Biomedical Engineering Reference
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Phe
L y s
Phe
Lys
GCP
Lys
Lys
GCP
no stereoselectivity
Glu
Glu
Glu
Glu
Glu
Glu
D/L-Ala
stereoselective
recognition
Fig. 4 Screening of a tetrapeptide library revealed that stereoselectivity is observed at positions
where receptor and substrate are locked by strong electrostatic interactions at both sides of the
stereogenic center: D -Ala: K
5,000 M 1 ; L -Ala: K
900 M 1 ( right ). When this is not the case
¼
¼
7,900 M 1 ; L -Ala: K
8,500 M 1 ( left )
no stereoselectivity can be witnessed: D -Ala: K
¼
¼
10 4 M 1 in buffered water at pH 6
by means of screening a combinatorial library with 512 peptide-based artificial
receptors (9) containing a guanidiniocarbonyl pyrrole unit (GCP) as the headgroup [ 9 ].
The rigid, planar GCP group allows for efficient binding of carboxylates in aqueous
media by forming a hydrogen bond-enforced salt bridge with the C-terminus of the
oligopeptide. The guanidinium group in 9 is connected to the pyrrole core by an acetyl
group, thus decreasing the p K a value of free guanidine from 13.5 to approximately
6-7 [ 10 ]. As a consequence, the potency of its action as a hydrogen donor was
increased dramatically. Furthermore, the intramolecular hydrogen bond between the
carboxyl and guanidinium group preorganizes the binding motif in the correct confor-
mation for oxoanion binding. By combinatorially varying the amino acid hinge, which
is attached to the GCP moiety, the receptor could be fine-tuned for the substrate.
The best artificial receptor corresponds to the sequence R 1
(EKAA, 10) with binding constants of up to K
R 2
lysine and R 3
¼
phenylalanine side chains (11). The interaction between the C-terminus of the peptidic
substrate 10 with the GCP unit is the main driving force for complex formation.
Additionally, the attached linear tripeptide chain interacts with the substrate by forming
a hydrogen bond-mediated
¼
¼
-sheet with the peptidic substrate. An additional salt bridge
is formed between the glutamate and the lysine ammonium group. In order to verify the
substrate selectivity, the library was also screened against the inverse substrate
sequence N-Ac- D -Ala- D -Ala- L -Lys- D -Glu-OH (AAKE), which resulted in binding
constants that were lower by approximately one order of magnitude [ 7 ]. Thus, despite
the fact that the complex stability is mainly based on a directed ion pair, the binding
constants are strongly dependent on the amino acid sequence of the substrate. For
binding of a polar substrate in water the binding constants achieved are very good.
In another set of experiments, the best receptor for the EKAA sequence (GCP-
KKF) was tested against a rather small combinatorial tetrapeptide library consisting
of 320 members [ 11 ]. With only three amino acids present in the library (Ala, Lys,
and Glu) all substrates were closely related. Two amino acids just differed in their
absolute configuration ( D / L -Ala). Binding constants were determined via fluores-
cence spectroscopy in buffered water at pH 6.1 and ranged from K
b
50 M 1
to
<
10 4 M 1 . More importantly, a sequence-dependent stereoselectivity of the
receptor could be observed, as depicted in Fig. 4 , when the D / L -Ala amino acid of
3
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