Self-assembly of amylin(20-29) amide-bond derivatives into helical ribbons and peptide nanotubes rather than fibrils

Article abstract of DOI:10.1002/chem.200501374

Uncontrolled aggregation of proteins or polypeptides can be detrimental for normal cellular processes in healthy organisms. Proteins or polypeptides that form these amyloid deposits differ in their primary sequence but share a common structural motif: the (anti)parallel β sheet. A well-accepted approach for interfering with -sheet formation is the design of soluble β-sheet peptides to disrupt the hydrogen-bonding network; this ultimately leads to the disassembly of the aggregates or fibrils. Here, we describe the synthesis, spectroscopic analysis, and aggregation behavior, imaged by electron microscopy, of several backbone-modified amylin(20-29) derivatives. It was found that these amylin derivatives were not able to form fibrils and to some extent were able to inhibit fibril growth of native amylin(20-29). However, two of the amylin peptides were able to form large supramolecular assemblies, like helical ribbons and peptide nanotubes, in which β-sheet formation was clearly absent. This was quite unexpectedWiley-VCH since these peptides have been designed as soluble β-sheet breakers for disrupting the characteristic hydrogen-bonding network of (anti)parallel β sheets. The increased hydrophobicity and the presence of essential amino acid side chains in the newly designed amylin(20-29) derivatives were found to be the driving force for self-assembly into helical ribbons and peptide nanotubes. This example of controlled and desired peptide aggregation may be a strong impetus for research on bionanomaterials in which special shapes and assemblies are the focus of interest.

Full text of DOI:10.1002/chem.200501374

DOI: 10.1002/chem.200501374
Self-Assembly of Amylin(20–29)Amide-Bond Derivatives into Helical
A
Ribbons and Peptide Nanotubes rather than Fibrils
Ronald C. Elgersma,[a] Tania Meijneke,[a] George Posthuma,[b] Dirk T. S. Rijkers,[a] and
Rob M. J. Liskamp*[a]
Abstract: Uncontrolled aggregation of
proteins or polypeptides can be detri-
mental for normal cellular processes in
healthy organisms. Proteins or polypep-
tides that form these amyloid deposits
differ in their primary sequence but
share a common structural motif: the
(anti)parallel b sheet. Awell-accepted
approach for interfering with b-sheet
formation is the design of soluble b-
sheet peptides to disrupt the hydrogen-
bonding network; this ultimately leads
to the disassembly of the aggregates or
fibrils. Here, we describe the synthesis,
spectroscopic analysis, and aggregation
behavior, imaged by electron microsco-
py, of several backbone-modified
amylin(20–29) derivatives. It was found
that these amylin derivatives were not
able to form fibrils and to some extent
were able to inhibit fibril growth of
since these peptides have been de-
signed as soluble b-sheet breakers for
disrupting the characteristic hydrogen-
bonding network of (anti)parallel b
sheets. The increased hydrophobicity
and the presence of essential amino
acid side chains in the newly designed
AHCTREUNG
native amylin(20–29). However, two of
G
the amylin peptides were able to form
large supramolecular assemblies, like
helical ribbons and peptide nanotubes,
in which b-sheet formation was clearly
absent. This was quite unexpected
amylin(20–29) derivatives were found
N
to be the driving force for self-assem-
bly into helical ribbons and peptide
nanotubes. This example of controlled
and desired peptide aggregation may
be a strong impetus for research on
bionanomaterials in which special
shapes and assemblies are the focus of
interest.
Keywords: amyloids · helical struc-
tures · peptides · protein modifica-
tions · self-assembly
Introduction
loid polypeptide (IAPP) in the form of amylin fibrils which
are present in the pancreatic islets. Since peptide–peptide,
peptide–protein, and protein–protein interactions are ubiq-
uitous, it is fair to expect that, in the future, diseases will be
uncovered where protein aggregation is a (co)causitive
factor, especially when locally high peptide/protein concen-
trations favor possible intermolecular aggregation, as is the
case for the example with insulin.[6] Insights into the mecha-
nism of aggregation by structure–aggregation–activity stud-
ies, might not only shed more light on the structural parame-
ters, which play key roles in these processes, but also lead to
compounds capable of interfering with aggregation.[7–9]
Uncontrolled peptide/protein aggregation leading to precipi-
tation of proteins is a major cause of a number of diseases[1]
for which there is no therapy available as yet. The most
well-known diseases of these diseases are Alzheimerꢀs dis-
ease,[2] Parkinsonꢀs disease,[3] transmissible spongiform en-
cephalopathies (scrapie, bovine spongiform encephalopathy
(BSE), and Creutzfeldt–Jakob disease),[4] and diabetes
type II.[5] The latter is characterized by deposits of islet amy-
[a] R. C. Elgersma, T. Meijneke, Dr. Ir. D. T. S. Rijkers,
Prof. Dr. R. M. J. Liskamp
For interference with amyloid formation by inhibition of
b sheets, small molecules including small peptides and pepti-
domimetics are naturally preferred as possible future drugs
for inhibition of fibril growth or resolubilization of fibrils in
aggregation diseases and considerable activity is currently
taken place in this area of research.[7–9] Although we have
shown that mutation of a single amide bond, into the corre-
sponding ester, peptoid, or N-butylated amino acid residue,
Department of Medicinal Chemistry
Utrecht Institute for Pharmaceutical Sciences
Utrecht University, P.O. Box 80082
3508 TB Utrecht (The Netherlands)
Fax : (+31)30-253-6655
E-mail: r.m.j.liskamp@pharm.uu.nl
[b] Dr. G. Posthuma
Department of Cell Biology
Center for Electron Microscopy
University Medical Center, 3508 GAUtrecht (The Netherlands)
at position 28 of human IAPP
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amyloid formation,[10a] these modified human IAPP
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FULL PAPER
derivatives were not, however, able to inhibit/retard fibril
formation of full-length amylin (37 residues)[11,12] or to resol-
ubilize preformed amylin fibrils.[10b] However, amide bond
modification at alternate posi-
drogen-bonding network (Scheme 1, top).[16] In general, the
formation of (anti)parallel b-pleated sheets is responsible
for a decreased solubility of many proteins. However, by
tions has been used successfully
by Meredith and co-workers
with (Ab(16–22): Ac-Lys-Leu-
A
Val-Phe-Phe-Ala-Glu-NH2)
peptides[13] in order to obtain
aggregation inhibitors. There-
fore, we have designed amylin-
A
amide bonds at positions 24, 26,
and 28 have been modified by
N-butylation or by incorpora-
tion of peptoid- or ester-bond
moieties. Thus, at crucial posi-
tions the NH hydrogen-bond
donors of the amide bond are
no longer present and bulky
Scheme 1. Rationale for the design of b-sheet-breaker peptides based on the amylin(20–29) sequence.
A
substituents have been introduced on to the nitrogen atom.
Alternatively, NH hydrogen-bond donors were replaced by
oxygen atoms through the preparation of suitable depsipep-
tides. These newly designed amylin derivatives did not form
amyloid fibrils but surprisingly gave rise to the formation of
helical ribbons and peptide nanotubes by self-assembly.
In general, these and other backbone modifications
assume a central position in our research program.[14] Back-
bone modifications are both very important and highly inter-
esting because the peptide side chains are maintained for
molecular recognition, while changes in the geometrical con-
straints (f, y, and w dihedral angles, absence or presence of
hydrogen-bond donors/acceptors) are crucial for the result-
ing supramolecular assembly. It will be the balance between
these two structural moieties—side chains versus back-
bone—in peptides and peptidomimetics that will determine
the outcome with respect to the biological activity and/or
material properties.
using a simple model as a basis for preventing or at least in-
terfering with this hydrogen-bond formation (Scheme 1,
bottom), we, and others, have shown that specifically de-
signed peptides are capable of delaying and/or inhibiting b-
sheet formation and thus fibril formation.[9,10a] In principle,
such peptides, denoted as b-sheet-breaker peptides, could be
used as a new approach for therapeutic intervention in amy-
loid formation. Along these lines, we found that a single
amino acid substitution in the amylin(20–29) sequence,
R
namely, Ser28!NNle (NNle=N-butylglycine), was respon-
sible for complete inhibition of fibril formation and also de-
layed fibril formation of native amylin
preformed fibrils of native amylin(20–29) or full-length
amylin did not redissolve in the presence of amylin(20–
29)Ser28!NNle. This contrasted with results described in
A
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the literature with N-methylated Ab
U
G
There are many examples of controlled and desired pro-
tein aggregation mechanisms in living organisms. If these
protein aggregation mechanisms, especially those involved
in certain diseases, lead to typical morphological changes,
they may have a significant impact on research into bio-
dogenic peptide that could redissolve preformed Ab
fibrils,[13] was applied to our amyloid peptide model, amylin-
(20–29). At three alternate positions[17] (Scheme 1) the
A
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amide bonds of the native peptide 6 were replaced by ester
moieties (depsipeptides), N-butylated amino acid residues,
N-butylated glycine residues (peptoids) by preparation of
nanomaterials,[15] in which special shapes and assemblies are
within the focus of interest. The described amylin deriva-
tives open up promising possibilities for the further study of
the process of aggregation as well as for carrying out struc-
ture–activity-relationship studies in order to design bionano-
materials based on self-assembly of amyloid-derived pep-
tides.
human IAPP(20–29) derivatives 7–12 (Scheme 2). In the
T
cases of the peptide–peptoid hybrids 9, 11, and 12, the influ-
ence of the a-amino acid side chains on the amyloidogenic
character of the peptide was also investigated.
Synthesis of the peptides: Unfortunately, direct solid-phase
synthesis of the depsipeptide 7 and the N-butylated peptide
8 gave unsatisfactory results.[10b] It was thought that intro-
duction of the complete building blocks comprising the dep-
sipeptide moiety and the peptoid moiety, that is, Fmoc-Ala-
Ilec-OH (2; Fmoc=9H-fluoren-9-ylmethoxycarbonyl, Ilec=
2S-hydroxy-3S-methylpentanoic acid) and Fmoc-Ala-
Results and Discussion
Rationale for design: Amyloid fibrils ultimately result from
the assembly of antiparallel oriented peptides, in which the
amide bonds of the peptide backbone can form an ideal hy-
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R. M. J. Liskamp et al.
(NBu)Ile-OH (5), respectively, would be more successful
(Scheme 3).
For the synthesis of Fmoc-Ala-Ilec-OH (2), the l-a-hy-
droxy acid equivalent of l-isoleucine was first synthesized
by diazotization of the amino acid according to the method
of Shin et al.[18a] followed by esterification with allylbromide;
a-hydroxy acid allyl ester 1 was obtained in 61% overall
yield. Fmoc-Ala-OH was coupled to this with DCC/DMAP
in 68% yield[18c] and the allyl ester was removed with Pd-
A
give the Fmoc-protected dipeptide acid 2 in 71% yield.
For N-butylation of isoleucine, the corresponding benzyl
ester (65% yield) was synthesized and this was followed by
introduction of the sulfonamide moiety with p-nitrobenzene-
sulfonyl chloride in the presence of TEAas base to give
3
in 66% yield. After a Mitsunobu reaction[20] with 1-butanol/
PPh3/DIAD in THF, with a nearly quantitative yield,[21] and
removal of the p-nitrobenzenesulfonyl moiety by thiopheno-
late, the secondary amine 4 was obtained in 67% yield.
Coupling of Fmoc-Ala-OH to H-N(Bu)Ile-OBzl was very
difficult due to severe steric hindrance. Coupling reagents 3-
(3-dimethylaminopropyl)-1-ethylcarbodiimide
aza-1-hydroxy-1H-benzotriazole (HOAt),[22] N-[(dimethyl-
amino)-1H-1,2,3-triazole[4,5-b]-pyridin-1-ylmethylene]-N-
methylmethanaminium hexafluorophosphate N-oxide
(EDCI)/7-
A
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[24]
(HATU)/HOAt/TEA,[23] and neat sym-collidine/HATU
did not give the desired dipeptide. However, the use of tri-
phosgene, as described by Falb et al.,[25] via the in situ acid
chloride gave—after hydrogenolysis—the required Fmoc-
Ala-(NBu)Ile-OH (5) building block in 35% (over two
steps).
Peptides 6 and 10 were synthesized automatically by using
Fmoc/tBu solid-phase peptide-synthesis protocols.[26] Pep-
tide–peptoid hybrids 9, 11, and 12 were assembled on the
solid phase as described by Kruijtzer et al.[27]
The synthesis of depsipeptide 7 is depicted in Scheme 4.
After treatment of Fmoc-Ser(tBu)-NH-Rink amide ArgoGel
G
with piperidine to remove the Fmoc group, O-tritylglycolic
acid was coupled with BOP/HOBt/DIPEAin NMP. This
coupling was complete after 2 h according to the Kaiser
test[28] and the trityl group was then removed by diluted
Scheme 2. Structures of the amylin
study.
A
Scheme 3. Synthesis of depsidipeptide Fmoc-Ala-Ilec-OH (2) and N-butylated dipeptide Fmoc-Ala-N(Bu)Ile-OH (5). DCC=N,N’-dicyclohexylcarbodi-
imide, DMAP=4-(N,N-dimethylamino)pyridine, Tos=toluene-4-sulfonyl, Bzl=benzyl, pNBS-Cl=4-nitrobenzenesulfonyl chloride, TEA=triethylamine,
DIAD=diisopropyl azodicarboxylate, THF=tetrahydrofuran, DMF=N,N-dimethylformamide, BTC=bis(trichloromethyl)carbonate=triphosgene.
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Amylin(20–29) Derivatives
A
FULL PAPER
Scheme 4. Solid-phase synthesis of depsipeptide 7. Rink=4-[(2’,4’-dimethoxyphenyl)aminomethyl]phenoxy, NMP=N-methylpyrrolidone, HBTU=2-
(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, HOBt=N-hydroxybenzotriazole, DIPEA=N,N-diisopropylethylamine, Trt=
trityl=triphenylmethyl, Glyc=glycolic acid, BOP=benzotriazol-1-yl-oxy-tris-(dimethylamino)phosphonium hexafluorophosphate, TFA=trifluoroacetic
acid, TIS=triisopropylsilane, DCM=dichloromethane, DIC=N,N’-diisopropylcarbodiimide, SPPS=solid-phase peptide synthesis.
TFA. The resulting free hydroxy group was coupled to
Fmoc-Leu-OH with DIC/HOBt/DMAP in NMP[29] and the
coupling yield (76%) was determined by an Fmoc determi-
nation.[30] Next, Fmoc-group removal was effected with pi-
peridine and dipeptide building block 2 was introduced with
HATU/HOAt/DIPEA.[23] After deprotection of the resulting
product, O-trityl glycolic acid was coupled. Finally, removal
of the trityl group was followed by attachment of Fmoc-
Phe-OH through a carbodiimide-mediated coupling[18c] in
74% yield. Three additional deprotection/coupling cycles
completed the solid-phase synthesis and, after cleavage and
simultaneous deprotection with TFA, depsipeptide 7 was ob-
tained in an overall yield of 33%.
was coupled, with HATU/HOAt as the coupling reagents in
the presence of DIPEAas base in NMP. Five additional de-
protection/coupling cycles completed the solid-phase synthe-
sis and, after cleavage and simultaneous deprotection, pep-
tide–peptoid hybrid 8 was obtained in an overall yield of
31%.
Amyloid fibril formation: Native amylin(20–29) (6;
N
10 mgmLÀ1) was dissolved in 0.1% TFA/H2O and this led to
rapid gel formation. The presence of amyloid fibrils was
verified by transmission electron microscopy (TEM; data
not shown)[11,12] and b-sheet formation was confirmed by the
amide I absorption at approximately 1630 cmÀ1 in the Fouri-
er transform infrared (FTIR) spectrum (Table 1), which was
in agreement with literature data and our earlier experi-
ments.[10a,13]
Synthesis of the N-butylated-Gly24, -Ile26, and -Ser28
peptide 8 began with the preparation of Fmoc-Ser
A
ACHTREUNG
switching of the Fmoc group for an oNBS group, the result-
ing sulfonamide NH group could be subjected to a Mitsuno-
bu reaction in a site-specific N-alkylation method.[31] This re-
action was monitored by the bromophenol blue (BPB)
test.[32] Then, the oNBS group was removed by treatment
with b-mercaptoethanol in the presence of DBU as base.
The resulting secondary amine was treated with Fmoc-Leu-
OH/BTC in sym-collidine/dioxane as described by Falb
et al.,[25] with modifications described by Jung and co-work-
ers.[33] The coupling efficiency (70%) was calculated from an
Fmoc determination.[30] After Fmoc removal, dipeptide 5
The influence of substitution of three alternate amide
bonds by ester moieties on amyloid fibril formation was
studied with depsipeptide 7. Asolution of this peptide also
formed a gel, a result that was rather unexpected since we
had previously found that a single amide-bond replacement
(on position 28) significantly postponed gel formation as
compared to that with 6.[10a] However, typical amyloid fibrils
were not observed by TEM, a result that was confirmed by
FTIR spectroscopy since the absorption at 1625–1630 cmÀ1
,
typical for b-sheets, was absent.[34] Instead of amyloid fibrils,
large helical ribbons[35] and even tubelike supramolecular
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R. M. J. Liskamp et al.
Scheme 5. Solid-phase synthesis of N-butylated peptide 8. oNBS-Cl=2-nitrobenzenesulfonyl chloride, DCE=1,2-dichloroethane, DBU=1,8-
diazabicyclo[5.4.0]undec-7-ene.
A
Table 1. Physicochemical properties of the amylin derivatives.
Peptide
Gelation
Fibrils
Morphology
FTIR [cmÀ1
]
6
7
8
9
10
11
12
13
yes: <10 min, turbid
yes: <10 min, turbid
no: clear solution
no: clear solution
no: clear solution
no: clear solution
no: clear solution
yes: <10 min, turbid
yes
no
no
no
no
no
no
yes
length: 1; width: 10 nm
1631 (s), 1670 (m)
1665 (s), 1642 (m), 1740 (w)
1673 (s), 1637 (m)
1669 (s)
1664 (s)
1677 (s), 1642 (m)
1678 (s)
helical tapes; length: 7.4 mm; width: 170 nm
helical tapes; length: 1.5 mm; width: 250 nm
–
–
–
–
length: 1; width: 10 nm
1629 (s)
structures[36] were observed (Figure 1). Although never de-
scribed for nor studied in depsipeptides, this morphology of
helical ribbons has been observed before,[35] as intermediate
nanostructures in the self-assembly of amyloid fibrils[37] or as
final-stage folding assemblies.[38]
As expected, peptide 8 with three N-butylated amino acid
residues formed a clear solution in 0.1% TFA/H2O and gel
formation did not occur. The N-alkylated peptide 8 was de-
signed as a water-soluble b-sheet mimic to disrupt any b-
sheet formation, according to the model in Scheme 1. Fibril
formation, as judged by TEM and FTIR spectroscopy, was
completely absent. Surprisingly, despite the absence of any
visible gel formation, helical ribbons, similar to those
formed by depsipeptide 7, were also observed by TEM in
this case (Figure 2).
TEM or FTIR spectroscopy. In contrast to depsipeptide 7
and N-alkylated peptide hybrid 8, however, this amylin de-
rivative did not assemble into helical supramolecular struc-
tures. In order to obtain some insight into the molecular
basis of this behavior, peptide 10 was prepared, in which
Ile26 and Ser28 were replaced by a glycine residue. This de-
rivative might shed some light on the role of the side chain
and/or amide in the formation of supramolecular assemblies.
It was found earlier that replacement of Ser28 by glycine, as
in peptide 13 (Table 2), did not affect fibril formation.[10a]
However, the additional replacement of Ile26 by glycine
completely abrogated fibril formation. Apparently, side-
chain-to-side-chain interactions also play an important role
in the formation of amyloid fibrils (6 versus 9 and 10) or
other self-assembled structures (7 and 8).[39]
Similarily, peptide–peptoid hybrid 9 rapidly dissolved in
0.1% TFA/H2O and, here too, gel formation was absent. In
addition, no amyloid fibrils could be detected by either
Inhibition of aggregation behavior: The aggregation behav-
ior of native amylin(20–29) (6) was monitored in the pres-
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Amylin(20–29) Derivatives
A
FULL PAPER
Figure 1. TEM image of amylin(20–29) derivative 7. Scale bar: 1 mm.
G
Arrow a: single strand; arrow b: two strands starting to intertwine;
arrow c: helical ribbon; arrow d: (closed) peptide tube; arrows e and f:
two helical tapes with pitches of 500 and 330 nm, respectively.
ence or absence of a potential inhibitory sequence. Astock
solution of 6 was diluted in phosphate buffer at pH 7.4 and
the increase of turbidity was monitored at 400 nm, as shown
in Figure 3. Peptide 6 instantly increased the turbidity of the
solution and, after 30 min, a plateau was reached. In the
presence of an equal amount (w/w) of depsipeptide 7, a
small lag phase was observed and reaching the plateau was
postponed to 60 min (Figure 3 A). This plateau was also ap-
proximately 20% lower than that observed in the absence
of 7. In case of the trialkylated peptide 8 with 6, the lag
phase was absent and the plateau was now reached after
45 min and was only slightly lower (10%) than the plateau
reached by 6. Peptides 7 and 8 did not increase the turbidity
of the solution by themselves (data not shown), but they
Figure 2. TEM images of amylin(20–29) derivative 8. Scale bar: 2 mm (A
A
were not able to inhibit fibril formation of amylin(20–29)
G
and B), 0.5 mm (C). A) The arrows point at the filaments of which a
ribbon consists; B) an almost closed peptide tube; C) enlargement of the
joining of 5–7 filaments to form a peptide nanotube.
(see above). This may be the result of their tendency to
form other supramolecular assemblies, that is, helical rib-
bons. As expected, the peptoid–peptide hybrid 9 inhibited
fibril formation of 6 by almost 85%. These results were con-
firmed by electron microscopy of the amylin
G
turbidity assay (Figure 3B). However, amylin(20–29) deriva-
N
tor mixtures. In an aged (2 weeks) mixture of peptide 6 with
7 or 8, fibrils were observed. By contrast, in a mixture of 6
and peptoid–peptide hybrid 9, fibrils remained absent (data
not shown).
tives 15 (Ser28!Pro) and 16 (Ser28!NNle) significantly in-
hibited fibril formation of 6, since both mutations were re-
sponsible for the absence of any fibril formation in this
assay. These observations were also confirmed by electron
Our previously synthesized b-sheet breaker peptides
microscopy and no fibrils were observed when amylin
29)Ser28!Pro (15) or amylin(20–29)Ser28!NNle (16)
were mixed with native amylin(20–29) (data not shown).
None of the b-sheet-breaker peptides based on amylin-
(20–29) were able to inhibit fibril formation of full-length
amylin(1–37) and preformed fibrils were also unaffected by
the addition of peptide derivatives 7–9, 11, and 12. Appa-
(20–
based on amylin
A
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position 28 was replaced by either glycolic acid, proline, or
N-butyl glycine were also evaluated in this assay. As was de-
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scribed earlier, the Ser28!Glyc modification in amylin
C
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R. M. J. Liskamp et al.
tures that give rise to helical ribbons and peptide nanotubes.
Several examples of self-assembly into helical ribbons/pep-
tide nanotubes by designed b-sheet model peptides are de-
scribed in the literature. However, according to FTIR data
(see above) and CD spectroscopy (Figure 4), depsipeptide 7
&
*
*
:
Figure 4. CD spectra of the amylin
A
9.
and N-alkylated peptide 8 do not form b sheets. Therefore,
in our case, aggregation other than that responsible for the
formation of b sheets must be now responsible for the for-
mation of helical ribbons and peptide nanotubes. The in-
creased hydrophobicity of peptides 7 and 8 might be a driv-
ing force for the self-assembly into the observed helical rib-
bons.
&
Figure 3. Aggregation curves. A)
:
6
(10 mgmLÀ1); &: 6+8
(10 mgmLÀ1);
6+7 (10 mgmLÀ1);
:
6+9 (10 mgmLÀ1); B)
:
6
&
*
:
*
(10 mgmLÀ1); &: 6+14 (10 mgmLÀ1); : 6+15 (10 mgmLÀ1); : 6+16
*
*
(10 mgmLÀ1).
Replacement of a backbone
amide with an ester moiety
(depsipeptide 7) eliminated the
Table 2. Sequences of the amylin derivatives synthesized in this study.
Peptide sequence
Mass
[M+H]+ found (calcd)
Rt
[min]
hydrogen-bond donor (NH
group) and resulted in a weaker
hydrogen-bond acceptor (ester
carbonyl group).[41] However,
the backbone conformation in
the depsipeptide, in terms of f
G
ACHTREUNG
H-Ser-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser-Ser-NH2 (6)
H-Ser-Asn-Asn-Phe-Glyc-Ala-Ilec-Leu-Glyc-Ser-NH2 (7)
1008.65 (1008.50)
981.65 (981.45)
1176.90 (1176.70)
1090.85 (1090.63)
922.60 (922.44)
1090.80 (1090.27)
688.85 (687.91)
reference [10a]
reference [10a]
reference [10a]
reference [10a]
17.15
18.65
21.40
20.12
15.77
19.72
22.48
n.d.[a]
n.d.[a]
n.d.[a]
n.d.[a]
H-Ser-Asn-Asn-Phe-NNle-Ala-
N
N
H-Ser-Asn-Asn-Phe-NNle-Ala-NNle-Leu-NNle-Ser-NH2 (9)
H-Ser-Asn-Asn-Phe-Gly-Ala-Gly-Leu-Gly-Ser-NH2 (10)
H-Ser-Asn-Asn-Phe-NNle-Ala-Ile-Leu-NNle-Ser-NH2 (11)
H-Phe-NNle-Ala-Ile-Leu-NNle-Ser-NH2 (12)
H-Ser-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Gly-Ser-NH2 (13)
H-Ser-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Glyc-Ser-NH2 (14)
H-Ser-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Pro-Ser-NH2 (15)
H-Ser-Asn-Asn-Phe-Gly-Ala-Ile-Leu-NNle-Ser-NH2 (16)
and
y dihydral angles, re-
mained virtually unaffected and
the trans ester conformation, as
is the case in native amide
bonds, was still strongly prefer-
red.[41] Possibly, as a result of
this amide to ester substitution,
7 did not aggregate into fibrils
[a] n.d.=not determined.
rently, the highly amyloidogenic amylin
A
involving a hydrogen-bond pattern but formed (anti)parallel
b-sheet-like tapes, which in turn self-assembled into helical
ribbons due to the intrinsic chirality of the depsipeptide.
N-alkylation of three amide bonds in 8 or 9 also removed
three NH hydrogen-bond donors. In contrast to the situation
in 7, the resulting tertiary amides significantly influenced
the conformation of the backbone, since, for example, turns
can be introduced by proline, N-alkyl amino acid, and N-
alkyl glycine (peptoid) moieties and ciscoid conformations
involving tertiary amide bonds are more preferred. Incorpo-
not optimal for efficient molecular recognition of amylin
ACHTREUNG
37) fibrils, as recently described by Gazit and co-workers,[40]
who found that amino acid residues at the N terminus are
important for binding to full-length amylin.
Self-assembled ribbons and peptide nanotubes: Although
peptides 7 and 8 were designed as b-sheet-breaker peptides
for the disruption of the characteristic hydrogen-bond pat-
tern (Scheme 2), they themselves form self-assembled struc-
3720
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Chem. Eur. J. 2006, 12, 3714 – 3725

Amylin(20–29) Derivatives
A
FULL PAPER
ration of these building blocks is a well-accepted approach
to the design of b-sheet-breaker peptides.[9,10a,13,16] A s was
confirmed by FTIR spectroscopy from the absence of the
1625–1633 cmÀ1 peak, peptides 8 and 9 did not form b-sheet-
aggregated fibers. The absence of b sheets was also con-
firmed by CD spectroscopy (Figure 4). However, here too,
the increased hydrophobicity of peptide 8 might be responsi-
ble for the formation of the supramolecular assemblies
shown in Figure 2. Absence of any supramolecular struc-
tures formed by peptide–peptoid hybrid 9 could be ex-
plained by the increased flexibility of this hybrid, as com-
pared to trisalkylated peptide 8, because the side chains on
the a-carbon atoms in two amino acid residues are absent.
This is corroborated by the observation that signals in the
1H NMR spectrum of 9 occurred as doublets and broadening
of the HPLC elution profiles was observed with the pres-
ence of peptoid moieties in the peptide backbone. Addition-
ally, the absence of any secondary structure was confirmed
by CD spectroscopy (Figure 4).
Experimental Section
Instruments and methods: The peptides were synthesized on an Applied
Biosystems 433Apeptide synthesizer. Analytical HPLC runs were car-
ried out on a Shimadzu HPLC system and preparative HPLC runs were
performed on a Gilson HPLC workstation. Liquid chromatography/elec-
trospray-ionization mass spectrometry was measured on a Shimadzu
LCMS-QP8000 single-quadrupole bench-top mass spectrometer operat-
ing in the positive-ionization mode. Electron microscopy was performed
on a Jeol 1200 EX transmission electron microscope. Fourier transform
infrared spectra were measured on a BioRad FTS 6000 spectrophotome-
ter. Circular dichroism spectra were measured on an OLIS RSM 1000
CD spectrometer. 1H NMR spectra were recorded on a Varian G-300
(300 MHz) spectrometer and chemical shifts (d) are given in ppm relative
to tetramethylsilane (TMS). 13C NMR spectra were recorded on a Varian
G-300 (75.5 MHz) spectrometer and chemical shifts are given in ppm rel-
ative to CDCl3 (77.0 ppm). The 13C NMR spectra were recorded by using
the attached proton test (APT) sequence. Retention factor (Rf) values
were determined by thin-layer chromatography (TLC) on Merck precoat-
ed silica gel 60 F254 plates. Spots were visualized by UV quenching, ninhy-
drin,
or
Cl2/N,N,N’,N’-tetramethyl-4,4’-diaminodiphenylmethand
(TDM).[43] Melting points were measured on a Büchi Schmelzpunktbes-
timmungsapparat and are uncorrected. Elemental analyses were done at
the Kolbe Mikroanalytisches Labor (Mülheim an der Ruhr, Germany).
Morphology: In Figure 1, different folding stages of depsi-
peptide 7 can be observed. Arrow a points to a single strand
and two strands intertwine at the position b to form a helical
ribbon at position c. Ultimately, this self-assembly leads to
the formation of a (closed) peptide tube at position d. Ar-
rows e and f point to two helical tapes with different pitches
(500 and 330 nm, respectively). Aribbon consists of several
filaments, as is clearly shown in Figure 2 A(arrows). Figure
2B shows an almost closed peptide tube and Figure 2C is an
enlargement of the joining of 5–7 filaments to form a pep-
tide nanotube. The (almost) closed peptide tubes may be ex-
plored as insulation or as versatile precursors for nano-
Chemicals and reagents: ArgoGel Fmoc-Rink-Amide resin functional-
ized with the 4-((2’,4’-dimethoxyphenyl)aminomethyl)phenoxyacetamido
moiety (the Rink amide linker)[44] was used in all the syntheses. The cou-
pling reagents HBTU[45] and BOP[46] were obtained from Biosolve.
[23]
HATU and HOAt[23] were obtained from Applied Biosystems. HOBt
was from Advanced ChemTech and Na-9-fluorenylmethyloxycarbonyl
amino acids were obtained from MultiSynTech. The side-chain protecting
groups were chosen as tert-butyl for serine and trityl for asparagine. Pep-
tide-grade DCM, DCE, tert-butyl methylether (MTBE), NMP, and TFA
and HPLC-grade acetronitrile were purchased from Biosolve. Piperidine,
DMAP, DIPEA, TEA, and triphenylphosphine were obtained from
Acros Organics. TIS, 1,2-ethanedithiol (EDT), and HPLC-grade TFA
were obtained from Merck. DIAD, triphosgene, DIC, pNBS-Cl, and
oNBS-Cl were purchased from Aldrich. Glycolic acid, sym-collidine, b-
mercaptoethanol, and DBU were purchased from Fluka.
A
2-Hydroxy-3-methyl-pentanoic acid allyl ester (1): Isoleucine (5 g,
38 mmol) was dissolved in 2.5n H2SO4 (25 mL) and cooled to 08C. Aso-
lution of NaNO2 (3.9 g, 57.1 mmol) in H2O (20 mL) was added dropwise
over 1 h and the obtained reaction mixture was stirred for 2 h at 08C and
then 16 h at room temperature. The reaction mixture was then extracted
with diethyl ether (3”75 mL). The combined organic layers were washed
with brine (50 mL) and dried with MgSO4 and concentrated under re-
duced pressure. 2-Hydroxy-3-methyl-pentanoic acid was obtained as a
colorless oil (4.63 g, 92%): [a]2D0 =À13.9 (c=1.0 in CHCl3; literature
value:[18a] À21.6 (c=1.0 in CHCl3)); Rf =0.24 (CH2Cl2/MeOH 95:5), 0.76
mation of nanochannels. Multilayered peptides[42] may even
provide adequate insulation to develop neuron mimics. Fur-
thermore, nanochannels, -tubes, or -devices may be useful
for drug delivery purposes.[15a,b]
Conclusion
1
(CHCl3/MeOH/AcOH 95:20:3); H NMR (300 MHz, CDCl3): d=4.20 (d,
We have found that by modifying the peptide amide linkage
it is possible to dramatically alter the aggregation behavior
of a peptide causing fibril formation involving b sheets. Syn-
thetically challenging depsipeptides and N-alkylated pep-
tides—both categories are not generally accessible as yet—
as well as peptoid–peptide hybrids were designed and syn-
thesized. Their behavior with respect to the formation of
special supramolecular assemblies, that is, helical ribbons
and peptide nanotubes, was quite unexpected and cannot be
rationalized by assuming the formation of the common hy-
drogen-bonding pattern of b sheets. Also, subtle side-chain-
to-side-chain interactions were found to play a decisive role
in the formation of either fibrils or helical ribbons. The self-
assembly of these modified amylin derivatives into struc-
tures other than amyloid fibrils makes them of high value
for the design of peptide-based nanomaterials.
1H, aCH), 2.02 (d, 1H, OH), 1.89 (m, 1H, bCH), 1.46–1.24 (m, 2H,
gCH2), 1.02 (d, 3H, g’CH3 Ilec), 0.92 ppm (t, 3H, dCH3); 13C NMR
(75 MHz, CDCl3): d=179.2, 74.6, 38.7, 23.6, 15.2, 11.6 ppm.
K2CO3 (5.2 g, 36.9 mmol, 1.5 equiv) and allylbromide (4.35 mL,
49.2 mmol, 2 equiv) were added to a solution of 2-hydroxy-3-methyl-pen-
tanoic acid (3.35 g, 24.6 mmol) in acetone (100 mL). The obtained reac-
tion mixture was stirred for 16 h at room temperature. The solvent was
then evaporated in vacuo and the residue was redissolved in EtOAc
(100 mL), washed with 5% NaHCO3 (2”50 mL) and brine (50 mL),
dried over Na2SO4, and concentrated under reduced pressure. Allyl ester
1 was obtained as a colorless oil (2.80 g, 66%): Rf =0.46 (CH2Cl2/MeOH
98:2); 1H NMR (300 MHz, CDCl3): d=5.98–5.88 (m, 1H, CH allyl),
5.39–5.26 (dd, 2H, CH2 allyl), 4.69 (d, 2H, CH2 allyl), 4.11 (d, 1H, aCH),
2.77 (s, 1H, OH), 1.84 (m, 1H, bCH) 1.41–1.23 (m, 2H, gCH2), 0.99 (d,
3H, g’CH3 Ilec), 0.90 ppm (t, 3H, dCH3); 13C NMR (75 MHz, CDCl3):
d=174.7, 131.4, 119.1, 74.7, 66.0, 39.1, 23.7, 15.4, 11.7 ppm.
2-[2-(9H-Fluoren-9-ylmethoxycarbonylamino)-propionyloxy]-3-methyl-
pentanoic acid (Fmoc-Ala-Ilec-OH)(2) : DCC (1.65 g, 8 mmol), Fmoc-
Chem. Eur. J. 2006, 12, 3714 – 3725
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3721

R. M. J. Liskamp et al.
Ala-OH (2.63 g, 8 mmol), and DMAP (78 mg, 7.5 mol%) were added to
a solution of allyl ester 1 (1.38 g, 8 mmol) in CH2Cl2 (75 mL). The ob-
tained reaction mixture was stirred for 16 h at room temperature. Next,
DCU was removed by filtration and the solvent was evaporated in vacuo.
The residue was redissolved in EtOAc (150 mL) and the organic layer
was washed with 1n KHSO4 (3”75 mL), 5% NaHCO3 (3”75 mL), and
brine (3”100 mL), dried (Na2SO4), and concentrated under reduced
pressure. The residue was purified by column chromatography (CH2Cl2/
MeOH 99:1) and Fmoc-Ala-Ilec-OAllyl was obtained as a white solid
(2.48 g, 67%): M.p.=72–758C; Rf=0.60 (CH2Cl2/MeOH 98:2); 1H NMR
(300 MHz, CDCl3): d=7.77–7.28 (m, 8H, arom. CH), 5.97–5.83 (m, 1H,
CH allyl), 5.38–5.24 (m, 3H, CH2 allyl/NH), 5.00 (d, 1H, aCH Ilec), 4.64
(d, 2H, OCH2 allyl), 4.49 (t, 1H, aCH Ala), 4.37 (d, 2H, CH2 Fmoc),
4.22 (t, 1H, CH Fmoc), 2.03/1.83 (2”s, 1H, bCH Ilec), 1.52 (d, 3H, bCH3
Ala), 1.53–1.21 (brm, 2H, gCH2 Ilec), 0.99 (d, 3H, g’CH3 Ilec), 0.91 ppm
(q, 3H, dCH3 Ilec); 13C NMR (75 MHz, CDCl3): d=172.9, 168.9, 155.6,
143.9, 141.2, 131.4, 127.7, 127.0, 125.1, 119.9, 119.0, 76.9, 67.0, 66.0, 65.8,
49.4, 47.1, 39.1, 36.5, 24.4, 23.7, 18.6, 15.4, 11.7 ppm; EI-MS: (50 eV): m/z
(%): 466.50 (52) [M+H]+, 488.35 (100) [M+Na]+.
d=8.00 (d, 2H, arom. CH), 7.87 (d, 2H, arom. CH), 7.34 (m, 3H, arom.
CH), 7.18 (m, 2H, arom. CH), 4.88 (s, 2H, CH2 benzyl), 4.27 (d, 1H,
aCH Ile), 3.46–3.07 (m, 2H, N-CH2 butyl), 1.93 (m, bCH Ile), 1.81–1.17
(m, 6H, CH2 butyl (2”2H)/gCH2 Ile), 0.97–0.86 ppm (m, 9H, CH3 butyl/
g’CH3/dCH3 Ile); 13C NMR (75 MHz, CDCl3): d=169.9, 149.6, 145.5,
134.4, 128.8, 128.6, 128.6, 128.4, 123.8, 66.8, 64.8, 45.6, 34.9, 32.6, 25.2,
20.2, 15.5, 13.5, 10.8 ppm.
K2CO3 (3.85 g) and then thiophenol (1.1 mL) were added to a solution of
pNBS-N(Bu)Ile-OBzl (4.95 g) in DMF (40 mL) and the obtained reac-
tion mixture was stirred for 90 min. The reaction mixture was then dilut-
ed by the addition of H2O (80 mL) and the aqueous phase was extracted
with diethyl ether (3”100 mL). The combined organic layers were
washed with H2O (2”60 mL), 5% NaHCO3 (2”60 mL), and brine
(60 mL), dried (Na2SO4), and concentrated in vacuo. The residue was pu-
rified by column chromatography (CH2Cl2/MeOH 99:1) to obtain H-
N(Bu)Ile-OBzl (4) as a slightly brownish oil (1.72 g, 67%): Rf =0.41
(CH2Cl2/MeOH 97:3); 1H NMR (300 MHz, CDCl3): d=7.36 (m, 5H,
arom. CH), 5.16 (s, 2H, CH2 benzyl), 3.11 (d, 1H, aCH Ile), 2.59–2.37
(dm, 2H, N-CH2 butyl) 1.67 (m, 1H, bCH Ile), 1.60–1.07 (m, 6H, CH2
butyl (2”2H)/gCH2 Ile), 0.90–0.84 ppm (m, 9H, CH3 butyl/g’CH3/dCH3
Ile); 13C NMR (75 MHz, CDCl3): d=175.3, 135.9, 128.5, 128.4, 128.3,
66.2, 66.1, 48.3, 38.4, 32.3, 25.7, 21.9, 20.3, 15.5, 11.4 ppm; EI-MS:
(50 eV): m/z (%): 278.25 (100) [M+H]+.
Fmoc-Ala-Ilec-OAllyl (930 mg, 2 mmol) was dissolved in CH2Cl2 (25 mL)
and purged with argon for 5 min. Phenylsilane (1.2 mL, 5 equiv) and a
catalytic amount of Pd0
(PPh3)4 were then added under argon and the re-
G
action mixture was stirred overnight at room temperature. Subsequently,
the solvent was removed under reduced pressure and the residue was re-
dissolved in EtOAc (50 mL). The organic layer was washed with 1n
KHSO4 (2”25 mL) and brine (25 mL), dried (Na2SO4), and concentrated
in vacuo. After purification by column chromatography (CH2Cl2/MeOH
92:8), 2 was obtained as a white foam (600 mg, 71%): M.p.=131–1338C;
Rf =0.46 (CH2Cl2/MeOH 95:5); 1H NMR (300 MHz, CDCl3): d=7.78–
7.26 (m, 8H, arom. CH), 5.46 (d, 1H, NH), 4.97 (s, 1H, aCH Ilec), 4.45
(t, 1H, aCH Ala), 4.38 (d, 2H, CH2 Fmoc), 4.21 (t, 1H, CH Fmoc), 2.05
(m, 1H, bCH Ilec), 1.49–1.21 (m, 5H, bCH3 Ala/gCH2 Ilec), 0.98 (d, 3H,
g’CH3 Ilec), 0.90 ppm (t, 3H, dCH3 Ilec); 13C NMR (75 MHz, CDCl3):
d=173.1, 171.9, 156.0, 143.6, 141.1, 133.9, 127.6, 126.9, 124.9, 119.8, 66.8,
49.7, 46.9, 36.2, 24.2, 17.9, 15.2, 11.4 ppm; EI-MS: (50 eV): m/z (%):
448.35 (100) [M+Na]+; elemental analysis: calcd (%) for C24H27NO6
(425.47): C 67.75, H 6.40, N 3.29; found: C 67.25, H 6.48, N 3.08.
Fmoc-Ala-N(Bu)Ile-OH (5): Fmoc-Ala-OH·H2O (329 mg, 1 mmol) was
coevaporated with chloroform and toluene (2”10 mL) and subsequently
dissolved in dry THF (20 mL). Triphosgene (0.33 mmol) and sym-colli-
dine (1.25 equiv) were added to this solution and the reaction mixture
was stirred for 2 min to obtain the acid chloride. Asolution of
4
(1 mmol) in dry THF (20 mL) was then added dropwise and the obtained
reaction mixture was stirred for 1 h. After removal of the solvent, the res-
idue was dissolved in EtOAc (50 mL) and subsequently washed with 1n
KHSO4 (50 mL), 5% NaHCO3 (50 mL), and brine (50 mL). The organic
phase was dried (Na2SO4) and concentrated in vacuo. The residue was
dissolved in EtOH (25 mL), then CHCl3 (2 mL) and subsequently Pd/C
(100 mg) were added and the reaction mixture was stirred for 16 h in the
presence of H2. Subsequently, the reaction mixture was filtered over
Hyflo and concentrated under reduced pressure. The residue was purified
by column chromatography (CH2Cl2/MeOH 95:5) and 5 was obtained as
a white foam in 34% yield over two reaction steps (159 mg): Rf =0.26
(EtOAc/hexane/AcOH 2:1:0.01); 1H NMR (300 MHz, CDCl3): d=7.78–
7.30 (m, 8H, arom. CH), 5.67 (d, 1H, NH), 4.70 (t, 1H, aCH Ala), 4.38
(m, 2H, CH2 Fmoc), 4.22 (t, 1H, CH Fmoc), 3.62–3.13 (m, 2H, N-CH2
butyl), 2.47 (d, 1H, aCH Ile), 1.67 (t, 1H, bCH Ile), 1.49–1.08 (m, 6H,
CH2 butyl (2”2H, gCH2 Ile), 1.01–0.88 ppm (m, 9H, CH3 butyl/g’CH3/
dCH3 Ile); 13C NMR (75 MHz, CDCl3): d=175.2, 172.3, 155.6, 143.8,
143.6, 141.2, 127.6, 127.0, 125.1, 119.9, 70.0, 68.4, 67.0, 50.1, 47.4, 47.0,
32.9, 31.4, 25.1, 19.9, 19.1, 16.0, 13.6, 10.9 ppm; EI-MS: (50 eV): m/z (%):
503.40 (100) [M+Na]+; elemental analysis: calcd (%) for C28H36N2O5
(480.60): C 69.98, H 7.55, N 5.83; found: C 69.79, H 7.48, N 5.78.
3-Methyl-2-(4-nitrobenzenesulfonylamino)-pentanoic acid benzyl ester
(3): H-Ile-OH (13.1 g, 100 mmol) was suspended in toluene (250 mL);
benzylalcohol (12.9 mL, 125 mmol) and then TosOH·H2O (20.9 g,
110 mmol) were added. The reaction mixture was refluxed for 16 h in a
Dean–Stark apparatus and subsequently concentrated in vacuo. The resi-
due was triturated with diethyl ether, filtered, and dried in a desiccator.
The obtained tosylate was dissolved in EtOAc (150 mL); the organic
layer was washed with a saturated solution of NaHCO3 (100 mL), dried
(Na2SO4), and evaporated in vacuo. The residue was dissolved in CH2Cl2
(75 mL) and then TEA(5.2 mL, 2 equiv) was added, followed by pNBS-
Cl (4.1 g, 1 equiv). After being stirred for 16 h at room temperature, the
reaction mixture was concentrated under reduced pressure. The residue
was redissolved in EtOAc (100 mL), washed with 1n KHSO4 (3”75 mL)
and brine (50 mL), and dried (Na2SO4), and then the solvent was re-
moved in vacuo. Compound 3 was obtained as a yellowish solid (6.10 g,
66%): M.p.=79–838C; Rf =0.55 (EtOAc/hexane 7:3); 1H NMR
(300 MHz, CDCl3): d=8.17 (d, 2H, arom. CH), 7.94 (d, 2H, arom. CH),
7.33 (m, 3H, arom. CH), 7.17 (m, 2H, arom. CH), 5.32 (d, 1H, NH), 4.92
(dd, 2H, CH2 benzyl), 3.90 (q, 1H, aCH Ile), 1.88 (m, 1H, bCH Ile),
1.38–1.11 (m, 2H, gCH2 Ile), 0.95–0.86 ppm (m, 6H, g’CH3/dCH3 Ile);
13C NMR (75 MHz, CDCl3): d=170.7, 145.4, 134.5, 128.9, 128.6, 128.5,
128.3, 124.1, 67.4, 60.6, 38.4, 24.3, 15.5, 11.3 ppm.
Peptide synthesis (general procedure): Peptides 6 and 10 were synthe-
sized by using the FastMoc protocol on a 0.25 mmol scale[26] on Argogel
Fmoc-Rink-Amide resin to obtain the C-terminally amidated peptide.[44]
Each synthetic cycle consisted of Na-Fmoc removal by a 10 min treat-
ment with 20% piperidine in NMP, a 6 min NMP wash, a 45 min cou-
pling step with preactivated Fmoc amino acid (1.0 mmol) in the presence
of DIPEA(2 equiv), and a 6 min NMP wash. Na-Fmoc amino acids were
activated in situ with 1.0 mmol HBTU/HOBt (0.36m in NMP) in the
presence of DIPEA(2.0 mmol). The peptides were detached from the
resin and deprotected by treatment with TFA/H2O/EDT/TIS
(85:8.5:4.5:2) for 3 h. The peptides were precipitated with MTBE/hexane
(1:1) at À208C and finally lyophilized from tert-butanol/H2O (1:1).
2-Butylamino-3-methyl-pentanoic acid benzyl ester (4): Asolution of sul-
fonamide 3 (4.42 g, 10.9 mmol) and PPh3 (2.85 g, 10.9 mmol) in dry THF
(25 mL) was cooled to À608C. nBuOH (1 mL) and then DIAD
(2.15 mL) were added dropwise and the reaction mixture was allowed to
react for 16 h. After removal of the solvent by evaporation, the obtained
residue was dissolved in EtOAc. The precipitate was removed by filtra-
tion and the EtOAc layer was evaporated to dryness. After recrystalliza-
tion, pNBS-N(Bu)Ile-OBzl was obtained in a nearly quantitative yield
(5.0 g): M.p.=87–908C; Rf =0.62 (CH2Cl2); 1H NMR (300 MHz, CDCl3):
Depsipeptide 7: Fmoc-Ser(tBu)NH-Rink-Amide resin (0.10 mmol) was
G
washed with CH2Cl2 (3”2 min, 10 mL) and NMP (3”2 min, 10 mL) and
treated with 20% piperidine/NMP (3”8 min, 10 mL) to remove the
Fmoc group. After washing the resin with NMP (3”2 min, 10 mL),
CH2Cl2 (3”2 min, 10 mL), and NMP (3”2 min, 10 mL), Trt-Glyc-OH[10a]
(127 mg, 0.40 mmol) was coupled to the a-amino group with BOP
(177 mg, 0.40 mmol)/DIPEA(70 mL, 0.40 mmol) in NMP (10 mL). The
3722
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Chem. Eur. J. 2006, 12, 3714 – 3725

Amylin(20–29) Derivatives
A
FULL PAPER
coupling was monitored by the Kaiser test[28] and was complete after 2 h.
Subsequently, the resin was washed with NMP (3”2 min, 10 mL), CH2Cl2
(3”2 min, 10 mL), and NMP (3”2 min, 10 mL) and the trityl functionali-
ty was removed by treatment with TFA/TIS/CH2Cl2 (1:5:94, 5”2 min,
20 mL). After washing the resin with CH2Cl2 (5”2 min, 10 mL) and
NMP (3”2 min, 10 mL), Fmoc-Leu-OH (141 mg, 0.40 mmol) was cou-
pled to the a-hydroxy group with DIC (153 mg, 0.80 mmol)/HOBt (245,
1.6 mmol)/DMAP (49 mg, 0.40 mmol) in NMP (10 mL) for 16 h. The
resin was washed with NMP (3”2 min, 10 mL) and CH2Cl2 (3”2 min,
10 mL) to remove the excess reagents; the coupling yield of Fmoc-Leu-
OH, as calculated from an Fmoc determination,[30] was 76%. After re-
moval of the Fmoc group, Fmoc-Ala-Ilec-OH (85 mg, 0.20 mmol) was
coupled with HATU (76 mg, 0.20 mmol)/HOAt (27 mg, 0.20 mmol)/
DIPEA(70 mL, 0.40 mmol) in NMP (10 mL). The coupling reaction was
monitored by the Kaiser test and was complete after 90 min. The Fmoc
group was removed by treatment with 20% piperidine in NMP and the
resin was washed as described above. Trt-Glyc-OH was then coupled to
the a-amino group with BOP/HOBt/DIPEAin NMP under the same
conditions as those described earlier. Subsequently, the trityl group was
removed by acid and Fmoc-Phe-OH was coupled to the primary hydroxy
functionalityin the presence of DIC/HOBt/DMAP in NMP. The coupling
yield was determined to be 74% (0.19 mmolgÀ1). After this amino acid,
the peptide sequence was completed as described in the general proce-
dure.
TFAin CH 3CN/H2O 95:5). The purities were evaluated by analytical
HPLC on an Adsorbosphere XL C8 column (90 Š pore size, 5 mm parti-
cle size, 0.46”25 cm) at a flow rate of 1.0 mLminÀ1 by using a linear gra-
dient 100% buffer A ! 100% buffer B in 30 min.
Peptide characterization: The peptides were characterized by mass spec-
trometry. The mass of each analogue was measured and the observed
monoisotopic [M+H]+ values were correlated with the calculated
[M+H]+ values by using the MacBioSpec program (Perkin–Elmer Sciex
Instruments, Thornhill, ON, Canada). The values are given in Table 2.
Gelation experiments: Each peptide sample (10 mg) was dissolved in
0.1% TFA/H2O (1 mL) at 258C. The aggregation state was determined
by eye at regular time intervals by tilting the test tube and checking if
the solution still flowed. If no flow was observed, gelation was said to
have taken place.
Transmission electron microscopy: Apeptide gel/solution aged for three
weeks (10 mL) was placed on a carbon-coated copper grid. After 15 min,
any excess of peptide was removed by washing the copper grid with a
drop of demi-water. This was repeated five times. Finally, the samples
were stained by methylcellulose/uranyl acetate and dried in air. The sam-
ples were visualized under a Jeol 1200 EX transmission electron micro-
scope operating at 60 kV. The magnification ranged from 20000–100000
times.
Fourier transform infrared spectroscopy: Apeptide gel/solution aged for
three weeks (100 mL) was lyophilized and subsequently resuspended in
D2O (150 mL) and lyophilized. This treatment was repeated twice. The
lyophilized peptides were dried over P2O5 in high vacuum for 24 h. A
peptide sample was mixed with KBr and pressed into a pellet. IR spectra
were recorded on a BioRad FTS6000 spectrometer. The optical chamber
was flushed with dry nitrogen for 5 min before data collection started.
The interferograms from 1000 scans with a resolution of 2 cmÀ1 were
averaged and corrected for H2O and KBr.
N-Butylated-Gly24, -Ile26, and -Ser28 peptide 8: Fmoc-Ser
A
ACHTREUNG
2 min, 10 mL) and NMP (3”2 min, 10 mL) and treated with 20% piperi-
dine/NMP (3”8 min, 10 mL) to remove the Fmoc group. After washing
the resin with DMF (5”2 min, 10 mL) and DCE (5”2 min, 10 mL) the
a-amino functionality was treated with oNBS-Cl in the presence of sym-
collidine as base in DCE (10 mL) for 2 h. After washing the resin with
CH2Cl2 (6”2 min, 10 mL), the sulfonamide moiety was allowed to react
with triphenylphosphine (319 mg), 1-butyl alcohol (222 mL), and DIAD
(239 mL) in DCE (10 mL) for 3 h. The resin was then washed with
CH2Cl2 (3”2 min, 10 mL) and DMF (6”2 min, 10 mL). The oNBS group
was removed by treatment with 0.5m 2-mercaptoethanol in DMF (5 mL)
in the presence of DBU (182 mL) for 75 min. The resin was washed with
DMF (6”2 min, 10 mL) and CH2Cl2 (3”2 min, 10 mL). Subsequently,
Fmoc-Leu-OH was coupled to the N-butyl amine by using the BTC
method.[25, 33] First, Fmoc-Leu-Cl was prepared in situ by adding triphos-
gene (60 mg, 0.20 mmol) to a solution of Fmoc-Leu-OH in dioxane
(10 mL) in the presence of sym-collidine (212 mL, 1.6 mmol) as base.
After being stirred for 60 s, the reaction mixture was transferred to the
resin and allowed to react for 1 h. This treatment was repeated once. The
resin was then washed with DMF (3”2 min, 10 mL), CH2Cl2 (3”2 min,
10 mL), and DMF (3”2 min, 10 mL) and the yield was determined to be
70% (0.24 mmolgÀ1). After capping of the resin with Ac2O/DIPEA/
HOBt in NMP (10 mL), the resin was treated with 20% piperidine/NMP
to remove the Fmoc group and the resin was washed. Subsequently,
Fmoc-Ala-N(Bu)Ile-OH (170 mg, 0.3 mmol) was coupled for 16 h by
using HATU (133 mg, 0.35 mmol)/HOAt (48 mg, 0.35 mmol)/DIPEA
(122 ml, 1.4 mmol) in NMP (10 mL). After coupling of this dipeptide, the
synthesis was continued by coupling Fmoc-NNle-OH with the peptoid-
coupling protocol.[27] Finally, the synthesis was completed as described
above in the general procedure.
Circular dichroism spectroscopy: CD spectra were measured at 1.0 nm in-
tervals over the range 195–250 nm as the average of 20 runs and by using
a spectral band width of 2.0 nm. Cuvettes of 0.5 mm thermostated at
208C were used, with the optical chamber continually flushed with dry
N2 gas. The spectra were measured in 0.1% TFAin H O. The concentra-
2
tions (1 mgmLÀ1) were determined on the basis of the calculated molecu-
lar mass of the purified lyophilized peptides. Apeptide sample was dis-
solved in 0.1% TFAin H 2O and stored for 4 days at 48C prior to analy-
sis.
Aggregation assay: The procedure for this assay was based on refer-
ence [47]. Astock solution (10 m m) of hIAPP(20–29) (6) in dimethylsulf-
E
oxide (DMSO) was prepared and this stock solution (25 mL) was added
to an equimolar quantity of lyophilized inhibitor peptide (7–11). After
immediate mixing, the obtained DMSO mixture was incubated for
15 min. The DMSO mixture was then diluted into a phosphate buffer
(225 mL, pH 7.4, 100 mm NaCl, 1.8 mm NaH2PO4, 8.2 mm Na2HPO4) and
the turbidity (absorbance at 400 nm) was measured over 150 min at room
temperature. These aggregation assays were performed twice in three in-
dependent experiments. Turbidity measurements were performed on a
Bio-TEK mQuant plate reader.
Acknowledgements
Peptoid–peptide hybrids 9, 11, and 12: The Fmoc/tBu-based solid-phase
synthesis of peptoids and peptoid–peptide hybrids as described by Kruijt-
zer et al.[27] was used. In short, Fmoc-NNle-OH was coupled to the a-
amino group of the preceding amino acid in the presence of HBTU/
HOBt and DIPEAin NMP for 45 min. After removal of the Fmoc
group, the next amino acid residue was coupled to the secondary amine
with HATU/HOAt/DIPEA in NMP for 90 min.
These investigations were supported by the Council for Chemical Scien-
ces of the Netherlands Organization for Scientific Research (CW-NWO).
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Received: November 4, 2005
Published online: March 10, 2006
Chem. Eur. J. 2006, 12, 3714 – 3725
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3725