Active transport of amino acids by a guanidiniocarbonyl-pyrrole receptor

Article abstract of DOI:10.1002/chem.201000509

Herein we report the synthesis and characterization of a transporter 9 for N-acetylated amino acids. Transporter 9 is a conjugate of a guanidiniocarbonyl pyrrole cation, one of the most efficient carboxylate binding motifs reported so far, and a hydrophobictris (dodecylbenzyl) group, which ensures solubility in organic solvents. In its protonated form, 9 binds N-acetylated amino acid carboxylates in wetorganic solvents with association constantsin the range of 10⁴m^-1 as estimated by extraction experiments. Aromatic amino acids are preferred due to additional cation-α-interactions of the amino acid side chain with the guanidiniocarbonyl pyrrole moiety. U-tube experiments established efficient transport across a bulk liquid chloroform phase with fluxes approaching 10^-6 molm^-2s^-1. In experiments with single substrates, the release rate of the amino acid from the receptor-substrate complex at the interface with the receiving phase is rate determining. In contrast to this, in competition experiments with several substrates, the therm-odynamic affinity to 9 becomes decisive. As 9 can only transport anions in its protonated form and has a pK_a value of approximately 7, pH-driven active transport of amino acids is also possible. Transport occurs as a symport of the amino acid carboxylate and a proton.

Full text of DOI:10.1002/chem.201000509

DOI: 10.1002/chem.201000509
Active Transport of Amino Acids by a Guanidiniocarbonyl–Pyrrole Receptor
Christian Urban and Carsten Schmuck*[a]
Abstract: Herein we report the synthe-
sis and characterization of a transport-
er 9 for N-acetylated amino acids.
Transporter 9 is a conjugate of a guani-
diniocarbonyl pyrrole cation, one of
the most efficient carboxylate binding
motifs reported so far, and a hydropho-
bic tris(dodecylbenzyl) group, which
ensures solubility in organic solvents.
In its protonated form, 9 binds N-acet-
amino acids are preferred due to addi-
tional cation-p-interactions of the
amino acid side chain with the guanidi-
niocarbonyl pyrrole moiety. U-tube ex-
periments established efficient trans-
port across a bulk liquid chloroform
single substrates, the release rate of the
amino acid from the receptor–substrate
complex at the interface with the re-
ceiving phase is rate determining. In
contrast to this, in competition experi-
ments with several substrates, the ther-
modynamic affinity to 9 becomes deci-
sive. As 9 can only transport anions in
its protonated form and has a pKa
value of approximately 7, pH-driven
active transport of amino acids is also
possible. Transport occurs as a symport
of the amino acid carboxylate and a
proton.
phase
with
fluxes
approaching
10ꢀ6 molmꢀ2 sꢀ1. In experiments with
Keywords: active transport · amino
acids · guanidinium cations · mem-
brane proteins · molecular recogni-
tion
ACHTUNGTRENNUNGylated amino acid carboxylates in wet
organic solvents with association con-
stants in the range of 104 mꢀ1 as estimat-
ed by extraction experiments. Aromatic
Introduction
metal–ligand binding.[2] Even though a variety of efficient
host molecules for amino acids have been developed over
the last years,[3] efficient artificial transporters are still rare.
One problem is that very often receptors which bind amino
acids in aqueous solvents are charged, containing, for exam-
ple, ammonium or guanidinium cations or metal ions.[4]
Hence, they possess limited solubility in a lipid membrane
or the nonpolar bulk organic phase used for transport ex-
periments. In transporters realized so far, such binding
motifs have, therefore, been combined with nonpolar hydro-
phobic groups (e.g., such as steroids or aromatic units) to in-
crease their solubility in the organic phase.
One of the first examples for amino acid transport was re-
ported by Lehn who showed in 1973 that amino acids and
dipeptides can be transported from one aqueous phase
through a chloroform phase into another aqueous phase by
lipophilic quaternary ammonium compounds, such as Ali-
quat 336 (methyltricapryl ammonium chloride, also used as
a phase-transfer catalyst).[5] Substrate binding in this case
was simply due to unspecific electrostatic interactions and
transport efficiency was thus determined by the lipophilicity
of the amino acid side chain: Phenylalanine and tryptophan
were transported faster than, for example, alanine or gly-
cine. Tsukube then introduced crown ethers and their open
chain analogues, lariat ethers, as amino acid transporters,
which have been used extensively in this context either
The transport of amino acids and amino acid derivatives
across a lipid membrane is a crucial part of metabolism in
biological systems. As polar molecules they are not capable
of crossing the membrane by passive diffusion and thus re-
quire a membrane transporter to do so. Accordingly, the de-
velopment of artificial transporters has become an active
area of research since the advent of supramolecular chemis-
try about 30 years ago.[1] Transport of any compound from
one aqueous phase (source phase) across a membrane or a
bulk liquid organic phase to another aqueous phase (receiv-
ing phase) requires a host molecule (an artificial receptor)
capable of efficiently binding the substrate under aqueous
conditions (or at least at the water/organic interface). There-
fore, binding of the amino acid to the receptor must be ener-
getically similar to solvation by water molecules, otherwise
the substrate cannot be pulled out of the aqueous phase by
complex formation. Thus the strongest noncovalent interac-
tions are required, such as electrostatic interactions or
[a] Dr. C. Urban, Prof. Dr. C. Schmuck
Universitꢀt Duisburg-Essen, Institut fꢁr Organische Chemie
Universitꢀtsstrasse 7, 45141 Essen (Germany)
Fax : (+49)201-183-4256
E-mail: carsten.schmuck@uni-due.de
9502
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 9502 – 9510

FULL PAPER
alone or in combination with additional binding sites ever
since (e.g., also by Zinic and Gokel, Voyer, Reetz, or more
recently by Hosgçren).[6] Enantioselective transport of
amino acid derivatives was accomplished with chiral crown
ether derivatives. By using the high affinity of crown ethers
for potassium ions, active transport of amino acids can also
be achieved when the transport of the amino acid is coupled
to a counter-transport of the alkali metal cation. Charged
hosts have also been used for amino acid transport. For ex-
ample, in early work Rebek used a Kempꢃs triacid derived
acridinium–carboxylate zwitterionic host to extract free
zwitterionic amino acids from aqueous solutions into chloro-
form.[7] Transport was also reported, but suffered from the
hydrolytic instability of the host. Another zwitterionic trans-
porter based on a sapphyrin–lasalocid conjugate was report-
ed by Sessler.[8] This system preferentially binds aromatic
amino acids in their zwitterionic form by double-ion-pair
formation and transports them over a bulk liquid mem-
brane. Negatively charged N-protected amino acid carboxy-
lates can be transported by cationic transporters. For exam-
ple, Davis developed a steroidal guanidinium receptor,
which enantioselectively transported N-acetylated amino
acids through a bulk liquid membrane in a U-tube experi-
ment.[9] Another elegant guanidinium cation based trans-
porter is a crown ether conjugate developed by de Mendoza
which facilitates passive transport of underivatized zwitter-
ionic amino acids under neutral conditions.[10] The disad-
vantage of classical guanidinium cation based transporters is
the high basicity of the guanidinium group, which prevents
deprotonation under ambient conditions (the pKa of the
guanidinium cation is ca. 13.5). Hence, the transporter is
always protonated. Transport of N-acylated amino acid car-
boxylates by guanidinium-based receptors thus requires the
counter-transport of another anion (most often halide) from
the receiving phase back into the starting phase. Using a
corresponding halide gradient in the back direction allows
active transport of the amino acid to the receiving phase,
but the overall transport always is a counter-transport. In
this context we present here the first example, to the best of
our knowledge, of an artificial symporter 9, which allows for
active transport of N-acylated amino acid carboxylates by
using a pH gradient as the driving force.
amino acids by this class of cationic receptors. Also stereose-
lective complexation of small oligopeptides in aqueous solu-
tion was achieved.[13] We, therefore, reasoned that this
highly efficient binding motif could also be used for the
transport of N-acetylated amino acids across a bulk liquid
membrane. However, this was so far hampered by the insol-
ubility of this receptor class in nonpolar organic solvents.
We have, therefore, now developed an amphiphilic deriva-
tive 9, which efficiently transports N-acylated amino acid
carboxylates across a bulk liquid membrane of chloroform
and also allows an active symport of the carboxylate and a
proton by using a pH gradient as the driving force.
To increase the solubility of the guanidiniocarbonyl pyr-
role receptor in organic solvents, a lipophilic tris(dodecyl-
benzyl) group was attached.[14] It contains three long alkyl
chains, which should ensure sufficient solubility in nonpolar
organic solvents, while also preventing the receptor from en-
tering the aqueous phase. To connect this moiety to the gua-
nidiniocarbonyl pyrrole cation moiety without affecting its
anion binding properties, we used an orthogonally protected
pyrrole triester scaffold recently developed by us.[15] The
synthesis of the transporter 9 thus started with the triacid
derivative 2 (Scheme 1). Relative to the literature protocol,
we could further improve the yield of 2 by adjusting the
conditions for the oxidation of 1 by excluding light and lim-
iting the use of diethyl ether, thus reducing unwanted radi-
cal side reactions. By using our initially reported amino acid
receptor[11] as a blueprint, we then attached l-Val-NH2 to
the pyrrole before introducing the protected guanidine. The
ester functionality in the propionic acid side chain can be se-
lectively hydrolyzed by using LiOH in THF/water mixtures.
To this free carboxylic acid group, the lipophilic amine 7
was coupled by using HCTU as the coupling reagent.
Amine 7 was synthesized by starting from the corresponding
hydroxyl compound, which can be obtained in two steps
from commercially available gallic acid ethyl ester according
to a literature procedure.[16] The hydroxyl compound was
first transformed into the bromide with phosphorous tribro-
mide. Subsequently the bromide was reacted with sodium
azide and the resulting azide was reduced with lithium alu-
minum hydride to yield the desired amine 7 in 71% over all
steps. Finally all protecting groups were cleaved off by using
TFA in CH2Cl2. Lyophilization with aqueous HCl then
yielded transporter 9 as the chloride salt.
Results and Discussion
Extraction experiments: To examine the ability of 9 to trans-
fer anionic N-acylated amino acids into an organic phase,
first extraction experiments were performed. Receptor 9
(1 mm) was dissolved in chloroform and used to extract the
N-acetylated amino acids valine, tyrosine, and tryptophane
(10 mm) from aqueous buffered solutions of pH 6. This pH
was chosen to ensure that 9 will be protonated as needed
for anion binding, while at the same time the amino acids
will still be present as the carboxylate. This allows ion-pair
formation between the receptor and the amino acid sub-
strate in the organic phase. The amino acid uptake into the
organic phase was quantified by using HPLC with an RP-8
Receptor design and synthesis: A few years ago we intro-
duced the guanidiniocarbonyl pyrrole cation as a highly effi-
cient oxoanion binding site, which also allows complex for-
mation even in aqueous solutions.[11] Substrate binding is
due to the formation of a hydrogen bond directed ion pair
with the anion. One reason for the improved anion binding
efficiency of the guanidiniocarbonyl cation relative to
simple guanidinium cations is its lower pKa value of approxi-
mately 6–7.[12] Hence, the guanidinium NHs are significantly
more acidic and thus better hydrogen bond donors. We ob-
served side-chain selective binding of anionic N-acetylated
Chem. Eur. J. 2010, 16, 9502 – 9510
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9503

C. Schmuck and C. Urban
Scheme 1. Synthesis of transporter 9. DCM=dichloromethane, PyBOP=(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate,
NMM=N-methylmorpholine, DMAP=4-dimethylaminopyridine, TFA=trifluoroacetic acid, HCTU=O-(6-chloro-1-hydrocibenzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate.
column and UV detection at 220 nm. All three amino acids
were transferred into the organic phase in approximately
equimolar amounts to 9 (Table 1).
The differences in the extraction efficiency and the rela-
tive order relate to the hydrophobicity of the amino acids[17]
Table 1. Distribution constants of N-acetylated amino acids between
aqueous BIS-TRIS buffer at pH 6 and chloroform and calculated binding
constants with receptor 9.[a]
[b]
Substrate
Kex
Extraction efficiency [%][c]
Kass [mꢀ1
]
Ac-Val-OH
Ac-Tyr-OH
Ac-Trp-OH
0.149
0.046
0.071
126
92
106
4.3ꢄ103
1.5ꢄ104
1.4ꢄ104
Figure 1. Extraction of an anionic substrate Sꢀ by the cationic receptor 9
from an aqueous phase into an organic phase.
[a] Conditions for the extraction: 1 mL of 100 mm BIS-TRIS buffer at
pH 6 with a substrate concentration of 10 mm was extracted with 1 mL of
a 1 mm solution of receptor 9 in chloroform. [b] Distribution constant
K
ex =corg/caq. [c] Concentration of substrate in the organic phase as a per-
centage of receptor concentration. Note that the efficiency can be more
than 100%, because free substrate is also distributed between the two
phases.
corgðRSÞ
Kass
¼
corgðSꢀÞcorgðRHþÞ
and their intrinsic solubility in chloroform, which were also
determined in control experiments. The valine derivative
was extracted the most, followed by tryptophane and then
tyrosine. From the extraction experiments, also the binding
constants of the amino acids to 9 were calculated based on
the binding and exchange equilibria shown in Figure 1.
According to the law of mass action the association con-
stant Kass is defined as follows:
corg(RS) is the concentration of receptor–substrate complex
in the organic phase, corg(Sꢀ) is the concentration of free
amino acid (present as the carboxylate) in the organic phase
and corgACHTUNGTRNEUNG
(RH+) the concentration of the free cationic receptor
in the organic phase. The concentration of free substrate in
the organic phase depends on its distribution between the
aqueous and organic phase according to the Nernst distribu-
tion law:
9504
www.chemeurj.org
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 9502 – 9510

Active Transport of Amino Acids
FULL PAPER
corgðSꢀÞ ¼ KexcaqðSꢀÞ
(Figure 2), in which the aromatic indole ring indeed p-stacks
with the planar guanidiniocarbonyl pyrrole cation. The top
face is shielded by the tris(dodecylbenzyl) group, whereas
the carboxylate forms an hydrogen bond enforced ion pair
with the cation as known from related receptors.
Kex is the distribution constant and was measured by extrac-
tion experiments of the N-acetylated amino acids in the ab-
sence of 9 (data given in Table 1) and caq(Sꢀ) is the concen-
tration of the amino acid in the aqueous phase, which was
determined experimentally by HPLC measurements. The
amount of receptor–substrate complex in the organic phase
could not be measured directly, only the overall amount of
free and bound substrate corgACTHNUTRGNEUNG
(Sꢀ+RS) could be determined
by subtracting the amount of substrate in the aqueous phase
caq(Sꢀ) from the total concentration c0(Sꢀ).
corgðSꢀ þ RSÞ ¼ corgðSꢀÞ þ corgðRSÞ ¼ c0ðSꢀÞꢀcaqðSꢀÞ
Combining these formulas, the association constant Kass can
be calculated as follows:
c0ðSꢀÞꢀcaqðSꢀÞꢀKexcaqðSꢀÞ
Kass
¼
kexcaqðSꢀÞ½c0ðRÞꢀc0ðSꢀÞ þ caqðSꢀÞ þ KexcaqðSꢀÞꢁ
This formula is derived on the assumption that the receptor
and the receptor–substrate complex are only present in the
organic phase and not in the aqueous phase (Figure 1). That
this is a valid assumption could be proven by UV measure-
ments of the aqueous phase, which traced at most a minute
amount of 9 (<1%) in the aqueous phase. Hence, com-
pound 9 due to the large lipophilic tris(dodecylbenzyl)
group is nearly insoluble in water despite its charge. The as-
sociation constants calculated by using this formula are
given in Table 1. The aromatic amino acids tryptophan and
tyrosine have similar binding constants of Kass =1.4ꢄ104 and
1.5ꢄ104 mꢀ1, respectively. They are bound about three times
Figure 2. Calculated energy-minimized structure of the complex between
l-N-Ac-Trp (yellow) and 9 (grey) (chloroform solvation model). The
alkyl chains of the trisACTHNUTRGENUG(N dodecyl) benzyl group were omitted for clarity.
Transport experiments: After we successfully established
that receptor 9 is soluble in organic solvents, but not in
water and efficiently binds amino acid carboxylates even in
the presence of water, we measured the transport capacity
of 9 in a U-tube experiment. The experimental setup is
shown in Figure 3.
stronger than the nonaromatic amino acid valine (Kass
=
4.3ꢄ103 mꢀ1). Similar association constants of approximatley
103–104 mꢀ1 were obtained by using UV/Vis spectroscopy in-
stead of HPLC to determine the concentration of the amino
acids in the aqueous phase. Hence, the amino acid carboxy-
lates are bound very efficiently by 9. In accordance with the
less-polar solvent (wet chloroform), the binding constants
are approximatley one order of magnitude larger than previ-
ously reported data for amino acid binding by guanidiniocar-
bonyl pyrrole receptors in aqueous DMSO.[11b]
The preference for aromatic side chains corresponds to
the binding properties of guanidiniocarbonyl pyrrole recep-
tors in general, as already observed in earlier studies.[11a]
Cation-p-stacking interactions[18] between the aromatic side
chain and the planar guanidiniocarbonyl pyrrole cation fur-
ther stabilize the complex in addition to the ion-pair forma-
tion. Experiments with the l- and d-enantiomers of the
amino acids (data not shown) did not reveal any stereoselec-
tivity; the binding constants were identical within the error
margin of this experiment (ꢂ10%). A simple force-field cal-
culation confirmed the idea of cation-p-interactions. A Mon-
teCarlo conformational search (Macromodel V8,[19] amber*
force field, GB/SA solvation model for chloroform,
50.000 steps) revealed an energy-mimimized structure
Figure 3. Setup of the U-tube experiment for the determination of trans-
port rates.
As the source phase, 3.0 mL of a 50 mm solution of the N-
acetylated amino acid in 100 mm BIS-TRIS buffer at pH 6
were used and as the transport layer 3.5 mL of a 1 mm solu-
Chem. Eur. J. 2010, 16, 9502 – 9510
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9505

C. Schmuck and C. Urban
Table 2. Results of the transport experiments.
tion of receptor 9 in chloroform were used. The receiving
phase consisted of 3.0 mL of 100 mm BIS-TRIS buffer at
pH 8. These conditions assured that the substrates were
transported in the direction of the receiving phase, due to
the different protonation states and hence anion binding
properties of 9 at these two pH values. The receptor 9 has a
pKa of approximately 7 and is, therefore, protonated at pH 6
at the interface to the source phase, whereas 9 will be de-
protonated at the interface with the receiving phase (pH 8).
However, anion binding under these conditions occurs only
by the cationic receptor and not by the deprotonated neutral
one. As shown in Figure 4, the transport is a symport of the
negatively charged amino acid carboxylate and a proton.
Substrate
Flux (single)
Flux (comp.)
Flux (control)
[a]
[b]
[molmꢀ2 sꢀ1
]
[molmꢀ2 sꢀ1
]
[molmꢀ2 sꢀ1
]
Ac-Val-OH
Ac-Phe-OH
Ac-Ala-OH
Ac-Trp-OH
Ac-Tyr-OH
1.11ꢄ10ꢀ6
4.67ꢄ10ꢀ7
1.92ꢄ10ꢀ7
8.05ꢄ10ꢀ8
6.24ꢄ10ꢀ8
1.54ꢄ10ꢀ7
2.03ꢄ10ꢀ7
2.30ꢄ10ꢀ8
2.10ꢄ10ꢀ7
5.65ꢄ10ꢀ8
1.23ꢄ10ꢀ7
4.54ꢄ10ꢀ8
n/a
4.62ꢄ10ꢀ8
2.20ꢄ10ꢀ9
[a] Conditions: the source phase was 3 mL of 100 mm BIS-TRIS buffer at
pH 6 with a substrate concentration of 50 mm, the organic phase was
3.5 mL of a 1 mm solution of receptor 9 in chloroform, the receiving
phase was 3 mL of 100 mm BIS-TRIS buffer at pH 8. [b] Conditions: the
source phase was 3 mL of 100 mm BIS-TRIS buffer at pH 6 with sub-
strate concentrations of 2 mm each, the organic phase was 3.5 mL of a
1 mm solution of receptor 9 in chloroform, the receiving phase was 3 mL
of 100 mm BIS-TRIS buffer at pH 8.
Figure 4. Transport of acetylated amino acid carboxylates (S) by receptor
9 (R). Only the protonated form RH+ of the receptor can bind the
anionic substrate Sꢀ and transport it across the bulk liquid membrane.
The experiments were conducted with the N-acetylated l-
amino acids of alanine, valine, tyrosine, phenylalanine, and
tryptophan. At first transport was measured for each amino
acid alone. Samples from the receiving phase were taken
over a period of one to two weeks and were analyzed by
HPLC analysis on a RP-8 column with UV detection at
220 nm. Prior to the experiments, the aqueous source phase
and the organic phase were equilibrated by shaking the two
phases together for 5 min. Otherwise an induction period
was observed before the transport started. Each experiment
was performed twice along with a control experiment. For
the control experiments, the exact same setup and proce-
dure, but without the receptor 9, was used. The data given
are the average values of both runs. To test for enantioselec-
tive transport, the experiments were also carried out with
the d-enantiomers (data not shown), but as in the extraction
experiments the differences were found to be within the
error range, which was estimated to be in a range of ꢂ10%.
The results are summarized in Table 2 and shown in
Figure 5.
As the data show, receptor 9 is capable of efficiently
transporting the amino acids studied across the chloroform
phase. The efficiency (as, for example, expressed by the flux
values), significantly depends on the amino acid and de-
creases in the order Val>Phe>Ala>Trp >Tyr (Figure 5,
red columns). The valine derivative was transported fastest,
with a very high flux of 1.11ꢄ10ꢀ6 molmꢀ2 sꢀ1. In compari-
son, the tyrosine derivative was transported 18 times slower
with a flux of 6.24ꢄ10ꢀ8 mol mꢀ2 sꢀ1 (Table 2). Whereas the
flux determined in the control experiments reflects the hy-
drophobicity of the amino acids, the transport rates in the
Figure 5. Comparison of the flux values of the single, competitive, and
the control experiment.
presence of a receptor showed a different behavior.[17] The
valine, phenylalanine, alanine, and tyrosine derivatives are
roughly in the expected relative order, whereas tryptophan
is transported slower than would be expected based on its
hydrophobicity. Even more interesting is that these trans-
port rates are not in accord with the relative order of associ-
ation constants as determined in the extraction experiments
(Table 1). Tryptophan and tyrosine are both bound signifi-
cantly more strongly by 9 than valine, but are transported
more than one order of magnitude slower. This shows that
the transport rate under this experimental setup is not deter-
mined by the affinity of the substrate to the transporter. The
transporter is present in only 2.3 mol% relative to the
amount of amino acid in the source phase. The rate-deter-
mining step is, therefore, not the binding of the substrate
but its release upon contact with the receiving phase. The
stronger a substrate binds to 9, the higher is the energy bar-
rier for its release from the receptor–substrate complex, in
accord with the Bell–Evans–Polanyi principle. Therefore the
most stable complexes show the slowest exchange rates and
thus the slowest overall transport rate.
We also performed a competition experiment, in which all
five amino acids were present at the same time (Figure 5,
light grey columns). In this experiment, quite different
values for flux and selectivity were found. The individual
9506
www.chemeurj.org
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 9502 – 9510

Active Transport of Amino Acids
FULL PAPER
overall transport rates are slower than in the first experi-
ment, due to the much smaller concentration of each amino
acid in the source phase (2 mm instead of 50 mm, respective-
ly). Furthermore, the relative transport rate decreases in the
order Trp>Phe>Val >Tyr >Ala. Tryptophan is now trans-
ported the fastest and approximately nine times faster than
the slowest substrate alanine. This change in the relative
order of the transport efficiency shows that under competi-
tive conditions the binding strength is now the determining
factor for transport and not the release rate. With direct
competition for the receptor, the substrates with higher
binding constants are preferred. The substrates with weaker
binding affinity cannot compete for the receptor at the inter-
face of the source phase and accordingly cannot be trans-
ported across the organic phase. For example, tryptophan,
which has the highest affinity for receptor 9, consequently
also has the highest transport rate in the competitive experi-
ment, whereas in the single experiments it showed one of
the slowest transport rates of all substrates. The opposite is
true for valine, which due to its significantly weaker binding
affinity has no chance in the competitive experiment, as it
cannot beat the other substrates. But in the individual ex-
periment its faster release from the complex at the interface
with the receiving phase is decisive.
crease in the concentration of the substrate in the receiving
phase relative to the source phase and thus creates a differ-
ence in chemical potential that thermodynamically favors
the back transport of the substrate. pH gradient driven
active transport of the amino acid can occur as long as the
sum of the chemical potentials of both species is still nega-
tive. As shown in Figure 6, in our experimental setup active
Active transport: By using equal concentrations of amino
acid in both the source and the receiving phase, active trans-
port against the concentration gradient was investigated by
using phenyl alanine as the substrate. And indeed efficient
transport with a concentration change of approximately
15 mm within 15 days was observed. The measured transport
velocity of 4.91ꢄ10 ꢀ7 molmꢀ2 sꢀ1 is similar to the rate mea-
sured in the single experiment. The driving force for this
transport against the concentration gradient of the amino
acid stems from the proton gradient between the source
(pH 6) and receiving phase (pH 8). As the transport scheme
in Figure 4 shows, transport of the substrate is coupled to
the transport of a proton in the same direction. Hence, sym-
port of the amino acid carboxylate and a proton occurs and
overall the uncharged N-acetylated protonated amino acid
is thus transported. Thereby the thermodynamically favora-
ble transport of the proton along the pH gradient drives the
unfavorable transport of the substrate. This active transport
can occur as long as the overall chemical potential for this
symport is favorable. In general, any concentration gradient
between the source and receiving phase creates a difference
in the chemical potential Dm and thus a thermodynamic
driving force for transport as long as the concentrations are
not equal and Dm did not yet reach zero.
Figure 6. Change in the chemical potential during transport. c=Cou-
pled transport of proton and substrate, a=50 mm substrate in both
phases, and g=100 mm buffer (pH 6 to 8).
transport can occur until the concentration of the amino
acid in the receiving phase has increased to 74.4 mm, thus
approximately half of the amino acid has been transported
from the source phase to the receiving phase.
Conclusion
We here have presented a new transporter 9, which allows
pH gradient driven active transport of N-acetylated amino
acids. Transporter 9 combines the efficient anion binding
properties of a guanidiniocarbonyl pyrrole cation with the
hydrophobic character of a tris(dodecylbenzyl) group, thus
ensuring solubility of 9 in a nonpolar organic phase. Due to
the lowered pKa of 9 relative to a simple guanidinium
cation, 9 can both be protonated and deprotonated around a
neutral pH. However, only the protonated, cationic form of
9 binds anions. Hence, by using a pH gradient between the
source and receiving phase, symport of the amino acid car-
boxylate and a proton occurs. In transport experiments of
single amino acids (with an excess of substrate over the
transporter), the transport rates are determined by the re-
lease rate of the substrate from the complex at the interface
with the receiving phase. The substrates that are bound to
the weakest are, therefore, transported the fastest and vice
versa. When several substrates have to compete for the
c2
Dm ¼ RTIn
c1
As the concentration of H3O+ ions in both phases
changes over the course of the experiment due to the sym-
port of substrate and proton so does the difference in the
chemical potential. Accordingly, the transport leads to an in-
Chem. Eur. J. 2010, 16, 9502 – 9510
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9507

C. Schmuck and C. Urban
1H NMR (400 MHz, CDCl3): d=1.05 (t, 3JHꢀH =8.28 Hz, 6H; CHCH-
transporter in a competition experiment, the thermodynamic
stability of the receptor–substrate complex determines the
transport rate. As receptor 9 prefers aromatic amino acids
due to additional cation-p-interactions within the complex,
tryptophan and phenylalanine show the fastest transport
rates. In all experiments transport is very efficient with
fluxes in the order of 10ꢀ6 molmꢀ2 sꢀ1 (similar to the most ef-
ficient artificial transporters reported in the literature so
far). The use of a pH gradient from pH 6 in the source
phase to 8 in the receiving phase also enabled active trans-
port of the amino acid carboxylate against a concentration
gradient. The transport of the substrate is coupled to the si-
multaneous transport of a proton (symport), which provides
the thermodynamic driving force.
A
ACHTNUGERTN(NUGN CH3)3), 2.24 (s, 3H; pyrrole-CH3), 2.26–2.31 (m,
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
A
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
role-C), 161.0, 162.4, 171.0, 174.2 ppm (carbonyl-C); IR: (solid): n˜3316
(w), 3189 (w), 2961 (w), 1734 (w), 1673 (s), 1619 (s), 1534 (w), 1433 (m),
1368 (w), 1309 (m), 1247 (m), 1149 (s), 1099 (w), 975 (w), 837 (s), 781
(m), 664 (m), 619 cmꢀ1 (w); HRMS (pos. ESI): m/z: calcd for
C20H31N3NaO6: 432.2107 [M+Na]+; found: 432.2105.
5-[(R)-1-Carbamoyl-2-methylpropylcarbamoyl]-4-[2-(methoxycarbon
ACHTUNGTRENNUNGyl)-
A
3
0.896 mmol) was dissolved in TFA/CH2Cl2 3:1 (20 mL) and was stirred at
room temperature for 5 h. The solvent was removed under reduced pres-
sure and the residue was suspended in water and lyophilized. Compound
4 was obtained (308 mg, 0.872 mmol, 97%) as a colorless solid. M.p.
2058C; 1H NMR (400 MHz, CDCl3): d=0.88 (d, 3JHꢀH =6.76 Hz, 6H;
Experimental Section
CHCHACHTUNGRTENNUG ACHTUGNTREN(NUGN CH3)2), 2.16 (s, 3H;
(CH3)2), 2.02 (q, 3JHꢀH =6.82 Hz, 1H; CHCH
3
General: 1H and 13C NMR spectra were recorded on a Bruker Avance-
400 spectrometer at 258C. Chemical shifts are reported relative to residu-
al undeuterated solvent peaks. Melting points were determined on a
Bꢁchi melting point apparatus SMP-20. IR spectra were obtained from a
Jasco FTIR 410 on a diamond ATR crystal. Flash column chromatogra-
phy was carried out on MP Biomedicals GmbH silica gel MP-Silica 32–
63, 60 ꢅ. Reagents were purchased from Aldrich, Fluka, Lancaster, Iris
Biotech, or Sigma and were used without further purification. All sol-
vents were distilled prior to use; water for chromatography, extraction,
and transport experiments was taken from a TKA MicroPure water
system. HPLC spectra were recorded on a system consisting of a Shimad-
zu LP-6A Liquid Chromatograph pump with a SCL-6B System Control-
ler, a Rheodyne 7125 with 20ml sample loop and a SPD-6A Spectropho-
tometric Detector. HPLC columns were Supelcosil LC-8 25 cmꢄ4.6 mm,
5 mm and Astec Chirobiotic T.
pyrrole-CH3), 2.41 (t, JHꢀH =7.74 Hz, 2H; pyrrole-CH2CH2), 2.90 (t, 2H,
3
3JHꢀH =7.76 Hz; pyrrole-CH2), 3.52 (s, 3H; OCH3), 4.24 (t, 1H, JHꢀH
=
3
4.14 Hz; CHCH
(CH3)2), 6.99 (s, 1H), 7.54 (s, 1H), 8.19 (d, JHꢀH
=
8.32 Hz, 1H; amide-NH), 11.88 ppm (s, 1H; pyrrole-NH); 13C NMR
(100 MHz, CDCl3): d=10.0 (pyrrole-CH3), 18.8 (CHCH
(pyrrole-CH2), 30.6 (CHCH(CH3)2), 34.7 (pyrrole-CH2CH2), 51.8
(OCH3), 58.3 (CHCH(CH3)2), 120.1, 124.7, 126.0, 128.6 (pyrrole-C),
ACHTUGNTRNEN(UNG CH3)2), 20.4
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
160.7, 162.9, 173.8, 174.1 ppm (carbonyl-C); IR (solid): nꢆ=>=3315
(m), 2959 (w), 1676 (s), 1615 (s), 1576 (w), 1540 (w), 1487 (m), 1436 (m),
1305 (m), 1272 (s), 1242 (m), 1196 (w), 1169 (s), 981 (w), 898 (w), 834
(w), 720 cmꢀ1 (m); HRMS (pos. ESI): m/z: calcd for C16H23N3NaO6:
376.1479 [M+Na]+; found: 376.1484.
2-Mono-tert-butoxycarbonyl (Boc)-guanidinocarbonyl-5-[(R)-1-carbamo-
yl-2-methylpropylcarbamoyl]-3-methylpyrrole-4-propionic acid methyl
ester (5):
A solution of compound 4 (680 mg, 1.93 mmol), HCTU
3-(2-Methoxycarbonylethyl)-4-methyl-1H-pyrrole-2,5-dicarboxylic acid-5-
tert-butyl ester (2): Compound 1 (2.00 g, 7.11 mmol) and K2CO3 (4.00 g,
28.9 mmol) were suspended under Ar in anhydrous Et2O (50 mL) under
the exclusion of light and cooled down to 08C. Freshly distilled sulfuryl
chloride (2.33 mL, 24.9 mmol) was added slowly through a septum with a
syringe. The mixture was warmed slowly to room temperature and stirred
for 2 h at room temperature. The solvent was removed at room tempera-
ture under reduced pressure and the oily residue was stirred for 1 h in a
solution of NaOAc (5.00 g, 61.0 mmol) in water/dioxane (1:1, 100 mL) at
1108C. The solution was cooled down to 08C and adjusted to pH 2 with
concentrated hydrochloric acid. The solution was extracted with Et2O
(3ꢄ50 mL). The combined organic phases were extracted with a half-sa-
turated aqueous solution of NaHCO3 (3ꢄ50 mL) and the combined
aqueous solutions were cooled down to 08C. This aqueous solution was
then acidified slowly and under vigorous stirring with concentrated hy-
drochloric acid to pH 2. The precipitate was filtered off and washed with
cold water (3ꢄ50 mL) to give 5 (1.79 g, 5.76 mmol, 81%) as a white
solid. M.p. 1698C; 1H NMR (400 MHz, DMSO): d=1.52 (s, 9H; C-
(865 mg, 2.10 mmol) and Boc-guanidine (334 mg, 2.10 mmol) in CH2Cl2/
DMF 5:1 (24 mL) with NMM (1 mL) was stirred for 1 h at room temper-
ature. Water (20 mL) was added, the phases were separated and the
aqueous phase was extracted with CH2Cl2 (3ꢄ20 mL). The combined or-
ganic phases were dried with Na2SO4, the solid was filtered, and the sol-
vent was removed under reduced pressure. The resulting solid was puri-
fied by flash column chromatography (silica gel, CH2Cl2/MeOH 20:1) to
give
(decomp.); 1H NMR (400 MHz, CDCl3): d=1.04 (dd, 3JHꢀH =6.76,
4JHꢀH =2.76 Hz, 6H; CHCH
(CH3)2), 1.49 (s, 9H; C(CH3)3), 2.35 (s, 3H;
pyrrole-CH3), 2.26–2.38 (m, 1H; CHCH
(CH3)2), 2.67 (t, 3JHꢀH =7.02 Hz,
2H; pyrrole-CH2CH2), 3.01 (m, 2H; pyrrole-CH2), 3.64 (s, 3H; OCH3),
5 (536 mg, 1.08 mmol, 56%) as a colorless solid. M.p. 1498C
A
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
4.44 (t, 1H, 3JHꢀH =8.02 Hz; CHCH
ACTHNUGTRNEUGN(CH3)2), 6.26 (s, 1H), 6.54 (s, 1H),
8.39 (d, 3JHꢀH =8.20 Hz, 1H; amide-NH), 8.71 (s, 1H), 10.3 ppm (s, 1H;
pyrrole-NH); 13C NMR (100 MHz, CDCl3): d=10.7 (pyrrole-CH3), 18.9
(CHCH
G
N
ACHTUNGTRENNUNG(CH3)2),
34.1 (pyrrole-CH2CH2), 52.1 (OCH3), 59.5 (CHCHAHCTUNGTRENNNUG
ACHTUNGTRENNUNG
(CH3)3), 2.17 (s, 3H; pyrrole-CH3), 2.44 (t, 3JHꢀH =8.10 Hz, 2H; pyrrole-
3
(pyrrole-C), 162.1, 174.9 (carbonyl-C). IR (solid): n˜ =3391 (w), 1725 (w),
1672 (w), 1625 (s), 1541 (m), 1434 (m), 1309 (m), 1238 (s), 1147 (s), 835
(s), 625 (m), 611 cmꢀ1 (w); HRMS (pos. ESI): m/z: calcd for
C22H34N6NaO7: 517.2381 [M+Na]+; found: 517.2378.
CH2CH2), 2.90 (t, 2H, JHꢀH =8.10 Hz; pyrrole-CH2), 3.57 (s, 3H; OCH3),
11.2 ppm (s, 1H; pyrrole-NH); 13C NMR (100 MHz, CDCl3): d=9.7 (pyr-
role-CH3), 19.7 (pyrrole-CH2), 28.0 (C
51.2 (OCH3), 80.5 (C(CH3)3), 119.3, 122.4, 125.0 (pyrrole-C), 159.9, 161.8,
172.7 ppm (carbonyl-C).
5-[(R)-1-Carbamoyl-2-methylpropylcarbamoyl]-4-[2-(methoxycarbon
ACHTUNGTRENNUNGethyl]-3-methylpyrrole-2-tert-butylester (3): A solution of compound 2
ACHTUNGNERT(UNNG CH3)3), 34.3 (pyrrole-CH2CH2),
ACHTUNGTRENNUNG
2-Mono-Boc-guanidinocarbonyl-5-[(R)-1-carbamoyl-2-methylpropylcar-
bamoyl]-3-methylpyrrol-4-propionic acid (6): A mixture of compound 5
(250 mg, 0.505 mmol) and lithium hydroxide (36.5 mg, 1.52 mmol) in
THF/H2O 5:1 (24 mL) was stirred for 5 h at room temperature. The sol-
vent was evaporated under reduced pressure. HCl (1m) was added to the
residue until pH 4 was reached. The precipitate was filtered off and
AHCTUNGTERGyNNUN l)-
(500 mg, 1.61 mmol), l-Val-NH2 (369 mg, 2.41 mmol), HCTU (797 mg,
1.92 mmol), and DMAP (30 mg, 0.245 mmol) in CH2Cl2/DMF 5:1
(25 mL) with NMM (1 mL) was stirred for 24 h at room temperature.
The solvent was removed under reduced pressure to give an oily residue.
Flash column chromatography (silica gel, CH2Cl2/MeOH 15:1+1%
HOAc) yielded a colorless solid (643 mg, 1.57 mmol, 98%). M.p. 2028C;
lyophilized to give 6 (150 mg, 0.312 mmol, 62%) as a colorless solid. M.p.
3
2168C (decomp.); 1H NMR (400 MHz, CDCl3): d=0.91 (d, JHꢀH
=
6.72 Hz, 6H; CHCH
ACHTUTGNRENNGU(CH3)2), 1.49 (s, 9H; CCAHTUNGTREN(NUNG CH3)3), 2.03–2.11 (m, 1H;
CHCH
AHCTUNGTRENNUNG
9508
www.chemeurj.org
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 9502 – 9510

Active Transport of Amino Acids
FULL PAPER
3
pyrrole-CH2CH2), 2.86–2.92 (m, 2H; pyrrole-CH2), 4.30 (t, 1H, JHꢀH
=
=
left to stand for another 10 min or until the foam had disappeared. Sam-
ples of 50 mL were taken out of the aqueous phase and either directly in-
jected into the HPLC with an UV detector or in the case of tryptophan
and tyrosine diluted tenfold and then injected. Spectra were recorded at
220 nm. For the injection, the 20 mL sample loop was used to measure ex-
actly equal quantities. The experiments were carried out twice and the
average value was determined. Solutions of defined substrate concentra-
tion were used for calibration of the UV-detector.
3
7.64 Hz; CHCHACHTUNGTRENNUNG(CH3)2), 7.03 (s, 1H), 7.48 (s, 1H), 8.18 (d, JHꢀH
8.60 Hz, 1H; amide-NH), 8.81 (s, 1H), 9.58 (s, 1H), 11.66 ppm (s, 1H;
pyrrole-NH); 13C NMR (100 MHz, CDCl3): d=10.2 (pyrrole-CH3), 18.3
(CHCH
N
U
ACHTUGNRTNE(NUNG CH3)2),
34.4 (pyrrole-CH2CH2), 57.7 (CHCH
N
ACHTUNGTRENNUNG
role-C), 160.2, 173.1, 174.3 ppm (carbonyl-C); IR (solid): n˜ =3318 (w),
2965 (w), 1670 (m), 1626 (s), 1537 (s), 1409 (m), 1285 (s), 1147 (s), 1091
(w), 845 (w), 754 (m), 619 cmꢀ1 (m); HRMS (pos. ESI): m/z: calcd for
C21H32N6NaO7: 503.2225 [M+Na]+; found: 503.2230.
U-tube transport experiments: The form of the U-tubes was as seen in
Figure 3. The stirring bar was 7 mm long and 2 mm in diameter. The or-
ganic phase consisted of 3.5 mL of a 1 mm solution of receptor 9 in
chloroform or pure chloroform, respectively, for the control experiments.
The stirring bar was rotated at 1250 rpm. In the single substrate experi-
ments, the source phase consisted of 3 mL of a 50 mm solution of the re-
spective substrate in 100 mm BIS-TRIS buffer, which was adjusted to
pH 6 by the addition of 0.1 mm HCl and 0.1 mm NaOH. The receiving
phase consisted of 3 mL of 100 mm BIS-TRIS buffer that was adjusted to
pH 8. In the active-transport experiments, the source phase consisted of
3 mL of a 50 mm solution of Ac-Phe-OH in 100 mm BIS-TRIS buffer at
pH 6 and the receiving phase consisted of 3 mL of a 50 mm solution of
Ac-Phe-OH in 100 mm BIS-TRIS buffer at pH 8. In the competitive ex-
periments, the source phase consisted of 3 mL of a solution 2 mm in con-
centration of each of the employed substrates Ac-Ala-OH, Ac-Val-OH,
Ac-Phe-OH, Ac-Tyr-OH, and Ac-Trp-OH. The receiving phase consisted
of 3 mL of 100 mm BIS-TRIS buffer that was adjusted to pH 8. Prior to
the experiments, the organic phase was equilibrated with the source
phase by manually shaking the two solutions for 5 min in a 10 mL glass
vial. The two solutions were then left to stand to separate and afterwards
they were filled into the U-tube. The U-tubeꢃs upper ends were sealed
and the experiment was started by stirring. To measure the concentration
of substrate, samples of 50 mL were taken of the aqueous phases and in-
jected into the HPLC with a UV detector or in the case of tryptophan
and tyrosine diluted tenfold and then injected. Spectra were recorded at
220 nm. For the injection, the 20 mL sample loop was used to measure ex-
actly equal quantities. The experiments were carried out twice and the
average value was determined. Solutions of defined substrate concentra-
tion were used for calibration of the UV detector.
2-Mono-Boc-guanidinocarbonyl-3-methyl-4-ethyl-pyrrol-5-[(R)-3-methyl-
2-carbamoyl]butylamide (8): A solution of 6 (110 mg, 0.229 mmol), 7
(190 mg, 0.288 mmol), and HCTU (119 mg, 0.288 mmol) in CH2Cl2/DMF
2:1 (15 mL) with NMM (0.5 mL) was stirred at room temperature for
3 d. Water (15 mL) was added and the phases were separated. The aque-
ous phase was extracted with CH2Cl2 (3ꢄ15 mL), the combined organic
phases were dried with MgSO4, and the solid was filtered off. The solvent
was evaporated under reduced pressure and the residue was purified by
flash column chromatography (silica gel, cyclohexane/ethyl acetate 1:1+
1% HOAc) to give 8 (153 mg, 0.136 mmol, 59%) as a colorless solid.
M.p. 928C; 1H NMR (400 MHz, CDCl3): d=0.88 (t, 3JHꢀH =6.82 Hz, 9H;
CH2CH3), 1.04 (d, 3JHꢀH =6.72 Hz, 6H; CHCH
49H; CH2 d-l), 1.39–1.47 (m, 6H; OCH2CH2CH2), 1.49 (s, 8H; C
1.67–1.80 (m, 6H; OCH2CH2), 2.25–2.33 (m, 1H; CHCH(CH3)2), 2.35 (s,
3H; pyrrole-CH3), 2.45–2.66 (m, 2H; pyrrole-CH2CH2), 2.95–3.13 (m,
2H; pyrrole-CH2), 3.87–3.91 (m, 6H; OCH2), 4.19–4.30 (m, 2H; benzyl-
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
CH2), 4.46 (t, 1H, 3JHꢀH =7.58 Hz; CHCH
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
105.9 (phenyl-Co), 133.1 (pyrrole-C), 137.7 (phenyl-Ci,p), 153.4 (phenyl-
Cm), 161.8, 172.9, 175.0 ppm (carbonyl-C); IR (solid): nꢆ=>=3330 (w),
2917 (s), 2850 (s), 1673 (m), 1628 (s), 1536 (m), 1465 (w), 1437 (m), 1369
(w), 1320 (m), 1287 (m), 1217 (m), 1150 (s), 1115 (s), 1092 (w), 1018 (w),
852 (w), 816 (w), 754 (m), 720 (m), 655 (w), 624 cmꢀ1 (m); HRMS (pos.
ESI): m/z: calcd for C64H112N7O9: 1122.8516 [M+H]+; found: 1122.8516.
5-[(R)-1-Carbamoyl-2-methylpropylcarbamoyl]-3-methyl-4-{2-[3,4,5-tris-
ACHTUNGTRENNUNG(dodecyl-oxy)phenyl]methylcarbamoyl}ethyl-pyrrole-2-carbonylguanidini-
um chloride (9): A solution of 8 (40.0 mg, 35.7 mmol in CH2Cl2/TFA 4:1
(20 mL) was stirred at room temperature for 4 h. The solvent was evapo-
rated under reduced pressure. The resulting solid was lyophilized four
Acknowledgements
Ongoing financial support of our work from the DFG (Deutsche For-
schungsgemeinschaft) and the FCI (Fonds der Chemischen Industrie) is
gratefully acknowledged.
times with 5% HCl to give 9 (37.7 mg, 35.6 mmol, 100%) as a colorless
3
solid. M.p.: 2378C; 1H NMR (400 MHz, CDCl3): d=0.87 (t, JHꢀH
=
6.82 Hz, 9H; CH2CH3), 1.05 (s, 6H; CHCHACHTNUTRGEN(UNG CH3)2),1.15–1.37 (s, 48H;
CH2 d-l), 1.38–1.48 (s, 6H; OCH2CH2CH2), 1.67–1.80 (m, 6H;
OCH2CH2), 2.24 (s, 3H; pyrrole-CH3), 2.50–2.64 (s, 2H; pyrrole-
CH2CH2), 2.85–3.10 (m, 2H; pyrrole-CH2), 3.82–3.95 (m, 6H; OCH2),
4.07–4.17 (m, 1H; CHCHACTHNUTRGNEUNG(CH3)2), 4.18–4.35 (m, 2H; benzyl-CH2), 6.42
(s, 2H; phenyl-CH), 6.76 (s, 1H), 8.33 (s, 2H), 8.88 (s, 1H), 11.3 (s, 1H),
11.5 ppm (s, 1H); 13C NMR (100 MHz, CDCl3): d=10.3 (pyrrole-CH3),
[1] a) T. W. Bell, Curr. Opin. Chem. Biol. 1998, 2, 711–716; b) J. M.
Boon, B. D. Smith, Curr. Opin. Chem. Biol. 2002, 6, 749–756;
c) B. A. McNally, W. M. Leevy, B. D. Smith, Supramol. Chem. 2007,
19, 29–37; d) S. Licen, F. D. Riccardis, I. Izzo, P. Tecilla, Curr. Drug
Discovery Technol. 2008, 5, 86–97; e) A. P. Davis, D. N. Sheppard,
B. D. Smith, Chem. Soc. Rev. 2007, 36, 348–357.
[2] a) G. V. Oshovsky, D. N. Reinhoudt, W. Verboom, Angew. Chem.
2007, 119, 2418–2445; Angew. Chem. Int. Ed. 2007, 46, 2366–2393;
b) T. Rehm, C. Schmuck, Chem. Commun. 2008, 801–813.
[3] For some selected recent examples of artificial receptors for amino
acid derivatives, see: a) C. Schmuck, L. Geiger, J. Am. Chem. Soc.
2005, 127, 10486–10487; b) C. Schmuck, U. Machon, Chem. Eur. J.
2005, 11, 1109–1118; c) C. Schmuck, V. Bickert, Org. Lett. 2003, 5,
4579–4581; d) M. E. Bush, N. D. Bouley, A. R. Urbach, J. Am.
Chem. Soc. 2005, 127, 14511–14517; e) S. Bartoli, T. Mahmood, A.
Malik, S. Dixon, J. D. Kilburn, Org. Biomol. Chem. 2008, 6, 2340–
2345; f) I. Alfonso, M. I. Burguete, F. Galindo, S. V. Luis, L. Vigara,
J. Org. Chem. 2009, 74, 6130–6142; g) A. Ragusa, S. Rossi, J. M.
Hayes, M. Stein, J. D. Kilburn, Chem. Eur. J. 2005, 11, 5674–5688;
h) C. Miranda, F. Escart, L. Lamarque, M. J. Yunta, P. Navarro, E.
Garcia-Espana, M. L. Jimeno, J. Am. Chem. Soc. 2004, 126, 823–
14.3 (CH2CH3), 19.4 (pyrrole-CH2), 19.7 (CHCH
26.3 (g-CH2), 29.5, 29.8, 30.3, 30.5, 30.6 (d-i CH2), 32.1 (pyrrole-
CH2CH2), 44.9 (benzyl-CH2), 60.8 (CHCH(CH3)2), 69.1, 73.9 (a-CH2),
ACHTNUGTRNEN(NUG CH3)2), 22.8 (l-CH2),
AHCTUNGTRENNUNG
112.2 (phenyl-C), 128.5, 142.0 (pyrrole-C), 160.4, 172.6, 176.8 ppm (car-
bonyl-C); IR (solid): n˜ =3306 (w), 2922 (s), 2852 (s), 1639 (m), 1488 (w),
1464 (m), 1431 (m), 1377 (w), 1285 (s), 1202 (s), 1118 (s), 835 (w), 799
(w), 755 (w), 720 cmꢀ1 (m); HRMS (pos. ESI): m/z: calcd for
C59H104N7O7: 1022.7992 [M+H]+; found: 1022.7992.
Extraction experiments: The extraction experiments were carried out in
4 mL glass vials. The organic phase consisted of 1 mL of a solution of re-
ceptor 9 in chloroform or pure chloroform, respectively, for the control
experiment. The aqueous solutions consisted of 10 mm solutions of the
acetylated amino acids valine, tryptophan, and tyrosine in 100 mm BIS-
TRIS buffer. The solutions were adjusted to pH 6 by the addition of
0.1 mm HCl and 0.1 mm NaOH. The different phases were put together
in the glass vials and shaken manually for 10 min. Afterwards, they were
Chem. Eur. J. 2010, 16, 9502 – 9510
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9509

C. Schmuck and C. Urban
833; i) H. Wang, W.-H. Chan, A. W. M. Lee, Org. Biomol. Chem.
2008, 6, 929–934; j) A. V. Yakovenko, V. I. Boyko, V. I. Kalchenko,
L. Baldini, A. Casnati, F. Sansone, R. Ungaro, J. Org. Chem. 2007,
72, 3223–3231. For work before 1998, see literature cited in refer-
ence [8].
Ayling, M. N. Perez-Payan, A. P. Davis, J. Chem. Soc. Perkin Trans.
1 2001, 1329–1341; c) A. P. Davis, L. J. Lawless, Chem. Commun.
1999, 9–10.
[10] a) P. Breccia, M. V. Gool, R. Perez-Fernandez, S. Martin-Santamara,
F. Gago, P. Prados, J. de Mendoza, J. Am. Chem. Soc. 2003, 125,
8270–8284; b) A. Galan, D. Andreu, A. M. Echavarren, P. Prados, J.
De Mendoza, J. Am. Chem. Soc. 1992, 114, 1511–1512.
[11] a) C. Schmuck, Chem. Commun. 1999, 843–844; b) C. Schmuck,
Chem. Eur. J. 2000, 6, 709–718.
[12] S. Niebling, S. K. Srivastava, C. Hermann, P. R. Wich, C. Schmuck,
S. Schlꢁcker, Chem. Commun. 2010, 46, 2133–2135.
[13] C. Schmuck, P. Wich, Angew. Chem. 2006, 118, 4383–4387; Angew.
Chem. Int. Ed. 2006, 45, 4277–4281.
[4] For recent reviews on anion binding by artificial receptors, see:
a) K. A. Schug, W. Lindner, Chem. Rev. 2005, 105, 67–113;
b) R. J. T. Houk, S. L. Tobey, E. V. Anslyn, Top. Curr. Chem. 2005,
255, 199–220; c) P. Blondeau, M. Segura, R. Perez-Fernandez, J. de
Mendoza, Chem. Soc. Rev. 2007, 36, 198–210; d) C. Schmuck,
Coord. Chem. Rev. 2006, 250, 3053–3067; e) S. Kubik, Chem. Soc.
Rev. 2009, 38, 585–605; f) I. Stibor, P. Zlatuskova, Top. Curr. Chem.
2005, 255, 31–63.
[5] J.-P. Behr, J.-M. Lehn, J. Am. Chem. Soc. 1973, 95, 6108–6110.
[6] a) H. Tsukube, J. Chem. Soc. Perkin Trans. 1 1982, 2359–2363;
[14] R. van de Coevering, P. C. A. Bruijnincx, C. A. van Walree, R. J. M.
Klein Gebbink, G. van Koten, Eur. J. Org. Chem. 2007, 2931–2939.
[15] C. Schmuck, D. Rupprecht, C. Urban, N. Walden, Synthesis 2006,
89–96.
ˇ
ˇ
´
´
b) M. Zinic, L. Frkanec, V. Skaric, J. Trafton, G. W. Gokel, J. Chem.
Soc. Chem. Commun. 1990, 1726–1728; c) M. Pietraszkiewicz, M.
´
Kozbiał, O. Pietraszkiewicz, J. Membr. Sci. 1998, 138, 109–113; d) I.
[16] J. van Herrikhuyzen, A. Syamakumari, A. P. H. J. Schenning, E. W.
Meijer, J. Am. Chem. Soc. 2004, 126, 10021–10027.
[17] a) A. Radzicka, R. Wolfenden, Biochemistry 1988, 27, 1664–1670;
b) K. M. Biswas, D. R. DeVido, J. G. Dorsey, J. Chromatogr. A 2003,
1000, 637–655.
[18] J. C. Ma, D. A. Dougherty, Chem. Rev. 1997, 97, 1303–1324.
[19] F. Mohamadi, N. G. J. Richards, W. C. Guida, R. Liskamp, M.
Lipton, C. Caufiled, G. Chang, T. Hendrickson, W. C. Still, J.
Comput. Chem. 1990, 11, 440–467.
Aydin, T. Aral, M. Karakaplan, H. Hosgoeren, Tetrahedron: Asym-
metry 2009, 20, 179–183; e) M. Reetz, J. Huff, J. Rudolph, K. Tçll-
ner, A. Deege, R. Goddard, J. Am. Chem. Soc. 1994, 116, 11588–
11589; f) N. Voyer, B. Guꢇrin, Chem. Commun. 1997, 2329–2330.
[7] J. Rebek, B. Askew, D. Nemeth, K. Parris, J. Am. Chem. Soc. 1987,
109, 2432–2434.
[8] J. L. Sessler, A. Andrievsky, Chem. Eur. J. 1998, 4, 159–167.
[9] a) B. BaragaÇa, A. G. Blackburn, P. Breccia, A. P. Davis, J. de Men-
doza, J. M. P. Carrillo, P. Prados, J. Riedner, J. G. de Vries, Chem.
Eur. J. 2002, 8, 2931–2936; b) L. J. Lawless, A. G. Blackurn, A. J.
Received: February 26, 2010
Published online: May 17, 2010
9510
www.chemeurj.org
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 9502 – 9510