A click fluorophore sensor that can distinguish CuII and Hg II via selective anion-induced demetallation

Article abstract of DOI:10.1002/chem.201002477

A cyclam-based fluorescent sensor featuring a novel triazole pendant arm has been synthesised using click chemistry. The sensor is highly responsive to both Cu^II and Hg^II in neutral aqueous solution and displays excellent selectivity

Full text of DOI:10.1002/chem.201002477

DOI: 10.1002/chem.201002477
A Click Fluorophore Sensor that Can Distinguish CuII and HgII via Selective
Anion-Induced Demetallation
Yu Heng Lau, Jason R. Price, Matthew H. Todd,* and Peter J. Rutledge*[a]
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FULL PAPER
Abstract: A cyclam-based fluorescent
sensor featuring a novel triazole pend-
ant arm has been synthesised using
click chemistry. The sensor is highly re-
sponsive to both CuII and HgII in neu-
tral aqueous solution and displays ex-
cellent selectivity in the presence of
various competing metal ions in 50-fold
excess. The addition of specific anions
and effective method for distinguishing
solutions containing CuII, HgII or a
mixture of both ions, even in doped
seawater samples. X-ray crystal struc-
tures of both the HgII sensor complex
and a model CuII complex show that
pendant triazole coordination occurs
through the central nitrogen atom
(N2), providing to the best of our
knowledge the first reported examples
of this unusual coordination mode in
macrocycles. Fluorescence, mass spec-
trometry and 1H NMR experiments
reveal that the mechanism of anion-in-
duced fluorescence revival involves
either displacement of pendant coordi-
nation or complete removal of the HgII
from the macrocycle, depending on the
anion.
Keywords: anions · azamacrocycles ·
chemosensors · click chemistry ·
fluorescence
2À
such as IÀ and S2O3 causes a com-
plete revival of fluorescence only in
the case of HgII, providing a simple
Introduction
Chemical sensing is an active area of research driven by the
vast potential for industrial, environmental and biological
application.[1] A range of chemosensors have been reported
in the literature for the detection of many different chemical
species including metal ions, anions and organic molecules.[2]
Despite their apparent structural diversity, the majority of
these sensors are comprised of two main components: a
domain which binds the target molecule, and a reporter
which signals the binding event through an observable
change.[3] Given this modular design, click chemistry pro-
vides a convenient synthetic method for uniting the two
components.[4] The copper(I)-catalysed azide–alkyne cyclo-
addition reaction is particularly suited to the synthesis of
metal ion sensors as the resultant 1,2,3-triazole acts as both
a covalent linker and a potential coordination site for metal
ions.[5] The vast majority of chelating triazoles coordinate
through the side nitrogen atom (N3),[5a] with DFT calcula-
tions showing that N3 has a greater electron density than
the central nitrogen atom (N2).[6] In the Cambridge Struc-
tural Database, there are very few reports of click-generated
triazoles which exhibit exclusive N2 coordination.[7]
Previous work involving our group has used N-monofunc-
tionalised cyclam ligands with 1,2,3-triazole pendant arms as
sensors (see below). Compound 1 is a selective ZnII sensor,
signalling metal coordination with a six-fold increase in fluo-
rescence emission of the pendant naphthalimide.[8] The de-
velopment of heterogeneous sol–gel sensors based on 1 has
also been explored.[9] An alternative approach has involved
compound 2, with the pendant biotin molecule acting as a
binding site for avidin, and the loss of triazole coordination
at the metal in the presence of avidin signalled by changes
in the EPR spectrum.[10]
We now outline the synthesis of a cyclam-based fluores-
cent sensor 3 featuring a novel triazole pendant, linked to
the macrocycle via the N1 position rather than C4. The ra-
tionale behind the design of this ligand was to alter the ring
size of the coordinating pendant arm relative to 1 as well as
changing the fluorophore, thereby modifying the selectivity
and fluorescent response to metal ion binding. The result is
a system that coordinates via N2 and responds selectively to
CuII and HgII (in stark contrast to the zinc sensor 1). Detec-
tion of CuII is important as it is both an essential biological
trace element and an environmental pollutant,[11] whilst HgII
is well known for its high toxicity even at low concentra-
tions.[12]
Whilst numerous cyclam-based fluorescent sensors have
been developed in recent years, many have only been stud-
ied in organic solvents or organic/aqueous mixtures, limiting
their practical application.[13] Another common challenge
when designing sensors is poor selectivity for specific metal
ions, including the inability to distinguish between CuII and
HgII due to their similar fluorescence quenching proper-
ties.[13d,e,14] Our new sensor is able to operate in neutral
aqueous solution and in the presence of excess competing
[a] Y. H. Lau, Dr. J. R. Price, Dr. M. H. Todd, Dr. P. J. Rutledge
School of Chemistry, The University of Sydney
NSW 2006 (Australia)
Fax : (+61)2-9351-3329
E-mail: matthew.todd@sydney.edu.au
peter.rutledge@sydney.edu.au
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201002477.
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M. H. Todd, P. J. Rutledge et al.
metal ions. The sensor can be used to distinguish CuII and
HgII effectively, even when both are present as a mixture,
through the addition of specific anions. This sensor-and-
anion system represents a new method for detecting these
metals simultaneously and independently.
Results and Discussion
Synthesis: Model compound 7 was prepared to test the fea-
sibility of the synthetic route and investigate the coordina-
tion properties of the novel triazole pendant (Scheme 1). In-
itial attempts to alkylate tri-Boc-protected cyclam 4 by re-
fluxing with stoichiometric toluenesulfonic acid-2-azidoethyl
ester proved low yielding (29%), with the majority of start-
ing materials being reclaimed, consistent with previous ob-
servations that 4 is a poor nucleophile for unstabilised pri-
mary electrophiles.[15] Using the tosylate in excess (ca.
16 equiv) afforded the alkylated cyclam 5 in an excellent
89% yield, with the unreacted azide easily reclaimed by
chromatography. Reaction of 5 with phenylacetylene under
standard click conditions yielded the triazole 6 in good
yield, and this was deprotected using trifluoroacetic acid to
give the model compound 7 in near quantitative yield.
Figure 1. ORTEP representation of the single-crystal X-ray structure of 8
with 50% thermal ellipsoids.[16] The non-coordinated perchlorate anion
has been deleted for clarity. The pendant triazole is coordinating through
the central nitrogen atom (N2) to form a six-membered metallocycle.
The cyclam ring is in the expected trans-III configuration.
metal centre via N2 rather than N3, forming a six-mem-
bered metallocycle (Figure 1).
Sensor 3 was synthesised via an analogous route in excel-
lent yield (Scheme 2). The CuII and HgII complexes 10a and
10b were then prepared by adding the corresponding metal
perchlorate salt.
X-ray diffraction quality crystals of 10b were grown by
slow diffusion of ether into a nitromethane solution of 10b,
with the resulting structure indicating coordination via N2
also occurs in the case of HgII (Figure 2).
Scheme 1. i) Toluenesulfonic acid-2-azidoethyl ester (ca. 16 equiv),
Na2CO3, MeCN, reflux, 72 h, 89%; ii) phenylacetylene, CuSO4·5H2O
(5 mol%), Na ascorbate (10 mol%), tBuOH/H2O 1:1, 16 h, 76%; iii)
TFA (20%) in CH2Cl2, RT, 6 h, 99%; iv) Cu
1 h, 74%.
ACHTUGNTRENUN(GN ClO4)2·6H2O, EtOH, reflux,
To investigate the coordina-
tion properties of 7, the CuII
complex
8 was prepared by
treatment with copper(II) per-
chlorate, and X-ray diffraction
quality crystals were grown by
slow diffusion of diethyl ether
into an acetonitrile solution of
8. As expected, the pendant tri-
azole adopts a different coordi-
nation mode to previously re-
ported azamacrocyclic pendant
triazoles.[8–10] In complex 8, the
Scheme 2. i) 7-Ethynyl-4-methyl-2H-chromen-2-one, CuSO4·5H2O (5 mol%), Na ascorbate (10 mol%),
tBuOH/H2O 1:1, 16 h, 86%; ii) TFA (20%) in CH2Cl2, RT, 6 h, 97%; iii) a) Cu(ClO4)2·6H2O, EtOH, reflux,
(ClO4)2·3H2O, MeOH, RT, 16 h, 58%.
AHCTUNGTRENNUNG
triazole is coordinated to the 1 h, 72%; b) Hg
ACHTUNGTRENNUNG
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A Click Fluorophore Sensor
FULL PAPER
ly, sensor 3 is only able to operate by a fluoresence quench-
ing mechanism because it appears to be less prone to intra-
molecular PET than sensor 1, although further investiga-
tions are currently being carried out to determine whether
the modified pendant connectivity or the fluorophore itself
is responsible for the different sensing and fluorescence
properties.
Fluorescence titrations of sensor 3 with CuII and HgII con-
firm the expected 1:1 binding stoichiometry, with association
constants of 2.2
G
E
for CuII and HgII, respectively, using a non-linear curve fit-
ting analysis (see Supporting Information).[18] The detection
limits were calculated to be 3.5ꢁ10À7 and 4.3ꢁ10À7 m, re-
spectively.[19] Screening the response of 3 to CuII and HgII at
different pH indicates optimum performance at neutral pH
(Figure 4). Presumably, the coordination of metal ions at
low pH is less favoured due to protonation of the amine
groups in the cyclam ring. At high pH, the lone pairs of the
non-protonated amine groups act to quench the fluores-
cence of the coumarin fluorophore by PET,[20] thus attenuat-
ing the full quenching response normally seen upon addition
of CuII or HgII.
Figure 2. ORTEP representation of the single-crystal X-ray structure of
10b with 50% thermal ellipsoids.[16] The anions and nitronate solvent
molecules have been deleted for clarity. The pendant is triazole coordi-
nating through the central nitrogen atom (N2). The cyclam ring is in the
trans-I configuration.
Metal-ion sensing: To assess the metal-ion sensing ability of
compound 3 in aqueous solution, a wide range of metal ions
were screened, of which only CuII and HgII caused a signifi-
cant change in fluorescence emission (Figure 3). In contrast
to the previously reported ZnII sensor 1 which exhibits an
increase in fluorescence due to inhibition of photoinduced
electron transfer (PET), CuII and HgII trigger a quenching
response with sensor 3 which can be attributed to well-
known paramagnetic and heavy metal effects.[17] Interesting-
^
Figure 4. Fluorescence emission of ligand 3 ( ) at different pH, and ca.
5 min after adding 1 equiv of CuII or HgII. Maximal response to CuII ( )
&
and HgII
(
~
) occurs around neutral pH.
To investigate the effectiveness of sensor 3 in the presence
of competing metal ions, 50 equivalents of various metal
ions were added to a 10 mm solution of 3, followed after
about 5 min by the addition of one equivalent of CuII or
HgII (Figure 5). In almost all cases, the quenching response
was completely unaffected by the competing metal ions, a
welcome result given the ability of cyclam to bind a range of
transition-metal ions.[21] In competition experiments between
CuII and HgII, neither ion was able to displace the other
from the ligand, which can be attributed to their similar
binding constants and the kinetic macrocyclic effect.[21] No-
tably, a 50-fold excess of ZnII caused some quenching of
fluorescence which was not affected by the subsequent addi-
tion of CuII or HgII, suggesting that a reasonably stable com-
plex forms with ZnII. However, this quenching response was
significantly slower (equilibration over ca. 1 h) than the re-
sponse to CuII and HgII. In a modified competition experi-
ment, premixed solutions containing ZnII (1 and 50 equiv)
Figure 3. Fluorescence emission spectra of 3 (10 mm) in HEPES buffer
(10 mm, pH 7.4) ca. 5 min after the addition of various metal ions
(1 equiv). Only CuII and HgII trigger a significant response.
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M. H. Todd, P. J. Rutledge et al.
pendant group or complete re-
moval of HgII from the macro-
cycle (Scheme 3).[22] It was rea-
soned that CuII should bind to
the cyclam macrocycle if com-
plete removal of HgII was oc-
curring. The addition of one
equivalent of CuII after anion
addition triggered the full
quenching response in all cases
except ClÀ (Figure 8). This
result suggests demercuration
of the complex, although this
does not rule out the possibility
of lowered binding affinity for
HgII due to displacement of
Figure 5. Investigating the selectivity of ligand 3 in the presence of competitive metal ions. Metal ions
(50 equiv) were added to 3 (10 mm) in HEPES buffer (10 mm, pH 7.4), followed after ca. 5 min by addition of
either CuII or HgII (1 equiv). In all cases except ZnII, the excess competing ions do not significantly affect the
response to CuII or HgII.
and either CuII or HgII (1 equiv) were added to a solution of
ligand 3 (Figure 6). The response to HgII was not significant-
ly affected by the presence of even 50 equivalents of ZnII,
whilst the CuII response tolerated one equivalent of ZnII,
but was affected slightly by a 50-fold excess.
Figure 7. Fluorescence emission spectra upon addition of anions
(150 equiv) after adding HgII (1 equiv) to 3 in HEPES buffer (10 mm,
pH 7.4). The original fluorescence spectra of ligand 3 and HgII complex
2À
10b are also shown. ClÀ, BrÀ, IÀ, SCNÀ and S2O3 cause some revival of
fluorescence emission.
Figure 6. Modified competition experiments to further investigate the in-
terference by ZnII. Pre-mixed solutions containing ZnII (1 or 50 equiv)
with either CuII or HgII (1 equiv) were added to a solution of 3 (10 mm) in
HEPES buffer (10 mm, pH 7.4) and the fluorescence was measured after
ca. 5 min. The slow response of 3 to ZnII allows CuII and HgII to be de-
tected in the presence of ZnII.
Effect of coordinating anions: The tolerance of this system
to a range of competitive anions was also screened by
adding a large excess (150 equiv) of various sodium salts to
the CuII and HgII complexes of 3 (Figure 7). Whilst none of
the anions tested had any effect on the fluorescence emis-
sion of the CuII complex (see Supporting Information), addi-
2À
tion of ClÀ, BrÀ, IÀ, SCNÀ and S2O3 caused some revival of
fluorescence emission for the HgII complex. In particular,
the quenching response of HgII was nullified by less than
2À
five equivalents of IÀ and S2O3
.
Fluorescence, mass spectrometry and NMR experiments
were carried out to determine whether the anion-induced
fluorescence revival was due to anion displacement of the
Scheme 3. Two possible mechanisms for anion-induced fluorescence re-
vival: a) displacement of pendant coordination or b) complete removal
of HgII from the cyclam macrocycle.
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A Click Fluorophore Sensor
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Figure 8. Fluorescence revival upon addition of anions (5 equiv of IÀ and
S2O32À, 150 equiv of BrÀ, ClÀ, SCNÀ) to the HgII complex 10b in HEPES
buffer (10 mm, pH 7.4), then subsequent addition of CuII (1 equiv). In all
cases except ClÀ, CuII is able to cause significant fluorescence quenching,
indicating that HgII is completely removed from the macrocycle.
pendant coordination, with HgII retained inside the macro-
cycle until CuII is introduced. The observed formation of
precipitates with BrÀ, IÀ and SCNÀ in more concentrated
solutions is also consistent with macrocycle demercuration,
as the corresponding inorganic HgII salts are highly insoluble
in aqueous solution.[23] No precipitate was observed for
S2O32À, as HgII thiosulfate complexes are water-soluble.[24]
Mass spectrometry (ESI+) experiments on solutions con-
taining HgII complex 10b and each of the anions were con-
sistent with the fluorescence results (see Supporting Infor-
mation). The mass spectra of complex 10b mixed with BrÀ,
Figure 9. 1H NMR spectra in D2O of a) ligand 3; b) complex 10b; c)
complex 10b and ClÀ; d) complex 10b and S2O32À. Cyclam ring CH2
peaks are underlined. Pendant triazole/dye peaks are labelled with aster-
isks. Comparing b) and c), the upfield shift in pendant peaks indicates
displacement of triazole coordination. Comparing b) and d), the upfield
shift in the cyclam peaks indicates removal of HgII from the macrocycle.
can be utilised as a simple method of distinguishing between
solutions containing each metal ion and a mixture of both
ions (Figure 10). Fluorescence quenching indicates the pres-
ence of either CuII or HgII; no fluorescence change upon
subsequent addition of thiosulfate is indicative of CuII,
whilst a large increase is indicative of HgII. If both CuII and
HgII are present in solution, the initial fluorescence quench-
ing response lies between those of CuII and HgII individually,
and the addition of thiosulfate causes a drop in fluorescence
due to substitution of CuII for HgII in the macrocycle.
À
IÀ, SCNÀ and S2O3 do not show a peak for the intact HgII
complex, and instead have one dominant peak that corre-
sponds to the free ligand 3 (m/z 454 [M+H]+). In contrast,
the mass spectrum of complex 10b without additional
anions shows only a peak for the intact complex (m/z 754,
À
[M2+ +ClO4 ]+), and no free ligand signal. The mass spec-
trum for the sample containing 10b and ClÀ shows both free
ligand 3 and complex 10b, indicating that ClÀ does not com-
pletely remove HgII from the macrocycle.
To test the efficacy of the sensor in a real world context,
the quenching experiments were then repeated using CuII
1H NMR spectra of the anion-treated mixtures were also
obtained. Comparing the spectra of free ligand 3 and HgII
complex 10b, a downfield shift is apparent in all the peaks
due to the deshielding effect of HgII (Figure 9). This shift is
most pronounced in the peaks corresponding to the hydro-
gens of the cyclam ring and the triazole, due to their prox-
imity to the metal centre. When ClÀ is added, minimal
changes are observed for the cyclam peaks, whilst the dye
and triazole peaks shift upfield, consistent with retention of
the metal in the macrocycle and movement of the pendant
2À
away from the metal centre. In contrast, when S2O3 is
added, all the peaks shift upfield and the spectrum reverts
back to one similar to the uncomplexed ligand. Hence, it
can be concluded that ClÀ displaces pendant ligand coordi-
2À
nation, whereas BrÀ, IÀ, SCNÀ and S2O3 strip HgII from
the cyclam ring.
Figure 10. Fluorescence emission of 3 in HEPES buffer (10 mm, pH 7.4)
upon addition of CuII, HgII or a mixture of both (1 equiv of each metal
2À
ion), followed by S2O3 (5 equiv). No change is observed with CuII only,
whilst an increase in fluorescence is seen for HgII, and a decrease in fluo-
rescence for the mixture of CuII and HgII. The effect of varying the stoi-
chiometry of the mixture is detailed in the Supporting Information.
Distinguishing between CuII and HgII: The different respons-
es of the CuII and HgII complexes 10a and 10b to anions
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M. H. Todd, P. J. Rutledge et al.
and HgII doped seawater samples from Darling Harbour in
Sydney, Australia. Despite the wide range of cations and
anions present in seawater, the sensor was still effective for
detecting CuII and HgII, exhibiting similar fluorescence
changes to the experiments conducted in distilled water (see
Supporting Information). The only significant difference was
a reduced amount of quenching in response to HgII, attribut-
ed to pendant displacement by the high concentration of
ClÀ in seawater.
to maintain a constant ligand concentration, and fitting the titration
curves to a 1:1 binding model using a non-linear curve fitting analysis.[18]
Details of the X-ray crystallographic studies are given in the Supporting
Information. CCDC 789681 (8), 789855 (10b) contain the supplementary
crystallographic data for this paper. These data can be obtained free of
charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif
1H NMR peaks are assigned according to the scheme defined in Support-
ing Information (Section 1).
For metal complexes which exhibit a complex mixture of isotopes, the
mass of the most abundant species for each ion fragment has been re-
ported; M2+ is used to denote the divalent metal–ligand cation.
Synthetic procedures
Conclusion
SAFETY NOTE: Perchlorate salts of metal complexes with organic li-
gands are potentially explosive. Only small amounts of material should
be prepared and these should be handled with caution.
Click chemistry has been used to synthesise a fluorescent
sensor which responds to both CuII and HgII. This sensor has
desirable properties for practical applications, including se-
lectivity for detecting CuII and HgII in the presence of excess
competing metal ions, as well as optimum functionality in
neutral aqueous solution and even seawater. Furthermore,
we have studied the responses of the CuII and HgII com-
plexes to the addition of competing anions, and utilised
their different responses to distinguish CuII from HgII. The
HgII complex and CuII model complex are the first two re-
ported examples of N2 triazole coordination in macrocycles.
The contrasting selectivity of 3 for CuII and HgII com-
pared to the previously reported ZnII sensor 1 presumably
hinges on the different length of the pendant arm (there are
three bridging atoms between the tertiary amine and tria-
zole ligands in 3 vs two atoms in 1) and the different elec-
tronics of triazole coordination through N2 and N3. Further
work is being carried out to determine whether the differ-
ence in pendant dye, triazole coordination mode or other
factors are responsible for the different fluorescence charac-
teristics (quenching vs PET turn-on) of sensors 3 and 1. This
work will enhance our understanding of the photophysical
processes behind the fluorescence responses, and allow the
design of new azamacrocyclic sensors with novel fluorescent
properties.
11-(2-Azidoethyl)-1,4,8,11-tetraazacyclotetradecane-1,4,8-tricarboxylic
acid tri-tert-butyl ester (5): Toluenesulfonic acid-2-azidoethyl ester
(1.26 g, 5.22 mmol) and Na2CO3 (2.02 g, 19.1 mmol) were added to a so-
lution of 1,4,8,11-tetraazacyclotetradecane-1,4,8-tricarboxylic acid tri-tert-
butyl ester 4 (413 mg, 0.825 mmol) in acetonitrile (25 mL). After reflux-
ing for 72 h, the reaction mixture was filtered and concentrated in vacuo.
Purification by column chromatography (2% to 5% MeOH in CH2Cl2)
gave azide 5 as a pale yellow oil (413 mg, 89%). 1H NMR (200 MHz,
CDCl3): d = 3.47–3.18 (m, 14H, CH2BocNCH2, Hf), 2.69–2.55 (m, 4H,
Hc, He), 2.45 (t, 2H, J = 5.7 Hz, Hb), 1.86 (qn, 2H, J = 7.4 Hz, Hd), 1.68
(qn, 2H,
J = CACHTNGUTRENNUGN
7.4 Hz, Ha), 1.44 ppm (s, 27H, (CH3)3); 13C NMR
(50 MHz, CDCl3): d = 155.2, 155.0, 79.0, 54.0, 53.7, 51.9, 48.7, 48.2–46.1
(several peaks), 45.4, 28.1, 26.4 ppm; IR (ATR, CDCl3): n=2973, 2097,
1689, 1463, 1412, 1391, 1247, 1163 cmÀ1; HRMS (ESI): m/z: calcd for
C27H51N7O6Na: 592.3793, found 592.3785 [M+Na]+.
11-(2-(4-Phenyl-1H-1,2,3-triazol-1-yl)ethyl)-1,4,8,11-tetraazacyclotetrade-
cane-1,4,8-tricarboxylic acid tri-tert-butyl ester (6): Compound 5 (376 mg,
0.660 mmol) and phenylacetylene (74 mL, 0.68 mmol) were dissolved in
tBuOH/H2O 1:1 (10 mL), and CuSO4·5H2O (5 mol%, 8 mg dissolved in
1 mL H2O) and sodium ascorbate (10 mol%, 13 mg dissolved in 1 mL
H2O) were added. After stirring at RT for 16 h, 5% NaHCO3 solution
(5 mL) was added and the reaction mixture was extracted with CH2Cl2
(4ꢁ40 mL). The organic extracts were dried over MgSO4, concentrated
in vacuo and purified by flash chromatography (ethyl acetate/hexane 1:1)
to give triazole
6 as a white foam (335 mg, 76%). M.p. 71–778C;
1H NMR (200 MHz, CDCl3): d = 7.89–7.79 (m, 2H, ArH), 7.81 (s, 1H,
Ht), 7.48–7.30 (m, 3H, ArH), 4.41 (t, 2H, J = 6.5 Hz, Hf), 3.38–3.08 (m,
12H, CH2BocNCH2), 2.98 (t, 2H, J = 6.4 Hz, He), 2.67 (t, 2H, J =
5.1 Hz, Hc), 2.51 (t, 2H, J = 5.6 Hz, Hb), 1.83–1.60 (m, 4H, Ha, Hd), 1.46
(s, 18H,
(CH3)3); 13C NMR (50 MHz,
CACHTNGUTREUNN(G CH3)3), 1.44 ppm (s, 9H, CCAHTUNGTRENNNUG
CDCl3): d = 155.3, 155.2, 155.1, 147.1, 130.4, 128.5, 127.8, 125.2, 120.2,
79.3, 79.2, 54.7, 53.5, 52.0, 48.3–46.0 (several peaks), 45.5, 28.5, 28.2,
26.4 ppm; IR (ATR, CDCl3): n =2976, 2935, 1689, 1466, 1413, 1391,
1246, 1163 cmÀ1; HRMS (ESI): m/z: calcd for C35H58N7O6: 672.4443,
found 672.4435 [M+H]+.
Experimental Section
General information
1-(2-(4-Phenyl-1H-1,2,3-triazol-1-yl)ethyl)-1,4,8,11-tetraazacyclotetrade-
cane (7): Ester 6 (335 mg, 0.499 mmol) was dissolved in CH2Cl2 (12 mL)
and trifluoroacetic acid (3 mL) was added dropwise. After stirring at RT
for 6 h, the reaction mixture was concentrated under reduced pressure.
The oily residue was dissolved in H2O (5 mL) and 10% aqueous Na2CO3
solution was added until effervescence ceased. The solution was extracted
with chloroform (5ꢁ40 mL), the extracts dried over Na2SO4 and concen-
trated in vacuo to give the free amine 7 as a white solid (335 mg, 99%).
M.p. 86–898C; 1H NMR (200 MHz, CDCl3): d = 8.10 (s, 1H, Ht), 7.85
(dd, 2H, J = 8.2, 1.4 Hz, ArH), 7.48–7.29 (m, 3H, ArH), 4.56 (t, 2H, J
All reagents were purchased from Sigma–Aldrich and used without fur-
ther purification. Toluenesulfonic acid-2-azidoethyl ester,[25] 7-ethynyl-4-
methyl-2H-chromen-2-one[26] and tri-Boc-protected cyclam 4[27] were pre-
pared according to literature procedures.
Fluorescence emission spectra were recorded at 258C on a Varian Cary
Eclipse Fluorimeter with excitation at 327 nm. Unless otherwise speci-
fied, emission was recorded at 389 nm. Quantum yields were calculated
using l-tryptophan as a standard reference (F=0.13 in H2O, pH 6).[28]
Fluorescence experiments were carried out by adding a small (2–10 mL)
amount of the relevant ion (2–100 mm) in HEPES buffer (10 mm, pH 7.4)
to a stock solution of ligand 3 (2 mL, 10 mm). For pH studies, a stock solu-
tion of ligand 3 was made in HEPES buffer (10 mm) and the pH adjusted
with HClO4 or NaOH prior to addition of metal ions. Binding constants
were obtained by titrating metal ion solutions (0.5–100 mm) containing
ligand 3 (10 mm) into a stock solution of ligand 3 (2 mL, 10 mm) in order
=
6.2 Hz, Hf), 2.93 (t, 2H, 6.2 Hz, He), 2.78–2.66 (m, 6H,
J =
CH2NHCH2), 2.58–2.36 (m, 8H, CH2NHCH2, Hc), 2.31 (t, 2H, J
=
5.5 Hz, Hb), 1.92 (brs, 3H, NH), 1.76–1.61 ppm (m, 4H, Ha, Hd);
13C NMR (50 MHz, CDCl3): d = 146.9, 130.6, 128.7, 127.9, 125.4, 121.5,
54.0, 52.9, 51.0, 50.5, 48.3, 47.9, 47.4, 47.1, 47.0, 46.5, 28.0, 25.9 ppm; IR
2856
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Chem. Eur. J. 2011, 17, 2850 – 2858

A Click Fluorophore Sensor
FULL PAPER
(ATR, CDCl3): n=3500–2700 (br), 2816, 1687, 1465, 1131 cmÀ1; HRMS
(ESI): m/z: calcd for C20H34N7: 372.2870, found 372.2870 [M+H]+.
He), 2.97 (t, 2H, J = 6.3 Hz, He), 3.56–2.58 (m, 16H, CH2NHCH2, Hb,
Hc), 2.47 (d, 3H, J = 1.1 Hz, CH3), 1.94–1.51 ppm (m, 4H, Ha, Hd);
13C NMR (75 MHz, CD3CN): d = 161.1, 154.9, 153.8, 148.2, 133.3, 127.2,
126.7, 122.5, 121.5, 116.0, 114.5, 59.4, 55.8, 55.6, 54.8, 54.7, 52.8, 49.6, 48.5,
47.2, 46.5, 29.0, 26.4, 18.6 ppm; IR (ATR, acetone): n=3260, 2891, 1705,
1621, 1453, 1366, 1088, 942, 623 cmÀ1; LRMS (ESI): m/z: 753.9 (100)
[M2+ +ClO4À]+, 346.9 (72) [M2+ +HCl]2+, 326.5 (67) [M2+]; elemental
analysis calcd (%) for C24H35Cl2N7O10Hg: C 33.79, H 4.14, N 11.49;
found: C 33.64, H 3.96, N 11.21; quantum yield: F=0.031 (H2O, pH 7).
[Cu(7)]
ACHTUNGTRENNUNG
EtOH (3 mL), a solution of CuAHCTNUGTRENNNUG
EtOH (1 mL) was added dropwise. After refluxing for 1 h, the reaction
mixture was cooled on ice and the solvent was decanted. The solid resi-
due was triturated with ice-cold EtOH (3ꢁ2 mL) and diethyl ether (3ꢁ
3 mL), then dried under a flow of nitrogen to give the complex 8 as a
purple powder (54.2 mg, 74%). Purple block-shaped crystals suitable for
X-ray diffraction were obtained by slow diffusion of diethyl ether into an
acetonitrile solution of the complex. M.p. 184–1878C; IR (ATR, ace-
tone): n=3239, 2880, 1703, 1458, 1363, 1089, 772, 697, 623 cmÀ1; LRMS
(ESI): m/z (%): 547.3 (100) [M2+ +TFAÀ]+, 533.3 (80) [M2+ +ClO4À]+,
433.1 (88) [M2+ÀH+]+; elemental analysis calcd (%) for
C20H33Cl2N7O8Cu: C 37.89, H 5.25, N 15.47; found: C 37.54, H 5.30, N
15.29.
Acknowledgements
We thank the University of Sydney for financial support and the award
of a postgraduate scholarship to Y.H.L. and Mingfeng Yu for cyclam.
11-(2-(4-(4-Methyl-2-oxo-2H-chromen-7-yl)-1H-1,2,3-triazol-1-yl)ethyl)-
1,4,8,11-tetraazacyclotetradecane-1,4,8-tricarboxylic acid tri-tert-butyl
ester (9): Compound 9 was prepared in a similar manner to compound 5
using 4 (583 mg, 1.02 mmol) and 7-ethynyl-4-methyl-2H-chromen-2-one
(189 mg, 1.03 mmol). Purification by flash chromatography (ethyl ace-
tate/hexane 3:2) gave triazole 9 as a pale yellow foam (660 mg, 86%).
M.p. 93–1018C; 1H NMR (200 MHz, CDCl3): d = 7.96 (s, 1H, Ht), 7.88
(dd, 1H, J = 8.3, 1.6 Hz, Hx), 7.74 (d, 1H, J = 1.4 Hz, Hz), 7.66 (d, 1H,
J = 8.3 Hz, Hy), 6.30 (d, 1H, J = 1.2 Hz, COCH), 4.44 (t, 2H, J =
6.5 Hz, Hf), 3.40–3.12 (m, 12H, CH2BocNCH2), 3.01 (t, 2H, J = 6.6 Hz,
He), 2.69 (t, 2H, J = 5.4 Hz, Hc), 2.54 (t, 2H, J = 5.6 Hz, Hb), 2.46 (d,
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3H, J = 1.1 Hz, CH3), 1.86–1.61 (m, 4H, Ha, Hd), 1.46 (s, 18H, C
ACHTUNGTRENNUNG
1.44 ppm (s, 9H, C =
R
155.1, 155.0, 154.9, 153.4, 151.8, 145.2, 133.9, 124.8, 121.5, 120.9, 118.9,
114.1, 112.7, 79.1, 79.0, 54.5, 53.3, 51.8, 48.3–45.0 (several peaks), 28.4,
28.0, 26.2, 18.0 ppm; IR (ATR, CDCl3): n=2975, 2933, 1736, 1688, 1621,
1464, 1414, 1390, 1242, 1161 cmÀ1
; HRMS (ESI): m/z: calcd for
C39H59N7O8Na: 776.4317, found 776.4323 [M+Na]+.
1-(2-(4-(4-Methyl-2-oxo-2H-chromen-7-yl)-1H-1,2,3-triazol-1-yl)ethyl)-
1,4,8,11-tetraazacyclotetradecane (3): Compound 3 was prepared in a
similar manner to compound 7 using 9 (736 mg, 0.976 mmol), trifluoro-
acetic acid (4 mL) and CH2Cl2 (16 mL) to give the free amine 3 as a
yellow solid (430 mg, 97%). M.p. 76–818C; 1H NMR (200 MHz, CDCl3):
d = 8.28 (s, 1H, Ht), 7.87 (dd, 1H, J = 8.2, 1.6 Hz, Hx), 7.77 (d, 1H, J =
1.6 Hz, Hz), 7.66 (d, 1H, J
=
8.2 Hz, Hy), 6.30 (d, 1H, J = 1.1 Hz,
COCH), 4.59 (t, 2H, J = 6.3 Hz, Hf), 2.97 (t, 2H, J = 6.3 Hz, He), 2.82–
2.16 (m, 19H, CH2NHCH2, Hb, Hc, CH3), 1.80–1.64 ppm (m, 4H, Ha, Hd);
13C NMR (50 MHz, CDCl3): d = 160.0, 153.3, 151.8, 144.9, 133.9, 124.8,
122.1, 120.8, 118.8, 114.1, 112.6, 54.1, 52.4, 51.2, 49.9, 47.9, 47.6, 47.3, 46.9,
46.7, 27.8, 25.7, 18.1 ppm; IR (ATR, CDCl3): n=3279, 3135, 2920, 2815,
1718, 1620, 1463, 1389, 1240, 1158 cmÀ1; HRMS (ESI): m/z: calcd for
C24H36N7O2: 454.2925, found 454.2926 [M+H]+; quantum yield: F=0.10
(H2O, pH 7).
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N
ACHTUNGTRENNUNG
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8
using
3
ACHTUNGTRENNUNG
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0.0918 mmol) to give the complex 10a as a purple powder (46.9 mg,
72%). M.p. 220–2228C; IR (ATR, acetone): n=3233, 2885, 1704, 1621,
1456, 1365, 1226, 1089, 942, 623 cmÀ1; LRMS (ESI): m/z (%): 615.1 (100)
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Chem. Eur. J. 2011, 17, 2850 – 2858
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Received: August 27, 2010
Published online: February 8, 2011
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ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 2850 – 2858