UV-visible and 1H-15N NMR spectroscopic studies of colorimetric thiosemicarbazide anion sensors

Article abstract of DOI:10.1039/c4ob02091j

Four model thiosemicarbazide anion chemosensors containing three N-H bonds, substituted with phenyl and/or 4-nitrophenyl units, were synthesised and studied for their anion binding abilities with hydroxide, fluoride, acetate, dihydrogen phosphate and chlo

Full text of DOI:10.1039/c4ob02091j

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Biomolecular Chemistry
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UV-visible and 1H–15N NMR spectroscopic studies
of colorimetric thiosemicarbazide anion sensors†
Cite this: DOI: 10.1039/c4ob02091j
Kristina N. Farrugia,a Damjan Makuc,b,c Agnieszka Podborska,d
Konrad Szaciłowski,d,e,f Janez Plavecb,c and David C. Magri*a
Four model thiosemicarbazide anion chemosensors containing three N–H bonds, substituted with phenyl
and/or 4-nitrophenyl units, were synthesised and studied for their anion binding abilities with hydroxide,
fluoride, acetate, dihydrogen phosphate and chloride. The anion binding properties were studied in
DMSO and 9 : 1 DMSO–H2O by UV-visible absorption and 1H/13C/15N NMR spectroscopic techniques and
corroborated with DFT studies. Significant changes were observed in the UV-visible absorption spectra
with all anions, except for chloride, accompanied by dramatic colour changes visible to the naked eye.
These changes were determined to be due to the deprotonation of the central N–H proton and not due
to hydrogen bonding based on 1H/15N NMR titration studies with acetate in DMSO-d6–0.5% water. Direct
evidence for deprotonation was confirmed by the disappearance of the central thiourea proton and the
formation of acetic acid. DFT and charge distribution calculations suggest that for all four compounds the
central N–H proton is the most acidic. Hence, the anion chemosensors operate by a deprotonation
mechanism of the central N–H proton rather than by hydrogen bonding as is often reported.
Received 30th September 2014,
Accepted 17th November 2014
DOI: 10.1039/c4ob02091j
www.rsc.org/obc
been the use of receptor units, such as conjugated ureas and
thioureas, as potential hydrogen bond donors.12–16
Introduction
Molecular chemosensors for the detection of anions have
Hydrogen bonding between receptors and anions is corre-
gained significant interest in the last two decades.1,2 The topic lated with the acidity of the receptor protons.1 The introduc-
has become an established area of research in the field of supra- tion of electron-withdrawing substituents, notably on a phenyl
molecular chemistry.3–9 This increased interest is owing to the substituent, enhances the acidity of the anion binding
significant role that anionic species play in biological systems subunit. However, as the acidity of the receptor protons
and environmental ecosystems.10,11 The standard paradigm has increases, the likelihood of deprotonation also increases. The
result is a dichotomy between the realm of supramolecular
chemistry involving hydrogen bonding and the realm of acid–
base indicator chemistry involving deprotonation.8,17 The
possibility of a competition between hydrogen bonding and
aDepartment of Chemistry, Faculty of Science, University of Malta, Msida,
MSD 2080, Malta. E-mail: david.magri@um.edu.mt; Fax: +(356) 2340 3320;
deprotonation is now recognized by researchers in the field,
which is dependent on many parameters including the basicity
of the anion, the stability of the conjugate base, and the
solvent in addition to the acidity of the anion receptor.18
Pyrrolylamidothiourea-based anion receptors undergo a
Tel: +(356) 2340 2276
bSlovenian NMR Centre, National Institute of Chemistry, Hajdrihova 19, SI-1000
Ljubljana, Slovenia
cEN→FIST Centre of Excellence, Trg Osvobodilne fronte 13, SI-1000 Ljubljana,
Slovenia
dAGH University of Science and Technology, Faculty of Non-Ferrous Metals, al.
Mickiewicza 30, 30-059 Kraków, Poland
eJagiellonian University, Faculty of Chemistry, ul. Ingardena 3, 30-060 Kraków,
Poland
fAcademic Centre of Materials and Nanotechnology, AGH University of Science and
−
color change in DMSO with F−, AcO−, C6H5COO− and H2PO4
resulting in dramatic spectral changes in the UV-vis
spectra.19–21 X-ray crystallography was used by the Gale group
to confirm that deprotonation occurs at the thiourea N–H
proton next to the pyrrole moiety after the addition of only one
equivalent of anion. Gunnlaugsson and co-workers have
reported naphthalimide thiosemicarbazide anion chemo-
sensors with striking colour changes from yellow/green to red/
purple.22 The interactions are considered to result from com-
plexation, with the exception of excess fluoride. However, most
recently, Gunnlaugsson has reported a pyridine-based thio-
Technology, al. Mickiewicza 30, 30-059 Kraków, Poland
†Electronic supplementary information (ESI) available: 1H NMR and 1H–13C,
1H–15N NMR gHSQC and gHMBC spectra for 1–4, UV-visible titration spectra for
1 and 4 in 9 : 1 DMSO–water, UV-visible titration spectrum profiles of 3 and 4 in
DMSO, addition of an excess of anions with 3 in 9 : 1 DMSO–water, 1H NMR
titration spectra of 3, 1H–15N gHSQC spectra of 3 & 4 with excess acetate, 1H–13C
gHSQC and 1H–13C gHMBC NMR spectra of 3 and 4 upon addition of 2 equi-
valents of AcO− in DMSO-d6–0.5% H2O and charge distribution calculations for
1–4. See DOI: 10.1039/c4ob02091j
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semicarbazide derivative that undergoes deprotonation in aceto- hydrazine 6 with phenyl isothiocyanate 7 or 4-nitrophenyl iso-
nitrile.23 The titration profiles were dependent on the basicity of thiocyanate 8 in a one-step reaction by refluxing in dry CH3CN
the anion with OH−, F− and AcO− requiring two equivalents, for 2 hours. All four products 1–4 were collected as solids after
−
2−
while H2PO4 and SO4 requiring significantly higher molar trituration from CHCl3 in 57–85% yields. The compounds were
equivalents to reach titration completion. These observations fully characterised using spectroscopic techniques including
are thought to result from an initial unstable hydrogen bond 1H, 13C and 15N NMR, IR and HRMS (see the Experimental
complex, followed by the deprotonation of the central thiourea section for data).
N–H proton as concluded from X-ray crystallography data.22,23
Previous studies by the groups of Jiang24 and Lin25 also
UV-visible spectrophotometric studies
include work on thiosemicarbazides. Jiang reported the anion
UV-visible spectroscopic studies of receptors 1–4 were carried
sensing ability in acetonitrile while Lin conducted studies
out in competitive media of 9 : 1 DMSO–H2O with the sodium
in DMSO and aqueous 95 : 5 DMSO–H2O. In both solvent
salts of OH−, F−, AcO−, H2PO4− and Cl−. Compound 1, lacking
systems, a bathochromic shift was observed on addition of
a
nitrophenyl electron-withdrawing substituent, exhibits
basic anions including fluoride, acetate and dihydrogen phos-
phate with concomitant colour changes. The presence of water
did not alter the anion–receptor interaction significantly in
DMSO.25 Furthermore, in both studies, the spectral changes
were assigned to a 1 : 1 hydrogen bond complex based on
1H NMR titrations, among other arguments. However, a recent
study on thiosemicarbazide anion sensors with electron-with-
drawing substituents suggests that the interaction is due to
deprotonation as tested with hydroxide.23
almost no absorption between 280 and 700 nm. Upon addition
of OH−, F−, and AcO−, 1 displays a broad shoulder appearing
at ca. 335 nm accompanied by a slight colour change from
colourless to faint yellow (Fig. 1a). No significant changes were
observed in the UV-visible spectra with either H2PO4 or Cl−
−
indicating that the receptor is not significantly interacting
with these anions in the competitive solvent medium
(Fig. S1†). Equilibrium constants of 4.2 and 4.4 were deter-
mined for 1 in the presence of OH− and F−. However, the
broadness of the absorption shoulder prevented the determi-
nation of a stability constant for the other anions tested.
The 4-nitrophenyl substituted molecule 2 exhibits a λmax at
352 nm (log ε = 4.09) in 9 : 1 DMSO–H2O. Upon increasing con-
centrations of OH−, F−, AcO− and H2PO4−, the absorption
band at 352 nm decreases slightly at the expense of new absor-
bance bands at 308 and 392 nm, the latter tailing out to ca.
700 nm (Fig. 2). The changes are accompanied by two dis-
In this study we examine the anion recognition of a series
of simple model thiosemicarbazide-based chemosensors 1–4
in aqueous DMSO and DMSO to gain further insight into the
mechanism of action between hydrogen bonding and deproto-
nation. The four diarylthiosemicarbazide anion sensors differ
in the number and position of the electron-withdrawing
4-nitrophenyl substituent. We examine the anion binding/
deprotonation properties of 1–4 using UV-visible absorption
spectroscopy and 1H NMR titration experiments with anions of
−
varying basicity including OH−, F−, AcO−, H2PO4 and Cl−. In
1
1
addition we employ two-dimensional H/15N and H/13C NMR
correlation spectroscopy to probe the chemistry of the three
N–H bonds in order to gain a better understanding of the
mechanism of action.
Results and discussion
Synthesis
The synthesis of the chemosensors 1–4 is shown in Scheme 1.
The compounds were synthesised from commercially available
chemicals by reacting phenyl hydrazine 5 or 4-nitrophenyl
Fig. 1 Colour changes observed for 2.7
× M 1–4 in 9 : 1
10−5
Scheme 1 Synthesis of the thiosemicarbazide anion sensors 1–4 in dry
CH3CN on refluxing for 2 hours.
DMSO–H2O on addition of up to 4 equivalents of OH−, F−, AcO−, Cl−
and H2PO4− as sodium salts.
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Fig. 2 UV-visible absorption spectra of 2.7 × 10−5 M 2 in 9 : 1 DMSO–H2O upon addition of various anions as sodium salts: (a) OH− (0–4 equiva-
lents), (b) F− (0–4 equivalents), (c) AcO− (0–4 equivalents) and (d) H2PO4− (0–55 equivalents.).
tinguishable isosbestic points at 338 and 362 nm, and a colour
The anion titration profiles in 9 : 1 DMSO–H2O for com-
change from almost colourless to brown-yellow (Fig. 1b). pounds 2–4 with OH−, F−, AcO− and H2PO4 are shown in
Further analysis of the spectral changes clearly shows that all Fig. 4. The addition of 2 equivalents of OH−, F−, AcO− were
four anions result in similar UV-vis absorption profiles requir- required to reach the plateau in the case of 2 and 3 (Fig. 4a
−
−
ing two equivalents of anions to reach the plateau (Fig. 4a). No and 4b, respectively) while with H2PO4 up to 50 equivalents
change was observed upon the addition of Cl−.
of the anion were necessary. In contrast, titrations with 4 in
In 9 : 1 DMSO–H2O compounds 3 and 4 exhibit an intense 9 : 1 DMSO–H2O (Fig. 4c) and 3 and 4 in DMSO only required
absorption band at ca. 367 nm (log ε = 4.3) and a weaker band the addition of 1 equivalent of OH−, F−, AcO− and H2PO4
−
at ca. 527 nm (log ε = 3.8). The addition of OH−, F−, AcO− and (Fig. S4†).
−
H2PO4 to 3 or 4 resulted in similar spectral changes. The
Fig. 1 highlights the dramatic colour changes observed for
absorbance spectra of
3 with and without subsequent the nitro-containing compounds 2–4 in the presence of basic
additions of anions are shown in Fig. 3. Compound 3 exhibi- anions. For all three compounds, the observed colour change
ted a new band at 305 nm and the band at 527 nm increases is identical for OH−, F−, AcO− and H2PO4−, suggesting the
significantly resulting in isosbestic points at 327 and 418 nm. same mechanism of action with this group of anions. No
Similarly for compound 4, titration with the four most basic colour change was observed with the weakly basic anion Cl−.
anions resulted in a significant increase centred at 528 nm The titration profiles confirm that with compound 4 only one
with two clear isosbestic points at 296 and 392 nm (Fig. S2†). equivalent of the anion (hydroxide, fluoride, acetate or dihy-
Distinct colour changes from colourless to intense purple were drogen phosphate) is necessary to reach the plateau.19 Dis-
observed for 3 (Fig. 1c) and color changes from pale pink to sociation constants for OH−, F−, AcO− and H2PO4− with 4 were
dark reddish-pink were observed for 4 (Fig. 1d). UV-vis titration experimentally identical with a value of log βA− of 5.2. Upon
experiments with compounds 3 and 4 in DMSO provided a addition of OH−, F− and AcO−, compounds 2 and 3 were near
virtual equivalent set of spectra with a slight red-shift of 5 and the plateau after one equivalent of the anion, although a
7 nm in the charge transfer band maximum at 532 and second equivalent is required to reach the asymptote. In con-
535 nm, respectively, on addition of basic anions (Fig. S3†).
trast, at least 40 equivalents of dihydrogen phosphate are
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Fig. 3 UV-visible absorption spectra of 2.7 × 10−5 M 3 in 9 : 1 DMSO–H2O upon addition of various anions as sodium salts: (a) OH− (0–4 equiva-
lents), (b) F− (0–4 equivalents), (c) AcO− (0–4 equivalents) and (d) H2PO4− (0–40 equivalents.).
required to arrive at the end point. Dissociation constants to 443 nm, while the absorption band at 305 nm increases in
log βA− of 5.0 were determined for both 2 and 3 in the presence intensity. Accompanying these changes is a shift in the iso-
−
of F−, AcO− and OH− while with H2PO4 the values were an sbestic point from 418 to 431 nm and a colour change from
order of magnitude lower at 4.0 and 4.2, respectively. A purple to orange (Fig. S5†). These colour changes are attribu-
summary of the dissociation constants is provided in Table 1.
ted to two sequential deprotonation reactions, a phenomenon
It is worth noting that the addition of a large excess of OH− which was reported for naphthalimide-based thiosemicarba-
results in distinct colour changes with 3. After four equivalents zides on addition of F−.22 However, in our case, addition of
of hydroxide, the solution is purple with a maximum at excess fluoride resulted only in an increase in the baseline
527 nm. However, on continuous addition of excess hydroxide absorption at ca. 360 nm with no colour change. In contrast, no
(up to 80 equivalents) the band decreases and gradually shifts colour or UV-vis spectroscopic change is observed on addition
of 80 equivalents of acetate and dihydrogen phosphate.
1H NMR titration studies
Table 1 Dissociation constants (log βA−) of compounds 1–4a
The 1H, 13C and 15N resonances of 1–4 were assigned based on
9 : 1 DMSO–H2Ob
100% DMSOc
the analysis of 1D proton and carbon spectra as well as 1H–13C
1
and H–15N correlations based on 2D HSQC and HMBC spec-
Anions
1
2
3
4
3
4
tral analyses. The 1H NMR spectra are given in Fig. S6–S9† and
the 1H–15N gHSQC and gHMBC spectra are given in
Fig. S10–17.† 26 Particular attention was paid to the absolute
assignment of the three N–H nitrogen and hydrogen atoms by
15N NMR spectroscopy.27,28 The aryl protons fall within a
OH−
F−
4.2 0.1 5.0 0.1 5.0 0.2 5.3 0.2 4.9 0.1 5.4 0.2
4.4 0.2 5.0 0.2 5.0 0.3 5.1 0.3 5.1 0.1 5.3 0.1
AcO−
NId
5.1 0.2 5.0 0.1 5.4 0.1 5.1 0.3 5.3 0.1
4.0 0.1 4.2 0.1 5.2 0.2 5.1 0.1 5.4 0.2
H2PO4 NId
−
a Determined by the UV-visible absorption titration technique.
Estimated errors are given as the range calculated from two titration chemical shift range of 6.7–7.5 ppm for 1 and within 6.7 and
experiments. b Sodium salts were used. c Tetrabutylammonium salts
8.2 ppm for 2–4. The aromatic N–H proton, H1, is observed in
the range of 8.1–9.3 ppm while the two thiourea protons, H2
were used. d The band is a shoulder and does not allow for accurate
identification and determination of log βA− (NI: not identified). No
anion–receptor interaction is observed.
and H3, are observed to be further downfield at 9.7–10.3 ppm
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Fig. 4 Titration profiles in 9 : 1 DMSO–H2O at a concentration of 2.7 × 10−5 M (a) 2 at 392 nm, (b) 3 at 527 nm and (c) 4 at 525 nm with 0–4 equiva-
–
lents of anions as sodium salts: OH−
(
), F−
(
), AcO−
(
) and H2PO4
(
).
and 9.8–10.4 ppm, respectively. The chemical shifts are consist- 4 and 3 upon addition of TBAOAc.) The two thiourea N–H
ently in the order of σH3 > σH2 > σH1. The assignment of these protons H2 and H3 appear at 10.29 and 10.37 ppm, respect-
three characteristic proton resonances was assisted using ively, while the aromatic N–H1 proton appears at 9.28 ppm. As
1H–15N gHSQC and gHMBC NMR correlation experiments. 15N can be seen from Fig. 5, the main resonance shifts were
chemical shifts for the three thiourea N–H nitrogen atoms were observed between 0 and 1 equivalent of added AcO−. Negli-
determined indirectly by 2D correlation studies. The aromatic gible chemical shift changes are observed for the aromatic C–
N–H nitrogen atom, N1, was observed between 96 and 106 ppm, H protons, whereas considerable changes occur for the N–H
while the two thiourea N–H nitrogen atoms, N2 and N3, were protons. After the addition of 1 equivalent of added AcO−, only
observed in the regions of 131–139 ppm and 124–127 ppm, two N–H proton signals were observed corresponding to H1
respectively. The chemical shift of the nitro groups was consist- and H3.
ently observed at 370 ppm. 13C NMR spectra are indicative of
The combination of 2D 1H–15N/1H–13C gHSQC and gHMBC
the thiourea carbonyl, which appears at 181 ppm while the experiments allowed for the unequivocal assignment of the
remaining aromatic carbon signals are clustered between 112 N–H protons in 3 and 4 in the presence of acetate (Fig. S19–
and 154 ppm. A summary of the diagnostic 1H, 13C and 15N S26†). It was determined that the central thiourea N–H proton
NMR chemical shifts is given in Table 2.
H2 disappeared, while the remaining thiourea N–H proton H3
1H NMR titrations with 1.0 × 10−2 M of 3 and 4 in the pres- and the aromatic N–H proton H1 are shielded (ΔδH3
=
ence of AcO−, added as the TBA salt, were performed in 1.34 ppm) and deshielded (ΔδH1 = 0.56 ppm) to δ 9.03 and
DMSO-d6–0.5% water and the anion–receptor interactions of 9.84 ppm, respectively (Fig. S19†). These changes are indicative
the thiosemicarbazides protons were closely monitored. At of deprotonation of the thiourea unit by AcO− supporting the
higher concentrations, colour changes were immediate upon conclusion that a deprotonation mechanism occurs. Noticeable
addition of only 0.1 equivalents of the anion. (Fig. 5 and changes in the 15N NMR chemical shifts upon addition of the
Fig. S18† show the changes observed in the 1H NMR spectra of acetate to 3 and 4 are shown in Fig. S18 and S19,† respectively.
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Table 2 Selected 1H, 13C and 15N chemical shifts (δ) of compounds 1–4 in DMSO-d6
1H
13C
15Na
N1
96
97
106
106
Compound
H1
H2
9.71
10.11
9.89
H3
9.83
10.33
9.94
Ar–H
CvS
Ar–C
N2
N3
NO2
b
1
2
3
4
8.07
8.15
9.20
9.28
6.71–7.52
6.74–8.24
6.82–8.15
6.84–8.21
181.2
180.9
181.5
181.2
113.1–148.0
113.3–147.7
111.6–154.1
111.9–153.8
134
139
131
135
124
125
126
127
—
370
371
370c
10.29
10.37
a 15N NMR δ values were determined by 1H–15N correlations from gHMBC and gHSQC spectra. b Compound 1 lacks a NO2 functionality.
c Accounts for two superimposed 15N resonances.
Fig. 5 Stacked 1H NMR spectra of 0.01 M 4 upon addition of AcO− in DMSO-d6–0.5% water at 298 K. Numbers on the left correspond to the equi-
valence of anions added. The assignments of the 1H resonances are shown for the receptor before and after the addition of increasing amount of
TBAAcO. The asterisk * marks the resonance due to protonation of acetate.
More specifically, the aromatic N–H1 nitrogen atom was in acetonitrile by UV-visible spectroscopy determined the
− 22
strongly deshielded from δ 106 to 158 ppm (ΔδN1 = 52 ppm), selectivity for 3 and 4 in the order of AcO− > F− > H2PO4
.
In
while the thiourea N–H3 nitrogen was only slightly deshielded both studies hydrogen bonding was proposed as the anion
from δ 127 to 131 ppm (ΔδN3 = 4 ppm) in 4. 15N chemical shift sensing mechanism. However, in both studies no titrations
changes have been reported in several previous NMR spectro- with hydroxide were reported.
scopic studies on anion–receptor interactions.26 Typically, the
Together with the resonances in the 1H NMR spectra,
nitrogen nuclei are deshielded by up to 10 ppm when the which are confidently assigned to deprotonation of the indi-
respective NH donor group is involved in hydrogen-bond inter- cators, a broad signal at 11.95 ppm is observed consistent with
actions in anion–receptor complexes. However, a strongly the acidic proton of acetic acid resulting from the addition of
deshielded aromatic N–H1 nitrogen atom is a characteristic up to 1.0 equivalent of AcO−. Beyond the addition of 1 equi-
feature resulting from deprotonation of an unsaturated valent of acetate, no further chemical shift changes were
system.29
observed. The identity of the signal at 11.95 ppm was con-
1
In general, the anion selectivity trend in DMSO and 9 : 1 firmed by conducting a separate H NMR experiment with an
−
DMSO–H2O was determined to be OH− ≈ F− ≈ AcO− > H2PO4
.
authentic sample of 10 mM glacial acetic acid in DMSO-d6. To
−
In the case of 4, no selectivity for H2PO4 was observed. Our the best of our knowledge, this is the first time that direct evi-
findings differ from those in the literature.24,25 The Lin group dence for deprotonation by NMR with AcO− has been reported
determined from UV-visible absorption changes that the with a thiosemicarbazide. Our findings provide convincing evi-
selectivity of 3 in DMSO and 95 : 5 DMSO–H2O is in the order dence in favour of a deprotonation mechanism rather than a
AcO− > F− ≈ H2PO4−. Similarly, the studies by the Jiang group complexation mechanism.
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Fig. 6 Depiction of the calculated HOMOs and LUMOs for compounds 1–4.
DFT studies
more, charge distribution calculations indicate that the
N–H2 hydrogen atom is the most acidic in all cases. Therefore,
there is excellent agreement between the experimental and cal-
culated spectra for the deprotonation mechanism as directly
observed by 1H and 15N NMR spectroscopy.
The DFT calculations also support the 2D NMR correlation
studies. In all cases the N–H2 proton is the most acidic, as in
the neutral molecules it bears the highest positive Mulliken
charge (Table 3).32 Furthermore, an increase of the N–H
The frontier molecular orbitals for the chemosensors 1–4 are
shown in Fig. 6. With the exception of 3, the HOMO is mostly
delocalized over the arylhydrazine and the thiourea portions of
the molecules and the LUMO is mostly delocalized on the
other aryl ring. The addition of the nitro group in 2 does not
result in a significant change in the spatial distribution of the
frontier molecular orbitals. The similarity in the frontier mole-
cular orbitals of 1 and 4 indicates that the overall effect of two
nitro substituents on both phenyl rings is a net cancellation of
the substituent effect at both terminals of the molecules. The
frontier molecular orbitals of 3 are different from the other
three molecules. The nitro group on the hydrazine side sub-
stantially lowers the energy difference and thus changes the
Table 3 Mulliken charges (in atomic units) on N–H1, N–H2 and N–
H3 hydrogen atoms in molecules 1–4 calculated at the B3LYP/
6-311++G(d,p) level of theory with tight convergence criteria
distribution of the energy levels. The DFT calculations clearly
illustrate the internal charge transfer (ICT) character within
the molecular systems.30,31 The interchange of HOMO and
LUMO in the case of 2 also results in a much lower transition
energy and higher oscillator strength in this case. Further-
Compound
N–H1
N–H2
N–H3
1
2
3
4
0.207
0.192
0.202
0.207
0.278
0.318
0.307
0.314
0.261
0.270
0.272
0.273
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proton acidity upon introduction of NO2 functionalities is 2.50 ppm and 39.52 ppm for 1H and 13C NMR, respectively.
clearly evident. In all studied cases, the protons closest to the Infrared (IR) spectra were recorded on a Shimadzu IRAffinity-1
thiocarbonyl moiety are the most sensitive, which may be a spectrophotometer calibrated versus polystyrene at 1601 cm−1
.
result of a push–pull interaction between the electron donor Solid samples were dispersed in KBr and recorded as clear
and electron acceptor (Fig. S27†).
discs. Ultra-violet absorption spectra were recorded at room
temperature using a Jasco V-650 spectrophotometer with
1.0 cm quartz cuvettes. Electrospray time-of-flight (ES-TOF)
mass spectra were obtained on a Waters LC Premier instru-
ment. Chemical ionization (CI) used ammonia as the proton
source. Melting points were measured on a Gallenkamp
melting point apparatus.
Conclusions
In conclusion, we have synthesised and studied a series of
model thiosemicarbazide-based colorimetric sensors for
anions 1–4 in DMSO and 9 : 1 DMSO–H2O with anions of
varying basicity including OH−, F−, AcO− and H2PO4−. The
receptors 1–4 possess acidic properties suitable for recognising
anionic species through directional hydrogen bonds or anion-
induced deprotonation of the thiosemicarbazide functionality.
UV-visible absorption spectroscopy revealed that upon
addition of these more basic anions, a significant red shift is
observed in the ICT absorption band, which is accompanied
by dramatic colour changes visible to the naked eye. 1H/15N
NMR studies revealed a significant chemical shift in the 15N
NMR spectrum with formation of acetic acid at 11.95 ppm in
the proton NMR spectra on titration with the acetate. These
results suggest that the mechanism of action of 3 and 4 in
DMSO-d6–0.5% water with AcO− involves deprotonation of the
thiosemicarbazide moiety, in particular at the central thiourea
N–H proton, in agreement with the DFT calculations. To the
1H NMR titration experiments and 2D NMR experiments
including 1H–15N heteronuclear single quantum correlation
(HSQC) and heteronuclear multiple bond correlation (HMBC)
were performed on a DD2 Agilent Technology NMR spectro-
meter at a frequency of 297.80 MHz, 74.89 MHz and
30.18 MHz for 1H, 13C and 15N NMR, respectively. Chemical
shifts are referenced to the residual solvent signal of DMSO-d6
as noted above while 15N chemical shifts were referenced rela-
tive to ammonia (δ 0.00 ppm).
Syntheses
Compounds 1–4 were prepared according to literature pro-
cedures with slight modification.20 Due to incomplete or
unreported literature data, the compounds were fully charac-
terised. The general procedure was as follows: in a 100 mL
round-bottom flask one equivalent of isothiocyanate and one
equivalent of hydrazine were dissolved in ca. 20 mL of dry
CH3CN and refluxed with magnetic stirring for ca. 2 hours.
The reaction was monitored via TLC with silica gel (60F 254,
Sigma-Aldrich) on aluminum using 1 : 1 hexane–ethyl acetate
as the eluent and observed under 254 nm UV light. On com-
pletion the solution was removed using a rotary evaporator
resulting in a crude residue, which was purified by trituration
with chloroform, and collected by suction filtration.22
best of our knowledge, this is the first time that H/15N NMR
1
titration spectra have been used to gain insight into the chem-
istry of AcO− with thiosemicarbazides. It is worth emphasising
that 2D 1H–13C/15N gHSQC and gHMBC NMR spectroscopy
was essential for correctly assigning the chemical shifts of the
three N–H proton units in addition to providing insight into a
deprotonation mechanism.
N,2-Diphenylhydrazinecarbothioamide (1). Phenyl hydra-
zine 5 (265 mg, 2.45 mmol) and phenyl isothiocyanate 7
(329 mg, 2.44 mmol) in dry acetonitrile yielded the desired
product as a white crystalline solid in 57% yield. Rf: 0.50; m.p.:
Experimental
Chemicals
1
4-Nitrophenyl isothiocyanate (Sigma-Aldrich), phenyl isothio-
cyanate (Sigma-Aldrich), 4-nitrophenylhydrazine (BDH) and
phenylhydrazine (Scharlau) were used as received. Dimethyl-
sulfoxide-d6 (99.8 atom% D) from Roth was used as received.
Acetonitrile (HPLC grade, Sigma-Aldrich) was distilled over
calcium hydride and stored in a glass bottle over 3 Å molecular
sieves in a desiccator. Other chemicals were used as received
without further purification.
172–173 °C; H NMR (250 MHz, DMSO-d6, ppm): 9.83 (1H, s,
thiourea–NH3), 9.71 (1H, s, thiourea–NH2), 8.07 (1H, s, Ar–
NH1), 7.52 (2H, d, J = 7.9 Hz, Ar–H), 7.20–7.32 (4H, m, Ar–H),
7.08–7.16 (1H, m, Ar–H), 6.75–6.84 (3H, m, Ar–H); 13C NMR
(63 MHz, DMSO-d6, ppm): 181.2 (CvS), 148.0, 139.2, 128.9,
127.9, 125.0, 124.7, 119.8, 113.1; 15N NMR (30 MHz, DMSO-d6,
ppm): 134 (1N, thiourea-N2H), 124 (1N, thiourea–N3H), 96 (1N,
Ar–N1H); IR (KBr, cm−1): 3252, 3205, 3148, 1599, 1568, 1505,
1481, 1333, 1281, 1209, 1111, 891, 847, 748, 689, 650; HRMS
(ESI-TOF): Calculated C13H14N3S [M − H]− 242.0752, Found
Instrumentation
The compounds were characterised using 1H and 13C NMR 242.0762.
spectra recorded on a Bruker AM250 NMR spectrometer N-(4-Nitrophenyl)-2-diphenylhydrazinecarbothioamide (2).
equipped with frequency of Phenyl hydrazine 5 (264 mg, 2.45 mmol) and 4-nitrophenyl iso-
1H/13C dual probe at
a
a
250.10 MHz and 62.90 MHz, respectively. The acquisition data thiocyanate 8 (418 mg, 2.32 mmol) in dry acetonitrile yielded a
were processed on a Bruker Aspect 3000 computer using WIN yellow crystalline solid in 52% yield. Rf: 0.51; m.p.: 170–172 °C;
NMR software. The NMR spectra are reported in parts per 1H NMR (250 MHz, DMSO-d6, ppm): 10.33 (1H, s, thiourea–
million (ppm) relative to the dimethylsulfoxide-d6 peak at NH3), 10.11 (1H, s, thiourea–NH2), 8.13–8.24 (3H, m, Ar–NH1
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and 2Ar–H), 8.03 (2H, d, J = 9.1 Hz, Ar–H), 7.24 (2H, t, J = specific wavelength.33 Plotting log[(Amax − A)/(A − Amin)] versus
7.8 Hz, Ar–H), 6.75–6.90 (3H, m, Ar–H); 13C NMR (63 MHz, the −log [A−], the log βA− was derived from the slope of the
DMSO-d6, ppm): 180.9 (CvS), 147.7, 145.7, 143.1, 128.9, 123.9, resulting plot. Titrations were repeated at least twice until the
123.5, 120.1, 113.3; 15N (30 MHz, DMSO-d6, ppm): 371 (1N, results were reproducible.
Ar–NO2), 139 (1N, thiourea–N2H), 125 (1N, thiourea–N3H), 97
1H NMR titration experiments
(1N, Ar–N1H); IR (KBr, cm−1): 3250, 3203, 3190, 3145, 1600,
1H NMR titration experiments with anion sensors 3 and 4 were
performed in DMSO-d6/0.5% water at 298 K. To 0.6 mL of
10 μM sensor solution, increasing concentrations of TBAAcO
salt solution were progressively added and subsequently
measured by 1H NMR spectroscopy. Measurements were per-
formed in the same NMR tube on addition of 0.0, 0.2, 0.4, 0.6,
1.0, 1.5, 2.0, 3.0 and 5.0 molar anion equivalents. Individual
resonances in the 1H NMR titration profile were assigned on
the basis of the chemical shifts, signal integrations, multi-
plicity, and via 1H–13C/15N gHSQC and gHMBC correlation
studies. Stacked NMR spectral plots were produced using
MestReNova software version 6.
Theoretical modeling was performed with Gaussian 09 Rev.
D.01 (Gaussian, Inc.).34 The final geometry was obtained by
using DFT with the B3LYP (the Becke three-parameter-Lee–
Yang–Parr) functional and the 6-311++G(d,p) basis set. Mole-
cular orbitals and surfaces were plotted using GaussView soft-
ware. Electronic transitions were calculated by using time-
dependent DFT with the B3LYP functional and the 6-311++G
(d,p) basis set and the polarizable continuum model (PCM)
using the integral equation formalism variant (IEFPCM) with
DMSO as the solvent.
1568, 1540, 1330, 1280, 1209, 1111, 891, 846, 748, 688, 648;
HRMS (ES-CI-TOF): Calculated C13H14N4SO2 [M
289.0759, Found 289.0751.
+
H]+
2-(4-Nitrophenyl)-N-phenylhydrazinecarbothioamide
(3).
4-Nitrophenyl hydrazine 6 (254 mg, 1.66 mmol) and phenyl
isothiocyanate 7 (225 mg, 1.67 mmol) in dry acetonitrile
yielded a brown-orange powder in 85% yield. Rf: 0.35; m.p.:
1
218–221 °C; H NMR (250 MHz, DMSO-d6, ppm): 9.94 (2H, m,
2thiourea–NH3), 9.20 (1H, s, Ar–NH1), 8.15 (2H, d, J = 9.1 Hz,
Ar–H), 7.45 (2H, d, J = 7.5 Hz, Ar–H), 7.32 (2H, t, J = 7.5 Hz, Ar–
H), 7.09–7.20 (1H, m, Ar–H), 6.82 (2H, d, J = 9.1 Hz, Ar–H); 13C
NMR (63 MHz, DMSO-d6, ppm): 181.5 (CvS), 154.1, 139.0,
138.9, 127.9, 125.7, 125.5, 125.1, 111.6; 15N (30 MHz, DMSO-
d6, ppm): 371 (1N, Ar–NO2), 131 (1N, thiourea–N2H), 126 (1N,
thiourea–N3H), 105.6 (1N, Ar–N1H); IR (KBr, cm−1): 3319, 3163,
3148, 3084, 1609, 1595, 1549, 1489, 1447, 1339, 1248, 1231,
1178, 1111, 1028, 931, 910, 843, 775, 752, 731, 696, 623; HRMS
(ESI-TOF): Calculated C13H14N4SO2 [M + H]+: 289.0759, Found
289.0764.
N,2-Bis(4-nitrophenyl)hydrazinecarbothioamide (4). 4-Nitro-
phenyl hydrazine 6 (249 mg, 1.63 mmol) and 4-nitrophenyl iso-
thiocyanate 8 (295 mg, 1.64 mmol) in dry acetonitrile yielded a
1
bright yellow solid in 58% yield. Rf: 0.27; m.p.: 208–211 °C; H
NMR (250 MHz, DMSO-d6, ppm): 10.20–10.48 (2H, m,
thiourea–NH2 and NH3), 9.28 (1H, s, Ar–NH1), 8.21–8.27 (4H,
m, Ar–H), 7.93 (2H, d, J = 9.5 Hz, Ar–H), 6.84 (2H, d, J = 9.1 Hz,
Ar–H); 13C NMR (63 MHz, DMSO-d6, ppm): 181.2 (CvS), 153.8,
145.6, 143.3, 139.3, 125.8, 124.6, 123.7, 111.9; 15N (30 MHz,
DMSO-d6, ppm): 370 (2N, Ar–NO2), 135 (1N, thiourea–N2H),
127 (1N, thiourea–N3H), 106 (1N, Ar–N1H); IR (KBr, cm−1):
3308, 3281, 3144, 3020, 1595, 1553, 1497, 1412, 1331, 1265,
1223, 1111, 934, 856, 843, 748, 707, 634; HRMS (ESI-TOF) Cal-
culated C13H12N5SO4 [M + H]+: 334.0610, Found: 334.0602.
Acknowledgements
We acknowledge the financial support from the University of
Malta, the Strategic Educational Pathways Scholarship (STEPS)
part-financed by the European Social Fund (ESF) under Oper-
ational Programme II, and the European Cooperation in
Science and Technology (COST Action CM1005 “Supramolecu-
lar Chemistry in Water”). Our appreciation is extended to Prof.
Robert M. Borg for assistance with the acquisition of the NMR
data. We also gratefully acknowledge the financial support of
the Slovenian Research Agency, Ministry of Higher Education,
Science and Technology of Republic Slovenia (program no. P1-
0242), ENFIST Centre of Excellence. DFT calculations were per-
formed at the Academic Computer Centre CYFRONET AGH
within computational grant no. MEiN/SGI3700/UJ/085/2006.
UV-visible absorption spectrometric titrations
UV-visible absorption titrations were performed in 9 : 1 DMSO–
H2O at room temperature with sodium salts. Freshly pre-
pared solutions of 3 × 10−5 M sensor were used. Aliquots of
aqueous stock solutions of anions were added (OH−, F−, AcO−,
H2PO4 and Cl−) diluted with distilled water. Experiments
−
in DMSO were performed at room temperature with tetra-
butylammonium (TBA) salts. The solutions were prepared with
3.4 × 10−5 M of sensor in DMSO with varying volumes of
anions.
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