Synthesis and application of new 3-amino-2-pyridone based luminescent dyes for ELISA

Article abstract of DOI:10.1016/j.dyepig.2020.109072

A series of new 3-amino-4-arylpyridin-2(1H)-one derivatives was synthesized on the basis of oxazolo[5,4-b]pyridin-2(1H)-ones. Their photophysical properties and antioxidant activity were studied. Effective luminophores with an photoluminescence yield of up to 0.76 and substances exhibiting antioxidant activity exceeding ascorbic acid were revealed. It was found that the obtained luminescent dyes are horseradish peroxidase substrates and can be used to develop a rather sensitive ELISA method. The detection limits of hydrogen peroxide and horseradish peroxidase in the system containing the tested dyes lie in the nanomolar concentration range.

Full text of DOI:10.1016/j.dyepig.2020.109072

Dyes and Pigments 187 (2021) 109072
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: http://www.elsevier.com/locate/dyepig
Synthesis and application of new 3-amino-2-pyridone based luminescent
dyes for ELISA
,
c
Anton Shatsauskasa, Yuri Shatalinb, Victoria Shubinab, Yuri Zablodtskiic, Sergei Chernenkoa
,
,
*
Anna Samsonenkoc, Anastasiya Kostyuchenkoc, Alexander Fisyuka
a Laboratory of New Organic Materials, Omsk State Technical University, 11 Mira Ave, 644050, Omsk, Russia
b Institute of Theoretical and Experimental Biophysics of the Russian Academy of Sciences, Institutskaja Str., 3, 142290, Pushchino, Russia
c Department of Organic Chemistry, Omsk F.M. Dostoevsky State University, 55a Mira Ave, 644077, Omsk, Russia
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of new 3-amino-4-arylpyridin-2(1H)-one derivatives was synthesized on the basis of oxazolo[5,4-b]
pyridin-2(1H)-ones. Their photophysical properties and antioxidant activity were studied. Effective lumino-
phores with an photoluminescence yield of up to 0.76 and substances exhibiting antioxidant activity exceeding
ascorbic acid were revealed. It was found that the obtained luminescent dyes are horseradish peroxidase sub-
strates and can be used to develop a rather sensitive ELISA method. The detection limits of hydrogen peroxide
and horseradish peroxidase in the system containing the tested dyes lie in the nanomolar concentration range.
Enzyme immunoassay
3-Aminopyridin-2(1H)-One
Luminescent dye
Antioxidant activity
Horseradish peroxidase
1. Introduction
immobilized peroxidase is required. Sometimes this leads to a misrep-
resentation of the analysis results due to the complex kinetics of the
Currently, sensitive methods for detecting viral infection are of
particular importance due to pandemic caused by the coronavirus
COVID-19. Methods of enzyme-linked immunosorbent assay (ELISA) are
widely used to diagnose human viral diseases. This group of methods is
based on the detection of a complex formed as a result of the interaction
of antigens with antibodies conjugated with a tag enzyme. The presence
and quantity of antigens is judged by the color change of the enzyme-
sensitive substance (substrate) with which the complex is treated. One
of the most frequently used enzymes in ELISA catalyzing substrate
oxidation is horseradish peroxidase (HRP) [1–11]. This is due to its low
cost, high catalytic activity, stability, and ease of detection of the reac-
tion products. The most frequently used type of HRP substrates are
substances, the oxidation of which results in the formation of colored
compounds. Examples of such organometallic compounds include
5-aminosalicylic acid [12], 3,3′,5,5′-tetramethylbenzidine (TMB) [13],
2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) [14] and
others. As a rule, these peroxidase substrates have a lower sensitivity
compared to substrates whose oxidation products fluoresce [15]. Ex-
amples of such substrates are dihydroxyphenoxazine derivatives
(10-acetyl-3,7-dihydroxyphenoxazine, Amplex Red) and tyramide [16].
However, at a low concentration of the target molecules, a long time of
incubation of substrates in the presence of hydrogen peroxide and
enzymatic reaction. For a more accurate assessment, it is necessary to
take into account the inhibition and inactivation of peroxidase over
time, as well as the side reactions of the fluorescent substrate, etc. The
use of substrates that lose their fluorescent properties during oxidation
process can make it possible to more accurately estimate the amount of
immobilized peroxidase from the descending kinetic dependence of the
fluorescent dye oxidation.
Earlier, we have developed methods for the synthesis of 4-aryl-3-
amonopyridin-2(1H)-ones [17–25], which turned out to be effective
luminophores. When studying the antioxidant activity of these com-
pounds, we noticed that they are easily oxidized, they lose luminescent
properties upon oxidation and therefore are of interest as dyes for ELISA.
Data on amino-substituted derivatives of 3-amino-4-arylpyridin-2
(1H)-ones, their methods of preparation and luminescent properties are
scarce.
The aim of this work is to synthesize 3-amino-4-arylpyridin-2(1H)-
one derivatives, to study their photophysical properties, and also to
search for luminescent dyes among them, which can be used as new
luminescent HRP substrates in ELISA.
* Corresponding author.
E-mail address: fisyuk@chemomsu.ru (A. Fisyuk).
https://doi.org/10.1016/j.dyepig.2020.109072
Received 20 October 2020; Received in revised form 5 November 2020; Accepted 6 December 2020
Available online 16 December 2020
0143-7208/© 2020 Elsevier Ltd. All rights reserved.

A. Shatsauskas et al.
Dyes and Pigments 187 (2021) 109072
2. Results and discussion
upon heating resulted in the opening of the five-membered ring to form
carbamates 10, 11 and ureas 12–16 in 56–72% and 60–71% yields,
respectively. Amide 17 was obtained by acylation of 3-amino-2-pyri-
done 4 as described in ref. [18].
2.1. Synthesis of 3-amino-4-arylpyridin-2(1H)-one derivatives
Recently, we have developed a simple method for the synthesis of
oxazolo[5,4-b]pyridin-2(1H)-ones 1–3 [20,25]. These compounds, as
well as the N-alkylation products 7a–d, 7i obtained by interaction of
oxazolo[5,4-b]pyridin-2(1H)-one 1 with alkyl halides, were used as
starting materials for the preparation of 4-phenyl-3-amonopyridin-2
(1H)-ones 8a–c, 8e, 8i, 9. The reaction of (2-bromoethyl)-5-methyl-7--
Thus, we obtained a series of 3-amino-4-arylpyridin-2(1H)-ones
bearing various substituents at the nitrogen atom. Some of them are
readily soluble in water (8a, 10–16), alcohols (4–6, 8a–c, 8e, 8h, 8j,
9–16) and non-polar solvents (4–6, 8a–c, 8e, 8h, 8j, 9).
2.2. Photophysical properties of 3-amino-4-arylpyridin-2(1H)-one
derivatives and their antioxidant activity
phenyloxazolo[5,4-b]pyridin-2(1H)-one 7d (R
=
CH2Br) with
2-(2-(2-methoxyethoxy)ethoxy)ethan-1-ol in the presence of NaH in
DMF furnished podand 7j. Heating oxazolo[5,4-b]pyridin-2(1H)-ones
1–3, 7a–c, 7j with alkali in water or water–dioxane (1:1) mixture led to
hydrolysis of the oxazole ring and the formation of 3-amino-4--
arylpyridin-2(1H)-ones 4–6, as well as their N-substituted derivatives
8a–c, 8j.
In order to select the most promising substrates for ELISA, the pho-
tophysical properties and antioxidant activity of the synthesized com-
pounds were studied. UV and fluorescence spectra were recorded for
alcohol solutions of compounds 4–6, 8a–c, 8e–h, 8j, 9–17, the light
absorption coefficient and the quantum fluorescence yield were calcu-
lated. In the absorption spectra of 3-amino-4-arylpyridin-2(1H)-ones
derivatives 4–6 there are two main absorption bands with maxima in
Methyl-3-(methylamino)-4-phenylpyridin-2(1H)-one 8h and 3-
(ethyl(methyl)amino)-6-methyl-4-phenylpyridin-2(1H)-one 9 were ob-
tained by reduction of oxazolo[5,4-b]pridin-2(1H)-ones 1 and 7i with
sodium borohydride in the presence of trifluoroacetic acid or AlCl3 in
THF (Scheme 1). The reaction of 3-amino-6-methyl-4-phenylpyridin-2
<251 and 320–351 nm regions, corresponding to π–π* and n–π* tran-
sitions. The emission maxima lie in the 420–451 nm range, and the
luminescence quantum yield reaches 0.76 (4). The luminescence
quantum yield decreases when the phenyl substituent at the C4 atom is
replaced by substituted aryl (5, Φfl = 0.50) or thiophene moiety (6, Φfl
= 0.37). Substituent at the nitrogen atoms (8a–c, 8e–h, 8j, 9) also leads
to a decrease in the quantum yield to 0.43–0.63 (8a–c, 8e–h) and to 0.05
in the case of the presence of two alkyl groups (9). For carbamates 10, 11
and ureas 12–16, a hypsochromic shift is observed in the absorption and
luminescence spectra. The absorption maxima in UV and luminescence
spectra are in the 317–323 and 420–440 nm region, respectively.
The quantum yields of compounds 10–17 also lie in the range of
(1H)-one
4
and aromatic aldehydes (benzaldehyde, thio-
phenecarbaldehyde, and furfural) with NaBH4 in the presence of formic
acid led to the formation of 3-alkylamino-4-arylpyridin-2(1H)-ones
8e–g in 78–87% yield. Previously, these compounds have been prepared
through a two-step process including the isolation of intermediate Schiff
bases [22].
Since oxazolo[5,4-b]pyridin-2(1H)-ones 1–3, 7 underwent the alkali-
induced ring opening, we studied their reactions with nucleophiles.
Thus, the reactions of compound 4 with alcohols, ammonia, or amines
Scheme 1. Synthesis of 3-amino-4-arylpyridin-2(1H)-one derivatives.
2

A. Shatsauskas et al.
Dyes and Pigments 187 (2021) 109072
0.11–0.31. It should be noted that, for all the studied compounds, a
sufficiently large Stokes shift (98–114 nm) is observed, and in the case of
dialkyl amine 9 it reaches 172 nm (Table 1). UV spectra of compounds
8a–c, 8e–h, 8j, 9–17 are presented in Supplementary Material
(Table SB1, Figures SB1–SB5).
luminescent properties, compounds 4–6, 8a, 8e, 8j differing in the
structure of the aryl moiety and the substituent at the nitrogen atom
were selected for further studies. It was found that the concentration
dependence of the fluorescence intensity of these compounds in water
remains linear up to 100 μM (Fig. 1).
The antioxidant activity (AOA) of 3-amino-4-arylpyridin-2(1H)-ones
derivatives 8a–c, 8e, 8h, 8j, 9–17 was studied by a modified FRAP
method based on the spectrophotometric determination of the
Fe2+–2,2′-bipyridyl complex obtained by reduction of ferric iron with an
antioxidant in the presence of 2,2′-bipyridyl [31,32] for 15 min. The
AOA of the tested fluorescent dyes (FD) was assessed relative to ascorbic
acid (AA) according to the calibration graph (SM, Table SC1–SC3,
Figures SC1–SC3). The results obtained are shown in Table 1 and are
expressed as the equivalent antioxidant activity of ascorbic acid
([FD]/[AA]). It was shown that the AOA of 3-amino-2-pyridones 4–6,
8a–c, 8e–h, 8j is comparable to activity of ascorbic acid, and some
compounds are superior in activity to ascorbic acid. It should be noted
that the introduction of an electron-withdrawing substituent to the
amino group leads to a drop in AOA, which for compounds 10–17 is
0.02–0.12. The activity of N-dialkyl-substituted 3-amino-2-pyridone 9
also turned out to be low (0.24). Among the 3-amino-4-arylpyridin-2
(1H)-one derivatives, which have better antioxidant activity and
No oxidation of aqueous solutions of 3-amino-4-arylpyridin-2(1H)-
ones 4–6, 8a, 8e, 8j was observed when standing in air for 24 h at room
temperature. The results obtained indicate the absence of self-
quenching, aggregation, and sufficient stability of these compounds,
which makes it possible to calibrate their aqueous solutions.
2.3. Oxidation of 3-amino-4-arypyridin-2(1Н)-ones with H2O2 in the
presence of horseradish peroxidase
Most of the simplest derivatives of phenol and aniline can be
oxidized by hydrogen peroxide in the presence of HRP [33]. It could be
assumed that 3-amino-4-arylpyridin-2(1H)-ones derivatives will be
oxidized in a similar way (Scheme 2).
Indeed, the luminescent properties of solutions of the studied dyes
4–6, 8a, 8e, 8j in the presence of hydrogen peroxide at a concentration
of 500 μM did not change practically. Whereas in the presence of HRP
and hydrogen peroxide, a decrease in the fluorescence intensity of
Table 1
Photophysical properties and antioxidant activity of compounds 4–6, 8a–c, 8e–h, 8j, 9–17.
◦
N
Fluorescent dye
Ar, X or R1,R2
UV–Visa
Photoluminescenceb
АОА = mol(FD)/mol
(AA)c
max λ
ε
, 103 [l/
λex[nm]
λ em
Stokes shift
Quantum yield
max
[nm] abs
cm*mol]
8.9
9.8
8.3
6.9
8.8
7.7
8.8
7.8
7.0
9.3
9.7
4.3
8.1
6.1
4.8
5.5
6.4
5.5
5.2
7.0
[nm]
(Φfl)b
[nm]
101
[eV]
4
5
6
Ph
334
334
351
338
337
336
339
338
337
340
329
320
317
319
319
317
318
323
318
333
335;
350
335;
350
350;
355
340;
350
340;
350
325;
335
330;
340
330;
340
330;
340
340;
350
330;
340
320;
330
310;
320
310;
320
310;
320
310;
320
310;
320
310;
320
310;
320
330;
340
435
437
451
447
448
449
447
448
452
445
443
492
424
427
420
424
428
440
425
431
0.86
0.88
0.78
0.89
0.91
0.93
0.89
0.90
0.94
0.86
0.93
1.35
0.99
0.99
0.96
0.97
1.00
1.02
0.98
0.85
0.76 ± 0.03
0.50 ± 0.02
0.37 ± 0.01
0.53 ± 0.02
0.55 ± 0.02
0.58 ± 0.02
0.39 ± 0.01
0.44 ± 0.01
0.44 ± 0.01
0.43 ± 0.02
0.62 ± 0.03
0.05 ± 0.00
0.11 ± 0.00
0.14 ± 0.01
0.12 ± 0.00
0.21 ± 0.01
0.28 ± 0.01
0.31 ± 0.01
0.31 ± 0.01
0,16 ± 0.01
0.94 ± 0.04
0.97 ± 0.02
1.17 ± 0.03
0.91 ± 0.01
0.94 ± 0.01
0.95 ± 0.03
1.13 ± 0.05
1.16 ± 0.04
1.11 ± 0.04
0.95 ± 0.03
1.14 ± 0.01
0.24 ± 0.01
0.09 ± 0.01
0.06 ± 0.01
0.02 ± 0.01
0.04 ± 0.01
0.07 ± 0.01
0.05 ± 0.01
0.06 ± 0.01
0.12 ± 0.01
C6H3-3,4-(OMe)2
Thienyl
103
100
109
111
113
108
110
115
105
114
172
107
108
101
107
110
117
107
98
8a
8b
8c
8e
8f
H, CH2CH2OH
H,
(CH2CH2O)2Me
H,
(CH2CH2O)3Me
H, CH2-2-Furyl
H, CH2-2-Thienyl
H, Bn
8g
8h
8j
H, Me
H,
(CH2CH2O)4Me
Me, Et
9
10
11
12
13
14
15
16
17
EtO
i-PrO
NH2
NHMe
NHBn
NHPh
N(CH2CH2)2O
Me
a
EtOH was used as a solvent (10ꢀ 7–10ꢀ 5 mol/l).
b
c
The fluorescence quantum yield was calculated relative to the standard (quinine sulfate in 0.5 M H2SO4, Фfl = 0.546) [26–30].
The antioxidant activity (AOA) of the studied compounds (FD) was assessed by the FRAP method relative to the standard ascorbic acid (AA) [31,32].
3

A. Shatsauskas et al.
Dyes and Pigments 187 (2021) 109072
method [34]. This parameter is the minimum amount of the analyte in
the sample, the signal from which can be reliably distinguished from the
background. The calculation of the detection limits for hydrogen
peroxide in the HRP-catalyzed oxidation of compounds 4–6, 8a, and 8e
was performed on the basis of the data obtained by titrating 1 μM so-
lutions of the tested compounds with a hydrogen peroxide solution (SM,
Figure SD1). Since the fluorescence intensity of the solution quickly
reached a stationary level during titration after the addition of each new
aliquot of peroxide (Fig. 3), the limit of detection for H2O2 was calcu-
lated using the standard deviation of the fluorescence signal [35,36].
To determine the limit of detection for HRP, HRP at various con-
centrations was added to solutions containing fluorescent dyes 4–6, 8a,
8e, and H2O2. The mixtures were incubated at 37 ◦C for 15 min, then the
fluorescence was recorded. As shown in Fig. 5, there was a linear
dependence of the change in the concentration of the dye on the con-
centration of HRP. However, the kinetics of oxidation of dyes at different
concentrations of HRP was different. In this regard, the RMSE method
was used to determine the limit of detection for HRP [37]. The results
are collected in Table 2.
Fig. 1. The concentration dependence of fluorescence intensity of compounds
4–6, 8a, 8e, 8j.
In conclusion, the detection limits of hydrogen peroxide and horse-
radish peroxidase in systems containing the tested compounds are
approximately the same as for the dyes used in ELISA kits, and lie in the
range of nanomolar concentrations. Therefore, the obtained compounds
can be used to develop a rather sensitive method for the determination
of hydrogen peroxide and, in the future, to modify ELISA method.
solutions of these compounds was observed, which indicates their
oxidation. The oxidation rate of the tested compounds linearly depended
on the enzyme concentration (Fig. 2). This indicates that the studied
compounds are HRP substrates.
To determine the stoichiometry of HRP-catalyzed oxidation of 3-
amino-4-arylpyridin-2(1H)-ones 4–6, 8a, 8e, 8j with hydrogen
peroxide, the following experiments were carried out: a solution of
3. Materials and methods
hydrogen peroxide was successively added to 1 and 100 μM solutions of
3.1. General information
tested compounds. The final concentration of hydrogen peroxide varied
from 10 nM to 1
second.
μM in the first case and from 1 μM to 100 μM in the
All chemicals and solvents were purchased from commercial sup-
pliers and used as granted. Crude products were purified by column
chromatography with silica gel (100–200 mesh) or recrystallization. The
reaction progress and purity of the products were monitored by thin
layer chromatography on Sorbfil UV-254 plates which were visualized
In both cases, the fluorescence intensity rapidly decreased and
reached a certain constant value after adding each new aliquot, which
indicates a rapid consumption of hydrogen peroxide. At the same time,
the relative fluorescence intensity (I/I0) of compounds 4–6, 8a, and 8e
linearly depended on the concentration of hydrogen peroxide (as well as
the molar ratio of hydrogen peroxide/dye) (SM, Figure SD1). As an
example, Fig. 3 shows the change in the fluorescence intensity of com-
pound 6 during titration with hydrogen peroxide and the obtained I/I0
= f([H2O2]) dependence for this compound.
The dependence of the change in the molar concentration of com-
pounds 4–6, 8a, and 8e on the molar concentration of hydrogen
peroxide added to the system was also linear (Fig. 4). The slope of the
tangent, equal to the desired stoichiometric ratio [FD]/[H2O2], was
practically the same for these compounds and was close to 1 (Fig. 4).
At the same time, the amount of hydrogen peroxide required for the
oxidation of compound 8j was higher than those of compounds
mentioned above (Fig. 4). In addition, for compound 8j at a concen-
tration of 100
μ
M, two linear regions can be distinguished, whereas at a
concentration of 1
μ
M, one linear curve was observed. Apparently, the
oxidation of compound 8j involved not only the heterocycle, but also the
substituent at the nitrogen atom, which makes its use as a horseradish
peroxidase substrate inappropriate. The coefficients of the linear
regression equations for the above relationships for compounds 4–6, 8a,
and 8e are given in Supplementary Material (SM, Table SD1).
Fig. 2. Dependence of the initial rate of oxidation of fluorescent dyes (FD) on
the horseradish peroxidase concentration (HRP).
Limit of detection (LOD) is one of the important parameters of ELISA
Scheme 2. Oxidation of 3-amino-4-arypyridin-2(1Н)-ones.
4

A. Shatsauskas et al.
Dyes and Pigments 187 (2021) 109072
Fig. 3. Change in the relative fluorescence intensity of dye 6 upon successive addition of hydrogen peroxide. Left – titration curve; right – relative intensity of dye
fluorescence depending on the concentration of added hydrogen peroxide. C0(6) = 1 M.
μ
Fig. 4. Changes in the concentration of fluorescent dyes depending on the concentration of hydrogen peroxide. The initial concentration of the dyes was 1
and 100 M (right).
μM (left)
μ
Table 2
Limits of detection for hydrogen peroxide and horseradish peroxidase.
Fluorescent dye
LODH2O2, nM
LODHRP, nM
4
8.97 ± 1.01
7.78 ± 3.75
3.17 ± 0.70
11.45 ± 4.61
6.84 ± 2.79
2.45 ± 0.45
3.40 ± 1.06
1.58 ± 0.44
2.92 ± 0.63
7.45 ± 2.19
5
6
8а
8e
were obtained in the J-modulation mode.
The IR spectra were recorded on Infralum FT-801 spectrometer from
KBr pellets or thin layer.
The elemental analyses were carried out on a Carlo Erba 1106 CHN
analyzer. The melting points were determined on a Reach devices RD-
MP. UV–Vis spectra were taken in EtOH or DMSO (10ꢀ 7-10ꢀ 5 mol/l)
using a UV/VIS/NIR Spectrometer Lambda 750 (PerkinElmer), while
fluorescence spectra were recorded on Cary Eclipse (Agilent) fluores-
cence spectrometer. Fluorescence excitation spectra were recorded to
determine the excitation wavelength at which the maximum fluorescent
response is observed. Subsequent fluorescence emission spectra were
recorded at excitation wavelengths determined in this manner. The
quantum yield of examined compounds was determined relative to
quinine sulfate in 0.5 M H2SO4, Фfl = 0.546, using comparative method
[26–30].
Fig. 5. Changes in the concentration of fluorescent dyes (FD) depending on the
concentration of horseradish peroxidase (HRP). The initial concentration of
fluorescent dyes was 100 μM, and the concentration of hydrogen peroxide was
50 μM.
with UV light (254, 365 nm). All chemicals were of analytical grade and
purchased from Sigma-Aldrich Chemical Co.
The 1H and 13C NMR spectra were recorded on a Bruker Avance
Instrument operating at 400 MHz and 100 MHz for 1H and 13C nuclei,
respectively. Chemical shifts are referenced relative to TMS or the re-
sidual signals of protonated solvents as an internal standard: chloroform
(δ = 7.26 (1) in 1H NMR, δ = 77.00 (3) in 13C NMR), DMSO‑d6 (δ = 2.49
(5) in 1H NMR, δ = 39.5 (7) in 13C NMR). Most of the 13C NMR spectra
The antioxidant activity of the tested compounds was determined by
the modified FRAP method [31,32]. Antioxidant activity was assessed
relative to a standard substance (ascorbic acid) according to a calibra-
tion graph by measuring the optical density of aqueous solutions con-
taining 1.0 ml of an alcohol solution of 2,2′-bipyridyl (c =1.2∙10ꢀ 2
5

A. Shatsauskas et al.
Dyes and Pigments 187 (2021) 109072
mol/l), 1.0 ml of a FeNH4(SO4)2⋅12H2O solution (c = 6.0∙10ꢀ 3 mol/l),
and 0.5–1.00 ml of a solution of the test compound (c = 0.1 mg/ml) (SM,
Table SC1-SC3, Figures SC1-SC3). Before studying antioxidant activity
of the tested compounds, the possibility of complexation of these re-
agents with Fe2+ ions was studied. These compounds do not form
complexes with Fe2+, which is confirmed by the absence of significant
deviations from additivity in the spectra of solutions containing the re-
agent and the Fe2+ salt (in the ratio of Fe2+:R = 1:3).
The calculation of the concentrations of compounds was carried out
according to the following formulas:
[H2O2] = [H2O2]0⋅Va/(V0+Va);
[FD] = [FD]0 ⋅ I/I0;
where [H2O2] и [H2O2]0 – final and initial concentrations of hydrogen
peroxide, respectively, μM; V0 – initial volume of a solution containing
FD and HRP; Va – additive volume of H2O2; [FD] and [FD]0 – final and
initial concentration of the fluorescent dye, respectively, μM; I – fluo-
3.2. Synthesis
rescence intensity after adding hydrogen peroxide; I0 – fluorescence
intensity before addition of hydrogen peroxide.
Procedures for the syntheses of 7-aryl[1,3]oxazolo[5,4-b]pyridin-2
(1H)-ones 1–3, 3-amino-4-arylpyridine-2(1H)-ones 4–6, 1-alkyl-5-
methyl-7-phenyl[1,3]oxazolo[5,4-b]pyridin-2(1H)-ones 7b, 7j, and N-
(6-methyl-2-oxo-4-phenyl-1,2-dihydropyridin-3-yl)acetamide 16 have
been reported earlier in Ref. [18,20,25,30]. Synthesis methods and
spectral data for all the synthesized compounds are presented in SM
(Section A).
Next, a graph was plotted as a function of the molar concentration of
the compound oxidized after the addition of hydrogen peroxide versus
the molar concentration of hydrogen peroxide added to the system. The
stoichiometry of the HRP-catalyzed oxidation of the tested compounds
with hydrogen peroxide was determined from the slope of the obtained
curves.
3.3.3. Determination of the limits of detection
3.3. Study of HRP-catalyzed oxidation of luminescent dyes with hydrogen
peroxide
To determine the limit of detection of H2O2 by fluorimetry using
fluorescent dyes 4–6, 8a, 8e, we used the data obtained by titrating 1 μM
dye solutions with a hydrogen peroxide solution. A graph was plotted as
a function of the relative intensity (I/I0) of fluorescence versus the molar
All solutions were prepared in distilled water, additionally purified
on a Milli-Q device (Millipore, USA). HRP (CAT #P8125) was purchased
from Sigma-Aldrich (USA). HRP was dissolved in 20 mM phosphate-
buffered saline, pH 7.4 (A403/A275 = 2.8–3.0), and its concentration
was determined spectrophotometrically (ε403 = 100 mMꢀ 1 cmꢀ 1) [38].
Hydrogen peroxide (30%) was purchased from Sigma-Aldrich (USA) and
its concentration was confirmed spectrophotometrically (ε240 = 39.4
ratio H2O2/FD. LODH2O2 was calculated using the formula: LODH2O2
=
3*SD/k1 [34], where SD is the standard deviation of the fluorescence
signal, k1 is the slope of the regression line obtained for the experimental
data.
To determine the limits of detection for HRP, HRP at various con-
centrations was added to solutions containing fluorescent dye (4–6, 8a,
8e) and H2O2 (SM, section D). The mixtures were incubated at 37 ◦C for
15 min, then fluorescence was recorded on a Tecan Infinite F200
microplate reader (Austria) in 96 well plates using excitation and
emission wavelengths of 360 ± 12.5 and 465 ± 17.5 nm, respectively.
Changes in the fluorescence intensity of compounds were converted to
changes in compound concentrations. Next, a graph was plotted as a
function of the molar concentration of the compound oxidized after the
addition of hydrogen peroxide versus the molar concentration of HRP
M
ꢀ 1 cmꢀ 1 [39]).
Stock solutions of fluorescent dyes (4–6, 8a, 8e, 8j) (100 mM) were
prepared in DMSO and their concentrations were determined spectro-
photometrically (using the extinction coefficients presented in Table 1).
Fresh solutions of fluorescent dyes, HRP, and H2O2 were prepared
before each experiment.
3.3.1. Oxidation of fluorescent dyes
Oxidation of the tested compounds was evaluated by the decrease in
the fluorescence intensity with time. Measurements were performed in
20 mM phosphate buffered saline (PBS) in the absence and presence of
hydrogen peroxide at 37 ◦C on a Tecan Infinite F200 microplate reader
(Australia) in 96 well plates (Greiner 655076). The excitation wave-
length was 360 ± 12.5 nm, and the emission wavelength was 465 ±
added to the system. LODHRP was calculated as follows [37]: LODHRP
=
3⋅RMSE/k2, where RMSE – root mean square error of linear regression;
k2 – linear regression slope.
The calculation of standard deviations, root mean square errors of
the model and slope coefficients of linear regression was carried out
using the LibreOffice 6.4 Calc and QtiPlot 0.9.8.9 software packages.
17.5 nm. The final concentration of fluorescent dyes was 100
μ
M, the
final concentration of hydrogen peroxide was 500
μM. In both cases, no
oxidation reaction of fluorescent dyes was observed. To study the effect
of HRP on the oxidation of fluorescent dyes, HRP was added to a mixture
Declaration of competing interest
containing fluorescent dyes (at a final concentration of 100 μM) and
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
H2O2 (at a final concentration of 50 μM). The final concentration of the
enzyme varied from 9 to 145 nM. The initial rates were determined from
the slope of the initial linear region of the kinetic curves. At least five
independent experiments were performed and the mean values of the
initial rates were calculated. Next, a graph was plotted of the depen-
dence of the average values of the initial rates (converted into changes in
the concentration of the fluorescent dyes) on the molar concentration of
HRP.
Acknowledgements
This work was supported by the Russian Science Foundation (Grant
no. 19-13-00273).
Appendix A. Supplementary data
3.3.2. Stoichiometry of HRP-catalyzed oxidation of fluorescent dyes
Stock solutions of the fluorescent dyes (4–6, 8a, 8e, 8j) (100 mM)
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.dyepig.2020.109072.
were used to prepare working solutions (1 μM and 100 μM) in PBS (pH
7.4). Then the dyes were titrated with hydrogen peroxide in the presence
of HRP (SM, section D). The oxidation of the tested compounds was
evaluated by the decrease in the fluorescence intensity. Measurements
were performed on a Cary Eclipse spectrofluorimeter (Varian, Australia)
using standard 1 cm quartz cells at room temperature using the wave-
lengths shown in Table 1.
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