Design, synthesis and characterization of a novel fluorescent probe for nitric oxide based on difluoroboradiaza-s-indacene fluorophore

Article abstract of DOI:10.1016/j.saa.2004.05.041

Nitric oxide (NO) is a gaseous, free radical, which plays a role as an intracellular second messenger and a diffusible intercellular messenger. To obtain evidence for NO function in vivo, 1,3,5,7-tetramethyl-8-(4′-aminophenyl-N-(2′′-amino)-phenzyl) -difluoroboradiaza-s-indacene (TMAPABODIPY) was designed and synthesized as a fluorescent probe for nitric oxide, which features high photostability and no pH dependency over a wide pH range. The fluorescence of TMAPABODIPY itself is very strong. When TMAPABODIPY traps NO in the presence of dioxygen, the weak fluorescent triazole form is obtained, which offers the advantages of specificity, and sensitivity for direct detection of NO. The relationship was obtained between the quenching fluorescence intensity and NO concentration in the range 0.02-4.0 μmol l^-1. The detection limit is 5 nmol l ^-1 (S/N = 3). The proposed method has been used to monitor the release of NO from S-nitrosocysteine, a NO-releasing agent.

Full text of DOI:10.1016/j.saa.2004.05.041

Spectrochimica Acta Part A 61 (2005) 1045–1049
Design, synthesis and characterization of a novel fluorescent probe
for nitric oxide based on difluoroboradiaza-s-indacene fluorophore
Xian Zhanga,∗, Hua-shan Zhangb
a
School of Chemical Engineer and Pharmocy, Wuhan Institute of Chemical Technology, Wuhan, Hubei 430073, PRChina
b
Department of Chemistry, Wuhan University, Wuhan 430072, PRChina
Received 10 March 2004; accepted 14 May 2004
Abstract
Nitric oxide (NO) is a gaseous, free radical, which plays a role as an intracellular second messenger and a diffusible intercellular messenger.
To obtain evidence for NO function in vivo, 1,3,5,7-tetramethyl-8-(4ꢀ-aminophenyl-N-(2ꢀꢀ-amino)-phenzyl)-difluoroboradiaza-s-indacene
(TMAPABODIPY) was designed and synthesized as a fluorescent probe for nitric oxide, which features high photostability and no pH
dependency over a wide pH range. The fluorescence of TMAPABODIPY itself is very strong. When TMAPABODIPY traps NO in the presence
of dioxygen, the weak fluorescent triazole form is obtained, which offers the advantages of specificity, and sensitivity for direct detection
of NO. The relationship was obtained between the quenching fluorescence intensity and NO concentration in the range 0.02–4.0 ␮mol l−1
.
The detection limit is 5 nmol l−1 (S/N = 3). The proposed method has been used to monitor the release of NO from S-nitrosocysteine, a
NO-releasing agent.
© 2004 Elsevier B.V. All rights reserved.
Keywords: 1,3,5,7-Tetramethyl-8-(4ꢀ-aminophenyl-N-(2ꢀꢀ-amino)-phenzyl)-difluoroboradiaza-s-indacene; Fluorescent probe; Nitric oxide
1. Introduction
Techniques for the detection of NO based on chemi-
luminescence, or using electrochemical probes have been
developed [6–8]. Although these techniques allow detection
of NO production from tissues, they do not allow imaging
of NO production at cellular level. Therefore, we set out
to design molecular probes that would allow real time
visualization of NO production within cells. For studies of
intracellular concentrations of small molecules and ions,
fluorescent probes are widely used and they also offered the
most attractive prospect for this application as a non-invasive
and potentially highly sensitive method.
Several fluorescent probes for NO were developed based
on which aromatic vicinal diamines can react with NO in
presence of dioxygen to produce the corresponding triazenes.
Theyinclude2,3-diaminonaphthaleneanditsderivate[9–10],
diaminofluorescein [11], and diaminorhodamine [12]. These
methods can obtain much higher sensitivity than other fluo-
rescent methods. Diaminofluorescein and diaminorhodamine
have been already used to image NO in cells. But the final de-
tection of 2,3-diaminonaphthalene was performed in alkaline
Nitric oxide (NO) acts as a biological second messenger,
and plays an important role in different functions of the hu-
man body. NO has different, concentration-dependent roles
in many tissues, such as in the vasculature, immune system
and neurotransmission [1–3]. In vivo and in vitro studies
have indicated that NO at low concentration is an important
molecule, essential for normal bone physiological, however,
high levels of NO have been demonstrated to be inhibitory
for bone and cartiage cell function [4–5]. To understand more
clearly the role of NO in physiology, better methods are re-
quired to determine the exact concentration of NO that are
beneficial or harmful for specific cell types. Moreover, it is
necessary to get more accurate information on the cellular
localization of NO production.
∗
Corresponding author. Tel.: +86 27 87196017; fax: +86 27 87196017.
E-mail address: xianzhang9@263.net (X. Zhang).
1386-1425/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2004.05.041

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X. Zhang, H.-s. Zhang / Spectrochimica Acta Part A 61 (2005) 1045–1049
medium, which does not fit for biological sample. Further-
more, diaminofluorescein is unstable in strong light and can
only be applied in a small pH range, while diaminorhodamine
has relatively low sensitivity.
Difluoroboradiaza-s-indacenes (boron-dipyrromethene,
BODIPY®) as a fluorophore, has favourable photophysical
and optoelectronic properties. Its derivatives have found nu-
merous applications in biochemistry and molecular biology
[13–15]. In previous work, we have synthesized 1,3,5,7-tet-
ramethyl-8-(3ꢀ,4ꢀ-diaminophenyl)-difluoroboradiaza-s-inda-
cence [16] and 8-(3ꢀ,4ꢀ-diaminophenyl)-difluoroborad-
iaza-s-indacence [17]. Now, we newly designed and syn-
thesized a fluorescent NO probe 1,3,5,7-tetramethyl-8-(4ꢀ-
aminophenyl-N-(2ꢀꢀ-amino)-phenzyl)-difluoro-boradiaza-s-
indacene (TMAPABODIPY), to detect NO in biological
systems as a means to examine the physiological functions
of NO. TMAPABODIPY itself has very strong fluorescence,
due to two amines groups as electron-donating group. When
it reacts with NO to produce the corresponding triazole, the
fluorescence of the fluorophore is quenched. Based on this
finding, a simple method with high sensitivity and selectivity
for the detection of NO has been developed.
Scheme 1. Synthetic scheme of TMAPABODIPY.
The mixture of 2,4-dimethylpyrrole 0.95 g (10 mmol) and
p-bromo-benzaldehyde 0.92 g(5 mmol) in30 ml CH2Cl2 was
purged with N2 for 30 min. Several drops trifluoroacetic acid
was added to initiate the condensation. After 1.5 h, thin layer
chromatography (TLC) silica (dichloromethane, CH2Cl2)
showed that all of the aldehyde had been consumed. The
reaction solution was then washed with 100 ml of 0.1 mol l−1
NaOH solutions and 200 ml of water, dried (Na2SO4) and
filtered. The solvent was evaporated on a rotary evaporator.
The resultant product was used immediately. The product
was redissolved in 50 ml of toluene. 2,3-Dichloro-5,6-
dicyanobenzoquinone (DDQ) (1.0 g, 4.49 mmol) was added
in the solution. After 10 min, triethylamine (7 ml) was
added to the black reaction mixture followed immediately
by BF3-etherate (7 ml of neat BF3-etherate). The mixture
was stirred for 1.5 h, poured into water, and extracted with
CH2Cl2. The CH2Cl2 solution was washed three times with
100 ml portions of water and dried (Na2SO4). The solvent
was evaporated on a rotary evaporator.
The phenylenediamine (1.2 g, 10 mmol) was dissolved in
drymethanol(50 ml)andheatedtoreflux. Theaboveobtained
compound was slowly added and the mixture refluxed for 1 h.
After cooling, the mixture was poured into ether (200 ml) and
the hydrochloride salt precipitated and filtered off. The salt
was dissolved in 20 ml of ethanol and 1 mol l−1 of NaOH
solution was used to adjust the solution to the neutral. The
mixture solution was extracted by ethyl acetate. The extracted
solvent was evaporated on a rotary evaporator. The product
was purified by silica gel chromatography with ethyl acetate
and petroleum ether (3:1). TLC showed that the product puri-
fied had only one point. Mass spectra: m/z: 435(M+ + 1). IR
(KBr, ν (cm−1)): 3425, 3203 (–NH2), 2924.0, 2853 (–CH3),
1083.7 (B–F), 1654.0 (–C N).
2. Experimental
2.1. Apparatus and reagents
Unless otherwise specified, all reagents were of analytical
reagent grade from Shanghai Chemical Reagent Co. (Shang-
hai, China). 2,3-Dichloro-5,6-dicyanobenzo-quinone (DDQ)
was purchased from Acros Organce Co. (Belgium). All so-
lutions were prepared in doubly distilled water.
TMAPABODIPY was synthesized in our laboratory and
its 0.2 mmol l−1 stock solution was prepared with ethanol,
and stable for at least 1 month when stored in refrigera-
tor. Working solution was appropriately diluted. KH2PO4–
Na2HPO4 buffer was prepared by mixing 0.78 mmol l−1
KH2PO4 solution and 0.78 mmol l−1 Na2HPO4 solution to
appropriate pH value.
Mass spectra were obtained by means of a VG ZAB-3F
GC–MS instrument (Manchester, UK). FTIR spectra were
obtained for the products in KBr disks by means of a Bruker
(Karlsruhe, Germany) IFS48 instrument. Fluorescence spec-
tra were recorded with a Shimadzu (Kyoto, Japan) RF-5000
spectrofluorimeter. Absorption spectra were recorded with a
Shimadzu (Kyoto, Japan) UV-1601 spectrophotometer. pH
was determined by means of a DF-801 accurate acidometer
(Zhongshan University, Guangzhou, China).
2.2. Synthesis of TMAPABODIPY
2.3. Preparation of NO solution and S-nitrosocysteine
solution
The synthetic route employed to obtain the new dye is
outlined in Scheme 1.
2,4-Dimethypyrrole was synthesized according to the lit-
erature [18].
NO solution was prepared according to the literatures
[19,20] and working solution was appropriately diluted.

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1047
S-nitrosocysteine solution (0.15 mmol l−1) was prepared
as described by Field et al. [21]. The solution was diluted
with methanol to the desired concentration.
2.4. Fluorometric analysis
Relative fluorescence quantum efficiencies were obtained
by comparing the peak area under the corrected emission
spectrum of the test sample at 492 nm excitation with that
of a solution of fluorescein in 0.1 mol l−1 NaOH solution
that has a quantum efficiency of 0.85 according to the liter-
ature [11]. The slit width was 1.5 nm for both excitation and
emission.
Scheme 2. Reaction of TMAPABODIPY with NO.
of dioxygen to form a new compound, TMAPABODIPY-T.
The fluorescence maximum excitation and emission wave-
lengths of TMAPABODIPY-T were at 508 and 520 nm,
with a red shift in comparison with TMAPABODIPY,
exhibiting the extension of the conjugated system compared
to TMAPABODIPY (Fig. 2).
The phenomenon of TMAPABODIPY reacted with
NO is the same to that of 1,3,5,7-tetramethyl-8-(3ꢀ,4ꢀ-
diaminophenyl)-difluoroboradiaza-s-indacence with NO. In
2.5. Procedure
The 0.3 ml of 2×10−4 mol l−1 TMAPABODIPY solution
and 2.0 ml of pH 7.4 sodium phosphate buffers were trans-
ferred to a 10 ml test-tube. Then a solution containing NO
with a gas-tight syringe was added. The resulting solution
was diluted to 5 ml with water and stood for 35 min at 40 ◦C.
After 35 min, the solution was diluted to the mark with water.
The relative fluorescence intensity was measured at 510.0 nm
with excitation at 500.0 nm. The slit width for emission and
excitation was both 5 nm.
2.6. Sample analysis procedure
A series of 0.3 ml of 2.0 × 10−4 mol l−1 TMAPABODIPY
solution was transferred to 10 ml test-tubes. Then 2.0 ml pH
7.4 sodium phosphate buffers and 0.5 ml of 0.15 mmol l−1
S-nitrosocysteine solutions were added, respectively. The re-
sulting solutions were diluted to 5 ml with water and stood
for different time at 40 ◦C. Every 5 min, the whole solution
was diluted to the mark with water. The measure procedure
is same to that above described.
Fig. 1. Fluorescence spectra of TMAPABODIPY and TMAPABODIPY-T
CTMAPABODIPY = 1.00 × 10−6 mol l−1, CNO = 2.00 × 10−6 mol l−1, pH
7.4, 1 is excitation spectrum of TMAPABODIPY at 510 nm emission wave-
length, 2 is excitation spectrum of TMAPABODIPY-T at 520 nm emission
wavelength, 1ꢀ is emission spectrum of TMAPABODIPY at 500 nm excita-
tion wavelength, 2ꢀ is emission spectrum of TMAPABODIPY-T at 508 nm
excitation wavelength,. The slit of excitation and emission are both 5 nm.
3. Result and discussion
3.1. The spectra properties of TMAPABODIPY and its
product
It was reported that o-phenylenediamine group react with
NO in the presence of dioxygen to produce the corresponding
triazole under neutral conditions [22]. Based on this, the re-
action scheme of TMAPABODIPY with NO in the presence
of dioxygen is shown in Scheme 2.
The fluorescence spectra of TMAPABODIPY and its
reactive product with NO in the presence of dioxygen, the
triazole (TMAPABODIPY-T) are shown in Fig. 1. The
fluorescence of TMAPABODIPY itself is very strong at
λex/λem = 500/510 nm. For its stoke shift is very small, the
fluorescence quantum efficiency is high. The fluorescence
was decreased greatly by addition of NO solution, which
indicated TMAPABODIPY reacted with NO in the presence
Fig. 2. Effect of pH on the fluorescence intensity of TMAPABODIPY and
TMAPABODIPY-T, CTMAPABODIPY = 6.0 × 10−6 mol l−1, CNO = 2.00 ×
10−5 mol l−1, (ꢀ) TMAPABODIPY, ( ) TMAPABODIPY-T.

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X. Zhang, H.-s. Zhang / Spectrochimica Acta Part A 61 (2005) 1045–1049
Fig. 4. The pH effect of the final determination (CTMAPABODIPY = 6.0 ×
10−5 mol l−1, CNO = 2.00 × 10−6 mol l−1, reaction time: 30 min, reaction
temperature: 30 ◦C).
Fig. 3. Effect of TMAPABODIPY concentration (CNO
=
2.00
×
10−6 mol l−1, pH 7.40, reaction time: 30 min, reaction temperature: 30 ◦C).
our previous literature, we have explained this phenomenon
in details [17].
Fig. 3 shows the effect of pH on the fluorescence intensity
of TMAPABODIPY and its product. The fluorescence inten-
sity of TMAPABODIPY is stable above pH 4. While under
pH 4, it greatly decreases. Maybe in strong acidic condition,
amine groups of TMAPABODIPY are protonated and make
fluorescence intensity decrease. The triazole is stable from
pH 2 to 12 for 12 h.
Fig. 5. Effect of buffer volume (CTMAPABODIPY = 6.0 × 10−5 mol l−1, CNO
= 2.00 × 10−6 mol l−1, pH 7.40, reaction time: 30 min, reaction temperature:
30 ◦C).
3.2. Optimization of reaction conditions of
TMAPABODIPY with NO
The conditions for the reaction of TMAPABODIPY were
optimized. Several factors influencing the reaction were stud-
ied, including reagent concentration, amount and pH range of
KH2PO4–Na2HPO4 buffer, reaction time and temperature.
TMAPABODIPY concentration affected the quenching
fluorescence intensity due to TMAPABODIPY itself having
strong the fluorescence. Fig. 3 shows the effect of TMA-
PABODIPY concentration on the quenching fluorescence
intensity. The maximum quenching fluorescence intensity
was obtained at range of TMAPABODIPY concentrations
of 5.0–8.0 ␮mol l−1. We used 6.0 ␮mol l−1 of TMAPABOD-
IPY as optimal condition.
The effect of the buffer pH on the quenching fluorescence
intensity was study by using KH2PO4–Na2HPO4 buffer from
pH 4.7 to 12. It was found that the quenching fluorescence
intensity kept stable at pH 7.4–12. In order to fit biologi-
cal sample, pH 7.4 was chosen for the determination of NO
(Fig. 4).
The variation of the buffer volume from 1.0 to
3.5 ml had the effect shown in Fig. 5. When 2.0 ml of
KH2PO4–Na2HPO4 buffer was added, the quenching fluo-
rescence intensity reached the maximum. Therefore, 2.0 ml
of was used in the procedure.
Temperatures and times are two critical factors for the
reaction. The effects of varied times and temperatures on
the reaction of TMAPABODIPY with NO in the presence
of dioxygen are demonstrated in Fig. 6. Both high and low
temperature gave the weaker quenching fluorescence inten-
Fig. 6. Effect of reaction and temperature (CTMAPABODIPY = 6.0 ×
10−5 mol l−1, CNO = 2.00 × 10−6 mol l−1, pH 7.40, (ꢁ) 30 ◦C, (ꢂ) 40 ◦C,
(ꢀ) 50 ◦C).
sity compared with 40 ◦C. At a high temperature (50 ◦C), the
fluorescence of TMAPABODIPY was decreased, while the
fluorescence of the triazole was unchanged. So the quenching
fluorescence intensity decreased. It was found that the reac-
tion was much faster at high temperature (40–50 ◦C) com-
pared with low temperature (30 ◦C). However, the reaction
at high temperatures (40–50 ◦C) got to balance more quickly
than at low temperature (30 ◦C). At 40 ◦C, the quenching flu-
orescence intensity was almost unchanged after 30 min of
the reaction. Therefore, the reaction was performed at 40 ◦C
for 35 min. After formation, the product was stable for at
least 12 h.

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1049
Table 1
Effect of foreign ions
releasing agent. Fig. 7 showed the time course of the decrease
of the fluorescence intensity at 510 nm the probe. When S-
nitrosocysteine was added to the probe solution, the fluores-
cence at 510 nm decreased gradually. The time course of the
fluorescence decrease corresponded to that of NO production
from S-nitrosocysteine. This finding indicates that the fluo-
rescence decrease was caused by TMAPABODIPY reaction
with NO.
The sensitivity of this method is enough for the deter-
mination of NO in biological body. The half-life of NO in
the physiological body is around 5–15 s, and NO releasing
is a continuous procedure. Therefore, the proposed method
can monitor NO releasing off line. But the proposed method
needs reaction time much longer, it cannot determinate NO
releasing in situ for a relatively long time needed.
Foreign ion Tolerance (␮g ml−1
)
Foreign ion Tolerance (␮g ml−1
)
Ca2+
Mg2+
Al3+
Zn2+
100
10
20
40
40
Fe3+
2a
20
100
20
2
2−
CO3 −
NO3
BSA−
NO2
2−
SO4
a
In the presence of 0.01 mg ml−1 EDTA.
References
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Fig. 7. The time course of the decrease of the fluorescence intensity at
510 nm of the fluorescence probe (CTMAPABODIPY = 6.0 × 10−5 mol l−1).
A spontaneous NO-releasing reagent, S-nitrosocysteine was added to probe
solution at a final concentration of 7.5 ␮mol l−1 at 0 min.
3.3. Linearity, sensitivity and precision
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curve was obtained in the NO concentration range of
0.02–4.0 ␮mol l−1. The detection limit was found to be
5 nmol l−1 with a signal-to-noise ratio of 3. The linear regres-
sion equation and the coefficient are Y = 300.91X − 10.78,
where Y is the fluorescence quenching intensity, X is NO
concentration (␮mol l−1), r = 0.9978. The relative standard
deviation for 10 replication measurement of 0.2 ␮mol l−1 NO
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3.4. Effect of foreign ions
Using 2.0 ␮mol l−1 of nitric oxide, under the chosen con-
ditions, the interference from foreign ions was investigated.
The tolerance limit of foreign ions was taken as the maxi-
mum amount causing an error ≤ 5% in the fluorescence of
the sample. The results are shown in Table 1. From the result
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in the determination of NO in biological specimen.
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3.5. Sample analysis
S-nitrosocysteine is thermodynamically and photolyti-
cally unstable compounds, which is a spontaneous NO-