Synthesis of bicyclic dioxetanes bearing a 3-hydroxy-4-isoxazolylphenyl moiety: New CIEEL-active dioxetanes emitting light with remarkable high-efficiency in aqueous medium

Article abstract of DOI:10.1016/S0040-4039(02)02162-7

Bicyclic dioxetanes bearing a 3-hydroxy-4-isoxazolylphenyl moiety (3a-c) were synthesized. All these dioxetanes (3a-c) underwent base-induced CIEEL-decay to afford light with high efficiency in an NaOH/H₂O system as well as in a TBAF (tetrabutylammonium fluoride)/acetonitrile system. Among them, a dioxetane bearing a 3-hydroxy-4-[5-(trifluoromethyl)isoxazol-3-yl]phenyl moiety (3a) emitted light in the aqueous system with the highest efficiency which parallels that attained in an aprotic solvent system. Fluorescence study and the AM1 calculations for 3a and the parent CIEEL-active dioxetanes (1) suggested that one important factor affecting chemiluminescence efficiency in an aqueous system should be the hydrogen-bonding at the carbonyl oxygen site of an oxyanion of hydroxyarenecarboxylate as the emitter produced by the CIEEL-decay.

Full text of DOI:10.1016/S0040-4039(02)02162-7

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 8955–8958
Synthesis of bicyclic dioxetanes bearing a
3-hydroxy-4-isoxazolylphenyl moiety: new CIEEL-active
dioxetanes emitting light with remarkable high-efficiency in
aqueous medium
Masakatsu Matsumoto,* Toshimitsu Sakuma and Nobuko Watanabe
Department of Materials Science, Kanagawa University, Tsuchiya, Hiratsuka, Kanagawa 259-1205, Japan
Received 26 August 2002; revised 4 September 2002; accepted 27 September 2002
Abstract—Bicyclic dioxetanes bearing a 3-hydroxy-4-isoxazolylphenyl moiety (3a–c) were synthesized. All these dioxetanes (3a–c)
underwent base-induced CIEEL-decay to afford light with high efficiency in an NaOH/H2O system as well as in a TBAF
(tetrabutylammonium fluoride)/acetonitrile system. Among them, a dioxetane bearing a 3-hydroxy-4-[5-(trifluoromethyl)isoxazol-
3-yl]phenyl moiety (3a) emitted light in the aqueous system with the highest efficiency which parallels that attained in an aprotic
solvent system. Fluorescence study and the AM1 calculations for 3a and the parent CIEEL-active dioxetanes (1) suggested that
one important factor affecting chemiluminescence efficiency in an aqueous system should be the hydrogen-bonding at the carbonyl
oxygen site of an oxyanion of hydroxyarenecarboxylate as the emitter produced by the CIEEL-decay. © 2002 Elsevier Science
Ltd. All rights reserved.
The intramolecular CIEEL (chemically initiated elec-
tron exchange luminescence)1,2 of hydroxyarene-sub-
stituted dioxetanes has received a great deal of
attention from the viewpoint of mechanistic interests
related to bioluminescence and application to modern
biological analysis using chemiluminescence.3–5 An
adamantylidene–dioxetane (1) is a typical example for
such CIEEL-active dioxetanes and its phosphate-pro-
tected form is now used in chemiluminescence
bioassays.3,6,7 The base-induced CIEEL from 1 occurs
to emit light effectively in an aprotic solvent, such as
DMSO and acetonitrile, while its chemiluminescence
efficiency (FCIEEL) decreases markedly in aqueous
medium (FCIEEL in H2O/FCIEEL in DMSO=1/
39000).6–9 This significant defect has been improved
to some extent (ca. 400 times enhancement) by
adding a surfactant or by tethering a fluorescer to a
dioxetane.5,10 However, further development of high-
sensitive biological analysis using chemiluminescence
would require realizing new CIEEL-active dioxetanes
emitting light with high efficiency even in aqueous
medium (Fig. 1).
Figure 1.
Keywords: singlet oxygenation; 1,2-dioxetane; CIEEL; aqueous medium.
* Corresponding author. E-mail: matsumo@chem.kanagawa-u.ac.jp
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)02162-7

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M. Matsumoto et al. / Tetrahedron Letters 43 (2002) 8955–8958
with a 3-methoxy-4-[5-(trifluoromethyl)isoxazol-3-yl]-
phenyl (7a) (yield based on 6=55%). Demethylation of
7a with EtSNa in hot DMF gave a precursor (4a) in
84% yield. Dihydrofurans (4b–d) were similarly synthe-
sized in 33–63% yields based on the oxime. When a
solution of 4a (100 mg) and tetraphenylporphin (TPP)
(1 mg) in CH2Cl2 (5 mL) was irradiated with a 940-W
Na lamp under an oxygen atmosphere at 0°C for 1 h,
the desired dioxetane (3a) was produced in 87% isolated
yield as pale yellow granules after chromatographic
purification with SiO2/Et2O–hexane (1:20).13 The other
dioxetanes (3b: R=H) and (3c: R=t-butyl) were
obtained by similar singlet oxygenation of the corre-
sponding dihydrofurans (4b and 4c) in isolated yield of
32 and 88%, respectively. It should be noted here that
the singlet oxygenation of a methyl analog (4d) gave
little of the desired dioxetane (3d) but complex mixture
derived presumably from the oxygenation of the isoxa-
zole ring. All dioxetanes (3a–c) synthesized here were
quite stable enough to permit handling at room
temperature.
A very recent study on the CIEEL of dioxetanes bear-
ing a 4-hydroxybenzoxazole moiety (2a and 2b) has
revealed that (1) a marked drop of chemiluminescence
efficiency (FCIEEL) for the CIEEL-decay in an aqueous
system is attributed to synergistic effect of the
decreased chemiexcitation yield (FS) and the decreased
fluorescence yield (Ffl) of the emitter; and (2) the expan-
sion of the p-system by introducing an aryl group at the
2-positon of the benzoxazole moiety leads to apprecia-
ble improvement of chemiexcitation yield as well
fluorescence yield of the emitter, though the chemilu-
minescence efficiency is yet considerably lower in an
aqueous system than in the DMSO system
[FCIEEL(H2O)/FCIEEL(DMSO)=1/35–1/63].9 These facts
prompted us to design new CIEEL-active dioxetanes
bearing a 4-aryl-substituted 3-hydroxyphenyl moiety,
and it was found that 3-hydroxy-4-isoxazolylphenyl-
substituted dioxetanes (3), especially a dioxetane bear-
ing
a
3-hydroxy-4-[5-(trifluoromethyl)isoxazol-3-yl]-
phenyl moiety (3a), exhibit CIEEL-decay with remark-
able high chemiluminescence efficiency even in an
aqueous system.
When a dioxetane (3a) was treated with a large excess
of tetrabutylammonium fluoride (TBAF) in acetonitrile
at 25°C,14 decomposition of 3a occurred smoothly to
follow pseudo-first-order kinetics independent of the
TBAF concentration and to emit blue light (maximum
wavelength: uCmIaExEL=481 nm, chemiluminescence
Dioxetanes bearing
a 3-hydroxy-4-isoxazolylphenyl
moiety (3) were synthesized by singlet oxygenation of
the corresponding dihydrofurans (4), which were pre-
pared from 4-acetyl-3-methoxyphenyl-substituted dihy-
drofuran (5)11 or its 3-hydroxy analog by several steps.
The synthetic process of 3a (Fig. 2) as a representative
was as follows: an oxime (6) of 5 was lithiated12 with
BuLi (2 equiv.) at −78°C in THF and was successively
quenched with S-ethyl trifluoroethanethioate to give an
intermediary hydroxyisoxazoline, of which dehydration
with TsOH/toluene afforded a dihydrofuran substituted
efficiency:FCIEEL=0.44,15 half-lifeofCIEEL-decay:t1/2
=
11000 s). Similar treatment of the other dioxetanes
(3b and 3c) with TBAF/acetonitrile caused also the
CIEEL-decay to afford light whose chemiluminescent
properties are summarized together with those for 3a
and the parent dioxetane (1) in Table 1. Comparing the
chemiluminescent properties for 3-hydroxy-4-isoxa-
Figure 2. Reagents and conditions: (i) NH2OH/EtOH; (ii) BuLi/CF3COSEt/THF/−78°C; (iii) TsOH/toluene; (iv) EtSNa/DMF; (v)
hw/TPP/O2.
Table 1. Base-induced chemiluminescent decomposition of dioxetanes bearing a 3-hydroxy-4-(isoxazol-3-yl)phenyl group (3)
Dioxetane
TBAF/acetonitrilea
FCIEEL t1/2 (s)
NaOH/H2Ob
t1/2 (s)
CIEEL
max
CIEEL
max
CIEEL
FCwIaEteErL/F
acetonitrile
u
(nm)
u
(nm)
FCIEEL
3a
3b
3c
1
481
477
474
466d
0.44
0.39
0.36
0.12e
11000 (1400)c
650
380
13e
473
477
477
466d
0.24
0.064
13000 (1200)c
2800
1500
1/1.8
1/6.1
1/24
1/16000
0.015
7.5×10−6,f
180
a A solution of a dioxetane in acetonitrile (1.0×10−5 M, 1 mL) was added to a TBAF solution in acetonitrile (1.0×10−2 M, 2 mL) at 25°C.
b A solution of a dioxetane in acetonitrile (1.0×10−3 M, 0.1 mL) was added to a NaOH solution in water (0.1 M, 2.9 mL) at 25°C.
c A figure in parenthesis means half-life of the CIEEL-decay at 45°C.
d Ref. 17.
e Ref. 7.
f Ref. 6.

M. Matsumoto et al. / Tetrahedron Letters 43 (2002) 8955–8958
8957
CIEEL
max
zolylphenyl-substituted dioxetanes (3a–c) with those for
1, which bears an unsubstituted 3-hydroxyphenyl, one
realizes that chemiluminescence efficiency (FCIEEL) is
3–4 times higher for 3 than for 1, while half-life of the
CIEEL-decay is prolonged markedly for 3. It is note-
worthy that heating at 45°C accelerated the CIEEL-
u
of the CIEEL emission for 3a in TBAF/acetoni-
trile.16 Using this fluorescence yield (Ffl) and chemilu-
minescence yield (FCIEEL
shown in Table 1,
)
singlet-chemiexcitation yield (Fs=FCIEEL/Ffl) is esti-
mated to be 0.63 in TBAF/acetonitrile. The fluores-
cence of 8a was also very effective even in NaOH/H2O
(Ffl=0.50). Using this value, singlet-chemiexcitation
yield (Fs) for 3a is estimated roughly to be 0.48 in an
aqueous system, though the fluorescence maximum
wavelength of 3a (umfl ax=466 nm) was observed in a
little shorter region than the case of the CIEEL emis-
sion (uCmIaExEL=473 nm). These results show that both
the singlet-chemiexcitation efficiency for the CIEEL of
3a and the fluorescence efficiency of the emitter (8a) are
unprecedentedly high for the CIEEL of hydroxyarene-
substituted dioxetanes in an aqueous system.
decay rate considerably for 3a with little change of
FCIEEL and u
(t1/2 at 45°C=1400 s).
CIEEL
max
Next, we carried out NaOH-induced decomposition of
the present dioxetanes (3a–c). Treatment of 3a (1×10−3
M in acetonitrile, 0.1 mL) with NaOH (0.1 M in H2O,
2.9 mL) gave blue light (uCmIaExEL=473 nm, t1/2=13000 s
at 25°C) with remarkable high efficiency (FCIEEL
=
0.24), which parallels that attained in TBAF/acetoni-
trile system, and exceeds FCIEEL of the parent dioxetane
(1) in aprotic medium (acetonitrile). The CIEEL-decay
rate of 3a in an aqueous system was also accelerated on
heating (t1/2 at 45°C=1200s). The other analogs (3b
and 3c) gave also light effectively in NaOH/H2O
though their FCIEEL was inferior to that of 3a (see
Table 1).
Hydrogen-bonding effects are presumably responsible
for the significant decrease of chemiluminescent
efficiency (FCIEEL) of phenolate-substituted dioxetanes
in an aqueous system.4,9,10 Of the two factors (Fs and
Ffl) relating to FCIEEL directly, Ffl of the produced
emitter would be influenced by hydrogen-bonding,
though it is not quite clear how hydrogen-bonding
affects singlet-chemiexcitation process. Qualitative com-
parison of the degree of hydrogen-bonding to an
oxyanion of hydroxyarenecarboxylates as the emitter
should lead to rationalizing the significant difference in
chemiluminecence efficiency between 3 and 1 in an
aqueous system. The semiempirical AM1 calculations
are relevant to attaining this purpose, as has been used
by Adam to rationalize the solvatochromism related to
the CIEEL of 1 in an aqueous system.17 Thus, we
selected four typical CIEEL-active dioxetanes 1, 2a, 2b,
and the present dioxetane (3a), for which the order of
FCIEEL is 1<2aꢀ2bꢀ3a, and carried out the AM1
calculations for their emitters simplified as a methyl
ester, namely, oxyanions of an ester (9) for 1, an ester
(10a) for 2a, an ester (10b) for 2b, and an ester (11a) for
8a. As shown in Table 2, the large HOMO coefficients
on the phenolate oxygen atom guarantee appreciable
hydrogen bonding at this site in the ground state for all
CIEEL
max
The maximum wavelength (u
) of the CIEEL emis-
sion for 3a in TBAF/acetonitrile coincided with fluores-
cence maximum wavelength (uflmax) of the corresponding
spent reaction mixture, from which a free form of
ketoester (8a) was isolated exclusively (Fig. 3). The
fluorescence of the authentic ketoester (8a), prepared
from 3a by thermolysis, was very effective (Ffl=0.70)
and its maximum wavelength (uflmax) coincided also with
Figure 3.
Table 2. HOMO and LUMO coefficients for oxyanions of methyl hydroxyarenecarboxylates (9, 10a, 10b and 11a)

8958
M. Matsumoto et al. / Tetrahedron Letters 43 (2002) 8955–8958
four oxyanions, while small LUMO coefficients there
suggest far weaker hydrogen-bonding at the same site
in the excited state. On the other hand, significant
difference is shown in the HOMO and the LUMO
coefficients on the ester carbonyl oxygen atom among
the four oxyanions. For all oxyanions, the hydrogen
bonding should be markedly weak at the carbonyl
oxygen atom in the ground state due to the very small
HOMO coefficients on this site. In contrast, the LUMO
coefficients on the carbonyl oxygen atom are signifi-
cantly different among 9, 10a, 10b, and 11a, and they
decrease in the order 9>10a>10bꢁ11a so that the
hydrogen-bonding at this site should be appreciable for
9 whereas minimal for 11a in the excited state. This
order coincides with the order of the chemilumines-
cence efficiency, 1<2aꢀ2bꢀ3a, in an aqueous medium.
Conclusively, the AM1 calculations suggest that the
hydrogen-bonding at the carbonyl oxygen site of the
ester rather than the phenolate oxygen site in an oxyan-
ion of the phenolic ester as the emitter plays an impor-
tant role to decrease its fluorescence efficiency which
affects the chemiluminescence efficiency for the CIEEL-
active dioxetanes in an aqueous system.
2. Catalani, L. H.; Wilson, T. J. J. Am. Chem. Soc. 1989,
111, 2633–2639.
3. Schaap, A. P.; Chen, T.-S; Handley, R. S.; DeSilva, R.;
Giri, B. P. Tetrahedron Lett. 1987, 28, 1155–1158.
4. Beck, S.; Koster, H. Anal. Chem. 1990, 62, 2258–2270.
5. Mayer, A.; Neuenhofer, S. Angew. Chem., Int. Ed. Engl.
1994, 33, 8535–8538.
6. Adam, W.; Bronstein, I.; Edwards, B.; Engel, T.; Rein-
hardt, D.; Schneider, F. W.; Trofimov, A. V.; Vasil’ev, R.
F. J. Am. Chem. Soc. 1996, 118, 10400–10407 and refer-
ences cited therein.
7. Trofimov, A. V.; Mielke, K.; Vasil’ev, R. F.; Adam, W.
Photochem. Photobiol. 1996, 63, 463–467.
8. Matsumoto, M.; Arai, N.; Watanabe, N. Tetrahedron
Lett. 1996, 37, 8535–8538.
9. Matsumoto, M.; Mizoguchi, Y.; Motoyama, T.; Watan-
abe, N. Tetrahedron Lett. 2001, 42, 8869–8872.
10. Schaap, A. P.; Akhavan, H.; Romano, L. J. Clin. Chem.
1989, 35, 1863–1864.
11. Matsumoto, M.; Sakuma, T.; Watanabe, N. Lumines-
cence 2001, 16, 275–280.
12. Whitesell, J. K.; Whitesell, M. A. Synthesis 1983, 517–
536.
13. Selected data for 3a: pale yellow granules melted at
126.0–128.0°C (from hexane, CH2Cl2); 1H NMR (400
MHz, CDCl3) lH 1.02 (s, 9H), 1.17 (s, 3H), 1.39 (s, 3H),
3.85 (d, J=8.3 Hz, 1H), 4.60 (d, J=8.3 Hz, 1H), 7.28
(dd, J=8.3 and 1.5 Hz, 1H), 7.39 (d, J=1.5 Hz, 1H),
7.59 (d, J=1.5 Hz, 1H), 7.91 (d, J=8.3 Hz, 1H), 10.67 (s,
1H); 13C NMR (100 MHz, CDCl3) lC 18.5, 25.1, 27.0,
36.8, 45.7, 80.5, 105.4, 110.3, 115.9, 117.9, 118.7, 119.7,
126.0, 127.5, 138.7, 141.8, 157.3, 162.7; IR (KBr) 3144,
2975, 2898, 1613, 1550; MS (m/z, %) 413 (M+, 1), 381
(13), 366 (20), 357 (28), 273 (33), 256 (100), 228 (13).
Anal. calcd for C20H22F3NO5: C, 58.11; H, 5.36; N, 3.39.
Found: C, 58.16; H, 5.38; N, 3.74%.
Dioxetanes bearing
a 3-hydroxy-4-isoxazolylphenyl
moiety (3a–c) synthesized here, especially 3a, were
shown to exhibit remarkably high chemiluminescence
efficiency even in an aqueous system. The AM1 calcula-
tions of the emitters for 1, 2 and 3a suggested that the
hydrogen-bonding at the carbonyl oxygen site should
affect Ffl. Further study on the CIEEL of 3 is expected
to provide a clue to examining the relation of the
chemiexcitation process to the hydrogen bonding,
which remains unsolved.
Acknowledgements
14. A solution of 3a in acetonitrile (1.0×10−5 M, 1 mL) was
added to a TBAF solution in acetonitrile (1.0×10−2 M, 2
mL) at 25°C.
The authors gratefully acknowledge financial assistance
provided by a grant-in-aid for Scientific Research by
the Ministry of Education, Science, Sports and Culture.
15. Chemiluminescence efficiency (FCIEEL) was based on the
reported value for tert-butyldimethylsilyl ether of 1:
F
CIEEL=0.29 in DMSO (Ref. 7).
16. Quinine bisulfate was used as the fluorescence standard.
17. Adam, W.; Bronstein, I.; Trofimov, A. V. J. Phys. Chem.
A 1998, 102, 5406–5414.
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