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Development of a novel chemiluminescent probe, 4-(5',6'-Dimethoxybenzothiazolyl) phthalhydrazide
ANALYTICA
CHIMICA
ACIA ELSEVIER
Analytica Chimica Acta 309 (1995) 211-219
Development of a novel chemiluminescent probe, 4-t 5’,6’-dimethoxybenzothiazolyl) phthalhydrazide Junichi Ishida a, Maki Takada a, Syuuji Hara a, Kazumi Sasamoto b, Kenyu Kina b, Masatoshi Yamaguchi ap* a Faculty of Pharmaceutical Sciences, Fukuoka Universiry, Nanakuma, Johnan-ku, Fukuoka 814-80, Japan b Dojindo Laboratories, Tabaru 2025-5, Mashiki-machi, Kamimashiki-gun, Kumamoto 861-23, Japan
Received 24 October 1994; revised 20 December 1994; accepted 22 January 1995
Abstract 4-(5’,6’-Dimethoxybenzothiazolyl)phthalhydrazide (DBPH) was synthesized as a new, highly sensitive chemiluminescent reagent. The reagent consists of phthalhydrazide and benzothiazole moieties and generates strong chemiluminescence by reaction with hydrogen peroxide in the presence of potassium hexacyanoferrate(II1) or peroxidase. The chemiluminescence intensity of DBPH was about 2U times greater than that of luminol. The detection limit of DBPH was 1.3 X lo-” M [0.65 fmol per well (200 pl), signal to blank ratio = 21. The reaction of DBPH was also applied to the determination of hydrogen peroxide by flow injection. The detection limit is 0.5 pmol for a lOO-/*I injection. Keywords: Flow injection; Chemiluminescence; Hydrogen peroxide; 4-(5’,6’-Dimethoxybenzothiazolyl)phthalhydrazide
1. Introduction In recent years, chemiluminescence has become an attractive detection method for analytical determinations because of its high sensitivity [1,2]. Luminol chemiluminescence is popular, and is based on the reaction of luminol with hydrogen peroxide in the presence of potassium hexacyanoferrate(II1) or peroxidase as a catalyst. We have studied new, luminol type chemiluminescence reagents which have a higher efficiency than luminol. One of our strategies is to introduce a
* Corresponding
author.
0003-2670/95/$09.50 0 1995 Elsevier Science B.V. AU rights reserved SSDI 0003-2670(95)00082-S
strongly fluorescent group into the reagent. We have reported [3] 6-isothiocyanatobenzo[ glphthalazine1,4(2H,3H)-dione (IPO), 3-propyl-7,&dihydropyridazino[4,5-g]quinoxaline-2,6,9(1 H)-trione (PDIQ) and 3-benzyl-7,8_dihydropyridazino[4,5glquinoxaline-2,6,9(1 Hl-trione (BDIQ) as the new chemiluminescent reagents. They produce chemiluminescence 1.7-5.5 times as intense as luminol. In this study, a novel luminol related compound, 4-(5’,6’-dimethoxybenzothiazolyllphthalhydrazide (DBPH), has been synthesized (Fig. 11, in the hope that it would be a more sensitive chemiluminescent reagent than those mentioned above. This reagent has a phthalhydrazide moiety as a reaction site and 5,6-dimethoxybenzothiazole as an emission site, which is a highly fluorescent compound developed
212
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Chimica Acta 309 (1995) 21 l-219
in our laboratory [4]. DBPH was found to produce strong chemiluminescence by reaction with hydrogen peroxide in the presence of potassium hexacyanoferrate(lI1) or peroxidase (Fig. 2). The optimum chemiluminescence reaction conditions were investigated by a manual method. Furthermore, the reagent was evaluated for the determination of hydrogen peroxide, in comparison with lumino1 by flow injection analysis (FIA).
2. Experimental
spectometry (FAB-MS) measurements were made on a JEOL JMS-AX 505W instrument. Uncorrected melting points were obtained on a Yamato MP-21 melting point apparatus. Chromatography was carried out on Kieselgel 60 (Merck, Darmstadt). Chemiluminescence intensities and reaction time courses were measured by a Microluminoreader MLR-100 (Corona) equipped with a Corona DP-50 autopipetter. Uncorrected fluorescence spectra and intensities were measured with a Hitachi 650-60 spectrofluorimeter in 10 X 10 mm quartz cells; a spectral bandwidth of 5 nm was used in both the excitation and emission monochromator.
2.1. Apparatus 2.2. Chemicals ‘H Nuclear magnetic resonance (NMR) spectra were measured on a Bruker AC-2000 spectrometer at 200 MHz using tetramethylsilane as an internal standard. The splitting patterns were designated as follows: s, singlet; d, doublet; m, multiplet; br, broad. IR spectra were taken in KBr disks on a Hitachi 270-30 spectrometer. Fast atom bombardment mass
and solutions
Peroxidase (POD) and microperoxidase (MP, MP11 grade) were purchased from Sigma; these solutions were stored in a refrigerator and used within one week. A stock solution of DBPH (1 X 10m3 M) was prepared in dimethylsulfoxide. A mixed solution of DBPH and MP for FIA was prepared by dilution
N Ph R 0
2 (R = CH3) 3 (R = CHBn) 4 (R = CHO)
CHSO
CH90
CH,O
CH,O 6
5
7
1 Fig. 1. Synthesis of DBPH.
J. Ishida et al. /Analytica
of the stock solutions with 10 mM phosphate buffer (pH 8.0). Luminol was purchased from Nacalai Tesque (Kyoto).
Di(4,5-dimethoxy-2-nitrophenyl)sulfide
4-Methyl-N-phenylphthalimide
(2) was as detailed in the literature
4-Dibromomethyl-N-phenylphthalimide
[51.
was performed
and
according
to the
imide (7)
(4)
A suspension containing 3 (3.30 g, 8.4 mmol) and silver carbonate (6.0 g, 21.8 mmol) in a 1:l (v/v) mixture (150 ml) of tetrahydrofuran and Hz0 was heated at reflux for 24 h. The reaction mixture was concentrated in vacua to a small volume. Then, it was diluted with tetrahydrofuran (500 ml) and filtered through a celite pad. The filtrate was concentrated to give a crude product which was purified by silica gel chromatography with benzene containing 5% of ethyl acetate to give 0.84 g (40%) of aldehyde 4 as a slightly yellow powder. m.p. 179-180” C. IR Vnl,X (cm -‘): 1724 (CHO), 1712 (imide C=O). ‘H NMR (CDCI,): 7.43-7.54 (m, 5H, =NPh), 8.14 (d,
Fig. 2. Suggested
The synthesis literature [41.
(5) (6)
4-(5’,6’-Dimethoxybenzothiazolyl)-N-phenylphthal-
(3)
A solution containing compound 2 (6.99 g, 29.5 mmol), N-bromosuccinimide (10.48 g, 58.9 mmol) and benzoyl peroxide (0.18 g) in Ccl, (175 ml) was refluxed for 16 h. The reaction mixture was filtered and the filtrate was concentrated in vacua to leave a yellowish residue. Silica gel chromatography with benzene afforded 3.39 g (29%) of 3 as an off-white powder. m.p. 178-180” C. IR vmax (cm-‘): 1710 (imide C=O). ‘H NMR (CDCI,): 6.73 (s, lH, CHBr,), 7.36-7.53 (m, 5H, =NPh), 7.96 (s, 2H, H-3 and H-6), 8.19 (s, lH, H-5). FAB-MS: m/z = 396 [M + l]+. 4-Formyl-N-phenylphthalimide
lH, .I = 7.8 Hz, H-6), 8.33 (dd, lH, J = 7.6, 1.4 Hz, H-5) 8.46 (s, lH, H-3) 10.21 (s, lH, CHO). FABMS: m/z = 252 [M + l]+. 4,5-Dimethoxy-2-nitrobromobenzene
2.3. Synthesis of DBPH
The synthesis
213
Chimica Acta 309 (1995) 21 I-21 9
Hydrochloric acid (1.5 ml) was added dropwise at 40-50” C to a stirred suspension containing disulfide 6 (100 mg, 0.23 mmol) and tin powder (0.40 g) in ethanol (15 ml); the mixture was refluxed for 1 h, during which time all of the starting material dissolved. The reaction mixture was filtered after being cooled to room temperature. To this filtrate was added aldehyde 4 (155 mg, 0.62 mmol) and the mixture was refluxed for 1 h. The initial suspension turned clear and then a precipitate formed almost instantly. After the reaction, the solids were collected and dried in vacua. Chromatography with CHCl, containing 35% of methanol gave 68 mg (35%) of compound 6 as a light yellow crystalline solid. m.p. 294-295” C. Fluorescence (CHCI,): A,, = 520 nm (excited at A,, = 400 nm). ‘H NMR (CDCI,): 4.01 (s, lH, OCH,), 4.02 (s, lH, OCH,), 7.35 (s, lH, H-4’), 7.43-7.55 (m, 5H, =NPh), 7.58 (s, lH, H-7’), 8.05 (d, lH, H-3, J= 8.5 Hz, H-6). 8.49 (dd, lH, J = 8.4, 1.9 Hz, H-5), 8.57 (d, lH, J = 1.7 Hz, H-3). FAB-MS: IR vmax (cm -I): 1722 (imide C=O). m/z = 417 [M + l]+. 4-(5’,6’-Dimethoxybenzothiazolyl)phthalhydrazide
(1) Hydrazine hydrate (0.30 ml) was added to a suspension of compound 5 (70.5 mg, 0.17 mmol) in a
chemiluminescence
reaction of DBPH.
J. Ishida et al. /Analytica
214
Chimica Acta 309 (I 995) 21 I-219
1:l (v/v) mixture (3 ml) of ethanol and dioxane. The mixture was refluxed for 30 min. The precipitate was collected by filtration, washed excessively with ethanol and dioxane, and dried in vacua over P,O,, yielding 61.3 mg (100%) of hydrazide 1 as an offwhite powder. m.p. > 300” C. ‘H NMR (DMSO-d,): 3.88 (s, lH, OCH,), 3.89 (s, lH, OCH,), 4.58 (br, 14H, Hydrazide), 7.68 (s, lH, H-4’) 7.75 (s, lH, H-7’), 8.19 (d, lH, J = 8.5 Hz, H-6), 8.41 (dd, lH,
J = 8.4, 1.9 Hz, H-5), 8.63 (d, lH, J = 1.7 Hz, H-3). IR VIII,, (cm-‘): 3328 (lactam NH), 1634 (lactam C=O). FAB-MS: m/z = 3.56 [M + l]+. 2.4. Procedure for properties
(1
examining
chemiluminescent
A 50-~1 portion of the reagent (DBPH) solution 10e7 M) was placed into a well of a black
x
(A)
Hydrogm
prroxl6r
(mhl)
K3Fe(CN)O
(mu)
0 0
1
2
Sodium
hydroxlde
3
(M)
Fig. 3. Effects of (A) hydrogen peroxide, (B) potassium hexacyanoferrate(II1) and (C) sodium hydroxide concentrations on integrated chemiluminescence intensities (a run time of 20.5 s). (1) DBPH (or (2) luminol) (1 X lo-’ M) was treated according to the procedure with various concentrations of hydrogen peroxide, potassium hexacyanoferrate(II1) and sodium hydroxide.
J. Ishida et al. /Analytica
215
Chimica Acta 309 (I 995) 21 l-21 9
microplate. The black microplate was inserted into the microluminoreader. (i) Chemiluminescence with potassium hexacyanoferrate(II1). The chemiluminescence reaction was initiated by simultaneous automatic injections of 50 ~1 of hydrogen peroxide solution (l-40 mM) and 100 ~1 of potassium hexacyanoferrate(II1) (l-80 mM) in sodium hydroxide (0.5-3.0 M). (ii) Chemiluminescence with POD. The chemiluminescence reaction was initiated by automatic injec-
tions of 50 ~1 of the hydrogen peroxide solution (l-40 mM) and 100 ml of POD (l-55 U) in 10 mM phosphate buffer (pH 6.0-9.0). Chemiluminescence intensities were monitored immediately after the automatic injection. 2.5. FIA with chemiluminescence detection The mixture of DBPH (2 X 10m5 M) and microperoxidase (1.2 X 10m5 M) in 10 mM phosphate
(8) 2OOow
0 0
10
20
Time
(8)
20
10
Time
(8)
Fig. 4. Effects of (A) hydrogen peroxide, (B) potassium hexacyanoferrate(II1) and (C) sodium hydroxide concentrations on the time courses reaction of DBPH. Curves: (A, mM) 1, 1; 2, 5; 3, 10; 4, 20; 5, 30; 6, 40. (B, mM) 1, 1; 2, 5; 3, 10; 4, 20, 5, 30; 6, of the chemiluminescence 40; 7, 80. (C, M) 1, 0.5; 2, 0.8; 3, 1.0; 4, 1.2; 5, 1.5; 6, 2.0; 7, 3.0.
216
J. lshida et al./Analytica
Chimica Acta 309 (1995) 211-219
buffer (pH 8.0) was delivered by a Jasco 880-PU pump equipped with a Rheodyne 7125 syringe-loading sample injector valve (loo-~1 loop). Hydrogen peroxide solution was injected into the flowing reagent solution. The flow-rate was 0.5 ml min- ‘. The temperature was ambient (23 f 2” Cl. The generated chemiluminescence was monitored by an ATT0 (Tokyo) AC-2220 luminomonitor equipped with a 60-~1 flow cell. Stainless-steel tubing (0.5 mm i.d.1 was used for the FIA system.
3. Results and discussion 3.1. Chemiluminescent properties of DBPH DBPH was evaluated as a chemiluminescent reagent by comparing it with luminol at the same concentration (1 X 10e7 M). DBPH produces chemiluminescence by reaction with hydrogen peroxide in the presence of potassium hexacyanoferrate(II1) or peroxidase.
(A) r”
600000
‘,
6OG000 *
Woo00
..
5owoo-
4ooow
.,
400000-
3ooom
-’
3OOwo-
I
E
W
a :
r f
E
J
200oDo~
b
lOcmQ-
= E 0
200000-
100000~
2 047.
,
I)
10
.
,
,
20
30
Hydrogen
prroxlde
.
.,
.
40
50
0
(mu)
10
7
20
POD
30
40
50
60
(unltr/l00~l)
sooooo”
4owoo
,.
1ooooo -*
O..,T 5
. 6
, 7
.
I
6
,
1
.
9
PH Fig. 5. Effects of (A) hydrogen peroxide, (B) POD units and (C) pH of the phosphate run time of 20.5 s). Curves: 1, DBPH, 2, luminol.
buffer on integrated
chemiluminescence
intensities (a
J. Ishida et al. /Analytica
217
Chimica Acta 309 (1995) 21 I-21 9
reaction of DBPH. The chemiluminescence was initiated by addition of hydrogen peroxide and potassium hexacyanoferrate(II1) to the compound solution. The chemiluminescence intensity reached a maximum at < 1.5 s after the addition and then decreased rapidly. The hydrogen peroxide concentration did not influence the time course of the chemiluminescence reaction, but it was affected by changes of the concentration of potassium hexacyanoferrate(II1) and sodium hydroxide. In both cases, the rate of the chemiluminescence reaction was much faster as their concen-
In the case of chemiluminescence using potassium hexacyanoferrate(II1) as catalyst, the concentrations hydrogen peroxide, potassium of hexacyanoferrate(II1) and sodium hydroxide influenced the chemiluminescence intensity (Fig. 3); 10 mM hydrogen peroxide, 5 mM potassium hexacyanoferrate(II1) and 1.5 M sodium hydroxide gave almost the maximum intensity. Fig. 4 shows the effect of the concentrations of hydrogen peroxide, potassium hexacyanoferrate(II1) and sodium hydroxide on the time course of the chemiluminescence
W
2
4
DBPH
0
6
(x10-5
M)
3
2
1
MP
(x10-5
M)
G)
6
9
10
11
PH Fig. 6. Effects of (A) DBPH cow.,
(B) microperoxidase
cow. and (C) pH of the phosphate
buffer on peak height.
218
J. Ishida et al. / Analytica Chimica Acta 309 (I 995) 21 l-21 9
tration increased. Luminol showed almost the same decay curves as those of DBPH. The calibration curve for DBPH was linear (r = 0.999) up to at least 1 X lop6 M under the optimum reaction conditions. The limit of detection for DBPH was 1.3 X lo- ’ 1 M (0.65 fmol per well), which gave a chemiluminescence intensity of twice the blank. This value was about 20 times lower as that of luminol. The precision was established by repeated determinations (n = 15) using DBPH solutions of 1 X 10e8 and 1 X lo-’ M. The relative standard deviations were 6.3 and 4.2%, respectively. In the case of chemiluminescence using peroxidase as a catalyst, the concentrations of hydrogen peroxide and peroxidase and the pH value of the reaction medium influenced the chemiluminescence intensity (Fig. 5); 20 mM hydrogen peroxide, 22 U of peroxidase and a pH of 7.0 gave the maximum intensity. DBPH produces the maximum chemiluminescence intensity at neutral pH. The calibration graph for DBPH was linear (I = 0.998) up to at least 1 X lop6 M under the optimum reaction conditions. The limit of detection for DBPH was 5.9 X lo-” M (3.0 fmol per well). In the chemiluminescence of luminol, the 3aminophthalate ion produced during the reaction has been proved to be the light emitter. Therefore, in the case of DBPH, the corresponding phthalate ion (Fig. 2) is expected to be the light-emitting species. The fluorescence properties (fluorescence excitation and emission maxima and the relative intensity) of this species was measured in comparison with luminol after the chemiluminescence reaction was completed under the optimum reaction conditions in each case (Table 1). The fluorescence from DBPH after the reaction was about 500 times more intense than that from luminol, but the chemiluminescence from DBPH is 20 times more intense as described above.
Table 1 Fluorescence excitation (Ex) and emission (Em) maxima and relative intensities (RF11 of the reagents after the chemiluminescence reaction Reagent
Ex (nml
Em (nm)
RFIa
Luminol DBPH
316 361
450 448
100.0 51344.5
aFluorescence
intensity of luminol was taken as 100.0.
ao-
Tube
length
(cm)
Fig. 7. Effect of the tubing length between the injector chemiluminescence detector on the peak height.
and
It is known that the efficiency of the chemiluminescent compound is partly dependent on the fluorescence intensity of the light-emitting species which produced during the reaction. However, other factors such as energy transfer relate to the chemiluminescence production. Therefore, the multiplication of factors including fluorescence efficiency made the chemiluminescence from DBPH 20 times intense than that from luminol. 3.2. Measurement of hydrogen peroxide by FIA The optimum chemiluminescence reaction conditions for FIA were examined using 5 X lo-’ M hydrogen peroxide. As a result (Fig. 6) 20 PM DBPH, 12 PM microperoxide and 10 mM phosphate buffer (pH 8.0) gave maximum peak height ratios for the test and blank solutions. Thus, these concentrations and pH were employed for the FIA method. The length of the reaction tubing affected the peak height ratio (Fig. 7). The greatest ratio were achieved at a lo-cm tube. The relationship between the peak heights and the amount of hydrogen peroxide was linear up to at least 60 pmol per loo-p1 injection volume (Fig. 8). The precision was established by repeated determinations (n = 15) using a hydrogen peroxide test solution (20 and 50 pmol/lOO ~1). The relative standard deviations were 5.3 and 0.8%, respectively. The detection limit was 0.5 pmol per 100 ~1 injection
.I. Ishida et al. /Analytica Chimica Acta 309 (1995) 2X1-21 9
219
luminescence by reaction with hydrogen peroxide in the presence of potassium hexacyanoferrate(II1) or peroxidase. The chemiluminescence intensity of DBPH was about 20 times as strong as that of luminol. The reaction was applied to the determination of hydrogen peroxide by FIA. The detection limit was 0.5 pmol for a loo-p1 injection. DBPH is likely to be a useful chemiluminescence probe for highly sensitive analyses, especially for chemiluminescence immunoassays.
Acknowledgements
Fig. 8. Recorder responses for replicated injections of hydrogen peroxide at pH 8.0. Number on the signals indicates the concentration (pmol per injection) of hydrogen peroxide.
volume heights almost blank duced, nation
at a ratio of 2 for the chemiluminescent peak for the test and blank. This sensitivity was the same as that obtained with luminol. If the chemiluminescence from DBPH can be rethe reagent should be useful for the determiof hydrogen peroxide.
4. Conclusions DBPH was newly synthesized as a sensitive chemiluminescent reagent. It produced strong chemi-
We are grateful to Professor M. Nakamura (Fukuoka University) for useful discussion and Miss Y. Takeuchi for her skilful assistance. The financial support of the Grant-in-Aid for Scientific Research (No. 06772117) from the Ministry of Education, Science and Culture of Japan is gratefully acknowledged.
References 111T.E.A. Ahmed and A. Townshend,
Anal. Chim. Acta, 292 (1994) 169. 121K. Robards and P.J. Worsfold, Anal. Chim. Acta, 266 (1992) 147. [31 J. Ishida, M. Takada, T. Yakabe and M. Yamaguchi, Dyes and Pigments, (1994) in press. [41 S. Hara, M. Nakamura, F. Sakai, H. Nohta, Y. Ohkura and M. Yamaguchi, Anal. Chim. Acta, 291 (1994) 189. 151 K. Sasamoto and Y. Ohkura, Chem. Pharm. Bull., 39 (1991) 411.
CHIMICA
ACIA ELSEVIER
Analytica Chimica Acta 309 (1995) 211-219
Development of a novel chemiluminescent probe, 4-t 5’,6’-dimethoxybenzothiazolyl) phthalhydrazide Junichi Ishida a, Maki Takada a, Syuuji Hara a, Kazumi Sasamoto b, Kenyu Kina b, Masatoshi Yamaguchi ap* a Faculty of Pharmaceutical Sciences, Fukuoka Universiry, Nanakuma, Johnan-ku, Fukuoka 814-80, Japan b Dojindo Laboratories, Tabaru 2025-5, Mashiki-machi, Kamimashiki-gun, Kumamoto 861-23, Japan
Received 24 October 1994; revised 20 December 1994; accepted 22 January 1995
Abstract 4-(5’,6’-Dimethoxybenzothiazolyl)phthalhydrazide (DBPH) was synthesized as a new, highly sensitive chemiluminescent reagent. The reagent consists of phthalhydrazide and benzothiazole moieties and generates strong chemiluminescence by reaction with hydrogen peroxide in the presence of potassium hexacyanoferrate(II1) or peroxidase. The chemiluminescence intensity of DBPH was about 2U times greater than that of luminol. The detection limit of DBPH was 1.3 X lo-” M [0.65 fmol per well (200 pl), signal to blank ratio = 21. The reaction of DBPH was also applied to the determination of hydrogen peroxide by flow injection. The detection limit is 0.5 pmol for a lOO-/*I injection. Keywords: Flow injection; Chemiluminescence; Hydrogen peroxide; 4-(5’,6’-Dimethoxybenzothiazolyl)phthalhydrazide
1. Introduction In recent years, chemiluminescence has become an attractive detection method for analytical determinations because of its high sensitivity [1,2]. Luminol chemiluminescence is popular, and is based on the reaction of luminol with hydrogen peroxide in the presence of potassium hexacyanoferrate(II1) or peroxidase as a catalyst. We have studied new, luminol type chemiluminescence reagents which have a higher efficiency than luminol. One of our strategies is to introduce a
* Corresponding
author.
0003-2670/95/$09.50 0 1995 Elsevier Science B.V. AU rights reserved SSDI 0003-2670(95)00082-S
strongly fluorescent group into the reagent. We have reported [3] 6-isothiocyanatobenzo[ glphthalazine1,4(2H,3H)-dione (IPO), 3-propyl-7,&dihydropyridazino[4,5-g]quinoxaline-2,6,9(1 H)-trione (PDIQ) and 3-benzyl-7,8_dihydropyridazino[4,5glquinoxaline-2,6,9(1 Hl-trione (BDIQ) as the new chemiluminescent reagents. They produce chemiluminescence 1.7-5.5 times as intense as luminol. In this study, a novel luminol related compound, 4-(5’,6’-dimethoxybenzothiazolyllphthalhydrazide (DBPH), has been synthesized (Fig. 11, in the hope that it would be a more sensitive chemiluminescent reagent than those mentioned above. This reagent has a phthalhydrazide moiety as a reaction site and 5,6-dimethoxybenzothiazole as an emission site, which is a highly fluorescent compound developed
212
J. Ishida et al. /Analytica
Chimica Acta 309 (1995) 21 l-219
in our laboratory [4]. DBPH was found to produce strong chemiluminescence by reaction with hydrogen peroxide in the presence of potassium hexacyanoferrate(lI1) or peroxidase (Fig. 2). The optimum chemiluminescence reaction conditions were investigated by a manual method. Furthermore, the reagent was evaluated for the determination of hydrogen peroxide, in comparison with lumino1 by flow injection analysis (FIA).
2. Experimental
spectometry (FAB-MS) measurements were made on a JEOL JMS-AX 505W instrument. Uncorrected melting points were obtained on a Yamato MP-21 melting point apparatus. Chromatography was carried out on Kieselgel 60 (Merck, Darmstadt). Chemiluminescence intensities and reaction time courses were measured by a Microluminoreader MLR-100 (Corona) equipped with a Corona DP-50 autopipetter. Uncorrected fluorescence spectra and intensities were measured with a Hitachi 650-60 spectrofluorimeter in 10 X 10 mm quartz cells; a spectral bandwidth of 5 nm was used in both the excitation and emission monochromator.
2.1. Apparatus 2.2. Chemicals ‘H Nuclear magnetic resonance (NMR) spectra were measured on a Bruker AC-2000 spectrometer at 200 MHz using tetramethylsilane as an internal standard. The splitting patterns were designated as follows: s, singlet; d, doublet; m, multiplet; br, broad. IR spectra were taken in KBr disks on a Hitachi 270-30 spectrometer. Fast atom bombardment mass
and solutions
Peroxidase (POD) and microperoxidase (MP, MP11 grade) were purchased from Sigma; these solutions were stored in a refrigerator and used within one week. A stock solution of DBPH (1 X 10m3 M) was prepared in dimethylsulfoxide. A mixed solution of DBPH and MP for FIA was prepared by dilution
N Ph R 0
2 (R = CH3) 3 (R = CHBn) 4 (R = CHO)
CHSO
CH90
CH,O
CH,O 6
5
7
1 Fig. 1. Synthesis of DBPH.
J. Ishida et al. /Analytica
of the stock solutions with 10 mM phosphate buffer (pH 8.0). Luminol was purchased from Nacalai Tesque (Kyoto).
Di(4,5-dimethoxy-2-nitrophenyl)sulfide
4-Methyl-N-phenylphthalimide
(2) was as detailed in the literature
4-Dibromomethyl-N-phenylphthalimide
[51.
was performed
and
according
to the
imide (7)
(4)
A suspension containing 3 (3.30 g, 8.4 mmol) and silver carbonate (6.0 g, 21.8 mmol) in a 1:l (v/v) mixture (150 ml) of tetrahydrofuran and Hz0 was heated at reflux for 24 h. The reaction mixture was concentrated in vacua to a small volume. Then, it was diluted with tetrahydrofuran (500 ml) and filtered through a celite pad. The filtrate was concentrated to give a crude product which was purified by silica gel chromatography with benzene containing 5% of ethyl acetate to give 0.84 g (40%) of aldehyde 4 as a slightly yellow powder. m.p. 179-180” C. IR Vnl,X (cm -‘): 1724 (CHO), 1712 (imide C=O). ‘H NMR (CDCI,): 7.43-7.54 (m, 5H, =NPh), 8.14 (d,
Fig. 2. Suggested
The synthesis literature [41.
(5) (6)
4-(5’,6’-Dimethoxybenzothiazolyl)-N-phenylphthal-
(3)
A solution containing compound 2 (6.99 g, 29.5 mmol), N-bromosuccinimide (10.48 g, 58.9 mmol) and benzoyl peroxide (0.18 g) in Ccl, (175 ml) was refluxed for 16 h. The reaction mixture was filtered and the filtrate was concentrated in vacua to leave a yellowish residue. Silica gel chromatography with benzene afforded 3.39 g (29%) of 3 as an off-white powder. m.p. 178-180” C. IR vmax (cm-‘): 1710 (imide C=O). ‘H NMR (CDCI,): 6.73 (s, lH, CHBr,), 7.36-7.53 (m, 5H, =NPh), 7.96 (s, 2H, H-3 and H-6), 8.19 (s, lH, H-5). FAB-MS: m/z = 396 [M + l]+. 4-Formyl-N-phenylphthalimide
lH, .I = 7.8 Hz, H-6), 8.33 (dd, lH, J = 7.6, 1.4 Hz, H-5) 8.46 (s, lH, H-3) 10.21 (s, lH, CHO). FABMS: m/z = 252 [M + l]+. 4,5-Dimethoxy-2-nitrobromobenzene
2.3. Synthesis of DBPH
The synthesis
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Chimica Acta 309 (1995) 21 I-21 9
Hydrochloric acid (1.5 ml) was added dropwise at 40-50” C to a stirred suspension containing disulfide 6 (100 mg, 0.23 mmol) and tin powder (0.40 g) in ethanol (15 ml); the mixture was refluxed for 1 h, during which time all of the starting material dissolved. The reaction mixture was filtered after being cooled to room temperature. To this filtrate was added aldehyde 4 (155 mg, 0.62 mmol) and the mixture was refluxed for 1 h. The initial suspension turned clear and then a precipitate formed almost instantly. After the reaction, the solids were collected and dried in vacua. Chromatography with CHCl, containing 35% of methanol gave 68 mg (35%) of compound 6 as a light yellow crystalline solid. m.p. 294-295” C. Fluorescence (CHCI,): A,, = 520 nm (excited at A,, = 400 nm). ‘H NMR (CDCI,): 4.01 (s, lH, OCH,), 4.02 (s, lH, OCH,), 7.35 (s, lH, H-4’), 7.43-7.55 (m, 5H, =NPh), 7.58 (s, lH, H-7’), 8.05 (d, lH, H-3, J= 8.5 Hz, H-6). 8.49 (dd, lH, J = 8.4, 1.9 Hz, H-5), 8.57 (d, lH, J = 1.7 Hz, H-3). FAB-MS: IR vmax (cm -I): 1722 (imide C=O). m/z = 417 [M + l]+. 4-(5’,6’-Dimethoxybenzothiazolyl)phthalhydrazide
(1) Hydrazine hydrate (0.30 ml) was added to a suspension of compound 5 (70.5 mg, 0.17 mmol) in a
chemiluminescence
reaction of DBPH.
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1:l (v/v) mixture (3 ml) of ethanol and dioxane. The mixture was refluxed for 30 min. The precipitate was collected by filtration, washed excessively with ethanol and dioxane, and dried in vacua over P,O,, yielding 61.3 mg (100%) of hydrazide 1 as an offwhite powder. m.p. > 300” C. ‘H NMR (DMSO-d,): 3.88 (s, lH, OCH,), 3.89 (s, lH, OCH,), 4.58 (br, 14H, Hydrazide), 7.68 (s, lH, H-4’) 7.75 (s, lH, H-7’), 8.19 (d, lH, J = 8.5 Hz, H-6), 8.41 (dd, lH,
J = 8.4, 1.9 Hz, H-5), 8.63 (d, lH, J = 1.7 Hz, H-3). IR VIII,, (cm-‘): 3328 (lactam NH), 1634 (lactam C=O). FAB-MS: m/z = 3.56 [M + l]+. 2.4. Procedure for properties
(1
examining
chemiluminescent
A 50-~1 portion of the reagent (DBPH) solution 10e7 M) was placed into a well of a black
x
(A)
Hydrogm
prroxl6r
(mhl)
K3Fe(CN)O
(mu)
0 0
1
2
Sodium
hydroxlde
3
(M)
Fig. 3. Effects of (A) hydrogen peroxide, (B) potassium hexacyanoferrate(II1) and (C) sodium hydroxide concentrations on integrated chemiluminescence intensities (a run time of 20.5 s). (1) DBPH (or (2) luminol) (1 X lo-’ M) was treated according to the procedure with various concentrations of hydrogen peroxide, potassium hexacyanoferrate(II1) and sodium hydroxide.
J. Ishida et al. /Analytica
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microplate. The black microplate was inserted into the microluminoreader. (i) Chemiluminescence with potassium hexacyanoferrate(II1). The chemiluminescence reaction was initiated by simultaneous automatic injections of 50 ~1 of hydrogen peroxide solution (l-40 mM) and 100 ~1 of potassium hexacyanoferrate(II1) (l-80 mM) in sodium hydroxide (0.5-3.0 M). (ii) Chemiluminescence with POD. The chemiluminescence reaction was initiated by automatic injec-
tions of 50 ~1 of the hydrogen peroxide solution (l-40 mM) and 100 ml of POD (l-55 U) in 10 mM phosphate buffer (pH 6.0-9.0). Chemiluminescence intensities were monitored immediately after the automatic injection. 2.5. FIA with chemiluminescence detection The mixture of DBPH (2 X 10m5 M) and microperoxidase (1.2 X 10m5 M) in 10 mM phosphate
(8) 2OOow
0 0
10
20
Time
(8)
20
10
Time
(8)
Fig. 4. Effects of (A) hydrogen peroxide, (B) potassium hexacyanoferrate(II1) and (C) sodium hydroxide concentrations on the time courses reaction of DBPH. Curves: (A, mM) 1, 1; 2, 5; 3, 10; 4, 20; 5, 30; 6, 40. (B, mM) 1, 1; 2, 5; 3, 10; 4, 20, 5, 30; 6, of the chemiluminescence 40; 7, 80. (C, M) 1, 0.5; 2, 0.8; 3, 1.0; 4, 1.2; 5, 1.5; 6, 2.0; 7, 3.0.
216
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Chimica Acta 309 (1995) 211-219
buffer (pH 8.0) was delivered by a Jasco 880-PU pump equipped with a Rheodyne 7125 syringe-loading sample injector valve (loo-~1 loop). Hydrogen peroxide solution was injected into the flowing reagent solution. The flow-rate was 0.5 ml min- ‘. The temperature was ambient (23 f 2” Cl. The generated chemiluminescence was monitored by an ATT0 (Tokyo) AC-2220 luminomonitor equipped with a 60-~1 flow cell. Stainless-steel tubing (0.5 mm i.d.1 was used for the FIA system.
3. Results and discussion 3.1. Chemiluminescent properties of DBPH DBPH was evaluated as a chemiluminescent reagent by comparing it with luminol at the same concentration (1 X 10e7 M). DBPH produces chemiluminescence by reaction with hydrogen peroxide in the presence of potassium hexacyanoferrate(II1) or peroxidase.
(A) r”
600000
‘,
6OG000 *
Woo00
..
5owoo-
4ooow
.,
400000-
3ooom
-’
3OOwo-
I
E
W
a :
r f
E
J
200oDo~
b
lOcmQ-
= E 0
200000-
100000~
2 047.
,
I)
10
.
,
,
20
30
Hydrogen
prroxlde
.
.,
.
40
50
0
(mu)
10
7
20
POD
30
40
50
60
(unltr/l00~l)
sooooo”
4owoo
,.
1ooooo -*
O..,T 5
. 6
, 7
.
I
6
,
1
.
9
PH Fig. 5. Effects of (A) hydrogen peroxide, (B) POD units and (C) pH of the phosphate run time of 20.5 s). Curves: 1, DBPH, 2, luminol.
buffer on integrated
chemiluminescence
intensities (a
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Chimica Acta 309 (1995) 21 I-21 9
reaction of DBPH. The chemiluminescence was initiated by addition of hydrogen peroxide and potassium hexacyanoferrate(II1) to the compound solution. The chemiluminescence intensity reached a maximum at < 1.5 s after the addition and then decreased rapidly. The hydrogen peroxide concentration did not influence the time course of the chemiluminescence reaction, but it was affected by changes of the concentration of potassium hexacyanoferrate(II1) and sodium hydroxide. In both cases, the rate of the chemiluminescence reaction was much faster as their concen-
In the case of chemiluminescence using potassium hexacyanoferrate(II1) as catalyst, the concentrations hydrogen peroxide, potassium of hexacyanoferrate(II1) and sodium hydroxide influenced the chemiluminescence intensity (Fig. 3); 10 mM hydrogen peroxide, 5 mM potassium hexacyanoferrate(II1) and 1.5 M sodium hydroxide gave almost the maximum intensity. Fig. 4 shows the effect of the concentrations of hydrogen peroxide, potassium hexacyanoferrate(II1) and sodium hydroxide on the time course of the chemiluminescence
W
2
4
DBPH
0
6
(x10-5
M)
3
2
1
MP
(x10-5
M)
G)
6
9
10
11
PH Fig. 6. Effects of (A) DBPH cow.,
(B) microperoxidase
cow. and (C) pH of the phosphate
buffer on peak height.
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tration increased. Luminol showed almost the same decay curves as those of DBPH. The calibration curve for DBPH was linear (r = 0.999) up to at least 1 X lop6 M under the optimum reaction conditions. The limit of detection for DBPH was 1.3 X lo- ’ 1 M (0.65 fmol per well), which gave a chemiluminescence intensity of twice the blank. This value was about 20 times lower as that of luminol. The precision was established by repeated determinations (n = 15) using DBPH solutions of 1 X 10e8 and 1 X lo-’ M. The relative standard deviations were 6.3 and 4.2%, respectively. In the case of chemiluminescence using peroxidase as a catalyst, the concentrations of hydrogen peroxide and peroxidase and the pH value of the reaction medium influenced the chemiluminescence intensity (Fig. 5); 20 mM hydrogen peroxide, 22 U of peroxidase and a pH of 7.0 gave the maximum intensity. DBPH produces the maximum chemiluminescence intensity at neutral pH. The calibration graph for DBPH was linear (I = 0.998) up to at least 1 X lop6 M under the optimum reaction conditions. The limit of detection for DBPH was 5.9 X lo-” M (3.0 fmol per well). In the chemiluminescence of luminol, the 3aminophthalate ion produced during the reaction has been proved to be the light emitter. Therefore, in the case of DBPH, the corresponding phthalate ion (Fig. 2) is expected to be the light-emitting species. The fluorescence properties (fluorescence excitation and emission maxima and the relative intensity) of this species was measured in comparison with luminol after the chemiluminescence reaction was completed under the optimum reaction conditions in each case (Table 1). The fluorescence from DBPH after the reaction was about 500 times more intense than that from luminol, but the chemiluminescence from DBPH is 20 times more intense as described above.
Table 1 Fluorescence excitation (Ex) and emission (Em) maxima and relative intensities (RF11 of the reagents after the chemiluminescence reaction Reagent
Ex (nml
Em (nm)
RFIa
Luminol DBPH
316 361
450 448
100.0 51344.5
aFluorescence
intensity of luminol was taken as 100.0.
ao-
Tube
length
(cm)
Fig. 7. Effect of the tubing length between the injector chemiluminescence detector on the peak height.
and
It is known that the efficiency of the chemiluminescent compound is partly dependent on the fluorescence intensity of the light-emitting species which produced during the reaction. However, other factors such as energy transfer relate to the chemiluminescence production. Therefore, the multiplication of factors including fluorescence efficiency made the chemiluminescence from DBPH 20 times intense than that from luminol. 3.2. Measurement of hydrogen peroxide by FIA The optimum chemiluminescence reaction conditions for FIA were examined using 5 X lo-’ M hydrogen peroxide. As a result (Fig. 6) 20 PM DBPH, 12 PM microperoxide and 10 mM phosphate buffer (pH 8.0) gave maximum peak height ratios for the test and blank solutions. Thus, these concentrations and pH were employed for the FIA method. The length of the reaction tubing affected the peak height ratio (Fig. 7). The greatest ratio were achieved at a lo-cm tube. The relationship between the peak heights and the amount of hydrogen peroxide was linear up to at least 60 pmol per loo-p1 injection volume (Fig. 8). The precision was established by repeated determinations (n = 15) using a hydrogen peroxide test solution (20 and 50 pmol/lOO ~1). The relative standard deviations were 5.3 and 0.8%, respectively. The detection limit was 0.5 pmol per 100 ~1 injection
.I. Ishida et al. /Analytica Chimica Acta 309 (1995) 2X1-21 9
219
luminescence by reaction with hydrogen peroxide in the presence of potassium hexacyanoferrate(II1) or peroxidase. The chemiluminescence intensity of DBPH was about 20 times as strong as that of luminol. The reaction was applied to the determination of hydrogen peroxide by FIA. The detection limit was 0.5 pmol for a loo-p1 injection. DBPH is likely to be a useful chemiluminescence probe for highly sensitive analyses, especially for chemiluminescence immunoassays.
Acknowledgements
Fig. 8. Recorder responses for replicated injections of hydrogen peroxide at pH 8.0. Number on the signals indicates the concentration (pmol per injection) of hydrogen peroxide.
volume heights almost blank duced, nation
at a ratio of 2 for the chemiluminescent peak for the test and blank. This sensitivity was the same as that obtained with luminol. If the chemiluminescence from DBPH can be rethe reagent should be useful for the determiof hydrogen peroxide.
4. Conclusions DBPH was newly synthesized as a sensitive chemiluminescent reagent. It produced strong chemi-
We are grateful to Professor M. Nakamura (Fukuoka University) for useful discussion and Miss Y. Takeuchi for her skilful assistance. The financial support of the Grant-in-Aid for Scientific Research (No. 06772117) from the Ministry of Education, Science and Culture of Japan is gratefully acknowledged.
References 111T.E.A. Ahmed and A. Townshend,
Anal. Chim. Acta, 292 (1994) 169. 121K. Robards and P.J. Worsfold, Anal. Chim. Acta, 266 (1992) 147. [31 J. Ishida, M. Takada, T. Yakabe and M. Yamaguchi, Dyes and Pigments, (1994) in press. [41 S. Hara, M. Nakamura, F. Sakai, H. Nohta, Y. Ohkura and M. Yamaguchi, Anal. Chim. Acta, 291 (1994) 189. 151 K. Sasamoto and Y. Ohkura, Chem. Pharm. Bull., 39 (1991) 411.