Synthesis, characterization and anion recognition studies of new fluorescent alkyl bis(naphthylureylbenzamide)-based receptors

Article abstract of DOI:10.1016/j.tet.2019.130815

The synthesis of new alkyl (C₃ to C₈) bis(naphthylureylbenzamide)-based receptors and the study of their interaction with anions by UV–Vis, molecular fluorescence and ¹H NMR were performed. The results suggest both ureylbe

Full text of DOI:10.1016/j.tet.2019.130815

Journal Pre-proof
Synthesis, characterization and anion recognition studies of new fluorescent alkyl
bis(naphthylureylbenzamide)-based receptors
Luis Miguel Lopéz-Martínez, José García-Elías, Adrián Ochoa-Terán, Hisila
Santacruz Ortega, Karen Ochoa-Lara, César Ulises Montaño-Medina, Anatoli K.
Yatsimirsky, José Z. Ramírez, Victoria Labastida-Galván, Mario Ordoñez
PII:
S0040-4020(19)31222-0
https://doi.org/10.1016/j.tet.2019.130815
TET 130815
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 17 October 2019
Revised Date: 15 November 2019
Accepted Date: 19 November 2019
Please cite this article as: Lopéz-Martínez LM, García-Elías José, Ochoa-Terán Adriá, Ortega HS,
Ochoa-Lara K, Montaño-Medina CéUlises, Yatsimirsky AK, Ramírez JoséZ, Labastida-Galván
V, Ordoñez M, Synthesis, characterization and anion recognition studies of new fluorescent alkyl
bis(naphthylureylbenzamide)-based receptors, Tetrahedron (2019), doi: https://doi.org/10.1016/
j.tet.2019.130815.
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Synthesis, characterization and anion recognition studies of new
fluorescent alkyl bis(naphthylureylbenzamide)-based receptors
Luis Miguel Lopéz-Martínez1,2, José García-Elías3, Adrián Ochoa-Terán*1, Hisila
Santacruz Ortega4, Karen Ochoa-Lara*4, César Ulises Montaño-Medina1, Anatoli K.
Yatsimirsky5, José Z. Ramírez,1 Victoria Labastida-Galván6 and Mario Ordoñez6
1 Centro de Graduados e Investigación en Química, Tecnológico Nacional de México/IT
de Tijuana, Tijuana, B.C. México.
2 Universidad Estatal de Sonora (UES), Hermosillo, Sonora, México.
3
Unidad Académica de Ciencias de la Tierra, Universidad Autónoma de Zacatecas,
Zacatecas, México.
4
Departamento de Investigación en Polímeros y Materiales. Universidad de Sonora.
Hermosillo, Sonora. México
5 Facultad de Química, Universidad Nacional Autónoma de México. Ciudad de México.
México.
6
Centro de Investigaciones Químicas-(IICBA), Universidad Autónoma del Estado de
Morelos, Cuernavaca, México.
* Correspondence e-mail: ochoa@tectijuana.mx, karenol@polimeros.uson.mx
Abstract
The synthesis of new alkyl (C3 to C8) bis(naphthylureylbenzamide)-based receptors and
1
the study of their interaction with anions by UV-Vis, molecular fluorescence and H
NMR were performed. The results suggest both ureylbenzamide units participate in the
complexion with anions due to their inherent flexibility, which promotes a cooperative
binding effect. The position of the urea and amide groups, as well as, the alkyl chain
length, have an important influence in the fluorescent response; meta receptors present
an ON1/OFF/ON2 response, while ortho receptor have an OFF/ON response. The most
significant changes were observed with dihydrogen phosphate and hydrogen
1
pyrophosphate due to high affinity for these anions. The H NMR studies show
1

differences in the interaction sites depending the relative position of the urea and amide
groups, as wells as, the anion type. Finally, a theoretical analysis of complexes by DFT
support the experimental results.
Keywords: Anion recognition; fluorescent receptors; bis-urea; benzamide
Introduction
The selective recognition of anions has been studied over the last two decades and is
subject of important research in the supramolecular chemistry field focused to selective
recognition of anions with biological importance.1,2 Anions play an important role in a
range of biological processes.3, 4 They are essential in the formation of a variety of
enzyme-substrate and enzyme-cofactor complexes, as well as, in the interaction between
proteins and nucleic acids.5,6 On the other hand, the poor regulation of anion transport
mechanisms results in serious complications and diseases, such as cystic fibrosis.7
Consequently, it is important to develop receptors or sensors that can detect the
presence of various anionic species and regulate their transport in biological systems.8
One way in which host molecules can recognize anionic guests is based on electrostatic
interactions.9 However, host molecules that operate based on this principle often suffer
lack of selectivity due to the non-directional properties of electrostatic interactions.5
The chromogenic and fluorogenic receptors that include urea groups as a binding
subunit have proved to be particularly excellent candidates for recognition of anions,
such as fluoride, acetate, dihydrogen phosphate, among others.3,10,11 A very important
motif for anion recognition is the hydrogen bonding because of its directionality, which
is extremely useful in design of selective hosts for anions with different geometries.12,13
Most of the receptors belonging to this class of urea derivatives are neutral and contain
a subunit capable to donate H-bonds to the anion, therefore, anion recognition occurs
through the H-bonding interaction of urea N-H hydrogens, to give a stable anion-
receptor complex.1,4,9 The negative charge of anion stabilizes the excited state of the
chromophore reducing the energy of urea-to-chromophore charge-transfer transition.14,
15
On the other hand, deprotonation of one N-H fragment is accompanied by a drastic
electron rearrangement in the receptor, which results in a significant change of
1
absorption spectrum, as well as, the pattern of H NMR signals, which makes the
process unambiguously discernible.2,9,14 Also, some receptors that contain thiourea
groups as hydrogen bond donors offers a greater opportunity for anion recognition than
2

urea, due to an enhanced acidity of the N-H bonds, that readily deprotonates with a
number of anionic species.1,11,12,16 These findings demonstrate considerable selectivity
for specific anions, even in protic media.10 The urea (or thiourea) moiety has been used
as a host attached to o-phenylene,7,17 m-phenylene, p-phenylene,6,11 naphthalene,18
anthracene, pyrene19,20 and anthraquinone.5,21 However, anion selective systems able to
operate in aqueous media remains as considerable challenge.10
The synthesis of urea-based receptors capable of interact selectively with anions is an
important subdiscipline in supramolecular chemistry. The anionic recognition by this
type of receptors is mainly based on hydrogen bond donation and the functionality and
directionality provided by the N-H group.22 These characteristics play an important role
in selective recognition of anions with different geometries. An example was reported
by Amendola et al., where selective binding of urea with acetate was described as a "Y"
type arrangement, through two hydrogen bonds provided by the urea in the receptor
giving rise to the anionic recognition. In addition, the selectivity is also related to the
energy of receptor-anion interaction.23,24 The strongest interactions of hydrogen bonds
are established with the most basic anions such as fluoride, carboxylates and inorganic
oxoanions. According to a recent point of view, all hydrogen bonds are considered as
proton transfer reactions.25-27 Therefore, for a receptor containing acid N-H groups, the
selectivity is related mainly with the anion basicity or pKa for the conjugate acid. The
greater basicity of the anion generates a stronger hydrogen bonding interaction.28
Moreover, Dos Santos et al. reported receptors with urea and acetamide groups in a
different relative position (ortho, meta and para). This investigation showed that the
position affects selectivity and stoichiometry of the complexes.29 In receptors with meta
and para relative position, the anionic recognition is carried out only through the urea
group, while the amide group participates only when there is an allosteric change and
the conformation is favored to establish a hydrogen bond with the anion, forming a
complex with 1:2 stoichiometry. In addition, the receptor with a relative ortho position
forms complexes with 1:1 stoichiometry, and there is cooperativity from the amide in
the complexes.29
The bis(urea) receptors have become good hosts for the recognition of anions with
different size and geometry, this is attribute to the donation of hydrogens by the N-H
groups present in the receptor. However, these receptors also fulfil other important
characteristics to increase the viability of molecular recognition as the high structural
3

complexity and the preorganization. In the bis(urea) receptors, more binding sites are
available to induce cooperativity. This structural level allows the host to adopt different
conformations in the complexation process to reach all available sites. In addition, these
receptors exhibit a limited number of stable conformations for binding with the host,
acquiring the most stable form.30,31 In case of halides, which are spherical anions, the
nature of the hydrogen bond is the same, while carboxylates show a "Y" type interaction
with 1:1 stoichiometry. Formation of hydrogen bonds does not change according to the
X-ray diffraction structures shown by Boiocchi et al.25, 26 Hay et al. reported through
theoretical calculations that the geometry of the hydrogen bond can have seven possible
viable forms in complexes with tetrahedral anions.30
In this work, the synthesis of alkyl bis(naphthylureylbenzamide)-based receptors and
the study of their binding properties towards different anions were performed (Figure
1). All receptors contain two naphthylureylbenzamide units connected through an alkyl
chain with different length (C3 to C8) and position (ortho or meta position). According
to the aforementioned, the electronic absorption and emission properties, as well as, the
1
pattern of the H NMR signals, must be sensitive to the H-bond acceptor behavior and
the basicity of the interacting anion. This research was carried out to increase our
knowledge in the relationship between selectivity and the number of available binding
sites, as well as, to detect the possibility of cooperativity and allosteric effects in these
receptors. Therefore, the results obtained should be of importance for the rational design
of new receptors with improved selectivity.
Figure 1. New alkyl bis(naphthylureylbenzamide)-based o(3-8)N and m(3-8)N
receptors.
4

Experimental
Materials and equipment
All reagents and solvents were obtained from commercial suppliers and used without
1
13
further purification. NMR spectra (400 MHz for H, 100 MHz for C) were recorded
on a Bruker Avance 400 spectrometer in DMSO-d6 at a probe temperature of 23 °C,
with TMS as internal standard. Electronic impact mass spectra were obtained by direct
insertion in an Agilent 5975C mass spectrometer, and the intensities were reported as a
relative percentage to the base peak after the corresponding m/z value. FAB mass
spectra and HRMS were recorded in MStation JMS-700 JOEL. UV-Vis absorption
spectra were obtained using a Cary 100 conc UV-Visible spectrophotometer.
Fluorescence emission spectra were obtained using a Cary Eclipse fluorescence
spectrophotometer.
Anion recognition study by UV-Vis titrations
The effect of anions upon the absorbance was examined adding 6 µL of a 1
TBAX solution to a known volume (3 mL) of a 1
10-5 M receptor solution. The
×
10-3 M
×
receptors and TBAX stock solutions were prepared in spectroscopic grade acetonitrile.
A quartz cuvette was filled with 3 mL of the receptor stock solution, then 6 µL aliquots
of TBAX stock solution were added successively with a calibrated micropipette, thus
the ratio of the total concentration [TBAX]t/[L]t was in a desired range. The resulting
complex solutions were stirred for 3 min and the spectra were recorded. The absorption
spectrum was confirmed to be unchanged with time at each measurement.
Anion recognition study by fluorometric titrations
The effect of anions upon the emission was examined adding 7.5 µL of a 1
TBAX solution to a known volume (3 mL) of a 1
10-5 M receptor solution. A quartz
×
10-3 M
×
cuvette was filled with 3 mL of the receptor stock solution, and then 7.5 µL aliquots of
TBAX stock solution were added successively with a calibrated micropipette, thus the
ratios of the total concentration [TBAX]t/[L]t was in a desired range. The resulting
complex solutions were stirred for 3 min and the spectra were recorded. The emission
intensity was confirmed to be unchanged with time at each measurement.
5

Anion recognition study by NMR titrations
The effect of anions upon the chemical shifts of the receptor signals was examined
adding 2 µL of a 0.3 M TBAX stock solution to a known volume (0.5 mL) of a 6
M receptor solution contained in a NMR tube. The addition was limited to 0.056 mL,
then the dilution effect was insignificant. For the studies of anion recognition, a 6
10-3
×
10-3
×
M receptor solution was prepared in DMSO-d6, and then 2 µL aliquots of a 0.3 M
TBAX stock solution was added with a calibrated micropipette, thus the [TBAX]/[L]
1
ratio was in a desired range. The kinetic study by H NMR of anion recognition at
relationship 1:5 was obtained at temperatures of 277 K.
Molecular modelling
The molecular optimization of the receptors and complexes geometries were performed
by DFT, the PBE0 functional and the DZVP base were use in Gaussian 09
computational program.32
Synthesis
Synthesis of alkyl bis(2-aminobenzamide) compounds
In a single neck round bottom flask, the isatoic anhydride (2.9 g. 18.3 mmol) was
suspended in 100 mL of dry tetrahydrofuran (THF) with stirring under Ar atmosphere.
Then, a condenser was connected to the reaction flask and 8.3 mmol of alkyl diamine
(3, 4, 6 and 8 carbons) were added to the mixture. The reaction was refluxed and stirred
for 24 h. Then, the solvent was removed by heating under reduced pressure and the
product was washed and filtered as many times as needed to give an analytically pure
solid compound.
N,N'-(propane-1,3-diyl)bis(2-aminobenzamide) (o3). White solid. Yield: 40.8%. M. p.
1
158-160 °C. FT-IR: 3470, 3355, 3294, 3055, 2967, 2931, 1625, 1576, 1531 cm-1. H
NMR (DMSO-d6, 400 MHz): δ 8.21 (s, 2 H), 7.47 (d, J = 7.7 Hz, 2 H), 7.13 (t, J = 7.2
Hz, 2 H), 6.69 (d, J = 7.7 Hz, 2 H), 6.51 (t, J = 7.2 Hz, 2 H), 6.37 (s, 4 H), 3.27 (brs, 4
H), 1.81 - 1.61 (m, 2 H). 13C NMR (DMSO-d6, 100 MHz): δ 168.8, 149.4, 131.4, 127.8,
116.2, 114.8, 114.5, 36.5, 29.2. MS (FAB+): 313 [M+H]+. HRMS (FAB+): Calculated
for C17H21N4O2: 313.1665; Found: 313.1599.
N,N'-(butane-1,4-diyl)bis(2-aminobenzamide) (o4). White solid. Yield: 72.2%. M. p.
1
190-192 °C. FT-IR: 3469, 3557, 3284, 3056, 2930, 2860, 1621, 1578, 1534 cm-1. H
6

NMR (DMSO-d6, 400 MHz): δ 8.19 (t, J = 4.9 Hz, 2 H), 7.46 (d, J = 7.85 Hz, 2 H), 7.12
(t, J = 7.4 Hz, 2 H), 6.68 (d, J = 7.85 Hz, 2 H), 6.49 (t, J = 7.4 Hz, 2 H), 6.36 (brs, 4 H),
13
3.40 - 3.18 (m, 4 H), 1.59 - 1.49 (m, 4 H). C NMR (DMSO-d6, 100 MHz): δ 168.7,
149.4, 131.3, 127.9, 116.2, 114.9, 114.4, 38.4, 26.7. MS (FAB+): 326 [M+H]+. HRMS
(FAB+): Calculated for C18H23N4O2: 327.1821; Found: 327.1820.
N,N'-(hexane-1,6-diyl)bis(2-aminobenzamide) (o6). White solid. Yield: 54.8%. M. p.
1
158-160 °C. FT-IR: 3473, 3668, 3292, 3057, 2926, 2858, 1622, 1581, 1538 cm-1. H
NMR (DMSO-d6, 400 MHz): δ 8.16 (t, J = 4.4 Hz, 2 H), 7.45 (d, J = 7.9 Hz, 2 H), 7.11
(t, J = 7.4 Hz, 2 H), 6.67 (d, J = 7.9 Hz, 2 H), 6.50 (t, J = 7.4 Hz, 2 H), 6.34 (brs, 4 H),
13
3.28 - 3.10 (m, 4 H), 1.59 - 1.41 (m, 4 H), 1.41 - 1.22 (m, 4 H). C NMR (DMSO-d6,
100 MHz): δ 169.2, 150.0, 131.9, 128.5, 116.7, 115.6, 115.0, 39.2, 29.7, 26.8. MS
(FAB+): 355 [M+H]+. HRMS (FAB+): Calculated for C20H27N4O2: 355.2134; Found:
355.2120.
N,N'-(octane-1,8-diyl)bis(2-aminobenzamide) (o8). White solid. Yield: 36.1%. M. p.
1
164-166 °C. FT-IR: 3475, 3670, 3292, 3057, 2926, 2858, 1622, 1581, 1538 cm-1. H
NMR (DMSO-d6, 400 MHz): δ 8.15 (t, J = 4.9 Hz, 2 H), 7.45 (d, J = 7.9 Hz, 2 H), 7.11
(t, J = 7.4 Hz, 2 H), 6.67 (d, J = 7.9 Hz, 2 H), 6.49 (t, J = 7.4 Hz, 2 H), 6.35 (brs, 4 H),
3.19 (q, J = 6.6 Hz, 4 H), 1.56 - 1.41 (m, 4 H), 1.36 - 1.19 (m, 8 H). 13C NMR (DMSO-
d6, 100 MHz): δ 169.2, 150.0, 131.9, 128.5, 116.7, 115.6, 115.0, 39.3, 29.6, 29.3, 27.0.
MS (FAB+): 383 [M+H]+. HRMS (FAB+): Calculated for C22H31N4O2: 383.2447;
Found: 383.2469.
Synthesis of N,N'-alkyl bis(2-(3-(naphthalen-1-yl)ureyl)benzamide) compounds
In a single neck round bottom flask, 1.0 equivalent of alkyl bis(2-aminobenzamide) (o3,
o4, o6 or o8) was dissolved in 100 mL of dry THF with stirring under Ar atmosphere
(15 min). Then, a condenser was connected to the reaction flask and 2.2 equivalent of 1-
naphthyl isocyanate was added to the mixture. The reaction was refluxed and stirred for
24 h. Then, the solvent was removed by heating under reduced pressure and the product
was washed and filtered as many times as needed to give an analytically pure solid
compound.
N,N'-(propane-1,3-diyl)bis(2-(3-(naphthalen-1-yl)ureyl)benzamide) (o3N). Pearl solid.
Yield: 80.3%. M. p. 218-220 °C. FT-IR: 3298, 3051, 3079, 2827, 2852, 1646, 1543 cm-
7

1. 1H NMR (DMSO-d6, 400 MHz): δ 10.16 (s, 2 H), 9.58 (s, 2 H), 8.74 (t, J = 5.5 Hz, 2
H), 8.23 (d, J = 8.3 Hz, 2 H), 8.10 (d, J = 8.3 Hz, 2 H), 7.90 (d, J = 7.8 Hz, 2 H), 7.75
(d, J = 7.3 Hz, 2 H), 7.67 (d, J = 8.0 Hz, 2 H), 7.62 (dd, J = 7.8, 1.3 Hz, 2 H), 7.53 -
7.38 (m, 8 H), 7.06 (t, J = 7.3 Hz, 2 H), 3.45 - 3.37 (m, 4 H), 1.90 - 1.79 (m, 2 H). 13C
NMR (DMSO-d6, 100 MHz): δ 168.3, 153.3, 139.3, 134.2, 133.7, 131.0, 128.0, 127.7,
127.4, 125.7, 125.4, 123.9, 122.3, 121.0, 120.7, 120.2, 36.7, 28.7. MS (FAB+): 651
[M+H]+. HRMS (FAB+): Calculated for C39H35N6O4: 651.2720; Found: 651.2710.
N,N'-(butane-1,4-diyl)bis(2-(3-(naphthalen-1-yl)ureyl)benzamide) (o4N). Pearl solid.
Yield: 58.7%. M. p. 242-244 °C. FT-IR: 3296, 3254, 3069, 2933, 2861, 1735, 1646,
1
1606, 1543 cm-1. H NMR (DMSO-d6, 400 MHz): δ 10.28 (s, 2 H), 9.62 (s, 2 H), 8.69
(t, J = 5.4 Hz, 2 H), 8.19 (d, J = 8.3 Hz, 2 H), 8.15 (s, 2 H), 7.92 (s, 2 H), 7.77 - 7.67 (m,
4 H), 7.62 (dd, J = 7.8, 1.3 Hz, 2 H), 7.58 - 7.35 (m, 8 H), 7.02 (t, J = 7.4 Hz, 2 H), 3.35
- 3.27 (m, 4 H), 1.70 - 1.52 (m, 4 H). 13C NMR (DMSO-d6, 100 MHz): δ 168.1, 153.5,
139.5, 134.3, 133.8, 131.0, 128.1, 127.7, 125.8, 125.6, 125.5, 124.1, 122.6, 122.2,
121.0, 120.5, 28.9, 26.3. MS (FAB+): 665 [M+H]+. HRMS (FAB+): Calculated for
C40H37N6O4: 665.2876; Found: 665.3036.
N,N'-(hexane-1,6-diyl)bis(2-(3-(naphthalen-1-yl)ureyl)benzamide) (o6N). Pearl solid.
Yield: 35.1%. M. p. 220-222 °C. FT-IR: 3298, 3250, 3073, 2930, 2862, 1737, 1645,
1
1606 cm-1. H NMR (DMSO-d6, 400 MHz): δ 10.24 (s, 2 H), 9.61 (s, 2 H), 8.64 (t, J =
4.8 Hz, 1 H), 8.18 (d, J = 8.3 Hz, 2 H), 8.14 (d, J = 8.8 Hz, 2 H), 7.91 (m, 2 H), 7.72 (d,
J = 7.3 Hz, 2 H), 7.69 (d, J = 8.0 Hz, 2 H), 7.59 (d, J = 7.0 Hz, 2 H), 7.56 - 7.36 (m, 8
H), 7.03 (t, J = 7.5 Hz, 1 H), 3.27 (q, J = 6.3 Hz, 4 H), 1.65 - 1.45 (m, 4 H), 1.44 - 1.25
13
(m, 4 H). C NMR (DMSO-d6, 100 MHz): δ 168.9, 154.2, 140.2, 135.0, 134.5, 131.7,
128.8, 128.4, 128.4, 126.5, 126.2, 124.8, 123.3, 122.7, 121.7, 121.3, 41.1, 40.9, 27.2.
MS (FAB+): 693 [M+H]+. HRMS (FAB+): Calculated for C42H41N6O4: 693.3189;
Found: 693.3144.
N,N'-(octane-1,8-diyl)bis(2-(3-(naphthalen-1-yl)ureyl)benzamide) (o8N). Pearl solid.
Yield: 64.4%. M. p. 216-218 °C. FT-IR: 3332, 3269, 3053, 2922, 1693, 1622, 1614,
1603, 1596, 1526 cm-1. 1H NMR (DMSO-d6, 400 MHz): δ 10.23 (s, 2 H), 9.61 (s, 2 H),
8.62 (t, J = 5.3 Hz, 1 H), 8.20 - 8.12 (m, 4 H), 7.95 - 7.89 (m, 2 H), 7.73 (d, J = 7.5 Hz,
2 H), 7.69 (d, J = 8.3 Hz, 2 H), 7.59 (dd, J = 7.8, 1.5 Hz, 2 H), 7.63 - 7.39 (m, Hz, 4 H),
7.47 (t, J = 8.0 Hz, 2 H), 7.44 (dt, J = 8.5, 4.5 Hz, 2 H), 7.04 (dt, J = 7.8, 1.3 Hz, 2 H),
8

3.25 (q, J = 6.5 Hz, 4 H), 1.52 (s, 4 H), 1.41 - 1.22 (m, 8 H). 13C NMR (DMSO-d6, 100
MHz): δ 168.1, 153.4, 139.4, 134.3, 133.8, 130.9, 128.1, 127.7, 125.8, 125.6, 125.4,
124.0, 122.6, 122.3, 121.1, 121.0, 120.4, 28.9, 28.7, 26.5. MS (FAB+): 721 [M+H]+.
HRMS (FAB+): Calculated for C44H45N6O4: 721.3502; Found: 721.3603.
Synthesis of alkyl bis(3-nitrobenzamide) compounds
In a single neck round bottom flask, 3.4 g (18.3 mmol) of 3-nitrobenzoyl chloride were
dissolved in 183 mL of dry THF with stirring under Ar atmosphere for 15 min. Then,
8.3 mmol of alkyl diamine (3, 4, 6 and 8 carbons) and 0.3 mL of triethylamine were
added to the solution. The reaction was stirred for 24 h. Then, the solvent was removed
by heating under reduced pressure and the product was washed and filtered as many
times as needed to give an analytically pure solid compound.
N,N'-(propane-1,3-diyl)bis(3-nitrobenzamide) (A). White solid. Yield: 90.5%. M. p.
1
210-212 °C. FT-IR: 3363, 3098, 2945, 2874, 1637, 1548, 1524, 1471 cm-1. H NMR
(DMSO-d6, 400 MHz): δ 8.93 (t, J = 5.5 Hz, 2 H), 8.68 (t, J = 2.0 Hz, 2 H), 8.38 (ddd, J
= 8.0, 2.0, 1.0 Hz, 2 H), 8.31 (td, J = 8.0, 2.0 Hz, 2 H), 7.78 (t, J = 8.0 Hz, 2 H), 3.46 -
13
3.36 (m, 4 H), 1.87 (qnt, J = 6.9 Hz, 2 H). C NMR (DMSO-d6, 100 MHz): δ 164.0,
147.7, 135.9, 133.5, 130.0, 125.7, 121.8, 37.4, 28.7. MS (FAB+): 373 [M+H]+. HRMS
(FAB+): Calculated for C17H17N4O6: 373.1148; Found: 373.1143.
N,N'-(butane-1,4-diyl)bis(3-nitrobenzamide) (B). White solid. Yield: 80.3%. M. p. 242-
1
244 °C. FT-IR: 3361, 3091, 2937, 2861, 1637, 1551, 1523, 1478 cm-1. H NMR
(DMSO-d6, 400 MHz): δ 8.87 (t, J = 5.4 Hz, 2 H), 8.67 (t, J = 2.0 Hz, 2 H), 8.37 (ddd, J
= 0.7, 2.0, 8.1 Hz, 1 H), 8.29 (td, J = 1.2, 8.1 Hz, 2 H), 7.77 (t, J = 8.1 Hz, 2 H), 3.39 -
13
3.26 (m, 4 H), 1.66 - 1.56 (m, 4 H). C NMR (DMSO-d6, 100 MHz): δ 163.8, 147.6,
135.9, 133.5, 130.0, 125.6, 121.7, 33.6, 26.4. MS (FAB+): 387 [M+H]+. HRMS (FAB+):
Calculated for C18H19N4O6: 387.1305; Found: 387.1310.
N,N'-(hexane-1,6-diyl)bis(3-nitrobenzamide) (C). White solid. Yield: 59%. M. p. 192-
1
194 °C. FT-IR: 3309, 3100, 2935, 2873, 1629, 1577, 1530, 1478 cm-1. H NMR
(DMSO-d6, 400 MHz): δ 8.83 (t, J = 5.4 Hz, 2 H), 8.67 (t, J = 2.0 Hz, 2 H), 8.37 (ddd, J
= 8.1, 2.3, 1.0 Hz, 2 H), 8.28 (td, J = 8.1, 1.3 Hz, 2 H), 7.77 (t, J = 8.1 Hz, 2 H), 3.38 -
13
3.24 (m, 4 H), 1.63 - 1.47 (m, 4 H), 1.46 - 1.30 (m, 4 H). C NMR (DMSO-d6, 100
9

MHz): δ 163.8, 147.7, 135.9, 133.5, 130.0, 125.6, 121.8, 39.3, 28.8, 26.1. MS (FAB+):
415 [M+H]+. HRMS (FAB+): Calculated for C20H23N4O6: 415.4260; Found: 415.1628.
N,N'-(octane-1,8-diyl)bis(3-nitrobenzamide) (D). White solid. Yield: 59%. M. p. 178-
1
180 °C. FT-IR: 3312, 3100, 2932, 2872, 1629, 1577, 1530, 1477 cm-1. H NMR
(DMSO-d6, 400 MHz): δ 8.81 (s, 2 H), 8.66 (s, 2 H), 8.28 (d, J = 7.3 Hz, 2 H), 8.37 (d,
J = 8.1 Hz, 2 H), 7.77 (t, J = 7.3 Hz, 2 H), 1.55 (m, 4 H), 1.43 - 1.17 (m, 8 H). 13C NMR
(DMSO-d6, 100 MHz): δ 161.9, 148.2, 134.1, 130.5, 126.2, 122.3, 114.2, 33.3 29.4,
29.2, 26.9 MS (FAB+): 443 [M+H]+. HRMS (FAB+): Calculated for C22H27N4O6:
443.1931; Found: 443.1889.
Synthesis of alkyl bis(3-aminobenzamide) compounds
In a single neck round bottom flask, Pd/C at 10% and the corresponding alkyl bis(3-
nitrobenzamide) (A, B, C o D) were placed. The flask was purged with an Ar
atmosphere. Then, dry MeOH (10 mL × 1 mmol) was added. The Ar atmosphere was
replaced by a hydrogen atmosphere and the reaction was stirred for 24 h. Then, the
mixture was filtered to separate the catalyst and the solvent was removed by heating
under reduced pressure, to give an analytical pure product.
N,N'-(propane-1,3-diyl)bis(3-aminobenzamide) (m3). Brown solid. Yield: 97.8%. M. p.
1
98-100 °C. FT-IR: 3324, 3226, 3063, 2931, 1622, 1579, 1528, 1487 cm-1. H NMR
(DMSO-d6, 400 MHz): δ 8.29 (t, J = 5.7 Hz, 2 H), 7.08 (t, J = 7.7 Hz, 2 H), 7.05 (s, 2
H), 6.96 (d, J = 7.7 Hz, 2 H), 6.69 (ddd, J = 7.7, 2.4, 1.0 Hz, 2 H), 5.22 (brs, 4 H), 3.35 -
13
3.20 (m, 4 H). C NMR (DMSO-d6, 100 MHz): δ 167.5, 149.1, 136.0, 129.1, 116.8,
114.6, 113.2, 37.2, 29.8. MS (FAB+): 313 [M+H]+. HRMS (FAB+): Calculated for
C17H21N4O2: 313.1665; Found: 313.1599.
N,N'-(butane-1,4-diyl)bis(3-aminobenzamide) (m4). Orange solid. Yield: 69.2%. M. p.
120-122 °C. FT-IR: 3433, 3332, 3222, 3057, 2943, 2866, 1647, 1583, 1547, 1520 cm-1.
1H NMR (DMSO-d6, 400 MHz): δ 8.23 (t, J = 5.5 Hz, 2 H), 7.04 (t, J = 7.5 Hz, 2 H),
7.01 (s, 2 H), 6.94 (ddd, J = 7.5, 2.7, 1.1 Hz, 2 H), 6.67 (ddd, J = 7.5, 2.4, 1.3 Hz, 2 H),
5.21 (brs, 4 H), 3.32 - 3.12 (m, 4 H), 1.52 (brs, 4 H). 13C NMR (DMSO-d6, 100 MHz):
166.9, 148.6, 135.7, 128.5, 116.2, 114.2, 112.8, 26.8, 23.9.
10

MS (FAB+): 327 [M+H]+. HRMS (FAB+): Calculated for C18H23N4O2: 327.1821;
Found: 327.1820.
N,N'-(hexane-1,6-diyl)bis(3-aminobenzamide) (m6). Pink solid. Yield: 80.3%. M. p.
100-102 °C. FT-IR: 3390, 3324, 3218, 3054, 2928, 2854, 1625, 1582, 1533, 1489 cm-1.
1H NMR (DMSO-d6, 400 MHz): δ 8.18 (t, J = 5.6 Hz, 2 H), 7.00 (t, J = 7.6 Hz, 2 H),
7.01 (s, 2 H), 6.92 (td, J = 7.6, 0.5 Hz, 2 H), 6.66 (ddd, J = 7.6, 2.0, 1.0 Hz, 2 H), 5.19
(s, 4 H), 3.31 - 3.08 (m, 4 H), 1.63 - 1.40 (m, 4 H), 1.31 (brs, 4 H). 13C NMR (DMSO-
d6, 100 MHz): δ 166.9, 148.6, 135.8, 128.5, 116.2, 114.2, 112.8, 39.0, 29.2, 26.3. MS
(FAB+): 355 [M+H]+. HRMS (FAB+): Calculated for C20H27N4O2: 355.2134; Found:
355.2143.
N,N'-(octane-1,8-diyl)bis(3-aminobenzamide) (m8). Beige solid. Yield: 90.4%. M. p.
120-122 °C. FT-IR: 3393, 3349, 3208, 3054, 2926, 2853, 1625, 1583, 1538, 1490 cm-1.
1H NMR (DMSO-d6, 400 MHz): δ 8.17 (t, J = 5.5 Hz, 2 H), 7.15 - 6.85 (d, J = 7.8 Hz, 2
H), 7.01 (s, 2 H), 6.92 (dd, J = 7.8, 1.2 Hz, 2 H), 6.66 (td, J = 7.8, 1.1 Hz, 1 H), 5.19
13
(brs, 4 H), 3.19 (q, J = 6.5 Hz, 4 H), 1.58 - 1.39 (m, 4 H), 1.28 (s, 8 H). C NMR
(DMSO-d6, 100 MHz): δ 166.9, 148.6, 135.8, 128.5, 116.2, 114.2, 112.9, 29.2, 28.8,
26.5. MS (FAB+): 383 [M+H]+. HRMS (FAB+): Calculated for C22H31N4O2: 383.2447;
Found: 383.2469.
Synthesis of N,N'-alkyl bis(3-(3-(naphthalen-1-yl)ureyl)benzamide) compounds
In a single neck round bottom flask, 2.2 equivalents of 1-naphthyl isocyanate were
added to a stirred solution of an alkyl bis(3-aminobenzamide) (1 equivalent of m3, m4,
m6 or m8) in dry THF under Ar atmosphere. Then, the reaction mixture was stirred for
24 h at RT and the solvent was removed by heating under reduced pressure. The
product was washed and filtered as many times as needed to give an analytically pure
solid compound.
N,N'-(propane-1,3-diyl)bis(3-(3-(naphthalen-1-yl)ureyl)benzamide) (m3N). Purple
solid. Yield: 67.9%. M. p. 218-220 °C. FT-IR: 3271, 3054, 2956, 2935, 1720, 1634,
1585, 1528 cm-1. 1H NMR (DMSO-d6, 400 MHz): δ 9.21 (s, 2 H), 8.78 (s, 2 H), 8.48 (t,
J = 5.5 Hz, 2 H), 8.13 (d, J = 8.4 Hz, 2 H), 8.02 (d, J = 7.4 Hz, 2 H), 7.94 (d, J = 2.1 Hz,
2 H), 7.98 - 7.89 (m, 2 H), 7.70 (dd, J = 7.9, 0.9 Hz, 2 H), 7.65 (d, J = 8.2 Hz, 2 H), 7.60
(dt, J = 7.1, 1.0 Hz, 2 H), 7.54 (dt, J = 7.0, 0.7 Hz, 2 H), 7.48 (t, J = 7.9 Hz, 2 H), 7.47
11

(d, J = 7.8 Hz, 2 H), 7.40 (t, J = 8.4 Hz, 2 H), 3.45 - 3.36 (m, 4 H), 1.81 (qnt, J = 6.8
13
Hz, 2 H). C NMR (DMSO-d6, 100 MHz): δ 166.2, 139.8, 135.4, 134.0, 133.6, 128.7,
128.3, 125.9, 125.8, 125.7, 123.0, 121.2, 120.5, 120.2, 117.5, 117.2, 37.0, 29.2. MS
(FAB+): 651 [M+H]+. HRMS (FAB+): Calculated for C39H35N6O4: 651.2720; Found:
651.2735.
N,N'-(butane-1,4-diyl)bis(3-(3-(naphthalen-1-yl)ureyl)benzamide) (m4N). Beige solid.
Yield: 54.7%. M. p. 234-236 °C. FT-IR: 3271, 3054, 2961, 2936, 1637, 1585, 1537 cm-
1
1. H NMR (DMSO-d6, 400 MHz): δ 9.21 (s, 2 H), 8.78 (s, 2 H), 8.46 (t, J = 5.6 Hz, 2
H), 8.13 (d, J = 8.6 Hz, 2 H), 8.02 (d, J = 7.6 Hz, 2 H), 7.98 - 7.90 (m, 4 H), 7.70 (dd, J
= 8.1, 1.2 Hz, 2 H), 7.65 (d, J = 8.3 Hz, 2 H), 7.61 - 7.49 (m, 4 H), 7.49 - 7.43 (m, 4 H),
13
7.38 (t, J = 8.1 Hz, 2 H), 3.33 - 3.23 (m, 4 H), 1.60 (brs, 4 H). C NMR (DMSO-d6,
100 MHz): δ 166.1, 152.8, 139.7, 135.5, 134.1, 133.7, 133.6, 128.6, 128.3, 125.9, 125.8,
125.7, 125.6, 123.0, 121.2, 120.3, 117.4, 117.3, 117.2, 38.9, 26.6. MS (FAB+): 651
[M+H]+. HRMS (FAB+): Calculated for C39H35N6O4: 651.2720; Found: 651.2768.
N,N'-(hexane-1,6-diyl)bis(3-(3-(naphthalen-1-yl)ureyl)benzamide) (m6N). Pink solid.
Yield: 59.7%. M. p. 222-224 °C. FT-IR: 3282, 3081, 3054, 2932, 2856, 1637, 1629,
1586, 1543 cm-1. 1H NMR (DMSO-d6, 400 MHz): δ 9.20 (s, 2 H), 8.78 (s, 2 H), 8.43 (t,
J = 5.6 Hz, 2 H), 8.13 (d, J = 8.6 Hz, 2 H), 8.02 (dd, J = 7.7, 1.1 Hz, 2 H), 7.98 - 7.88
(m, 4 H), 7.69 (ddd, J = 8.1, 2.2, 1.0 Hz, 1 H), 7.65 (d, J = 8.1 Hz, 2 H), 7.61 - 7.49 (m,
6 H), 7.49 - 7.42 (m, 4 H), 7.38 (t, J = 7.8 Hz, 2 H), 3.27 (q, J = 6.8 Hz, 4 H), 1.55 (s, 4
H), 1.44 - 1.29 (m, 4 H). 13C NMR (DMSO-d6, 100 MHz): δ 166.4, 153.1, 140.0, 135.9,
134.6, 133.9, 133.9, 128.6, 126.2, 126.1, 126.1, 126.0, 125.9, 123.3, 123.1, 121.6,
121.5, 120.7, 120.5, 117.7, 117.6, 117.5, 39.4, 29.3, 26.5. MS (FAB+): 693 [M+H]+.
HRMS (FAB+): Calculated for C42H41N6O4: 693.3189; Found: 693.3278.
N,N'-(octane-1,8-diyl)bis(3-(3-(naphthalen-1-yl)ureyl)benzamide) (m8N). Pink solid.
Yield: 69.0%. M. p. 188-190 °C. FT-IR: 3278, 3053, 2930, 2854, 1636, 1628, 1586,
1
1543 cm-1. H NMR (DMSO-d6, 400 MHz): δ 9.20 (s, 2 H), 8.78 (s, 2 H), 8.41 (t, J =
5.6 Hz, 2 H), 8.13 (d, J = 8.1 Hz, 2 H), 8.02 (dd, J = 7.6, 1.0 Hz, 2 H), 7.98 - 7.88 (m, 4
H), 7.69 (ddd, J = 8.1, 2.2, 1.0 Hz, 3 H), 7.65 (d, J = 8.3 Hz, 2 H), 7.63 - 7.49 (m, 4 H),
7.49 - 7.41 (m, 4 H), 7.38 (t, J = 7.8 Hz, 2 H), 3.25 (q, J = 6.1 Hz, 4 H), 1.61 - 1.42 (m,
13
4 H), 1.32 (s, 8 H). C NMR (DMSO-d6, 100 MHz): δ 165.9, 152.7, 139.6, 135.4,
134.1, 134.0, 133.5, 133.5, 128.5, 128.2, 125.8, 125.7, 125.6, 125.5, 122.8, 122.7,
12

121.2, 121.1, 120.3, 117.3, 117.2, 117.1, 39.0, 28.9, 28.6, 26.3. MS (FAB+): 721
[M+H]+. HRMS (FAB+): Calculated for C44H45N6O4: 721.3502; Found: 721.3503.
Results and discussion
Synthesis
The synthetic routes for o(3-8)N and m(3-8)N receptors are shown in Scheme 1. First,
2.2 equivalents of isatoic anhydride were treated with 1.0 equivalent of alkyl diamine
(3, 4, 6 and 8 carbons) to obtain the alkyl bis(2-aminobenzamide)s o(3-8). Then, the
alkyl bis(2-aminobenzamide)s o(3-8), were treated with 2.2 equivalents of 1-naphthyl
isocyanate to afford the o(3-8)N receptors. In other synthetic route, 2.2 equivalents of
m-nitrobenzoyl chloride were treated with 1.0 equivalent of alkyl diamine to obtain the
corresponding alkyl bis(3-nitrobenzamide)s A-D. Then, a nitro-reduction of A-D
compounds under H2 atmosphere gave the alkyl bis(3-aminobenzamide)s m(3-8), and
finally the addition of 2.2 equivalents of 1-naphthyl isocyanate led to m(3-8)N
receptors.
All receptors in ortho and meta position were characterized by infrared spectroscopy
13
(FTIR), nuclear magnetic resonance (1H and C NMR) and fast atom bombardment
mass spectrometry (FAB-MS), and their chemical composition was verified by high-
resolution mass spectrometry (HRMS). The IR spectra showed vibrations corresponding
to the urea and amide N-H stretching between 3250 and 3300 cm−1. Also, two stretching
vibrations for urea and amide carbonyls were found at 1640-1720 and 1620-1650 cm−1,
respectively. The typical signal patterns of an ortho and meta substituted aromatic ring
1
for o(3-8)N and m(3-8)N receptors were observed in the H NMR spectra obtained in
DMSO-d6. In addition, the signals for the naphthyl group in o(3-8)N and m(3-8)N
receptors were assigned correctly.
The 1H NMR spectra of ortho receptors show singlet signals at 10.29 and 9.60 ppm for
urea hydrogens Hf,f´ and Hg,g´, respectively, and a triplet signal in the range of 8.74-8.62
ppm for amide hydrogen He,e´; whereas in meta receptors, the urea and amide hydrogens
signals are located at 9.20 (Hf,f´) and 8.78 (Hg,g´) and 8.48-8.41 (He,e´) ppm, respectively.
This difference in chemical shifts for urea and amide hydrogens signals is caused by
13

structural features; the relative position of urea and benzamide groups and the resonance
effect provided by the chromophore, are the main factors. The major effect was
observed over the urea hydrogen (Hg,g´) directly attached to the chromophore. On the
other hand, the signal Hf,f´ is the most shifted downfield, due to the resonance effect in
the benzamide. Finally, the HRMS analysis confirmed the chemical composition for all
compounds.
Scheme 1. Synthesis routes for alkyl bis(naphthylureylbenzamide)-based o(3-8)N and
m(3-8)N receptors.
Anion recognition by UV-Vis
The m(3-8)N and o(3-8)N receptors have broad absorption bands due to the presence of
an amino conjugated naphthyl group, with maximums at 296 and at 301-306 nm,
respectively. These bands correspond to an auxochrome π-π* electronic transition. The
14

supramolecular interaction of receptors was evaluated by titration with
tetrabutylammonium salts, TBAX, where X = acetate (AcO), benzoate (BzO),
dihydrogen phosphate (HP), fluoride (F) and hydrogen pyrophosphate (HPP) by UV-
Vis in acetonitrile. The results showed significant changes in the absorption bands of
m(3-8)N receptors. Figure 2a shows the absorption spectra of m6N with TBAF at
different concentration. The absorption band at 296 nm shifted to 305 nm with an
increase in absorbance (hyperchromic effect). In addition, two isosbestic points located
in 275 and 294 nm are present. These changes indicate the supramolecular interaction
between the receptor and the anion. All m(3-8)N receptors underwent a similar behavior
towards TBAX salts (Figures S1-S4). The major response was observed with HPP. The
spectral changes and sensibility of m(3-8)N towards the different anions were in the
order HPP > F ≈ HP > AcO > BzO, as seen in the absorbance profiles (Figure 2b). This
behavior is directly related to the negative charges number and basicity of the anion.
Interestingly, receptors o(3-8)N did not show significant changes with any of the
selected anions for this study (Figures S5-S8). The behavior of o(3-8)N with TBAX
salts is similar to the observed with a previously reported N-benzyl mono
ureylbenzamide receptor.4 The anion interacts with the urea hydrogens and a rotation of
the amide carbonyl occurs, allowing the interaction of the anion and amide hydrogen.
However, the conformational changes avoids the electronic conjugation from urea to
naphthyl group, and the absorption band do not change during the titration. The
coplanarity of the naphthyl-urea-benzamide system is conserved in m(3-8)N receptors
when interact with anions, and the electronic transitions are affected.
15

Figure 2. (a) UV-Vis spectra obtained in the titration of m6N [1×
10-5 M] with TBAF in
acetonitrile. (b) Relative absorbance profiles obtained by titration of m6N with different
TBAX salts, λ = 305 nm.
It was possible to estimate the association constants K for the complexes using the
absorbance data. A non-linear fit was used for a 1:1 interaction model, following the
Equation 1 reported by Thordarson.33
Eqn. (1)
ꢔ
1
ꢐ
1
ꢐ
ꢊ
ꢎꢗ
ꢑ
ꢋ ꢍ
ꢌ
ꢋ ꢍ
+ ꢏ
ꢋ ꢍ
ꢋ ꢍ
ꢋ ꢍ ꢋ ꢍ
ꢓ − 4 ꢌ ∗ ꢏ
+
−
ꢒ ꢌ + ꢏ
+
ꢎ
ꢎ
ꢎ
ꢎ
ꢎ
ꢀꢁꢂꢃꢄꢅ = ꢀꢁꢂꢆ + 0.5∆ꢀꢁꢂꢇ
ꢋ ꢍ
ꢌ
ꢉ
ꢈ
ꢖ
ꢕ
ꢎ
16

where Aobs is the complexed receptor absorbance, AH is the free receptor absorbance,
ꢀAbs∞ is the maximum absorbance change induced by the presence of a given guest,
[G]T is the total concentration of guest, [H]T is the total concentration of receptor, and K
is the association constant. Table 1 lists the association constants for the complexes
calculated from absorbance data using Equation 1 for a 1:1 interaction model. The K
values were in the range of 105 to 108. More refined calculations using HypSpec34-36
program were performed, to analyze the data that did not fit with the Equation 1. The
program confirmed the K values for 1:1 complexes, and provided the K values for 2:1
and 1:2 complexes (Table 1). The formation of 2:1 complexes was very interestingly
considering that the typical behavior of the bis-urea receptors is the formation of 1:1 and
1:2 complexes. The presence of 2:1 complexes with AcO and HPP indicate that the
anions may serve as template for the self-assembly of two receptor molecules at low
concentration of anion, but the 1:1 complex is predominant when the anion
concentration increases. Interestingly, only the 2:1 stoichiometry is detected for m8N-
HPP complex (Figures S9 and S10). The K values were determined only for meta
receptors due to the ortho receptors do not show significant change in the presence of
TBAX salts.
Table 1. Association constants (K) for m(3-8)N and anion complexes
determined from UV-Vis data.
m3N
K (M-1)
b1.9×1012
b3.1×106
b9.3×1010
b4.1×105
b3.1×109
a1.5×107
a2.8×107
ND
m4N
K (M-1)
b1.7×1014
b1.1×107
ND
m6N
K (M-1)
ND
m8N
K (M-1)
ND
X
Ratio: R/A
R2A
RA
AcO
a3.4×105
a3.7×105
RA2
RA
ND
ND
8.3×105
2.7×105
ND
a5.3×106
a2.7×105
b2.5×1017
b6.3×108
4.7×105
ND
a7.1×105
a2.2×106
b1.1×1011
ND
BzO
RA2
RA
ND
F
a2.7×105
a4.0×106
b6.3×1016
b3.2×108
HP
RA
R2A
RA
HPP
ND
ND: Not determined. According to the data obtained, a non-linear adjustment was
not achieved, and the association constants could not be determined. R: Receptor.
a
b
A: Anion. Determined by Eqn. 1. Determined by HypSpec refined calculation
program. K values have M-2 unit for 2:1 and 1:2 complexes and M-1 for 1:1
complexes.
17

The K values are consistent with the experimental results, the highest values correspond
to the m(3-8)N complexes with HPP, followed by F and HP. However, not all the K
values were determined due to the strong anion-receptor interaction. In general, there is
a tendency of K to decrease as the alkyl chain length increases. The K value changes as
the geometry and size of the anion change, but the charge of the anion has a significant
effect. The relative position of the amide and urea groups is determinant in the anion
recognition and the association stability due to the steric effect. Moreover, the shape,
size and basicity of the anion have a great influence in the recognition process and
stability of the complexes.
Anion recognition by fluorescence
The fluorescence spectra of m(3-8)N and o(3-8)N receptors, as well as, their respective
titrations with TBAX salts were obtained in acetonitrile (Figure S11-S18). All receptors
showed broad fluorescence bands due to the presence of a naphthyl fluorophore
attached to the urea group. The emission band for m(3-8)N receptors has a maximum
intensity at 370 nm and o(3-8)N receptors at 398 nm. The m(3-8)N receptors are more
fluorescent than o(3-8)N receptors, due to a greater electron delocalization from the
urea towards the naphthyl group, whereas the o(3-8)N receptors display an inductive
effect from the benzamide system over urea and naphthyl groups, causing a poor
electron mobility towards the fluorophore.
All m(3-8)N receptors show a fluorescent response towards the anions under study. In
most cases, the fluorescence emission spectra showed a decrease in intensity as the
TBAX salt was added, indicating the interaction with the anion. The Figure 3(a) shows
the fluorescence spectra obtained in the titration of m6N with TBAF. The fluorescence
profiles showed an ON/OFF response in the order AcO > F > HP ≈ BzO (Figure 3b).
18

Figure 3. (a) Emission spectra obtained in the titration of m6N [1×
10-5 M] with TBAF
in acetonitrile. (b) Relative fluorescence profiles of m6N with different TBAX salts, λex
= 302 nm, λem = 370 nm.
The fluorescence spectra of m(3-8)N receptors with HPP showed a different behavior
from those observed with other TBAX salts (Figures 3b and 5). These receptors behave
as ON1/OFF/ON2 fluorescent probes in presence of HPP. First, there is a decrease in
fluorescence intensity of m(3-8)N receptors when the addition of HPP reaches 0.5
equivalents (2:1 receptor-anion). Then, a partial increase in the intensity of a new
emission band is observed, reaching its maximum intensity when 1.5 equivalents of
HPP are added. Interestingly, this ON1/OFF/ON2 response in fluorescence is more
19

evident in receptors with a shorter spacer as m3N and m4N. Figure 4 shows the
fluorescence spectra of m4N at different concentrations of HPP.
This fluorescent response may be due to a more than one electronic process involved in
the anionic recognition. The quenching of fluorescence is attributed to an electron-
transfer (eT) process from the carbonyl oxygen of the urea, which assumes a partial
negative charge in the interaction with the anion, facing the excited naphthyl group.14
Electron density is transferred to the oxygen from the coordinated anion through H-
bonds in this stage. Then a new band at 375 nm appears and shows an increase of
fluorescence intensity after adding more than 0.5 equivalents. The increase of the new
fluorescence band intensity is attributed to a charge transfer (cT) process, which occurs
by deprotonation of urea. The red shift of the emission band (7 nm approx.) in m(3-8)N
is induced by the complexation, and reflects the stabilization of the excited state by the
H-bond interaction with the anion.37 The fluorescence profiles of m(3-8)N show an
ON1/OFF/ON2 response with HPP in the order m3N > m4N > m6N > m8N (Figure 5).
These results evidence the formation of 2:1 complexes with HPP, as well as, by
electronic spectroscopy. The changes of fluorescence intensity indicate that the
closeness of both ureylbenzamide units has an important influence over the
spectroscopic response.
Figure 4. Emission spectra obtained in the titration of m4N [1×
10-5 M] with TBAHPP
in acetonitrile.
20

Figure 5. Relative fluorescence profiles obtained for m(3-8)N receptors with TBAHPP
salt, λex = 302 nm, λem = 370 nm.
The data obtained from the fluorescence titrations were used to determine the
association constants K of the supramolecular complexes. A non-linear fit was used for
a 1:1 interaction model, this method is based on the same principle of Equation 1, and
the difference is the use of fluorescence data, as depicted in Equation 2.
Eqn. (2)
ꢔ
1
ꢐ
1
ꢐ
ꢊ
ꢎꢗ
ꢑ
ꢋ ꢍ
ꢌ
ꢋ ꢍ
+ ꢏ
ꢋ ꢍ
ꢋ ꢍ
ꢋ ꢍ ꢋ ꢍ
ꢓ − 4 ꢌ ∗ ꢏ
+
−
ꢒ ꢌ + ꢏ
+
ꢎ
ꢎ
ꢎ
ꢎ
ꢎ
ꢘꢃꢄꢅ = ꢘꢆ + 0.5∆ꢘꢇ
ꢋ ꢍ
ꢌ
ꢉ
ꢈ
ꢖ
ꢕ
ꢎ
where Fobs is the fluorescence intensity for the complex, FH is the fluorescence intensity
of the free receptor, ꢀF∞ is the maximum fluorescence intensity change induced by the
presence of a given guest, [G]T is the total concentration of the guest, [H]T is the total
concentration of the receptor, and K is the association constant.
Table 2 lists the K values obtained from the emission data using Equation 2 for a 1:1
interaction model, which were in the range of 103 and 106. More refined calculations
using the HypSpec program were performed to analyze the data which did not fit with
the Equation 2. The program confirmed the K values for 1:1 complexes, and provided
the K values for 2:1 complexes with AcO and m(3-8)N receptors. There is a difference
in the K values and stoichiometry estimated from the fitting of UV-Vis and fluorescence
data for some complexes. This is hard to explain, but one plausible explanation is
21

related with the fact of that 2:1, 1:1 and 1:2 complexes have the same emission band,
and as the 2:1 complex is formed first, the transformation to 1:1 and 1:2 complexes is
not detected. It is believe that K values calculated from the UV-Vis data are more
accurate, but the fitting of fluorescence data confirms the formation of 2:1 complexes.
It is evident that magnitude of K is influenced by the length of the spacer alkyl chain.
For example, there is a clear decrease of the K value in the complexes with HP as the
alkyl chain length increases, and there is a similar tendency with the rest of anions.
Moreover, the K values for supramolecular complexes of m(3-8)N and HPP could not
be determined due to the high dispersion in the data, while the complexes with o(3-8)N
receptors showed values in the range of 104 and 107.
Table 2. Association constants (K) for anion interaction with m(3-8)N and o(3-6)N
receptors using the fluorescence data.
m3N
m4N
m6N
m8N
o3N
o4N
o6N
X
K
K
K
K
K
K
K
R/A
R2A
RA
R2A
RA
RA
RA
RA
b5.7×1011 b3.3×1011 b1.0×1011 b1.0×1012
AcO
a3.8×106
b1.0×1011
b1.2×106
a1.0×106
a1.7×106
ND
a2.8×106
b1.4×105
b1.5×106 a2.0×104 a4.0×105 a3.1×104
ND
BzO
a1.0×106
a7.1×106
a1.3×106
ND
a3.8×105
a4.3×106
a3.7×105
ND
a2.1×105
a3.7×105 a5.8×103
a2.0×105
a1.4×105 a5.2×104
ND
F
ND
a1.3×104
ND
ND
HP
ND
HPP
a4.1×104 a8.3×104 b2.8×107
ND
ND: Not determined. According to the obtained data a non-linear adjustment was not achieved,
a
and therefore the association constants could not be determined. R: Receptor. A: Anion.
Determined by Eqn. 2. b Determined by HypSpec refined calculation program. K values have M-2
unit for 2:1 and 1:2 complexes and M-1 for 1:1 complexes.
The o(3-8)N receptors show an interesting OFF/ON fluorescent response in the
titrations with TBAX salts. A shift of the emission band to shorter wavelength (20 nm
approx.) is observed in the course of the titration. This change is more evident in o3N
and o4N with HP and HPP salts (Figure 6 and Figure S14). This response is no longer
observed as the length of the alkyl chain increases (Figures S13-S16).
22

Figure 6. (a) Emission spectra obtained in the titration of o3N [1×
10-5 M] with
TBAHPP in acetonitrile. (b) Relative fluorescence profiles obtained with different
TBAX salts, λex = 334 nm, λem = 398 nm.
The excitation and emission bands of o3 and o3N have the same maximum excitation
and emission wavelengths (λexc= 334 nm y λem= 398 nm), although the fluorescence
intensity is lower for o3N (Figure 7). In addition, it was observed that the emission band
of o3N-HPP complex has an emission band similar to m3N, where the fluorescence is
due to the naphthyl fluorophore. These observations suggested a photoinduced electron
transfer (PET) process between the naphthyl group (NAPH) and the o-aminobenzamide
fragment (OABA). In a previous work, it was found that a PET process occurs if the
OABA is conjugated with another fluorophore.38
23

Figure 7. Relative fluorescence profiles obtained for the receptors: o3, o3N, m3N, and
the o3N-HPP supramolecular complex.
A theoretical study was carried out with DFT, a PBE0 functional and a DZVP base
using the Gaussian 09 program.32 The geometry of the receptor was optimized and the
energy of the molecular orbitals (MOs) was calculated.32 As a result, it was found that
the MOs of naphthylurea flourophore (HOMO-LUMO)NAPH have higher energy than the
MOs of the o-aminobenzamide florophore (HOMO-LUMO)OABA (Figure 8). The
(OABA-NAPH) dyad is irradiated at 334 nm and the OABA fluorophore is excited
1
generating the (OABA)*-NAPH species. A photoinduced electron transfer (PET) from
HOMONAPH to the HOMOOABA is possible due to this MO is located in a superior
position. Then, the OABA -NAPH specie is formed, and the energy emission is
inactivated reducing the fluorescence intensity. The PET is inactivated in the complex
and the emission of NAPH fluorophore is possible.
LUMOOABA
-1.43 eV
LUMONAPH
-1.18 eV
24

HOMOOABA
-6.57 eV
HOMONAPH
-6.08 eV
Figure 8. Molecular orbitals of o3N calculated by DFT.
The MO's energy in the receptor changes when it complexes with anions. The HOMO is
located in the two naphthylurea units and the LUMO in the o-aminobenzamide units in
the free receptor. Instead, the HOMO is located in the naphthylurea units and the
LUMO orbital in the naphthyl group in the complexes; then a S1→S0 transition or
energy emission occurs in the naphthylurea fragment. This result is consistent with the
emission wavelength observed for the complexes. Also, the MO's energy increases with
the basicity and charge of the anion (Figure 9).
a)
b)
c)
d)
4.89 eV
0.80 eV
-1.43 eV
0.75 eV
-3.49 eV
0.74 eV
-6.08 eV
-3.39 eV
Figure 9. Distribution of the MOs in a) free o3N, b) o3N-Ac, c) o3N-HP and d) o3N-
HPP complexes.
Anion recognition study by 1H NMR
1
The H NMR titrations of receptors were performed to establish the interaction sites
with different anions. For this purpose, it was decided to analyze two “short receptors”
(m3N and o3N) and two “long receptors” (m6N and o6N) in order to detect an effect
over the anion recognition process by the alkyl chain length. The main idea was to
analyze the interaction of these receptors in a non-competitive solvent, which was
25

clearly possible at low concentration (0.01 mM) in acetonitrile. However, these
receptors are insoluble in acetonitrile at high concentracion (6 mM), thus the use of a
1
polar solvent as DMSO was necessary in the studies by H NMR. The m3N and m6N
receptors display spectral changes with all anions. The most affected hydrogens belong
to the urea group, Hf,f´ and Hg,g´, at 9.24 and 8.80 ppm, respectively. The recognition of
AcO and BzO anions by m3N and m6N show a similar behavior (Figures S20-S22). The
Hf,f´ and Hg,g´ signals shift to downfield about 2.67 and 2.48 ppm, respectively, after the
addition of six equivalents of salt to a m3N solution in DMSO-d6 (Figure 10). The
amide signal He,e´ at 8.52 ppm is not affected by AcO and BzO. Therefore, the amide is
not involved in the anion recognition. Moreover, the singlet at 7.94 ppm which
corresponds to Hd,d´ hydrogen shows a 0.18 ppm downfield shift. A doublet at 8.12 ppm
corresponding to Hp,p´ shows a 0.19 ppm downfield shift. A 0.24 ppm up-field shift of
Hh,h´ signal is due to the loss of the carbonyl effect over this hydrogen once the complex
is formed. This chemical shift is caused by a change in the configuration and
conjugation of the naphthyl with the urea group, giving a protective effect over Hh,h´
hydrogen (Figure 10).
1
Figure 10. Partial H NMR spectra obtained in the titration of m3N [6 mM] with
TBAAcO in DMSO-d6.
26

The geometry of m3N, m3N-AcO and m6N-AcO were optimized using DFT, a PBE0
functional and a DZVP base, in order to visualize the interaction of these receptors with
acetate (Figure 11). The ureylbenzamide units adopt a quasi-orthogonal position to
minimize the distance of AcO and urea hydrogens in both complexes. Also, the
naphthyl groups are oriented towards the acetate adopting an “endo”orientation to
conserve the coplanatity of the naphtyl-urea-benzamide structure. The amide hydrogens
are oriented to the anion, but the distance is unfavorable for an interaction. These
geometries are consistent with the NMR experiments, Hf,f´, Hg,g, Hd,d´, Hh,h´ and Hp,p´
signals are the most affected in the interaction with AcO.
a)
b)
c)
Figure 11. Optimized geometries of m3N (a), m3N-AcO complex (b) and m6N-AcO
complex (c) by DFT.
1
The H NMR spectra of m3N and m6N with F show a downfield shift of Hf,f´ and Hg,g´
signals. Noteworthy, there is a significant shift of He,e´ signal. The chemical shift of He,e´
signal is constant until one molar equivalent of F added, then the signal shows a
downfield shift about 1.16 ppm, evidencing the participation of the amide hydrogen in
the recognition of a second F unit (Figure 12). The Hd,d´ hydrogen gradually shifts to up-
27

field when F is added from zero to one equivalent, evidencing the electron-enrichment
in the aromatic ring. Then, the signal shifts downfield indicating the interaction of this
hydrogen with F. A previously reported N-benzyl mono ureylbenzamide receptor does
not show a change in the amide signal, only the interaction with the urea hydrogens is
observed.4 Based on the changes of these signals, it is proposed the formation of a 1:1
complex where only the urea groups participate (Figure 13a). Then, the closeness of
amide hydrogens in the 1:1 complex favors the interaction with a second F unit, forming
a 1:2 complex, where Hd,d´ hydrogens also participate (Figure 13b). In fact, the shift or
broadening of the signals are less sensitive in m6N-F complex (Figure S23) indicating
an important influence of the alkyl chain length in the recognition process of F. In
addition, the Hp,p´ signal is shifted 0.47 ppm downfield from its initial position due to
the polarization of the aromatic C-H bond in the recognition of fluoride. The Hh,h´, Hk,k´
y Hl,l´ signals corresponding to the naphthyl group also show different chemical
environment produced by electronic and conformation changes.
Figure 12. Partial 1H NMR spectra obtained in the titration of m3N [6 mM] with TBAF
in DMSO-d6.
a)
b)
28