Chemiluminescence of methoxycarbonylphenyl 10-methyl-10λ4 -2,7-disubstituted acridine-9-carboxylate derivatives

Article abstract of DOI:10.1016/j.jphotochem.2020.112851

Methoxycarbonylphenyl 10-methyl-10λ⁴-2,7-disubstituted acridine-9-carboxylate derivatives were synthesized, and both their chemiluminescence intensities at pH 7?10 and their chemical stabilities were clarified. The products and generation efficiency of the chemiluminescence reactions were evaluated. 4-(Methoxycarbonyl)phenyl 10-methyl-10λ⁴-2,7-dimethylacridine-9-carboxylate gave relatively strong chemiluminescence intensities at pH 7?10 and was applied to the measurement of hydrogen peroxide.

Full text of DOI:10.1016/j.jphotochem.2020.112851

Journal of Photochemistry & Photobiology, A: Chemistry 403 (2020) 112851
Contents lists available at ScienceDirect
Journal of Photochemistry & Photobiology, A: Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Chemiluminescence of methoxycarbonylphenyl 10-methyl-10λ4
-2,7-disubstituted acridine-9-carboxylate derivatives
,
Manabu Nakazonoa *, Shinkoh Nanbub, Takeyuki Akitaa, Kenji Hamasea
a Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan
b Faculty of Science and Technology, Sophia University, 7-1 Kioi-Cho, Chiyoda-ku, Tokyo, 102-8554, Japan
A R T I C L E I N F O
A B S T R A C T
Methoxycarbonylphenyl 10-methyl-10λ4-2,7-disubstituted acridine-9-carboxylate derivatives were synthesized,
and both their chemiluminescence intensities at pH 7ꢀ 10 and their chemical stabilities were clarified. The
products and generation efficiency of the chemiluminescence reactions were evaluated. 4-(Methoxycarbonyl)
phenyl 10-methyl-10λ4-2,7-dimethylacridine-9-carboxylate gave relatively strong chemiluminescence intensities
at pH 7ꢀ 10 and was applied to the measurement of hydrogen peroxide.
Keywords:
Acridinium ester
Chemiluminescence
pH
Hydrogen peroxide
Electron-donating group
1. Introduction
acridinium esters were introduced, and the CL intensities of acridinium
esters also decreased at pH 9ꢀ 10 [14]. By removing this disadvantage i.
Chemiluminescent compounds can emit light without stimulation by
an external a light source [1,2,3,4,5–8]. Excited chemiluminescence
(CL) species are produced by chemical reaction. Various chemilumi-
nescent compounds have been developed, such as acridinium ester,
luminol (5-Amino-2,3-dihydro-1,4-phthalazinedione), lophine (2,4,
5-triphenyl imidazole), and indole derivatives, and some of them have
been used as analytical reagents (Fig. 1). In particular, luminol de-
rivatives [9] and acridinium ester derivatives [10,11] are useful CL
compounds. These compounds emit strong light under alkaline condi-
tions, for example by the addition of an alkali such as sodium hydroxide
or potassium hydroxide.
e. the decrease in CL intensity at higher pH, it is anticipated that acri-
dinium esters that can be used for highly sensitive assays under both
neutral and alkaline conditions might be developed.
Aside from the substitution on the phenyl moeity, in the CL of acri-
dinium esters, substitution on the acridine moiety also affects the CL
reaction. For example, a weakly electron-donating group, such as a
methyl group, slowed the CL reaction [10]. Thermochemiluminescent
acridine-containing 1,2-dioxetanes were developed, and the introduc-
tion of methyl groups at the 2,7-positions on the acridine moiety caused
a decrease in the activation energy of the thermochemiluminescence
reaction [15]. Thus, we have focused on the introduction of substituents
on the acridine moiety to develop acridinium esters that have new CL
properties. By suitable substitution at the 2,7-positions on the acridine
moiety, the CL reactions and intensities under alkaline conditions might
be improved. To further clarify the role of the substituents, it is of in-
terest to study both electron-donating and electron-withdrawing groups.
In this study, we selected methyl or methoxy as the electron-donating
group and bromo as the electron-withdrawing group, and introduced
these groups at the 2,7-positions of the acridine moiety in 4-(methox-
Acridinium esters react with hydrogen peroxide to produce fluores-
cent 10-methyl-9(10 H)-acridone via a dioxetanone (Fig. 1) [12]. Acri-
dinium esters have relatively strong CL intensities among
chemiluminescent compounds, and we previously synthesized various
acridinium esters and evaluated their CL properties. The introduction of
electron-withdrawing groups (COOCH3, CN, NO2) to the 4-position of
the phenyl moiety intensified the CL of acridinium esters under neutral
conditions. In doing so, methoxycarbonylphenyl 10-methyl-10-
λ4-acridine-9-carboxylates that show strong CL intensities under neutral
conditions were identified and utilized for the measurement of hydrogen
peroxide and glucose under neutral conditions [13]. However, the CL
intensities decreased dramatically at pH 9ꢀ 10. In other work, various
functional groups at the 2,4-positions on the phenyl moiety in
ycarbonyl)phenyl
10-methyl-10λ4-acridine-9-carboxylate,
tri-
fluoromethanesulfonate salt or 3,4-(dimethoxycarbonyl)-phenyl 10-
methyl-10λ4-acridine-9-carboxylate, trifluoromethanesulfonate salt. We
synthesized these acridinium esters, clarified their CL behavior, and
applied them to the measurement of hydrogen peroxide concentrations
* Corresponding author.
E-mail address: nakazono@phar.kyushu-u.ac.jp (M. Nakazono).
https://doi.org/10.1016/j.jphotochem.2020.112851
Received 20 May 2020; Received in revised form 11 August 2020; Accepted 13 August 2020
Available online 24 August 2020
1010-6030/© 2020 Elsevier B.V. All rights reserved.

M. Nakazono et al.
Journal of Photochemistry & Photobiology, A: Chemistry 403 (2020) 112851
Fig. 1. Representative chemiluminescent compounds and their chemiluminescence species under alkaline conditions.
Scheme 1. Synthesis of 1aꢀ 5b.
2

M. Nakazono et al.
Journal of Photochemistry & Photobiology, A: Chemistry 403 (2020) 112851
under optimum CL conditions. During the course of these studies it was
revealed that 4-(Methoxycarbonyl)phenyl 10-methyl-10λ4-2,7-dime-
thylacridine-9-carboxylate gave relatively strong CL intensities at pH
7ꢀ 10. To gain further insights into the influence of the different sub-
stituents, the charge on the carbon at the 9-position on the acridine
moiety was calculated. The relationships between reaction time and
reaction products on the CL of the acridinium ester were investigated.
Hz, 2 H), 8.84 (d, J =10 Hz, 2 H). 13C NMR (125.7 MHz, DMSO-d6)
125.3, 129.1, 137.5, 140, 153.6, 159.4, 163.7, 165.9. HRFAB-MS (m/z)
calculated for C25H22NO6 [M]+ 432.1447, found 432.1449. Anal Calcd
for C26H22F3NO9S: C, 53.70; H, 3.81; N, 2.41 Found: C, 53.52; H, 3.81;
N, 2.34.
2.3.3. 4-(Methoxycarbonyl)phenyl 10-methyl-10λ4-2,7-dibromoacridine-
9-carboxylate, trifluoromethanesulfonate salt (4a)
2. Experimental
Yellow solid (10 mg, 14 % yield), m.p. 262ꢀ 265 ◦C. 1H NMR (500
MHz, Chloroform-d) 3.99 (s, 3 H), 5.17 (s, 3 H), 7.57 (d, J =9 Hz, 2 H),
8.28 (d, J =8.5 Hz, 2 H), 8.5 (m, 4 H), 8.76 (d, J =9.5 Hz, 2 H). 13C NMR
(125.7 MHz, DMSO-d6) 72.7, 112.5, 125.8, 128, 139.1, 154.2, 165.8,
169.8. HRFAB-MS (m/z) calculated for C23H16Br2NO4 [M]+ 527.9446,
found 527.9450. Anal Calcd for C24H16Br2F3NO7S: C, 42.44; H, 2.37; N,
2.06 Found: C, 42.15; H, 2.34; N, 1.99.
2.1. Chemicals
All reagents were obtained commercially and were used as received
without further purification. Compounds 1a, 1b, and PMAC were pre-
pared by a previously reported method [13]. 2,7,10-Trimethyl-9(10
H)-acridone was also synthesized as reported previously [16].
2.3.4. 4-(Methoxycarbonyl)phenyl 10-methyl-10λ4-2,7-diphenylacridine-
9-carboxylate, trifluoromethanesulfonate salt (5a)
2.2. Apparatus
Red solid (27 mg, 12 % yield), m.p. 254ꢀ 257 ◦C. 1H NMR (600 MHz,
Acetone-d6) 3.95 (s, 3 H), 5.36 (s, 3 H), 7.6 (m, 2 H), 7.64 (m, 4 H), 7.89
(d, J =8.4 Hz, 2 H), 8.03 (m, 4 H), 8.26 (dd, J = 1.8, 6.6 Hz, 2 H), 8.84 (s,
2 H), 8.99 (dd, J = 1.8, 9.6 Hz, 2 H), 9.2 (d, J =9.6 Hz, 2 H). 13C NMR
(150.9 MHz, DMSO-d6) 40.7, 52.9, 114.3, 121.4, 121.8, 122.7, 123.4,
124, 125.2, 126.4, 127.3, 127.9, 128.2, 129, 129.5, 130, 131.4, 131.8,
132.3, 137.1, 138.7, 140, 141.1, 141.7, 145.3, 153.7, 162.9, 165.8, 171.
HRFAB-MS (m/z) calculated for C35H26NO4 [M]+ 524.1862, found
524.1890. Anal Calcd for C36H26F3NO7S: C, 64.19; H, 3.89; N, 2.08
Found: C, 62.08; H, 3.98; N, 1.94.
The melting points were uncorrected. The 1H-NMR spectra were
recorded on 500 and 600 MHz spectrometers. The 13C-NMR spectra
were recorded on 125.7 and 150.9 MHz spectrometers. HRMS spectra
were obtained by fast atom bombardment. Chemiluminescence was
measured using a Lumat LB 9507 (Berthold, Bad Wildbad, Germany)
luminometer. The fluorescence and CL spectra were obtained using a FP-
6500 fluorometer (JASCO, Tokyo, Japan). A conventional reversed-
phase HPLC system was conducted using a JASCO (Tokyo, Japan) PU-
980 pump, a UV-970 detector, and a CAPCELL PAK C18 (5 μm, 250
mm x 4.6 mm i.d.) column. The HPLC conditions were as follows: mobile
phase, 75 % acetonitrile-H2O; flow rate, 0.83 mL/min; detection
wavelength, 280 nm.
2.3.5. 3,4-(Dimethoxycarbonyl)phenyl 10-methyl-10λ4-2,7-
dimethylacridine-9-carboxylate, trifluoromethanesulfonate salt (2b)
Yellow solid (0.20 g, 62 % yield), m.p. 274ꢀ 277 ◦C. 1H NMR (600
MHz, DMSO-d6) 2.74 (s, 6 H), 3.9 (s, 3 H), 3.91 (s, 3 H), 4.93 (s, 3 H),
8.05 (m, 2 H), 8.19 (d, J =2.4 Hz, 1 H), 8.34 (s, 2 H), 8.37 (dd, J = 1.8,
9.6 Hz, 2 H), 8.83 (d, J =9 Hz, 2 H). 13C NMR (150.9 MHz, DMSO-d6)
21.4, 53.4, 53.5, 120.1, 122.8, 123.1, 125.2, 125.4, 130, 131.5, 134.6,
140.6, 140.9, 141.6, 143.8 151.8, 163.3, 166.6, 167.1. HRFAB-MS (m/z)
calculated for C27H24NO6 [M]+ 458.1604, found 458.1605. Anal Calcd
for C28H24F3NO9S: C, 55.35; H, 3.98; N, 2.31 Found: C, 54.68; H, 3.96;
N, 2.25.
2.3. General procedure for the synthesis of 2aꢀ 5a and 2bꢀ 5b (Scheme
1)
To a solution of acridine-9-carbonyl chloride (0.1 g, 0.33 mmol) in
dry dichloromethane (5 mL) under nitrogen gas was added 4-dimethyla-
minopyridine (0.02 g, 0.16 mmol), the corresponding phenols (0.66
mmol), and triethylamine (0.4 mL). The mixture was stirred at ambient
temperature overnight. Chloroform (100 mL) and aqueous 10 % (w/v)
sodium chloride (100 mL) were added to the solution. The organic layer
was dried with anhydrous sodium sulfate. The filtrate was evaporated in
vacuo, and the residue was purified by column chromatography (silica
2.3.6. 3,4-(Dimethoxycarbonyl)phenyl 10-methyl-10λ4-2,7-
dimethoxyacridine-9-carboxylate, trifluoromethanesulfonate salt (3b)
Yellow solid (0.01 g, 4% yield), m.p. 228ꢀ 230 ◦C. 1H NMR (500
MHz, DMSO-d6) 3.87 (s, 6 H), 4.11 (s, 6 H), 4.93 (s, 3 H), 7.59 (d, J =3
Hz, 2 H), 8.02 (m, 2 H), 8.12 (m, 3 H), 8.83 (d, J =10.5 Hz, 2 H). 13C
NMR (125.7 MHz, DMSO-d6) 125.4, 130.3, 134.4, 137.5, 139.6, 151.9,
159.4, 163.5, 166.8, 166.9. HRFAB-MS (m/z) calculated for C27H24NO8
[M]+ 490.1502, found 490.1503. Anal Calcd for C28H24F3NO11S: C,
52.58; H, 3.78; N, 2.19 Found: C, 51.41; H, 3.89; N, 2.08.
gel, 40ꢀ 50 μm, chloroform-methanol or ethyl acetate-hexane solution)
to produce the desired acridine-9-carboxylates. The acridine-9-
carboxylates were N-methylated by methyl trifluoromethanesulfonate
in dichloromethane and were purified by column chromatography (sil-
ica gel, particle size 40ꢀ 50 μm, chloroform-methanol) to produce the
desired acridine-9-carboxylates, trifluoromethanesulfonate salt ().
2.3.1. 4-(Methoxycarbonyl)phenyl 10-methyl-10λ4-2,7-dimethylacridine-
9-carboxylate, trifluoromethanesulfonate salt (2a)
2.3.7. 3,4-(Dimethoxycarbonyl)phenyl 10-methyl-10λ4-2,7-
Yellow solid (0.17 g, 65 % yield), m.p. 284ꢀ 287 ◦C. 1H NMR (600
MHz, DMSO-d6) 2.75 (s, 6 H), 3.93 (s, 3 H), 4.93 (s, 3 H), 7.94 (m, 2 H),
8.21 (m, 2 H), 8.3 (s, 2 H), 8.37 (m, 2 H), 8.84 (d, J =8 Hz, 2 H). 13C NMR
(150.9 MHz, DMSO-d6) 21.4, 52.9, 120.1, 123, 123.1, 125.1, 129.1,
131.6, 140.6, 140.9, 141.6, 144.1, 153.4, 163.5, 165.8. HRFAB-MS (m/
z) calculated for C25H22NO4 [M]+ 400.1549, found 400.1549. Anal
Calcd for C26H22F3NO7S: C, 56.83; H, 4.04; N, 2.55 Found: C, 55.83; H,
4.04; N, 2.48.
dibromoacridine-9-carboxylate, trifluoromethanesulfonate salt (4b)
Yellow solid (50 mg, 14 % yield), m.p. 242ꢀ 245 ◦C. 1H NMR (500
MHz, DMSO-d6) 3.44 (s, 3 H), 3.78 (s, 6 H), 7.13 (m, 4 H), 7.54 (dd, J =
2.5, 9 Hz, 2 H), 7.75 (d, J =8 Hz, 1 H), 7.81 (d, J =2.5 Hz, 2 H). 13C NMR
(125.7 MHz, DMSO-d6) 72.7, 112.5, 125.7, 129.2, 134.1, 139.2, 152.5,
166.7, 166.8, 169.8. HRFAB-MS (m/z) calculated for C25H18Br2NO6
[M]+ 585.9501, found 585.9527. Anal Calcd for C26H18Br2F3NO9S: C,
42.36; H, 2.46; N, 1.90 Found: C, 41.47; H, 2.55; N, 1.79.
2.3.8. 3,4-(Dimethoxycarbonyl)phenyl 10-methyl-10λ4-2,7-
2.3.2. 4-(Methoxycarbonyl)phenyl 10-methyl-10λ4-2,7-
diphenylacridine-9-carboxylate, trifluoromethanesulfonate salt (5b)
Yellow solid (30 mg, 11 % yield), m.p. 250ꢀ 253 ◦C. 1H NMR (600
MHz, Acetone-d6) 3.93 (s, 6 H), 5.36 (s, 3 H), 7.6 (m, 6 H), 8.03 (m, 7 H),
8.89 (d, J =1.8 Hz, 2 H), 8.98 (dd, J = 1.8, 9.6 Hz, 2 H), 9.19 (d, J =9.6
dimethoxyacridine-9-carboxylate, trifluoromethanesulfonate salt (3a)
Orange solid (0.02 g, 9% yield), m.p. 250ꢀ 253 C. 1H NMR (500
◦
MHz, DMSO-d6) 3.91 (s, 3 H), 4.12 (s, 6 H), 4.93 (s, 3 H), 7.54 (d, J =3
Hz, 2 H), 7.86 (d, J =9 Hz, 2 H), 8.12 (dd, J = 3, 10 Hz, 2 H), 8.2 (d, J =9
3

M. Nakazono et al.
Journal of Photochemistry & Photobiology, A: Chemistry 403 (2020) 112851
Hz, 2 H). 13C NMR (150.9 MHz, DMSO-d6) 40.5, 53.5, 121.3, 122.7,
123.7, 124.1, 124.6, 125.3, 125.5, 126.4, 127.3, 128, 128.3, 129.4, 130,
130.3, 131.5, 132.4, 134.1, 137.2, 138.7, 140, 141, 141.7, 145, 152,
162.7, 166.8. HRFAB-MS (m/z) calculated for C37H28NO6 [M]+
582.1917, found 582.1918. Anal Calcd for C38H28F3NO9S: C, 62.38; H,
3.86; N, 1.91 Found: C, 62.19; H, 3.95; N, 1.89.
2.4. Chemiluminescence measurement of 1aꢀ 5b
To 50
added 100
100 mM Gly-NaOH for pH 9 and pH 10). After the mixture was allowed
μ
L of a 10 nM solution of 1aꢀ 5b in dimethyl sulfoxide was
μ
L of a buffer solution (100 mM Trisꢀ HCl for pH 7 and pH 8,
to stand for 20 s, the CL reaction was initiated by adding 100 μL of
0.01ꢀ 10 mM aqueous hydrogen peroxide solution to the luminometer
using an automatic injection system. The CL emission was measured for
1 min, and the integral photon counts were used to evaluate the CL
intensity.
2.5. Chemiluminescence measurement of luminol
To 50
added 100
was allowed to stand for 20 s, the CL reaction was initiated by adding
μ
L of a 10 nM solution of luminol in dimethyl sulfoxide was
μ
L of 0ꢀ 10 mM NaOH aqueous solution. After the mixture
100 μL of 1 mM aqueous hydrogen peroxide solution to the luminometer
using an automatic injection system. The CL emission was measured for
1 min, and the integral photon counts were used to evaluate the CL
intensity.
2.6. Chemiluminescence measurement of phenyl 10-methyl-10λ4-
acridine-9-carboxylate (PMAC)
To 50
added 100
100 mM Gly-NaOH for pH 9 and pH 10). After the mixture was allowed
μ
L of a 10 nM solution of PMAC in dimethyl sulfoxide was
μ
L of a buffer solution (100 mM Trisꢀ HCl for pH 7 and pH 8,
to stand for 20 s, the CL reaction was initiated by adding 100 μL of 1 mM
aqueous hydrogen peroxide solution to the luminometer using an
automatic injection system. The CL emission was measured for 1 min,
and the integral photon counts were used to evaluate the CL intensity.
2.7. Chemical stability of 1aꢀ 5b in dimethyl sulfoxide at 25◦C for 3h
A 10 nM dimethyl sulfoxide solution of 1aꢀ 5b was allowed to stand
Fig. 2. Time-courses of the chemiluminescence development of 1a¡3a at pH 7
and pH 10. (A) pH 7: (a) 1a; (b) 2a; (c) 3a. (B) pH 10: (d) 2a; (e) 1a. The
concentration of 1aꢀ 3a and hydrogen peroxide was 10 nM and 1 mM,
respectively.
at 25 ◦C for 3 h. Then, to 50
μL of the 10 nM dimethyl sulfoxide solution
of compounds 1aꢀ 5b was added 100
μL of a buffer solution (100 mM
Trisꢀ HCl, pH 8, 100 mM Gly-NaOH for pH 9). After the mixture was
Natural bond orbital (NBO) version 3 and Mulliken analyses were
employed to find the charge distribution of each compound. All ab initio
calculations were performed by Gaussian 16 [22].
allowed to stand for 20 s, the CL reaction was initiated by adding 100 μL
of 1 mM aqueous hydrogen peroxide solution to the luminometer using
an automatic injection system. The CL emission was measured for 1 min,
and the integral photon counts were used to evaluate the CL intensity.
2.10. Relationship between reaction time and reaction products on the
chemiluminescence of 1a and 2a at pH 7ꢀ 10
2.8. Standard curves of hydrogen peroxide using 1aꢀ 5b
To 50
added 100
100 mM Gly-NaOH for pH 9 and pH 10). The CL reaction was initiated
μ
L of a 0.1 mM solution of 1a and 2a in dimethyl sulfoxide was
To 50
added 100
Gly-NaOH for pH 9). After the mixture was allowed to stand for 20 s, the
μ
L of a 10 nM solution of 1aꢀ 5b in dimethyl sulfoxide was
μ
L of a buffer solution (100 mM Trisꢀ HCl for pH 7 and pH 8,
μ
L of a buffer solution (100 mM Trisꢀ HCl for pH 8, 100 mM
by adding 100 μL of 100 mM aqueous hydrogen peroxide solution. After
CL reaction was initiated by adding 100
μL of 0ꢀ 10 mM aqueous
the mixture was allowed to stand for 20ꢀ 600 s, a 20 μL portion of the
hydrogen peroxide solution to the luminometer using an automatic in-
jection system. The CL emission was measured for 1 min, and the inte-
gral photon counts were used to evaluate the CL intensity.
mixture solution was subjected to HPLC.
3. Results and discussion
2.9. Calculation of the charge on the carbon at the 9-position in the
acridine moiety of 1aꢀ 3a
3.1. Synthesis of 1aꢀ 5b
The calculations were performed by the complete active space self-
consistent field (CASSCF(4e,4o)), the density functional theory (DFT)-
based B3LYP [17,18], and M062X [19] in Dunning’s basis sets [20,21].
As shown in Scheme 1, isatin derivatives were prepared from 4,4′-
diphenylamine derivatives and oxalyl chloride in dichloromethane. The
isatins were converted to acridine-9-carboxylic acids in the presence of
4

M. Nakazono et al.
Journal of Photochemistry & Photobiology, A: Chemistry 403 (2020) 112851
Fig. 3. Chemiluminescence intensities of 1aꢀ 5b at pH 7ꢀ 10. The concentration of 1aꢀ 5b and hydrogen peroxide was 10 nM and 1 mM, respectively. The CL
emission was measured for 1 min, and the RLU integrated for 1 min were used to evaluate the CL intensity.
potassium hydroxide [23]. The esterification reaction of
Table 1
acridine-9-carbonyl chloride and phenols proceeded in the presence of
Comparison of the chemiluminescence intensities of 2a and luminol.
triethylamine, 4-dimethylaminopyridine and dichloromethane. Methyl
pH
CL intensity (RLU x1 min) of 2a
RCL intensity of 2a to luminola
trifluoromethanesulfonate was used for the N-methylation of acridine
moieties in dichloromethane. Notably, the yields (62 %, 65 %) of 2a and
2b were much higher than those of the other compounds.
7
130525
844730
954033
285931
1.6
11
8
9
12
10
3.6
3.2. Chemiluminescence measurement and stability of 1aꢀ 5b
a
The maximum chemiluminescence intensity of 10 nM luminol was obtained
in the concentration of 1 mM sodium hydroxide and hydrogen peroxide. The
chemiluminescence intensity (RLU x1 min) of luminol was 79244. RCL intensity
shows relative chemiluminescence intensity, and the chemiluminescence in-
tensity of luminol was taken as 1.
The time-courses of the CL development of 1aꢀ 3a at pH 7 and pH 10
are shown in Fig. 2. At pH 7, the CL of 1aꢀ 3a was long-lasting and 1a
exhibited the strongest CL intensity i.e. the maximum relative light units
(RLUs) of 2a and 3a, which were electron-donating groups introduced at
the 2,7-positions on the acridine moiety, were lower than that of 1a. The
time required to reach the maximum RLU was within approximately 30 s
after the addition of hydrogen peroxide. Interestingly, at pH 10 the RLU
of 2a was much greater than that of 1a and the maximum RLU of 2a was
maintained for 60 s. As will be described below, compound 2a gave
similar results at pH 9.
%, and 83 %, respectively (Fig. S1, Table S1). The introduction of
methoxy and bromo at the 2,7-positions in the acridine moiety
decreased the stabilities of acridinium esters.
Luminol has strong CL intensity under alkaline conditions and has
been known as a highly sensitive CL compound. To explore further, the
CL intensities of 2a (10 nM) and luminol (10 nM) were compared, and
those of 2a at pH 7ꢀ 10 were approximately 2- to 12-fold stronger than
that of luminol (Table 1, Fig. S2). Moreover, compared to the CL in-
tensities of phenyl 10-methyl-10λ4-acridine-9-carboxylate (Scheme 1,
R1ꢀ 3=H) at pH 7ꢀ 10, those of 2a were 6-, 9-, 2- and 0.3-fold (Table S2).
Fig. 3 shows the effect of pH on the CL development of 1aꢀ 5b. As
shown in Fig. 3, compounds 1a, 2a, 1b, and 2b had strong CL intensities
at pH 7 and pH 8. However, the CL intensities of 2a at pH 9 and pH 10
were 13- and 8-fold stronger, respectively, than that of 1a. Similarly, the
CL intensities of 2b at pH 9 and pH 10 were 12- and 6-fold stronger,
respectively, than that of 1b. Thus, apparent increases in CL intensities
at pH 9 and pH 10 were observed. The CL intensities of 3a and 3b were
highest at pH 9, indicating that the introduction of the methoxy
electron-donating group at the 2,7-positions on the acridine moiety
shifted the optimum pH to alkaline. However, although the highest CL
intensities were observed at pH 9 for 2a, 3a and 3b, the CL intensities of
3a and 3b were noticeably lower. In 3a and 3b, the CL reaction may
have been slowed by introducing a strongly electron-donating methoxy
group. Compounds 4a, 5a, 4b, and 5b exhibited low CL intensities at pH
9 and pH 10. The introduction of bromo and phenyl group at the 2,7-po-
sitions on the acridine moiety had not increased the CL intensities at
higher pH. The change of CL intensities of 2bꢀ 5b (3,4-(dimethox-
ycarbonyl)phenyl derivatives) was very similar to that of 2aꢀ 5a (4-
(methoxycarbonyl)phenyl derivatives) at pH 7ꢀ 10. Compared to the CL
intensities of 1a and 1b at pH 7 and pH 8, that of 2aꢀ 5a and 2bꢀ 5b at
pH 7 and pH 8 decreased, respectively.
3.3. Chemiluminescence assay of hydrogen peroxide
It is difficult to develop a highly sensitive assay of hydrogen peroxide
at pH 7ꢀ 10 using a CL compound. A relatively strong CL intensity at
each pH is needed to determine the hydrogen peroxide concentration. In
1aꢀ 5b, the hydrogen peroxide concentration that gave the maximum
CL intensity was determined at each pH (Fig. S3). Standard curves for
hydrogen peroxide at pH 7ꢀ 10 using 2a are shown in Fig. 4. The linear
calibration range of hydrogen peroxide was 10 ꢀ 1000 μM (regression
equation: y = 863,786 x + 28296, R = 0.997) at pH 8 using 2a. A long
range of detectability and satisfactory reproducibility using 2a were
obtained at pH 7ꢀ 10. On the other hand, 3a and 2b can be used to
measure hydrogen peroxide at pH 9 and pH 8ꢀ 9, respectively.
The detection limit (defined as the mean value of blank + 5
σ) of
hydrogen peroxide was 10 μM, 3 μM, 2 μM, and 5 μM at pH 7ꢀ 10,
The stabilities of 1aꢀ 5b (10 nM) in dimethyl sulfoxide (25 ◦C, 3 h)
were approximately 80 %, 76 %, 54 %, 59 %, 77 %, 87 %, 72 %, 49 %, 39
respectively. We note here that peroxalate CL (hydroxybenzyl alcohol-
incorporated copolyoxalate and rubrene) [24], luminol-hybrid
5

M. Nakazono et al.
Journal of Photochemistry & Photobiology, A: Chemistry 403 (2020) 112851
Fig. 4. Standard curves of hydrogen peroxide at pH 7ꢀ 10 using 2a. (a) pH 9; (b) pH 8; (c) pH 10; (d) pH 7. The concentration of 2a was 10 nM. The CL emission was
measured for 1 min, and the RLU integrated for 1 min were used to evaluate the CL intensity.
Mg-Alꢀ CO3 [25], CL resonance energy transfer (luminol and quantum
dot) [26], and adamantylidene-dioxetane [27] were previously devel-
oped for CL detection of hydrogen peroxide. The detection limits of
3.4. The charge on the carbon at the 9-position in the acridine moiety of
1aꢀ 3a
hydrogen peroxide were 0.1
μM, 0.1
μM, 0.3
μ
M, and 7.1 nM respec-
In the CL of acridinium esters, hydroperoxide anion (ꢀ OOH) reacts
with carbon at the 9-position in the acridine moiety. The carbon reac-
tivity at the 9-position in the acridine moiety of 1aꢀ 3a and hydroper-
oxide anion should be evaluated at natural bond orbital (NBO) version 3
and Mulliken population analyses. Since it would better for ionic sys-
tems to add diffuse functions to normal basis sets, augmented
correlation-consistent polarized valance double ζ-functions (AVDZ, aug-
cc-pVDZ) were employed. Complete active space self-consistent field
(CASSCF) calculation were furthermore performed on 4 valence-active
oribtals with 4 electrons (4e, 4o) to make sure of the property of MOs
obtained by density functional theory (DFT) calculations. It was how-
ever impossible to compute CASSCF(4e, 4o) at AVDZ basist set because
our computer-resources were not enough. But CASSCF results agree well
with DFT results at VDZ level. The positive charge on the carbon at the 9-
position in the acridine moiety of 1a was larger than that of 2a or 3a
(Table 2, Fig. S6). This was seen especially in the result for B3LYP/AVDZ
[16]. Thus, the introduction of electron-donating groups such as methyl
and methoxy groups at the 2,7-positions in the acridine moiety
decreased the carbon charge at the 9-position in the acridine moiety.
Therefore, the reactivity of 1a with hydroperoxide anion is higher than
tively using these CL methods. Thus, it is necessary to improve the
sensitivity of the acridinium esters investigated in the current work. The
stability of this CL method for the detection of hydrogen peroxide was
investigated. The relative standard deviation was 4.6 % (30 repeated
measurements) using this CL method (Fig. S5), and that was 2.1 % (85
repeated measurements) using previous CL method [26].
The CL spectra of 1a and 2a were measured at pH 7ꢀ 10 (Fig. S4).
The maxima wavelengths of CL of 1a and 2a were 430 nm and 444 nm,
respectively. The CL spectra of 1a and 2a were similar to the fluores-
cence emission spectra of 10-methyl-9(10 H)-acridone (10M9A) and
2,7,10-trimethyl-9(10 H)-acridone (2,7,10-TM9A), respectively.
Finally, the tolerance limits of coexistent substances were evaluated
[28]. The tolerance limits of K+, glucose, Mg2+, ascorbic acid, Fe3+ and
glutathione in the determination of 20 μM hydrogen peroxide at pH 7.4
using 2a were 200 mM, 20 mM, 10 mM, 0.5 mM, 0.25 mM, and 0.005
mM respectively, indicating that glutathione could influence the deter-
mination of hydrogen peroxide in cells.
Table 2
The electron population analysis of 1aꢀ 3a.
entry
1a
2a
3a
Analysis
NBO
Mulliken
NBO
Mulliken
NBO
Mulliken
Computational method
CASSCF(4e, 4o)/VDZ
B3LYP/VDZ
0.066
0.018
0.028
0.022
0.032
0.292
0.274
1.522
0.232
1.413
0.057
0.012
0.020
0.016
0.025
0.307
0.280
0.237
0.242
0.882
0.004
0.256
ꢀ 0.010
ꢀ 0.003
ꢀ 0.006
0.000
0.257
B3LYP/AVDZ
ꢀ 0.820
0.213
M062X/VDZ
M062X/AVDZ
ꢀ 0.124
6

M. Nakazono et al.
Journal of Photochemistry & Photobiology, A: Chemistry 403 (2020) 112851
Fig. 5. Possible chemiluminescence reaction of 1a and 2a.
that of 2a or 3a, and the CL reaction may be faster than that observed for
that the amount of 2,7,10-TM9A was smaller than that of 10M9A. After
M4HB was produced, the formation of 2,7,10-TM9A may be slower than
that of 10M9A. As shown in Fig. 3, the CL intensities of 1a decreased
apparently at pH 9 and pH 10. In contrast, the CL intensities of 2a
increased. The CL intensity of 2a at pH 9 was 13-fold stronger than that
of 1a (Fig. 3). The reason for this will be considered here based on the CL
of 1a and 2a at pH 9 and a 60 s reaction time. The amounts of M4HB
produced in the CL of 1a and 2a were approximately the same (Fig. 6.
(a), (c)). The generation efficiencies of 10M9A (from 1a) and 2,7,
10-TM9A (from 2a) were 0.61 and 0.4, respectively (Fig. 6 (b), (d)).
The possible CL reactions of 1a at pH 9 and pH 10 are shown in Fig. 7.
Two possible CL reactions can be speculated. Hydroxide reacts with the
carbon at 9-position on the acridine moiety at pH 9 and pH 10 (pseu-
dobase formation). The hydrolysis proceeds, and 10M9A is produced. Or
the pseudobase is formed, hydroperoxide anion reacts with the carbon of
carbonyl group in the phenyl ester, and 10M9A is produced (Fig. 7 (a)).
The formation of pseudobase initiates nonchemiluminescent pathway
[30,32,33,35]. Hydroperoxide anion reacts with the carbon at 9-posi-
tion on the acridine moiety at pH 9 and pH 10. The hydrolysis pro-
ceeds, and 10M9A is produced (Fig. 7 (b)). In contrast, the dioxetanone
could be mainly formed in the CL reaction of 2a at pH 9 and pH 10
(Fig. 5). Therefore, the CL intensity of 2a increases, which was observed
here (Fig. 2 and Fig. 3).
the other compounds.
3.5. Relationships between reaction time and reaction products on
chemiluminescence of 1a and 2a
The possible CL reactions of 1a and 2a are shown in Fig. 5. Hydrogen
peroxide reacts with the carbon at the 9-position on the acridine moiety,
and the dioxetane intermediate is formed. The dioxetane intermediate
forms a very unstable dioxetanone ring, splitting off M4HB. The dioxe-
tanone decomposes, yielding the acridone 10M9A and 2,7,10-TM9A
[29–35]. 10M9A and 2,7,10-TM9A are CL species. The products of the
CL reaction of 1a and 2a at pH 7ꢀ 10 were investigated using
reversed-phase HPLC (Fig. S7). On the CL reaction, the peaks of methyl
4-hydroxybenzoate (M4HB), 10M9A, and 2,7,10-TM9A were observed
at pH 7ꢀ 10. The main products of the CL reactions of 1a and 2a were
M4HB, 10M9A, and 2,7,10-TM9A. The relationships between reaction
time and reaction products on the CL of 1a and 2a are shown in Fig. 6.
When M4HB production increased, 10M9A and 2,7,10-TM9A produc-
tion also increased. M4HB, 10M9A, and 2,7,10-TM9A were produced
rapidly at pH 9 and pH 10. Hydroperoxide anion (HOOꢀ ) reacts spon-
taneously with the carbon at the 9-position on the acridine moiety under
alkaline conditions, and 1a and 2a could be converted to these com-
pounds. A comparison of the amounts of 10M9A and 2,7,10-TM9A
produced in the CL reactions of 1a and 2a (Fig. 6 (b), (d)) revealed
7

M. Nakazono et al.
Journal of Photochemistry & Photobiology, A: Chemistry 403 (2020) 112851
Fig. 6. Relationships between reaction time and reaction products on chemiluminescence of 1a and 2a at pH 7ꢀ 10. (a) M4HB, 1a; (b) 10M9A, 1a; (c) M4HB, 2a; (d)
2,7,10-TM9A, 2a. The concentration of 1a and 2a was 0.1 mM. The concentration of hydrogen peroxide was 100 mM.
Fig. 7. Possible chemiluminescence reaction of 1a at pH 9 and pH 10.
8

M. Nakazono et al.
Journal of Photochemistry & Photobiology, A: Chemistry 403 (2020) 112851
dipicolylaminomethyl groups: Interactions with zinc ion and ATP, Spectrochim.
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All the authors are acknowledged about the submission of this
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Declaration of Competing Interest
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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.
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atoms revisited. Systematic basis sets and wave functions, J. Chem. Phys. 96 (1992)
6796–6806.
This work was the result of the use of research equipment shared in a
MEXT Project for the promotion of the public utilization of advanced
research infrastructure(Program supporting the introduction of the new
sharing system), Grant Number JPMXS0422300120. We thank Prof.
Takashi Ito (Graduate school of medical sciences, Kyushu University)
and Prof. Satoshi Okada (Graduate school of medical sciences, Kyushu
University) for helpful discussion in measurement of chem-
iluminescence spectra.
[22] Gaussian 16., Revision C.01.01, M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E.
Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, G. A. Petersson, H.
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