Amino acid binding in water by a new guanidiniocarbonyl pyrrole dication: The effect of the experimental conditions on complex stability and stoichiometry

Article abstract of DOI:10.1016/j.tetlet.2004.12.120

The synthesis and binding properties of a new guanidiniocarbonyl pyrrole dication 2 are reported, which efficiently binds alanine carboxylate with log K_ass = 3.9 in buffered water. Due to the increased charge density in this dication, the binding constant is five times larger than for the parent guanidiniocarbonyl pyrrole monocation 1 (log K = 3.2). However, the experimental conditions for determining the binding constant significantly influence both complex stability and stoichiometry. With increasing amount of substrate added during the titration, the overall complex stability decreases due to the increasing ionic strength of the solution. Furthermore, the formation of 1:2 complexes between 2 and 7 becomes increasingly important. Therefore, for the comparison of binding data it has to be assured that exactly the same experimental conditions are used for their determination.

Full text of DOI:10.1016/j.tetlet.2004.12.120

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 1295–1298
Amino acid binding in water by a new guanidiniocarbonyl pyrrole
dication: the effect of the experimental conditions on
complex stability and stoichiometry
Carsten Schmuck* and Svea Graupner
Institut fu¨r Organische Chemie, Universita¨t Wu¨rzburg, Am Hubland, 97074 Wu¨rzburg, Germany
Received 17 November 2004; revised 20 December 2004; accepted 22 December 2004
Available online 13 January 2005
Abstract—The synthesis and binding properties of a new guanidiniocarbonylpyrrole dication 2 are reported, which efficiently binds
alanine carboxylate with logKass = 3.9 in buffered water. Due to the increased charge density in this dication, the binding constant is
five times larger than for the parent guanidiniocarbonyl pyrrole monocation 1 (logK = 3.2). However, the experimentalconditions
for determining the binding constant significantly influence both complex stability and stoichiometry. With increasing amount of
substrate added during the titration, the overall complex stability decreases due to the increasing ionic strength of the solution. Fur-
thermore, the formation of 1:2 complexes between 2 and 7 becomes increasingly important. Therefore, for the comparison of bind-
ing data it has to be assured that exactly the same experimental conditions are used for their determination.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
acetonitrile.5 Therefore, our guanidiniocarbonylpyr-
roles also allow the complexation of carboxylates even
in aqueous solvents.1a–c,5,6 Hence, this new recognition
motif has already found versatile use in various fields
of supramolecular3 and bioorganic chemistry.1a–c
Today, there are only a few artificial receptors known,
that allow the complexation of an amino acid or a small
peptidic substrate in water.1,2 One reason for this is that
the strength of H-bonds, often successfully used for
molecular recognition in organic solvents, decreases rap-
idly with increasing polarity of the solvent making the
design of receptors for aqueous media challenging.
Therefore most systems reported so far either use hydro-
phobic cavities for substrate binding or rely on the much
stronger metal–ligand binding interaction, for example.
We currently explore how additional ionic interactions
enhance the binding affinity of simple hydrogen bonding
motifs.3 In this context, we have introduced a new and
highly efficient receptor for the complexation of carb-
In an earlier investigation we had studied the origin of
these highly efficient complexation properties of the
guanidiniocarbonyl pyrroles by comparing complex sta-
bilities within a series of systematically varied receptors.5
This study revealed that the ion pairing is the main
attractive interaction providing roughly 50% of the
binding energy. The H-bond between the amide NH at-
tached to position 5 of the pyrrole ring and the bound
carboxylate is responsible for another 25% of the total
binding energy whereas the pyrrole NH seems to be of
only minor importance. As charged hydrogen bonds
oxylates and amino acid carboxylates, the guanidinio-
4
carbonylpyrroels.
Due to the increased acidity of the
acylguanidinium moiety and the additionalH-bonds,
these complexes are much stronger than those of simple
guanidinium cations, which only form stable ion pairs in
organic solvents of low polarity such as chloroform or
Keywords: Amino acids; Guanidinium cations; Supramolecular che-
mistry; Molecular recognition.
*
Corresponding author. Tel.: +49 931 8885326; fax: +49 931
8884625; e-mail: schmuck@chemie.uni-wuerzburg.de
0040-4039/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.12.120

1296
C. Schmuck, S. Graupner / Tetrahedron Letters 46 (2005) 1295–1298
are normally stronger than neutral ones,7 we were inter-
ested to see whether a second positive charge at the
position of the amide NH would further increase the
complex stability. For that reason, we chose receptor
2, a bis-cation, as a new receptor prototype to test this
hypothesis.
2. Results
Receptor 2 was synthesized according to Scheme 1.
Starting from the pyrrole aldehyde 3,8 the methylester
functionality was cleaved with LiOH and the resulting
carboxylic acid 4 was subsequently coupled with
N-Boc-guanidine.9 A reductive amination of 5 with
H-Val-NH2 using NaBH3CN provided after deprotec-
tion with HClthe desired receptor 2, isolated as the
bis-chloride salt.
Figure 1. Changes in the UV spectra for the complexation of Ac-L-
Ala-OÀ (7) by receptor 2 in water at pH = 6.
The complexation properties of 2 were first probed quali-
tatively by NMR studies in 40% H2O in DMSO, the
same solvent mixture in which the monocation 1 was
previously studied.5 Upon the addition of N-Ac-L-Ala-
OÀ (NMe4+-salt) 7 to a solution of 2 significant
complexation induced shift changes for protons of both
substrate and receptor are observed indicating a mole-
cular interaction between anionic guest and cationic
receptor in this solvent mixture. A quantitative analysis
of these shift changes was however not possible as the
data could neither be fitted adequately with a simple
1:1 or 1:2 (2:7) binding modelnor a combination of
both.
nium moiety. As hydrogen bonding to an anion can be
regarded as a beginning deprotonation, it is not surpris-
ing that the UV absorption of the guanidiniocarbonyl
pyrrole decreases upon carboxylate binding. A quantita-
tive analysis of these changes in the UV spectra was per-
formed using a nonlinear least-squares fitting with a 1:1
association model10 using the Specfit/32 software pro-
gram from Spectrum Software Associates. The 1:1 com-
plex stoichiometry under these experimental conditions
was confirmed by a modified Job plot analysis (Fig. 2).11
According to this UV titration, the new bis-cationic
receptor 2 binds N-acetylaalnine carboxyalte
2 with
an association constant of Kass = 7.95 · 103 MÀ1
(logK = 3.9) in water. Under exactly the same condi-
tions the mono-cationic receptor 1 binds 7 with only
Kass = 1.60 · 103 MÀ1 (logK = 3.2). Hence, the addi-
tionalpositive charge in bis-cation 2 favors the binding
of the negatively charged substrate relative to the mono-
cation 1 by a factor of five.7 A charged H-bond at posi-
tion 5 of the pyrrole is indeed superior to a neutral
amide NH at the same position.
We therefore performed UV titration studies in buffered
water. Aliquots of the amino acid carboxylate 7
(NMe4+-salt) were added to a solution of receptor 2
(chloride salt) in water (bis–tris-buffer, 4.7 mM,
pH = 6.0, [2]o = 4.8 · 10À5 M). Changes in the UV spec-
trum of the receptor were recorded and used to deter-
mine the binding constants as the absorbance of the
pyrrole moiety at k = 294 nm decreases upon complex
formation (Fig. 1). This decrease in absorbance is in
good agreement with the pH-dependance of the UV
spectrum of receptor 2. In basic solution (pH > 9) the
absorption of the guanidiniocarbonylpyrroles decreases
significantly due to the deprotonation of the guanidi-
Figure 2. Modified Job plot extracted from the titration data
indicating the change of complex stoichiometry with increasing
amounts of added substrate.
Scheme 1. Synthesis of the bis-cationic receptor 2 via reductive
amination of aldehyde 5 with H-Val-NH2.

C. Schmuck, S. Graupner / Tetrahedron Letters 46 (2005) 1295–1298
1297
However, in comparing binding data for individual
receptors one has to be very cautious. Depending on
the conditions under which the binding constants were
determined significant deviations can occur, which
might lead to misinterpretations. If the binding is pri-
marily electrostatic in nature as in these cases, the over-
all solvent composition significantly influences the
complexation. Especially the total ionic strength of the
solution plays a major role. However, differences in
the ionic strength cannot only be due to different buffer
concentrations14a but also can have their origin in the
titration itself. Depending on how many equivalents of
the ionic substrate are added to the receptor, the calcu-
lated binding constants can vary. The more substrate is
added, the smaller the overall association constant is due
to the increasing Ôsalt effectÕ caused by the substrate
itself. For example, for receptor 2, the binding constant
varies from K = 7950 MÀ1 with 5 equiv of 7 added to
K = 5900 MÀ1 at 20 equiv of 7 even though Job plots
indicate the predominant formation of 1:1 complexes
in both cases. If larger amounts of substrate are added
the apparent association constant further decreases but
now complexes of higher stoichiometry also form in sig-
nificant amounts (vide infra; Fig. 2). However, also the
addition of large amounts of NaCl causes a similar de-
crease in the observed association constant. If 10 mM
NaClis added, no association constant can be deter-
Figure 3. Calculated energy minimized structure for the complex
between 7 (yellow) and 2 (gray). Hydrogen bonds are shown in red.
Nonpolar hydrogens are omitted for clarity.
bound by both of the cationic guanidinium NHs form-
ing bifurcated H-bonds whereas the other is primarily
bound by the ammonium cation with assistance by the
terminalcarboxamide NH ( Fig. 3). The binding motif
in 2 is hence different from the one found for the
mono-cationic guanidiniocarbonylpyrroel
1,5 where
mined using UV titration anymore indicative of an asso-
10
the guanidinium moiety forms bidentate hydrogen
bonds to both carboxylate oxygens. The higher positive
charge of the ammonium group in 2 compared to the
neutralamide NH in 1 causes the shifting of the carb-
oxylate toward this H-bond donor.
ciation constant K 6 1000 MÀ1
.
But with too little
substrate added the data set is not significant enough
to allow a calculation of the binding constant as the ex-
tent of complexation is then not sufficiently large.10 One
has to assure therefore, that only binding constants ob-
tained under exactly the same experimental conditions,
as done here, are used for comparisons. Otherwise any
data interpretation especially of small effects has only
limited value.
The five times higher binding affinity of receptor 2 rela-
tive to 1 is in good agreement with its higher positive
charge density.7,13 Though this electrostatic effect is
probably somewhat counterbalanced by the higher flexi-
bility of 2 making complexation entropically less favor-
able causing in total only to an increase by
approximately a factor of five in complex stability. Also
the bis-cationic receptor 2 is likely much more solvated
in water as is mono-cation 1.13 Therefore, the desolva-
tion costs are probably also larger especially in water.
This desolvation, which is necessary before complexa-
tion can take place, sometimes even causes ion pair
interactions to be endothermic and hence entropy driven
in polar solutions.14 In this respect an increase in com-
plex stability by a factor of five in water is a significant
improvement demonstrating how a clustering of electro-
static interactions, as weak as they individually are in
water, can lead to an overall stronger complexation.
Furthermore, also the complex stoichiometry can vary
upon the addition of larger amounts of substrate.
Whereas for the bis-cationic receptor 2 in water upon
the addition of 10 equiv of carboxylate 7, a Job plot
from the titration data11 clearly demonstrates the 1:1
complex stoichiometry, 1:2 (2:7) and probably even
higher complexes are formed with increasing equivalents
of substrate (Fig. 2).
If one does not consider these two effects (decrease of K
due to the increasing salt effect and changes in stoichio-
metry during titration, respectively) upon setting up an
experiment, the calculated binding constant can be
misleading. We found that for our systems the addition
of 10 equiv of substrate is a good compromise to assure
a significant amount of clean 1:1 complexation as
needed for data analysis with a still neglectable salt
effect.
Furthermore, this also underlines that suitable binding
sites at position 5 of our guanidiniocarbonylpyrroel
motif can have a significant effect on the complex stabil-
ity opening the door for further improvements of the
receptor design.
To get an idea about the structure of the complex
between 2 and 7, we performed molecular mechanics
calculations. According to
a
MD simulation
3. Conclusion
(MacromodelV 8.0, Amber* force fiedl , GB/SA water
solvation treatment, 100 ps simulation time, 1.5 fs time
steps at 300 K)12 one of the carboxylate oxygens is
We have presented here the synthesis and evaluation
of the binding properties of a new prototype of a

1298
C. Schmuck, S. Graupner / Tetrahedron Letters 46 (2005) 1295–1298
2000, 6, 185–216; (c) Schneider, H. J. Angew. Chem. 1993,
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bis-cationic guanidiniocarbonylpyrroel receptor 2. Due
to the additionalpositive charge 2 has superior binding
properties compared to an analogous mono-cationic
receptor 1 underlining the importance of electrostatic
interactions for guest binding in water.
3. Schmuck, C.; Wienand, W. J. Am. Chem. Soc. 2003, 125,
452–459.
4. Schmuck, C. Chem. Eur. J. 2000, 6, 709–718.
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Best, M. D.; Tobey, S. L.; Anslyn, E. V. Coord. Chem.
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1 2002, 841–864; (d) Schmidtchen, F. P.; Berger, M. Chem.
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Acknowledgements
This work was supported by the Deutsche Forschungs-
gemeinschaft (SCHM 1501/2-2) and the Fonds der
Chemischen Industrie (FCI).
6. Schmuck, C.; Geiger, L. Curr. Org. Chem. 2003, 7, 1485–
1502.
7. Dietrich, B.; Fyles, D. L.; Fyles, T. M.; Lehn, J.-M. Helv.
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Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.tetlet.
2004.12.120. Details of the synthesis of receptor 2.
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