii
iii
+
1
4
5a (73%)
5b (59%)
5c (76%)
Scheme 1. Synthesis of the compounds. (i) KOtBu, DMF, rt, 1 h; (ii) KOtBu, DMF, 70 ꢀC, 18 h; (iii) HBr/HOAc; (iv) NaBH4, Pd/C.
We wondered what the simplest chromophore and binding
* Corresponding author. Tel.: þ1 919 515 4252; fax: þ1 919 515 8920.
E-mail address: chris_gorman@ncsu.edu (C.B. Gorman).
Summer Undergraduate Research Student from Zhejiang University, Hangzhou,
group for mercuric ion could be. Furthermore, we asked, which
synthetic approaches could most efficiently incorporate simple but
effective chemical functionalities into these molecules. To these
y
China.
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.03.063

4294
Y. Wan et al. / Tetrahedron 65 (2009) 4293–4297
Figure 1. ORTEP diagrams of 3b, 3c, 5b, and 5c.
ends, we show here that a simple reaction sequence can produce
a viable, small molecule mercury sensor. The way the molecule
binds to mercury via deprotonation is novel and potentially useful
in further sensor design paradigms.
Upon addition of mercuric ions, both the fluorescence and ab-
sorbance of the compounds changed. This change was most notable
in molecule 3c, so all further studies are reported on this molecule.
Qualitatively (Fig. 2), 3c exhibited very high selectivity toward
mercury ion, with an obvious color change from colorless to dark
yellow, as well as significant quenching of fluorescence. No other
cation examined exhibited the same color change or fluorescence
quenching. Also, the more conjugated 5c provided no advantage
(e.g., larger change in absorbance or fluorescence upon addition of
mercury ion) suggesting that extended conjugation is not required
for these type of molecules in this application. Finally, commer-
cially available 2-amino isoquinoline mixed with mercuric ion as
above displayed no color changes. The pendant phenyl group is
thus necessary for the colorimetric response.
Quantitatively, a ca. 40 nm red shift in the UV absorption peak
was observed when 3c was added to mercuric ion. A poor fit was
found, however, when absorbance changes were graphed as
a function of mercuric ion concentration using a Benesi–Hildebrand
plot.10 A Job plot indicated an unexpected 2:1 binding stoichio-
metry between 3c and mercuric ion in acetonitrile.11 This initially
puzzling observation had a simple explanation, however. Ammonia
is known to bind to mercuric ion in a fashion that consumes 2 equiv
of ammonia per mercuric ion to form a 1:1 complex. The second
2. Results and discussion
The synthesis of the molecules studied here involves three,
straightforward steps from commercial starting materials
(Scheme 1). It has been shown that isoquinoline derivatives can be
synthesized via acid-promoted cyclization,4–9 and this methodology
could be adapted to the synthesis of 3b.
Interestingly, this methodology could be extended to a double
cyclization to form 5b in reasonable yield. Note that the benzylic CH
group spontaneously tautomerized to NH to result in an entirely
aromatic system. To our knowledge, this is the first example of
cyclization/aromatization of this kind. This type of cascade cycli-
zation has potential for the preparation of larger, fused aromatic
molecules, and this chemistry is under further exploration for that
purpose. To determine the effect of the bromine atom on the
photophysical properties of these molecules, the dehalogenated
molecules 3c and 5c were prepared via simple reduction.
Crystals of 3b, 3c, 5b, and 5c, suitable for X-ray diffraction were
obtained (Fig. 1). These data confirmed the closure of multiple ar-
omatic rings. The isoquinoline region of 3b and 3c was planar.
Molecules 5b and 5c were notably nonplanar.
Table 1
Photophysical data (solutions in CH3CN)
The absorbance, fluorescence, and quantum yields of the four
molecules were measured. Each has a long wavelength absorbance
with a reasonable extinction coefficient (>1000 Mꢁ1 cmꢁ1) that is
potentially suitable as a colorimetric sensor. Also, all compounds
fluoresce with a quantum yield that is much higher in the absence
of the heavy bromine atom (Table 1).
Compound
Absorbance lmax (nm)
3max (Mꢁ1 cmꢁ1))
Fluorescence
lmax (nm) (Q)
(
3b
3c
5b
5c
380 (3269)
370 (3497)
350 (3827)
346 (3208)
447 (0.02)
436 (0.87)
450 (0.03)
432 (0.15)

Y. Wan et al. / Tetrahedron 65 (2009) 4293–4297
4295
Figure 2. Color change and fluorescence quenching (under UV light) when 3c binds to Hg2þ ion, compared to binding to other cations. (Top) Photograph of acetonitrile solutions of
2 mM 3c plus 2 mM of different metal cations under day light. (Bottom) Photograph of 0.1 mM 3c plus 0.1 mM of different metal cations in acetonitrile under a UV lamp.
equivalent of ammonia merely serves as a base, and the resulting
complex is a mercuric–amido complex (Scheme 2).
When the same Job plot experiment was rerun in a 99:1 mixture
of acetonitrile and phosphate buffer at pH 7.0, a 1:1 binding stoi-
chiometry was determined. Presumably in this case, the phosphate
acted as the proton acceptor and an amido complex was formed.
Ancker et al. reported an X-ray structure of a neutral, 1:1 complex
between HgCl and 2-(trimethylsilyl amino)-6-methyl pyridine,
supporting this idea.12
A similar red shift in the absorbance and fluorescence quenching
upon addition of Hg2þ to 3c in 99:1 acetonitrile/buffer was observed
(Fig. 3). From a Benesi–Hildebrand plot, a Ka of 5.0ꢂ104 Mꢁ1 was
measured for the absorbance change upon binding and from
a Stern–Volmer plot, a Ka of 1.8ꢂ104 Mꢁ1 was measured. Thus,
a reasonable agreement was found between these two values. These
values are also reasonably large, making 3c a good candidate as
a small molecule mercuric ion sensor.
Molecule 3c showed a good, naked eye detection limit of ca.
0.1 ppm (0.5 mM) in both organic (CH3CN) and semi-aqueous
(DMSO/H2O 4:1) media (S/N¼3:1). At this concentration, only a 3:1
ratio of signal to noise was measured. Moreover, it showed a high
2+
Hg
N
NH2
N
NH2
Hg2+
3c
+ 3c
1+
Hg
+
N
NH3
N
NH
(3c•Hg)+
+
(3c•Hg)+
2 eq. Hg2+ req'd.
Only 1 eq.
Hg2+ req'd.
Figure 3. (Top) Overlay plot of UV–vis absorption spectra in 99:1 acetonitrile/buffer as
Hg2þ was added to 3c. Increases and decreases in relative absorbance are shown with
vertical arrows. (Bottom) Invariant concentration Job plot of molecule 3c and Hg2þ
.
Scheme 2. Rationalization of the 2:1 and 1:1 binding stoichiometries.

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Y. Wan et al. / Tetrahedron 65 (2009) 4293–4297
selectivity for binding with Hg2þ. As can be observed qualitatively
in Figure 2, little change in color or fluorescence was observed
when 3c was mixed with other cations. The second strongest
complex measured was between 3c and Zn2þ with a Ka value of
MeOH), light yellow crystals of 2-(cyano(phenyl)methyl)benzoni-
trile (3a), were obtained in 97% yield (780 mg). Alternatively,
compound 3a could be obtained from recrystallization in hot
ethanol (6.6 g, 71% yield). 1H NMR (CDCl3): 5.50 (s, 1H), 7.26–7.67
(m, 9H). 13C NMR (CDCl3): 40.1, 112.1, 117.1, 118.5, 127.8, 129.0, 129.2,
129.6, 133.7, 134.0, 134.3, 139.7. Mp: 84–87 ꢀC. Elemental Analysis
calcd: C, 82.55; H, 4.62; N, 12.84. Found: C, 82.41; H, 4.64; N, 12.85.
6.6ꢂ102 Mꢁ1, which is >20 times smaller than that with Hg2þ
.
Addition of all other cations gave an undetectable absorbance
change in the UV–vis spectrum of 3c. Furthermore, in a mixed ion
sensing experiment, where 3c and Hg2þ were mixed with the same
concentration of Liþ, Naþ, Kþ, Agþ, Cd2þ, Pb2þ, Co2þ, Ca2þ, Sr2þ
,
4.3. Synthesis of 1-bromo-4-phenylisoquinolin-3-amine (3b)
Ba2þ, La3þ, and Tl3þ, the changes in absorbance were the same as in
the absence of these added cations.
A solution of 7 mL of 45% HBr in acetic acid was added to 626 mg
(2.87 mmol) of 3a in a flask. The solution turned yellow upon the
addition of HBr, and was stirred at room temperature for another
2 h. A yellow solid was precipitated out by the addition of 10 mL
diethyl ether, and this solid was collected via filtration and washed
with more ether. This solid was dissolved in 40 mL ethyl acetate,
neutralized by shaking with aqueous saturated sodium hydrogen
carbonate and dried over anhydrous sodium sulfate. After removal
of solvent and column purification with DCM/MeOH, yellow crys-
tals of 1-bromo-4-phenylisoquinolin-3-amine (3b) were obtained
in 72% yield (620 mg). Alternatively, compound 3b could be
obtained from recrystallization in hot ethanol (4.0 g, 60% yield). 1H
NMR (CDCl3): 4.40 (s, 2H), 7.24–7.59 (m, 9H), 8.13 (d, J¼8.7 Hz, 1H).
13C NMR (CDCl3): 123.8, 124.1, 128.4, 128.9, 129.7, 130.7, 131.1, 135.0,
139.2, 151.0, 154.5. Mp 164–168 ꢀC. Elemental Analysis Calcd: C,
60.22; H, 3.71; N, 9.36. Found: C, 60.24; H, 3.65; N, 9.39.
3. Conclusions
In conclusion, molecule 3c is a simple, conveniently prepared,
colorimetric mercury ion sensor. Comparison to 3b, 5b, and 5c
indicates that, from a structural perspective, it possesses the
minimum number of features required. Indeed, when commer-
cially available 2-amino isoquinoline was mixed with mercuric ion
as above, no color changes were observed. This observation sug-
gests that changes in relative twist of the pendant, rotatable arene
ring of 3c are likely important to the color and fluorescence
changes upon binding. Thus, this molecule represents a reasonable
optimization of synthetic steps and structural features for a simple
mercuric ion sensor.
4. Experimental
4.4. Synthesis of 4-phenylisoquinolin-3-amine (3c)
4.1. General methods
To a mixture of 2.0 mL of 4 N potassium hydroxide and 2.0 mL
methanol in Schlenk flask, 107 mg (0.36 mmol) of 3b was added
slowly under nitrogen protection. To this solution, a mixture of
60 mg (0.028 mmol) 5% Pd on activated carbon and 60 mg
(1.59 mmol) sodium borohydride was added, and the suspension
was stirred at room temperature overnight. The solution was fil-
tered and the solids were washed with tetrahydrofuran (THF). The
resulting solution was dried over anhydrous sodium sulfate. After
removal of solvent and column purification with DCM/MeOH,
All chemicals were purchased from Aldrich or Acros. Acetoni-
trile was dried by distillation over CaH2.
1H NMR spectra were referenced to residual 1H shift in CDCl3
(7.24 ppm). CDCl3 (77.0 ppm) was used as the internal reference for
13C NMR. The following abbreviations were used to explain the
multiplicities: s¼singlet, d¼doublet, t¼triplet, q¼quartet, br s¼
broad singlet.
Reactions were monitored by thin-layer chromatography (TLC)
on commercial silica precoated plates with a particle size of 60 Å.
Developed plates were viewed by UV lamp (254 nm). Flash chro-
matography was performed using 230–400 mesh silica gel. Mi-
crowave heating was performed by a microwave reactor of model
The Discover instrument by CEM Corp., Matthews, N.C., operating
at a power of 300 W. Temperature was measured via an internal IR-
sensor. Specified reaction times refer to total heating time. Ramp
times are specified for each individual solvent and temperature. All
reactions were performed in sealed vessels.
a
light yellow solid, 4-phenylisoquinolin-3-amine (3c), was
obtained in 79% yield (741 mg). 1H NMR (CDCl3): 4.36 (s, 2H), 7.23–
7.59 (m, 8H), 7.83 (d, J¼8.4 Hz, 1H), 8.8418.91 (s, 1H). 13C NMR
(CDCl3): 111.7, 112.9, 123.2, 124.1, 128.1, 128.2, 129.6, 130.6, 130.8,
136.0, 137.5, 151.5, 152.0. Mp 144–147 ꢀC. Elemental Analysis Calcd:
C, 81.79; H, 5.49; N, 12.72. Found: C, 81.21; H, 5.57; N, 12.48. HRMS:
Calcd: 220.100. Found: 220.100.
4.5. Synthesis of molecule 5a
Crystallographic data (excluding structure factors) for the
structures in this paper have been deposited with the Cambridge
Crystallographic Data Centre as supplementary publication nos.
CCDC 708184–708187. Copies of the data can be obtained, free of
charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ,
UK, (fax: þ44 (0)1223 336033 or e-mail: deposit@ccdc.cam.ac.uk).
To a mixture of 2-chloro-benzonitrile (768 mg, 5.58 mmol), 2-
cyanophenyl acetonitrile (1.21 g, 8.38 mmol), and potassium tert-
butoxide (1.94 g, 17.3 mmol) in a Schlenk flask, 12 mL anhydrous
THF was added through a syringe. The solution was then refluxed
under dinitrogen at 70 ꢀC with stirring for 18 h. A solution of
aqueous saturated ammonium chloride (30 mL) was added to
quench excess potassium tert-butoxide. The solution was extracted
with DCM and dried over anhydrous sodium sulfate. After removal
of solvent and column purification with DCM/MeOH, a red solid
(5a), was obtained in 73% yield (0.99 g). 1H NMR (CDCl3): 5.89 (s,
1H), 7.52–7.78 (m, 8H). 13C NMR (CDCl3): 39.8, 112.9, 116.4, 129.8,
129.9, 133.9, 134.4, 136.7. Mp 171–176 ꢀC. Elemental Analysis Calcd:
C, 79.00; H, 3.73; N, 17.27. Found: C, 78.73; H, 3.67; N, 17.25.
4.2. Synthesis of 2-(cyano(phenyl)methyl)benzonitrile (3a)
To a solution of potassium tert-butoxide (823 mg, 7.35 mmol) in
10 mL dimethyl formamide (DMF) immersed in an ice bath, a mix-
ture of 2-chloro-benzonitrile (1) (656 mg, 4.77 mmol) and 2-phenyl-
acetonitrile (2) (430 mg, 3.67 mmol) in 6 ml DMF was added
dropwise. The mixture was stirred at room temperature for 1 h,
after which 20 mL saturated aqueous ammonium chloride was
added to quench excess potassium tert-butoxide. The solution was
extracted with diethyl ether, washed with deionized water, and
then dried over anhydrous sodium sulfate. After removal of solvent
and column purification with dichloromethane/methanol (DCM/
4.6. Synthesis of molecule 5b
A mixture of 10 mL 45% HBr in acetic acid and 10 mL DCM was
added to 435 mg (1.79 mmol) of 5a in a flask, and stirred at room

Y. Wan et al. / Tetrahedron 65 (2009) 4293–4297
4297
temperature overnight. Then 100 mL of diethyl ether was added,
and a yellow solid precipitated out and was filtered. This solid was
redissolved in 50 mL THF, neutralized by addition of aqueous, sat-
urated sodium hydrogen carbonate, and dried over anhydrous so-
dium sulfate. After removal of solvent and column purification with
DCM/MeOH, a light yellow solid (5b), was obtained in 59% yield
(341 mg). 1H NMR (DMSO): 5.76 (s, 2H), 7.63–7.99 (m, 4H), 8.36 (d,
J¼8.1 Hz, 1H), 8.48 (d, J¼9.3 Hz, 1H), 8.86 (q, J¼8.7 Hz, 2H). 13C NMR
(DMSO): 106.8, 119.1, 124.8, 125.0, 125.3, 126.3, 126.9, 127.3, 129.2,
131.4, 132.2, 133.1, 134.3, 144.7, 150.6, 157.7. Mp decomp. Elemental
Analysis Calcd: C, 59.28; H, 3.11; N, 12.96. Found: C, 59.44; H, 3.06;
N, 12.81.
[Hg2þ] were plotted against their corresponding mercury con-
centrations, in which F0 is the initial fluorescence without mer-
cury ion, F are fluorescence intensities at corresponding mercury
concentrations. The value of Ka was calculated as absolute value of
the Y-intercept.14
The binding ratios were calculated using Job plots. Stock solu-
tions of 0.25 mM Hg2þ and 0.25 mM 3c were prepared in 99:1
acetonitrile/buffer. The first series of samples was prepared con-
taining 2 mL 3c stock solution, and different volumes of mercury
stock solution (0.1 mL increments). The second series of samples
was prepared containing 2 mL mercury stock solution, and differ-
ent volumes of 3c stock solution (added in 0.1 mL increments).10
The corresponding absorbance was measured and the values
A were plotted against [3c]/([3c]þ[Hg2þ]), where A is the corre-
sponding absorbance of different fraction of molecule 3c in the
mixed solutions. The peak of this plot shows the binding ratio, at
which neither molecule 3c nor mercury ion is in excess.
4.7. Synthesis of molecule 5c
To a mixture of 2.0 mL of 4 N potassium hydroxide and 2.0 mL
methanol in Schlenk flask, 76 mg (0.23 mmol) of 5b in 1 mL of THF
was added slowly under nitrogen protection. To this solution,
a mixture of 48 mg (0.044 mmol) 5% Pd on activated carbon and
90 mg (2.38 mmol) sodium borohydride was added, and the sus-
pension was stirred at room temperature for 3 days. The solution
was filtered and the solids were washed with tetrahydrofuran
(THF). The resulting solution was dried over anhydrous sodium
sulfate. After removal of solvent and column purification with DCM/
MeOH, a light yellow solid (5c), was obtained in 76% yield (439 mg).
1H NMR (CDCl3): 4.11 (s, 2H), 7.46 (t, J¼6.9 Hz, 1H), 7.56 (t, J¼7.5 Hz,
1H), 7.72 (q, J¼7.5 Hz, 2H), 7.96 (d, J¼7.8 Hz, 1H), 8.06 (d, J¼7.8 Hz,
1H), 8.70 (d, J¼8.7 Hz, 1H), 8.81 (d, J¼8.4 Hz, 1H), 9.10 (s, 1H). 13C
NMR (CDCl3): 108.5, 119.3, 124.1, 124.8, 125.2, 126.3, 127.0, 127.1,
129.1, 130.9, 131.3, 133.4, 134.3, 151.0, 153.8, 157.1. Mp decomp.
HRMS: Calcd: 245.095. Found: 245.095.
Acknowledgements
We would like to thank Dr. Lin He for suggestions on fluores-
cence measurements. This work was supported by the Department
of Energy (Grant DE-FG02-05ER46238). Mass spectra were
obtained at the NCSU Department of Chemistry Mass Spectrometry
Facility. Funding was obtained from the North Carolina Bio-
technology Center, and the NCSU Department of Chemistry.
Supplementary data
Additional photophysical data. Supplementary data associated
with this article can be found in the online version, at doi:10.1016/
j.tet.2009.03.063.
4.8. Photophysical measurements
Quantum yields of 3b, 3c, 5b, and 5c were measured by using 2-
aminopyridine as standard.13
References and notes
Binding constants were measured from absorbance changes
using the Benesi–Hildebrand method. Stock solutions of 2 mM
Hg2þ and 0.1 mM 3c were prepared in 99:1 acetonitrile/buffer. A
series of samples were prepared containing 1 mL mercury stock
solution, different volumes of 3c stock solution (added in 0.1 mL
increments) and enough additional solvent to make a total volume
of 2 mL. The corresponding absorbance was measured and the
values 1/A were plotted against 1/[3c], in which A is the absorbance
at corresponding concentration of molecule 3c. Ka was calculated as
the ratio of the Y-intercept/slope for a linear fit of these data.10
Binding constants were also measured from fluorescence
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changes via a Stern–Volmer plot. Stock solutions of 50
and 50 M 3c were prepared in 99:1 acetonitrile/buffer. A series of
m
M Hg2þ
m
samples were prepared containing 1 mL 3c stock solution, differ-
ent volumes of 3c stock solution (added in 0.1 mL increments) and
enough additional solvent to make a total volume of 2 mL. The
corresponding fluorescence emission was measured from 375 to
600 nm (excited at 370 nm), and different values of (F0/Fꢁ1)/