Novel dansyl-appended calix[4]arene frameworks: Fluorescence properties and mercury sensing

Article abstract of DOI:10.1039/b815379e

Covalently-attached fluorophores may impart enhanced chemosensing capabilities to calixarene frameworks. Synthesis and characterization of six novel dansyl-appended calix[4]arenes, namely, H/Dan₄, NO ₂/Dan₄, H/(OH)₂

Full text of DOI:10.1039/b815379e

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Novel dansyl-appended calix[4]arene frameworks: fluorescence properties
and mercury sensing†
Shubha Pandey,a,b Amir Azam,*a Siddharth Pandey*b and H. M. Chawla*b
Received 3rd September 2008, Accepted 29th September 2008
First published as an Advance Article on the web 18th November 2008
DOI: 10.1039/b815379e
Covalently-attached fluorophores may impart enhanced chemosensing capabilities to calixarene
frameworks. Synthesis and characterization of six novel dansyl-appended calix[4]arenes, namely,
H/Dan4, NO2/Dan4, H/(OH)2Dan2, H/(Ester)2(Dan)2, t-Bu/(OH)2Dan2, and t-Bu/(Ester)2Dan2,
containing two or four dansyl moieties are reported. Among these, fluorescence intensity of NO2/Dan4
is observed to decrease significantly in the presence Hg2+ in the solution. Based on the decrease in
fluorescence, a limit of detection for Hg2+ of 20 ppb is obtained. NO2/Dan4 as a chemosensing agent for
Hg2+ shows excellent selectivity and adequate reversibility. Complexation of NO2/Dan4 with Hg2+ is
investigated using fluorescence spectroscopy and is observed to be 2:1. The formation constant of
(NO2/Dan4)2Hg2+ is estimated to be 5.2( 0.8) ¥ 1010 M-2 at ambient conditions. These observations are
traced to the fact that while all other dansyl-appended calix[4]arenes show cone conformation in the
solution, NO2/Dan4 is in the 1,3-alternate conformation. Stokes shift versus solvent orientational
polarizability for NO2/Dan4 also indicates the difference in the ground- to excited-state dipole moment
of this compound to be the maximum among all six, rendering it most sensitive to its environment.
Fluorescence emission of NO2/Dan4 in nonpolar chloroform, polar-aprotic acetonitrile, and
polar-protic ethanol is observed to be different than that of the rest of the dansyl-appended
compounds as well.
maxima, appearance and/or disappearance of fluorescence bands,
Introduction
changes in the excited-state intensity or anisotropy decay kinetics,
Calix[4]arenes and their derivatives have been of continuous
among others, may form the basis of the recognition event. In
interest to the scientific community due to their widespread
general, one of two approaches is used in such investigations;
applications; the most important perhaps being their behavior
either the calix[4]arene-based host is suitably modified by co-
as unique and versatile hosts.1–6 A variety of guests with differing
valent attachment of a judiciously selected fluorophoric moiety
size, shapes, properties, and characteristics have been recognized
(or moieties), or the fluorescence from the guest and/or some
by different calix[4]arenes and their derivatives.1 It is established
that the versatility of these hosts as far as either the recognition
of more types and classes of guests is concerned or increasing the
effectiveness of overall recognition process for a particular guest is
of primary interest can be expanded immensely due to enormous
derivatization possibilities of calixarene molecular frameworks. In
this context, facile derivatization at the lower and/or the upper
rim of a calixarene molecular framework may result in improved
efficiency as well as versatility toward guest recognition.1 Due
to this, calixarenes routinely serve as an ideal platform for the
development of a variety of chemosensors for the recognition of
cations, anions, and small organic molecules.2,3,5
Fluorescence-based investigations of complexation and sens-
ing involving calix[4]arenes and related compounds are routine
due to inherently high sensitivity associated with fluorescence-
based techniques.7,8 Reduction or enhancement in steady-state
fluorescence intensity or anisotropy, shifts in fluorescence band
external analyte becomes the basis of analysis.2,7 Needless to
say, researchers are actively involved in exploring both these
avenues to come up with calixarene-based effective and versatile
chemosensors. However, the former of the two approaches has
garnered more attention from organic chemists perhaps due to its
well-documented effectiveness and more potential applications.
Inadequate fluorescence from most guests of interest and un-
availability of suitable externally-added fluorophores are also to
blame for this bias. Further, due to the availability of a variety of
derivatization strategies associated with the calixarene molecular
framework, the design and development of novel fluorophore-
appended calixarene-based fluorescence chemosensors with en-
hanced sensitivity and increased selectivity is both exciting and
useful. Due to enormous possibilities, the development of fluores-
cent molecular sensors based on calixarenes is a challenge, as they
can offer high sensitivity and potential applications in the field of
analytical, pharmaceutical, and biological chemistry.6,9–12
Several common and popular fluorophoric moieties have
been utilized to design and synthesize fluorophore-appended
calixarene-based fluorescence chemosensors in the past.13
Among others, dansylated calixarenes (fluorogenic calixarenes
with dansyl/dansyl-appended calixarenes) have received ever-
increasing (albeit limited) attention for the recognition and sensing
of heavy, transition, and alkali metals, such as, Hg2+, Pb2+, Cd2+,
aDepartment of Chemistry, Jamia Millia Islamia, New Delhi, 110028, India
bDepartment of Chemistry, Indian Institute of Technology Delhi, Hauz Khas,
New Delhi, 110016, India. E-mail: sipandey@chemistry.iitd.ac.in; Tel: +91-
11-26596503
† Electronic supplementary information (ESI) available: Fig. S1–S5, details
of the calculation of formation constant, and 1H NMR and 13C NMR of
all the new compounds. See DOI: 10.1039/b815379e
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Cr3+, Cu2+, Na+, etc.5,11–13 The dansyl group, when covalently
bound to a host molecule, offers many attractive and pertinent
features due partly to (i) its strong fluorescence (absorption bands
in the near-UV and intense fluorescence in the visible region),14 (ii)
relatively long emission wavelength,5a and (iii) its solvatochromic
nature (i.e., sensitivity to the polarity of the medium owing to
the presence of a twisted intramolecular charge-transfer (TICT)
excited state).15–17
In recent years considerable effort has been devoted to the se-
lective and efficient detection of heavy metal ions, especially Hg2+,
as mercury and its salts have high toxicity, and they are widely
used in industry and hence are widespread in the environment.11–13
As a result, there has been much effort (and numerous reports)
on the design and development of fluorescent chemosensors for
detection of Hg2+.11,12 However, very few offer acceptable sensitiv-
ity, adequate selectivity and long-term reversibility for potential
applications in biological and environmental monitoring. In this
paper, we report the synthesis of a series of novel dansyl-appended
fluorescent calix[4]arenes containing two or four covalently at-
tached dansyl moieties at the lower rim of the calix[4]arene
molecular framework possessing other functionalities at various
positions (Scheme 1). The potential of these dansyl-appended
calix[4]arenes as fluorescence chemosensors for metal ions is also
explored and presented. Spectroscopic properties relevant to their
behavior as fluorescence chemosensing agents for metal ions are
used to explain such behavior of these novel compounds. In this
context, the fluorescence spectroscopic behavior of these two or
four dansyl-appended calix[4]arenes is compared with those of
mono-dansylated reference compounds (Scheme 2). One of our
Scheme 2 Synthesis of reference compounds, H/Dan1, t-Bu/Dan1, and
NO2/Dan1.
novel dansyl-appended calix[4]arenes, NO2/Dan4 (Scheme 1), is
observed to offer very high sensitivity, adequate selectivity, and
acceptable reversibility toward sensing Hg2+ in solution.
Results and discussion
Synthesis and characterization of novel dansyl-appended
compounds
5,11,17,23-Tetra-tert-butyl-25,26,27,28-tetrahydroxy-calix[4]-
arene (1) and 25,26,27,28-tetra-hydroxy-calix[4]arene (2) were
synthesized according to the reported literature procedure.18
5,11,17,23-Tetra-tert-butyl-25,27-di(ethoxycarbonyl-methoxy)-
26,28-dihydroxy-calix[4]arene (1a) and 25,27-di(ethoxycarbonyl-
methoxy)-26,28-dihydroxy-calix[4]arene (2a) were synthesized by
refluxing the corresponding calix[4]arenes (1 and 2, respectively)
with ethyl bromoacetate in the presence of K2CO3 in acetonitrile
for 15 hours.18
Scheme 1 Synthesis of novel dansyl-appended calix[4]arenes. Reagents and conditions: (i) ethyl bromoacetate, K2CO3, reflux; (ii) 100% HNO3, glacial
acetic acid, stirring, ice-bath; (iii) dansyl chloride, stirring, Et3N.
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Compound 1 was converted to its tetra-nitro derivative (3)
via ipso-nitration.19a,b The reaction of compounds 2 and 3
with equimolar amounts per hydroxyl group of the reagents
(1:4:4; OH:dansyl chloride:Et3N) resulted in tetra-dansylated
derivatives: 25,26,27,28-tetra(N-(5-dimethylaminonaphthalene-
1-sulfonyl))calix[4]arene (H/Dan4) and 5,11,17,23-tetra-nitro-25,
26,27-tetra(N-(5-dimethylaminonaphthalene-1-sulfonyl))calix[4]-
arene (NO2/Dan4), respectively (Scheme 1). Corresponding
reactions of 1, 2, 1a, and 2a with dansyl chloride in the presence
of triethylamine (Et3N) as base (1:2:2 molar ratio) resulted
in di-dansylated calix[4]arene derivatives: 5,11,17,23-tetra-tert-
butyl-25,27-bis(N-(5-dimethylaminonaphthalene-1-sulfonyl))-26,
28-dihydroxycalix[4]arene (t-Bu/(OH)2Dan2), 25,27-bis(N-(5-
dimethylaminonaphthalene-1-sulfonyl))-26,28-dihydroxycalix[4]-
arene (H/(OH)2Dan2), 5,11,17,23-tetra-tert-butyl-25,27-di(etho-
xycarbonyl-methoxy)-26,28-bis(N -(5-dimethylaminonaphtha-
lene-1-sulfonyl))calix[4]arene (t-Bu/(Ester)2Dan2), and 25,27-
di (ethoxycarbonyl-methoxy)-26,28-bis(N-(5-dimethylaminona-
phthalene-1-sulfonyl))calix[4]arene (H/(Ester)2Dan2), respec-
tively (Scheme 1). It is interesting to note that when 1 was reacted
with four equivalents of dansyl chloride and triethylamine,
instead of the expected tetra-dansylated product, a di-dansylated
derivative of tert-butyl calix[4]arene was obtained. Possible steric
hindrance due to the presence of four each of tert-butyl and dansyl
groups in close proximity may explain this outcome. To compare
the spectroscopic properties of our dansylated calix[4]arenes,
model compounds t-Bu/Dan1, H/Dan1,14 and NO2/Dan1 were
synthesized by the base-catalyzed acylation of p-tert-butylphenol,
phenol, and p-nitrophenol, respectively, with dansyl chloride and
triethylamine (Scheme 2). These model compounds were further
purified by column chromatography and re-crystallized from
CHCl3/CH3OH.
The structures of novel dansylated calix[4]arene deriva-
tives [H/Dan4, NO2/Dan4, t-Bu/(OH)2Dan2, H/(OH)2Dan2,
t-Bu/(Ester)2Dan2, and H/(Ester)2Dan2] and model compounds
[t-Bu/Dan1, H/Dan1,14 and NO2/Dan1] were established by
the analysis of their 1H and 13C NMR spectra as well
as MALDI-TOF MS analysis. The 1H NMR spectra of
t-Bu/(OH)2Dan2, H/(OH)2Dan2, H/Dan4, t-Bu/(Ester)2Dan2,
H/(Ester)2Dan2 show two sets of doublets for bridging methylene
protons. The presence of a pair of doublets for ArCH2Ar protons
1
in the H NMR spectra of synthesized dansylated calix[4]arene
derivatives (except NO2/Dan4) and only one signal for methylene
carbon in the range of 29–32 ppm in their 13C NMR spectra
suggested that these dansylated calix[4]arene derivatives were in
a symmetrical cone conformation in solution.19c For example,
1
the H NMR spectrum of t-Bu/(OH)2Dan2 shows two pairs of
doublets for the axial and equatorial protons at 2.92 and 3.85 ppm,
respectively (Fig. 1), and one distinct signal at 31.5 ppm for the
methylene carbon in 13C NMR spectrum (ESI: Fig. S1). The–
N(CH3)2 signals appeared at 2.82 ppm in 1H NMR and 45.3 ppm
in 13C NMR spectrum. The tert-butyl group was represented by
1
two singlets at 1.17 and 0.74 ppm in H NMR spectrum and at
32.4 ppm in C13 NMR spectrum. All aromatic protons for dansyl
group as well as p-tert-butyl-calix[4]arene appeared in the range
of 8.59 to 6.52 ppm. The signal for the -OH proton appeared at
1
1.49 ppm, which is exchangeable with D2O. In contrast, the H
NMR spectrum of NO2/Dan4 shows a singlet at 3.37 ppm for
the methylene bridged ArCH2Ar protons instead of two pairs of
doublets as observed in the case of other dansylated calix[4]arene
derivatives (Fig. 2). Appearance of a singlet for methylene bridged
protons indicates that NO2/Dan4 is present in the 1,3-alternate
conformation,12,19d,e which is further confirmed by the appearance
of one signal at 34.3 ppm in 13C NMR spectrum (ESI: Fig. S2).
Fig. 1 Molecular structure and 1H NMR spectrum of t-Bu/(OH)2Dan2 at 25 ◦C after D2O exchange. The peak labeled * represents residual D2O after
exchange.
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Fig. 2 Molecular structure and 1H NMR spectrum of NO2/Dan4 at 25 ◦C in CDCl3 (* represents signal due to residual solvent).
These d values from NMR results are in good agreement with
those reported for other 1,3-alternate compounds.12b,19d,e
Temperature-dependent 1H NMR was also carried out on
NO2/Dan4 in CDCl3 as 3, the parent compound of NO2/Dan4,
shows a pair of doublet at 23 ◦C which start to coalesce into a
singlet at temperature above 23 ◦C.19a From the low-temperature
1H NMR spectrum it is clearly confirmed that the singlet for
ArCH2Ar of NO2/Dan4 remains as a singlet when the temperature
was decreased from +48 ◦C to -25 ◦C (ESI: Fig. S3 and S4), which
is a characteristic of the 1,3-alternate conformation.19d,e Existence
of NO2/Dan4 in the 1,3-alternate instead of the cone conformation
may be explained in part on the basis of the steric hindrance due
to the presence of four nitro and four dansyl groups; the weak
interaction between -NO2 and -N(CH3)2 of the dansyl moiety due
to electron-withdrawing nature of NO2 group may contribute to
this as well. The different conformation of NO2/Dan4 as compared
to our other dansyl-appended calix[4]arenes may result in its
unique sensing behavior (vide infra).
Dansyl-appended calix[4]arenes as fluorescence chemosensors
Due to the solvatochromic nature of the dansyl-based
fluorophores20,21 and well-documented host nature of the
calix[4]arene framework11 to which the dansyl moieties are at-
tached, we decided to explore the potential of our novel dansyl-
appended calix[4]arenes as possible fluorescence chemosensing
agents for metal ion sensing. For this purpose, dilute solutions of
dansyl-appended calix[4]arenes (~10 mM) in acetonitrile (ACN)
were excited at an optimum excitation wavelength of 351 nm;20 the
resulting fluorescence was subsequently measured in the presence
of varying concentration of metal ions.
Interestingly, as shown in Fig. 3, our investigation indicated
a significant decrease in fluorescence intensity of NO2/Dan4
upon addition of Hg2+; a 74% reduction in fluorescence intensity
was observed upon addition of 8 mM Hg2+. Clearly, NO2/Dan4
Fig. 3 Quenching of NO2/Dan4 fluorescence intensity in the presence
of Hg2+ in acetonitrile at ambient conditions ([NO2/Dan4] = 1 ¥ 10-5 M,
lexcitation = 351 nm). Inset shows change in fluorescence (DF) upon addition
of Hg2+ for 0 ≤ [Hg2+] ≤ 8 mM.
shows tremendous sensing/recognition ability toward Hg2+. If the
reduction in NO2/Dan4 fluorescence intensity (i.e., DF = F0 - F,
where F0 and F are the fluorescence intensities in the absence and
presence of Hg2+, respectively) as up to 8 mM Hg2+ is added to the
host solution is assumed to vary linearly with [Hg2+] (inset Fig. 3),
limit of detection (LOD) for Hg2+ can be calculated for NO2/Dan4
as fluorescence chemosensing agent using calibration sensitivity
(m) of DF versus [Hg2+] in the aforementioned range.21 For the
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purpose of LOD calculation, minimum change in the signal (DFmin
)
metal cation. These results imply significantly high selectivity
associated with host NO2/Dan4 toward the sensing of Hg2+. It is
important to mention that under identical conditions fluorescence
response of all of our other dansyl-appended calix[4]arenes as
well as the model compounds are not affected to the same
extent as Hg2+ is added to the solution. As shown in Fig. 5,
the % reduction in fluorescence intensity upon addition of 8 mM
Hg2+ is observed to be insignificant for t-Bu/(OH)2Dan2 and
t-Bu/(Ester)2Dan2, but were found to be ca. 38%, 31%, and 20%
for H/Dan4, H/(OH)2Dan2, and H/(Ester)2Dan2, respectively.
Though the% reduction in fluorescence intensities of H/Dan4,
H/(OH)2Dan2, and H/(Ester)2Dan2 upon addition of 8 mM Hg2+,
respectively, may not be deemed insignificant, it is certainly not
as dramatic as that of NO2/Dan4. It is clear that fluorescence of
NO2-substituted dansyl-appended calix[4]arene has the highest
sensitivity to the presence of Hg2+ followed by a much lower
sensitivity to the unsubstituted and almost no sensitivity to
the tert-butyl-substituted dansyl-appended calix[4]arenes. At this
point, we would like to propose the conformational dissimilarity
of NO2/Dan4 (it shows a 1,3-alternate conformation, vide supra)
with that of our other dansyl-appended calix[4]arenes (they exist
in cone conformation) to be the reason for this unique behavior.
This is further supported by a previous literature report implying
that calixarene derivatives in the 1,3-alternate conformation show
superior metal binding capabilities as compared to their cone
counterparts.12b
due to the presence of Hg2+ was taken to be 3¥s0, where s0 is
the standard deviation of F0 for twelve replicate measurements.22
3 ¥ s0
m
Thus, the LOD is calculated using
, and it is
LOD
=
]
2+
[Hg
observed to be ~0.1 mM (or ~20 ppb) Hg2+. Such low LOD clearly
demonstrates the high sensitivity of NO2/Dan4 toward Hg2+.
Next, we investigated the change in fluorescence intensity of
NO2/Dan4 in the presence of other metal ions besides Hg2+ under
identical conditions as above (Fig. 4). Although, as discussed ear-
lier, the fluorescence of NO2/Dan4 was severely decreased by Hg2+,
interestingly, no significant change in NO2/Dan4 fluorescence
intensity was observed in the presence of 8 mM Fe2+, Cu2+, Ni2+,
Zn2+, CO2+, Pb2+, Cr3+, Cd2+, Na+, Li+, and K+, respectively. These
results clearly imply the high selectivity of NO2/Dan4 toward Hg2+
m
m
DF
M
n+
n+
M
recognition. Selectivity coefficients,
,
k
=
2+
=
n+
M
,Hg
DF
2+
2+
Hg
Hg
were calculated for all metal ions investigated for possible
interference.23 Almost no change in the fluorescence intensity of
NO2/Dan4 is observed in the presence of Fe2+, Pb2+, and K+,
respectively (Fig. 4), suggesting no interference from these metal
n+
2+
n+
2+
ions (i.e., kM
= 0). Values of kM
for other metal ions are
, Hg
, Hg
calculated and plotted in Fig. 4 (inset). A careful observation of
+
2+
this data reveals kMn
for most metal ions to be small enough
, Hg
to pose any significant interference with the detection of Hg2+.
We also carried out interference studies by measuring reduction
in fluorescence signal of NO2/Dan4 by Hg2+ in the presence of
other metal cations (ESI: Fig. S5). The data implies that even
in the presence of other metal cations, the efficiency of Hg2+ in
quenching NO2/Dan4 fluorescence is significant, and in many
cases, even similar to that observed in the absence of any other
Fig. 5 Percentage reduction in the fluorescence intensity of NO2/Dan4,
t-Bu/(OH)2Dan2, H/Dan4, H/(OH)2Dan2, t-Bu/(Ester)2Dan2, and
H/(Ester)2Dan2 in the presence of 8 mM Hg2+ in acetonitrile at ambient
conditions (lexcitation = 351 nm).
Fig. 4 Percentage reduction in the fluorescence intensity of NO2/Dan4
(1 ¥ 10-5 M) in the presence of 8 mM Mn+ in acetonitrile at ambient
conditions (lexcitation = 351 nm). Inset shows selectivity coefficients
Reversibility is universally recognized as one of the most desired
properties of a modern day chemosensor.20 To further explore the
properties of NO2/Dan4 as a chemosensing agent for Hg2+, we
m
DF
n+
n+
M
M
k
=
2+
=
for NO2/Dan4.
n+
M
,Hg
m
DF
2+
2+
Hg
Hg
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carried out a set of experiments to determine the reversibility of
this system. For this purpose, we used ethylenediaminetetraacetic
acid (EDTA), an external additive, as the formation constant of
EDTA:Hg2+ complex is very high (Kf = 6.3 ¥ 1021 at 20 ◦C).24 We
observed that the decrease in fluorescence intensity of NO2/Dan4
due to the addition of Hg2+ was completely recovered by the
addition of EDTA of the same concentration as Hg2+ (data not
shown). Further addition of Hg2+ again reduced the fluorescence
intensity of NO2/Dan4 in the solution. This cycle was repeated
several times with the same final outcome. This clearly establishes
the reversible nature of our chemosensing agent. In summary, we
have demonstrated the sensing capability of our dansyl-appended
calix[4]arene molecular framework, NO2/Dan4, for Hg2+ to be
highly sensitive, adequately selective and reversible.
Stoichiometry and efficiency of NO2/Dan4: Hg2+ interaction
F - F
F
0
Fluorescence data of NO2/Dan4 in the presence of varying [Hg2+]
(at higher concentration regime) was further analyzed to obtain
key information concerning the interaction between the host and
the guest. A detailed Stern–Volmer analysis2,20 of the fluorescence
data ruled out the possibility of dynamic or static quenching or a
Fig. 6
versus xNO
from fluorescence reduction of NO2/Dan4
2 /Dan4
in the presence of Hg2+ (lexcitation = 351 nm) clearly showing the stoichiome-
try of the complexation to be 2 NO2/Dan4 per Hg2+ at ambient conditions.
1
F2
[Hg2+
DF
]
0
Inset shows the plot of
versus
, where [Hg2+]0 is the analytical
concentration of Hg2+ in the solution (for detail, see ESI).
F
0
-1
F
0
combination of both (
versus [Hg2+] as well as
versus
F
[Hg2+
]
F
constant (e) and the refractive index (n) of the cybotactic region
through the Lippert-Mataga equation21
[Hg2+] are observed to be far from linear) suggesting interaction
or complexation between NO2/Dan4 and [Hg2+] to be not of the
Df
m - m 2 +constant
(2)
SS =
(
)
F - F
F
E
G
hca3
0
1:1 type. A plot of
xNO
versus the mole fraction of the host (i.e.,
where h is Planck’s constant, c is speed of light, a is the cavity radius
swept out by the fluorophore, and mE and mG are the fluorophore
excited-state dipole moment and fluorophore ground-state dipole
moment, respectively. The term Df is empirical in nature and is
called solvent orientational polarizability:
4 ) (please refer to Fig. 6) clearly shows the stoichiometry
/Dan
2
of the complexation to be NO2/Dan4:[Hg2+] = 2:1. Based on our
data, formation of the complex of the type (NO2/Dan4)2Hg2+ may
be proposed as the result of the recognition of Hg2+ by NO2/Dan4.
Based on the equilibrium
2 NO2/Dan4 + Hg2+ ꢀ (NO2/Dan4)2Hg2+
È
Í
Î
˘
˙
˚
2
e -1
2e +1
n -1
È
˘
Df =
-
(3)
2n2 +1
Í
Î
˙
˚
where
In order to assess the dansyl cybotactic region in different
compounds, we collected emission and excitation maxima of
all our dansyl-appended compounds in seven different solvents:
tetrahydrofuran (THF), ethanol (EtOH), acetic acid, toluene,
CHCl3, dichloromethane (DCM), and acetonitrile (ACN) at
ambient conditions. Stokes shifts are calculated from the fluo-
rescence spectra and are presented in Fig. 7. While for single
dansyl-containing model compounds it may be assumed that the
fluorescence behavior of dansyl depends on the solvent only, for
dansyl-appended compounds it may not be so. The molecular
architecture around dansyl moiety may have significant affect on
its fluorescence behavior. A careful examination of Fig. 7 reveals
several interesting outcomes. It is convenient to note that average
SS of all Dan4 compounds is observed to be lower than those
for Dan2 and Dan1 compounds (8530 295 cm-1 versus 9042
537 cm-1 versus 9013 611 cm-1). Clearly, while the presence of
two dansyl moieties on a calix[4]arene framework does not alter
the dipolarity of the dansyl cybotactic region in comparison to
that of mono-dansylated compounds, introduction of two more
dansyl reduces the dipolarity of the dansyl microenvironment.
This may suggest the presence of interaction between the dansyl
moieties when four of them are crowding the one calix[4]arene
[(NO2 / Dan4 )2 Hg2+
[NO2 / Dan4 ]e2q [Hg2+
]
eq
(1)
Keq = Kf =
]
eq
1
F2
[Hg2+
]
0
a plot of
versus
is observed to show good linear
DF
behavior (refer to inset Fig. 6 and ESI), where DF and F are the
decrease in fluorescence intensity and the fluorescence intensity
at the wavelength of analysis, respectively, and [Hg2+]0 is the
analytical concentration of Hg2+ in the solution. Using the values
of the slope and the y-intercept along with independently obtained
relationship between [NO2/Dan4] and F, the Keq (or the Kf ) for
the complexation of NO2/Dan4 with Hg2+ is calculated to be
5.2( 0.8) ¥ 1010 M-2 at ambient conditions (see ESI for details).
A Keq (or Kf ) = 5.2( 0.8) ¥ 1010 M-2 represents a fairly efficient
complexation between NO2/Dan4 and Hg2+ that manifests itself
in a favorable LOD for Hg2+ by this novel host (vide supra).
Fluorescence of dansyl-appended compounds: Stokes shifts
The Stokes shift (SS), difference between the electronic excitation
and emission maxima in cm-1, for a solvatochromic fluorescence
probe is observed to depend empirically upon the dielectric
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framework. Further, in general, the average SS of the compounds
are in agreement with the dipolarity of the solvents. Specifically,
the maximum average SS decreases in the order: acetic acid >
ACN > THF > EtOH > DCM > CHCl3 > toluene. In model
compounds, except in toluene, the decreasing order of the SS is
t-Bu/Dan1 > NO2/Dan1 > H/Dan1. This must be due to the nature
of the substituents on the model compound framework (i.e.,
t-Bu versus NO2 versus H). If the cavity radius swept by dansyl is
considered similar for all three, the difference (mE - mG) appears to
be maximum for t-Bu/Dan1. However, the fact that a similar trend
is not observed for multi dansyl-appended calix[4]arenes with
different functionalities (see Fig. 7) further indicates interaction
of dansyl moiety with other dansyl moiety (or moieties) and/or
with the molecular architecture around the dansyl.
sensitive the fluorophore (or fluorophoric moiety) is toward its
immediate microenvironment. We may propose that a relatively
large difference in the ground to excited state dipole moment
imparts the unique chemosensing behavior to NO2/Dan4.
Fluorescence of dansyl-appended compounds: relative fluorescence
intensities
The reason for the solvatochromism of dansyl and similar
fluorophores can be traced to at least two major reasons. The
first reason is the change in fluorophore’s dipole moment on
optical excitation as a result of general solvent effect depending
on static dielectric constant and refractive index of the solvent
milieu according to Lippert-Mataga equation (vide supra).20
A
For each compound, SS in different solvents is plotted against Df
of the solvent (eqn 3). Fair-to-good linear regression coefficients
indicate compliance with Lippert-Mataga empirical relationship
(eqn 2). The most important information is afforded by the slope
of the Lippert-Mataga equation (inset Fig. 7). To our convenience,
the slope for NO2/Dan4 is observed to be significantly higher
than those for all other compounds. This provides an explanation
and lends support to our chemosensing results (vide supra),
which indicated the better sensitivity of NO2/Dan4 fluorescence
in the presence of Hg2+ as compared to our other dansyl-
appended compounds. The slope of the Lippert-Mataga equation
second contribution to their solvatochromism arises from the
fact that they possess two excited states; locally excited (LE)
and twisted intramolecular charge transfer (TICT). On excitation,
the less polar, semi-planar state forms initially, which is very
similar in structure to the ground state, giving rise to structured
fluorescence spectrum. This LE state can undergo intramolecular
electron/charge transfer from the donor site -N(CH3)2 to the
acceptor site -SO2- with a corresponding twist of the donor residue
about the bond that connects it to the acceptor. Subsequently, in
the TICT state, the donor and acceptor are orthogonal to one
another, and this state is much more polar in comparison to the
LE state. The fluorescence corresponding to this TICT state is
usually structure-less and is shifted bathochromically with respect
to the LE state. The propensity to form the TICT state and thus
shift the spectrum is influenced by the solvent dielectric properties
which help to mediate charge transfer and the ability of the donor
residue to rotate within the fluorophore. The ability of the donor
2
m - m
(
)
. Considering similar cavity radius (a) for dansyl
E
G
is
hca3
moiety in all compounds investigated, a maximum (mE - mG)
for NO2/Dan4 indicates the difference in the transition dipole
moment from ground to excited electronic state to be maximum
for this compound. In general, the larger the (mE - mG), the more
(mE - mG )2
Fig. 7 Stokes shifts of novel dansyl-appended calix[4]arenes in various solvents at ambient conditions. Inset shows
(i.e., the slope of the
hca3
Stokes shifts versus orientational polarizability of solvents plot according to eqns 2 and 3) for six novel dansyl-appended calix[4]arenes as well as three
reference compounds. The color of the bar corresponding to a compound in the inset represents the Stokes shift of the same compound in each of the
solvents.
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Fig. 8 Fluorescence spectra of six novel dansyl-appended calix[4]arenes and three reference compounds in nonpolar chloroform (left panels),
polar-aprotic acetonitrile (middle panels), and polar-protic ethanol (right panels) at ambient conditions (lexcitation = 351 nm). Relative and normalized
fluorescence intensities are represented on the y-axis in the upper and lower panels, respectively.
to rotate depends, in part, on any physical impediments to donor
rotation.21
the presence of ester groups appears to help the TICT process.
To our surprise, NO2/Dan4 shows appreciable TICT fluorescence
as compared to the model compound NO2/Dan1. This must
be attributed to the electron-withdrawing capability of the NO2
groups located on calix[4]arene framework. Exceptional behavior
of NO2/Dan4 is again demonstrated nonetheless.
We collected fluorescence emission spectra of our dansyl-
appended calix[4]arenes in relatively nonpolar CHCl3, polar-
aprotic ACN, and polar-protic EtOH at ambient conditions
(Fig. 8). Most importantly, in the fluorescence data collection
the absorbance of the solutions of different compounds at the
excitation wavelength are identical, such that the same number
of photons of the given energy is used in the excitation process
irrespective of the identity of the compound. Fig. 8 (left panels)
presents fluorescence emission spectra of all the compounds in
CHCl3 at ambient conditions. While relative fluorescence is shown
in the top panel, the bottom panel presents normalized fluores-
cence intensity with respect to the highest energy band for each of
the compounds. Structured LE fluorescence from dansyl moiety
occurs at lower wavelengths while the broad structure-less TICT
band appears bathochromically shifted for all the compounds. It
is convenient to note that, except for NO2-containing dansylated
compounds, the fluorescence intensity due to TICT is maximum
for model compounds, H/Dan1 and t-Bu/Dan1. It is easy to
comprehend, as charge-transfer would be unhindered in these
compounds. For calix[4]arene compounds appended with two
or more dansyl moieties, reduction in the efficiency of TICT is
represented in the reduced lower energy fluorescence from these
compounds (e.g., the TICT fluorescence intensity follows the
trend H/Dan1 > H/(Ester)2Dan2 > H/(OH)2Dan2 ~ H/Dan4 and
t-Bu/Dan1 > t-Bu/(Ester)2Dan2 > t-Bu/(OH)2Dan2). Apparently,
The TICT is expected to be facilitated in ACN, a polar-
aprotic solvent. It is clear from the fluorescence spectra of the
compounds in ACN (Fig. 8, middle panels) that, in general, the
structured higher energy LE fluorescence is replaced by broad and
bathochromically shifted fluorescence bands in most compounds.
Excited-state intramolecular interactions among dansyl groups
are again clearly indicated by the decreased fluorescence from
two or more dansyl-containing calix[4]arenes as compared to
the model compounds. It is easy to comprehend that the similar
order of TICT fluorescence is observed in ACN as that in CHCl3;
even the increased polarity of ACN could not enhance the TICT
process in NO2/Dan1. It is clear again that TICT is restricted
in NO2-containing compounds which confirms our earlier results
(vide supra). Fig. 8 (right panels) presents fluorescence of dansyl-
appended compounds in EtOH at ambient conditions. In most
cases, spectra have become broader as compared to the spectra
in CHCl3 and ACN. While the model compound H/Dan1
shows significant fluorescence; the fluorescence signal decreases
considerably for two or more dansyl-containing calix[4]arenes,
again indicating the possibility of excited-state energy transfer
between the dansyl moieties within the calixarene molecular
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framework. Interestingly, NO2-containing compounds show much
reduced fluorescence in EtOH (top panel Fig. 8). We may conclude
that fluorescence from dansyl-appended calix[4]arenes clearly
demonstrate excited-state interaction between dansyl moieties,
and further that the fluorescence from NO2/Dan1 and NO2/Dan4
show different trends from those shown by other compounds.
These differences lend further support to the observation of
selective chemosensing of Hg2+ by NO2/Dan4.
Spectral response from appropriate blanks was subtracted before
data analysis. All the measurements were taken in triplicate
and averaged. Dilution correction was also carried out where
necessary. Melting points were determined on an electrothermal
melting point apparatus obtained from M/S Toshniwal and were
uncorrected.
Synthesis of starting material
p-Tert-butylcalix[4]arene (1), calix[4]arene (2), 5,11,17,23-
tetra(p-tert-butyl)-25,27-di(ethoxycarbonylmethoxy)-26,28-dihy-
droxy-calix[4]arene (1a), 25,27-di(ethoxycarbonylmethoxy)-
26,28-dihydroxy-calix[4]arene (2a), and 5,11,17,23-tetranitro-
25,26,27,28-tetrahydroxy-calix[4]arene (3) were synthesized as
described previously.18,19 The analytical data for compounds 1, 2,
1a, 2a, and 3 were found to be same as reported earlier.18,19
Conclusion
Among the six novel dansyl-appended calix[4]arene frameworks
bearing two and four dansyl groups, fluorescence of one of
the compounds, NO2/Dan4, is demonstrated to be remarkably
sensitive to the presence of Hg2+ in the solution; the detection
limit is estimated to be ~20 ppb. The compound shows high
selectivity and adequate reversibility as a chemosensing agent for
Hg2+. The reason for this recognition behavior is proposed to be
the 1,3-alternate conformation of NO2/Dan4 as compared to the
cone conformation of all other dansyl-appended calix[4]arenes.
The stoichiometry of NO2/Dan4 : Hg2+ complexation is observed
to be 2:1 with a formation constant of 5.2( 0.8) ¥ 1010 M-2
at ambient conditions. Stokes shift measurements of dansyl-
appended calix[4]arenes indicate the NO2/Dan4 cybotactic region
to be the most sensitive, as the change in the dipole moment
from ground state to excited state is found to be maximum
for this compound. The fluorescence intensity of NO2/Dan4 in
chloroform, acetonitrile, and ethanol is observed to show different
trends from other dansyl-appended calix[4]arenes. In conclusion,
the synthesis, characterization, and interesting properties of novel
dansyl-appended calix[4]arene molecular frameworks have been
established for their potential utilization in various chemical
applications.
General procedure for the synthesis of dansylated calix[4]arene
derivatives
To a solution of corresponding calix[4]arene (1, 2, 1a, 2a, and 3)
in MeCN (20mL), Et3N and dansyl chloride (equimolar to per
hydroxyl group) were added under N2 atmosphere with vigorous
stirring. The reaction mixture was stirred at room temperature for
96 hours. In most of the cases a light yellow precipitate formed,
that was filtered, redissolved in CHCl3, and washed with water
and extracted with CHCl3. The organic layer was collected and
evaporated to dryness under reduced pressure to yield dansylated
calix[4]arene derivatives as yellow solid that was further purified by
column chromatography and recrystallized from CHCl3/CH3OH.
Compound t-Bu/(OH)2Dan2. Pale yellow solid, yield: 66%,
mp 231–233 ◦C; UV (lmax, CH3CN): 272, 355 nm. IR (KBr
pellet, cm-1): 3571, 1578, 1365, 1307, 1262, 1190, 1083. 1H NMR
(300 MHz, CDCl3, d in ppm): 8.59 (2H, d, Dan-H), and 8.47
(2H, d, Dan-H), 8.14 (2H, d, Dan-H), 7.54 (4H, m, Dan-H),
7.14 (2H, d, Dan-H), 6.86 and 6.52 (8H, 2 s, Ar-H), 3.85 (d, 4H,
ArCH2Ar), 2.92 (d, 4H, ArCH2Ar), 2.82 (s, 12H, N(CH3)2), 1.17,
0.74 (2 s, 36H, C(CH3)3), 1.49 (bs, 2H, OH, D2O exchangeable).
13C NMR (75 MHz, CDCl3, d in ppm): 151.3, 149.9, 149.0, 142.3,
133.0, 131.8, 129.9, 129.3, 127.9, 125.9, 125.0, 122.8, 119.9, 115.9,
45.3, 32.4, 31.5. HRMS (ESI-TOF) m/z: calcd. 1114.5200, found
1114.5498 [M+].
Experimental
Materials and instrumentation
All the reagents used in the study were purchased from Sigma–
Aldrich or Merck and were chemically pure. The solvents used
were dried and distilled. HPLC grade solvents were used for flu-
orescence spectroscopic measurements. Column chromatography
was performed on silica gel (60–120 mesh) obtained from Merck.
1H and 13C NMR spectra were recorded on a 300 MHz Bruker
DPX 300 instrument using tetramethylsilane (TMS) at 0.00 as
Compound H/(OH)2Dan2. Pale yellow solid, yield: 56%, mp
271–272 ◦C; UV (lmax, CH3CN): 272, 356 nm. IR (KBr pel-
1
let, cm-1): 3549, 1577, 1365, 1311, 1243, 1184, 1063. H NMR
1
an internal standard. Low temperature H NMR spectra were
(300 MHz, CDCl3, d in ppm): 8.67 (2H, d, Dan-H), and 8.65 (2H,
d, Dan-H), 8.18 (2H, d, Dan-H), 7.67 (4H, m, Dan-H), 7.22 (2H,
d, Dan-H), 6.93 (d, 4H, Ar-H), 6.64 (8H, m, Ar-H), 3.95 (d, 4H,
ArCH2Ar), 3.03 (d, 4H, ArCH2Ar), 2.89 (s, 12H, N(CH3)2), 1.57
(bs, 2H, OH, D2O exchangeable). 13C NMR (75 MHz, CDCl3,
d in ppm): 151.3, 149.9, 149.0, 142.3, 133.8, 132.1, 130.2, 129.4,
129.0, 128.6, 126.9, 122.9, 119.5, 116.0, 45.3, 31.5; HRMS (ESI-
TOF) m/z: calcd. 891.2774, found 891.2812 [M+].
also recorded on the same instrument as mentioned above. FAB
mass spectra were recorded on a JEOL SX 102/DA-6000 Mass
spectrometer/Data System using Argon/Xenon (6 kV, 10 mA)
as the FAB gas while IR spectra were recorded on a Nicolet
Prote´ge´ 460 spectrometer in KBr disks. Fluorescence spectra were
acquired on a spectrofluorometer purchased from Horiba-Jobin
Yvon, Inc. model FL 3–11 Fluorolog-3 modular spectrofluorom-
eter with single Czerny-Turner grating excitation and emission
monochromators having 450 W Xe arc lamp as the excitation
source and PMT as the detector. A Perkin-Elmer Lambda bio-
20 double beam spectrophotometer with variable bandwidth was
used for acquisition of the uv-vis molecular absorbance. All
the data were acquired using 1-cm2 path length quartz cuvettes.
Compound H/Dan4. Pale yellow solid, yield: 70%, mp 260–
◦
262 C; UV (lmax, CH3CN): 272, 339 nm. IR (KBr pellet, cm-1):
1575, 1366, 1310, 1184, 1133, 1061. 1H NMR (300 MHz, CDCl3,
d in ppm): 8.84 (4H, d, Dan-H), 8.81 (4H, d, Dan-H), 8.35 (4H, d,
Dan-H), 7.84 and 7.71 (8H, t, Dan-H), 7.38 (4H, d, Dan-H), 7.09
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(d, 4H, Ar-H), 6.83 (m, 8H, Ar-H), 4.10 (d, 4H, ArCH2Ar), 3.19
(d, 4H, ArCH2Ar), 3.06 (s, 24H, N(CH3)2). 13C NMR (75 MHz,
CDCl3, d in ppm): 152.5, 151.5, 144.7, 133.9, 132.1, 130.2, 129.8,
129.4, 129.0, 128.6, 128.4, 126.9, 122.9, 119.6, 116, 45.4, 31.6
HRMS (ESI-TOF) m/z: calcd.1356.3717, found 1356.3727 [M+].
N(CH3)2). 13C NMR (75 MHz, CDCl3, d in ppm): 152.3, 149.8,
144.7, 134.9, 133.7, 130.9, 130.3, 123.2, 122.6, 119.8, 116.2, 45.7;
HRMS (ESI-TOF) m/z: calcd. 327.5991, found 327.5912 [M+].
Compound NO2/Dan1. Pale yellow solid, yield: 85%, mp 98–
◦
100 C; UV (lmax, CH3CN): 272, 356 nm. IR (KBr pellet, cm-1):
1577, 1522, 1345, 1315, 1235, 1175, 1101, 1065. 1H NMR
(300 MHz, CDCl3, d in ppm): 8.57 (H, d, Dan-H), 8.36 (H, d,
Dan-H), 8.03 (2H, d, Ar-H), 7.65 (2H, t, Dan-H), 7.39 (2H, t,
Dan-H), 7.10(2H, d, Dan-H), 7.19 (2H, d, Ar-H), 2.84 (s, 6H,
N(CH3)2).13C NMR (75 MHz, CDCl3, d in ppm): 153.9, 152.1,
146.0, 132.6, 131.4, 129.4, 125.2,122.8, 118.8, 115.8, 45.3; HRMS
(ESI-TOF) m/z: calcd. 373.5991, found 373.5901 [M+].
Compound t-Bu/(Ester)2Dan2. Pale yellow solid, yield: 60%,
mp 210–211 ◦C; UV (lmax, CH3CN): 272, 348 nm. IR (KBr
pellet, cm-1): 1761, 1581, 1362, 1304, 1184, 1122, 1065. 1H NMR
(300 MHz, CDCl3, d in ppm): 8.62 (2H, d, Dan-H), 8.23 (4H,
m, Dan-H), 7.55 (2H, t, Dan-H), 7.39 (2H, t, Dan-H), 7.12 (2H,
d, Dan-H), 6.87 and 6.26 (8H, 2 s, Ar-H), 4.72 (s, OCH2CO),
4.26 (q, 4H, OCH2CH3), 4.23 (d, 4H, ArCH2Ar), 2.88 (s, 12H,
N(CH3)2), 2.62 (d, 4H, ArCH2Ar), 1.35 (t, 6H, CH3), 1.23, 0.73
(2 s, 36H, C(CH3)3). 13C NMR (75 MHz, CDCl3, d in ppm): 169.7,
153.0, 151.2, 147.6, 145.4, 142.6, 133.18, 132.3, 131.3, 129.4, 128.3,
125.9, 124.9, 123.2, 119.8, 115.1, 70.13, 60.5, 45.3, 32.3, 31.5,
14.2; HRMS (ESI-TOF) m/z: calcd 1309.5933, found 1309.5298
[M+ + Na].
Acknowledgements
Shubha Pandey thanks CSIR, India for Senior Research fellow-
ship. Help from Mr. Yogesh Kumar with HRMS data collection
and analysis is greatly appreciated. Mr. Munna Lal is recognized
for his support in NMR data acquisition. Siddharth Pandey
thanks Royal Society of Chemistry for Travel Award for Inter-
national Authors (2007).
Compound H/(Ester)2Dan2. Pale yellow solid, yield: 45%,
mp 222–224 ◦C; UV (lmax, CH3CN): 272, 347 nm. IR (KBr
pellet, cm-1): 1710, 1577, 1366, 1310, 1259, 1185, 1067. 1H NMR
(300 MHz, CDCl3, d in ppm): 8.65 (2H, d, Dan-H), 8.25 (2H, d,
Dan-H), 8.09 (2H, d, Dan-H), 7.53 (4H, m, Dan-H), 7.19 (2H, d,
Dan-H), 6.74–6.27 (12H, m, ArH), 3.94–3.69 (8H, m, OCH2CH3
and OCH2CO), 3.36 (d, 4H, ArCH2Ar), 2.89 (s, 12H, N(CH3)2),
2.52 (d, 4H, ArCH2Ar), 1.25 (t, 6H, CH3), 13C NMR (75 MHz,
CDCl3, d in ppm): 169.4, 165.6,156.7, 151.4, 145.4, 135.9, 134.5,
132.3, 131.6, 130.8, 129.5, 128.7, 127.7, 125.3, 123.2, 122.7, 119.8,
115.4, 70.5, 60.8, 45.4, 31.9, 14.2; HRMS (ESI-TOF) m/z: calcd.
1062.7941, found 1062.7570 [M+].
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Compound t-Bu/Dan1. Pale yellow solid, yield: 80%, mp 95–
97 ◦C; UV (lmax, CH3CN): 272, 352 nm. IR (KBr pellet, cm-1):
1575, 1368, 1302, 1263, 1150, 1065, 1020. H NMR (300 MHz,
CDCl3, d in ppm): 8.54 (H, d, Dan-H), 8.51 (H, d, Dan-H), 8.04
(H, d, Dan-H), 7.61 (2H, t, Dan-H), 7.41 (2H, t, Dan-H), 7.10(H,
d, Dan-H), 7.19(2H, d, Ar-H), 6.74 (2H, d, Ar-H), 2.84 (s, 6H,
N(CH3)2), 1.15 (s, 9H, C(CH3)3).13C NMR (75 MHz, CDCl3, d
in ppm): 151.4, 149.6, 147.0, 131.4, 130.7, 129.3, 126.0, 122.6 121.6,
119.2, 115.2, 45.1, 34.1; HRMS (ESI-TOF) m/z: calcd. 406.1453,
found 406.1446 [M++ Na].
1
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◦
101 C; UV (lmax, CH3CN): 272, 363 nm. IR (KBr pellet, cm-1):
1
1577, 1365, 1310, 1265, 1143, 1065, 1020. H NMR (300 MHz,
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(H, d, Dan-H), 7.56 (2H, t, Dan-H), 7.35 (2H, t, Dan-H), 7.09(H,
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