Optimizing the readout of lanthanide-DOTA complexes for the detection of ligand-bound copper(I)

Article abstract of DOI:10.3390/molecules22050802

The CuAAC 'click' reaction was used to couple alkyne-functionalized lanthanide-DOTA complexes to a range of fluorescent antennae. Screening of the antenna components was aided by comparison of the luminescent output of the resultant sensors using data normalized to account for reaction conversion as assessed by IR. A maximum 82-fold enhanced signal:background luminescence output was achieved using a Eu(III)-DOTA complex coupled to a coumarin-azide, in a reaction which is specific to the presence of copper(I). This optimized complex provides a new lead design for lanthanide-DOTA complexes which can act as irreversible 'turn-on' catalytic sensors for the detection of ligand-bound copper(I).

Full text of DOI:10.3390/molecules22050802

molecules
Communication
Optimizing the Readout of Lanthanide-DOTA
Complexes for the Detection of
Ligand-Bound Copper(I)
Jill R. Hanna, Christopher Allan, Charlotte Lawrence, Odile Meyer, Neil D. Wilson
and Alison N. Hulme *
EaStCHEM School of Chemistry, The University of Edinburgh, Joseph Black Building, David Brewster Road,
Edinburgh EH9 3FJ, UK; s0789206@ed-alumni.net (J.R.H.); C.Allan-3@sms.ed.ac.uk (C.A.);
s0569419@ed-alumni.net (C.L.); odile.meyer1@ac-strasbourg.fr (O.M.); s0094548@ed-alumni.net (N.D.W.)
* Correspondence: Alison.Hulme@ed.ac.uk; Tel.: +44-131-650-4711
Academic Editor: Margaret A. Brimble
Received: 22 March 2017; Accepted: 8 May 2017; Published: 14 May 2017
Abstract: The CuAAC ‘click’ reaction was used to couple alkyne-functionalized lanthanide-DOTA
complexes to a range of fluorescent antennae. Screening of the antenna components was aided by
comparison of the luminescent output of the resultant sensors using data normalized to account for
reaction conversion as assessed by IR. A maximum 82-fold enhanced signal:background luminescence
output was achieved using a Eu(III)-DOTA complex coupled to a coumarin-azide, in a reaction which
is specific to the presence of copper(I). This optimized complex provides a new lead design for
lanthanide-DOTA complexes which can act as irreversible ‘turn-on’ catalytic sensors for the detection
of ligand-bound copper(I).
Keywords: CuAAC click reaction; DOTA complexes; luminescence; Cu(I) sensor; picolinyl azides;
coumarin azides
1. Introduction
The misregulation of copper in humans is linked with serious neurodegenerative disorders
such as Menkes and Wilson’s diseases [1–3], amyotrophic lateral sclerosis [4,5], and Alzheimer’s
disease [ ]. To avoid the accumulation of excess copper, cells control copper pools rigorously through
6,7
a range of uptake, storage, and trafficking mechanisms; this maintains a low concentration of labile
copper ions, while simultaneously preserving sufficient cellular copper stores for function. It is
thought that there are essentially no ‘free’ copper ions within the cell [
considerable progress in the development of fluorescent sensors for both copper(I) and copper(II) in
recent years [ 10], most of these sensors rely on the detection of copper(I) through complexation of the
8]. Although, there has been
9,
‘free’ metal ion giving rise to a concomitant change in spectroscopic properties of the sensor molecule
in either a turn-on or turn-off sense. In an alternative strategy, we [11] and others [12] have reported
reaction-based probes [13], which rely upon the irreversible formation or breaking of covalent bonds
rather than the formation of supramolecular complexes. These ‘reactive’ probes can be used to detect
the presence of ligand-bound copper(I); however, to date these sensors have been limited by either
a modest signal output (10-fold increase in signal over background) [11], or the limit of detection
(20 µM) [12]. Clearly, the signal output which a turn-on catalytic sensor must generate in order to be
‘useful’ depends upon the specific application, but in the current study we set a goal of attaining a
signal enhancement of 102–103 fold.
Lanthanide chelates offer considerable advantages over the use of standard fluorescent dyes
for detection in vivo, especially when there is significant autofluorescence [14]. Our sensor design
Molecules 2017, 22, 802; doi:10.3390/molecules22050802
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(Figure 1) relies upon the copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) coupling of
two components—a lanthanide-DOTA complex or , and a fluorescent azide antenna —to give
a luminescent complex or . Since we [11 15] and others [16 19] first introduced the use of
bioorthogonal DOTA-alkyne complexed lanthanides such as and for ‘click’ chemistry they have
found a range of applications; from metal ion reporting [11 20], to MR contrast agents [16 17 21 23].
1
2
3
4
5
,
–
1
2
,
, , –
It was envisaged that a dramatic improvement in their application to copper(I) sensing might be
achieved through optimization of the energy transfer process. This might be achieved through:
(i) screening of the fluorescent azides
at the core of the sensor itself (1a Ln = Eu, 1b Ln = Tb etc.); (iii) alteration of the linker length
between the DOTA core and the fluorescent antenna (e.g., using DOTA-alkyne complexes 1a 2a, etc.);
3 used as the antenna; (ii) an investigation of the lanthanides
,
and (iv) reversal of the component reactivity, i.e., DOTA-azide complexes reacting with fluorescent
alkynes (not shown). Because the CuAAC reaction of DOTA complexes such as 2a and 2b has been
reported to be sluggish [18
,19,24], rendering them unsuitable for use as sensors, we chose to focus this
study on approaches (i) and (ii).
Figure 1. Sensor design for the detection of ligand-bound copper(I).
2. Results and Discussion
2.1. Synthesis of Sensor Components
Lanthanide DOTA complexes 1a and 1b were prepared by standard methods through
functionalization of 1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid tri-tert-butyl ester
(DO3tBu) [25] with propargyl chloroacetamide [26], followed by deprotection of the acid functionalities
and complexation with either Eu(OTf)3 or Tb(OTf)3 (Supplementary Material (SM) Scheme S1) [11].
Although lanthanides have previously been shown to be sensitised by a variety of fluorophores [27
with a view to potential future biological applications of our sensor, we restricted ourselves to four
classes of fluorophore azide ( ) which have been shown to be cell-permeable and to have potential in
two-photon excitation studies (Figure 2).
–31],
3
1.
Picolinate-derivatized ligands have previously been shown to act as sensitizers for europium and
terbium ions [32 35], to undergo cellular entry via diffusion, and to be suitable for two photon
–
excitation studies. Thus picolinate azides were prepared from pyridine-2,6-dicarboxylic
6–9
acid dimethyl ester through ready adaptation of the synthetic route to the 10-coordinate
N,N,N0,N0-tetrakis[(6-carboxypyridin-2-yl)methyl]ethylenediamine (tpaen) ligand reported by
Mazzanti et al. [33
,36,37] (SM Scheme S2; three steps (28% overall), four steps (15% overall),
three steps (27% overall), and four steps (22% overall) respectively).

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2.
3.
4.
Lanthanide complexes based on coumarin derivatives were pursued due to the known membrane
permeability of coumarin azides [38], and previous reports of strong fluorescence activation of
lanthanides by coumarin [39–42]. Coumarin azides 10 [43] and 11 [44] were both readily prepared
in one step from commercially available starting materials (in 72% and 82% yields, respectively).
A derivative of carbostyril 124, azide 12, was chosen because lanthanides complexed with ligands
functionalised with carbostyril 124 have been shown to have long-lifetimes, good water solubility,
and measurable brightness [45–47]. Diazotization of carbostyril 124, followed by addition of
sodium azide, yielded 12 (65%) [48].
Finally, for comparison with our previously reported CuAAC reaction [11], dansyl azide 13 was
prepared in two steps from dansyl chloride (70% overall) [49].
Figure 2. Fluorescent azides screened in this study.
2.2. Component Coupling by the CuAAC Reaction
In our original work we made use of glutathione ligands for the CuAAC reaction, as complexation
of Cu(I) to the carboxylate anion of glutathione (GS−), to give a GS−-Cu(I) complex, is thought
to provide a pooling mechanism for Cu(I) in living cells [11,12]. However, this complex was
found to be quite sensitive to environmental conditions and, in order to conduct a component
screen, alternative catalyst systems were sought. The CuAAC reaction may be catalyzed by a
range of ligand-bound copper(I) sources [50–52]. Notably, the readily accessible, stable ligand
tris-(benzyltriazolemethyl)amine (TBTA) has been shown to bind strongly to the copper(I) ion [53],
making it a good model for ligand-bound copper(I). Thus, to screen for alkyne/azide pairings with the
most intense luminescent read-out, lanthanide-alkyne complex 1a or 1b was premixed with TBTA in
tBuOH:H2O (2:1), sodium ascorbate was added followed by copper(II) sulfate, finally the fluorescent
azide (1 equivalent) was added and the mixture stirred at room temperature for 16 h. To quench the
reaction, the solution was gently shaken with a metal scavenger resin to remove the copper. Removal of
both the scavenger resin and solvent gave crude products in which the formation of the luminescent
DOTA complex could be confirmed by ESI-MS.
The optimum wavelength for excitation of each sensor complex was determined from the
excitation spectrum of the fluorophore component (Table 1). Dilution experiments (performed
on 19-Eu) indicated that a standard concentration of 100
µM in water would give the sharpest
luminescence emission spectra. The europium luminescence arising from 5Do to 7FJ transitions was
evaluated at 593 or 615 nm, depending on which offered the greatest increase in signal; whilst terbium
luminescence was measured at 545 nm as this provides the most intense terbium transition.

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Table 1. Sensor Output after CuAAC Reaction a.
Emission/cps b (wavelength/nm)
Entry
Alkyne
Azide
Sensor
λ
ex/nm
1
2
3
4
5
6
7
8
9
10
11
12
13
1a
1b
1a
1b
1a
1b
1a
1b
1a
1b
1a
1b
1a
1b
1b
6
6
7
7
8
8
9
9
10
10
11
11
12
12
13
14-Eu
14-Tb
15-Eu
15-Tb
16-Eu
16-Tb
17-Eu
17-Tb
18-Eu
18-Tb
19-Eu
19-Tb
20-Eu
20-Tb
21-Tb
325
325
325
325
300
300
300
300
345
345
325
325
345
345
350
100
240
800
338
610
845
1300
791
5371
6078
56,592
10,000
16,665
25,000
4530
(615)
(545)
(615)
(545)
(615)
(545) c
(615)
(545) c
(593)
(545)
(593)
(545)
(593)
(545)
(545)
14
15
a
Reagents and Conditions: CuSO4 (10 mol%), NaAsc (20 mol%), TBTA (10 mol%), tBuOH:H2O (2:1), rt,
b
16 h; Relative emission at specified wavelength (100
µM in H2O, time delay = 0.076 ms, slits = 10 nm,
sample window = 5 ms, number of flashes = 20); c Decrease in signal from background.
2.3. Initial Analysis of the Sensor Design by Component
To facilitate comparison of the different sensors, the increase in lanthanide luminescence intensity
between a background spectrum (1:1 mix of alkyne and azide components, 100 µM in water) and
the crude lanthanide sensor was expressed as a “fold increase” (Figure 3a,b, SM Tables S1–S4).
These data indicate that—in terms of both the absolute signal brightness, and fold-increase over
background—sensor 19-Eu, arising from the CuAAC coupling of DOTA-alkyne complex 1a with
coumarin azide 11, offers the optimum readout. These experiments also highlighted that CuAAC
coupling of picolinate-derived ligands to DOTA-alkyne complex 1a gave complexes (14-17)-Eu in
which the europium was not efficiently sensitised. This is perhaps not surprising, because it has already
been shown that, in general, sensitization of terbium by picolinate-derived ligands is more efficient [33].
However in the case of complexes 16-Tb and 17-Tb (which combine terbium alkyne complex 1b with
the di-picolinate ligands
of the azide and alkyne components, accompanied by a significant decrease in signal (4–4.5-fold) on
CuAAC coupling of the components. This suggests that the component azides and actually bind to,
8 and 9), surprisingly high background signals were observed for the 1:1 mix
8
9
and sensitise, the terbium metal rather well; but that when they are separated from the terbium ion
through CuAAC coupling to the DOTA core, this sensitization is significantly reduced.

Molecules 2017, 22, 802
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80
60
40
20
0
-20
(a)
(b)
(c)
Figure 3. Change in Signal Output after CuAAC Reaction: (
Back (orange): Eu) vs. Signal increase over background (fold); (
(blue) and background 1a 11 (1:1) (orange), showing a 69-fold increase in emission (measured at 100 µM
a) Sensor-Ln complex (Front (blue): Tb;
b
) Luminescence of crude sensor 19-Eu
:
in H2O, time delay = 0.076 ms, slits = 10 nm, sample window = 5 ms, number of flashes = 20); (
c
) FT-IR
−1
analysis of azide peak at 2100 cm for crude sensor 19-Eu (blue) with normalization of absorbance to
−1
carbonyl band at 1612 cm in a 1:1 mixture of 1a:11 (orange).
2.4. Normalization of Luminescence Output Data by IR
In comparing the output data, it became clear that not all of the reactions had reached completion
within the standard 16 h timeframe used for the CuAAC coupling reaction. This was evidenced by MS
data which indicated the presence of both starting material(s) and coupled product (e.g., SM Figure S1
for 20-Tb), and is not surprising given the use of TBTA which is known to have relatively poor
acceleration characteristics in the CuAAC reaction [50–52]. In seeking a rapid analysis method to
determine the absolute increase in signal for each coupled product over the background signal,
we were attracted to the use of vibrational spectroscopies such as IR to determine the extent to which
the reaction had proceeded in the crude reaction mixture. In situ IR monitoring, using the azide
absorption at ~2100 cm−1 for quantification, has previously been used to reveal mechanistic insights
into the CuAAC reaction [54]. The utility of this technique to determine click reaction conversions,
based on IR absorption levels corresponding to residual azide, has also been reported in a variety of
contexts [55–60]. IR monitoring is particularly appropriate in this instance, as both the sensor complex
and the lanthanide-DOTA core are paramagnetic; rendering reaction monitoring and assessment of
product purity by NMR challenging.
The presence of carbonyl bands in both the initial complex (1a or 1b) and the CuAAC coupled
product (
CuAAC reaction, led to an expectation that normalization and hence quantification of the conversion
data could be achieved [56 58]. In order to demonstrate that there were no unexpected absorption peaks
4, Figure 1), which were not expected to shift in either position or intensity following the
,
in the azide region of the coupled product, a sample of one of the complexes was purified by HPLC
(SM Figure S2 for 19-Tb); the IR spectrum of the purified material showed the complete absence of
signal at 2115 cm−1 which had previously been ascribed to the presence of unreacted azide in the crude
material. In addition, experiments with the comparatively unreactive Eu-DOTA complex 2a showed
that, under identical CuAAC conditions when there is no coupling reaction, the fluorophore azide
absorptions remain at their original intensities. On this basis, IR spectra for the crude product mixtures
from four of the most promising reactions were normalized (using the carbonyl bands indicated in
Table 2) against composite spectra generated for the appropriate 1:1 mixtures of initial azide and
alkyne components (Figure 3c, SM Tables S5 and S6). The percentage of unreacted azide remaining
in the crude product mixtures was determined and was used to estimate the percentage conversion
of each of these reactions. These conversion values were then used to adjust the maximal emission
output of the lead sensors (Figure 4) to allow rapid determination of the ‘optimum’ sensor design;
the maximal output for sensor 19-Eu was determined to be an 82-fold increase over background using

Molecules 2017, 22, 802
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this normalization process. This lead complex was purified by HPLC (SM Figure S3) and its UV-vis
(SM Figure S4), MS (SM Figure S5) and 1H-NMR (SM) spectra were acquired.
Figure 4. Structures of lead sensors 19-Eu/Tb and 20-Eu/Tb.
Table 2. Normalized Values for Signal Increase for Sensors 19 and 20.
Output a
(fold)
Conversion
(%)
Normalized Output c
(fold)
Wavenumber b
Entry
Sensor
−1
(cm
)
1
2
3
4
19-Eu
19-Tb
20-Eu
20-Tb
69
57
30
29
b
1612 (1612)
1614 (1615)
1628 (1624)
1614 (1626)
84
85
83
68
82
67
36
43
a
Signal increase over background; Carbonyl bands used to normalize data for % conversion calculation:
1:1 component mixture (product spectra); c Sensor output normalized to 100% conversion for CuAAC reaction.
2.5. Metal Ion Specificity for Formation of the Optimum Complex 19-Eu
Metal ion specificity for the optimum sensor was confirmed through coupling of DOTA alkyne 1a
and coumarin azide 11 to give 19-Eu in three solutions, one containing Na(I), K(I), Fe(II), Ni(II), Zn(II),
and Cu(II) (10 mol% each); a second containing CuSO4 (10 mol%), sodium ascorbate (20 mol%) and
TBTA (10 mol%); and a third in which there were no metal ions present. Each solution was stirred for
16 h at room temperature and, following metal ion extraction as described in Section 2.2, was excited at
325 nm. The lanthanide luminescence was measured at 593 nm; the mixed ion solution (without Cu(I))
showed no increase in signal over background (at ~800 cps), whilst the reaction conducted in the
presence of Cu(I) showed the expected increase in signal output to ~60,000 cps. These experiments
confirmed that this CuAAC coupling reaction has the potential to selectively detect ligand-bound
copper(I) in the presence of other biologically relevant metal ions.
3. Materials and Methods
3.1. General Procedure for the Synthesis of CuAAC Coupled Complexes 14–21
To Eu-DOTA complex 1a, or Tb-DOTA complex 1b (1 eq; dissolved at 20 mM concentration)
in BuOH:H2O (2:1) was added TBTA (0.1 eq) and the mixture was allowed to stir for 15 min.
t
Sodium ascorbate (0.2 eq; 0.1 M aq.) was added and the mixture was allowed to stir for 15 min
followed by the addition of copper(II) sulfate (0.1 eq; 0.1 M aq.). After a further 15 min of stirring,
the appropriate azide was added (1 eq) and the solution was allowed to stir under nitrogen at room
temperature for 16 h. QuadraPure-IDA® metal scavenger resin was added and the mixture was gently
shaken at room temperature overnight, during which the blue colour of the solution faded. The resin
was removed by filtration and the solvent was then removed in vacuo to give the crude triazole sensor.

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3.2. Luminescence Measurements on Crude CuAAC Coupled Complexes 14–21
Excitation and emission fluorescence spectra were measured using a Horiba Jobin Yvon
Fluoromax-P instrument. Lanthanide emission spectra were measured using a time-delayed setting on
the same instrument.
Solutions of the crude mixtures of (14
–21)-Eu/Tb were prepared (100
µM, H2O). The lanthanide
luminescence intensity of each solution was measured at the stated
λmax of the antenna component
(Table 1) using the following settings: time delay = 0.076 ms, slits = 10 nm, sample window = 5 ms,
number of flashes = 20. The output was recorded as the relative emission (cps) at the wavelength (nm)
corresponding to the most intense lanthanide transition (Table 1). A fold-response was calculated
by comparing this emission (in cps) to that of a solution of the sensor components (100 µM each,
1:1 mixture in H2O) stirred at rt for 16 h, measured under identical conditions at the same wavelength.
3.3. Normalization of Output of CuAAC Coupled Complexes 19 and 20 by IR
IR were measured on a Perkin Elmer Paragon 100 FT-IR machine, and ASCII files of the resultant
spectra used for subsequent data handling in Excel.
Samples were prepared as KBr die (at a final concentration of 0.5 mg azide, alkyne or crude
complex in 120 mg KBr). The reproducibility of sample preparation was confirmed by preparing
triplicate dies of pure sensor 19-Tb. A spectrum of the 1:1 alkyne:azide starting component mixtures
was generated from the reference spectra of the two starting materials using the principle of additivity
of the two absorbers present. Normalization of absorbance between reference and crude product
spectra was carried out at the wavenumber shown, allowing for a slight shift (<10 cm−1) in the C=O
stretch between the two spectra (Table 2). Normalized luminescence output data (expressed as a fold
increase) were calculated by estimating the % remaining azide in the normalized IR spectrum of the
crude product mixture (Beer Lambert Law) and using this to determine the % reaction conversion by
which the measured output could be proportionately adjusted.
3.4. Data for Purified Lead Complex 19-Eu
UV-vis (nm)
λ
max = 219, 325; 1H-NMR
δ
(600 MHz, D2O) 32.76 (1H, s), 31.26 (1H, s), 30.60 (1H, s), 30.46
0.37 (1H, s),
7.64 (1H, s),
14.23 (1H, s), 14.40
(1H, s), 7.67 (1H, s), 6.92-6.63 (3H, m), 5.08 (2H, s), 4.85 (2H, s), 3.82 (3H, s), 0.06 (1H, s),
−
−
−
2.57 (1H, s),
7.95 (1H, s),
−
2.67 (1H, s),
11.04 (1H, s),
−
3.33 (1H, s),
−
4.55 (1H, s),
−
−
5.85 (1H, s),
−
7.18 (1H, s),
−
−
−
11.33 (1H, s), 11.66 (1H, s),
−
12.22 (1H, s),
−
−
(1H, s),
−
14.85 (1H, s),
−
15.67 (2H, s),
−
16.86 (1H, s); m/z (ESI+, H2O) 823 ([153EuM + H]+, 74), 821
([151EuM + H]+, 44), 412 ([153EuM + 2H]2+, 100), 411 ([151EuM + 2H]2+, 80).
4. Conclusions
The detection of copper(I) species in a native biological setting (i.e., not one in which copper
has been artificially introduced at non-physiological conditions) has the potential to enhance our
understanding of a range of diseases, including those in which copper metabolism is misregulated
(such as Menkes and Wilson’s diseases) and those in which copper is thought to be either causative
or related to disease progression (such as Alzheimer’s disease). However, this detection is reliant on
the design of sensors which are capable of reacting with predominantly ligand-bound copper, with a
signal output which is not perturbed by the intrinsic properties of the biological milieu (e.g., inherent
cellular fluorescence). For these reasons, sensors which are constructed using the exquisitely metal-ion
selective CuAAC reaction [61] and which produce luminescent read-outs are particularly promising.
By screening a range of potential antenna, using classes of fluorophore which are known to be
cell-permeable, we have identified a sensor pairing which gives nearly two orders of magnitude signal
increase over background, overcoming previous limitations in sensor design. Pairing this knowledge
with recent advancements in ligand acceleration of the CuAAC reaction [62] could enable the very
rapid detection of ligand-bound copper in an intracellular environment.

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Supplementary Materials: The following are available online: Schemes S1 and S2 and synthetic details for
preparation of DOTA complexes 1a and 1b, and azides 13 63 64]; spectroscopic data for complexes 14 21
Tables S1–S4: luminescence spectra for complexes 14 21; Figure S1: ESI mass spectrum for crude reaction product
6–
[
,
– ;
–
20-Tb; Figure S2: HPLC chromatogram of crude reaction product 19-Tb; Tables S5 and S6: normalized IR spectra
for complexes 18 and 19; 1H and 13C-NMR spectra for azides
1H-NMR spectra for purified complex 19-Eu.
7, 8 and 9; HPLC chromatogram, UV-vis, MS and
Acknowledgments: We thank Patricia Richardson and Kenneth G. McKendrick for helpful discussions. We thank
the EC FP6 (Marie Curie EST Fellowship to JRH; Contract: MEST-CT-2005-020744) and EPSRC (Grant Ref.
EP/C539001) for funding.
Author Contributions: A.N.H. and J.R.H. conceived and designed the experiments; J.R.H., C.A., C.L., O.M. and
N.D.W. performed the experiments; A.N.H. and J.R.H. analyzed the data; A.N.H. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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