Synthesis and enhanced chemiluminescence of new cyclomaltooligosaccharide (cyclodextrin) -bound 6-phenylimidazo[1,2- a]pyrazin-3(7H)-one

Article abstract of DOI:10.1016/S0008-6215(97)10059-3

In order to provide chemiluminescent substrates that have high light- emitting efficiency in aqueous solution, the structural design on 6- phenylimidazo[1,2-a]pyrazin-3(7H)-one compounds was studied in the covalent attachment of a light-producing chromophore to a cyclomaltooligosaccharide (cyclodextrin). The synthesis of cyclodextrin-bound 6-phenylimidazo[1,2- a]pyrazin-3(7H)-one compounds was achieved by the formation of an amido bond between a 6-phenylimidazo[l,2-a]pyrazin-3(7H)-one and a mono-6-amino-6- deoxycyclodextrin. The properties of oxygen-induced chemiluminescence of the synthesized cyclodextrin-bound light-emitting chromophores were investigated. The light-emitting efficiency in pH 8.3 phosphate buffer was remarkably dependent on the kind of bound cyclodextrin and the binding site between the chromophore and cyclodextrin. The light-emitting efficiency of a cyclodextrin-bound compound in which cyclomaltoheptaose (β-cyclodextrin) had been covalently attached to the 2-position of the imidazo[1,2-a]pyrazin- 3(7H)-one ring system showed an up to 11-fold enhancement over that of a non- cyclodextrin chromophore, whereas attachment to cyclomaltohexaose (α- cyclodextrin) resulted in no enhancement. Moreover, this study indicated that the strategy that involves covalently attaching a light-producing chromophore onto a cyclodextrin for the enhancement of chemiluminescence is more efficient than the use of an aqueous solution containing very large amounts of cyclodextrin.

Full text of DOI:10.1016/S0008-6215(97)10059-3

Ž
.
Carbohydrate Research 306 1998 177–187
Synthesis and enhanced chemiluminescence of new
ž
/
cyclomaltooligosaccharide cyclodextrin -bound
w
x
ž /
6-phenylimidazo 1,2-a pyrazin-3 7H -one
)
Katsunori Teranishi , Atsuko Komoda, Makoto Hisamatsu,
Tetsuya Yamada
Faculty of Bioresources, Mie UniÕersity, Kamihama, Tsu, Mie 514, Japan
Received 2 June 1997; accepted 3 October 1997
Abstract
In order to provide chemiluminescent substrates that have high light-emitting efficiency in
w
x
Ž
.
aqueous solution, the structural design on 6-phenylimidazo 1,2-a pyrazin-3 7H -one com-
pounds was studied in the covalent attachment of a light-producing chromophore to a
Ž
.
cyclomaltooligosaccharide cyclodextrin . The synthesis of cyclodextrin-bound 6-phenylim-
w
x
Ž
.
idazo 1,2-a pyrazin-3 7H -one compounds was achieved by the formation of an amido bond
w
x
Ž
.
between a 6-phenylimidazo 1,2-a pyrazin-3 7H -one and a mono-6-amino-6-deoxycyclo-
dextrin. The properties of oxygen-induced chemiluminescence of the synthesized cyclodex-
trin-bound light-emitting chromophores were investigated. The light-emitting efficiency in pH
8.3 phosphate buffer was remarkably dependent on the kind of bound cyclodextrin and the
binding site between the chromophore and cyclodextrin. The light-emitting efficiency of a
Ž
.
cyclodextrin-bound compound in which cyclomaltoheptaose b-cyclodextrin had been cova-
w
x
Ž
.
lently attached to the 2-position of the imidazo 1,2-a pyrazin-3 7H -one ring system showed
an up to 11-fold enhancement over that of a non-cyclodextrin chromophore, whereas
Ž
.
attachment to cyclomaltohexaose a-cyclodextrin resulted in no enhancement. Moreover, this
study indicated that the strategy that involves covalently attaching a light-producing chro-
mophore onto a cyclodextrin for the enhancement of chemiluminescence is more efficient
than the use of an aqueous solution containing very large amounts of cyclodextrin. q 1998
Elsevier Science Ltd. All rights reserved.
Keywords: Cyclomaltooligosaccharide; Cyclodextrin; Cyclodextrin-bound light-producing compound; Chemilumi-
nescence; Enhanced chemiluminescence; MCLA
1. Introduction
The chemiluminescent reaction of Cypridina lu-
ciferin without enzyme in dimethyl sulfoxide and in
) Corresponding author.
0008-6215r98r$19.00 q 1998 Elsevier Science Ltd All rights reserved.

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)
178
K. Teranishi et al.rCarbohydrate Research 306 1998 177–187
the presence of oxygen was first reported in 1966 by
w x
lytical system should not affect the subject except to
promote luminescence. It is noteworthy to construct a
novel chemiluminescent system that promotes greater
chemiluminescent activity. In this paper, we report
the first example of the synthesis of light-producing
compounds, in which MCLA, a chemiluminescent
chromophore, is covalently bound to one cyclodextrin
molecule, and show that the chemiluminescent inten-
sity of the cyclodextrin-bound luminescent chro-
mophore is effectively enhanced in an aqueous sol-
vent.
Johnson et al. 1 . Diethylene glycol dimethyl ether,
Ž
.
containing a trace amount of acetate buffer pH 5.6
was later reported as an efficient solvent by Goto et
w x
al. 2 . In this solvent system the quantum efficiency
of Cypridina chemiluminescence is about 10% that of
Cypridina bioluminescence, which was reported as
w x
ca. 0.28 3 , whereas the quantum efficiency in
dimethyl sulfoxide was reported to be below 0.0005
w x
1 . Thereafter, it has been shown that several synthe-
sized analogues of Cypridina luciferin, such as 2-
methyl-6-indolyl-, 2-methyl-6-phenyl-, and 2-methyl-
Ž
.
w
x
Ž
.
6- p-methoxyphenyl -imidazo 1,2-a pyrazin-3 7H -one
Ž
. Ž .
MCLA 1 , generate light and the emitter 3 via
2. Results and discussion
formation of a singlet-excited amide 2 by oxidation
w
x
with triplet oxygen in buffer solutions 4,5 , as shown
in Scheme 1, and with superoxide anion in aqueous
The synthetic route used is shown in Scheme 2.
w x
The coupling of amino pyrazine 4 8 and a-keto-
w
x
solvents 6–8 . However, the chemiluminescent quan-
tum efficiency of Cypridina luciferin and its ana-
logues in an aqueous solvent has been less than 1%
that of Cypridina bioluminescence.
glutaric acid gave amino diacide 5 with a 30% yield.
Compound 5 was treated with acetic anhydride in
Ž
.
tetrahydrofuran THF at room temperature to give
the yellow cyclized compound 6 in a 30% yield.
Chemiluminescence offers several potential advan-
tages for analytical applications. Many studies on the
enhancement of chemiluminescence for analysis have
dealt with the regulated medium, such as using a
micellar surfactant or cyclomaltooligosaccharide
Then,
6 and mono-6-amino-6-deoxycyclomal-
Ž
.
Ž .
tohexaose mono-6-amino-6-deoxy-a-cyclodextrin
Ž .
w
x
7 13 or mono-6-amino-6-deoxy-b-cyclodextrin 8
w
x
Ž
13 were coupled in the presence of 1-ethyl-3- 3-di-
methylaminopropyl carbodiimide hydrochloride
.
Ž
.
Ž
.
cyclodextrin . It has been shown that cyclodextrin
enhances the chemiluminescent intensity of lumines-
w x
WSC to afford cyclodextrin-bound luminescent
compounds 9 and 10 in 47% and 48% yields, respec-
tively. Cyclodextrin-bound luminescent compound 14,
which possesses a different binding mode, was pre-
pared by the route shown in Scheme 3. Compound 12
was prepared in a 72% yield by the condensation of
cent compounds, such as lucigenin 9 , peroxyoxalate
w
x
w x
10 , isoluminol 11 , and Cypridina luciferin ana-
logues 12 in aqueous solvents. Cypridina luciferin
w
x
Ž . Ž
.
analogue MCLA 1 1.7 mM in 0.01 M aqueous
solutions of cyclomaltoheptaose b-cyclodextrin or
Ž
.
Ž
compound 11 which was obtained by the treatment
of compound 4 with pyridine hydrochloride at 210
Ž
.
heptakis 2,6-di-O-methyl -b-cyclodextrin showed
chemiluminescence with 0.0034 or 0.010 quantum
efficiency, respectively. The study showed that the
cavity of b-cyclodextrin may be most closely fitted
to MCLA, but that very large amounts of cyclodex-
trin are needed for the aqueous chemiluminescent
system. For analysis by chemiluminescence, the ana-
.
8C with sodium hydride and ethyl 4-bromobutyrate
Ž
.
in N,N-dimethylformamide Me2 NCHO . A mixture
of compound 12 and pyruvic aldehyde was heated in
a water–1,4-dioxane mixture in the presence of HCl
to afford compound 13 in 34% yield. Condensation
of compound 13 with 8 was carried out by treatment
Scheme 1.

(
)
K. Teranishi et al.rCarbohydrate Research 306 1998 177–187
179
Scheme 2.
with WSC in pyridine similar to the procedure used
for the preparation of 10.
The chemiluminescent reaction consists of many
reaction steps such as binding with an oxygen
is influenced by the molecular state of the light-pro-
Ž
.
ducing chromophore. Electronic spectra Fig. 1 of 9,
10 and 14 show the molecular states of the chro-
mophores in these compounds in 30 mM phosphate
Ž .
Ž
.
molecule, generation of intermediate s having high
buffer pH 8.3 . The binding with b-cyclodextrin in
10 and 14 influences the spectra in comparison to 1
as shown in Fig. 1, whereas 9 is similar in shape and
intensity to 1. These results show that the molecular
conditions of the light-producing chromophores in 10
energy, formation of an excited singlet state, and
emission of light, and efficiencies in these reaction
steps determine the quantum yield of chemilumines-
cence. Efficiency in binding with an oxygen molecule
Scheme 3.

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)
180
K. Teranishi et al.rCarbohydrate Research 306 1998 177–187
Fig. 1. Electronic spectra of 1, 9, 10 and 14 in 30 mM
. Ž
10; -P-P-P 14. Concentration of luminescent compound is
10 mM.
Fig. 2. Induced circular dichroism spectra of 9, 10 and 14
Ž . Ž . Ž .
in 30 mM phosphate buffer pH 8.3 . —– 9; PPPPP 10;
Ž
.
Ž
.
Ž
.
phospate buffer pH 8.3 .
1; --- 9; PPPPP
Ž
.
Ž
.
-P-P-P 14. Concentration of luminescent compound is 10
mM.
and 14 are different from those of 1 and 9 and that
reactivities to oxygen are not the same. Only when
the cyclodextrin forms the inclusion complex with an
achiral chromophore does it show circular dichroism
in the absorption area of the chromophore. The mag-
nitude of intensity and pattern of the induced circular
light-emitting chromophore cannot be deeply in-
cluded in the cavity. The cavity of a-cyclodextrin is
not sufficiently deep to include the light-emitting
chromophore in contrast with that of b-cyclodextrin,
and the imidazopyrazinone moiety of the light-pro-
ducing chromophore may not have hydrophobic char-
acter for the inclusion. In comparison with the case of
10, the ICD spectrum of 14 exhibits positive bands
only below 380 nm. This observation suggests that
the moiety of the light-emitting chromophore in 14 is
included in the b-cyclodextrin cavity differently with
10. It is possible that the light-emitting chromophore
of 14 could not be sufficiently included into the
cavity of cyclodextrin in contrast to 10, because the
imidazopyrazinone ring that is located at the tip of
the chromophore of 14 seems to be more hydrophilic
than the anisole group.
Ž
.
dichroism ICD spectrum indicates the inclusion
mode. Fig. 2 shows ICD spectra of 9, 10 and 14 in 30
Ž
.
mM phosphate buffer pH 8.3 without oxygen. The
ICD spectrum of 10 exhibits positive bands below
500 nm, whereas 9 shows too small negative bands in
a similar region. The structural features of the cy-
clodextrin compounds 9, 10 and 14 could be reflected
in the ICD spectra, and the light-emitting chro-
mophore in 10, which exhibits strong positive bands,
must be included deeply in the b-cyclodextrin cavity.
Since compound 9 exhibits a very weak band, the
Table 1
a
Chemiluminescent properties of MCLA and cyclodextrin-bound MCLA in phosphate bufferb,c
y2
y2
y2
y2
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
No. of compound
FCL =10
t1r2 min
FR =10
FF =10
FS =10
Ž
.
1 MCLA
9
0.17
0.21
1.9
0.51
0.29
1.1
80
170
32
85
80
27
39
49
41
31
53
5
13
17
23
27
22
13
4
23
5
3
9
10
14
1qb-cyclodextrind
1qb-cyclodextrine
45
aConcentration of compounds is 10 mM.
b
Ž
.
30 mM phosphate buffer pH 8.3 at 25 8C.
cAbbreviations include the following:
dConcentration of b-cyclodextrin is 1 mM.
eConcentration of b-cyclodextrin is 10 mM.
FCL sQuantum efficiency of chemiluminescence.
t1r2 sHalf time of chemiluminescence.
FR sFormation yield of the corresponding amide.
FF sQuantum efficiency of fluorescence of the corresponding amide.
FS sQuantum efficiency of excited singlet state formation calculated from the equation FCL sFR =FS =FF.

(
)
K. Teranishi et al.rCarbohydrate Research 306 1998 177–187
181
Ž .
Because intermediate s having high energy such
cies. In the present case, it was observed that the
fluorescent spectrum of the emitter is not superim-
posable on the corresponding chemiluminescent spec-
trum.
as dioxetane and singlet excited state molecule are
too unstable and short-lived to be detected with con-
ventional apparatus, the influence of these short-lived
Ž .
molecule s by bound cyclodextrin cannot be directly
The chemiluminescent quantum efficiency and
chemiluminescent half life of 9, 10 and 14 were
analyzed in particular. Generally, one of the chemilu-
Ž
.
minescent properties, the chemiluminescent quantum
tested in 30 mM phosphate buffer pH 8.3 in the
presence of oxygen. The results are summarized in
Ž
.
efficiency FCL , is given by the following equation;
Ž
Ž .
FCL sFR =FS =FF FR: formation yield of emit-
ter, FS: quantum efficiency of excited singlet state
Table 1, in comparison with MCLA 1 . The chemi-
luminescent quantum efficiency of b-cyclodextrin-
bound compound 10 was about 11 times greater than
that of MCLA, whereas the quantum efficiency of
a-cyclodextrin-bound compound 9 was practically
the same as that of MCLA. A decrease in chemilumi-
nescent half-life was shown in the chemilumines-
cence of 10. These observations suggest, in the case
of b-cyclodextrin, that an intramolecular inclusion of
the chemiluminescent emitter by the cyclodextrin
cavity has occurred, thus causing the enhancement of
formation of the emitter, FF: quantum efficiency of
.
fluorescence of the emitter . FCL and FR are easily
determined by experiments. Veritable quantum effi-
ciency of excited-singlet-state formation and quantum
efficiency of fluorescence, however, cannot be deter-
mined, because the same singlet excited-state
Ž .
Ž .
molecule s as the singlet excited-state intermediate s
formed in the course of a chemiluminescent reaction
cannot be duplicated for analysis of these efficien-
Scheme 4.

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)
182
K. Teranishi et al.rCarbohydrate Research 306 1998 177–187
Fig. 5. Electronic spectra of 3, 16, 17 and 20 in 30 mM
. Ž
17; -P-P-P 20. Concentration of luminescent compound is
10 mM.
Fig. 3. Chemiluminescent spectra of 1, 9, 10 and 14 in 30
. Ž
Ž
.
Ž
.
Ž
.
Ž
.
Ž .
1; --- 9;
phospate buffer pH 8.3 .
3; --- 16; PPPPP
mM phosphate buffer pH 8.3 .
PPPPP 10; -P-P-P 14; — –— –— — 1q b-
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
cyclodextrin 10 mM . Concentration of luminescent com-
pound is 10 mM.
b-cyclodextrin in 10 and 14 has a larger effect than
a-cyclodextrin supports the theory that singlet ex-
cited emitters of 10 and 14 are included into b-
cyclodextrin as discussed in the section on the chemi-
luminescent quantum yield.
luminescence due to the hydrophobic environment; in
the case of a-cyclodextrin, the cavity size is probably
insufficient to accommodate the luminescent
Ž .
emitter s . It is noted that the quantum efficiency of
Ž
.
Amido compounds 16, 17 and 20 see Scheme 4
10 was greater than that of MCLA in using cyclodex-
w
x
are products of the chemiluminescent reactions of 9,
10 and 14, respectively, and are considered to be the
light emitters according to the comparison of their
chemiluminescent spectra and the fluorescent spectra
trin solutions reported by Toya 12 . The quantum
efficiency of another b-cyclodextrin-bound com-
pound 14 was only three times that of MCLA. This
observation indicates that the binding site of the
Ž
.
Ž .
Fig. 4 of the corresponding amides. They were
chemiluminescent emitter s to cyclodextrin is impor-
synthesized from the amino compounds 4 or 12 as
shown in Scheme 4, for several analyses. As shown
in Table 1, the cyclodextrin-bound compound 10
afforded 1.8 times higher formation yield of the
amide 17 than 1. In the case of using b-cyclodextrin
tant for effective enhancement. Binding a b-cyclo-
dextrin to MCLA has some effect on the chemilumi-
nescent spectra as shown in Fig. 3. The luminescent
maxima of 10 and 14 are 440 and 428 nm, respec-
tively, which are shorter than that of 1, whereas
a-cyclodextrin-bound compound 9 shows no effect.
The shift in luminescent maximum is similarly ob-
served with MCLA in b-cyclodextrin solution. That
Ž
.
10 mM solution, the formation yield of 3, which
was generated from 1 by the chemiluminescent reac-
tion, was twice as high as that of the non-b-cyclo-
dextrin similar to the case of 10. These results indi-
cate that using covalent and noncovalent cyclodextrin
Fig. 4. Fluorescent spectra of 3, 16, 17 and 20 in 30 mM
Fig. 6. Induced circular dichroism spectra of 16, 17 and 20
Ž
. Ž
.
Ž
.
Ž
.
Ž . Ž . Ž .
in 30 mM phosphate buffer pH 8.3 . --- 16; PPPPP 17;
phosphate buffer pH 8.3
3; --- 16; PPPPP
Ž
.
Ž
.
17; -P-P-P 20. Concentration of luminescent compound is
10 mM.
-P-P-P 20. Concentration of luminescent compound is 10
mM.

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)
K. Teranishi et al.rCarbohydrate Research 306 1998 177–187
183
1
is one of the methods for the effective oxidation of
MCLA. Fluorescent quantum efficiencies of the
amides were measured under the same conditions as
the chemiluminescent reaction, and the results are
shown in Table 1. The cyclodextrin-bound amido
compounds 16, 17 and 20 gave higher fluorescent
quantum efficiencies than the amide 3. The binding
of cyclodextrin to the amide 3 has an effect on its
spectrometer. H NMR spectra were determined on a
JEOL JNM-A500 spectrometer at 20 8C using tetra-
methylsilane or tert-butanol as an internal reference.
Ž
.
Chemical shift values are reported in d ppm rela-
tive to an internal standard of Me4Si, and coupling
constants J are in Hz. FAB mass spectra positive-
Ž .
.
Ž
Ž
.
ion mode or SIMS spectra positive-ion mode were
measured with a JEOL DX-303 or HITACHI M-
80LCAPI instrument using glycerol as a matrix. Ele-
mental analyses were measured with a Yanaco CHN-
Corder MT-3 instrument. Chemiluminescent intensity
time curves were obtained using an Aloka Lumines-
cence Reader BLR-3, and chemiluminescent spectra
and fluorescent spectra were obtained using a Hitachi
F-2000 spectrofluorometer. ICD spectra were ob-
tained using a JASCO J-720M spectrometer. All
chemicals not otherwise mentioned were purchased
Ž
.
fluorescent spectrum, electronic spectrum Fig. 5 ,
Ž
.
and ICD spectrum Fig. 6 . The fluorescent maxima
of 16, 17 and 20 are shorter than that of 3 and show
large hypsochromic effects. Bathochromic shifts in
the electronic spectra are observed with 16, 17 and
20. The ICD spectra of 16, 17 and 20 show strong
positive bands in contrast with the ICD spectra of 9,
10 and 14. The influence on the fluorescent quantum
efficiencies and these spectra suggests that all the
chromophores of amides 16, 17 and 20 are strongly
included in the cyclodextrin cavity, because the amido
molecules are more hydrophobic than the imidazopy-
razinone molecules. Quantum efficiency in the singlet
excited-state formation of 17 was increased over that
of 3, whereas those of 16 and 20 had an opposite
effect. The b-cyclodextrin-bound compound 10 has
an ability for increasing all the efficiencies in chemi-
luminescence.
Ž
.
from Tokyo Kasei Kogyo Tokyo, Japan or Aldrich
Ž
.
Chemical Co. Milwaukee, WI, USA in chemically
pure grade and were used as such. Mono-6-amino-6-
deoxy-a-cyclodextrin 13 , mono-6-amino-6-deoxy-
b-cyclodextrin 13 , 1 8 , 3 8 , and 4 8 were
prepared according to the published method.
2 - 5 - 4 - Methoxyphenyl - 2 - pyrazinyl amino-
pentanedioic acid 5 .—Pd–C 5%, 2.0 g was added
to a solution of 4 5.0 g, 25 mmol and a-keto-
w
x
w x
w
x
w x
w x
[
(
)
]
Ž .
Ž
.
Ž
.
Ž
.
Ž
.
In summary, this study shows the first example of
glutaric acid 7.3 g, 50 mmol in ethanol 100 mL ,
and the mixture was stirred under a hydrogen atmo-
sphere at 55 8C for 5 h. The reaction mixture was
filtered through Celite, and the filtrate was evapo-
rated under reduced pressure to dryness. The product
Ž
.
cyclodextrin-bound covalently bound chemilumi-
nescent compounds whose luminescence is enhanced
by the intramolecular inclusion into b-cyclodextrin in
an aqueous solvent. This strategy to increase the
chemiluminescent quantum efficiency is more effi-
cient than the use of ordinary cyclodextrin solutions
alone in typical host–guest interactions.
Ž
was crystallized from methanol to afford 5 2.5 g,
30% : mp 190–191 8C; UV MeOH lmax nm ´
.
Ž
Ž
.
Ž .
.
Ž
.
Ž
.
Ž
.
354 7580 , 285 26,700 , and 221 6720 ; IR KBr :
n 3370, 1740, 1600 and 1500 cmy
;
1H NMR
1
Ž
.
Ž
.
Ž
CD3OD : d 2.0–2.6 m, 4 H, CH2CH2 , 3.77 s, 3
.
Ž
.
Ž
H, OCH3 , 4.55 m, 1 H, HOOCCHN , 6.92 d, 2 H,
J 9.0 Hz, ArH , 7.70 d, 2 H, J 9.0 Hz, ArH , 8.00
Ž
.
Ž
.
3. Experimental
.
Ž
.
s, 1 H, CH pyrazine , and 8.30 s, 1 H, CH pyrazine ;
Anal. Calcd for C16H17N3O5: C, 58.00; H, 5.17; N,
General methods.—HPLC analysis and isolation
were carried out using a JASCO Gulliver HPLC
system with a MD-910 three-dimensional UV–vis
12.68. Found: C, 58.33; H, 5.26; N, 12.32. SIMS:
w
x
mrz 332 Mq1 .
[
(
)
[
]
3 - 6 - 4 - Methoxyphenyl imidazo 1, 2 - a pyrazin-
(
)
Ž
]
Ž .
detector.
A
Fuji Silysia Chromatorex-ODS
3 7 H -one-2-yl propionic acid 6 .—Acetic anhy-
Ž
.
.
DU0005MT column 4.6 mm=150 mm was used
for the analysis. Analytical TLC was performed on
Merck Kieselgel 60F254 precoated glass plates of
dride 4.5 mL of 1.0 M in tetrahydrofuran was added
to a solution of 5 1.0 g, 3.0 mmol in tetrahydrofu-
Ž
.
Ž
.
ran 60 mL at room temperature, and the reaction
mixture was stirred at room temperature for 3 days
under argon. The reaction mixture was filtered, and
the resulting crystals were washed with ethyl acetate
and dried under reduced pressure to give cyclized
Ž
.
0.25 mm layer thickness. All melting point mp
values were measured with a Yanagimoto Seisakusho
apparatus and are uncorrected. IR spectra were taken
with a Shimadzu IR-470 infrared spectrometer, and
UV spectra were obtained with a Shimadzu UV-3100
Ž
.
compound 6 0.28 g, 30% yield : mp 213–214 8C;

(
)
184
K. Teranishi et al.rCarbohydrate Research 306 1998 177–187
Ž
.
Ž .
Ž
.
Ž
.
Ž
UV MeOH lmax nm ´ 431 6900 , 356 4810 ,
d 2.6–4.2 m, H b-cyclodextrin, CH2CH2, and
Ž
.
Ž
.
.
Ž
.
.
Ž
and 264 18,800 ; IR KBr : n 3450, 1690, 1610,
OCH3 , 4.8–5.1 m, H-1 b-cyclodextrin , 7.26 d, 2
H, J 8.0 Hz, ArH , 7.41 s, 1 H, CH pyrazine , 7.60
d, 2 H, J 8.0 Hz, ArH , and 8.05 s, 1 H, CH
pyrazine ; Anal. Calcd for C58H84N4O37 P5H2O: C,
1
1560, and 1510 cmy ; H NMR CD3OD : d 2.83 t,
1
Ž
.
Ž
.
.
Ž
.
.
.
Ž
Ž
.
Ž
2 H, J 7.3 Hz, CH2 , 3.11 t, 2 H, J 7.3 Hz, CH2 ,
Ž
Ž
.
3.87 s, 3 H, OCH3 , 7.07 d, 2 H, J 8.6 Hz, ArH ,
Ž
.
Ž
7.62 d, 2 H, J 8.6 Hz, ArH , 7.83 s, 1 H, CH
45.85; H, 6.24; N, 3.69. Found: C, 45.35; H, 6.33; N,
.
Ž
.
w
x
)
pyrazine , and 7.90 brs, 1 H, CH pyrazine ; Anal.
Calcd for C16H15N3O4: C, 61.34; H, 4.83; N, 13.41.
Found: C, 61.52; H, 5.44; N, 13.26. SIMS: mrz 314
3.12. FABMS: mrz 1429 Mq1 .
(
Ž
.
2-Amino-5- 4-hydroxyphenyl pyrazine 11 .—A
Ž
.
mixture of 2 2.5 g, 12 mmol and pyridine hydro-
w
x
Ž
.
Mq1 .
chloride 12.5 g, 0.11 mol was heated at 210 8C for
30 min. After cooling, the mixture was dissolved in
ethyl acetate, neutralized with 10% NaHCO3, and
extracted with ethyl acetate three times. The extracts
were washed with saturated NaCl, dried over anhy-
drous Na2SO4, and evaporated to dryness. The residue
was crystallized from a mixture of ethyl acetate and
I
I
[[
(
)
[
6 -Deoxy-6 - 6- 4-methoxypheny imidazo 1,2-
]
(
)
]
]
a pyrazin - 3 7 H - one - 2 - yl propionyl amino-
cyclomalthohexaose 9 .—Pyridine 4.0 mL was
added dropwise to a mixture of 6 0.10 g, 0.32
mmol , WSC 0.49 g, 2.6 mmol , and 6 -amino-6 -
deoxycyclomaltohexaose 0.62 g, 0.64 mmol at about
y100 8C under argon gas, and the solution was
deoxygenated by bubbling argon gas into the mixture.
The mixture was stirred at room temperature for 2 h
and evaporated under reduced pressure to dryness.
The residue was dissolved with water and filtered
through a membrane filter. The filtrate was subjected
to high-performance liquid chromatography HPLC
on an ODS column Fuji Silysia Chromatorex-ODS
DU0005MT column, 20 mm=250 mm using aceto-
nitrile–water mixture as eluent to afford 9 0.19 g,
47% yield ; yellow powder, mp 189 8C dec, from
acetonitrile–water ; UV 0.03 M phosphate buffer,
pH 8.3, with little oxygen l
and 262 24,300 ; IR KBr : n 3400 and 1630 cm
Ž
)
Ž
.
Ž
I
I
.
Ž
.
Ž
.
Ž
.
hexane to give a powder 1.7 g, 74% yield ; mp
210–211 8C; UV MeOH lmax nm ´ 352 6040 ,
Ž
.
Ž .
Ž
.
Ž
.
Ž
.
and 280 20,800 ; IR KBr : n 3450, 3300, 3200,
1625, 1605, 1595, 1510, 1485, 1440, and 1390 cmy
;
1
1
Ž
.
Ž
.
H NMR CD3OD : d 6.85 d, 2 H, J 8.0 Hz, ArH ,
7.65 d, 2 H, J 8.0 Hz, ArH , 7.95 s, 1 H, CH
pyrazine , and 8.25 s, 1 H, CH pyrazine ; Anal.
Calcd for C10H9N3O: C, 64.16; H, 4.85; N, 22.45.
Found: C, 64.13; H, 5.01; N, 22.72.
Ethyl 4- 2-amino-5-pyrazinyl phenoxybutyrate 12 .
—Sodium hydride 0.27 g, 60% NaH, 6.8 mmol was
added to a solution of 11 1.0 g, 5.3 mmol in
Ž
.
Ž
Ž
.
.
Ž
.
Ž
.
Ž
(
)
Ž
.
.
Ž
Ž
.
.
Ž
Ž
.
.
.
Ž . Ž
nm ´ 410 6000
.
Ž
.
Me2 NCHO 20 mL , and the mixture was stirred at 5
max
y
1
Ž
.
Ž
;
8C for 30 min. To the mixture was added ethyl
1
Ž
.
Ž
Ž
.
H NMR 0.02 M TFA–D2O : d 2.73 m, 1 H,
CH2CH2 , 2.83 m, 1 H, CH2CH2 , 3.05–3.30 m, 2
H, CH2CH2 , 3.05–3.30 m, 2 H, H a-cyclodextrin ,
2.7–5.2 m, H a-cyclodextrin , 3.86 s, 3 H, OCH3 ,
4-bromobutyrate 1.9 mL, 13 mmol , and the mixture
was stirred at 20 8C for 12 h. The reaction mixture
was dissolved in a mixture of dichloromethane and
water and extracted with dichloromethane three times.
The extracts were evaporated to dryness. The residue
was chromatographed on a silica gel column ethyl
acetate–hexane to give 12 1.2 g, 75% ; mp 96–97
8C; UV MeOH lmax nm ´ 351 7280 , and 281
.
Ž
.
Ž
.
Ž
.
.
Ž
.
Ž
Ž
.
Ž
4.86 d, 1 H, J 3.0 Hz, H-1 a-cyclodextrin , 4.99 d,
.
Ž
Ž
1 H, J 3.0 Hz, H-1 a-cyclodextrin , 7.05 d, 2 H, J
8.5 Hz, ArH , 7.63 d, 2 H, J 8.5 Hz, ArH , 7.95 s,
1 H, CH pyrazine , and 8.46 s, 1 H, CH pyrazine ;
Anal. Calcd for C52 H74N4O32 P4H2O: C, 46.64; H,
6.17; N, 4.18. Found: C, 46.01; H, 6.52; N, 4.06.
.
Ž
.
Ž
.
Ž
.
.
Ž
.
Ž
.
Ž .
Ž
.
Ž
.
Ž
.
25,200 ; IR KBr : n 3400, 3300, 3200, 1715, 1640,
1
1505, and 1470 cmy ; H NMR CDCl3 : d 1.26 t,
1
Ž
.
Ž
w
x
. Ž
FABMS mrz 1267 Mq1 .
3 H,
J
7.3 Hz, CH3CH2 , 2.14 m, 2 H,
CH2CH2CH2O , 2.54 t, 2 H,
C H2CH 2CH 2O , 4.06 t,
CH2CH2CH2O , 4.15 q, 2 H, J 7.3 Hz, CH3CH2O ,
4.56 brs, 2 H, NH2 , 6.96 d, 2 H, J 8.6 Hz, ArH ,
7.79 d, 2 H, J 8.6 Hz, ArH , 8.04 d, 1 H, J 1.2 Hz,
I
I
[[
(
)
[
.
.
Ž
6 -Deoxy-6 - 6- 4-methoxypheny imidazo 1,2-
J
J
7.3 Hz,
6.1 Hz,
.
]
(
)
]
]
Ž
a pyrazin-3 7 H -one-2-yl propionyl amino-cyclomal-
toheptaose 10 .—This compound was prepared from
0.10 g 0.32 mmol of 6, WSC 0.50 g, 2.6 mmol ,
and 6 -amino-6 -deoxy-cyclomaltoheptaose 0.72 g,
0.63 mmol as described for the synthesis of 9, giving
0.092 g 48% yield ; yellow powder, mp 175 8C dec,
from acetonitrile–water ; UV 0.03 M phosphate
buffer, pH 8.3, with little oxygen l
2 H,
Ž
.
.
Ž
Ž
.
Ž
.
Ž
Ž
.
Ž
.
.
I
I
Ž
Ž
.
.
Ž
CH pyrazine , and 8.39 d, 1 H, J 1.2 Hz, CH
Ž
.
Ž
.
pyrazine ; Anal. Calcd for C16H19N3O3: C, 63.77; H,
6.36; N, 13.94. Found: C, 64.21; H, 6.72; N, 13.76.
.
Ž
.
Ž .
w
x
nm ´ 418
SIMS: mrz 302 Mq1 .
max
.
Ž
.
Ž
.
Ž
Ž
.
[ [
[
]
(
)
2700 , 340 5200 , and 268 14,100 ; IR KBr :n
4- 4- 2-Methylimidazo 1,2-a pyrazin-3 7 H -one-6-
1
3400 and 1640 cmy ; H NMR 0.02 M TFA–D2O :
1
Ž
.
]
]
Ž
.
Ž
yl phenoxy butyric acid 13 .—A mixture of 12 1.0

(
)
K. Teranishi et al.rCarbohydrate Research 306 1998 177–187
185
.
Ž
.
g, 3.3 mmol , pyruvic aldehyde 0.76 mL, 5.0 mmol ,
Found: C, 59.92; H, 5.26; N, 13.90. SIMS mrz 302
Ž
.
Ž
.
w
x
5 N HCl 6.6 mL , 1,4-dioxane 20 mL , and water
10 mL was heated at 100 8C for 1 h. After cooling,
the mixture was evaporated to dryness. The residue
was chromatographed on an ODS column Fuji Silysia
Chromatorex-ODS DM1020T, acetonitrile–water to
give 13 0.37 g, 34% ; mp 205 8C dec ; UV MeOH
Mq1 .
X
I
I
Ž
.
[
(
)
6 - Deoxy - 6 - N - 5 - 4 - methoxyphenyl - 2 -
]
Ž
.
pyrazinyl succinamidocyclomaltohexaose 16 .—Pyr-
Ž
Ž
.
Ž
idine 4.0 mL was added to a mixture of 15 0.10 g,
0.33 mmol , WSC 0.51 g, 2.7 mmol , and 6 -amino-
I
.
.
Ž
.
I
Ž
.
Ž
.
Ž
.
Ž
.
6 -deoxycyclomlatohexaose 7 0.65 g, 0.67 mmol at
room temperature, and the mixture was stirred at
room temperature for 3.5 h. The pyridine was evapo-
rated to dryness under reduced pressure. The residue
was dissolved with water and filtered through a mem-
brane filter. The filtrate was chromatographed on an
Ž . Ž . Ž .
lmax nm
´
430 4640 , 355 3300 , and 263
y
1
Ž
.
Ž
.
12,100 ; IR KBr : n 3450, 1720, and 1570 cm
;
1 H NMR CD3OD :
d
Ž
2.10 m,
2
H,
Ž
.
Ž
.
.
Ž
HOOCH2CH2CH2O , 2.45 s, 3 H, CH3 , 2.51 t, 2
H, J 7.3 Hz, HOOCH2CH2CH2O , 4.10 t, 2 H, J
6.1 Hz, HOOCH2CH2CH2O , 7.08 d, 2 H, J 7.9
Hz, ArH , 7.62 d, 2 H, J 7.9 Hz, ArH , 7.63 brs, 1
H, CH pyrazine , 7.91 brs, 1 H, CH pyrazine ; Anal.
Calcd for C17H17N3O4: C, 62.38; H, 5.23; N, 12.84.
Found: C, 62.81; H, 5.66; N, 12.44. SIMS: mrz 328
.
Ž
.
Ž
Ž
ODS column Fuji Silysia Chromatorex-ODS
.
Ž
.
.
Ž
.
DM1020T column, 15 mm=150 mm using aceto-
nitrile–water mixtures as eluent to afford 16 0.25 g,
56% yield ; mp 293 8C dec, from acetonitrile–water ;
UV 0.03 M phosphate buffer, pH 8.3 l
Ž
.
Ž
.
Ž
.
Ž
.
Ž .
nm ´
max
w
x
Ž
.
Ž
.
Ž
.
Mq1 .
334 12,200 , 291 13,200 , and 279 14,300 ; IR
I
I
[[ [
[
]
Ž
.
6 -Deoxy-6 - 4- 2-methylimidazo 1,2-a pyrazin-
3 7 H -one-6-yl phenoxy butyryl amino-cyclomalto-
heptaose 14 .—This compound was prepared from
0.10 g 0.31 mmol of 13, WSC 0.50 g, 2.6 mmol ,
and 6 -amino-6 -deoxy-cyclomaltoheptaose 0.72 g,
0.63 mmol as described for the synthesis of 9, giving
0.15 g 33% yield ; mp 205 8C decomposition ; UV
0.03 M phosphate buffer, pH 8.3, with little oxygen
KBr : n 3400, 2950, 1640, 1545, 1500, 1470, 1415,
1
1355, 1250, and 1150 cmy ; H NMR CD3OD : d
1
(
)
]
]
]
Ž
.
Ž
.
Ž
.
Ž
2.5–2.9 m, 4 H, CH2CH2 , 3.0–4.0 m, H a-
cyclodextrin , 3.85 s, 3 H, OCH3 , 4.8–5.0 m, H-1
a-cyclodextrin , 7.04 d, 2 H, J 9.0 Hz, ArH , 7.94
d, 2 H, J 9.0 Hz, ArH , 8.75 s, 1 H, CH pyrazine ,
and 9.35 s, 1 H, CH pyrazine ; Anal. Calcd for
C51H74N4O32 P5H2O: C, 45.54; H, 6.29; N, 4.16.
Found: C, 45.46; H, 6.53; N, 4.01. FABMS mrz 1255
Ž
.
Ž
.
.
Ž
.
Ž
I
I
Ž
.
Ž
.
.
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
Ž
l
Ž
Ž
.
Ž .
Ž
.
Ž
.
nm ´ 273 16,400 , 345 6300 , and 396
max
1
4000 ; IR KBr : 3400, and 1640 cm 1; H NMR
y
.
Ž
.
w
x
Mq1 .
X
I
I
.
Ž
[
(
)
D2O :
d
2.0–5.3 m,
H
b-cyclodextrin,
6 - Deoxy - 6 - N - 5 - 4 - methoxyphenyl - 2 -
.
Ž
.
]
Ž
.
CH2CH2CH2O, and CH3 , 7.17 d, 2 H, J 8.5 Hz,
pyrazinyl succinamido - cyclomaltohepttaose 17 .—
.
Ž
Ž
Ž
ArH , 8.02 d, 2 H, J 8.5 Hz, ArH , 8.21 s, 1 H, CH
This compound was prepared from 0.10 g 0.33
mmol of 15, WSC 0.51 g, 2.7 mmol , and 6 -
amino-6 -deoxy-cyclomaltoheptaose 8 0.75 g, 0.66
mmol as described for the synthesis of 16, giving
0.22 g 0.43% yield ; mp 305 8C dec, from aceto-
nitrile–water ; UV 0.03 M phosphate buffer, pH 8.3
I
.
Ž
.
.
Ž
.
pyrazine , and 8.73 s, 1 H, CH pyrazine ; Anal.
Calcd for C59H86N37O4 P5H2O: C, 46.21; H, 6.31;
N, 3.65. Found: C, 46.00; H, 6.80; N, 3.21. FABMS:
I
Ž
.
w
x
Ž
.
Ž
mrz 1443 Mq1 .
[ (
)
]
.
Ž
.
5- 4-Methoxyphenyl -2-pyrazinyl succinamic acid
Ž
.
Ž
.
Ž . Ž . Ž .
nm ´ 333 13,000 , 294 15,300 , and 280
15 .—A mixture of 4 0.50 g, 2.5 mmol , succinic
anhydride 1.2 g, 12 mmol , and Me2 NCHO 10 mL
was heated at 120 8C for 3 h. The Me2 NCHO was
removed under reduced pressure and the resulting
solids were dissolved in ethyl acetate and the product
l
max
Ž
.
Ž
.
Ž
.
Ž
.
15,900 ; IR KBr :n 3400, 2950, 1650, 1540, 1350,
1
1160, and 1030 cmy ; H NMR CD3OD : d 2.5–2.9
1
Ž
.
Ž
.
Ž
m, 4 H, CH2CH2 , 3.1–4.0 m, b-cyclodextrin and
OCH3 , 4.8–5.0 m, H 1 b-cyclodextrin , 7.06 d, 2
H, J 8.5 Hz, ArH , 7.94 d, 2 H, J 8.5 Hz, ArH ,
8.76 s, 1 H, CH pyrazine , and 9.35 s, 1 H, CH
.
Ž
.
Ž
Ž
.
Ž
.
was crystallized to give compound 15 0.61 g, 81%
yield ; mp 238–239 8C; UV MeOH lmax nm ´
.
Ž
.
Ž .
Ž
.
Ž
Ž
.
Ž
.
Ž
.
.
334 15,200 , 291 18,850 , and 279 19,900 ; IR
pyrazine ; Anal. Calcd for C57H84N4O37 P7H2O: C,
Ž
.
KBr : n 3450, 3200, 3100, 1690, 1605, 1560, 1495,
44.36; H, 6.40; N, 3.63. Found: C, 44.10; H, 6.56; N,
1475, 1440, 1415, 1360, 1310, 1255, and 1170 cmy
;
1
w
x
3.36. FABMS: mrz 1417 Mq1 .
1
Ž
.
Ž
[
(
)
]
H NMR Me2SO-d6 : d 2.52 t, 2 H, J 6.7 Hz,
4 - 4 - 2 - acetamido- 5 - pyrazinyl phenoxy butyric
.
Ž
.
Ž
Ž
.
Ž
.
CH2 , 2.67 t, 2 H, J 6.7 Hz, CH2 , 3.82 s, 3 H,
acid 19 .—Acetyl chloride 0.16 mL, 2.3 mmol
.
Ž
.
Ž
Ž
.
OCH3 , 7.06 d, 2 H, J 9.0 Hz, ArH , 8.04 d, 2 H,
J 9.0 Hz , 8.92 s, 1 H, CH pyrazine , 9.31 s, 1 H,
CH pyrazine , and 10.88 s, 1 H, COOH ; Anal.
was added to a solution of 12 0.45 g, 1.5 mmol in a
mixture of dichloromethane 4.5 mL and pyridine
4.5 mL at 0 8C, and the mixture was stirred at 20 8C
.
Ž
.
Ž
.
Ž
.
.
Ž
Ž
.
Calcd for C15H15N3O4: C, 59.80; H, 5.02; N, 13.95.
for 1 h. The mixture was cooled to 0 8C and neutral-

(
)
186
K. Teranishi et al.rCarbohydrate Research 306 1998 177–187
Ž
.
Ž
.
ized with 10% NaHCO3. The mixture was extracted
with dichloromethane three times, and the organic
extracts were dried over anhydrous Na2SO4 and
evaporated to dryness. The residue was dissolved in a
buffer pH 8.3 , 30 mM phosphate buffer pH 8.3
Ž
.
containing b-cyclodextrin 10 mM or Me2 NCHO at
25 8C. The reaction mixture was placed in the pho-
tometer, and the chemiluminescent intensity time
curve was obtained at 25 8C. The chemiluminescent
quantum efficiencies were determined on the basis of
Ž
.
Ž
.
mixture of 1,4-dioxane 20 mL and water 6.7 mL ,
Ž
and to the solution was added 1 N NaOH 1.8 mL,
1.8 mmol at 0 8C. The mixture was stirred at 20 8C
.
w
x
Toya’s report 12 . The fluorescent quantum efficien-
cies of the corresponding amides were determined on
the basis of quinine sulfate. Chemiluminescent spec-
tra were obtained as follows: The luminescent solu-
tion above was placed in the spectrofluorometer, and
the spectrum was obtained without emission light.
Yields of the amido compounds generated by the
chemiluminescent reaction were obtained follows: an
HPLC system was employed for analyzing the lumi-
nescence-spent products and the yields of the amides
were determined by comparison with the correspond-
ing synthesized amides. Elution conditions for amide
16: solvent, 20:80 acetonitrile–water; flow rate, 0.8
mLrmin; for amide 17: solvent, 15:85 acetonitrile–
water; flow rate, 0.8 mLrmin; and for amide 20:
solvent, 10:90 acetonitrile–water; flow rate, 0.8
mLrmin.
for 8 h. The reaction mixture was dissolved in a
Ž
.
mixture of ethyl acetate and water pH 3 and ex-
tracted with ethyl acetate three times. The extracts
were washed with saturated NaCl solution, dried over
anhydrous Na2SO4, and evaporated to dryness. The
product was crystallized from a mixture of methanol
Ž
.
and ethyl acetate to give 19 0.29 g, 62% yield ; mp
Ž
.
Ž .
240 8C decomposition; UV MeOH lmax nm ´
334 15,500 , 291 19,600 , and 279 20,300 ; IR
Ž
.
.
Ž
.
Ž
.
Ž
KBr : n 3450, 3050, 1695, 1605, 1560, 1470, 1425,
1
1355, 1295, 1250, 1185, and 1160 cmy1; H NMR
Ž
.
Ž
M e 2 S O -d 6
:
d
.
1 .9 7
m ,
2
H ,
.
Ž
HOOCCH2CH2CH2O , 2.15 s, 3 H, NHCOCH3 ,
2.40 t, 2 H, J 7.3 Hz, HOOCCH2CH2CH2O , 4.05
Ž
H, J 9.0 Hz, ArH , 8.02 d, 2 H, J 9.0 Hz, ArH ,
8.92 s, 1 H, CH pyrazine , 9.31 s, 1 H, CH pyra-
zine , and 10.78 s, 1 H, COOH ; Anal. Calcd for
Ž
.
.
Ž
t, 2 H, J 6.7 Hz, HOOCCH2CH2CH2O , 7.05 d, 2
.
Ž
.
.
Ž
Ž
.
.
Ž
Acknowledgements
The NMR instrument used in this research was
installed at Cooperative Research Center of Mie Uni-
versity.
described for the synthesis of 16, giving 0.20 g
References
Ž
.
Ž
0.83% yield ; mp 287 8C decomposition from wa-
.
Ž
.
ter ; UV 0.03 M phosphate buffer, pH 8.3 l max nm
w x
1 F.H. Johnson, H.-D. Stachel, E.C. Taylor, and O.
Ž .
Ž
.
Ž
.
Ž
.
´ 333 11,100 , 293 12,300 , and 280 13,200 ; IR
Ž
.
Shimomura, in F.H. Johnson and Y. Haneda Eds. ,
Bioluminescence in Progress, Princeton Univ. Press,
1966, pp 67–68.
Ž
.
;
KBr : n 3400, 2950, 1640, 1540, 1155, and 1030
cmy
1 H NMR D2O :
d
2.02 m, 1 H,
1
Ž
.
Ž
.
Ž
C O C H C H 2 C H O , 2 .2 5
COCH2CH2CH2O , 2.27 s, 3 H, NCOCH3 , 2.41
Ž
cyclodextrin , 4.9–5.2 m, H 1 b-cyclodextrin , 7.03
Ž
ArH , 8.72 s, 1 H, CH pyrazine ,and 9.27 s, 1 H,
CH pyrazine ; Anal. Calcd for C58H86N4O37 P5H2O:
m ,
1
.
H ,
2
2
w x
2 T. Goto, S. Inoue, and S. Sugiura, Tetrahedron Lett.,
.
Ž
Ž
.
1968 3873–3876.
.
Ž
w x
3 F.H. Johnson, O. Shimomura, Y. Saiga, L.C. Gersh-
m, 2H, COCH2CH2CH2O , 2.8–4.2 m, H b-
man, G.T. Reynolds, and J.R. Waters, Cell. Comp.
.
Ž
.
Ž
.
Physiol., 60 1962 85–103.
4 T. Goto and H. Fukatsu, Tetrahedron Lett., 1969
.
Ž
d, 2 H, J 8.5 Hz, ArH , 7.96 d, 2 H, J 8.5 Hz,
w x
Ž
.
.
Ž
.
Ž
4299–4302.
.
w x
5 H. Sawada, K. Masuyama, and M. Nakayama, Yuka-
C, 45.79; H, 6.36; N, 3.68. Found: C, 45.63; H, 6.59;
Ž
.
Ž
.
gaku, 39 1990 47–49; Chem. Abstr., 113 1990
w
x
5554.
N, 3.30. FABMS mrz 1431 Mq1 .
w x
6 T. Goto and T. Takagi, Bull. Chem. Soc. Jpn., 53
Procedure for chemiluminescence measurement.
—Chemiluminescent intensity time curves were ob-
tained as follows: 20 mL of 0.5 mM 1 dissolved in
methanol or 0.5 mM 9, 10 or 14 dissolved in distilled
water was added to 0.98 mL of 30 mM phosphate
Ž
.
1980 833–834.
w x
7 A. Nishida, H. Kimura, M. Nakano, and T. Goto,
Ž
.
Clin. Chim. Acta., 179 1989 177–182.
w x
8 Y. Toya, T. Kayano, K. Sato, and T. Goto, Bull.
Ž
.
Chem. Soc. Jpn., 65 1992 2475–2479.

(
)
K. Teranishi et al.rCarbohydrate Research 306 1998 177–187
187
w x
Ž
.
w
x
Ž
.
9 M.L. Grayeski and E.J. Woolf, J. Lumin., 33 1985
12 Y. Toya, Nippon Nogeikagaku Kaishi, 66 1992
Ž .
742–747; Chem. Abstr. 117 1992 23473.
115–121.
w
w
x
Ž
.
w
x
10 E.J. Woolf and M.L. Grayeski, J. Lumin., 39 1987
13 K. Hamasaki, H. Ikeda, A. Nakamura, A. Ueno, F.
19–27.
Toda, I. Suzuki, and T. Osa, J. Am. Chem. Soc., 115
x
Ž
.
Ž
.
11 H. Karatani, Chem. Lett., 1986 377–380.
1993 5035–5040.