Dioxetane formation and chemiluminescent emission upon the combination of a vinylphenol derivative with naphthalene endoperoxide

Article abstract of DOI:10.1039/c6ra28079j

We designed a chemiluminescent system using nanoparticles. We prepared mesoporous nanoparticles bearing naphthalene endoperoxide as a singlet oxygen generator in the pore and vinyl phenol derivative as an emission unit. The combination of these functional molecules gave highly efficient and bright chemiluminescence through the formation of a 1,2-dioxetane intermediate.

Full text of DOI:10.1039/c6ra28079j

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Dioxetane formation and chemiluminescent
emission upon the combination of a vinylphenol
Cite this: RSC Adv., 2017, 7, 9472
derivative with naphthalene endoperoxide†
a
a
a
b
*
*
Yui Umehara, Aoi Son, Teruyuki Kondo and Kazuhito Tanabe
Received 12th December 2016
Accepted 25th January 2017
We designed a chemiluminescent system using nanoparticles. We prepared mesoporous nanoparticles
bearing naphthalene endoperoxide as a singlet oxygen generator in the pore and vinyl phenol derivative
as an emission unit. The combination of these functional molecules gave highly efficient and bright
chemiluminescence through the formation of a 1,2-dioxetane intermediate.
DOI: 10.1039/c6ra28079j
rsc.li/rsc-advances
Chemiluminescent technology offers considerable potential for the emission of chemiluminescence without any additives or
biological imaging, and various systems have been developed to other triggers. Furthermore, we prepared mesoporous nano-
characterize biological functions and properties.1,2 In partic- particles bearing NEP within their pores (MSN-NEP) and
ular, the combination of luciferase and luciferin, which conrmed that these particles induced the oxidation of VPA and
produces red emission through the consumption of ATP, allows led to chemiluminescence. The present system is a promising
biological processes to be visualized in living cells and approach to chemiluminescence-based bio-imaging without the
animals.3–12 However, the practical use of conventional chemi- need for either gene or cellular manipulation.
luminescent techniques has been limited because they have
Initially, we prepared VPA and characterized its properties as
several drawbacks, such as requirements for complex gene an emission unit, because it is known to be a representative
technology and cell manipulation to generate specic enzymes precursor of a chemiluminescent emission unit.13 On the other
in the subjects, resulting in relatively low versatility. On the hand, NEP, as a donor of 1O2, was prepared from 1,4-dime-
other hand, small-molecule-based chemiluminescent systems thylnaphthalene by the photosensitization of methylene blue.
that are based on distorted dioxetane derivatives have been To evaluate the chemiluminescence of VPA, we combined VPA
developed.13–15 However, their suitability for biological imaging and NEP and monitored the emission. As shown in Fig. 2A, we
is also limited because of the instability of the molecules and observed chemiluminescent emission at around 450 nm that
the difficulties involved in further modication of the core was typical of oxidized VPA, indicating that VPA was sponta-
structure. Therefore, much effort has recently been made to neously converted into the corresponding dioxetane derivative
identify simpler chemiluminescent systems.
in the presence of 1O2 generated from the thermal degradation
Herein, we report a novel chemiluminescent system in which of NEP. The luminescence of VPA increased as the pH was
dioxetane derivatives are formed in situ. We developed this increased, suggesting that deprotonation of the hydroxyl group
system using a combination of naphthalene endoperoxide on the aromatic unit is a key for the emission of VPA, which is
(NEP), which generates singlet oxygen (1O2) through thermol- consistent with previous reports.13 We also conrmed that the
ysis, and vinylphenols bearing an adamantyl group (VPA) as an emission of VPA in the presence of NEP could be seen with the
emission unit. The latter is converted into dioxetane derivatives naked eye, as shown in Fig. 2B.
1
through the [2 + 2] cycloaddition of O2 (Fig. 1). Mixing of VPA
To evaluate this reaction in detail, we next analyzed the
with NEP leads to the spontaneous formation of dioxetane and products obtained from [2 + 2] cycloaddition of VPA and 1O2
generated from NEP by means of GC-MS. Although the forma-
tion of dioxetane derivative of VPA was not detected, the
measurement of MS spectra indicated the formation of methyl
3-hydroxybenzoate (molecular weight: 152), which was formed
aDepartment of Energy and Hydrocarbon Chemistry, Graduate School of Engineering,
Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan. E-mail: teruyuki@scl.
kyoto-u.ac.jp; Fax: +81-75-383-2504; Tel: +81-75-383-7055
bDepartment of Chemistry and Biological Science, College of Science and Engineering, by the degradation of dioxetane derivative (Fig. S1†). This result
Aoyama Gakuin University, 5-10-1 Fuchinobe, Chuo-ku, Sagamihara, 252-5258, Japan.
strongly supports the occurrence of cycloaddition to form the
E-mail: tanabe.kazuhito@chem.aoyama.ac.jp; Fax: +81-42-759-6493; Tel: +81-42-
dioxetane derivative.
759-6229
It is well known that 1O2 is a strong oxidant that can act on
† Electronic supplementary information (ESI) available: Detail of the
a huge variety of compounds including organic molecules,
experimental procedure and MS spectra of VPA, absorption spectra of MSN-NEP
polymers as well as biomolecules. Thus, control of its activity is
and HPLC prole of VPA before and aer the reaction. See DOI:
10.1039/c6ra28079j
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Fig. 1 Schematic outline of chemiluminescence system using VPA and thermal 1O2 generation from NEP.
Therefore, we designed a system in which NEP was incorporated
within the pores of mesoporous silica nanoparticles (MSN-
NEP). The synthesis of MSN-NEP is outlined in Scheme 1.
Acid 1 was coupled with an ethylenediamine derivative to form
amide 2. Removal of the Boc group in 2 under acidic conditions
gave naphthalene derivative 3 possessing a primary amino
group. Coupling of MSN bearing carboxylic acid groups (MSN-
CO2H) with 3 formed nanoparticles possessing naphthalene
units (MSN-NA). The photooxidation of MSN-NA in the presence
of methylene blue as a photochemical generator of 1O2 fur-
nished MSN-NEP. The formation of endoperoxide at the naph-
thalene unit was conrmed by measurement of the absorption
spectrum. As shown in Fig. 3A, the absorption at around 300 nm
in MSN-NA, which was assigned to absorption by the naph-
thalene unit, decreased as the photooxidation time increased,
which indicated that NEP was formed in pores. In order to
identify the thermal generation of singlet oxygen (1O2) from
MSN-NEP, we measured the phosphorescence of 1O2 formed
from MSN-NEP. We conducted photo-irradiation of MSN-NEP to
generate heat for the formation of 1O2 and measured the
Fig. 2 (A) Chemiluminescent spectra of VPA (1.5 mM) in the presence
of NEP (20 mM). The spectra were recorded under various pH
conditions. (B) Visualization of chemiluminescence emission of VPA in
the presence of NEP (pH 11.0).
critical for the practical use of the present system in bio-
imaging. It has been proposed that 1O2 species generated in
the pores of nanoparticles are conned within the pores and
inactivated before leaking out because of their short lifetime.16
1
emission of O2. We observed the emission at 1270 nm, which
1
was identied as the phosphorescence of O2. In addition, we
Scheme 1 Synthesis of MSN-NEP. Reagents and conditions. (a) EDCI, HOBt, DMF, 74%; (b) HCl, 15%; (c) 3, EDCI, HOBt, DMF; (d) hn (665 nm),
methylene blue.
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Fig. 3 (A) Absorption spectra of MSN-NA before or after oxidation by 1O2. MSN-NA (1 mg mLÀ1) was photoirradiated at 665 nm for 0 (solid line) or
30 min (dotted line) in the presence of methylene blue (100 mM). (B) Time dependence of phosphorescence emission (1270 nm) of 1O2 generated
from MSN-NEP (black) or NEP (red). (C) Chemiluminescent spectra of VPA (1.5 mM) in the presence of MSN-NEP (1 mg mLÀ1). After the mixture of
VPA and MSN-NEP was left to stand for 90 min, the spectra were recorded under basic conditions (pH 11.0).
compared the lifetime of 1O2 and conrmed that 1O2 generated
from MSN-NEP showed longer lifetime (9.1 ms) than that from
Acknowledgements
NEP (4.8 ms) (Fig. 3B). These results and the evidence that the
lifetime of 1O2 is affected by their surrounding environment
such as solvents17 led the conclusion that MSN-NEP thermally
generated 1O2 in their pores. We also conrmed that heating of
MSN-NEP at 40 ꢀC for 5 min resulted in an increase in
absorption at around 300 nm due to the release of 1O2 (Fig. S2†).
The number of naphthalene units incorporated on the surface
of MSN was estimated to be 136 nmol mgÀ1, which was calcu-
lated from the absorption at 290 nm.
We sincerely thank Professor Tadashi Suzuki (Aoyama Gakuin
University) for the measurement of emission and lifetime of
singlet oxygen. This work is partly supported by the Innovative
Techno-Hub for Integrated Medical Bio-imaging Project of the
Special Coordination Funds for Promoting Science and Tech-
nology, and by Grant-in-Aid for Scientic Research (for K. T.
Grant number 23113508, for A. S. Grant number 16K12876),
from the Ministry of Education, Culture, Sports, Science and
Technology (MEXT), Japan.
We then evaluated the reaction of VPA in the presence of
MSN-NEP under basic conditions and compared the reaction
efficiency between MSN-NEP and NEP upon reaction with VPA.
HPLC analysis of the reaction showed that consumption
amount of VPA in the presence of MSN-NEP was about half of
that in the presence of NEP (Fig. S3†). Subsequently, we moni-
tored the emission of VPA aer the reaction for 1.5 h in the
presence of MSN-NEP. Although the emission intensity was
relatively weak, we observed chemiluminescence at around
450 nm, as shown in Fig. 3C. These results indicate that 1O2 was
generated within the pores of MSN and reacted with VPA to
form a dioxetane derivative, which degraded with the concom-
itant emission.
In conclusion, we prepared a novel chemiluminescent
system using a vinyl phenol derivative (VPA) and naphthalene
endoperoxide (NEP). In this system, VPA and NEP act as an
emission unit and thermal 1O2 generator, respectively. The
combination of VPA and NEP resulted in the formation of
a dioxetane derivative via the [2 + 2] cycloaddition of VPA and
1O2; subsequent decomposition of a dioxetane derivative led
to bright chemiluminescence. In addition, we prepared mes-
oporous silica nanoparticles bearing NEP within the pores
(MSN-NEP) and conrmed that selective oxidation of VPA
occurred within the pores to give a chemiluminescence.
Although both the efficiency of the reaction and the emission
intensity need to be improved, this approach may lead to
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