Inhibition of the prototropic tautomerism in chrysazine by p-sulfonatocalixarene hosts

Article abstract of DOI:10.1039/c8ob00978c

This study explores the interesting effect of p-sulfonatocalix[n]arene hosts (SCXn) on the excited-state tautomeric equilibrium of Chrysazine (CZ), a model antitumour drug molecule. Detailed photophysical investigations reveal that conversion of CZ from its more dipolar, excited normal form (N*) to the less dipolar, tautomeric form (T*) is hindered in SCXn-CZ host-guest complexes, which is quite unexpected considering the nonpolar cavity of the hosts. The atypical effect of SCXn is proposed to arise due to the partial inclusion or external binding of CZ with the hosts, which facilitates H-bonding interactions between CZ and the sulfonate groups present at the portals of the hosts. The intermolecular H-bonding subsequently leads to weakening of the pre-existing intramolecular H-bond network within CZ, and thus hinders the tautomerizaion process. Our results suggest that rather than the binding affinity, it is the orientation of CZ in the SCXn-CZ complexes, and its proximity to the portals of the host that plays a predominant role in influencing the tautomeric equilibrium. These observations are supported by quantum chemical calculations. Thermodynamic studies validate that SCXn-CZ interaction is essentially enthalpy driven and accompanied by small entropy loss, which is consistent with the binding mechanisms.

Full text of DOI:10.1039/c8ob00978c

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Inhibition of the Prototropic Tautomerism in Chrysazine by p-
Sulfonatocalixarene Hosts
Received 00th January 20xx,
Accepted 00th January 20xx
a
b
a,b,*
a,b,*
Poojan Milan Gharat, Dilip Kumar Maity, Haridas Pal, and Sharmistha Dutta Choudhury
DOI: 10.1039/x0xx00000x
This study explores the interesting effect of p-sulfonatocalix[n]arene hosts (SCXn) on the excited-state tautomeric
equilibrium of Chrysazine (CZ), a model antitumour drug molecule. Detailed photophysical investigations reveal that
conversion of CZ from its more dipolar, excited normal form (N*) to the less dipolar, tautomeric form (T*) is hindered in
SCXn-CZ host-guest complexes, which is quite unexpected considering the nonpolar cavity of the hosts. The atypical effect
of SCXn is proposed to arise due to the partial inclusion or external binding of CZ with the hosts, which facilitates H-
bonding interactions between CZ and the sulfonate groups present at the portals of the host. The intermolecular H-
bonding subsequently leads to weakening of the pre-existing intramolecular H-bond network within CZ, and thus hinders
the tautomerizaion process. Our results suggest that rather than the binding affinity, it is the orientation of CZ in the SCXn-
CZ complexes, and its proximity to the portals of the host that plays a predominant role in influencing the tautomeric
equilibrium. These observations are supported by quantum chemical calculations. Thermodynamic studies validate that
SCXn-CZ interaction is essentially enthalpy driven and accompanied by small entropy loss, which is consistent with the
binding mechanisms.
www.rsc.org/
2
,22
molecules. In many instances, proton transfer is found to be
more favourable in the excited state than in the ground state,
due to modulation in the acid-base characteristics of
Introduction
Prototropic tautomerism refers to the dynamic equilibrium
between two stable forms (tautomers) of a molecule by the
repositioning or movement of proton(s) from one molecular
2
-5,13,16-25
molecules upon electronic excitation.
Being an essential process in various biochemical systems,
prototropic tautomerism of many different kinds of molecules
1
-5
site to another. This phenomenon plays important roles in
numerous biological processes like, enzymatic catalysis, drug
5
,16,19
are extensively studied in both homogeneous
and
. The effects of
and additives like, metal
2
0,21,26-30
6
-9
microheterogeneous solvent media
35,36
activity and nucleic acid chemistry. The ability of DNA bases
to tautomerize is well-recognized to be the origin of
5
,31,32
33,34
solvent,
2
ions
temperature,
pH,
3,37,38
18,39-41
9
-11
and macrocycle cavitands,
are investigated to
spontaneous point mutations.
Since tautomerization is
determine the factors that can influence tautomeric equilibria,
for their better applications. The studies with macrocyclic
cavitands are especially significant, because host molecules
can trap suitable prototropic guest molecules within their
cavity and thus mimic the confined pockets existing in
often accompanied by changes in optical properties, this
process also finds applications in various fields of science and
technology such as, optical switches, laser technology, sensing
3
,12-15
and molecular data processing.
The necessary criterion for
a
molecule to depict
4
2,43
biomolecular assemblies.
Understanding the effect of host-
prototropic tautomerism is that it should possess both proton
donating and proton accepting substituents. The proton-
donating and accepting groups may be vicinal and
intramolecularly H-bonded, in which case the relocation of the
guest interactions on tautomeric equilibria provides a means
to visualize how biological environments are able to selectively
transform one tautomer form of a molecule to another or
3
,4,16-18 choose the desired tautomeric form, by inducing or inhibiting
the tautomerization process.
proton occurs directly through the pre-existing H-bond.
Alternatively, the two substituents may be distal, in which case
the proton relocation is mediated through solvent H-bonded
This paper describes the effect of two p-
sulfonatocalix[n]arene hosts on the prototropic tautomerism
of a model anthracycline antitumour drug, 1-8-dihydroxy-9-10-
anthraquinone or Chrysazine (CZ), based on ground state
absorption, steady-state fluorescence and time-resolved
fluorescence measurements. The photophysics of CZ is quite
interesting because it has two equivalent proton donating
hydroxyl groups that are intramolecularly H-bonded from
either side with the central proton accepting carbonyl group
5
,19-21
bridges
or through doubly H-bonded dimers of the
a
Radiation & Photochemistry Division, Bhabha Atomic Research Centre, Mumbai
00085, India.
4
b
Homi Bhabha National Institute, Training School Complex, Anushaktinagar,
Mumbai 400094, India.
E-mail: hpal@barc.gov.in, sharmidc@barc.gov.in
†Electronic Supplementary Information (ESI) available: Figs. S1-S8 Notes S1, S2
and Tables S1-S3. See DOI: 10.1039/xxxxxxxxxx
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4
,44-47
(Scheme 1).
In the ground state, CZ exists exclusively in a the hosts, as a result of which there is a strong tendency for
single tautomeric form that is designated as the normal form intermolecular H-bonding interactions between the hydroxyl
(
N). However, upon photoexcitation in the first excited singlet groups of CZ and the sulfonate groups present at the portals of
) state, the dye undergoes rapid proton transfer from either the host, and a subsequent weakening of the pre-existing
(S
1
of the two hydroxyl groups to the carbonyl group, to yield the intramolecular H-bond network of CZ. These propositions are
4
,44-47
excited tautomer form (T*).
In accordance with this well supported by FTIR and quantum chemical calculations.
mechanism, the absorption spectrum of CZ shows the Our study further reveals that the SCXn-CZ host-guest
signature of a single species (the normal or N form), whereas interaction is in essence an enthalpy driven process and
the steady-state emission spectrum shows two distinct accompanied by a small overall loss in entropy.
emission bands. The lower wavelength emission band (LWEB)
represents the excited state of the normal form (N*) and the
Experimental
higher wavelength band (HWEB) represents the excited
tautomeric form (T*). Due to the strong pre-existing
intramolecular H-bonding in CZ, its excited state
tautomerization is reported to be very facile and efficient. The
prototropic tautomerism remains operative in a variety of
The dye, CZ, was procured from Sigma-Aldrich and was
recrystallised from acetonitrile. The hosts, SCX4 and SCX6,
were purchased from TCI Mark, Tokyo and were used as
received. A concentrated stock solution of CZ was prepared in
ethanol and small aliquots of this stock solution were added to
4
,44-47
solvents and under different temperature conditions.
water for preparing aqueous solutions of CZ with
a
concentration around 3 μM. The interaction of CZ with SCX4
and SCX6 was studied by adding different weighed amounts of
the corresponding hosts to the aqueous solution of CZ.
H
H
O
O
O
−
1
Nanopure water with a conductivity of less than 0.1 μS cm
was obtained from a Millipore Elix-3/A10 water purification
system. The solutions were freshly prepared prior to the
experiments. The slight decrease in the pH of the solutions on
addition of the SCXn hosts was disregarded in the present
study, because pH change within the range of 3-8 does not
have any effect on the absorption and emission spectral
characteristics of the dye (Fig. S1, ESI†). Hence, any spectral
changes of CZ in the presence of the hosts are attributed to
specific host-guest interactions.
O
Chrysazine (CZ)
N form)
(
Scheme 1 Molecular structures of the hosts (SCX4 and SCX6) and the guest, Chrysazine
CZ).
(
The two host molecules used in the present work, namely
p-sulfonatocalix[4]arene (SCX4) and p-sulfonatocalix[6]arene
SCX6), are homologues of the p-sulfonatocalix[n]arene series
Absorption spectra were recorded with a Jasco UV-vis
spectrophotometer (model V-650). Steady-state fluorescence
spectra were obtained with a spectrofluorimeter (model FS5,
(
of macrocycles. These cup shaped container molecules are
comprised of p-hydroxybenzenesulfonate units linked with
8-50
4
Edinburgh
Instruments).
Time-resolved
fluorescence
methylene groups (Scheme 1).
They have an interior
measurements were carried out using a time-correlated single
photon counting (TCSPC) instrument (Horiba Jobin Yvon, UK),
where samples were excited by the light pulses from a nano-
LED source (406 nm; repetition rate: 1 MHz) and the
fluorescence was detected using a PMT based detection
module (model TBX4). The instrument response function (IRF)
of the present setup is about 185 ps. Measurements were
carried out at magic angle configuration to eliminate the
contribution of the rotational depolarization of CZ on the
observed fluorescence decays. The fluorescence decay traces
were analyzed by the reconvolution method, considering
mono-exponential function. The quality of the fit was judged
hydrophobic cavity with two asymmetrical hydrophilic portals.
Their high water solubility, flexible π-electron rich cavities and
ability to provide anchoring points of sulfonate groups at one
of the portals, endows them with versatile complexation
4
2,48-53
properties for different guest molecules.
The host-guest
binding in these systems occur primarily due to favorable
electrostatic, hydrophobic, H-bonding, π-stacking and CH-π
4
8,51
interactions.
constitutions but differ in their cavity dimensions,
Since SCX4 and SCX6 have similar chemical
51,54,55
these
two hosts were particularly chosen to determine whether
cavity size has any influence on the binding interaction and
consequently on the prototropic tautomerism of the guest, CZ.
Our results show that both SCX4 and SCX6 form complexes
with CZ and also inhibit the otherwise facile excited state
tautomerization of CZ. Surprisingly, it is observed that
although SCX6 (which is the macrocycle with the larger cavity
size) has a relatively weaker binding affinity with CZ as
compared to SCX4, the former host is capable of inhibiting the
prototropic tautomerism of CZ to a much larger extent than
the latter host. All of these results are rationalized by
considering the partial inclusion or external binding of CZ with
2
by the reduced chi-square (χ ) value and the distribution of the
weighted residuals among the data channels. For a good fit,
2
the χ value was close to unity and the weighted residuals
5
6
were distributed randomly among the data channels. FTIR
spectra were recorded with an IR-Affinity-1 Spectrometer
(Shimadzu), using diamond ATR setup.
Unless otherwise stated, all measurements were carried
out at ambient temperature, 25 ± 1°C. For the binding studies
at different temperatures, a temperature controller (Quantum
2
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4
,44,46
Northwest TC125) was used to adjust the temperature of the normal form (N) of CZ.
solutions (within ± 1°C).
Quantum chemical calculations were carried out using the along with a slight red shift in the absorption maximum, and
With increase in the host
concentration, there is marginal increase in the absorbance
5
7
general ab initio quantum package, Gaussian 16. The gradual saturation in the absorption changes, for both SCX4-CZ
minimum energy ground-state structures of the hosts, SCX4 and SCX6-CZ systems. The changes in the absorption features
and SCX6, the guest, CZ, as well as those of the 1:1 host-guest are indicative of interaction between the dye and the hosts.
complexes were determined after considering several initial However, as the changes in the absorption characteristics are
guess geometries, for optimization under isolated gas phase not that large, these were not used further for any
condition. The optimized gas phase structures were allowed to quantitative estimation of binding affinities.
relax further in water medium applying a macroscopic solvent
1
0
0
.8
.9
.0
1
1
(A)
model. The model considers a self-consistent reaction field
SCRF) method based on solute electron density (SMD). All
3
2
1
0000
0000
0000
0
T*
(
electronic structure calculations were carried out applying
B97D density functional and adopting Dunning correlated
atomic basis functions, namely, cc-pVDZ as well as Pople style
basis set, 6-311++G(d,p). The DFT functional considered in the
present study is known to take into account the long range
dispersion correction, which plays a crucial role in shaping the
structures of host-guest complexes.
3
10
N*
500 600 700
Wavelength (nm)
500
600
700
Results and discussion
Wavelength (nm)
(
A)
1.8
T*
1
0.03
0.02
0.01
0.00
1
(B)
30000
20000
10000
0
5
0
0
.9
.0
9
3
N*
500 600 700
Wavelength (nm)
1
400
450
500
Wavelength (nm)
5
00
600
700
Wavelength (nm)
0
.03
.02
.01
.00
(B)
Fig. 2 (A) Emission spectra of CZ (3 µM) in water with 0, 2.0, 3.4, 4.4, 6.7, 9.4, 12.5,
7.2, 23.3 and 31.0 mM SCX4 (1-10). Inset shows the fluorescence intensity of CZ
5
1
normalized at 515 nm in the presence of 0, 17.2 and 31.0 mM SCX4. (B) Emission
spectra of CZ (3 µM) in water with 0, 1.0, 2.2, 3.2, 5.3, 8.1, 11.2, 15.3 and 20.5 mM
SCX6 (1-9). Inset shows the fluorescence intensity of CZ normalized at 515 nm in the
presence of 0, 11.2 and 20.5 mM SCX6. Excitation wavelength was 415 nm.
0
1
0
0
Considering that fluorescence is a more sensitive
parameter than absorbance, we next investigated the
fluorescence characteristics of CZ in the presence of the hosts.
It may be mentioned that in many previous studies on host-
guest interactions, considerable changes have been noticed in
the emission characteristics of the dyes, although under similar
4
00
450
500
Wavelength (nm)
Fig. 1 Absorption spectra of CZ (3 µM) in water with increasing host concentrations; (A) conditions, the changes in the absorption characteristics may
4
9,51,58-60
SCX4, (1-5)/mM: 0, 5.5, 10.5, 21, 26 and (B) SCX6, (1-5)/mM: 0, 4.6, 7.6, 15.2, 18.
not be that significant.
Based on the changes in the
fluorescence characteristics, especially that in the intensities,
-1
2
7
Fig. 1 shows the absorption spectra of CZ in water with binding affinities over a wide range (~10 -10 M ) have been
4
9,59-63
increasing concentrations of the hosts, SCX4 and SCX6. The reliably estimated for a variety of host-guest systems.
absorption spectrum of CZ in aqueous solutions has a peak at
The steady-state fluorescence spectra of CZ in aqueous
30 nm. According to previous literature reports, this solution, with increasing concentration of SCX4 and SCX6, are
4
absorption band is ascribed to the S
0
to S
1
transition of the presented in Figs. 2A and 2B, respectively. In pure water, CZ
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shows a weak lower wavelength emission band (LWEB) at intensity with increasing polarity, which is in accordance with
around 515 nm and a strong higher wavelength emission band the greater dipolar character of the N* form of CZ. Therefore,
(HWEB) with maximum at 585 nm. As mentioned before, the based on a comparison of the dipolar natures of the two
LWEB is due to the emission from excited-state normal form tautomer forms, it was anticipated that the encapsulation of
*
N ) while, the HWEB is attributed to the excited-state CZ within the hydrophobic cavity of SCXn hosts would facilitate
(
*
tautomeric form (T ) of the dye.
4,44,46
its tautomerization from the more polar N* form to the
On addition of the SCXn hosts, two kinds of changes are relatively less polar T* form. That the SCXn-CZ systems behave
observed in the emission spectra of CZ. First, the emission in an opposite manner and hinder the prototropic
intensity is gradually quenched with increasing concentration tautomerism of the bound dye from the N* to T* form, is an
of the hosts. Second, the nature of the fluorescence spectra of interesting deviation from the normal expectation.
CZ systematically changes such that at the highest
Excitation spectra were recorded to understand the origin
concentration of the hosts, the emission contribution of HWEB of the two emission bands, LWEB (515 nm) and HWEB (585
*
T emission), which is initially much stronger than the LWEB nm) for the bound CZ dye. As in the case of free CZ, for the
(
*
N emission) becomes almost at par with LWEB.
(
SCXn-CZ systems as well, a single excitation band is observed,
The decrease in the fluorescence intensity of CZ with which closely resembles the absorption spectrum (Fig. S4,
increasing concentration of SCXn is justified, because the ESI†). This indicates that for both CZ and SCXn-CZ, the
quinoid moiety of CZ is a good electron acceptor, while tautomeric forms (N* and T*) arise from the same initially
phenoxy moieties of the hosts are known to be good electron excited normal form of the dye.
donors. Consequently, the host can quench the fluorescence To decipher the mechanism by which the SCXn hosts affect
intensity of the dye due to electron transfer or charge transfer the prototropic tautomerism of CZ in such a unique manner, so
4
9,51
type of interactions.
as to inhibit the transformation of N* to T*, we looked into the
In accordance with the decrease in the steady-state binding affinities of CZ with SCX4 and SCX6. The binding
fluorescence intensity, we also observed a gradual decrease in constants for the SCXn-CZ systems were calculated by
the fluorescence decay time of CZ with increasing considering the changes in the fluorescence intensity of CZ at
concentration of SCXn. Representative decay traces for the both the LWEB (515 nm) and the HWEB (585 nm), with varying
*
SCX4-CZ system monitored at both LWEB (515 nm, N
host concentrations. The host-guest binding stoichiometry was
*
emission) and HWEB (585 nm, T emission) are shown in Fig. considered to be 1:1, as indicated from the inflection observed
S2, ESI†. The corresponding fluorescence decay times are at 0.5 mole fraction of the host in the Job plots for the SCX4-CZ
presented in Table S1, ESI†. It may be mentioned that the de- and SCX6-CZ systems (Fig. S5, ESI†), and from the geometry
excitation rates for both N* and T* forms of the dye are quite optimization studies (discussed later). The formation of 1:1
similar (τ
equilibrium between the two forms of the excited dye at the (Fig. S6, ESI†).
time scales probed by TCSPC measurements (Note S1, The host-guest complex formation of SCXn with CZ can be
expressed as,
f
=0.29 ns for the free dye), indicating a kinetic SCXn-CZ complexes is also reasonably supported by ESI-MS
4
,44,46
ESI†).
K
eq
Apart from the fluorescence quenching, the change in the
relative intensities of the LWEB and HWEB in the emission
spectrum of CZ (cf. Fig. 2), is quite an interesting observation.
C
N +H
N
(1)
where H represents the SCXn host, N is the normal form of CZ
It suggests that the existing equilibrium between the N* and (which is the only form that exists in the ground state) and CN
T* forms of CZ is disturbed when the dye is bound to the SCXn
hosts. More specifically, the emission contribution of the T*
form (HWEB) is observed to be lower for the SCXn-CZ systems
as compared to CZ alone. This is illustrated more clearly in the
insets of Fig. 2, which depict the representative emission
is the normal form of the complexed dye in the ground state. If
is not very large and the host concentration, [H] , is
K
eq
0
much higher than the dye concentration, [N] , then one can
0
assume that the equilibrium host concentration in the solution
spectra of the SCXn-CZ systems with their intensities is effectively the same as the actually added host
normalized at the LWEB (N* form). It can be seen that with concentration, i.e., [H]≈[H] . In this situation, the equilibrium
increasing SCXn concentration, as the population of SCXn-CZ constant can be expressed as,
0
complexes increases in the solution, there is a noticeable
decrease in the relative contribution of the T* form (HWEB)
over the N* form. It is thus evident that the binding interaction
of SCXn with CZ hinders the formation of T* from N*.
[
C ]
N
K
=
(2)
eq
[N][H]
0
Further, considering that excited-state lifetimes of guest dyes
are generally much shorter (sub-ns for CZ, cf. Table S1, ESI†)
than the exchange rate between the free and host-bound
According to literature reports, the N* form of CZ has a
,44,64
4
higher dipole moment than the T* form.
This is also
6
5
forms of the dye (occurs in microseconds), the fluorescence
intensity at any host concentration is a composite of the
fluorescence intensity contributions from the complexed and
uncomplexed forms of the dye. Accordingly, the fluorescence
intensity of CZ at 515 nm can be expressed as,
evident from the emission spectra of CZ recorded in solvents
of different polarities (Fig. S3, ESI†). It is clear that while the
position of the HWEB (T* form) remains unaffected by the
polarity of the solvent medium, the LWEB (N* form) shows a
systematic redshift along with marginal increase in the relative
4
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N]
ARTICLE
[
[C ]
∞
N
[N ]
0
fluorescence intensity changes of either the normal or the
tautomer forms of CZ), are quite similar within experimental
error. This result is expected because only the normal form of
the dye exists in the ground state, and therefore the binding of
SCXn occurs exclusively with the normal form. It is only in the
excited state that the free and the bound forms of CZ convert
to the corresponding tautomeric forms.
0
I
=I
+I
N
(3)
N(obs)
N
[
N ]
0
0
Where
I
is the initial fluorescence intensity of the neutral
N
∞
form of CZ in the absence of the host and
I
is the
N
extrapolated fluorescence intensity when the neutral form is
completely complexed with SCXn (Note S2, ESI ). The change
†
Table 1 Binding parameters for the interaction of CZ with the SCXn hosts.
in the fluorescence intensity at 515 nm, ꢀIN , can thus be
0
T
0
N
a
∞
T
∞
b
/ I
system
parameters
parameters
I
/ I
I
correlated with the equilibrium constant as,
N
obtained by fitting obtained by fitting
K
[H]
0
∞
eq
+Keq[H]0
to eq. 4
to eq. 5
ꢀ
I
= ꢀI
(4)
N
N
1
-1
-1
SCX4-CZ
SCX6-CZ
K
eq = 135 M
K
eq = 118 M
2.1
2.1
0.6
0.3
∞
∞
∞
ꢀI
= -10038 (arb. ꢀI = -27117 (arb.
Where, ꢀI is the extrapolated difference in the emission
N
T
N
unit)
unit)
intensity at 515 nm in the presence of zero and infinite host
-1
-1
K
eq = 79 M
K
eq = 81 M
∞
∞
concentrations (Note S2, ESI
†
Similarly, the fluorescence intensity changes at 585 nm
).
ꢀI = -2878 (arb.
ꢀI = -12038 (arb.
N
T
unit)
unit)
a
b
corresponding to the tautomer form, ꢀIT , can be correlated
Calculated from the experimental data for the free dye Calculated from fitted
parameters for the SCXn-CZ complexes.
K
[H]
0
as, ꢀIT = ꢀI ∞T 1
eq
+Keq[H]0
(5)
In the present host-guest systems, in addition to the
binding equilibrium, the two other equilibria that exist due to
the prototropic tautomerism of the free and bound forms of
excited CZ are expressed as follows.
∞
where, ꢀI is the extrapolated difference in the emission
T
intensity at 585 nm in the presence of zero and infinite host
concentrations (Note S2, ESI
†
).
KT
N
T
(6)
(7)
0
0
4000
8000
(A)
(B)
K'
T
C
C
ꢀ
-10000
N
T
-
-
Here
K
and K' are the equilibrium constants for the
T
T
-
20000
transformation of the excited normal form,
T∗
N∗
, to the excited
0
.00
0.01
[
0.02
0.03
C)
0.00
0.01
0.02
0.03
tautomer form,
respectively.
, of free CZ and SCXn-CZ complex,
SCX4] (M)
[SCX4] (M)
0
-4000
-8000
0
1000
2000
(
(D)
Considering that the absorption spectrum of CZ does not
change to a very large extent in the presence of the hosts (cf.
Fig. 1) and assuming that both N* and T* forms of CZ are
-
-
0 0
I
equally quenched by SCXn, it can be derived that
K
∝I
T
T N
0.00
0.01
0.02
0.00
0.01
0.02
'
∞ ∞
I
(Note S2, ESI
and K ∝I
†). From the experimentally
T
T
N
[
SCX6] (M)
[SCX6] (M)
observed intensity data and the fitted parameters (Table 1),
Fig. 3 The binding isotherms for SCX4-CZ system following the variation in the
0
0
value for free dye is determined to be about 2.1,
fluorescence intensity at, (A) 515 nm (B) 585 nm, and for SCX6-CZ system following the the
I
I
T
N
variation in the fluorescence intensity at, (C) 515 nm (D) 585 nm.
∞
∞
values for SCX4-CZ and SCX6-CZ systems are
while the
I
I
T
N
Figs. 3A and B show the variations in the fluorescence
intensity of SCX4-CZ complex at the LWEB (515 nm) and HWEB
determined to be about 0.6 and 0.3, respectively. These values
provide approximate estimates for the tautomerization
equilibria of the free and bound dye, and confirm that when CZ
is bound to the SCXn hosts, the transformation of the N* form
of the dye to the T* form is indeed hindered very significantly.
Surprisingly, it is found that although the binding constant of
(585 nm) with increasing SCX4 concentration, along with the
fitted curves according to equation (4) and equation (5),
respectively. Similar plots for the SCX6-CZ system are shown in
Figs. 3C and D. Reasonably good fits are obtained in all the
cases. The fitted parameters for both the systems are
presented in Table 1.
-1
SCX4-CZ system is much higher (Keq~127±9 M ; average of the
values obtained from equations 4 and 5, cf. Table 1) than that
-1
of SCX6-CZ (K =80±2 M ; average of the values obtained from
It may be noted that the values of the binding constants
eq
(Keq) estimated by equations 4 and 5, (by monitoring the
equations
4
and 5, cf. Table 1), the prototropic
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Journal Name
tautomerization from N* to T* is inhibited to a much larger simple hydrophobic effects, it is the competition between
extent by the SCX6 host than SCX4.
intramolecular H-bonding within CZ and intermolecular H-
The fact that the binding of CZ with the SCXn hosts inhibits bonding of CZ with the sulfonate groups of the hosts, that is
the conversion of N* to T* rather than promoting it, together mainly responsible for inhibition of the prototropic
with the result that the SCX6 host, which has a lower binding tautomerism in CZ by SCXn. As depicted in Scheme 2, in the
free dye, the hydroxyl groups of either ring A or ring C can take
part in the tautomerization reaction along the pre-existing
intramolecular H-bonds with the carbonyl group (a and a’;
both H-bonds being equivalent), thereby converting the N*
form to the T* form in a facile manner. On binding to the
hosts, it can be envisaged that there will be a strong tendency
for CZ to form intermolecular H-bonds with the sulfonate
groups present at the portals of the host. The formation of
new H-bonds by the hydroxyl group of CZ that is suitably
located in the vicinity of a sulfonate group of the host
affinity for CZ than SCX4, is able to arrest the conversion of N*
to T* more efficiently compared to SCX4, suggests that it is not
the inclusion of the guest into the hydrophobic host cavity per
se, that affects the prototropic equilibrium. Therefore, it is
necessary to consider other factors to explain these interesting
and unusual observations made with the SCXn-CZ systems.
Taking into account the relative dimensions of the guest,
5
1,54,55
and the hosts (Table S2, ESI†),
it may be anticipated that
CZ cannot penetrate deep within the cavity of either of the
SCXn hosts. The guest dye possibly remains close to the
periphery of the cavitands, where the sulfonate groups of
SCXn can provide suitable anchoring points for stabilization of
the host-guest complexes through H-bonding interactions.
However, since the upper rim diameter of SCX4 cavity is
smaller than that of SCX6, a tight host-guest binding is more
likely for SCX4-CZ system compared to SCX6-CZ, leading to a
higher binding constant value for the former.
(depicted as b for the SCXn-CZ complex in Scheme 2), will quite
expectedly lead to a weakening of the pre-existing
intramolecular H-bonding of CZ. Consequently, the
tautomerization of CZ from the N* form to the T* form may be
anticipated to reduce considerably in SCXn-CZ systems (cf.
Scheme 2) compared to that of free CZ, as observed in the
present study.
(A) SCX4-CZ
2’
1
?
E=-26.7 kcal mol-1
2
H(2)-O(1): 1.678 Å; H(2)-O(S): 2.930 Å
’
’
H(2 )-O(1): 1.657 Å; H(2 )-O(S): 3.758 Å
B) SCX6-CZ
E=-34.6 kcal mol-1
H(2)-O(1): 1.779 Å; H(2)-O(S): 2.399 Å
(
?
’
’
H(2 )-O(1): 1.655 Å; H(2 )-O(S): 3.865 Å
Fig.
4 Geometry optimized structures of the SCX4-CZ and SCX6-CZ host-guest
complexes along with their corresponding stabilization energies and H-bond distances
for the H atoms in the two hydroxyl groups of CZ. O(S) represents the oxygen atom of
the host sulfonate group that is nearest to each of the indicated H atoms in CZ.
Scheme 2 Schematic of intramolecular (a, a') and intermolecular (b) H-bonding in CZ
and SCXn-CZ, respectively, and the tautomerization equilibria in each case.
Since the low solubility of CZ in aqueous medium precluded
us from obtaining any NMR signals, we resorted to quantum
chemical calculations to understand the binding modes of CZ
with the hosts and substantiate our propositions regarding the
structures of the SCXn-CZ complexes. Such calculations are
widely used to understand and complement host-guest
These propositions are indeed corroborated by the
quantum chemical calculations, where a clear trend is
observed in the bond lengths for the intramolecular H-bonds
5
1,66-69
interaction studies in many cases.
The most stable of CZ vis a vis the intermolecular H-bonding in SCXn-CZ. From
geometry optimized structures obtained for the 1:1 SCXn-CZ the geometry optimized structures of the SCXn-CZ complexes,
complexes are shown in Fig. 4. It is clearly seen that CZ is partly it is revealed that the intramolecular H-bond distances
accommodated within the cavity of SCX4, but remains between the central carbonyl group of CZ (O(1) ) and the H-
CZ
externally bound at the portals of SCX6, suggesting that the atoms of the two hydroxyl groups on either side (H(2)CZ and
dye cannot experience much of the hydrophobic environments
in either of the host-guest complexes.
H(2')CZ), are not of equal length in the bound dye, although
these H-bonds are equivalent in free CZ. Due to the availability
of another H-bond accepting oxygen atom (belonging to the
sufonate group of the host) in the vicinity of the complexed
Based on the aforementioned results that CZ resides in the
vicinity of the portals instead of being embedded deeply
within the host cavities, it is rationalised that rather than
6
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dye, the H-atom of one of its hydroxyl groups forms a new driven by favorable enthalpy changes accompanied by small
alternate H-bond with the host and this leads to the elongation entropy losses.
of the corresponding intramolecular H-bond. Thus, for both
0
host-guest complexes (cf. Fig. 4) it is noticed that the H(2)CZ-
4.8
4.5
4.2
(
A)
O(S)SCXn distance is less than H(2')CZ-O(S)SCXn and accordingly,
H(2)CZ-O(1)CZ bond length is greater than H(2')CZ-O(1)CZ bond
length. Interestingly, the calculations also reveal that the
elongation in the intramolecular H-bond of CZ occurs to a
much larger extent in SCX6-CZ (1.655 Å to 1.779 Å) than in
SCX4-CZ (1.657 Å to 1.678 Å), as the H-atom of the
corresponding hydroxyl group of CZ and the O-atom of the
sulfonate group of the host are more closely placed in the
former than in the latter (intermolecular H-bond distances are
0
.0030
0.0032
-1
-
-
4000
1/T
(
K
)
8000
2.399 Å and 2.939 Å, respectively). This result is in accordance
0.00
0.01
0.02
SCX4] (M)
0.03
with the experimental observation that tautomerization of CZ
is hindered to a larger extent in SCX6-CZ than in SCX4-CZ
despite the binding constant value being higher for the latter.
Therefore, the specific orientations of the host-guest systems
and the relative propensities of intramolecular H-bonding
within CZ and the intermolecular H-bonding between CZ and
SCXn, essentially determines the prototropic tautomerism of
CZ in the studied host-guest complexes.
[
4
.4
4.0
.6
0
(B)
3
0
.0030
0.0032
-1
-
800
1/T(K )
The formation of SCXn-CZ intermolecular H-bonds is
further substantiated by FTIR studies. The OH bending mode of
1
-1
CZ which appears around 1400 cm for the free dye is shifted
-1
toward lower frequency (~1370 cm ) in the presence of both
SCX4 and SCX6 (Fig. S7, ESI ), which is a signature of
intermolecular H-bonding in the host-guest systems.
-1600
†
7
0,71
0.00
0.01
0.02
To have an idea about the thermodynamics of the host-
guest interaction, we estimated the binding constants at
[
SCX6] (M)
various temperatures ranging from 30°C to 60°C. The Fig. 5 Binding isotherms for (A) SCX4-CZ and (B) SCX6-CZ systems at 30°C ( ), 40°C ( ),
temperature dependent binding isotherms obtained by 50°C ( ) and 60°C ( ). Insets show the corresponding van’t Hoff plots.
monitoring the emission intensity changes at the LWEB (515
nm) for SCX4-CZ and SCX6-CZ, are shown in the Figs. 5A and B.
The binding isotherms obtained by monitoring the emission
intensity changes at the HWEB (585 nm) for both the systems
The negative enthalpy change originates from H-bonding,
7
3-80
electrostatic and van der Waals interactions,
and suggests
that a combination of these favourable noncovalent forces
drives the complexation between CZ and the hosts. The
entropy change, on the other hand, originates from the
interplay between entropic gain due to release of well-ordered
water molecules initially surrounding the individual host and
guest molecules (desolvation effect), and the entropic loss due
to decrease in the motional freedoms upon mutual
are shown in Fig. S8, ESI†. Since the ratio between the
intensities of the normal and tautomeric forms of free CZ
remains invariant with temperature, it was assumed that the
same would be true for the bound dye as well. Accordingly,
the binding constants estimated from the fitted curves using
7
3-78
equations 4 and 5, are listed in Table S3, ESI
†
. It is observed complexation (configurational effect).
In the present
that the Keq values decrease systematically with increase in the systems, the latter effect seems to have a predominant
temperature. The entropy and enthalpy changes for the contribution, resulting in an overall entropy loss. Since, SCXn
are known to be flexible molecules that can adopt different
binding of CZ to SCX6 and SCX4 were determined following the
7
8
’ 72
van t Hoff equation (equation 8), and the corresponding plots
conformations, the loss in motional freedom on binding with
the guest is quite understandable. The larger entropy loss for
SCX6-CZ system than for SCX4-CZ (cf. Table 2) is in accordance
with the greater flexibility of the higher homologue of the
host. The enthalpy change is also observed to be higher for the
are shown in the insets of Fig. 5 (and insets of Fig. S8, ESI
0
†
).
ꢀ
H
ꢀS0
lnK = −
+
(8)
eq
RT
R
The calculated thermodynamic parameters presented in Table SCX6-CZ system than for SCX4-CZ, which correlates well with
(obtained from the temperature dependent changes in Keq), the internal energy changes obtained from quantum chemical
indicate that interactions between SCXn and CZ are mainly calculations (cf. Fig. 4). Overall, however, the Gibbs free energy
2
0
0
0
change, ΔG =ΔH -TΔS , is estimated to be higher for the SCX4-
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CZ complex (ΔG ~-14 kJ mol ) than the SCX6-CZ complex (ΔG
Journal Name
0
-1
0
Centre, Mumbai. We thank Prof. A. Datta and Dr. S. Rakshit, IIT
-1
~-11.8 kJ mol ), which is in agreement with the higher binding Bombay, for helping us with the ESI-MS measurements and Dr.
constant for the former complex than the latter.
J. A. Kuttan, BARC for helping us with the FTIR studies. P. M.
Gharat acknowledges the BARC-Mumbai University joint
research initiative for his research fellowship.
Table 2 Thermodynamic parameters for the SCXn-CZ systems (T=298 K).
system
SCX4-CZ
SCX6-CZ
parameters obtained from van’t Hoff analysis
K
eq determined by
fitting to eq. 4
K
eq determined by
fitting to eq. 5
Notes and references
0
-1
0
-1
ΔH = -18.8 kJ mol
ΔH = -21.7 kJ mol
1
J. Elguero, A. R. Katritzky and O. V. Denisko, Prototropic
0
-1
0
-1
TΔS = -6.1 kJ mol
TΔS = -6.4 kJ mol
0
-1
-1
0
-1
-1
tautomerism of heterocycles: Heteroaromatic tautomerism-General
ΔG = -12.7 kJ mol
ΔG = -15.3 kJ mol
0
0
overview and methodology., Elsevier, 2000.
ΔH = -24.4 kJ mol
ΔH = -23.8 kJ mol
0
-1
0
-1
2
3
J. Waluk, Acc. Chem. Res., 2003, 36, 832.
TΔS = -12.5 kJ mol
TΔS = -12.2 kJ mol
0
-1
0
-1
J. Zhao, S. J. Y. Chen, H. Guo and P. Yang, Phys. Chem. Chem.
ΔG = -11.9 kJ mol
ΔG = -11.6 kJ mol
Phys., 2012, 14, 8803.
A. Manna, M. Sayed, A. Kumar and H. Pal, J. Phys. Chem. B
2014, 118, 2487.
4
,
,
Conclusions
5
1
6
7
S. Dutta Choudhury and H. Pal, J. Phys. Chem. B, 2016, 120
1970.
J. P. Richard, Biochemistry, 2012, 51, 2652.
S. Z. Fisher, C. M. Maupin, M. Budayova-Spano, L. Govindasamy,
Our study reveals that the facile prototropic transformation of
CZ is considerably inhibited when the dye is bound to SCXn
hosts. This result is contradictory to usual expectations
because binding of CZ within the relatively hydrophobic host
cavity should have facilitated its tautomerization from N* form
C. Tu, M. Agbandje-McKenna, D. N. Silverman, G. A. Voth and R.
*
McKenna, Biochemistry, 2007, 46, 2930.
to the T* form instead of inhibiting the process, as T is less
*
dipolar in character than the N form. Furthermore, it is
8
8
9
L. Guasch, M. L. Peach and M. C. Nicklaus, J. Org. Chem., 2015,
, 9900.
0
observed that the tautomerization equilibrium of CZ is more
severely hindered in the SCX6-CZ system than in SCX4-CZ,
although the binding affinity of the former host-guest pair is
lower. All of these unusual effects are proposed to arise due to
the specific intermolecular H-bonding interactions of CZ with
the portals of SCXn hosts, rather than due to the inclusion of
the dye within the host cavities. The formation of new
intermolecular H-bonds between the hydroxyl groups of CZ
and the sulfonate groups present at the portals of the hosts,
effectively disrupts the pre-existing intramolecular H-bond
network in CZ, and consequently hinders the tautomerization
process. Moreover, the proximity of CZ near the portals of the
larger host SCX6, allows the intermolecular H-bonding to be
more efficient in the SCX6-CZ system than in SCX4-CZ. As a
result, the tautomerism from N* to T* form of CZ is reduced to
a larger extent in the former case, despite its lower binding
affinity. The present results emphasize that it is not only the
localized binding pockets provided by biological environments,
but also the orientations and specific interactions, that must
be considered in interpreting the course of any tautomeric
equilibria occuring in natural systems. These additional
parameters influence the tautomerization of vital
biomolecules and thereby impart selectivity to various
biochemical processes.
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Organic & Biomolecular Chemistry
Page 10 of 10
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DOI: 10.1039/C8OB00978C
Graphical Abstract
This study reveals the unusual inhibition of excited-state prototropic tautomerism of Chrysazine
by p-sulfonatocalix[4,6]arene hosts.