Self-assembled peptide tubelets with 7 A pores

Article abstract of DOI:10.1002/chem.200500478

Here we report the preparation and structural characteristics of self-assembling peptide tubelets composed of 32-membered rings formed of alternating cx-amino acids and cis-3-aminocyclohexanecarboxylic acids. The tubelets possess a partial hydrophobic cor

Full text of DOI:10.1002/chem.200500478

FULL PAPER
DOI: 10.1002/chem.200500478
Self-Assembled Peptide Tubelets with 7 Š Pores
Manuel Amorín, Luis Castedo, and Juan R. Granja*[a]
Abstract: Here we report the preparation and structural characteristics of self-as-
sembling peptide tubelets composed of 32-membered rings formed of alternating
a-amino acids and cis-3-aminocyclohexanecarboxylic acids. The tubelets possess a
partial hydrophobic core environment, provided by the projection of the cyclohex-
ane C2 methylene moiety into the lumen, and a Van der Waals pore diameter of
about 7 Š.
Keywords: amino acids · dimeriza-
tion · nanotubes · peptides · self-
assembly
Introduction
acids,[8] alternating a,e-amino acids,[9] and oligoureas.[10]
These design choices are not purely artistic, because the
functional characteristics of the nanotubes made by this
strategy depend on the nature of both their interior and ex-
terior surfaces. The hydrophilic nature of the peptide nano-
tube interior has been an important feature in designing
transmembrane ion channels and pores.[6b,11] The possibility
of extending this class of nanotubes to include tubes with
hydrophobic inner surfaces, a taskthat has not yet been
widely performed because it requires peptide main-chain re-
placements that should not adversely affect the conforma-
tional requirements for the self-assembly process, has re-
cently been shown in workon dimers that self-assemble
from cyclic peptides composed of alternating d-a-amino
acids and (1R,3S)-3-aminocyclohexanecarboxylic acids (d-a-
Aas and l-g-Achs, respectively), with the hydrophobic core
environment being provided by the projection of one of the
cyclohexane methylene moieties into the lumen.[12,13] In that
work, each cyclic monomer consisted of three [l-g-Ach-d-a-
Aa] units (1a, Scheme 1) and the approximate Van der
Waals internal diameter of the resulting dimeric “tubelets”
was 4.3 Š. For analogous tubelets composed of 32-mem-
bered rings formed of four [l-g-Ach-d-a-Aa] units (1b) or
four [d-g-Ach-l-a-Aa] units, a Van der Waals pore diameter
of about 7 Š can be calculated. Here we report the prepara-
tion and structural characteristics of tubelets confirming
these expectations.
In recent years, considerable effort has been devoted to the
synthesis of organic and inorganic nanotubes,[1] spurred by
their potential utility in a wide variety of chemical, biologi-
cal, and materials science applications.[1b,2,3] Proposed de-
signs include hollow bundles of rod-like units, helically
wound linear species, rolled-up sheets, helically juxtaposi-
tioned truncated wedges, and stacked rings. In particular,
this last design in principle facilitates control of what in
most applications would be one of the critical parameters of
the nanotubes, their internal diameter.
One of the most promising implementations of the
stacked ring approach is the self-assembling peptide nano-
tube (SPN).[4] The elementary components of SPNs are
cyclic peptides, the chiralities of the amino acids of which
allow the ring to adopt a quasiplanar conformation.[4–7] In
this conformation, peptide backbone NH and C=O groups
project perpendicularly from the plane of the ring on either
side and are therefore able to form hydrogen bonds with
those of neighboring rings, so constructing a nanotube. Spe-
cifically, self-assembling nanotubes have been designed
based on cyclic peptides of alternating d,l-a-amino acids,[4,5]
b-amino acids,[6] alternating a,b-amino acids,[7] d-amino
[a] M. Amorín, Prof. Dr. L. Castedo, Prof. Dr. J. R. Granja
Departamento de Química Orgµnica e Unidade Asociada ó C.S.I.C.
Facultade de Química, Universidade de Santiago
15782 Santiago de Compostela (Spain)
Scheme 1 (left) shows how molecules of 1b can stackto
constitute a nanotube. As each constituent cyclic peptide
has its g-Ach NH and C=O groups on one face (the g face)
and its d-a-Aa NH and C=O groups on the other (the a
face) and because the HN···C=O spacing of the a- and g-
amino acids is different, the orientations of the rings must
alternate so that a faces bond to a faces and g faces to g
Fax : (+34)981-595-012
E-mail: qojuangg@usc.es
Supporting Information for this article (1H and 13C NMR spectra of
1
l-Boc-MeN-g-Ach-OFm; H and 13C NMR, NOESY and/or ROESY,
and FTIR spectra of peptides 2b, 3b, 6a, 6b) is available on the
WWW under http://www.chemeurj.org/ or from the author.
Chem. Eur. J. 2005, 11, 6543 – 6551
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6543

Scheme 1. Center: A generic cyclo[(d-a-Aa-l-g-Ach)n] peptide. Left: Structure of a nanotube self-assembled therefrom by Ach-to-Ach (g–g) and Aa-to-
Aa (a–a) hydrogen bonding between antiparallel rings. Right: Dimers of derivatives with N-methylated d-a-Aa (top) or N-methylated l-g-Ach (bottom)
illustrating g–g and a–a bonding, respectively. For clarity, amino acid side chains have been omitted in the representations of the nanotube and dimers.
faces, in each case through the formation of a b-sheet-like
set of hydrogen bonds between antiparallel rings. In this
study, we investigated the stabilities of these two types of in-
terring interaction (a–a and g–g) by preparing and charac-
terizing tubelets formed of rings in which the stacking of
more than two rings was blocked by N-methylation of either
the a-amino acid (for formation of g–g-bound tubelets) or
the g-amino acid (for formation of a–a-bound tubelets).[14]
form containing 1 equivalent of (+)-1-phenylethanamine
gave (1R,3S)-N-Boc-3-aminocyclohexanecarboxylic acid (l-
Boc-g-Ach-OH) with an enantiomeric purity greater than
95%.[16] The N-methylated amino acid Boc-MeN-g-Ach-OH
was then obtained by treatment of Boc-g-Ach-OH with
sodium hydride and methyl iodide in THF, and the fluoren-
yl-protected ester Boc-MeN-g-Ach-OFm was obtained by
treatment of the resulting Boc-MeN-g-Ach-OH with 9-
fluorenylmethanol (Scheme 2).
The cyclopeptides were prepared following the synthetic
strategy shown in Scheme 3, by starting from the fluorenyl-
protected ester of the Boc-protected N-methylamino acid,
that is, d-Boc-MeN-Ala-OFm for the cyclic-peptide precur-
sors of g–g-bound tubelets and l-Boc-MeN-g-Ach-OFm for
the cyclic-peptide precursors of a–a-bound tubelets. The
corresponding dipeptides, tetrapeptides, and octapeptides
were prepared by using standard solution-phase peptide-syn-
thesis protocols, and treatment of the linear unprotected oc-
tapeptides with TBTU and DIEA at 6 mm concentrations in
dichloromethane led to formation of the desired cyclic prod-
ucts in good yields (50–75%).
Results and Discussion
The cis-3-aminocyclohexanecarboxylic acids (Boc-g-Ach and
Boc-MeN-g-Ach; Boc=tert-butoxycarbonyl, MeN=N-methyl)
needed for these studies were prepared by hydrogenation of
the sodium salt of m-aminobenzoic acid in the presence of
alkaline Raney nickel at 90–100 atm and 1508C[15] followed
by treatment with tert-butoxycarbonyl anhydride to provide
the racemic acid Boc-g-Ach-OH in 64% yield (Scheme 2).
Resolution with successive recrystallizations from chloro-
The N-methylated cyclopeptide cyclo[(d-MeN-Ala-l-g-
Ach)4] (2b) was characterized by NMR and FTIR spectros-
1
copy and mass spectrometry. Its H NMR spectrum shows it
to have a flat, all-trans conformation in polar and nonpolar
solvents. In nonpolar solvents, such as deuteriochloroform,
the spectrum reflects two species that are moderately slow
at exchanging on the NMR timescale, and the peaks repre-
senting these species are sharp enough at 273 K to show
that both have JNH–gH constants of 7.4 Hz, a value indicative
of the flat all-trans backbone conformation. The concentra-
tion dependence of the ratio of these two species is consis-
tent with one being monomeric 2b and the other being the
dimer 4b (Scheme 1),[17] with the association constant in
CDCl3 at 273 K being 340mÀ1.[18] The b-sheet nature of the
Scheme 2. Synthesis of the fluorenyl-protected ester of cis-N-Boc-N-
methyl-3-aminocyclohexanecarboxylic acid (Boc-MeN-g-Ach-OFm): a) H2
(95 atm), Raney Ni, NaOH, H2O, 1508C, 80%; b) (Boc)2O, DIEA, H2O/
dioxane, 80%; c) resolution with (+)-1-phenylethylamine, CHCl3/
hexane; d) NaH, MeI, THF, 88%; e) FmOH, EDC, HOBt, DMAP,
CH2Cl2, 89%. DIEA=N,N-diisopropylethylamine, THF=tetrahydrofur-
an, Fm=9H-fluoren-9-ylmethyl, EDC=3-(3-dimethylaminopropyl)-1-
ethylcarbodiimide, HOBt=1-hydroxy-1H-benzotriazole, DMAP=4-di-
methylaminopyridine.
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Self-Assembled Peptide Tubelets
FULL PAPER
oxide (DMSO), its 1H NMR spectrum reflects a slowly inter-
converting conformer equilibrium;[23] however, in nonpolar
solvents the spectrum consists of a single set of signals and
exhibits no concentration dependence (JNH–aH =8.6 Hz), with
the chemical shift of the Phe NH proton remaining constant
(d=8.57 ppm) at concentrations down to 1 mm. Addition of
35% or more of methanol, or heating to 333 K, does not
alter this NH signal by more than about 0.1 ppm. Since the
FTIR spectrum in CHCl3 shows b-sheet-like hydrogen bond-
ing (amide I, IIII, and A bands at n˜ =1623, 1525, and
3312 cmÀ1, respectively),[19] compound 3b must therefore be
entirely dimerized as 5b, and the a–a interaction must
therefore be considerably stronger than the g–g interaction.
This difference is probably due to the backbone conforma-
tion being better fitted for a–a than for g–g interactions and
to the NH group of the a-amino acid being more polarized
than that of the g-amino acid.[22]
Conclusive proof of the dimerization of 2b to 4b and 3b
to 5b was provided by X-ray diffractometry of single crys-
tals obtained by crystallization from CHCl3/CCl4 (2b) or
MeOH (3b). In both cases the colorless prismatic crystals
were composed of units in which two antiparallel cycloocta-
peptide rings were linked in b-sheet fashion by eight hydro-
gen bonds (Figure 1),[24] although in the case of 3b the unit
cell contained two nonequivalent dimers: one in which the
range of hydrogen-bond N···O distances, 2.91–3.01 Š, was
similar in breadth to that observed in 2b (2.84–2.93 Š) and
another in which the N···O distances were more irregular,
ranging from 2.86–3.12 Š (Figure 1 f). In the peptide rings
of both dimers of 3b, all four Phe C(O)–Ca–N angles are
very similar (ꢀ1108), thereby resulting in circular molecules
(Figure 1e) and dimer lumina with approximate Van der
Waals diameters of 6.6 Š (Hb-Ach–Hb-Ach), but in those of the
dimer of 2b, probably because of lattice packing forces, the
Ala C(O)–Ca–N angle alternated between 107.68 and 112.98
in one monomer and between 110.38 and 111.68 in the
other, thereby resulting in elliptical molecules (Figure 1a)
and dimer lumina with approximate minimum and maxi-
mum diameters (Ca-Ala–Ca-Ala) of 8.06 and 12.05 Š for one
cyclopeptide and 8.50 and 12.00 Š for the other. The cavities
of the dimers 4b and 5b have Van der Waals volumes of ap-
proximately 232 and 286 Š3,[25] respectively. In both cases
they contain disordered solvent molecules, a fact that estab-
lishes the ability of 4b to retain hydrophobic molecules
(CHCl3 and/or CCl4) and the ability of 5b to retain hydro-
philic molecules (MeOH, H2O);[26,27] in particular, the fact
that the observed electron density for water molecules in
the cavity of 5b is the time average for molecules at several
overlapping sites suggests that these molecules are loosely
bound and can easily move around within the cavity. It may
be noted that all the hydrogen-bonding amide groups in 4b
are slightly tilted towards the center of the cavity (Fig-
ure 1b); this may explain why the enthalpy of dimerization
was only 38.2 kJmolÀ1, instead of about 46 kJmolÀ1 as
would be suggested by the value for the corresponding d,l-
cyclohexapeptide dimer.[12a] Finally, the body-centered struc-
ture of the crystals of 4b and 5b, together with the annular
Scheme 3. General synthetic strategy for cyclic peptide preparation:
a) FmOH, EDC, HOBt, DMAP, CH2Cl2; b) 50% TFA in CH2Cl2; c) Boc-
Aa-OH, HATU, DIEA, CH2Cl2; d) 20% piperidine in CH2Cl2; e) HBTU,
DIEA, CH2Cl2; f) TBTU, DIEA, CH2Cl2. TFA=trifluoroacetic acid,
HATU=2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-
phosphate, HBTU=2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroni-
um hexafluorophosphate, TBTU=2-(1H-benzotriazole-1-yl)-1,1,3,3-tetra-
methyluroniumtetrafluoroborate.
hydrogen bonding in the dimer is shown by the downfield
shift of the Ala CaH signal from d=5.31 ppm in the NMR
spectrum of the monomer to d=5.59 ppm in that of the
dimer (the d shift of the g-Ach CgH signal from 3.59 to
3.82 ppm probably has the same significance). The b-sheet
nature is also indicated by the FTIR spectrum in CHCl3,
which shows an amide I band at n˜ =1627 cmÀ1 and an ami-
de IIII band at n˜ =1540 cmÀ1. Amide A FTIR bands near n˜ =
3315 cmÀ1 suggest an intermonomer distance of 4.85–
4.90 Š.[19]
The strength of the hydrogen bonds in 4b is shown by the
NMR signals for its NH groups lying considerably downfield
of those of 2b, at d=8.12 ppm as against d=6.76 ppm.
However, the hydrogen bonding in 4b is weaker than in the
dimer (4a) of the hexapeptide cyclo[(d-MeN-Ala-l-g-
Ach)3]:[12] Vanꢁt Hoff plots for the range 243–283 K afford
values of À38.2 kJmolÀ1 for DH2o98 and À94.2 kJÀ1 molÀ1 for
DSo298. These results show that although the dimerization pro-
cess is enthalpy driven,[20] as in the case of the hexapeptide,
the energy per hydrogen bond is only 1.27 kJmolÀ1 in 4b as
against 2.20 kJmolÀ1 for the hexapeptide dimer (4a), for
which
DHo298 =À34.1 kJmolÀ1
and
DSo298
=
À69.8 kJÀ1 molÀ1.[12] The difference in energy per bond is
due partly to the more negative DSo298 value of 4b, which is
attributable to the greater flexibility of its larger ring,[21] and
partly to a disproportionately small increase in negative
DHo298, which is discussed below in relation to the crystallo-
graphic characterization of 4b.[22]
To investigate the stability of a–a-bound tubelets, we pre-
pared compound 3b. In polar solvents, such as dimethyl sulf-
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6545

J. R. Granja et al.
We hypothesized that hydro-
gen bonding between mutually
opposed Ser hydroxy groups
would favor the formation of
7b.[28]
As expected, the 1H NMR
spectrum of 6b in chloroform
showed two sets of signals,
both of which reflect b-sheet-
like hydrogen bonding be-
tween
all-trans
monomers
(Figure 2, bottom left). The
ratio between these two spe-
cies was 6:1 and did not
depend on peptide concentra-
tion or temperature, thereby
showing both forms to be com-
pletely dimerized.[29] Unexpect-
edly, however, 2D NOESY ex-
periments showing NHSer
–
NHPhe and HbSer–HbPhe cross-
peaks (Figure 2, bottom right)
implied that the major form of
the dimer was 7b*. Hypothe-
sizing that the predominance
of 7b* might be due to steric
hindrance between the mutual-
ly opposed phenyl moieties of
7b or to weakhydrogen bonds
between phenyl (Phe) and hy-
droxy (Ser) groups in 7b*,[30]
we examined the dimerization
behavior of cyclopeptide 6a,
from which 6b had been pre-
pared. Although steric hin-
drance between phenyl groups
must be similar for 7a* and
7a, and although there is no
Figure 1. Crystal structures of dimers of a–c) cyclo[(d-MeN-Ala-l-g-Ach)4] (2b) and d–f) cyclo[(d-Phe-l-MeN-g-
Ach)4] (3b), as viewed along (a and e) or perpendicular to (b and f) the dimer axis or along the crystallograph-
ic axis (c and d).
À
possibility of O H···Ph hydro-
nature of the dimers themselves, creates channels along the
crystallographic b (2b) and c (3b) axes that are composed
of two alternating types of segment with a period of about
12.5 Š for 2b and 10.5 Š for 3b; this spacing is due to the
dimer pore and the space surrounded by the external surfa-
ces of four coplanar dimers (Figure 1c,d). In the case of 4b
these channels are straight, while those of 5b are more sinu-
ous.
gen bonding in 7a*, it was again the “staggered” arrange-
ment (7a*) that predominated in a similar ratio. Examina-
tion of the chemical shifts of signals for the NH groups of
7b and 7b* suggests that, although the NHPhe hydrogen
bond is stronger in 7b (d(NHPhe)=8.65 ppm) than in 7b* (d-
(NHPhe)=8.49 ppm), the NHSer hydrogen bond is much
weaker (d(NHSer)=8.14 ppm in 7b; d(NHSer)=8.96 ppm in
7b*), so that the overall balance seems to favor the stag-
À
To investigate whether self-assembly might be significant-
ly affected by interactions between amino acid side chains
on adjacent cyclopeptides, we prepared cyclo[(l-Ser-d-MeN-
g-Ach-l-Phe-d-MeN-g-Ach)2] (6b; Figure 2). This cyclopep-
tide has twofold symmetry instead of the fourfold symmetry
of 2b and 3b, and can accordingly, in principle, form two
kinds of a–a-bonded dimer with stabilities that might differ
because of differences in between-monomer side-chain in-
teractions: one in which Phe faces Phe and Ser faces Ser
(7b), and the other in which Phe faces Ser (7b*; Figure 2).
gered form. The weakness of the NHSer OSer bond is in
keeping with Ser having a somewhat lower propensity to
form b sheets than Phe;[31] in all the other dimerizing hydro-
À
À
gen bonds of 7b and 7b* (NHSer
O
Phe, NHPhe
OSer, and
À
NHPhe
OPhe) at least one Phe is involved. It may be pointed
out that the above interpretation suggests that eclipsed a–a
bonding of the kind modeled by 7b results in the compo-
nent cyclopeptides becoming buckled, with the Phe–Phe dis-
tances being shorter than the Ser–Ser distances.
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Chem. Eur. J. 2005, 11, 6543 – 6551

Self-Assembled Peptide Tubelets
FULL PAPER
Figure 2. Top: Cyclooctapeptides 6a and 6b and the two dimers that can be formed from each: in one dimer the Phe of one monomer faces the Phe of
the other, while in the second dimer Phe faces the other a-amino acid (Ser or benzylated Ser). For clarity, amino acid side chains have been omitted.
Bottom left: 1H NMR spectrum of 6b showing the NH signals of both 7b and 7b*. Bottom right: NOESY spectrum showing NHSer(7b*)–NHPhe(7b*)
and HbPhe–HbSer cross-peaks.
Experimental Section
Conclusion
General: HBTU, TBTU, HATU, Boc-phenylalanine, serine, and N-meth-
Like their hexapeptide analogues, cyclooctapeptides com-
posed of four [l-g-Ach-d-a-Aa] or four [d-g-Ach-l-a-Aa]
units can form dimers in which antiparallel rings are linked
by a b-sheet-like set of eight hydrogen bonds. This suggests
that the class of a,g-SPNs extends at least to members com-
posed of 32-membered rings with pore diameters of around
7 Š. The finding of both hydrophobic and hydrophilic sol-
vent molecules in the cavities of the dimers synthesized in
this worksupports the possibility that a,g-SPNs could be
used for inclusion of both hydrophobic and hydrophilic mol-
ecules. As has been pointed out elsewhere, the fact that C2
of g-Ach intrudes into the cavity (where it must contribute
to hydrophobicity) means that a,g-SPNs, unlike SPNs based
on a- or b-amino acids, might be endowed with functional-
ized inner surfaces affording greater selectivity as ion chan-
nels, catalysts, receptors, or molecule containers.
ylalanine were purchased from Novabiochem or Advanced ChemTech.
All reagents and solvents were used as received unless otherwise noted.
1H NMR spectra were recorded on Varian Inova-750 MHz, Varian Mer-
cury-300 MHz, Varian Inova-400 MHz, Bruker AMX-500 MHz, or
Bruker WM-250 MHz spectrometers. Chemical shifts (d) were reported
in parts per million (ppm) relative to tetramethylsilane (d=0.00 ppm).
1H NMR splitting patterns are designated as singlet (s), doublet (d), trip-
let (t), or quartet (q). All first-order splitting patterns were assigned on
the basis of the appearance of the multiplet. Splitting patterns that could
not be easily interpreted are designated as multiplet (m) or broad (br).
13C NMR spectra were recorded on Varian Mercury-300 MHz and
Bruker WM-250 MHz spectrometers. Carbon resonances were assigned
by using DEPT spectra obtained with phase angles of 1358. Mass spectra
were obtained by using Bruker Autoflex MALDI-TOF and Micromass
Autospec mass spectrometers. Crystallographic data were collected in a
FR591-KappaCCD2000 Bruker-Nonius diffractometer. FTIR measure-
ments were made on a JASCO FT/IR-400 spectrophotometer with solu-
tions at a concentration of 5–10 mm in CHCl3 and placed in an NaCl so-
lution IR cell. Column chromatography was performed on EM Science
silica gel 60 (230–400 mesh). Solvent mixtures for chromatography are re-
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6547

J. R. Granja et al.
ported as v/v ratios. Reversed-phase HPLC was carried out on C18 col-
umns with the use of water/acetonitrile/TFA gradients between 99:1:0.1
and 10:90:0.1, and HPLC was carried out on phenomenex maxsil-10
silica column with CH2Cl2/MeOH gradients between 100 and 90:10. THF
was distilled from sodium/benzophenone under argon immediately prior
to use. CH2Cl2 and pyridine to be used as reaction solvents were distilled
from CaH2 over argon immediately prior to use.
cis-3-Aminocyclohexanecarboxylic acid (g-Ach-OH):[15] A solution of 3-
aminobenzoic acid (11.3 g, 82.5 mmol) and NaOH (3.3 g, 82.5 mmol) in
H2O (200 mL) in a pressure bottle was treated with Raney Ni (7.0 g).
The resulting mixture was hydrogenated (90–100 atm) at 1508C for 1 h in
a Parr apparatus. The mixture was then filtered over celite, and the cata-
lyst was washed with H2O. Acidification of the resulting solution to pH 2
with 10% HCl and concentration under vacuum gave a residue that was
desalted by Dowex AG50W-X4 chromatography (1m pyridine) to pro-
with EDC (112 mg, 0.584 mmol), HOBt (79 mg, 0.584 mmol), 9-fluorenyl-
methanol (92 mg, 0.467 mmol), and DMAP (70 mg, 0.584 mmol). After
being stirred for 1 h at room temperature, the solution was washed with
10% citric acid and saturated NaHCO3. The combined organic layers
were dried over Na2SO4, filtered, and concentrated under reduced pres-
sure. The crude material was purified by flash chromatography (50–80%
CH2Cl2 in hexane) to give l-Boc-MeN-g-Ach-OFm (151 mg, 89%) as a
yellow oil; Rf =0.95 (10% MeOH in CH2Cl2); 1H NMR (250 MHz,
CDCl3, 258C): d=7.70 (d, J(H,H)=7.2 Hz, 2H), 7.53 (d, J(H,H)=
7.2 Hz, 8H), 7.38–7.20 (m, 4H), 4.36 (d, J(H,H)=6.5 Hz, 2H), 4.14 (t, J-
(H,H)=6.7 Hz, 1H), 3.99 and 3.74 (m, 1H), 2.67 (s, 3H), 2.43 (m, 1H),
1.42 ppm (s, 9H); 13C NMR (CDCl3, 62.83 MHz): d=174.9 (C=O), 155.5
(C=O), 143.7 (C), 141.3 (C), 127.7–119.9 (CH), 79.3 (C), 66.6 (CH2), 52.7
(CH), 46.9 (CH), 42.7 (CH), 32.2 (CH2), 29.2 (CH2), 28.4 (CH3), 28.2
(CH3), 28.1 (CH2), 24.5 ppm (CH2); FAB+-MS: m/z (%): 436 (7) [MH+],
336 (100) [MHÀBoc+]; HRMS: calcd: 436.24878 [MH+]; found:
436.24997.
vide g-Ach-OH (9.73 g, 80%) as
a white solid; Rf =0.48 (MeOH);
1H NMR (250 MHz, D2O, 25 8C): d=3.04 (m, 1H), 2.20–1.70 (m, 5H),
1.30–0.90 ppm (m, 4H).[32]
Boc-[l-g-Ach-d-MeN-Ala]-OFm: solution of d-Boc-MeN-Ala-OFm
A
(160 mg, 0.570 mmol) in TFA/CH2Cl2 (1:1) was stirred at room tempera-
ture for 10 min. After removal of the solvent, the residue was dried
under high vacuum for 2 h and then dissolved in dry CH2Cl2 (40 mm). l-
Boc-g-Ach-OH (138 mg, 0.570 mmol), HATU (229 mg, 0.601 mmol), and
DIEA (400 mL, 2.280 mmol) were successively added. After being stirred
for 30 min at room temperature, the solution was poured into a separa-
tion funnel and washed with 10% citric acid and saturated NaHCO3. The
organic layers were dried over Na2SO4 and concentrated under reduced
pressure, and the crude material was purified by flash chromatography
(0–5% MeOH in CH2Cl2) to give the desired dipeptide (231 mg, 80%)
(1R,3S)-N-Boc-3-aminocyclohexanecarboxylic acid (l-boc-g-Ach-OH):
Boc2O (7.0 g, 32 mmol) and DIEA (14.7 mL, 84 mmol) were added to a
solution of cis-3-aminocyclohexanecarboxylic acid (4.0 g, 28 mmol) in
water (25 mL) and dioxane (25 mL). After being stirred at room temper-
ature for 3 h, the solution was acidified to pH 2 and extracted with
CH2Cl2. The combined organic layers were dried (Na2SO4), filtered, and
concentrated, and the resulting oil was crystallized from CH2Cl2/hexane
(2:1) to give racemic Boc-g-Ach-OH (3.4 g initially and 2.1 g in a second
crystallization, 80%); Rf =0.85 (MeOH); m.p. 136–1388C. The racemic
Boc-g-Ach-OH was redissolved by crystallization from CHCl3/hexane
(2:1) in the presence of (+)-1-phenylethanamine (1 equiv). The resulting
white crystals were washed with CHCl3/hexane (2:1), and the mixture
was poured into a separation funnel, dissolved in CH2Cl2, and washed
with 10% citric acid. This operation was repeated 2–3 times. The com-
bined organic layers were dried (Na2SO4), filtered, and concentrated, and
as
a
white foam; 1H NMR (300 MHz, CDCl3, 258C): d=7.72 (d,
J(H,H)=7.3 Hz, 2H), 7.50 (t, J(H,H)=9.2 Hz, 2H), 7.40–7.23 (m, 4H),
5.14 (q, J(H,H)=7.2 Hz, 1H), 4.68 (d, J(H,H)=7.3 Hz, 1H), 4.50 (m,
2H), 4.12 (t, J(H,H)=5.7 Hz, 1H), 3.46 (m, 1H), 2.62 (s, 3H), 2.44 (m,
H), 1.42 (s, 9H), 1.22 ppm (d, J(H,H)=7.3 Hz, 3H); 13C NMR
(75.40 MHz, CDCl3, 258C): d=174.6 (C=O), 171.5 (C=O), 155.0 (C=O),
143.4 and 143.2 (C), 141.2 and 141.1 (C), 127.6–119.7 (CH), 78.8 (C), 66.4
and 65.9 (CH2), 54.6 and 51.9 (CH), 48.7 (CH), 46.7 and 46.6 (CH), 39.5
and 39.2 (CH), 35.8 and 35.2 (CH2); 32.8 and 32.6 (CH2), 30.9 (CH3),
28.6 and 28.2 (CH3), 27.8 (CH2), 24.2 (CH2), 15.4 and 14.1 ppm (CH3);
FAB +-MS: m/z (%): 507 (18) [MH+]; 407 (100) [MHÀBoc+]; HRMS:
calcd: 507.28590 [MH+]; found: 507.28780.
Boc-[d-Phe-l-MeN-g-Ach]-OFm: This compound was prepared in the
same way as dipeptide Boc-[l-g-Ach-d-MeN-Ala]-OFm by using l-Boc-
MeN-g-Ach-OFm (145 mg, 0.334 mmol) and d-Boc-Phe-OH (88.5 mg,
0.334 mmol) to afford the desired dipeptide (164 mg, 84%); 1H NMR
(250 MHz, CDCl3, 258C): d=7.76 (t, J(H,H)=6.5 Hz, 2H), 7.57 (t,
J(H,H)=7.0 Hz, 2H), 7.41–7.16 (m, 9H), 5.43 (d, J(H,H)=8.2 Hz, 1H),
4.79 (dd, J(H,H)=7.8, 13.7 Hz, 1H), 4.44 (m, 2H), 4.18 (dd, J(H,H)=
6.8, 13.6 Hz, 1H), 3.25 (m, 1H), 2.96 (m, 2H), 2.70 and 2.44 (s, 3H),
1.42 ppm (s, 9H); 13C NMR (62.83 MHz, CDCl3, 258C): d=174.4 (C=O),
171.1 (C=O), 154.8 (C=O), 143.4 and 143.4 (C), 141.1 (C), 136.5 and
136.2 (C), 129.3–119.7 (CH), 79.3 and 79.3 (C), 66.8 and 65.8 (CH2), 54.9
(CH), 51.7 and 51.3 (CH), 46.8 and 46.7 (CH), 42.4 and 42.1 (CH), 40.4
and 40.1 (CH2), 31.8 and 31.1 (CH2), 29.5 and 29.4 (CH2), 28.8 (CH3),
28.1 and 27.2 (CH3), 27.8 (CH2), 24.0 ppm (CH2); FAB+-MS: m/z (%):
583 (100) [MH+], 483 (70) [MHÀBoc+]; HRMS: calcd: 583.31273
[MH+]; found: 583.31460.
the resulting oil was crystallized from CH2Cl2/hexane (2:1);[16] [a]D20
=
À50.5 (c=1 in MeOH); 1H NMR (250 MHz, CDCl3, 258C): d=5.56 (m,
1H, NH), 4.47 (m, 1H), 3.44 (m, 1H), 1.42 ppm (s, 9H); 13C NMR
(62.83 MHz, CD3OD, 258C): d=178.7 (C=O), 157.6 (C=O), 79.8 (C),
50.2 (CH), 43.5 (CH), 36.5 (CH2), 33.4 (CH2), 29.4 (CH2), 28.9 (CH3),
25.4 ppm (CH2); elemental analysis: calcd (%): C 58.99, H 8.68, N 5.73;
found: C 58.53, H 9.03, N 5.75%; FAB+-MS: m/z (%): 487 (17) [M2H+],
387 (18) [M2H+ÀBoc], 244 (66) [MH+], 188 (94) [MH+ÀCH2=C(CH3)2],
144 (100) [MH+ÀBoc].
(1R,3S)-N-Boc-N-methyl-3-aminocyclohexanecarboxylic acid (l-Boc-
MeN-g-Ach-OH): A solution of l-Boc-g-Ach-OH (1.38 g, 5.68 mmol) in
dry THF (50 mL) was treated with NaH (680 mg, 60% in mineral oil,
17.1 mmol). The resulting mixture was stirred at 08C for 30 min, and
then methyl iodide (1.06 mL, 17.1 mmol) was added. After the mixture
had been stirred overnight at room temperature, an additional three
equivalents of NaH (680 mg, 17.1 mmol) and methyl iodide (1.06 mL,
17.1 mmol) were added at 08C if starting material was detected by TLC,
and the resulting mixture was stirred for 3 h at room temperature. After
being quenched with water, the solution was concentrated to remove the
THF and the resulting aqueous solution was washed with diethyl ether,
acidified to pH 3 by addition of HCl (10%), and finally extracted with
CH2Cl2. The combined organic layers were dried over Na2SO4 and con-
centrated under reduced pressure. The crude material was crystallized
from CH2Cl2/hexane to give the l-Boc-MeN-g-Ach-OH (1.29 g, 88%) as a
Boc-[l-Phe-d-MeN-g-Ach]-OFm: This compound was prepared in the
same way as dipeptide Boc-[l-g-Ach-d-MeN-Ala]-OFm by using d-Boc-
MeN-g-Ach-OFm (1.335 g, 3.07 mmol) and l-Boc-Phe-OH (0.814 g,
3.07 mmol) to afford the desired dipeptide (1.61 g, 90%); FAB+-MS:
m/z (%): 583 (69) [MH+], 483 (100) [MHÀBoc+].
white solid; Rf =0.58 (10% MeOH in CH2Cl2); m.p. 141–1438C; [a]D20
=
À51.7 (c=1 in MeOH); 1H NMR (250 MHz, CDCl3, 258C): d=4.01 and
3.77 (m, 1H), 2.69 (s, 3H), 2.42 (m, 1H), 1.42 ppm (s, 9H); 13C NMR
(62.83 MHz, CDCl3, 258C): d=180.5 (C=O), 155.6 (C=O), 79.6 (C), 54.0
and 52.6 (CH), 42.4 (CH), 31.9 (CH2), 29.1 (CH2), 28.4 (CH3), 28.2(CH3),
28.0 (CH2), 24.4 ppm (CH2); FAB+-MS: m/z (%): 515 (6) [2MH+], 258
(67) [MH+], 158 (86) [MHÀBoc+]; HRMS: calcd: 258.17053 [MH+];
found: 258.17042.
Boc-[l-Ser(Bn)-d-MeN-g-Ach]-OFm (Bn=benzyl): This compound was
prepared in the same way as dipeptide Boc-[d-g-Ach-l-MeN-Ala]-OFm
by using d-Boc-MeN-g-Ach-OFm (1.055 g, 2.43 mmol) and l-Boc-
Ser(Bn)-OH (0.717 g, 2.43 mmol) to afford the desired dipeptide (1.290 g,
90%); 1H NMR (250 MHz, CDCl3, 258C): d=7.71 (d, J(H,H)=7.3 Hz,
2H), 7.54 (d, J(H,H)=7.1 Hz, 2H), 7.39–7.18 (m, 9H), 5.51 (m, 1H),
4.85 (m, 1H), 4.48–4.37 (m, 4H), 4.23 (m, 1H), 3.80 (m, 1H), 3.57 (m,
(1R,3S)-(9H-fluoren-9-yl)-methyl N-Boc-N-methyl-3-aminocyclohexane-
carboxylate (l-boc-Men-g-Ach-OFm): A solution of l-Boc-g-MeN-Ach-OH
(100 mg, 0.389 mmol) in dry CH2Cl2 (10 mL) was treated successively
6548
www.chemeurj.org
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2005, 11, 6543 – 6551

Self-Assembled Peptide Tubelets
FULL PAPER
2H), 2.86 and 2.78 (s, 3H), 2.47 (m, 1H), 1.42 ppm (s, 9H); 13C NMR
(62.83 MHz, CDCl3, 258C): d=174.5 and 174.1 (C=O), 170.2 and 170.0
(C=O), 155.0 and 154.9 (C=O), 143.5 (C), 141.1 (C), 137.6 and 137.3 (C),
128.1–119.8 (CH), 79.5 and 79.5 (C), 73.1 and 73.0 (CH2), 71.1 and 70.0
(CH2), 65.9 and 65.8 (CH2), 54.7 (CH), 51.6 and 51.1 (CH), 50.1 and 49.8
(CH), 49.8 and 46.7 (CH), 42.2 and 42.1 (CH), 32.2 and 31.2 (CH2), 29.5
and 28.5 (CH2), 29.4 (CH3), 28.2 and 27.6 (CH3), 27.6 (CH2), 24.1 ppm
(CH2); FAB+-MS: m/z (%): 613 (48) [MH+], 513 (100) [MHÀBoc+];
HRMS: calcd: 613.32776 [MH+]; found: 613.32670.
Boc-[(l-g-Ach-d-MeN-Ala)2]-OFm: A solution of Boc-[l-g-Ach-d-MeN-
Ala]-OFm (1.012 g, 2.0 mmol) in TFA/CH2Cl2 (1:1) was stirred at room
temperature for 10 min. After removal of the solvent, the residue was
dried under high vacuum for 2 h to give TFA·H-[l-g-Ach-d-MeN-Ala]-
OFm, which was used without further purification.
temperature for 10 min. After removal of the solvent, the residue was
dried under high vacuum for 2 h and used without further purification.
The linear peptide was dissolved in CH2Cl2 (25 mL) and treated with
TBTU (53 mg, 0.165 mmol). This was followed by dropwise addition of
DIEA (105 mL, 0.6 mmol). After 12 h, the solvent was removed under
reduced pressure, and the crude residue was purified by reversed-phase
HPLC to afford 2b (125 mg, 50%) as a white solid; 1H NMR (400 MHz,
DMSO, 258C): d=7.54 (d, J(H,H)=8.2 Hz, 1H), 4.86 (d, J(H,H)=
7.2 Hz, 1H), 3.63 (m, 1H), 2.90 (s, 3H), 2.69 (m, 1H), 1.17 ppm (d,
J(H,H)=7.2 Hz, 3H); 13C NMR (100.53 MHz, DMSO, 258C): d=183.9
(C=O), 179.6 (C=O), 60.9 (CH), 56.4 (CH), 49.5–48.5 (CH), 44.0 (CH2),
41.8 (CH2), 40.0 (CH3), 37.5 (CH2), 33.4 (CH2), 24.2 ppm (CH3); FTIR
(CHCl3): n˜ =3315 (amide A), 2934, 2859, 1672, 1627 (amide I), 1540 cmÀ1
(amide IIII); FAB+-MS: m/z: 841 [MH+]; HRMS: calcd: 841.55514
[MH+]; found: 841.55643.
A
solution of dipeptide Boc-[l-g-Ach-d-MeN-Ala]-OFm (1.012 g,
cyclo[(d-Phe-l-MeN-g-Ach)4] (3b): This compound was prepared in the
same way as 2b from Boc-[(d-Phe-l-MeN-g-Ach)4]-OFm (201 mg,
0.140 mmol) to give, after HPLC purification, 3b (112 mg, 70%);
1H NMR (250 MHz, CDCl3, 258C): d=8.57 (d, J(H,H)=8.6 Hz, 1H),
7.17 (m, 5H), 5.30 (dd, J(H,H)=8.0 and 13.6 Hz, 1H), 4.45 (m, 1H), 3.0–
2.7 (m, 3H), 2.64 ppm (s, 3H); 13C NMR (75.40 MHz, CDCl3, 258C): d=
175.1 (C=O), 171.3 (C=O), 136.5 (C), 129.6 (CH), 128.2 (CH), 126.8
(CH), 51.4 (CH), 49.9 (CH), 43.3 (CH), 40.4 (CH2), 30.8 (CH2), 30.1
(CH2), 29.5 (CH3), 28.5 (CH2), 24.7 ppm (CH2); FTIR (CHCl3): n˜ =3312
(amide A), 2935, 2864, 1660, 1623 (amide I), 1525 cmÀ1 (amide IIII);
FAB +-MS: m/z: 1145 [MH+]; HRMS: calcd: 1145.68034 [MH+]; found:
1145.68176.
cyclo[(l-Phe-d-MeN-g-Ach-l-Ser(Bn)-d-MeN-g-Ach)2] (6a): This com-
pound was prepared in the same way as 2b from Boc-[(l-Phe-d-MeN-g-
Ach-l-Ser(Bn)-d-MeN-g-Ach)2]-OFm (188 mg, 0.125 mmol) to give, after
HPLC purification, 6a (112 mg, 75%); 1H NMR (750 MHz, CDCl3,
258C): d=8.67 (d, J(H,H)=8.0 Hz, 1H), 8.59 (d, J(H,H)=9.2 Hz, 1H),
7.22–7.16 (m, 10H), 5.39 (dd, J(H,H)=6.9 and 12.0 Hz, 1H), 5.25 (dd,
J(H,H)=9.1 and 15.6 Hz, 1H), 4.50 (m, J(H,H)=15.3 Hz, 2H), 4.39 (s,
2H), 3.64 (m, 1H), 3.54 (m, 1H), 3.18 (m, 1H), 3.08 (m, 1H), 2.98 (s,
3H), 2.88 (m, 1H), 2.80 (s, 3H), 2.46 ppm (m, 1H); 13C NMR
(62.83 MHz, CDCl3, 258C): d=175.9 (C=O), 174.5 (C=O), 171.5 (C=O),
169.9 (C=O), 137.6 (C), 137.2 (C), 127–126 (CH), 73.2 (CH2), 71.6 (CH2),
51.9 (CH), 51.0 (CH), 49.8 (CH), 48.5 (CH), 44.2 (CH), 42.4 (CH), 38.9
(CH2), 31.8 (CH2), 31.0 (CH2), 30.3 (CH2), 29.9 (CH3), 29.6 (CH3), 28.7
(CH2), 28.4 (CH2), 24.6 ppm (CH2); FTIR (CHCl3): n˜ =3311 (amide A),
2934, 2861, 1660, 1625 (amide I), 1523 cmÀ1 (amide IIII); FAB+-MS: m/z:
1206 [MH+]; HRMS: calcd: 1205.70147 [MH+]; found: 1205.70536.
cyclo[(l-Phe-d-MeN-g-Ach-l-Ser-d-MeN-g-Ach)2] (6b): A solution of pep-
tide 6a (49 mg, 0.041 mmol) in ethanol (1.5 mL) was treated with 10%
Pd/C (10–30 mg) and stirred at room temperature under a hydrogen at-
mosphere overnight. The resulting mixture was filtered through a celite
pad and washed with ethanol to afford, after concentration under re-
duced pressure, 6b (41 mg, 100%); 1H NMR (750 MHz, CDCl3, 258C):
d=8.96 (brs, 1H), 8.49 (d, J(H,H)=9.8 Hz, 1H), 7.17 (m, 5H), 5.27 (brs,
1H), 5.19 (brs, 1H), 4.52 and 4.47 (m, 2H), 3.79 and 3.70 (m, 2H), 3.28
(m, 1H), 3.05 (m, 4H), 3.94 (m, 1H), 2.80 (s, 3H), 2.42 ppm (m, 1H);
13C NMR (75.40 MHz, CDCl3, 258C): d=177.9 (C=O), 174.2 (C=O),
171.8 and 171.6 (C=O), 168.6 and 168.6 (C=O), 136.8 and 136.8 (C),
129.8–126.7 (CH), 67.0 (CH2), 53.3 (CH), 52.2 (CH), 51.2 (CH), 50.0
(CH), 44.4 (CH), 42.5 (CH), 39.3 (CH2), 32.2 and 32.1 (CH2), 30.8 (CH2),
29.9 and 29.8 (CH3), 29.1 and 28.7 (CH2), 24.7 ppm (CH2); FTIR
(CHCl3): n˜ =3317 (amide A), 2935, 2863, 1660, 1620 (amide I), 1524 cmÀ1
(amide IIII); FAB+-MS: m/z: 1026 [MH+]; HRMS: calcd: 1025.60757
[MH+]; found: 1025.60908.
1H NMR Assignments of cyclic peptides: 1H NMR spectra (CDCl3) of
peptides were assigned from the corresponding double-quantum-filled
2D COSY (2QF-COSY) and/or NOESY and ROESY spectra acquired
at the concentration and temperature indicated (see Supporting Informa-
tion). Mixing times (ꢀ250 ms or 400 ms) were not optimized. Due to
conformation averaging on the NMR timescale, peptides with Cn se-
quence symmetry (n=2 or 4) generally display Cn symmetrical 1H NMR
2.0 mmol) in 20% piperidine in CH2Cl2 (5 mL) was stirred at room tem-
perature for 30 min, and then the solvent was removed. The residue was
dissolved in CH2Cl2, and the solution was washed with 10% citric acid,
dried over Na2SO4, filtered, and concentrated to give Boc-[l-g-Ach-d-
MeN-Ala]-OH, which was used without further purification.
The previously prepared dipeptides Boc-[l-g-Ach-d-MeN-Ala]-OH and
TFA·H-[l-g-Ach-d-MeN-Ala]-OFm were dissolved in dry CH2Cl2
(25 mL), and HBTU (758 mg, 2.00 mmol), and DIEA (1.354 mL,
0.80 mmol) were successively added. After being stirred 30 min at room
temperature, the solution was washed with 10% citric acid and saturated
NaHCO3, dried over Na2SO4, and concentrated under reduced pressure.
The resulting material was purified by flash chromatography (0–5%
MeOH in CH2Cl2) to give Boc-[(l-g-Ach-d-MeN-Ala)2]-OFm (1.156 g,
81%) as a white foam; FAB+-MS: m/z (%): 717 (42) [MH+], 617 (100)
[MHÀBoc+]; HRMS: calcd: 717.42273 [MH+]; found: 717.42222.
Boc-[(d-Phe-l-MeN-g-Ach)2]-OFm: This compound was prepared in the
same way as tetrapeptide Boc-[l-g-Ach-d-MeN-Ala]2-OFm from dipeptide
Boc-[d-Phe-l-MeN-g-Ach]-OFm (280 mg, 0.481 mmol) to afford Boc-[(d-
Phe-l-MeN-g-Ach)2]-OFm (380 mg, 91%); FAB+-MS: m/z (%): 869 (10)
[MH+], 769 (100) [MHÀBoc+]; HRMS: calcd: 869.48532 [MH+]; found:
869.48466.
Boc-[l-Phe-d-MeN-g-Ach-l-Ser(Bn)-d-MeN-g-Ach]-OFm: This compound
was prepared in the same way as tetrapeptide Boc-[(l-g-Ach-d-MeN-
Ala)2]-OFm from dipeptides Boc-[l-Phe-d-MeN-g-Ach]-OFm (387 mg,
0.663 mmol)
and
Boc-[l-Ser(Bn)-d-MeN-g-Ach]-OFm
(392 mg,
0.663 mmol) to afford Boc-[l-Phe-d-MeN-g-Ach-l-Ser(Bn)-d-MeN-g-Ach]-
OFm (477 mg, 80%); FAB+-MS: m/z (%): 899 (14) [MH+], 799 (100)
[MHÀBoc+]; HRMS: calcd: 899.49589 [MH+]; found: 899.49656.
Boc-[(l-g-Ach-d-MeN-Ala)4]-OFm: This compound was prepared in the
same way as tetrapeptide Boc-[(l-g-Ach-d-MeN-Ala)2]-OFm from tetra-
peptide Boc-[(l-g-Ach-d-MeN-Ala)2]-OFm (303 mg, 0.492 mmol) to
afford Boc-[(l-g-Ach-d-MeN-Ala)4]-OFm (512 mg, 91%); FAB+-MS: m/z
(%): 1138 (80) [MH+], 1038 (100) [MHÀBoc+]; HRMS: calcd:
1137.70002 [MH+]; found: 1137.69933.
Boc-[(d-Phe-l-MeN-g-Ach)4]-OFm: This compound was prepared in the
same way as Boc-[(l-g-Ach-d-MeN-Ala)4]-OFm by using tetrapeptide
Boc-[(d-Phe-l-MeN-g-Ach)2]-OFm (174 mg, 0.20 mmol) to afford Boc-
[(d-Phe-l-MeN-g-Ach)4]-OFm (201 mg, 73%); MALDI-TOF MS: m/z
calcd for C87H108N8O11Na [MNa+]: 1463.8; found: 1462.4.
Boc-[(l-Phe-d-MeN-g-Ach-l-Ser(Bn)-d-MeN-g-Ach)2]-OFm: This com-
pound was prepared in the same way as Boc-[(l-g-Ach-d-MeN-Ala)4]-
OFm from tetrapeptide Boc-[l-Phe-d-MeN-g-Ach-l-Ser(Bn)-d-MeN-g-
Ach]-OFm (477 mg, 0.531 mmol) to afford Boc-[(l-Phe-d-MeN-g-Ach-l-
Ser(Bn)-d-MeN-g-Ach)2]-OFm (541 mg, 68%); FAB+-MS: m/z (%): 1502
(10) [MH+]; 1402 (100) [MHÀBoc+]; HRMS: calcd: 1402.79364
[MHÀBoc+]; found: 1402.79118.
cyclo[(l-g-Ach-d-MeN-Ala)4] (2b): A solution of Boc-[(l-g-Ach-d-MeN-
Ala)4]-OFm (170 mg, 0.15 mmol) in 20% piperidine in CH2Cl2 (2 mL)
was stirred at room temperature for 30 min. After removal of the solvent,
the residue was dissolved in CH2Cl2, and the mixture was washed with
10% citric acid, dried over Na2SO4, filtered, and concentrated. The re-
sulting material was dissolved in TFA/CH2Cl2 (1:1) and stirred at room
Chem. Eur. J. 2005, 11, 6543 – 6551
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
6549

J. R. Granja et al.
spectra for monomeric species and Dn symmetrical spectra for dimeric
species.
[4] a) N. Khazanovich, J. R. Granja, D. E. McRee, R. A. Milligan, M. R.
Ghadiri, J. Am. Chem. Soc. 1994, 116, 6011–6012; b) J. D. Hartger-
ink, J. R. Granja, R. A. Milligan, M. R. Ghadiri, J. Am. Chem. Soc.
1996, 118, 43–50; c) K. Rosenthal-Aiman, G. Svensson, A. UndØn,
J. Am. Chem. Soc. 2004, 126, 3372–3373.
[5] a) M. R. Ghadiri, J. R. Granja, R. A. Milligan, D. E. McRee, N. Kha-
zanovich, Nature 1993, 366, 324–327; b) T. D. Clark, J. M. Buriak, K.
Kobayashi, M. P. Isler, D. E. McRee, M. R. Ghadiri, J. Am. Chem.
Soc. 1998, 120, 8949–8962.
[6] a) D. Seebach, J. L. Matthews, A. Meden, T. Wessels, C. Baerlocher,
L. B. McCusker, Helv. Chim. Acta 1997, 80, 173–182; b) T. D. Clark,
L. K. Buehler, M. R. Ghadiri, J. Am. Chem. Soc. 1998, 120, 651–
656.
Measurement of solution association constants by variable-concentration
1H NMR spectroscopy: The HPLC-purified peptide was dissolved in
CDCl3 and the association constant, Ka, was estimated with a nonlinear
regression fitting program by using the concentration-dependent shifts of
the NH amide group between 28.6”10À3–1.7”10À3. The set of observed
shifts (dobs) for each titration experiment was adjusted to the equation
[P]t =Iobs/Ka[2Isat(1ÀIobs/Isat)2],[18] where P corresponds to the set of con-
centrations and Ka is the calculated value.
Vanꢀt Hoff analysis of dimerization: The HPLC-purified cyclic peptide
2b was dissolved in CDCl3 at concentrations of 28.6, 14.3, 7.1, 7.7, 3.6,
1
and 1.78 mm. H NMR (Vanꢁt Hoff) spectra of the resulting samples were
[7] I. L. Karle, B. K. Handa, C. H. Hassall, Acta Crystallogr. Sect. B
1975, 31, 555–560.
acquired at intervals of 10 K in the temperature range 233–313 K. Single-
point determinations of the Ka value were estimated at each temperature
by using nonlinear regression to fit the equation shown above[15] to
d(NH)C data obtained from 1H NMR titrations at total monomer con-
centrations C. A plot was then made of 1/T (in K) versus ln Ka, from
which the following thermodynamic parameters were established for the
[8] a) D. Gauthier, P. Baillargeon, M. Drouin, Y. L. Dory, Angew.
Chem. 2001, 113, 4771–4774; Angew. Chem. Int. Ed. 2001, 40, 4635–
4638; b) S. Leclair, P. Baillargeon, R. Skouta, D. Gauthier, Y. Zhao,
Y. L. Dory, Angew. Chem. 2004, 116, 353–357; Angew. Chem. Int.
Ed. 2004, 43, 349–353.
dimerization of 2b: DHo298 =À38.2 kJmolÀ1 and DSo298 =À94.2 JKÀ1 molÀ1
.
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CCDC-265524 (2b) and CCDC-265525 (3b) contain the supplementary
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charge from the Cambridge Crystallographic Data Centre via
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Acknowledgements
This workwas supported by the Ministerio de Ciencia y Tecnología
(Grant nos.: SAF2001-3120 and SAF2004-01044) and the Xunta de Gali-
cia (Grant nos.: PGIDT02BTF20901PR and PGIDT04BTF209006PR).
M.A. also thanks the Ministerio de Ciencia y Tecnología for an FPU
grant.
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Received: April 28, 2005
Published online: August 17, 2005
Chem. Eur. J. 2005, 11, 6543 – 6551
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
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