Anion sensing by Phenazine-based urea/thiourea receptors

Article abstract of DOI:10.1016/j.tetlet.2008.09.033

The novel colorimetric receptors 2,3-bis-N-(9,10-diaza-anthracen-1-yl)-N′-phenylurea and 2,3-bis-N-(9,10-diaza-anthracen-1-yl)-N′-phenylthiourea have been prepared by the reaction of 2,3-diaminophenazine with phenylisocyanate and phenylisothiocyanate, respectively, in quantitative yields. The interaction and colorimetric sensing properties of receptor = 2 and 3 with different anions were investigated by naked eye, UV-vis and fluorescence spectroscopy in DMSO. The receptors effectively and selectively recognized biologically important F^-, CH₃COO^-, H₂PO₄^- in the presence of other anions, such as Cl^-, Br^-, I^- and HSO₄^- in DMSO.

Full text of DOI:10.1016/j.tetlet.2008.09.033

Tetrahedron Letters 49 (2008) 6646–6649
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Anion sensing by Phenazine-based urea/thiourea receptors
*
Shive Murat Singh Chauhan , Tanuja Bisht, Bhaskar Garg
Bioorganic Research Laboratory, Department of Chemistry, University of Delhi, Delhi 110 007, India
a r t i c l e i n f o
a b s t r a c t
The novel colorimetric receptors 2,3-bis-N-(9,10-diaza-anthracen-1-yl)-N0-phenylurea and 2,3-bis-N-
(9,10-diaza-anthracen-1-yl)-N0-phenylthiourea have been prepared by the reaction of 2,3-diaminophen-
azine with phenylisocyanate and phenylisothiocyanate, respectively, in quantitative yields. The interac-
tion and colorimetric sensing properties of receptor = 2 and 3 with different anions were investigated by
naked eye, UV–vis and fluorescence spectroscopy in DMSO. The receptors effectively and selectively rec-
Article history:
Received 9 June 2008
Revised 3 September 2008
Accepted 8 September 2008
Available online 11 September 2008
ognized biologically important Fꢀ, CH3COOꢀ, H2PO4 in the presence of other anions, such as Clꢀ, Brꢀ, Iꢀ
ꢀ
Keywords:
Anion sensing
Diaminophenazine (DAP)
Host–guest chemistry
Urea/thiourea
ꢀ
and HSO4 in DMSO.
Ó 2008 Elsevier Ltd. All rights reserved.
UV–vis spectroscopy
The design and synthesis of new anion binding agents is an
important area of current research interest due to the growing
awareness of the environmental and clinical importance of anions.
The different sizes and shapes of anions are responsible for the
slow growth in the development of anion binding agents as com-
pared to cation binding agents.1–3 The ‘naked-eye’ detection of an-
ions without the use of any spectroscopic instrumentation is an
important area in the design and fabrication of new anion recep-
tors. Naked-eye or optical detectable receptors have been devel-
oped by the covalent attachment of chromogenic or fluorogenic
groups. Two strategies of anion detections have been developed,
one based on cationic ligands such as polyammonium, guanidium,
quaternary ammonium and lewis acids and the second based on
neutral ligands such as amides, ureas, thioureas, calix[4]pyrroles
and related ligands.4–10 The anion binding portion, that is, the urea
and thiourea groups have been attached covalently to o-phenyl-
enediamine,11,12 calix[4]arenes,13–15 anthraquinones,16–20 acri-
done,21 and related chromophores. The planar electron-rich
heterocyclic diamine, 2,3-diaminophenazine (DAP) is an interest-
ing chromophore with intense luminescence and significant muta-
genic and genotoxic behavior. This compound is very useful in
analytical chemistry for fluorimetric determinations of laccase
activity22 and in chemotherapeutics as a DNA cleavage agent.23
Herein, we report the synthesis of two novel colorimetric anion
receptors having bisurea/thiourea binding sites and phenazine as
a chromogenic signaling subunit and their effective and selective
‘naked-eye’ differentiation between fluoride, acetate and dihydro-
gen phosphate in DMSO.
The reaction of o-phenylenediamine with ferric chloride in
refluxing aqueous solution gave 2,3-diaminophenazine. Further,
the reaction of 2,3-diaminophenazine with two moles of phenyl-
isocyanate or phenylisothiocyanate in DMF/THF (4:1,v/v) gave
2,3-bis-N-(9,10-diaza-anthracen-1-yl)-N0-phenylurea 2 and 2,3-
bis-N-(9,10-diaza-anthracen-1-yl)-N0-phenylthiourea 3, respec-
tively (Scheme 1). The spectroscopic analysis results were consis-
tent with the proposed structures of the receptors. FT-IR spectra
of receptors
2 and 3 showed broad –NH bands at 3340–
3380 cmꢀ1. Further, a strong band at 1635 cmꢀ1 in the IR spectrum
of 2 was assigned to C@O stretching.
The naked-eye detection experiment was carried out initially in
DMSO by addition of the corresponding anion (5 ꢁ 10ꢀ6
–
5 ꢁ 10ꢀ2 M) to solutions of receptors 2 and 3 (5 ꢁ 10ꢀ5 M). The
addition of tetrabutyl ammonium fluoride anions into DMSO solu-
tions of 2 and 3 resulted in yellow to red color changes, due to the
appearance of a broad new band centered at 512 nm and 516 nm
in the UV–vis spectra of receptors 2 and 3, respectively, suggesting
that fluoride interacts with the receptors 2 and 3 more strongly
due to its higher electronegativity and smaller size compared to
the other halides. Moreover, the color change of thiourea 3 was
much more sensitive to Fꢀ than that of urea 2. One tenth equiva-
lent of Fꢀ was enough to induce an observable color change from
yellow to pink in 3. Further addition of Fꢀ changed the color of
the solution from pink to dark red. The most remarkable effect
was the selective color change induced in 2 and 3 towards dihydro-
gen phosphate and acetate. Receptor 2 gave a well-defined orange
color on addition of H2PO4ꢀ and AcOꢀ (5 ꢁ 10ꢀ4 M). In contrast, the
solution of thiourea 3 in the presence of dihydrogen phosphate and
acetate turned red at a low concentration (5 ꢁ 10ꢀ5 M) (Fig. 1). The
color changes are most probably due to the formation of hydrogen
* Corresponding author. Tel./fax: +91 11 27666845.
E-mail address: smschauhan@chemistry.du.ac.in (S. M. S. Chauhan).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.09.033

S. M. S. Chauhan et al. / Tetrahedron Letters 49 (2008) 6646–6649
6647
X
X
N
N
NH
NH
HN
HN
NH2
NH2
N
N
NH2
NH2
i, ii
iii
1
2, X = O (76 %)
3, X = S (53 %)
Scheme 1. The synthesis of receptors 2 and 3. Reagents and conditions: (i) anhydrous ferric chloride, H2O, rt, 12 h; (ii) concentrated aq ammonia, 15 min, rt, 23%; (iii) phenyl
isocyanate or phenylisothiocyanate (excess), DMF/THF (4:1, v/v), reflux, 48 h.
Figure 1. Left panel: color changes of receptor 2 (5.0 ꢁ 10ꢀ5 M) in DMSO upon addition of tetrabutylammonium anions (5.0 ꢁ 10ꢀ2 M); (a) = free receptor, (b) = fluoride,
(c) = chloride, (d) = bromide, (e) = iodide, (f) = hydrogen sulfate, (g) = dihydrogen phosphate, (h) = acetate; right panel: color changes of receptor 3 (5.0 ꢁ 10ꢀ5 M) in DMSO
upon addition of tetrabutylammonium anions (5.0 ꢁ 10ꢀ3 M); (a) = free receptor, (b) = fluoride, (c) = chloride, (d) = bromide, (e) = iodide, (f) = hydrogen sulfate, (g) = dihy-
drogen phosphate, (h) = acetate.
bond interactions between the urea or thiourea groups and the
corresponding anions.24,25 Both the receptors were found to be
insensitive to addition of a large excess (5 ꢁ 10ꢀ2 M) of chloride,
bromide, iodide and hydrogen sulfate. The anion sensing ability
is reflected in quantitative terms in the UV–vis absorption spectra
of receptors 2 and 3. The UV–vis spectra of urea 2 and thiourea 3
changed dramatically on addition of fluoride, dihydrogen phos-
phate and acetate anions. Figure 2 shows the absorption spectra
of 2 and 3 in the presence of fluoride anions. On addition of fluoride
anions in DMSO (5.0 ꢁ 10ꢀ4 M), the characteristic absorption peaks
of 2 and 3 at 400 nm decreased gradually with a strong red shift
and a new peaks at 512 nm and 516 nm were produced, respec-
tively. At the same time a clear isobestic point at 440 nm for recep-
tor 2 and three clear isobestic points at 295 nm, 345 nm and
440 nm were observed for 3 (Fig. 2). The absorption intensity of
3ꢂFꢀ complex at 516 nm of was greater than the intensity of 2ꢂFꢀ
complex at 512 nm, when less than 2 equiv of Fꢀ was added. This
result shows that Fꢀ binds more tightly with thiourea 3 than with
urea 2, indicating the formation of stronger hydrogen bonds be-
tween the acidic N-H groups of thiourea 3 and Fꢀ.
a
b
0.280
0.26
1.20
0.24
0.35
0.30
0.25
1.1
1.0
0.22
0.20
0.18
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.9
A
0.8
0.7
A
0.20
A 0.6
0.5
0.02
0.15
0.10
0.002
350.0 380 400 420 440 460 480 500 520 540 560 580 600.0
nm
0.4
0.3
0.2
0.05
0.1
0.00
0.000
220.0 250
300
350
400
nm
450
500
550 600.0
350.0
400
450
500
nm
550
600
Figure 2. UV–vis spectral changes observed for 2 and 3 in DMSO at room temperature. Left: UV–vis titrations of urea 2 (5.0 ꢁ 10ꢀ4 M) upon addition of tetrabutylammonium
fluoride (from 0.0, 5.0 ꢁ 10ꢀ5 to 5.0 ꢁ 10ꢀ4 M) right: UV–vis titrations of thiourea 3 (5.0 ꢁ 10ꢀ4 M) upon addition of tetrabutylammonium fluoride (from 0.0, 5.0 ꢁ 10ꢀ5 to
7.5 ꢁ 10ꢀ4 M) (inset expanded region from 350 nm to 600 nm).

6648
S. M. S. Chauhan et al. / Tetrahedron Letters 49 (2008) 6646–6649
Further the UV–vis spectral change of urea 2 on addition of
dihydrogen phosphate was significantly different from that with
fluoride. The kmax slowly moved from 400 to 425 nm, when
10 equiv of dihydrogen phosphate ions was added. Addition of
more than 10 equiv of phosphate did not result in any further spec-
tral changes. This behavior of urea 2 was also reflected in the naked
eye detection with a yellow to orange color change. However, the
spectral changes of thiourea 3 in response to dihydrogen phos-
phate and acetate were similar to those with fluoride addition.
The addition of other halides and hydrogen sulfate ions did not
cause any spectral changes with both urea 2 and thiourea 3. The
UV–vis spectral changes of thiourea 3 in the presence and absence
of different anions are depicted in Figure 3. Fluorescence spectro-
scopy studies were also carried out in order to evaluate the ability
of the receptors to operate as a fluorescent anion sensor. A 2.5 ml
solution of 2,3-bis-N-(9,10-diaza-anthracen-1-yl)-N0-phenylurea
(2, 2.0 ꢁ 10ꢀ4 M) in DMSO in a quartz cell was titrated with an
increasing volume of tetrabutylammonium fluoride (Fig. 4). The
fluorescence spectra of 2 showed a characteristic peak at 610 nm.
A large quenching (>85%) in the intensity of the 610 nm band
was observed on addition of 1.0 equiv of Fꢀ anions indicating that
on formation of the hydrogen-bonded complex between Fꢀ and 2,
the excited state was modified considerably leading to the quench-
ing of fluorescence. On continuous addition of Fꢀ to a solution of 2
the peak was red shifted to 645 nm. A clear isobestic point was
observed at 625 nm. The changes observed in the fluorescence
spectra on adding more than 1 equiv of Fꢀ aliquots to 2 were
insignificant, which is in good agreement with the results of the
UV–vis titration.
83
73
63
53
43
33
23
13
3
-7550
570
590
610
630
650
670
690
nm
Figure 4. Fluorescence spectra of 2 (5.0 ꢁ 10ꢀ4 M) in the presence of n-Bu4 N+Fꢀ
(from 0.0, 5.0 ꢁ 10ꢀ5 to 7.5 ꢁ 10ꢀ4 M) in DMSO at room temperature; kex = 450 nm.
Table 1
Association constants (Kass)a for
2 and 3 ) and anionic substrates both at
(Mꢀ1
5.0 ꢁ 10ꢀ4 M in DMSO solution at 25 °Cb
Receptor
Association constant Kass (Mꢀ1
)
Fꢀ
Clꢀ
Brꢀ
Iꢀ
HSO4
H2PO4
AcOꢀ
ꢀ
ꢀ
2
3
5.6 ꢁ 104
n.dc
75
n.d.c
n.d.c
n.d.c
n.d.c
4.8 ꢁ 103
4.1 ꢁ 105
9.5 ꢁ 103
9.1 ꢁ 105
n.d.c
350
6.2 ꢁ 105
a
Determined from absorption spectroscopic titrations.
All errors are 10%.
Changes in the UV–vis spectra were insufficient to calculate the binding
b
c
All the titration curves gave a satisfactory fit to a 1:1 binding
model as confirmed by the continuous variation method. The
constants.
selectivity trends in the binding affinities of the anions for 2
and 3 followed the order of Fꢀ > AcOꢀ > H2PO4 ꢃ Brꢀ > Clꢀ
>
ꢀ
anions. These receptors may find use in various sensing
applications as well as in other situations, such as anion transport
and purification.
ꢀ
ꢀ
HSO4 > I . The observed binding sequence was not completely
consistent with the anion basicity. The quantitative titration exper-
iments allowed us to determine the apparent binding constants of
urea 2 and thiourea 3 with different anions as described in the lit-
erature.12 The respective Kass values are summarized in Table 1.
In summary, we have developed the new structurally simple
sensors 2 and 3 having bis-urea/thiourea binding sites anchored
to phenazine signaling subunits that not only allow for the easy
Synthesis of 2,3-bis-N-(9,10-diaza-anthracen-1-yl)-N0-phenylurea/
thiourea: Phenylisocyanate (273.7 mg, 2.3 mmol) was added drop-
wise to a solution of 2,3-diaminophenazine (420 mg, 2 mmol) in
dry DMF and THF (4:1, v/v, 25 ml) with stirring under inert atmo-
sphere at reflux at 80 °C for 48 h. The reaction mixture was cooled
at room temperature and the solvent was removed under reduced
pressure leaving 5 ml of the solvent. Crushed ice was poured into
the reaction mixture and precipitated was filtered. After several
colorimetric detection of Fꢀ and H2PO4 ions, but also are amena-
ꢀ
ble to ‘color tuning’ depending upon the type and amount of
F-
-
Host HSO4
Cl-
a
b
0.750
0.70
0.65
0.60
0.55
0.50
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
3.50
3.2
3.0
2.8
2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
H2PO-4
Br-
A
A
0.2
0.03
0.033
235.0
300
350
400
450
500
550 600.0
350.0 380 400 420 440 460 480 500 520 540 560 580 600.0
nm
nm
Figure 3. (a) Absorption spectra of 3 (5 ꢁ 10ꢀ4 M) in the presence and absence of different anions (5.0 ꢁ 10ꢀ4 M) in DMSO. (b) Expanded region from 350 nm to 600 nm.

S. M. S. Chauhan et al. / Tetrahedron Letters 49 (2008) 6646–6649
6649
washings with THF and acetonitrile (5 ꢁ 10 ml), the desired com-
References and notes
pound 2 was crystallized by hot methanol/DMSO to obtain as pale
yellow crystals.
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Data for receptor 2: yield: 76%, mp: 238 °C, UV–vis: kmax
(5 ꢁ 10ꢀ5 M, DMSO)/nm: 275 (1.32), 408 (0.378), IR (KBr): mmax
/
cmꢀ1 3340 (–NH), 1635 (C@O), 1552, 1438, 1353, 1221, 750; 1H
NMR: dH (300 MHz; DMSO-d6; Me4Si): 9.27 (2H, s, NH), 8.62 (2H,
s, NH), 8.58 (2H, s, 1,4-CHphenazine), 8.12 (2H, d, J = 7.5 Hz, 5,8-
CHphenazine), 7.79 (2H, m, 6,7-CHphenazine), 7.42 (4H, d, J = 8.7 Hz,
o-phenyl), 7.29 (4H, m, m-phenyl), 6.91 (2H, m, p-phenyl); 13C
NMR: d (75 MHz; DMSO-d6): 151.9, 143.9, 141.8, 140.8, 140.2,
139.7, 129.1, 128.8, 128.5, 127.3, 116.5; HR-MS for C26H20N6O2Na:
calcd: 471.1540, found: 471.1535.
The compound 3 was prepared by using above procedure using
2,3-diaminophenazine (420 mg, 2 mmol) and phenylisothiocya-
nate (310.5 mg, 2.3 mmol) with stirring under inert atmosphere
at reflux at 80 °C for 50 h. Compound 3 was crystallized by hot
methanol/DMSO.
Data for receptor 3: yield: 53%; mp: >300 °C; UV–vis: kmax
(5 ꢁ 10ꢀ5 M, DMSO)/nm: 265 (1.23), 400 (0.293); IR(KBr): mmax
/
cmꢀ1 3377 (NH), 1610, 1566, 1498, 1313, 1226, 753; 1H NMR: dH
(300 MHz; DMSO-d6; Me4Si): 10.35 (2H, s, NH), 9.42 (2H, s, NH),
9.01 (2H, s, 1,4-CHphenazine), 8.82 (2H, d, J = 7.4 Hz, 5,8-CHphenazine),
8.19 (2H, m, 6,7-CHphenazine), 7.75 (4H, d, J = 8.7 Hz, o-phenyl), 7.58
(4H, m, m-phenyl), 7.21 (2H, m, p-phenyl). 13C NMR: d (75 MHz;
DMSO-d6): 179.1, 144.1, 142.3, 140.2, 139.2, 138.5, 128.7, 128.6,
128.2, 127.9, 118.3; HR-MS for C26H20N6S2Na: calcd: 503.1083,
found: 503.1080.
Acknowledgments
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The authors are thankful to the Department of Science and
Technology (Govt. of India) for Project No. SR/S1/OC-54/2003, the
Council of Scientific and Industrial Research and University of
Delhi for financial assistance.