5-Amino-2-aryl-1,2,3-triazol-4-carboxylic acids: Synthesis, photophysical properties, and application prospects

Article abstract of DOI:10.1016/j.dyepig.2020.108343

To enlarge the highly active fluorophore family of small molecular heterocycles, we present an effective strategy for the synthesis of novel 2-aryl-1,2,3-triazol-4-carboxylic acids (ATAs). The behavior of ATAs in different solvents showed that these new 1

Full text of DOI:10.1016/j.dyepig.2020.108343

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5-Amino-2-aryl-1,2,3-triazol-4-carboxylic acids: Synthesis, photophysical properties,
and application prospects
Nikita E. Safronov, Timur O. Fomin, Artem S. Minin, Lozan Todorov, Irena Kostova,
Enrico Benassi, Nataliya P. Belskaya
PII:
S0143-7208(20)30223-0
DOI:
https://doi.org/10.1016/j.dyepig.2020.108343
DYPI 108343
Reference:
To appear in: Dyes and Pigments
Received Date: 28 January 2020
Revised Date: 6 March 2020
Accepted Date: 6 March 2020
Please cite this article as: Safronov NE, Fomin TO, Minin AS, Todorov L, Kostova I, Benassi E, Belskaya
NP, 5-Amino-2-aryl-1,2,3-triazol-4-carboxylic acids: Synthesis, photophysical properties, and application
prospects, Dyes and Pigments (2020), doi: https://doi.org/10.1016/j.dyepig.2020.108343.
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5-Amino-2-aryl-1,2,3-triazol-4-carboxylic acids: Synthesis, photophysical properties,
and application prospects
Nikita E. Safronov,a Timur O. Fomin,a Artem S. Minin,a,e Lozan Todorov d Irena Kostova,d Enrico
,
Benassi,*c Nataliya P. Belskaya*a,b
aUral Federal University, 19 Mira Str., Yekaterinburg 620002, Russian Federation
bPostovsky Institute of Organic Synthesis, Ural Branch of Russian Academy of Science, 22 S.
Kovalevskaya Str., Yekaterinburg, 620219, Russian Federation
cShihezi University, 280 N 4th Rd, Shihezi, Xinjiang 832000, China
dDepartment of Chemistry, Faculty of Pharmacy, Medical University, Dunav Str. 2, 1000 Sofia, Bulgaria
eM. N. Mikheev Institute of Metal Physics, Ural Branch of Russian Academy of Science, 18 S.
Kovalevskaya Str., Yekaterinburg, 620219, Russian Federation
*Corresponding authors: E-mail: n.p.belskaya@urfu.ru, ebenassi3@gmail.com
Abstract
To enlarge the highly active fluorophore family of small molecular heterocycles, we present an effective
strategy for the synthesis of novel 2-aryl-1,2,3-triazol-4-carboxylic acids (ATAs). The behavior of ATAs
in different solvents showed that these new 1,2,3-triazole derivatives had bright blue fluorescence with
excellent quantum yields, large Stokes shifts, and prolonged fluorescence lifetimes. Their photophysical
and physicochemical properties showed extra sensitivity to both structural changes and the
microenvironment, usually accompanied by marked changes in quantum yield. The responses of the
ATAs to varying pH were different depending on the structure of the amino group at the triazole ring C5
atom. pKa values of 7.65–8.08 were obtained for ATAs bearing a tert-amino group. In contrast, the pKa
values of ATAs bearing secondary alkylamines were in the range of 3.20–3.52. Quantum mechanical
methods were employed to understand the underlying transitions and explain the obtained experimental
data. Among the synthesized compounds, aggregation-induced emission enhancement (AIEE) behavior
was observed, with potential applications as sensors for monitoring and controlling pH. The reversibility
of ATAs between acid and alkaline pH values, as well as their toxicity, supports their real-time use in
biological research.
Keywords: Triazole carboxylic acid, fluorescence, aggregation-induced emission enhancement,
solvatochromism, acidity, reversibility

1. Introduction
Carboxylic acids are among the most common substances in nature, biology, medicine, and chemistry.
They are important and prevalent building blocks that can be transformed into various acid derivatives,
including esters, amides, hydrazides, and heterocycles, with different physicochemical properties and
biological actions and decreased side effects; this enables their use in organic synthesis, medicines,
biochemistry, and the chemical industry [1-11]. The synthetic and natural heterocyclic derivatives of
carboxylic acid structures are a privileged class of compounds, especially azoles and azines, which have
numerous applications in medicinal research and pharmacology [7, 12-14]. Among these heterocycles,
N(1) and N(2)-substituted 1,2,3-triazoles have become popular as attractive targets for organic synthesis
in the last decade [15-26]. This has been due to their ubiquitous use in two important practical chemistry
applications, namely, as biologically active compounds [27-39] and in materials science [40-44].
The most famous and widely used strategy for constructing triazole cores is based on the 1,3-dipolar
cycloaddition of substituted azides to alkynes, including metal-catalyzed variants [15-20, 27-39], which is
mainly limited to the construction of isomeric 2-aryl-1,2,3-triazoles [21-26]. Despite various synthetic
methods being reported in the literature, no method is more common than the catalytic cycloaddition of
azides. However, the best known synthetic approaches have a finite scope and limitations. Therefore,
improving the selectivity and enhancing the effectiveness of 2-aryl-1,2,3-triazole synthesis remains a
challenge. A promising approach to the preparation of new 2-aryl-1,2,3-triazoles is the synthesis of
functionalized derivatives and their subsequent modification. The diversity of triazole structural
modifications is important to increase the possibility of tuning their biological, physicochemical, and
photophysical properties for specific applications. Functional fluorophores can be used to label molecules,
giving them optical properties, and as separate indicators to determine intrinsically non-fluorescent
species and parameters, such as pH, oxygen, metal ions, and other analytes.

Scheme 1. Literature examples of 2-substituted triazole acid synthesis.
In the last decade, our efforts toward the synthesis of triazole derivatives [21-23] have led to the
development of a convenient method for 2-aryl-1,2,3-triazole construction using a two-step sequence of
nucleophilic addition and oxidation reactions with separation of the intermediate amidine, which has
subsequently been developed into a one-pot process. This method allows the triazole cycle to be saturated
with substituents and functionalities. Using the proposed one-pot strategy, 2-aryltriazoles are available
with cyano, amino, and alkylamino groups, and carboamides with alkyl- and tert-cycloalkylamino groups,
crowning the heterocycle core. This set of functionalities, namely, an aromatic cycle, CN/COOEt/COAr
groups, and amino groups, is important for the physicochemical, photophysical, and biological properties
of these compounds and for further modification through different transformations. For example, the
cyano group is a convenient precursor block that can be readily transformed into amines, aldehydes,
ketones, amides, carboxyl derivatives, and heterocycles, among others [45, 46]. Reports on triazoles with
carboxylic acid fragments are notably scarce. For 2-aryltriazole derivatives bearing carboxylic acids,
known examples were obtained by the hydrolysis of ethers (I and II) and amides (III). These methods have
some limitations and disadvantages, which are connected to the multistep approaches to the starting ester
or amide formation, low structural diversity, and long hydrolysis times. This method was used to
synthesize 5-methyl-2-(2H-1,2,3-triazol-2-yl)benzoic acid, which is a key intermediate for suvorexant [12,
14, 26].

To enlarge the family of highly fluorescent 1,2,3-triazoles, we have developed a synthetic approach
for new carboxylic acid derivatives and investigated their photophysical properties and sensitivity to
external stimuli, such as acids, bases, and pH variation.
Scheme 2. Synthetic route to targeted 5-amino-2-aryl-2H-1,2,3-triazole-4-carboxylic acid 1.
2. Experimental
2.1. Instruments and Materials
1Н NMR and 13C NMR spectra were recorded with a Bruker Avance II spectrometer (Karlsruhe,
1
13
Germany) (400 MHz for H, 100 MHz for C). Chemical shifts are reported in parts per million (ppm)
1
relative to TMS in H NMR and to the residual solvent signals in 13C as external reference. Coupling
constant (J) values are given in Hertz (Hz). Signal splitting patterns are described as a singlet (s), doublet
(d), triplet (t), quartet (q), sextet (sext), quintet (quin), multiplet (m), broad (br), doublet of doublets (dd),
doublet of triplets (dt), or AA′XX′ spin systems of para-substituted benzene with two different
substituents. Mass spectra were recorded with a Shimadzu GCMS-QP 2010 “Ultra” mass spectrometer
(Kyoto, Japan) using the electron impact (EI) ionization technique (40–200 °C, 70 eV). The abbreviation
[M]+ refers to the molecular ion. Spectra of exact mass were acquired on a quadrupole orthogonal
acceleration time-of-flight mass spectrometer (Synapt G2 HDMS, Waters, Milford, USA). Samples were
infused at 3 µL/min, and spectra were obtained in positive (or negative) ionization mode with a resolution
of 15000 (FWHM) using leucine enkephalin as lock mass. The Fourier-transform infrared (FT-IR) spectra
were obtained using a Bruker Alpha (NPVO, ZnSe) spectrometer (Ettlingen, Germany). Elemental
analyses were carried out using a CHNS/O analyzer Perkin-Elmer 2400 Series II instrument (Shelton, CT
USA). Melting points for compounds were determined on a Stuart SMP3 apparatus (Staffordshire, UK).
X-ray crystallographic analysis was performed with an Xcalibur diffractometer with CCD using a
standard procedure (MoK-irradiation, graphite, ω-scanning with steps of 1°). CCDC-1973263 (for 1e) is
given in the ESI and supplementary crystallographic data for this paper. These data can be obtained free of
charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

UV-Vis absorption spectra were recorded on a Perkin-Elmer Lambda 35 UV-Vis spectrophotometer
(Shelton, USA). Fluorescence of the sample solutions was measured using a Hitachi F-7000
spectrophotometer (Tokyo, Japan). The absorption and emission spectra were recorded in DMSO, DMSO-
water mixture (v/v, 9/1), EtOH, MeCN, DCM, dioxane, and toluene using 10.00 mm quartz cells. The
excitation wavelength was at the long wavelength absorption maxima. Atmospheric oxygen contained in
solutions was not removed. The concentrations of the compounds in the solution were 5.0 × 10-5 M and
5.0 × 10-6 M for absorption and fluorescence measurements, respectively. The relative fluorescence
quantum yields (QY) were determined using quinine sulfate in 0.1 M H2SO4 as a standard (ΦF = 0.546).
Absolute quantum yields for solid state and time-resolution study were recorded on a Horiba FlouroMax 4
spectrofluorometer (Kyoto, Japan) with a Quanta-φ integrating sphere using FluorEssence 3.5 software.
The pH measurements were carried out on a Milwaukee MW101 pH meter (Milwaukee, USA).
To assess the cytotoxic effect of substances, we used a green monkey epithelial cell culture (Vero)
obtained from the cell culture bank of the Institute of Cytology RAS (St. Petersburg, Russia,). The cell
cultures were maintained in culture flasks (Eppendorf, Germany) in DMEM (Sigma-Aldrich, USA)
supplemented with 10% fetal calf serum (Biolot, Russia) and 0.5% gentamicin (Biolot, Russia) in an
incubator with an atmosphere containing 5% carbon dioxide.
The reactions were monitored by analytical thin-layer chromatography (TLC) on aluminium-backed
silica-gel plates (Sorbfil UV–254). Visualization of the components was accomplished by short-
wavelength UV light (254 nm). Solvents were dried and distilled according to common procedures. All
solvents were of spectroscopic grade. Full experimental details for the synthesis of compounds 2a-j and 1,
as well as their NMR spectra, photophysical characterization, and quantum mechanical calculation data,
are reported in the Electronic Supplementary Information (ESI, Experimental section, Figs. S1-S10).
Malononitrile, arylamines, dialkyl- and monoalkylamines 4a-f were obtained from Acros Organics
and used without further purification.
Arylhydrazones 3a-d were prepared according to procedures reported in the literature [22-23]. NMR,
IR, and mass spectrometry data for the new compounds are presented in the Electronic Supporting
Information (ESI).
3. Results and discussion
3.1. Synthesis of 2-aryl-4-cyano-1,2,3-triazoles
The synthesis of the starting triazoles 2a–r was performed using the one-pot procedure outlined in
Scheme 3 [22].

Scheme 3. Synthesis of 5-amino-2-aryl-2H-1,2,3-triazole-4-carbonitriles 2a–r.
According to the published method, a series of corresponding triazoles 2a–r were obtained in good to
excellent yields. This method expanded the scope of previously proposed one-pot techniques for the
synthesis of functionalized 2-aryl-1,2,3-triazoles, and included in this transformation are a number of new
hydrazones, 3. The structures of the newly obtained 2-aryl-1,2,3-triazoles 2d, g, h, j, m-o, q and r were
13
characterized by 1H and C NMR spectroscopy and high-resolution mass spectrometry (HRMS), with all
data presented in the Electronic Supplementary Information (ESI, Figs. S1-S10).
The hydrolysis of cyano groups can be performed under strong acid or strong base conditions [45].
Initially, we optimized the reaction conditions of the carboxylic acid synthesis using 2-aryl-4-cyano-1,2,3-
triazole 2a as the model substrate (Table 1). When 2a (0.2 mmol) was reacted with H2SO4, no product was
detected during 10 h, independent of the temperature and the quality of acid used. Reflux under acidic
conditions in dioxane/water or ethanol/water as mixed solvents gave a mixture of the starting compound
and trace amounts of the intermediate amide and carboxylic acid after 10 h. Basic conditions gave better
results. In the presence of aqueous NaOH solution, and depending on the reflux time, carboxamide 5a or
acid 1a were obtained selectively from 2a. Adding 10–15 equiv. of NaOH and refluxing for 1h led to the
formation of amide 5a, while longer reflux periods (1.5–5 h) and 20–40 equiv. of NaOH led to complete

hydrolysis of the cyano group to a carboxylic acid group and triazole acid formation (ATA) in excellent
yields (Table 1, entries 6 and 7). The optimized conditions obtained from this short preliminary
investigation are shown in Table 1, entry 7.
Table 1. Acid and alkaline hydrolysesa of 2-methoxyphenyl-4-cyanotriazole 2a.
Acid/Base,
COOH
N
CN
CONH2
N
N
N
EtOH/H2O
N
N
N
N
N
N
N
N
Reflux
MeO
MeO
MeO
1a
2a
5a
Entry Acid/Base
Conc, %
Volume, Solvent
mL
T, °C
Time, h Compd
Yield,b %
H2SO4
H2SO4
H2SO4
H2SO4
NaOH aq
NaOH aq
NaOH aq
100
100
100
100
10
10
10
10
10
15
15
15
EtOH
25
78
78
100
78
78
78
10
10
10
10
1
-
-
-
-
1
2
3
4
5
6
7
2a
EtOH : H2O (4 : 1)
EtOH : H2O (4 : 1)
Dioxane : H2O (4 : 1)
EtOH : H2O
EtOH : H2O
EtOH : H2O
2a + 5ac
2a+5ac
2a+5ac+1ac
5a
95d
90d
93d
20
40
5
1.5
1a
1a
b
aConditions: Triazole 2a (0.55 mmol), acid or base, and solvent (5 mL) for 10 h at the indicated temperature. Yield determined after
separation by liquid column chromatography. cCompounds present in reaction mixture in trace amounts and could not be separated. dReaction
was complete, as confirmed by total consumption of starting compound.
Under the optimized reaction conditions, the scope of this method for 1,2,3-triazole carboxylic acid
synthesis was explored. As shown in Scheme 4, various 2-aryl-1,2,3-triazolocarbonitriles 2b–r bearing
electron-donating and electron-withdrawing substituents reacted smoothly in this procedure, affording the
corresponding carboxylic acids 1b–r in good to excellent yields. The partial hydrolysis of nitriles 2e, 2i,
and 2l for 1.0–2.5 h afforded amides 5b–d with good yields.

Scheme 4. Synthesis of 5-amino-2-aryl-2H-1,2,3-triazole-4-carboxylic acids 1b–r and amides 5b–d.
Isolated yields are shown
.
New 2-aryltriazoles 1d, g, h, j, k, m-o, q, r and 5a–d were fully characterized by infrared (IR)
1
13
spectroscopy, H and C NMR spectroscopy, mass spectrometry, and elemental analysis (see ESI, Figs.
S11–S32). From the IR spectra, carbonyl group stretching in acid derivatives 1 produced a single
absorption peak in the range of 1670–1660 cm−1. The ν(NH) stretching mode appeared at approximately
3600–3500 cm−1 (weak), along with the ν(OH) vibrations. Lowering of the C=O stretching mode can be
caused by intramolecular hydrogen bonding and the presence of a strongly electronegative N atom
weakening the C=O bond. The molecular ion peaks in the mass spectra of compounds 1a–r and 5a–d
corresponded to the calculated molecular masses and appeared with an intensity of 100%.
Compound 1e was crystallized as a solvate with DMSO (1:1) (Fig. 1). In the crystal, the DMSO
molecules were fixed by intermolecular OH–O-type hydrogen bonds with participation of the triazole
COOH group and DMSO SO group. The COOH group and aryl substituent on the triazole were
positioned approximately in the heterocycle plane (Fig. 1b). The piperidino group had a chair
conformation with the heterocycle positioned (pseudo)equatorially. The N-atom of the piperidino group
showed significant intramolecular conjugation with the triazole ring (Fig. 1a). The crystal packing was

stacked with a distance of approximately 3.6 Å (Fig. 1d). Several intermolecular interactions with a length
range of 2.419–2.803 Å increased the rigidity of the molecular packing (Fig. 1e).
a
b
d
c
e
Fig. 1. ORTEP view of 1,2,3-triazole 1e with thermal ellipsoids at the 50% probability level: (a) Front
view; (b) side view; (c) molecular cell; and (d,e) molecular packing (RWDW(NH) = 2.6 Å, RWDW(OH) =
2.5 Å, RWDW(NO) = 2.9 Å).
As-synthesized ATAs 1a–r and 2-aryl-1,2,3-triazole-4-carboxamides 5a–d were white and slightly
yellow powders, respectively. These compounds were soluble in most organic solvents and DMSO-water
(9/1, v/v), and insoluble in pure water.
3.2. Optical properties of ATAs 1a-r in solution
Initially, the UV–Vis and fluorescence spectra were recorded in DMSO solution at room temperature. The
observed photophysical parameters are summarized in Table S1 (ESI), and the absorption and emission
spectra are shown in Fig. 2. and Fig. S33 (ESI). The absorption spectra of the ATAs exhibited strong
absorption bands corresponding to π→π*. Absorption and emission bands showed a small blue-shift (in
the range of 7–13 nm for absorption and 12–26 nm for emission) for compounds bearing electron-
donating substituents, with a corresponding ~1.2–3.2-fold increase in quantum yield (QY).

a
b
c
d
e
f
g
h
Fig. 2. (a,c,e,g) Absorption and (b,d,f,h) fluorescence spectra of ATAs (a,b,e,f) 1e–h and (c,d,g,h) 1l–o in
(a–d) DMSO and (e–h) DMSO-water (1/9, v/v). For absorption measurements, c = 5 × 10−5 M; for
fluorescence spectra, c = 5 × 10−6 M and λex = λabs.
a
c
b
d
Fig. 3. Photographs of ATAs 1a–r in (a,b) DMSO solution and in (c,d) DMSO-water mixture
(v/v, 1/9) under irradiation (hand-held UV lamp at 365 nm).

The optic properties of compounds 1d, e, h, l and 1o were recorded in different solvents, with increased
polarity from toluene to DMSO observed during excitation (Figs. 4, 5; ESI, Table S1). As polar solvents
stabilized the polarization of ATAs in the excited state (ES) more than in the ground state (GS), the
emission maximum showed a larger shift to long wavelengths (2–8 nm vs. 3–24 nm) and a corresponding
decrease in their effectivities (up to 3.0-fold). The ATAs with electron-accepting substituent group (CF3)
on the aromatic ring (17–24 nm) were more sensitive to the solvent polarity than those with electron-
donating MeO group (3-9 nm). The largest QY was observed for compound 1e (96.1–96.9%) in nonpolar
1,4-dioxane and toluene, while decreased QYs were observed in polar DMSO (for ATA 1e, 74.9% in
DMSO). ATAs 1a–r showed large Stokes shifts in DMSO (4750–7367 cm-1) and MeCN (up to 7689 cm-
1) indicated that emissions from ATAs were predominantly based on intramolecular charge transfers
without reabsorption of the emitted photons [46]. A large Stokes shift is a rare property in small-molecule
fluorophores but is very useful for their use in imaging applications.
1d
1e
a
b
c
d
1h
1o
e
f
g
h
Fig. 4. (a,c,e,g) Absorption (c = 5 × 10−5 М) and (b,d,f,h) emission (c = 5 × 10−6 М) spectra of 2-aryl-
1,2,3-triazoles 1e (a,b), 1d (c,d), 1h (e,f), and 1o (g,h) (c = 5 × 10−6 М) measured in different solvents at
room temperature.

a
b
Fig. 5. Photographs of solutions of ATA 1d in different solvents under (a) daylight and (b) irradiation
(hand-held UV lamp at 365 nm). (1) Toluene, (2) 1,4-Dioxane, (3) EtOH, (4) DCM, (5) MeCN, (6)
DMSO, (7) DMSO-Water (1/9, v/v).
Interesting results were obtained in the DMSO-water mixture (1/9, v/v) in compare with pure DMSO.
The absorption maxima of ATAs were blue-shifted by ~10–22 nm, while the maximum emission was
shifted by only ~1–14 nm (see ESI, Tables S1). Furthermore, the Stokes shifts had the largest magnitudes
in this mixed solvent (6345–9021 cm-1). However, the ATAs showed different changes in emission
efficiency, with ATAs 1a–d and 1i–j showing ~1.2–3.5-fold decreases in QY, and ATAs 1k–r showing
~3.5-16.0-fold decreases in QY, compared with their solutions in pure DMSO. In contrast, compounds 1e–
h bearing a piperidine substituent exhibited the opposite trend in QY. As shown in Table S1 (Entries 15,
22, 24 and 26), the QYs increased ~1.01–1.48-fold in the DMSO-water mixture. The high emission
efficiency of most acids 1 in solvents of various natures was attributed to the formation of many
intramolecular noncovalent interactions (NCIs), which increased the structural rigidity of these
compounds, as observed by X-ray diffraction (Fig. 1) and optimized the geometries obtained by quantum
mechanical calculations (see ESI, Fig. S35).
A decreased emission efficiency in polar solvents is a common property of triazole acids and might be
attributed to strengthened intermolecular interactions with solvent molecules and weakened intramolecular
and intermolecular NCIs between neighboring ATA molecules. This leads to altered rigidity and decreases
the QY values. For polar protic solvents (DMSO-water, EtOH), there is stronger competition between two
types of hydrogen bonds, and a new system of rigidity is created, providing unique photophysical
characteristics.
3.3. Aggregation-induced enhancement of the emission effect
The emissions data for acids 1e–h showed an increase in QY in the DMSO-water mixture compared with
pure DMSO (see ESI, Table S1). Therefore, these compounds might show aggregation-induced emission
enhancement (AIEE) behavior and form aggregates in the DMSO-water mixture, leading to decreased
energy loss during geometry relaxation after the vertical transition [47-52]. To verify this assumption, we

investigated changes in the emission characteristics in DMSO-H2O mixtures of different ratios (Fig. 6;
ESI, Table S2 and Fig. S34). ATAs 1e–h showed good fluorescence in pure DMSO (QY, 41%–77%). The
fluorescence emission showed no significant change in the maxima position, but the intensity increased
with an increasing water fraction, reaching 90% for ATA 1e. The lesser increase of QY (by 1%) was
observed for compound 1f from the piperidine ATA series (Table S1, Entries 21 and 22).
a
b
c
Fig. 6. Emission intensities (QY) (a) of ATAs 1e, 1g, 1h, and 1l versus water fraction (from 0% to 90%)
in the DMSO-water mixture; (b) QY changes of ATAs 1a, e-h, l (5 × 10−6 M) in DMSO solution and (c) in
the DMSO-water mixture (v/v, 1/9) in different concentrations. (the experiment was repeated several
times).
In contrast to the AIEE behavior of ATAs 1e–h, compounds 1a–d and 1i–r exhibited an aggregation-
induced quenching (AIQ) effect. However, the sensitivity to water content was different. ATAs 1a–d and
1i–k bearing tert-cycloalkylamino substituents (pyrrolidine, azepane, and 4-phenylpiperazine) showed
less of a decrease in QY (1.2–4.9-fold) compared with triazoles 1l–r (bearing N-methyl and N-
cyclohexylamino substituents), which showed a significant drop in intensity (7–16-fold). A detailed
investigation of AIEE (ESI, Fig. S34 and Table S2) showed a decreasing QY fluorescence for ATA 1l in
DMSO with an increasing water fraction from 0 to 90% (v/v). In pure DMSO, ATA 1l exhibited a high
fluorescence intensity of 96%. With increasing water content, a marked decrease in fluorescence intensity
was observed along with a slight red-shift in the emission band (5 nm) (ESI, Table S2). A blue shift was
observed in the absorption maxima (12 nm). The fluorescence intensity was quenched by 82%, reaching
14%. The different aggregation behaviors of ATAs 1e–h might be attributed to morphological changes in
the aggregates owing to different conformations and packing models that arise from attaching the
piperidine ring and, in addition, is dependent on the substituent at the para-position of the aromatic cycle.

To gain a better understanding of how aggregation influences the fluorescence of ATAs 1, we recorded
emission spectra of ATAs 1a, 1e-f and 1l in DMSO and DMSO-water mixture (v/v, 1/9) at different
concentrations (Fig. 6b and 6c). Notably, the emission intensities of ATAs 1a, 1f, and 1l decreased with
increasing concentration by 1.1–1.4-fold and 1.03–1.4-fold in DMSO and DMSO-water, respectively,
without shifting the emission wavelength (ESI, Table S3). By contrast, acids 1e, 1g and 1h showed the
opposite trend. Their emission intensities increased with increasing concentration by 1.1–1.5-fold and
1.02–1.1-fold in DMSO and DMSO-water, respectively. The emission wavelength of ATAs 1e-h showed
slight shifting of 3–4 nm.
In addition, almost all ATAs showed weak solid-state emission in the blue region (410–458 nm) (ESI,
Table S4). The most intense fluorescence was observed for ATAs 1b, 1e–h, and 1p (QY, 1%–11%).
Among them, ATAs 1e, 1g, and 1h showed an AIEE effect; in contrast, acid 1l did not exhibit this
phenomenon but had a negligible emission in the crystalline state. These results demonstrated the
existence of AIEE and emphasized the influence of the alkylamino group structure at C(5) of the triazole
ring on the photophysical properties [52].
The mechanism of enhanced emission intensity might be associated with several relaxation channel
pathways for the excited states to nonradioactively decay to the ground state, as follows: (i) Restriction of
intramolecular rotation (RIR); (ii) J-aggregate formation (JAF); (iii) twisted intramolecular charge transfer
(TICT); and (iv) excited-state intramolecular proton transfer (ESIPT) [47-52]. RIR is known to have a
large-scale scope for AIE/AIEE effects to arise. There are no limitations on the fluorophore polarity (it can
be polar or nonpolar), the packing can be ordered or random, compound spectra might be unchanged or
blue/red shifted, and the size of the Stokes shift might be large or small. The scope of JAF is much
narrower than that of RIR, having been observed for specific dyes bearing strong polar or functional
groups with donor–acceptor (D–A) interactions, specific and highly ordered packing, and exhibiting
redshifts in absorption or emission spectra and very small Stokes shifts. As shown in the previous
discussion and Tables S1–S4, some of these requirements are not suitable for the photophysical
characteristics of ATAs 1e–h. Therefore, RIR processes owing to the piperidine chair conformation are
proposed to be the major reason for the observed AIEE phenomena. It should also be noted that the
substituent in the para-position of the aromatic cycle can enhance or weaken the AIEE behavior. The best
AIEE effect was observed for the fluoromethylphenyl fragment at the C2 triazole atom.
The other two pathways (TICT and ESIPT) were not taken into consideration because these ATA
compounds do not contain structural elements needed for their realization.
3.4. Quantum mechanical calculations

Initially, conformational analysis was performed for ATAs 1a, 1c, and 1l in DMSO and buffer (pH 7.4)
solutions to determine the most stable rotamers (ESI, Table S7), with the results used to calculate the
optimized geometry, absorption spectrum, emission spectrum, highest occupied molecular orbital
(HOMO)–lowest unoccupied molecular orbital (LUMO) distribution, and molecular electrostatical
potential (MEP). Geometry analysis of ATAs 1a, 1c, and 1l (ESI, Fig. S35) showed that the triazole
molecules were planar due to the formation of a series of NCIs, as confirmed by reduced density gradient
(RDG) analysis (ESI, Fig. S36) [53-59]. Tables S8–S12 (ESI) show that the calculated bond lengths and
bond angles were very similar in the DMSO and buffer solutions in the GS. The main differences were the
small increase in torsion angle between the N(2) aromatic cycle the and triazole ring (θ), and the
weakening of all intramolecular hydrogen bonds in the buffer solution. The bond lengths of 1a and 1b
decreased after light absorption and subsequent geometry relaxation. The shortest C2–N3 bond (decreased
by 4%–5%) in the triazole structure in the ES (ESI, Tables S9, S11, S12) was between the aromatic ring
and triazole nitrogen atom. The dihedral angle decreased to 1.7–1.8°, and the molecules became almost
planar. However, the intramolecular hydrogen bond lengths increased in the ES by 2.5%–4.6%.
The calculated absorption and fluorescence characteristics of ATAs 1a, 1c, and 1l in DMSO
are shown in Table 2. Time-dependent density functional theory (TD-DFT) calculation results
indicated that: (i) The calculated absorption and emission λmax values were in good agreement with
the experimental values; and (ii) for all compounds, the S0
LUMO character, while S1 S0 had a dominant LUMO–HOMO character. The oscillator strength
corresponding to the S0 S1 transition (f01) showed moderate values, while a small decrease was observed
for the reverse transition (S1 S0).
→S1 transition had a dominant HOMO–
→
→
→
Table 2. Computed absorption wavelength (λa) and oscillator strength (f01), emission wavelength (λe) and
oscillator strength (f10), Stokes shift (∆ν), modulus of electric dipole moments in the ground state (µ0),
vertical FC excited state (µ1v), relaxed excited state (µ1r), and angles formed by the dipole moment vectors
(
θ0,1v and θ0,1r) for the most stable rotamer of ATAs 1a and 1c in DMSO.
Entry Compd
λ01, nm
328
339
f01
µ0, D
4.5
3.1
cH-L
λem, nm
397
426
f10
cL-H
µ1v,
7.8
8.2
4.7
D
µ1r,
8.1
8.6
4.8
D
θ0,1v,
°
θ0,1r,°
1
2
3
1a
1c
1l
0.6584
0.6032
0.7137
0.6890
0.6881
0.6856
0.5723 0.6962
0.5233 0.6962
0.6857 0.6911
20.0
18.7
21.8
130.7
111.6
52.0
342
2.6
412
ATAs 1a and 1c had similar dipole moments in the GS, which were increased by approximately two-fold
in the ES. As shown in Table 2, the ratio of ES dipole moments to GS dipole moments was too large for all

investigated molecules. Therefore, charge separation increased upon vertical excitation, with the dipole
moment vector markedly changing direction during excitation and relaxation.
The molecular orbitals (MOs) involved in the electron transition from the HOMO to the LUMO are
presented in Fig. 7. This data shows that the HOMO and LUMO were inherently
π and π* orbitals and
that the S0→S1 transition was dominant. As shown in Fig. 7, the HOMO orbital was distributed over the
whole molecule, while the LUMO orbital was shifted to the triazole and aromatic rings and completely
leaves the alkylamino group (electron-donating group). The HOMO and LUMO diagrams show the
intramolecular charge transfer (ICT), confirming previously reported solvatochromic data.
GS
ES
HOMO
LUMO
HOMO
LUMO
1a
1c
1l
Fig. 7. Frontier molecular orbitals (FMOs), HOMO and LUMO, in the ground and excited states for
ATAs 1a, 1c, and 1l in DMSO and water (|Isovalue(MO)| = 0.02 a.u.; |Isovalue( )| = 0.0004 a.u.).
ρ
The MEP shape showed more active sites for electrophilic attack, nucleophilic attack, or weak
intermolecular bonds formation [60]. The potential values are defined by different colors, with negative
potentials (red) associated with oxygen atoms of COOH groups (Fig. 8) and positive potentials (blue and
dark blue) located at the hydrogens of OH-group acid functions and spread over the hydrogen atoms in
pyrrolidine. Excitation clearly led to strengthening and enlargement of both the negative and positive
potentials, as well as electron density separation. For example, the triazole ring N(3) atom became highly

negative. Therefore, these data provide important information for predicting or explaining the reactivity
and sensitivity to the microenvironment of the compounds under investigation. These sites are responsible
for interactions between neighboring molecules of probes, their interactions with solvent molecules, or
attack by external stimuli.
Entry
1
S0
S1v
1a
S1r
2
3
1c
1l
Fig. 8. Plot of MEPs of ATAs 1a, 1c, and 1l, calculated by TD-DFT at the ω-B97X-D / 6−311++G** //
IEF-PCM(UFF) level of theory, for ground and excited states in dioxane. Map colours: Red (negative
potential), blue (positive potential). Elements: Hydrogen (white), carbon (grey), nitrogen (blue), oxygen
(red), sulfur (yellow), chlorine (green). (Range, –0.05 to 0.05; density |Isovalue| = 0.0004 a.u.).
3.5. Fluorescence lifetime
The fluorescence lifetime determines the time available for a chromophore to diffuse or interact with its
environment or any external stimuli in its excited state, and is, therefore, an important parameter that can
expand the applications of fluorescent molecules [62]. The fluorescence lifetimes (⟨τ⟩f) of ATAs 1a–r
were measured in DMSO and DMSO-water (1/9, v/v), and in toluene for compounds 1a, e and 1g, and the
radiative (kr) and nonradiative (knr) rate constants were also calculated [47-52, 61]. These experimental
and calculated data are presented in Tables S5 and S6 (ESI). The fluorescence decay of ATAs 1a–r was
well described by the biexponential in DMSO solution, the usual triexponential in DMSO-water (1/9, v/v),

and even the tetra-exponential for compound 1a in toluene. These data may be explained by the lower
solubility of ATAs in toluene and the formation of different aggregates. It is known that the emission life-
time of the aggregates (1) may be improved by multi-exponent decay and (2) their life-time increases
owing to the restriction of the intramolecular rotation of the separated molecular segments. However,
analysis of the data in Tables S5 and S6 shows that the more stable ES values were observed in DMSO
compared to DMSO-water, where aggregate formation was more probable. The further detailed
experimental investigation and quantum chemical calculation results established the more complicated
behavior of the ATAs in ES. The investigated compounds showed a wide range of fluorescence lifetime
values (1.21–8.48 ns), but it is important that most of the obtained values were quite large. The longest
fluorescence lifetimes were observed for ATAs 1a-h and 1m in DMSO (5.99–8.48 ns), which decreased
for the same triazoles in DMSO-water (2.59–6.26 ns) and toluene (4.09–5.34 ns).
ATAs 1a–r were classified into two groups according to the stability of the S1 state. In the first group,
ATAs 1a–i with pyrrolidine, piperidine, and azepane at the triazole ring C(5) position showed that kr > knr
or that kr and knr have equal values (ESI, Table S6). For example, ATA 1e had a kr value that was 23 times
higher than the corresponding knr (toluene), which was in accordance with the highest QY of this
compound (96.1%). In the second group, only ATAs 1l and 1m showed kr > knr (DMSO), while the other
ATAs in this series with NHMe and NH-cyclo-C6H12 substituents showed knr > kr. Notably, ATAs 1a–k
from the first group showed higher kr values in DMSO-water than in pure DMSO solution. However, the
second group (ATAs 1l–r) showed the opposite dependence, with the kr value in DMSO-water being
higher (2.4–21.0-fold) than in pure DMSO.
A significant increase in the ATAs’ fluorescence lifetime might be explained by the effect of
thermally activated delayed fluorescence (TADF) [47-52, 62-67]. This results in emission with the same
spectral distribution as normal fluorescence, but with a much longer decay time because the molecules
stay in the triplet state before moving back to S1. The usual transition to the excited triplet state often leads
to quenching by molecular oxygen or nonradiative decay. TADF proposes an alternative productive
pathway through reverse intersystem crossing (RISC) and subsequent emission from S1 to S0. Notably,
along with an increase in lifetime, TADF leads to an increase in fluorescence intensity. A high
fluorescence intensity is typical for ATAs.
The probability of ISC processes depends on whether the singlet and triplet states overlap. If the
S0→S1 transition is an n→π-type transition, intersystem crossing is often efficient. Therefore, ICT might
be an essential process for investigated triazoles 1, which have structures bearing several nitrogen atoms.
The energy gaps between the two nearest S1 and Tn levels were calculated, as shown in Table S13 (ESI).
When the singlet–triplet energy splitting (∆EST) is quite small (<0.5 [64] and even <0.3 eV [65]), ISC

processes might actively occur between S1 and the nearest triplet state. Two, three, or even five triplet-
excited states with energies less than S1 were found to be present in triazole molecules in the excited state
solution in DMSO. T2 (1a–d, 1j, 1l–r), T3 (1e–h,i), and T5 (1k) states might favor the reverse process
occurring.
However, it was not possible to establish any evident correlation between the ∆EST and fluorescence
lifetime, QY, or charges in GS and ES. This might be due to the main influence being back transmission.
The reverse intersystem crossing T1→S1 can occur when the energy difference between S1 and T1 is quite
small and the lifetime of T1 is sufficiently long. This requires a decreased gap between the HOMO and
LUMO and strict HOMO–LUMO separation, which is usually determined by the donor and acceptor parts
of the fluorophore. The ICT was observed in DMSO solution during excitation of the ATA molecules
(Fig. 7), but it might not have been sufficient for singlet–triplet energy splitting to afford substantial RISC
efficiency. The ordered packing and high planarity of the ATA geometry (Fig. 1; ESI, Fig. S35) should
provide good overlap of the FMOs. In contrast, the rigidity can weaken vibrations and strengthen
molecular interactions to stabilize the excited triplet state and inhibit the nonradiative relaxation pathway
[62]. To elucidate this unusual behavior of ATAs 1, more specific theoretical (quantum mechanical
calculations) and experimental (spectral) investigations are currently in progress.
Fluorophores that have longer emission lifetimes are of great interest in photonics, OLEDs,
bioimaging, and biosensors. Small soluble TADF molecules have considerable importance in the
development of solution-processed TADF OLEDs, but such investigations remain rare [62-67].
3.6. Optical response of ATAs to pH in DMSO-buffer solution
The absorption and emission spectra of ATAs 1 were measured in DMSO-buffer (1/9, v/v) solutions at
different pH values (2.2, 3.0, 4.0, 5.0, 6.0, 7.0, 7.4, 8.0, 8.5; 9.0, 11.0, and 12.0) (Fig. 9; ESI, Figs. S37–47
and Tables S14–24). As observed in the absorption spectra of compounds 1a, e, g–i, l–p, and 1r, with
increasing pH, the maximum absorption bands of all investigated ATAs were blue-shifted by 2–15 nm,
accompanied by isosbestic points at 272–294 and 337–365 nm, which were attributed to the transition
between the acid form and base form. The maximum absorption intensity increased with increasing pH
throughout the pH range studied. However, a clear relationship between the increase in absorption
intensity and change in pH could not be established because it was random and intermittent.
As shown in Fig. 9, the fluorescence intensity of ATAs 1a and 1e, g–i was very low at highly acidic
pH (pH 2.0) and remained constant up to pH 6.0 for ATAs 1g, e-i, and pH 7.0 for 1a. A further increase in
pH led to a sharp increase in the emission intensity up to pH 8.0 for ATAs 1a, e and 1g, and pH 9.0 for
ATAs 1h and 1i. In contrast, the fluorescence intensities of ATAs 1l–p, r showed QY maxima in acidic

media (pH 1.0–2.2), which sharply decreased up to pH 5.0. The relationship between QY and pH is more
ordered than the relationship between the molar extinction coefficient and pH. A sigmoidal curve forms
when increasing the QY and the pH for compounds 1a, e, g-i, and the opposite curve behavior is found for
compounds 1l-p,r showing decreasing QYs upon increasing pH. Therefore, we can see that the
investigated compounds may be classified into two groups according to their change in QY at different pH
values.
Acid ionization constants were determined from emission titration curves using the nonlinear fitting
method expressed by the Henderson–Hasselbalch equation (log[Imax−I)/(I−Imin)] = pH−pKa) [68-75]. As a
result, we obtained two different sets of pKa values in the range of 7.65–8.08 for ATAs 1a, 1e, and 1g–i,
and 3.20–3.52 for ATAs 1l-p and 1r. Based on the linear regression equations (Figs. S37–S46(d)), a good
relationship between fluorescence intensity at maximum emission and pH was observed at pH-sensitive
intervals for ATAs 1e (Fig. 9h), 1h (Fig. S40d), 1i (Fig. S41d), 1l (Fig. 9l) and 1m (Fig. 9p). These results
clearly showed the application prospects of ATAs for pH detection. Furthermore, the pH ranges in which
ATAs 1 gave an effective fluorescence response were pH 6–8 and 3–5, which is suitable for bioassay
applications.
a
b
c
d
e
f
g
h

i
j
k
l
m
n
o
p
Fig. 9. (a,e,i,m) Absorption spectra, (b,f,j,n) fluorescence, (c,g,k,o) changes in relative intensity with pH,
and (d,h,l,p) linear relationships/approximation of fluorescence intensity for (a–d) ATAs 1a, (e–h) 1e, (i–
l) 1l, and (m–p) 1p.
These interesting effects of pH on the photophysical properties of ATA solutions drove us to further
extend this work to study the pH reversibility of the synthesized compounds, which is very important for
the real-time monitoring of pH changes in living cells [69-72, 74].
Reversibility tests of ATAs 1e and 1l were conducted with small volumes of NaOH and HCl (1 M).
Time-course analysis (30 s, 1 min, and 3 min) showed that the compounds responded almost immediately.
The ATA solution pH was modulated back and forth between 6.0 and 9.0 for ATA 1e and between 2 and
5 for ATA 1l. As shown in Fig. 10a, after five cycles, the fluorescence intensity of ATA 1e showed
excellent reversibility in the different pH solutions. Meanwhile, ATA 1l showed good recurrence to pH 5
and less recurrence to pH 2 (5%) (Fig. 10b). This good reversibility endowed the synthesized ATAs with
good pH stability and reversible ‘on–off’ photoswitching.

a
b
Fig. 10. Fluorescent intensity reversible changes of ATAs 1e (a) and 1l (b) between pH 9-6 (1e) and pH 2-
5 (1l).
To understand the pH sensing mechanism of the ATAs, 1H NMR experiments were performed under
acidic (trifluoroacetic acid, TFA) and alkaline (trimethylamine, TEA) conditions in DMSO-d6. As shown
in Fig. 11 and Tables S25–S29 (ESI), upon addition of TFA, compared with the original NMR spectrum,
the proton chemical shifts in the ortho-Ar position and the MeO-group proton signal showed a distinct
downfield shift of 0.05–0.06 and 0.03–0.04 ppm, respectively, without any noticeable change in the
position of the proton signals of other proton groups. The signal for NH-protons (NH-Me and NHCH)
disappeared from the NMR spectra. These observations suggested that ATAs 1e, 1l, and 1p were
protonated in the same manner, through attack at a triazole N-atom. Symmetric changes in the position of
signals for ortho-protons in the aromatic cycle indicated a higher probability of hydrogen addition to the
N(2) atom and the formation of protonated form A (Scheme 4). However, this form is less stable than C
and D owing to the disturbance of heterocycle aromaticity. The MEP analysis (Fig. 8) showed that the
more active site for electrophilic attack in the GS and ESI is atom N(3), which protonation lead to the
formation of form D.

Scheme 5. Acid/base behavior of ATAs 1
.
ortho-HAr
MeO
ortho-HAr MeO
ortho-HAr
MeO
a
b
c
Fig. 11. Shifts in proton signals of 1H NMR spectra for ATAs 1e (a), 1l (b) and 1p (c) treated with TFA.
Upon adding TEA to the solutions of ATA 1e, 1l, and 1p, the largest downfield shift was observed for
NH proton signals in ATAs 1l and 1p (upfield shift of 0.54–0.71 ppm, Tables S27, S29 (ESI)) in
accordance with the change in electronic distribution owing to ionization of the COOH group (Scheme 4,
deprotonated form F).
3.7. Cytotoxicity of ATAs
Spectral investigation clearly showed the application prospects of the ATAs in biological experiments. An
important factor in the further promotion of ATAs in this field is their cytotoxicity. The cytotoxicity of
ATAs was assessed using the MTT test [71]. Vero cells were reseeded in a 96-well culture plate
(Eppendorf, Germany) and cultured until 70%–80% confluence was achieved. Next, a solution of the test
substance at different concentrations was added to the cells and incubated for three days. The cells were
then washed with Hanks' solution, after which a fresh nutrient medium containing 0.5 mg/mL MTT (Dia-
M, Russia) was added, and the cells were incubated for 2 h. The nutrient medium was then removed,

DMSO (Panreac, USA) was added to the cells, and the color intensity was analyzed using a DS2 plate
spectrophotometer (Dynex, USA). At the substance concentrations studied, no statistical cytotoxic effect
was observed (Fig. 12).
Fig. 12. Effects of ATAs 1a, 1e, and 1l with varied concentrations on the viability of green monkey
epithelial (Vero) cells. The viability of the cells with the same solvent (without ATAs added) is defined as
100% (Control). The results are means
± standard deviation of five separate measurements.
4. Conclusions
In summary, we have presented a simple and convenient approach to the synthesis of new functional 2-
aryl-1,2,3-triazole derivatives. The ATAs showed a bright-blue emission with high QY, while maintaining
a large Stokes shift and prolonged fluorescence lifetime. The ATAs showed optic characteristics that were
strongly dependent on their structure. Therefore, the structural units defined their behavior in different
solvents and DMSO-water mixtures in response to external stimuli (acid and base, pH variation), acidity,
and their ability to show optic phenomena (AIEE). The important structural unit was the aromatic ring
substituent, which influenced the ATA molecule electron density distribution and showed an λem ranging
from 415 to 451 nm and a QY for 1a-r of 20.6–96.0% (DMSO). The structure of the amino group at the
C(5) triazole ring allowed the ATAs to be classified into two groups with different properties. All
investigated ATAs showed marked changes in fluorescence QY during pH variation, which accompanied
protonation of the triazole ring under acidic conditions and deprotonation of the carboxylic function under
basic conditions. Experiments on the reversibility from acid to base conditions showed that ATAs are
novel ‘on–off’ probes that might be new practical tools for assessing pH change in real-time. In addition

to the outstanding properties of these 2-aryl-1,2,3-triazole derivatives, this investigation discovered many
new interesting aspects of their optical properties, which require further study.
Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgments
We thank RFBR for financially supporting this work through the project No 19-03-00720A. The
Siberian Branch of the Russian Academy of Sciences (SB RAS) Siberian Supercomputer Centre is
gratefully acknowledged for providing supercomputer facilities.
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