Cyclam-based "clickates": Homogeneous and heterogeneous fluorescent sensors for Zn(II)

Article abstract of DOI:10.1021/ic901939x

In an effort to improve upon the recently reported cyclam based zinc sensor 1, the "click"-generated 1,8-disubstituted analogue 2 has been prepared. The ligand shows a 2-fold increase in its fluorescence emission compared to 1 exclusively in the presence

Full text of DOI:10.1021/ic901939x

Inorg. Chem. 2010, 49, 3789–3800 3789
DOI: 10.1021/ic901939x
Cyclam-Based “Clickates”: Homogeneous and Heterogeneous Fluorescent
Sensors for Zn(II)
Emiliano Tamanini,† Kevin Flavin,† Majid Motevalli,† Silvia Piperno,‡ Levi A. Gheber,‡ Matthew H. Todd,*,§ and
Michael Watkinson*,†
†The Joseph Priestly Building, School of Biological and Chemical Sciences, Queen Mary University of London,
Mile End Road, London, E1 4NS, U.K., ‡Department of Biotechnological Engineering, Ben Gurion University Negev,
IL-84105 Beer Sheva, Israel, and §School of Chemistry, University of Sydney, NSW 2006, Australia
Received October 2, 2009
In an effort to improve upon the recently reported cyclam based zinc sensor 1, the “click”-generated 1,8-disubstituted
analogue 2 has been prepared. The ligand shows a 2-fold increase in its fluorescence emission compared to 1
exclusively in the presence of Zn(II) that is typical of switch-on PET fluorescent sensors. Single crystal X-ray diffraction
of complexes of model ligand 10 reveals that the configuration adopted by the macrocyclic framework is extremely
sensitive to the metal ion to which it coordinates. For Zn(II), Mg(II), and Li(I) the metal ions adopt an octahedral
geometry with a trans III configuration of the cyclam ring. In contrast for Ni(II) the ligand adopts the rare cis V
configuration, while for Cu(II) a clear preference for five-coordinate geometry is displayed with a trans I configuration of
the macrocyclic ring being observed in two essentially isostructural compounds prepared via different routes. The
ligand displays an increased selectivity for Zn(II) compared to 1 in the majority of cases with excellent selectivity upheld
over Na(I), Mg(II), Ca(II), Mn(II), Ni(II), Co(II), and Fe(III). In contrast for Cu(II) and Hg(II) little improvement was
observed for 2 compared to 1 and for Cd(II) the selectivity of the new ligand was inferior. In the light of these findings
and the slower response times for ligand 2, our original “click”-generated cyclam sensor system 1 was employed in a
proof of concept study to prepare a heterogeneous sol-gel based material which retains its PET response to Zn(II).
The versatile nature of the sol-gel process importantly allows the simple preparation of a variety of nanostructured
materials displaying high surface area-volume ratio using fabrication methods such as soft lithography, electrospin-
ning, and nanopipetting.
Introduction
and catalytic roles in enzymes as well as its important role in
neural signal transmission, “mobile” pools of zinc exist in
certain mammalian organs such as the brain and pancreas,
which are carefully regulated. Unsurprisingly it is increas-
ingly recognized that the disruption of these pools of zinc is
associated with a number of disease states which include type
I and II diabetes,3 neural function,4 particularly Alzheimer’s
disease5 and certain cancers.6 Furthermore, zinc is now
recognized as an important factor in the regulation of
apoptosis.7 Consequently much effort has been directed
toward the preparation of highly specific sensors for the
Because of the essential and varied role of metal cations in
biological systems, allied to concerns over the role of certain
elements as environmental pollutants, much effort has fo-
cused in recent years on the development of effective sensors
for a number of these ions.1 Arguably greatest interest has
focused on the development of sensors for the detection of
zinc(II).2 This reflects the fact that zinc is the second most
abundant transition metal in the human body. Although over
90% of zinc is classified as “static”, playing crucial structural
roles in transcription factors and related proteins, structural
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*To whom correspondence should be addressed. E-mail: m.todd@
chem.usyd.edu.au (M.H.T.), m.watkinson@qmul.ac.uk (M.W.).
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r
2010 American Chemical Society
Published on Web 03/18/2010
pubs.acs.org/IC

3790 Inorganic Chemistry, Vol. 49, No. 8, 2010
Tamanini et al.
Figure 1. Click-generated fluorescent probes for Zn(II).
detection of Zn(II) both in cellular media and in vivo.2,8 The
vast majority of the sensors reported to date have focused on
imaging the spectroscopically silent d10 ion by preventing
fluorophore quenching by photoinduced electron transfer
(PET).9 This is because upon coordination of the functiona-
lized ligand to the metal ion fluorescence is switched on (since
the quenching mechanism is turned off) which allows the
rapid and facile visualization of this elusive metal ion in
biological samples. While many sensors for Zn(II) have been
reported, there continues to be extensive interest in the field.10
This is because the need remains for highly selective, non-toxic,
and water-soluble sensors able to operate at physiological pH,
which are also able to sense and quantify zinc levels over the
broad concentration range known to be present in biology.
Our recent interests in sensing have focused on the use of
macrocyclic based ligand systems.11 While oxygen rich
macrocycles have mainly found use as ligands for alkali
and alkali earth metals, azamacrocycles are more suited
toward coordinating the softer transition metals. Among
the azamacrocycles, cyclen is probably the most used for this
purpose, and it has proven to be a good ligand in a number of
PET-based probes for the zinc ion.12 Surprisingly, there are
very few examples of analogous small molecule cyclam-based
fluorescent sensors for Zn(II) that have been shown to
display selectivity for zinc.13 We recently reported the synthe-
sis of the cyclam-based fluorescent Zn(II) sensor 1 (Figure 1),
which was easily assembled via the Cu(I)-mediated Huisgen
[3 þ 2] “click” cycloaddition between a propargyl-functiona-
lized cyclam ionophore and an azide-functionalized naphtha-
limide fluorophore.11a While the “click” reaction has attrac-
ted increasing interest in recent years because of its potency in
the effective and facile “ligation” of different chemical moie-
ties, the resultant triazole has generally only been used as a
junction in the final structures.14 More recently, however,
several systems have appeared in which the heterocycle plays
an active role in the functionality of the final product;11a,b,14g-14i,15
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Article
Inorganic Chemistry, Vol. 49, No. 8, 2010 3791
in the case of 1 serving as a scorpionate ligand reminiscent of
the histidine ligands that bind zinc ions in the active site of a
range of enzymes such as carbonic anhydrase. Our papers were
the first to report the synthesis of azamacrocycles bearing
pendant triazole motifs. In the absence of metal ions the
fluorescence of 1 was quenched via PET from the azamacro-
cycle receptor to the excited state of the naphthalimide fluor-
ophore (Figure 1). On addition of Zn(II) PET was inhibited
resulting in a typically strong switch-on enhancement of the
fluorescent emission. The sensor upheld excellent selectivity
over a range of biologically relevant metal ions including
Na(I), Ca(II), Mg(II), and Fe(III), although full fluorescence
response was never recovered, and the system was unable to
detect zinc in the presence of an excess of either Cu(II) or
Hg(II). This system also proved to be cell-permeable and
capable of detecting biologically available zinc in mammalian
cells, sensing the Zn(II) flux that exists during apoptotic cell
death without the need for the addition of extracellular Zn(II).
We envisaged that the performance of 1 could be improved
through the development of new “clickates” integrating more
than one triazole within the macrocyclic framework. Besides
the inherent novelty of these triazole-appended azamacro-
cycles as architectures for coordination chemistry, the mod-
ularity of the click reaction allows for a range of systematic
changes to be readily made to the ligand structure, for
example, (i) the incorporation of two different fluorophores
within the same ligand might allow ratiometric sensing; (ii) the
generation of an excimer emission through the interaction of
two fluorophores (vide infra), or (iii) the incorporation of two
identical fluorophores to increase the intensity of the fluore-
scence response of the original sensor. We initially targeted
the last of these through the second generation fluorescent
Zn(II) probe 2, which presents two identical fluorophores
“clicked” onto diametrically opposed cyclam nitrogens. This
ligand should preferentially accommodate cations in an
octahedral environment with the click-generated triazoles
occupying the metal’s axial positions when the cyclam ring
adopts the thermodynamically favored trans (III) geometry.16
Although it is well established that the stability constants of
cyclam ligands of this type with sterically undemanding
pendant arms are higher for Cu(II) over Zn(II),16b,c we hoped
that the larger triazole appended fluorophores in 2 might also
provide a kinetic barrier to prevent the coordination of copper
over zinc should significant conformational rearrangement of
the ligand be required upon metal binding. Although compe-
titive quenching of fluorescence by Cu(II) is a problem for
putative zinc sensors in vitro,10,11a it is not of general biolo-
gical relevance as it has been estimated that intracellular free
Cu(II) is limited to less than one free copper ion per cell,17a
although high concentrations of both Cu(II) and Zn(II) can
occur in biology, for example, in amyloid plaques.17b
tions were carried out using commercial grade solvents. Flash
chromatography was carried out using BDH silica gel. 1H and
13C NMR spectra were recorded on a JEOL JMN-EX270
NMR spectrometer at 270 and 67.5 MHz respectively. 1H
chemical shifts are reported in parts per million (ppm) relative
to the residual proton signal of the deuterated solvents. Cou-
pling constants (J) are reported in hertz (Hz). 13C chemical
shifts are reported in parts per million relative to the carbon
signal of the solvents. IR spectra were recorded with a Shimad-
zu FTIR-8300. Fluorescence emission spectra were recorded
with a Jobin Yvon Horiba FluoroMax-3 in a 1 cm path length
cell. Electrospray Ionization mass spectroscopy was obtained
from the EPSRC National Mass Spectrometry Service, Uni-
versity of Wales, Swansea with either a Waters ZQ400 or a
Micromass Quattro II. Accurate masses were recorded with
either a Finnigan MAT900 or MAT 95 using polyethylenimine
as reference.
1,8-Di-prop-2-ynyl-1,4,8,11-tetraaza-tricyclo[9.3.1.14,8]hexa-
decane Bromide Salt (4). To a solution of 318 (556 mg, 2.5 mmol)
in acetonitrile (50 mL) was added propargyl bromide (80% in
toluene, 1.4 mL, 12.4 mmol), and the reaction mixture was
stirred at room temperature (rt) overnight. The white precipitate
formed was filtered, washed with cold acetonitrile, and dried in
vacuo to give the desired product 4 as a white powder (926 mg,
78% yield): mp = 102-104 °C; 1H NMR (D2O): δ 5.48-5.36
(m, 2n), 4.83 (s, 1n), 4.58-4.28 (m, 6n), 3.92-3.76 (m, 2n),
3.70-3.24 (m, 12n), 3.22-3.06 (m, 2n), 3.00-2.80 (m, 4n),
2.72-2.38 (m, 4n), 2.06-1.86 (m, 3n); the presence of different
conformers and possibly diastereoisomers in solution makes it
difficult to assign the multiplicity and the integral values in the
1H NMR. The integrals are reported as “n” which represents the
proportion between the integrals of different signals in the
spectrum (see Supporting Information);
13C NMR (D2O): δ 82.5, 82.0, 77.8, 76.4, 75.9, 70.0, 61.3, 53.1,
51.3, 51.0, 50.3, 48.5, 47.2, 45.1, 40.8, 21.9, 19.8; MS (ESI) m/z:
381 [(M - Br)þ], 151 [(M - 2Br)2þ]; HRMS (ES) calcd. for
C18H30N479Br [(M - Br)þ] 381.1648, found. 381.1644.
1,8-Di-prop-2-ynyl-1,4,8,11tetraaza-cyclotetradecane (5). To
the bisaminal intermediate 4 (926 mg, 1.9 mmol) was added 3 M
NaOH (80 mL), and the resulting suspension was stirred at rt for
3 h. The product was extracted with CH2Cl2 (3 ꢀ 60 mL), the
combined organic phases were dried (MgSO4) and concentrated
in vacuo to afford the desired product 5 as a colorless glue in
quantitative yield (540 mg); νmax (CH2Cl2)/cm-1: 3302, 2134; 1H
NMR (CDCl3): δ 3.40 (d, J = 2.2, 4H), 2.90-2.50 (m, 18H),
2.14-2.80 (m, 2H), 1.75-1.62 (m, 4H); 13C NMR (CDCl3): δ
78.1, 72.9, 53.9, 51.3, 49.8, 47.1, 41.2, 25.7; MS (ESI) m/z: 277.2
[(MþH)þ]; HRMS (ES) calcd. for C16H29N4 [(MþH)þ]
277.2387, found. 277.2390.
4,11-Di-prop-2-ynyl-1,4,8,11tetraaza-cyclotetradecane-1,8-
dicarboxylic acid Di-tert-butyl Ester (6). To a solution of 5
(552 mg, 2.0 mmol) in CH2Cl2 (20 mL) were added TEA (834 μL,
6.0 mmol), di-tert-butyl dicarbonate (958 mg, 4.4 mmol) and
DMAP (49 mg, 20 mol %), and the reaction mixture was stirred
at rt for 16 h. The reaction was quenched by addition of a
saturated solution of NaHCO3 (20 mL) and extracted with
CH2Cl2 (3 ꢀ 50 mL). The combined organic phases were dried
(MgSO4) and concentrated in vacuo. The crude material was
purified by flash chromatography on silica gel (EtOAc, 100%)
to afford pure 6 as a colorless solid (950 mg, 84% yield): mp =
153-155 °C ; νmax (CH2Cl2)/cm-1: 3302, 1678; 1H NMR
(CDCl3): δ 3.42-3.20 (m, 12H), 2.68-2.58 (m, 4H), 2.56-2.44
(m, 4H), 2.14 (s, 2H), 1.76-1.60 (m, 4H), 1.43 (s, 18H); 13C
NMR (CDCl3): δ 155.8, 79.4, 78.7, 72.8, 52.9, 51.2, 46.5, 43.4,
Experimental Section
General Information. All reagents were purchased from
Sigma-Aldrich and used without further purification. All reac-
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1975. (b) Kurosaki, H.; Bucher, C.; Espinosa, E.; Barbe, J.-M.; Guilard, R. Inorg.
Chim. Acta 2001, 322, 145–149.

3792 Inorganic Chemistry, Vol. 49, No. 8, 2010
Tamanini et al.
43.0, 28.5, 26.9; MS (ESI) m/z: 477.3 [(MþH)þ]; HRMS (ES)
calcd. for C26H45N4O4 [(MþH)þ], 477.3435, found. 477.3435.
4,11-Bis-[1-(2-ethyl-1,3-dioxo-2,3-dihydro-1H-benzo[de]iso-
quinolin-6-yl)-1H-[1,2,3]triazol-4-ylmethyl]-1,4,8,11-tetraaza-
cyclotetradecane-1,8-dicarboxylic Acid Di-tert-butyl Ester (8).
To a solution of 6 (174 mg, 375 μmol) in THF/H2O (7/3, 10 mL)
dried (MgSO4), filtered and concentrated in vacuo to give 10 as a
colorless semisolid (178 mg, 87% yield): νmax (CH2Cl2)/cm-1
:
3433, 1461, 1358; 1H NMR (CDCl3): δ 7.41 (s, 2H), 7.30-7.10
(m, 10H), 5.40 (s, 4H), 3.59 (s, 4H), 2.74-2.32 (m, 16H),
1.76-1.60 (m, 4H); 13C NMR (CDCl3): δ 144.4, 134.9, 129.0,
128.6, 127.9, 122.8, 53.9, 52.6, 51.6, 49.1, 48.2, 47.0, 25.3; MS
(ESI) m/z: 543.5 [(MþH)þ]; HRMS (ES) calcd. for C30H43N10
[(MþH)þ], 543.3667, found 543.3670.
were added 711a (220 mg, 825 μmol), CuSO4 5H2O (9.3 mg,
3
10 mol %), and sodium ascorbate (15 mg, 20 mol %) under N2.
The solution was stirred at rt overnight. Saturated NH4Cl
(20 mL) was added, and volatiles were evaporated in vacuo.
The aqueous residue was extracted with CH2Cl2 (3 ꢀ 30 mL),
and the combined organic portions were dried (MgSO4), fil-
tered, and concentrated in vacuo. The crude material was
purified by flash chromatography on silica gel (EtOAc: Pet.
spirit 9:1 ramping to EtOAc 100%) to give 8 as a yellow solid
General Procedure for the Synthesis of Metal Complexes with
Ligand 10. To a solution of 10 (100 mg, 0.18 mmol) in MeOH
(5 mL) was added 1 equiv of the metal ion as a perchlorate salt.
The solution was stirred overnight at rt. The solid formed was
removed by filtration, washed with a small amount of cold
MeOH, and dried in vacuo. Single crystals suitable for X-ray
diffraction were obtained by slow diffusion of toluene vapor
into a solution of the metal complex in acetonitrile.
(234 mg, 62% yield): mp = 142-143 °C; νmax (CH2Cl2)/cm-1
:
3054, 1665, 1650; 1H NMR (CDCl3): δ 8.68-8.30 (m, 4H),
8.20-7.86 (m, 4H), 7.84-7.58 (m, 4H), 4.30-4.14 (m, 4H),
3.98-3.76 (m, 4H), 3.60-3.20 (m, 8H), 2.80-2.40 (m, 8H),
2.10-1.60 (m, 4H), 1.50-1.14 (m, 24H); 13C NMR (CDCl3): δ
163.2, 162.7, 155.8, 146.7, 138.9, 131.8, 130.3, 129.4, 128.6, 128.4,
126.2, 125.1, 123.4, 122.7, 79.4, 77.4, 55.1, 52.2, 50.3, 50.1, 47.3,
46.8, 35.7, 28.5, 13.3; MS (ES) m/z: 1009.6 [(MþH)þ]; HRMS
(ES) calcd. for C54H65N12O8 [(MþH)þ], 1009.5043, found
1009.5043.
[Zn(10)](ClO4)2 was obtained as described above from Zn-
(ClO4) 6H2O as a colorless crystal in 62% yield. HRMS (ES)
3
calcd. for C30H42N10O4ClZn (M - ClO4)þ, 705.2365, found
705.2361.[Ni(10)](ClO4)2 was obtained as described above from
Ni(ClO4) 6H2O as a pink crystal in 71% yield. HRMS (ES)
3
calcd. for C30H42N10O4ClNi (M - ClO4)þ, 699.2427, found
699.2431.[Cu(10)](ClO4)2 was obtained as described above from
Cu(ClO4) 6H2O as deep blue crystals in 80% yield. HRMS (ES)
3
calcd. for C30H42N10O4ClCu (M - ClO4)þ, 704.2370, found
704.2370.[Cu(10)](PF6)2 was obtained as previously described.21
6-Bromo-2-(3-hydroxypropyl)-1H-benzo[de]isoquinoline-1,3(2H)-
dione (12). To a stirred solution of 11 (500 mg, 1.8 mmol) in
dioxane (20 mL) was added 3-aminopropan-1-ol (163 mg,
2.16 mmol), and the solution was heated at reflux overnight
under a nitrogen atmosphere. The reaction mixture was cooled
to rt, and the product precipitated by the addition of water
(40 mL). The precipitate was collected by filtration, washed with
water, and dried in vacuo to provide 12 as a gold-yellow solid
(416 mg, 70% yield): vmax (CH2Cl2)/cm-1 2307, 1701, 1655,
1419, 1269; 1H NMR (CDCl3): δ 8.57 (d, J = 7.2, 1H), 8.48 (d,
J = 8.7, 1H), 8.32 (d, J = 7.9, 1H), 7.96 (d, J = 7.9, 1H), 7.78
(m, 1H), 4.28 (t, J = 6.2, 2H), 3.57 (t, J = 5.4, 2H), 3.06 (bs, 1H),
1.95 (m, 2H); 13C NMR (CDCl3): δ = 164.1 (2 ꢀ CdO), 133.4,
132.2, 131.4, 131.1, 130.6, 130.5, 128.8, 128.1, 122.6, 121.7, 58.9,
37.0, 30.9; MS (ESI) m/z: 336.1, 334.1 [(MþH)þ for 81Br and
79Br respectively], 329.2; HRMS (ES) calcd. for C15H1379BrNO3
[(MþH)þ] 334.0073, found 334.0078.
4,11-Bis-[1-(2-ethyl-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoqu-
inolin-6-yl)-1H-[1,2,3]triazol-4-ylmethyl]-1,4,8,11-tetraaza-cyclo-
tetradecane (2). To 8 (150 mg, 149 μmol) was added TFA (20%
in CH2Cl2, 5 mL) at rt, and the reaction mixture was stirred for
3 h. A saturated solution of NaHCO3 (10 mL) was added, and
the product was extracted with CH2Cl2 (3 ꢀ 20 mL). The
combined organic phases were dried (MgSO4), filtered, and
the volatiles were removed in vacuo to give 2 as a yellow solid
(107 mg, 89% yield): mp = 95-97 °C; νmax (CH2Cl2)/cm-1
:
1
3054, 1664; H NMR (CDCl3): δ 8.62-8.52 (m, 4H), 8.12 (d,
J = 8.4, 2H), 8.00 (s, 2H), 7.80-7.68 (m, 4H), 4.18 (q, J = 7.0,
4H), 3.88 (s, 4H), 2.80-2.50 (m, 16H), 1.87 (bs, 4H), 1.27 (t, J =
7.0, 6H); 13C NMR (CDCl3): δ 163.4, 162.9, 144.3, 138.2, 132.0,
130.5, 129.4, 129.0, 128.6, 126.4, 125.2, 123.8, 123.5, 122.9, 53.9,
53.5, 51.8, 49.9, 47.7, 47.5, 35.8, 25.9, 13.3; MS (ESI) m/z: 809.6
[(MþH)þ]; HRMS (ES) calcd. for C44H49N12O4 [(MþH)þ],
809.3994, found 809.3997.
4,11-Bis-(1-benzyl-1H-[1,2,3]triazol-4-ylmethyl)-1,4,8,11tetra-
aza-cyclotetradecane-1,8-dicarboxylic Acid Ditert-butyl Ester
(9). To a solution of 6 (200 mg, 420 μmol) in THF/H2O (7/3,
10 mL) were added benzyl azide (115 μL, 924 μmol), Cu-
6-Azido-2-(3-hydroxypropyl)-1H-benzo[de]isoquinoline-1,3(2H)-
dione (13). To a stirred solution of 12 (400 mg, 1.19 mmol) in N-
pyrrolidinone (6 mL) was added sodium azide (389 mg, 5.98 mmol),
and the mixture heated at 110 °C for 1 h. The reaction mixture
was cooled to rt, washed with brine, and the organic phase dried
(MgSO4). The solvent was removed in vacuo to afford 13 as a
dark orange solid (191 mg, 54%): vmax (CH2Cl2)/cm-1 2125,
1697, 1654, 1589, 1419; 1H NMR (CDCl3): δ = 8.57 (dd, J =
7.2, 1.2, 1H), 8.51 (d, J = 7.9, 1H), 8.39 (dd, J = 8.4, 1.2, 1H),
7.70 (dd, J = 8.4, 7.3, 1H), 7.40 (d, J = 7.9, 1H), 4.29 (t, J = 6.2,
2H), 3.56 (t, J = 5.2, 2H), 3.15 (bs, 1H), 1.97-1.92 (m, 2H); 13C
NMR (CDCl3): δ = 164.5, 164.1, 143.4, 132.5, 132.0, 129.0,
126.8, 124.2, 122.1, 118.3, 114.7, 58.8, 36.8, 30.9 (presumably
because of overlapping signals not all signals were resolved); MS
(ESI) m/z: 297.2 [(MþH)þ], 256.1, 105.0, 60.3; HRMS (ESI)
calcd. for C15H13N4O3 [(MþH)þ] 297.0982, found 297.0978.
Tri-tert-butyl-11-((1-(2-(3-hydroxypropyl)-1,3-dioxo-2,3-dihydro-
1H-benzo[de]isoquinolin-6-yl)-1H-1,2,3-triazol-4-yl)methyl)-1,4,8,11-
tetraazacyclotetradecane-1,4,8-tricarboxylate (15). To a stirred
solution of 14 (180 mg, 0.335 mmol) in H2O/THF (3/1, 10 mL)
SO4 5H2O (10.5 mg, 10 mol %), and sodium ascorbate (16.6
3
mg, 20 mol %) under N2. The solution was stirred at rt over-
night. Saturated NH4Cl (20 mL) was added, and volatiles were
evaporated in vacuo. The aqueous residue was extracted with
CH2Cl2 (3 ꢀ 30 mL), and the combined organic portions were
dried (MgSO4), filtered, and concentrated in vacuo. The crude
material was purified by flash chromatography on silica gel
(EtOAc: Pet. spirit 9:1 ramping to EtOAc 100%) to give the
desired product 9 as a pale yellow solid (280 mg, 90% yield):
1
mp = 60-62 °C; νmax (CH2Cl2)/cm-1: 1678, 1419; H NMR
(CDCl3): δ 7.42-7.12 (m, 12H), 5.43 (s, 4H), 3.64 (s, 4H),
3.38-3.10 (m, 8H), 2.58-2.46 (m, 4H), 2.42-2.32 (m, 4H),
1.72-1.56 (m, 4H), 1.34 (s, 18H); 13C NMR (CDCl3): δ 155.7,
145.2, 135.0, 129.1, 128.6, 127.9, 122.4, 79.2, 77.4, 53.9, 53.7,
51.9, 50.4, 46.9, 46.5, 28.5, 27.2; MS (ESI) m/z: 743.7 [(MþH)þ];
HRMS (ES) calcd. for C40H59N10O4 [(MþH)þ], 743.4715,
found. 743.4714.
1,8-Bis-(1-benzyl-1H-[1,2,3]triazol-4-ylmethyl)-1,4,8,11-tetra-
aza-cyclotetradecane (10). To 9 (280 mg, 377 μM) was added
TFA in (20% in DCM, 5 mL), and the reaction mixture was
stirred at rt overnight. The reaction was quenched with satu-
rated NaHCO3 solution (10 mL), and the product was extracted
with DCM (3 ꢀ 30 mL). The combined organic phases were
under N2 were added 13 (100 mg, 0.335 mmol), CuSO4 5H2O
3
(4.2 mg, 0.017 mmol), and sodium ascorbate (6.6 mg, 0.033
mmol), and the mixture was stirred overnight. The reaction
mixture was quenched with saturated NH4Cl (10 mL), the
volatiles were removed in vacuo, and the residue extracted with
DCM (3 ꢀ 30 mL). The combined organic portions were dried

Article
Inorganic Chemistry, Vol. 49, No. 8, 2010 3793
(MgSO4) and concentrated in vacuo. The crude material was
purified by silica gel column chromatography (8/2, EtOAc:Pet.
spirit 8:2 ramping to EtOAc:MeOH 9:1) to afford 13 as an
(Nanonics, Jerusalem, Israel) with a flat scanner and two coaxial
optical microscopes that allowed examination of the sample
simultaneously from above and below. Nanofountain pen
probes, which are cantilevered nanopipettes (300-600 nm aper-
ture diameter) were used for deposition of single microdots of
sol solution on the substrate. Following deposition, microarrays
were cured at 80 °C for 12 h to remove solvent and stabilize the
structures.
orange-yellow solid (280 mg, 64% yield): vmax (CH2Cl2)/cm-1
:
3055, 1662, 1647; 1H NMR (CDCl3): δ 8.73-8.70 (m, 2H),
8.36-8.28 (m, 1H), 7.91-7.86 (m, 3H), 4.39-4.35 (m, 2H), 3.95
(m, 2H), 3.65-3.61 (m, 2H), 3.36 (m, 12H), 2.74-2.72 (m, 2H),
2.59-2.55 (m, 2H), 2.03-1.64 (m, 6H), 1.39 (m, 30H); 13C NMR
(CDCl3): δ 163.2, 163.6, 155.8, 155.6, 138.5, 132.4, 131.1, 130.0,
129.0, 128.7, 126.4, 125.1, 123.6, 123.4, 122.6, 79.7, 59.2, 47.6,
45.6, 37.3, 31.0, 28.5; MS (ESI) m/z: 835.5 [(MþH)þ], 501.4;
HRMS (ESI) calcd. for C43H63N8O9 [(MþH)þ] 835.4713, found
835.4707.
X-ray Crystallography. Data were collected at 120 K using a
Nonius Kappa CCD area detector diffractometer mounted at
the window of molybdenum rotating anode (50 KV, 90 mA, λ =
˚
0.71069 A). The crystal-to-detector distance was 30 mm, and φ
and Ω scans (2.0° increments, 12 s exposure time) were carried
out to fill the Ewald sphere. Data collection and processing were
carried out using the COLLECT,33 DENZO,34 and MaXus35
softwares, and empirical absorption correction was applied
using SORTAV.36 The structures were solved by heavy-atom
method using DIRDIF99,37 and refined by full-matrix least-
squares on F2 using the SHELXL-9738 program. All non-
hydrogen atoms were refined anisotropically with hydrogen
atoms included in idealized positions with thermal parameters
riding on those of parent atom (N-H on the cyclam rings were
refined freely, except for [Zn(10)](ClO4)2 and [Mg(10)](ClO4)2
which were idealized). The programs ORTEP-339 and PLA-
TON40 were used for drawing the molecules. WINGX41 was
used to prepare material for publication. All data relating to
these single crystal X-ray structures have been deposited at the Cam-
bridge Crystallographic Database: CCDC 730863 ([Cu(10)]-
(ClO4)2), 730864 ([Cu(10)](PF6)2), 730865 ([Li(10)](ClO4)2),
730866 ([Mg(10)](ClO4)2), 730867 ([Ni(10)](ClO4)2), and 730868
([Zn(10)](ClO4)2).
6-(4-((1,4,8,11-Tetraazacyclotetradecan-1-yl)methyl)-1H-1,2,3-
triazol-1-yl)-2-(3-hydroxypropyl)-1H-benzo[de]isoquinoline-1,3(2H)-
dione (16). A solution of TFA (20% in DCM, 10 mL) was added
to 15 (182 mg, 0.218 mmol), and the reaction mixture was stirred
at rt for 2 h. The reaction mixture was quenched with saturated
NaHCO3 (10 mL), and the product extracted with CH2Cl2 (3 ꢀ
20 mL). The organic layer was dried (MgSO4), filtered and
concentrated in vacuo to afford 16 as an orange-yellow solid
which was used without further purification (114 mg, 98%): vmax
(CH2Cl2)/cm-1: 3055, 1662, 1593; 1H NMR (CDCl3): δ
8.71-8.68 (m, 2H), 8.23-8.20 (m, 1H), 7.98-7.94 (m, 2H),
7.88-7.82 (m, 1H), 4.47 (t, J = 6.2, 1H), 4.35 (t, J = 6.1, 2H),
3.61 (t, J = 5.7, 2H), 3.31-2.88 (m, 14H), 2.30-2.21 (m, 2H),
2.04-1.95 (m, 5H); 13C NMR (CDCl3): δ 164.1, 163.7, 163.6,
163.2, 162.1, 144.6, 138.1, 132.5, 131.0, 129.6, 129.0, 128.8,
126.5, 125.1, 124.4, 123.8, 122.6, 119.0, 114.6, 66.2, 59.2, 56.9,
54.9, 49.3, 49.0, 48.4, 47.9, 47.0, 45.7, 45.5, 37.4, 31.0, 27.0, 25.6,
23.1; MS (ESI) m/z: 535.4 [(MþH)þ]; HRMS (ESI) calcd. for
C28H39N8O3 [(MþH)þ] 535.3140, found 535.3139.
AFM Imaging. AFM images were taken in contact mode
using a Nanonics NSOM/AFM 100 system (Nanonics, Jerusa-
lem, Israel) and “Ultrasharp” contact probes from MikroMasch
(0.03 N/m nominal spring constant) at ∼ 1 Hz line-scan velocity.
Preparation of Sol-Gel Based Micro/Nanostructured Mate-
rials. Sol-Gel Preparation. Aliquots of 32% HCl (aq) (0.027
mL) and ethanol (5.7 mL) were added to water (0.475 mL).
Tetraethoxysilane (TEOS) (1.71 mL) was added slowly to this
solution, and the mixture allowed to stir for 10 min. Ligand 16
(10 mg, 0.019 mol) in ethanol (2 mL) was slowly added to the
reaction mixture which was then allowed to stir for 24 h. This
produced a solution with a molar composition of 1 TEOS: 15
EtOH: 0.1 HCl: 3 H2O while the Si/fluorescent ligand molar
ratio was 400.
Results and Discussion
Ligand Synthesis. The most convenient way to access
1,8-disubstituted cyclam ligands is via the Guilard pro-
cedure18 which allows the facile and completely selective
alkylation of the diametrically opposite cyclam nitrogen
atoms. The synthesis (Scheme 1) starts from cyclam bis-
aminal 3 which is easily obtained by treatment of cyclam
with formaldehyde in water following well-known proce-
dures.19 Treatment of 3 with propargyl bromide in acet-
onitrile afforded the 1,8-dialkylated product 4 in 78%
yield. Removal of the aminal groups was effected in
quantitative yield by basic hydrolysis in 3 M NaOH
solution. While click reactions have recently been per-
formed on unprotected azamacrocyclic ligands,15d,20 this
requires either prior metalation of the macrocycle or an
excess of copper for the reaction to proceed, implying Cu-
cyclam complexes formed in situ are inactive catalysts.
Consequently we elected to Boc-protect the secondary
amine groups to prevent coordination of copper to the
azamacrocycle, thus also obviating the need for its sub-
sequent removal. The double click reaction between 6
and azido naphthalimide 7 proceeded smoothly, and
the desired product 8 was isolated in 62% yield after
Preparation of Zinc-Selective Sol-Gel Based Micropattern by
Soft Lithography. A polydimethylsiloxane (PDMS) stamp was
fabricated using a silicon grid as template. The dimensions of the
stamp prepared were 5 μm (micropattern cross-section), spaced
5 μm apart, with a height of 1.2 μm. To produce the micro-
structure, the stamp was immersed in a drop of the sol solution,
and subsequently pressed against a glass substrate (cleaned by
immersion in piranha solution, followed by acetone and dried
under a stream of nitrogen) for 45 s. The templated substrate
was then cured at 80 °C for 12 h following the removal of the
stamp.
Preparation of Zinc-Selective Sol-Gel Based Nanofibers. The
prepared sol (3 mL) was modified by addition of polyethylene
oxide (PEO) (180 mg) to elevate the solution viscosity. For
electrospinning, each sol was transferred to a 1 mL syringe and
delivered to the stainless steel needle manually. The needle was
connected to a high voltage direct current (DC) power supply
(Gamma High Voltage Research-ES 20KV). Glass micrsocope
slides, wrapped with aluminum foil were used as the counter
electrode and mounted at distances between 5 and 15 cm from
the needle. Continuous hybrid organic/inorganic nanofibers
were electrospun at rt using a driving voltage of 20 kV and
collected on the glass substrates, which were subsequently cured
at 80 °C for 12 h to remove solvent and stabilize the fibers.
Preparation of Zinc-Selective Sol-Gel Based Microarray.
Array deposition on glass substrates was performed under
ambient conditions using a Nanonics NSOM/AFM 100 system
(19) (a) Gabe, E. J.; Le Page, Y.; Prasad, L. Acta Crystallogr. 1982, B38,
2752–2754. (b) Bailly, T.; Leroux, Y.; Manouni, E. D.; Neuman, A.; Prange, T.;
Burgada, R. C. R. Acta Sci. 1996, Ser IIB, 151-154.
€
€
(20) Antoni, P.; Malkoch, M.; Vamvounis, G.; Nystrom, D.; Nystrom,
A.; Lindgren, M.; Hult, A. J. Mater. Chem. 2008, 18, 2545–2554.
ꢀ
(21) El Ghachtouli, S.; Cadiou, C.; Dechamps-Olivier, I.; Chuburu, F.;
Aplincourt, M.; Roisnel, T. Eur. J. Inorg. Chem. 2006, 3472–3481.

3794 Inorganic Chemistry, Vol. 49, No. 8, 2010
Tamanini et al.
Scheme 1. Synthesis of Sensor 2 and Model System 10a
a Reagents and conditions: (a) Propargyl bromide, CH3CN, rt; (b) 3 M NaOH, rt, 3 h; (c) Boc2O, TEA, cat. DMAP, CH2Cl2; (d) 7, CuSO4 5H2O (10 mol
%), Na ascorbate (20 mol %), H2O/THF, rt; (e) TFA (20% in CH2Cl2); (f) 7, CuSO4 5H2O (10 mol %), Na ascorbate (20 mol %), H2O/THF, rt.
3
3
chromatography. In the final step, Boc protecting groups
were readily removed by treatment of 8 with 20% TFA in
CH2Cl2 to afford sensor 2 in an excellent yield of 89%. In
addition the model “double-click” ligand 10 was also pre-
pared in an analogous manner to investigate the coordi-
nation chemistry of the new ligand system, an approach
that we have successfully adopted previously.11a Thus
model ligand 10 was prepared by a double click reaction
between Boc-protected cyclam 6 and benzyl azide. Re-
moval of the Boc groups with TFA in CH2Cl2 afforded
the desired product 10 in excellent yield (78% over two
steps).
Coordination Chemistry of Model Ligand 10. To inves-
tigate the coordination geometry of 2 with various metals,
complexes of model ligand 10 were prepared by treatment
of its methanolic solution with 1 equiv of the perchlorate
salt of a number of different metal ions (see Experimental
Section). Single crystals suitable for X-ray diffraction
were obtained for [Zn(10)](ClO4)2, [Ni(10)](ClO4)2, and
[Cu(10)](ClO4)2 by slow vapor diffusion of toluene into
acetonitrile solutions of the complexes (Figure 2). In
addition we employed a known procedure for synthesis
of copper(II) complexes of cyclam in which the macro-
cycle adopts the trans (III) configuration by the aerial
oxidation of a copper(I) complex to give [Cu(10)](PF6)2,21
with crystals suitable for single crystal X-ray diffraction
being obtained as before. During these studies we also
isolated and crystallographically characterized two other
species, [Li(10)]ClO4 and [Mg(10)](ClO4)2, presumably
through adventitious metalation of the macrocycle dur-
ing the synthesis (see Supporting Information). The
crystal structures of the other cationic complexes are
presented in Figure 2 together with the crystallographic
data in Table 1, and selected bond lengths and angles in
Table 2.
siderably with three of the five possible configurations
being exhibited: the standard and expected16b,c trans (I)
configuration with copper(II); the trans (III) configura-
tion with zinc(II), in which the metal adopts an octahedral
coordination geometry with the two triazole rings occu-
pying the opposite axial sites (the magnesium(II) and
lithium(I) complexes are essentially isostructural); while
for nickel(II) the more unusual cis (V) configuration was
observed.22 This shows that for copper(II) there is a very
clear preference for five coordinate geometry and the
trans (I) configuration of the macrocycle with only one
heterocycle chelating the metal even when procedures
previously shown to result in complexes with trans (III)
configurations were employed. The structures are inter-
mediate between square based pyramidal and trigonal
bipyramidal geometry (τ = 0.50 for [Cu(10)](ClO4)2 and
0.49 for [Cu(10)](PF6)2)23 and are comparable to a num-
ber of previously reported complexes of this type.16b,c
Efficacy of 2 in Zinc Sensing. Given the very different
coordination modes exhibited for 10 together with the
already high selectivity of 1 for zinc we were optimistic
that its analogue 2 would also provide excellent selectivity
for zinc over a range of other metal ions. The fluorescence
of a 10 μM solution of 2 in H2O/CH3CN (7:3) buffered at
pH 7 (50 mM HEPES buffer) showed an emission band at
about 416 nm after excitation at 350 nm, mirroring the
behavior of the monosubstituted system 1 (Figure 3).
Upon addition of 1 equiv of Zn(II) a 12.7-fold increase
of the emission maximum was observed. The [1:1] stoi-
chiometry of the complex in solution was confirmed by
(22) (a) Donnelly, M. A.; Zimmer, M. Inorg. Chem. 1999, 38, 1650–1658.
(b) McRobbie, G.; Valks, G. C.; Empson, C. J.; Khan, A.; Silversides, J. D.;
Pannecouque, C.; De Clercq, E.; Fiddy, S. G.; Bridgeman, A. J.; Young, N. A.;
Archibald, S. J. Dalton Trans. 2007, 5008–5018. (c) Silversides, J. D.; Allan, C.
C.; Archibald, S. J. Dalton Trans. 2007, 971–978.
We were pleased to see that the configuration adopted
by the cyclam ring in the metal complexes varied con-
(23) Addison, A. W.; Rao, T. N.; Reddjik, J.; van Rijn, J.; Verschoor,
G. C. J. Chem. Soc., Dalton Trans. 1984, 1349–1356.

Article
Inorganic Chemistry, Vol. 49, No. 8, 2010 3795
Figure 2. Crystal structures of (a) [Zn(10)](ClO4)2, (b) [Ni(10)](ClO4)2, (c) [Cu(10)](ClO4)2, (d) [Cu(10)](PF6)2; a generic atomlabelingschemeis presented
in Table 2.
Table 1. X-ray Crystallographic Dataa
[Zn(10)](ClO4)2
[Ni(10)](ClO4)2
[Cu(10)](ClO4)2
[Cu(10)](PF6)2
empirical formula
formula weight
crystal size (mm)
crystal system
C30H42Cl2N10O8Zn
807.01
C30H42Cl2N10NiO8
800.35
0.22 ꢀ 0.12 ꢀ 0.06
monoclinic
P21/c
C32H45Cl2CuN11O8
846.23
C30H42CuF12N10P21.5C7H8
1034.42
0.40 ꢀ 0.36 ꢀ 0.14
0.18 ꢀ 0.16 ꢀ 0.15
0.14 ꢀ 0.12 ꢀ 0.06
monoclinic
P21/n
triclinic
P1
triclinic
P1
space group
unit cell dimensions
˚
a (A)
10.2539(2)
16.8869(5)
11.0525(2) A
17.866(2)
10.0482(12)
21.869(2)
90
110.773(5)
90
3670.7(7)
4
1.448
10.9250(3)
14.1486(4)
14.2506(3)
63.385(2)
77.978(2)
78.005(2)
1909.57(9)
2
12.9815(7)
13.1070(6)
14.3786(7)
88.645(3)
70.461(3)
85.132(3)
2297.3(2)
2
˚
b (A)
˚
c (A)
˚
R (deg)
β (deg)
γ (deg)
90
115.1890(10)
90
1731.83(7)
2
3
˚
volume (A )
Z
F (calcd) (Mg/m3)
absorption coefficient
1.548
0.929
1.472
0.776
1.495
0.637
0.736
(mm-1
F(000)
)
840
1672
882
1068
θ range (deg)
index ranges
3.16-27.50
3.05-27.59
2.93-27.63
2.95-25.00
-13 e h e 13
-21 e k e 21
-12 e l e 14
22080
-23 e h e 23
-13 e k e 13
-26 e l e 28
29015
-14 e h e 14
-18 ek e 18
-18 e l e 18
34217
-15 e h e 15
-15 e k e 15
-17 e l e 17
32521
reflections collected
independent reflections
max. and min transmission
data/restraints/parameters
goodness-of-fit on F2
3940 [R(int) = 0.0503]
0.8810 and 0.7076
3940/0/220
1.035
R1 = 0.0702, wR2 =
0.1973
8353 [R(int) = 0.0537]
0.9572 and 0.8549
8353/0/468
1.087
R1 = 0.0699, wR2 =
0.1306
8724 [R(int) = 0.0532]
0.8925 and 0.8730
8724/0/496
1.066
R1 = 0.0567, wR2 =
0.1076
8022 [R(int) = 0.0800]
0.9628 and 0.9161
8022/5/569
1.058
R1 = 0.0833, wR2 =
0.1804
final R indices [I >2σ(I)]
R indices (all data)
R1 = 0.0853, wR2 =
0.2087
R1 = 0.1110, wR2 =
0.1530
R1 = 0.0758, wR2 =
0.1175
R1 = 0.1160, wR2 =
0.2060
largest diff. peak and hole
)
1.028 and -1.568
0.708 and -0.503
0.626 and -0.459
1.269 and -1.008
-3
˚
(e.A
a
2
˚
All data were collected using MoKR radiation (λ = 0.71073 A) at 120(2) K and were refined by full-matrix least-squares on F .
titrating a 300 μM solution of Zn(ClO4)2 into a 10 μM
solution of 2 (Figure 3) and Kd estimated (because of the
very high stability of the complex) as approximately 107
M-1 (see Supporting Information).11a To compare the
photophysical properties of 2 with the previously reported
system, the fluorescence of 1 and its response upon the
addition of zinc ion were monitored under the same condi-
tions. This revealed the emission intensity of 2 to be roughly
double that of 1, indicating that there are no self-quenching
processes taking place between the two fluorophores as

3796 Inorganic Chemistry, Vol. 49, No. 8, 2010
Tamanini et al.
˚
Table 2. Selected Bond Lengths (A) and Angles (deg) for the Metal Complexes
with esds and Schematic Guide to Atom Numbering
[Zn(10)]
(ClO4)2
[Ni(10)]
(ClO4)2
[Cu(10)]
(ClO4)2
[Cu(10)]
(PF6)2
M-N(1)
M-N(2)
M-N(3)
M-N(4)
M-N(5)
M-N(6)
2.091(3)
2.158(3)
2.091(3)
2.158(3)
2.217(3)
2.217(3)
2.115(4)
2.113(2)
2.071(3)
2.104(2)
2.150(3)
2.051(3)
174.19(13)
2.021(3)
2.085(2)
1.998(3)
2.070(2)
2.046(5)
2.076(5)
2.028(5)
2.040(5)
2.274(2)
2.247(5)
N(1)-M-N(5) 88.71(11)
Figure 4. Fluorescence emission of 2 (10 μM) in the presence of a
number of other metal ions (10 μM) at pH 7.0 in HEPES buffer/CH3CN
7:3 about 25 min after the addition of the metal ions.
N(2)-M-N(5) 100.10(11) 101.80(13)
N(3)-M-N(5) 91.29(11)
N(4)-M-N(5) 79.90(11)
N(1)-M-N(6) 91.29(11)
N(2)-M-N(6) 79.90(11)
N(3)-M-N(6) 88.71(11)
N(4)-M-N(6) 100.10(11) 99.18(13)
N(5)-M-N(6) 180.00(14) 89.24(13)
87.01(14)
79.67(13)
88.62(13)
81.65(13)
104.49(10) 103.55(19)
79.12(9) 78.81(17)
quenching or excimer formation is observed, and we
can only speculate that this difference is a result of the
reduced conformational flexibility of our system which
prevents the two fluorophores engaging in such strong
short-range interactions. Thus the coordination of the
metal, rather than the dynamic conformation of the
ligand, dominates the fluorescence response.
172.48(14) 107.14(10) 106.84(19)
99.89(17)
98.55(9)
To test the selectivity of 2 for Zn(II), aqueous solutions
of a number of other biologically relevant divalent metals
were added to a solution of sensor 2 in H2O (HEPES)/
CH3CN (7:3). This resulted in a negligible enhancement
of the fluorescence emission of the solutions for all of the
metals tested (Figure 4). Additionally for Cd(II), selectiv-
ity appears to be superior compared to the single fluor-
ophore sensor 1 as the fluorescence of the Cd(II) complex
is well within the average response of all of the other metal
complexes and is stable at that level for at least 4 h. The
emission maximum for Cd(II) does increase slightly after
a 24 h period (see Supporting Information) to reach a
value which is five times lower than that of the Zn(II)
complex, which clearly does not compromise the excellent
response selectivity of 2 toward Zn(II). As previously
observed for 1, the fluorescence of 2 is slightly quenched
by the addition of Cu(II) and Hg(II), but this is not a
concern for potential biological applications of the Zn
sensor owing to the very low free intracellular concentra-
tions of these ions.
Competitive binding studies were performed by adding
1 equiv of Zn(II) to solutions of 2 containing a 5-fold
excess of competing metal ions after about 25 min. The
maximum emission of the solutions before and after the
addition of zinc ion were recorded and plotted against the
emission intensity of a sample containing [Zn(2)]2þ. Ex-
cellent selectivity for Zn(II) over the biologically relevant
macro-metals such as Naþ, Ca2þ, and Mg2þ was ob-
served, with the fluorescence of the solution completely
restored upon addition of Zn2þ, Figure 5. It is evident that
2 also shows exceptional selectivity over most of the first-
row transition metals added (Mn2þ, Ni2þ, Fe3þ, and
Co2þ). Although this selectivity is broadly comparable
to that observed for 1 the maximal emission observed for
[Zn(1)]2þ was never recovered in the presence of the
transition metal ions, indicating that some of the ligand
remained coordinated to the competing metal. In contrast
Figure 3. Fluorescence titration of a 10 μM solution of 2 in HEPES
buffer/CH3CN (7:3, pH = 7) with a 300 μM solution of Zn(ClO4)2 in
water after 25 min. Inset: emission intensity of the solution upon addition
of different concentrations of Zn2þ (0, 2, 4, 6, 8, 10, 12, 14, and 18 μM). λex
= 350 nm.
observed for a similar system reported by Valeur et al.24 in
which two identical coumarins were attached to a diaza-18-
crown-6.
Structurally related 1,8-disubstituted cyclams deriva-
tized with anthracenes and pyrenes, reported by Chang
et al.25, showed no fluorescence response for Zn(II); the
fluorescence response of these systems is extremely sensi-
tive to solvent and generally relies on the quenching of an
excimer emission upon binding of certain metal ions to
the cyclam ligand, giving a major change in conformation
that destroys the excimer. In the case of 2 no self-
(24) Bourson, J.; Pouget, J.; Valeur, B. J. Phys. Chem. 1993, 97, 4552–
4557.
(25) (a) Youn, N. J.; Chang, S.-K. Tetrahedron Lett. 2005, 46, 125–129.
(b) Moon, S.-Y.; Youn, N. J.; Park, S. M.; Chang, S.-K. J. Org. Chem. 2005, 70,
2394–2397. (c) Park, S. M.; Kim, M. H.; Choe, J.-I.; No, K. T.; Chang, S.-K.
J. Org. Chem. 2007, 72, 3550–3553.

Article
Inorganic Chemistry, Vol. 49, No. 8, 2010 3797
Figure 5. Changes in the fluorescence intensity of 2 upon addition of Zn(II) (10 μM) to a solution of 2 (10 μM) in HEPES buffer/CH3CN (7:3) (pH 7)
containing different competing ions (50 μM).
Scheme 2. Synthesis of Sol-Gel Precursor 16a
a Reagent and conditions: (a) 3-aminopropan-1-ol, dioxane, Δ; (b) NaN3, N-pyrrolidinone, Δ; (c) CuSO4 5H2O (5 mol %), Na ascorbate (10 mol %),
3
H2O/THF, rt; (d) TFA (20% in CH2Cl2); (e) NaH, DMF, -20 °C then 3-chloropropyl-triethoxysilane.
for 2 the maximum emission seen for [Zn(2)]2þ is always
recovered. As for 1, Cd(II), Hg(II), and Cu(II) appear to
form such thermodynamically stable complexes with
ligand 2 that the addition of Zn(II) to a solution of these
complexes only results in a partial increase of the emission
intensity of the final mixture. It is therefore apparent that
the inherent thermodynamic stability of cyclam com-
plexes is not affected by the incorporation of the two
large “click” generated triazole pendant arms and that, as
for other putative zinc sensors, application of this system
would be limited to common biological environments
where the concentrations of Cu(II) are negligible.17a
Fabrication of Nanostructured Zinc Sensor Materials.
Luminescent chemical sensing by sol-gel derived materi-
als is an attractive area as a result of the versatility and
flexibility associated with this method of preparation.26
The physical properties of these materials, such as stabi-
lity, optical transparency, flexibility, and permeability are
also very appealing, and we viewed ligand systems 1 and 2
as excellent candidates for the generation of such materi-
als. Moreover, the sol-gel process also allows the facile
preparation of a variety of nanostructured materials
displaying high surface area-volume ratio using fabrica-
tion methods such as soft lithography, electrospinning,
and nanopipetting. We were further encouraged by the
recent report of the application of siloxane functionalized
ligands in the preparation of sol-gel derived silica nano-
particles which were efficacious in the detection of Zn-
(II).27 Given the excellent selectivity for Zn(II) upheld by
1, the slower complexation rate of Zn(II) by 2, and the fact
that 2 displays only slightly enhanced selectivity/recovery
for Zn(II) over other divalent metals over 1 in homo-
geneous solution, we elected to investigate the efficacy of
analogues of 1 as luminescent sol-gel derived materials.
Initially we targeted 17 as a suitable sol-gel precursor
as outlined in Scheme 2, as we assumed that Boc depro-
tection would also occur under the strongly acidic sol-gel
conditions employed. Thus, commercially available 11
was readily converted to derivatized alcohol 12 by reaction
(27) Teolato, P.; Rampazzo, E.; Arduini, M.; Mancin, F.; Tecilla, P.;
Tonellato, U. Chem.;Eur. J. 2007, 13, 2238–2245.
(28) Armelao, L.; Bottaro, G.; Quici, S.; Cavazzini, M.; Concetta Raffo,
M.; Barigelletti, F.; Accorsi, G. Chem. Commun. 2007, 2911–2913.
(26) Tao, S.; Li, G.; Yin, J. J. Mater. Chem. 2007, 17, 2730–2736.

3798 Inorganic Chemistry, Vol. 49, No. 8, 2010
Tamanini et al.
Figure 6. (a) Visualization of the fluorescence response of the sol-gel coated glass slides, and (b) typical fluorescence emission response of the sol-gel
coated glass slides upon soaking the coated slides in a 75 μM solution of ZnSO4 for 24 h.
with 3-aminopropan-1-ol in refluxing dioxane overnight.
Nucleophilic aromatic substitution with sodium azide, as
used to prepare 7, gave 13 in good overall yield. The click
reaction of 13 with the previously reported azide 14 using
our standard protocol provided 15 in satisfactory yield. Our
attempts to react 15 with (3-chloropropyl)-triethoxysilane
provided 17 as judged by NMR spectroscopy, but this
material was also contaminated by a number of other
unidentified materials. We therefore elected to use the
approach recently reported by Barigelletti and co-workers
to effect the covalent incorporation of fluorescent molecules
into sol-gel matrixes which was reported to produce
homogeneous, transparent, and luminescent silica based
layers.29 The global Boc deprotection of 15 proceeded
essentially quantitatively using 20% TFA to give 16 which
was subsequently incorporated into the siloxane network
without further purification under the appropriate proces-
sing conditions.
The sol solution was prepared by acid catalyzed copo-
lymerization of tetraethoxysilane in an aqueous/ethanolic
medium in the presence of 16. Sol-gel films were laid by
spin-coating the solution onto clean glass substrates at a
range of speeds (500-1500 r.p.m.). Following the curing
procedure, the derivatized macrocycle was shown to
retain its fluorescent response to Zn(II) after soaking
the slides in a Zn(II) solution and a representative set of
coated glass slides are presented (Figure 6a). The change
in fluorescence is evident from Figure 6b; however, longer
response times were required with the emission intensity
increasing gradually over a period of time, presumably as
a result of slower zinc diffusion through the matrix. The
detection limit of the films at this stage is about 75 μM
Zn(II) as this was the lowest concentration that gave
consistent and reproducible results in which a real flor-
escence response rather than noise could be observed.
There are a number of reasonably straightforward
methods of overcoming this problem such as enhancing
the porosity by variation of the processing conditions or
by addition of a co-porogen,29 which we intend to in-
vestigate in due course. However, our aim at this stage
was to demonstrate the versatility and ease with which
this processing technique could be used in the fabrication
of zinc sensitive micro- and nanostructured materials,
such as nanoarrays and nanofibres which have potential
for application in devices for the one-off section of zinc.
Such structures possess an increased surface to volume
ratio, compared with their bulk counterparts, leading to
increased accessibility to the recognition sites and thus
high sensitivity. For these reasons they should provide
materials with improved response times and sensitivity to
minute amounts of sample. Furthermore, as such materi-
als can in principle be fabricated into highly ordered
arrays we felt that this represented an opportunity to
demonstrate the principle of preparation of prototype
sensor arrays. On the basis of this we investigated whether
nanostructured materials could be prepared by soft litho-
graphy,30 electrospinning,31 and using a “nanofountain
pen”32 (see Experimental Section). From the images
presented in Figure 7 it can clearly be seen that the target
materials can be prepared with exquisite control. In the
case of the electrospun fibers it proved necessary to add
polyethylene oxide to the sol mixture to provide a solu-
tion with sufficient viscosity, although alteration of the
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Article
Inorganic Chemistry, Vol. 49, No. 8, 2010 3799
Figure 7. Sol-gel nanostructures fabricated from 16 prepared using (a, b) soft lithography, (c) by electrospinning, and (d, e) by nanofountain pen,
visualized by fluorescence microscopy (a, c, and d) and AFM (b and e).
processing conditions should allow the direct use spin-
ning of the sol-solution in the absence of any additives.29
system for binding Zn(II) is comparable to the best PET
based zinc sensors reported to date. This is reflected in
competitive binding experiments with a range of metal
cations, which also show almost complete recovery of the
maximal fluorescent response. As is the case for many
putative zinc sensors in the presence of an excess of Cu(II),
Hg(II), and Cd(II), the addition of Zn(II) results in only a
small recovery of fluorescence. For the majority of biological
applications such deficiencies are not important as neither
Conclusions
We have reported the synthesis of a new class of cyclam-
based “clickates” capable of the selective detection of Zn(II)
over a number of other divalent metals which provides a 2-
fold increase in the fluorescence response compared to our
previously reported system 1. The selectivity of this ligand

3800 Inorganic Chemistry, Vol. 49, No. 8, 2010
Tamanini et al.
Hg(II) nor Cd(II) occur in significant concentrations in
biology, and it has been recently estimated that free Cu(II)
is limited to less than one free Cu(II) atom per cell.17a
Nonetheless, in cases where Cu(II) concentrations are com-
parable to Zn(II), for example, in amyloid plaques,17b further
developments are required to improve sensor selectivities.
Although the response of the new ligands was superior to 1 in
that it provided an enhanced fluorescence response and
improved recovery of fluorescence in the presence of other
divalent metals, its response to zinc was significantly slower.
We therefore chose to prepare sol-gel derived materials
using a derivative of ligand 1 and demonstrated that the
sol-gel material retains its sensitivity for Zn(II). We also
show in a proof of principle study that the sol-gel precursor
16 can be used in the fabrication of a range of highly ordered
nanostructured materials. As a result of these finding we
propose that it will be possible to prepare highly structured
nanoarrays for the selective detection, and ultimately quan-
titation, of zinc invivo and invitro. Investigations along these
lines are currently ongoing in our laboratories.
Acknowledgment. We thank EPSRC (GR/T17014/01)
for funding through the Life Sciences initiative (E.T.) and
the EC for support through the Marie Curie Research
Training Network “Nanomaterials for application sen-
sors, catalysis, and emerging technologies (NASCENT)”,
FP6-033873-2 (contract number MRTN-CT-2006-033873).
We are also grateful to the EPSRC for the provision of the
National Mass Spectrometry Service (University of Wales,
Swansea), and The National X-ray Crystallographic Service
(University of Southampton).
Supporting Information Available: Figures S1-S27, Tables S1
and S2, and crystallographic data in the form of CIF files. This
material is available free of charge via the Internet at http://
pubs.acs.org.