Turn-on fluorescent probes for nitric oxide sensing based on the ortho-hydroxyamino structure showing no interference with dehydroascorbic acid

Article abstract of DOI:10.1039/c3cc49555h

A new family of fluorescent synthetic molecular probes for nitric oxide sensing based on ortho-hydroxyamino-triarylpyrylium salts is presented. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c3cc49555h

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Turn-on fluorescent probes for nitric oxide
sensing based on the ortho-hydroxyamino
structure showing no interference with
dehydroascorbic acid†
Cite this: Chem. Commun., 2014,
50, 3579
Received 17th December 2013,
Accepted 7th February 2014
Alicia Beltran,a M. Isabel Burguete,a Daniel R. Abanades,b Dolores Perez-Sala,b
´
´
´
DOI: 10.1039/c3cc49555h
Santiago V. Luis*a and Francisco Galindo*a
www.rsc.org/chemcomm
A new family of fluorescent synthetic molecular probes for nitric medium,7 using enzymes and capillary electrophoresis8 and multi-
oxide sensing based on ortho-hydroxyamino-triarylpyrylium salts is derivatization image analysis.9 In this regard, notable detection
presented.
limits have been reached using a contactless correction method
(10 nM).7 It should be noted that not taking into account this
Nitric oxide (NO) has captured the attention of many researchers important biological interfering species may lead researchers in
during the last decade. Interest in NO derives from its known biomedical areas to wrongly interpret the recorded data. However,
biological activity, in particular its pivotal role in vasodilatation or developing a more selective probe, not reactive with DHA, thus
its functions as a neurotransmitter and an antimicrobial agent.1
avoiding the aforementioned correction methods, continues to be an
Fluorescence techniques have become very popular for NO important target of current interest in the field.
sensing because of their high sensitivity and spatiotemporal resolu-
Herein we present the development of a new family of synthetic
tion when combined with microscopy.2 Thus, the construction of molecular probes for NO in an oxygenated medium based on the
small fluorescent sensors suitable for specific NO detection in pyrylium cation which meets the essential criteria, namely: (1) they
living systems has received great attention. A number of fluorescent are sensitive to NO/O2 without showing significant interference with
probes have been reported to date. The most common approach for other reactive nitrogen (RNS) and oxygen species (ROS), and espe-
NO detection involves the use of o-diamino aromatics under cially without showing interference with dehydroascorbic acid, and
aerobic conditions. These molecules react with N2O3 (formed from (2) they are obtained by a simple methodology that allows efficient
NO and O2), to yield fluorescent triazole derivatives.3
synthesis of the product in only two synthetic steps and in high
Another family of probes, in this case reacting directly with NO, yields from readily available starting products. Besides, the synthetic
is based on transition-metal complexes.4 An increasing number of procedure allows for a straightforward introduction of structural
other strategies have been described in the literature.5
variants favoring a fine tuning of the different spectroscopic proper-
Furthermore, although the past several years have witnessed ties (excitation and fluorescence emission wavelengths, emission
great progress in the development of ortho-diamine fluorescent quantum yields, and fluorescence lifetimes, etc.).
probes, they still have some shortcomings. One of the most impor-
The key factor to accomplish the above-mentioned features
tant drawbacks, is that dehydroascorbic acid (DHA) reacts with is the utilization of the ortho-hydroxyamino structure instead of
o-diamino aromatics and turns on the fluorescence of such probes the traditional ortho-diamino one.
in an analogous way as N2O3 does. As pointed out by Sweedler and
The synthesis of compounds 2a–d started with the preparation of
coworkers, dehydroascorbic acid reacts with ortho-diamine probes to the intermediates 1a–d with an ortho-hydroxynitro substructure
give fluorescent adducts which are indistinguishable from those (Fig. 1). Intermediates 1a–d were easily prepared by adding BF3Á
arising from the reaction with N2O3.6 To solve this problem various OEt2 to a solution of 4-hydroxy-3-nitrobenzaldehyde and the corre-
correction strategies have been developed, including freezing of the sponding acetophenone in anhydrous toluene. The solution was
refluxed for 2 h. After cooling to room temperature, acetone was
a
added and the solution was poured into excess ether. A precipitate
´
´
´
Universitat Jaume I, Departamento de Quımica Inorganica y Organica, Avda. Sos
´
Baynat s/n, 12071, Castellon, Spain. E-mail: francisco.galindo@uji.es, luiss@uji.es was formed which was then filtered, washed with ether and dried
b
´
Centro de Investigaciones Biologicas, C.S.I.C., Ramiro de Maeztu, 9,
under vacuum. A series of analyses confirmed that the precipitate
was the hydroxynitro pyrylium salts 1a–d. Compounds 2a–d were
obtained by reduction of the nitro group to an amino group by using
SnCl2Á2H2O in dichloromethane and excess of hydrochloric acid
under nitrogen atmosphere. The reaction mixture was refluxed for
28040 Madrid, Spain
† Electronic supplementary information (ESI) available: Synthetic procedures and
characterizations, fluorometric and absorption studies, and X-ray crystallo-
graphic and biological studies. CCDC 977073–977075. For ESI and crystallo-
graphic data in CIF or other electronic format see DOI: 10.1039/c3cc49555h
This journal is ©The Royal Society of Chemistry 2014
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Fig. 1 Synthesis of the pyrylium probes 2. Reagents and conditions:
(a) BF3ÁOEt2 in toluene (2 h, reflux) and (b) SnCl2ÁH2O, HCl in CH2Cl2 (3 h, reflux).
Fig. 2 X-ray structures of compounds 1a and 2a.
3 h and extracted with dichloromethane. Analyses of the purified
products by HRMS, 1H NMR and 13C NMR spectroscopy confirmed
the identity of 2a–d. Furthermore, for compounds 1a and 2a crystals
suitable for X-ray diffraction were obtained (Fig. 2). As it can be seen,
the keto (deprotonated) form of 2a is obtained in the solid state.10
All pyrylium salts 2a–d were sensitive to the presence of an
excess of NO in aerated water, their absorption spectra changing
dramatically after bubbling gaseous NO. Besides, compound 2c
displayed a remarkable fluorescence turn-on response with a
83-fold increase in the intensity of the fluorescence signal relative
to 1c, excited under the same conditions, (Fig. 3). Photophysical
properties of compounds 2a–d and reaction products of 2a–d with
NO/O2 are summarized in Table 1.
The corresponding analytical limits of detection (LOD) for 2a–d
were calculated according to the IUPAC method (blank + 3s),11 and
are presented in Table 2. As can be seen in Table 2, the LOD for 2c is
2.1 mM whereas the LOD for the rest of the probes are higher. As
micromolar concentrations of NO are typically formed in cultures of
macrophages1 for instance, the obtained LOD values suggest that
our probes could be useful for measurements in these cell types.
Besides, a good linear correlation (R2 = 0.995) between the fluores-
cence intensity and the NO concentration was also found for
compound 2c.
Fig. 3 Spectroscopic studies using 2c as a probe for NO. On the top:
emission spectra of compound 2c (10 mM) in aerated water at pH 7.2 in the
presence of increasing amounts of NO (from 0.1 mM to 1000 mM), (lexc
=
470 nm). On the bottom: emission intensity at l = 550 nm of compound
2c (10 mM) in aerated water at pH 7.2 in the presence of NO, other RNS/
ROS and DHA/HAA (50 equiv.). Also shown photographs of solutions of 2c
before and after exposure to NO (under visible and UV light).
Table 1 Photophysical properties of compounds 2a–d
Compound
labs (nm) (log e)
lemis (nm)
2a
2b
2c
2d
Before NO
After NO
Before NO
After NO
Before NO
After NO
Before NO
After NO
365 (4.0), 549 (4.2)
418 (4.0)
379 (3.6), 550 (3.6)
430 (3.9)
407 (3.8), 550 (3.8)
465 (3.8)
411 (3.6), 540 (3.6)
433 (3.2)
475
484
485
503
527
543
480
523
Table 2 Limits of detection (LOD) for compounds 2(a–d) towards NO
(aerated aqueous solution)
Compound
Enhancement factor
LOD (mM)
2a
2b
2c
2d
8
31
83
5
12.2
6.3
2.1
As stated above, the high specificity of the probe is imperative for
its application. In this regard, we screened a wide array of possible
46.7
competitiÀve RNS and ROS plus other species (50 equivalents): H2O2,
ClOÀ, O2
,
ꢀOH, O2, NO3À, NO2À, ONOOÀ, HNO, ascorbic acid
1
(HAA) and dehydroascorbic acid (DHA). As shown in Fig. 3, no 2a–d in the presence of NO/air, a sample of 2a was allowed to
detectable responses for these reagents were observed under the react with an excess of NO in aerated water and the isolated
1
same conditions. Moreover, DHA was found not to diminish the product was analyzed by H NMR, 13C NMR, HRMS and FTIR
sensitivity of 2c towards NO (Fig. S15, ESI†).
spectroscopy. Surprisingly, no product was found to have an
In order to get deeper knowledge about the mechanism increased molecular weight other than one with M+ = 325,
involved in the fluorescence turn-on response of compounds 12 133, suggesting that the reaction with N2O3 leads to
3580 | Chem. Commun., 2014, 50, 3579--3581
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Work at UJI was financially supported by the MINECO
(CTQ2009-09953, CTQ2012-38543-C03-01), GV (PROMETEO/
2012/020) and UJI (P1 1B-2012-41). A.B. is grateful for the
support of a FPU fellowship. Technical assistance from the
`
SCIC and from D. Recatala with X-ray structures is acknowl-
edged. Work at DPS laboratory was supported by grants from
MINECO (SAF2009-11642 and SAF2012-36519), RD12/0013/0008
´
from ISCIII and CTS-6603 from Junta de Andalucıa.
Notes and references
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Fig. 4 Fluorescence of probe 2c in murine macrophages. RAW264.7 cells
were cultured in the presence of the indicated agents. After incubation
with the 2c probe at 37 1C for 30 min, cells were visualized by confocal
microscopy. Scale bar: 20 mm.
deamination of the probe.12 As a matter of fact, crystals of the
reaction product suitable for X-ray diffraction were obtained,
confirming the loss of the amine group (Fig. S19, ESI† shows the
deaminated product, also in the keto form after loss of a proton
from the hydroxy group). The presence of a hydroxy group
neighboring the amine functionality must be the key factor for
this reaction to occur. In order to confirm this hypothesis, model
compound 4, bearing only NH2, was synthesized and tested
towards NO/O2 and no fluorescence enhancement was recorded.
We have also assessed the ability of compound 2c for imaging
NO in live macrophages (RAW264.7) by means of confocal fluores-
cence microscopy. Cells incubated in the presence of a vehicle
showed negligible fluorescence. Non-activated macrophages incu-
bated with the probe showed a weak background. Macrophages
(activated with LPS and IFN-g to produce NO) showed a clear
increase in fluorescence with a predominantly cytosolic distribution
(Fig. 4). This increase was blunted when cells were activated in the
presence of the NOS inhibitor L-NMMA. As a control for these
assays, the accumulation of nitrite in the cell supernatants was
measured, reflecting the increase in NO generation upon macro-
phage activation, which was reduced by 50% in the presence of
L-NMMA (Fig. S20, ESI†). Taken together, these results suggest that
the increase in fluorescence observed in activated cells is due to
NOS-generated NO.13 It is worth noting that under these conditions
only intracellular fluorescence was detected.
¨
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Hung, W.-F. Liaw and Y.-M. Wang, Inorg. Chem., 2012, 51, 5400.
In summary, we have developed a new family of probes,
based on pyrylium salts and the ortho-hydroxyamino structure,
which are sensitive to NO (aerated solution). Deamination of
the probe takes place leading to a highly fluorescent product.
The new probes are sensitive to NO at the micromolar level,
present no interference with DHA and are suitable for the
imaging of NO in live RAW264.7 cells by confocal microscopy.
´
˜
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This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 3579--3581 | 3581