High affinity sulfate binding in aqueous media by cyclic peptides with thiourea arms

Article abstract of DOI:10.1039/c2cc37686e

Tripodal thioureas based on a cyclic peptide scaffold have been synthesised and their anion binding properties evaluated. These receptors show high affinity towards sulfate ions in aqueous solution. This journal is

Full text of DOI:10.1039/c2cc37686e

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High affinity sulfate binding in aqueous media by
cyclic peptides with thiourea arms†
Cite this: DOI: 10.1039/c2cc37686e
Victoria J. Dungan, Huy Tien Ngo, Philip G. Young and Katrina A. Jolliffe*
Received 22nd October 2012,
Accepted 15th November 2012
DOI: 10.1039/c2cc37686e
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Tripodal thioureas based on a cyclic peptide scaffold have been syn- the sulfate oxygen atoms.8 Receptor 1 has spacers three methylene
thesised and their anion binding properties evaluated. These receptors units long between the cyclic peptide backbone and the thiourea
show high affinity towards sulfate ions in aqueous solution.
substituents. We envisioned that the importance of the backbone
amide protons in sulfate binding might be heightened if the
thiourea sites were brought closer to these hydrogen bond donors,
thereby decreasing the conformational freedom of the receptor and
providing a better match to the size of sulfate ions. Therefore,
receptors 2 and 3 (Fig. 1) in which these spacers are reduced to two
or one methylene units, respectively were prepared from the known
appropriately functionalised trisamine scaffolds10 upon treatment
with para-tbutylphenylisothiocyanate.
The anion binding properties of 2 and 3 were quantitatively
determined by 1H NMR spectroscopic titrations with a range of
anions as their tetrabutylammonium salts in CDCl3 to allow
direct comparison with the previously reported data for 1.
Apparent stability constants (Table 1) were determined by
following the downfield shifts of both of the thiourea NH
proton signals and fitting the titration data to a 1 : 1 binding
model11 (see ESI† for fitted titration data). As was previously
observed for 1, under these conditions fast exchange processes were
observed on titration of all anions except sulfate, which exhibited
behaviour indicative of slow exchange on the NMR timescale.
The numerous roles that anions play in biology and the environ-
ment has led to substantial current interest in the development of
small molecule receptors with high affinity and selectivity for specific
anion targets.1 Many potential applications require receptors to bind
and discriminate between anions in aqueous media but this
remains a significant challenge.2 There are relatively few reports of
electroneutral receptors reliant on hydrogen bonding interactions
for anion binding that show high anion affinity and selectivity in
aqueous solution, particularly in the case of hydrophilic anions such
as sulfate.3–5 One approach to the design of such receptors relies on
the appropriate positioning of hydrogen-bond donors around a
cavity or cleft to effectively shield the anion from the aqueous
environment in a manner which mimics that of the sulfate binding
protein.5 Tripodal receptors of this type have been found to provide
good selectivity for tetrahedral anions.6 We have recently reported
the use of Lissoclinum-type cyclic peptide scaffolds7 as a core for the
development of tripodal anion receptors.8 In addition to the thiourea
groups, these scaffolds provide additional hydrogen bond donors in
the form of the cyclic peptide amide protons resulting in extra
interactions with sulfate ions in comparison to other anionic
guests.8,9 To facilitate this binding mode and develop receptors
capable of binding sulfate ions with higher affinity and selectivity in
competitive solvents we have now prepared receptors in which the
thiourea binding sites are moved closer to the cyclic peptide scaffold.
We report here the results of this study.
In previous studies we observed that receptor 1 bound a range of
anions in organic solvents (CDCl3 and CDCl3/10% d6-DMSO) with
high sulfate selectivity. This was attributed to a unique binding
mode for sulfate in which additional hydrogen bonding interactions
are possible between the cyclic peptide amide protons and one of
School of Chemistry (F11), The University of Sydney, NSW 2006, Australia.
E-mail: kate.jolliffe@sydney.edu.au; Fax: +61 2 9351 3329; Tel: +61 2 9351 2297
† Electronic supplementary information (ESI) available: Experimental details
and spectroscopic data for new compounds and anion titration data. See DOI:
10.1039/c2cc37686e
Fig. 1 Structures of receptors 1–7.
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Table 1 Apparent stability constants (Ka, MÀ1) of 1–3 towards various anionsa,b
Table 2 Apparent stability constants (Ka, MÀ1) of 1–7 towards sulfate ions in
increasing amounts of H2O in d6-DMSOa
1d
2
3
3
Anionsc
CDCl3
CDCl3
CDCl3
(CDCl3 : d6-DMSO 85 : 15)
0.5% H2O
10% H2O
20% H2O
25% H2O
ClÀ
BrÀ
IÀ
298
104
36
900
150
75
>104
>104
2100
>104
2680
>104
>104 f
2280
330
—
b
b
1
2
3
4
5
6
7
>104 c
>104 c
>104 c
>104 c,d
>104 c
>104
>104
>104
>104 c
>104 d
>104
>104
>104
—
—
>104
—
b
g
>104
—
b
À
À
1.3 Â 103 d
141d
>104
221
NO3
74
980
670
e
g
>104
HSO4
1700
189
>104 f
—
—
TsOÀ
540
970
209
2À
>104 f
>104 f
>104 c
>104
>104
SO4
a
a
Determined at 300 K. Data was fitted to a 1 : 1 binding model.
Determined at 300 K. Data was fitted to a 1 : 1 binding model.
Ka values are an average obtained from monitoring NHa and NHb
Ka values are an average obtained from monitoring NHa and NHb.
b
b
(see Fig. 2. for labels). Errors o15%. Addition of acetate and hydrogen
Errors o15%. Receptor not sufficiently soluble to allow
c
c
phosphate resulted in deprotonation of the thiourea groups. Anions
determination of Ka. Titration displayed slow exchange on the
d
e
d
added as their tetrabutylammonium salts. Data from ref. 8. Peak
broadening prevented an association constant from being determined.
NMR timescale. Data from ref. 9.
f
g
Titration displayed slow exchange on the NMR timescale. Negligible
changes in chemical shift were observed.
geometry for hydrogen bonding interactions with all nine of the
receptor hydrogen bond donors (Fig. 2).
Restricted rotation of the receptor arms was observed upon anion
binding, indicating that these are fixed in place as a result of
hydrogen bonding between the thiourea protons and anionic
guests. Binding affinity for all anions increases as the distance
between the cyclic peptide scaffold and thiourea sites is shor-
tened, to the point where 3 binds almost all anions investigated
with apparent stability constants too high to measure by 1H NMR
in CDCl3, suggesting that this receptor provides better shielding
of guests from the solvent than 1 and 2, with additional inter-
actions occuring between all anions and the amide protons.
Evidence for these additional interactions comes from the observed
downfield shifts of the amide NH proton signals; for the same
anion these increased in magnitude on shortening the side chain
arms from three to one methylene units (e.g. for binding of
bromide ions, maximum Dd = 0.6 ppm for 3 compared to
0.1 ppm for 1), indicating the closer proximity of these hydrogen
bond donors to the anionic guests in 3. Notably, the observed
downfield shifts were three to four times larger on addition of
sulfate, compared to all other anions, to 1–3 (Dd = 1.62 ppm on
addition of sulfate to 3). For compound 3, fitting the chemical shift
data for the amide NH signal to a 1 : 1 binding model gave
apparent stability constants consistent with those obtained from
the thiourea NH protons (within experimental error). Increasing the
solvent polarity by adding 15% d6-DMSO to the CDCl3 allowed the
relative affinity of 3 for different anions to be ascertained. All three
receptors 1–3 exhibited selectivity for sulfate. This can be attributed
to the divalent charge of this anion in addition to the optimum
Given the high affinities observed for sulfate binding by receptors
1–3 together with our previous observations that the related cryp-
tand-like cyclic peptide 4 binds sulfate in aqueous media,9 we
examined binding of this ion by 1–3 in more competitive media.
Table 2 summarises the apparent stability constants found for
2À
titrations performed with SO4
in 0.5, 10, 20, and 25% v/v
H2O–DMSO-d6 mixtures. Addition of other anions to 1–3 under
these conditions did not result in significant changes in their
1H NMR spectra. In 0.5% v/v H2O–DMSO-d6, slow exchange pro-
cesses were observed upon addition of sulfate to all three receptors.
New signals for each proton environment in 1–3 appeared upon the
addition of sulfate, with all three NH signals undergoing significant
downfield shifts indicative of the formation of hydrogen bonds to
the anion (Fig. 3). A steady increase in the population of the new
signals was observed as the titration progressed until, after one
equivalent of anion had been added, the original signals could
no longer be observed, indicating very strong 1 : 1 binding (Ka >
104 MÀ1). In all cases, moving to more competitive media resulted in
fast exchange processes on the NMR timescale as evidenced by time-
averaged signals for each proton environment of 1–3. Fitting of this
data to a 1 : 1 binding model gave apparent stability constants of
>104 MÀ1 in all cases. Notably, for receptor 1 in 0.5% v/v
H2O–DMSO-d6, at higher sulfate concentrations additional binding
equilibria are apparent, as we have previously observed for this
receptor in CDCl3.8 However, on increasing the water content of the
Fig. 3 1H NMR spectra obtained on addition of sulfate ions to 3 in
Fig. 2 Proposed binding mode between 3 and sulfate.
0.5% v/v H2O–DMSO-d6.
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solvent these additional processes are suppressed. Such solvent together with effective shielding of the guest from the solvent. The
dependent behaviour has also been observed with indole-based development of receptors of this type with increased water solubility,
sulfate receptors.4 While receptors 2 and 3 were soluble in up to yet still capable of shielding guests from the surrounding solvent will
20% v/v H2O–DMSO-d6 mixtures, receptor 1 was not soluble in enable the full potential of these compounds to be realised and is
mixtures containing >10% H2O. To prepare an analogue of 1 with currently underway in our laboratories.
increased water solubility, compound 5 was prepared in the same
manner as described above, replacing the para-tbutylphenyl isothio-
cyanate with para-(trifluoromethyl)-phenyl isothiocyanate. Receptor
5 had improved solubility in aqueous mixtures and was found to
bind sulfate in a similar manner to 1 with Ka > 104 MÀ1 in 25% v/v
H2O–DMSO-d6. No apparent stability constants could be obtained
for any of the receptors in solvent mixtures containing >25% v/v H2O
as result of their low solubility. However, the observed high affinity
and selectivity for sulfate are particularly remarkable when com-
pared to those of the related cryptand-like receptor 4 (in 25% v/v
H2O–DMSO-d6 Ka for SO42À = 141 MÀ1; Ka for ClÀ = 83 MÀ1).9 While
the additional preorganisation of cryptand-type receptors is well-
established to provide improved binding affinities and selectivities,13
for these cyclic peptide derivatives the opposite is true. This may be a
result of better shielding of the anionic guest from the solvent by the
tripodal receptors as a result of hydrophobic interactions between
the aromatic substituents in 1–3 and 5, as suggested by the
significant and complex changes in the chemical shifts of the
aromatic protons of these receptors, which are observed upon
We thank the Australian Research Council for financial support.
Notes and references
‡ The methyl groups on the scaffold were omitted to enhance water
solubility.
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2À
ability evaluated.‡ Both compounds were found to bind SO4 with
1 : 1 stoichiometry, although in 0.5% v/v H2O–DMSO-d6, 6 displayed
more complex behaviour at high sulfate concentrations, as described
above for 1. Notably, while the affinity of 4 for SO42À decreases by an
order of magnitude on increasing the water content of the DMSO-d6
solution from 20% to 25% (Table 2),9 the apparent binding affinity
of tripodal 6 for SO42À under the same conditions remains constant
and in 25% v/v H2O–DMSO-d6 is of a similar magnitude to that of
the cryptand derivative, suggesting improved shielding from the
solvent by the tripodal receptor. For compound 7, in which the
thiourea binding sites are closer to the scaffold than those of 6,
affinity for SO42À (Ka > 104 MÀ1) remains too high to quantify in this
solvent mixture, confirming that reducing the space between the
sidechain and backbone hydrogen-bond donors results in increased
binding affinity in more polar solvent mixtures. Significant upfield
shifts (for 7, maximum Dd = 0.22 ppm in 10% v/v H2O–DMSO-d6) of
the aromatic protons of both 6 and 7 were observed upon addition
of sulfate suggesting that interactions are maintained between the
aromatic groups in the benzyl derivatives.
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In summary, reducing the distance between the thiourea and
cyclic peptide backbone hydrogen bond donors results in increased
affinity for a range of anions for this class of receptor. Notably, in
mixtures of water and DMSO all six compounds exhibit remarkably 12 All curve fitting was performed using the Equilibria program, C. E.
Marjo, University of New South Wales Analytical Centre, Sydney,
Australia, http://www.sseau.unsw.edu.au/Index.htm, 2009.
1
high selectivity and affinity for sulfate ions with apparent stability
constants too high to quantify by H NMR (Ka > 104 MÀ1). This is
13 J. W. Steed and J. L. Atwood, Supramolecular Chemistry, John Wiley &
attributed to a good fit between the host and guest geometries
Sons, Ltd, Chichester, 2000.
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