Fluorescent molecular rotors as sensors for the detection of thymidine phosphorylase

Article abstract of DOI:10.1016/j.bmc.2020.115881

Three new fluorescent molecular rotors were synthesized with the aim of using them as sensors to dose thymidine phosphorylase, one of the target enzymes of 5-fluorouracil, a potent chemotherapic drug largely used in the treatment of many solid tumors, tha

Full text of DOI:10.1016/j.bmc.2020.115881

Bioorg. Med. Chem. 29 (2021) 115881
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Fluorescent molecular rotors as sensors for the detection of
thymidine phosphorylase
a
,
c
a,
c,
*
b
b
,
1
a
Manuela Petaccia , Luisa Giansanti
, James N. Wilson , Heajin Lee , Sara Battista ,
c
Giovanna Mancini
a
Dipartimento di Scienze Fisiche e Chimiche, Universit a` degli Studi dell’Aquila, Via Vetoio 10, 67100 Coppito (Aq), Italy
Department of Chemistry, University of Miami, 1301 Memorial Drive, Coral Gables, FL 33124, USA
CNR – Istituto per i Sistemi Biologici, Via Salaria km 29.300, 00016 Monterotondo Scalo (RM), Italy
b
c
A R T I C L E I N F O
A B S T R A C T
Keywords:
Three new fluorescent molecular rotors were synthesized with the aim of using them as sensors to dose thymidine
phosphorylase, one of the target enzymes of 5-fluorouracil, a potent chemotherapic drug largely used in the
treatment of many solid tumors, that acts by hindering the metabolism of pyrimidines. 5-Fluorouracil has a very
narrow therapeutic window, in fact, its optimal dosage is strictly related to the level of its target enzymes that
vary significantly among patients, and it would be of the utmost importance to have an easy and fast method to
detect and quantify them.
Molecular Rotor
Thymidine Phosphorylase
5
-Fluoruracil
Fluorescent Sensor
Spacer Length
The three molecular rotors developed as TP sensors differ in the length of the alkylic spacer joining the ligand
unit, a thymine moiety, and the fluorescent molecular rotor, a [4-(1-dimethylamino)phenyl]-pyridinium bro-
mide. Their ability to trigger an optical signal upon the interaction with thymidine phosphorylase was investi-
gated by fluorescent measurements.
1
. Introduction
with similar body surface area.7,8 It is thus evident that a reliable, and
possibly inexpensive, method to rapidly and easily detect the presence of
Thymidine phosphorylase, TP, thymidylate synthase and dihy-
dropyrimidine dehydrogenase, DPD, are the target enzymes of
–fluorouracil (5-FU), a chemotherapeutic agent that interferes with the
these enzymes in patients before and during the treatment with 5-FU is a
serious medical issue.
5
In general, a chemical sensor requires the assembly of one or more
receptor units together with a transducer part in a defined geometry to
allow the fast and reliable identification of the presence or the absence
of specific target molecules. Usually the sensing element and the
transducer are distinct components packaged together in direct spatial
contact in the same unit. In certain chemical sensors the sensing element
and the transducer responsible of signaling the recognition event are
parts of the same molecule,9 so they are in close proximity because
metabolism of pyrimidines and that is used in the treatment of many
solid malignant tumors (breast, colon, and skin cancer). 5-FU has a very
narrow therapeutic window and only approximately 25% of patients are
treated with the optimal dose of it, whereas in the other cases patients
are overdosed or underdosed with a consequent increase of the toxic side
effects or a reduction of the therapeutic efficacy, respectively.1,2 The
high variability of the efficacy of the treatment is strictly related to the
variability of the level of the target enzymes of 5-FU in patients
covalently linked though electronically independent1
0–12
When the
tissues.3 In addition, 5–8% of patients are affected by DPD deficiency
–5
6
response signal is based on fluorescence these sensors are called
extrinsic fluorescent probes or conjugates13 with the interaction of the
analyte with the receptor inducing a change in the fluorophore sur-
roundings and, as a consequence, in its emission.
and for them the administration of the drug can be fatal because DPD is
directly involved in 5-FU catabolism. At present 5-FU is typically dosed
after evaluating body surface area of each, but it is well known that this
estimation bring to different 5-FU systemic exposure for patient because
levels of 5-FU in plasma can significantly vary also between subjects
We previously described a fluorescent sensor for the detection of TP
based on liposomes containing both an amphiphile tail-tagged with a
*
Corresponding author.
E-mail address: luisa.giansanti@univaq.it (L. Giansanti).
1
Present address: Department of Chemistry, Tampa University, 401 W. Kennedy Blvd. Tampa, FL 33606.
https://doi.org/10.1016/j.bmc.2020.115881
Received 9 October 2020; Received in revised form 12 November 2020; Accepted 13 November 2020
Available online 21 November 2020
0
968-0896/© 2020 Elsevier Ltd. All rights reserved.

M. Petaccia et al.
Bioorganic & Medicinal Chemistry 29 (2021) 115881
pyrene moiety and a 5-FU derivative, where the fluorescence signal
occurred upon the interaction of 5-FU derivative with TP, due to the
variation of excimer/monomer ratio of pyrene residues.14 In that case
the sensor elements were not covalently linked. Here we report the
synthesis and the characterization of new extrinsic fluorescent molecu-
lar probes (1, 2 and 3, Chart 2) designed to detect the target enzymes of
as a function of the aminoacidic composition of the enzymatic pocket
and of its conformation. The ability of the three MRs to trigger an optical
signal upon the interaction with the target enzyme was investigated by
fluorescent measurements.
2. Experimental section
5
-FU. The evaluation of binding and sensor capabilities was carried out
on TP because it is the only commercially available among the three
target enzymes.
2.1. Instrumentation
The three newly synthesized fluorescent molecules differ for the
length of the alkyl spacer joining the receptor unit, a thymine moiety,
and the fluorophore residue responsible of signaling the recognition
event, a [4-(1-dimethylamino)phenyl]-pyridinium bromide, a molecular
rotor (MR). Among MRs, the fluorescence properties of p-(dimethyla-
1H and 13C NMR spectra were carried out on a Varian NMR 500 MHz;
δ in ppm relative to the residual solvent peak of CDCl
ppm for 1H and 13C, respectively; J in Hz.
3
at 7.26 and 77.0
Steady-state fluorescence experiments were carried out on
Fluoromax-4 Horiba-Jobin Yvon spectrofluorimeter.
a
1
5–18
mino)-benzene derivatives have been extensively studied.
probes 1, 2 and 3, belonging to this class of MRs, exhibit the so-called
turn-on” emission as a function of their chemical microenvironment.
The
High-resolution electrospray ionisation mass spectrometry (HRE-
SIMS) spectra were recorded using a Micromass Q-TOF Micromass
spectrometer (Waters) in the electrospray-ionization mode.
“
The presence of the electron donating dimethyl amino group coupled
opposite to the electron withdrawing N-methylpyridinium ring yields a
molecule with strong charge transfer character in the planar locally
excited (LE) state. Another feature that contributes to the high sensi-
tivity of these compounds is the ability of the molecule to twist from
coplanarity, that is, to form a dihedral angle φ between the aniline and
pyridinium rings (Figure 1) significantly different from zero.
2.2. Materials
Phosphate–buffered saline (PBS, 0.01 M phosphate buffer; 0.0027 M
KCl; 0.137 M NaCl; pH 7.4), TP recombinant from Escherichia coli and all
reagents employed for the synthesis of 1–3 were purchased from Sigma-
Aldrich and were used without further purification.
The minimum excited-state energy is reached when the aniline
◦
(
donor) and pyridinium ring (acceptor) planes are rotated 90 relative to
2.2.1. Synthesis of pyrimidines 1b, 2b and 3b
each other (Figure 1). In this geometrical condition the energy gap be-
tween the twisted intramolecular charge transfer (TICT) state and the
α
, -Alkyldibromide 1a (2a, 3a) (1 eq) was added to a solution of
ω
thymine (4 eq) dissolved in DMF (“purum” grade bottle) (26 eq) fol-
lowed by anhydrous potassium carbonate (2 eq). The mixture was stir-
red for 48 h at room temperature. The resulting slurry was filtered
through a Celite pad and the cake washed two times with DMF (5 mL).
The solvent was removed under reduced pressure to give 1b (2b, 3b) as
a white solid that was purified by column chromatography on silica gel
using CH C1 /ETOAc 9:1 as eluent (yield 37% for 1b, 45% for 2b and
ground state (GS) becomes dramatically small and the relaxation from
this twisted state is practically radiationless.1
7,19
Polar solvents
conceivably help to stabilize the TICT state resulting in a shift to lower
emission energies as well as lead to emission quenching. On the other
hand, apolar solvents do not particularly favor the formation of TICT
state with consequent increase of emission intensity.18 The twisting is
also supposed to be limited in viscous solvents or in geometrically
confined environments hence resulting in a sharp increase of emission.20
The binding of the new fluorescent molecules with the binding site of the
target enzymes, promoted by the presence of thymine, should place the
MR in a less polar and restrictive environment compared to the free
molecule in aqueous solvent. In this configuration turn-on emission of
the MR should occur. This approach offers the advantage of minimizing
background emission from unbound MR, because the emission in
aqueous solvent is turned-off.
2
2
1
55% for 3b). Compound 1b: H NMR (CDCl ) δ ppm 1.86 (d, 3H, J = 1.5
3
Hz), 2.51 (t, 3H, J = 2 Hz), 4.16 (t, J = 8 Hz, 2H), 4.67 (t, J = 8 Hz, 2H),
1
3
7.57 (d, J = 1 Hz, 1H) C NMR (500 MHz, CDCI3) 8 12.44, 42.45,
1
66.17, 116.93, 151.01, 158.43, 161.55. Compound 2b: H NMR (CDCl )
3
δ ppm 1.31 (m, J = 1.2, 4H), 2.05 (s, 3H), 3.44 (t, J = 8.5 Hz, 2H), 3.88
1
3
(t, J = 8.5 Hz, 2H), 7.53 (s, 1H), 11.2 (s, 1H). C NMR (500 MHz,
CDCI3) δ ppm 12.4, 30.1, 35.9, 48.9, 110.9, 139.2, 150.8, 163.7.
Compound 3b: 1H NMR (CDCl ) δ ppm 1.67–1.71 (m, 3H, J = 6 Hz),
3
1.74–1.79 (m, 3H, J = 6 Hz), 2.50 (s, 3H) 3.55 (t, J = 6.5 Hz, 2H), 3.65
1
3
The different length of the alkylic spacer of the three new fluorescent
MRs 1–3 might affect the ability to show the turn-on emission upon the
binding with TP because the fluorophore should be located in environ-
ments characterized by different local polarity and/or steric hindrance
(t, J = 6.5 Hz, 2H), 7.54 (s, 1H), 11.23 (s, 1H). C NMR (CDCl ) δ ppm
3
12.43, 27.67, 29.64, 34.98, 46.73, 109, 141.81, 151.37, 164.74.
2.2.2. Synthesis of 4-[4-(1-Dimethylamino)phenyl]-pyridine (DMAPP)
Palladium (II) acetate (5% mol) and triphenylphosphine (10% mol)
were added to a mixture of 4-bromopyridine HCl (1.2 eq), then a solu-
tion of 4-(dimethylamino)phenylboronic (1 eq), and potassium car-
bonate (3 eq) in dimethylformamide (DMF, 1.5 mL) was added. The
reaction mixture was kept at reflux overnight, then filtered through a
celite plug and concentrated. The crude product was purified by flash
chromatography (silica, CH
2
Cl
2
/ETOAc = 99:1 → 95:5) to give the
1
product, DMAPP, as a tan solid (30%). H NMR (CDCl
3
): δ ppm 8.56 (d,
J = 4.9 Hz, 2H), 7.61 – 7.55 (m, 2H), 7.46 (dd, J = 4.7, 1.5 Hz, 2H), 6.82
–
1
6.76 (m, 2H), 3.02 (s, 6H) 13C NMR (CDCl
28.3, 131.2, 147.2, 149.7, 155.8.
3
): δ ppm 41.3, 112.7, 120.6,
2
.2.3. Synthesis of MRs 1, 2 and 3
4
-[4-(1-Dimethylamino)phenyl]-pyridine (1 eq) was combined with
1
eq of 1b (2b, 3b) and 0.5–4.0 mL of DMF. The reaction mixture was
◦
heated at 80 C for 48 h to observe the formation of a yellow precipitate.
After cooling the reaction mixture, the crude product was isolated by
suction filtration and rinsed with EtOAc. The crude product was crys-
tallized from MeOH/EtOAc to yield 32% for 1, 62% for 2 and 75% for 3
Fig. 1. Intramolecular twisting of MR of 1, 2 and 3.
2

M. Petaccia et al.
Bioorganic & Medicinal Chemistry 29 (2021) 115881
of yellow powders.
(2H, t, J = 7.5 Hz), 2.85 (2H, s,), 1.73 (3H, s) 1.87 (2H, t, J = 7.5 Hz),
3.07 (s, 6H), 3.7 (2H, t, J = 7.5 Hz) 4.48 (2H, t, J = 7.5 Hz), 6.86 (2H, d,
J = 5.0 Hz), 7.53 (1H,s), 8.03 (2H, d, J = 5.0 Hz), 8.33 (2H, d, J = 6.5
1
Spectral data relative to compound 1 H NMR (DMSO‑d
s, 6H), 3.2–3.8 (2H,m), 4.67 (2H, t, J = 7.5 Hz), 6.85 (2H, d, J = 5.0
Hz), 7.53 (1H,s), 8.03 (2H, d, J = 5.0 Hz), 8.32 (2H, d, J = 6.5 Hz), 8.8
6
): δ ppm 3.07
(
1
3
Hz), 8.79 (2H, d, J = 7.0 Hz), 11.25 (1H,s). C NMR (DMSO‑d ): δ ppm
6
1
3
(
2H, d, J = 7.0 Hz) 10.93 (1H, s) C NMR (DMSO‑d
6
): δ ppm 12.4, 41.3,
8.2, 52.9, 110.9, 112.7, 125.9, 128.4, 130.2, 139.2, 146.4, 150.8,
55.1, 163.7. HRESIMS m/z found 430.1020 (calcd for C32 44NO
12.4, 25.6, 28.0, 34.8, 40.1, 40.2, 40.4, 40.6, 47, 49.1, 58.8, 109.0,
112.6, 119.2, 121.4, 130.1, 141.9, 144.1, 151.4, 153.5, 154.4, 164.8.
4
1
H
3
HRESIMS m/z found 458.1338 (calcd for C32
H44NO
3
[M]+, 458.1336).
[
M]+, 430.1016).
Spectral data relative to compound 2 H NMR (DMSO‑d
1
6
): δ ppm. 1.74
2
.3. Absorbance and emission characterization of MRs 1, 2 and 3
(
2H,s), 2.49 (3H,s), 3.07 (6H, s), 3.7 (2H, t, J = 7.5 Hz) 4.48 (2H, t, J =
7
8
.5 Hz), 6.85 (2H, d, J = 5.0 Hz), 7.53 (1H,s), 8.03 (2H, d, J = 5.0 Hz),
13
Absorbance spectra of 10
μ
M solutions of 1, 2 and 3 in methanol,
.32 (2H, d, J = 6.5 Hz), 8.8 (2H, d, J = 7.0 Hz), 11.27 (1H,s). C NMR
): δ (ppm) 12.4, 24.5, 41.3, 48.9, 55.2, 110.9, 112.7, 125.9,
28.4, 130.2, 139.2, 146.4, 150.8, 155.1, 155.3, 163.7. HRESIMS m/z
44NO
[M]+, 444.1233).
◦
octanol, water and PBS were recorded at 25 C (λmax, abs = 423 nm).
(
DMSO‑d
6
Emission spectra of solutions 1
μM of 1, 2 and 3 were recorded (λex =
1
4
23 nm, λem = 440–700 nm) in the same experimental conditions. The
found 444.1231 (calcd for C32
H
3
1
fluorescence quantum yields (Q) of 1 µM solution of 1, 2 and 3 were
Spectral data relative to compound 3 H NMR (DMSO‑d
6
): δ ppm 1.99
◦
measured in methanol, octanol, chloroform and PBS at 25 C, on
Scheme 1. Synthetic pattern for the preparation of the 1–3 derivatives.
3

M. Petaccia et al.
Bioorganic & Medicinal Chemistry 29 (2021) 115881
solutions with absorbance lower than 0.05 to minimize inner filter ef-
fects. Perylene in cyclohexane was used as a reference for quantum
e. PBS and methanol, characterized by high polarity and low viscosity,
octanol, characterized by high viscosity and low polarity, and chloro-
2
1
24,25
yields. Photoluminescence quantum yield (Q) of amphiphile solutions
was determined by comparing the wavelength-integrated intensity of an
unknown sample to that of a cyclohexane solution of perylene used as a
standard. The quantum yield of the unknown sample was calculated
according to equation (1):
form that features a low viscosity and the lowest polarity.
1–3
fluorescent MRs are organic cationic dyes that display solvatochromic
photophysical properties. Their absorption maxima are blue-shifted in
polar media, ranging from 423 nm in PBS or alcohols to 436 nm in
chloroform (Table 1). As expected the presence and the length of the
alkyl chain do not significantly influence the absorption properties of
the fluorophore in all the solvents investigated. In Figure 2 the absorp-
tion and emission spectra of compound 3 in different solvents are re-
ported as an example.
2
Q = Q I
R R
OD n
I
(1)
R
OD n2
R
where I is the integrated intensity, n is the refractive index, and OD is
the optical density. The subscript R refers to the reference fluorophore of
known Q.
The red-shift and the lower intensity in fluorescence emission
observed in octanol with respect to chloroform is probably due to its
higher polarity (
μ = 1.76 D and μ = 1.04D for octanol and chloroform,
respectively).23
2
.4. Binding of compounds 1, 2, 3 with TP
On the other hand, the emission of 1–3 is quenched in polar and
scarcely viscous solvents (Table 1). These results are in good agreement
with those reported in the case of MRs containing the p–(dimethy1a-
mino) group.26
In order to study the interaction of compounds 1, 2 and 3 with TP
enzyme, emission fluorescent spectra (λexc = 430 nm) were run in the
presence of a fixed TP concentration (3 µM) varying the concentration of
MR (0.18–1.8 mM). The plot of the maximum fluorescent emission (λmax
As a whole, absorbance and emission spectroscopies indicate that the
three fluorescent MRs exhibit similar photophysical behavior, including
high sensitivity to the medium polarity as shown by their ability to ac-
cess the non-fluorescent TICT state and, hence, to reduce their emission
signal.
=
517 nm) vs TP enzyme concentration gave the corresponding binding
curve and the binding constant (K ) was determined from curve fitting.
d
3
. Results and discussion
3
.3. Fluorescence of the MRs upon binding with TP
3
.1. Synthesis of the fluorescent molecular MRs 1–3
The ability of the MRs 1–3 to give a flurescent signal due to TICT
MRs 1, 2 and 3 were prepared according to Scheme 1.
process induced by the binding with TP was assessed by fluorescence
experiments. A fluorescent signal switched on in the presence of TP in
the case of the MR 1 (Figure 3a) whereas the presence of the enzyme did
not induce any fluorescent signal in the case of MRs 2 and 3, as shown in
Figure 3b in the case of compuond 3 reported as example.
Compounds 1–3 were prepared by alkylating 4-[4-(1-dimethyla-
mino)phenyl]-pyridine, DMAPP, with synthetic products 1b, 2b and
3
b obtained by reacting the corresponding αω-dibromoalkanes (1a, 2a
and 3a) with 4 equivalents of thymine in the presence of a stoichio-
metric amount of K CO according to a procedure reported in litera-
2
3
In the case of 1 the emission induced by the presence of TP is
centered at ~ 517 nm, value of wavelength very similar to the value
observed in the case of the emission spectrum of the MR in octanol. On
the other hand, in the case of 2 and 3 no fluorescence is observed in the
presence of TP. This finding suggests either that no binding occurs or
that upon the binding of the enzyme the MR is located in a different
environment with respect to 1 characterized by different polarity and/or
viscosity. Considering that in the case of 2 and 3 the difference with 1 is
one or two methylenes in the alkyl spacer, respectively, is unlikely that
these molecules can fold in such a way that their binding to the active
site of TP is hindered. It is more reliable to hypothesize that, despite the
interaction with the enzyme, the probe is not properly geometrically
confined; as a consequence, its twisting is not limited, or at least not
enough, to block the molecules in a configuration that results in a turn-
on emission. At this purpose, we decided to carry out molecular dy-
namics simulations of the complexes of 1, 2 or 3 with TP in aqueous
solution in thermal conditions which will be reported in a fortcoming
study. Preliminary results, reported in detail in the Supplementary In-
formation, show a markedly different behaviour of 1 with respect to 2
and 3. More precisely, only the former, along the whole simulation time,
2
2
ture. The yield of both reactions is higher as the length of the alkyl
spacer increases. Probably the short alkyl spacer, that characterizes
compounds 1, involves steric hindrance in the nucleophilic sub-
stitutions. Molar ratios were optimized in order to reduce the amount of
2
3
dialkylated product. DMAPP was prepared as previously reported by a
Suzuki cross coupling of 4-bromopyridine with 4-(dimethylamino)phe-
nylboronic acid.
3
.2. Characterization of the fluorescent molecular MRs 1–3
All the photophysical features of the novel amphiphilic fluorescent
MRs were investigated in different solvents. Absorbance and emission
spectra were recorded in solvent with different polarity and viscosity, i.
2
7
appears as stably confined within the initial configuration hence
qualitatively explaining, although not fully elucidating, the different
Table 1
Photochemical properties of fluorescent MRs 1, 2 and 3.
MRs
SOLVENTS
METHANOL
PBS
OCTANOL
(λ =
423)
CHLOROFORM
ε
(λ =
Q
ε
(λ =
Q
ε
Q
ε
(λ =
Q
4
25)
423)
436)
-
1
ꢀ 1
-1 ꢀ 1
-1 ꢀ 1
-1 ꢀ 1
cm M
cm
M
cm
M
cm
M
1
2
3
31,000
35,000
32,000
-
-
-
25,000
26,000
23,000
-
-
-
28,000
29,000
29,900
0.63
0.62
0.69
37,000
38,500
38,600
0.87
0.95
0.95
Chart 2. Molecular structure of fluorescent molecular sensors 1, 2, 3.
4

M. Petaccia et al.
Bioorganic & Medicinal Chemistry 29 (2021) 115881
Fig. 2. Absorbance and emission spectra of 3 in different solvents.
Fig. 3. (a) Fluorescent spectra of samples containing different amounts of compound 1 (left) and 3 (right) in the presence and in the absence (black trace) of 3
μM TP.
spectroscopic behaviour in the presence of the protein.
To evaluate the K of the binding of the MR with the target enzyme
This result indicates that the functionalization of thymine at the N3
d
position does not affect its ability to interact with the target enzyme (i.e.
functionalization does not hamper the access of thymine to the binding
site of TP). Therefore is reasonable to assume that also 2 and 3, though
featuring a longer alkyl spacer, bind to the active site of TP without
being embedded in a chemical microenvironment that induces the
occurrence of a fluorescent signal.
the intensity of the emission of increasing amounts of the MR was
investigated in the presence of a given amount of TP. The plot of the
intensity of the maximum at 517 nm versus the concentration of 1
(
Figure 4) shows that the intensity of the fluorescent signal increases as a
function of the amount of 1 up to a threshold value that corresponds to
the saturation of the binding site of the enzyme.
The obtained K
d
value, 200 µM, is in good agreement with the data
4. Conclusions
2
8
reported in the literature concerning the binding of thymine to TP.
Three new fluorescent amphiphilic MRs 1, 2 and 3, differing in the
length of the alkyl spacer joining the receptor unit, a thymine moiety,
and a MR as fluorophore were synthesized and characterized. Their
potentiality as extrinsic molecular fluorescent sensors to detect the
target enzyme TP was investigated. The study of the interaction of 1–3
with TP revealed that only compound 1 is able to exhibit a “turn-on”
fluorescent signal upon the binding with the enzyme through the elim-
ination of a non-radiative quenching in the less polar environment of the
enzyme binding site. On the other hand, the other two MR 2 and 3 does
not give any fluorescent signal in the presence of the target enzyme. As a
consequence, the relaxation from the excited state proceeds mainly via
the nonradiative TICT pathway. The finding that one of the newly syn-
thesized MRs is able to detect the presence of TP enlarges the prospec-
tive of exploitation of MRs in the design of molecular sensors and
constitutes a proof of principle that could be extended to the fast and
easy detection of biological markers.
Fig. 4. Plot of the intensity of fluorescence band of 1 in the presence of 3
TP, at 517 nm, as a function of concentration of 1.
μ
M
5

M. Petaccia et al.
Bioorganic & Medicinal Chemistry 29 (2021) 115881
Declaration of Competing Interest
11 Meng LZ, Mei GX, He YB, Zeng ZY. Synthesis and Anion Recognition Properties of
Two Novel Tetraamide Calix[4](aza)crowns for Optical Anion Sensor. Acta Chim
Sinica. 2005;63:416–420.
The authors declared that there is no conflict of interest.
1
1
1
2
3
4
Lee JY, Kim SK, Jung JH, Kim JS. Bifunctional Fluorescent Calix[4]arene
Chemosensor for Both a Cation and an Anion. J Org Chem. 2005;70:1463–1466.
Kashyap B, Dutta K, Das DK, Phukan P. Structurally Simple Ferrocene Derivatives for
Selective Cadmium Sensing. J. Fluoresc. 2014;24(3):975–981.
Acknowledgements
Petaccia M, Giansanti L, Leonelli F, La Bella A, Gradella Villalva D, Mancini G.
Fluorescent Lipid Based Sensor for the Detection of Thymidine Phosphorylase as
Tumor Biomarker. Sens. Actuators B Chem. 2017;245:213–220.
The authors thank Prof. Massimiliano Aschi for his support in theo-
retical calculations.
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