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Glycomimetic Cyclic Peptides Stimulate Neurite Outgrowth.

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DOI: 10.1002/anie.200601579
Glycomimetic Cyclic Peptides Stimulate Neurite
Dirk Bchle, Gabriele Loers, Eckhart W. Guthhrlein,
Melitta Schachner, and Norbert Sewald*
Carbohydrates are involved in a broad spectrum of physiological and pathological processes, many of which rely on
molecular recognition.[1] Oligosaccharides that are exposed
on the surface of, for example, tumor cells or pathogens can
be regarded as potential therapeutic targets, either for direct
application or for vaccination.[2] Despite the tremendous
methodological advances in oligosaccharide synthesis,[3, 4]
carbohydrate-based therapeutics or vaccines require non[*] Dr. D. Bchle, Dr. E. W. Guth!hrlein, Prof. Dr. N. Sewald
Department of Chemistry, Organic and Bioorganic Chemistry
University of Bielefeld
Universittsstrasse 25, 33615 Bielefeld (Germany)
Fax: (+ 49) 521-106-8094
Dr. G. Loers, Prof. Dr. M. Schachner
Zentrum f>r Molekulare Neurobiologie
Universitt Hamburg
Martinistrasse 52, 20246 Hamburg (Germany)
[**] Support from the Erika und Conrad W. Schnyder-Stiftung, NRW
Graduate School in Bioinformatics and Genome Research at
Bielefeld University (PhD grant to E.W.G.), the Deutsche Forschungsgemeinschaft (SFB 613), and the Fonds der Chemischen
Industrie is gratefully acknowledged.
Supporting information for this article is available on the WWW
under or from the author.
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2006, 45, 6582 –6585
routine multistep syntheses. Moreover, oligosaccharides
mostly display unfavorable ADME (absorption, distribution
metabolism, excretion) parameters.[5]
Peptides that functionally and/or structurally mimic
oligosaccharides provide a straightforward and elegant alternative for interfering with the interaction between complex
carbohydrates and their receptors;[6] cyclic peptides are
especially privileged because of their increased metabolic
Phage display techniques either use an oligosaccharide
receptor or monoclonal antibodies specific for an oligosaccharide antigen for retrieval of glycomimetic peptides. We
chose the human natural killer cell glycan HNK-1[7, 8] as a
target for our investigations (Figure 1). Its consensus epitope
Figure 2. Cyclic-peptide scan and d-amino acid scan provide different
series of linear and cyclic hexapeptides derived from the peptide
sequences TFKLSETTLEYY and TFQLSTRTLPFS. Cyclic hexapeptides
can be regarded as pairwise pseudosymmetric with respect to the
geometry of the two complementary b turns. Therefore, in many cases
only three of six peptides were examined.
Figure 1. The HNK-1 trisaccharide consensus epitope.
consists of a 3’-sulfated glucuronic acid attached to N-acetyl
lactosamine and is found on glycolipids and glycoproteins.
HNK-1 and its binding partners are involved in many
developmental processes in the nervous system.[9] Neurite
outgrowth from motor neurons in culture is enhanced in the
presence of a substrate that contains HNK-1, while neurite
outgrowth from sensory neurons remains unaffected.[8] HNK1 is indeed expressed along the routes that motor axons follow
as they regrow after peripheral nerve damage.[10] Hence,
HNK-1 and its mimetics have the potential to promote motorneuron-specific reinnervation. As HNK-1 is difficult to isolate
from natural sources and is equally difficult to synthesize
chemically, the isolation and structural optimization of
glycomimetics, for example, peptides imitating the HNK-1
oligosaccharide are viable and intriguing possibilities for
potential clinical applications.
A library comprising 26 cyclic hexapeptides (see Figure 2
and the Supporting Information) was synthesized on the basis
TFQLSTRTLPFS, which were discovered by randomized
peptide phage display against the monoclonal antibody 412.[11]
The parent sequences were divided virtually into sets of
overlapping hexapeptides, providing a total of six hexapeptide sequences. Each of these was taken as the basis for the
synthesis of cyclic hexapeptide sublibraries, and a d-amino
acid scan was performed with the cyclic hexapeptides derived
from the six linear basis peptide epitopes. In each discrete
member of these cyclic hexapeptide sublibraries, one amino
acid was incorporated as its d enantiomer,[12] providing
diastereomeric cyclic peptides. While a cyclic hexapeptide
displays several interconverting conformations, the presence
of a building block with conformational bias (e.g. proline, damino acids) locks the overall conformation of the peptide. dAmino acids are predominantly found in position i + 1 of a
bII’ turn. Consequently, the peptide presents the functionalities of putative recognition elements in predictable threeAngew. Chem. Int. Ed. 2006, 45, 6582 –6585
dimensional arrays, determined by the relative position of the
d-amino acid (spatial screening[13]). A cyclic peptide has less
conformational flexibility than its linear parent and, hence,
fewer degrees of freedom. If binding to a receptor is still
possible for the cyclic peptide, the loss of entropy in the
binding event is smaller than in the binding of the linear
The peptides were functionally characterized by surface
plasmon resonance with respect to binding to the immobilized
412 antibody. Striking effects were observed in the
TFKLSETTLEYY series (Figure 3). Clear sequence- and
conformation-dependent effects occurred in the middle
epitope LSETTL (Figure 3 a). The d-Leu-containing cyclic
peptide c-(LSETTl) binds to the antibody with KD = 67 mm,
which is 12 times stronger than the binding of the linear
dodecapeptide and four times stronger than that of the linear
An alanine scan revealed the importance of the ETTl
sequence. Peptide c-(lSETTL), where the d-amino acid is
shifted by one position relative to c-(LSETTl), represents the
mismatch case with an improper display of the side chains and
entropically disfavored interaction. Interestingly, the two
cyclic hexapeptides c-(LSETtL) and c-(LsETTL), which can
be regarded as pseudo-symmetric with respect to the geometry of the two complementary b turns, display similar
affinities to the antibody (Figure 3 a). Another affine peptide,
c-(RTLPFS) (KD = 73 mm), was discovered in the
TFQLSTRTLPFS series (Figure 3 b). It does not require the
incorporation of a d-amino acid for turn stabilization because
proline also induces turns. The cyclic derivatives of the Cterminal hexapeptide RTLPFS all display considerably higher
affinity to the antibody than the linear hexapeptide (64-fold)
and the linear dodecapeptide (sixfold) do.
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 3. KD values of the peptides derived from the basis sequences
for binding to the antibody 412 as determined by surface plasmon
resonance. White bars: basis sequence; hatched bars: randomized
sequence; gray bars: linear hexapeptides; black bars: cyclic hexapeptides. In exceptional cases, the KD values are given on top of the bars.
For bars reaching the maximum displayed value (10 000 mm), no
binding was detected at all. Error bars are not visible because of the
logarithmic scale. a) TFKLSETTLEYY; b) TFQLSTRTLPFS.
Selected peptides were investigated with respect to their
ability to stimulate neurite outgrowth of murine and human
motor neurons (Figure 4 a). For this purpose enriched motor
neuron preparations were isolated from embryos and cultured on glass coverslips coated with poly-l-ornithine (PLO)
alone, which served as a negative control, or with PLO and
the HNK-1 mimetics. Laminin is known to stimulate outgrowth of neurites[14, 15] and was used as a positive control.
Neurite outgrowth of both murine and human motor neurons
(Figure 4 a) was stimulated by the glycomimetic cyclic peptides c-(LSETTl) and c-(RTLPFS). The two cyclic hexapeptides also increased survival of motor neurons by approximately 30 % after eight days in vitro when compared to
experiments with PLO alone (Figure 4 b). Both effects,
stimulation of neurite outgrowth and increase of survival,
were not only much stronger than with the linear dodecapeptide sequences but also significantly depended on the
conformation. Peptide c-(lSETTL), which can be assumed
Figure 4. Neurite outgrowth (Figure 4 a; mean values standard error
of the mean (SEM)) and survival rates (Figure 4 b; mean values
SEM) of murine (black bars) and human (white bars) motor
neurons plated onto substrate-coated poly-l-ornithine (PLO) in the
presence of glycomimetic peptides, laminin, and serum, respectively.
Neurite outgrowth and survival are quoted relative to the negative
control PLO (= 100 %). The experiments were performed three times
for neuritogenesis and twice for neuronal survival.
to adopt a different turn geometry than that of c-(LSETTl)
because of the different position of the d-amino acid, did not
display any significant functional activity. Preincubation of
the 412 antibody with the HNK-1 presenting murine glycoprotein L1 led to a significant decrease in binding to c(LSETTl) and c-(RTLPFS), which suggests that the two
peptides represent functional mimics of the HNK-1 oligosaccharide.
The solution conformations of c-(LSETTl) and c(RTLPFS) were determined by NMR spectroscopy in combination with molecular dynamics simulations. Peptide c(LSETTl) populates a single conformer cluster characterized
by a bII’ turn with d-Leu in position i + 1 and a comple-
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2006, 45, 6582 –6585
Figure 5. Left: Solution conformation of c-(LSETTl) in [D6]DMSO.
Right: Solution conformation of c-(RTLPFS) in [D6]DMSO, major
conformer. Blue N, red O, white H, gray C.
mentary bI-like turn around -Ser-Glu-Thr-Thr- (Figure 5).
The bII’ turn is supported by strong NOEs between Ha(dLeu) and HN(Leu), as well as between HN(Leu) and HN(Ser),
while strong NOEs between HN(Glu) and HN(Thr4) as well as
between HN(Thr4)and HN(Thr5) support classification of the
complementary turn as bI-like.
Peptide c-(RTLPFS) exists as a 2:1 mixture of conformers
with trans and cis configurations at the Leu–Pro peptide bond.
The major conformer contains only trans peptide bonds and
adopts a structure comprising a g turn with Arg in position i +
1, which is stabilized by a hydrogen bond between HN(Thr)
and CO(Ser). This g turn is nested during the trajectory with a
b turn containing Arg in the i + 1 position. This is supported
by a strong NOE between HN(Thr) and HN(Leu). Proline
occupies position i + 1 of a g turn which is nested with a bturn-like conformation that cannot be classified as one of the
ideal turn types (Figure 5). The minor conformer comprises a
cis peptide bond at Leu–Pro and is much more flexible than
its all-trans configured correlate.
As expected for carbohydrate mimetics, both peptides
contain several amino acids with hydroxylated side chains
(Ser, Thr). Additionally, the backbone amide groups of the
peptide may act as intermolecular hydrogen-bond donors and
acceptors. Unpolar amino acids are present in both peptides
to establish hydrophobic interactions with the receptor(s). c(LSETTl) essentially requires the Glu residue for affinity to
the antibody 412, which presumably imitates a negatively
charged residue of the HNK-1 epitope. Interestingly, c-
Angew. Chem. Int. Ed. 2006, 45, 6582 –6585
(RTLPFS) does not contain an acidic side chain but a basic
Arg residue instead.
Compounds able to enhance neurite regeneration after
damage may hold promise, for example, in the treatment of
paraplegic patients. The results obtained by transformation of
the basis linear peptide sequences TFKLSETTLEYY and
TFQLSTRTLPFS as mimetics of the HNK-1 trisaccharide
into subsets of cyclic hexapeptides in a spatial screening
approach clearly show that it is possible to increase the
biological activity of glycomimetic peptides by backbone
Received: April 21, 2006
Published online: September 8, 2006
Keywords: bioorganic chemistry · glycomimetics · neuroactive
substances · neurochemistry · peptides
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