Synthesis of 4-(7-Diethylaminocoumarin-3-yl)benzeneisocyanate (DACB-NCO): A highly sensitive fluorescent derivatization reagent for alcohols in high-performance liquid chromatography [1]

Article abstract of DOI:10.1002/jhet.5570380202

4-(7-Diethylaminocoumarin-3-yl)benzeneisocyanate (DACB-NCO) was synthesized as a new fluorescent derivatization reagent for alcohols for use in high-performance liquid chromatography (hplc). Saturated alcohols (C₆-C₂₂) were derivatiz

Full text of DOI:10.1002/jhet.5570380202

Mar-Apr 2001
Synthesis of 4-(7-Diethylaminocoumarin-3-yl)benzeneisocyanate
(DACB-NCO): A Highly Sensitive Fluorescent Derivatization Reagent
for Alcohols in High-Performance Liquid Chromatography [1]
Haruko Takechi*, Yasuo Goto, Hajime Takahashi and Minoru Machida
333
Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido,
Ishikari-Tobetsu, Hokkaido 061-0293, Japan
Received June 7, 2000
4-(7-Diethylaminocoumarin-3-yl)benzeneisocyanate (DACB-NCO) was synthesized as a new fluorescent
derivatization reagent for alcohols for use in high-performance liquid chromatography (hplc). Saturated
alcohols (C -C ) were derivatized in good yields into the corresponding fluorescent DACB-carbamic esters
6
22
by treating with DACB-NCO. The DACB-carbamic esters of these alcohols were clearly separated on a
reversed-phase hplc column (Inertsil ODS-2, mobile phase: methanol-water, excitation wavelength 402 nm;
emission wavelength 488 nm). The detection limit (S/N = 3) of cetyl alcohol, as a test compound, was 5
fmol/10 µl.
J. Heterocyclic Chem., 38, 333 (2001).
Various fluorescent derivatization reagents have been
proposed for the determination of alcohols by high-perfor-
mance liquid chromatography (hplc) [2-11]. In the
previous paper, we have shown that 3-aryl-7-dialkyl-
aminocoumarin was one of the promising candidates as a
fluorogenic group, and developed the derivatization
reagent, 4-(7-diethylamino-coumarin-3-yl)benzoyl
cyanide (DACB-CN) for alcohols [2]. Although
DACB-CN was one of the most sensitive fluorescent
derivatization reagents (detection limit: 1-2 femtomol per
injection volume), this reagent required the derivatization
reaction in open vial because of low reactivity to alcohols
in the reaction medium of highly diluted solution. The
present paper deals with the preparation of 4-(7-diethyl-
aminocoumarin-3-yl)benzeneisocyanate (DACB-NCO, 3)
having an isocyanate moiety as the reacting group, and its
fundamental studies on its properties as a new
derivatization reagent for hplc analysis of alcohols.
(ε = 38,200) and 482 nm, respectively. DACB-NCO is
stable in the crystalline state for at least a year in daylight
at room temperature.
Preparation of Standard Samples (DACB-Carbamic
Esters, 4a-k) and their Spectroscopic Properties.
The standard samples of DACB-carbamic esters (4a-k)
were obtained by the reaction of a series of saturated
alcohols (C -C ) with an equimolar amount of
6
22
DACB-NCO in acetonitrile (room temperature for 2.0
hours) in the presence of 4-dimethylamino-pyridine
(DMAP) (4 equivalents). The structures of DACB-
carbamic esters (4a-k) were determined on the basis of
spectral and analytical data (Table 1). All the DACB-
derivatives (4a-k) had absorption maxima (λmax) and
fluorescence maxima (F.λmax) at 402 nm and 487-489
nm, respectively, with much the same values of molar
absorptivity and fluorescence intensity in ethanol
(Table 1). DACB-carbamic esters (4a-k) were stable in
ethanol solution for at least a year in a refrigerator.
Synthesis of DACB-NCO (3).
DACB-NCO (3) was synthesized by the route shown in
Scheme 1. The absorption maximum and the fluorescence
maximum of 3 in acetonitrile appeared at 401 nm
The fluorescence properties of DACB-carbamic ester of
myristyl alcohol (4e) were examined in several solvent
systems often used in reversed-phase chromatography

334
H. Takechi, Y. Goto, H. Takahashi and M. Machida
Vol. 38
Table 1
Physical Properties of DACB-Carbamic Esters (4a-k)
[a]
[b]
Mp(°C)
Formula
Molecular Formula Analysis (%)
Absorption
RFI (488 nm)
(Ex 403 nm)
Compound
Calcd
H
Found
H
ε (λmax nm)
C
N
C
N
4a
4b
4c
4d
4e
4f
4g
4h
4i
158-160
158-160
146-147
137-138
132-134
134-135
131-132
135-136
129-130
119-120
130-131
C
C
C
C
C
C
C
C
C
C
C
H
N O
71.53
72.38
73.14
73.81
74.41
74.69
74.96
75.21
75.45
75.90
76.32
7.39
7.81
8.18
8.52
8.82
8.96
9.09
9.21
9.33
9.56
9.76
6.42
6.03
5.69
5.38
5.11
4.98
4.86
4.74
4.63
4.43
4.24
71.54
72.28
72.91
73.77
74.43
74.76
75.05
75.23
75.30
75.98
76.44
7.50
7.71
8.26
8.57
8.85
9.10
9.12
9.40
9.26
9.65
9.82
6.52
6.02
5.65
5.30
5.31
4.84
5.01
4.77
4.74
4.44
4.24
38,600 (402)
38,600 (402)
38,100 (402)
38.900 (402)
38,500 (402)
38,500 (402)
38,900 (402)
38,200 (402)
38,500 (402)
38,600 (402)
38,600 (402)
1.01
1.02
1.00
1.01
1.00
1.00
0.98
1.00
1.02
1.02
1.01
26 32
2
4
4
4
4
4
4
4
4
4
4
4
H
N O
28 36
2
H
N O
2
30 40
H
N O
32 44
2
H
N O
2
34 48
H
N O
35 50
2
H N O
36 52 2
H
N O
2
37 54
H
N O
38 56
2
4j
4k
H N O
40 60 2
H
N O
42 64
2
-5
[a] Concentration in ethanol: 2.0 X 10 M; [b] Relative fluorescence intensity: the compound 4c is arbitrarily taken as 1.00. Concentration in
-6
ethanol: 4.5 X 10
M
(Table 2). As the solvent polarity decreases, both the
absorption maxima and the fluorescence maxima shift
slightly toward the blue. However, the variations in the
shift values of the F.λmax and fluorescence intensities of
4e in each solvent are so small that it is possible to separate
and detect various alcohols by using a gradient elution
technique.
Table 2
Absorption and Fluorescence Properties
of 4e in Various Solvent Systems
[a]
[b]
Absorption
Fluorescence
Solvent
[c]
λmax
(nm)
ε
Ex
(nm)
F.λmax
(nm)
RFI
Optimal Conditions for Hplc Analysis of Alcohols.
ethanol
methanol
acetonitrile
methanol:water
(90:10, v/v)
402
403
399
406
38,500
38,800
39,100
38,800
403
403
399
407
488
489
481
491
1.00
1.06
1.04
1.06
In order to examine the optimal conditions for hplc
analysis, the derivatization yield of cetyl alcohol, a test
compound, with DACB-NCO was estimated by comparing
the fluorescence intensity of the product (4g) at 488 nm
with that of an internal standard 4e. The derivatization
reactions [cetyl alcohol (0.01 µmol); DACB-NCO (0.05
µmol); I.S (0.01 µmol)] in chloroform (70 µl), were
examined in sealed vials at 40, 60, 80, 100, and 120° for
0-120 minutes, respectively. At 60°, the peak area increased
slowly; higher temperatures allowed the fluorescence to
develop more rapidly. The peak heights reached a
maximum and constant after heating at 120° for 10
minutes. Further, the derivatized products were obtained in
good yields in acetonitrile (91%) and chloroform (88%).
Chloroform was employed as a solvent due to the insolu-
bility of DACB-NCO. As for base catalysts, DMAP gave
good results in the preparative scale reaction at room
temperature, but the derivatization reaction at 120°
proceeded in the absence of DMAP. Furthermore, the
respective various concentrations of DACB-NCO for
derivatization of cetyl alcohol were examined. The results
indicated that a 3-5-fold molar excess of reagent for cetyl
alcohol is suitable. Consequently, the derivatization of
alcohols was carried out under the following conditions:
heating at 120° for 30 minutes in chloroform with 5-fold
molar excess of DACB-NCO, in the absence of base
catalysts in sealed vials.
-5
[a] Concentration in ethanol: 2.0 X 10 M; [b] Concentration in ethanol:
4.5 X 10 M; [c] Relative fluorescence intensity: the fluorescence
intensity in ethanol is arbitrarily taken as 1.00.
-6
Regarding the calibration curve, a linear relationship
between the ratio of the peak areas of cetyl alcohol DACB-
carbamic ester (4g) to that of the internal standard (4e) and
the amount of cetyl alcohol was observed in the range of
50 fmol - 100 pmol/injection volume (10 µl) of the alcohol
(linear correlation coefficient: 0.999), and the detection
limit in this case was 5 fmol/10 µl (S/N=3).
Next, the simultaneous determination of saturated
alcohols (C -C ) was examined. As shown in Figure 1,
peaks corresponding to the DACB-derivatives of a series
of saturated alcohols (C -C ) (4a-k) were completely
separated by gradient elution with methanol-water as the
mobile phase within 30 minutes (Figure 1).
6
22
6
22
The precision of analysis was established by repeated
determinations (n = 7) using a mixture of ten alcohols
(5 pmol each per 10 µl). The yields of the fluorescent
derivatives under these conditions are 78% (4a for hexyl
alcohol), 86% (4b for octyl alcohol), and 88-98% for long
chain alcohols (4c,d,f-k; C -C ). Further, the relative
10 22

Mar-Apr 2001
Synthesis of 4-(7-Diethylaminocoumarin-3-yl)benzeneisocyanate (DACB-NCO)
335
excess. The reaction of DACB-NCO in sealed vials
allowed for the determination of lower molecular weight
alcohols (the number of carbons: C or above) than those
6
examined in the case of DACB-CN.
DACB-NCO
Comparison of the Spectroscopic Properties of DACB-
Carbamic Ester (4a) with those of DACB-Ester (5), MAC-
Carbamic Ester (6), OBM-Ester (7) and 9-AN-Ester (8).
4d
4e
4f
4g
4h
To confirm the utility of 3-aryl-7-dialkylaminocoumarin
as a fluorogenic group, we compared the spectroscopic
properties of both DACB-carbamic ester (4a) and DACB-
ester (5) with those of ethyl 7-dimethylaminocoumarin-3-
carbamate (MAC-carbamic ester, 6) [3b], 2-(5-ethoxy-
carbonyl-2-oxazolyl)-5,6-methylenedioxybenzofuran
(OBM-ester, 7) [7b], and methyl anthracene-9-carboxylate
(9-AN-ester, 8) [6]. As shown in Table 3, the quantum
yields of 4a (0.70) and 5 (0.75) in ethanol were
approximately equal to that of 6 (0.72) and 7 (0.83). Since
the fluorescence sensitivity is proportional to both the
molar absorptivity (ε) and quantum yield (φ), the
fluorescence sensitivity is generally defined as ε x φ
(Table 3) [12]. The relative fluorescence sensitivity of
DACB-ester (5) is stronger than that of OMB-ester (7) and
24 times as strong as that of 9-AN-ester (8) because of the
large molar absorptivity (42,200) of 5. On the other hand,
the fluorescence sensitivity of carbamic ester 4a is nearly
equal to that of benzofuran derivative (7) and hexyl
3,4-dihydro-6,7-dimethoxy-4-methyl-3-oxo-quinoxaline-
2-carbamate [8b]. These results show that DACB-CN and
DACB-NCO having a 3-phenyl-7-diethylaminocoumarin
moiety as a fluorogenic group are excellent derivatization
reagents for use in hplc with fluorescence detection.
4c
4b
4i
4j
4a
4k
0
5
10
15
20
25
30
Retention Time (min)
Figure 1. Chromatogram of the DACB-Carbamic Esters of Saturated
Alcohols (4a-k). The derivatization was carried out as described in the
experimental section. Each peak area corresponds to 5 pmol alcohol.
pH Dependence of Absorption and Fluorescence Spectra
of 3-Phenyl-7-diethylaminocoumarin Derivative (9).
standard deviations were 8% for hexyl alcohol, 6% for
octyl alcohol, 3% for decyl alcohol, and did not exceed 1%
Since 7-diethylamino-3-phenylcoumarin, the
fluorogenic group in DACB-ester and DACB-carbamic
ester, contains the basic diethylamino functional group, the
absorption and fluorescence spectra of these derivatives
are dependent on the pH of the solution. To investigate the
effect of pH, for this fluorogenic group, 7-(diethylamino)-
3-[4-(hydroxymethyl)phenyl]-2H-1-benzopyran-2-one (9)
[13] was used because of its solubility in phosphate buffer
solutions. Figure 2 shows the pH dependence of
absorbance at 409 nm and the fluorescence intensity at 498
nm of 9. Apparently the diethylamino group of 9 is not
protonated above pH 4, and the results obtained are in
agreement with those of N-(7-dimethylamino-4-methyl-3-
coumarinyl)succinimide (DACS) [14].
for long chain alcohols (C -C ).
12 22
In addition, the reactivities of DACB-NCO (3) toward
secondary and tertiary hydroxy groups were also
examined. Dihydrocholesterol (a secondary alcohol) was
also derivatized in good yield (88%) under the same
condition, however tertiary butyl alcohol was almost inert
(< 5%) toward this derivatization reagent.
As shown in a previous paper [2], the reactivity of
DACB-CN was somewhat lower for hydroxy group of
alcohols, so that derivatization with DACB-CN was
carried out under the conditions where the solvent is
distilled away during the reaction in open vials. Because at
least 20-fold molar excess of DACB-CN is required for a
quantitative derivatization of alcohols, tailing is observed
on the chromatogram resulting from carboxylic acid
generated by the hydrolysis of DACB-CN during the
derivatization reaction. Because DACB-NCO (3) is more
reactive toward the hydroxy group than DACB-CN only a
slight excess of reagent is required, rather than the 3-5-fold
Conclusion.
The newly developed fluorescent derivatization
reagent, DACB-NCO, for alcohols has proved to be
satisfactory with respect to sensitivity and reactivity.
Thus, DACB-NCO as well as DACB-CN would be
applicable to hplc analysis of trace amounts of primary

336
H. Takechi, Y. Goto, H. Takahashi and M. Machida
Vol. 38
Table 3
Absorption and Fluorescence Properties of Derivatized Compounds
[a]
[b]
Absorption
λmax (nm)
Fluorescence
Ex (nm)
[c]
[d]
Compound
ε
F.λmax (nm)
φ
φ x ε
RFS
DACB-Carbamic ester (4a)
DACB-Ester (5)
MAC-Carbamic ester (6)
OMB-Ester (7)
402
413
378
350
362
38,600
42,200
23,700
32,900
7,800
403
413
378
352
363
489
488
480
443
447
0.70
0.75
0.72
0.83
0.17
27000
31700
17100
27300
1300
(21)
(24)
(13)
(21)
(1)
9-AN-Ester (8)
-5
-6
[a] Concentration in ethanol: 2.0 x 10 M; [b] Concentration in ethanol: 4.5 x 10 M; [c] Quantum yield; [d] Relative fluorescence sensitivity: the
compound 8 is arbitrarily taken as 1.00.
EXPERIMENTAL
All melting points were determined on a Yamato melting point
apparatus (Yanaco MP-J3) and are uncorrected. Ir were recorded
1
on JASCO FT/IR-300 spectrometer. The H nmr spectra were
acquired on either a JEOL JMN-LA-300 or JEOL JNM-EX-400
spectrometer. Ms were determined using a Shimadzu GC
MS-9100-MK gas chromatograph-mass spectrometer with a
direct inlet system. Absorption and fluorescence spectra were
measured with a Hitachi 288 dual-wavelength spectrophotometer
and a Hitachi F-4500 fluorescence spectrophotometer, which is
equipped with a R928F photomultiplier (200-900 nm).
Fluorescence quantum yields were determined according to the
method of Parker and Rees [15], as reported previously [16], in
0.2
0.1
0
1
-2
which quinine sulfate in 5 x 10 M sulfuric acid was used as a
0.5
standard. The hplc system consisted of a Hitachi L-6200 pump, a
Rheodyne Model 7125 injector valve, a Hitachi F-1040
fluorescence spectrophotometer, a Hitachi D-2500 chromato-
integrator and a Gasukuro Kogyo Model-545 degassing unit. The
column was Inertsil ODS-2 (150 x 4.6 mm i.d.; particle size,
5 µm; Gasukuro Kogyo, Tokyo).
0
ph
Synthesis of 4-[7-(Diethylamino)-2-oxo-2H-1-benzopyran-3-
yl]benzeneisocyanate [4-(7-Diethylaminocoumarin-3-yl)-
benzeneisocyanate (DACB-NCO, 3)].
Figure 2. pH Dependence of the Absorption and Fluorescence Spectra of
9 in 0.1 M Phosphate Buffer.
A mixture of 4-[7-(diethylamino)-2-oxo-2H-1-benzopyran-3-
yl]benzoyl chloride (1, 1.78 g, 5.0 mmoles) prepared from
carboxylic acid [2] and oxalyl chloride in the usual manner, and
hydrazine monohydrate (0.5 ml, 10 mmoles) in THF (200 ml) was
stirred for 1.5 hours at room temperature. The resulting precipitates
alcohols
and certain secondary alcohols such as steroids
in biological fluids, with satisfactory accuracy and
reliability at a femtomol level.

Mar-Apr 2001
Synthesis of 4-(7-Diethylaminocoumarin-3-yl)benzeneisocyanate (DACB-NCO)
337
were collected and recrystallized from chloroform-hexane to give
2-(5-Ethoxycarbonyl-2-oxazolyl)-5,6-methylenedioxybenzo-
4-[7-(diethylamino)-2-oxo-2H-1-benzopyran-3-yl]benzene-
furan (OMB-ester, 7).
carbohydrazide (2) (1.45 g, 82%). Yellow needles, mp 224-226°; ir
Compound 7 was prepared according to the reported method
[7]. Pale yellow prisms (from ethyl acetate-hexane), mp
174-175°, lit mp 186° [7].
-1 1
(nujol): 3340, 3280, 1705 cm ; H nmr (dimethyl-d sulfoxide, 400
6
MHz): δ 1.14 (t, J = 7Hz, 6H, N(CH CH ) ), 3.46 (q, J = 7Hz, 4H,
2
3 2
N(CH CH ) ), 4.75 (bs, 2H, N-NH ), 6.57 (s, 1H, ArH), 6.75
2
3 2
2
(d, J = 9Hz, 1H, ArH), 7.53 (d, J = 9Hz, 1H, ArH), 7.80 (d, J = 7Hz,
2H, ArH), 7.87 (d, J = 7Hz, 2H, ArH), 8.18 (s, 1H, ArH), 9.82
Methyl 9-Anthracenecarboxylate (9-AN-ester, 8).
A mixture of the 9-anthracenecarboxylic acid (225 mg,
1.0 mmole) and (trimethylsilyl)diazomethane (2.5 ml,
5.0 mmoles) in methanol (6 ml) was refluxed overnight. The
reaction mixture was evaporated to dryness in vacuo, and the
residue was treated with ethyl acetate and water. The organic
layer was washed with brine, dried over magnesium sulfate and
evaporated. The residue was chromatographed on silica gel (ethyl
acetate-hexane, 1:4, v/v) to give 8 (223 mg, 95%). Pale yellow
prisms (from ethyl acetate-hexane), mp 110-111°; ir (nujol): 1730
(s, 1H, CONH); high-resolution ms: m/z Calcd. for C H N O :
20 21
3 3
+
351.1583 (M ). Found: 351.1554.
To a suspension of 2 (1.05 g, 3.0 mmoles) in water (24 ml) was
added acetic acid (30 ml). Sodium nitrite (207 mg, 3.0 mmoles) in
water (15 ml) was then added slowly to the solution under ice-
cooling and the reaction mixture was stirred at same temperature for
30 minutes. The precipitated solid was filtered, washed with cold
water and dried in vacuo. The resulting carbonyl azide was heated at
90° in toluene (120 ml) for 2 hours. The insoluble part was filtered
off and the filtrate was concentrated to one-third of its original
volume. Subsequently hexane was added to the concentrated
solution to give precipitates. The precipitates were collected by
filtration and recrystallized from ethyl acetate-hexane to give
DACB-NCO (3) (0.67 g, 67 %). Yellow prisms, mp 142-143°; ir
-1
1
cm ; H nmr (deuteriochloroform, 400 MHz): δ 4.18 (s, 3H,
OCH ), 7.4-7.6 (m, 4H, ArH), 8.0-8.1 (m, 4H, ArH), 8.52 (s, 1H,
3
+
ArH); ms: m/z 236 (M ).
Anal. Calcd. for C
H O : C, 81.34; H, 5.12. Found: C,
16 12 2
81.50; H, 5.17.
-1 1
(nujol): 2265, 1705 cm ; H nmr (deuteriochloroform, 400 MHz): δ
Derivatization Procedure and Hpcl Conditions.
1.23 (t, J = 7Hz, 6H, N(CH CH ) ), 3.43 (q, J = 7Hz, 4H,
2
3 2
Stock solutions of alcohol (1.0 mM), DACB-NCO (3, 5.0
mM), and internal standard (4e, 1.0 mM) were prepared in
chloroform. To a 10 µl of a test solution of alcohol were sequen-
tially added 50 µl of DACB-NCO and 10 µL of 4e solutions. The
vial was tightly capped and heated at 120° for 30 minutes. After
cooling, the reaction mixture was diluted with acetonitrile to
5 ml, and then an aliquot (10 µl) of the mixture was injected into
the liquid chromatograph. The eluent from the column was
monitored with a fluorophotometric detector at an excitation
wavelength of 402 nm and an emission wavelength of 488 nm.
The eluent flow-rate was 1.0 ml/minute.
N(CH CH ) ), 6.53 (d, J = 2Hz, 1H, ArH), 6.59 (dd, J = 9, 2Hz, 1H,
2
3 2
ArH), 7.12 (d, J = 7Hz, 2H, ArH), 7.31 (d, J = 9Hz, 1H, ArH), 7.67
+
(d, J = 7Hz, 2H, ArH), 7.68 (s, 1H, ArH); ms: m/z 334 (M ).
Anal. Calcd. for C
H N O : C, 71.84; H, 5.43; N, 8.38.
20 18 2 3
Found: C, 71.59; H, 5.63; N, 8.18.
Syntheses of Alkyl 4-[7-(Diethylamino)-2-oxo-2H-1-benzopyran-
3-yl]phenyl Carbamates [DACB-Carbamic Esters (4a-k)].
General Procedure.
A mixture of DACB-NCO (3, 100 mg, 0.3 mmole), an alcohol
(C -C ) (0.3 mmole), and DMAP (147 mg, 1.2 mmoles) in
6
22
Calibration Curve for Formation of 4g
acetonitrile (9 ml) was stirred at room temperature for 2 hours.
The solvent was evaporated in vacuo and the residue was
chromatographed on silica gel (benzene-hexane-ethyl acetate,
10:3:1, v/v) to give the corresponding DACB-carbamic esters
(4a-k). Compounds 4a-k were obtained in 64-86% yields.
Physical properties and spectral data for DACB-carbamic esters
are listed in Table 1.
Stock solutions of cetyl alcohol (0.25 µM-5.0 mM), DACB-
NCO (3, 5.0 mM), and internal standard (4e, 1.0 µM-1.0 mM)
were prepared in chloroform. The derivatization reaction and
detection were carried out according to the standard procedure
described above.
Simultaneous Determination of Saturated Alcohols (4a-d,4f-k).
Syntheses of Fluorescent Derivatized Compounds (5-8).
Stock solutions of ten alcohols (C -C ) (0.25 mM each),
6
22
Ethyl 4-[7-(diethylamino)-2-oxo-2H-1-benzopyran-3-yl]-
benzoate (DACB-ester, 5) was prepared according to the reported
method [2]. Yellow needles (from ethyl acetate-hexane), mp
DACB-NCO (3, 2.5 mM), and internal standard (4e, 0.25 mM)
were prepared in chloroform. The derivatization reaction and
detection were carried out according to the standard procedure
described above. Simultaneous determination was attained by
gradient elution with methanol/water (methanol concentration
in the mobile phase: 95%, 0-8 minutes; 95-100%, 8-30
minutes).
1
144-145°; H nmr (deuteriochloroform, 400 MHz): δ 1.23 (t, J =
7Hz, 6H, N(CH CH ) ), 1.41 (t, J = 7Hz, 3H, OCH CH ) 3.44
2
3 2
2
3
(q, J = 7Hz, 4H, N(CH CH ) ), 4.39 (q, J = 7Hz, 2H,
2
3 2
OCH CH ), 6.54 (d, J = 2Hz, 1H, ArH), 6.61 (dd, J = 9, 2Hz, 1H,
2
3
ArH), 7.34 (d, J = 9Hz, 1H, ArH), 7.78 (s, 1H, ArH), 7.80 (d, J =
9Hz, 2H, ArH), 8.08 (d, J = 9Hz, 2H, ArH).
Fluorescence Quantum Yields of 4a and 5-8.
Anal. Calcd. for C
H NO : C,72.31; H, 6.34; N, 3.83.
22 23 4
Absorbances of the solution were kept below 0.2 at the
excitation wavelength. The quantum yields of all compounds
(4a, 5-8) were obtained using a 350, 370, and 390 nm
excitation wavelength, respectively. In all compounds, since
individual variation of the values measured with different
excitation wavelengths was small, each average of their values
was taken in Table 3.
Found: C, 72.31; H, 6.30; N, 3.94.
Ethyl 7-Diethylaminocoumarin-3-carbamate (MAC-carbamic
ester, 6).
Compound 6 was prepared according to the reported method
[3b]. Pale yellow prisms (from ethanol), mp 183-184°, lit mp
187° [3b].

338
H. Takechi, Y. Goto, H. Takahashi and M. Machida
Vol. 38
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pH Dependence of the Absorption and Fluorescence Spectra of 9.
A stock solution of 9 [13] (1.0 mM) was prepared in
ethylene glycol dimethyl ether. For measurement of absorp-
tion and fluorescence spectra, the stock solution was diluted
with various 0.1 M phosphate buffer solutions (pH; 1.0, 1.5,
2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 7.0, 9.0) to 50 µM and 5 µM,
respectively. Absorbances and fluorescence intensities were
monitored at 409 nm and 498 nm (excitation at 407 nm),
respectively.
Acknowledgement.
This work was supported in part by a Grant-in-Aid for the
High Technology Research Program from the Ministry of
Education, Science, Sports and Culture of Japan.
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