Strongly Chemiluminescent Acridinium Esters under Neutral Conditions: Synthesis, Properties, Determination, and Theoretical Study

Article abstract of DOI:10.1021/acs.joc.6b02748

Various novel acridinium ester derivatives having phenyl and biphenyl moieties were synthesized, and their optimal chemiluminescence conditions were investigated. Several strongly chemiluminescent acridinium esters under neutral conditions were found, and then these derivatives were used to detect hydrogen peroxide and glucose. Acridinium esters having strong electron-withdrawing groups such as cyano, methoxycarbonyl, and nitro at the 4-position of the phenyl moiety in phenyl 10-methyl-10λ⁴-acridine-9-carboxylate trifluoromethanesulfonate salt showed strong chemiluminescence intensities. The chemiluminescence intensity of 3,4-dicyanophenyl 10-methyl-10λ⁴-acridine-9-carboxylate trifluoromethanesulfonate salt was approximately 100 times stronger than that of phenyl 10-methyl-10λ⁴-acridine-9-carboxylate trifluoromethanesulfonate salt at pH 7. The linear calibration ranges of hydrogen peroxide and glucose were 0.05-10 mM and 10-2000 μM using 3,4-(dimethoxycarbonyl)phenyl 10-methyl-10λ⁴-acridine-9-carboxylate trifluoromethanesulfonate salt at pH 7 and pH 7.5, respectively. The proposed chemiluminescence reaction mechanism of acridinium ester via a dioxetanone structure was evaluated via quantum chemical calculation on density functional theory. The proposed mechanism was composed of the nucleophilic addition reaction of hydroperoxide anion, dioxetanone ring formation, and nonadiabatic transition due to spin-orbit coupling around the transition state (TS) to the triplet state (T₁) following the decomposition pathway. The TS which appeared in the thermal decomposition would be a rate-determining step for all three processes.

Full text of DOI:10.1021/acs.joc.6b02748

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Article
Strongly Chemiluminescent Acridinium Esters under Neutral
Conditions: Synthesis, Property, Determination and Theoretical Study
Manabu Nakazono, Yuji Oshikawa, Mizuho Nakamura, Hidehiro Kubota, and Shinkoh Nanbu
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b02748 • Publication Date (Web): 07 Feb 2017
Downloaded from http://pubs.acs.org on February 8, 2017
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Strongly Chemiluminescent Acridinium Esters under Neutral
Conditions: Synthesis, Property, Determination and Theoretical
Study
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Manabu Nakazono,†,* Yuji Oshikawa,† Mizuho Nakamura,‡ Hidehiro Kubota§
and Shinkoh Nanbu‡
†Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku,
Fukuoka 812-8582, Japan
‡Faculty of Science and Technology, Sophia University, 7-1 Kioi-Cho, Chiyoda-ku, Tokyo 102-
8554, Japan
§ATTO Corporation, 3-2-2 Motoasakusa, Taito-ku, Tokyo 111-0041, Japan
*Corresponding author. Tel: +81-92-642-6597.
E-mail address: nakazono@phar.kyushu-u.ac.jp.
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Table of Contents
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ABSTRACT
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Various novel acridinium ester derivatives having phenyl and biphenyl moieties were
synthesized and their optimal chemiluminescence conditions were investigated. Several strongly
chemiluminescent acridinium esters under neutral conditions were found and then these
derivatives were used to detect hydrogen peroxide and glucose. Acridinium esters having strong
electron-withdrawing groups such as cyano, methoxycarbonyl and nitro groups at the 4-position
of the phenyl moiety in phenyl 10-methyl-104-acridine-9-carboxylate, trifluoromethane-
sulfonate salt showed strong chemiluminescence intensities. The chemiluminescence intensity
of 3,4-dicyanophenyl 10-methyl-104-acridine-9-carboxylate, trifluoromethanesulfonate salt was
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approximately 100 times stronger than that of phenyl 10-methyl-104-acridine-9-carboxylate,
trifluoromethanesulfonate salt at pH 7. The linear calibration ranges of hydrogen peroxide and
glucose were 0.0510 mM and 102000 M using 3,4-(dimethoxycarbonyl)phenyl 10-methyl-
104-acridine-9-carboxylate, trifluoromethanesulfonate salt at pH 7 and pH 7.5, respectively.
The proposed chemiluminescence reaction mechanism of acridinium ester via a dioxetanone
structure was evaluated via quantum chemical calculation on density functional theory. The
proposed mechanism was composed of the nucleophilic addition reaction of hydroperoxide anion,
the dioxetanone ring formation and nonadiabatic transition due to spin-orbit coupling around the
transition state (TS) to the triplet state (T1) following the decomposition pathway. The TS which
appeared in the thermal decomposition would be a rate-determining step for all three processes.
KEYWORDS: chemiluminescence, acridinium ester, hydrogen peroxide, glucose, neutral
condition
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INTRODUCTION
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Chemiluminescence (CL) assays have been utilized for determining various kinds of
compounds qualitatively and quantitatively.111 CL compounds have been developed as useful
luminescent reagents. In particular, luminol (3-aminophthalhydrazide) derivatives,1221
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acridinium ester derivatives2230 and dioxetane derivatives3137 are essential compounds in highly
sensitive CL assays. Recently, luminol was applied to CL imaging in vivo by CL resonance
energy transfer (CRET).38 Acridinium ester derivatives have been frequently selected in enzyme
immunoassays.3941 Intramolecular chemically initiated electron exchange luminescence
mechanism and CRET were applied to acridinium-substituted 1,2-dioxetanes.42 CL sensing of
fluoride ions using a self-immolation mechanism was developed based on dioxetane CL.43 The
CL property and mechanism of 2-coumaranones were clarified by experimental and theoretical
study.44
Acridinium esters react with hydrogen peroxide to produce fluorescent 10-methyl-9-acridone
via a dioxetanone structure (Scheme 1).45,46a Acridinium ester derivatives have relatively strong
CL intensity among CL compounds and various derivatives can be synthesized in a facile
manner. 10-Carboxymethylacridinium derivatives were synthesized, when methyl group (an
electron-donating group) was introduced at the 2 and 6-position of the phenyl moiety, strong CL
intensity was obtained.23 9-Acridine carboxylic esters of hydroxamic and sulphohydroxamic
acids had strong CL intensity at pico molar levels.25 Acridinium dimethylphenyl esters
containing N-sulfopropyl groups in the acridinium ring were highly sensitive CL compounds.39b
2',6'-dimethyl-4'-(N-succinimidyloxycarbonyl)phenyl-10-methyl-acridinium-9-carboxylate-1-
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Scheme 1. Chemiluminescence mechanism of acridinium esters.
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propanesulfonate inner salt was used for capillary CL determination of sympathomimetic
drugs.47 These CL compounds were dissolved in acetonitrile or dimethylformamide, the CL was
measured in sodium hydroxide and hydrogen peroxide solution. In general, most CL compounds
require strong alkaline conditions and the CL signals are short lasting. A little was studied about
acridinium esters having novel CL property under neutral conditions. Di-ortho-bromophenyl
acridinium ester generated CL signal under neutral conditions.48,49 If compounds exhibiting
strong CL under neutral conditions could be identified, it would be possible to develop novel and
useful CL assays. For example, in the CL measurement of an enzyme activity producing
hydrogen peroxide under neutral conditions, the enzyme activity could be measured without the
addition of alkali. Therefore, a method is needed to synthesize acridinium esters that can readily
react with hydrogen peroxide under neutral conditions. In a previous study, the introduction of
electron-withdrawing groups such as bromide to the 4-position of the phenyl moiety in phenyl
10-methyl-104-acridine-9-carboxylate, trifluoromethanesulfonate salt was shown to increase the
CL intensity of acridinium esters.46a Electron-withdrawing groups could polarize the electron
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density toward the phenyl ring and thereby reduce the electron density at the C9 position of the
acridine moiety. Hydroperoxide anion (HOO-) reacts with the C9 position in a facile manner and
light is produced.46a First, therefore, we decided to focus on cyano, trifluoromethyl,
methoxycarbonyl and nitro groups. We thus considered that the detailed effects of electron-
donating groups and electron-withdrawing groups on the 4-position of the phenyl moiety in
phenyl 10-methyl-104-acridine-9-carboxylate, trifluoromethanesulfonate salt should also be
evaluated. In a related experiment, we investigated the increase of CL intensity by the increase
of acridinium moieties. Second, the crystal structure of [1,1'-biphenyl]-4-yl 10-methyl-104-
acridine-9-carboxylate, trifluoromethanesulfonate salt was previously cleared and the two phenyl
rings of the biphenyl moiety was oriented at a dihedral angle of 42.9°.50 We predicted that the
twist structure of the biphenyl moiety would cause rapid cleavage of the biphenoxy moiety in the
dioxetane intermediate, and thereby increase the production of 10-methyl-9-acridone and the
level of CL intensity. Thus, various derivatives of [1,1'-biphenyl]-4-yl 10-methyl-104-acridine-
9-carboxylate, trifluoromethanesulfonate salt were synthesized.
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Useful CL methods for determining various analytes are developed by measurement of
enzymatically generated hydrogen peroxide. For example, glucose reacts with glucose oxidase
(EC1.1.3.4) in the presence of oxygen, and hydrogen peroxide is generated. The quantification
of glucose can be achieved by the reaction of hydrogen peroxide and CL compound. In this
study, various novel acridinium ester derivatives were designed and synthesized, and their CL
was measured. Finally, novel acridinium esters, which appear to have strong CL intensity under
neutral conditions, were developed and utilized for the CL determination of hydrogen peroxide
and glucose. Furthermore, a theoretical study was performed to clarify the charge distributions
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of C9 and N10 positions of acridine moiety and energy diagram on the proposed CL mechanism
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by ab initio chemical calculations.
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RESULTS and DISCUSSION
Synthesis of Compounds 126. Acridine-9-carbonyl chloride and biphenols were prepared
by previously described methods.51,52 The esterification reaction of acridine-9-carbonyl chloride
Table 1. Synthesis and chemical structure of compounds 117.
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and phenols proceeded promisingly in the presence of triethylamine, 4-dimethylaminopyridine
and tetrahydrofuran. Methyl trifluoromethanesulfonate was used for the N-methylation of
acridine moieties in dichloromethane (Table 1). As shown in Table 2, 4-iodophenol was
transformed by arylboronic acids through Suzuki-Miyaura coupling and compounds 1820 were
synthesized in a facile manner. In the synthesis of compound 26, 1,1':4',1''-terphenyl-4-ol was
prepared by 4-bromo-4'-hydroxyphenyl and phenylboronic acid in the presence of
tetrakis(triphenylphosphine)palladium, potassium carbonate and dimethoxyethane.53 In the
purification of compounds 126 by column chromatography, the crude products were excluded
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by chloroform-methanol solution (v/v, 20/1) and compounds 126 were eluted by using
chloroform-methanol solution (v/v, 5/1, 4/1 or 2/1).
Table 2. Synthesis and chemical structure of compounds 1826.
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Chemiluminescence Property of Compounds 126. Compounds 3, 4 and 913 had
particularly strong CL intensities at pH 7. The CL of compounds 3 and 1013 was long-lasting.
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Figure 1. (A) Time-course of the chemiluminescence development of compounds 1, 3, 9, 12, 13
and luminol in 100 mM Tris-HCl buffer at pH 7: (a) luminol (b) compound 1 (c) compound 9 (d)
compound 3 (e) compound 13 (f) compound 12. The concentration of compounds 1, 3, 9, 12, 13
and luminol was 10 nM. The concentration of hydrogen peroxide was 5 mM. (B)
Chemiluminescence of compound 3 (100 M) in 100 mM phosphate buffer at pH 7.
In compound 3, the time required to the maximum photon count was 15 s after the addition of
hydrogen peroxide (Figure 1A). In contrast, the CL intensities of compound 1 and luminol were
very weak at pH 7. Compared to the CL curves of compounds 3, 9, 12 and 13, increasing the
number of electron-withdrawing groups on the phenyl moiety caused the increase of CL intensity.
Compound 3 produced CL in phosphate buffer at pH 7 and blue light emission was observed
(Figure 1B). The effect of pH on the CL development of compounds 117 is shown in Figure 2.
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Novel acridinium esters that can produce strong CL were found under neutral conditions (pH 7
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and pH 8). Most of compounds 117 had maximum CL intensities at pH 8. However, the CL
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Figure 2. Effect of pH on the chemiluminescence development of compounds 117. To 10
nM solution of compounds 117 in dimethyl sulfoxide was added buffer solution (100 mM Tris-
HCl for pH 7 and pH 8, 100 mM Gly-NaOH for pH 9 and pH 10). The CL reaction was
initiated by adding 5mM aqueous hydrogen peroxide solution to the luminometer. The CL
emission was measured for 1 min and the integral photon counts were used to evaluate the CL
intensity.
intensities of compounds 1 and 5 increased under alkaline conditions (pH 9 and pH 10). In
compounds 217, the CL intensities appeared to be greater than that of compound 1 at pH 7
(Figure 3). The CL intensity of compound 12 was approximately 100 times stronger than that of
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compound 1 at pH 7. The introduction of strong electron-withdrawing groups such as cyano,
methoxycarbonyl and nitro groups at the 4-position of the phenyl moiety increased the CL
intensity and that of electron-donating groups such as methyl and methoxy groups decreased the
CL intensity. Quantum yields of acridinium esters are correlated with the pKa of the leaving
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Figure 3. Relative chemiluminescence intensity of compounds 117 at pH 7 and pH 8. The
concentration of compounds 117 was 10 nM. The concentration of hydrogen peroxide was 5
mM. Chemiluminescence was measured for 1 min. The CL intensity of compound 1 at each pH
7 and pH 8 was taken as 1.
group.54 The pKa values of phenols having electron-withdrawing groups at 4-position of phenol
moiety are 7.18.5.55 When electron-withdrawing groups to the 4-position of the phenyl moiety
are introduced, the pKa of the leaving group decreases.55 Based on the CL mechanism of
acridinium ester (Scheme 1), probably the phenyl moiety of compounds 3, 4 and 813 is readily
left and 10-methyl-9-acridone is produced. In 100 mM phosphate buffer (pH 7), the relative CL
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intensities of compounds 1, 3, 4, 8, 9, 10, 11 and 13 were similar to those in 100 mM Tris-HCl
(Figure S1 in supporting information). Acridinium esters exist in equilibrium with their
corresponding pseudo-bases (Figure 4) in aqueous solution.39a The pseudo-base is
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Figure 4. Equilibrium between acridinium esters and their pseudo-bases.
nonchemiluminescent compound. When the pseudo-base formation is suppressed, the CL
intensity may increase. The absorption spectra of compounds 1 and 3 were measured in Tris-
HCl solution (pH 7 and pH 8). The absorption maxima of pseudo-bases were observed at
approximately 280 nm56 and the apparent difference of these spectra was not observed (Figure
S4 in supporting information). In compound 3, the pseudo-base formation is not suppressed
under neutral conditions. Thus, pseudo-base formation could not be crucial factor in the strong
CL intensity of compound 3 under neutral conditions. In comparing the CL intensities of
compound 1 and compound 14, it appeared that the twist structure of the biphenyl group was not
the cause of the increase of CL intensity. The stabilities of compounds 1, 3, 4 and 1013 (10
nM) in dimethyl sulfoxide (25 °C, 3 h) were approximately 80%, 90%, 90%, 80%, 85%, 70%
and 94%. Most of compounds 1826 had maximum CL intensities at pH 9 (Figure S2 in
supporting information). The introduction of various functional groups to the biphenyl moiety
(Table 2) appeared to be reasonable for the change of CL intensity. Specifically, the introduction
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of a methoxycarbonyl (compound 22) and a nitro group (compound 25) at the 4-position of the
biphenyl moiety caused an increase of CL intensity (Figure S3 in supporting information). The
CL intensity of compound 25 at pH 8 was approximately 5 times stronger than that of compound
14. Unfortunately, the CL intensity of compound 26 did not increase (Table S1 in supporting
information). Taken together, these results demonstrate that the introduction of electron-
withdrawing groups to the 4-position of the phenyl and biphenyl moieties is crucial for
increasing the CL intensity of acridinium esters.
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Standard Curves for Hydrogen Peroxide at pH 7 and pH 8 using Compounds 3, 4 and 13.
Compounds 3, 4 and 13 having high chemical stability were used for determination of hydrogen
peroxide. The CL intensities of compounds 117 increased with the increase of hydrogen
peroxide concentration (5100 mM) at pH 7. Standard curves for hydrogen peroxide at pH 7
and pH 8 using compounds 3, 4 and 13 were shown in Figure 5. The linear calibration ranges of
hydrogen peroxide were 0.0525 mM (regression equation: y = 121812x + 68861, R=0.997) at
pH 7 and 0.055 mM (regression equation: y = 613613x + 126736, R=0.996) at pH 8 using
compound 3. The relative standard deviation was mostly within 4.8% (n=3). The detection
limits (S/N=2) of hydrogen peroxide were 50 M at pH 7 and pH 8, respectively. The linear
calibration ranges of hydrogen peroxide were 0.140 mM (regression equation: y = 33036x +
52387, R=0.997) at pH 7 and 0.055 mM (regression equation: y = 340474x + 41759, R=0.999)
at pH 8 using compound 4. The relative standard deviation was mostly within 5.8% (n=3). The
detection limits (S/N=2) of hydrogen peroxide were 100 M at pH 7 and 50 M at pH 8. The
linear calibration ranges of hydrogen peroxide were 0.0510 mM (regression equation: 188851x
+ 12236, R=0.999) at pH 7 and 0.0251 mM (regression equation: y = 1120300x + 21398,
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R=0.999) at pH 8 using compound 13, respectively. The relative standard deviation was mostly
within 4.3% (n=3). The detection limits (S/N=2) of hydrogen peroxide were 50 M at pH 7 and
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Figure 5. Standard curves of hydrogen peroxide in 100 mM Tris-HCl buffer at pH 7 and pH 8
using compounds 3, 4 and 13: (A) pH 7 (a) compound 13 (b) compound 3 (c) compound 4 (B)
pH 8 (d) compound 13 (e) compound 3 (f) compound 4. The concentration of compounds 3, 4
and 13 was 10 nM. Chemiluminescence was measured for 1 min.
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25 M at pH 8, respectively. A long range of detectability and satisfactory reproducibility for
the detection of hydrogen peroxide were obtained at pH 7 and pH 8. Similar standard curves
were obtained at pH 7 and pH 8 in 100 mM phosphate buffer (Figure S5 in supporting
information). In particular, the linear calibration ranges of hydrogen peroxide were 0.015 mM
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(R=0.993) at pH 7 and 51000 M (R=0.999) at pH 8 using compound 13, respectively. The
relative standard deviation was mostly within 5.4% (n=3). The detection limits (S/N=2) of
hydrogen peroxide were 10 M at pH 7 and 5 M pH 8, respectively. Compounds 3, 4 and 13
were thus shown to be suitable for the CL determination of hydrogen peroxide under neutral
conditions.
Standard Curves for Glucose using Compound 13. Compound 13 was very stable, used for
the determination of hydrogen peroxide and applied for highly sensitive detection of glucose
(Scheme 2). The linear calibration range of glucose was 102000 M (regression equation: y =
Scheme 2. Chemiluminescence determination of glucose using compound 13 at pH 7.5.
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175.93x + 8131.4, R=0.999) at pH 7.5 using compound 13 (Figure 6). The relative standard
deviation was mostly within 4.1% (n=3). The detection limit (defined as the mean value of blank
+ 5) of glucose was 5 M. This CL method of glucose showed similar sensitivity and linearity
compared to the previous CL methods such as 1-ethyl-3-methylimidazolium ethylsulfate/Cu2+-
luminol-glucose oxidase, luminol-H2O2-horseradish peroxide-glucose oxidase and the CuII-
catalyzed CL of lucigenin using synthetic imidazolium-based ionic liquid derivatives.5759 Thus,
this CL method can detect glucose at relatively high sensitivity without the addition of alkali and
catalyst.
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Figure 6. Standard curve of glucose at pH 7.5 using compound 13. The concentration of
compound 13 was 10 nM. Chemiluminescence was measured for 1 min.
Theoretical Analysis. The potential energy of the S0 state for compound 1 has been
theoretically explored in detail by Błażejowski et al.56,60; the energy diagrams on the electronic
ground state (S0) has been determined for 10-methyl-9-(phenoxycarbonyl)acridium cations
substituted with various alkyl groups in the phenyl fragment. However, the final products were
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assumed to be the electronic ground states for N-methylacridone (NMA) in their work. To make
acridone molecule emit a photon, the electronically excited states of NMA have to be found at
the end of this reaction in the proposed CL reaction mechanism by McCapra and Nelson.45,46a In
the present work, the energetics was mainly explored by density functional theory (DFT) based
B3-LYP functional at the correlation-consistent polarized-valence double-zeta (cc-pVDZ) basis
function61 for (i) the thermally accessible dioxetanone to reach an electronic excited state of
acridone molecule in S0, (ii) nonadiabatic transition through spin-orbit coupling between S0 and
T1, and (iii) the final decomposition to reach acridone in T1. The first triplet state (T1) is
electronically excited state from the viewpoint of singlet calculations based on the DFT approach,
but since our interest is in the electronically ground state in triplet states, the DFT approach for
the triplet ground state (T1) was assumed to be reasonable for searching the reaction-path, rather
than TDFT approach. Polarized continuum model was also employed to reproduce the solvent
effect caused by dimethyl sulfoxide (DMSO). Each energy listed in Figure 7 was confirmed by a
single-point calculation at symmetry adapted cluster / configuration interaction (SAC-CI)
method with using the optimized geometry by the DFT calculation employing the same PCM by
DMSO (Table S2 in supporting information).
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Since the mechanism of the nucleophilic addition of hydroperoxide anion was proposed by
McCapra and Nelson et al.45,46a and theoretically discussed by Błażejowski et al.,56,60 the charges
of acridinium ester were evaluated by natural bond orbital (NBO) analysis. The atomic charge of
N10 position of compound 1 shows the negative charge which is about -0.343 (-0.303) at B3-
LYP (M06-2X); the hydroperoxide anion could be repulsive to N10 side.
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Figure 7. The energy diagram on the chemiluminescence mechanism proposed by McCapra
and Nelson et al.45,46a (A) compound 1 (B) compound 3.
Figure 7A shows the energy diagram started from the dioxetanone ring-structure (min1) for
compound 1, which was a stable dioxetane intermediate found by Błażejowski et al.56,60 and then
the transition state (TS) search was performed with the initial coordinate given by this min1
structure, because the driving force for this reaction would assumed to be thermal decomposition.
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The TS structure appeared at TS1 to be close to the dioxetane intermediate (min1) as was
expected; the C-C bond distance in the dioxetane structure was slightly stretched, compared with
min1. And then the reaction path (IRC1) on the electronic ground state (S0) was obtained from
the intrinsic reaction coordinate (IRC) analysis with the TS structure (TS1). The reaction path
(IRC1) determined by the IRC-analysis from TS1 was found to be correlated to the interaction
structure between the S0 and lowest triplet (T1) states at int1 (Figure 8), which clearly shows the
triplet state lying closely to S0. Furthermore, the S1 state was much higher than T1 at int1 (Table
S2(a) in supporting information); the vertical excitation energy of S1 was 2.02 eV at the S0 –T1
closest point (int1) by SAC-CI calculation.
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As discussed in Ref. 62, the inter-system crossing (ISC, nonadiabatic transition in other
words) mechanism was explored due to the spin-orbit coupling between S0 and T1 states along
the reaction path of IRC1. The potential energy curve of the T1 state was found to be lying
closely to S0 at int1. Thus, the nonadiabatic transition between S0 and T1 could happen due to the
spin-orbit coupling effect at the vicinity to T1.63 If we here assumed the nonadiabatic transition
between S0 and T1, the molecule could be expected to hop to T1 and be dissociated into 10-
methyl-9-acridone and the fragment anion in T1; it follows that the IRC-analysis was performed
again on T1 with the int1 structure instead of IRC1. The obtained reaction path (IRC2) is found
to be correlated with the decomposition product including the triplet state of 10-methyl-9-
acridone, compared to IRC1; the phosphorescence could be anticipated by this process.
Furthermore, the transition probability due to the spin-orbit coupling was estimated by Zhu-
Nakamura formula;63 the spin-orbit coupling elements were computed by complete active space
(CAS) SCF calculation by ab initio program package Molpro 2012 at cc-pVDZ basis set.64
Using 8 cpu-cores of AMD Opteron 2382 and 8 GB ram (Tempest9D2 model, CONCURRENT
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SYSTEM ltd.) in Sophia University, it took about four months to obtain all results. However,
spin-orbit coupling elements were obtained by complete active space (CAS) SCF methods at cc-
pVDZ basis set. The active space was determined by (6e, 4o); the electron configurations of
CASSCF were totally 32,758. The probability was 96% at the int1 structure by the available
excess-energy proposed by Błażejowski et al.56,60 In general, the transition probability is
considered to be small for singlet–triplet transition, but there are several evidences discussed on
nonadiabatic transition probability between singlet and triplet states;65 the probabilities shown in
several molecular systems are actually almost unity for intersystem crossing (ISC).
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Figure 8. The intrinsic reaction coordinate determined with the initial molecular geometry of
TS1 (IRC1). The employed step-width was 0.05 Bohr around TS.
Finally, the phosphorescence might happen, because the 10-methyl-9-acridone staying in T1
could emit photon for deactivating the excited 10-methyl-9-acridone. The phosphorescence
process is discussed in the later paragraph.
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We also carried out the same theoretical analysis on the compound 3 as shown in Figure 7B;
the height of TS from min2 to TS2 was 0.05 eV, which was much smaller than the case of the
compound 1 (0.76 eV). The 4-cyanophenol moiety in compound 3, moreover, leaved along
IRC3 and the stable dioxetanone was produced at min3. The potential energy of S0 was found to
be lying closely to the triplet state (T1) at this min3. Since the S1 state was much higher than T1
(see Table S2(b)), the transition by ISC to the triplet state could be crucial on the CL of
compound 3.
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The potential energy of the S0 state for compound 1 has been theoretically explored by
Błażejowski,56,60 but the exploration of electronically excited states is still necessary for the
photoemission process of 10-methyl-9-acridone at the end of this reaction. It’s because the
electronically excited state (S1 or T1) of 10-methyl-9-acridone should appear as the thermal
reaction product through S0; there should be two possible paths by nonadiabatic transition to S1
or T1. If we are considering that the nonadiabatic transition of S0 - S1 will happen, the energy
separation (E) between these states should be less than 1.0 eV. Figure 7 and Table S2 however
suggest that this S0 - S1 transition would not occur because of E > 2.0 eV. On the other hand, if
the nonadiabatic transition of S0 - T1 will happen, it would happen because of E < 1.0 eV and
our estimated probability by 96% at the int1 structure, but phosphorescence process should be
explored at the end of the reaction. The acridinium 4-cyanophenyl ester of compound 3 should
be also explored in the same manner, because the reaction path on S0 was found to be quite
different from compound 1 as shown in Figure 7. As clearly seen in Table S2, the S1 state is
much higher than T1 at each molecular conformation except at products (i.e. 10-methyl-9-
acridone). Although we were not able to perform the molecular geometry-optimization and IRC-
calculation on each conformation at SAC-CI level, the obtained triplet states reasonably agree
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with the trend of DFT results, especially at the geometry of the S0 - T1 closest point at int1 and
min3. Here we are assuming the photoemission from the T1 state lying closely to the S1 state
(4.15 eV at SAC-CI) at the equilibrium geometry of 10-methyl-9-acridone, the expected
emission energy would be 2.43 eV at DFT (3.88 eV at SAC-CI). This emission energy from T1
quite agrees with the experimental spectrum which features the maximum speak at 430 nm (2.88
eV). However, since the transition dipole moment between S1 and S0 is 1.25 a0 at the equilibrium
conformation of 10-methyl-9-acridone, the acridone would produce luminescence on the way of
the final dissociation processes in IRC2 and IRC4, together with vibronic allowed transitions
described by the intensity borrowing mechanism (due to spin-orbit mixing) between these two
excited states (S1 and T1) lying closely to each other. The photoemission would happen in the
middle of these decomposition processes.
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On the other hand, the direct search for the minimum energy conical intersection (MECI)
between S0 and T1 for both compounds 1 and 3 were performed to make sure of our discussion;
we carried out the state-averaged CAS(6e,4o)SCF optimizations of a singlet-triplet conical
intersection in vacuo.64 The result is shown in Figure S6 (supporting information) for compound
1, which is similar to the int1 structure shown in Figure 7A. Concerning compound 3, we also
found the MECI structure at around the min3 structure shown in Figure 7B. Thus, the thermal
decomposition of acridanyl dioxetanes would happen along our proposal. It is furthermore found
that the solvent effect (DMSO) could not cause the different paths in compounds 1 and 3, albeit
indirectly. As a result of these factors and the energy difference between TS1 and TS2 (barrier-
height, E=0.76 and 0.05 eV), we could conclude that the p-cyanophenolate anion is more
stabilized than the phenolate anion in thermal decomposition processes, which leads to the
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difference between the paths for compounds 1 and 3. Compound 3 could show the stronger
fluorescence intensity than compound 1.
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Furthermore, the spin-orbit coupling was also evaluated at this CASSCF level with the MECI
structure for compound 1. The potential energies of S0 and T1 states were shifted due to the spin-
orbit coupling, as shown in Table S3 (supporting information); the shifted energy range is
approximately 80 cm-1. Since the value of atomic carbon is 43.4 cm-1,66 this shifted range is
obviously larger than the normal carbon atom. Probably the carbon atoms are strongly affected
by the oxygen atoms. Thus, our result could reasonably support the reaction pathway through
the triplet state (T1).
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CONCLUSION
Various acridinium ester derivatives were synthesized and the chemiluminescence intensities
were cleared. Acridinium esters that show strong chemiluminescence intensities under neutral
conditions were identified. The introduction of electron-withdrawing groups at the 4-position of
the phenyl and biphenyl moiety was very effective for increasing chemiluminescence intensity;
the effect would induce the polarizability not only at the specific positions in acridine moiety, but
also the strong polarizability as a whole molecule. 3,4-(Dimethoxycarbonyl)phenyl 10-methyl-
104-acridine-9-carboxylate, trifluoromethanesulfonate salt was useful compound for the
determination of hydrogen peroxide and glucose under neutral conditions.
The
chemiluminescence reaction mechanism has been proposed by McCapra and Nelson, et al.45,46a
The proposed mechanism was mainly evaluated via quantum chemical calculation on density
functional theory with the polarizable continuum model. The actual proposed mechanism was
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composed of the nucleophilic addition reaction of hydroperoxide anion, the highly strained
dioxetanone ring formation and the decomposition of the ring molecule. Natural bond orbital
(NBO) charge analysis shows that the atomic charge of C9 position in acridine moiety was
obviously positive than the other atoms, and was quite attractive to a nucleophilic reagent such as
hydroperoxide anion. The decomposition reaction would happen after the formation of the
dioxetane intermediate; there was a transition state (TS) on the way of this decomposition, and it
would be a rate-determining step in the whole process. Since the first triplet state (T1) was lying
closely to S0 at around TS, nonadiabatic transition of S0→T1 could happen, but the photoemission
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of the mixed state from T1 and S1 states of 10-methyl-9-acridone was expected due to the
intensity borrowing mechanism at the end of the reaction. This mechanism would be consistent
with the experimental result. This study should provide significant information toward the
development of novel chemiluminescent acridinium esters.
EXPERIMENTAL SECTION
General Information. Melting points were uncorrected. 1H and 13C NMR spectra were
recorded on a 500 MHz and 125.7 MHz spectrometers. HRMS spectra were obtained by
electrospray ionization time-of-flight (ESI-TOF) system or fast atom bombardment (FAB).
General Procedure for the Synthesis of Compounds 117. To a solution of acridine-9-
carbonyl chloride (0.12 g, 0.5 mmol) in dry tetrahydrofuran (10 mL) under nitrogen gas was
added 4-dimethylaminopyridine (0.01 g, 0.08 mmol), the corresponding phenols (0.75 mmol)
and triethylamine (2 mL). The mixture was refluxed for 2.5 h. Chloroform (150 mL) and water
(100 mL) were added to the solution. The organic layer was dried with anhydrous sodium
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sulfate. The filtrate was evaporated in vacuo, and the residue was purified by column
chromatography (silica gel, chloroform or ethyl acetate-hexane solution) to produce the desired
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acridine-9-carboxylates.
The acridine-9-carboxylates were N-methylated by methyl
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trifluoromethanesulfonate in dichloromethane, which were purified by column chromatography
(silica gel, chloroform-methanol) to produce the desired acridine-9-carboxylates,
trifluoromethanesulfonate salt.
Phenyl 10-methyl-104-acridine-9-carboxylate, trifluoromethanesulfonate salt (1). Yellow
solid (0.01 g, 13%); mp 233235 °C; 1H NMR (500 MHz, Acetone-d6): 5.25 (s, 3H), 7.48 (m,
1H), 7.63 (m, 2H), 7.69 (m, 2H), 8.26 (m, 2H), 8.64 (m, 2H), 8.75 (d, J = 8.5 Hz, 2H), 9.06 (d, J
13
= 9.5 Hz, 2H); C NMR (125.7 MHz, DMSO-d6):  119.9, 121.8, 122.2, 127.5, 129.8, 130.1,
139.1, 141.9, 146.8, 149.5, 163.5; HRMS ESI (m/z) calculated for C21H16NO2 [M]+ 314.1181,
found 314.1177.
p-Tolyl 10-methyl-104-acridine-9-carboxylate, trifluoromethanesulfonate salt (2). Yellow
1
solid (0.03 g, 14%); mp 232234 °C; H NMR (500 MHz, Acetone-d6):  2.43 (s, 3H), 5.24 (s,
3H), 7.43 (d, J = 8 Hz, 2H), 7.55 (d, J = 8.5 Hz, 2H), 8.25 (m, 2H), 8.64 (m, 2H), 8.72 (d, J = 8.5
Hz, 2H), 9.05 (d, J = 9.5 Hz, 2H); 13C NMR (125.7 MHz, DMSO-d6)  20.4, 119.9, 121.5, 122.2,
127.5, 129.7, 130.4, 136.9, 139.1, 141.9, 146.9, 147.3, 163.6; HRMS ESI (m/z) calculated for
C22H18NO2 [M]+ 328.1338, found 328.1361.
4-Cyanophenyl 10-methyl-104-acridine-9-carboxylate, trifluoromethanesulfonate salt
1
(3). Yellow solid (0.05 g, 23%); mp 278280 °C; H NMR (500 MHz, Acetone-d6):  5.25 (s,
3H), 7.98 (d, J = 8.5 Hz, 2H), 8.08 (d, J = 8.5 Hz, 2H), 8.25 (m, 2H), 8.64 (m, 2H), 8.78 (d, J =
8.5 Hz, 2H), 9.07 (d, J = 9 Hz, 2H); 13C NMR (125.7 MHz, DMSO-d6):  110.6, 118, 119.9,
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122.3, 123.5, 127.5, 129.8, 134.6, 139.2, 141.9, 145.9, 152.5, 162.8; HRMS ESI (m/z) calculated
for C22H15N2O2 [M]+ 339.1134; found 339.1139.
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4-(Trifluoromethyl)phenyl 10-methyl-104-acridine-9-carboxylate, trifluoromethane-
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sulfonate salt (4). Yellow solid (0.03 g, 13%); mp 252255 °C; H NMR (500 MHz, Acetone-
d6):  5.26 (s, 3H), 7.98 (m, 4H), 8.26 (m, 2H), 8.65 (m, 2H), 8.79 (d, J = 8.5 Hz, 2H), 9.07 (d, J
= 9.5 Hz, 2H); 13C NMR (125.7 MHz, DMSO-d6):  119.9, 121.9, 122.3, 122.7, 123.1, 124.9,
127.1, 127.5, 128, 128.3, 129.2, 129.8, 139.2, 141.9, 146.1, 152.2, 163; HRMS ESI (m/z)
calculated for C22H15F3NO2 [M]+ 382.1055; found 382.1055.
4-Methoxyphenyl 10-methyl-104-acridine-9-carboxylate, trifluoromethanesulfonate salt
1
(5). Yellow solid (0.04 g, 25%); mp 265268 °C; H NMR (500 MHz, Acetone-d6)  3.88 (s,
3H), 5.24 (s, 3H), 7.14 (d, J = 9 Hz, 2H), 7.6 (d, J = 9.5 Hz, 2H), 8.25 (m, 2H), 8.63 (m, 2H),
8.71 (d, J = 8.5 Hz, 2H), 9.05 (d, J = 9 Hz, 2H); 13C NMR (125.7 MHz, DMSO-d6)  55.7, 114.9,
119.4, 119.8, 121.9, 122.2, 122.8, 127.5, 129.7, 139.1, 141.9, 142.8, 147, 158, 163.8; HRMS
ESI (m/z) calculated for C22H18NO3 [M]+ 344.1287, found 344.1294.
4-Bromophenyl 10-methyl-104-acridine-9-carboxylate, trifluoromethanesulfonate salt
1
(6). Yellow solid (0.11 g, 63%); mp 248250 °C; H NMR (500 MHz, Acetone-d6)  5.24 (s,
3H), 7.7 (d, J = 9 Hz, 2H), 7.8 (d, J = 9 Hz, 2H), 8.24 (t, J = 8 Hz, 2H), 8.63 (m, 2H), 8.74 (d, J
= 9 Hz, 2H), 9.05 (d, J = 9.5 Hz, 2H); 13C NMR (125.7 MHz, DMSO-d6)  119.9, 120, 122.3,
124.2, 127.5, 129.7, 132.9, 139.1, 141.9, 146.4, 148.6, 163.2; HRMS ESI (m/z) calculated for
C21H15BrNO2 [M]+ 392.0286, found 392.0290.
4-Iodophenyl 10-methyl-104-acridine-9-carboxylate, trifluoromethanesulfonate salt (7).
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Yellow solid (0.05 g, 26%); mp 267270 °C; H NMR (500 MHz, Acetone-d6):  5.25 (s, 3H),
7.56 (d, J = 9 Hz, 2H), 7.99 (d, J = 8.5 Hz, 2H), 8.24 (t, J = 7 Hz, 2H), 8.64 (m, 2H), 8.74 (d, J =
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9 Hz, 2H), 9.05 (d, J = 9.5 Hz, 2H); 13C NMR (125.7 MHz, DMSO-d6):  92.7, 119.9, 122.3,
124.3, 127.5, 129.7, 138.8, 139.2, 141.9, 146.4, 149.3, 163.2; HRMS ESI (m/z) calculated for
C21H15INO2 [M]+ 440.0147; found 440.0140.
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4-Formylphenyl 10-methyl-104-acridine-9-carboxylate, trifluoromethanesulfonate salt
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(8). Yellow solid (0.14 g, 57%); mp 255258 °C; H NMR (500 MHz, Acetone-d6):  5.26 (s,
3H), 7.96 (d, J = 8.5 Hz, 2H), 8.2 (d, J = 6.5 Hz, 2H), 8.26 (m, 2H), 8.65 (m, 2H), 8.79 (d, J = 9
Hz, 2H), 9.07 (d, J = 9.5 Hz, 2H), 10.15 (s, 1H); 13C NMR (125.7 MHz,DMSO-d6):  119.9,
122.3, 122.9, 127.5, 129.8, 131.4, 135.2, 139.2, 141.9, 146.1, 153.6, 163, 192.1; HRMS ESI
(m/z) calculated for C22H16NO3 [M]+ 342.1130; found 342.1126.
4-(Methoxycarbonyl)phenyl 10-methyl-104-acridine-9-carboxylate, trifluoromethane-
1
sulfonate salt (9). Yellow solid (0.07 g, 30%); mp 250253 °C; H NMR (500 MHz, Acetone-
d6):  3.96 (s, 3H), 5.25 (s, 3H), 7.88 (d, J = 8.5 Hz, 2H), 8.25 (m, 4H), 8.65 (m, 2H), 8.77 (d, J =
9 Hz, 2H), 9.07 (d, J = 9.5 Hz, 2H); 13C NMR (125.7 MHz, DMSO-d6):  52.4, 119.9, 122.3,
122.4, 127.5, 128.8, 129.8, 131.2, 139.2, 141.9, 146.2, 152.9, 163, 165.3; HRMS ESI (m/z)
calculated for C23H18NO4 [M]+ 372.1236; found 372.1222.
4-(Ethoxycarbonyl)phenyl 10-methyl-104-acridine-9-carboxylate, trifluoromethane-
1
sulfonate salt (10). Yellow solid (0.01 g, 4%); mp 258260 °C; H NMR (500 MHz, Acetone-
d6):  1.38 (t, J = 7 Hz, 3H), 4.39 (q, J = 7 Hz, 2H), 5.26 (s, 3H), 7.86 (d, J = 9 Hz, 2H), 8.25 (m,
4H), 8.65 (t, J = 8 Hz, 2H), 8.77 (d, J = 8.5 Hz, 2H), 9.07 (d, J = 10 Hz, 2H); 13C NMR (125.7
MHz, DMSO-d6):  14.1, 61.1, 119.9, 122.3, 122.4, 127.5, 129.1, 129.8, 131.1, 139.2, 141.9,
146.2, 152.8, 163, 164.8; HRMS ESI (m/z) calculated for C24H20NO4 [M]+ 386.1392; found
386.1392.
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4-Nitrophenyl 10-methyl-104-acridine-9-carboxylate, trifluoromethanesulfonate salt (11).
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6
7
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Yellow solid (0.07 g, 46%); mp 232235 °C; H NMR (500 MHz, Acetone-d6):  5.26 (s, 3H),
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8.05 (d, J = 9 Hz, 2H), 8.26 (m, 2H), 8.5 (d, J = 9 Hz, 2H), 8.65 (m, 2H), 8.8 (d, J = 9 Hz, 2H),
9.07 (d, J = 9.5 Hz, 2H); 13C NMR (125.7 MHz, DMSO-d6)  119.9, 122.3, 123.5, 125.7, 127.5,
129.8, 139.2, 141.9, 145.8, 146.3, 153.8, 162.7. HRMS ESI (m/z) calculated for C21H15N2O4
[M]+ 359.1032, found 359.1052.
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3,4-Dicyanophenyl 10-methyl-104-acridine-9-carboxy-late, trifluoromethanesulfonate
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salt (12). Ocherous solid (0.03 g, 12%); mp 252254 °C; H NMR (500 MHz, Acetone-d6): 
5.26 (s, 3H), 8.24 (m, 2H), 8.38 (s, 2H), 8.57 (t, J = 1.5 Hz, 1H), 8.65 (m, 2H), 8.84 (d, J = 8 Hz,
2H), 9.07 (d, J = 9.5 Hz, 2H); 13C NMR (125.7 MHz, DMSO-d6): =113, 113.9, 115, 115.3,
116.7, 119.8, 122.4, 127, 127.7, 128.1, 128.3, 129.3, 129.7, 136.2, 139.2, 141.9, 145.2, 152,
162.4; HRMS FAB (m/z) calculated for C23H14N3O2 [M]+ 364.1086; found 364.1090.
3,4-(Dimethoxycarbonyl)phenyl
10-methyl-104-acridine-9-carboxylate,
trifluoro-
1
methanesulfonate salt (13). Yellow solid (0.03 g, 13%); mp 268271 °C; H NMR (500 MHz,
Acetone-d6):  3.93 (s, 6H), 5.27 (s, 3H), 8.04 (m, 3H), 8.25 (m, 2H), 8.66 (m, 2H), 8.83 (d, J =
8.5 Hz, 2H), 9.08 (d, J = 9.5 Hz, 2H); 13C NMR (125.7 MHz, DMSO-d6):  52.9, 113, 119.8,
122.3, 125, 126.9, 127.7, 129.6, 129.8, 134.2, 139.2, 141.9, 145.9, 151.2, 162.9, 166.1, 166.5;
HRMS FAB (m/z) calculated for C25H20NO6 [M]+ 430.1291; found 430.1274.
[1,1'-Biphenyl]-4-yl 10-methyl-104-acridine-9-carboxy-late, trifluoromethanesulfonate
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salt (14). Yellow solid (0.06 g, 23%); mp 250253 °C; H NMR (500 MHz, DMSO-d6):  4.96
(s, 3H), 7.42 (t, J = 7 Hz, 1H), 7.51 (t, J = 8 Hz, 2H), 7.76 (d, J = 7.5 Hz, 2H), 7.85 (d, J = 9 Hz,
2H), 7.91 (d, J = 9 Hz, 2H), 8.18 (t, J = 7 Hz, 2H), 8.55 (t, J = 8 Hz, 2H), 8.66 (d, J = 8.5 Hz,
2H), 8.94 (d, J = 9 Hz, 2H); 13C NMR (125.7 MHz, DMSO-d6):  119.9, 122.3, 126.9, 127.5,
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127.8, 128.3, 129, 129.8, 139, 139.2, 139.5, 141.9, 146.7, 148.9, 163.5; HRMS ESI (m/z)
calculated for C27H20NO2 [M]+ 390.1494; found 390.1514.
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Naphthalen-1-yl 10-methyl-104-acridine-9-carboxylate, trifluoromethanesulfonate salt
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(15). Yellow solid (0.04 g, 50%); mp 245248 °C; H NMR (500 MHz, Acetone-d6):  5.29 (s,
3H), 7.62 (m, 2H), 7.74 (t, J = 8 Hz, 1H), 8.01 (d, J = 8.5 Hz, 1H), 8.06 (d, J = 8.5 Hz, 1H), 8.11
(d, J = 8 Hz, 2H), 8.31(m, 2H), 8.68 (m, 2H), 8.84 (d, J = 8 Hz, 2H), 9.1 (d, J = 9.5 Hz, 2H); 13C
NMR (125.7 MHz, DMSO-d6):  118.9, 120, 122.4, 125.4, 125.7, 127.1, 127.2, 127.3, 127.6,
128.3, 129.9, 134.3, 139.2, 141.9, 144.9, 146.6, 163.4; HRMS ESI (m/z) calculated for
C25H18NO2 [M]+ 364.1338; found 364.1342.
2-Oxo-2H-chromen-4-yl
10-methyl-104-acridine-9-carboxylate,
trifluoromethane-
1
sulfonate salt (16). Yellow solid (0.01 g, 4%); mp 238241 °C; H NMR (500 MHz, Acetone-
d6):  3.63 (s, 3H), 6.29 (s, 1H), 6.78 (m, 1H), 7.1 (m, 3H), 7.28 (d, J = 8.5 Hz, 1H), 7.33 (d, J =
9 Hz, 2H), 7.48 (m, 2H), 7.56 (m, 1H), 7.6 (m, 2H); 13C NMR (125.7 MHz, DMSO-d6):  91,
115.7, 116.3, 117.3, 119.6, 120.7, 121.5, 123.2, 123.9, 128, 128.9, 132.7, 138.9, 140.8, 141.8,
151.3, 153.5, 161.8, 165.6, 166.3; HRMS ESI (m/z) calculated for C24H16NO4 [M]+ 382.1079;
found 382.1088.
(3-Oxo-1,3-dihydroisobenzofuran-1,1-diyl)bis(4,1-phenylene)
bis(10-methyl-104-
acridine-9-carboxylate), ditrifluoromethanesulfonate salt (17). Yellow solid (0.13 g, 21%);
1
mp 280283 °C; H NMR (500 MHz, DMSO-d6):  4.95 (s, 6H), 7.60 (d, J = 9 Hz, 1H), 7.66
(m, 3H), 7.73 (d, J = 9 Hz, 1H), 7.77 (m, 2H), 7.84 (m, 3H), 7.96 (m, 2H), 8.03 (m, 1H), 8.13 (m,
4H), 8.26 (d, J = 9 Hz, 1H), 8.29 (d, J = 9 Hz, 1H), 8.53 (m, 3H), 8.61 (d, J = 8.5 Hz, 3H), 8.93
(d, J = 9.5 Hz, 3H); 13C NMR (125.7 MHz, DMSO-d6): =89.9, 119.4, 119.9, 121.4, 122, 122.3,
122.4, 122.5, 124.3, 124.7, 124.8, 126, 127.4, 128.4, 128.5, 129.1, 129.8, 130.5, 131.5, 135.5,
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