Cyclotriveratrylene (CTV) as a new chiral triacid scaffold capable of inducing triple helix formation of collagen peptides containing either a native sequence or Pro-Hyp-Gly repeats

Article abstract of DOI:10.1002/1521-3765(20021018)8:20<4613::AID-CHEM4613>3.0.CO;2-R

A new triacid scaffold is described based on the cone-shaped cyclo-triveratrylene (CTV) molecule that facilitates the triple helical folding of peptides containing either a unique blood platelet binding collagen sequence or collagen peptides composed of Pro-Hyp-Gly repeats. The latter were synthesized by segment condensation using Fmoc-Pro-Hyp-Gly-OH. Peptides were coupled to this CTV scaffold and also coupled to the Kemp's triacid (KTA) scaffold. After assembly of peptide H-Gly-[Pro-Hyp-Gly]₂-Phe-Hyp-Gly Glu(OAll)-Arg-Gly-Val-Glu(OAll)-Gly [Pro-Hyp-Gly]₂-NH₂ (13) by an orthogonal synthesis strategy to both triacid scaffolds, followed by deprotection of the allyl groups, the molecular constructs spontaneously folded into a triple helical structure. In contrast, the non-assembled peptides did not. The melting temperature (T_m) of (+/-) CTV[CH₂C- (O)N(H)Gly-[Pro-Hyp-Gly]₂-Phe-Hyp-Gly-Glu-Arg- Gly-Val-Glu-Gly-[Pro-Hyp-Gly]₂-NH₂]₃ (14) is 19 °C, whereas KTA[Gly-Gly-[Pro-Hyp-Gly]₂-Phe-Hyp-Gly-Glu-Arg- Gly-Val-Glu-Gly-[Pro-Hyp-Gly]₂-NH₂]₃ (15) has a T_m of 20 °C. Thus, it was shown for the first time that scaffolds were also effective in stabilizing the triple helix of native collagen sequences. The different stabilizing properties of the two CTV enantiomers could be measured after coupling of racemic CTV triacid to the collagen peptide, and subsequent chromatographic separation of the diastereomers. After assembly of the two chiral CTV scaffolds to the model peptide H-Gly-Gly-(Pro-Hyp-Gly)₅-NH₂ (24), the (+)- enantiomer of CTV 28b was found to serve as a better triple helix-inducing scaffold than the (-)-enantiomer 28a. In addition to an effect of the chirality of the CTV scaffold, a certain degree of flexibility between the CTV cone and the folded peptide was also shown to be of importance. Restricting the flexibility from two to one glycine residues resulted in a significant difference between the two collagen mimics 20a and 20b, whereas the difference was only slight when two glycine residues were present between the CTV scaffold and the peptide sequence in collagen mimics 30a and 30b.

Full text of DOI:10.1002/1521-3765(20021018)8:20<4613::AID-CHEM4613>3.0.CO;2-R

FULL PAPER
Cyclotriveratrylene (CTV) as a New Chiral Triacid Scaffold Capable of
Inducing Triple Helix Formation of Collagen Peptides Containing either a
Native Sequence or Pro-Hyp-Gly Repeats
Erik T. Rump,[a, b, c] Dirk T. S. Rijkers,[b] Hans W. Hilbers,[b] Philip G. de Groot,[a] and
Rob M. J. Liskamp*[b]
Abstract: A new triacid scaffold is de-
scribed based on the cone-shaped cyclo-
triveratrylene (CTV) molecule that fa-
cilitates the triple helical folding of
peptides containing either a unique
blood platelet binding collagen se-
quence or collagen peptides composed
of Pro-Hyp-Gly repeats. The latter were
synthesized by segment condensation
using Fmoc-Pro-Hyp-Gly-OH. Peptides
were coupled to this CTV scaffold and
also coupled to the Kemp×s triacid
(KTA) scaffold. After assembly of pep-
tide H-Gly-[Pro-Hyp-Gly]2-Phe-Hyp-Gly-
Glu(OAll)-Arg-Gly-Val-Glu(OAll)-Gly-
[Pro-Hyp-Gly]2-NH2 (13) by an orthog-
onal synthesis strategy to both triacid
scaffolds, followed by deprotection of
the allyl groups, the molecular con-
structs spontaneously folded into a triple
helical structure. In contrast, the non-
assembled peptides did not. The melting
temperature (Tm) of (‡/ À ) CTV[CH2C-
(O)N(H)Gly-[Pro-Hyp-Gly]2-Phe-Hyp-
Gly-Glu-Arg-Gly-Val-Glu-Gly-[Pro-Hyp-
Gly]2-NH2]3 (14) is 198C, whereas
KTA[Gly-Gly-[Pro-Hyp-Gly]2-Phe-Hyp-
Gly-Glu-Arg-Gly-Val-Glu-Gly-[Pro-Hyp-
Gly]2-NH2]3 (15) has a Tm of 208C. Thus,
it was shown for the first time that
scaffolds were also effective in stabiliz-
ing the triple helix of native collagen
sequences. The different stabilizing
properties of the two CTV enantiomers
could be measured after coupling of
racemic CTV triacid to the collagen
peptide, and subsequent chromato-
graphic separation of the diastereomers.
After assembly of the two chiral CTV
scaffolds to the model peptide H-Gly-
Gly-(Pro-Hyp-Gly)5-NH2 (24), the (‡)-
enantiomer of CTV 28b was found to
serve as a better triple helix-inducing
scaffold than the (À)-enantiomer 28a. In
addition to an effect of the chirality of
the CTV scaffold, a certain degree of
flexibility between the CTV cone and
the folded peptide was also shown to be
of importance. Restricting the flexibility
from two to one glycine residues result-
ed in a significant difference between
the two collagen mimics 20a and 20b,
whereas the difference was only slight
when two glycine residues were present
between the CTV scaffold and the pep-
tide sequence in collagen mimics 30a
and 30b.
Keywords: circular dichroism
helical structures ¥ protein folding
protein models solid-phase
synthesis
¥
¥
¥
Introduction
Synthetic collagen structures that mimic the structure and
conformation of native collagens are of special interest for
studying cell-matrix interactions. The triple helical conforma-
tion of collagen molecules is of crucial importance since it is
[a] Dr. E. T. Rump, Prof. Dr. P. G. de Groot
Department of Haematology
University Medical Center
thought to be essential for recognition by its ligand.[1]
A
Utrecht (The Netherlands)
[b] Prof. Dr. R. M. J. Liskamp, Dr. E. T. Rump, Dr. Ir. D. T. S. Rijkers,
H. W. Hilbers
synthetic collagen mimic that meets this structural feature can
therefore serve as a valuable tool to identify the exact binding
sites that interact with their receptors. This has been shown for
collagen molecules that include recognition sites for for
example integrins and metalloproteinases.[2, 3] The exact
nature of collagen protein interactions are now beginning
to be elucidated, as was recently shown by the first high-
resolution crystal structure of a synthetic collagen peptide
interacting with an integrin fragment.[4]
Department of Medicinal Chemistry
Utrecht Institute for Pharmaceutical Sciences
Utrecht University, PO Box 80082
3508 TB Utrecht (The Netherlands)
Fax : (‡31) 30 2536655
E-mail: r.m.j.liskamp@pharm.uu.nl
[c] Dr. E. T. Rump
Present address: bioMerieux B.V. Boseind 15
5281 RM Boxtel (The Netherlands)
The collagen triple helix is composed of three separate
peptide chains, which are staggered by one residue and
Supporting information for this article is available on the WWW under
http://www.chemeurj.org/ or from the author.
Chem. Eur. J. 2002, 8, No. 20
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R. M. J. Liskamp et al.
FULL PAPER
intertwine to form a right-handed triple helix.[5] The primary
structure of these peptides is characterized by trimeric
repeating units X-Y-G, where X and Y can be any amino
acid. One trimer, Pro-Hyp-Gly (Hyp ˆ hydroxyproline) or
POGis found most prominently in native collagen, and
peptides composed solely of these trimers give rise to the most
stable triple helices.
Results
Collagen peptides containing short naturally occurring or
native sequences do not fold into triple helices at low
temperatures. Triple helix formation can be induced by a
high content of the imino acids hydroxyproline (O) and
proline, and by increasing the length of the composing
peptides. Stabilization of the triple helix can be achieved by
assembly of the three composing peptides to a scaffold
molecule. To study the assembly of a native collagen sequence
with two Pro-Hyp-Gly (POG) repeats at both termini, a new
triacid scaffold based upon a CTVunit that is compound 4 was
synthesized (Scheme 1). The Kemp×s triacid (KTA[Gly-OH]3)
scaffold 2 was chosen for comparison. The CD spectra and the
stabilizing characteristics of these two triacid scaffold struc-
tures were compared.
Previous work by the group of Goodman[7] already showed
the stabilizing properties of the KTA[Gly-OH]3 template 2
for collagen structures composed solely of Gly-Pro-Hyp
trimers or alkyl- and aryl peptoid-containing peptides. So
far, assembly on KTA 1 of collagen peptides containing native
sequences has not been reported. In our approach, the
collagen peptides were assembled on KTA or CTV scaffolds
2 and 4 (Scheme 1) in solution, and therefore protection of the
functional groups in the amino acids side chains was required.
The collagen peptide, containing the native collagen se-
quence, was synthesized using Fmoc chemistry on the solid
phase using a Rink amide linker. The stabilizing POG
sequences were conveniently introduced by segment conden-
sation using Fmoc-POG-OH. This tripeptide was synthesized
starting from Boc-Pro-OH (5) and H-Hyp-OMe. The imino
acids were coupled with BOP resulting in the dipeptide Boc-
Pro-Hyp-OMe[10] 6 (Scheme 2). After saponification of the
methyl ester and coupling of H-Gly-OMe, Boc-Pro-Hyp-Gly-
OMe (8) was obtained. Re-
In order to increase the thermal stability of synthetic
collagen mimics,[6] different branching protocols with the aim
to connect all three peptides of the collagen molecule have
been applied. Branching can be achieved by the use of
synthetic scaffold molecules. This has been shown for either
collagen peptides with Pro-Hyp-Gly repeats or native colla-
8]
gen sequences with a much lower content of imino acids.[6
Goodman et al. used the Kemp×s triacid (KTA, 1) as a
template to assemble peptide peptoid structures.[9] This
triacid scaffold is currently the most stabilizing collagen triple
helix template, since it was shown to induce triple helical
folding of the very short nona-peptide H-(GPO)3-NH2.
However this scaffold was only used to study triple helices
of model peptides.
In this study we describe a new chiral triacid scaffold, based
upon a cone-shaped cyclotriveratrylene (CTV, 3) structure.
Both the model and native collagen sequences were attached
to this scaffold. The latter were synthesized using an
orthogonal protecting group strategy. This new scaffold was
compared to the Kemp×s triacid scaffold. Analysis of the
collagen mimics was performed by CD spectroscopy. The
influence of the spacer length between the scaffold molecule
and the collagen peptide as well as the effect of the two
enantiomers of the CTVunit upon the triple helix stability was
also assessed.
moval of the Boc group and
ester hydrolysis was achieved
by acidic treatment, followed
HO
OH
HO
O
O
by introduction of the Fmoc
protection group to give
O
HN
HO
OH
OH
NH
NH
O
O
O
O
Fmoc-POG-OH (9) in 31%
overall yield over five steps.
As described by Ottl et al.,[11]
there is no need for protec-
tion of the hydroxy function
in hydroxyproline, and the
use of this tripeptide building
block significantly improved
the purity and yield of colla-
gen peptides containing POG
repeats.
1. HCl•H-Gly-OMe,
EDCI, HOBt, TEA
O
O
Me
Me
Me
Me
2. dioxane, MeOH,
H2O, NaOH
Me
2: KTA[Gly-OH]3
Me
1: KTA
O
OMe
HO
OMe
N+Bu4
O
O
1. BrCH2CO2Et,
Cs2CO3, CH3CN
O Bu4N+
O
2. Bu4NOH, MeOH,
CH2Cl2
The new CTV triacid scaf-
fold 4[12] was prepared start-
ing from the CTV triol 3
(Scheme 1) by coupling with
a-bromo ethylacetate, fol-
lowed by saponification of
the CTV triester with Bu4-
NOH.[16] The reference tem-
MeO
OH
O
MeO
O
O
OMe
HO
OMe
O Bu4N+
4: (+/-) CTV[CH2CO2 Bu4N+]3
3: (+/-) CTV
Scheme 1. Synthesis of (‡/-)-CTV triacid scaffold and KTA(Gly-OH)3 scaffold.
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Collagen Peptides
4613 4621
peptide chains seemed to have no effect on this deprotection
step. Final purification of the product by gel filtration and RP-
HPLC yielded the two desired assembled collagen peptides 14
and 15 (Scheme 3, Table 1). The correct composition of the
collagen structures was verified by MS. As a reference peptide
for triple helical folding, the acetylated peptide 16 (Scheme 3,
Table 1) was prepared. This was synthesized starting from the
allyl-protected peptide 13. The allyl protection groups were
removed, and subsequently the N-terminus was acetylated
using AcOSu.[13]
N
OH
dioxane, MeOH
4N NaOH
HCl•H-Hyp-OMe
N
N
Boc
N
BOP, Et3N
Boc
Boc
OH
O
MeO
O
O
5
6
HCl•H-Gly-OMe, EDCl
N
OH
OH
N
HOBt, DIPEA, DMF
N
O
Boc
O
HO
O
O
HN
8
7
CD analysis of the collagen peptide assembled to either the
CTV-triacid scaffold 4, or KTA(Gly-OH)3 (2), showed that
both scaffolds are capable of assisting in the folding of the
peptides into a triple helix. Moreover, assembly using the
scaffolds increased the stability of the helix significantly
compared with the non-assembled peptide. As is depicted in
Figure 1, the CD spectrum exhibited a positive band at 223 nm
and a large negative band at 200 nm for the KTA-assembled
compound 15, and 224 nm and 200 nm for the CTV-assembled
compound 14, respectively. A high ratio of these two bands
(ratio positive over negative ™Rpn∫ values) and a cooperative
melting transition curve is indicative for the proper triple
helical conformation.[6] Rpn values are 0.15 for the KTA-
assembled compound 15 and 0.14 for the CTV assembled
compound 14, measured in 10mm HOAc. In H2O, the values
are 0.15 and 0.15, respectively.
OMe
O
a) 1N HCl
b) Fmoc-OSu, Et3N
N
OH
Fmoc
N
O
O
Fmoc-Pro-Hyp-Gly-OH (9)
HN
(Fmoc-POG-OH)
OH
O
Scheme 2. Synthesis of Fmoc-Pro-Hyp-Gly-OH (Fmoc-POG-OH).
plate structure, KTA(Gly-OH)3 (2), was synthesized from
KTA 1 by a procedure described by Feng et al.[7]
The synthesis of H-Gly-[Pro-Hyp-Gly]2-Phe-Hyp-Gly-
Glu(OAll)-Arg-Gly-Val-Glu(OAll)-Gly-[Pro-Hyp-Gly]2-
NH2 (13) is depicted in Scheme 3. Subsequently, the peptide
was coupled at its N-terminus to both scaffolds 2 and 4 using
BOP as a coupling reagent. The CTV triacid scaffold 4 was
coupled as its Bu4N salt, which is soluble in DMF.[16] After
coupling, the allyl protection groups were removed by treat-
ment with [Pd(PPh3)4]. The close proximity of the three
Melting curves were determined by monitoring the ellip-
ticity at the maximal wavelength (positive peak) as a function
of the temperature. Calculation of the first derivative of the
melting curve leads to the melting temperature (Tm). The Tm
1. 20% piperidine, NMP
MeO N(H)Fmoc
Fmoc(H)N
FmocN(H)-[Pro-Hyp-Gly]2-N(H)
2. Fmoc-Pro-Hyp-Gly-OH (9),
HBTU, DIPEA
H
10
N
Argogel
MeO
O
3. repeat steps 1 and 2
O
1. 20% piperidine, NMP
2. Fmoc-Xxx-OH, HBTU,
FmocN(H)Phe-Hyp-Gly-Glu(OAll)- Arg-Gly-Val-Glu(OAll)-Gly-[Pro-Hyp-Gly]2-N(H)
11
HOBt, DIPEA, Xxx = Gly
3. repeat steps 1 and 2 for
Xxx = Glu(OAll), Val, Gly, Arg, Glu(OAll),
Gly, Hyp, Phe
1. 20% piperidine, NMP
FmocN(H)Gly-[Pro-Hyp-Gly]2-Phe-Hyp-Gly-Glu(OAll)- Arg-Gly-Val-Glu(OAll)-Gly-[Pro-Hyp-Gly]2-N(H)
2. Fmoc-Pro-Hyp-Gly-OH, HBTU,
HOBt, DIPEA
12
3. repeat steps 1 and 2
4. 20% piperidine, NMP
5. Fmoc-Gly-OH
1. 20% piperidine, NMP
H-Gly-[Pro-Hyp-Gly]2-Phe-Hyp-Gly-Glu(OAll)-Arg-Gly-Val-Glu(OAll)-Gly-[Pro-Hyp-Gly]2-NH2
2. TFA/TIS/H2O
13
(+/-) CTV[CH2C(O)N(H)Gly-[Pro-Hyp-Gly]2-Phe-Hyp-Gly-Glu-Arg-Gly-Val-Glu-Gly-[Pro-Hyp-Gly]2-NH2]3
1. 4, BOP, DMF
or 2, BOP, DMF
14
or
13
2. [Pd(PPh3)4],
DMF, NMM,
HOAc
KTA[Gly-Gly-[Pro-Hyp-Gly]2-Phe-Hyp-Gly-Glu-Arg-Gly-Val-Glu-Gly-[Pro-Hyp-Gly]2-NH2]3
15
1. [Pd(PPh3)4],
DMF, NMM,
HOAc
Ac-Gly-[Pro-Hyp-Gly]2-Phe-Hyp-Gly-Glu-Arg-Gly-Val-Glu-Gly-[Pro-Hyp-Gly]2-NH2
13
2. AcOSu, DMF
16
Scheme 3. Synthesis of the collagen peptides containing the a2b1 integrin recognition sequence GFOGERGVE.
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R. M. J. Liskamp et al.
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Table 1. Electrospray (ES) and MALDI-TOF mass spectrometry, and CD data of peptides.[a]
[Pro-Hyp-Gly]2-NH2]3 (14) two
peaks with equal mass eluting
very close to each other could
be separated. CD analysis at
58C of both compounds re-
vealed that only one of the
products had the expected tri-
Compounds
Calcd mass (m/z)
1084.5 [M‡2H]2‡
MS results Tm [8C] Rpn
13 H-G-(POG)2FOGE(All)RGVE(All)G(POG)2-NH2
1084.5
1698.4
1358.7
1328.1
1065.9
1411.0
1453.7
1467.1
1510.8
1587.0
1607.7
1233.0
n.d.
< 5
n.d.
n.d.
14a (‡ or À)CTV-[G-(POG)2FOGERGVEG(POG)2-NH2]3 1698.3 [M‡4H]4‡
14b other diastereomer
1358.8 [M‡5H]5‡
1328.2 [M‡5H]5‡
1065.5 [M‡2H]2‡
19; 23[b] 0.14
15 KTA-[GG-(POG)2FOGERGVEG(POG)2-NH2]3
16 Ac-G-(POG)2FOGERGVEG(POG)2-NH2
19 H-G-(POG)5-NH2
22 Ac-G-(POG)5-NH2
24 H-GG-(POG)5-NH2
20; 21[b] 0.15
< 5
n.d.
9.6
n.d.
9.2
37
50
55
58
55
58
62
n.d.
n.d.
0.12
n.d.
0.11
0.10
0.10
0.12
0.12
0.12
0.11
0.14
‡
1410.7 [M‡H]
ple
helical
conformation,
‡
1453.7 [M‡H]
‡
whereas the other product did
not. The CD spectrum of this
latter product resembled the
CD spectrum of the acetylated
single chain peptide, and no
temperature-dependent transi-
tion could be measured (Fig-
ure 2). Since the CTV triacid
scaffold that we used was a
racemic mixture of (‡) and
(À)-CTV, we most likely sepa-
rated the diastereomeric colla-
gen mimics originating from the
1467.7 [M‡H]
‡
26 Ac-GG-(POG)5-NH2
1510.7 [M‡H]
20a (‡ or À)CTV-[G-(POG)5-NH2]3
1586.7 [M‡3H]3‡
1607.7 [M‡3Na]3‡
1233.0 [M‡4H]4‡
20b other diastereomer
25a (‡ or À)CTV-[GG-(POG)5-NH2]3
25b other diastereomer
30a (À) CTV-[GG-(POG)5-NH2]3
30b (‡) CTV-[GG-(POG)5-NH2]3
21 KTA-[GG-(POG)5-NH2]3
1248.8 [M‡3Na‡H]4‡ 1248.8
1248.8 [M‡3Na‡H]4‡ 1249.3
1243.5 [M‡2Na‡2H]4‡ 1243.5
1151.8 [M‡4H]4‡
1152.5
[a] Melting temperatures (Tm) were determined in 10 mm AcOH at a concentration of 0.5 mgmLÀ1. Rpn: Ratio of
the positive value (at 223 or 224 nm, see text) over the negative value (at ꢀ200 nm). [b] Same measurements in
H2O instead of AcOH. n.d. : not determined.
for the CTV-assembled peptide is 19.38C and 20.38C for the
KTA-assembled compound, both measured in 10mm HOAc
(Figure 1). The melting temperatures in H2O are 23.0 and
21.48C, respectively. In contrast, the acetylated single chain
peptide did not adopt a triple helical conformation at 58C,
since no thermal transition could be measured; this indicates
that the presence of other amino acids–as are present in this
native sequence–than those of the POGrepeats (see below)
hamper the formation of triple helices.
two CTV enantiomers. Apparently, there is a difference in
assisting the correct folding of the collagen peptide into a
triple helix between the two CTV enantiomers. The orienta-
tion of the side chains of the CTV core is for one enantiomer
presumably more favorable for adopting the right-handed
triple helix conformation.
To further explore the different stabilizing properties of the
two CTV enantiomers, as well as the difference in stabilizing
properties between the CTV triacid and KTA(Gly-OH)3
scaffolds, we synthesized collagen model peptides H-Gly-
(Pro-Hyp-Gly)5-NH2 (19) and H-Gly-Gly-(Pro-Hyp-Gly)5-
During the HPLC purification of CTV[CH2C(O)N(H)Gly-
[Pro-Hyp-Gly]2-Phe-Hyp-Gly-Glu-Arg-Gly-Val-Glu-Gly-
Figure 1. A) CD spectrum of KTA[GG(POG)2FOGERGVEG(POG)2-NH2]3 (15), at a concentration of 0.5 mgmLÀ1 in 10 mm HOAc at 58C. B) CD
spectrum of CTV[G(POG)2FOGERGVEG(POG)2-NH2]3 (14), at a concentration of 0.5 mgmLÀ1 in 10 mm HOAc at 58C. C) Melting temperature
measurement of KTA[GG(POG)2FOGERGVEG(POG)2-NH2]3 (15). D) Melting temperature measurement of CTV[G(POG)2FOGERGVEG(POG)2-
NH2]3 (14).
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Collagen Peptides
4613 4621
equivalent (KTA[Gly-Gly-(Pro-Hyp-Gly)5-NH2]3), revealed
that, alike the native collagen peptide attached to these
scaffolds, the thermal stability of both compounds measured
in water is almost equal (i.e., Tm ˆ 58 and 628C, respectively).
The other, slightly less stable CTV[Gly-Gly(Pro-Hyp-Gly)5-
NH2]3 structure 25a or 25b, did also show a temperature-
dependent transition, which was somewhat lower (Tm ˆ
558C). A shortening of the spacer to only one glycine residue,
caused a marked decrease in the thermal stability of the CTV-
assembled peptides. The Tm of the most stable CTV[Gly(Pro-
Hyp-Gly)5-NH2]3 decreased to 508C, (Table 1) whereas the Tm
of the other diastereomer dropped to 378C (Table 1). All
these thermal transitions were fully reversible upon cooling to
48C.
To determine the absolute configuration of the CTV
enantiomer, present in the two CTV diastereomers, it was
decided to couple the diastereomers separately to the model
peptide. The required CTV enantiomers were obtained by
resolution of the racemic CTV triol 3, which was achieved by
chromatographic separation of the camphanic acid triesters of
CTV 27 as described by Canceill et al.[14, 15] The optically
active CTV triols 28a and 28b were converted into the
corresponding triacid scaffolds 29a and 29b, respectively, as
depicted in Scheme 5.
Figure 2. CD spectrum of (À)CTV[G(POG)2FOGERGVEG(POG)2-
NH2]3, at a concentration of 0.5 mgmLÀ1 in 10mm HOAc at 58C (top).
CD spectrum of Ac-(POG)2FOGERGVEG(POG)2-NH2, at a concentra-
tion of 0.5 mgmLÀ1 in 10mm HOAc at 58C (bottom).
After coupling of the chiral CTV templates to H-Gly-Gly-
(Pro-Hyp-Gly)5-NH2 leading to 30a and 30b (Figures 3 and
Figure 4), respectively, and concomitant CD analysis, the
melting temperatures were determined. As expected, the Tm
values did match perfectly the Tm values of the compounds
that were purified from the mixture of diastereomers. Based
upon these data we could assign (‡)-CTV as the best triple
helix stabilizing CTV triacid scaffold leading to collagen
mimic 30b, with a Tm of 588C (Table 1). The (À)-CTV
assembled peptide in collagen mimic 30a showed a Tm of
558C (Table 1). The orientation of the side chains of (‡)-CTV
seem to accomodate the right-handed folding of the super-
helix the best. The orientation of the side chains of (À)-CTV
might be less favorable, and at least two glycine residues were
NH2 (24) (Scheme 4, Table 1). The model peptides were
successfully coupled in solution to racemic CTV triacid using
BOP to give 20ab and 25ab, respectively. KTA(Gly-OH)3 was
coupled to the model peptide H-Gly-(Pro-Hyp-Gly)5-NH2 to
give 21. The influence of the spacer length (Gly and Gly-Gly)
between the CTV triacid scaffold and the peptide was studied.
A certain degree of flexibility of the spacer is needed to allow
for one residue staggering of the three peptide chains, which is
necessary for a triple helical formation. After synthesis of
CTV[Gly-(Pro-Hyp-Gly)5-NH2]3, starting from racemic CTV
triacid, both diastereomers could be separated by RP-HPLC.
A comparison of the CD data of the most stable CTV[Gly-
Gly(Pro-Hyp-Gly)5-NH2]3 molecule and the KTA-assembled
1. 20% piperidine, NMP
Fmoc(H)N
FmocN(H)-[Pro-Hyp-Gly]5-N(H)
2. Fmoc-Pro-Hyp-Gly-OH, HBTU,
HOBt, DIPEA, NMP
17
3. repeat steps 1 and 2 four times
1. 20% piperidine, NMP
1. 20% piperidine, NMP
2. TFA/TIS/H2O
17
FmocN(H)-Glyn-[Pro-Hyp-Gly]5-N(H)
H-Glyn-[Pro-Hyp-Gly]5-NH2
2. Fmoc-Gly-OH, HBTU,
DIPEA
18 (n=1)
23 (n=2)
19 (n=1)
24 (n=2)
4, BOP, separation
(+)- and (-)-CTV[CH2C(O)N(H)Gly-[Pro-Hyp-Gly]5-NH2]3
20a,b
19
2, BOP
KTA[Gly-Gly-[Pro-Hyp-Gly]5-NH2]3
19
19
21
AcOSu
Ac-Gly-[Pro-Hyp-Gly]5-NH2
22
4, BOP, separation
(+)- and (-)-CTV[CH2C(O)N(H)Gly-Gly-[Pro-Hyp-Gly]5-NH2]3
25a,b
24
24
AcOSu
Ac-Gly-Gly-[Pro-Hyp-Gly]5-NH2
26
Scheme 4. Synthesis of the collagen peptides containing the model peptides Gn(POG)5.
Chem. Eur. J. 2002, 8, No. 20 ¹ 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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R. M. J. Liskamp et al.
FULL PAPER
NH2
Gly
needed to allow the three as-
sembled peptides to fold into a
right-handed triple helix.
NH2
NH2
Gly
NH2
Gly
Gly
NH2
Gly
NH2
Hyp
Hyp
Gly
Hyp
Hyp
NH2
Gly
NH2
Gly
Pro
Gly
Gly
Hyp
Hyp
Pro
Gly
Gly
NH2
Gly
5
5
Pro
Gly
Gly
Pro
Gly
Gly
5
5
Pro
Gly
Gly
Pro
Gly
Gly
5
Discussion
5
Hyp
Hyp
Hyp
Pro
Gly
Gly
Pro
Gly
Gly
NH
HN
5
5
A synthetic collagen mimic can
serve as a valuable tool to study
the interaction of collagens
with other proteins in more
detail at the molecular level.
Therefore, a synthetic protocol
to synthesize small and stable
native collagen structures was
developed. Using one new and
one known scaffold molecule,
small native collagen sequences
were induced to fold into a
Pro
Gly
Gly
NH
HN
5
O
O
NH
HN
O
O
O
O
O
OMe
MeO
O
HN
NH
O
OMe
MeO
O
O
O
NH
O
O
MeO
OMe
O
Me
Me
Me
30a
30b
21
Figure 3. (À)-CTV[Gly-Gly-(Pro-Hyp-Gly)5-NH2]3 (30a), (‡)-CTV[Gly-Gly-(Pro-Hyp-Gly)5-NH2]3 (30b) and
KTA[Gly-Gly-(Pro-Hyp-Gly)5-NH2]3.
triple helical conformation. This was accomplished by assem-
bly of three collagen peptides on triacid scaffold structures
and extension of the native sequence with POGrepeats. The
peptides were prepared on the solid phase by an orthogonal
synthesis strategy. Cleavage from the resin left the carboxylic
Cl
OMe
*R
O
O
O
O
O
*R
O
O
*R
Cl
1. Separation
2. LiAlH4
MeO
3
O
TEA
O
OMe
R*
27
O
CH3O
OH
HO
CH3O
CH3O
OH
OH
CH3O
HO
OH
OCH3
H3CO
28a (-) CTV
28b (+) CTV
m
1. BrCH2CO2Et,
Cs2CO3, MeCN
2. Bu4NOH, MeOH,
CH2Cl2
Bu4N+O
O Bu4N+
O
O
Bu4N+O
O
O
O
Bu4N+
O
Bu4N+O
CH3O
O Bu4N+
O
O
CH3O
O
O
CH3O
CH3O
O
O
O
OCH3 H3CO
Figure 4. Molecular models constructed using MacroModel[29] of
(À)-CTV[Gly-Gly-(Pro-Hyp-Gly)5-NH2]3 (30a, left), (‡)-CTV[Gly-Gly-
(Pro-Hyp-Gly)5-NH2]3 (30b, right) clearly showing the triple helix struc-
ture.
29a
29b
m
Scheme 5. Optical Resolution of the CTV triacid scaffold.
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4618

Collagen Peptides
4613 4621
acid moieties of the amino acid side chains protected. This
enabled coupling of the amino-terminus to the triacid
scaffolds.
to allow the correct folding. The different stabilizing proper-
ties of the two CTV diastereomers may be explained by the
orientation of the side chains of the CTV cone, which
probably point in the opposite direction. A certain flexibility
is therefore likely necessary for both one amino acid residue
staggering and right-handed folding of the triple helix. This
was also shown by comparing the stability of the two CTV
triacid scaffolds coupled to H-G(POG)5-NH2. In case of (À)-
CTV coupled to H-G(POG)5-NH2, the melting temperature
was 378C compared with 508C for the other CTV diaster-
eomer (Table 1). If the flexibility was limited to one glycine,
the first residues of the peptide will probably have to function
as the spacer to compensate for the less ideal orientation of
the side chains. Since the difference in Tm for the two
CTV[G(POG)5-NH2]3 diastereomers is significantly larger
than for both CTV[GG(POG)5-NH2]3 diastereomers, the two
glycines can probably also compensate partly for the less ideal
orientation of the side chains of (À)-CTV in order to induce
the right-handed superhelix formation. The large difference in
Tm for the two CTV-collagen derived (CTV[G(POG)5-NH2]3)
peptides also correlates with the large difference in thermal
stability found for CTV[G(POG)2FOGERGVEG(POG)2-
NH2]3.
The two peptides used in this study differ also in their triple
helical twist. High resolution crystal structures of collagen
peptides show that peptides composed of repetitive POG
triplets generate a seven-fold (75) symmetry, whereas the
native collagen peptides, possessing imino-acid-poor regions,
generate a ten-fold (107) symmetry.[25, 28] This sequence
dependent helical twisting may thus also effect our conclu-
sions, since we compare the triple helical folding of both a
native sequence and an imino-acid-rich peptide Gn(POG)5.
However, since we only observe a small difference in thermal
stability between the KTA(G-OH)3 template and the (‡)-
CTV triacid scaffold, it is not expected that the helical twisting
effects the stability data.
Comparison of the structures of the two triacid scaffolds
showed that the CTV template is much larger. The distance
between the three CTV-cone side chains is approximately 8
10 ä. For KTA the distance between these three function-
alities is much shorter and more close to the interchain
distances of the three peptides in the triple helix (ꢀ3 ä). This
could explain that for the CTV triacid as a scaffold, the full
length of the G2 spacer was necessary for a proper folding of
the whole peptide into a triple helix.
The moderate to low yields of the collagen mimics were
mostly due to difficult RP-HPLC purifications. HPLC puri-
fication of collagen structures can be difficult, as was also
shown by others. RP-HPLC of such structures can result in
peak broadening resulting from aggregation or conforma-
tional effects.[26] Especially collagen structures composed of
POGrepeats are prone to partial denaturation by the HPLC
system.[27] It was shown that conformations may differ at
different HPLC conditions. In our case, the collagen struc-
tures containing the native sequences could be purified
somewhat easier by RP-HPLC than the collagen structures
composed of only POGrepeats. The latter structures gave
broad peaks. In addition, purification of collagen mimics
which were synthesized from the racemic CTV triacid scaffold
The necessity for stabilization of collagen peptides con-
taining native sequences is mostly due to the destabilizing
effect of non-imino acids on the collagen triple helix. As was
illustrated by studies of the group of Brodsky,[17] replacement
of two imino acids in a POGmultimer by two non-imino acids
residues, commonly found in the native collagen molecule,
resulted in a dramatic destabilization of the triple helix.
The idea of an organized assembly of three peptide chains
to increase the stability of the collagen triple helix has been
described by several groups. A strong reduction of entropy
loss involved in triple helix formation is thought to be the
driving force for the increased triple helical stability. Assem-
bly has been achieved using a template structure such as the
Kemp×s triacid, the tripodal amine TREN or di-amino
21]
acids.[7, 18 Tanaka et al.[20] constructed a collagen molecule
by crosslinking both the C- and N-terminus by a Lys-Lys
dimer template. Alternatively, the three peptides can be
assembled by disulfide bridges, which also allowed assembly
of heterotrimeric constructs.[22] Directed self-assembly, with-
out covalent linkages between the peptides, has been achieved
by peptide amphiphiles.[23]
A comparison of the various stabilizing strategies is
difficult, since no consensus sequences were used. In this
study we showed the stabilizing properties of a new triacid
scaffold, compared with the known Kemp×s triacid, using two
different kinds of collagen peptides. One peptide we used
contains the a2b1 integrin recognition sequence GFO-
GERGVE (residues 502 510 of human type I collagen
a1(I) chain[24]). The other peptides were composed of POG
repeating units. The high imino acid content in these peptides
resulted in a different handedness (seven-fold symmetry) of
the helix compared with the natural collagen molecule (ten-
fold symmetry).[25] Both scaffolds significantly increased the
Tm of the triple helical structures, compared with non-
assembled peptides (Table 1). The KTA(G-OH)3 was already
used for studying the structure and stability of collagen and
collagen-like triple helices.[7] The CTV triacid scaffold, which
has C3 symmetry, has not been used before to induce collagen
structures. Our data demonstrated that the CTV triacid can
indeed stabilize the triple helical folding of a collagen peptide.
This was shown by CD melting curves of the triple helical
structure of either an assembled peptide containing a native
collagen motif, or an assembled collagen model peptide. Due
to the different eluting properties of the two CTV diaster-
eomers after coupling to the peptides, we were able to study
the effect of each of both diastereomers.
Our data showed that (À)-CTV was less potent than (‡)-
CTV in assisting the correct folding of the collagen peptides.
In case of the native collagen motif, no folding into a triple
helical structure could be measured for (À)-CTV. However,
due to the high thermal stability of the triple helix of the
model peptide H-Gn(POG)5-NH2 (n ˆ 1,2), a triple helical
structure of (À)-CTV could be measured (Table 1). The
remarkable difference in stability of the CTV triacid diaster-
eomers coupled to either H-G(POG)5-NH2 or H-GG(POG)5-
NH2 illustrated the necessity for a certain degree of flexibility
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R. M. J. Liskamp et al.
FULL PAPER
13C NMR (CDCl3, 75 MHz): d ˆ 24.01, 28.20, 29.15, 36.22, 40.91, 46.79,
was even more difficult, due to the different retention times of
the two CTV diastereomers.
54.69, 51.86, 57.94, 58.35, 70.19, 79.84, 154.80, 170.15, 172.01, 172.39; ES-
‡
MS: m/z: calcd for C18H29N3O7 ‡ Na : 421.2; found: 422.4.
Fmoc-Pro-Hyp-Gly-OH (9): Boc-Pro-Hyp-Gly-OMe (5.1 g; 10.1 mmol)
was dissolved in 1n HCl (100 mL), and the mixture was stirred for 3 d.
Subsequently, the mixture was concentrated in vacuo, and the residue was
dissolved in CH3CN/H2O (80 mL, 1:1 v/v). The pH was adjusted to pH 9
9.5 using TEA. A solution of of Fmoc-OSu (4.3 g; 12.7 mmol) in CH3CN
(75 mL) was then added in one portion. Stirring was continued for 1 h, and
the pH of the solution was maintained at pH 8.5 9. Subsequently 2n HCl
was added until pH 7.5, and the solvents were removed in vacuo. The
residue was dissolved in 2% NaHCO3 and was washed three times with
EtOAc. The aqueous phase was acidified to pH 3 with 2n HCl, and the
mixture was stored overnight at 48C, after which a white precipitate was
formed. The precipitate was dried, and crystallized from hot CH3CN to give
Fmoc-Pro-Hyp-Gly-OH (4.03 g, 63%). M.p. 179 1828C (lit.:[11] 177
1818C); Rf ˆ 0.19 (CHCl3/MeOH/AcOH 40:10:1 v/v/v); RP-HPLC (C18,
300 ä, gradient of 10 60% B in 50 min): tR ˆ 29 min; ES-MS: m/z: calcd
Conclusion
We have introduced a new chiral scaffold that induces the
folding of collagen peptides into a triple helical structure.
Although both enantiomers are capable of folding collagen
peptides into a triple helix, CD data shows that the (‡)-CTV
enantiomer is a better triple helix-stabilizing scaffold than
(À)-CTV. In addition to POGrepeats, the CTV scaffold was
also effective in stabilizing the triple helix of native collagen
sequences, comparable to the ability of KTA. The latter was
demonstrated for the first time in this paper. Thus, the CTV
scaffold seems a valuable addition to the presently available
limited number of rigid scaffolds, which can be used for the
organization of large peptide sequences as is described here as
well as smaller peptides.[30]
‡
‡
for C27H29N3O7 ‡ H : 508.54; found: 508.55, 530.35 [M‡Na] .
Synthesis and analysis of linear peptides: All peptides were synthesized as
C-terminal amides by solid phase peptide synthesis on an ABI 433A
peptide synthesizer using Argogel Rink-amide resin and the Fastmoc
0.25 mmol protocol. All amino acids were obtained from Alexis or
Novabiochem and are of the l-configuration. Removal of the Fmoc group
was monitored at 301 nm. Sequences of Pro-Hyp-Gly were introduced by
segment condensation using 2 equiv of Fmoc-Pro-Hyp-Gly-OH. Cleavage
of the peptide from the resin was carried out with TFA/TIS/H2O (92.5:2.5:5
v/v/v). To obtain the acetylated compounds, the cleaved peptide was
treated with 1.5 equiv of AcOSu in DMF/DIPEA. Removal of the allyl
protection group was performed in solution by treatment for 72 h with
[Pd(PPh3)4] and DMF/AcOH/NMM (50:10:1 v/v/v) under Ar atmos-
phere,[12] followed by applying the reaction mixture to a PD10 (sephadex
G25) gel filtration column. All peptides were purified by preparative RP-
HPLC (C18, 300 ä), using a gradient from A (0.1% TFA in H2O) to 80%
B (0.085% TFA in 95% CH3CN/H2O). Analysis was performed by ES or
MALDI-TOF MS.
Experimental Section
Synthesis of Fmoc-POG-OH (9)
Boc-Pro-Hyp-OMe (6): HCl ¥ H-Hyp-OMe (3.9 g; 26.9 mmol, 1.05 equiv)
was dissolved in dry DMF (90 mL), and the solution was cooled to 08C.
Subsequently Et3N (11.6 mL; 83.3 mmol), Boc-Pro-OH (5.5 g; 25.6 mmol)
and BOP (11.3 g; 25.6 mmol) were added. After 4 h, the cooling bath was
removed, and the mixture was stirred for 2 d at RT. Next, the mixture was
concentrated in vacuo, and the residue was dissolved in EtOAc, and was
washed once (successive washing with aqueous solutions resulted in severe
loss of product, due to the aqueous solubility of this compound) with 5%
KHSO4, 5% NaHCO3 and brine. The product was purified by column
chromatography (EtOAc) to give Boc-Pro-Hyp-OMe as a colorless oil
(6.0 g, 68%). [a]D ˆ À109.5 (c ˆ 1, MeOH) [lit.:[10] À112.6 (c ˆ 0.8,
MeOH); Rf ˆ 0.15 (EtOAc); 1H NMR (CDCl3, 300 MHz): d ˆ 1.40, 1.43
(2 Â s, 9H, Boc), 1.79 2.58 (m, 6H, Pro-g, Pro-b, Hyp-b), 3.35 3.82, 4.00,
4.04, 4.20 4.71 (m, 7H, Pro-d, Pro-a, Hyp-d, Hyp-a, Hyp-g), 3.72 (s, 3H,
OMe); 13C NMR (CDCl3, 75 MHz): d ˆ 24.00, 28.29, 29.03, 37.30, 46.81,
52.01, 54.53, 57.33, 57.39, 70.11, 79.84, 154.62, 171.28, 172.79.
Purification and analysis of assembled peptides: After coupling of the
peptides to the triacid scaffolds, the reaction mixture was dissolved in H2O/
CH3CN (4:1 v/v) and was dialyzed (membrane cutoff 3500) against H2O
(3 Â 2 L). Allyl protection groups of the assembled peptides were removed
by treatment for 72 h with [Pd(PPh3P)4] as described above. Final
purification was performed by preparative RP-HPLC (C18, 300 ä), using
a gradient from A (0.1% TFA in H2O) to 60% B (0.085% TFA in 95%
CH3CN/H2O). Analysis was performed by ES or MALDI-TOF MS.
Boc-Pro-Hyp-OH (7): Boc-Pro-Hyp-OMe (6.0 g; 18.3 mmol) was dis-
solved in dioxane/MeOH/4n NaOH (14:5:1 v/v/v) and the mixture was
stirred for 16 hours at RT. The mixture was concentrated in vacuo and the
residue was dissolved in H2O. The aqueous solution was acidified to pH 2
with 2n KHSO4, followed by successive extractions with EtOAc. After
removal of the solvents in vacuo, Boc-Pro-Hyp-OH was obtained as a white
foam (4.6 g, 80%). [a]D ˆ À66.2 (c ˆ 1, CHCl3); Rf ˆ 0.10 (CH2Cl2/MeOH
9:1); 1H NMR (CDCl3, 300 MHz): d ˆ 1.39, 1.43 (2  s, 9H, Boc), 1.76 2.40
(m, 6H, Pro-g, Pro-b, Hyp-b), 3.34 3.91 (m, 4H, Pro-d, Hyp-d), 4.36 4.72
(m, 3H, Pro-a, Hyp-a, Hyp-g); 13C NMR (CDCl3, 75 MHz): d ˆ 24.08,
28.36, 29.11, 36.82, 46.99, 54.75, 57.61, 58.16, 70.1, 80.51, 154.98, 172.47,
CD spectroscopy: Spectra were recorded on a OLIS spectropolarimeter
using a 0.01 mm pathlength quartz cuvette. The cuvette was placed in a
thermally controlable holder. Samples were dissolved in H2O or in 10 mm
AcOH at a concentration of 0.5 mgmLÀ1 and were stored at 48C at least
24 h prior to recording the measurements. Spectra were obtained by
averaging 10 scans. Melting temperatures were obtained by data collection
of the positive maximum at 220 225 nm at a heating rate of 0.258CminÀ1
and 15 min equilibration at the temperature of data collection.
(À)-2,7,12-Trimethoxy-3,8,13-[tris(acetoxyethyl)oxy]-10,15-dihydro-5H-
tribenzo[a,d,g]cyclononene: The synthesis of the cyclotriveratrylene triol
(racemic mixture) and the optical resolution of this triol has been described
by Canceill et al.[13, 14] The thus obtained (‡)-triol (125 mg; 0.31 mmol) was
dissolved in dry CH3CN (30 mL), and the mixture was cooled to 48C.
Subsequently Cs2CO3 (600 mg; 1.84 mmol) was added, and after stirring for
5 min bromoacetic ethyl ester (120 mL) was added. The mixture was
allowed to warm to room temperature, and was stirred for 2 d. The solvent
was removed without heating (to avoid racemisation of CTV) in vacuo, and
the residue was dissolved in EtOAc and 1n HCl. The EtOAc layer was
washed with brine and dried over anhydrous Na2SO4. After removal of the
solvent in vacuo, again without heating, the residue was purified by column
chromatography (CH2Cl2/Et2O, gradient from 5 to 10% Et2O) to give the
desired compound (93 mg, 46%). [a]D ˆ À118 (c ˆ 1, CHCl3); 1H NMR
(CDCl3, 200 MHz): d ˆ 1.23 (t, 9H), 3.52 (d, 3H), 3.88 (s, 9H, OCH3), 4.19
(m,6H), 4.59 (d, 6H), 4.71 (d, 3H), 6.87 (d, 6H, arom H); 13C NMR
(CDCl3, 75 MHz): d ˆ 14.08, 36.33, 55.98, 61.08, 67.56, 113.74, 118.00,
131.42, 134.03, 145.94, 148.70, 169.40.
‡
173.81; ES-MS: m/z: calcd for C15H24N2O6 ‡ Na : 350.2; found: 351.1.
Boc-Pro-Hyp-Gly-OMe (8): Boc-Pro-Hyp-OH (4.6 g; 14.0 mmol), HCl ¥
H-Gly-OMe (2.0 g; 15.9 mmol) and HOBt ¥ H2O (2.3 g; 15.0 mmol) were
dissolved in DMF (50 mL). Subsequently, DIPEA (2.7 mL) was added, and
the mixture was cooled on ice. EDCI (3.0 g; 15.6 mmol) was then added,
and after cooling for another 4 h the solution was stirred at room
temperature overnight. The solvents were then removed in vacuo, and
the residue was suspended in CH2Cl2 (350 mL) and filtered. The CH2Cl2
solution was washed twice with 5% NaHCO3 (10 mL). The organic layer
was dried over anhydrous Na2SO4. After removal of the solvent in vacuo,
the residue was purified by column chromatography (CH2Cl2/MeOH,
gradient from 2% to 5% MeOH) to give Boc-Pro-Hyp-Gly-OMe as a
white foam (5.1 g, 90%). Rf ˆ 0.22 (CH2Cl2/MeOH 9:1 v/v); 1H NMR
(CDCl3, 300 MHz): d ˆ 1.40, 1.44 (2  s, 9H, Boc), 1.76 2.54 (m, 6H, Pro-
g, Pro-b, Hyp-b), 3.36 4.80 (m, 9H, Gly-a, Pro-d, Hyp-d, Pro-a, Hyp-a,
Hyp-g), 3.69, 3.70 (2 Â s, 3H, OMe), 7.76, 8.50 (2 Â m, 1H, Gly-NH);
4620
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Chem. Eur. J. 2002, 8, No. 20

Collagen Peptides
4613 4621
(‡)-2,7,12-Trimethoxy-3,8,13-[tris(acetoxyethyl)oxy]-10,15-dihydro-5H-
tribenzo[a,d,g]cyclononene: The procedure was as described for the (À)-
enantiomer. Yield: 70 mg (50%). [a]D ˆ ‡88 (c ˆ 1, CHCl3); 1H NMR
(CDCl3, 200 MHz): d ˆ 1.23 (t, 9H), 3.52 (d, 3H), 3.88 (s, 9H, OCH3), 4.19
(m, 6H), 4.59 (d, 6H), 4.70 (d, 3H), 6.87 (d, 6H, ar H); 13C NMR (CDCl3,
75 MHz): d ˆ 14.08, 36.33, 55.98, 61.08, 67.56, 113.74, 118.00, 131.42, 134.03,
145.94, 148.70, 169.40.
[9] M. Goodman, Y. Feng, G. Melacini, J. P. Taulane, J. Am. Chem. Soc.
1996, 118, 5156 5157.
[10] A. Lewis, J. Wilkie, T. J. Rutherford, D. Gani, J. Chem. Soc. Perkin
Trans. 1 1998, 3777 3793.
[11] J. Ottl, H. J. Musiol, L. Moroder, J. Pept. Sci. 1999, 5, 103 110.
[12] A cyclotribenzylene (CTB) derivative was used as a scaffold for a
three-helix template-assembled synthetic protein: A. S. Causton, J. C.
Sherman, Bioorg. Med. Chem. 1999, 7, 23 27.
[13] S. A. Kates, N. A. Sole, C. R. Johnson, D. Hudson, G. Barany, F.
Alberico, Tetrahedron. Lett. 1993, 34, 1549 1552.
[14] J. Canceill, A. Collet, G. Gottarelli, J. Am. Chem. Soc. 1984, 106,
5997 6003.
[15] J. Canceill, A. Collet, J. Gbard, G. Gottarelli, G. P. Spada, J. Am.
Chem. Soc. 1985, 107, 1299 1308.
[16] G. Veriot, J.-P. Dutasta, G. Matouzenko, A. Collet, Tetrahedron 1995,
51, 398 400.
[17] J. A. Ramshaw, N. K. Shah, B. Brodsky, J. Str. Biol. 1998, 122, 86 91;
A. V. Persikov, J. A. Ramshaw, B. Brodsky, Biopolymers 2000, 55,
436 450.
[18] W. Henkel, T. Vogl, H. Echner, W. Voelter, C. Urbanke, D. Schleuder,
J. Rauterberg, Biochemistry 1999, 38, 13610 13622.
[19] H. Hojo, Y. Akamatsu, K. Yamauchi, M. Kinoshita, S. Miki, Y.
Nakamura, Tetrahedron 1997, 53, 14263 14274.
CTV[G(POG)2FOGERGVEG(POG)2-NH2]3: The lineair peptide
H-G(POG)2FOGE(OAll)RGVE(OAll)G(POG)2-NH2]3 (25 mg, 12 mmol),
which was prepared as desribed above, and the NBu4 salt of CTV triacid[16]
(2.33 mg; 4 mmol) were dissolved in DMF (0.75 mL). Subsequently Et3N
(7.5 mL) was added, and the mixture was cooled to 48C. After 5 min, BOP
(9 mg; 20.4 mmol) in DMF (100 mL) was added, and the mixture was stirred
at RT. After 48 h the mixture was concentrated in vacuo and the residue
was dissolved in H2O/CH3CN (5 mL). After HPLC purification, the
separate diastereomers (2 mg each) were obtained. ES-MS: m/z: calcd for
‡
C
303H426N78O102 ‡ 5H : 1358.8; found: 1358.7 (diastereomer 1) and m/z:
‡
calcd for C303H426N78O102 ‡ 4H : 1698.3; found: 1698.4 (diastereomer 2).
All other assembled peptides were synthesized as described above and are
listed in Table 1
Acknowledgements
[20] Y. Tanaka, K.Suzuki, T. Tanaka, J. Pept. Sci. 1998, 51, 413 419.
[21] M. Goodman, J. Kwak, E. Locardi, In Peptides for the new millennium
(Eds.: G. B. Fields, J. P. Tam, G. Barany), Kluwer, Dordrecht, 2000,
pp. 352 354.
This project was supported by grant PL963517 of the European Commis-
sion.
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Received: April 22, 2002 [F4032]
Chem. Eur. J. 2002, 8, No. 20
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