Synthesis and Evaluation of 1,8-Disubstituted-Cyclam/Naphthalimide Conjugates as Probes for Metal Ions

Article abstract of DOI:10.1002/open.201600010

Fluorescent molecular probes for metal ions have a raft of potential applications in chemistry and biomedicine. We report the synthesis and photophysical characterisation of 1,8-disubstituted-cyclam/naphthalimide conjugates and their zinc complexes. An ef

Full text of DOI:10.1002/open.201600010

DOI: 10.1002/open.201600010
Synthesis and Evaluation of 1,8-Disubstituted-Cyclam/
Naphthalimide Conjugates as Probes for Metal Ions
[a]
[a]
[a]
[b]
[a]
Joseph K.-H. Wong, Sandra Ast, Mingfeng Yu, Roman Flehr, Andrew J. Counsell,
[a, c]
[a]
[a]
[a]
Peter Turner,
Patrick Crisologo, Matthew H. Todd,* and Peter J. Rutledge*
Fluorescent molecular probes for metal ions have a raft of po-
tential applications in chemistry and biomedicine. We report
the synthesis and photophysical characterisation of 1,8-disub-
stituted-cyclam/naphthalimide conjugates and their zinc com-
plexes. An efficient synthesis of 1,8-bis-(2-azidoethyl)cyclam
has been developed and used to prepare 1,8-disubstituted tri-
azolyl-cyclam systems, in which the pendant group is connect-
ed to triazole C4. UV/Vis and fluorescence emission spectra,
zinc binding experiments, fluorescence quantum yield and life-
time measurements and pH titrations of the resultant bis-
naphthalimide ligand elucidate a complex pattern of photo-
physical behaviour. Important differences arise from the inclu-
sion of two fluorophores in the one probe and from the varia-
tion of triazole substitution pattern (dye at C4 vs. N1). Intro-
ducing a second fluorophore greatly extends fluorescence life-
times, whereas the altered substitution pattern at the cyclam
amines exerts a major influence on fluorescence output and
metal binding. Crystal structures of two key zinc complexes
evidence variations in triazole coordination that mirror the so-
lution-phase behaviour of these systems.
1. Introduction
The development of fluorescent molecular probes for biologi-
cally significant metal ions has attracted considerable attention
[
1–8]
in recent years.
Many such probes incorporate a naphthali-
mide fluorophore (1H-benzo[de]isoquinoline-1,3(2H)-dione,
Figure 1), owing to the favourable properties of this system,
which include high photostability, bright fluorescence and
[
9–14]
long Stokes shifts.
Figure 1. Cyclam–naphthalimide conjugates studied as fluorescent probes:
the original mono-naphthalimide compound 1, in which the dye is connect-
We have previously reported a series of macrocyclic molecu-
lar probes based on a cyclam–triazole–naphthalimide recep-
[15]
ed to triazole N1; the ‘reversed’ probe 2 with the dye connected to tria-
[16]
[17]
zole C4; bis-naphthalimide 3 with original connectivity; and the new
bis-naphthalimide 4, which has the reversed connectivity in this work.
[
15]
tor–linker–fluorophore system, with mono-naphthalimides 1
and 2 and the symmetrical bis-naphthalimide 3. In these
and related systems,
[
16]
[17]
[18–25]
the triazole linker was generated by
I
[26–27]
a Cu-catalysed azide–alkyne cycloaddition reaction.
The
cyclam core, while also installing the coordinating triazole as
[
5]
versatility of this click reaction allows the quick modular at-
tachment of a variety of fluorophores to the tetradentate
a potential fifth metal-binding site.
In the original cyclam–triazole–naphthalimide probe 1, the
fluorescent dye is connected to triazole N1: ligand 1 was made
[
15]
[
a] J. K.-H. Wong, Dr. S. Ast, Dr. M. Yu, A. J. Counsell, Dr. P. Turner, P. Crisologo,
Prof. Dr. M. H. Todd, Prof. Dr. P. J. Rutledge
School of Chemistry, The University of Sydney
Sydney, NSW 2006 (Australia)
by clicking a naphthalimide-azide with a propargyl cyclam.
This system demonstrated high selectivity for zinc(II) over
other metal cations and a sixfold enhancement of fluorescence
intensity in response to zinc binding in aqueous solution
(1.0 mm HEPES buffer at pH 7). It was rationalised that, in the
free ligand, photoinduced electron transfer (PET) from the
cyclam/triazole unit to the naphthalimide fluorophore quench-
es fluorescence; when zinc binds, this PET is retarded and the
fluorescence output is enhanced. Ligand 1 also showed mod-
erate fluorescence quenching in response to copper(II) and
E-mail: peter.rutledge@sydney.edu.au
matthew.todd@sydney.edu.au
[
b] Dr. R. Flehr
Institute for Chemistry, University of Potsdam
Karl-Liebknecht St. 24—25, 14476 Potsdam (Germany)
[
c] Dr. P. Turner
Crystal Structure Analysis Facility, The University of Sydney
Sydney, NSW 2006 (Australia)
[
15,22]
Supporting Information and the ORCID identification number(s) for the
author(s) of this article can be found under http://dx.doi.org/10.1002/
open.201600010.
mercury(II).
The related probe 2 was made by combining an ethynyl-
naphthalimide with an azidoethyl-cyclam, so the dye is con-
ꢀ
2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
[16]
nected to triazole C4 in the resulting ligand. This simple re-
This is an open access article under the terms of the Creative Commons
Attribution License, which permits use, distribution and reproduction in
any medium, provided the original work is properly cited.
versal of triazole connectivity wrought a tenfold brighter signal
in response to zinc(II), as it enables an alternative mode of
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fluorescence quenching; twisted intramolecular charge transfer
(
TICT) quenching predominates in the excited state of 2, and is
[16,22,28]
interrupted by zinc coordination.
The emission wave-
length and Stokes shift of ligand 2 vary significantly with sol-
vent polarity: lem =458 nm in aqueous solution versus 437 nm
[
16,22,28]
in acetonitrile.
A solvent-dependent shift in emission
wavelength like this—specifically a redshift in a solvent of
higher polarity—is consistent with an increase in dipole
moment in the excited state and a TICT quenching mechanism
[
29,30]
with ligand 2.
Incorporating a second triazolyl-naphthalimide unit by com-
bining the naphthalimide-azide with a symmetrical bis-prop-
argyl cyclam afforded ligand 3, which displayed metal-ion re-
sponses similar to 1: significant fluorescence enhancement in
the presence of zinc(II) and moderate quenching with
copper(II) or mercury(II) bound. A 12.7-fold increase in the
emission maximum of 3 was observed upon adding one equiv-
alent of zinc(II)—double the enhancement reported with
probe 1. However, for reasons of solubility, ligand 3 was evalu-
ated only in a mixed solvent system of water and acetonitrile
[17]
(
7:3, buffered at pH 7 with 50 mm HEPES buffer).
We have recently observed significant solvent dependence
[16]
in the fluorescence properties of ligand 2, as noted above,
and wished to revisit the fluorescence response of ligand 3 in
light of this observed solvatochromaticity. Moreover, what hap-
pens when the structural properties of 2 and 3 are combined
and two C4-linked naphthalimides are incorporated in a 1,8-
disubstituted cyclam-triazole-naphthalimide? Herein, we report
the synthesis and characterisation of the ‘reversed’ bis-naph-
thalimide probe 4, plus a more detailed photophysical investi-
gation of bis-naphthalimide 3, to determine the intrinsic fluo-
rescence properties of these probes and the basis of their re-
sponse to metal-ion binding.
Scheme 2. Synthesis of reversed bis-naphthalimide probe 4: a) CuSO
sodium ascorbate, THF/H O (7:3), 508C, 16 h, 93%; b) TFA/CH Cl /H
90:5:5), rt, 1 h, 81%.
4 2
·5H O,
2
2
2
2
O
(
Cyclam 5 was first combined with formaldehyde to generate
[
31]
bridged derivative 6 by following a literature method. At-
tempts to alkylate 6 directly with toluenesulfonic acid-2-
azidoethyl ester (TsOCH CH N ), built on the established route
2
2
3
[
16,18]
to 2,
were unsuccessful; unreacted starting material 6 was
recovered. So, 6 was instead alkylated with tert-butyl bromoa-
[
31]
cetate, adapting Pandya’s procedure, to give the known 1,8-
trans tert-butyl ester salt 7. A high-yielding, one-pot procedure
was developed to induce cleavage of the bis-aminal bridges of
2
. Results and Discussion
2.1. Synthesis of 1,8-Disubstituted-Cyclam Derivatives
7
and Boc protection of the liberated secondary amines, using
A reliable route to compound 3 has been developed previous-
aqueous sodium hydroxide and di-tert-butyl dicarbonate to
generate intermediate 8. The tert-butyl esters were reduced by
[16–17]
ly and was used to prepare ligand 3 for this work;
com-
pound 4 has not been reported previously, and an effective
synthetic path to this probe was developed as part of the cur-
rent study (Schemes 1 and 2).
using LiAlH
, giving diol 9 in high purity and near-quantitative
4
yield (99%).
It had been envisaged that conversion of diol 9 to a bis-me-
sylate followed by azide displacement would give the desired
bis-azide intermediate 10. However, this sequence of reactions
proved highly unreliable with yields ranging from 6 to 60%
and averaging about 30% over the two steps. Several by-prod-
ucts were observed by using TLC during the mesylation reac-
tion, with some evidence that the reaction conditions were
promoting chlorination of 9 (OHs replaced by Cls) and partial
Boc deprotection. Treating diol 9 with diphenylphosphoryl
[
32]
azide (DPPA) and sodium azide in refluxing THF generated
the desired bis-azide 10 in high yield (75%). Attempts to use
DPPA alone—without sodium azide—were not successful and
led to recovery of starting material 9, whereas experiments
using the corresponding diphenylphosphoryl chloride with
sodium azide proceeded, but only with poor yield (ca. 25%).
Scheme 1. Synthesis of bis-azido cyclam intermediate 10: a) formaldehyde,
H
2
O, 08C, 3 h, 89%; b) tert-butyl bromoacetate, MeCN, rt, 16 h, 81%; c) 2.5m
NaOH, MeOH, rt, 1 h (i), Boc O, 2.5m NaOH, MeOH, rt, 16 h (ii), 87% over
, THF, 08C, 1 h, 99%; e) DPPA, NaN , DBU, THF,
2
steps (i) and (ii); d) LiAlH
reflux, 16 h, 75%.
4
3
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To complete the synthesis of 4 (Scheme 2), bis-azide 10 was
[33]
reacted with N-ethyl-4-ethynyl-1,8-naphthalimide 11
under
modified click conditions used previously to make ligands 1, 2
[
15–17]
and 3;
this afforded Boc-protected bis-naphthalimide 12
in excellent yield (93%). The Boc groups were removed by
[34]
using a TFA/CH Cl /H O (90:5:5) solvent system to give the
2
2
2
TFA salt of ligand 4 in high yield (81%). The salt was converted
to the free amine 4, immediately prior to spectroscopic experi-
ments, using Ambersep 900 hydroxide form resin and a proce-
Figure 2. Fluorescence enhancement factors of A) probe 3 (2 mm,
ex =350 nm) with different metal ions (20 mm) in HEPES buffer (10 mm,
[21]
dure that has been reported previously.
l
Phenyl–triazolyl–cyclam derivatives 13 and 14 were pre-
pared as simplified analogues of 3 and 4, respectively, for crys-
tallographic analysis (Scheme 3). Compound 13 was made by
pH 7.4); B) probe 4 (2 mm, lex =357 nm) with different metal ions (20 mm) in
HEPES buffer (10 mm, pH 7.4).
2.3. Metal Binding Experiments in Acetonitrile
The fluorescence response of ligands 3 and 4 to metal binding
changes markedly in acetonitrile. In this solvent system, both
II
ligands exhibit a significant turn-on response to Zn , and more
detailed mechanistic studies were undertaken in this medium.
The UV/Vis absorption spectrum of probe 3 in acetonitrile
shows the expected naphthalimide absorption maximum cen-
tred at 343 nm and a shoulder at 333 nm (black line, Fig-
ure 3A). For reversed probe 4, the absorption maximum is cen-
tred at 362 nm with a weak shoulder at 348 nm (black line, Fig-
ure 3B). This redshift of 19 nm relative to 3 can be attributed
to the extended aromatic system that results from the reversed
triazole connectivity, in which the naphthalimide is connected
Scheme 3. Synthesis of model compounds 13 and 14: a) PhN
sodium ascorbate, THF/H O (7:3), 508C, 16 h, 61%; b) TFA/CH
90:5:5), rt, 16 h, 98%; c) Ambersep 900 hydroxide form resin, EtOH, rt,
3
, CuSO
4 2
·5H O,
2
2
Cl /H
2
2
O
(
1
0 min (i), Zn(ClO
4
)
2
·6H
·5H
2
O, EtOH, reflux, 16 h (ii), 41% over steps (i) and (ii);
O, sodium ascorbate, THF/H O (7:3), 508C, 16 h, 74%;
d) PhCꢀCH, CuSO
4
2
2
e) HCl, dioxane, rt, 16 h (i), Ambersep 900 hydroxide form resin, MeOH, rt,
0 min (ii), HPLC (0.1% TFA in H O and MeCN) (iii), 69% over steps (i)–(iii);
1
2
f) Ambersep 900 hydroxide form resin, EtOH, rt, 10 min (i), Zn(ClO
EtOH, reflux, 16 h (ii), 68% over steps (i) and (ii).
4 2 2
) ·6H O,
uniting the doubly protected, doubly propargylated cyclam
[17,35]
15
and phenylazide to afford Boc-protected ligand 16, fol-
lowed by TFA-mediated deprotection; compound 14 was syn-
thesised from the cyclam-derived bis-azide 10 and phenylace-
tylene to afford Boc-protected ligand 17, followed by HCl-
mediated deprotection and HPLC purification. Ligands 13 and
14 were each converted to the corresponding zinc(II) com-
plexes by adapting the method reported previously for related
[
34]
systems.
2.2. Metal Binding Experiments in Aqueous Solution
The response of probes 3 and 4 to a wide variety of metal ions
was first evaluated in aqueous solution (10 mm HEPES buffer
I
II
II
II
II
II
II
III
II
I
at pH 7.4); the ions Ag , Ba , Ca , Cd , Co , Cu , Fe , Fe , Hg , K,
I
II
II
I
II
II
I
II
Li, Mg , Mn , Na , Ni , Pb , Rb and Zn were all screened with
each ligand (Figure 2). Both ligands proved largely unrespon-
sive under these conditions; both 3 and 4 gave only a weak
II
fluorescence enhancement in the presence of Zn (red line,
II
II
Figure 2) and no discernible response to either Cu or Hg . This
is an intriguing contrast to the mono-naphthalimide ligands
II
Figure 3. Changes in UV/Vis absorption spectra upon addition of Zn :
A) probe 3 and B) probe 4 (50 mm ligand solution in MeCN, with addition of
Zn(ClO ) solution (5 mm in MeCN), added in steps of 0.1 equivalents up to
4 2
total addition of 1.1 equivalents).
1
and 2, both of which exhibit a strong fluorescence enhance-
II
ment in the presence of Zn (ca. six fold) and significant fluo-
II
II [15–16,22]
rescence quenching in the presence of Cu and Hg .
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[22]
to triazole C4 (versus N1 in probe 3). Monitoring changes in
the absorption spectra of ligand 3 in response to increasing
II
Zn concentrations reveals an isosbestic point at 350 nm and
significant changes in the naphthalimide band shape, as well
as a slight increase in total absorbance. As the concentration
II
of Zn approaches one equivalent, the shoulder at 333 nm
transforms into a distinct peak as the new local maximum,
giving rise to a final spectrum that contains two peaks at 333
II
and 343 nm. In contrast, the addition of Zn to probe 4 triggers
a discernible blueshift of the absorption maximum from 362 to
3
48 nm (Figure 3B), accompanied by an increase in naphthali-
mide absorbance. These changes in absorption profile give rise
to an isosbestic point at 359 nm. The shape of these spectra
also changes throughout the titration, indicating a conforma-
tional change of the chromophore when the ligand complexes
II
Zn .
These differences in the absorption spectra of the zinc(II)
complexes of probes 3 and 4 indicate differences in the coordi-
nation behaviour of the two ligands. This could be rationalised
by invoking coordination of both triazoles to the bound metal
ion in the case of ligand 4, but only ‘one-armed’ coordination
with ligand 3, with the second pendant chromophore hanging
free. To test this hypothesis, further studies were undertaken
to investigate the excited state of these ligands, and to charac-
terise the zinc complexes crystallographically.
Figure 4. Changes in fluorescence spectra in response to increasing zinc(II)
concentrations: A) probe 3 (lex =350 nm) and B) probe 4 (lex =359 nm).
2.4. Fluorescence Emission Spectra
4 2
(2 mm ligand solution in MeCN with addition of Zn(ClO ) (400 mm solution in
Both probes 3 and 4 were titrated with increasing concentra-
tions of Zn in acetonitrile while exciting at their respective iso-
MeCN) in steps of 0.1 equivalent up to total addition of 1.1 equivalents).
II
sbestic points (350 and 359 nm) and monitoring the fluores-
cence emission (Figure 4). For ligand 3, the addition of one
equivalent of Zn brought a 12.5-fold fluorescence enhance-
of the zinc(II) titration of probe 3 (Figure 4A) represents a mix-
ture of both free and coordinated chromophore and, therefore,
fails to achieve maximal fluorescence enhancement.
II
ment with a blueshift of the emission maximum, from 428 nm
II
for 3 to 397 nm for 3·Zn ; these spectra show an isosbestic
The inconsistent changes in fluorescence intensity of 3
throughout the zinc(II) titration can also be rationalised by
considering the presence of multiple transitions in the excited
state. The changes observed in the emission profile and the
blueshift of the peak maxima underline the presence of at
least two chromophore-based transitions in excited ligand 3.
The presence of two transitions of very similar energy has
been shown previously by theoretical calculations of a triazol-
point at 448 nm and a weak shoulder at 428 nm (Figure 4A).
With ligand 4, on the other hand, adding one equivalent of
II
Zn produced a 22-fold fluorescence enhancement accompa-
nied by a blueshift of the emission maximum, from 433 nm for
II
4
to 420 nm for 4·Zn ; these spectra displayed an isosbestic
point at 446 nm, formed as a result of changes to the band
shape (Figure 4B).
[
36]
Plotting the integrated emission spectra versus equivalents
yl-naphthalimide chromophore with N1-connectivity.
That
II
of Zn added confirms a 1:1 stoichiometry for the complexa-
this is seen only for ligand 3—and not with 4—presumably re-
sults from the different naphthalimide/triazole connectivity, as
well as the strong electron-withdrawing character of the naph-
thalimide itself. Thus, generating an electron-rich centre at the
naphthalimide core through the formation of an N-naphthali-
mide bond leads to strong internal charge transfer (ICT).
II
tion of 4 with Zn in acetonitrile (Figure S1). Interestingly, the
corresponding plot of integrated fluorescence intensities of 3
II
versus Zn concentration does not show a similarly clear trend
in complex formation (Figure S2). However, the 1:1 stoichiome-
II
try of the 3·Zn complex was confirmed by plotting the ab-
sorption intensity at the 343 nm maximum (Figure S3). This dis-
crepancy in outcome between the two spectroscopic methods
indicates the existence of multiple excited-state species in
ligand 3. Revisiting the shape of the fluorescence emission
2
.5. Fluorescence Quantum Yields and Lifetimes
Fluorescence quantum yields (F) and lifetimes (t) were deter-
II
II
spectrum of 3·Zn reveals that the shoulder at 428 nm could,
mined for ligands 3 and 4, and their zinc complexes 3·Zn and
4·Zn (Table 1), to better understand the differences in the
spectroscopic features of these ligands and their different re-
sponses to zinc(II) complexation.
II
in fact, result from non-coordinated chromophore overlapping
partially with the zinc(II) complex formed upon coordination of
the other pendent arm. As a consequence, the final spectrum
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3
.3 times higher than that of uncomplexed 3 (0.026), whereas
Table 1. Overview of spectral properties of probes 1, 2, 3 and 4.
II
F for 4·Zn (0.473) is more than 30 times higher than that of
free ligand 4 (0.015). The zinc complexes of the mono-substi-
tuted ligands 1 and 2 in acetonitrile also have higher F values
than the free ligands: F for 2·Zn (0.550) is more than 60 times
higher than that of free ligand 2 (0.009), whereas the F in-
crease of 1·Zn (0.030) relative to free ligand 1 (0.003) is exactly
tenfold. As seen in water, the quantum yield of ligand 3 (0.026)
in acetonitrile is close to that of 1·Zn (0.030), indicating
a much weaker fluorescence quenching mechanism in free
ligand 3 compared to free ligand 1, even in the non-protic sol-
vent. Thus, it is apparent that appending a second fluorophore
affects the efficiency of fluorescence quenching in ligand 3,
but not in ligand 4, in which the naphthalimide is attached to
triazole C4, and there is a two-carbon spacer between cyclam
and the triazole.
[
b]
l
abs [nm]
l
em [nm]
F
t
[ns]
HEPES buffer
II
1
347
347
358
357
344
343
358
358
416
407
458
435
424
0.003 0.16
0.065 0.33
0.140 1.00
0.760 2.02
II
1·Zn
2
2·Zn
3
3·Zn
4
4·Zn
II
II
II
II
0.066 3.36 (420 nm), 6.53 (460 nm)
[a]
II
424, 467 0.068 3.51 (420 nm), 6.64 (460 nm)
477
472
0.027 3.90
0.033 3.84
MeCN
1
342
342
357
357
406
398
437
437
0.003 0.22
0.030 0.60
0.009 0.82
0.550 3.30
II
1·Zn
2
2·Zn
3
3·Zn
4
4·Zn
II
II
II
[a]
333 , 343 428
333, 343 397, 428 0.087 3.36 (400 nm), 5.89 (420 nm)
362
348
0.026 3.63 (400 nm), 6.29 (420 nm)
[a]
[a]
To investigate the nature of the excited state species in free
ligand 3 and its zinc(II) complex, the fluorescence lifetime t
was monitored at two different wavelengths. The aim of this
experiment was to determine if there are two very close transi-
tions in the excited chromophore of this system, if—in the
433
420
0.015 1.52 (420 nm), 2.19 (440 nm)
0.473 3.61 (420 nm), 3.88 (440 nm)
[
a] Shoulder. [b] Averaged fluorescence t from the bi-exponential decay
profile.
II
complex 3·Zn —only one of the pendant arms coordinates to
the metal, or if a combination of both effects is at play. For
ligand 3 in acetonitrile, this meant exciting at its emission max-
imum (ca. 400 nm) and at the shoulder (ca. 420 nm), which
gave rise to two different fluorescence lifetimes; t is 3.63 ns
when exciting at 400 nm, but almost double that (6.29 ns)
when exciting at 420 nm. This is strong evidence for the pres-
ence of two transitions in the excited fluorophore of 3. The
In aqueous buffer, the quantum yield of the free ligand 3
0.066) is more than twice that of ligand 4 (0.027). More strik-
(
ingly, the F of ligand 3 is 22 times higher than that of the cor-
responding mono-naphthalimide ligand 1 (0.003); not only is
the F of uncomplexed 3 considerably higher than that of un-
II
complexed 1, it also matches the value measured for the 1·Zn
II
complex (0.065), indicating the absence of an efficient fluores-
cence-quenching process in free ligand 3. In contrast, the
quantum yield of ligand 4 (0.027) is significantly smaller than
that of the corresponding mono-naphthalimide 2 (0.140), and
fluorescence lifetimes of the complex 3·Zn are very similar to
those of the free ligand: 3.36 ns at 400 nm and 5.89 ns at
420 nm. This is in contrast to the quantum yield and steady-
state emission measurements, where significant changes are
apparent in response to zinc binding. This apparent contradic-
tion supports the proposal that one fluorophore remains unaf-
fected by zinc coordination—that is not coordinated to the
metal—and suggests that this uncoordinated triazolyl-naph-
thalimide unit is masking the t of the complexed fluorophore
II
much below that of the 2·Zn complex (0.760).
The quantum yields of the zinc(II) complexes in aqueous so-
lution reflect the observations made in steady-state emission
studies above; zinc binding to ligands 3 and 4 triggers much
smaller changes in fluorescence emission (0.066!0.068 and
II
0
.027!0.033, respectively) compared to the corresponding
in 3·Zn . The corresponding experiment in aqueous buffer re-
mono-naphthalimide ligands 1 and 2 (0.003!0.065 and
.140!0.760, respectively). As discussed above, the difference
in quantum yield between mono-substituted ligand 1 and
veals a similar result, that is, different lifetimes are observed at
different excitation wavelengths, and the t values recorded in
aqueous solution similar to those measured in acetonitrile:
3.36 and 6.53 ns for the free ligand, 3.51 and 6.64 ns for the
zinc(II) complex.
0
II
1
·Zn results from suppression of PET upon zinc binding,
II
whereas the variation between 2 and 2·Zn arises primarily
[22]
through a TICT mechanism. For bis-naphthalimide probes 3
and 4, however, the situation is not so clear. It appears that
there are different effects happening in parallel, which also in-
terfere with each other.
For comparison, the fluorescence lifetimes of ligand 4 were
also monitored at two different wavelengths in acetonitrile,
and two similar values were observed: t is 1.52 ns exciting at
420 nm and 2.19 ns at 440 nm. The lifetimes of the complex
II
In acetonitrile, the quantum yield of free ligand 3 (0.026) is
again higher than that of 4 (0.015), but by a lesser margin, and
only around nine times higher than that of ligand 1 (0.003), re-
flecting qualitatively the results found in water. However, in or-
ganic solvent, the quantum yield of 4 (0.015) is higher than
that of the mono-naphthalimide analogue 2 (0.009); a direct
contrast to the aqueous conditions. Zinc binding to the bis-
naphthalimide ligands triggers much more significant fluores-
4·Zn are roughly double those of the free ligand: 3.61 ns at
420 nm and 3.88 ns at 440 nm. The changes upon metal bind-
ing are qualitatively in agreement with the differences in
steady-state emission and quantum yield measurements for 4
II
II
versus 4·Zn , but in contrast to 3 versus 3·Zn .
Interestingly, attaching the second triazolyl-naphthalimide
unit to cyclam leads to longer fluorescence lifetimes for bis-
substituted probes 3 and 4 compared to the mono-substituted
analogues 1 and 2. Appending the second chromophore
II
cence enhancements in acetonitrile: F for 3·Zn (0.087) is
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brings an order of magnitude increase in t in the case of 3
compared to 1 (a 21-fold increase in aqueous solvent, 16.5-fold
in acetonitrile) and roughly doubled t for 4 relative to ligand 2
(
3.9 times larger in aqueous solvent, 1.9 times in acetonitrile).
The lifetimes of the zinc(II) complexes are also longer for the
bis-substituted probes 3 and 4, though less significantly so.
Thus, with ligand 3, multiple effects are operating in the free
ligand and its zinc(II) complex. The intrinsically high quantum
yield of the free ligand and the fact that only one of the two
triazoles coordinates to the metal upon zinc binding (vide
infra) both contribute to the absence of a significant response
to zinc(II) in aqueous buffer, and the relatively poor turn-on re-
sponse in acetonitrile. The presence of two very close transi-
tions in the excited state is a possible explanation for the
much higher quantum yield of 3 compared to both the mono-
substituted analogue 1 and the structurally related bis-naph-
thalimide derivative 4 in both solvents. With ligand 4, however,
the quantum yield is not intrinsically high, nor is mono-triazole
coordination evident. So, the question remains: why is a more
significant fluorescence turn-on response to zinc binding not
seen for ligand 4 in water?
2.6. pH Dependence of Zinc Response
To address the question raised above, pH-dependent measure-
ments were performed. Emission spectra of 3 and 4 in water
were recorded at a range of pH values, before and after the
II
addition of Zn ; the spectra were integrated and plotted
against pH (Figure 5). Interestingly, both ligands as well as
II
their Zn complexes exhibited contrasting behaviour.
The fluorescence intensity of ligand 3 is greater at lower pH
values, when the cyclam nitrogen atoms are protonated; the
fluorescence enhancement at pH 2 is about threefold com-
pared to that at pH 10. When zinc(II) is added to the mix, the
Figure 5. A) Variation of fluorescence enhancement for 3 (black squares) and
II
3·Zn (red circles) with pH; B) fluorescence enhancement factors for 4 (black
II
squares) and 4·Zn (red circles) at different pHs. Error bars represent the
standard deviation over 3 replicates.
II
fluorescence output of 3·Zn is greater than 3 alone at pH>
II
6
.5. At pH 9 (pH of maximal fluorescence), 3·Zn exhibits an
almost fourfold fluorescence enhancement over the free ligand
. Below pH 6.5, the addition of zinc(II) has no significant effect
fluorophore, and mapped the influence of pH on metal bind-
ing. These collected observations are consistent with the pos-
tulation that PET from the amines in an uncomplexed, non-
protonated cyclam unit quenches fluorescence of the append-
ed fluorophore, whereas protonation or metal coordination in-
hibits this PET and—at least partially—restores fluorescence.
Crucially, the pH at which ligand 4 begins to show a discerni-
ble response to metal-ion binding (ca. 8.0) is a full pH unit
above the equivalent value for ligand 3 (ca. 7.0), and both of
these values are significantly higher than those measured pre-
viously for the corresponding mono-naphthalimide ligand
3
on the fluorescence output of 3, suggesting that zinc complex-
ation is inhibited in this pH range.
Ligand 4 also displays higher fluorescence enhancement at
lower pH values. The fluorescence enhancement is greater
with 4 than that with 3 at the corresponding acidic pH values,
and this enhancement decreases more substantially at higher
pH values. For ligand 4, the fluorescence enhancement at
pH 10 is about 5.5-fold weaker than that at pH 2. Again, the
addition of zinc(II) has little or no effect on the fluorescence
output of 4 at acidic pH values, up to pH 7.4. Above 7.4, for-
[
15]
1 (4.5), and coumarin analogue of ligand 2 (4.5), which con-
II
[18]
mation of the 4·Zn complex does trigger an increased fluores-
tains the same ion-binding motif as 2. Thus, it appears that
cence output, to a maximal twofold enhancement over the
free ligand at pH 9.
the low fluorescence quantum yield of ligand 4 relative to 2 in
HEPES buffer at pH 7.4, and the weak response of 4 to zinc(II)
binding under these conditions, are caused by the higher pKa
of the cyclam-amines in the bis-naphthalimide system com-
pared to those in the mono-naphthalimide.
The variation of fluorescence intensity with pH seen with li-
gands 3 and 4 is consistent with previous observations when
[
15]
[18]
using ligand 1, the coumarin analogue of 2, and the an-
thracenyl-cyclam fluoro-ionophores studied by Fabbrizzi
[
37]
et al.
Fabbrizzi et al. characterised fluorescence changes
versus pH in cyclam ligands bearing an appended anthracene
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2
.7. Crystal Structure Determination
reveals that only one of the triazole-phenyl arms of the bis-sub-
stituted system coordinates to the metal centre. It does so
with N3 of the pendant 1,2,3-triazole [N(5) in the crystallo-
graphic numbering scheme] coordinating to the metal ion at
an equatorial position. The equatorial coordination bond
lengths are 2.049(3) Š [Zn(1)ÀN(1)], 2.056(3) Š [Zn(1)ÀN(3)],
and 2.043(3) Š [Zn(1)ÀN(5)] and are close to the calculated
The significant change in absorption behaviour of 3 in the
presence of increasing concentrations of zinc(II) highlights the
possibility of additional conformational changes in the ground
state. Attempts to study these conformational changes using
1
H NMR spectroscopy were hampered by the different solubili-
[
45]
ty profiles of the free ligands 3 and 4 compared to the com-
ideal of 2.07 Š.
The axial positions of [Zn(13)](ClO ) are occupied by macro-
II
II
plexes 3·Zn and 4·Zn , and the fact that the NMR spectra of
the zinc complexes are complicated by the aza-macrocycle
adopting multiple geometries upon metal binding (see Sec-
tions 14 and 15 in the Supporting Information). Thus, single-
crystal X-ray diffraction studies of the analogous 1,8-disubsti-
tuted cyclam–triazole–phenyl model systems 13 and 14 were
undertaken. Ball-and-stick depictions of the resultant structures
4
2
cyclic nitrogens N(2) and N(4), with an N(2)ÀZn(1)ÀN(4) angle
of 174.06(12)8 and bond lengths of 2.287(3) Š [Zn(1)ÀN(2)] and
2.196(3) Š [Zn(1)ÀN(4)]. These metrics are consistent with pre-
vious observations that MÀN distances are generally longer for
[
46]
tertiary than for secondary amines in 3d-metal complexes.
The observed Zn–triazole interaction is consistent with the
prominent shift in the absorption maximum observed as the
Zn titration of 3 proceeds (Figure 3A), owing to a change in
the composition of the combined transitions at 333 and
343 nm, which arise from the presence of both mono-coordi-
nated and free naphthalimide side-chain species.
[
Zn(13)](ClO ) and [Zn(14)](ClO ) are shown in Figure 6; crys-
4 2 4 2
tallographic data (Table S1), selected bond lengths (Table S2)
and ORTEP depictions (Figure S4 and S5) are presented in the
[40]
Supporting Information.
The [Zn(13)](ClO4)2 complex (Figure 6A) adopts a strained
trigonal bipyramidal pentacoordinate geometry around the
zinc(II) centre, typical of previously reported zinc(II) cy-
Compound [Zn(14)](ClO4)2 contains the reversed triazole
connectivity. This complex exhibits the more unusual cis-V con-
figuration of the macrocycle, with the zinc(II) adopting a dis-
torted octahedral coordination geometry. The structure dis-
plays mutual cis coordination of both pendant triazole-phenyl
arms, consistent with the photophysical observations made
using the corresponding naphthalimide ligand 4 (Figure 3B).
The reversed connectivity imposes a difference in the nature of
triazole coordination; in contrast to the triazole N3 coordina-
tion observed in [Zn(13)](ClO ) and reflecting greater ligand
[
17,41]
clams.
The orientation of the macrocyclic N-substituents
indicates that complex [Zn(13)](ClO ) adopts the trans-I con-
figuration; this being the preferred stereochemistry of pen-
4
2
[
42]
[43,44]
tacoordinate cyclam complexes.
Importantly, this structure
4
2
flexibility, in [Zn(14)](ClO ) , the triazole coordinates through
4
2
N2. We have previously found the same behaviour in copper(II)
[
18]
and mercury(II) complexes of related compounds.
We have also previously reported a crystal structure for the
bis-triazolyl-benzyl pendant analogue of [Zn(13)](ClO ) —
4
2
where benzyl replaces phenyl—in which the macrocycle
adopts a trans-III configuration and both triazole pendants are
[
17]
coordinated to the metal through triazole N3. Moreover, the
two triazoles occupy the opposite axial sites within an octahe-
dral coordination sphere. Comparison with the structures of
[
Zn(13)](ClO ) and [Zn(14)](ClO ) reported here suggests that
4 2 4 2
the electronics of the triazole are important in determining the
mode of coordination, as much as the nature of the linker be-
tween the macrocycle and the triazole (i.e. whether n=1 or 2
in the cyclam–(CH ) –triazole connection).
2
n
Further discussion of these crystal structures and their rela-
tionship to others previously described elsewhere is presented
in the Supporting Information (Section 7).
3. Conclusions
An efficient route to 1,8-bis-(2-azidoethyl)cyclam 10 has been
developed and used to prepare 1,8-disubstituted cyclam sys-
tems containing the ‘reversed’ triazole topology, that is, dye/
pendant group connected to triazole C4 (versus N1 in the
‘original’ systems). Photophysical characterisation of bis-naph-
thalimide ligands 3 and 4 and their responses to metal ions
Figure 6. Ball-and-stick depictions of the crystal structures of
A) [Zn(13)](ClO and B) [Zn(14)](ClO (exogenous perchlorates omitted for
clarity) generated with X-Seed and POV-Ray. Non-hydrogen-bonding hy-
drogen atoms omitted.
4
)
2
4 2
)
[
38]
[39]
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ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

reveal complex behaviour and important differences between
the two, and between these ligands and the corresponding
mono-naphthalimides 1 and 2. Notably, introducing the
second chromophore greatly extends fluorescence lifetimes of
dicarbonate (2.98 g, 13.7 mmol) in MeOH (5 mL) and stirring was
continued at rt for 16 h. MeOH was removed, and the product was
filtered and washed with H O (100 mL) to afford the Boc-protected
2
product 8 as an off-white solid (2.13 g, 87%). m.p.: 103–1048C.
1
H NMR (400 MHz, CDCl ): d=1.44 (s, 18H), 1.45 (s, 18H), 1.66–1.75
3
3
relative to 1, and 4 relative to 2. Also, differences in the ba-
(
4
2
m, 4H), 2.65 (t, 4H, J 5.5), 2.79 (t, 4H, J 5.5), 3.24 (s, 4H), 3.28 (br s,
sicity of the triazole and cyclam nitrogen atoms with changing
substitution exert a significant influence on fluorescence
output and metal binding. Single-crystal X-ray structures of
zinc complexes of bis-phenyl-triazolyl analogues 13 and 14
show that, although both triazoles coordinate to the metal in
the ‘reversed’ compound 14, only one is bound to zinc in 13,
which are consistent with the photophysical behaviour of
these systems in solution.
13
H), 3.40 ppm (br s, 4H). C NMR (100 MHz, CDCl ): d=27.2, 28.2,
3
8.5, 46.4, 47.1, 52.3, 53.4, 57.3, 79.2, 80.8, 155.7, 170.7 ppm. LRMS
+
(ESI+): m/z 629.5 ([M+H] , 100%). HRMS (ESI+): m/z Calcd. for
C
nmax cm : 2976, 2932, 1732, 1691, 1410, 1366, 1250, 1155.
+
+
H
32
N
O
[M+H]
629.4484, found 629.4481. FTIR (ATR)
61
4
À1
8
Di-tert-butyl 4,11-bis(2-hydroxyethyl)-1,4,8,11-tetraazacyclo-
tetradecane-1,8-dicarboxylate, 9
LiAlH (1.0m, 4.40 mL, 4.40 mmol) was added to a chilled solution
of 8 (694 mg, 1.10 mmol) in THF (10 mL). The reaction mixture was
stirred at 08C for 1 h and quenched by the sequential slow addi-
4
Experimental Section
Safety note: Sodium azide, organic azides and perchlorate salts of
metal complexes with organic ligands are potentially explosive.
Only small amounts of material should be prepared and these
should be handled with caution.
tion of EtOAc (1 mL) and H O (1 mL). Rochelle salt (sat., 10 mL) was
2
added and the volatiles were removed. The product was extracted
with EtOAc (2”25 mL), and the extracts were combined, dried
(
Na SO ) and concentrated under reduced pressure to afford the
2 4
General experimental procedures are detailed in the Supporting
Information.
bis-alcohol 9 as a yellowish oil (532 mg, 99%). The crude product
was used in subsequent reactions without purification. H NMR
1
(
400 MHz, CDCl ): d=1.46 (s, 18H), 1.77 (qn, 4H, J 6.9), 2.49 (t, 4H,
3
J 6.2), 2.57 (t, 4H, J 5.2), 2.62 (t, 4H, J 6.0), 3.25–3.40 (m, 8H),
1
,4,8,11-Tetraazatricyclo[9.3.1.1(4,8)]hexadecane, 6
13
3
4
4
.57 ppm (t, 4H, J 5.2). C NMR (100 MHz, CDCl ): d=27.5, 28.4,
3
7.2, 47.5, 52.1, 53.9, 56.9, 59.0, 79.6, 155.9 ppm. LRMS (ESI+): m/z
Formaldehyde (37%, 2.23 mL, 27.5 mmol) was added to a chilled
solution of cyclam 5 (1.00 g, 4.99 mmol) in H O (50 mL). The reac-
tion mixture was stirred at 08C for 3 h. The product was filtered
and washed with H O (100 mL) to yield 6 as a white solid (1.00 g,
+
+
89.1 ([M+H] , 41%), 577.1 ([M+Na] , 100%). HRMS (ESI+): m/z
2
+
+
Calcd. for C H N O [M+H] 489.3647, found 489.3650. FTIR
24
49
4
6
À1
(
ATR) nmax cm : 3418, 2972, 2934, 2813, 1672, 1479, 1415, 1366,
2
[47]
1
1249, 1158, 1046.
8
9%). m.p.: 105–107 (lit. 106–1088C) . H NMR (500 MHz, CDCl ):
3
d=1.13–1.21 (m, 2H), 2.24 (dt, 1H, J 12.9, 5.2), 2.29 (dt, 1H, J 13.0,
5
2
2
.2), 2.38 (d, 4H, J 10.0), 2.62 (td, 4H, J 12.6, 3.6), 2.81–2.87 (m, 4H),
.90 (d, 2H, J 10.9), 3.14 (d, 4H, J 9.9), 5.44 ppm (dt, 2H, J 10.9,
Di-tert-butyl 4,11-bis(2-azidoethyl)-1,4,8,11-tetraazacyclo-
tetradecane-1,8-dicarboxylate, 10
+
+
.1). LRMS (ESI ): m/z 225.1 ([M+H] , 100%). The spectroscopic
[35,47]
data were in agreement with those in the literature.
DPPA (1.33 mL, 6.19 mmol) and DBU (771 mL, 5.16 mmol) were
added to a solution of 9 (1.26 g, 2.58 mmol) in THF (5 mL) under
N . The reaction mixture was stirred for 5 min and sodium azide
2
1
,8-Bis(2-(tert-butoxy)-2-oxoethyl)-1,4,8,11-tetraazatricy-
(
1.01 g, 15.5 mmol) was added. The reaction mixture was stirred at
clo[9.3.1.1(4,8)]hexadecane-1,8-diium Dibromide, 7
reflux for 16 h. H O (75 mL) was added and the product was ex-
2
tracted with EtOAc (3”50 mL). The extracts were combined, dried
tert-Butyl bromoacetate (1.87 mL, 12.7 mmol) was added to a solu-
tion of 6 (1.13 g, 5.06 mmol) in MeCN (10 mL). The reaction mixture
was stirred at rt for 16 h. The product was isolated by centrifuga-
(
Na SO ) and concentrated under reduced pressure, and the resi-
2 4
due was purified by automated flash column chromatography
25 g cartridge, 10% EtOAc in petroleum ether (PE) over 1 column
(
tion and washed with MeCN (20 mL) to yield 7 as a white solid
1
volume (CV), 10% to 100% over 12 CV, 100% over 1 CV) to afford
(
[
2.51 g, 81%). m.p.: 1858C (decomp.). H NMR (400 MHz,
1
the bis-azide 10 as a pale yellow oil (1.04 g, 75%). H NMR
D ]DMSO): d=1.49 (s, 18H), 1.70–1.85 (m, 2H), 2.31–2.48 (m, 4H),
6
(
500 MHz, CDCl ): d=1.46 (s, 18H), 1.68–1.80 (m, 4H), 2.49 (t, 4H, J
3
2
2
.72 (d, 2H, J 15.2), 3.03–3.14 (m, 2H), 3.25 (d, 2H, J 13.6), 3.36 (d,
H, J 15.2), 3.50–3.61 (m, 2H), 3.64 (d, 2H, J 9.7), 3.81 (d, 2H, J
5
3
.8), 2.57–2.65 (m, 4H), 2.63 (t, 4H, J 6.1), 3.26 (t, 4H, J 5.8), 3.23–
.33 (m, 4H), 3.33–3.45 ppm (m, 4H). C NMR (100 MHz, CDCl3):
13
1
5
2
1.4), 4.35 (t, 2H, J 13.9), 4.43 (d, 2H, J 16.7), 4.59 (d, 2H, J 16.7),
1
3
d=27.2, 28.5, 46.3, 47.1, 49.3, 52.8, 53.5, 55.1, 79.4, 155.7 ppm.
.23 ppm (d, 2H, J 9.6). C NMR (100 MHz, [D ]DMSO): d=19.2,
6
+
LRMS (ESI+): m/z 539.4 ([M+H] , 100%). HRMS (ESI+): m/z Calcd.
7.6, 46.4, 47.7, 50.6, 57.3, 59.9, 76.5, 84.3, 163.5 ppm. LRMS (ESI+):
+
+
+
for C H N O
[M+H] 539.3776, found 539.3774. FTIR (ATR)
m/z 429.0 [M-2CH -2Br+3H] . The NMR data were in agreement
with those in the literature; the melting point of this compound
has not been reported previously.
24 47 10
À1
4
2
[31]
nmax cm : 2972, 2934, 2822, 2098, 1690, 1470, 1410, 1391, 1250,
1157.
Di-tert-butyl 4,11-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,8,11-
tetraazacyclotetradecane-1,8-dicarboxylate, 8
Di-tert-butyl 4,11-bis(2-(4-(2-ethyl-1,3-dioxo-2,3-dihydro-1H-
benzo[de]isoquinolin-6-yl)-1H-1,2,3-triazol-1-yl)ethyl)-
1,4,8,11-tetraazacyclotetradecane-1,8-dicarboxylate, 12
NaOH (2.5m, 10.0 mL, 25.0 mmol) was added to a solution of 7
(
2.40 g, 3.91 mmol) in MeOH (10 mL). The reaction mixture was
A mixture of copper sulfate pentahydrate (9.27 mg, 371 mmol) and
stirred at rt for 1 h before the addition of a solution of di-tert-butyl
sodium ascorbate (14.7 mg, 74.3 mmol) in H O (3 mL) was added to
2
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ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

1
3
a solution of 10 (200 mg, 371 mmol) and N-ethyl-4-ethynyl-1,8-
(t, 4H, J 7.8), 7.85 (d, 4H, J 7.7), 8.53 ppm (s, 2H). C NMR
naphthalimide 11 (222 mg, 891 mmol) in THF (7 mL) under N . The
(100 MHz, [D ]DMSO, 323 K) d=26.4, 27.8, 45.7, 46.0, 48.7, 51.2,
2
6
reaction mixture was stirred at 508C for 16 h. NH Cl (sat., 25 mL)
52.6, 78.0, 119.7, 121.4, 128.2, 129.6, 136.6, 144.5, 154.7 ppm. LRMS
(ESI+): m/z 715.2 ([M+H] , 100%). HRMS (ESI+): m/z Calcd. for
4
+
was added and the solvent was removed. The product was extract-
ed with CH Cl (2”50 mL), and the extracts were combined, dried
+
+
C H N O
[M+H] 715.4402, found 715.4400. FTIR (ATR)
2
2
38 55 10
4
À1
(
Na SO ) and concentrated under reduced pressure. The residue
nmax cm : 2971, 2930, 2812, 1687, 1503, 1468, 1414, 1366, 1233,
1156, 1042, 759. NMR spectra were acquired at 323 K due to broad-
ening of signals at room temperature (300 K).
2
4
was purified by flash column chromatography (EtOAc:PE=3:1 to
EtOAc) to afford the clicked product 12 as an orange gum
1
(
357 mg, 93%). H NMR (400 MHz, CDCl ): d=1.34, (t, 6H, J 7.1),
3
1
4
4
8
.46 (s, 18H), 1.58 (qn, 4H, J 6.8), 2.36–2.47 (m, 4H), 2.55–2.65 (m,
H), 2.90–3.01 (m, 4H), 3.13–3.29 (m, 8H), 4.24 (q, 4H, J 7.1), 4.30–
.41 (m, 4H), 7.68–7.75 (m, 2H), 7.93–7.99 (m, 2H), 7.97 (s, 2H),
.56 (d, 2H, J 7.1), 8.61 (d, 2H, J 7.6), 9.07 ppm (d, 2H, J 8.5).
1,8-Bis((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)-1,4,8,11-
tetraazacyclotetradecane, 13
13
The Boc-protected bis-phenyl 16 (68 mg, 95 mmol) was dissolved in
C NMR (100 MHz, CDCl ): d=13.3, 26.9, 28.5, 35.6, 46.6, 47.0, 48.8,
3
a mixture of TFA (4.5 mL), H O (0.25 mL) and CH Cl (0.25 mL), and
5
1
2.7, 54.0, 55.0, 79.8, 122.5, 122.8, 124.4, 127.0, 127.3, 128.8, 129.0,
30.6, 131.4, 132.7, 134.0, 145.6, 155.8, 163.7, 163.9 ppm. LRMS
2
2
2
stirred at rt for 16 h. The volatiles were removed in vacuo and lyo-
+
+
philisation afforded the TFA salt of 13 as an off-white hygroscopic
(
ESI+): m/z 1037.5 ([M+H] , 100%), 1059.5 ([M+Na] , 15%).
1
+
+
solid (75 mg, 98%). H NMR (400 MHz, [D ]DMSO, 323 K): d=2.30–
HRMS (ESI+): m/z Calcd. for C H N NaO [M+Na] 1059.5175,
6
5
6
68 12
À1
8
2.40 (m, 4H), 3.19 (t, 4H, J 5.5), 3.31 (t, 4H, J 5.5), 3.60 (br s, 4H),
found 1059.5181. FTIR (ATR) nmax cm : 3398, 2963, 1652, 1575,
260, 1089, 1033.
3
.71 (br s, 4H), 4.24 (s, 4H), 7.80–8.00 (m, 10H), 8.51 ppm (s, 2H).
1
1
3
C NMR (100 MHz, [D ]DMSO, 323 K): d=23.0, 45.6, 47.6, 49.1, 51.9,
6
5
5
5.0, 121.4, 123.0, 130.2, 130.6, 136.7, 145.1 ppm. LRMS (ESI+): m/z
+
15.5 (free base [M+H] , 100%). HRMS (ESI+): m/z Calcd. for
6,6’-(1,1’-((1,4,8,11-Tetraazacyclotetradecane-1,8-diyl)bis-
+
+
C H N
free base [M+H] 515.3354, found 515.3357. FTIR (ATR)
28
39 10
À1
(ethane-2,1-diyl))bis- (1H-1,2,3-triazole-4,1-diyl))bis(2-
nmax cm : 2841, 1675, 1503, 1466, 1195, 1133, 1053, 799, 761, 721.
ethyl-1H-benzo[de]isoquinoline-1,3(2H)-dione), 4
Anal.: Calcd. for C H N ·2.5CF CO H·0.5H O: C 49.01, H 5.17, N
28
38 10
3
2
2
1
7.32; found C 49.19, H 5.05, N 17.27. NMR spectra were acquired at
The Boc-protected bis-naphthalimide 12 (52 mg, 50 mmol) was dis-
3
23 K due to broadening of signals at room temperature (300 K).
solved in a mixture of TFA (4.5 mL), H O (0.25 mL) and CH Cl
2
2
2
(
0.25 mL), and stirred at rt for 1 h. The volatiles were removed in
vacuo, and the residue was triturated with EtOAc (2”3 mL) and
[
Zn(13)](ClO4)2
lyophilised to afford the TFA salt of 4 as a yellow solid (48 mg,
1
8
1%). m.p.: 1268C (decomp.). H NMR (500 MHz, CD OD): d=1.29
3
The TFA salt of 13 (20 mg, 26 mmol) was stirred in a suspension of
Ambersep 900 hydroxide form resin in EtOH (4 mL) for 10 min. The
resin was filtered off and Zn(ClO ) ·6H O (27 mg, 26 mmol) was
added. The reaction mixture was stirred at reflux for 16 h. The
product was isolated by centrifugation, washed with EtOH (2”
5 mL) and dried in vacuo. The product was then re-dissolved in
MeCN (5 mL) and the solution filtered through a 0.2 mm PTFE sy-
ringe filter. The solvent was removed and the product was lyophi-
lised to afford the zinc complex of 13 as a white solid (8.5 mg,
41%). m.p.: 2828C (decomp.). LRMS (ESI+): m/z 576.9 ([M+H] ,
100%).
5
1502, 1461, 1093, 764, 691, 623. Anal.: Calcd. for
C H Cl N O Zn·0.5H O C 42.68, H 4.99, N 17.78; found C 42.88, H
(
3
t, 6H, J 7.1), 2.05–2.13 (m, 4H), 2.88 (t, 4H, J 5.2), 3.00 (t, 4H, J 4.8),
.26 (t, 4H, J 5.9), 3.32–3.37 (m, 4H), 3.36 (t, 4H, J 5.1), 4.14 (q, 4H,
J 7.1), 4.72 (t, 4H, J 5.9), 7.56 (dd, 2H, J 8.5, 7.3), 7.78 (d, 2H, J 7.7),
.24 (d, 2H, J 7.6), 8.30 (d, 2H, J 7.1), 8.47 (s, 2H), 8.62 ppm (d, 2H,
4
2
2
8
1
3
J 8.4). C NMR (125 MHz, CD OD): d=13.5, 24.5, 36.4, 45.7, 47.6,
3
5
1
J
0.3, 52.5, 52.9, 117.8 (q, J
28.3, 129.2, 129.5, 131.2, 131.8, 132.9, 134.7, 146.4, 162.4 ppm (q,
289.5), 123.0, 123.5, 127.1, 128.0,
CÀF
+
36.0), 164.5, 164.8. LRMS (ESI+): m/z 837.3 (free base [M+H] ,
CÀF
+
1
00%). HRMS (ESI+): m/z Calcd. for C H N O free base [M+
+
4
6
53 12
4
+
À1
H] 837.4307, found 837.4297. FTIR (ATR) nmax cm : 1695, 1656,
589, 1454, 1373, 1202, 1139, 1066. Anal.: Calcd. for
C H N O ·3CF CO H·H O: C 52.17, H 4.80, N 14.04; found C 52.32,
+
+
HRMS (ESI+): Calcd. for C H N Zn
77.2490, found 577.2489. FTIR (ATR) nmax cm : 2925, 2878, 1597,
[M-2ClO -H]
28
37 10
À1
4
1
46
52 12
4
3
2
2
H 4.77, N 14.03.
28
38
2
10
8
2
4
.94, N 17.48.
Di-tert-butyl 4,11-bis((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)-
,4,8,11-tetraazacyclotetradecane-1,8-dicarboxylate, 16
1
Di-tert-butyl 4,11-bis(2-(4-phenyl-1H-1,2,3-triazol-1-yl)ethyl)-
,4,8,11-tetraazacyclotetradecane-1,8-dicarboxylate, 17
1
A mixture of copper sulfate pentahydrate (13.0 mg, 0.0521 mmol)
and sodium ascorbate (20.6 mg, 0.104 mmol) in H O (3 mL) was
A mixture of copper sulfate pentahydrate (11.1 mg, 0.0445 mmol)
2
added to
.520 mmol) and phenyl azide (127 mg, 1.24 mmol) in THF (7 mL)
under N . The reaction mixture was stirred at 508C for 16 h. NH Cl
a
solution of bis-propargyl cyclam 15 (280 mg,
and sodium ascorbate (17.7 mg, 0.0893 mmol) in H O (3 mL) was
added to a solution of 10 (240 mg, 0.446 mmol) and phenyl acety-
lene (117 mL, 1.07 mmol) in THF (7 mL). The reaction mixture was
2
0
2
4
(
sat., 25 mL) was added and the solvent was removed. The product
stirred at 508C for 16 h. NH Cl (sat., 25 mL) was added and the sol-
4
was extracted with CH Cl (2”50 mL), and extracts were combined,
vent was removed. The product was extracted with EtOAc (2”
25 mL), and the extracts were combined, dried (Na SO ) and con-
2
2
dried (Na SO ) and concentrated under reduced pressure. The resi-
2
4
2
4
due was purified by automated flash chromatography (10 g car-
tridge, 25% EtOAc in PE over 2 CV, 25% to 100% over 4 CV, 100%
centrated under reduced pressure. The residue was purified by au-
tomated flash chromatography (10 g cartridge, 10% EtOAc in PE
over 1 CV, 10% to 100% over 6 CV, 100% over 5 CV) to afford the
over 8 CV) to afford the clicked product 16 as an off-white solid
1
1
(
236 mg, 61%). m.p.: 168–1708C. H NMR (400 MHz, [D ]DMSO,
clicked product 17 as a yellowish gum (246 mg, 74%). H NMR
6
3
2
23 K) d=1.30 (s, 18H), 1.66–1.80 (m, 4H), 2.44–2.52 (m, 4H), 2.53–
.62 (m, 4H), 3.27–3.39 (m, 8H), 3.78 (s, 4H), 7.46 (t, 2H, J 7.4), 7.56
(400 MHz, CDCl ): d=1.35–1.50 (m, 4H), 1.46 (s, 18H), 2.31 (t, 4H, J
6.6), 2.47–2.59 (m, 4H), 2.80 (t, 4H, J 5.9), 3.00–3.22 (m, 8H), 4.10–
3
ChemistryOpen 2016, 5, 375 – 385
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383
ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

4
7
4
1
7
.25 (m, 4H), 7.27–7.34 (m, 2H), 7.37–7.44 (m, 4H), 7.73 (s, 2H),
[1] D. W. Domaille, E. L. Que, C. J. Chang, Nat. Chem. Biol. 2008, 4, 168–175.
1
3
[
[
2] E. M. Nolan, S. J. Lippard, Acc. Chem. Res. 2009, 42, 193–203.
3] Z. Xu, J. Yoon, D. R. Spring, Chem. Soc. Rev. 2010, 39, 1996–2006.
.88 ppm (d, 4H, J 7.5). C NMR (100 MHz, CDCl ): d=26.7, 28.5,
3
6.4, 47.2, 48.5, 52.6, 54.2, 55.3, 79.7, 121.2, 125.6, 128.1, 128.9,
+
[4] E. Tomat, S. J. Lippard, Curr. Opin. Chem. Biol. 2010, 14, 225–230.
[5] Y. H. Lau, P. J. Rutledge, M. Watkinson, M. H. Todd, Chem. Soc. Rev. 2011,
30.7, 147.1, 155.9 ppm. LRMS (ESI+): m/z 743.0 ([M+H] , 35%),
+
+
65.2 ([M+Na] , 100%). HRMS (ESI+): m/z Calcd. for C H N O
4
4
0
59 10
À1
4
0, 2848–2866.
+
[
M+H] 743.4715, found 743.4716. FTIR (ATR) nmax cm : 2973,
817, 1684, 1466, 1414, 1365, 1248, 1230, 1161, 766, 696.
[
[
[
6] E. J. New, Dalton Trans. 2013, 42, 3210–3219.
7] Z. Liu, W. He, Z. Guo, Chem. Soc. Rev. 2013, 42, 1568–1600.
8] D. J. Hare, E. J. New, M. D. de Jonge, G. McColl, Chem. Soc. Rev. 2015, 44,
2
5
941–5958.
1
,8-Bis(2-(4-phenyl-1H-1,2,3-triazol-1-yl)ethyl)-1,4,8,11-
[
9] A. P. de Silva, H. Q. N. Gunaratne, J.-L. Habib-Jiwan, C. P. McCoy, T. E.
Rice, J.-P. Soumillion, Angew. Chem. Int. Ed. Engl. 1995, 34, 1728–1731;
Angew. Chem. 1995, 107, 1889–1891.
tetraazacyclotetradecane, 14
The protected bis-phenyl 17 (243 mg, 0.327 mmol) was dissolved
in dioxane (2 mL) and a solution of HCl in dioxane (4.0m, 1.00 mL,
[
[
10] T. Gunnlaugsson, T. C. Lee, R. Parkesh, Org. Biomol. Chem. 2003, 1,
265–3267.
3
4
1
.00 mmol) was added. The reaction mixture was stirred at rt for
6 h. The volatiles were removed in vacuo and the residue was tri-
11] R. Parkesh, T. Clive Lee, T. Gunnlaugsson, Org. Biomol. Chem. 2007, 5,
310–317.
turated with EtOAc (2”3 mL). The HCl salt was neutralised with
excess Ambersep 900 hydroxide form resin in MeOH (3 mL) and
[12] S. Y. Kim, J.-I. Hong, Tetrahedron Lett. 2009, 50, 2822–2824.
[13] R. M. Duke, E. B. Veale, F. M. Pfeffer, P. E. Kruger, T. Gunnlaugsson, Chem.
Soc. Rev. 2010, 39, 3936–3953.
purified by HPLC (0% to 100% MeCN in H O containing 0.1% TFA
2
[14] M. Kumar, N. Kumar, V. Bhalla, Chem. Commun. 2013, 49, 877–879.
over 30 min) to afford the TFA salt of 14 as a white solid (173 mg,
1
[15] E. Tamanini, A. Katewa, L. M. Sedger, M. H. Todd, M. Watkinson, Inorg.
Chem. 2009, 48, 319–324.
6
9%). m.p.: 2138C (decomp.). H NMR (400 MHz, D O): d=1.65–
2
1
.80 (m, 4H), 2.35–2.49 (m, 8H), 2.49–2.60 (m, 4H), 2.90–3.05 (m,
[
[
16] S. Ast, P. J. Rutledge, M. H. Todd, Eur. J. Inorg. Chem. 2012, 5611–5615.
17] E. Tamanini, K. Flavin, M. Motevalli, S. Piperno, L. A. Gheber, M. H. Todd,
M. Watkinson, Inorg. Chem. 2010, 49, 3789–3800.
8
7
H), 4.57 (t, 4H, J 5.7), 7.36–7.44 (m, 2H), 7.44–7.52 (m, 4H), 7.76–
1
3
.82 (m, 4H), 8.39 ppm (s, 2H). C NMR (100 MHz, D O): d=23.1,
2
4
1
2.8, 43.0, 46.0, 47.0, 49.0, 49.7, 123.2, 125.2, 128.7, 129.2, 129.4,
[18] Y. H. Lau, J. R. Price, M. H. Todd, P. J. Rutledge, Chem. Eur. J. 2011, 17,
+
47.6 ppm. LRMS (ESI+): m/z 543.1 (free base [M+H] , 100%).
2850–2858.
+
+
HRMS (ESI+) m/z Calcd. for C H N
43 10
free base [M+H]
[19] M. Yu, Q. Yu, P. J. Rutledge, M. H. Todd, ChemBioChem 2013, 14, 224–
3
0
À1
5
1
43.3667, found 543.3665. FTIR (ATR) nmax cm : 2856, 1682, 1424,
199, 1172, 1119, 1073, 829, 770, 718, 695. Anal.: Calcd. for
C H N ·3CF CO H·3H O: C 48.70, H 5.45, N 15.78; found C 48.50,
229.
[20] M. Yu, S. Ast, Q. Yu, A. T. S. Lo, R. Flehr, M. H. Todd, P. J. Rutledge, Plos
One 2014, 9, e100761.
30
42 10
3
2
2
[
[
[
[
[
[
21] M. Yu, J. K. H. Wong, C. Tang, P. Turner, M. H. Todd, P. J. Rutledge, Beil-
stein J. Org. Chem. 2015, 11, 37–41.
22] S. Ast, S. Kuke, P. J. Rutledge, M. H. Todd, Eur. J. Inorg. Chem. 2015, 58–
H 5.35, N 16.06.
66.
[Zn(14)](ClO4)2
23] M. Yu, N. H. Lim, S. Ellis, H. Nagase, J. A. Triccas, P. J. Rutledge, M. H.
Todd, ChemistryOpen 2013, 2, 99–105.
24] M. Yu, T. M. Ryan, S. Ellis, A. I. Bush, J. A. Triccas, P. J. Rutledge, M. H.
Todd, Metallomics 2014, 6, 1931–1940.
25] M. Yu, G. Nagalingam, S. Ellis, E. Martinez, V. Sintchenko, M. Spain, P. J.
Rutledge, M. H. Todd, J. A. Triccas, J. Med. Chem. 2016, 59, 5917–5921.
26] V. V. Rostovtsev, L. G. Green, V. V. Fokin, K. B. Sharpless, Angew. Chem.
Int. Ed. 2002, 41, 2596–2599; Angew. Chem. 2002, 114, 2708–2711.
27] J. E. Hein, V. V. Fokin, Chem. Soc. Rev. 2010, 39, 1302–1315.
28] J. Canning, S. Ast, M. A. Hossain, H. Chan, P. J. Rutledge, A. Jamalipour,
Opt. Mater. Express 2015, 5, 2675–2681.
The TFA salt of 14 (94 mg, 0.11 mmol) was stirred in a suspension
of Ambersep 900 hydroxide form resin in EtOH (4 mL) for 10 min.
The resin was filtered and Zn(ClO ) ·6H O (40 mg, 0.11 mg) was
4
2
2
added. The reaction mixture was stirred at reflux for 16 h. The
product was isolated by centrifugation, washed with EtOH (2”
5
mL) and dried in vacuo. The product was then re-dissolved in
MeCN (5 mL) and the solution filtered through a 0.2 mm PTFE sy-
ringe filter. The solvent was removed and the product was lyophi-
lised to afford the zinc complex of 14 as a white solid (58 mg,
[
[
6
1
7
1
8%). m.p.: 2148C (decomp.). LRMS (ESI+): m/z 705.2 ([M+H]+,
[29] W. Rettig, E. A. Chandross, J. Am. Chem. Soc. 1985, 107, 5617–5624.
[30] W. Rettig, Angew. Chem. Int. Ed. Engl. 1986, 25, 971–988; Angew. Chem.
1986, 98, 969–986.
+
+
00%). HRMS (ESI+): Calcd. for C H ClN O Zn
05.2365, found 705.2354. FTIR (ATR) nmax cm : 3260, 2875, 1453,
[M-ClO4]
3
0
42
10
À1
4
[31] D. N. Pandya, J. Y. Kim, J. C. Park, H. Lee, P. B. Phapale, W. Kwak, T. H.
358, 1331, 1067, 976, 930, 838, 768, 695, 621. Anal.: Calcd. for
Choi, G. J. Cheon, Y.-R. Yoon, J. Yoo, Chem. Commun. 2010, 46, 3517–
C H Cl N O Zn C 44.65, H 5.25, N 17.36; found C 44.57, H 5.19, N
1
30
42
2
10
8
3
519.
32] A. Allam, L. Dupont, J.-B. Behr, R. Plantier-Royon, Eur. J. Org. Chem.
012, 817–823.
33] M. Sawa, T.-L. Hsu, T. Itoh, M. Sugiyama, S. R. Hanson, P. K. Vogt, C.-H.
Wong, Proc. Natl. Acad. Sci. USA 2006, 103, 12371–12376.
7.27.
[
[
[
2
Acknowledgements
34] M. Yu, J. R. Price, P. Jensen, C. J. Lovitt, T. Shelper, S. Duffy, L. C. Windus,
V. M. Avery, P. J. Rutledge, M. H. Todd, Inorg. Chem. 2011, 50, 12823–
We thank the Australian Research Council (DP120104035) and
the National Health and Medical Research Council (APP1084266)
for funding. M.Y. was supported by a University of Sydney Inter-
national Scholarship (USydIS) and J. K.-H.W. was supported by an
Australian Postgraduate Award (APA) from the Australian Gov-
ernment.
1
2835.
[35] G. Royal, V. Dahaoui-Gindrey, S. Dahaoui, A. Tabard, R. Guilard, P. Pul-
lumbi, C. Lecomte, Eur. J. Org. Chem. 1998, 1971–1975.
36] S. Ast, T. Fischer, H. Müller, W. Mickler, M. Schwichtenberg, K. Rurack, H.-
J. Holdt, Chem. Eur. J. 2013, 19, 2990–3005.
37] L. Fabbrizzi, M. Licchelli, P. Pallavicini, A. Perotti, A. Taglietti, D. Sacchi,
Chem. Eur. J. 1996, 2, 75–82.
[
[
[
[
38] L. J. Barbour, J. Supramol. Chem. 2001, 1, 189–191.
39] Persistence of Vision Pty. Ltd., 2004, Persistance of Visionꢁ Raytracer.
Persistence of Vision Pty. Ltd., Williamstown, Victoria, Australia http://
www.povray.org.
Keywords: chromophores
probes · macrocyclic compounds · photophysics
·
click triazoles
·
fluorescent
ChemistryOpen 2016, 5, 375 – 385
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384
ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

[
40] CCDC 1443384 and 1483555 contain the supplementary crystallograph-
ic data for ([Zn(13)](ClO ) and ([Zn(14)](ClO ), respectively. These data
[45] L. Y. Martin, L. J. DeHayes, L. J. Zompa, D. H. Busch, J. Am. Chem. Soc.
1974, 96, 4046–4048.
4
)
2
4 2
)
can be obtained free of charge from The Cambridge Crystallographic
Data Centre.
[46] E. Kent Barefield, Coord. Chem. Rev. 2010, 254, 1607–1627.
[47] R. W. Alder, E. Heilbronner, E. Honegger, A. B. McEwen, R. E. Moss, E. Ole-
firowicz, P. A. Petillo, R. B. Sessions, G. R. Weisman, J. Am. Chem. Soc.
1993, 115, 6580–6591.
[
41] L. G. Alves, M. Souto, F. Madeira, P. Ad¼o, R. F. Munhµ, A. M. Martins, J.
Organomet. Chem. 2014, 760, 130–137.
[
[
[
42] B. Bosnich, C. K. Poon, M. L. Tobe, Inorg. Chem. 1965, 4, 1102–1108.
43] X. Liang, P. J. Sadler, Chem. Soc. Rev. 2004, 33, 246–266.
44] P. J. Connolly, E. J. Billo, Inorg. Chem. 1987, 26, 3224–3226.
Received: February 2, 2016
Published online on August 3, 2016
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ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim