Dynamic combinatorial development of a neutral synthetic receptor that binds sulfate with nanomolar affinity in aqueous solution

Article abstract of DOI:10.1039/c1cc13451e

Using dynamic combinatorial disulfide chemistry we have developed a new generation of neutral synthetic receptors for anions, based on a macrobicyclic peptide structure. These receptors show an exceptional affinity and selectivity for sulfate ions in aque

Full text of DOI:10.1039/c1cc13451e

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COMMUNICATION
Dynamic combinatorial development of a neutral synthetic receptor that
binds sulfate with nanomolar affinity in aqueous solutionw
Zaida Rodriguez-Docampo,a Eugenia Eugenieva-Ilieva,b Carsten Reyheller,b
Ana M. Belenguer,a Stefan Kubik*b and Sijbren Otto*c
Received 10th June 2011, Accepted 12th July 2011
DOI: 10.1039/c1cc13451e
Using dynamic combinatorial disulfide chemistry we have
developed a new generation of neutral synthetic receptors for
anions, based on a macrobicyclic peptide structure. These
receptors show an exceptional affinity and selectivity for sulfate
ions in aqueous solution [log Ka = 8.67 in 41 mol% (67 volume%)
acetonitrile in water]. The high affinity depends on a delicate
balance between rigidity and flexibility in the structure of the
receptor.
Often, the addition of even small amounts of water eliminates
binding.4 Cyclopeptide 1, developed by one of us, is a notable
exception: this receptor binds sulfate and halides in highly
aqueous media.9 The 2 : 1 sandwich complexes formed by two
units of 1 with the anion can be further stabilized by covalently
linking the two cyclopeptide rings.10 Selection of the optimal
linkers using dynamic combinatorial chemistry has yielded
neutral anion receptors, 2b and 2d, which exhibit micromolar
affinities for sulfate in mixtures of water and acetonitrile.10d To
the best of our knowledge, these are the highest affinities
reported for neutral anion receptors in aqueous solution prior
to the present study. The extraordinary affinity is partly due to
reinforced molecular recognition.11 In the conformation in
which the bis(cyclopeptide) binds the anion, hydrophobic
interactions between the two cyclopeptide units contribute to
the overall complex stability. We have previously estimated
that these interactions enhance binding affinity by two orders
of magnitude.12
Molecular recognition plays a central role in biology, spurring
attempts to develop synthetic host–guest systems that operate
in aqueous solution.1 However, obtaining synthetic receptors
that bind their guests with good affinity and selectivity in water
is a considerable challenge, as water molecules are strong
competitors for most noncovalent interactions. Where nature
has solved this problem long ago, chemists still struggle;
synthetic receptors that operate in water are, on average, six
orders of magnitude less efficient in binding guests than
biomolecules.1a Yet, high affinities are essential for use of
synthetic receptors in biological contexts.2 We have already
made significant progress in developing receptors that strongly
bind hydrophobic cations in water.3 We now report a new
generation of hosts for hydrophilic anions, which are much
more challenging targets as anions are usually more strongly
solvated in aqueous solution than cations.4
One of the hallmarks in the recognition of anions in biology
is the sulfate-binding protein (SBP) that presents a neutral
binding pocket for the anionic guest.5 Several neutral synthetic
anion receptors have been developed, many of which are
inspired by the binding motif of SBP.6,7 While good binding
affinities have been obtained in organic solvents, only few
systems retain detectable anion binding in aqueous solution.8
We have since extended our studies to target receptors, in
which the two cyclopeptide rings are connected via two linkers.
Such macrobicyclic receptors should be significantly better
preorganized for complex formation. Access to these complex
architectures is difficult when using traditional synthesis, but
relatively straightforward when using a dynamic combinatorial
strategy (Scheme 1). Dynamic combinatorial chemistry13 relies
on equilibrium mixtures that respond to the addition of
template molecules (i.e. anionic guests) by amplifying strongly
binding library members at the expense of the other
a Department of Chemistry, University of Cambridge, Lensfield Road,
Cambridge CB2 1EW, UK
b Fachbereich Chemie-Organische Chemie, Technische Universitat
Kaiserslautern, Erwin-Schrodinger-Straße, D-67663 Kaiserslautern,
¨
¨
Germany. E-mail: kubik@chemie.uni-kl.de; Fax: +49 631 205 3921
c Centre for Systems Chemistry, Stratingh Institute, University of
Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
E-mail: s.otto@rug.nl; Fax: +31 50 363 4296
w Electronic supplementary information (ESI) available: Synthesis of
3a, preparation of the DCLs, isolation and characterization of
receptors 2c, 3c and 3d and ITC (displacement) binding assays. See
DOI: 10.1039/c1cc13451e
c
9798 Chem. Commun., 2011, 47, 9798–9800
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Determining the binding constants of sulfate and selenate
with receptors 3c and 3d required the use of displacement
techniques15 as binding of these anions was too strong to be
accurately measured by direct titration. We first performed
titrations of the relatively weakly binding iodide anion. After
binding was saturated we titrated in salts containing the more
strongly binding anions (sulfate or selenate) and recorded the
heat associated with displacing the iodide anion from the
binding site of the receptor. The results of the ITC binding
studies are summarized in Table 1. For comparison we also
include data obtained for the corresponding receptors linked
by only one spacer 2c (prepared for the present study) and 2d
(reported previously).10d
Scheme 1 Anion-induced amplification of a macrobicyclic receptor
from a dynamic combinatorial library of disulfides.
compounds in the mixture. We have used dynamic combina-
torial libraries (DCLs) based on disulfide exchange.14 Such
libraries can be generated from thiols which readily oxidize
when exposed to oxygen from the air at neutral pH. Disulfide
exchange is mediated by residual thiolate anions and can be
halted by addition of an acid.
Table 1 clearly shows that our new anion receptors possess
exceptionally high anion affinities; the highest reported so far
in aqueous media for neutral synthetic receptors. They are also
the strongest binders of all synthetic receptors developed
through dynamic combinatorial chemistry reported to date.
In addition, our results provide valuable information about
effects that control binding affinities and selectivities in these
receptors. The singly linked bis(cyclopeptide) receptors 2c and
2d have comparable anion affinities, almost independent of
whether the spacer is flexible (as in 2c) or rigid (as in 2d). In
contrast, for the doubly linked receptors 3c and 3d the nature
of the spacer has a pronounced effect on anion binding. While
the expectation is that by increasing the preorganization of the
receptor through the introduction of a second linker the
binding affinity should increase, this effect is only modest
(4.3 kJ molꢀ1 in the case of sulfate binding) for aromatic
linker d. The corresponding effect for the more flexible
aliphatic linker c is much larger: affinity for sulfate increases
by 11 kJ molꢀ1, highlighting the importance of flexibility in
achieving high affinities. Note that for both sulfate and iodide
binding, the enhanced binding affinity upon introduction of a
second spacer is solely an entropic effect and accompanied by
an unfavorable change in the enthalpy of binding. It is unlikely
that the entropic gain upon introducing a second spacer is
associated with anion desolvation. We have shown previously
that, even for the singly linked receptors, the anion binding
pocket of the receptor is well shielded from the solvent and
that the bound anion is completely desolvated.12 Assuming
that also in the doubly-linked bis(cyclopeptides) 3c and 3d the
bound anion is completely desolvated, this suggests that
Bis(cyclopeptide) 3a was prepared from a monocyclic
peptide with alternating L-proline and 6-aminopicolinic acid
subunits in which two proline rings contain 4S-thiobenzoate
substituents. The monocyclic peptide was synthesized by
assembling the linear precursor in solution from appropriately
functionalized dipeptides followed by cyclization under pseudo
high-dilution conditions.w
DCLs were prepared by mixing bis(cyclopeptide) 3a with
various dithiol spacers in acetonitrile–water mixturesz, using
sulfate salts or salts of other anions as templates. After allowing the
libraries to equilibrate for six days the composition of the
DCLs in the presence and absence of different anions was
analyzed by HPLC-MS. Fig. 1 shows the results for the two
most successful spacers cy and d with the anions that produced
the biggest amplifications: sulfate, iodide and selenate.
Significant amplifications of macrobicycles 3c and 3d were
observed in all cases, most notably with sulfate. Receptors 3c
and 3d were isolated by preparative HPLC from sulfate-
templated libraries. Unlike the previous generation of
receptors, it was necessary to remove most of the sulfate prior
to injection by precipitation with BaCl2 and filtration, other-
wise the receptors retained the sulfate anion during chromato-
graphy. This already suggested that these compounds exhibit
a strong affinity for sulfate, which was confirmed using
isothermal titration calorimetry (ITC).
Table 1 Association constants, Gibbs binding energies, enthalpies
and entropies of various anions to receptors 2c, 2d, 3c, and 3da
Salt
KI
Receptor
log Ka
DG1b
DH1b
TDS1b
2c
3c
2d
3d
2c
3c
2d
3d
3c
3d
4.89
6.04
4.75
5.08
6.78
8.67
6.83
7.59
8.04
6.60
ꢀ27.9
ꢀ34.4
ꢀ27.1
ꢀ28.9
ꢀ38.6
ꢀ49.5
ꢀ39.0
ꢀ43.3
ꢀ46.0
ꢀ37.7
ꢀ18.7
ꢀ12.7
ꢀ13.4
ꢀ7.2
2.8
9.2
21.7
13.7
21.7
41.4
59.1
42.7
49.3
Na2SO4
9.6
3.7
6.0
16.0
Na2SeO4
62.0
c
c
Fig. 1 HPLC analysis (260 nm) for DCLs containing bis(cyclopeptide)
3a (0.67 mM), spacer c (1.33 mM, left) or d (1.33 mM, right) and
various salts (10 mM) in CH3CN/H2O 2 : 1 (v/v) at pH 9 after 6 days
of equilibration.
a
b
Recorded in CH3CN/H2O 2 : 1 (v/v) at 298 K. Energies in
c
kJ molꢀ1
.
The relatively weak affinity of SeO24ꢀ relative to Iꢀ did
not allow obtaining accurate values for the binding enthalpy and entropy.
c
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introducing a second spacer gives rise to more favorable
receptor desolvation. A well ordered array of water molecules
has recently been observed in the solid state inside the cavity
of a triply-linked bis(cyclopeptide).10e If the cavities of
bis(cyclopeptides) 3c and 3d contain similarly well-ordered
water molecules in the absence of anions, complex formation
should indeed benefit significantly from the release of these
solvent molecules. In addition, the more favorable entropic
terms observed for the doubly-linked bis(cyclopeptides) with
respect to the singly-linked analogs could also reflect a smaller
loss of conformational flexibility when 3c and 3d interact with
anions, i.e. the doubly linked receptors are better preorganized.
The more favorable entropic contribution to anion binding
resulting from introduction of the second linker is partly
compensated by a reduced binding enthalpy. This reduction
could result from the second linker preventing the two cyclo-
peptide rings from adopting the optimal mutual arrangement
for anion binding and/or from a larger enthalpy required for
desolvation of 3c and 3d.
1 (a) K. N. Houk, A. G. Leach, S. P. Kim and X. Y. Zhang, Angew.
Chem., Int. Ed., 2003, 42, 4872; (b) G. V. Oshovsky, D. N. Reinhoudt
and W. Verboom, Angew. Chem., Int. Ed., 2007, 46, 2366.
2 D. A. Uhlenheuer, K. Petkau and L. Brunsveld, Chem. Soc. Rev.,
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297, 590; (b) L. Vial, R. F. Ludlow, J. Leclaire, R. Perez-Fernandez
´ ´
and S. Otto, J. Am. Chem. Soc., 2006, 128, 10253; (c) K. R. West,
R. F. Ludlow, P. T. Corbett, P. Besenius, F. M. Mansfeld,
P. A. G. Cormack, D. C. Sherrington, J. M. Goodman,
M. C. A. Stuart and S. Otto, J. Am. Chem. Soc., 2008, 130, 10834.
4 S. Kubik, Chem. Soc. Rev., 2010, 39, 3648.
5 B. L. Jacobson and F. A. Quiocho, J. Mol. Biol., 1988, 204, 783.
6 Progress in anion coordination chemistry is regularly reviewed by
P. Gale. For the most recent review, see: (a) P. A. Gale, Chem. Soc.
Rev., 2010, 39, 3746; For reviews on anion coordination through
hydrogen bonds, see: (b) S. O. Kang, M. A. Hossain and
K. Bowman-James, Coord. Chem. Rev., 2006, 250, 3038;
(c) S. Kubik, Chem. Soc. Rev., 2009, 38, 585.
7 For recent papers on synthetic receptors for sulfate, see:
(a) J. I. Kim, H. Juwarker, X. Liu, M. S. Lah and K.-S. Jeong,
Chem. Commun., 2010, 46, 764; (b) A. Galstyan, P. J. S. Miguel and
B. Lippert, Chem.–Eur. J., 2010, 16, 5577; (c) I. Ravikumar and
P. Ghosh, Chem. Commun., 2010, 46, 6741; (d) B. A. Moyer,
F. V. Sloop Jr., C. J. Fowler, T. J. Haverlock, H.-A. Kang,
L. H. Delmau, D. M. Bau, M. A. Hossain, K. Bowman-James,
J. A. Shriver, N. L. Bill, D. E. Gross, M. Marquez, V. M. Lynch
and J. L. Sessler, Supramol. Chem., 2010, 22, 653; (e) C. Jia, B. Wu,
S. Li, X. Huang and X.-J. Yang, Org. Lett., 2010, 12, 5612;
(f) P. A. Gale, J. R. Hiscock, C. Z. Jie, M. B. Hursthouse and
M. E. Light, Chem. Sci., 2010, 1, 215; (g) M. Wenzel, Q. W. Knapp
and P. G. Plieger, Chem. Commun., 2011, 47, 499; (h) C. Jia, B. Wu,
S. Li, X. Huang, Q. Zhao, Q.-S. Li and X.-J. Yang, Angew. Chem.,
Int. Ed., 2011, 50, 486.
The properties of receptors 3c and 3d thus depend on a
delicate balance between rigidity and flexibility.16 Rigidifi-
cation of the receptor into a macrobicyclic structure by
incorporating two spacers enhances affinity and selectivity,
when compared to the corresponding receptors with zero or
one spacer. The best results were obtained when using two
flexible linkers c as compared to the more rigid linker d. These
results illustrate the challenge posed by rigidification: the
positioning of the atoms involved in binding needs to be
exactly right for optimal interactions, which is extremely
difficult to achieve when this positioning is dictated by
covalent bonds. Nature’s solution to this problem is probably
to shape binding sites using noncovalent interactions
(i.e. through protein folding), which allow small conforma-
tional adjustments to be made more easily. In our covalently
assembled system, the more flexible linker that is more tolerant
toward conformational adjustments (i.e. induced fit) during
complex formation gives rise to the better receptor. The
importance of the right degree of flexibility16 in the covalent
framework is further underlined by the recent work in which
two cyclopeptide subunits were covalently linked using click
chemistry.10e In this system, the increase in the number of
linkers between the two cyclopeptides from one to three has
only a moderate impact on sulfate binding since the linkers
adopt energetically unfavorable conformations in the
complexes. We are currently investigating whether even better
receptors can be obtained through a dynamic combinatorial
approach targeting triply-linked bis(cyclopeptides).
8 (a) E. Fan, S. A. Van Arman, S. Kincaid and A. D. Hamilton,
J. Am. Chem. Soc., 1993, 115, 369; (b) R. C. Jagessar, M. Shang,
W. R. Scheidt and D. H. Burns, J. Am. Chem. Soc., 1998,
120, 11684; (c) P. Anzenbacher Jr., K. Jursıkova and
´ ´
J. L. Sessler, J. Am. Chem. Soc., 2000, 122, 9350; (d) I. E. D.
Vega, S. Camiolo, P. A. Gale, M. B. Hursthouse and M. E. Light,
Chem. Commun., 2003, 1686; (e) R. Yang, W.-X. Liu, H. Shen,
H.-H. Huang and Y.-B. Jiang, J. Phys. Chem. B, 2008, 112, 5105;
(f) C. Caltagirone, J. R. Hiscock, M. B. Hursthouse, M. E. Light
and P. A. Gale, Chem.–Eur. J., 2008, 14, 10236; (g) J. Suk and
K.-S. Jeong, J. Am. Chem. Soc., 2008, 130, 11868.
9 (a) S. Kubik, R. Goddard, R. Kirchner, D. Nolting and J. Seidel,
Angew. Chem., Int. Ed., 2001, 40, 2648; (b) S. Kubik and
R. Goddard, Proc. Natl. Acad. Sci. U. S. A., 2002, 99, 5127.
10 (a) S. Kubik, R. Kirchner, D. Nolting and J. Seidel, J. Am. Chem.
Soc., 2002, 124, 12752; (b) C. Reyheller, B. P. Hay and S. Kubik,
New J. Chem., 2007, 31, 2095; (c) C. Reyheller and S. Kubik, Org.
Lett., 2007, 9, 5271; (d) S. Otto and S. Kubik, J. Am. Chem. Soc.,
2003, 125, 7804; (e) T. Fiehn, R. Goddard, R. W. Seidel and
S. Kubik, Chem.–Eur. J., 2010, 16, 7241.
11 S. Otto, Dalton Trans., 2006, 2861.
12 Z. Rodrıguez-Docampo, S. I. Pascu, S. Kubik and S. Otto, J. Am.
´
Chem. Soc., 2006, 128, 11206.
13 For reviews, see: (a) B. L. Miller, Dynamic Combinatorial Chem-
istry in Drug Discovery, Bioorganic Chemistry, and Materials
Science, Wiley, Hoboken, NJ, 2010; (b) J. H. N Reek, S Otto,
Dynamic Combinatorial Chemistry, Wiley-VCH, Weinheim, 2010;
(c) P. T. Corbett, J. Leclaire, L. Vial, K. R. West, J.-L. Wietor,
J. K. M. Sanders and S. Otto, Chem. Rev., 2006, 106, 3652;
(d) R. A. R. Hunt and S. Otto, Chem. Commun., 2011, 47, 847.
14 S. Otto, R. L. E. Furlan and J. K. M. Sanders, J. Am. Chem. Soc.,
2000, 122, 12063.
This work has been supported by the EU (Marie Curie EST
8303 ChemBioCam, Marie Curie RTN DCC and COST D31),
the EPSRC, the Royal Society and the Deutsche Forschungs-
gemeinschaft. We are also very grateful to Dr Luminita
Damian from Microcal Europe (UK).
15 B. W. Sigurskjold, Anal. Biochem., 2000, 277, 260.
16 Achieving the right balance between rigidity and flexibility is a
longstanding problem in supramolecular chemistry. See for example:
(a) J.-M. Lehn, Angew. Chem., Int. Ed., 1988, 27, 89;
(b) F. Eblinger and H.-J. Schneider, Angew. Chem., Int. Ed.,
1998, 37, 826; (c) H.-J. Schneider, Angew. Chem., Int. Ed., 2009,
48, 3924.
Notes and references
z Addition of organic cosolvent was required to ensure sufficient
solubility of cyclopeptides 2 and 3.
y Symmetric spacer c was used instead of b as the latter gave rise to
complex mixtures of stereoisomeric products.
c
9800 Chem. Commun., 2011, 47, 9798–9800
This journal is The Royal Society of Chemistry 2011