Synthesis and conformational analysis of 18-membered Aib-containing cyclohexapeptides

Article abstract of DOI:10.1016/j.tet.2004.12.012

The synthesis and conformational analysis of two Aib-containing cyclic hexapeptides, cyclo(Gly-Aib-Leu-Aib-Phe-Aib) 1 and cyclo(Leu-Aib-Phe-Gly-Aib- Aib) 2, is described. The linear precursors of 1 and 2 were prepared using solution phase techniques, and

Full text of DOI:10.1016/j.tet.2004.12.012

Tetrahedron 61 (2005) 1871–1883
Synthesis and conformational analysis of 18-membered
Aib-containing cyclohexapeptides
*
Tatjana Jeremic, Anthony Linden, Kerstin Moehle and Heinz Heimgartner
¨
¨
Institute of Organic Chemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland
Received 11 October 2004; revised 29 November 2004; accepted 2 December 2004
Available online 13 January 2005
Abstract—The synthesis and conformational analysis of two Aib-containing cyclic hexapeptides, cyclo(Gly-Aib-Leu-Aib-Phe-Aib) 1 and
cyclo(Leu-Aib-Phe-Gly-Aib-Aib) 2, is described. The linear precursors of 1 and 2 were prepared using solution phase techniques, and the
cyclization efficiency of three different coupling reagents (HATU, PyAOP, DEPC) was examined. The success of the cyclization was found
to be reagent dependent. Solid-state conformational analysis of 1 and 2 was performed by X-ray crystallography and has revealed some
unusual features as all three Aib residues of 1 assume nonhelical conformations. Furthermore, the residue Aib4 adopts an extended
conformation (fZK175.9(3)8, jZC178.6(2)8), which is, to the best of our knowledge, the first observation of an Aib residue adopting an
extended conformation in a cyclopeptide. The structure of 1 is also a rare example in which an Aib residue occupies the (iC1) position of a
type II0 b-turn, stabilized by a bifurcated hydrogen bond. The cyclic peptide 2 adopts a more regular conformation in the solid state,
consisting of two fused b-turns of type I/I0, stabilized by a pair of intramolecular hydrogen bonds. In addition, the conformational study of the
cyclic peptide 1 in DMSO-d6 by NMR spectroscopy and molecular dynamics simulations revealed a structure, which is very similar to its
structure in the crystalline state.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Our previous successful synthesis of cyclic hexapeptides
containing several Aib (a-aminoisobutyric acid) residues
and two Gly residues in positions 1 and 4 of the peptide
backbone7,8 prompted us to investigate the cyclization of
hexapeptides containing only one Gly residue as the turn-
inducing element. Here, we describe the synthesis of two
cyclic hexapeptides cyclo(Gly-Aib-Leu-Aib-Phe-Aib) (1)
and cyclo(Leu-Aib-Phe-Gly-Aib-Aib) (2), composed of
three protein amino acids, i.e. Gly, Leu, Phe and three
a-aminoisobutyric acids. The crystal structures of both
cyclic peptides were examined by X-ray diffraction in order
to study the influence of the Aib residues on the
conformation of the backbone of the cyclic hexapeptides.
A NMR-based structure determination of 1 in solution was
also performed in the present study.
Cyclic peptides continue to be challenging targets for
chemical synthesis.1 As the synthesis of linear peptides
generally proceeds well, the key step for the chemical
synthesis of cyclic peptides is usually the cyclization
reaction. In particular, the cyclization of small peptides of
less than seven amino acid residues is often difficult.2
Incorporation of turn-inducing elements such as Gly, Pro,
D-amino acids and N-alkylated amino acids into the peptide
backbone is known to improve cyclization yields.3
Although conformational constraints are usually introduced
into peptides through cyclization, cyclic peptides can still
possess a remarkable flexibility.4,5 Thus, the incorporation
of sterically hindered C(2)-tetrasubstituted a-amino acids
into the peptide backbone leads to more rigid compounds. In
addition, cyclic penta- and hexapeptides are often chosen
for the synthesis of model cyclopeptides, since larger cyclic
peptides already exhibit greater flexibility.6 Conformation-
constrained cyclic peptides may have enhanced metabolic
stability, receptor selectivity, and bioavailability, all of
which may lead to useful medicinal properties.
Keywords: Cyclic peptides; Peptide synthesis; a-Aminoisobutyric acid;
Peptide conformation.
* Corresponding author. Tel.: C41 1 6354282; fax: C41 1 6356812;
e-mail: heimgart@oci.unizh.ch
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.12.012

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T. Jeremic et al. / Tetrahedron 61 (2005) 1871–1883
Scheme 1.
Scheme 2.

T. Jeremic et al. / Tetrahedron 61 (2005) 1871–1883
Table 1. Conditions used for the cyclization of Aib-containing hexapeptides 5 and 8
1873
Cyclic peptide
Cyclization method
Reaction time
Yield (%)
Cyclo(Gly-Aib-Leu-Aib-Phe-Aib) 1
HATU/HOAt (3 equiv)
PyAOP/HOAt (5 equiv)
DEPC (10 equiv)
HATU/HOAt (3 equiv)
DEPC (10 equiv)
PyAOP/HOAt (3 equiv)
1 day
24
31
53
16
33
48
3 days
6 days
3 days
6 days
3 days
Cyclo(Leu-Aib-Phe-Gly-Aib-Aib) 2
2. Results and discussion
times of up to six days were used. This time, the cyclic
hexapeptide 1 was obtained in good yield, while cyclo-
peptide 2 was isolated in moderate yield. As is evident from
these results, the success of the cyclization is dependent
upon the choice of the cyclization reagent. However, 1 and 2
were obtained in remarkably similar overall moderate
(30%) to good yields (50%). Comparing the best cyclization
protocols for 1 and 2 it was surprising that the lactamization
between the less hindered pair H2N-Gly and Aib-CO
proceeded only slightly better than that between NH2 of
the sterically demanding Leu residue and Aib-CO. It
appears that the conformation or (and) sequence of the
linear precursor played a more important role than the size
of the residue at the N-terminus.
2.1. Preparation and cyclization of linear hexapeptides
The linear hexapeptides Z-Gly-Aib-Leu-Aib-Phe-Aib-OtBu
(5) and Z-Leu-Aib-Phe-Gly-Aib-Aib-N(Me)Ph (8) were
synthesized by solution-phase methods as shown in
Schemes 1 and 2. A [2C2C2]-fragment condensation
was chosen in the case of 5. At first, the dipeptide Z-Phe-
Aib-OtBu (3) was prepared by coupling Z-Phe-OH with
HCl$H-Aib-OtBu using PyAOP as the coupling reagent.
Then, 3 was N-deprotected to give H-Phe-Aib-OtBu by
means of catalytic hydrogenation. The PyAOP-mediated
coupling of the latter with Z-Leu-Aib-OH9 afforded
tetrapeptide Z-Leu-Aib-Phe-Aib-OtBu (4) in high yield.
Removal of the Z protecting group of 4, and coupling of the
resulting H-Leu-Aib-Phe-Aib-OtBu with Z-Gly-Aib-OH in
the presence of PyAOP afforded the linear hexapeptide 5.
The cyclic structures of 1 and 2 were established by standard
two-dimensional NMR techniques. The assignment of all H-
and C-signals was possible by using 2D HSQC and HMBC
spectra. A combination of these two types of spectra
allowed the complete assignment of the amide NH, CO and
C(a) signals of all residues, as well as enabling the signals
of different Aib residues to be distinguished. In addition,
selected ROESY correlations observed in DMSO-d6
solution are shown in Figure 1.
For the synthesis of the linear hexapeptide 8, a convergent
[3C3] strategy was employed. Thus, the tripeptide Z-Leu-
Aib-Phe-OtBu (6) was prepared by coupling Z-Leu-Aib-OH
with the hydrochloride of H-Phe-OtBu using PyBOP as the
coupling reagent. Treatment of 6 with TFA in CH2Cl2,
followed by the reaction with H-Gly-Aib-Aib-N(Me)Ph,
which was obtained by deprotection of Z-Gly-Aib-Aib-
N(Me)Ph (7),7 led, in the presence of PyAOP, to the
hexapeptide 8 in moderate yield.
The linear hexapeptides Z-Gly-Aib-Leu-Aib-Phe-Aib-OtBu
(5) and Z-Leu-Aib-Phe-Gly-Aib-Aib-N(Me)Ph (8) were
then deprotected at the N- and C-terminus and treated with
the coupling reagents HATU, PyAOP and DEPC to
investigate the cyclization tendency of each peptide. All
of the cyclization reactions were performed in diluted DMF
solutions (10K4–10K3 M) using a large excess of coupling
reagent and base (DIEA). The yields of the cyclization
reactions are shown in Table 1. In the first attempts we used
HATU as the activating agent, since it has proven to be
versatile and highly efficient.2 However, the cyclohexa-
peptides 1 and 2 were obtained only in relatively low yields.
One explanation for the less efficient macrolactamization
than expected could be that HATU participated in a side
reaction at the amino terminus to give a guanidino
derivative. This side reaction is known to occur when an
excess of the aminium salt based coupling reagents is
used.10 To avoid this problem, phosphonium reagents such
as PyBOP and PyAOP are recommended.11 Thus, PyAOP
together with HOAt was employed in the cyclization step,
leading to 1 in moderate yield while 2 was obtained in good
yield. Next, the macrolactamization ability of the organo-
phosphorus reagent DEPC was tested. Because of the slower
reaction rate under the DEPC/DIEA conditions, reaction
Figure 1. Selected ROESY correlations for compounds 1 and 2 in DMSO-
d6.
2.2. Solid state conformation
Cyclic peptides are frequently found among natural
products and exhibit a wide range of biological activities.12
Therefore, their conformations have been studied exten-
sively both in the solid state and in solution, since the
chemical properties and biological activities of such
structures are known to be closely related to their molecular
conformation.13–15 The crucial determinants of the confor-
mation of cyclic peptides are the turns (b, g) and
intramolecular hydrogen bonds. Cyclic hexapeptides have
been used as model peptides for b-turns since these
peptides, due to geometric factors, generally adopt a
conformation with two b-turns, stabilized by a pair of two
intramolecular hydrogen bonds between residues i and
iC3.16 Turns do not necessarily contain hydrogen bonds,

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T. Jeremic et al. / Tetrahedron 61 (2005) 1871–1883
but the lack of hydrogen bonds generally results in distorted
or unstable structures.
f(Aib2)ZK52.9(7)8, j(Aib2)ZK31.2(7)8, f(Aib4)Z
C50.0(7)8, j(Aib4)ZC44.9(7)8), those of Aib3 of
cyclo[Gly-(RS)-Phe(2Me)-Aib-Aib-Gly] do not correspond
with the helical conformational space (fZK159.7(2)8,
jZC166.3(2)8). In the asymmetric unit of the crystal
structure of the third cyclopentapeptide, i.e. cyclo[Gly-(R)-
Phe(2Me)-Pro-Aib-Phe],20 there are two independent
molecules with quite different conformations. One of the
molecules forms a b-turn with a Aib4/Gly1 hydrogen
bond, whereas the other molecule is characterized by a
g-turn (NH(Aib4)/CO(Pro3) hydrogen bond) and an
a-turn (NH(Phe5)–CO(Gly1) hydrogen bond). In the
molecule containing a b-turn, Aib4 shows f, j values
that belong to the nonhelical conformational space (fZ
C154.8(4)8, jZK51.0(4)8).
By extensive crystallographic studies, a-amino isobutyric
acid (Aib) has been shown to favor left- or right-handed
310/a-helical conformations in a wide variety of acyclic
peptides of differing lengths and sequences.17–20 Indeed,
Aib residues with very few exceptions almost invariably
adopt conformations with f and j values near G(60G20)8
and G(30G20)8, respectively. In addition, theoretical
calculations show the presence of minima in
a
semiextended region of the f, j space (fZG(60G20)8,
jZG(120G20)8).17
There are relatively few reports concerning crystallographic
studies of Aib residues incorporated into cyclic peptides,21–25
so the structural information about the conformational
preferences of Aib residues in cyclic molecules is rather
scarce. The cyclic tetrapeptide dehydrochlamydocin21 and
the cyclic pentapeptide cyclo(Phe-Phe-Aib-Leu-Pro)22 both
have the Aib residue at the center of a g-turn with
unexpected values of the torsion angles f, j that lie in the
nonhelical conformational space. The torsion angles (f, j)
of the Aib residues in the crystal structures of cyclo(Gly-
Aib-Gly)2,23 [Aib5,6-D-Ala8]cyclolinopeptide A,24 and
As a part of our investigation of the synthesis of cyclic
hexapeptides containing Aib residues,7,26 we have been
interested in the conformations of these cyclic molecules in
order to estimate if the incorporation of several Aib residues
into cyclic hexapeptide structures stabilizes certain types of
turns. Previously, we have investigated the cyclization and
conformation of hexapeptides containing two or three Aib
residues, and two Gly residues in positions 1 and 4 of the
peptide backbone.7 The crystal structures of two cyclic
peptides, cyclo(Gly-Aib-Aib-Gly-Aib-Phe) and cyclo(Gly-
(S)-Phe(2Me)-Aib-Gly-Aib-Phe), showed that these
molecules have two fused b-turns. The observed b-turns
were stabilized by intramolecular hydrogen bonds between
the NH of Gly1 and the C]O of Gly4 and between the NH
of Gly4 and C]O of Gly1. Different types of b-turn
conformations, i.e. I, I0 and III0, have been observed
depending on the sequence, with Aib residues occupying
positions (iC1) and/or (iC2) of the turns.
25
cyclo(Pro-Phe-Phe-Aib-Leu)2 all lie well inside the 310/
a-helical region of the conformational space, as is observed
in linear peptides.
In our research group, we have synthesized several Aib-
containing cyclopentapeptides,19,20 and the crystal-
structures of three of them have been established by X-ray
crystallography. The structures of cyclo[Gly-(RS)-
Phe(2Me)-Aib-Aib-Gly]19 and cyclo[Gly-Aib-(RS)-
Phe(2Me)-Aib-Gly]20 are very similar and have a b-turn,
which is stabilized by a hydrogen bond between NH of Gly1
and CO of Phe(2Me)3 and CO of Aib3, respectively.
Whereas in the structure of cyclo[Gly-Aib-(RS)-Phe(2Me)-
Aib-Gly] the torsion angles of both Aib residues
show values typical for the helical region (cyclo[Gly-
The solid-state conformations of the new cyclohexapeptides
cyclo(Gly-Aib-Leu-Aib-Phe-Aib) (1) and cyclo(Leu-Aib-
Phe-Gly-Aib-Aib) (2) were examined by X-ray crystal-
lography. Crystals of 1 suitable for the X-ray analysis were
obtained from a mixture of MeOH/i-PrOH/CHCl3 and
acetone, and those of 2 were grown from MeOH/EtOH/
i-PrOH and water. The ORTEP plots27 of the
molecules with the atom numbering schemes are presented
Aib-(R)-Phe(2Me)-Aib-Gly]:
j(Aib2)ZC42.9(6)8, f(Aib4)ZK47.2(7)8, j(Aib4)Z
K46.4(6)8; cyclo[Gly-Aib-(S)-Phe(2Me)-Aib-Gly]:
f(Aib2)ZC50.4(7)8,
Figure 2. ORTEP plot27 of the molecular structure of 1 (50% Probability ellipsoids, arbitrary numbering of atoms, only one of the disordered arrangements of
the Leu side chain is shown).

T. Jeremic et al. / Tetrahedron 61 (2005) 1871–1883
1875
Figure 3. ORTEP plot27 of the molecular structure of 2 (50% Probability ellipsoids, arbitrary numbering of atoms, solvent molecules omitted for clarity).
Table 2. Backbone torsion angles [8] for the crystal structures of 1 and 2
1
Gly1
Aib2
Leu3
Aib4
Phe5
Aib6
f
j
u
K170.0(3)
C119.6(3)
C178.2(2)
C55.2(3)
K130.4(3)
K164.7(2)
K90.7(3)
C42.6(3)
C166.1(2)
K175.9(3)
C178.6(2)
K171.8(2)
K45.4(4)
C127.2(3)
C169.1(2)
C76.9(4)
K7.7(4)
K178.1(3)
2
Leu1
Aib2
Phe3
Gly4
Aib5
Aib6
f
j
u
K113.0(3)
K171.7(2)
K165.6(2)
K57.1(3)
K34.4(3)
K171.4(2)
K113.3(3)
C22.8(3)
K178.3(2)
C117.8(3)
C169.4(2)
C170.3(2)
C56.4(3)
C41.2(3)
C172.5(2)
C93.8(3)
K15.3(3)
K173.8(2)
in Figures 2 and 3. The isopropyl part of the Leu side chain
of 1 is disordered over two almost equally occupied
conformations, while the asymmetric unit of 2 contains
one molecule of the cyclic peptide plus two water
molecules, one disordered EtOH molecule and one
disordered i-PrOH molecule. Two approximately equally
occupied positions were modelled for each of the disordered
solvent molecules. The backbone torsion angles of the
cyclopeptides are summarized in Table 2 and the hydrogen
bonding parameters in Tables 3 and 4.
helical region is less pronounced at Aib6. The residue Aib2
adopts a rare semiextended conformation with torsion
angles f, j almost identical to those previously reported
for a cyclic hexapeptide having a disulfide linkage.28
Interestingly, very similar f, j values were also observed
for one Aib residue in the linear tetrapeptide Boc-Leu-Aib-
Phe-Aib-OMe, which was reported to form a continuous
hydrogen-bonded supramolecular helix.29 The residue Aib4
adopts a fully extended conformation, so far known to be
characteristic of the higher homologs of Aib, like a,a-
diethylglycine (Deg),30–32 and a,a-dipropylglycine
(Dpg).33,34 The torsion angles f and j of the Phe and
Aib6 residues in the Aib4-Phe5-Aib6-Gly1 sequence show
values close to those for a type II b-turn with an
intramolecular 1)4 hydrogen bond (N(1)–H.O(12))
Surprisingly, all three Aib residues of cyclo(Gly1-Aib2-
Leu3-Aib4-Phe5-Aib6) (1) assume conformations in the
nonhelical region of the Ramachandran diagram (Table 2).
The deviation of the backbone conformation from the
Table 3. Hydrogen bonding parameters for cyclo(Gly-Aib-Leu-Aib-Phe-Aib) (1)
˚
Distance [A] D-H
˚
˚
Donor D-H
Acceptor A
Distance [A] H.A
Distance [A] D.A
Angle [8] D-H.A
N(1)–H(1)
N(4)–H(4)
N(7)–H(7)
N(10)–H(10)
N(10)–H(10)
N(13)–H(13)
N(16)–H(16)
O(12)
O(3i)
0.81(3)
0.84(3)
0.94(3)
0.83(3)
0.83(3)
0.87(3)
0.94(3)
2.34(3)
2.30(3)
2.00(3)
2.17(3)
2.38(3)
2.11(3)
1.95(4)
3.126(4)
3.131(4)
2.877(3)
2.604(3)
3.083(3)
2.972(3)
2.867(3)
163(3)
172(3)
154(3)
113(3)
143(3)
171(3)
163(3)
O(18ii)
O(12)
O(6)
O(6iii)
O(9iv)
Primed atoms refer to the molecule in the following symmetry-related positions: i2Kx, K1/2Cy, 1/2Kz; ii2Kx, 1/2Cy, 1/2Kz; iii1Kx, 1/2Cy, 1/2Kz; iv1K
x, K1/2Cy, 1/2Kz.

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T. Jeremic et al. / Tetrahedron 61 (2005) 1871–1883
Table 4. Hydrogen bonding parameters for cyclo(Leu-Aib-Phe-Gly-Aib-Aib) (2)
˚
Distance [A] D-H
˚
˚
Donor D-H
Acceptor A
Distance [A] H.A
Distance [A] D.A
Angle [8] D-H.A
N(1)–H(1)
N(4)–H(4)
N(7)–H(7)
N(7)–H(7)
O(12)
O(44i)
O(38a)
O(38b)
O(3)
O(43)
O(42a)
O(42b)
O(12)
O(12)
O(3)
0.87(4)
0.89(4)
0.89(4)
0.89(4)
0.93(4)
0.88(4)
0.73(3)
0.73(3)
0.84
0.84
0.84
0.84
0.98(6)
0.79(5)
0.87(6)
0.95(5)
2.24(4)
1.90(4)
2.21(4)
2.18(4)
2.18(4)
1.91(4)
2.48(3)
2.38(3)
1.91
2.26
2.15
1.96
1.83(6)
1.96(5)
1.89(6)
1.80(5)
3.042(3)
2.782(3)
2.92(1)
2.87(1)
3.078(3)
2.787(3)
3.10(1)
2.99(1)
2.75(2)
2.87(2)
2.79(2)
2.74(2)
2.805(3)
2.750(3)
2.754(3)
2.734(3)
154(3)
171(3)
136(3)
135(3)
161(3)
176(3)
145(3)
141(3)
173
129
132
154
174(4)
177(4)
171(5)
168(4)
N(10)–H(10)
N(13)–H(13)
N(16)–H(16)
N(16)–H(16)
O(38a)–H(381)
O(38b)–H(382)
O(42a)–H(421)
O(42b)–H(422)
O(43)–H(431)
O(43)–H(432)
O(44)–H(441)
O(44)–H(442)
O(3)
O(15ii)
O(18iii)
O(6iv)
O(9)
Primed atoms refer to the molecule in the following symmetry-related positions: iK1Cx, y, z; ii1Kx, K1/2Cy, 2Kz; iii1Cx, y, z; iv1Kx, 1/2Cy, 1Kz.
involving the NH of Gly1 and the C]O of Aib4 (Tables 2
and 3). In contrast, the backbone conformation of the
sequence Gly1-Aib2-Leu3-Aib4 cannot be strictly
categorized. It could best be described as a distorted
type II0 b-turn, with values for fiC1 (C55.2(3)8), jiC1
(K130.4(3)8) and fiC2 (K90.7(3)8) being close to the
ideal values (fiC1ZC(60G30)8; jiC1ZK(120G30)8,
fiC2ZK(80G30)8) for this type of turn, and jiC2
(C42.6(3)8) deviating largely from the ideal value
(jiC2Z0G508) (Table 2). The residue Aib2 is obviously
forced to assume the conformation of a D-amino acid as it
prefers the (iC1) position of a b-turn of type II0.16 As a
consequence of the large deviation of jiC2 from ideality, no
1)4 intramolecular hydrogen bond is observed between
the NH of Aib4 and C]O of Gly1. However, the extended
conformation of Aib4 gives rise to an intramolecular
hydrogen bond between the NH and C]O groups within
this residue (N(10)–H.O(12), Table 3), which is unusual
and has only infrequently been inferred from crystal
structure data of some dipeptides.17 Furthermore, the NH
group of Aib4 acts not only as a donor for C]O of Aib4, but
also as a donor for the carbonyl group of Aib2, and is thus
involved in inverse bifurcation.35
conformation of only one of the Aib residues, Aib6, shows
slight deviation from the helical region of the confor-
mational space. The values of torsion angles f and j reveal
the presence of a type I b-turn across Leu1-Aib2-Phe3-Gly4
and a type I0 b-turn spanning the residues Gly4-Aib5-Aib6-
Leu1 (Table 2).
2.3. Solution conformational analysis
The conformation of the cyclic hexapeptide cyclo(Gly-Aib-
Leu-Aib-Phe-Aib) (1) in DMSO-d6 solution has been
determined by 1H NMR spectroscopy. The structure
calculation was performed by restrained molecular
dynamics in torsion angle space by applying the simulated
annealing protocol implemented in the program DYANA.36
The NOE intensities were calibrated with the tools of the
program, and yielded an input of 45 upper-distance limits
(11 intra-residual, 29 sequential and 5 medium/long-range)
(Tables 5–7). The final calculation was started with 100
randomized conformers, and a bundle of 20 DYANA
conformers with the lowest target function was selected for
structure analysis and visualization with the program
MOLMOL.37 The results of DYANA calculations for 1
are shown in Figure 4, and the observed average backbone
torsion angles (f, j) are listed in Table 8.
The cyclic peptide cyclo(Leu1-Aib2-Phe3-Gly4-Aib5-Aib6)
(2) was found to possess a more regular structure with two
fused b-turns stabilized by two intramolecular hydrogen
bonds, one between C]O of Leu1 and NH of Gly4 (N(10)–
H.O(3), Table 4) and the other between C]O of Gly4 and
NH of Leu1 (N(1)–H.O(12)). Furthermore, the
As is evident from Figure 4 and Table 8, 1 is well structured
in solution and with a mean RMSD value of the backbone
˚
atoms of 0.3 A very similar to the backbone conformation
found in the crystal structure, although some torsion angles
Table 5. Intraresidual upper distance restraints derived from integration of ROESY cross-peak volumes for the cyclic peptide cyclo(Gly-Aib-Leu-Aib-Phe-
Aib) (1)
˚
Distance [A]
Residue
Atom
Residue
Atom
Gly1
Leu3
Leu3
Leu3
Leu3
Leu3
Phe5
Phe5
Phe5
Phe5
Phe5
HN
HN
HA
HA
HA
HA
HN
HN
HN
HA
HA
Gly1
Leu3
Leu3
Leu3
Leu3
Leu3
Phe5
Phe5
Phe5
Phe5
Phe5
HA1
HB2
HB2
HB3
QD1
QD2
HA
HB2
HB3
HB2
HB3
2.87
2.77
2.68
2.62
3.64
5.69
2.83
2.99
3.27
2.65
2.71

T. Jeremic et al. / Tetrahedron 61 (2005) 1871–1883
1877
Table 6. Sequential upper distance restraints derived from integration of ROESY cross-peak volumes for the cyclic peptide cyclo(Gly-Aib-Leu-Aib-Phe-Aib)
(1)
˚
Distance [A]
Residue
Atom
Residue
Atom
Gly1
Gly1
Gly1
Gly1
Gly1
Gly1
Aib2
Aib2
Aib2
Aib2
Aib2
Aib2
Aib2
Leu3
Leu3
Leu3
Leu3
Leu3
Aib4
Aib4
Aib4
Aib4
Phe5
Phe5
Phe5
Phe5
Phe5
Phe5
Phe5
HN
HN
HN
HN
Aib2
Aib6
Aib6
Aib6
Aib2
Aib2
Leu3
Leu3
Leu3
Leu3
Leu3
Leu3
Leu3
Aib4
Aib4
Aib4
Aib4
Aib4
Phe5
Phe5
Phe5
Phe5
Aib6
Aib6
Aib6
Aib6
Aib6
Aib6
Aib6
HN
HN
QB1
QB2
HN
HN
HN
HN
HA
QD1
QD2
QD1
QD2
HN
HN
QB1
HN
HN
HA
QD
HN
QD
HN
HN
QB2
HN
HN
QB2
QB2
4.69
3.24
4.74
5.34
2.83
2.71
3.42
4.55
6.38
7.56
7.56
7.57
7.57
3.11
2.62
5.88
3.79
3.83
6.04
8.66
4.10
8.67
4.07
2.40
6.54
4.82
4.29
8.29
8.67
HA1
HA2
HN
QB1
QB1
QB1
QB1
QB2
QB2
HN
HA
HA
HB2
HB3
QB1
QB1
QB2
QB2
HN
HA
HA
HB2
HB3
QD
QE
Table 7. Medium and long range upper distance restraints derived from integration of ROESY cross-peak volumes for the cyclic peptide 1
˚
Distance [A]
Residue
Atom
Residue
Atom
Gly1
Gly1
Gly1
Gly1
Aib4
HN
HN
HN
HN
QB1
Aib4
Aib4
Phe5
Phe5
Aib6
HN
HB1
HN
HA
HN
4.20
5.73
4.14
3.61
6.54
deviate significantly. In particular, the residue Aib4 assumes
almost identical, for an Aib residue unexpected, extended
conformations in both the solid state and in the solution. The
average conformer exhibits two b-turns, one type I-like
b-turn centered at Aib2-Leu3 and one type II-like b-turn
across Aib4-Phe5-Aib6-Gly1. The large 3J(HN, HC(a))
coupling constant of 9 Hz at Leu3 which correlates to a
torsion angle f around K1008 provide further support for
the occurrence of the type I b-turn in solution. An analysis
of the hydrogen-bonding patterns using the final NMR
coordinates shows a significant population of intramolecular
hydrogen bonding between the carbonyl group of Gly1 and
the NH of the Aib4 residue, which is contrary to the
observation found in the crystal structure where a hydrogen
bond is formed between the CO group of Aib4 and the NH
group of Gly1.
good yields. Since cyclo(Gly-Aib-Leu-Aib-Phe-Aib) (1)
and cyclo(Leu-Aib-Phe-Gly-Aib-Aib) (2) have been
obtained in similar overall cyclization yields, being 24–
53% for 1 and 16–48% for 2, the choice of coupling reagent
apparently played a more important role in the cyclization
than the sequence of the linear precursor. In addition, the
coupling reagents PyAOP and DEPC proved to be superior
to HATU. The structures of 1 and 2 were examined in the
solid state by X-ray crystallography in order to gain
information about the conformational preferences of Aib
residues incorporated into cyclic peptides. A detailed
comparison of the crystal structures of 1 and 2 with those
obtained previously7 for cyclo(Gly-Aib-Aib-Gly-Aib-Phe)
and cyclo(Gly-(S)-Phe(2Me)-Aib-Gly-Aib-Phe), reveals
severe conformational restraints imposed on the peptide
backbone of cyclic hexapeptide 1 consisting of alternating
Aib and proteinogenic amino acid residues. Thus, all three
Aib residues of 1 assume torsion angles well outside the
helical region of the conformational space, which is highly
uncommon. It appears that the conformational constraint is
less pronounced in the other three cyclopeptides having two
adjoining Aib residues or one Aib residue adjacent to
another a,a-disubstituted amino acid residue such as
Phe(2Me) (Phe(2Me)Za-methylphenylalanine). Each of
3. Conclusion
In conclusion, we have shown that it is possible to cyclize
hexapeptides containing three constrained Aib residues, two
rather large proteinogenic amino acid residues (Leu, Phe)
and only one Gly residue as a turn-inducing element, in

1878
T. Jeremic et al. / Tetrahedron 61 (2005) 1871–1883
1H (300 MHz) and 13C NMR (75.5 MHz) spectra: Bruker
ARX-300 instrument; 1H (600 MHz) and 13C NMR
(150.9 MHz) spectra of cyclic peptides: Bruker DRX-600
instrument; in (D6)DMSO at 300 K unless otherwise stated;
d in ppm, coupling constants J in Hz. ROESY spectra were
measured with a mixing time of 300 ms. Acquisition
parameters of the ROESY experiment of 1: F1: ND0 1,
TD 512, SFO1 600.1325 MHz, FIDRES 11.252340 Hz, SW
9.600 ppm, FnMODE undefined; F2: TD 2048, NS 16, SWH
5787.037 Hz, AQ 0.1770836 s, RG 32, d0 0.000003 s, D1
3.000000 s, d11 0.030000 s, d12 0.000020 s. Acquisition
parameters of the ROESY experiment of 2: F1: ND0 1, TD
512, SFO1 600.1325 MHz, FIDRES 10.783298 Hz, SW
9.200 ppm, FnMODE undefined; F2: TD 2048, NS 32, SWH
5530.974 Hz, AQ 0.1852796 s, RG 128, d0 0.000003 s, D1
3.000000 s, d11 0.030000 s, d12 0.000020 s. MS: Finnigan
SSQ-700 or MAT-90 instrument for CI; Finnigan TSQ-700
triple quadrupole spectrometer for ESI; m/z (rel.%).
Abbreviations: DEPC: diethylphosphorocyanidate, DIEA:
N-ethyl-N,N-diisopr0opylamine, HATU: O-(7-Azabenzo-
triazol-1-yl)-N,N,N ,N0-tetramethyluronium hexafluoro-
phosphate, HOAt: 1-hydroxy-7-azabenzotriazole, PyAOP:
(7-azabenzotriazol-1-yloxy)tris(pyrrolidino)phosphonium
hexafluorophosphate, PyBOP: (1H-benzotriazol-1-yloxy)-
tris(pyrrolidino) phosphonium hexafluorophosphate.
General Procedure A (GP A). To a solution of a Z-protected
peptide in MeOH was added Pd/C (10% on activated
charcoal) and the mixture was hydrogenated overnight
under atmospheric pressure using an H2-filled balloon. The
catalyst was removed by filtration through a pad of celite
and the solvent evaporated under reduced pressure. The
crude product was further purified by filtration through a
short column of SiO2, dried under vacuum and used directly
in the next reaction step.
Figure 4. Superimposition of the final 14 NMR structures for 1.
General Procedure B (GP B). To a solution of an
N-protected peptide acid (or N-protected amino acid) in
abs. CH2Cl2 (or CH2Cl2/MeCN mixture) were added the
amino component (1.0 or 1.1 equiv), PyAOP (or PyBOP,
1.1 equiv), and DIEA (2 equiv without and 3 equiv with
hydrochloride salts present). The mixture was stirred at rt
under N2 until the starting material was consumed (TLC).
The solvent was then evaporated, the residue was dissolved
in EtOAc and washed with 5% aq KHSO4 solution, 5% aq
NaHCO3 solution and brine. The organic layer was dried
(MgSO4), concentrated, purified by CC and dried under high
vacuum.
Table 8. Observed average backbone torsion angles for the cyclic peptide 1
as obtained from the final 14 NMR structures
Residue
f
j
Gly1
Aib2
Leu3
Aib4
Phe5
Aib6
K105.2
K63.8
K123.6
K162.9
K84.6
C80.9
C170.0
K26.5
C31.2
K165.3
C88.1
C21.1
these cyclic hexapeptides possesses only one Aib residue
that shows slight deviation of torsion angles from the helical
region of the conformational space.
General Procedure C (HATU-mediated Cyclization) (GP
C). The free linear hexapeptide was dissolved in abs. DMF
(0.7 or 1.5 mM) and cooled to 0 8C in an ice bath. To the
solution was added HATU (3 equiv), HOAt (3 equiv, 0.5 M
solution in DMF) and DIEA (1% v/v) under stirring. The
solution was kept at 0 8C for 2 h and at rt for 3 days. The
solvent was removed under reduced pressure, the residue
dissolved in EtOAc and washed with 1 M HCl solution,
water, and brine. The organic layer was dried over
anhydrous Na2SO4, filtered and concentrated. The crude
cyclopeptide was further purified by CC.
4. Experimental
4.1. General
Solvents were purified by standard procedures. Thin-layer
chromatography (TLC): Merck TLC aluminium sheets,
silica gel 60 F254. Column chromatography (CC): Uetikon-
Chemie ‘Chromatographiegel’ C-560. Mp: Bu¨chi 510
apparatus; uncorrected. IR Spectra: Perkin–Elmer-1600
General Procedure D (DEPC-mediated Cyclization) (GP
D). The free linear hexapeptide was dissolved in abs. DMF
FT-IR spectrophotometer; in KBr; absorptions in cmK1
.

T. Jeremic et al. / Tetrahedron 61 (2005) 1871–1883
1879
(1.5 mM) and the solution was cooled to 0 8C in an ice bath.
Then, a solution of 5 equiv of DEPC in abs. DMF (1 ml) was
added under stirring, and DIEA (1% v/v) was added slowly
over a period of 15 min. The solution was warmed to rt and
stirred. The addition of DEPC (2.5 equiv) was repeated after
2 and 4 days, and the reaction mixture was stirred for an
additional 2 days. The solvent was then evaporated under
reduced pressure, the residue was taken up in EtOAc and
washed with 5% aq KHSO4 solution, 5% aq NaHCO3
solution, and brine. The organic phase was then dried
(MgSO4) and concentrated to give the crude cyclohexa-
peptide, which was purified by CC.
H-Phe-Aib-OtBu (0.328 g, 1.07 mmol), using PyAOP
(0.56 g, 1.1 mmol) and DIEA (0.276 g, 2.14 mmol) in abs.
CH2Cl2 (10 ml) according to GP B. Reaction time: 20 h at
rt. Purification by CC (SiO2, EtOAc/hexane 20:1) yielded
0.556 g (81%) of tetrapeptide 4 as a white foam. IR: 3325s,
3064w, 3032m, 2979m, 2960m, 2936m, 2872w, 1735s,
1665s, 1535s, 1469m, 1455m, 1385m, 1367m, 1258s,
1222s, 1148s, 1081w, 1029m, 940w, 850w, 788w, 752m,
698m. 1H NMR (CD3OD): 7.34–7.16 (m, 10 arom. H); 5.12
(s, PhCH2O); 4.48–4.46, 4.05–3.95 (2m, CH(2) of Leu and
CH(2) of Phe); 3.32–3.29 (m, 1H of CH2(3) of Phe); 2.94
(dd, JZ14.2, 10.6 Hz, 1H of CH2(3) of Phe); 1.80–1.51 (m,
CH2(3) and CH(4) of Leu); 1.44, 1.43, 1.32, 1.16 (4s, 4Me
of 2Aib and Me3C); 0.96, 0.92 (2d, JZ6.6 Hz, 2Me(5) of
Leu). 13C NMR (CD3OD): 176.4, 175.3, 174.9, 172.5 (4s, 4
CO); 158.9 (s, CO (urethane)); 139.1, 137.9 (2s, 2 arom. C);
130.1, 129.4, 129.3, 129.0, 128.6, 127.5 (6d, 10 arom. CH);
81.9 (s, Me3C); 67.7 (t, PhCH2O); 57.7, 57.6 (2s, 2 C(2) of
2Aib); 55.9 (d, C(2) of Phe); 41.3, 37.8 (2t, C(3) of Phe and
C(3) of Leu); 28.1 (q, Me3C); 25.7 (d, C(4) of Leu); 25.4,
24.9, 24.7, 23.1, 22.1 (5q, 4Me of 2Aib and 2Me(5) of Leu);
C(2) of Leu not detectable. ESI-MS (NaICMeOH): 661
(100, [MCNa]C). Anal. calcd for C35H50N4O7 (638.80): C
65.81, H 7.89, N 8.77; found: C 65.66, H 8.04, N 8.70.
General Procedure E (PyAOP-mediated Cyclization) (GP
E). The free linear hexapeptide was dissolved in abs. DMF
(0.6–1.0 mM) under stirring. Then, PyAOP (3 or 5 equiv),
HOAt (3 or 5 equiv, 0.5 M solution in DMF) and DIEA
were added at rt and the solution was stirred at rt for an
additional 3 days. The solvent was then removed under
reduced pressure, the residue dissolved in EtOAc and
washed with 10% citric acid solution, 5% NaHCO3 solution,
and water. The organic layer was dried (Na2SO4), filtered,
and concentrated to afford a yellow oil which was purified
by CC.
4.2. Preparation of cyclo(Gly-Aib-Leu-Aib-Phe-Aib) (1)
4.2.3. tert-Butyl N-[(benzyloxy)carbonyl]-glycyl-
dimethylglycyl-(S)-leucyl-dimethylglycyl-(S)-phenyl-
alanyl-dimethylglycinate (Z-Gly-Aib-Leu-Aib-Phe-Aib-
OtBu) (5). Z-Leu-Aib-Phe-Aib-OtBu (4) (0.527 g,
0.82 mmol) was N-deprotected according to GP A (H2,
55 mg Pd/C, 10 ml MeOH, overnight). The crude product
was filtered through a short SiO2-column with EtOAc/
MeOH (17:1) and dried under vacuum to afford 0.4 g (96%)
of H-Leu-Aib-Phe-Aib-OtBu as a white foam. This material
(0.4 g, 0.79 mmol) was coupled with Z-Gly-Aib-OH7
(0.234 g, 0.79 mmol) by following GP B, using PyAOP
(0.521 g, 1.0 mmol) and DIEA (0.255 g, 1.6 mmol) in abs.
CH2Cl2 (10 ml). Reaction time: 20 h at rt. CC (SiO2,
EtOAc/hexane/MeOH 10:7:1) gave 0.576 g (93%) of
hexapeptide 5 as a white foam. IR: 3322s, 3065w, 3033w,
2982m, 2959m, 2873w, 1664s, 1534s, 1456m, 1387m,
1368m, 1261m, 1151s, 1082w, 1051w, 979w, 852s, 740w,
699m. 1H NMR (CD3OD): 7.35–7.15 (m, 6 arom. H); 5.17–
5.05 (m, PhCH2O); 4.42–4.39, 4.15–3.70 (2m, CH(2) of
Phe, CH(2) of Leu and CH2(2) of Gly); 3.35–2.80 (m,
CH2(3) of Phe); 1.90–1.47 (m, CH2(3) and CH(4) of Leu);
1.45, 1.44, 1.38, 1.18 (4s, 6Me of 3Aib and Me3C); 0.94,
0.89 (2d, JZ6.3 Hz, 2Me(5) of Leu). 13C NMR (CD3OD):
177.1, 177.0, 174.9, 174.5, 172.8, 172.1 (6s, 6 CO); 159.5 (s,
CO (urethane)); 139.3, 137.9 (2s, 2 arom. C); 130.1, 129.5,
129.3, 129.1, 128.6, 127.5 (6d, 10 arom. CH); 81.8 (s,
Me3C); 67.8 (t, PhCH2O); 57.9, 57.8, 57.7 (3s, 3 C(2) of
3Aib); 56.5, 54.1 (2d, C(2) of Phe and C(2) of Leu); 45.4,
39.9, 37.5 (3t, C(2) of Gly, C(3) of Phe and C(3) of Leu);
28.1 (q, Me3C); 26.1 (d, C(4) of Leu); 26.5, 26.0, 25.9, 25.5,
24.9, 24.3, 23.7, 21.5 (8q, 6Me of 3Aib and 2Me(5) of Leu).
ESI-MS (NaICMeOH): 804 (100, [MCNa]C).
4.2.1. tert-Butyl N-[(benzyloxy)carbonyl]-(S)-phenyl-
alanyl-dimethylglycinate (Z-Phe-Aib-OtBu) (3). Z-Phe-
OH (0.6 g, 2.0 mmol) was coupled with HCl$H-Aib-OtBu
(0.431 g, 2.2 mmol), using PyAOP (1.147 g, 2.2 mmol) and
DIEA (0.775 g, 6.0 mmol) in CH2Cl2/MeCN (6/4 ml)
according to GP B. Reaction time: 20 h at rt. Purification
of the crude product by CC (SiO2, EtOAc/hexane 15:10)
afforded 0.798 g (91%) of dipeptide 3. White powder. Mp
119.5–121.0 8C. IR: 3325s, 3236m, 3065m, 2979m, 2948m,
1727s, 1708s, 1660s, 1544s, 1498m, 1470m, 1455m,
1384m, 1370m, 1291s, 1260s, 1235s, 1215m, 1147s,
1085w, 1065m, 1044m, 1028m, 912w, 853w, 757m,
1
740m, 700s. H NMR: 8.23 (s, NH of Aib); 7.42 (d, JZ
8.95 Hz, NH of Phe); 7.34–7.19 (m, 10 arom. H); 4.94 (br s,
PhCH2O); 4.35–4.20 (m, CH(2) of Phe); 2.93–2.92, 2.77–
2.73 (2m, CH2(3) of Phe); 1.35 (s, Me3C); 1.33, 1.30 (2s,
2Me of Aib). 13C NMR: 172.7, 170.6 (2s, 2 CO); 155.6 (s,
CO (urethane)); 138.0, 136.9 (2s, 2 arom. C); 129.1, 128.1,
127.8, 127.5, 127.2, 126.1 (6d, 10 arom. CH); 79.3 (s,
Me3C); 64.9 (t, PhCH2O); 55.6 (d, C(2) of Phe); 55.3 (s,
C(2) of Aib); 37.6 (t, C(3) of Phe); 27.3 (q, Me3C); 24.6 (q,
2Me of Aib). ESI-MS (NaICMeOH): 463 (100, [MC
Na]C). Anal. calcd for C25H32N2O5 (440.54): C 68.16, H
7.32, N 6.36; found: C 68.06, H 7.20, N 6.25.
4.2.2. tert-Butyl N-[(benzyloxy)carbonyl]-(S)-leucyl-
dimethylglycyl-(S)-phenylalanyl-dimethylglycinate
(Z-Leu-Aib-Phe-Aib-OtBu) (4). Z-Phe-Aib-OtBu (3)
(0.475 g, 1.08 mmol) was N-deprotected by following GP
A (H2, 55 mg Pd/C, 15 ml MeOH, overnight). The crude
product was filtered through a short SiO2-column with
EtOAc/MeOH (15:1) and dried under vacuum to give
0.328 g (99%) H-Phe-Aib-OtBu as a white foam, which was
used directly in the next step.
4.2.4. Cyclo(Gly1-Aib2-Leu3-Aib4-Phe5-Aib6) (1). Z-Gly-
Aib-Leu-Aib-Phe-Aib-OtBu (5) (0.555 g, 0.71 mmol) was
N-deprotected according to GP A (H2, 60 mg Pd/C, 10 ml
MeOH, 20 h). Thus, 0.417 g (91%) of H-Gly-Aib-Leu-Aib-
Phe-Aib-OtBu were obtained as a white foam, which was
Z-Leu-Aib-OH9 (0.375 g, 1.07 mmol) was coupled with

1880
T. Jeremic et al. / Tetrahedron 61 (2005) 1871–1883
dissolved in abs. CH2Cl2 (20 ml), and TFA (20 ml) was
added at rt. The mixture was stirred for 6 h. Excess TFA was
removed under reduced pressure, followed by addition and
evaporation of two portions of CH2Cl2 (10 ml). Upon drying
under high vacuum, 0.461 g of the free linear hexapeptide
were obtained as its TFA salt in quantitative yield.
dimethylglycyl-(S)-phenylalaninate (Z-Leu-Aib-Phe-
OtBu) (6). Z-Leu-Aib-OH9 (0.25 g, 0.71 mmol) was
coupled with HCl$H-Phe-OtBu (0.202 g, 0.78 mmol) in
abs. CH2Cl2/MeCN (6/2 ml) according to GP B, using
PyBOP (0.371 g, 0.71 mmol) and DIEA (0.276 g,
2.14 mmol, overnight). Purification of the crude product
by CC (SiO2, CH2Cl2/MeOH 17:1, performed twice)
afforded 0.362 g (92%) of tripeptide 6 as a white foam.
IR: 3401m, 3368m, 3237m, 3033w, 2973m, 2951m, 1870w,
1720s, 1663s, 1651s, 1515s, 1457m, 1439m, 1389w,
1367m, 1246s, 1220m, 1166m, 1118w, 1040m, 861w,
846w, 780w, 755w, 743w, 700m. 1H NMR: 8.05 (br s, NH
of Aib); 7.52–7.48 (m, NH of Leu and NH of Phe); 7.33–
7.17 (m, 10 arom. H); 5.06–4.95 (m, PhCH2O), 4.34–4.31,
4.00–3.97 (2m, CH(2) of Phe and CH(2) of Leu); 2.96–2.93
(m, CH2(3) of Phe); 1.70–1.31 (m, CH2(3) and CH(4) of
Leu, 2Me of Aib and Me3C); 0.88–0.84 (m, 2Me(5) of Leu).
13C NMR: 173.6, 171.7, 170.1 (3s, 3CO); 156.0 (s, CO
(urethane)); 137.2, 136.8 (2s, 2 arom. C); 129.0, 128.1,
127.9, 127.6, 127.4, 126.3 (6d, 10 arom. CH); 80.5 (s,
Me3C); 65.2 (t, PhCH2O); 55.8 (s, C(2) of Aib); 54.2, 53.3
(2d, C(2) of Leu and C(2) of Phe); 40.0, 36.8 (2t, C(3) of
Leu and C(3) of Phe); 27.4 (q, Me3C); 24.0 (d, C(4) of Leu);
25.5, 23.8, 22.8, 21.4 (4q, 2Me of Aib and 2Me(5) of Leu).
ESI-MS (NaICMeOH): 576 (100, [MCNa]C). Anal. calcd
for C31H43N3O6 (553.69): C 67.24, H 7.83, N 7.59; found: C
67.23, H 7.82, N 7.54.
HATU-mediated cyclization: 0.121 g (0.17 mmol) of
H-Gly-Aib-Leu-Aib-Phe-Aib-OH$TFA were dissolved in
abs. DMF (112 ml) and subjected to cyclization according
to GP C, with HATU (0.194 g, 0.51 mmol), HOAt (69 mg,
0.51 mmol), and DIEA (1.2 ml). Reaction time: 1 day.
Purification by CC (SiO2, CH2Cl2/MeOH 10:1, EtOAc/
MeOH 15:1) afforded 23 mg (24%) of pure cyclohexa-
peptide 1.
DEPC-mediated cyclization: 0.121 g (0.17 mmol) of the
free linear peptide TFA salt were dissolved in abs. DMF
(112 ml) and the cyclization was performed according to GP
D using DEPC (0.139 g, 0.85 mmol) and DIEA (1.2 ml).
After 2 and 4 days of stirring, additional DEPC (69 mg,
0.425 mmol) was added. Reaction time: 6 days. The
obtained yellow oil was purified by CC (SiO2, EtOAc/
MeOH 15:1, performed twice) to provide 51 mg (53%) of
pure 1.
PyAOP-mediated cyclization: 0.121 g (0.17 mmol) of the
free linear peptide TFA salt were dissolved in abs. DMF
(170 ml) and treated with PyAOP (0.441 g, 0.85 mmol),
HOAt (0.116 g, 0.85 mmol), and DIEA (1.7 ml) following
GP E. Purification by CC (SiO2, CH2Cl2/MeOH 17:1,
performed twice) afforded 30 mg (31%) of pure 1. White
powder. Mp (dec.) 284–286 8C. IR: 3317s, 3061w, 2968m,
2871w, 1704m, 1650s, 1536s, 1457m, 1390m, 1367m,
4.3.2. Benzyl N-((S)-1-{[(1,1-dimethyl-2-{[1-(S)-benzyl-
2-({2-[(1,1-dimethyl-2-{[1,1-dimethyl-2-(methylphenyl-
amino)-2-oxoethyl]amino}-2-oxoethyl)amino]-2-oxo-
ethyl}amino)-2-oxoethyl]amino}-2-oxoethyl)amino]oxo-
methyl}-3-methylbutyl) carbamate (Z-Leu-Aib-Phe-
Gly-Aib-Aib-N(Me)Ph) (8). Z-Leu-Aib-Phe-OtBu (6)
(0.7 g, 1.26 mmol) was dissolved in CH2Cl2 (15 ml), TFA
was added (15 ml), and the mixture was stirred for 6 h at rt.
The solvent was then evaporated and the crude product
filtered through a short SiO2-column using CH2Cl2/MeOH
(12:1) to give 0.586 g (93%) of Z-Leu-Aib-Phe-OH as a
white foam, which was used directly in the next reaction.
1
1264m, 1219m, 1188m, 1080w, 1029w, 744w, 698m. H
NMR: 8.22 (s, NH of Aib2); 8.05 (s, NH of Aib6); 7.80 (d,
JZ8.9 Hz, NH of Leu3); 7.62 (d, JZ7.6 Hz, NH of Phe5);
7.55 (s, NH of Aib4); 7.27–7.16 (m, 5 arom. H of Phe5, NH
of Gly1); 4.33–4.27 (m, CH(2) of Leu3 and CH(2) of Phe5);
3.77 (dd, JZ17.0, 5.8 Hz, 1H of CH2(2) of Gly1); 3.70 (dd,
JZ17.0, 3.2 Hz, 1H of CH2(2) of Gly1); 2.94 (dd, JZ13.5,
7.7 Hz, 1H of CH2(3) of Phe5); 2.85 (dd, JZ13.5, 7.2 Hz,
1H of CH2(3) of Phe5); 1.62–1.47 (m, CH2(3) and CH(4) of
Leu3); 1.46, 1.38 (2s, 2Me of Aib4); 1.37, 1.29 (2s, 2Me of
Aib2); 1.26, 1.19 (2s, 2Me of Aib6); 0.88, 0.82 (2d, JZ
6.4 Hz, 2Me(5) of Leu3). 13C NMR: 174.2 (s, CO of Aib4);
174.0 (s, CO of Aib2); 173.7 (s, CO of Aib6); 171.3 (s, CO of
Leu3); 170.0 (s, CO of Phe5); 168.2 (s, CO of Gly1); 137.7
(s, 1 arom. C of Phe5); 129.3, 128.0, 126.2 (3d, 5 arom. CH
of Phe5); 56.4 (s, C(2) of Aib4); 56.2 (s, C(2) of Aib2); 56.0
(s, C(2) of Aib6); 55.1 (d, C(2) of Phe5); 50.9 (d, C(2) of
Leu3); 42.8 (t, C(2) of Gly1); 40.2 (t, C(3) of Leu3); 36.6 (t,
C(3) of Phe5); 27.0 (q, 1Me of Aib6); 26.9 (q, 1Me of Aib2);
25.9 (q, 1Me of Aib4); 24.3 (d, C(4) of Leu3); 23.47 (q, 1Me
of Aib2); 23.38 (q, Me(5) of Leu3); 23.14 (q, 1Me of Aib4);
23.09 (q, 1Me of Aib6); 21.1 (q, Me(5) of Leu3). ESI-MS
(NaICMeOH): 595 (100, [MCNa]C). Anal. calcd for
C29H44N6O6 (572.71): C 60.82, H 7.74, N 14.67; found: C
60.60, H 7.73, N 14.56.
Z-Gly-Aib-Aib-N(Me)Ph (7)7 (0.234 g, 0.5 mmol) was
N-deprotected following GP A (H2, 25 mg Pd/C, 6 ml
MeOH, overnight). The crude product was dried under
vacuum to give 0.159 g (95%) of H-Gly-Aib-Aib-N(Me)Ph,
which was used in the next reaction step without further
purification.
The coupling of Z-Leu-Aib-Phe-OH (0.215 g, 0.43 mmol)
with H-Gly-Aib-Aib-N(Me)Ph (0.159 g, 0.48 mmol) in abs.
CH2Cl2 (6 ml) was achieved according to GP B, using
PyAOP (0.26 g, 0.5 mmol) and DIEA (0.129 g, 1.0 mmol,
overnight). Purification by CC (SiO2, CH2Cl2/MeOH 20:1)
afforded 0.21 g (60%) of hexapeptide 8 as a white foam. IR:
3309s, 3062w, 3032m, 2957m, 2872w, 1662s, 1594m,
1533s, 1455s, 1389m, 1364m, 1332m, 1266m, 1221m,
1173m, 1118m, 1091m, 1045w, 1028w, 922w, 741w, 704m.
1H NMR (CD3OD): 7.40–7.16 (m, 15 arom. H); 5.14–4.99
(m, PhCH2O); 4.05–3.59 (m, CH(2) of Leu, CH(2) of Phe
and CH2(2) of Gly); 3.35–2.90 (m, MeN and CH2(3) of
Phe); 1.75–1.46 (m, CH2(3) and CH(4) of Leu, 4Me of
2Aib); 1.30, 1.27 (2s, 2Me of Aib); 0.96–0.91 (m, 2Me(5) of
Leu). 13C NMR (CD3OD): 177.0, 176.2, 176.1, 175.4,
4.3. Preparation of cyclo(Leu-Aib-Phe-Gly-Aib-Aib) (2)
4.3.1. tert-Butyl N-[(benzyloxy)carbonyl]-(S)-leucyl-

T. Jeremic et al. / Tetrahedron 61 (2005) 1871–1883
1881
174.4, 171.3 (6s, 6CO (amide)); 158.5 (s, CO (urethane));
139.3, 138.1 (2s, 3 arom. C); 130.2, 130.1, 129.9, 129.4,
129.0, 128.5, 128.4, 128.2, 127.6 (9d, 15 arom. CH); 67.6 (t,
PhCH2O); 58.6, 58.3, 57.7 (3s, 3 C(2) of 3Aib); 57.1, 55.3
(2d, C(2) of Leu and C(2) of Phe); 44.8, 41.3 (2t, C(3) of
Leu and C(2) of Gly); 41.27 (q, MeN); 36.4 (t, C(3) of Phe);
25.7 (d, C(4) of Leu); 26.3, 25.3, 24.7, 23.2, 22.1 (5q, 6Me
of 3Aib and 2Me(5) of Leu). ESI-MS (NaICMeOH): 837
(100, [MCNa]C). Anal. calcd for C44H59N7O8$1/3H2O
(819.99): C 64.45, H 7.33, N 11.96; found: C 64.35, H 7.36,
N 11.96.
CH2(3) of Leu1); 1.43 (s, Me of Aib6); 1.31, 1.30 (2s, 2Me
of Aib5); 1.23 (s, Me of Aib6); 1.12, 1.03 (2s, 2Me of Aib2);
0.899, 0.897 (2d, JZ6.4, 6.8 Hz, 2Me(5) of Leu1). 13C
NMR: 173.5 (s, CO of Aib2); 173.2 (s, CO of Aib5); 173.0
(s, CO of Aib6); 172.9 (s, CO of Leu1); 170.7 (s, CO of
Phe3); 169.1 (s, CO of Gly4); 138.6 (s, 1 arom. C of Phe3);
128.9, 127.9, 126.0 (3d, 5 arom. CH of Phe3); 56.9 (s, C(2)
of Aib6); 56.3 (s, C(2) of Aib5); 55.9 (s, C(2) of Aib2); 53.2
(d, C(2) of Phe3); 50.1 (d, C(2) of Leu1); 41.2 (t, C(2) of
Gly4); 40.4 (t, C(3) of Leu1); 35.5 (t, C(3) of Phe3); 28.0 (q,
Me of Aib6); 26.1 (q, Me of Aib5); 25.7 (q, Me of Aib2);
24.0 (d, C(4) of Leu1); 23.8 (q, Me(5) of Leu1); 23.7 (q, Me
of Aib2); 23.22 (q, Me of Aib6); 23.19 (q, Me of Aib5); 20.9
(q, Me(5) of Leu1). ESI-MS (NaICMeOH): 595 (100, [MC
Na]C). Anal. calcd for C29H44N6O6$1/2H2O (581.71): C
59.88, H 7.80, N 14.45; found: C 60.02, H 7.98, N 14.36.
4.3.3. cyclo(Leu1-Aib2-Phe3-Gly4-Aib5-Aib6) (2). Peptide
8 (0.42 g, 0.52 mmol) was dissolved in MeCN (3 ml) and
then 3 ml of 6 N HCl were added dropwise. The mixture
was stirred at rt overnight. The MeCN was evaporated under
reduced pressure and 2 N HCl (3 ml) was added. The
product was extracted with CH2Cl2, the organic layer was
dried (Na2SO4), filtered and concentrated under reduced
pressure. After drying under vacuum, 0.369 g (98%) of
Z-Leu-Aib-Phe-Gly-Aib-Aib-OH were obtained as a white
foam. Then, 0.318 g (0.44 mmol) of this compound were
N-deprotected according to GP A (H2, 35 mg Pd/C, 6 ml
MeOH, 20 h). After drying under vacuum, 0.241 g (93%) of
the free linear hexapeptide were obtained as a pale yellow
foam, which was used in the cyclization step without further
purification.
4.4. X-Ray crystal-structure determination of 1 and 2
All measurements were made on a Nonius KappaCCD area-
detector diffractometer38 using graphite-monochromated
˚
MoKa radiation (l 0.71073 A) and an Oxford Cryosystems
Cryostream 700 cooler. The data collection and refinement
parameters are given below39 and views of the molecules
are shown in Figures 2 and 3. The intensities were corrected
for Lorentz and polarization effects, but not for absorption.
Standard reflection intensities were not monitored.
Equivalent reflections, other than Friedel pairs, were
merged. The structures were solved by direct methods
using SIR92,40 which revealed the positions of all non-
hydrogen atoms.
HATU-mediated cyclization: 84 mg (0.14 mmol) of the free
linear hexapeptide were dissolved in abs. DMF (200 ml) and
subjected to macrolactamization according to GP C, with
HATU (0.162 g, 0.43 mmol), HOAt (58 mg, 0.43 mmol),
and DIEA (2 ml). Reaction time: 3 days. Purification by CC
(SiO2, CH2Cl2/MeOH 12:1, performed twice) afforded
13 mg (16%) of pure cyclohexapeptide 2 as a white foam.
The iso-propyl part of the Leu side chain in 1 is disordered.
Two positions were defined for the disordered atoms and
refinement of the site occupation factors yielded a value of
0.53(2) for the major conformation. Bond length and
similarity restraints were applied to all chemically
equivalent bond lengths and angles involving the disordered
atoms. Neighboring atoms within and between each
conformation of the disordered isopropyl group were also
restrained to have similar atomic displacement parameters.
DEPC-mediated cyclization: 88 mg (0.15 mmol) of the free
linear precursor were dissolved in abs. DMF (100 ml) and
the cyclization was performed according to GP D, using
DEPC (0.141 g, 0.75 mmol) and DIEA (1 ml). After 2 and 4
days, additional DEPC (60.5 mg, 0.375 mmol) was added to
the stirred mixture. Reaction time: 6 days. The obtained
yellow oil was purified by CC (SiO2, CH2Cl2/MeOH 14:1,
performed trice) to afford 28 mg (33%) of pure 2 as a white
foam.
The asymmetric unit of 2 contains one molecule of the
peptide plus two water molecules, one disordered EtOH
molecule and one disordered i-PrOH molecule. Two
positions were defined for each of the atoms of the two
disordered solvent molecules and the site occupation factors
of the major conformations refined to 0.51(2) and 0.50(2)
for the EtOH and i-PrOH molecules, respectively. Simi-
larity restraints were applied to the chemically equivalent
bond lengths within the disordered molecules and neigh-
boring atoms within and between each conformation of the
disordered molecule were also restrained to have similar
atomic displacement parameters.
PyAOP-mediated cyclization: 64 mg (0.11 mmol) of the
free linear precursor were dissolved in abs. DMF (180 ml)
and treated with PyAOP (0.169 g, 0.32 mmol), HOAt
(44 mg, 0.32 mmol) and DIEA (1.8 ml) by following GP
E. Purification by CC (SiO2, CH2Cl2/MeOH 12:1, per-
formed thrice) yielded 30 mg (48%) of pure 2 as a white
foam. IR: 3327s, 3030w, 2957m, 2871w, 1657s, 1534s,
1469m, 1455m, 1385m, 1365m, 1277m, 1225m, 1179m,
1030w, 945w, 820w, 748w, 702m. 1H NMR: 8.28 (s, NH of
Aib5); 8.14 (s, NH of Aib2); 7.43 (br s, NH of Gly4); 7.41 (d,
JZ9.7 Hz, NH of Leu1); 7.39 (s, NH of Aib6); 7.32 (d, JZ
9.0 Hz, NH of Phe3); 7.24–7.14 (m, 5 arom. H of Phe3);
4.59–4.55 (m, CH(2) of Phe3); 4.28–4.20 (m, CH(2) of Leu1
and 1H of CH2(2) of Gly4); 3.62–3.59 (m, 1H of CH2(2) of
Gly4); 3.31–3.28 (m, 1H of CH2(3) of Phe3); 2.93–2.89 (m,
1H of CH2(3) of Phe3); 1.88–1.83 (m, 1H of CH2(3) of
Leu1); 1.72–1.68 (m, CH(4) of Leu1); 1.60–1.55 (m, 1H of
The non-hydrogen atoms were refined anisotropically. The
amide H-atoms in both structures, and the water H-atoms in
2, were placed in positions indicated by a difference electron
density map and their positions were allowed to refine
together with individual isotropic displacement parameters.
All remaining H-atoms were placed in geometrically
calculated positions and refined using a riding model
where each H-atom was assigned a fixed isotropic

1882
T. Jeremic et al. / Tetrahedron 61 (2005) 1871–1883
displacement parameter with a value equal to 1.2Ueq of its
parent C-atom (1.5Ueq for the methyl groups). The
orientations of the hydroxy O–H vectors in the solvent
molecules of 2 were chosen so as to be directed towards the
nearest hydrogen bond acceptor atom. The refinement of
each structure was carried out on F2 using full-matrix
least-squares procedures, which minimised the function
Sw(F2oKF2c)2. Corrections for secondary extinction were
applied. For 1 and 2, 11 and two reflections, respectively,
were omitted from the final refinement. In each case, the
enantiomer used in the refinement was chosen to correspond
with the known S-configuration of the chiral centers derived
from precursor molecules.
peptide molecule to give a closed trimeric system. In each
case, this builds a loop with a graph set motif of R22(10). In
contrast, the water molecules form hydrogen bonds between
different peptide molecules and thereby link all of the
peptide and solvent molecules in the structure into an
infinite three-dimensional framework. Although there are
two symmetry-independent water molecules in the struc-
ture, each generates the same hydrogen-bonding pattern.
The path via one H-atom from each water molecule creates
a chain with a binary graph set motif of C22(7), while the path
via the other H-atom from each water molecule creates a
chain with a binary graph set motif of C22(10).
Crystal data for 1: C29H44N6O6, MZ572.70, colorless,
prism, crystal dimensions 0.10!0.12!0.25 mm, ortho-
rhombic, space group P212121, ZZ4, reflections for cell
determination 3164, 2q range for cell determination 4–508,
Neutral atom scattering factors for non-hydrogen atoms
were taken from Ref. 41 and the scattering factors for
H-atoms were taken from Ref. 42. Anomalous dispersion
effects were included in Fc;43 the values for f0 and f00 were
those of Ref. 44. The values of the mass attenuation
coefficients are those of Ref. 45. All calculations were
performed using the SHELXL97 program.46
˚
˚
˚
aZ9.7189(2) A, bZ10.0614(2) A, cZ31.9151(7) A, VZ
3
3120.8(1) A , TZ160 K, DXZ1.219 g cmK3, m(MoKa)Z
˚
0.0863 mmK1, 2q(max)Z508, total reflections measured
27,778, symmetry independent reflections 3146, reflections
with IO2s(I) 2265, reflections used in refinement 3135,
parameters refined 433; restraints 68, R(F) [IO2s(I)
reflections]Z0.0407, wR(F2) [all data]Z0.0870 (wZ
[s2(F2o)C(0.0332P)2]K1, where PZ(Fo2C2F2c)/3), goodness
of fit 1.000, secondary extinction coefficient 0.005(1), final
In 1, each N–H group of the peptide molecule acts as a
donor for hydrogen bonds. Two of the interactions, N(1)–H
and N(10)–H, are intramolecular hydrogen bonds. N(1)–H
interacts with the amide O(12)-atom that is diagonally
opposed in the peptide ring to give a loop with a graph set
motif47 of S(10). N(10)–H does not interact with a
diametrically opposed amide O-atom, but forms bifurcated
intramolecular hydrogen bonds with the amide O-atoms
(O(6) and O(12), respectively) of the two adjacent peptide
units. These two interactions have graph set motifs of S(7)
and S(5). N(4)–H forms an intermolecular hydrogen bond
with the amide O-atom of the same peptide unit of a
neighboring molecule and thereby links the molecules into
extended chains which run parallel to the [010] direction
and have a graph set motif of C(4). N(7)–H, N(13)–H and
N(16)–H form intermolecular hydrogen bonds with amide
O-atoms of almost diagonally opposed peptide units from
three different neighboring molecules. Each of these
interactions links the molecules into extended chains
which run parallel to the [010] direction and have a graph
set motif of C(10). Together, the intermolecular hydrogen
bonds link the molecules into extended two-dimensional
networks which lie parallel to the (001) plane.
K3
˚
D
max/s 0.001, Dr (max; min)Z0.18; K0.18e A
.
Crystal data for 2: C29H44N6O6$EtOH$i-PrOH$2H2O, MZ
714.89, colorless, prism, crystal dimensions 0.30!0.30!
0.35 mm, monoclinic, space group P21, ZZ2, reflections for
cell determination 4747, 2q range for cell determination
˚
˚
4–558,
aZ10.0827(1) A,
bZ12.5382(1) A,
cZ
3
˚
˚
15.7976(2) A, bZ96.4866(4)8, VZ1984.33(4) A , TZ
160 K, DXZ1.196 g cmK3, m(MoKa)Z0.0878 mmK1
,
2q(max)Z558, total reflections measured 44,105, symmetry
independent reflections 4757, reflections with IO2s(I)
4044, reflections used in refinement 4755, parameters
refined 573; restraints 164, R(F) [IO2s(I) reflections]Z
0.0459, wR(F2) [all data]Z0.1280 (wZ[s2(F2o)C(0.0817P)2
C0.2355P]K1, wherePZ(Fo2C2F2c)/3), goodnessoffit1.040,
secondary extinction coefficient 0.025(4), final Dmax/s 0.001,
K3
˚
Dr (max; min)Z0.42; K0.29e A
.
Acknowledgements
In 2, all available N–H and O–H donors in the structure are
involved in hydrogen bonds. The peptide molecule has two
intramolecular hydrogen N–H.O bonds which diagonally
cross the molecule to link the amide N–H donors with amide
O-atoms that are seven atoms further along the peptide
backbone. Each of these interactions has a graph set motif of
S(10), which, despite the cyclic nature of the peptide, is the
same as usually observed in open chain peptides. The
remaining four amide N–H donors form intermolecular
hydrogen bonds with the O-atoms from each of the four
symmetry-independent solvent molecules, so that the two
water molecules, the EtOH molecule and the i-PrOH
molecule each accept one hydrogen bond. Each of the
solvent O–H donors, in turn, forms an intermolecular
hydrogen bond with an amide O-atom of a peptide
molecule. The EtOH and i-PrOH molecules both act as
acceptors and donors of hydrogen bonds involving the same
We thank the analytical sections of our institute for spectra
and analyses. Financial support of the Swiss National
Science Foundation and F. Hoffmann-La Roche AG, Basel,
is gratefully acknowledged.
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