4-(2-Aminoethylamino)-7H-benz[de]benzimidazo[2,1-a]isoquinoline-7-one as a highly sensitive fluorescent labeling reagent for carnitine

Article abstract of DOI:10.1246/bcsj.74.173

4-(2-Aminoethylamino)-7H-benz[de]benzimidazo[2,1-a]isoquinoline-7-one, synthesized and identified by a HMBC study, can be used as a fluorescent labeling reagent for carnitine. The excitation and emission maxima in acetonitrile were observed at λ 454 and 508 nm, respectively. This reagent smoothly reacted with camitine in the presence of l-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride to afford the corresponding amide under mild conditions. The detection limit (S/N > 3) of camitine was ca. 12 fmol.

Full text of DOI:10.1246/bcsj.74.173

© 2001 The Chemical Society of Japan
173
Bull. Chem. Soc. Jpn., 74, 173–177 (2001)
4-(2-Aminoethylamino)-7H-benz[de]benzimidazo[2,1-a]isoquinoline-7-
one as a Highly Sensitive Fluorescent Labeling Reagent for Carnitine
Ken-ichi Nakaya, Toshiyuki Tanaka,† Yoshiaki Shirataki,†† Hisayoshi Shiozaki,†††
Kazumasa Funabiki, Katsuyoshi Shibata, and Masaki Matsui*
Department of Chemistry, Faculty of Engineering, Gifu University,Yanagido, Gifu 501-1193
†Gifu Prefectural Institute of Health and Environmental Sciences, 1-1 Naka Fudogaoka, Kakamigahara, Gifu 504-0838
††Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakato, Saitama 350-0295
†††Technology Research Institute of Osaka Prefecture, 2-7-1 Ayumino, Izumi, Osaka 594-1157
(Received August 28, 2000)
4-(2-Aminoethylamino)-7H-benz[de]benzimidazo[2,1-a]isoquinoline-7-one, synthesized and identified by a HMBC
study, can be used as a fluorescent labeling reagent for carnitine. The excitation and emission maxima in acetonitrile
were observed at λ 454 and 508 nm, respectively. This reagent smoothly reacted with carnitine in the presence of 1-[3-
(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride to afford the corresponding amide under mild conditions.
The detection limit (S/N > 3) of carnitine was ca. 12 fmol.
Carnitine, (4-(trimethylammonio)-3-hydroxybutanoate),
which acts as a carrier of long-chain fatty acids into the mito-
chondrial matrix, has been analyzed by high-performance liq-
uid chromatography (HPLC) using fluorescent labeling re-
agents. 9-Anthryldiazomethane (ADAM),1,2 4-bromophenacyl
trifluoromethanesulfonate,3 2-(2,3-naphthalimino)ethyl trifluo-
romethanesulfonate,3 N-(9-acridinyl)maleimide,4 9-fluorenyl-
methyl chloroformate,5–7 1-aminoanthracene,8 3-bromometh-
yl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone,9,10 2-(4-hy-
drazinocarbonylphenyl)-4,5-diphenylimidazole,11,12 N-[4-(2-
benzimidazolyl)phenyl]maleimide,13 pyrene-1-carbonyl cya-
nide,14 and 6-(4-aminophenyl)-3-cyano-4-[4-(dimethylami-
no)phenyl]-2-methylpyridine15 have been reported as the fluo-
rescent labeling reagents for carnitine. Fluorescent labeling re-
agents are required to have high sensitivity, appropriate excita-
tion and emission maxima, and a functional group smoothly re-
acting with target compounds. It is of significance to obtain
new fluorescent labeling reagents having a high detection limit
and good fluorescent spectral features. 7H-Benz[de]benzimi-
dazo[2,1-a]isoquinoline-7-ones have been prepared by the
condensation of 1,8-naphthalenedicarboxylic anhydrides with
o-phenylenediamines. This reaction can produce isomers. For
example, when 4-substituted 1,8-naphthalenedicarboxylic an-
hydride reacts with o-phenyenediamine, 3- and 4-substituted
7H-benz[de]benzimidazo[2,1-a]isoquinoline-7-ones can be
formed. Fluorescent 7H-benz[de]benzimidazo[2,1-a]isoquin-
oline-7-ones have been used as dyes and pigments for synthetic
fibers and plastics.16–24 Therefore, less attention has been paid
to the separation and identification of these isomers. We report
here the synthesis, identification, and application of novel 4-(2-
aminoethylamino)-7H-benz[de]benzimidazo[2,1-a]isoquino-
line-7-one as a fluorescent labeling reagent for carnitine.
Results and Discussion
The synthesis of the 4- and 3-(2-aminoethylamino)-7H-
benz[de]benzimidazo[2,1-a]isoquinoline-7-ones 5 and 5′ is
shown in Scheme 1. 4-Bromo-1,8-naphthalenedicarboxylic
anhydride (1) reacted with o-phenylenediamine (2) to give the
4- and 3-bromo derivatives 3 and 3′, which were carefully iso-
lated by column chromatography (SiO2, C6H6). The separated
components, 3 and 3′, reacted with ethylenediamine (4) to af-
ford the corresponding substitution products, 5 and 5′, respec-
tively.
The structure of 5 was confirmed by elemental analysis,
high-resolution MS, and NMR spectra. The 1H and 13C NMR
spectral data and CH long-range correlations in the HMBC
spectrum of 5 are shown in Table 1 and Fig. 1, respectively.
Normally, carbonyl-carbon is deshielded more strongly than
imino-carbon, due to a larger electron-withdrawing nature of
the oxygen atom.25 Therefore, the peaks at δ 159.7 and 149.3 in
the 13C NMR spectra were attributed to C-7 and C-13a, respec-
tively. Then, the significant 3J long-range correlations were ob-
served between H-1′/C-4, H-6/C-4, and H-6/C-7, as indicated
by the dotted allows in Fig. 1. Thus, compound 5 was identi-
fied as 4-(2-aminoethylamino)-7H-benz[de]benzimidazo[2,1-
a]isoquinoline-7-one.
The absorption and fluorescence spectra of 5 and 5′ in aceto-
nitrile are summarized in Table 2. Both the absorption (λmax
and emission maxima (λem) of 5 were more hypsochromic than
that of 5′. The MO (INDO/S) calculation supported that the 4-
)

174 Bull. Chem. Soc. Jpn., 74, No. 1 (2001)
Imidazole as Fluorescent Labeling Reagent
Scheme 1.
Table 1. 1H and 13C NMR spectral data of 5a)
No.
δH
δC
1
2
3
3a
4
8.65dd (7.3, 0.9)
7.65dd (8.2, 7.3)
8.57 dd (8.2, 0.9)
126.8
126.2
126.2
120.4
152.0
56.79 d (8.6)
104.3
6
6a
7
8a
9
10
8.36 d (8.6)
134.9
107.6
159.7
131.5
115.3
124.1
124.8
119.3
143.4
149.3
119.4
128.7
36.5
8.42 m
7.40 m
7.42 m
7.79 m
11
12
12a
13a
13b
13c
1ꢀ
3.37 t (6.4)
2.88 t (6.4)
2ꢀ
40.0
a) Measured in DMSO-d6 (500 and 125 MHz). All signals were as-
signed by 1H 1H COSY, HMQC, and HMBC spectra. The coupling
constants (parentheses) are in Hz
(2-aminoethylamino) derivative 5 (LUMO: –1.11 eV, HOMO:
–7.39 eV) was more hypsochromic than 5′ (LUMO: –1.13 eV,
HOMO: –7.26 eV). The introduction of the electron-donating
2-aminoethylamino group at the 3-position can raise the energy
Fig. 1. CH long range correlation in HMBC spectrum of 5.
The important correlations are shown in dotted allows.

K. Nakaya et al.
Table 2. Fluorescence spectra of 4- and 3-(2-aminoethyl-
Bull. Chem. Soc. Jpn., 74, No. 1 (2001) 175
HPLC analysis.
The reactivity of 5 with carnitine is summarized in Table 3.
Compound 5 smoothly reacted with carnitine in the presence of
1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochlo-
ride (EDC) under mild conditions.
The HPLC analysis of carnitine by 5 is shown in Fig. 3 Ma-
jor peaks were observed at retention times of 6.774 and 9.247
min. The former component was the labeling reagent 5. The
high resolution MS spectrum of the latter component showed
its MH+ ion peak (relative intensity: 100%) at m/z 472.2352,
amino)-7H-benz[de]benzimidazo[2,1-a]isoquinoline-
7-ones 5 and 5ꢀ
a)
a)
a)
Compound
λmax
εa)
λex
λem
RFIb)
nm
nm
nm
5
451
484
24200
12400
454
487
508
589
207
2
5ꢀ
a) Measured in acetonitrile. b) Relative fluorescence intensity mea-
sured in acetonitrile (1 × 10−5 mol dm−3, 25 ◦C, 6-(4-aminophen-
yl)-3-cyano-4-[4-(diethylamino)phenyl]-2-methylpyridine: λex
=
365nm, λem = 514 nm, RFI = 100; 7-(diethylamino)-4-methyl-
coumarin: λex= 372 nm, λem = 434 nm, RFI = 333).
Table 3. Reactivity of 5 with carnitine
Run Additive Reaction time Reaction temp. Reactivitya)
h
◦C
level of HOMO to produce a bathochromic shift in 5′.
Interestingly, the relative fluorescence intensity (RFI) of the
4-isomer 5 was much greater than that of the 3-isomer 5′. The
RFI of 5 was about two-times more intense than that of 6-(4-
aminophenyl)-3-cyano-4-[4-(diethylamino)phenyl]-2-meth-
ylpyridine, a previously reported fluorescent labeling reagent
for carnitine.15 The Stoke’s shift of 5 and 5′ were observed to
be 54 and 102 nm, respectively. This result suggests a more
rigid conformation of 5 in the excited state than that of 5′.
Since the HPLC analysis of amino acids, such as carnitine,
is usually performed in a reverse phase, the effect of water on
the RFI in 5 was examined. The result is shown in Fig. 2.
Though the RFI of the 6-(4-aminophenyl)-3-cyano-4-[4-(di-
ethylamino)phenyl]-2-methylpyridine decreased with the addi-
tion of water, that of 5 slightly increased up to a water concen-
tration of 70%, and then decreased. Compound 5 was suffi-
ciently soluble in aqueous acetonitrile used as the solvent in the
1
2
3
4
none
6
6
6
6
60
60
60
20
91
20
0
0
100
100
DCCb)
EDCc)
EDCc)
c)
5EDC
6
0.520
2
EDCc)
100
a) Reactivity was calculated on the basis of fluorescence intensity
by HPLC analysis (column: Inert SIL (4.6 mm × 150 mm); mo-
bile phase: hexane choroform = 7 : 3; flow rate: 0.8 ml min−1
;
detection: 254 nm). b) Dicyclohexylcarbodiimide. c) 1-[3-
(Dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride.
Fig. 2. Effect of added water on the relative fluorescence
intensity of 5. The relative fluorescence intensity was
Fig. 3. HPLC analysis of labeled carnitine. Column:
TSK gel ODS 80 TM (4.6 mm × 150 mm); mobile
phase: CH3CN–50 mM KH2PO4 (100 : 225) contain-
ing 10 mM of TFA, flow rate: 0.8 ml min−1; excitation:
454 nm, detection: 508 nm.
◦
measured at 25 C on 1 × 10−5 mol dm−3 of substrate
(5: λex = 454 nm, λem = 508 nm, 6-(4-aminophenyl)-
3-cyano-4-[4-(diethylamino)phenyl]-2-methylpyridine:
λex = 365nm, λem = 514 nm).

176 Bull. Chem. Soc. Jpn., 74, No. 1 (2001)
Imidazole as Fluorescent Labeling Reagent
completed, the product was extracted with chloroform (100 ml ×
2), purified by column chromatography (SiO2, CHCl3 then
CH3OH), and recrystallized from toluene. The physical and spec-
tral data are given below.
4-(2-Aminoethylamino)-7H-benz[de]benzimidazo[2,1-a]iso-
quinoline-7-one (5): Yield 42.6 mg (13%); mp 242–244 °C;
EIMS (70 eV) m/z (rel intensity) 328 (M+; 45), 298 (100), 285
(30), 270 (20); HRMS Calcd for C20H16N4O: 328.1323. Found: m/
z 328.1322. Found: C, 72.98; H, 5.11; N, 17.18%. Calcd for
C20H16N4O: C, 73.15; H, 4.91; N, 17.06%.
3-(2-Aminoethylamino)-7H-benz[de]benzimidazo[2,1-a]iso-
quinoline-7-one (5′): Yield 32.8 mg (10%); mp 128–129 °C;
1H NMR (CDCl3) δ 3.20 (t, J = 5.7 Hz, 2H), 3.44 (q, J = 5.7 Hz,
2H), 6.08 (br s), 6.81 (d, J = 8.4 Hz, 1H), 7.37–7.47 (m, 2H), 7.74
(dd, J = 8.3 and 7.3 Hz, 1H), 7.81–7.83 (m, 1H), 8.32 (d, J = 8.3
Hz, 1H), 8.55–8.57 (m, 1H), 8.71 (d, J = 8.4 Hz, 1H), 8.82 (d, J =
7.3 Hz, 1H); EIMS (70 eV) m/z (rel intensity) 328 (M+; 79), 298
(100), 285 (11), 270 (41). Found: C, 73.23; H, 5.03; N, 17.23%.
Calcd for C20H16N4O: C, 73.15; H, 4.91; N, 17.06%.
showing labeled carnitine (calcd for C27H30N5O3: 472.2349).
Conclusion
We have synthesized 4-(2-aminoethylamino)-7H-benz[de]-
benzimidazo[2,1-a]isoquinoline-7-one. This compound was
identified by a HMBC study. The excitation and emission
maxima in acetonitrile were observed at λ 454 and 508 nm, re-
spectively. This reagent smoothly reacted with carnitine in the
presence of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiim-
ide hydrochloride to afford the labeled product under mild con-
ditions. The detection limit (S/N > 3) of carnitine by this re-
agent was ca. 12 fmol, similar to that of ADAM. However,
both the excitation and emission maxima of 4-(2-amino-
ethylamino)-7H-benz[de]benzimidazo[2,1-a]isoquinoline-7-
one were more bathochromic than those of ADAM (λex: 365
nm, λem: 412 nm in CH3CN−CH3OH), being the preferred
properties for fluorescent labeling reagents.
HPLC Analysis of Carnitine. A methanol solution of car-
nitine (1 µg mL–1) was prepared. After evaporating one ml of this
solution, an acetonitrile−pyridine mixed solution (8:2, 1 mL–1) of
5 (12 nmol ) and EDC (6.2 nmol ) was added. The mixture was
stirred for 2 h at 20 °C. The solution was then analyzed by HPLC.
The analytical conditions in the HPLC are shown in Fig. 3.
Experimental
Melting points were measured with a Yanagimoto MP-S2 mi-
cro-melting-point apparatus. Fluorescence spectra were measured
with a Jasco FP-777 spectrofluorometer. NMR spectra were taken
on JEOL α-400 and α-500 spectrometers. MS spectra were re-
corded on Shimadzu QP-1000 and JEOL JMS-700 spectrome-
ters. Liquid chromatography was performed with a Jasco Triroter-
V instrument. DL-Carnitine hydrochloride was obtained from
NAKALAI TESQUE, Inc. 4-Bromo-1,8-naphthalenedicarboxylic
anhydride (1), o-phenylenediamine (2), and ethylenediamine (4)
were purchased from Tokyo Kasei Co., Ltd.
The authors are grateful to Messrs. Yutaka Takahashi and
Masao Nomoto of JEOL Ltd. for LCMS measurement.
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