Schiff bases containing a furoxan moiety as potential nitric oxide donors in plant tissues
Schiff bases containing a furoxan moiety as potential nitric oxide donors in plant tissues
Emilian Georgescu 0 1
Anca Oancea 1
Florentina Georgescu 1
Alina Nicolescu 1
Elena Iulia Oprita 1
Lucian Vladulescu 1
Marius-Constantin Vladulescu 1
Florin Oancea 1
Sergiu Shova 1
Calin Deleanu 1
0 Research Center Oltchim, RamnicuValcea, Romania, 2 National Institute of Research and Development for Biological Sciences , Bucharest , Romania , 3 Research Dept. , Teso Spec S.R.L., Fundulea, Calarasi , Romania , 4 aPetruPonio Institute of Macromolecular Chemistry, Romanian Academy , Aleea Grigore Ghica Voda, Iasi, Romania, 5 aC. D. Nenitescuo Centre of Organic Chemistry, Romanian Academy , Bucharest , Romania , 6 National Research & Development Institute for Chemistry & Petrochemistry ± ICECHIM, Bucharest, Romania, 7 Institute of Chemistry, Academy of Sciences , Chisinau , Republic of Moldova
1 Editor: Markus M Bachschmid, Boston University , UNITED STATES
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.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: This work was funded by the Romanian
Ministry of National Education ± National Authority
for Scientific Research UEFISCDI, www.uefiscdi.ro
Award Numbers PN-II-PT-PCCA-2013-4-0267 ±
SAFE-SEL [AO] and
PN-IIIP1-1.2-PCCDI-20170569 ± PROSPER [FO]. The funder provided
support in the form of salaries and research
materials [FO AO], but did not have any additional
Nitric oxide (NO) is a signaling molecule common to animals and plants [1±2]. In plants, NO
participates in important processes such as germination, flowering, stomatal closure [2±8],
activates disease resistance to pathogen attacks and possibly acts as direct anti-microbial agent
]. Plant defense responses to pathogen attacks is activated by a complex signal produced by
the accumulation of reactive oxygen species (ROS) and NO [3±6]. These findings stand for
eco-friendly means to control disease in plants. Many chemicals have been used as NO donors
or even for biological generation of NO in animals and plants [
]. Sodium nitroprusside
(SNP), S-nitrosoglutathione (GSNO), S-nitroso-N-acetylpenicillamine (SNAP) and so-called
NONOates (spermidine or diethylamine-NONOate) are among the most used nitric oxide
Searching for synthetic compounds acting as NO donors or as biological inducers of NO in
plants is important not only for understanding the NO mechanism in plants but also for field
applications. Our expertise on synthesis of new bioactive heterocyclic compounds [14±20] and
role in the study design, data collection and
analysis, decision to publish, or preparation of the
manuscript. The specific roles of these authors are
articulated in the ªauthor contributionsº section.
Competing interests: The authors declare no
competing interests. The affiliation of some
coauthors to commercial companies [EG FG LV
MCV] does not alter our adherence to PLOS ONE
policies on sharing data and materials.
interest in signaling compounds in plants [
] prompted us to obtain stable Schiff bases
containing a furoxan moiety as possible NO donors in plants. Furoxan, 1,2,5-oxadiazole
Noxide, is an important scaffold of many compounds that show typical NO-donor properties in
mammals, some furoxan derivatives being known as NO-donating pro-drugs [23±27]. Schiff
bases are resourceful intermediates in several enzymatic reactions  as well as for the design
of a large number of bioactive lead compounds [
]. Their biological properties include
], biocidal [
], antifungal [
], antiviral [
], and high antitumor
Various synthetic methods towards furoxan derivatives such as the cyclization of α-nitro
], the dimerization of nitrile N-oxides , the reaction of alkenes with
aqueous sodium nitrite in glacial acetic acid [40±42], and the reaction of styrene derivatives with
nitrosonium tetrafluoroborate (NOBF4), usually leading to both furoxan regioisomers, were
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.
Materials and methods
Melting points were measured on a BoeÈtius 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 1H, 13C, and 15N 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 (1H spectra and all H-C/H-N 2D experiments) or with a 5 mm four
nuclei direct detection z-gradient probe (13C spectra). Chemical shifts are reported in δ units
(ppm) and were referenced to internal TMS for 1H chemical shifts, to the internal deuterated
solvent for 13C chemical shifts (CDCl3 referenced at 77.0 ppm) and to liquid ammonia
(0.0 ppm) using nitromethane (380.2 ppm) as external standard for 15N chemical shifts.
Unambiguous 1D NMR signal assignments were made based on 2D NMR homo- and
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
XCALIBUR 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 [
], the structure was solved with the ShelXT [
] structure solution program using
Direct Methods and refined with the ShelXL [
] 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
Crotonic aldehyde, p-toluenesulfonyl hydrazide, 2,4,6-trimethylbenzenesulfonyl hydrazide,
4-phenyl-3-thiosemicarbazide, 4-(4-methylphenyl)-3-thiosemicarbazide, p-toluic hydrazide
were purchased from Sigma Aldrich and used further without purification.
Fluorescence measurements were recorded on a Zeiss AXIOÐOBSERVER 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
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Fluorescence indicator 2',7'-dichlorodihydrofluorescein diacetate (H2DCFA) was purchased
from Invitrogen Molecular Probes. Fluorescence
indicator4-amino-5-methylamino-2',7'difluorofluorescein diacetate (DAF-FM DA), chitosan, TWEEN 20, a nonionic detergent,
sulfanilamide and N-(1-naphthyl)ethylenediamine dihydrochloride for Griess reagent were
purchased from Sigma Aldrich.
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.
p-Toluenesulfonic acid (3-methyl-2-oxy-furazan-4-ylmethylene)hydrazide (3a). White
crystals, yield 86% (5.1 g), mp 163-165ÊC (from MeOH:H2O 1:1), IR (ν, cm-1): 3210, 1612,
1470, 1383, 1349, 1309, 1158, 1080, 1038. 1H NMR (400.1 MHz, DMSO-d6, δ (ppm)): 2.16
(3H, s, CH3-3), 2.40 (3H, s, CH3-12), 7.45 (2H, d, 8.0 Hz, H-11/13), 7.76 (2H, d, 8.0 Hz, H-10/
14), 7.93 (1H, s, H-6), 12.35 (1H, s, NH-8). 13C NMR (100.6 MHz, DMSO-d6, δ (ppm)): 9.2
(CH3-3), 21.0 (CH3-12), 111.2 (C-3), 127.2 (CH-10/14), 129.9 (CH-11/13), 134.9 (CH-6),
135.4 (C-9), 144.1 (C-12), 153.7 (C-4). 15N NMR (40.6 MHz, DMSO-d6, δ (ppm)): 175.4
(NH8), 340.2 (N-7), 357.8 (N-2), 373.1 (N-5). HRMS-ESI (m/z): [M-H]-for C11H11N4O4S, calcd.
295.0501, found 295.0518.
2,4,6-trimethylbenzenesulfonic acid (3-methyl-2-oxy-furazan-4-ylmethylene)hydrazide
(3b). Pale yellow solid, yield 74% (4.8 g), mp 177-179ÊC (from MeOH). IR (ν, cm-1): 3199,
2979, 2936, 1608, 1467, 1377, 1330, 1301, 1162, 1098, 1029. 1H NMR (400.1 MHz, DMSO-d6,
δ (ppm)): 2.04 (3H, s, CH3-3), 2.28 (3H, s, CH3-12), 2.60 (6H, s, CH3-10/14), 7.10 (2H, s,
H11/13), 7.94 (1H, s, H-6), 12.53 (1H, s, NH-8). 13C NMR (100.6 MHz, DMSO-d6, δ (ppm)):
8.7 (CH3-3), 20.4 (CH3-12), 22.5 (CH3-10/14), 111.1 (C-3), 131.7 (CH-11/13), 132.4 (C-9),
133.3 (CH-6), 139.3 (C-10/14), 142.9 (C-12), 153.3 (C-4). 15N NMR (40.6 MHz, DMSO-d6, δ
(ppm)): 177.5 (NH-8), 338.5 (N-7), 357.6 (N-2), 371.6 (N-5). X-Ray:C13H16N4O4S, (M =
324.36 g/mol): monoclinic, space group P21/n (no. 14), a = 7.8827(6) Å, b = 18.4644(14) Å,
c = 10.6000(8) Å, β = 110.634(9), V = 1443.9(2) Å3, Z = 4, T = 200.00(10) K, μ(MoKα) = 0.241
mm-1, Dcalc = 1.469 g/cm3, 5588 reflections measured (4.412 2Θ 50.046), 2552 unique
(Rint = 0.0303, Rsigma = 0.0558) which were used in all calculations. The final R1 was 0.0556
(I 2σ(I)) and wR2 was 0.1466 (all data).CCDC ± 1556711. HRMS-ESI (m/z): [M-H]- for
C13H15N4O4S, calcd.323.0814, found 323.0846.
4-methyl-benzoic acid (3-methyl-2-oxy-furazan-4-ylmethylene)hydrazide (5). White
solid, yield 71% (3.7 g), mp 212-214ÊC (from MeOH), IR (ν, cm-1): 3422, 3220, 3032, 1638,
1610, 1570, 1490, 1460, 1381, 1331, 1310, 1283, 1186, 1147, 1036. 1H NMR (400.1 MHz,
DMSO-d6, δ (ppm)): 2.40 (6H, bs, CH3-3 and CH3-13), 7.38 (2H, d, 7.1 Hz, H-12/14), 7.85
(2H, d, 7.1 Hz, H-11/15), 8.49 (1H, s, H-6), 12.33 (1H, s, NH-8). 13C NMR (100.6 MHz,
DMSO-d6, δ (ppm)): 9.1 (CH3-3), 21.1 (CH3-13), 111.7 (C-3), 127.8 (CH-11/15), 129.2
(CH12/14), 129.6 (C-10), 135.8 (CH-6), 142.6 (C-13), 154.1 (C-4), 163.2 (CO-9). 15N NMR (40.6
MHz, DMSO-d6, δ (ppm)): 174.0 (NH-8). HRMS-ESI (m/z): [M-H]- for C12H11N4O3, calcd.
259.0831, found 259.0851.
1-(3-methyl-2-oxy-furazan-4-ylmethylene)-4-phenyl-3-thiosemicarbazone (7a). Beige
solid, yield 69% (3.83 g), mp 184-186ÊC (from CHCl3/MeOH), IR (ν, cm-1): 3324, 3128, 2983,
1609, 1518, 1491, 1462, 1383, 1306, 1251, 1169, 1110, 1033. 1H NMR (400.1 MHz, DMSO-d6,
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δ (ppm)): 2.44 (3H, s, CH3-3), 7.24 (1H, t, 7.6 Hz, H-14), 7.40 (2H, t, 7.6 Hz, H-13/15), 7.59
(2H, d, 7.6 Hz, H-12/16), 8.24 (1H, s, H-6), 9.87 (1H, s, NH-10), 12.32 (1H, s, NH-8). 13C
NMR (100.6 MHz, DMSO-d6, δ (ppm)): 9.2 (CH3-3), 111.6 (C-3), 125.1 (CH-12/16), 125.6
(CH-14), 128.3 (CH-13/15), 131.4 (CH-6), 138.7 (C-11), 153.9 (C-4), 176.5 (CS-9). 15N NMR
(40.6 MHz, DMSO-d6, δ (ppm)): 129.1 (NH-10), 177.4 (NH-8), 334.1 (N-7), 357.6 (N-2),
372.3 (N-5). HRMS-ESI (m/z): [M-H]- for C11H10N5O2S, calcd. 276.0555, found 276.0537.
(7b). Yellow solid, yield 63% (3.67 g), mp 188-190ÊC (MeOH/Et2O), IR (ν, cm-1): 3412, 3350,
3127, 2966, 1612, 1540, 1517, 1458, 1379, 1304, 1259, 1205, 1170, 1120, 1024. 1H NMR (400.1
MHz, DMSO-d6, δ (ppm)): 2.31 (3H, s, CH3-14), 2.43 (3H, s, CH3-3), 7.19 (2H, d, 8.2 Hz,
H13/15), 7.45 (2H, d, 8.2 Hz, H-12/16), 8.23 (1H, s, H-6), 9.78 (1H, s, NH-10), 12.26 (1H, s,
NH8). 13C NMR (100.6 MHz, DMSO-d6, δ (ppm)): 9.2 (CH3-3), 20.5 (CH3-14), 111.6 (C-3), 125.1
(CH-12/16), 128.7 (CH-13/15), 131.2 (CH-6), 134.9 (C-14), 136.1 (C-11), 153.9 (C-4), 176.5
(CS-9). 15N NMR (40.6 MHz, DMSO-d6, δ (ppm)): 128.8 (NH-10), 177.4 (NH-8), 334.4 (N-7),
357.6 (N-2), 372.6 (N-5). HRMS-ESI (m/z): [M-H]- for C12H12N5O2S, calcd. 290.0712, found
Plant growth conditions and treatment. In our experiments we used Arabidopsis
thaliana plants, cultivated in laboratory in Arasystem . 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 21-23ÊC, 70% humidity, light intensity 150 μmol/m2 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 [
], 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 (H2DCFA) as fluorescent indicator. The
collected Arabidopsis leaves were washed with distilled water and incubated with 2.5 μMH2DCFA
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 H2DCFA ± 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-amino5-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 N2O3, generated by NO oxidation, and form a DAF-FM benzotriazole derivative which
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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 4ÊC. 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
Results and discussion
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 [
], 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, 1H, 13C and 15N 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 13C-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 15N- and
13C-NMR spectra for series of octahydroacridines [
]. The shifts induced by N-oxidation
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Fig 1. Synthesis of Schiff bases containing a furoxan ring.
to C-alpha (C3 in furoxan derivatives) and C-beta (C4 in furoxan derivatives) is consistent
with our previous studies , and with early data on simple furoxan derivatives [
Noxidation induces a significant shielding of the C-alpha and a slight deshielding of the C-beta
in the 13C-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
(O2−, OH· and H2O2) 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 [
]. 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 [
]. 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 [
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Fig 2. X-ray molecular structure of compound 3b. Thermal ellipsoids are drawn at 50% probability level.
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 [
], 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 (H2DCFA) [
]. 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:
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±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.
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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 [
]. 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
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
S1 File. Crystallographic information file (CIF) for the compound 3b.
S2 File. Fluorescence Microscopy and NMR information file.
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Access to research infrastructure developed in the ªPetruPoniº Institute of Macromolecular
Chemistry through the European Social Fund for Regional Development, Competitiveness
Operational Programme Axis 1, Project InoMatPol (ID P_36_570, Contract 142/10.10.2016,
cod MySMIS: 107464) is gratefully acknowledged.
Conceptualization: Emilian Georgescu, Florentina Georgescu, Florin Oancea, Calin Deleanu.
Data curation: Emilian Georgescu, Anca Oancea, Florentina Georgescu, Alina Nicolescu,
Elena Iulia Oprita, Lucian Vladulescu, Marius-Constantin Vladulescu, Florin Oancea,
Sergiu Shova, Calin Deleanu.
Formal analysis: Anca Oancea, Florentina Georgescu, Alina Nicolescu, Elena Iulia Oprita,
Florin Oancea, Sergiu Shova.
Funding acquisition: Anca Oancea, Florin Oancea.
Investigation: Emilian Georgescu, Calin Deleanu.
Methodology: Emilian Georgescu, Anca Oancea, Alina Nicolescu, Florin Oancea.
Resources: Anca Oancea, Calin Deleanu.
Writing ± original draft: Emilian Georgescu, Florentina Georgescu, Florin Oancea, Calin
Writing ± review & editing: Emilian Georgescu, Florentina Georgescu, Calin Deleanu.
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