Fluorescent indicators for nitric oxide based on rhodamine chromophore

Article abstract of DOI:10.1016/S0040-4039(99)02002-X

We have developed a novel fluorescent indicator for NO, DAR-M, which features long-wavelength excitation, high photostability and no pH dependency over a wide pH range.

Full text of DOI:10.1016/S0040-4039(99)02002-X

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 69–72
Fluorescent indicators for nitric oxide based on rhodamine
chromophore
Hirotatsu Kojima, Miki Hirotani, Yasuteru Urano, Kazuya Kikuchi, Tsunehiko Higuchi and
Tetsuo Nagano ∗
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Received 17 September 1999; revised 12 October 1999; accepted 18 October 1999
Abstract
We have developed a novel fluorescent indicator for NO, DAR-M, which features long-wavelength excitation,
high photostability and no pH dependency over a wide pH range. © 1999 Elsevier Science Ltd. All rights reserved.
Keywords: diamines; dyes; fluorescence; nitrogen oxides.
Direct detection of nitric oxide (NO) is important for the clarification of its many proposed biological
functions.1 We have reported diaminofluoresceins (DAFs) as fluorescent indicators for NO. By using the
diacetyl derivative (DAF-2 DA), which permeates into cells and is quickly transformed into water-soluble
DAF-2 by intracellular esterases, the temporal and spatial distribution of intracellular NO was visualized,
and its usefulness for functional studies was confirmed.2,3 However, some of the characteristics of DAF-2
are undesirable, such as low photostability, a wavelength range that overlaps autofluorescence in vivo, and
pH dependency. In particular, the pH dependency is a critical problem. It is said that NO has something
to do with the neurotoxicity after cerebral ischemia.1 The application of DAF-2 DA to brain tissues under
this condition is difficult, because intracellular pH falls below six during the ischemia.4
It was considered that DAF can be structurally divided into the diamine moiety as the reactive site
and the xanthene moiety as the fluorophore. Aromatic vicinal diamines can trap NO under aerobic
conditions to yield the corresponding triazole compounds. The change of the electron-donating ability
of the functional groups causes the switch of fluorescence, owing to photo-induced electron transfer.5
We supposed that this mechanism would also be applicable to the rhodamine fluorophore and that the
introduction of a diamine moiety into rhodamine instead of fluorescein might overcome the problems.
We designed and synthesized diaminorhodamines (DAR-1 and DAR-2, Scheme 1) from N,N-diethyl-
3-aminophenol and the corresponding acetoamido-nitrophthalic anhydride. The overall yields of DAR-1
and DAR-2 were 42% and 7.7%, respectively. DARs fluoresced after reaction with NO, as expected.
The absorbance and fluorescence properties are summarized in Table 1. The wavelengths involved are
∗
Corresponding author.
0040-4039/00/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(99)02002-X

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longer than those of DAFs. Although the quantum yields are relatively low, this does not represent a
practical disadvantage; indeed, the signal to noise ratio is, if anything, sometimes higher because of
the low background fluorescence at such a longer-wavelength, especially in the analysis of biological
samples.
Scheme 1. Synthesis of diaminorhodamines (DARs) and their products of reaction with NO
Photostability was examined in sunlight.7 The fluorescence intensity of DAR-1 T was not reduced at
all after 3 h, while that of DAF-2 T was reduced to 1% of the initial intensity.
The effect of pH on the fluorescence intensity of DAR-1 T and DAR-2 T was also examined. The
fluorescence intensity of rhodamine B, which is the chromophore of DARs, is stable above pH 4, since
it has no phenolic OH. However, we observed instability of the fluorescence intensity around neutral pH
as shown in Fig. 1. The instability may arise from a triazole proton, so, a methyl group was introduced
into DAR-1 with methyl iodide (DAR-M, Scheme 1). It was selectively introduced into the amine with
higher electron density.8
The absorbance and fluorescence properties of DAR-M (Table 1) were almost the same as those of
DAR-1 or DAR-2. The augmentation of the fluorescence intensity of DAR-M upon addition of NO is
shown in Fig. 2. Judging from NO standard curves calibrated with 10 µM DAR-M, the detection limit
was 10 nM. The sensitivity of DAR-M was 1.4 times higher than that of DAR-1. It is considered that this

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Table 1
Absorbance and fluorescence properties of dyes
*See Kojima et al.2
All data were obtained at 20°C in 0.1 M sodium phosphate buffer, pH 7.4. Relative quantum
efficiencies were obtained by comparing the area under the corrected emission spectrum of the test
sample at 535 nm excitation with that of a solution of rhodamine B in ethanol, which has a quantum
efficiency of 0.97 according to the literature6
Figure 1. Effect of pH on the fluorescence intensity of DAR-Ts and DAF-2 T. Triazole forms of DAR-1, DAR-2 and DAR-M
(final 1 µM) were added to sodium phosphate solution adjusted to various pH values. The pH was measured after mixing.
The fluorescence intensities of DAR-1 T (open circle), DAR-2 T (open square) and DAR-M T (closed circle) were determined
at 580, 575 and 575 nm with excitation at 565, 565 and 560 nm, respectively. The photomultiplier voltage was 950 V. The
curves were fitted to the following equations: (DAR-1 T) intensity=783/(1+102.13−pH) +42.7/(1+106.69−pH), R=0.956; (DAR-2 T)
intensity=899/(1+101.62−pH) −218/(1+107.27−pH), R=0.987; (DAR-M T) intensity=1000/(1+102.39−pH), R=0.951. DAF-2 T (open
triangle): The data are from Kojima et al.2. The photomultiplier voltage was 400 V
increase of sensitivity results from a higher rate of trapping of NO due to the electron-donating effect of
the methyl group. The fluorescence intensity of DAR-M T was stable above pH 4, as shown in Fig. 1.
In conclusion, we have developed an improved type of DAFs, DAR-M, which features longer-
wavelength excitation, higher photostability and no pH dependency above pH 4. Thus, DAR-M should
enable wider application of diamine compounds for detecting NO.

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Figure 2. Excitation (A) and emission (B) spectra for DAR-M at 37°C in 0.1 M sodium phosphate buffer (pH 7.4) with NO
concentrations ranging from 0 µM to 1.1 µM.NO solution was added to 10 µM DAR-M solution under aerobic conditions. The
spectra were obtained from an average of five repeated measurements after the addition of NO solution. (A), determined with
emission at 580 nm. (B), determined with excitation at 565 nm
Acknowledgements
This work was supported by JSPS Research Fellowships for Young Scientists and by Grants-in-Aid
for Scientific Research on Priority Areas, nos. 09304061, 10169215 and 10557243, from the Ministry of
Education, Science, Sport and Culture of Japan.
References
1. Kerwin Jr., J. F.; Lancaster Jr., J. R.; Feldman, P. L. J. Med. Chem. 1995, 38, 4343.
2. Kojima, H.; Nakatsubo, N.; Kikuchi, K.; Kawahara, S.; Kirino, Y.; Nagoshi, H.; Hirata, Y.; Nagano, T. Anal. Chem. 1998,
70, 2446.
3. Kojima, H.; Nakatsubo, N.; Kikuchi, K.; Urano, Y.; Higuchi, T.; Tanaka, J.; Kudo, Y.; Nagano, T. NeuroReport 1998, 9,
3345.
4. Chopp, M.; Frinak, S.; Walton, D. R.; Smith, M. B.; Welsh, K. M. A. Stroke 1987, 18, 919.
5. de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley, A. J. M.; McCoy, C. P.; Rademacher, J. T.; Rice, T. E.
Chem. Rev. 1997, 97, 1515.
6. Nishikawa, Y.; Hiraki, K. Analytical Methods of Fluorescence and Phosphorescence; Kyoritsu Publishing Company:
Tokyo, 1984; p. 76.
7. Bleaching of dyes (10 µM) was determined in 0.1 M sodium phosphate buffer (pH 7.4). The solutions in 30 ml vials were
placed in full sunlight on a fine day in November in Tokyo.
8. It is supported by data of the amino protons in the 1H NMR spectra. DAR-1 (300 MHz, DMSO-d6): δ 1.09 (t, 12H, J=7.2
Hz), 5.01 (br, 2H), 5.87 (br, 2H), 6.07 (d, 1H, J=7.5 Hz), 6.38–6.43 (m, 4H), 6.57 (d, 2H, J=8.6 Hz), 6.78 (d, 1H, J=7.5
Hz). DAR-M (300 MHz, DMSO-d6): δ 1.08 (t, 12H, J=6.8 Hz), 2.77 (s, 3H), 5.22 (br, 1H), 6.01 (br, 2H), 6.19 (d, 1H,
J=7.7 Hz), 6.40–6.44 (m, 4H), 6.56–6.63 (m, 3H).