Chiral fluorescent labeling reagent derived from Rhodamine B for Flurbiprofens

Article abstract of DOI:10.1246/bcsj.76.1405

A lactam-type enantiomeric fluorescent labeling reagent was prepared from Rhodamine B. This reagent showed its absorption and emission maxima at λ 557 and 580 nm in an acetonitrile-acetic acid (9:1) mixed solution, respectively. Racemic Flurbiprofens reacted with this reagent to produce diastereomeric esters, which were separated by non-chiral high-performance liquid chromatography (HPLC).

Full text of DOI:10.1246/bcsj.76.1405

ꢀ 2003 The Chemical Society of Japan
Bull. Chem. Soc. Jpn., 76, 1405–1408 (2003) 1405
Chiral Fluorescent Labeling Reagent Derived from Rhodamine B for
Flurbiprofens
ꢀ
Masaki Matsui, Takahiro Higeta, Masahiro Kimura, Kazumasa Funabiki, and Ken-ichi Nakayay
Department of Materials Science and Technology, Faculty of Engineering, Gifu University, Yanagido, Gifu 501-1193
yGifu Prefectural Institute of Health and Environmental Science, 1-1 Naka-fudohgaoka, Kakamigahara, Gifu 504-0838
(Received August 28, 2002)
A lactam-type enantiomeric fluorescent labeling reagent was prepared from Rhodamine B. This reagent showed its
absorption and emission maxima at ꢀ 557 and 580 nm in an acetonitrile–acetic acid (9:1) mixed solution, respectively.
Racemic Flurbiprofens reacted with this reagent to produce diastereomeric esters, which were separated by non-chiral
high-performance liquid chromatography (HPLC).
Fluorescent labeling reagents are required to have intense
and bathochromic fluorescence.1;2 Biologically active materi-
als having a chiral center in a molecule can be analyzed in
non-chiral columns by forming their diastereomers. HPLC
analyses of amino acids and peptides by benzoxadiazoles3{5
and carboxylic acids by anthracene-2,3-dicarboximide de-
rivatives6;7 have been reported. Xanthene dyes, such as fluo-
rescein and Rhodamine B, are known to show intense
fluorescence. Especially, Rhodamine dyes are interesting
and important compounds due to their strong and bathochro-
mic fluorescence. Therefore, they have been used as indicators
for calcium ion,8 labeling reagents for nucleic acids,9
proteins,10 muscle fibers,11 and DNA sequencing.12 Unfortu-
nately, no chiral fluorescent labeling reagents derived from
intensely fluorescent and bathochromic Rhodamine dyes have
been reported so far. We report here on the synthesis and
properties of a chiral fluorescent labeling reagent derived from
Rhodamine B for the analysis of Flurbiprofens.
Results and Discussion
Scheme 1 shows the synthesis of fluorescent labeling re-
agent 3 derived from Rhodamine B (1). Compound 1 reacted
with (R)-(ꢁ)-2-amino-1-propanol (2) to produce a lactam 3 in
a good yield. The optical purity (ee%) of this compound
Scheme 1.
Published on the web July 15, 2003; DOI 10.1246/bcsj.76.1405

1406 Bull. Chem. Soc. Jpn., 76, No. 7 (2003)
Chiral Fluorescent Labeling Reagent from Rhodamine B
Fig. 1. UV-vis absorption and fluorescence spectra of 3 (4)
in an acetonitrile–acetic acid mixed solution.
Fig. 3. Relationship among UV-vis absorption spectra,
fluorescence spectra, and temperature. An acetonitrile–
acetic acid (9:1) mixed solution of 4 (1 ꢂ 10ꢁ5 mol dmꢁ3
)
was allow to stand.
Table 1. UV-Vis Absorption and Fluorescence Spectral
Data of 4, 8, 9, and Rhodamine B
ꢀmax (")
nm (dm3 molꢁ1 cmꢁ1
ꢀex ꢀem
Compd
RFI
)
nm
nm
4a)
557 (104900)
353 (53100)
369 (27000)
545 (106000)
557
353
364
530
580
508
435
565
643
100
447
819
8b)
9b)
Rhodamine Bc)
a) Measured in an acetonitrile–acetic acid (9:1) mixed solution
on 1:0 ꢂ 10ꢁ5 mol dmꢁ3 of substrate at 25 ꢃC. b) Measuꢃred in
acetonitrile on 1:0 ꢂ 10ꢁ5 mol dmꢁ3 of substrate at 25 C. c)
Measured in methanol on 1:0 ꢂ 10ꢁ5 mol dmꢁ3 of substrate at
Fig. 2. Relationship between fluorescence intensity of 4
and reaction time. An acetonitrile–acetic acid (9:1) mixed
solution of 4 (1 ꢂ 10ꢁ5 mol dmꢁ3) was allow to stand at
ꢃ
25 C.
ꢃ
25 C.
65 ꢃC. These results indicate that only a small amount of open-
form 4 was converted into the lactam-form 3 with increasing
temperature. The remarkable decrease in the fluorescence in-
tensity can be attributed to the thermal deactivation of 4 in the
excited singlet state.
measured by HPLC (CHIROBIOTIC V (4.6 mm ꢂ 250 mm),
hexane–ethanol (85/15), 0.8 cmꢁ3 minꢁ1, detection: 254 nm)
was higher than 99%.
Compound
3 was colorless and non-fluorescent in
acetonitrile. However, compound 3 turned red and showed
fluorescence in an acetonitrile–acetic acid (9:1) mixed
solution. The UV-vis absorption and fluorescence spectra
are shown in Fig. 1. The absorption (ꢀmax) and emission
Table 1 summarizes the UV-vis absorption and fluorescence
spectra of 4, 6-(4-aminophenyl)-3-cyano-4-[4-(diethylamino)-
phenyl]-2-methylpyridine (8), 7-diethylamino-4-methylcou-
marin (9), and Rhodamine B. Compounds 8 and 9 were ref-
erence materials throughout our study on the fluorescent
labeling reagents.13{17 The Stokes shift of 4 was calculated
to be 23 nm, being very small, as normally observed for Rho-
damine dyes. The molar absorption coefficient (") of Rhoda-
maxima (ꢀem
) were observed at 557 and 580 nm,
respectively. The fluorescence intensity increased up to an
acetic acid ratio of 20%. Then, the intensity was almost con-
stant in the range of acetic acid ratios of 20–100%. Isosbestic
points were observed at 265, 285, 315, and 335 nm, indicating
that the formation of colored and fluorescent form 4 by the
addition of acetic acid was in equilibrium with the non-fluo-
rescent form 3, as shown in Scheme 1.
The relationship between the absorbance and the reaction
time in an acetonitrile–acetic acid (9:1) mixed solution is
shown in Fig. 2. When compound 3 was kept in the solution
at 25 ꢃC, the absorbance gradually increased, and became con-
stant after 10 min.
mine
B in methanol was calculated to be 106000
dm3 molꢁ1 cmꢁ1 at ꢀmax 545 nm, suggesting that most of lac-
tam form 3 were converted into the open-form 4 in an aceto-
nitrile–acetic acid mixed (9:1) solution. Both ꢀmax and ꢀem of
4 were most bathochromic among 4, 8, and 9. Furthermore,
the relative fluorescence intensity (RFI) of 4 was most intense
among them.
Since the HPLC analysis of water-soluble compounds is
usually performed in a reverse phase, the effect of water on
the RFI of 4 was examined. The result is shown in Fig. 4. The
RFI of 8 and 9 drastically decreased by the addition of water.
However, the decrease of RFI in 4 was only 16% even in a
water ratio 90%. This result indicates that compound 3 can act
The relationship among the absorbance, fluorescence inten-
sity, and temperature is shown in Fig. 3. No change in the
ꢃ
absorption spectra was observed in the range of 25 to 65 C.
Meanwhile, the fluorescence intensity drastically decreased at

M. Matsui et al.
Bull. Chem. Soc. Jpn., 76, No. 7 (2003) 1407
(S)- and (R)-Flurbiprofens, (R,S)- and (R,R)-7, were observed
at retention times of around 130 and 140 min, respectively.
The retention time of the (R,S) derivative was checked by
comparing that of the product obtained by the reaction of
(S)-5 with 3. The ratio between (R,S)- and (R,R)-7 was 55.8
to 44.2. Several pmol of racemic Flurbiprofens could be ana-
lyzed by this method.
Conclusion
An enantiomeric fluorescent labeling reagent for carboxylic
acids was prepared from Rhodamine B. The absorption and
emission maxima of this reagent was observed at ꢀ 557 and
580 nm in an acetonitrile–acetic acid (9:1) mixed solution,
respectively. Racemic Flurbiprofen smoothly reacted with this
reagent in the presence of EDC to produce the corresponding
diastereomeric esters, which were separated by non-chiral
HPLC.
Fig. 4. Effect of water on fluorescence intensity of 4, 8, and
9 (1 ꢂ 10ꢁ5 mol dmꢁ3). In the cases of 8 and 9, the ab-
scissa indicates the % ratio of (H2O/(H2O + MeCN)).
Experimental
Apparatus. Melting points were measured with a Yanagimoto
MP-S2 micro-melting-point apparatus. NMR spectra were taken
on a Jeol ꢁ-400 spectrometer. EIMS spectra were recorded on a
Shimadzu QP-1000 instrument. UV-vis and fluorescence spectra
were measured with Hitachi U-3500 and F-4500 spectrometers,
respectively. HPLC was performed with a Jeol Triroter-V instru-
ment.
Materials.
Rhodamine B (1) was purchased from Tokyo
Kasei Co., Ltd. (R)-(ꢁ)-2-Amino-1-propanol (2) was obtained
from ACROS Organics. Racemic and (S)-Flurbiprofens were pur-
chased from Aldrich Co., Ltd.
Synthesis of 3. To Rhodamine B (1) (120 mg, 0.25 mmol) was
added (R)-(ꢁ)-2-amino-1-propanol (2) (500 mg, 6.67 mmol). The
mixture was heated at 170 ꢃC for 24 h. After the reaction was
completed, the mixture was poured into brine. The product was
extracted with dichloromethane and recrystallized from ethyl
ꢃ
1
acetate. (106 mg, 79%). Mp 258.5–259.0 C; H NMR (CDCl3)
ꢂ 1.10 (d, J ¼ 7:1 Hz, 3H), 1.165 (t, J ¼ 7:1 Hz, 6H), 1.171 (t,
J ¼ 7:1 Hz, 6H), 3.21–3.24 (m, 1H), 3.34 (q, J ¼ 7:1 Hz, 4H),
3.35 (q, J ¼ 7:1 Hz, 4H), 3.48 (ddd, J ¼ 11:7, 8.7, and 4.6 Hz,
1H, a doublet peak (J ¼ 8:7 Hz) disappears in the presence of
D2O), 3.55 (d, J ¼ 11:7 Hz, 1H), 5.03 (d, J ¼ 8:7 Hz, 1H, dis-
appeared in the presence of D2O), 6.27–6.30 (m, 2H), 6.37 (dd,
J ¼ 9:5 and 2.8 Hz, 2H), 6.47 (dd, J ¼ 9:5 and 4.1 Hz, 2H), 7.05–
7.08 (m, 1H), 7.44–7.48 (m, 2H), 7.87–7.90 (m, 1H); EIMS (70
Fig. 5. HPLC analysis of labeled diastereomeric (R,S)- and
(R,R)-7. Column: Mightysil RP-18 GP (4.6 mm ꢂ 150
mm); Mobile phase: acetonitrile–tetrahydrofuran–0.5%
acetic acid (28:5:67); Flow rate: 0.8 cm3 minꢁ1; Excita-
tion: 562 nm, Detection: 582 nm.
25
eV) m=z (rel intensity) 499 (Mþ; 18), 469 (42), 468 (100). [ꢁ]D
(c ¼ 0:0455, CHCl3) ꢁ52:7ꢃ. Anal. Found: C, 74.23; H, 7.66; N,
8.13%. Calcd for C31H37N3O3: C, 74.52; H, 7.46; N, 8.41%.
HPLC Analysis of Racemic Flurbiprofens (5). To a dichlo-
romethane solution (0.6 cm3) of racemic Flurbiprofens 5 (1.2
mmol) were added a dichloromethane solution (0.6 cm3) of 4-(1-
pyrrolidinyl)pyridine (10 mmol dmꢁ3, 6 mmol), a dichloromethane
solution (0.6 cm3) of 3 (10 mmol dmꢁ3, 6 mmol), a dichloro-
methane solution (0.6 cm3) of EDC (10 mmol dmꢁ3, 6 mmol),
and aflatoxin G2 (10 mg dmꢁ3). Aflatoxin G2 was used as a
reference compound in the HPLC analysis. The mixture reacted
in a vial tube at 20 ꢃC for 7 h. After drying the reaction mixture, 2
cm3 of an acetonitrile–0.5% acetic acid (55:45) mixed solution
was added. The solution was then analyzed by HPLC (column:
Mightysil RP18 GP (4.6 mm ꢂ 150 mm); mobile phase: aceto-
nitrile–tetrahydrofuran–0.5% aqueous acetic acid (28:5:67), flow
rate: 0.8 cm3 minꢁ1; excitation: 562 nm, detection: 582 nm).
as a good fluorescent labeling reagent.
Finally, racemic Flurbiprofens (5), which acts as inhibitors
for cyclooxygenase, reacted with 3 in the presence of 4-(1-
pyrrolidinyl)pyridine and 1-[3-(dimethylamino)propyl]-3-
ethylcarbodiimide hydrochloride (EDC) to produce diastereo-
meric esters (R,R)- and (R,S)-6, which reacted with acetic acid
to form the corresponding open forms of (R,S)- and (R,R)-7, as
shown in Scheme 1. When an acetonitrile–acetic acid (9:1)
mixed solvent was used as a mobile phase, no good separation
of the diastereomers in HPLC was observed. Therefore, after
the formation of open-forms 7, the diastereomers were ana-
lyzed by HPLC. An acetonitrile–tetrahydrofuran–aqueous
0.5% acetic acid (28:5:67) mixed solvent was used as a mobile
phase. The HPLC analysis of racemic Flurbiprofens (5) is
shown in Fig. 5. The labeled diastereomers derived from

1408 Bull. Chem. Soc. Jpn., 76, No. 7 (2003)
Chiral Fluorescent Labeling Reagent from Rhodamine B
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