Synthesis and Characterization of Bis-1,2,3-Triazole Ligand and its Corresponding Copper Complex for the Development of Electrochemical Affinity Biosensors

Article abstract of DOI:10.1002/chem.202100250

The bis-triazole ligand and its corresponding copper complexes were synthesized and characterized for the first time and proposed as new labels for the development of electrochemical aptasensors. The bis-triazole ligand was prepared from methyl 1,6-heptad

Full text of DOI:10.1002/chem.202100250

Accepted Article
Accepted Article
Title: Synthesis and Characterization of Bis-1,2,3-Triazole Ligand
and its Corresponding Copper Complex for the Development of
Electrochemical Affinity Biosensors.
Authors: Hussein Kanso, Amani Ben Jrad, Nicolas INGUIMBERT,
Wassim Rammal, Christian Philouze, Fabrice Thomas,
Thierry NOGUER, and Carole Calas-Blanchard
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To be cited as: Chem. Eur. J. 10.1002/chem.202100250
Link to VoR: https://doi.org/10.1002/chem.202100250
01/2020

10.1002/chem.202100250
Chemistry - A European Journal
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Synthesis and Characterization of Bis-1,2,3-Triazole Ligand and
its Corresponding Copper Complex for the Development of
Electrochemical Affinity Biosensors.
Hussein Kanso,[a.b.c] Amani Ben Jrad,[a.b] Nicolas Inguimbert,[d] Wassim Rammal,[e] Christian Philouze,[f]
Fabrice Thomas,[f] Thierry Noguer,[a.b] and Carole Calas-Blanchard*[a.b]
[a]
[b]
Dr. H. Kanso, Dr. A. Ben Jrad, Prof. T. Noguer, and Prof. C. Calas-Blanchard
Biocapteurs-Analyses-Environnement
Université de Perpignan Via Domitia
F-66860, Perpignan, France.
E-mail: *Corresponding author: carole.blanchard@univ-perp.fr
Dr. H. Kanso, Dr. A. Ben Jrad, Prof. T. Noguer, and Prof. C. Calas-Blanchard
Laboratoire de Biodiversité et Biotechnologies Microbiennes
USR 3579 Sorbonne Universités (UPMC) Paris 6 et CNRS Observatoire Océanologique
F-66650, Banyuls-sur-Mer, France.
[c]
[d]
Dr. H. Kanso
Faculté de Médecine Dentaire
Université Libanaise, Campus Rafic Hariri, Hadath, Liban.
Prof. N. Inguimbert
Centre de Recherche Insulaire et Observatoire de l’Environnement (CRIOBE), USR CNRS 3278
Université de Perpignan Via Domitia
Bâtiment T, 58 avenue P. Alduy, 66860, Perpignan, France.
Prof. W. Rammal
Faculté des Sciences
Université Libanaise Section V
Nabatieh, Liban.
[e]
[f]
Dr. C. Philouze, and Prof. F. Thomas
Département de Chimie Moléculaire, UMR-5250 CNRS,
Université de Grenoble Alpes,
B.P. 53, 38041 Grenoble Cedex 9, France.
Supporting information for this article is given via a link at the end of the document.
Introduction
Biosensors are analytical devices that combine a sensitive
biological element with a physicochemical transducer. They are
very often regarded as portable and low-cost detection tools for
many applications in health, food and environmental fields. Even
if catalytic biosensors are widely developed, affinity biosensors
appear as attractive alternatives for the detection of many targets
that cannot be detected using enzymatic reactions. Affinity
biosensors are based on the highly selective interaction of the
recognition element with a specific target, the interaction being
transformed into a measurable signal by an appropriate optical or
electrochemical transducer.[1,2] According to each application and
specific condition, different types of recognition elements can be
used including antibodies, biologic receptors (hormone receptors),
and single-strand oligonucleotides, including aptamers.[3] A widely
used format in electrochemical affinity biosensors involves the
use of an enzymatic label that liberates an electroactive product
upon substrate addition. However, enzyme-labelled systems
suffer from several limitations like temperature sensitivity and
potential inactivation by natural inhibitors. In the last decade,
redox labels have been proposed as alternatives to enzymes in
electrochemical sensors.[4,5] Due to its reversible redox properties,
ferrocene has been very often used for designing sensitive
Abstract: Bis-triazole ligand and its corresponding copper complexes
were synthesized and characterized for the first time and proposed as
new labels for the development of electrochemical aptasensors. The
bis-triazole ligand was prepared from methyl 1,6-heptadiyne-4-
carboxylate and 2-(azidomethyl)phenol using classical CuAAC in
presence of different copper salts. X-Ray structure of bis-triazole
showed a symmetry center (C1). UV-Vis and X-band EPR spectra
showed that coordination capacity of bis-triazole ligand was improved
in presence of triethylamine due to deprotonation of triazole and
phenolate moieties. After complexation with copper, the obtained
complex was successfully attached to an anti-estradiol aptamer
through thiol-maleimide coupling, and the resulting labelled aptamer
was immobilized on
a
carbon screen-printed electrode by
carbodiimide coupling. The electrochemical response of the resulting
sensor was shown to decrease in the presence of estradiol,
demonstrating that the developed complexes can be applied for the
development of aptasensors.
1
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electrochemical sensors in various applications.[6-8] However, the
search for new redox labels is still a challenge, more specifically
for developing multiple sensors where several different labels are
required to ensure signal selectivity. For instance, Plaxco et al.
developed a multiplex DNA sensor using up to three sequence-
specific DNA probes, each of them being modified with a distinct
redox tracer including ferrocene (+0.18V vs Ag/AgCl),
anthraquinone (-0.54V vs Ag/AgCl) and methylene blue (-0.27V
vs Ag/AgCl). Such device allowed the simultaneous and specific
detection of three different DNA targets.[9]
characterized through their crystallographic structures, electronic
properties (UV-vis and EPR) and electrochemical activities. The
ligand was further functionalized with a maleimide-activated
spacer, allowing its further attachment to an anti-estradiol
aptamer.
Results and Discussion
In a previous study, we have described the development of novel
electrochemical immunosensors using oxovanadium salen and
salan complexes as redox markers.[10] These complexes showed
attractive properties including low equilibrium redox potentials,
close to 0V vs Ag/AgCl. These characteristics allowed developing
immunosensors for estradiol with high sensitivity and low
interference from other redox compounds.[10,11]
In this work, we propose a novel electrochemical marker based
on a bis-triazole ligand with high electrochemical response,
increased stability and solubility in aqueous phase.
General Procedure for Synthesis of Bis-triazole Ligands and
Complexes (Scheme 1). The methyl 1,6-heptadiyne-4-
carboxylate, compound 4, was synthesized according to literature
procedure (Figure S1-S2).[26] The 2-(azidomethyl)phenol,
compound 8, was prepared from two different methods. The first
method was based on the conversion of the amino group of 2-
(aminomethyl)phenol, compound 5, with the imidazole-1-sulfonyl
azide hydrochloride, compound 7, thanks to a diazo transfer
reaction (Figure S3).[27,28] The second method was based on the
conversion of the hydroxyl group of 2-hydroxy benzylalcohol,
compound 6, using sodium azide in presence of
trimethylphosphine and carbon tetrachloride (Figure S4).[29]
Yields of both chemical reactions were equal to 80 %. 2-
hydroxybenzylalcohol was slightly preferred as starting material
as the second method does not require the synthesis of
diazotransfer reagent.
Triazoles are heterocycles of synthetic origin possessing a high
degree of aromaticity. Their aromatic structure results in a high
dipole moment since the molecule is formed of 3 nitrogen atoms
having a great capacity to form hydrogen bonds. The 1,2,3-
triazoles contain both pyridine-type and pyrrole-type nitrogen and
thus possess acidic and basic properties.[12,13] Pyridine-type and
pyrrole type nitrogen are respectively σ-accepting/π-donating and
σ-donating/π-accepting compounds. Acid-base balance and
tautomerization are playing a crucial role in most of their biological,
chemical and physicochemical properties.[14] 1,2,3-triazole was
Bis-triazole ligand (compound 9, LN2O2) was prepared from
classical CuAAC using copper (I) iodide as catalyst, and
diisopropylethylamine (DIEA) base, or from copper sulfate as
catalyst and ascorbic acid as reductant, with yields of 70 and 75 %
respectively (Figure S5).
obtained through
a
Huisgen azide-alkyne 1,3-dipolar
cycloaddition.[15] Meldal et al. disclosed the use of copper (I) salts
to catalyze the regioselective azide-alkyne 1,3-dipolar
cycloaddition (CuAAC), allowing room-temperature synthesis of
1,4-disubstituted 1,2,3-triazole compounds.[16] Sharpless et al.
developed this reaction in parallel and proposed the first catalytic
mechanism.[17] This reaction is well-known to be reproducible,
quantitative, chemoselective, insensitive to conventional reaction
conditions, giving high yields and high efficiency with different
sources of copper (I). Because of its homology with the peptide
bond, 1,2,3-triazole moiety was extensively used in peptide
chemistry.[18-20]
π-donor and π-acceptor properties provide 1,2,3-triazoles good
coordination capacities with different transition metals through N2
and N3 nitrogen atoms. The quantum computation of the
electronic structure of the triazole ring based on Density
Functional Theory (DFT) suggests that the highest electron
density is located at the N3 position.[21,22] Hence, metallic
complexes based on triazole moieties have been successfully
applied for different catalytic reactions. Mn-triazole complexes
have shown efficient catalytic activity in the epoxidation of various
aliphatic terminal olefins.[23] Ni-triazole complexes have been also
used as catalysts for the polymerization of olefins, showing an
efficiency comparable to Schiff base complexes.[24,25]
In this work, we considered the use of triazole moiety as
complexing element for designing a novel electrochemical label
that offers better stability and hydrolysis resistance in aqueous
solution than Schiff base complexes. The new synthesized bis-
triazole ligand and its corresponding copper complex were
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Single crystals of compound 9 were obtained by slow evaporation
of
a solution of (10 mg) of the ligand in (4 mL of
CH2Cl2/cyclohexane 3/1). The ligand adopts an extended
conformation with the triazole-phenol moieties oriented in
opposite position with respect to the central ester group (Figure
1).
-----------------------------------------------------
Figure 1. X-ray crystal structure of compound 9, LN2O2 ligand. CCDC Deposition
Number 1939637.
UV-visible Spectroscopy.
The complexation of bis-triazole ligand (LN2O2) with copper (Cu2+
acetate monohydrate) was investigated by UV-vis spectroscopy
in methanol in absence or presence of triethylamine (TEA) to
facilitate phenolate formation (Figure 2). In the absence of TEA, a
growing hallmark band was observed at around 400 nm upon
gradual addition of copper (0.5, 1 molar equiv.) (Figure 2a). Based
on its energy and intensity (e ≈ 300 M-1 cm-1 at 1 molar equiv.
added) this band is assigned to a phenolate-to-copper charge
transfer (LMCT), inferring that the phenol arms are deprotonated
and coordinated to the metal. Deprotonation is facilitated by the
presence of an endogenous base (triazole) and the Lewis acidity
of the metal center, as reported for tripodal ligands.[30] This
hypothesis is further supported by the saturation observed at 0.5
molar equiv. added, which suggests the preferred formation of
complexes having (L:M) stoichiometry of 2, i.e., with the ligand
bearing one free and possibly protonated arm. A broad band of
low intensity is additionally detected at around 700 nm. Based on
its low energy and intensity (e < 150 M-1 cm-1), it is assigned to
copper dd transitions. This band matches that measured for
copper (II) acetate alone in solution (Figure 2 grey line) both in
terms of intensity and λmax, suggesting little perturbation of the
geometry upon complexation to H2LN2O2
.
The spectral evolution in the presence of TEA is depicted in
Figure 2b. It resembles the previous one except that the intensity
of the LMCT is twice as large, while the dd band did not show
such an increase. Hence each phenol moiety is deprotonated by
exogenous TEA in this case, allowing for copper complexation by
both arms.
Scheme 1. Synthesis of the ligands and their copper complexes.
Subsequent saponification of compound 9 allowed its conversion
into the corresponding acid, compound 15 (90% yield). The
carboxylic acid group was then coupled to the maleimide spacer,
compound
14,
using
the
oxyma
pure
reagent
(ethylcyano(hydroxyimino)acetate), yielding 77% of maleimide-
bis-triazole ligand (compound 16, maleimide-LN2O2, Figure S7).
The copper complexes CuLN2O2 (compound 10) and maleimide-
CuLN2O2 (compound 17) were prepared by mixing stoichiometric
amounts of Cu(OAc)2.H2O with the corresponding ligand (Figure
S8).
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case. It is higher than for tetrahedral salen complexes involving
an N2O2 donor set,[32] hence suggesting significant tetrahedral
distortion in the present case.
(a)
The EPR spectrum further evolves upon addition of larger
amounts of copper (II), showing two components at 1 molar equiv.
added. From simulation we determine the following spin
Hamiltonian parameters: gx = gy = 2.073, gz = 2.318 and Ax = Ay
= 30 MHz, Az = 510 MHz for the component 1 (main), gx = gy =
2.073, gz = 2.365 and Ax = Ay = 30 MHz, Az = 420 MHz for the
A
component
2 (minor). Note that both the overlap of the
resonances and line broadening precludes a precise evaluation
of the ratio, close to 1:2, while limiting the accuracy of the
simulation. Both the low Az and high gz of component 2 suggests
that it arises from weakly bound (or unbound) copper. On the
other hand, the hyperfine coupling constants of component 1 are
the same as at 0.5 molar equiv. copper added. The g tensor
somehow differs, indicative of distinct copper coordination sphere.
The larger gz value at 1 molar equiv. added reflects an enhanced
tetrahedral distortion in the complex. We tentatively assign this
behavior to the successive formation of complexes with
stoichiometries (L:M) of 2 (0.5 molar equiv. of copper added) and
next 1 (1 molar equiv. of copper added), both having a similar
N2O2 coordination sphere and featuring phenolate ligation.
λ / nm
(b)
A
λ / nm
Figure 2. UV-vis spectra of LN2O2 ligand (2 mM). Free form (red), and after
complexation with 0.5 equivalent (orange), 1 equivalent (black) and 1.5
equivalent (yellow) of Cu2+. 2 mM of copper acetate was used as blank (grey).
(a) obtained in the absence of TEA. (b) obtained in the presence of 2 equivalents
of TEA.
X-band EPR Spectroscopy.
The copper complexes were investigated by EPR spectroscopy
and the obtained results were correlated with those obtained in
UV-vis. Upon addition of 0.5 and next 1 molar equiv. of copper (II)
acetate to the ligand, with TEA, the X-band EPR spectrum shows
only a residual copper signal, which clearly differs from free metal
ion (Figure 3). The intensity is twice for the sample with 1 molar
equiv. of copper added, but far below that expected for 2 mM
solution of an isolated (S=1/2) system (Figure 3). Based on UV-
vis spectroscopy we ruled out the possibility that Cu(II) is reduced
in solution. Hence the copper (II) ion likely forms pairs in solution,
wherein the metal centers are magnetically interacting.
Consistently, both unprotected phenolates and acetates are
typical bridging ligands, which are known to mediate such
interaction.
Figure 3. X-band EPR spectrum of 2 mM MeOH solution of the ligand in the
presence of: (a) 1 molar equiv. of Cu(OAc)2 and 2 molar equiv. of TEA; (b) 0.5
molar equiv. of Cu(OAc)2 (c) 1 molar equiv. of Cu(OAc)2. Microwave Freq. 9.44
GHz, power 1.1 mW; Mod. Amp. 4 G, Freq. 100 KHz. T = 100 K. Black lines:
experimental spectra, red lines: simulations using the parameters given in the
text.
DFT Calculations.
When TEA is absent the intensity of the EPR resonances is higher,
suggesting formation of a larger amount of isolated copper (II)
systems. Spin integration reveals 2.5 and 8 times increases at 0.5
and 1 molar equiv. of copper added, respectively. At 0.5 molar
equiv. of copper added, the spectrum is axial, with the spin
Hamiltonian parameters gx = gy = 2.064, gz = 2.296 and Ax = Ay =
30 MHz, Az = 510 MHz. The Az value is much higher than that
typically observed for solutions of solvated copper (II),
demonstrating complex formation. Interestingly, this spectrum
resembles that obtained in the presence of TEA. The ratio gz/Az
(expressed in cm) is usually taken as a probe of distortion at the
copper center, with high values being indicative of large
distortion.[31] The ratio gz/Az is calculated at 135 cm in the present
Full geometry optimizations were performed with the Gaussian 9
program,[33] by using the B3LYP.[34, 35] The 6-31g* basis set was
used for the C, H, N, O atoms.[36] Frequency calculations were
systematically performed in order to ensure that the optimized
structure corresponds to a minimum and not a saddle point.
In order to gain insight onto the specific distortions induced by the
ligand in the monocopper complexes we used quantum chemistry.
Note that this study was restricted to mononuclear complexes
because the nature of the bridging ligand was not firmly
established in the copper pairs. Two models were computed, one
corresponding to two ligand halves bound to a single copper
center (A) and one complex (B) that correspond to compound 10
and allow to assess the influence of the bridge linking the two
4
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chelating units. We hypothesized a four-coordinate center (Figure
4) similar to salen complexes. Model A shows a distorted copper
center, whatever the position of the ligand (cis or trans), the cis
configuration being the most stable one. Optimized structures of
the (L:M) 2:1 complex with the full ligand, protonated or not, are
depicted in Figure S9 and S10. As expected, they show large
tetrahedral distortions at the metal center. Importantly, the extend
of distortion is almost similar in model A (cis) and B. The
tetrahedral distortions are thus due to the individual triazole-
phenolate units, the bridge having only little influence.
the square root of the scan rate confirmed that the process was
controlled by diffusion (Figure S12b).
Synthesis of Ligand-Aptamer and CuLN2O2-Aptamer
Conjugate.
An anti-estradiol(E2) aptamer[37] was selected to test the
effectiveness of CuLN2O2 for labeling such molecule. The
sequence of the E2-specific aptamer, as well as the mass spectra
of aptamer, ligand-aptamer conjugate (compound 18), and
CuLN2O2-aptamer conjugate (compound 19) are presented in
supporting information (Figure S13-S15).
LN2O2
CuLN2O2 2 mM
CuLN2O2 1 mM
4x10-6
2x10-6
0
-2x10-6
-4x10-6
-0.4
-0.2
0.0
0.2
0.4
0.6
0.8
Figure 4. DFT optimized structures for (left) the model A (cis) and (right) the
complex of stoichiometry (1:1) (L:M) (B) (B3LYP/6-31g*).
E/V
N2O2
Maleimide CuL
N2O2
2mM
4x10-6
2x10-6
0
CuL
2mM
Electrochemical Studies.
Cyclic voltammetry (CV) experiments were carried out to study
the electrochemical behavior of LN2O2 and its copper complex
CuLN2O2. Due to the insufficient water solubility of LN2O2, these
experiments were performed in CH2Cl2/MeOH (8/2 vol.)
containing 0.1 M tetrabutylammonium perchlorate (TBAP) as
supporting electrolyte. A glassy carbon electrode was used as
working electrode and a platinum sheet was used as counter
electrode. All potentials were expressed versus the silver/silver
chloride reference electrode.
-2x10-6
-4x10-6
Cyclic voltammograms of CuLN2O2 exhibit a one-electron redox
wave at Ec = -0.05 V and Ea = +0.5 V vs. Ag/AgCl (Figure 5a). This
-0.6 -0.4 -0.2 0.0
0.2
0.4
0.6
E/V
Figure 5. (a) Cyclic voltammograms of ligand in free state (LN2O2) and after
signal is assigned to
a
metal-centered redox process
corresponding to Cu2+/Cu+ redox couple. As expected, the current
intensity increased twice when CuLN2O2 concentration was
doubled from 1 to 2 mM. Cyclic voltammogram of copper acetate
(Cu(Ac)2) was tested as control to differentiate between the
electrochemical activity of free and complexed copper ion (Figure
S11). In experimental conditions, free Cu(Ac)2 exhibits two
oxidation waves at Ea1 = +0.2 V and Ea2 = +0.42 V, and two
overlapped reduction waves at Ec1 = +0.05 V and Ec2 = -0.02 V vs.
Ag/AgCl. These signals correspond to free metal redox process
corresponding to Cu+/Cu and Cu2+/Cu+ redox couples (Figure
S11). As shown in Figure 5b, the functionalization of copper
complex with a maleimide-activated spacer slightly affected
oxidation and reduction potentials. Peak-to-peak separation
between anodic and cathodic peak potentials (ΔE) was
decreased from 499 to 395 mV after maleimide modification. The
electrochemical process was investigated by carrying out cyclic
voltammetric experiments with 2 mM CuLN2O2 at increasing scan
rates ranging from 10 mV s-1 to 500 mV s-1 (Figure S12a). The
linear relationship observed between the anodic peak current and
complexation with copper (CuLN2O2), at concentrations of 1 mM and 2 mM. (b)
Cyclic voltammograms of
2 mM complexed ligand before and after
functionalization with a maleimide-activated spacer. Solvent: CH2Cl2 containing
0.1 M TBAP as supporting electrolyte. Reference Ag/AgCl. Scan rate = 0.05 V
s-1, T = 298 K.
The bis-triazole ligand 16 was conjugated to E2-aptamer through
the reaction of the 3’ modified thiol moiety of the aptamer with the
maleimide. Ligand-aptamer conjugate 18 was monitored by
HPLC coupled to HRMS with electrospray ionization in negative
ion mode, allowing the identification of
a
mass peak
corresponding to remaining excess of the ligand 16 at m/z 696.33
along with the expected ligand-aptamer conjugate 18 showing
peaks at m/z 1982.03 and 2378.83 that corresponds to the 6 and
5 charged species, respectively (Figure S14). The peak of the
native aptamer completely disappeared after the addition of a
slight excess of maleimide-LN2O2 CuLN2O2-aptamer conjugate 19
.
was obtained in the same conditions. Peaks at m/z 1991.19 and
2389.22 were attributed to the 6 and 5 charged species (Figure
S15). Two separate 5-times and 6-times charged derivatives were
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obtained including peaks of the ligand-aptamer conjugate 18 at
m/z 1982.03 and 2378.83 which correspond to metal dissociation.
The appearance of these different charged derivatives is certainly
due to the presence of the copper (II) in the coordination sphere.
Indeed, copper (II) can interfere with different species during the
ionization process inside the tandem mass spectrometry.
The results obtained after incubation with E2 allowed drawing a
calibration curve (Figure 7b) which displays a wide dynamic range
laying from 0.05 to 8 μM. Due to the low experimental error (5%)
associated to electrochemical detection, the limit of detection
(LOD) was defined as the E2 concentration inducing a 15%
decrease of the maximum signal. It was calculated to be 0.12 µM.
Electrochemical Sensor Assays
Without E2
0.05 µM E2
0.25 µM E2
0.5 µM E2
1 µM E2
8 µM E2
A screen-printed carbon electrode carrying carboxylic group on its
surface was activated using EDC/NHS allowing the
immobilization of the CuLN2O2-labelled aptamer through its amino
modified 5’-end (Figure 6a).[38] Controls were carried out by
immobilizing unmodified aptamers and aptamers bound to
uncomplexed ligand (LN2O2). Square Wave Voltammetry (SWV)
was selected as an appropriate method to investigate the
electrochemical signal resulting from labelled aptamer. Cu(Ac)2,
used as a control, did not display any electrochemical activity in
4x10-8
2x10-8
these conditions (Figure S12c). In the presence of CuLN2O2
-
labelled immobilized aptamer 19, SWV experiments showed a
significant peak at 0.4 V vs Ag/AgCl attributed to copper complex
electroactivity (Figure 7a). After incubation with estradiol (E2) at
concentrations ranging from 0.05 to 8 μM, this peak was shown
to gradually decrease (Figure 7a). Such behavior is explained by
the conformational change of the aptamer induced upon E2
binding, which prevents electron transfer from the CuLN2O2 label
to electrode surface (Figure 6b).[39] Before the recognition event,
the CuLN2O2 complex is in close vicinity of the electrode surface,
but once the aptamer-E2 self-assembly is achieved, the probe is
driven away from the electrode surface, resulting in the observed
decrease of current (Figure 6b).
These results are in agreement with a molecular dynamics
simulation study that highlighted that the 5’ and 3’ ends of E2-
aptamer are not interacting with E2 and can be thus used for
aptamer functionalization or immobilization. Furthermore, the
described results are consistent with previous experiment
involving a salan complex as electroactive label for the same
aptamer.[11,40]
0.2
0.3
0.4
0.5
E/V
100
50
0
0.01
0.1
1
10
Concentration of E2 / µM
Figure 7. (a) Square wave voltammograms obtained using CuLN2O2-labelled
immobilized aptamer (compound 19) in absence and in presence of E2 at
concentrations ranging from 0,05 to 8 µM. Step potential: 1 mV, modulation
amplitude: 10 mV, frequency: 50 Hz, interval time 0.02 s, scan rate: 53 mV.s-1.
(b) Calibration curve obtained for E2 detection fitted by the sigmoidal logistic
four parameter-equations, Origin Pro 2018: y = -5.62 + (104.76)/(1 +
(x/0.69)1.08); R = 0.99. All measurements were performed in triplicate in PBS
buffer at room temperature.
Figure 6. (a) Activation of carbon electrode surface by electrodeposition of 4-
carboxyphenyl groups and immobilization by carbodiimide coupling of 5’-
aminated aptamer. (b) Principle of electrochemical aptasensors based on the
recognition of the target by an aptamer, the E2 fixation induces a probe
distancing from the surface electrode respect to a ON/OFF aptasensor design.
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washed with H2O (2 × 100 mL) then saturated aqueous NaHCO3 (2 × 100
mL), dried over MgSO4 and filtered. A solution of HCl in EtOH [obtained by
the drop-wise addition of AcCl (21.3 mL, 300 mmol) to ice-cooled dry
ethanol (75 mL)] was added drop-wise to the filtrate with stirring, the
mixture chilled in an ice bath, filtered and the filter cake washed with EtOAc
(3 × 100 mL) to give compound 7 as white needles (12.21 g, 76%). 1H
NMR (400 MHz, D2O): δ=8.31 (s, 1H), 7.66 (s, 1H), 7.21 ppm (s, 1H)
(Figure S3). 13C NMR (100 MHz, D2O): δ=138.06, 130.60, 118.75 ppm.
Conclusion
This work shows the potential of bis-triazole derivative (LN2O2) as
a metal complexing ligand for the efficient electrochemical
labelling of aptamers. A complete and stable complexation of the
ligand was achieved using 1 equivalent of metal, in a typical
behavior for mononuclear copper (II) species. These results were
confirmed using EPR characterization that showed hyperfine
splitting signals due to interaction of electronic spin with copper
nuclear spin.
The complexed ligand CuLN2O2 was successfully linked to a SH-
functionalized anti-estradiol aptamer through maleimide coupling,
and the resulting labelled aptamer was immobilized on a screen-
printed carbon electrode through carbodiimide coupling. The
resulting sensor showed a characteristic signal at 0.4 V vs
Ag/AgCl, which was shut down upon binding of estradiol to
aptamer, demonstrating that the aptasensor behaves a signal
ON/ signal OFF system. These results undoubtedly open new
perspectives for developing electrochemical aptasensors for
other small targets based on binding-induced direct detection.
2-Hydroxybenzylazide, Compound 8. Method 1: Imidazole-1-sulfonyl
azide hydrochloride 7 (205 mg; 1 mmol) was added to 2-hydroxybenzyl
amine
5 (100 mg; 0.81 mmol), K2CO3 (112 mg; 2.4 mmol) and
CuSO4.5H2O (8.1 10-3 mmol; 2 mg) in MeOH (10 mL) and the mixture was
stirred at room temperature for 12 h. The mixture was concentrated, diluted
with H2O (15 mL), acidified with conc. HCl and extracted with ethyl acetate
(3 x 20 mL). The organic extracts were washed with brine (2 x 30 mL) and
dried over MgSO4, filtered and concentrated under reduced pressure.
Flash chromatography (EtOAc/cyclohexane 3/7) gave the desired azide 8
(96 mg g, 80%).
Method 2 followed a literature reported method[29]: A mixture of 2-hydroxy
benzylalcohol 6 (1.24 g, 10.0 mmol), sodium azide (0.78 g, 12 mmol) and
triphenylphosphine (2.62 g, 10 mmol) in a mixture of CCl4 and DMF (1:4,
20 mL) was heated with stirring (90 °C, 5 h). The resulting mixture was
removed from the heat and distributed between ether (50 mL)-water (25
mL). The ether layer was washed with additional portions of water (2 x 25
mL) and then separated. Upon cooling (0 °C), triphenylphosphine oxide
crystallizes. After separating the crystals, the ether was dried (MgSO4),
filtered and concentrated. The yellowish residue was subjected to flash
chromatography on silica (EtOAc/cyclohexanes 2/8) to afford 8 (1.20 g,
80%) as a light yellowish oil. 1H NMR (400 MHz, CDCl3): δ=7.20-7.18 (m,
2H), 6.92 (t, J = 7.6 Hz, 1H), 6.85 (d, J = 8 Hz, 1H), 5.51 (s, 1 H), 4.41ppm
(s, 2H). (Figure S4a). 13C NMR (100 MHz, CDCl3): δ=154.41, 130.20,
130.09, 121.74, 121.02, 116.18, 51.14 ppm. (Figure S4b).
Experimental Section
Dimethyl dipropargylmalonate, Compound 3. Dimethyl malonate 1 (4
mL, 34.4 mmol) was diluted in dry THF (10 mL) and added dropwise to a
stirred suspension NaH (60% in mineral oil, 3.44 g, 86 mmol) in THF (100
mL) at 0 oC. After 15 min propargyl bromide 2 (80% w/w in toluene, 7.42
mL, 68.8 mmol) was added dropwise. The mixture was stirred overnight at
room temperature. Complete disappearance of dimethyl malonate was
demonstrated by thin layer chromatography (TLC). After that, THF was
evaporated under vacuum and the reaction mixture was distributed
between diethyl ether and water (100/50), and the aqueous solution was
twice extracted with diethyl ether. The combined organic phase was
washed with brine and dried over magnesium sulfate. After the removal of
solvent, recrystallization from EtOAc/Hexane led to pure white solid (4.25
g, 60%). 1H NMR (400 MHz, [D6]DMSO): δ=3.68 (s, 6H), 3.04 (t, J = 2.56
Hz, 2H), 2.83 ppm (d, J = 2.56 Hz, 4H) (Figure S1a). 13C NMR (100 MHz,
[D6]DMSO): δ=169.09, 78.66, 75.32, 56.37, 53.77, 22.87 ppm (Figure S1b).
N2O2 bis-triazoles ligand (LN2O2), Compound 9. Method 1: Methyl 1,6-
heptadiyne-4-carboxylate
4
(70 mg, 0.45 mmol) and 2-
Hydroxybenzylazide 8 (170 mg, 1.12 mmol) were suspended in a 1:1
mixture of water and tert-butyl alcohol (8 mL). copper (II) sulfate
pentahydrate (8 mg, 0.02 mmol, in 200 µL of water) was added, followed
by Sodium ascorbate (40 mg, 0.2 mmol, 500 µL of freshly prepared from
ascorbic acid in water). The heterogeneous mixture was stirred 2 days at
50°C, at which point it cleared and TLC analysis indicated complete
consumption of the diazide. The reaction mixture was then diluted in ethyl
acetate (100 mL) and washed with HCl 0.5 N (20 mL) and brine. The
organic phase was dried over magnesium sulfate, filtered and
concentrated to leave brown oil. The residue was purified on silica gel
(dichloromethane (DCM) /MeOH, 9.5/0.5) and the product fractions were
concentrated. it was dried under vacuum to afford 151 mg (75 %) of pure
product as an off-white powder.
Methyl 1,6-heptadiyne-4-carboxylate, Compound 4. The synthesis of
methyl 1,6-heptadiyne-4-carboxylate followed a literature reported method.
[41,42] Dimethyl dipropargylmalonate 3 (2.5 g, 12 mmol) and NaCl (4.2 g, 72
mmol) were dissolved a mixture of DMSO (20 mL) and water (1 mL). The
reaction was refluxed for 1.5 h and subsequently cooled to room
temperature. The mixture distributed between ethyl acetate and water and
the aqueous layer was extracted twice with ethyl acetate. The combined
organic phases were washed with water and brine, dried over MgSO4,
filtered through silica gel and concentrate to provide a crude product, which
was purified by flash chromatography column using 20% ethyl acetate in
hexane, to give methyl 1,6-heptadiyne-4-carboxylate (1.26 g, 70%) as
pale-yellow oil. 1H NMR (400 MHz, CDCl3): δ=3.72 (s, 3H), 2.76 (q, J = 6.1
Hz, 1H), 2.62 (m, 4H), 2.008 ppm (s, 2H) (Figure S2a). 13C NMR (100 MHz,
[D6]DMSO): δ=172.86, 80.47, 70.59, 52.25, 43,04, 19,97 ppm (Figure S2b).
Method 2: To an ice cold and homogeneous solution of 4 (48 mg, 0.32
mmol) in dry acetonitrile was added DIEA (11 mL, 0.64 mmol), followed by
8 (96 mg, 0.64 mmol) under an N2 atmosphere. To the reaction mixture
under nitrogen, was added CuI (6 mg, 0.032 mmol) and stirred for 24h at
room temperature. The reaction was stirred under nitrogen for 6 h. The
mixture was diluted with ethyl acetate (EtOAc, 100 mL). The solution was
washed with saturated NH4Cl/NH4OH (9/1) then washed with 1M HCl (3 x
25 mL), brine (2 x 25 mL) and dried over MgSO4. The crude product was
either purified using column chromatography (DCM/MeOH 9.5/0.5) to
afford 9 (100 mg, 70%) as an off-white powder. 1H NMR (400 MHz, CDCl3):
δ=8.90 (s, 2H), 7.50 (s, 2H), 7.20-7.17 (m, 4H), 6.94 (d, J = 8 Hz, 2H), 6.85
(t, J = 7.3 Hz, 2H), 5.43 (s, 4 H), 3.42 (s, 3H), 3.07-2.80 ppm (m, 5H)
(Figure S5a). 13C NMR (100 MHz, [D6]DMSO): δ=174.68, 155.59, 144.28,
130.06, 130.02, 123.31, 122.64, 119.61, 115.79, 51.82, 48.75, 45.67,
27.73 ppm (Figure S5b). The purity of the ligand (LN2O2 compound 9) was
verified by ESI-MS, (Figure S5c – S5e).
Imidazole-1-sulfonyl Azide Hydrochloride, Compound 7.[28] Sulfuryl
chloride (6.4 mL, 77 mmol) was added drop-wise to an ice-cooled
suspension of NaN3 (5g, 77 mmol) in MeCN (50 mL) and the mixture stirred
overnight at room temperature. Imidazole (10 g, 380 mmol) was added
portion-wise to the ice-cooled mixture and the resulting slurry stirred for 3
h at room temperature. The mixture was diluted with EtOAc (100 mL),
7
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Maleimide spacer, Compound 13. N-Boc-1,6-hexanediamine
hydrochloride 12 (311 mg, 1,44 mmol) was added to a stirred solution of
4-Maleimidobutyric acid N-hydroxysuccinimide ester 11 (360 mg, 1,2
mmol) followed by DIPEA (3,6 mmol) in 6 mL of DCM and DMF mixture
(1:1 v/v) at room temperature.
Preparation of the copper complexes, Compound 10 and Compound
17. A general procedure was applied for the synthesis of metallic
complexes. To a stirred solution of ligand (1 equiv) in methanol (5 mL),
copper acetate monohydrate (1 equiv) dissolved in methanol (5 mL) was
added dropwise. The resulting solution was heated at reflux for 1 hour. The
resulting mixture was washed with diethyl ether and dried under vacuum.
Copper complex (CuLN2O2, compound 10) and maleimide-copper complex
(Maleimide-CuLN2O2, compound 17) were obtained as turquoise blue and
dark green solids, respectively.
The resulting mixture was stirred for 4 h at room temperature. The reaction
mixture was supplemented with ethyl acetate (100 mL) and then washed
successively with HCl (1 M, 1 × 20 mL), H2O (1 × 20 mL), NaHCO3 (1 M,
1 × 20 mL), H2O (1 × 20 mL) and brine (1 × 20 mL). The organic phase
was dried over anhydrous sulfate and purified on silica gel (DCM /MeOH,
9.5/0.5) and the product fractions were concentrated. it was dried under
vacuum to afford 370 mg (80 %) of pure product as white powder. 1H NMR
(400 MHz, [D6]DMSO): δ=7.72 (t, J = 5.8 Hz, 1H), 7.001 (s, 2H), 6.75 (t, J
= 5.5 Hz, 1H), 3.39 (t, J = 6.96 Hz, 2H), 2.98 (q, J = 6.6 Hz, 2H), 2.88 (q, J
= 6.6 Hz, 2H), 2.01 (t, J = 7.32 Hz, 2H), 1.69 ppm (m, 2H) (Figure S6).
Chemical formula: C19H31N3O5, exact mass: M: 381.23, MS (m/z): 382.02
[M + H]+, corresponding to maleimide spacer.
Due to the paramagnetic properties of the copper complex, the structure
of the complex, compound 10, was characterized by UV-vis and EPR
instead of RMN (Figure S8).
Electrochemical Studies
The electrochemical behavior of the complexes has been studied by cyclic
voltammetry (CV) in CH2Cl2 containing 0.1 M TBAP, tetrabutylammonium
perchlorate, as supporting electrolyte. A Ag/AgCl reference electrode with
double-junction system was used for all electrochemical experiments
(Metrohm AG, Switzerland). This reference electrode was filled either with
saturated solutions of KCl in water or LiCl in ethanol for experiments in
aqueous and organic medium, respectively.
Maleimide-N2O2 bis-triazole ligand, Compound 16:
1-
Saponification of compound 9.
Sodium hydroxide (4 mL, 4 M) was added dropwise to a 10 mL stirred
methanolic solution of LN2O2 9 (87 mg, 0.19 mmol). The solution was stirred
for 2 h at RT and monitored by TLC, until full consumption of the starting
material. The reaction mixture was diluted with water (10 mL) and acidified
with HCl to pH 3. The mixture was concentrated and extracted with ethyl
acetate (3 × 50 mL), and the organic phase was washed with brine, dried
over anhydrous sulfate, and evaporated to dryness, affording 74 mg (0.17
mmol, 90%) of saponified ligand compound 15.
The working and counter electrodes were made of glassy carbon
(Radiometer, Denmark) and platinum sheet electrode (Metrohm AG,
Switzerland) respectively. The electrodes were connected to
potentiostat/galvanostat Autolab (Metrohm AG, Switzerland).
a
Crystal Structure Analysis.
Deprotection of maleimide spacer, compound 14.
Crystals were mounted on a Kappa CCD Nonius diffractometer equipped
with graphite-monochromated Mo-Kα radiation (λ = 0.71073 Å) and a
cryostream cooler. The collected reflections were corrected for Lorentzian
and polarization effects but not for absorption. Crystal structural solution
(direct method) and refinement (by full-matrix least squares on F) was
performed using the teXsan analysis package. All non-hydrogen atoms
were refined with anisotropic thermal parameters. Hydrogen atoms were
generated in idealized positions, riding on the carrier atoms, with isotropic
thermal parameters.
63 mg (0.166 mmol) of maleimide spacer, compound 13, was dissolved in
10 mL of DCM and TFA (1:1 v/v). The reaction mixture was stirred at room
temperature for 2 h and monitored by TLC (ethylacetate/ cyclohexane, 7:3).
The reaction mixture was then evaporated under vacuum, and residual
TFA was eliminated by co-evaporation with cyclohexane.
2-
Addition of maleimide spacer to LN2O2
.
EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) (48 mg, 0.25 mmol)
and oxyma (35 mg, 0.25 mmol) were added to a stirred solution of
compound 15 (0.166 mmol) in 5 mL of DMF at room temperature.
Deprotected maleimide spacer, compound 14, (63 mg, 0.166 mmol) was
then added, followed by DIPEA (N,N-Diisopropylethylamine) (116 μL, 0.66
mmol). The resulting mixture was stirred overnight at room temperature.
The reaction mixture was supplemented with ethyl acetate (100 mL) and
then washed successively with acidified water (pH 4; 3 x 20 mL. The
organic phase was dried over anhydrous sulfate and purified on silica gel
(DCM /MeOH, 9.5/0.5) and the product fractions were concentrated. it was
dried under vacuum to afford 50 mg (44 %) of pure product as white
powder compound 16. 1H NMR (400 MHz, [D6]DMSO): δ=9.88 (s, 2H),
7.71 (q, J = 5.5 Hz, 2H), 7.64 (s, 2H), 7.14 (t, J = 7.7 Hz, 2H), 6.98 (s, 2H),
6.94 (d, J = 6 Hz, 2H), 6.84 (d, J = 8 Hz, 2H), 6.74 (t, J = 7.5 Hz, 2H), 5.42
(s, 4H), 2.51 (q, J = 6.76 Hz, 2H), 2.81 (m, 4H), 2.62 (m, 2H), 2.02 (t, J =
7.32), 1.69 (q, J = 7.16 Hz, 2H), 1.29 (t, J = 7.28, 3H), 1.14 ppm (m, 6H)
(Figure S7a and S7b). 13C NMR (100 MHz, [D6]DMSO): δ=170.95, 170.89,
154.97, 144.48, 134.36, 129.50, 129.39, 122.42, 122.00, 119.01, 115.22,
48.67, 46.31, 38.95, 38.72, 37.42, 33.15, 29.64, 29.31, 28.67, 26.54, 26.48,
24.71 ppm (Figure S7c).
Preparation of CuLN2O2-labelled Aptamer
The estradiol-specific aptamer (35 bases) was first linearized in sterile
water in order to avoid hybridization (90°C during 8 min and 0°C for 5 min).
It was then diluted to 1.1 mM in sterile water. To 10 µL of this aptamer
solution, 90 µL of 0.24 mM of desired compound (compound 16) in
methanol was added and incubated with stirring overnight at room
temperature. The solution was stored at 4°C. 20 µL of the obtained solution
was injected in the Liquid chromatography coupled to tandem mass
spectrometry (Figure S13 – S15).
Aptasensor Design.
First, the functionalization of the working electrode of a screen-printed
carbon electrode (SPCE) was carried out by electrochemical reduction of
diazonium salts (Figure 6). Briefly, a mixture of 0.1 mM NaNO2 and 2 mM
4-aminobenzoic acid was prepared in 1 mL of 0.5 M HCl solution.[43] The
mixture was left to react for 5 min at room temperature, and 100 µL were
deposited on the SPE surface. The electrochemical generation of surface
4-carboxyphenyl (4-CP) was then performed by linear sweep voltammetry
(LSV), by scanning the potential from 0.6 to -0.8 V vs Ag/AgCl. The
carboxylic groups of 4-CP-modified SPCE were then activated by
incubating the working electrode for 60 min in 5 mL of 100 mM MES buffer
The purity of the ligand (LN2O2 compound 9) was verified by ESI-MS,
(Figure S7e – S7f).
containing 100 mM of EDC and 25 mM of NHS (Figure 6). The CuLN2O2
-
8
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10.1002/chem.202100250
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labelled, LN2O2-labelled and label free aptamers were diluted in PBS to a
concentration of 20 μM. A heat treatment (8 min at 90 °C, 4 min at 0 °C
and 15 min at room temperature) was applied to ensure the linearization
of aptamer sequence. A drop of 20 μL of the different aptamers solutions
was placed on the working electrode and left to react during 4 hours at
room temperature and under humid atmosphere, allowing the formation of
a peptide bond between the 5’ terminal amine group of the aptamer and
the activated surface carboxylic group. The electrodes were then washed
with PBS buffer and square wave voltammograms (SWV) were recorded
between 200 mV to 600 mV (Step potential: 1 mV, modulation amplitude:
10 mV, frequency: 50 Hz, interval time 0.02 s, scan rate: 53 mV.s-1). The
detection step consisted in measuring these voltammograms after
incubation of a 50 μL solution of E2 (at concentrations ranging from 0.05
to 8 µM) in Tris buffer (20 mM, pH 7.6) on the electrode for 1 hour at room
temperature.
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Acknowledgements
The work was carried out in the framework of Capt’Avenir project
N° 2015006021. This project has received funding from Occitanie
region through the European Regional Development Fund
(ERDF).
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9
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Entry for the Table of Contents
A powerful alternative for biomolecule labelling, based on the electrochemical properties of a metallic complex, is presented. A bis-
triazole ligand and its corresponding copper complex were synthesized and characterized. The coupling of the copper complex with
an aptamer was realized and confirmed by mass spectroscopy analysis. The labelled aptamer was then immobilized on a screen-
printed carbon electrode for the design of an estradiol aptasensor.
10
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