A fluorogenic reagent, 7-phenylsulfonyl-4-(2,1,3-benzoxadiazolyl) isocyanate for alcohols, with development based on the empirical method for prediction

Article abstract of DOI:10.1021/ac9905120

During the course of our studies, we found the relationship between the fluorescence characteristics (the fluorescence intensity and the maximum excitation and emission wavelengths) of benzofurazan compounds and the sum and difference of Hammett substituent constants (σp) at the 4- and 7- positions. This prompted us to design a useful fluorogenic derivatization reagent having the benzofurazan skeleton for alcohols along this line of thought. Accordingly, the fluorogenic derivatization reagents, which have no fluorescence themselves, 7-N,N-dimethylaminosulfonyl-4-(2,1,3- benzoxadiazolyl) isocyanate (DBD-NCO), 7-phenylsulfonyl-4-(2,1,3- benzoxadiazolyl) isocyanate (PSBD-NCO), and 7-methylsulfonyl-4-(2,1,3- benzoxadiazolyl) isocyanate (MSBD-NCO), were synthesized. Among the derivatives derived from the three reagents, that from PSBD-NCO was most strongly fluorescent. PSBD-NCO reacted with 1-octanol within 4 h in acetonitrile solution in the absence of a catalyst at 60°C. The derivatives with four alcohols (1-octanol, 1-nonanol, 1-decanol, and 1-undecanol) were separated on a reversed-phase column and detected fluorimetrically at 490 nm with the excitation at 368 nm. The detection limits were at the 10-femtomole level. PSBD-NCO was superior to other fluorescent-labeling reagents with regard to the avoidance of the interfering peaks derived from the reagents themselves and degradation products in the chromatogram. The effectiveness of our approach is discussed in terms of the development of new fluorogenic reagents.

Full text of DOI:10.1021/ac9905120

Anal. Chem. 1999, 71, 5367-5371
A Fluorogenic Reagent,
7-Phenylsulfonyl-4-(2,1,3-benzoxadiazolyl)
Isocyanate for Alcohols, with Development Based
on the Empirical Method for Prediction
Seiichi Uchiyama,† Tomofumi Santa,† Sachiko Suzuki,‡ Hirochika Yokosu,‡ and Kazuhiro Imai*,†
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan,
and Tokyo Kasei Kogyo, 6-15-9 Toshima, Kita-ku, Tokyo, 114-0003, Japan
biological chemistry, since such relationships allow us to design
useful fluorescent derivatization reagents and probes.
During the course of our studies, we found the relation-
ship between the fluorescence characteristics (the fluo-
rescence intensity and the maximum excitation and
emission wavelengths) of benzofurazan compounds and
the sum and difference of Hammett substituent constants
(σp) at the 4 - and 7 - positions. This prompted us to design
a useful fluorogenic derivatization reagent having the
benzofurazan skeleton for alcohols along this line of
thought. Accordingly, the fluorogenic derivatization re-
agents, which have no fluorescence themselves, 7 -N,N-
dimethylaminosulfonyl-4 -(2 ,1 ,3 -benzoxadiazolyl) isocy-
anate (DBD-NCO), 7 -phenylsulfonyl-4 -(2 ,1 ,3 -benzoxa-
diazolyl) isocyanate (P SBD-NCO), and 7 -methylsulfonyl-
4 -(2 ,1 ,3 -benzoxadiazolyl) isocyanate (MSBD-NCO), were
synthesized. Among the derivatives derived from the three
reagents, that from P SBD-NCO was most strongly fluo-
rescent. P SBD-NCO reacted with 1 -octanol within 4 h in
acetonitrile solution in the absence of a catalyst at 6 0 °C.
The derivatives with four alcohols (1 -octanol, 1 -nonanol,
1-decanol, and 1-undecanol) were separated on a reversed-
phase column and detected fluorimetrically at 4 9 0 nm
with the excitation at 3 6 8 nm. The detection limits were
at the 1 0 -femtomole level. P SBD-NCO was superior to
other fluorescent-labeling reagents with regard to the
avoidance of the interfering peaks derived from the
reagents themselves and degradation products in the
chromatogram. The effectiveness of our approach is
disccussed in terms of the development of new fluorogenic
reagents.
In the previous research,1 we investigated the effects of the
substituent groups at the 4- and 7-positions on the fluorescence
characteristics of the benzofurazan (2,1,3-benzoxadiazole) com-
pounds and found the relationship between the fluorescence
characteristics of these compounds and the sum and difference
of Hammett substituent constants (σp)2,3 of the substituent groups
at the 4- and 7-positions. Using this relationship, we could predict
the fluorescence characteristics from the chemical structures of
4,7-disubstituted benzofurazan compounds.
The present project was focused on the design of useful
fluorescent reagents having the benzofurazan skeleton for alco-
hols, using the relationship obtained. Until now, the following
compounds are reported as fluorescent derivatization reagents for
alcohols: 3-chloroformyl-7-methoxycoumarin (3CMC),4 7-[(chloro-
carbonyl)-methoxy]-4-methylcoumarin (CMMC),5 pyrene-1-car-
bonylnitrile,6 1-anthroylnitrile,7 3,4-dihydro-6,7-dimethoxy-4-methyl-
3-oxo-quinoxaline-2-carbonyl chloride (DMEQ-COCl),8 3,4-dihydro-
6,7-dimethoxy-4-methyl-3-oxo-quinoxaline-2-carbonyl azide (DMEQ-
CON3),9 2-(5-chlorocarbonyl-2-oxazolyl)-5,6-methylenedioxybenzo-
furan (OMB-COCl),10 4-(N,N-dimethylaminosulfonyl)-7-(2-chloro-
formylpyrrolidin-1-yl)-2,1,3-benzoxadiazole (DBD-Pro-COCl),11 and
4-(N-chloroformylmethyl-N-methyl)-amino-7-N,N-dimethylamino-
sulfonyl-2,1,3-benzoxadiazole (DBD-COCl).12 However, these fluo-
rescent reagents are not always satisfactory with respect to the
(1) Uchiyama, S.; Santa, T.; Fukushima, T.; Homma, H.; Imai, K. J. Chem. Soc.,
Perkin Trans. 2 1 9 9 8 , 2165-2173.
(2) Hammett, L. P. J. Am. Chem. Soc. 1 9 3 7 , 59, 96-103.
(3) Hansch, C.; Leo, A.; Taft, R. W. Chem. Rev. 1 9 9 1 , 91, 165-195.
(4) Hamada, C.; Iwasaki, M. J. Chromatogr. 1 9 8 5 , 341, 426-431.
(5) Karlsson, K. E.; Wiesler, D.; Alasandro, M.; Novotny, M. Anal. Chem. 1 9 8 5 ,
57, 229-234.
(6) Goto, J.; Chikai, T.; Nambara, T. J. Chromatogr. 1 9 8 7 , 415, 45-52.
(7) Goto, J.; Komatsu, S.; Inada, M.; Nambara, T. Anal. Sci. 1 9 8 6 , 2, 585-586.
(8) Iwata, T.; Yamaguchi, M.; Hara, S.; Nakamura, M.; Ohkura, Y. J. Chromatogr.
1 9 8 6 , 362, 209-216.
There have been many reports on the effects of the skeleton
and the substituent groups of molecules on the fluorescence
characteristics (the fluorescence intensity and the maximum
excitation and emission wavelengths), yet general relationships
between the fluorescence characteristics and the chemical struc-
ture have hitherto not been revealed. Clarification of the relation-
ships between the chemical structure and the fluorescence
characteristics is of great value in the fields of the analytical and
(9) Iwata, T.; Yamaguchi, M.; Nakamura, M. J. Chromatogr. 1 9 8 7 , 421, 43-
50.
(10) Nagaoka, H.; Nohta, H.; Kaetsu, Y.; Saito, M.; Ohkura, Y. Anal. Sci. 1 9 8 9 ,
5, 525-530.
* To whom correspondence should be addressed. (tel.) 81-3-5841-4760; (fax)
81-3-5841-4885; (e-mail) kimai@mol.f.u-tokyo.ac.jp.
† The University of Tokyo.
(11) Toyo’oka, T.; Ishibashi, M.; Terao, T.; Imai, K. Analyst (Cambridge, U.K.)
1 9 9 3 , 118, 759-763.
(12) Imai, K.; Fukushima, T.; Yokosu, H. Biomed. Chromatogr. 1 9 9 4 , 8, 107-
113.
‡ Tokyo Kasei Kogyo.
10.1021/ac9905120 CCC: $18.00 © 1999 American Chemical Society
Published on Web 10/23/1999
Analytical Chemistry, Vol. 71, No. 23, December 1, 1999 5367

detectablity of alcohols, since the fluorescence of derivatives is
not strong, and the fluorescence of the reagent itself and the
degradation byproducts sometimes interfere with the quantifica-
tion of the small amount of substances. Further, the excitation
and emission wavelengths of the derivatives with these reagents
are short so that the quantification is often interfered by bioma-
trixes containing fluorescent substances. Thus useful derivatization
reagents for alcohols, to be developed, should ideally have the
following characteristics: (1) fluorogenic (reagent itself is not
fluorescent), (2) react with alcohols to form the strong fluorescent
derivatives, and (3) their derivatives are excited and emit at long
wavelengths.
In this article, we develop the fluorogenic derivatization
reagents having the benzofurazan skeleton for alcohols based on
the relationship obtained and examine the reactivity of the
reagents as well as the sensitivity of the derivatives.
EXPERIMENTAL SECTION
Materials. 1-Octanol, 1-nonanol, 1-decanol, and 1-undecanol
Figure 1. The design of fluorogenic derivatization reagents for
alcohols using the relationship between the fluorescence intensity of
4,7-disubstituted benzofurazan compounds and the Hammett sub-
stituent constants (σp) at the 4- and 7-positions.
were of guaranteed grade (Tokyo Kasei, Tokyo, Japan). Silica gel
60 was obtained from Merck (Darmstadt, Germany). Acetonitrile,
ethyl acetate, n-hexane, dichloromethane, and methanol were of
HPLC grade (Kanto Chemicals, Tokyo, Japan). Water was purified
using a Milli-Q reagent system (Millipore, Bedford, MA).
Apparatus. Melting points were measured on a Yanagimoto
Micro Point Apparatus (Tokyo, Japan) and uncorrected. Proton
nuclear magnetic resonance (1H-NMR) spectra were obtained on
a JEOL GSX-400 spectrometer (Tokyo, Japan) with tetramethyl-
silane as an internal standard in CDCl3 (abbreviations used: s )
singlet, d ) doublet, m ) multiplet). Mass spectra were measured
on a Hitachi M-1200 H mass spectrometer (atmospheric pressure
chemical ionization (APCI) system) (Tokyo, Japan). Fluorescence
spectra were measured on a Hitachi F-4010 fluorescence spec-
trometer (Tokyo, Japan).
Design of the Fluorogenic Derivatization Reagents for
Alcohols. Figure 1 showed the relationship between the fluores-
cence intensity of 4,7-disubstituted benzofurazan compounds and
the Hammett substituent constants (σp) at the 4- and 7-positions,
obtained in our previous research.1 In this figure, the abscissa (x
axis) and the ordinate (y axis) were the sum of σp at the 4- and
7-positions (x ) σp(4) + σp(7)) and the difference of σp between
the 4- and 7-position (y ) |σp(4) - σp(7)|), respectively. The
seventy 4,7-disubstituted benzofurazan compounds were classified
into three groups according to their relative fluorescence intensity
(RFI; fluorescence intensity of 4-amino-7-N,N-dimethylaminosul-
fonyl-2,1,3-benzoxadiazole (DBD-NH2) was arbitrarily taken as 1.0)
(RFI ) 0-1, having no or weak fluorescence (O); RFI ) 1-5,
having moderate fluorescence (2); RFI > 5, having strong
fluorescence (9)). The fluorescent compounds, represented as
closed squares and closed triangles, were concentrated in two
areas (A and B); in contrast the nonfluorescent compounds
scattered out of these two areas. Using this relationship, the
compounds with the coordinate (σp(4) + σp(7), |σp(4) - σp(7)|)
of the substituent groups at the 4- and 7- position in the area A or
B were predicted to be fluorescent. On the contrary, the
compounds with the coordinate outside the area A and B were
predicted to be nonfluorescent.
disubstituted benzofurazan compounds having the NCO group
at the 4-position were plotted on the line 1, since there was the
relationship y ) |x - 0.38|. The 4,7-disubstituted benzofurazan
compounds having the NHCOOMe group (substituent group after
the reaction of NCO with methanol; σp ) - 0.17) at the 4-position
were plotted on the line 2, since there was the relationship y )
|x + 0.34|. In this manner, the compounds having a certain
substituent group were plotted on one line. Next, in this graph,
the point of intersection of two lines reveals the 4,7-disubstituted
benzofurazan compounds having the substituent groups corre-
sponding to the two lines. The line, with which the point of
intersection of line 1 was out of the fluorescent area (A and B)
and the point of intersection of line 2 was in the fluorescent area
(A or B), was searched to determine the substituent group at the
7-position of the new fluorogenic reagent for alcohols. As a result,
the lines in the range of 3 were suited to this criteria, showing
that the substituent groups with a σp from 0.25 to 0.75 are
appropriate at the 7-position.
Synthesis. Diphosgene (trichloromethyl chloroformate, TCF)13,14
and benzofurazan isocyanate should be treated in draft with a
safety glove and glass.
7 -N,N-Dim ethylam inosu lfonyl-4 -(2 ,1 ,3 -benzoxadiazo-
lyl) isocyanate (DBD-NCO). 4-Amino-7-N,N-dimethylaminosul-
fonyl-2,1,3-benzoxadiazole (DBD-NH2)15 (244 mg, 1.0 mmol) was
suspended in 20 mL of dried ethyl acetate. After the addition of
720 µL of diphosgene, the mixture was stirred at 80 °C for 4 h.
The completion of the reaction was confirmed using NMR. The
reaction mixture was evaporated to dryness under reduced
pressure, and DBD-NCO was obtained as yellow powder. Im-
mediately, this powder was dissolved in 20 mL of acetonitrile, and
50 mmol/ L of DBD-NCO solution was obtained. δH 7.99 (1H, d,
J 7.6), 7.11 (1H, d, J 7.6), 2.96 (6H, s).
(13) Kurita, K.; Matsumura, T.; Iwakura, Y. J. Org. Chem. 1 9 7 6 , 41, 2070-2071.
(14) Nowakowski, J. J. Prakt. Chem./ Chem.-Ztg. 1 9 9 2 , 334, 187-189.
(15) Imai, K.; Uzu, S.; Nakashima, K.; Akiyama, S. Biomed. Chromatogr. 1 9 9 3 ,
7, 56-57.
At first, the isocyanate (NCO; σp ) 0.19) group was selected
as the reaction group at the 4-position. In Figure 1, the 4,7-
5368 Analytical Chemistry, Vol. 71, No. 23, December 1, 1999

7-Phenylsulfonyl-4-(2,1,3-benzoxadiazolyl) isocyanate (PS-
BD-NCO). 4-Amino-7-phenylsulfonyl-2,1,3-benzoxadiazole (PSBD-
NH2)16 (138 mg, 0.5 mmol) was suspended in 10 mL of dried ethyl
acetate. After the addition of 360 µL of diphosgene, the mixture
was stirred at 80 °C for 4 h. The completion of the reaction was
confirmed using NMR. The reaction mixture was evaporated to
dryness under reduced pressure, and PSBD-NCO was obtained
as yellow powder. Immediately, this powder was dissolved in 10
mL of acetonitrile, and 50 mmol/ L of PSBD-NCO solution was
obtained. δH 8.18-8.25 (3H, m), 7.53-7.64 (3H, m), 7.13 (1H, d,
J 7.6).
7-Methylsulfonyl-4-(2,1,3-benzoxadiazolyl) isocyanate (MS-
BD-NCO). 4-Amino-7-methylsulfonyl-2,1,3-benzoxadiazole (MSBD-
NH2)16 (107 mg, 0.5 mmol) was suspended in 10 mL of dried ethyl
acetate. After the addition of 360 µL of diphosgene, the mixture
was stirred at 80 °C for 4 h. The completion of the reaction was
confirmed using NMR. The reaction mixture was evaporated to
dryness under reduced pressure, and MSBD-NCO was obtained
as yellow powder. Immediately, this powder was dissolved in 10
mL of acetonitrile and 50 mmol/ L of MSBD-NCO solution was
obtained. δH 8.15 (1H, d, J 7.3), 7.18 (1H, d, J 7.3), 3.38 (3H, s).
7 -N,N-Dim ethylam inosulfonyl-4 -m ethoxyam ide-2 ,1 ,3 -
benzoxadiazole (DBD-NHCOOMe). 7-N,N-dimethylaminosul-
fonyl-4-methoxythioamide-2,1,3-benzoxadiazole (DBD-NHCSOMe)17
(10 mg, 0.032 mmol) was dissolved in acetonitrile (2 mL). After
the addition of 3% hydrogen peroxide solution in water (5 mL),
the mixture was stirred at 50 °C for 30 min. The reaction mixture
was evaporated to dryness under reduced pressure, and the
residue was chromatographed on silica gel with dichloromethane-
methanol (20:1) to afford DBD-NHCOOMe (3.6 mg, 38%) as
yellow powder. mp: 210 °C. δH 8.07 (1H, d, J 8.0), 8.03 (1H, d, J
8.0), 7.79 (1H, br), 3.91 (3H, s), 2.92 (6H,s). APCI-MS: m/ z 299-
((M - H)-).
7 -P henylsu lfonyl-4 -m ethoxyam ide-2 ,1 ,3 -benzoxadiaz-
ole (P SBD-NHCOOMe). 7-Phenylsulfonyl-4-(2,1,3-benzoxadiaz-
olyl) isocyanate (PSBD-NCO) solution in acetonitrile (50 mmol/
L) (500 µL, 0.025 mmol) was dissolved in methanol (20 mL). The
mixture was stirred at room temperature for 10 min and evapo-
rated to dryness under reduced pressure. The residue was
chromatographed on silica gel with dichloromethane-methanol
(20:1) to afford PSBD-NHCOOMe (4.5 mg, 54%) as yellow powder.
mp: 185-186 °C. δH 8.27 (1H, d, J 8.0), 8.18 (2H, d, J 7.6), 8.09
(1H, d, J 8.0), 7.78 (1H, br), 7.52-7.62 (3H, m), 3.92 (3H, s). APCI-
MS: m/ z 332((M - H)-).
7 -Methylsu lfonyl-4 -m ethoxyam ide-2 ,1 ,3 -benzoxadiaz-
ole (MSBD-NHCOOMe). 7-Methylsulfonyl-4-(2,1,3-benzoxadia-
zolyl) isocyanate (MSBD-NCS)16 (15 mg, 0.059 mmol) was
dissolved in methanol (10 mL). The mixture was stirred at 50 °C
for 30 min and evaporated to dryness under reduced pressure.
The residue was chromatographed on silica gel with dichlo-
romethane-methanol (20:1) to afford 7-methylsulfonyl-4-methox-
ythioamide-2,1,3-benzoxadiazole (MSBD-NHCSOMe) (12 mg,
71%) as yellow powder. mp: 176-178 °C (dec.). δH 9.07 (1H, br),
8.48 (1H, br), 8.19 (1H, d, J 8.0), 4.22 (3H, s), 3.35 (3H, s). APCI-
MS: m/ z 288((M + H)+). MSBD-NHCSOMe (12 mg, 0.042 mmol)
was dissolved in acetonitrile (2 mL). After the addition of 3%
hydrogen peroxide solution in water (5 mL), the mixture was
stirred at 50 °C for 30 min. The reaction mixture was evaporated
to dryness under reduced pressure, and the residue was chro-
matographed on silica gel with dichloromethane-methanol (20:
1) to afford MSBD-NHCOOMe (3.6 mg, 33%) as yellow powder.
mp: 165 °C. δH 8.19 (1H, d, J 8.0), 8.13 (1H, d, J 8.0), 7.86 (1H,
br), 3.92 (3H, s), 3.35 (3H, s). APCI-MS: m/ z 272((M + H)+).
7 -P henylsulfonyl-4 -octyloxyamide-2 ,1 ,3 -benzoxadiazole
(P SBD-NHCOOC8 H1 7 ). 7-Phenylsulfonyl-4-(2,1,3-benzoxadiaz-
olyl) isocyanate (PSBD-NCO) solution in acetonitrile (50 mmol/
L) (0.5 mL, 0.025 mmol) was dissolved in 1-octanol (4 mL). The
mixture was stirred at room temperature for 10 min and evapo-
rated to dryness under reduced pressure. The residue was
chromatographed on silica gel with ethyl acetate-n-hexane (1:2)
to afford PSBD-NHCOOC8H17 (9.5 mg, 88%) as yellow powder.
mp: 120-121 °C. δH 8.27 (1H, d, J 8.0), 8.18 (2H, d, J 7.6), 8.09
(1H, d, J 8.0), 7.77 (1H, br), 7.52-7.62 (3H, m), 4.47 (2H, t), 3.57
(2H, q), 1.27 (13H, m). APCI-MS: m/ z 432((M + H)+).
Measurement of Fluorescence Spectra. The solutions of
benzofurazan compounds (5 µM) were used for the measurement
of the fluorescence intensity and the maximum excitation and
emission wavelengths.
Time Course of the Reaction of P SBD-NCO with 1 -Oc-
tanol. To a vial (500-µL volume) were added 25 µL of PSBD-NCO
in acetonitrile (50 mmol/ L), 5µL of 1-octanol in acetonitrile (1
mmol/ L), and 470 µL of acetonitrile. The vial was capped and
heated at 60 °C for 5 h. At the fixed intervals, an aliquot (2 µL) of
the reaction mixture was subjected to HPLC. The reaction yield
was determined by comparison with the peak height of authentic
PSBD-NHCOOC8H17.
Calibration Curve for the Derivatives of P SBD-NCO with
1 -Octanol. To a vial (500-µL volume) were added 25 µL of PSBD-
NCO in acetonitrile (50 mmol/ L), 20 µL of 1-octanol in acetonitrile
(0.08, 0.4, 2, 10, 50, and 250 µmol/ L) and 455 µL of acetonitrile.
The vial was capped and heated at 60 °C for 4 h. An aliquot (5
µL) of the each reaction mixture was subjected to HPLC.
High-P erformance Liquid Chromatography. The high-
performance liquid chromatograph consisted of a Hitachi L-6300
pump, a Hitachi L-1080 fluorescence detector, and a Hitachi D-2500
integrator. The separation for the derivatives was studied on an
analytical column, TSKgel ODS-80Ts (150 × 4.6 mm i.d., 5 µm)
(TOSOH, Tokyo, Japan). The column temperature was ambient.
The eluent for the derivatives of alcohols with PSBD-NCO was
acetonitrile-water (4:1). The eluate was monitored with fluores-
cence detection (excitation at 368 nm, emission at 490 nm).
Derivatization of Alcohols with P SBD-NCO. To a vial (500-
µL volume) were added 25 µL of PSBD-NCO in acetonitrile (50
mmol/ L), 20 µL of mixed alcohols (250 µmol/ L each of 1-octanol,
1-nonanl, 1-decanol, and 1-undecanol) in acetonitrile, and 455 µL
of acetonitrile. The vial was capped and heated at 60 °C for 4 h.
After cooling in ice water, an aliquot (1 µL) of the reaction mixture
was subjected to HPLC.
RESULTS AND DISCUSSION
Design and Synthesis of New Fluorogenic Reagents for
(16) Toriba, A.; Santa, T.; Iida, T.; Imai, K. Analyst (Cambridge, U.K.) 1 9 9 9 ,
Alcohols. At first, the isocyanate (NCO) group was adopted as
the reaction group at the 4-position for alcohols, since the NCO
group reacts with alcohols quite easily without catalysts. The
124, 43-48.
(17) Uchiyama, S.; Santa, T.; Imai, K. J. Chem. Soc., Perkin Trans. 2 1 9 9 9 , 569-
576.
Analytical Chemistry, Vol. 71, No. 23, December 1, 1999 5369

Table 1. Fluorescence Characteristics of Three Derivatives of Methanol with Benzofurazan Isocyanates
R.F.I.a (λ ex(nm), λ em(nm))
solvents
methanol
acetonitrile
water
DBD-NHCOOMe
PSBD-NHCOOMe
MSBD-NHCOOMe
(c.f. NBD-NHMe)
(c.f. ABD-SMe)
4.84(371, 491)
408(371, 489)
25.3(366, 488)
120(461, 528)
5.10(384, 513)
0.59(371, 475)
451(366, 475)
25.5(363, 473)
192(458, 524)
26.2(385, 510)
1.10(369, 517)
41.9(372, 508)
2.46(363, 487)
14.5(478, 541)
2.40(390, 530)
a
R.F.I. ) Relative fluorescence intensity. Fluorescence intensity of DBD-NH2 was arbitrarily taken as 1.0.
Figure 2. Chemical structures of three benzofurazan isocyanates
and their reaction with alcohols.
Figure 3. Time course for the formation of a derivative of 1-octanol
with PSBD-NCO at 60 °C.
relationship between the fluorescence characteristics and the
Hammett substituent constants (σp) of the substituent groups at
the 4- and 7-positions suggests that a substituent group with a σp
value from 0.25 to 0.75 is required at the 7-position of the
fluorogenic reagent for alcohols. Among the substituent groups
with a σp value from 0.25 to 0.75, SO2NMe2 (σp ) 0.65), SO2Ph
(σp ) 0.68), and SO2Me (σp ) 0.72) groups were selected, since
these substituent groups are strong electron-accepting groups and
expected to increase the reactivity for alcohols. Therefore, 7-N,N-
dimethylaminosulfonyl-4-(2,1,3-benzoxadiazolyl) isocyanate (DBD-
NCO), 7-phenylsulfonyl-4-(2,1,3-benzoxadiazolyl) isocyanate (PSBD-
NCO), and 7-methylsulfonyl-4-(2,1,3-benzoxadiazolyl) isocyanate
(MSBD-NCO) were designed as the fluorogenic derivatization
reagents for alcohols (Figure 2).
DBD-NCO, PSBD-NCO, and MSBD-NCO were synthesized by
the reactions of 4-amino-7-N,N-dimethylaminosulfonyl-2,1,3-ben-
zoxadiazole (DBD-NH2), 4-amino-7-phenylsulfonyl-2,1,3-benzoxa-
diazole (PSBD-NH2), and 4-amino-7-methylsulfonyl-2,1,3-benzox-
adiazole (MSBD-NH2) with diphosgene, respectively. These
reagents, identified by NMR, were stored in dried acetonitrile,
since the solids of DBD-NCO, PSBD-NCO, and MSBD-NCO were
appreciably hydrolyzed by water in the atmosphere. The NMR
spectra showed that these reagents were stable for more than a
month in dried acetonitrile at 0 °C in the glass ampule (data not
shown). We could not obtain the fluorescence spectra of these
reagents because of the interference of a small amount of
fluorescent degradation products.
fluoresced strongly in comparison with DBD-NHCOOMe and
MSBD-NHCOOMe. The fluorescence intensity of PSBD-NH-
COOMe was larger than those of 4-methylamino-7-nitro-2,1,3-
benzoxadiazole (NBD-NHMe, the derivative of methylamine with
NBD-F)1 and 4-methylthio-7-aminosulfonyl-2,1,3-benzoxadiazole
(ABD-SMe, the derivative of methanethiol with ABD-F),1 the
strong fluorescent compounds having the benzofurazan skeleton.
These results showed that PSBD-NCO was the most suitable
reagent for alcohols with respect to the detectability of the
derivative. Therefore, PSBD-NCO was employed in the subse-
quent study.
Derivatization of 1-Octanol with P SBD-NCO. 1-Octanol was
selected as a representative of alcohols. The time course study of
the derivatization of 1-octanol (10 µM in acetonitrile) with PSBD-
NCO (2.5 mM in acetonitrile) was performed at 60 °C. As shown
in Figure 3, the reaction seems to have proceeded completely at
60 °C after 4 h (yield: 97.2%). This result also showed that the
derivative of 1-octanol with PSBD-NCO was stable in acetonitrile
at 60 °C. The derivatization of alcohols with the conventional
reagents proceeded at higher temperature (3CMC4(100 °C),
DMEQ-COCl8(100 °C), OMB-COCl10(100 °C), and DBD-COCl12-
(80 °C)), while the derivatization of 1-octanol with PSBD-NCO
proceeded at 60 °C, suggesting the latter’s superiority to the other
reagents with regard to the avoidance of decomposition of the
analytes. The calibration curve for the derivative of PSBD-NCO
with 1-octanol was linear over the range from 16 fmol to 50 pmol
per injection (r ) 0.999).
The Fluorescence Characteristics of DBD-NHCOOMe,
P SBD-NHCOOMe, and MSBD-NHCOOMe. We synthesized
7-N,N-dimethylaminosulfonyl-4-methoxyamide-2,1,3-benzoxadiaz-
ole (DBD-NHCOOMe), 4-methoxyamide-7-phenylsulfonyl-2,1,3-
benzoxadiazole (PSBD-NHCOOMe), and 4-methoxyamide-7-
methylsulfonyl-2,1,3-benzoxadiazole (MSBD-NHCOOMe) as
derivatives of methanol with the three reagents and measured
the fluorescence spectra of these derivatives in methanol, aceto-
nitrile, and water (Table 1). As shown in Table 1, all the three
derivatives fluoresced as expected. Especially, PSBD-NHCOOMe
Separation of the Derivatives of Alcohols with P SBD-NCO
on a Reversed-P hase Column. The chromatogram of the
derivatives thus obtained is shown in Figure 4. The elution was
in the order of the derivatives with 1-octanol, 1-nonanol, 1-decanol,
and 1-undecanol. Within five minutes, the two fluorescent by-
products were eluted; the major was identified as PSBD-NH2 (the
hydrolyzed product of PSBD-NCO) and the minor was presumed
to be a dimer (the reaction product of PSBD-NH2 with PSBD-
5370 Analytical Chemistry, Vol. 71, No. 23, December 1, 1999

CMMC (1-10 pmol),5 and DBD-COCl (38 fmol).12 Furthermore,
the excitation and emission wavelengths of PSBD-NHCOOMe
(λex/ λem ) 368 nm/ 490 nm) are longer than those of the
alcohols’ derivatives with OMB-COCl (357 nm/ 440 nm),10 3CMC
(355 nm/ 400 nm),4 CMMC (315 nm/ 400 nm),5 pyrene-1-carbonyl
nitrile (300 nm/ 410 nm),6 and DMEQ-CON3 (360 nm/ 440 nm),9
and the stokes shift of PSBD-NHCOOMe is also larger than those
of the derivatives with other reagents. These results showed that
PSBD-NCO was prominent with respect to the detectability of the
derivatives and the avoidance of interference from the biomatrixes.
In conclusion, considering the relationship between the
fluorescence intensity of the 4,7-disubstituted benzofurazan com-
pounds and the Hammett substituent constants (σp) at the 4- and
7-positions, new fluorogenic derivatization reagents for alcohols,
DBD-NCO, PSBD-NCO, and MSBD-NCO, were designed. The
derivatives of these three reagents fluoresced as expected, with
the derivative of PSBD-NCO most strongly fluorescing. PSBD-
NCO was superior to other reagents for alcohols with regard to
the sensitivity, the reactivity, and the absence of the interfering
peaks in the chromatogram. Thus, PSBD-NCO seemed to be
useful for the derivatization of a slight amount of biologically
important alcohols such as steroids and prostaglandins. These
results showed that our approach, employing the relationship
between the fluorescence characteristics and the chemical struc-
ture, was effective in the development of new fluorogenic reagents.
Further theoretical study is in progress for the precise prediction
of the fluorescence intensity of 4,7-disubstituted benzofurazan
compounds from the chemical structure.
Figure 4. Chromatogram of alcohols derivatized with PSBD-NCO:
(1) 1-octanol 10 pmol, (2) 1-nonanol 10 pmol, (3) 1-decanol 10 pmol,
(4) 1-undecanol 10 pmol; column, TSK gel ODS-80Ts (150 × 4.6
mm, i.d. 5 µm); eluent, acetonitrile - water (4:1); flow rate, 1.0 mL
min-1; detection, excitation 368 nm, emission 490 nm.
NCO). No other interfering peaks were detected. There was no
PSBD-NCO peak detected, because PSBD-NCO was presumed
to be nonfluorescent or the excess PSBD-NCO was hydrolyzed
to PSBD-NH2. The fluorescence intensity of PSBD-NH2 was
approximately 0.015% that of PSBD-NHCOOMe, and the excitation
and emission wavelengths of PSBD-NH2 (λex/ λem ) 428 nm/
544 nm) were different from those of PSBD-NHCOOMe. On the
contrary, other reagents (3CMC,4 pyrene-1-carbonyl nitrile,6 1-an-
throylnitrile,7 OMB-COCl10, and DBD-COCl12) produced many
fluorescent degradation products with the desired derivatives,
since the reagents themselves are fluorescent, and the fluores-
cence of the excess reagents and the degradation products
severely interfered with the quantification of the slight amount of
intended derivatives. These facts indicated that PSBD-NCO was
superior to the other reagents as a derivatization reagent for
alcohols. The detection limits (signal-to-noise ratio ) 3) for the
respective alcohols’ derivatives were 5.6-10.7 fmol, which are
equivalent to the derivatives of OMB-COCl (3-8 fmol),10 1-an-
throylnitrile (20 fmol),7 and DMEQ-CON3 (5 fmol)9 and superior
to those of DBD-Pro-COCl (sub-pmol),11 3CMC (2-40 pmol),4
ACKNOWLEDGMENT
We thank Dr. Chang-Kee Lim, MRC Toxicology Unit, Univer-
sity of Leicester for his valuable suggestion and discussion. We
also thank TOSOH for the gift of a TSK gel ODS-80Ts column.
Received for review May 13, 1999. Accepted September
16, 1999.
AC9905120
Analytical Chemistry, Vol. 71, No. 23, December 1, 1999 5371