Efficient complexation of N-acetyl amino acid carboxylates in water by an artificial receptor: Unexpected cooperativity in the binding of glutamate but not aspartate

Article abstract of DOI:10.1021/ja052699k

A new tris-cation 1 binds N-acetyl amino acid carboxylates in water even at millimolar salt concentrations with K_ass ≈ 10³ M^-1 due to a clustering of electrostatic interactions. Binding is efficient enough to allow a naked

Full text of DOI:10.1021/ja052699k

Published on Web 07/07/2005
Efficient Complexation of N-Acetyl Amino Acid Carboxylates in Water by an
Artificial Receptor: Unexpected Cooperativity in the Binding of Glutamate but
Not Aspartate
Carsten Schmuck* and Lars Geiger
Institute of Organic Chemistry, UniVersity of Wu¨rzburg, Am Hubland, 97074 Wu¨rzburg, Germany
Received April 26, 2005; E-mail: schmuck@chemie.uni-wuerzburg.de
The design of artificial receptors, which efficiently bind amino
acids under physiological conditions, still remains a challenging
task.1 Most amino acid receptors reported in the literature so far
need either hydrophobic1a-c and/or strong metal-ligand inter-
actions1f,g,2 to achieve substrate binding in water. We now present
for the first time a new tris-cationic receptor 1 which binds amino
acid carboxylates efficiently with Kass g 103 M-1 in water solely
based on electrostatic interactions. Furthermore, 1 shows an
unexpected cooperative 2:1 complex formation with N-acetyl
glutamatesbut not aspartate.
The receptor design is based on our guanidiniocarbonyl pyrroles,
which we introduced for the binding of carboxylates in polar
solvents.3 Their H-bond-enforced ion pairs with amino acid
carboxylate are much stronger than those of other organic cations
(e.g., the parent guanidinium cation),4 but still not strong enough
Figure 1. Binding isotherms for the amide NH of the amino acid
carboxylates 7-9 (c0 ) 1.5 mM, NMe4 salts) upon the addition of tris-
cation 1 (chloride salt) in 90% water/DMSO. The solid lines represent the
to allow efficient binding in water at millimolar salt concentrations.
We reasoned that the additional positive charges in 1 should further
curve fitting.
stabilize the complex. The synthesis of tris-cation 1 is shown in
Scheme 1. The pyrrole acid 2 is first coupled with tBoc-guanidine
3 to give 4. A reductive amination with amino acid 5 provided the
protected receptor 6. After deprotection and ion-exchange with HCl,
the water-soluble tris-cation 1 was obtained as the chloride salt.
The binding properties of this new tris-cation 1 were studied by
NMR titration experiments (for more details on the titrations and
the data analysis, see Supporting Information).5 In 40% water in
DMSO (v/v), the tris-cation 1 binds N-acetyl-L-alanine carboxylate
7 so effectively that the binding isotherm shows only a linear
increase up to a ratio of 1:1, indicating an association constant of
K g 105 M-1. Hence, the complex is at least 2 orders of magnitude
Figure 2. Calculated structure for the complex between 1 and 7 [nonpolar
hydrogens omitted for clarity, H-bond distances in Å].
more stable than with corresponding monocationic guanidiniocar-
bonyl pyrroles (K e 103 M-1 in 40% water in DMSO).3c,d Even in
90% water (10% DMSO was added for solubility reasons), 1 binds
alanine carboxylate 7 with a surprisingly high association constant
of K ) 2100 M-1, as obtained from a nonlinear curve fitting of
the binding isotherm (Figure 1). To the best of our knowledge,
tris-cation 1 is hence the first simple receptor that allows the
efficient complexation of an amino acid carboxylate in water solely
based on electrostatic interactions with K > 103 M-1 at millimolar
salt concentrations and, therefore, medium ionic strength.
Scheme 1. Synthesis of Tris-Cation 1
This surprisingly efficient binding probably results from the
clustering of electrostatic interactions in this binding motif.
However, the three charges do not contribute equally to the complex
stability, as can be seen by comparison of tris-cation 1 with the
parent guanidiniocarbonyl pyrrole monocations3c,d and a previously
reported dication.3a The significantly improved affinity of 1 must,
therefore, come from the terminal R-ammonium group.6 According
to the calculated energy-minimized structure7 for the complex
between tris-cation 1 and 7, this ammonium group can form an
additional chelate interaction with the two carboxylate oxygens
(Figure 2). On the basis of this complex structure, no stereoselective
substrate binding is expected, even though tris-cation 1 is chiral,
and indeed, N-acetyl-D-alanine carboxylate is bound with the same
affinity as its enantiomer (K ) 2040 M-1).
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10.1021/ja052699k CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
in the presence of tris-cation 1 due to complex formation and
increases again upon the addition of glutamate, a much better
binding substrate. The simple tris-cation 1 is, of course, not yet
selective enough for different amino acid carboxylates to allow their
distinction, but the introduction of additional binding sites, for
example, using the ester group in 1 should allow the design of
modified versions with improved selectivity for individual amino
acids. Such work is currently in progress.
In conclusion, we show here that a clustering of electrostatic
interactions as in tris-cation 1 allows the efficient complexation of
amino acid carboxylates in water. Additional hydrophobic or
metal-ligand interactions are not needed. Furthermore, even small
and flexible artificial receptors can show remarkable cooperativity,
thereby discriminating between structurally closely related guests.
Figure 3. Steric and/or electrostatic interactions (red) prevent the formation
of a 2:1 complex for aspartate but not glutamate.
Acknowledgment. This work was supported by the DFG and
the Fonds der Chemischen Industrie.
Supporting Information Available: Experimental details for the
synthesis of 1; binding data. This material is available free of charge
via the Internet at http://pubs.acs.org.
References
(1) For selected examples on the complexation of amino acids in aqueous
solvents, see: (a) Escuder, B.; Rowan, A. E.; Feiters, M. C.; Nolte, R. J.
M. Tetrahedron 2004, 60, 291-300. (b) Herna´ndez, J. V.; Mun˜iz, F. M.;
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Figure 4. Indicator-displacement assay in water ([CF] ) 10 µM, [1] ) 1
mM, [9] ) 0.5 mM in 2 mM bis-tris buffer, pH ) 6.3). Inset: Naked-eye
detection of glutamate in aqueous DMSO.
Tris-cation 1 should also be prone for the binding of multianionic
substrates. We, therefore, tested its affinity for N-acetyl-L-aspartate
8 and N-acetyl-L-glutamate 9. In 90% water/DMSO (v/v), aspartate
8 forms a 1:1 complex with tris-cation 1 with an association constant
of 480 M-1 (Figure 1).8 A Job plot confirmed the formation of a
1:1 complex. Surprisingly, the binding of N-acetyl glutamate 9,
which compared to aspartate 8 has only one additional methylene
group in the side chain, is completely different. Receptor 1 forms
a 2:1 complex with glutamate 9 (receptor:glutamate), and the
sigmoidal curvature of the binding isotherm indicates a positive
cooperativity effect in the formation of this 2:1 complex (Figure
1).9 The tris-cation 1 is hence capable of differentiating between
glutamate and aspartate, which is remarkable regarding their
structural similarity and flexibility. A possible explanation is
suggested in Figure 3. The smaller distance between the two
carboxylates in alanine compared to glutamate might prevent the
formation of 2:1 complexes due to unfavorable steric/electrostatic
interactions.
The positive cooperativity in the binding of 1 to glutamate is
also evident from the binding constants. A nonlinear curve fitting
of the titration data with a 2:1 association model provided K1 )
460 M-1 and K2 ) 3300 M-1. The binding constant for the second
association step is larger by a factor of 7 than the one for the first
step (K1 < K2), leading to the observed positive cooperativity. Such
allosteric binding processes are tremendously important in many
biological systems,9 but still difficult to achieve in small artificial
receptors.10
(2) Severin, K.; Bergs, R.; Beck, W. Angew. Chem., Int. Ed. 1998, 37, 1634-
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(3) (a) Schmuck, C.; Graupner, S. Tetrahedron Lett. 2005, 40, 1295-1298.
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1999, 843-844. (e) For a review, see: Schmuck, C.; Geiger, L. Curr.
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(6) The dication had a protonated amino methylene group next to the pyrrole
ring but binds amino acids only as efficiently as the monocations. Hence,
the second charge had no beneficial effect on complex stability.3a
(7) Macromodel V8.0, Amber* force field, GB/SA water solvation, MC
conformational search with 100.000 step and subsequent MD run at 300
K for 100 ps. Mohamadi, F.; Richards, N. G. J.; Guida, W. C.; Liskamp,
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(8) The weaker binding compared to that of alanine is probably due to the
more extensive solvation of dianions in water: Minofar, B.; Mucha, M.;
Jungwirth, P.; Yang, X.; Fu, Y. J.; Wang, X.-B.; Wang, L.-S. J. Am. Chem.
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(9) For reviews on cooperativity, see: (a) Williams, D. H.; Stephens, E.;
O’Brien, D. P.; Zhou, M. Angew. Chem., Int. Ed. 2004, 43, 6596-6616.
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(a) Kawai, H.; Katoono, R.; Nishimura, K.; Matsuda, S.; Fujiwara, K.;
Tsuji, T.; Suzuki, T. J. Am. Chem. Soc. 2004, 126, 5034-5036. (b)
Mazika, M.; Sicking, W. Tetrahedron Lett. 2004, 45, 3117-3121. (c)
Raker, J.; Glass, T. E. J. Org. Chem. 2001, 66, 6505-6512.
(11) Wiskur, S. L.; Ait-Haddou, H.; Lavinge, J. J.; Anslyn, E. V. Acc. Chem.
Res. 2001, 34, 963-972.
The binding affinity of tris-cation 1 for amino acid carboxylates
is large enough to allow their naked-eye detection using an indicator
displacement assay,11 as shown for glutamate as an example in
Figure 4. The fluorescence of carboxyfluorescein CF is quenched
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