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

Nitric oxide (NO) has captured the attention of many researchers during the last decade. Interest in NO derives from its known biological activity, in particular its pivotal role in vasodilatation or its functions as a neurotransmitter and an antimicrobial agent. Fluorescence techniques have become very popular for NO sensing because of their high sensitivity and spatiotemporal resolution when combined with microscopy. Thus, the construction of small fluorescent sensors suitable for specific NO detection in living systems has received great attention. A number of fluorescent probes have been reported to date. The most common approach for NO detection involves the use of o-diamino aromatics under aerobic conditions. These molecules react with N2O3 (formed from NO and O2), to yield fluorescent triazole derivatives. 3

Nitric oxide (NO) has captured the attention of many researchers during the last decade.Interest in NO derives from its known biological activity, in particular its pivotal role in vasodilatation or its functions as a neurotransmitter and an antimicrobial agent. 1 Fluorescence techniques have become very popular for NO sensing because of their high sensitivity and spatiotemporal resolution when combined with microscopy. 2 Thus, the construction of small fluorescent sensors suitable for specific NO detection in living systems has received great attention.A number of fluorescent probes have been reported to date.The most common approach for NO detection involves the use of o-diamino aromatics under aerobic conditions.These molecules react with N 2 O 3 (formed from NO and O 2 ), to yield fluorescent triazole derivatives. 3nother family of probes, in this case reacting directly with NO, is based on transition-metal complexes. 4An increasing number of other strategies have been described in the literature. 5urthermore, although the past several years have witnessed great progress in the development of ortho-diamine fluorescent probes, they still have some shortcomings.One of the most important drawbacks, is that dehydroascorbic acid (DHA) reacts with o-diamino aromatics and turns on the fluorescence of such probes in an analogous way as N 2 O 3 does.As pointed out by Sweedler and coworkers, dehydroascorbic acid reacts with ortho-diamine probes to give fluorescent adducts which are indistinguishable from those arising from the reaction with N 2 O 3 . 6To solve this problem various correction strategies have been developed, including freezing of the medium, 7 using enzymes and capillary electrophoresis 8 and multiderivatization image analysis. 9In this regard, notable detection limits have been reached using a contactless correction method (10 nM). 7It should be noted that not taking into account this important biological interfering species may lead researchers in biomedical areas to wrongly interpret the recorded data.However, developing a more selective probe, not reactive with DHA, thus avoiding the aforementioned correction methods, continues to be an important target of current interest in the field.
Herein we present the development of a new family of synthetic molecular probes for NO in an oxygenated medium based on the pyrylium cation which meets the essential criteria, namely: (1) they are sensitive to NO/O 2 without showing significant interference with other reactive nitrogen (RNS) and oxygen species (ROS), and especially without showing interference with dehydroascorbic acid, and (2) they are obtained by a simple methodology that allows efficient synthesis of the product in only two synthetic steps and in high yields from readily available starting products.Besides, the synthetic procedure allows for a straightforward introduction of structural variants favoring a fine tuning of the different spectroscopic properties (excitation and fluorescence emission wavelengths, emission quantum yields, and fluorescence lifetimes, etc.).
The key factor to accomplish the above-mentioned features is the utilization of the ortho-hydroxyamino structure instead of the traditional ortho-diamino one.
The synthesis of compounds 2a-d started with the preparation of the intermediates 1a-d with an ortho-hydroxynitro substructure (Fig. 1).Intermediates 1a-d were easily prepared by adding BF 3 Á OEt 2 to a solution of 4-hydroxy-3-nitrobenzaldehyde and the corresponding acetophenone in anhydrous toluene.The solution was refluxed for 2 h.After cooling to room temperature, acetone was added and the solution was poured into excess ether.A precipitate was formed which was then filtered, washed with ether and dried 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 SnCl 2 Á2H 2 O in dichloromethane and excess of hydrochloric acid under nitrogen atmosphere.The reaction mixture was refluxed for This journal is © The Royal Society of Chemistry 2014 3 h and extracted with dichloromethane.Analyses of the purified products by HRMS, 1 H NMR and 13 C 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. 10ll 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/O 2 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 macrophages 1 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 fluorescence intensity and the NO concentration was also found for compound 2c.
As stated above, the high specificity of the probe is imperative for its application.In this regard, we screened a wide array of possible competitive RNS and ROS plus other species (50 equivalents): H 2 O 2 , ClO À , O 2 À , OH, 1 O 2 , NO 3 À , NO 2 À , ONOO À , HNO, ascorbic acid (HAA) and dehydroascorbic acid (DHA).As shown in Fig. 3, no detectable responses for these reagents were observed under the same conditions.Moreover, DHA was found not to diminish the sensitivity of 2c towards NO (Fig. S15, ESI †).
In order to get deeper knowledge about the mechanism involved in the fluorescence turn-on response of compounds  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), (l exc = 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).deamination of the probe. 12As 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 NH 2 , was synthesized and tested towards NO/O 2 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 fluorescence microscopy.Cells incubated in the presence of a vehicle showed negligible fluorescence.Non-activated macrophages incubated 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 macrophage 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.
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.

Fig. 4
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.

Table 1
Photophysical properties of compounds 2a-d