Novel quinoxaline based chemosensors with selective dual mode of action: Nucleophilic addition and host-guest type complex formation

Article abstract of DOI:10.1039/c6ra14134j

New quinoxalinium salts 1-5 have been exploited as chemosensors via naked eye, UV-Vis absorption, fluorescence quenching and ¹H NMR experiments. New sensors 1-5 showed a dual mode, nucleophilic addition and a host-guest type complex towards ani

Full text of DOI:10.1039/c6ra14134j

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Novel Quinoxaline Based Chemosensors with Selective Dual Mode of Action: Nucleophilic
Addition and Host-Guest Type Complex Formation
a§
a§
b
c
c
d
Marium Ishtiaq, Iqra Munir, Mariya alꢀRashida, Maria, Khurshid Ayub, Jamshed Iqbal,
e,f
a
∗a
∗a
Ralf Ludwig, Khalid Mohammed Khan, Syed Abid Ali, and Abdul Hameed
a
H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological
Sciences, University of Karachi, Karachiꢀ75270, Pakistan
Department of Chemistry, Forman Christian College (A Chartered University), Ferozepur
b
Roadꢀ54600, Lahore, Pakistan
c
Department of Chemistry, COMSATS Institute of Information Technology, Abbottabad, KPK,
Pakistan 22060.
d
Center for Advanced Drug Research, COMSATS Institute of Information Technology,
Abbottabad 22060, Pakistan
e
LeibnizꢀInstitut für Katalyse e. V. an der Universität Rostock, AlbertꢀEinsteinꢀStr. 29a, 18059
Rostock, Germany
fDepartment of Physical Chemistry, University of Rostock, Dr.ꢀLorenzꢀWeg 1, 18059 Rostock,
Germany
Graphical Abstract
§
Shared first author(contributed equally)
∗
Corresponding Authors: S. A. Ali, eꢀmail: abid.ali@iccs.edu
A. Hameed Tel.: 0092ꢀ21ꢀ99261701ꢀ2; Fax: 0092ꢀ21ꢀ3481901; abdul.hameed@iccs.edu
1

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Abstract
New quinoxalinium salts 1ꢀ5 as chemosensors have been exploited via naked eye, UVꢀVis
1
absorption, fluorescence quenching and H NMR experiments. New sensors 1-5 showed dual
mode, nucleophilic addition and hostꢀguest type complex towards anion (Fꢁ, AcOꢁ and
ascorbate) detection. Small anions (Fꢁ/AcOꢁ) showed nucleophilic addition at C2 position of
quinoxalinium cation, while larger anion (ascorbate), revealed the formation of hostꢀguest type
complex due to steric hindrance posed by C3 of phenyl ring. Nucleophilic addition of small
anions (Fꢁ/AcOꢁ) leads to deꢀaromatization of quinoxalinium cation. However in the case of
larger anion, ascorbate, hostꢀguest type complex formation induces changes in
absorption/fluorescence signals of quinoxalinium moiety. This selective binding has been
1
confirmed on the basis of H NMR spectroscopic technique, whereupon nucleophilic addition of
a
small anions (Fꢁ/AcOꢁ) was confirmed by monitoring characteristic proton NMR signals of H
and methylene protons (CH ), which were clearly shifted in case of fluoride and acetate ions
2
addition confirming deꢀaromatization and nucleophilic addition. Whereas no such peak shifting
was observed in case of ascorbate ion addition confirming nonꢀcovalent addition of ascorbate.
Theoretical insight into the selectivity and complexation behavior of ascorbate ion with
quinoxaline moiety is gained through density functional theory (DFT) calculations. Moreover,
the absorption properties of these complexes are modeled theoretically, and compared with the
experimental data. In addition, thermal decomposition of sensor (1 and 2) has been studied by
the means of differential scanning calorimetry (DSC), thermogravimetry (TG), and differential
thermogravimetry (DTG) to signify their utility at variable temperature.
Keywords: Quinoxalinium, Fluoride (Fꢁ), Acetate (AcOꢁ), Ascorbate, Absorption/ Fluorescence,
Thermal studies, Density functional theory (DFT) calculations
Introduction
Anion sensing via chemosensors as receptors carries much importance in observing biological
processes, clinical diagnostics, and environmental monitoring. A number of compounds as
sensors have been reported for different anions such as halides, acetate, phosphate, and citrate
1
etc. in regulating their certain levels in biological processes and variable ecosystems. Among
2

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halides, fluoride (Fꢁ) continues to receive much attention due to its wide spread applications in
2
ꢀ4
dental care, and its role in certain enzyme inhibitions. Similarly, acetate ion (AcOꢁ) plays an
important role in many metabolic processes. It has also been known to serve as a tracer for
5
ꢀ7
certain malignancies. Acetate has been widely studied in prostate cancer and metastases.
Ascorbic acid (vitamin C) is an essential nutrient in humans and animals; it serves as an
8
, 9
important coꢀfactor in various enzymatic reactions.
Ascorbic acid is one of the important
biological molecules that are responsible for maintaining cell redox homeostasis. Imbalances in
redox status lead to oxidative stress and many pathophysiological disorders including aging and
1
0
cancer. Tumors have been known to maintain a reduced microenvironment, consequently
1
3
oxidized and reduced forms of hyperpolarized Cꢀlabeled ascorbic acid (vitamin C) have been
11
used as probes to examine tumor redox status. Therefore, vitamin C determination as analyte
presents an interesting biological aspect.
Ion detection, including small and large analytes, is an exciting domain to regulate their
concentration at a certain level in different biological systems, food, and drinking water
1
2ꢀ14
processes, industrial and clinical analysis etc.
Traditional methods of ion detection, such as
spectroscopy, chromatography and ion selective electrodes suffer from limitations when it comes
15
to biomedical research applications particularly in imaging of cells and tissues. The use of
fluorescent ion probes offers alleviation from these drawbacks and aims to provide a more
16ꢀ20
efficient means of cell imaging, combining selectivity with ease of detection.
Some selected
receptors (probes) for different ion detection have been presented in TableꢀS1. Although quite a
1
few fluorescent ion probes have been known , the interest in the quest to design new, even more
efficient probes with possible biological imaging applications continues to increase worldwide.
In the present study, we synthesized quinoxaline based compounds and employed them as
chemosensors for different analytes, including Fꢁ, AcOꢁ, and ascorbate. The anion sensing results
revealed dual mode (nucleophilic addition and hostꢀguest interaction) of quinoxaline based
probes for small and large analytes. Quinoxaline scaffold is an important biological template to
2
1
assemble different molecules of diverse applications especially in pharmaceutical industry. The
known medicinal significance and important role as pharmacophore in many drugs makes the
quinoxaline moiety a fascinating template for the synthesis of ion sensing molecules.
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Figure-1: Quinoxaline based probes 1-5 and Fꢁ, AcOꢁ, and ascorbate sensing (naked eye view).
Results and Discussion
The probes (receptors) are generally known to interact with analytes (anions) either via covalent
2
2
23, 24
binding, involving addition or substitution type reactions
, or via nonꢀcovalent interactions
A recent study with quinoline based
probe has described the deprotonation mechanism with fluoride and acetate anions to induce
25ꢀ27
that involve formation of hostꢀguest type complexes.
2
4
changes in observed absorption/fluorescence pattern. Another anion sensing study with 9ꢀ
ꢀ
alkoxy acridinium has demonstrated the nucleophilic addition of anions (F /AcOꢁ) at C9 position
of acridinium ring causing deꢀaromatization, consequently inducing drastic changes in
2
2
absorption/fluorescence signals of acridinium moiety. Our studies with quinoxalinium salts 1-5
revealed dual mode of action, that is; 1) via nucleophilic addition of small anions (Fꢁ/AcOꢁ), and
4

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2) via hostꢀguest type complex formation with larger anion (ascorbate). This different mode of
binding with small and large anions serves to establish selectivity among anion sensing.
Moreover, the basicity of anions (Fꢁ>AcOꢁ>ascorbate) further adds to the selective pattern of
receptors 1-5.
Nucleophilic addition of anions to quinoxalinium salts 1-5 at electron deficient position C2
seems feasible. For smaller anions (Fꢁ, AcOꢁ), there is less steric hindrance posed by C3 phenyl
ring. In order to further explore the reactivity and selectivity of quinoxalinium salts towards Fꢁ,
AcOꢁ, and ascorbate, five derivatives have been synthesized according to Schemeꢀ1. Synthesis
was carried out starting from commercially available acetophenones in three steps; i) oxidation,
ii) coupling with oꢀphenylenediamine, and iii) Nꢀalkylation to afford the desired quinoxalinium
salts 1-5, Oxidation of αꢀmethyl group of acetophenone was accomplished with selenium oxide.
The resultant dicarbonyl 7 was then coupled with diamine 8 to furnish corresponding
28
quinoxaline derivatives. NꢀAlkylation of quinoxaline analogues was carried out by using ethyl
º
iodide at 120ꢀ124 C in toluene to produce desired quinoxalinium salts 1-5 (Schemeꢀ1).
Alkylation reaction was found to be regioselective, even in the excess (>10 equiv.) of ethyl
iodide only N1 was alkylated. This offers additional benefit in anion sensing as nucleophile (the
analyte anion) will only attack C2 position of quinoxalinium salt.
Scheme-1: Synthetic layout of quinoxalineꢀbased probes 1-5.
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1
The addition of anions (Fꢁ, AcOꢁ and ascorbate) was also studied by H NMR spectroscopy. The
nucleophilic addition of Fꢁ/AcOꢁ to probe 1 and 2 was confirmed by the shifting of characteristic
1
H NMR signals, while in the case of ascorbate anion, no change in NMR peaks was observed
even after addition of excess (>30 equivalent) of ascorbate. This is due to the large size of
ascorbate molecule that interacts with probes via hostꢀguest mode to induce changes in the
1
a
absorption/fluorescence bands (Figureꢀ5). The characteristic H NMR signals (H and CH ) of
2
probe 2 were monitored after adding different equivalents of Fꢁ anion (1ꢀ5 equiv.). Upon
addition of 1 equivalent of fluoride ion, NMR spectra showed disappearance of singlet peak for
a
H proton at δ 9.71 with concomitant appearance of a new peak at δ 5.99 that confirms
nucleophilic addition of anion. Moreover, the CH proton signal (quartet) (originally at δ 5.19)
2
a
was also shifted upꢀfield as a multiplet at δ 3.72 (Figureꢀ2). Similar pattern of NMR peak (H
and CH ) shifting upon addition of Fꢁ ions was observed for probe 1, the only difference being
2
a
the complete disappearance of singlet signal for H proton (originally at δ 9.70), after the
addition of 5 equivalent of Fꢁ anions. Subsequently, the multiplet for methylene group (CH2)
shifted upꢀfield at δ 3.68 (FigureꢀS2). For acetate ion, the pattern of its nucleophilic addition to
ꢀ
C2 position of probe 2 was found to be identical to that of F ion addition, however, the complete
a
disappearance of H peak (originally at δ 10.27) was observed after addition of 5 equivalent of
ammonium acetate, which can be attributed to the difference in the size of the anion (AcOꢁ > Fꢁ)
1
(
Figureꢀ2). The photospectroscopic and H NMR results clearly demonstrate that small anions Fꢁ
and AcOꢁ follow the nucleophilic addition mode, whereas for the larger anion ascorbate,
observed absorption/fluorescence changes occurred due to collision quenching in hostꢀguest type
25, 29
complex.
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a
a
a
a
1
Figure-2: Study of anion (Fꢁ/AcOꢁ) addition to probe 2 via H NMR spectra; (A) a) without Fꢁ anion and b) with 1
equiv. of F ꢁsource (TBAF) in CD CN as solvent; (B) a) without AcOꢁ anion and b) with 5 equiv. of ammonium
3
acetate in DMSOꢀ d as solvent.
6
The spectroscopic properties of quinoxalinium probes 1-5 for anion (Fꢁ, AcOꢁ, ascorbate)
1
sensing via “switch off” response was studied by naked eye, UVꢀvisible, fluorescence and H
NMR analysis. A typical probe 2 having electronegative 4'ꢀfluoro substituent at C3 phenyl ring,
showed characteristic absorption bands at 262, 290, and 368 nm and blueꢀgreen fluorescence at
5
70 nm with a shoulder band at 480 nm in acetonitrile as solvent (Figureꢀ3). While the probe 1
bearing 4'ꢀmethyl substituent showed two slightly shifted bands at 242, 296 nm and a red shift at
74 nm (Figureꢀ4). In case of probe 1 a yellowꢀred fluorescence signal appeared at 670 nm.
Details of absorption/fluorescence signals for all probes 1-5 have been summarized in Tableꢀ1.
3
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Table-1: UVꢀVis absorption/fluorescence bands for probes 1-5 in acetonitrile as solvent.
Absorptions Bands
Fluorescence
(Ex: 278 nm)
570, 670
480, 570
550
Probes
(
nm)
296
290
290
292
290
1
2
3
4
5
242
262
269
245
260
374
368
354
360
365
550
550
The fluorescence sensing profile of probes 1-5 to determine biologically important anions (Fꢁ,
AcOꢁ, and ascorbate) was studied in acetonitrile/DMSO to overcome solubility issues.
Tetrabutylammonium fluoride (TBAF) was used as a source of Fꢁ, while ammonium acetate and
sodium Lꢀascorbate salts were used as sources for AcOꢁ and ascorbate anions respectively. The
solutions, made in DMSO for AcOꢁ and ascorbate determination, were further diluted with
ꢀ5
acetonitrile for absorbance/fluorescence (5 x 10 mM) studies. For all the investigated anions, a
decrease in fluorescence signal “switch off” was observed upon anion addition. Results showed
that with small anions (Fꢁ/AcOꢁ) fluorescence quenching occurred due to deꢀaromatization of
quinoxalinium ring (Figureꢀ2), while large analyte (ascorbate) quenches fluorescence via photoꢀ
2
9
induced electron transfer (PET) mechanism (Figureꢀ5). PET is an excited state electron transfer
1
2
process from donor to the excited state of the fluorophore moiety.
A gradual quenching of fluorescence emission intensity at 570 and 670 nm was observed for
representative probes 2 and 1 respectively after incremental addition (0.5, 1, 5…10 equivalent)
of Fꢁ anions (TBAF). With the addition of 5 equivalent of TBAF, fluorescence intensity
(excitation at 278) of both probes 2 (Figureꢀ3) and 1 (Figureꢀ4) was quenched to a large extent
with a concomitant increase at 475 nm (Figureꢀ4c). Upon excitation at 380 nm the fluorescence
quenching follows the same pattern for Fꢁ addition (0.5, 1, 5…10 equivalent), however, a shift of
fluorescence signal was observed, from 570 to 770 nm (Figureꢀ4d). Fluorescence quenching of
probe 1 with ascorbate anion (FigureꢀS3c,d in supporting information) follows the same pattern
as for Fꢁ; it was, however, found to be less efficient in case of AcOꢁ anion even after addition of
10 equivalent of ammonium acetate (FigureꢀS3a,b in supporting information). Interestingly, in
case of highly electronegative fluoride substituent containing probe 2, acetate sensing results
were found to be better where fluorescence intensity almost diminished in the presence of 10
equivalent of ammonium acetate (FigureꢀS1b). The UVꢀVis absorption peaks for both
quinoxalinium salts (1 and 2) were drastically decreased upon addition of Fꢁ anion from 0ꢀ5
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equivalent, as observed by color changes from light brown to light yellow which remained
constant after the addition of 5 equivalent of Fꢁ anion (Figureꢀ4/3). These characteristic changes
in color, intensity of absorption bands, and fluorescence emission strongly suggest the deꢀ
aromatization of quinoxaline ring as a result of small anion (Fꢁ/AcOꢁ) addition, or collision
2
2
quenching in the case of large analyte (ascorbate) via hostꢀguest type complex formation
Figureꢀ5). Ascorbate interaction via formation of hostꢀguest type complex with quinoxalinium
(
cation was further confirmed by adding methanol to the same fluorescent quenched solutions of
probes (in DMSO) containing ascorbate ions, whereby previously diminished absorbance was
replenished, as indicated by an increase in the absorbance at 378 nm (Figureꢀ5b).
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Figure-3: a) i) Naked eye view ii) under UV light at 254 nm; b) Absorption spectra of probe 2; c) Fluorescence
ꢀ5
spectra (excitation 278 nm); d) Fluorescence spectra (excitation 380 nm) (5 x 10 mM) – Upon systematic addition
0.5ꢀ5 equiv.) of TBAF as fluoride (Fꢁ) source in acetonitrile as solvent.
(
1
0

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Figure-4: a) i) Naked eye view ii) under UV light at 254 nm; b) Absorption spectra of probe 1; c) Fluorescence
ꢀ5
spectra (excitation 278 nm); d) Fluorescence spectra (excitation 380 nm) (5 x 10 mM) – Upon systematic addition
0.5ꢀ5 equiv.) of TBAF as fluoride (Fꢁ) source in acetonitrile as solvent.
(
1
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Figure-5: Ascorbate addition study with probe 2: a) Absorption spectra in CH CN indicating a decrease in
3
absorption intensity at 378 nm with systematic increase in addition of ascorbate ions (1ꢀ30 eq), and b) the reduced
absorption intensity at 378 nm is replenished upon subsequent addition of methanol, suggesting a reversible hostꢀ
ꢀ5
guest type interaction. Note: Stock solutions prepared in DMSO and then diluted in CH CN or MeOH (5 x 10 mM).
3
The absorption/fluorescence study was further extended with three more quinoxalium salts 3-5
having 4´ꢀ or 2´ꢀchloro, and 3´ꢀmethoxy substituents at phenyl ring, to observe their effect in
anion sensing process. Fluoride ion determination pattern with 4'ꢀchloro (probe 3) and 2'ꢀchloro
(
probe 4) substituted quinoxalium salts were found similar to the one described for probe 1 and
. Upon 5 equivalent addition of Fꢁ, all characteristic absorption peaks (Tableꢀ1) disappeared
FigureꢀS5). In fluorescence quenching experiments, probe 3 showed rapid decline, while the
2
(
probe 4 with 2'ꢀCl substituent showed more or less 50% fluorescence quenching which could be
due to the steric effects of 2'ꢀchloro substituent. Moreover, with AcOꢁ and ascorbate anion
1
2

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absorption/fluorescence quenching was less diminished even after adding 20 equivalent of
corresponding anions which strengthens the steric congestion factor for 2'ꢀchloro substituent
(FigureꢀS6). The probe 5 bearing 3'ꢀOMe group at phenyl ring showed decline in absorption
peaks with 5 equivalent addition of TBAF, while fluorescence quenching > 90% was obtained
after 10 equivalent addition of Fꢁ source. This could be due to steric factor of 3´ꢀmethoxy group
at phenyl ring in comparison to 4´ꢀposition probes. Consequently, the results of
absorption/fluorescence quenching with AcOꢁ and ascorbate anions were less prominent which is
in accordance with the fact that steric congestion around the reactive position has a significant
effect on anion sensing process (FigureꢀS7). Moreover, thermal studies of representative probes
(1 and 2) have been performed to elaborate stability and decomposition temperature of
quinoxalinium salts. Thermal analysis provides additional information about molecular
3
0ꢀ32
interactions and inclusion behaviour of chemosensors with guest molecules.
Computational Studies
Density functional theory (DFT) calculations have been performed to gain insight into the
complexation of ascorbate anion with the quinoxaline based probes, and its effect on the
UVꢀvis absorption spectra. The experimental data suggests that ascorbate ion replaces the
iodide through collision therefore, the ascorbate ion is held with the quinoxaline skeleton
through nonꢀbonding interactions. For nonꢀbonding interaction, the ascorbate ion can
orient on the quinoxaline skeleton in a variety of ways. Moreover the complexity further
increases with a number of conformations being possible for ascorbate ion. Fortunately,
the information regarding the stable conformations of ascorbic acid and ascorbate ion are
33, 34
already described in the literature.
According to these reports, global minimum
geometry is shown in (Figureꢀ6a). We have not only considered this global minimum
geometry in our study but, we have also considered another local minimum (shown in
Figureꢀ6b). This local minimum differs from the global minimum only in one
intramolecular nonꢀbonding interaction. The O6ꢀH acts as a hydrogen bond donor to O5
in the global minimum whereas the same O6ꢀH is a hydrogen bond donor to O3 in the
local minimum.
1
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Global minimum (6a)
local minimum (6b)
Figure-6: The global 6a and local minimum 6b geometries of ascorbate ion.
Considering these geometries for ascorbate ion, we have obtained a number of geometries for
nonꢀbonding interaction of ascorbate ion with the quinoxaline based cation. The optimized
geometries of these complexes (7a-7e) are shown in Figureꢀ7. The binding energies between two
fragments and relative energies with respect to the most stable isomer are given in Tableꢀ2. The
complex 7b (the isomer with the lowest energy) is not the complex with highest binding energy.
The highest binding energy is calculated for 7d.
Table-2: Binding energies, relative energies (with respect to 7b), and absorption properties of ascorbate quinoxaline
complexes 7a-7e, calculated theoretically at CAMꢀB3LYP/6ꢀ31G(d,p).
-1
UV-Vis 1
epsilon)
UV-vis 2
(epsilon)
763 (750)
763 (260)
761 (2400)
756 (50)
Entry
Erel
Ebind (kcal mol )
(
7
7
7
7
7
a
b
c
d
e
4.82
0.00
8.85
1.01
3.61
ꢀ82.64
ꢀ87.46
ꢀ81.92
ꢀ89.76
ꢀ87.17
367 (9100)
353 (11700)
350 (10700)
388 (9200)
368 (7000)
724 (50)
1
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Figure-7: Optimized geometries of ascorbate quinoxaline complexes 7a-7e.
All of these optimized complexes are characterized by two absorption bands: a quite intense
absorption band between 250 and 400 nm, and a very weak absorption band between 700ꢀ800
nm. The latter absorption band (although weak) is a characteristic band for nonꢀcovalent
interactions between ascorbate and quinoxaine moieties. This absorption band was missing in an
optimized geometry in which the ascorbate moiety binds covalently to the quinoxaline skeleton.
Existence of absorption bands between 650 and 800 nm in the experimental spectrum (See
supporting informationꢀS1) confirms the existence of nonꢀcovalent complex and also proves the
phenomenon of collision quenching.
After establishing through experiment and theory that the interaction between the ascorbate and
quinoxaline is nonꢀbonded in nature, attention is paid to the intensity of the absorption band
between 250 and 400 nm. From Figureꢀ8, it is quite obvious that the intensities of absorption
bands for 2a and 2b (iodo complexes) are relatively higher than the intensities for 7a to 7e
(ascorbate complexes). Comparison of experimental and theoretical data also suggests that a
number of geometries exist for 1:1 interaction between ascorbate and quinoxaline moieties. This
1
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inference is based on the appearance of a number of peaks between 700 and 800 nm (Figureꢀ
S1c). Each complex is believed to give rise to one peak in the region between 700 and 800 nm,
as evident from theoretical calculations.
The effect of increasing the concentration of ascorbate ion does not affect the UVꢀvis absorption
spectrum between 250–400 nm; however, the changes in the region between 600ꢀ800 nm are
indicative of changes in the geometries of the complexes. It might be possible that number of
ascorbate ions per unit of quinoxaline increases which significantly affects the absorptions in this
region.
Figure-8: Comparative theoretical absorption spectra for ascorbateꢀquinoxaline complexes (7a-7e) and iodo
quinoxaline (2a-2b), calculated at CAMꢀB3LYP/6ꢀ31G(d,p).
Conclusion
In conclusion, quinoxalium salts 1-5 have been explored as chemosensors for detection of three
biologically important anions (Fꢁ, AcOꢁ, and ascorbate) via naked eye, UVꢀvisible absorption,
1
fluorescence quenching, and H NMR spectroscopic techniques. In case of small anions
(Fꢁ/AcOꢁ), nucleophilic addition at C2 position leads to deꢀaromatization of quinoxaline ring,
while large ascorbate molecule formed a hostꢀguest type complex. This selective binding has
1
been confirmed on the basis of
H
NMR spectroscopic technique, whereupon
covalent/nucleophilic addition of small anions (Fꢁ/AcOꢁ) was confirmed by monitoring
a
characteristic proton NMR signals of H and methylene (CH ), which were clearly shifted in case
2
of fluoride ion and acetate ion addition confirming deꢀaromatization and nucleophilic addition.
Whereas no such peak shifting was observed in case of ascorbate ion addition confirming nonꢀ
1
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covalent addition of ascorbate. Moreover, the thermal stability studies of probes 1 and 2 have
been carried out to broaden the applicability range of these quinoxalineꢀbased probes (1-5) at
variable temperatures. Results from theoretical (DFT) calculations are supportive of collision
quenching mechanism. Computationally calculated UV/vis absorption intensities, for nonꢀ
bonded ascorbateꢀquinoxaline complexes are lower than the corresponding absorption intensities
for covalently bonded iodoꢀquinoxaline, which is consistent with the experimental finding.
Comparison of theoretical and experimental UVꢀvis spectra also suggests that a number of
ascorbate quinoxaline complexes exist under the experimental conditions. Moreover, the nature
of complexation is believed to change upon changing the concentration of ascorbate ion.
Materials and Methods (Chemistry)
Different starting materials which include substituted acetophenone (≥99%), selenium dioxide
(99.9%), 1,2ꢀdiaminobenzene (≥99%), tetrabutylammonium fluoride trihydrated (TBAF) (98%),
ethyl iodide (99%), ammonium acetate (≥98%), and Sodium Lꢀascorbate (≥98%) were purchased
from SigmaꢀAldrich and used without any purification unless otherwise stated. The analytical
grade solvents such as 1,4ꢀdioxane, ethyl acetate, diethyl ether and hexane were used as solvents
for preforming reactions, and also for purification purpose. Distilled water was used as solvent
for reaction workup purpose. All the reactions were performed in oven dried round bottom
flasks. Thin layer chromatography (TLC) was used to monitor reaction using silica gel 60
aluminiumꢀbacked plates 0.063ꢀ0.200 mm as the stationary phase with analytical grade solvents
such as ethyl acetate (EtOAc), diethyl ether, nꢀhexane etc. Chromatograms were visualized with
UV light (254 nm) or using different staining mixtures such as basic potassium permanganate,
vanillin, and bromocresol green. Infrared spectra IR (KBr discs) were recorded between 4000ꢀ
ꢀ1
5
00 cm on a Bruker Vectorꢀ22 spectrometer. TGA and DSC analyses were carried out on a TA
instrument, model number SDT Q600. Aluminum heating/sample pans were used. The sample
weight used was 3ꢀ6 mg. Decomposition profiles were obtained at different constant heating
ᵒ
rates, that is, 20 C/min. All analyses were carried out under nitrogen atmosphere (flow rate was
100 ml/min). The data was processed by use of Universal Analysis 2000 software, version 4.2E
1
(
TA Instruments, USA), and MS Excel 2013. Bruker spectrometers were used to record H NMR
13
spectra at 300, 400 or 500 MHz and C NMR at 75, 100 or 125 MHz as dilute solutions in the
º
appropriate deuterated solvent at 25 C.
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7

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All the chemical shifts were recorded on the δꢀscale (ppm) using residual solvents as an internal
1
13
1
13
standard (DMSO; H 2.50, C 39.43 and CHCl ; H 7.26, C 77.16). Coupling constant were
3
calculated in Hertz (Hz) and multiplicities were labelled as s (singlet), d (doublet), t (triplet), q
(quartet), quint (quintet) and the prefixes br (broad) or app (apparent) were used. Mass spectra
+
(
ESI ) were recorded at Finnigan MATꢀ321A, Germany by using electrospray (ES+), electron
impact (EI+) or FAB (Fast atom bombardment) techniques. Melting points of solids were
determined using a Stuart™ melting point apparatus SMP3 apparatus and are uncorrected All
the absorbance measurements were made on Ultrospec 4300 pro UV/visible spectrophotometer
.
(GE Healthcare, Germany) using quartz cells of 10ꢀmm path length. The fluorescence spectra
°
were scanned at 25 C on a Spectromax 5 Spectrofluorimeter (Molecular Devices, USA), using
TM
9
6 well standard fluorescent plates (Nunc , Germany). The emission range was selected as 300
to 800 nm, with an excitation at wavelength 278 and 380 nm. For anion sensing, stock solutions
ꢀ5
of probes 1-5 were made in CH CN or DMSO and then diluted CH CN (5 x 10 mM). Different
3
3
anion sources (TBAF, NH OAc, and sodium ascorbate) were added sequentially up to 30
4
equivalences.
General procedure for the synthesis of quinoxalinium salts (1-5)
Step I: Commercially available appropriate acetophenone (3.73 mmol, 1.0 equiv.) was dissolved
in a mixture of solvents; dioxane/water (10:1, v/v). To this solution, selenium dioxide was added
º
(
5.59 mmol, 1.5 equiv) at room temperature and then heated at 100 C for 3 h until no starting
material was observed by TLC analysis. The reaction mixture was cooled to room temperature
and then excess of selenium was removed by vacuum filtration. The resulting filtrate was
concentrated in vacuo and subsequently treated with excess of water (25 mL) at high temperature
º
(
100 C). It was then treated with activated charcoal to afford corresponding dicarbonyl
intermediate 7 as a white solid and was used without purification in the next step.
Step II: The above synthesized dicarbonyl intermediate was dissolved in dioxane (15 mL) and
treated with 1,2ꢀdiaminobenzene (4.48 mmol, 1.2 equivalent) at room temperature for 0.5 h until
the completion consumption of dicarbonyl intermediate, judged by TLC analysis. The reaction
mixture was diluted with water (30 mL) and then extracted with ethyl acetate (20 mL x 3). The
combined organic layers were dried over MgSO , filtered and evaporated in vacuo to get
4
corresponding quinoxaline as solid and used without purification in the next alkylation
2
8 1
reaction. H NMR showed > 95% purity of the compounds.
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8

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Step III: In an oven dried roundꢀbottomed flask, corresponding quinoxaline (200 mg, 0.909
mmol, 1 equivalent) in toluene (5 mL) was stirred at room temperature. Ethyl iodide (1 mL, 12.5
º
mmol) was then added and the resulting reaction mixture was heated under reflux (120ꢀ125 C)
until no starting material was observed by thin layered chromatography. The mixture was cooled
to room temperature to get precipitate of corresponding quinoxalinium salt. The resulting salt
was further washed with cold toluene or hexane, dried to afford (1-5) in 40ꢀ64% yield over three
1
13
steps. The final compounds 1-5 were characterized with H, CꢀNMR, IR, UV spectroscopy and
mass spectrometry.
1-Ethyl-3-(4'-methylphenyl)quinoxalin-1-ium iodide (1) Brick red colour, yield 55% (over 3
º
ꢀ1
steps, 192 mg), M.P. 198ꢀ200 C; IR (νmax, cm ): (KBr disc) 2988, 2928, 1607, 1535, 1445,
1
1
355, 1312, 1253, 1221, 1124, 1071, 1025, 875, 822, 760, 614, 477. H NMR (400 MHz,
2
CD CN): δH 9.72 (1H, s, C H), 8.44 (2H, app t, J = 8.8 Hz, ArH), 8.28 (2H, d, J = 8.4 Hz, ArH),
3
8
.22ꢀ8.20 (2H, m, ArH), 7.50 (2H, d, J = 8 Hz, ArH), 5.19 (2H, q, J = 7.2 Hz, CH CH ), 2.47
2 3
13
(
3H, s, ArCH ), 1.71 (3H, t, J = 7.6 Hz, CH CH ); C NMR (100 MHz, DMSOꢀd ): δC 153.8
3
2
3
6
(C), 144.7 (C), 142.5 (C), 140.4 (CH), 134.5 (CH), 133.7 (CH), 131.1 (C), 130.9 (CH), 130.1
(
CH x 2), 128.5 (C), 127.7 (CH x 2), 119.1 (CH), 54.1 (CH ), 21.1 (CH ), 15.1 (CH ). EIMS m/z
2
3
3
+
+
+
(
%), 249 (M ꢀI, 57), 220 (M ꢀIꢀC H 100); EIꢀHRMS (M −I) C H N Found 249.1393
2
5,
17 17
2
Calculated 249.1392.
1-Ethyl-3-(4'-fluorophenyl)quinoxalin-1-ium iodide (2) Brick red colour, yield 64% (over 3
º
ꢀ1
steps, 218 mg), M.P. 197ꢀ199 C; IR (νmax, cm ): (KBr disc) 3743, 2999, 2961, 1599, 1503,
1
1
447, 1356, 1306, 1220, 1158, 1122, 1063, 1025, 837, 753, 606, 536, 490. H NMR (400 MHz,
2
DMSOꢀd6): δH 10.28 (1H, s, C H), 8.69 (1H, dd, J = 4, 6 Hz, ArH), 8.69 (1H, dd, J = 3.6, 6.6 Hz,
ArH), 8.28 (2H, dd, J = 5.2, 8.4 Hz, ArH), 8.27ꢀ8.24 (2H, m, ArH), 7.58 (2H, t, J = 8.8 Hz,
1
3
ArH), 5.20 (2H, q, J = 7.2 Hz, CH CH ), 1.71 (3H, t, J = 7.2 Hz, CH CH ); C NMR (100 MHz,
2
3
2
3
DMSOꢀd6): δC 165.8 (C), 163.3 (C), 152.8 (C), 144.5 (C), 140.5 (CH), 134.8 (CH), 133.8 (CH),
131.1 (CH), 130.44/130.35 (CH x 2), 128.5 (C), 119.1 (CH), 116.7/116.5 (CH x 2), 54.1 (CH2),
+
+
+
1
5.1 (CH ). EIMS m/z (%), 253.1 (M ꢀI, 56), 224 (M ꢀIꢀC H 100); EIꢀHRMS (M −I)
3
2
5,
C H FN Found 253.1148 Calculated 253.1141.
16
14
2
1-Ethyl-3-(4'-chlorophenyl)quinoxalin-1-ium iodide (3) Dark brown colour, yield 47% (over 3
º
ꢀ1
steps, 170 mg), M.P. 135ꢀ137 C; IR (νmax, cm ): (KBr disc) 3850, 3733, 3049, 2923, 1645,
1
1
594, 1535, 1486, 1356, 1304, 1253, 1216, 1169, 1094, 1009, 955, 828, 756, 713, 548. H NMR
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9

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2
(
400 MHz, DMSOꢀd6): δH 10.29 (1H, s, C H), 8.70 (1H, d, J = 9.6 Hz, ArH), 8.53 (1H, d, J = 9.6
Hz, ArH), 8.40 (2H, d, J = 8.4 Hz, ArH), 8.28ꢀ8.26 (2H, m, ArH), 8.82 (2H, d, J = 8.4 Hz, ArH),
13
5
.20 (2H, q, J = 7.2 Hz, CH CH ), 1.71 (3H, t, J = 7.2 Hz, CH CH ); C NMR (75 MHz,
2 3 2 3
DMSOꢀd6): δC 152.8 (C), 144.5 (C), 140.7 (CH), 137.2 (C), 135 (CH), 133.8 (CH), 132.6 (C),
31.1 (CH), 129.6 (CH x 2), 129.5 (CH x 2), 128.6 (C), 119.1 (CH), 54.1 (CH ), 15.1 (CH ).
1
2
3
+
+
+
EIMS m/z (%), 269 (M ꢀI, 100), 240 (M ꢀIꢀC H 88); EIꢀHRMS (M −I) C H ClN Found
2
5,
16 14
2
269.0849 Calculated 269.0846.
1-Ethyl-3-(2'-chlorophenyl)quinoxalin-1-ium iodide (4) Brown colour, yield 40% (over 3
º
ꢀ1
steps, 140 mg), M.P. 131ꢀ135 C; IR (νmax, cm ): (KBr disc) 3853, 3744, 3065, 2927, 1651,
1
1
604, 1507, 1438, 1356, 1214, 1166, 1091, 1042, 901, 759. H NMR (400 MHz, DMSOꢀd ): δH
6
2
1
0.06 (1H, s, C H), 8.76 (1H, d, J = 8.8 Hz, ArH), 8.57 (1H, d, J = 8 Hz, ArH), 8.39ꢀ8.30 (2H,
m, ArH), 7.90 (1H, dd, J = 2.4, 7.6 Hz, ArH), 7.78 (1H, dd, J = 1.6, 7.2 Hz ArH), 7.73ꢀ7.65 (2H,
1
3
m, ArH), 5.22 (2H, q, J = 7.2 Hz, CH CH ), 1.69 (3H, t, J = 7.2 Hz, CH CH ); C NMR (100
2
3
2
3
MHz, DMSOꢀd6): δC 153.8 (C), 144.8 (C), 141.9 (CH), 135.9 (CH), 133.9 (CH), 133.4 (C),
132.2 (CH), 132.6 (CH), 131.8 (C), 131.3 (CH), 130.5 (CH), 128.8 (C), 128.1 (CH), 128.5 (C),
+
+
1
19.2 (CH), 54.1 (CH ), 15.1 (CH ). EIMS m/z (%), 253.1 (M ꢀI, 56), 224 (M ꢀIꢀC H 100); EIꢀ
2
3
2
5,
+
HRMS (M −I) C H FN Found 253.1148 Calculated 253.1141.
1
6
14
2
1-Ethyl-3-(3'-methoxyphenyl)quinoxalin-1-ium iodide (5) Reddish brown colour, yield 57%
º
ꢀ1
(
over 3 steps, 189 mg), M.P. 159ꢀ162 C; IR (νmax, cm ): (KBr disc) 3852, 3743, 3676, 2925,
1
1
696, 1535, 1457, 1357, 1305, 1262, 1219, 1169, 1119, 1046, 907, 758, 683, 578. H NMR (400
2
MHz, DMSO d6): δH 10.25 (1H, s, C H), 8.70ꢀ8.67 (1H, m, ArH), 8.55ꢀ8.52 (1H, m, ArH), 8.28ꢀ
8.25 (2H, m, ArH), 8.00 (1H, d, J = 8 Hz, ArH), 7.94 (1H, s, ArH), 7.63 (1H, t, J = 8 Hz, ArH),
7
.29 (1H, dd, J = 2, 8.4 Hz, ArH), 5.21 (2H, q, J = 7.2 Hz, CH CH ), 3.92 (3H, s, ArOCH ), 1.70
2
3
3
13
(
3H, t, J = 7.2 Hz, CH CH ); C NMR (100 MHz, DMSOꢀd ): δC 160.1 (C), 153.5 (C), 144.6
2
3
6
(C), 140.6 (CH), 135.1 (C), 134.9 (CH), 133.7 (CH), 131.2 (CH), 130.7 (CH), 128.5 (C), 120.1
(
CH), 119.1 (CH), 117.8 (CH), 113 (CH), 55.6 (ArOCH ), 54.1 (CH ), 15.1 (CH ). EIMS m/z
3
2
3
+
+
+
(
%), 265 (M ꢀI, 38), 236 (M ꢀIꢀC H 80); EIꢀHRMS (M −I) C H N O Found 235.1348
2
5,
17 19
2
Calculated 235.1341.
Computational methodology
2
0

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35
All calculations were performed using Gaussian 09 suit of programs. DFT methods
36,37
were used to investigate the geometries and electronic properties of molecules.
The
geometries of all molecules were optimized with Coulomb attenuated method (CAM), a
hybrid functional that merges the qualities of long range correction, of B3LYP for
38
calculation of excitation energies with 6ꢀ31G(d,p) basis set. The excited state
39ꢀ41
geometries were calculated with TDꢀDFT at TDꢀCAMꢀB3LYP/6ꢀ31G(d,p)
level of
theories, in gas phase. The solvent effect was studied through polarization continuum
model (PCM).
Acknowledgement
We are thankful to the Higher Education Commission (HEC) of Pakistan (Project No. 20ꢀ2073)
for providing financial support for this project. IM was the recipient of indigenous Ph. D.
scholarship from Higher Education Commission (HEC) of Pakistan.
Supplementary Information available:
General
experimental
procedure,
full
structural
characterization
data,
and
absorption/fluorescence spectral data for quinoxalium salts 1-5 are provided.
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