Schiff bases containing a furoxan moiety as potential nitric oxide donors in plant tissues

Stable Schiff bases containing a furoxan moiety are synthesized as single regioisomers by the reaction of 3-methyl-2-oxy-furazan-4-carbaldehydewith various amino compounds at room temperature. The structures of synthesized compounds were fully characterized by multinuclear NMR spectroscopy and X-ray crystallography. The effect of synthesized Schiff bases containing a furoxan moiety on biological generation of reactive oxygen species and nitric oxide in plant tissues was investigated for the first time by fluorescence microscopy and the released NO identified as nitrite with Griess reagent. There is a good correlation between the biological generation of NO determined by fluorescence microscopy and with Griess reagent. Some of the synthesized compounds exhibited both nitric oxide and reactive oxygen species generation abilities and represent potential NO donors in plant tissues.

Herein, we present the synthesis of stable Schiff bases containing a furoxan moiety obtained as single regioisomer and their effects as nitric oxide donors in plant tissues.

Analytical equipment
Melting points were measured on a Boëtius hot plate microscope and are uncorrected.
IR spectra were recorded on a Nicolet Impact 410 spectrometer, in KBr pellets. The NMR spectra have been recorded on a Bruker Avance III 400 instrument operating at 400.1, 100.6 and 40.6 MHz for 1 H, 13 C, and 15 N nuclei respectively. Samples were transferred in 5 mm Wilmad 507 NMR tubes and recorded with either a 5 mm multinuclear inverse detection z-gradient probe ( 1 H spectra and all H-C/H-N 2D experiments) or with a 5 mm four nuclei direct detection z-gradient probe ( 13 C spectra). Chemical shifts are reported in δ units (ppm) and were referenced to internal TMS for 1 H chemical shifts, to the internal deuterated solvent for 13 C chemical shifts (CDCl 3 referenced at 77.0 ppm) and to liquid ammonia (0.0 ppm) using nitromethane (380.2 ppm) as external standard for 15 N chemical shifts. Unambiguous 1D NMR signal assignments were made based on 2D NMR homo-and heterocorrelations.
High resolution MS spectra have been recorded on a Bruker Maxis II QTOF spectrometer with electrospray ionization (ESI) in the negative mode.
X-Ray crystallographic measurements were carried out with an Oxford-Diffraction XCALI-BUR E CCD diffractometer equipped with graphite-monochromated Mo-Kα radiation. The crystal was kept at 200.00(10) K during data collection. The unit cell determination and data integration were carried out using the CrysAlis package of Oxford Diffraction [44]. Using Olex2 [45], the structure was solved with the ShelXT [46] structure solution program using Direct Methods and refined with the ShelXL [47] refinement package using Least Squares minimization. These data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving. html (or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK).
Fluorescence measurements were recorded on a Zeiss AXIO-OBSERVER D1, equipped with a video digital camera AxioCamMRc using AxioVision Rel.4.6 software.
Spectrophotometric analysis were performed on the Elisa plate according the reported bioprotocol [48].

General procedure for Schiff bases containing a furoxan moiety 3, 5 and 7
A solution of 25 mmol (3.5 g) of 3-methyl-2-oxy-furazan-4-carbaldehyde 1 in 30 mL methanol was added dropwise to a stirred solution containing 20 mmol of an amino derivative (2, 4 and 6 respectively) in 30 mL methanol. After four hours stirring at ambient temperature, the solvent was partly removed under vacuum. The formed solid was filtered off, washed with cold ethanol (5 mL) and recrystallized.

Histochemical analysis
Plant growth conditions and treatment. In our experiments we used Arabidopsis thaliana plants, cultivated in laboratory in Arasystem [49]. Arabidopsis thaliana wild type seeds (provided by Lehke Seeds Texas, USA) have been seeded in sterilized soil and cultivated for six weeks in a special growth room, at 21-23˚C, 70% humidity, light intensity 150 μmol/m 2 and a photoperiode of 14/10. Each synthesized compound (0.5 mg, and 2.5 mg respectively) dissolved in ethanol was mixed with 0.25 g Tween 20 and demineralized water to prepare 50 mL of each test suspension/solution. The inductor suspensions were kept in spraying glass bottle, in the dark, at room temperature. The Arabidopsis leaves were sprayed with the inductor suspensions (at a rate of 1 mL/plant) andcollected after 24 hours. The leaves were washed with distilled water for histochemical analysis of ROS and NO by fluorescence microscopy, or worked-up according to the reported protocol [48], in order to determine NO releasing potential of new synthesized Schiff bases bearing a furoxan moiety with Griess reagent. As positive control in histochemical analysis by fluorescent microscopy we used plant treated with chitosan solution, 10 μg/mL, and 50 μg/mL respectively, in 0.5% acetic acid solution, buffered to pH 5.6 with NaOH 1 M. ROS and NO visualization by fluorescence microscopy. Intracellular ROS was visualized using 2',7'-dichlorodihydrofluorescein diacetate (H 2 DCFA) as fluorescent indicator. The collected Arabidopsis leaves were washed with distilled water and incubated with 2.5 μMH 2 DCFA solution (10 mMin DMSO), for 30 min, in the dark, at room temperature. Then the leave fragments were washed twice with distilled water and the H 2 DCFA -mediated fluorescence was detected (emission/excitation: 488/525 nm). As negative controls, Arabidopsis leaves untreated with inductor suspensions have been used. Intracellular NO was visualized using 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA)as fluorescent indicator. The collected Arabidopsis leaves were washed with distilled water and incubated with 10 μM DAF-FM diacetate (5mM in DMSO),for 15 min, in the dark, at room temperature. Then the leave fragments were washed twice with phosphate buffer saline (PBS) at pH 7.4 and the fluorescence of the reaction product of DAF-FM DA with NO was captured (emission/excitation: 488/525 nm). As negative controls, Arabidopsis leaves untreated with inductor suspensions have been used.
The NO specific dye, 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM), reacts with N 2 O 3 , generated by NO oxidation, and form a DAF-FM benzotriazole derivative which exhibits a green fluorescence. However, this dye is not cell permeant and its fluorescent derivatives are an indication of ROS (and NO) formation outside of the plant cells, on tissue level. The 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA) is a cell permeable dye. This dye is converted by cytosolic esterase to DAF-FM, which produces the benzotriazole fluorescent derivative inside the cell. The generation of ROS is a biological effect in plants, mainly due to the released NO, which are redox gasotransmitters. The reactive species formed in plants are NO and, most probably, on physiological conditions, peroxynitrite (due to NO reaction with ROS).
Determination of nitrite concentration in Aradidopsis thaliana leaves with Griess reagent. 50 μl of sulfanilamide 1% (w/v) solution in 5% (v/v) phosphoric acid and 50 μl of N-(1-naphthyl)ethylenediamine dihydrochloride 0.1% (w/v) solution were added to 50 μl Arabidopsis leaves extract supernatant. The leaves, treated with the same amounts of synthesized Schiff bases suspensions, collected after 24 hours, and controls washed with distilled water, were powdered with nitrogen liquid into a mortar. 100 mg of leaves powder was extracted for 30 min in 300 μl of 100 mM phosphate buffer, pH 7.4. The extract was centrifuged for 15 min at 10,000 x g and 4˚C. The resulted supernatant was used for indirect NO determination with sulfanilamide and N-(1-naphthyl)ethylenediamine dihydrochloride solutions, after incubation for 5-10 min at room temperature protected from light. The color appeared immediately as the Griess reagent is formed. The absorbance was directly measured in a plate reader with a filter between 520 nm and 550 nm. A nitrite standard curve was used to calculate the nitrite concentration in the samples and expressed as μM of nitrite anion. The detection of nitrite concentration in Aradidopsis thaliana leaves was performed according to the reported bioprotocol [48].

Synthesis of Schiff bases containing a furoxan moiety
Stable Schiff bases containing a furoxan moiety were synthesized in order to explore their chemical and biological properties. The synthetic procedure is based on the reactions of the 3-methyl-2-oxy-furazan-4-carbaldehyde with various amino compounds capable to produce stable Schiff bases bearing a furoxan ring. The intermediate 3-methyl-2-oxy-furazan-4-carbaldehyde (1), already described in literature [40], was easily obtained as single isomer from crotonic aldehyde and sodium nitrite in glacial acetic acid at room temperature. Therefore, by treating furoxan carbaldehyde 1 with phenylsulfonyl hydrazide derivatives 2a,b the corresponding phenylsulfonylhydrazones containing a furoxan moiety 3a,b were obtained (Fig 1i). Starting from the furoxan carbaldehyde 1 and p-toluic hydrazide 4 the corresponding Schiff base 5 bearing the furoxan ring was prepared (Fig 1ii). In the same way, the reaction of furoxan carbaldehyde 1 with thiosemicarbazides 6 led to the corresponding thio-semicarbazones 7a,b carrying a furoxan moiety (Fig 1iii). All reactions took place easily at room temperature and yields are in the range 63-86%.
The structures of all Schiff bases containing the furoxan moiety were assigned on the basis of chemical and spectral analysis (IR, 1 H, 13 C and 15 N NMR spectra). NMR data clearly indicated the presence of only one regioisomer bearing the external oxygen atom on the nitrogen in position 2 of the furoxan ring. The 2-versus5-N-oxidation of the furoxan ring in all compounds (3, 5, 7) is supported by similar chemical shifts for C3 and C4 in the 13 C-NMR spectra. Assigning the site of N-oxidation in various natural or biological active compounds is important both for structural and mechanistic purposes related to metabolisation of these compounds. We have also previously investigated the influence of N-oxidation on 15 N-and 13 C-NMR spectra for series of octahydroacridines [50,51]. The shifts induced by N-oxidation to C-alpha (C3 in furoxan derivatives) and C-beta (C4 in furoxan derivatives) is consistent with our previous studies [51], and with early data on simple furoxan derivatives [52]. The Noxidation induces a significant shielding of the C-alpha and a slight deshielding of the C-beta in the 13 C-NMR spectra.
The N-oxidation in position 2 of the furoxan ring in the case of derivative 3b has been also proven by X-ray crystallography (Fig 2).
Biological activity. Furoxan derivatives have been of considerable interest to chemists for years but they received relatively little attention from biologists despite their NO-releasing capacities. It is for the first time when Schiff bases containing a furoxan moiety were used as NO donors in plant experiments. We investigated the effect of synthesized compounds on ROS (O 2 − , OH• and H 2 O 2 ) and NO generation in plant tissues. Bio-molecules are rapidly damaged by reactive oxygen species produced under a pathogen attack or in abiotic stress conditions. It is known that both ROS and NO together are required to induce the activation of defense-related enzymes in plants [3]. The protection of plant cells at the sites of ROS and NO generation is ensured by both oxygen radical detoxifying enzymes and non-enzymatic antioxidants contained in plant cells [53,54]. The measurement of the ROS and NO levels in plant tissues is difficult due to very short physiological half-life and high reactivity of these radicals [12,55]. New nitric oxide donors in plants Both ROS and NO were detected by the fluorescence microscopy on Arabidopsisthaliana, a popular model organism for understanding themolecular biologyof many plant traits. Specific fluorescence indicators that are helpful to exactly define the sites of NO and ROS production were used. The presence of ROS and NO in Schiff bases bearing a furoxan moiety-treated Arabidopsis leaves was compared to untreated Arabidopsis leaves as a negative control. Chitosan, a fungal elicitor with known effect as NO and ROS inductor on Arabidopsis [56], was used as positive control at the same concentrations.
ROS induction was detected on Arabidopsis leaves treated with suspension of each synthesized Schiff bases containing a furoxan moiety at the concentration of 10 μg/mL, and 50 μg/ mL respectively, in the presence of the specific fluorescence indicator 2',7'-dichlorodihydrofluorescein diacetate (H 2 DCFA) [57]. Fluorescence microscopy images revealed the presence of ROS in Arabidopsis leaves treated with all Schiff bases having a furoxan moiety, at both concentrations, especially at higher concentration of compounds (50 μg/mL). Efficacy of Schiff bases bearing a furoxan moiety (3a,b, 5, 7a and 7b) on ROS generation pursues the series: 7a>3b!7b>5>3a.
NO donor properties of the synthesized Schiff bases bearing a furoxan moiety were determined on Arabidopsis leaves infiltrated with suspension of each synthesized compound at the same concentrations (10 μg/mL and 50 μg/mL respectively) in the presence of a specific and sensitive fluorescence indicator, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA) [57][58][59][60] and the DAF-FM DA-mediated fluorescence was measured. Strong fluorescence densities were observed at higher concentration (50 μg/mL) of Schiff bases bearing a furoxan moiety with their NO donor efficacy, following the series: 3b>7b>5>7a>3a. In order to assess the NO releasing potential in Arabidopsis leaves we used Griess reagent for indirect determination of NO through its oxidized nitrite form [48]. All Arabidopsis leaves treated with the same amounts of synthesized Schiff bases bearing a furoxan moiety were incubated at room temperature for 5-10 min. with Griess reagent, protected from light, and the absorbance was immediately measured in a plate reader with a filter between 520 nm and 550 nm. A nitrite standard curve was used to calculate the nitrite concentration in the samples and expressed as μM of nitrite anion ( Table 1).
The fluorescence data on NO releasing capacity of these compounds in Arabidopsisthaliana leaves correlate with spectrophotometric data obtained by indirectly assessing NO as nitrite anion with Griess reagent.
All data suggest that some of the synthesized Schiff bases containing a furoxan moiety are involved in ROS and NO production in Arabidopsis treated leaves. Among these, compounds 3b and 7b proved to be really active and are further tested in field trials.
Considering the long-lasting effect, most probably NO release is not only a result of furoxanes decomposition, being rather specific to plant tissue. The NO released from furoxanes could accumulate as S-nitrothiols / S-nitroso-glutathione NO-reservoirs and then slowly released and detected by fluorescence microscopy or with Griess reagent. Thus, plant cells could have a physiological reaction to the Schiff base containing a furoxan moiety. Further research is in progress to assess the mechanism of NO generation into plant tissues by these compounds.

Conclusions
Several Schiff bases containing a furoxan ring have been synthesized as single regioisomers starting from 3-methyl-2-oxy-furazan-4-carbaldehyde and various amino compounds capable to produce stable Schiff bases, in order to identify their chemical and biological properties. We detected for the first time ROS and NO releasing capacities in plant tissues using specific fluorescence indicators, and assessed the NO releasing potential of Schiff bases containing a furoxan ring treated Arabidopisis thaliana leaves. There is a good correlation between fluorescence data and indirect determination of NO biological releasing potential data in plant tissues. The results indicate that some of these compounds represent potential NO donors in plant tissues.