DOI: 10.1002/chem.200800432
Synthetic Receptors for the Differentiation of Phosphorylated Peptides
with Nanomolar Affinities
Andreas Grauer, Alexander Riechers, Stefan Ritter, and Burkhard Kçnig*[a]
Abstract: Artificial ditopic receptors
for the differentiation of phosphorylat-
ed peptides varying in i+3 amino acid
side chains were synthesized, and their
binding affinities and selectivities were
determined. The synthetic receptors
show the highest binding affinities to
phosphorylated peptides under physio-
logical conditions (HEPES, pH 7.5,
154 mm NaCl) reported thus far for ar-
tificial systems. The tight and selective
binding was achieved by high coopera-
tivity of the two binding moieties in
the receptor molecules. All receptors
interact with phosphorylated serine by
bis(ZnII–cyclen) complex coordination
and a second binding site recognizing a
carboxylate or imidazole amino acid
side chain functionality.
Keywords: chelates · cooperativity ·
emission spectroscopy · molecular
recognition · peptides
Introduction
tended the concept to a hybrid receptor[10] consisting of a
natural WW binding site (a protein module that binds pro-
line-rich ligands)[11] for phosphorylated peptides and a fluo-
rescent metal complex. Their hybrid receptor[12] shows en-
hanced micromolar binding affinity and selectivity towards
diphosphorylated peptides derived from sequences of the C-
terminal domain of the RNA polymerase II.
We report herein the design, synthesis and binding prop-
erties of synthetic ditopic receptors with nanomolar affinity
to phosphorylated peptides in buffered aqueous solution
under physiological pH. Their affinity depends on a second
amino acid residue present in the peptide, namely carboxyl-
ate or histidine.
Phosphorylation of proteins is a ubiquitous regulation mech-
anism in biology and plays a central role in controlling intra-
cellular signaling networks.[1] Protein phosphorylation regu-
lates enzyme activity and the reversible formation of signal-
ing complexes by specific recognition of phosphorylated
proteins by phosphoprotein binding domains.[2] To investi-
gate, monitor or specifically inhibit such phosphorylation-
dependent processes, analytical tools allowing a specific rec-
ognition of phosphorylated peptides and proteins are desira-
ble. Historically, phosphorylation detection methods relied
on radioisotopes or phosphoamino acid selective antibodies.
Chromatographic, staining and surface device techniques ex-
tended the available methods.[3] Several biosensors for pro-
tein kinase activity based on GFP-FRET probes[4–6] or syn-
thetic fluorophores[7] have been reported, which typically
signal their own phosphorylation or dephosphorylation.
However, the number of artificial systems for a specific rec-
ognition of phosphorylated peptides is still limited. Hamachi
et al.[8,9] recently reported fluorescent dinuclear zinc com-
plexes for the detection of peptide phosphorylation and ex-
As target peptides Flu-GpSAAEV-NH2 (1) and Flu-
GpSAAHV-NH2 (2) were selected from sequences of
human STAT (signal transducer and activator of transcrip-
tion) proteins[13] and prepared by standard solid-phase
synthesis methods. The peptides were N-terminally lab-
eled by fluorescein (Flu) to facilitate the binding studies. A
bisACHTNUTGRNEUNG
(ZnII–cyclen) triazine complex was used in the receptor
design as the binding site for phosphoserine. The high affini-
ty of this complex to phosphates was previously shown on
modified surfaces.[14] Peptide 1 presents, beside the phos-
phate ester, as an additional functional group for specific
molecular recognition a carboxylate in the sidechain of the
i+3 glutamic acid. Guanidinium moieties have been used as
binding sites for carboxylate binding.[15] Thus, to achieve se-
lective affinity for target peptide 1, both binding sites for
phosphoserine and the carboxylate side chain were connect-
ed by a peptidic linker giving compounds 3 and 4. Receptors
5 and 6 consist of two bis(ZnII–cyclen) triazine complexes
[a] Dipl.-Chem. A. Grauer, Dipl.-Chem. A. Riechers, Dr. S. Ritter,
Prof. Dr. B. Kçnig
Institute for Organic Chemistry
University of Regensburg, Universitꢀtsstr. 31
93040 Regensburg (Germany)
Fax : (+49)941-943-1717
Supporting information for this article is available on the WWW
8922
ꢁ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 8922 – 8927