Electro-organic synthesis and characterization of new dihydroxybenzene dinitrile derivatives with fluorescent properties

Article abstract of DOI:10.1248/cpb.56.749

Novel fluorescent molecules were synthesized by designing an environmentally friendly method involving the bulk electrolysis technique. This electrochemical treatment process helps protect the environment by minimizing the toxic waste component of effluent. The electrochemical oxidation of 3,6-dihydroxybenzene-1,2-dinitrile (DBD) in the presence of benzenesulfinic acids was studied in an aqueous solution (H₂O : AN, 90 : 10), which included an acetate buffer (pH=5.0). This research utilized a variety of experimental techniques, including cyclic voltammetry, controlled-potential electrolysis as well as spectroscopic identification of compounds produced as products. In addition, our fluorescent studies offered results in line with existing findings. At the wavelength of 205 nm, DBD and compound (6) were excited and their fluorescent emissions were monitored.

Full text of DOI:10.1248/cpb.56.749

June 2008
Chem. Pharm. Bull. 56(6) 749—752 (2008)
749
Electro-Organic Synthesis and Characterization of New
Dihydroxybenzene Dinitrile Derivatives with Fluorescent Properties
a
Abdolmajid Bayandori MOGHADDAM, Mohammad Reza GANJALI, ,a Rassoul DINARVAND,b
*
a
Maryam LATIFI,a and Parviz NOROUZI
a Center of Excellence in Electrochemistry, Faculty of Chemistry, University of Tehran; and b Medical Nanotechnology
Research Centre, Medical Sciences/University of Tehran; P. O. Box 14155–6451, Tehran, Iran.
Received September 9, 2007; accepted March 8, 2008; published online March 28, 2008
Novel fluorescent molecules were synthesized by designing an environmentally friendly method involving the
bulk electrolysis technique. This electrochemical treatment process helps protect the environment by minimizing
the toxic waste component of effluent. The electrochemical oxidation of 3,6-dihydroxybenzene-1,2-dinitrile (DBD)
in the presence of benzenesulfinic acids was studied in an aqueous solution (H2O : AN, 90 : 10), which included an
acetate buffer (pHꢀ5.0). This research utilized a variety of experimental techniques, including cyclic voltamme-
try, controlled-potential electrolysis as well as spectroscopic identification of compounds produced as products.
In addition, our fluorescent studies offered results in line with existing findings. At the wavelength of 205 nm,
DBD and compound (6) were excited and their fluorescent emissions were monitored.
Key words fluorescence spectroscopy; electro-organic synthesis; electrochemistry
Following the pioneering research of Kasha, Vavilov, Per- tive compounds with fluorescent properties using benzene-
rin, Jablonski, Weber, Stokes and Forster in 1951,1) fluores- sulfinic acids (as nucleophile). It is assumed that the synthe-
cence spectroscopy became a widely used research tool in sis of new dihydroxybenzene dinitrile derivatives with fluo-
pure biochemistry, biophysics and material sciences. How- rescent properties would be useful from a pharmaceutical
ever, in recent years a number of new applications using fluo- and biological viewpoint. However, following a literature
rescence spectroscopy have been developed. These develop- survey, it was discovered that this is the first research paper
ments have promoted it from being primarily a purely scien- concerning dihydroxybenzene dinitrile derivative electro-or-
tific tool to being a more routine analytical and diagnostic ganic reactions for synthesizing fluorescent molecules from
tool. The phenomenon of fluorescence is now exploited for such compounds. The attractive property of these new com-
accomplishing simple analytical assays in the fields of envi- pounds opens a new entrance in electrochemical applica-
ronmental science, clinical chemistry, cell identification— tions.
where it is a dominant method in flow cytometry—as well as
Voltammetric Studies In Fig. 1, the cyclic voltammo-
single cell imaging in medicine. Although there has been gram (CV) (a) was related to the electrochemical behavior of
rapid growth in the number of routinized fluorescence appli- benzenesulfinic acid (3). The CV (b) demonstrated one an-
cations, the principles have remained the same.2)
odic peak (A1) (at 0.501 V) and one corresponding cathodic
Fluorescence spectrometry is a conventional but highly peak (C1) (at 0.455 V). These peaks corresponded to the 3,6-
sensitive analytical method. The synthesis and the biological dihydroxybenzene-1,2-dinitrile (DBD) electro-oxidation to
applications of fluorescent compounds have previously been 3,6-dioxocyclohexa-1,4-diene-1,2-dicarbonitrile (2) and vice
investigated.3—8) It has been shown that the quinone/hydro- versa within a two-electron and two-proton process (DEpꢀ
quinone redox couple can interconvert reversibly by ex- 0.046 V). The CV (c) presented the CV obtained for a 0.1 mM
changing two protons and two electrons. This characteristics solution of DBD in the presence of 0.1 mM 3. It exhibited two
enables it to serve as an antenna or the control subunit, which anodic peaks at 0.504 V (A1) and 0.548 V (A2) vs. the refer-
affects the absorption or the emission optical properties of ence electrode, where the cathodic counterpart of the anodic
the compound. In such quinone optical molecular switches, peak A1 disappeared because of the reaction between 2 and
the interconversion of the four distinct states is caused by the 3, leading to the compound formation (5, Chart 1, Eq. 2).
multiplication of the two electrochromic redox states— The anodic peak A1 was associated with the DBD electro-ox-
quinone and hydroquinone.9—11) Furthermore, an important idation (CV (b) of Fig. 1 in comparison with that of the CV
pathway in the metabolism of dihydroxybenzene estrogens (c)). A2 was attributed to the compound electro-oxidation (5,
and catecholamines is the oxidation of their respective semi- Chart 1, Eq. 3). The CVs were scanned at different rates (Fig.
quinones and quinones. The biological activity function of 1, (II)). The C1 peak height rose with the scan rate increase.
such molecules is related to their ability to act both as oxi- In this graph, the C1 cathodic peak corresponded to the elec-
dants and electrophiles.12) Hydroquinone oxidation in aque- tro-reduction of 2. The plots of the peak current ratio
ous solutions is well documented.13—20) It involves a transfer (IPA 1 /IPC1) and the peak current function (IPA 1 /v1/2) vs. the dif-
of two electrons and two protons to create the associated ferent scan rates between 20 and 2500 mV·sꢁ1 (Fig. 1, (III))
quinone. The electrochemical synthesis between catechol, 4- were identified. The current function for the A1 peak
methylcatechol, 1,4-dihydroxybenzene and benzenesulfinic (IPA 1 /v1/2) reduced with the scan rate increase (with the ECE
acids was reported by S. M. Golabi and co-workers.21)
mechanism).22) A plot of the peak current ratio (IPC1/IPA 1 ) vs.
For the current work, we utilized the electrochemical the scan rate confirmed the reactivity of 2 towards 3, appear-
method to synthesize new dihydroxybenzene dinitrile deriva- ing as an increase in the height of the C1 cathodic peak at
∗ To whom correspondence should be addressed. e-mail: Ganjali@khayam.ut.ac.ir
© 2008 Pharmaceutical Society of Japan

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Vol. 56, No. 6
Fig. 2. CVs of 0.1 mmol 1 in the Presence of 2 (0.1 mmol) in the Course
of CPC at 480 mV, Scan Rate 80 mV·sꢁ1, Other Conditions as in Fig. 1
Chart 2
creased owing to the existence of smaller amounts of com-
pound 5 for electro-oxidation in the A1 peak potential to-
gether with DBD on the electrode surface. To determine the
number of transferred electrons, controlled-potential coulom-
etry (CPC) was performed in an aqueous solution, containing
0.1 mmol of DBD and 0.1 mmol of 3 at 480 mV vs. RE. Fig-
ure 2 depicts the obtained voltammograms during CPC. The
volume of electricity consumed in the CPC course was 21.5
coulombs (C), which was demonstrated by the consumption
of about 2eꢁ per DBD molecule. DBD electro-organic reac-
tions in the presence of p-toluenesulfinic acid (4) for com-
pound 6 formation proceeded in a way similar to that of 3.
However, Fig. 1 illustrated the ECE mechanism for the DBD
electro-oxidation in the presence of 3 and 4, consisting of
three consecutive steps; (i) the DBD electrochemical oxida-
tion (E step), (ii) the chemical reaction of 3 and 4 with 2 (C
step) and (iii) the electrochemical oxidation of the com-
pounds (E step). The EC part of this mechanism was used
with the application of chosen potential at 480 mV in con-
trolled-potential electrolysis for the electro-organic synthesis
of the compounds. It can be assumed that the oxidation
mechanisms on the electrode and in the body share similar
principles.23) Therefore cyclic voltammograms (Fig. 3) of
compound 6 were conducted. The CV of compound 6 exhib-
ited two anodic peaks at 174 mV and 559 mV together with
two corresponding cathodic peaks at 354 mV and 65 mV at
25 mV·sꢁ1. These peaks corresponded to compound 6 elec-
tro-oxidation of the related quinone within a two-electron
and two-proton process, with an initial deprotonation step
(Chart 2).24) Figure 3b shows the CV of compound 6 at
100 mV·sꢁ1. It is evident from this figure that the first oxida-
Fig. 1. (I) Comparative CVs of 0.1 mM 3 (a), DBD without (b) and with
(c) 3, at a GCE in Aqueous Solution (H2O : AN, 90 : 10), Including Acetate
Buffer (pHꢀ5.0), (II) as (I)-(c), (III) IPC1/IPA 1 and IPA 1 /v1/2 vs. Scan Rate
Chart 1
higher scan rates. It was evident that the A1 and A2 oxidation tion peak shifted to more positive potentials (appeared at
peaks converged at scan rates up to 40 mV·sꢁ1 and appeared 427 mV) in contrast to that of Fig. 3a. So, the oxidation
as the A1 anodic peak (Fig. 1, (II)). This phenomenon was peaks show a tendency to converge at higher scan rate.
due to the fact that at low scan rates, the time scale for the re-
Fluorescent Properties of DBD and the Compounds
action of 2 with 3 was longer and the compound formation Figure 4 (a) shows the UV–Vis spectrum for DBD with the
diminished during the scan rate increase. Therefore, IPA 1 de- lmax values of 205, 236 and 381 nm, while Fig. 4 (b) depicts

June 2008
751
Fig. 3. CVs of 0.1 mM Product 6 at GCE in Phosphate Buffer Solution (pHꢀ7.0)
Fig. 4. UV–Vis Spectrum of 0.125 mM (a) DBD and (b) Product 6 in Phosphate Buffer Solution (pHꢀ7.0)
Fluorescence spectrum of (c) DBD (0.125 mM, 39.25 mg/ml) and (d) product 6 (0.125 mM, 20 mg/ml). Excitation at 205 nm for the fluorescence measurements.
also enhanced by the acid dissociation reaction with in-
creased pH levels. Thus, this research studied the controlled-
potential macroscale electrolysis at pH 5.0 for the synthesis
of compounds 5 and 6 into new dihydroxybenzene dinitrile
derivatives with fluorescent properties for the purposes of
identifying new compounds with possible pharmaceutical
properties. This work provides a better understanding of
DBD electrochemical behavior in electro-organic reactions.
Experimental
Chemical Reagents 3,6-Dihydroxybenzene-1,2-dinitrile (Merck), ben-
zenesulfinic acid sodium salt (Aldrich), p-toluenesulfinic acid sodium salt
hydrate (Aldrich) were used without any further purification. For the prepa-
ration of the buffer solutions, the used salts were not purified (reagent grade
materials, Merck). All solutions were prepared with acetonitrile (Merck) and
deionized water.
Fig. 5. (A) DBD, (B) Product 6 and (C) p-Toluenesulfinic Acid with the
Concentration Values of 0.125 mM
the UV–Vis spectrum for compound 6 with the lmax values
of 205, 250 and 446 nm. In addition, to study the in vitro
photophysical properties of the compounds, fluorescence
spectral measurements were obtained in a buffer solution of
pH equal to 7.0 at 25°C. Figure 4 (c) shows the typical emis-
sion spectrum of DBD, while Fig. 4 (d) displays the typical
emission spectrum of compound 6. Similar results were ob-
tained for compound 5. It is evident from Figs. 4 (c) and (d)
that the fluorescence spectrum of compound 6 shifted to visi-
ble wavelengths in contrast to that of the starting material
(DBD). This phenomenon is clear in Fig. 5, which is associ-
ated with DBD (A), compound 6 (B) and p-toluenesulfinic
acid (C) at the concentration value of 0.125 mM.
In conclusion, the electro-organic reactions of 3,6-dihy-
droxybenzene-1,2-dinitrile (DBD) were studied in the pres-
ence of benzenesulfinic acids, involving the EC mechanism
reaction. These reactions comprised two steps; (i) electro-
chemical oxidation and (ii) the chemical reaction. The reac-
tion rate of the DBD dimerization increased with increase in
Measurements The NMR spectra were recorded on a Bruker FT-NMR-
500, while the IR spectra were recorded on a Shimadzu FT-IR-4300 Spec-
trophotometer. The MS spectra were obtained using an HP (Agilent Tech-
nology) GC-6890 combined with MS-5973 (EI at 20 eV and 70 eV). The flu-
orescence spectrophotometric studies were obtained using a Fluorescence
Spectrophotometer CARY Eclipse. The elemental analyses were performed
using a Heraeus CHN rapid analyzer. All electrochemical experiments were
performed by the Autolab potentiostat PGSTAT 30 (Eco Chemie B.V.,
Netherlands), equipped with the GPES 4.9 software. A glassy-carbon disk
electrode (2 mm in diameter) acted as the working electrode and a platinum
wire was employed as the counter electrode (1 mm in diameter and 2 cm in
length). In the controlled-potential bulk electrolysis, an assembly of three
carbon rods (0.8 cm in diameter and 5 cm in length) was used as the working
electrode (WE) with the total area value of 37.68 cm2, while a platinum
gauze was used as the counter electrode. Finally, the used reference elec-
trode for the WE potential measurement was an Ag|AgCl|KCl (sat.). All
electrodes were purchased from the AZAR Electrode Co. (Iran).
Electrochemical Synthesis of Compounds (5, 6) Using Bulk Electroly-
sis In the current trial 200 ml mixture of water–acetonitrile (90 : 10), con-
taining acetate buffer (pHꢀ5.0, cꢀ0.2 M), 1.5 mmol 3,6-dihydroxybenzene-
1,2-dinitrile (DBD, compound 1) and sulfinic acids (3, 4) (in equal concen-
tration), were electrolyzed at the pre-determined potential (480 mV) in an
undivided cell. For the less acetonitrile consumption as a nonaqueous sol-
pH, and the anionic formation of compounds 3 and 4 were vent, the high volume cell (200 ml) was used in the controlled-potential bulk

752
Vol. 56, No. 6
electrolysis. The electrolysis time for the electro-organic synthesis of 5 and 6
compounds was 24 h and 16 h respectively.19) Electrolysis stopped when thin
layer chromatography (TLC with ethyl acetate) monitored very low amounts
of the starting materials. Afterwards, the compounds were extracted by
washing with dichloromethane. This process was repeated several times.
TLC controlled both aqueous and nonaqueous media for the compound ex-
traction. Finally the dichloromethane was air-dried at room temperature. The
residual solids dissolved in the ethyl acetate following which 1 ml hexane (as
anti-solvent) was added to crystallize the compounds. Then, the compounds
were washed with hexane–ethyl acetate (4 : 1), air-dried and characterized by
means of FT-IR, 1H-, 13C-NMR and MS spectrometry. The yield of com-
pounds 5 and 6 was 48% and 63% respectively. The consumed charges were
2.23 and 2.19 F/mol for compounds 5 and 6 respectively.
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Products Characteristics 3,6-Dihydroxy-4-(phenylsulfonyl)benzene-
1,2-dinitrile (C14H8O4N2S, 5): mp 168—170 °C (dec.). FT-IR (KBr): nmax
(cmꢁ1): 3340—3093 (OH, br), 2245 (CN), 1695, 1600, 1481, 1460, 1392,
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536, 478. 1H-NMR (DMSO-d6, 500 MHz) d (ppm): 7.57 (2H, d, Jꢀ7.56 Hz,
sulfinic acid ring protons), 7.86 (2H, d, Jꢀ7.69 Hz, sulfinic acid ring pro-
tons), 7.93 (2H, m, sulfinic acid and dihydroxybenzene ring protons), 11.90
(2H, br, hydroxyl group protons). 13C-NMR (DMSO-d6, 500 MHz) d (ppm):
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SO2]ꢂ (50), 102 (10), 77 [Phenyl]ꢂ (100), 51 (45). Anal. Calcd for C,
55.99%; H, 2.69%; N, 9.33%. Found: C, 55.47%; H, 2.74%; N, 9.25%.
3,6-Dihydroxy-4-tosylbenzene-1,2-dinitrile (C15H10O4N2S, 6): mp 198—
200 °C (dec.). FT-IR (KBr): nmax (cmꢁ1): 3224—3001 (OH, br), 2252 (CN),
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1
746, 703, 682, 588, 528 and 480. H-NMR (DMSO-d6, 500 MHz) d (ppm):
2.41 (3H, s, Me), 7.45 (2H, d, Jꢀ8.15 Hz, sulfinic acid ring protons), 7.82
(2H, d, Jꢀ8.20 Hz, sulfinic acid ring protons), 7.93 (1H, s, dihydroxyben-
zene ring proton), 11.86 (2H, br, hydroxyl group protons). 13C-NMR
(DMSO-d6, 500 MHz) d (ppm): 21.99, 106.21, 106.37, 114.34, 122.94,
129.30, 130.59, 137.19, 137.48, 145.84, 151.35, 154.79. MS (70 eV): m/z:
315 [Mꢂ1]ꢂ (10), 314 [M]ꢂ (50), 297 [MꢁOH]ꢂ (5), 249 (25), 247 (30),
235 (15), 155 (15), 139 (20), 107 (15), 91 [Toluene]ꢂ (100), 77 [Phenyl]ꢂ
(35), 65 (60), 53 (25). Anal. Found: C, 56.91%; H, 3.27%; N, 8.86%. Calcd
C, 57.32%; H, 3.21%; N, 8.91%.
Acknowledgements The authors would like to thank Mr. G. Watson for 22) Bard A. J., Faulkner L. R., “Electrochemical Methods, Fundamentals
his help and the authors wish to express their appreciation for the useful
comments of the referees. Moreover, the financial support provided by the
University of Tehran Research Affairs is gratefully acknowledged.
and Applications,” 2nd ed., John Wiley & Sons, New York, 2001, pp.
496—500.
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