Synthesis of Aib-Pro oligopeptides by repeated azirine coupling with the Aib-Pro synthon

Article abstract of DOI:10.1002/hlca.201200136

A new synthesis of (Aib-Pro)_n oligopeptides (n=2, 3, and 4) via azirine coupling by using the dipeptide synthon methyl N-(2,2-dimethyl-2H- azirin-3-yl)-L-prolinate (1b; Fig. 1) is presented. The most important feature of the employed protocol is that no activation of the acid component is necessary, i.e., no additional reagents are required, and the coupling reaction is performed under mild conditions at room temperature. As an attempt to provide an answer to the question of the preferred conformation of the prepared molecules, we carried out experiments by using NMR techniques and X-ray crystallography. For example, in the case of the hexapeptide 11, it was possible to compare the conformations in the crystalline state and in solution. After the selective hydrolysis of the methyl ester p-BrBz-(Aib-Pro)₄-OMe (13) under basic conditions, the corresponding octapeptide acid was obtained, which was then converted into the octapeptide amide p-BrBz-(Aib-Pro) ₄-NHC₆H₁₃ (15) by using standard coupling conditions and activating reagents (HOBt/TBTU/DIEA) of the peptide synthesis. The conformation of this compound, as well as those of the tetrapeptides 14 and 18, was also established by X-ray crystallography and in solution by NMR techniques. In the crystalline state, a β-bend ribbon structure is the preferred conformation, and similar conformations are formed in solution. Copyright

Full text of DOI:10.1002/hlca.201200136

Helvetica Chimica Acta – Vol. 95 (2012)
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Synthesis of Aib-Pro Oligopeptides by Repeated Azirine Coupling with the
Aib-Pro Synthon
by Svetlana A. Stoykova1), Anthony Linden, and Heinz Heimgartner*
Organisch-Chemisches Institut der Universitꢀt Zꢁrich, Winterthurerstrasse 190, CH-8057 Zꢁrich
(phone: þ 41-44-635-4282; fax: þ 41-44-635-6812; e-mail: heimgart@oci.uzh.ch)
A new synthesis of (Aib-Pro)n oligopeptides (n ¼ 2, 3, and 4) via azirine coupling by using the
dipeptide synthon methyl N-(2,2-dimethyl-2H-azirin-3-yl)-l-prolinate (1b; Fig. 1) is presented. The most
important feature of the employed protocol is that no activation of the acid component is necessary, i.e.,
no additional reagents are required, and the coupling reaction is performed under mild conditions at
room temperature. As an attempt to provide an answer to the question of the preferred conformation of
the prepared molecules, we carried out experiments by using NMR techniques and X-ray crystallography.
For example, in the case of the hexapeptide 11, it was possible to compare the conformations in the
crystalline state and in solution. After the selective hydrolysis of the methyl ester p-BrBz-(Aib-Pro)4-
OMe (13) under basic conditions, the corresponding octapeptide acid was obtained, which was then
converted into the octapeptide amide p-BrBz-(Aib-Pro)4-NHC6H13 (15) by using standard coupling
conditions and activating reagents (HOBt/TBTU/DIEA) of the peptide synthesis. The conformation of
this compound, as well as those of the tetrapeptides 14 and 18, was also established by X-ray
crystallography and in solution by NMR techniques. In the crystalline state, a b-bend ribbon structure is
the preferred conformation, and similar conformations are formed in solution.
1. Introduction. – Peptaibol antibiotics, including the well-known alamethicin [2],
form an important class of linear peptides of fungal origin, which are characterized by a
high content of a,a-disubstituted glycines, e.g., a-aminoisobutyric acid (Aib) and
isovaline (Iva), an N-terminal Ac group, and a C-terminal b-amino alcohol [3]. Due to
the conformational constraints imposed on the peptide backbone by the presence of
a,a-dialkylated glycyl residues, Aib-containing peptides generally form helical
structures, either a, 310, or mixed a/310 helices, depending on the length of the peptide
and the number and location of Aib residues [4]. Helix-like structures have also been
described in the case of (Aib-Pro)n oligopeptides and (Xaa-Yaa-Aib-Pro) segments,
which give rise to the b-bend ribbon spiral [4a][5], a sub-class of 310-helices. It has been
suggested that the alternation of a conformationally restricted N-alkylated amino acid
residue such as Pro, which disrupts the conformation-stabilizing H-bonding schemes
observed in helices, and a helix-forming residue such as Aib, may give rise to the
formation of a b-bend ribbon [4a]. Helical structures are involved in the antimicrobial
properties of peptaibols, due to their ability to interact with biological membranes, to
modify their permeability and to form voltage-dependent trans-membrane ion
channels [6].
1
)
Part of the Ph.D. thesis of S. A. S., Universitꢀt Zꢁrich, 2004; presented in part at the Autumn
Meeting of the Swiss Chemical Society, Lausanne, 2001 [1].
ꢂ 2012 Verlag Helvetica Chimica Acta AG, Zꢁrich

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Fig. 1. 2H-Azirin-3-amines as Aib, Aib-Pro, and Aib-Hyp synthons
Since the discovery of 2,2-disubstituted 2H-azirin-3-amines of type 1a (Fig. 1) as
synthons for a,a-disubstituted glycines [7], we have reported their use in the synthesis
of several peptaibols and peptaibol segments [8], as well as sterically congested model
peptides [9], via the so-called ꢃazirine/oxazolone methodꢄ. Furthermore, it has been
demonstrated that the building blocks 1 may be used in a modified solid-phase peptide
synthesis [8h][10]. The key steps of this method are the spontaneous reaction of an
amino or peptide acid 2 with 1a leading to peptide amides 3 with the backbone
extended by an a-methylalanine (Aib), followed by the acid-catalyzed formation of an
intermediate oxazolone 4 and coupling of the next amino component to give 5
(Scheme 1). Alternatively, under hydrolytic conditions, selective hydrolysis of the
intermediates 4 leads to the acids 6, which can be used for the next ꢃazirine couplingꢄ.
Furthermore, methyl N-(2,2-dimethyl-2H-azirin-3-yl)-l-prolinates 1b and 1c have
been prepared and found to be suitable as dipeptide synthons for the sequence Aib-Pro
and Aib-Hyp (Hyp ¼ 4-hydroxyproline) [8b][11]. These building blocks have been
employed in the synthesis of the peptaibol antibiotic trichovirin I 1B and segments of
trichovirin I 4A [8b][8e], a segment of zervamicin II-2 [8d], and hypomurocin A1 [8f].
In the present study, we have used the Aib-Pro synthon 1b for the synthesis of some
terminally protected sequential peptides of the type p-BrBz-(Aib-Pro)n-OMe, p-BrBz-
(Aib-Pro)n-NHC6H13, and p-MeOBz-(Aib-Pro)n-OMe. We also present the results of
the conformational studies on these peptides in the solid state and in solution, and the
evidence for the b-bend ribbon structure, which has already been reported for
analogous peptides with other terminal groups [4a][5].
Scheme 1

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2. Results and Discussion. – 2.1. Synthesis of the Terminally Protected p-BrBz-(Aib-
Pro)n-OMe and p-BrBz-(Aib-Pro)n-NHC6H13. The dipeptide methyl ester 7 was
synthesized in good yield by treatment of 4-bromobenzoic acid (p-BrBzOH) with
azirine 1b (ꢃazirine couplingꢄ). A solution of p-BrBzOH (1 mol-equiv.) in THF was
cooled to 08, 1b (1.1 mol-equiv.) was added, and the mixture was stirred for 103 h at
room temperature. The product was purified by column chromatography to give 7 in
89% yield (Scheme 2). Base-catalyzed hydrolysis of 7 with LiOH · H2O (4 mol-equiv.)
in THF/MeOH/H2O gave the corresponding dipeptide acid 8 in excellent yield (96%).
The isolated product was pure enough to be used in the next coupling with another Aib-
Pro unit 1b. To a solution of 8 (1 mol-equiv.) in CH2Cl2 at 08 was added 1b (1.1 mol-
equiv.), and the mixture was stirred for 27 h at room temperature. After chromato-
graphic purification, the tetrapeptide p-BrBz-(Aib-Pro)2-OMe (9) was obtained
quantitatively (99%). Subsequent hydrolysis with LiOH · H2O (4 mol-equiv.) in
THF/MeOH/H2O afforded the corresponding acid 10 in 93% yield. By repeating the
sequence of azirine coupling and hydrolysis, the hexapeptides 11 (ester) and 12 (acid)
were prepared in high-to-excellent yields, whereas the azirine coupling of 12 with 1b
furnished the octapeptide ester 13 in only 24% yield.
Scheme 2

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In the case of the hexapeptide ester 11, suitable crystals for the X-ray crystal-
structure determination were obtained by slow evaporation of the solvent from a
solution of 11 in CH2Cl2/AcOEt. The molecular structure is shown in Fig. 2. The
absolute configuration (S,S,S) was confirmed independently by the diffraction experi-
ment and agrees with that expected from the synthesis of the compound. The
compound contains highly disordered or diffuse solvent molecules. The molecule 11
forms a helix which is held in place in the usual way by two intramolecular H-bonds.
N(7)ꢀH and N(13)ꢀH interact with the amide O-atoms that are seven atoms back
along the peptide backbone. Each of these interactions has the graph set [12] motif of
S(10). N(1)ꢀH, which is unable to form an intramolecular interaction because of its
position in the backbone, forms an intermolecular H-bond with the amide O(12’)-atom
near the middle of a different neighboring molecule. This interaction links the
molecules into extended chains which run parallel to the [010] direction and have a
graph set motif of C(14) (Fig. 3). As all possible H-bonds involving the NꢀH groups
have been detected, there are no potential donor interactions with any of the solvent
molecules.
Fig. 2. ORTEP Plot [13] of the molecular structure of 11 (arbitrary numbering of the atoms; 50%
probability ellipsoids; H-atoms bonded to C-atoms omitted for clarity)
Starting with the tetrapeptide acid 10, the corresponding hexylamide 14 was
prepared by a standard peptide-coupling method with 2-[(1H-benzotriazol-1-yl)oxy]-
1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) in the presence of 1-hydroxy-
benzotriazole (HOBt) and (ethyl)(diisopropyl)amine (DIEA, EtN(i-Pr)2) in CH2Cl2
at room temperature (Scheme 3). After usual workup, chromatographic purification
gave 14 in 90% yield.
Suitable crystals of 14 for the X-ray crystal-structure determination were grown
from a solution in CH2Cl2 by slow evaporation of the solvent. The molecular structure is
depicted in Fig. 4. The crystals are enantiomerically pure, and the absolute config-
uration (S,S) has been confidently determined independently by the diffraction
experiment. The asymmetric unit contains one peptide and two CH2Cl2 molecules, one
of which is highly disordered. Each NꢀH group of the peptide molecule acts as a donor
for H-bonds. Two of the interactions are intramolecular H-bonds which form a regular

Helvetica Chimica Acta – Vol. 95 (2012)
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Fig. 3. Packing diagram of 11 showing the H-bonding interactions (uninvolved H-atoms omitted for
clarity)
pattern along the peptide chain and lend the molecule a helical conformation: N(1)ꢀH
and N(6)ꢀH interact with the amide O-atoms that are seven atoms further along the
peptide backbone. Each of these interactions has the graph set motif of S(10). N(12)ꢀH
forms an intermolecular H-bond with the first amide O(1’)-atom at the opposite end of
a neighboring molecule. These interactions link the molecules into extended chains
which run parallel to the [001] direction and have a graph set motif of C(14) (Fig. 5).
In analogy to the sequence 9 ! 10 ! 14, the octapeptide ester 13 was transformed
into the corresponding hexyl amide 15 (Scheme 3). Saponification of 13 with LiOH ·
H2O (4 mol-equiv.) in THF/MeOH/H2O furnished the octapeptide acid in 89% yield,

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Scheme 3
Fig. 4. ORTEP Plot [13] of the molecular structure of 14 (arbitrary numbering of the atoms; 50%
probability ellipsoids; H-atoms bonded to C-atoms and solvent molecules omitted for clarity)
which was reacted with hexylamine by using TBTU/HOBt/DIEA. After chromato-
graphic purification of the crude reaction mixture, the desired octapeptide amide p-
BrBz-(Aib-Pro)4-NHC6H13 (15) was obtained in 44% yield.
Crystals of 15 suitable for the X-ray crystal-structure determination were obtained
from the mixture CH2Cl2/AcOEt/hexane by slow evaporation of the solvent. The
molecular structure is shown in Fig. 6. The crystals are enantiomerically pure, and the

Helvetica Chimica Acta – Vol. 95 (2012)
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Fig. 5. Packing diagram of 14 showing the H-bonding interactions (uninvolved H-atoms omitted for
clarity)
absolute configuration of the molecule has been confidently determined independently
by the diffraction experiment. This confirmed that the compound has the expected (S)-
configuration at each stereogenic center. The helical conformation of the molecule is
controlled by four intramolecular H-bonds. N(1)ꢀH, N(7)ꢀH, N(13)ꢀH, and N(19)ꢀH
interact with the amide O-atoms that are seven atoms further along the peptide
backbone. Each of these interactions has the graph set motif of S(10). N(25)ꢀH, which
is unable to form an intramolecular interaction because of its position in the backbone,
forms an intermolecular H-bond with the second amide O(5’)-atom from the opposite

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Fig. 6. ORTEP Plot [13] of the molecular structure of 15 (arbitrary numbering of the atoms; 50%
probability ellipsoids; H-atoms bonded to C-atoms omitted for clarity)
end of a neighboring molecule. This interaction links the molecules into extended
chains which run parallel to the [110] direction and have a graph set motif of C(23)
(Fig. 7)2).
2.2. Synthesis of the Terminally Protected p-MeOBz-(Aib-Pro)n-OMe (20). With the
aim of studying the potential influence of a MeO group in the para-position of the
aromatic ring on the peptide structure, we prepared the hexapeptide p-MeOBz-(Aib-
Pro)3-OMe (20) as described in the previous Sect. (Scheme 4). To a solution of p-
MeOBzOH (1 mol-equiv.) in THF at 08, a solution of 1b (1.1 mol-equiv.) in THF was
added, and the mixture was stirred for 42 h at room temperature. After chromato-
graphic purification, the crude dipeptide 16 was obtained as colorless crystals in 85%
yield. Saponification of the latter with LiOH · H2O (4 mol-equiv.) in THF/MeOH/H2O
gave the dipeptide acid 17 in 98% yield. Without further purification, 17 was coupled
with 1b (1.1 mol-equiv.) in CH2Cl2 (48 h, r.t.), to afford the tetrapeptide p-MeOBz-
(Aib-Pro)2-OMe (18) in 78% yield.
Suitable crystals for the X-ray crystal-structure determination were obtained by
slow evaporation of the solvent from a solution of 18 in CHCl3. The molecular structure
is depicted in Fig. 8. The asymmetric unit contains one peptide and one highly
disordered CH2Cl2 molecule plus a site which was assigned to a H2O molecule with only
one quarter site occupancy, although this site lies quite close to the CH2Cl2 molecule
and may actually be a further disordered position belonging to the CH2Cl2 molecule
(see Exper. Part). The central five-membered ring of the peptide molecule has a
disordered envelope conformation in which the envelope flap atom, C(26), flips to
either side of the ring. The crystals are enantiomerically pure, however, the absolute
configuration of the molecule has not been determined. The enantiomer used in the
2
)
Although the conformation of the molecule is clearly defined, the quality and precision of the
results are lower than normal, as evidenced by the high R factors. This can be attributed to
untreated disorder within the molecule. In particular, the alkyl end chain and the bromophenyl
group seem to be slightly disordered. However, attempts to model the disorder were unsuccessful,
and two significant peaks of residual electron density remain in the vicinity of the Br-atom.

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Fig. 7. Packing diagram of 15 showing the H-bonding interactions (uninvolved H-atoms omitted for
clarity)
refinement was based on the known (S)-configuration at C(5) and C(11). Each NꢀH
group of the molecule acts as a donor for H-bonds. N(7)ꢀH forms an intramolecular H-
bond with the amide O-atom that is seven atoms back along the peptide backbone, thus
producing a graph set motif of S(10). N(1)ꢀH forms an intermolecular H-bond with the

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Scheme 4
Fig. 8. ORTEP Plot [13] of the molecular structure of one of the two conformers of 18 (arbitrary
numbering of the atoms; 50% probability ellipsoids; H-atoms bonded to C-atoms and solvent molecules
omitted for clarity)
second last amide O(9’)-atom from the opposite end of a neighboring molecule. This
interaction links the molecules into extended chains which run parallel to the [001]
direction and have a graph set motif of C(11) (Fig. 9).

Helvetica Chimica Acta – Vol. 95 (2012)
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Fig. 9. Packing diagram of 18 showing the H-bonding interactions (uninvolved H-atoms omitted for
clarity; only one conformation of the disordered proline ring is shown)
Basic hydrolysis of 18 under the usual conditions led to the corresponding acid 19 in
98% yield, and the latter was coupled with 1b to give the hexapeptide p-MeOBz-(Aib-
Pro)3-OMe (20) in 88% yield (Scheme 4). Unfortunately, it was not possible to grow
suitable crystals for the X-ray crystal-structure determination.
2.3. Conformational Studies. 2.3.1. Octapeptide Amide p-BrBz-(Aib-Pro)4-NHC6H13
(15). The crystal structure and packing of 15 are shown in Figs. 6 and 7, respectively. As
expected, this molecule shows a helical structure of the b-bend type (see torsion angles
of the peptide backbone in Table 1). This sub-type of the well-known 310-helical
structure is stabilized by four 1 4 intramolecular H-bonds between the NH groups of
the Aib(3), Aib(5), and Aib(7) residues, and the NH of the hexylamide moiety and the
C¼O groups of the p-BrBz moiety, and the Pro(2), Pro(4), and Pro(6) C¼O groups,
respectively (Table 2).

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Table 1. Selected Main-Chain Torsion Angles [8] of 15
Angle Aib(1)
Pro(2)
Aib(3)
Pro(4)
Aib(5)
Pro(6)
Aib(7)
Pro(8)
f
y
w
ꢀ 54.1(6) ꢀ 65.7(5) ꢀ 46.3(5) ꢀ 83.4(4) ꢀ 54.1(5) ꢀ 71.5(4) ꢀ 54.2(5) ꢀ 66.4(4)
ꢀ 30.1(6)
ꢀ 5.7(6) ꢀ 42.2(5)
ꢀ 7.1(5) ꢀ 29.8(5) ꢀ 12.0(5) ꢀ 39.4(5) ꢀ 16.9(5)
ꢀ 175.2(5) ꢀ 179.2(4) ꢀ 170.9(4) ꢀ 175.3(3) ꢀ 173.0(4) 177.6(4) ꢀ 167.9(3) ꢀ 179.9(3)
Table 2. Intra- and Intermolecular H-Bonds of 15
DꢀH ··· Aa)
DꢀH [ꢅ]
H ··· A [ꢅ]
D ··· A [ꢅ]
DꢀH ··· A [8]
N(1)ꢀH(1 ··· O(8)
0.88
0.88
0.88
0.88
0.88
2.05
2.05
2.04
2.24
2.09
2.856(5)
2.907(4)
2.883(4)
3.070(5)
2.935(4)
152
165
159
158
160
N(7)ꢀH(7) ··· O(14)
N(13)ꢀH(13) ··· O(20)
N(19)ꢀH(19) ··· O(26)
N(25)ꢀH(25) ··· O(5’)
a) Primed atoms refer to the molecule in the symmetry-related position 1 þ x, 1 þ y, z.
The Pro side-chain torsion angles of p-BrBz-(Aib-Pro)4-NHC6H13 (15) are
compiled in Table 3. They show that both C(g)-endo and C(g)-exo pyrrolidine ring
puckerings are observed [14]. This finding is in agreement with the results obtained by
Toniolo et al. who reported that these two pyrrolidine ring conformations have also
been observed in the heptapeptide p-BrBz-Aib-(Pro-Aib)3-OMe and in the nona-
Table 3. Pro Side-Chain Torsion Angles [8] of 15
Angle
Pro(2)
Pro(4)
Pro(6)
Pro(8)
q
c1
c2
c3
4.9(5)
ꢀ 14.6(4)
ꢀ 1.6(4)
3.3(5)
ꢀ 25.9(6)
33.1(4)
ꢀ 39.1(4)
29.8(4)
ꢀ 20.4(5)
ꢀ 26.1(4)
37.1(6)
35.1(5)
ꢀ 35.6(5)
23.4(4)
39.4(4)
ꢀ 33.6(6)
17.9(5)
C(g)-exo
ꢀ 36.6(4)
20.5(5)
C(g)-exo
c4
ꢀ 9.3(4)
Puckering
C(g)-endo
C(g)-exo

Helvetica Chimica Acta – Vol. 95 (2012)
1337
peptide p-BrBz-Aib-(Pro-Aib)4-OMe [5c]. These findings rule out the suggestion that
this parameter would have a marked effect on the backbone conformation, although
some theoretical calculations have shown that the C(g)-endo state is the lowest-energy
conformation for the pyrrolidine ring in poly(Aib-Pro)n peptides, which adopt fairly
rigid helical structures stabilized by 1 4 intramolecular H-bonds [15].
Information about the three-dimensional structure of the octapeptide p-BrBz-(Aib-
Pro)4-NHC6H13 (15) in solution is available from NMR data. A simple indication for
the presence of different types of H-bonds is the dependence of the NH shifts on the
temperature and on the polarity of the solvent: NH groups involved in intramolecular
H-bonds should show a very small dependence, whereas the chemical shifts of solvent-
exposed NH H-atoms should be influenced more significantly [16][17]. For the peptide
15, the involvement of the NH groups in intramolecular H-bonds was evaluated on the
basis of the solvent dependence of d(NH) in CDCl3/(D6)DMSO (solvent-titration
experiment [18]) and the temperature dependence. The results obtained from the
solvent-titration experiment are presented in Fig. 10,a. Four NH groups are almost
Fig. 10. Chemical shifts of the NH resonances of 15 as a function a) of the (D6)DMSO concentration (in
% v/v) in CDCl3 and b) of the temperature in the range of 265 – 314 K
unaffected by an increase of the proportion of (D6)DMSO over the range from 0 to
12%. We assume that these are the NH signals of Aib(3), Aib(5), Aib(7), and the
hexylamine moiety, which form four 1 4 intramolecular H-bonds as in the crystalline
state. The Dd value of the NH H-atom of the Aib(1) residue is 0.353 ppm, indicating a
stronger solvent dependence. The temperature dependence of the NH resonances [19]
is displayed in Fig. 10,b. The experimentally obtained Dd/DT values in the temperature
range of 265 – 314 K are collected in Table 4. Whereas, the temperature coefficients of
NH of Aib(3), Aib(5), Aib(7), and NHC6H13 are small (ꢀ1.66 to ꢀ 2.33 ꢁ 10ꢀ3 ppm/
K), the coefficient of the NH H-atom of Aib(1) is ꢀ 7.46 ꢁ 10ꢀ3 ppm/K, which could be
interpreted as the result of the solvent exposure of this NH H-atom. From these results,
it can be concluded that 15 adopts in solution a helical structure analogous to that in the
crystal.
2.3.2. Octapeptide Ester p-BrBz-(Aib-Pro)4-OMe (13). As crystals of 13 suitable for
1
X-ray crystallography could not be obtained, H-NMR solvent-titration experiments
were carried out, and the temperature dependence of the NH absorptions was

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Helvetica Chimica Acta – Vol. 95 (2012)
Table 4. Temperature Coefficients (ꢀ Dd/DT [ppm/K]) for Amide NH of the Peptides 15, 13, 11, 14, and
18 in the Range of 265 – 314 K
NH(Aib(1))
NH(Aib(3))
NH(Aib(5))
NH(Aib(7))
NH(C6H13)
15
13
11
14
18
ꢀ 7.46 ꢁ 10ꢀ3
ꢀ 10.20 ꢁ 10ꢀ3
ꢀ 10.55 ꢁ 10ꢀ3
ꢀ 10.58 ꢁ 10ꢀ3
ꢀ 12.10 ꢁ 10ꢀ3
ꢀ 1.66 ꢁ 10ꢀ3
ꢀ 1.82 ꢁ 10ꢀ3
ꢀ 1.60 ꢁ 10ꢀ3
ꢀ 2.80 ꢁ 10ꢀ3
ꢀ 3.89 ꢁ 10ꢀ3
ꢀ 1.88 ꢁ 10ꢀ3
ꢀ 2.40 ꢁ 10ꢀ3
ꢀ 2.23 ꢁ 10ꢀ3
ꢀ 1.16 ꢁ 10ꢀ3
ꢀ 1.44 ꢁ 10ꢀ3
–
ꢀ 1.02 ꢁ 10ꢀ3
–
–
–
–
–
–
ꢀ 2.73 ꢁ 10ꢀ3
–
determined in order to obtain information about the three-dimensional structure of the
molecule in solution. As shown in Fig. 11,a, three amide NH groups are almost
unaffected by an increase of the concentration of (D6)DMSO in the range of 0 to 12%
(v/v). Based on the similarity with the results obtained for other described molecules,
we assume that these are the NH H-atoms of Aib(3), Aib(5), and Aib(7), which are
able to form three 1 4 intramolecular H-bonds. On the other hand, NH of Aib(1)
shows a small but significant dependence on the (D6)DMSO concentration. The
experimental values of the temperature dependent NH resonances are shown in
Fig. 11,b, and the values Dd/DT in the range of 265 – 314 K are compiled in Table 4.
Only NH of Aib(1) shows a significant dependence on the temperature. The coefficient
has been determined to be ꢀ 10.2 ꢁ 10ꢀ3 ppm/K, which established that this NH H-
atom is exposed to the solvent.
Fig. 11. Chemical shifts of the NH resonances of 13 as a function a) of the (D6)DMSO concentration (in
% v/v) in CDCl3 and b) of the temperature in the range of 265 – 314 K
2.3.3. Hexapeptide Ester p-BrBz-(Aib-Pro)3-OMe (11). The crystal structure and
packing of 11 are shown in Figs. 2 and 3, respectively. In Table 5, the most relevant
torsion angles of the peptide backbone are collected. The molecule also adopts a b-
bend ribbon structure, which is stabilized by two 1 4 intramolecular H-bonds
between the NH groups of the Aib(3) and Aib(5) residues, and the C¼O groups of the
p-BrBz-moiety and Pro(2), respectively (Table 6). In analogy to 15, both C(g)-endo
(Pro(4)) and C(g)-exo pyrrolidine ring puckerings (Pro(2) and Pro(6)) are observed.
The involvement of the NH groups of 11 in intramolecular H-bonds in solution was
evaluated on the basis of the temperature dependence of the NH chemical shifts in the

Helvetica Chimica Acta – Vol. 95 (2012)
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Table 5. Selected Main-Chain Torsion Angles [8] of 11
Angle
Aib(1)
Pro(2)
Aib(3)
Pro(4)
Aib(5)
Pro(6)
f
y
w
ꢀ 53.8(7)
ꢀ 28.9(7)
ꢀ 177.8(5)
ꢀ 69.4(7)
ꢀ 18.9(7)
178.6(5)
ꢀ 53.4(7)
ꢀ 46.8(7)
ꢀ 171.2(5)
ꢀ 81.2(6)
ꢀ 8.8(8)
ꢀ 56.9(7)
ꢀ 45.8(7)
ꢀ 169.7(5)
ꢀ 65.5(7)
ꢀ 40.5(9)
ꢀ 174.4(5)
ꢀ 177.8(5)
Table 6. Intra- and Intermolecular H-Bonds in 11
DꢀH ··· Aa)
DꢀH [ꢅ]
H ··· A [ꢅ]
D ··· A [ꢅ]
DꢀH ··· A [8]
N(1)ꢀH(1) ··· O(12’)
N(7)ꢀH(7) ··· O(21)
N(13)ꢀH(13) ··· O(5)
0.88
0.88
0.88
2.22
2.04
2.18
3.044(6)
2.901(6)
2.978(6)
155
167
150
a) Primed atoms refer to the molecule in the symmetry-related position 1 ꢀ x, y, ꢀ z.
temperature range of 265 – 314 K (Fig. 12). The temperature coefficients of the amide
NH resonances are collected in Table 4. The value for the NH group of Aib(1) is
ꢀ 10.55 ꢁ 10ꢀ3 ppm/K, which indicates that this H-atom is more or less freely exposed
to the solvent. On the other hand, NH(Aib(3)) and NH(Aib(5)) show temperature
dependences, which are ca. 10 times smaller. Therefore, it can be concluded that they
form intramolecular H-bonds.
Fig. 12. Chemical shifts of the NH resonances of 11 as a function of the temperature in the range of 265 –
314 K
2.3.4. Tetrapeptide Amide p-BrBz-(Aib-Pro)2-NHC6H13 (14). The crystal structure
and packing of 14 are depicted in Figs. 4 and 5. As expected, the molecule adopts a b-
bend ribbon structure, similar to the hexapeptide ester 11 (for relevant torsion angles,
see Table 7). There is evidence for two 1 4 intramolecular H-bonds between the NH
groups of the hexylamine residue and of Aib(3) and the C¼O groups of the p-BrBz
moiety and Pro(2), respectively (Table 8). Again, both types of pyrrolidine con-
formations are present (Pro(2): C(g)-exo; Pro(4): C(g)-endo).
Analogous 1H-NMR experiments as in the previous cases were conducted in order
to establish the H-bonding interactions and, on this basis, to propose the preferred

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Helvetica Chimica Acta – Vol. 95 (2012)
Table 7. Selected Main-Chain Torsion Angles [8] of 14
Angle
Aib(1)
Pro(2)
Aib(3)
Pro(4)
f
y
w
ꢀ 48.1(3)
ꢀ 38.7(3)
179.7(2)
ꢀ 63.7(2)
ꢀ 28.5(3)
ꢀ 178.0(2)
ꢀ 55.9(3)
ꢀ 48.9(2)
ꢀ 166.7(2)
ꢀ 81.1(2)
ꢀ 4.4(3)
179.2(2)
Table 8. Intra- and Intermolecular H-Bonds in 14
DꢀH ··· Aa)
DꢀH [ꢅ]
H ··· A [ꢅ]
D ··· A [ꢅ]
DꢀH ··· A [8]
N(1)ꢀH(1) ··· O(7)
N(6)ꢀH(6) ··· O(13)
N(12)ꢀH(12) ··· O(1’)
0.83(2)
0.83(2)
0.89(2)
2.17(2)
2.12(2)
1.96(2)
2.956(3)
2.912(2)
2.839(2)
158(2)
160(2)
170(2)
a
1
) Primed atoms refer to the molecule in the symmetry-related position 11ꢆ2 ꢀ x, 1 ꢀ y, ꢀ ꢆ2 þ z.
conformation of 14 in solution. The results obtained from the solvent-titration
experiment are presented in Fig. 13,a. It is obvious that two NH resonances are almost
unaffected by the polarity of the solvent; we suggest that these correspond to the NH
groups of Aib(3) and the hexylamine moiety, which form two intramolecular H-bonds,
as in the crystalline state. In contrast, the NH H-atom resonance of Aib(1) shows a
significant solvent dependence. The experimentally obtained Dd/DT values in the
temperature range of 265 – 314 K are collected in Table 4 (Fig. 13,b). The temperature
coefficient for NH of Aib(1) is ꢀ 10.58 ꢁ 10ꢀ3 ppm/K, which indicates that this NH H-
atom is exposed to the solvent. The corresponding values for NH(Aib(3)) and
NHC6H13 are ꢀ 2.8 ꢁ 10ꢀ3 and ꢀ 2.73 ꢁ 10ꢀ3 ppm/K, respectively, providing a strong
evidence that these NH groups are involved in intramolecular H-bonds.
Fig. 13. Chemical shifts of the NH resonances of 14 as a function a) of the (D6)DMSO concentration (in
% v/v) in CDCl3 and b) of the temperature in the range of 265 – 314 K
2.3.5. Tetrapeptide Ester p-MeOBz-(Aib-Pro)2-OMe (18). The crystal structure and
packing of 18 are shown in Figs. 8 and 9, respectively. The molecule adopts a helical
conformation (b-turn; Table 9) with one 1 4 intramolecular H-bond between the NH
group of the Aib(3) residue and the C¼O group of the p-MeOBz-moiety (Table 10).

Helvetica Chimica Acta – Vol. 95 (2012)
1341
Table 9. Selected Main-Chain Torsion Angles [8] of 18
Angle
Aib(1)
Pro(2)
Aib(3)
Pro(4)
f
y
w
ꢀ 54.2(5)
ꢀ 41.3(5)
ꢀ 169.7(4)
ꢀ 74.7(5)
ꢀ 15.9(6)
ꢀ 177.1(3)
53.6(5)
44.5(5)
169.4(4)
ꢀ 66.8(5)
ꢀ 38.6(7)
177.4(4)
Table 10. Intra- and Intermolecular H-Bons in 18
DꢀH ··· Aa)
DꢀH [ꢅ]
H ··· A [ꢅ]
D ··· A [ꢅ]
DꢀH ··· A [8]
N(1)ꢀH(1) ··· O(9’)
0.94(5)
0.86(3)
2.04(5)
2.11(4)
2.908(5)
2.963(5)
154(4)
169(4)
N(6)ꢀH(6) ··· O(15)
a) Primed atoms refer to the molecule in the symmetry-related position x, y, 1 þ z.
Table 11. Pro Side-Chain Torsion Angles [8] of the Two Conformers of 18
Angle
Pro(2) conform. A
Pro(4) conform. Aa)
Pro(2) conform. B
q
c1
c2
c3
ꢀ 9.3(5)
ꢀ 12.3(9)
28.2(12)
ꢀ 4.6(6)
26.1(6)
ꢀ 9.3(5)
27.0(7)
ꢀ 38.2(7)
ꢀ 34.1(8)
ꢀ 32.9(11)
34.5(7)
28.0(8)
c4
27.1(9)
ꢀ 18.4(6)
C(g)-endo
ꢀ 11.2(7)
C(g)-endo
Type of puckering
C(g)-exo
a) The disorder for Pro(4) could not be modelled satisfactorily. Therefore, the values for Pro(4) may be
partly an average of the two conformers.
The central five-membered ring of the peptide molecule has a disordered envelope
conformation, in which the envelope flap atom, C(26), flips to either side of the ring,
and, therefore, both C(g)-endo and C(g)-exo types of puckerings are found (Table 11).
As in all other cases, the preferred conformation of the tetrapeptide 18 in solution
1
was established by H-NMR experiments. The results obtained from the solvent-
titration experiment are presented in Fig. 14,a. One NH resonance is almost unaffected
when the concentration of (D6)DMSO was increased from 0 to 12%, and it is very
likely that this is the NH signal of Aib(3), which forms a 1 4 intramolecular H-bond
as in the crystalline state. The chemical shift of the other NH group was strongly
dependent on the (D6)DMSO concentration. The experimentally obtained coefficients
Dd/DT in the temperature range of 265 – 314 K (Fig. 14,b) are collected in Table 4. The
temperature coefficient of the NH H-atom of Aib(1) is ꢀ 12.1 ꢁ 10ꢀ3 ppm/K, i.e., this
NH H-atom is exposed to the solvent. The second NH group, with a temperature
coefficient of ꢀ 3.89 ꢁ 10ꢀ3 ppm/K, is assumed to be involved in an intramolecular H-
bond.
3. Conclusions. – The presented results show that methyl N-(2,2-dimethyl-2H-
azirin-3-yl)-l-prolinate (1b) is a suitable building block for the incorporation of the

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Helvetica Chimica Acta – Vol. 95 (2012)
Fig. 14. Chemical shifts of the NH resonances of 18 as a function a) of the (D6)DMSO concentration (in
% v/v) in CDCl3 and b) of the temperature in the range of 265 – 314 K
dipeptide unit Aib-Pro in a peptide backbone not only in the synthesis of segments of
naturally occurring petaibols, such as zervamicin II-2 [8d], trichovirin I 1B [8e], and
hypomurocin A1 [8f], but also in the cases of (Aib-Pro)n oligopeptides. Using the
advantages of the ꢃazirine couplingꢄ, we prepared highly constrained peptides,
containing consecutive (Aib-Pro) units, of the type p-BrBz-(Aib-Pro)n-OMe, p-
BrBz-(Aib-Pro)n-NHC6H13, and p-MeO-(Aib-Pro)n-OMe in order to study their
conformations in the solid state and in solution. The results revealed that the preferred
conformation is the b-bend ribbon structure, forming a sub-class of the 310-helices, in
agreement with the results obtained by Venkatachalapathi and Balaram [5a] and
Toniolo and co-workers [5c].
We thank the Analytical Sections of our institute for spectra and analyses, and the Swiss National
Science Foundation and F. Hoffmann-La Roche AG, Basel, for financial support.
Experimental Part
1. General. Solvents were purified by standard procedures. TLC: Merck TLC aluminum sheets, silica
gel 60 F254. Column chromatography (CC): Uetikon-Chemie, silica gel C-560 (0.04 – 0.063 mm, 230 – 400
mesh). M.p.: Bꢀchi Melting Point B-450 apparatus; uncorrected. IR Spectra: Perkin-Elmer, Spectrum one
FT-IR spectrophotometer; in KBr unless otherwise stated; n˜ in cmꢀ1. NMR Spectra: Bruker AC-300 (1H,
13C, DEPT) at 300 and 75 MHz, resp., or Bruker DRX-600 (1H, 13C, HSQC, HMBC, COSY) at 600 and
150 MHz, resp., in CDCl3 at 300 K unless otherwise stated; d in ppm, coupling constants J in Hz; 13C-
signal multiplicities from DEPT spectra. MS: Finnigan SSQ-700 (CI with NH3), or Finningan TSQ-700
instrument (ESI); m/z (rel. %).
2. General Procedures. General Procedure 1 (GP 1). To a soln. of a peptide acid, 4-bromobenzoic
acid (p-BrBz-OH) or 4-methoxybenzoic acid (p-MeOBz-OH) in dry THF or dry CH2Cl2, 2H-azirin-3-
amine 1b (1.1 mol-equiv.) was added, and the mixture was stirred at r.t. After completion of the reaction
(TLC), the soln. was concentrated in vacuo, and the residue was purified by CC (SiO2). The solvent was
evaporated, and the solid material was used without further purification.
General Procedure 2 (GP 2). To a soln. of the corresponding peptide methyl ester in THF/MeOH/
H2O 3 :1:1, LiOH · H2O (4 mol-equiv.) was added. The mixture was stirred at r.t. After completion of the
reaction (TLC), 1m HCl was added until pH 1 was reached, and the org. solvent was evaporated. The
residue was extracted with CH2Cl2 or AcOEt, the org. phases were dried (MgSO4), the solvent was
evaporated, and the residue was dried under h.v.
General Procedure 3 (GP 3). To a soln. of the peptide acid in dry CH2Cl2 were added 1-hydroxy-1H-
benzotriazole (HOBt, 1 mol-equiv.), 2-[(1H-benzotriazol-1-yl)oxy]-1,1,3,3-tetramethyluronium tetra-

Helvetica Chimica Acta – Vol. 95 (2012)
1343
fluoroborate (TBTU, 1 mol-equiv.), and (ethyl)(diisopropyl)amine (EtN(i-Pr)2, DIEA; 2 mol-equiv.).
A soln. of the amino component (1 mol-equiv.) was added dropwise, and the mixture was stirred at r.t.
After completion of the reaction (TLC), the soln. was washed with 1m HCl, sat. NaHCO3, and NaCl
soln., dried (MgSO4), evaporated, and purified by CC (SiO2). The solvent was evaporated, and the solid
material was used without further purification.
3. Starting Materials. The dipeptide synthon, methyl 1-(2,2-dimethyl-2H-azirin-3-yl)-l-prolinate (1b),
was prepared according to [8b], all other chemicals were commercially available (Fluka, Aldrich).
4. Synthesis of Aib-Pro Oligopeptides. Methyl N-(4-Bromobenzoyl)-2-methylalanylprolinate (p-
BrBz-Aib-Pro-OMe; 7). According to GP 1, p-BrBz-OH (253 mg, 1.26 mmol) in dry THF (10 ml) and 1b
(272 mg, 1.386 mmol); stirring for 103 h; CC (CH2Cl2/MeOH from 150 :1 to 20 :1): 446 mg (89%) of 7.
Colorless crystals. M.p. 2308. IR: 3296m, 3054m, 2982m, 2952m, 2869w, 1750s, 1663s, 1616s, 1590w, 1538s,
1482m, 1422s, 1383w, 1362m, 1313m, 1286w, 1205s, 1168s, 1153w, 1096w, 1069m, 1012m, 989w, 906m,
847m, 792w, 764m, 749w, 709w, 655w. 1H-NMR: 7.72 (br. s, NH); 7.70 – 7.64 (m, 2 arom. H); 7.75 – 7.50 (m,
2 arom. H); 4.61 – 4.57 (m, CH(a)(Pro)); 3.84 – 3.77 (m, 1 H of CH2(d)(Pro)); 3.73 (s, MeO); 3.67 – 3.59
(m, 1 H of CH2(d)(Pro)); 2.19 – 2.01 (m, CH2(b)(Pro)); 2.03 – 1.90 (m, CH2(g)(Pro)); 1.79, 1.77 (2s,
2 Me(Aib)). 13C-NMR: 172.8, 172.7 (2s, 2 C¼O); 164.5 (s, ArC¼O); 133.8 (s, 1 arom. C); 131.7 (d, 2 arom.
CH); 128.5 (d, 2 arom. CH); 126.0 (s, 1 arom. C); 61.1 (d, CH(a)(Pro)); 57.4 (s, C(a)(Aib)); 52.2 (q,
MeO); 48.3 (t, CH2(d))(Pro)); 27.7 (t, CH2(b)(Pro)); 25.8 (t, CH2(g)(Pro)); 23.0, 22.8 (2q, 2 Me(Aib)).
CI-MS: 528 (100, [M(81Br) þ (Pro-OMe) þ H]þ), 526 (90, [M(79Br) þ (Pro-OMe) þ H]þ), 448 (18), 399
(9, [M(81Br) þ H]þ), 397 (9, [M(79Br) þ H]þ), 130 (52, [(Pro-OMe) þ H]þ).
N-[N-(4-Bromobenzoyl)-2-methylalanyl]proline (p-BrBz-Aib-Pro-OH; 8). According to GP 2, 7
(400 mg, 1.01 mmol) in 15 ml of THF/MeOH/H2O, LiOH · H2O (169.5 mg, 4.04 mmol); stirring for 3 h:
371 mg (96%) of 8. The product was pure enough to be used in the next step without further purification.
IR: 3281m, 3068w, 2988m, 2927w, 1714s, 1640s, 1622s, 1590w, 1531m, 1482m, 1470w, 1416m, 1383w, 1365w,
1323m, 1240w, 1205s, 1165m, 1111w, 1095w, 1071m, 1011m, 907m, 881w, 845m, 793w, 759m, 709w, 683w,
625w. 1H-NMR ((D6)DMSO): 8.59 (s, NH); 7.84 – 7.81 (m, 2 arom. H); 7.69 – 7.66 (m, 2 arom. H); 4.24 –
4.21 (m, CH(a)(Pro)); 3.70 – 3.66 (m, 1 H of CH2(d)(Pro)); 3.23 – 3.17 (m, 1 H of CH2(d)(Pro)); 1.94 –
1.86 (m, CH2(b)(Pro)); 1.85 – 1.71 (m, CH2(g)(Pro)); 1.40 (s, 2 Me(Aib)). 13C-NMR ((D6)DMSO):
174.1, 171.5 (2s, 2 C¼O); 164.9 (s, ArC¼O); 133.5 (s, 1 arom. C); 131.7 (d, 2 arom. CH); 130.1 (d, 2 arom.
CH); 125.5 (s, 1 arom. C); 60.6 (d, CH(a)(Pro)); 56.7 (s, C(a)(Aib)); 47.8 (t, CH2(d))(Pro)); 27.9 (t,
CH2(b)(Pro)); 25.8 (t, CH2(g)(Pro)); 25.7, 24.9 (2q, 2 Me(Aib)). ESI-MS: 500 (96, [M(81Br) þ (Pro-
OH) þ H]þ), 498 (100, [M(79Br) þ (Pro-OH) þ H]þ), 385 (50, [M(81Br) þ H]þ), 383 (54, [M(79Br) þ
H]þ), 116 (100, [(Pro-OH) þ H]þ).
N-(N-{N-[N-(4-Bromobenzoyl)-2-methylalanyl]prolyl}-2-methylalanyl)proline Methyl Ester (p-
BrBz-(Aib-Pro)2-OMe; 9). According to GP 1, 8 (371 mg, 0.969 mmol) in dry CH2Cl2 (20 ml) and 1b
(209 mg, 1.066 mmol); stirring for 27 h, CC (CH2Cl2/MeOH from 150 :1 to 20 :1): 556 mg (99%) of 9.
M.p. 2678 (dec.). IR: 3300m, 2984m, 2927m, 2874m, 1746s, 1646s, 1589w, 1537s, 1482w, 1468w 1403m,
1378w, 1326w, 1302m, 1243w, 1201w, 1168s, 1111w, 1094m, 1071m, 1049w, 956w, 944w, 907w, 880w, 848s,
761s, 708w, 631w, 625m, 612w. 1H-NMR: 7.81 – 7.78 (m, 2 arom. H); 7.79 (s, NH); 7.64 – 7.61 (m, 2 arom. H,
NH); 4.60 – 4.55 (m, CH(a)(Pro)); 4.40 – 4.36 (m, CH(a)(Pro)); 3.93 – 3.86 (m, 1 H of CH2(d)(Pro));
3.76 – 3.71 (m, 1 H of CH2(d)(Pro)); 3.63 (s, MeO); 3.60 – 3.55 (m, 1 H of CH2(d)(Pro)); 3.26 – 3.23 (m,
1 H of CH2(d)(Pro)); 2.15 – 2.12 (m, 1 H of CH2(b)(Pro)); 2.10 – 1.91 (m, 3 H of 2 CH2(b)(Pro)); 1.84 –
1.78 (m, 4 H, 2 CH2(g)(Pro)); 1.66, 1.58, 1.54 (3s, 4 Me(Aib)). 13C-NMR: 173.2, 172.4, 172.3, 171.3 (4s,
4 C¼O); 165.8 (s, ArC¼O); 133.8 (s, 1 arom. C); 132.0 (d, 2 arom. CH); 129.1 (d, 2 arom. CH); 126.8 (s, 1
arom. C); 62.7, 60.6 (2d, 2 CH(a)(Pro)); 57.5, 56.6 (2s, 2 C(a)(Aib)); 51.9 (q, MeO); 48.2, 47.7 (2t,
2 CH2(d))(Pro)); 28.4, 28.0 (2t, 2 CH2(b)(Pro)); 26.0, 25.9 (2t, 2 CH2(g)(Pro)); 25.9, 25.1, 24.7, 24.5 (4q,
4 Me(Aib)). ESI-MS: 603 (18, [M(81Br) þ Na]þ), 601 (19, [M(79Br) þ Na]þ), 581 (10, [M(81Br) þ H]þ),
579 (10, [M(79Br) þ H]þ), 452 (100, [M(81Br) ꢀ (Pro-OMe) þ H]þ), 450 (92, [M(79Br) ꢀ (Pro-OMe) þ
H]þ), 367 (9, [M(81Br) ꢀ (Aib-Pro-OMe) þ H]þ), 365 (10, [M(79Br) ꢀ (Aib-Pro-OMe) þ H]þ).
N-(N-{N-[N-(4-Bromobenzoyl)-2-methylalanyl]prolyl}-2-methylalanyl)proline (p-BrBz-(Aib-
Pro)2-OH; 10). According to GP 2, 9 (503 mg, 0.869 mmol) in 20 ml of THF/MeOH/H2O, LiOH · H2O
(145.8 mg, 3.476 mmol); stirring for 22 h: 455 mg (93%) of 10. The isolated peptide acid was pure enough
to be used in the next step without further purification. 1H-NMR (CD3OD): 8.72, 8.04 (2s, 2 NH); 7.83 –

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Helvetica Chimica Acta – Vol. 95 (2012)
7.79 (m, 2 arom. H); 7.69 – 7.64 (m, 2 arom. H); 4.51 – 4.46 (m, CH(a)(Pro)); 4.44 – 4.40 (m,
CH(a)(Pro)); 3.82 – 3.74 (m, 1 H of CH2(d)(Pro)); 3.72 – 3.68 (m, 2 H of 2 CH2(d)(Pro)); 3.42 – 3.34
(m, 1 H of CH2(d)(Pro)); 2.22 – 2.05 (m, 2 H of 2 CH2(b)(Pro)); 1.98 – 1.75 (m, 6 H, CH2(b)(Pro),
2 CH2(g)(Pro)); 1.59, 1.56, 1.54, 1.50 (4s, 4 Me(Aib)). 13C-NMR (CD3OD): 173.8, 171.6, 171.2, 171.1 (4s,
4 C¼O); 165.5 (s, ArC¼O); 132.7 (s, 1 arom. C); 131.6 (d, 2 arom. CH); 129.8 (d, 2 arom. CH); 125.7 (s, 1
arom. C); 61.8, 60.2 (2d, 2 CH(a)(Pro)); 56.7, 55.7 (2s, 2 C(a)(Aib)); 47.9, 47.1 (2t, 2 CH2(d))(Pro)); 28.2,
27.7 (2t, 2 CH2(b)(Pro)); 25.5, 25.4 (2t, 2 CH2(g)(Pro)); 25.8, 25.5, 25.2, 24.5 (4q, 4 Me(Aib)). ESI-MS:
589 (32, [M(81Br) þ Na]þ), 587 (29, [M(79Br) þ Na]þ), 567 (13, [M(81Br) þ H]þ), 565 (13, [M(79Br) þ
H]þ), 452 (100, [M(81Br) ꢀ (Pro-OH) þ H]þ), 450 (98, [M(79Br) ꢀ (Pro-OH) þ H]þ), 367 (18,
[M(81Br) ꢀ (Aib-Pro-OH) þ H]þ), 365 (18, [M(79Br) ꢀ (Aib-Pro-OH) þ H]þ).
N-{N-[N-(N-{N-[-N-(4-Bromobenzoyl)-2-methylalanyl]prolyl}-2-methylalanyl)prolyl]-2-methyl}-
alanyl}proline Methyl Ester (p-BrBz-(Aib-Pro)3-OMe; 11). According to GP 1, 10 (455 mg, 0.805 mmol)
in dry CH2Cl2 (20 ml) and 1b (174 mg, 0.886 mmol); stirring for 96 h, CC (CH2Cl2/MeOH from 150 :1 to
20 :1): 553 mg (90%) of 11. M.p. 3028 (dec.). IR: 3272m, 2981m, 2945m, 2880w, 1749s, 1640s, 1588w,
1536s, 1480w, 1470w, 1406s, 1363w, 1198m, 1168s, 1110w, 1096w, 1071w, 1047w, 1009m, 853m, 810w, 760m,
1
709w, 664w, 611m. H-NMR: 8.32, 7.98 (2s, 2 NH); 7.87 – 7.84 (m, 2 arom. H); 7.67 – 7.63 (m, 2 arom. H,
NH); 7.66 (s, NH); 4.59 – 4.54 (m, 2 CH(a)(Pro)); 4.38 – 4.34 (m, CH(a)(Pro)); 4.00 – 3.86 (m, 2 H of
3 CH2(d)(Pro)); 3.83 – 3.76 (m, 2 H of 3 CH2(d)(Pro)); 3.61 (s, MeO); 3.27 – 3.18 (m, 2 H of
3 CH2(d)(Pro)); 2.27 – 2.07 (m, 4 H); 2.04 – 1.77 (m, 8 H); 1.64, 1.61, 1.59, 1.54, 1.49, 1.48 (6s, 6 Me(Aib)).
13C-NMR: 173.4, 172.8, 172.7, 172.7, 172.2, 171.9 (6s, 6 C¼O); 166.3 (s, ArC¼O); 132.1 (d, 2 arom. CH);
131.9 (s, 1 arom. C); 129.3 (d, 2 arom. CH); 126.9 (s, 1 arom. C); 62.5, 62.3, 60.6 (3d, 3 CH(a)(Pro)); 57.5,
56.5, 56.4 (3s, 3 C(a)(Aib)); 51.8 (q, MeO); 48.4, 48.1, 47.8 (3t, 3 CH2(d))(Pro)); 29.0, 28.2, 28.1 (3t,
3 CH2(b)(Pro)); 26.3, 26.1, 25.9 (3t, 3 CH2(g)(Pro)); 25.9, 25.1, 24.5, 24.4, 24.4, 24.1 (6q, 6 Me(Aib)).
ESI-MS: 785 (100, [M(81Br) þ Na]þ), 783 (90, [M(79Br) þ Na]þ), 634 (7, [M(81Br) ꢀ (Pro-OMe) þ H]þ),
632 (7, [M(79Br) ꢀ (Pro-OMe) þ H]þ).
Suitable crystals of 11 for the X-ray crystal-structure determination were grown from CH2Cl2/
AcOEt by slow evaporation of the solvent at r.t.
1-[N-(4-Bromobenzoyl)-2-methylalanyl]-N-{1-[2-(hexylcarbamoyl)pyrrolidin-2-yl]-2-methyl-1-oxo-
propan-2-yl}prolinamide (p-BrBz-(Aib-Pro)2-NHC6H13
; 14). According to GP 3, 10 (281 mg,
0.497 mmol), hexylamine (60.35 mg, 0.596 mmol), HOBt (67.2 mg, 0.497 mmol), TBTU (159.6 mg,
0.497 mmol), DIEA (128.5 mg, 0.994 mmol), dry CH2Cl2 (15 ml); stirring for 20 h, CC (CH2Cl2/MeOH
from 150 :1 to 20 :1): 290 mg (90%) of 14. M.p. 1328. IR: 3284m, 2983w, 2931m, 2872w, 1639s, 1589w,
1542s, 1483w, 1468w 1403m, 1378w, 1363w, 1316w, 1200w, 1174w, 1170m, 1010w, 922w, 851m, 761m, 728m.
1H-NMR: 8.03, 7.99 (2s, 2 NH); 7.87 – 7.84 (m, 2 arom. H); 7.75 (s, NH); 7.63 – 7.60 (m, 2 arom. H); 4.62 –
4.57 (m, CH(a)(Pro)); 4.55 – 4.51 (m, CH(a)(Pro)); 3.96 – 3.85 (m, 2 H of 2 CH2(d)(Pro)); 3.79 – 3.65
(m, 1 H of 2 CH2(d)(Pro)); 3.35 – 3.12 (m, 2 H); 3.06 – 2.96 (m, 1 H of 2 CH2(d)(Pro)); 2.33 – 2.11 (m,
2 H); 2.02 – 1.81 (m, 6 H); 1.76 – 1.51 (m, 8 H); 1.49, 1.43 (2s, 4 Me(Aib)); 0.88 – 0.84 (m, 3 H). 13C-NMR:
172.6, 172.6, 172.4, 172.3 (4s, 4 C¼O); 166.3 (s, ArC¼O); 131.9 (d, 2 arom. CH); 131.8 (s, 1 arom. C); 129.2
(d, 2 arom. CH); 126.9 (s, 1 arom. C); 62.4 (d, CH(a)(Pro)); 57.4, 56.5 (2s, 2 C(a)(Aib)); 54.9 (d,
CH(a)(Pro)); 48.3, 48.2 (2t, 2 CH2(d))(Pro)); 39.6 (t, CH2); 31.5, 29.2, 28.9, 28.8, 28.8, 26.6, 26.2, 25.8 (8t,
8 CH2); 26.4, 26.3, 24.4, 24.1 (4q, 4 Me(Aib)); 14.1 (q, Me). ESI-MS: 672 (15, [M(81Br) þ Na]þ), 670 (15,
[M(79Br) þ Na]þ), 650 (27, [M(81Br) þ H]þ), 648 (24, [M(79Br) þ H]þ) 452 (28, [M(81Br) ꢀ (Pro-
NHC6H13) þ H]þ), 450 (28, [M(79Br) ꢀ (Pro-NHC6H13) þ H]þ), 199 (5, [(Pro-NHC6H13) þ H]þ), 130
(100, [(Pro-OMe) þ H]þ).
Suitable crystals of 14 for the X-ray crystal-structure determination were grown from CH2Cl2 by
slow evaporation of the solvent at r.t.
N-{N-[N-(N-{N-[N-(4-Bromobenzoyl)-2-methylalanyl]prolyl}-2-methylalanyl)prolyl]-2-methylala-
nyl}proline (p-BrBz-(Aib-Pro)3-OH; 12). According to GP 2, 11 (500 mg, 0.657 mmol) in 30 ml of THF/
MeOH/H2O, LiOH · H2O (110 mg, 2.628 mmol), stirring for 24 h: 490 mg (99%) of 12.The isolated
peptide acid was pure enough to be used in the next step without further purification. 1H-NMR
((D6)DMSO): 12.1 (br. s, OH); 9.22 (s, NH); 7.98 – 7.94 (m, 2 arom. H); 7.93 (s, NH); 7.74 – 7.71 (m, 2
arom. H, NH); 7.59 (s, NH); 4.38 – 4.33 (m, 2 CH(a)(Pro)); 4.21 – 4.17 (m, CH(a)(Pro)); 3.78 – 3.59 (m,
3 H of 3 CH2(d)(Pro)); 3.59 – 3.55 (m, 2 H of 3 CH2(d)(Pro)); 3.18 – 3.14 (m, 1 H of 3 CH2(d)(Pro));

Helvetica Chimica Acta – Vol. 95 (2012)
1345
2.09 – 2.02 (m, 3 H); 1.87 – 1.63 (m, 9 H); 1.48, 1.45, 1.37, 1.30 (4s, 6 Me(Aib)). 13C-NMR ((D6)DMSO):
173.6, 172.7, 172.2, 172.0, 171.7, 171.1 (6s, 6 C¼O); 165.6 (s, ArC¼O); 132.4 (s, 1 arom. C); 131.4 (d, 2
arom. CH); 129.9 (d, 2 arom. CH); 125.6 (s, 1 arom. C); 61.9, 61.3, 60.1 (3d, 3 CH(a)(Pro)); 56.6, 55.7,
55.5 (3s, 3 C(a)(Aib)); 47.9, 47.4, 47.1 (3t, 3 CH2(d))(Pro)); 28.4, 28.1, 27.6 (3t, 3 CH2(b)(Pro)); 25.7, 25.2,
25.0 (3t, 3 CH2(g)(Pro)); 25.7, 25.2, 24.9, 24.3, 24.2, 23.9 (6q, 6 Me(Aib)). ESI-MS: 771 (34, [M(81Br) þ
Na]þ), 769 (23, [M(79Br) þ Na]þ), 749 (17, [M(81Br) þ H]þ), 747 (17, [M(79Br) þ H]þ), 634 (100,
[M(81Br) ꢀ (Pro-OH) þ H]þ), 632 (75, [M(79Br) ꢀ (Pro-OH) þ H]þ), 452 (20, [M(81Br) ꢀ (Pro-Aib-Pro-
OH) þ H]þ), 450 (20, [M(79Br) ꢀ (Pro-Aib-Pro-OH) þ H]þ).
N-[N-(N-{N-[N-(N-{N-[N-(4-Bromobenzoyl)-2-methylalanyl]prolyl}-2-methylalanyl)prolyl]-2-
methylalanyl}prolyl)-2-methylalanyl]proline Methyl Ester (p-BrBz-(Aib-Pro)4-OMe; 13). According to
GP 1, 12 (490 mg, 0.656 mmol) in dry THF (30 ml) and 1b (141 mg, 0.72 mmol); stirring for 120 h, CC
(CH2Cl2/MeOH from 150 :1 to 10 :1): 151 mg (24%) of 13. M.p. 3028 (dec.). IR: 3285m, 2984m, 2938m,
2876w, 1745m, 1643s, 1590w, 1536m, 1469w, 1407s, 1380w, 1363w, 1304w, 1245w, 1202m, 1171m, 1094w,
1
1071w, 1011m, 926w, 849w, 761m, 615w. H-NMR ((D6)DMSO): 8.98, 7.95 (2s, 2 NH); 7.90 – 7.87 (m, 2
arom. H); 7.81 (s, NH); 7.76 – 7.73 (m, 2 arom. H); 7.57 (s, NH); 4.41 – 4.31 (m, 3 CH(a)(Pro)); 4.25 – 4.21
(m, CH(a)(Pro)); 3.78 – 3.61 (m, 6 H of 4 CH2(d)(Pro)); 3.59 (s, MeO); 3.57 – 3.51 (m, 1 H of
CH2(d)(Pro)); 3.19 – 3.16 (m, 1 H of CH2(d)(Pro)); 2.13 – 1.99 (m, 4 H); 1.97 – 1.63 (m, 12 H); 1.48, 1.47,
1.38, 1.36, 1.30 (5s, 8 Me(Aib)). 13C-NMR ((D6)DMSO): 173.2, 172.7, 172.5, 172.1, 172.0, 171.8, 171.5,
171.4 (8s, 8 C¼O); 166.0 (s, ArC¼O); 132.9 (s, 1 arom. C); 131.6 (d, 2 arom. CH); 130.1 (d, 2 arom. CH);
126.0 (s, 1 arom. C); 62.3, 61.9, 61.7, 60.3 (4d, 4 CH(a)(Pro)); 56.9, 56.2, 56.0, 55.8 (4s, 4 C(a)(Aib)); 51.8
(q, MeO); 48.3, 47.9, 47.6, 47.5 (4t, 4 CH2(d))(Pro)); 28.9, 28.8, 28.5, 27.9 (4t, 4 CH2(b)(Pro)); 25.6, 25.4,
25.3, 25.2 (4t, 4 CH2(g)(Pro)); 26.1, 26.0, 25.9, 25.4, 24.5, 24.4, 24.3, 24.2 (8q, 8 Me(Aib)). ESI-MS: 967
(68, [M(81Br) þ Na]þ), 965 (47, [M(79Br) þ Na]þ), 816 (100, [M(81Br) ꢀ (Pro-OMe) þ H]þ), 814 (89,
[M(79Br) ꢀ (Pro-OMe) þ H]þ), 634 (76, [M(81Br) ꢀ (Pro-Aib-Pro-OMe) þ H]þ), 632 (83, [M(79Br) ꢀ
(Pro-Aib-Pro-OMe) þ H]þ), 452 (26, [M(81Br) ꢀ (Pro-Aib-Pro-Aib-Pro-OMe) þ H]þ), 450 (25,
[M(79Br) ꢀ (Pro-Aib-Pro-Aib-Pro-OMe) þ H]þ).
N-[N-(N-{N-[N-(N-{N-[N-(4-Bromobenzoyl)-2-methylalanyl]prolyl}-2-methylalanyl)prolyl]-2-
methylalanyl}prolyl)-2-methylalanyl]proline (p-BrBz-(Aib-Pro)4-OH). According to GP 2, 13 (120 mg,
0.127 mmol) in 18 ml of THF/MeOH/H2O, LiOH · H2O (21 mg, 0.508 mmol), stirring for 20 h: 118 mg
(89%) of the tetrapeptide acid. The isolated product was pure enough to be used in the next step without
further purification. 1H-NMR: 8.23, 8.17, 8.05, 8.01 (4s, 4 NH); 7.85 – 7.84 (m, 2 arom. H); 7.65 – 7.62 (m, 2
arom. H); 4.59 – 4.56 (m, 4 CH(a)(Pro)); 4.00 – 3.88 (m, 2 H of 4 CH2(d)(Pro)); 3.85 – 3.67 (m, 5 H of
4 CH2(d)(Pro)); 3.30 – 3.18 (m, 1 H of CH2(d)(Pro)); 2.84 – 2.04 (m, 4 H); 1.92 – 1.85 (m, 12 H); 1.65,
1.62, 1.60, 1.55, 1.52, 1.47, 1.46, 1.41 (8s, 8 Me(Aib)). 13C-NMR: 173.9, 173.8, 173.7, 172.9, 172.8, 172.8,
172.7, 172.5 (8s, 8 C¼O); 166.4 (s, ArC¼O); 131.9 (d, 2 arom. CH); 131.7 (s, 1 arom. C); 129.1 (d, 2 arom.
CH); 126.7 (s, 1 arom. C); 62.5, 62.2, 62.0, 61.9 (4d, 4 CH(a)(Pro)); 57.3, 56.4, 56.3, 56.3 (4s,
4 C(a)(Aib)); 48.5, 48.4, 48.2, 48.2 (4t, 4 CH2(d))(Pro)); 29.6, 29.3, 28.9, 28.5 (4t, 4 CH2(b)(Pro)); 26.1,
25.8, 25.8, 25.8 (4t, 4 CH2(g)(Pro)); 30.7, 26.1, 26.1, 26.1, 24.2, 23.9, 23.6, 23.4 (8q, 8 Me(Aib)).
1-[N-(N-{N-[N-(N-{N-[N-(4-Bromobenzoyl)-2-methylalanyl]prolyl}-2-methylalanyl)prolyl]-2-
methylalanyl}prolyl)-2-methylalanyl]-N-hexylprolinamide (p-BrBz-(Aib-Pro)4-NHC6H13; 15). Accord-
ing to GP 3, the tetrapeptide acid (103 mg, 0.111 mmol), hexylamine (13.5 mg, 0.133 mmol), HOBt
(15 mg, 0.111 mmol), TBTU (35.6 mg, 0.111 mmol), DIEA (28.7 mg, 0.222 mmol), dry CH2Cl2 (12 ml);
stirring for 19 h; CC (CH2Cl2/MeOH from 150 :1 to 10 :1): 49.5 mg (44%) of 15. M.p. 1328. IR: 3284s,
2983m, 2934m, 2873m, 1643s, 1590w, 1539s, 1469m, 1404s, 1378w, 1363w, 1306m, 1245w, 1201m, 1174m,
1
1146w, 1095w, 1071w, 1013m, 933w, 850m, 761m, 614w. H-NMR: 8.49, 8.07, 8.03 (3s, 3 NH); 7.91 – 7.88
(m, 2 arom. H); 7.77 (s, NH); 7.65 – 7.62 (m, 2 arom. H, NH); 4.61 – 4.58 (m, 3 CH(a)(Pro)); 4.51 – 4.46
(m, CH(a)(Pro)); 4.01 – 3.96 (m, 2 H of 4 CH2(d)(Pro)); 3.93 – 3.77 (m, 5 H of 4 CH2(d)(Pro)); 3.29 –
3.21 (m, CH2); 3.19 – 2.96 (m, 1 H, CH2(d)(Pro)); 2.24 – 2.15 (m, 4 H); 2.06 – 1.84 (m, 12 H); 1.81 – 1.73
(m, 8 H); 1.65, 1.63, 1.60, 1.56, 1.52, 1.51, 1.49, 1.47 (8s, 8 Me(Aib)); 0.88 – 0.84 (m, Me). 13C-NMR: 172.8,
172.8, 172.7, 172.6, 172.5, 172.4, 172.3, 172.2 (8s, 8 C¼O); 166.3 (s, ArC¼O); 131.9 (d, 2 arom. CH); 131.8
(s, 1 arom. C); 139.3 (d, 2 arom. CH); 126.8 (s, 1 arom. C); 62.5, 62.4, 62.3, 62.1 (4d, 4 CH(a)(Pro)); 57.5,
56.4, 53.8, 53.8 (4s, 4 C(a)(Aib)); 48.2, 48.1, 48.0, 47.9 (4t, 4 CH2(d))(Pro)); 39.4, 33.7, 31.5 (3t, 3 CH2);
29.3, 29.2, 29.0, 28.9 (4t, 4 CH2(b)(Pro)); 26.5 (t, CH2); 26.1, 26.0, 25.9, 25.6 (4t, 4 CH2(g)(Pro)); 22.5 (t,

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Helvetica Chimica Acta – Vol. 95 (2012)
CH2); 25.8, 25.7, 24.4, 24.3, 24.1, 24.0, 23.9, 23.8 (8q, 8 Me(Aib)); 14.0 (q, Me). ESI-MS: 1143 (5,
[M(81Br) þ (Pro-OMe) þ H]þ), 1141 (5, [M(79Br) þ (Pro-OMe) þ H]þ), 1036 (10, [M(81Br) þ Na]þ),
1034 (10, [M(79Br) þ Na]þ), 816 (100, [M(81Br) ꢀ (Pro-NHC6H13) þ H]þ), 814 (96, [M(79Br) ꢀ (Pro-
NHC6H13) þ H]þ), 634 (31, [M(81Br) ꢀ (Pro-Aib-Pro-NHC6H13) þ H]þ), 632 (29, [M(79Br) ꢀ (Pro-Aib-
Pro-C6H13) þ H]þ), 452 (5, [M(81Br) ꢀ (Pro-Aib-Pro-Aib-Pro-NHC6H13) þ H]þ), 450 (5, [M(79Br) ꢀ
(Pro-Aib-Pro-Aib-Pro-NHC6H13) þ Hþ).
Suitable crystals of 15 for the X-ray crystal-structure determination were grown from CH2Cl2/
AcOEt/hexane by slow evaporation of the solvent at r.t.
N-[N-(4-Methoxybenzoyl)-2-methylalanyl]proline Methyl Ester (p-MeOBz-Aib-Pro-OMe; 16).
According to GP 1, p-MeOBz-OH (109.6 mg, 0.72 mmol) in dry THF (15 ml) and 1b (155.2 mg,
0.792 mmol); stirring for 42 h; CC (CH2Cl2/MeOH from 150 :1 to 90 :1): 214 mg (85%) of 16. Colorless
crystals. M.p. 1658. IR: 3326m, 2987m, 2954m, 2839w, 1748s, 1653s, 1616s, 1577w, 1536s, 1504s, 1418s,
1377w, 1358m, 1317m, 1295w, 1256s, 1176s, 1110w, 1030m, 946w, 905m, 847m, 798w, 772m, 746w, 648w.
1H-NMR: 7.79 – 7.75 (m, 2 arom. H); 7.36 (s, NH); 6.93 – 6.88 (m, 2 arom. H); 4.62 – 4.59 (m,
CH(a)(Pro)); 3.84 (s, MeOC6H4); 3.82 – 3.76 (m, 1 H of CH2(d)(Pro)); 3.74 (s, MeO(Pro)); 3.65 – 3.59
(m, 1 H of CH2(d)(Pro)); 2.17 – 2.11 (m, CH2(b)(Pro)); 2.09 – 1.91 (m, CH2(g)(Pro)); 1.78, 1.75 (2s,
2 Me(Aib)). 13C-NMR: 172.9, 172.8 (2s, 2 C¼O); 165.2 (s, ArC¼O); 162.2 (s, 1 arom. C); 128.7 (d, 2
arom. CH); 127.1 (s, 1 arom. C); 113.7 (d, 2 arom. CH); 60.9 (d, CH(a)(Pro)); 57.1 (s, C(a)(Aib)); 55.4
(q, MeOC6H4); 52.1 (q, MeO(Pro)); 48.2 (t, CH2(d))(Pro)); 27.8 (t, CH2(b)(Pro)); 25.8 (t, CH2(g)(Pro));
23.6, 23.4 (2q, 2 Me(Aib)). ESI-MS: 478 (35, [M þ (Pro-OMe) þ H]þ), 349 (34, [M þ H]þ), 220 (14,
[M ꢀ (Pro-OMe) þ H]þ), 130 (100, [(Pro-OMe) þ H]þ). Anal. calc. for C18H24N2O5 (348.39): C 62.05, H
6.94, N 8.04; found: C 61.81, H 7.20, N 7.87.
N-[N-(4-Methoxybenzoyl)-2-methylalanyl]proline (p-MeOBz-Aib-Pro-OH; 17). According to
GP 2, 16 (100 mg, 0.287 mmol) in 15 ml of THF/MeOH/H2O, LiOH · H2O (48.2 mg, 1.148 mmol);
stirring for 20 h: 94 mg (98%) of 17. The product was pure enough to be used in the next step without
further purification. M.p. 1558. 1H-NMR: 7.82 – 7.79 (m, 2 arom. H); 7.13 (s, NH); 6.94 – 6.89 (m, 2 arom.
H); 4.68 – 4.64 (m, CH(a)(Pro)); 3.84 (s, MeOC6H4); 3.68 – 3.62 (m, 1 H of CH2(d)(Pro)); 3.56 – 3.51 (m,
1 H of CH2(d)(Pro)); 2.14 – 1.85 (m, CH2(b)(Pro), CH2(g)(Pro)); 1.68, 1.65 (2s, 2 Me(Aib)). 13C-NMR:
173.9, 173.5 (2s, 2 C¼O); 166.5 (s, ArC¼O); 162.7 (s, 1 arom. C); 129.1 (d, 2 arom. CH); 125.4 (s, 1 arom.
C); 113.9 (d, 2 arom. CH); 61.7 (d, CH(a)(Pro)); 57.2 (s, C(a)(Aib)); 55.4 (q, MeOC6H4); 48.4 (t,
CH2(d))(Pro)); 27.5 (t, CH2(b)(Pro)); 25.9 (t, CH2(g)(Pro)); 25.2, 24.5 (2q, 2 Me(Aib)). ESI-MS: 450
(40, [M þ (Pro-OH) þ H]þ), 357 (30, [M þ Na]þ), 335 (74, [M þ H]þ), 220 (100, [M ꢀ (Pro-OH) þ
H]þ), 135 (26, [M ꢀ (Aib-Pro-OH) þ H]þ), 116 (33, [(Pro-OH) þ H]þ). Anal. calc. for C17H22N2O5
(334.37): C 61.07, H 6.63, N 8.38; found: C 60.22, H 6.89, N 7.85.
N-(N-{N-[N-(4-Methoxybenzoyl)-2-methylalanyl]prolyl}-2-methylalanyl)proline Methyl Ester (p-
MeOBz-(Aib-Pro)2-OMe, 18). According to GP 1, 17 (94 mg, 0.281 mmol) in dry CH2Cl2 (15 ml) and 1b
(61 mg, 0.3091 mmol); stirring for 48 h, CC (CH2Cl2/MeOH from 170 :1 to 15 :1): 115 mg (78%) of 18.
M.p. 1328. IR: 3384m, 2986m, 2949m, 2877w, 1749s, 1639s, 1573w, 1542m, 1504m, 1406s, 1363m, 1298w,
1257m, 1177s, 1093w, 1025m, 924w, 900w, 849m, 791w, 773m, 751w, 729w, 699w, 610w. 1H-NMR: 7.85 – 7.83
(m, 2 arom. H); 7.67, 7.11 (2s, 2 NH); 6.99 – 6.95 (m, 2 arom. H); 4.67 – 4.62 (m, CH(a)(Pro)); 4.49 – 4.44
(m, CH(a)(Pro)); 3.87 (s, MeOC6H4); 3.84 – 3.70 (m, CH2(d)(Pro)); 3.68 (s, MeO(Pro)); 3.62 – 3.57 (m,
1 H of CH2(d)(Pro)); 3.31 – 3.25 (m, 1 H of CH2(d)(Pro)); 2.11 – 1.94 (m, 2 CH2(b)(Pro)); 1.90 – 1.72 (m,
2 CH2(g)(Pro)); 1.66, 1.61, 1.57 (3s, 4 Me(Aib)). 13C-NMR: 173.3, 172.3, 172.1, 171.0 (4s, 4 C¼O); 166.0
(s, ArC¼O); 162.7 (s, 1 arom. C); 128.9 (d, 2 arom. CH); 125.2 (s, 1 arom. C); 113.9 (d, 2 arom. CH); 62.5,
60.5 (2d, 2 CH(a)(Pro)); 57.1, 56.6 (2s, 2 C(a)(Aib)); 55.4 (q, MeOC6H4); 51.8 (q, MeO(Pro)); 47.9, 47.5
(2t, 2 CH2(d))(Pro)); 28.2, 27.9 (2t, 2 CH2(b)(Pro)); 25.8, 25.7 (2t, 2 CH2(g)(Pro)); 25.8, 25.0, 24.6, 24.6
(4q, 4 Me(Aib)). ESI-MS: 569 (45, [M þ K]þ), 553 (100, [M þ Na]þ), 531 (9, [M þ H]þ), 402 (74, [M ꢀ
(Pro-OMe) þ H]þ), 317 (10, [M ꢀ (Aib-Pro-OMe) þ H]þ), 220 (16, [M ꢀ (Pro-Aib-Pro-OMe) þ H]þ),
183 (13, C9H15N2Oþ2 ). Anal. calc. for C27H38N4O7 (530.62): C 61.12, H 7.22, N 10.56; found: C 59.77, H 7.51,
N 9.99.
Suitable crystals of 18 for the X-ray crystal-structure determination were grown from CHCl3 by slow
evaporation of the solvent at r.t.

Helvetica Chimica Acta – Vol. 95 (2012)
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N-(N-{N-[N-(4-Methoxybenzoyl)-2-methylalanyl]prolyl}-2-methylalanyl)proline (p-MeOBz-(Aib-
Pro)2-OH; 19). According to GP 2, 18 (100 mg, 0.188 mmol) in 10 ml of THF/MeOH/H2O, LiOH ·
H2O (32 mg, 0.752 mmol), stirring for 22 h: 95 mg (98%) of 19. The isolated peptide acid was pure
enough to be used in the next step without further purification. M.p. 1388. 1H-NMR (CD3OD): 8.61, 8.25
(2s, 2 NH); 8.01 – 7.98 (m, 2 arom. H); 7.14 – 7.11 (m, 2 arom. H); 4.64 – 4.59 (dd, J ¼ 9.1, 8.3,
CH(a)(Pro)); 4.56 – 4.52 (dd, J ¼ 9.0, 8.5, CH(a)(Pro)); 3.97 (s, MeOC6H4); 3.94 – 3.88 (m, 1 H of
CH2(d)(Pro)); 3.84 – 3.8 (m, 2 H of 2 CH2(d)(Pro)); 3.53 – 3.45 (m, 1 H of CH2(d)(Pro)); 2.33 – 2.12 (m,
2 CH2(b)(Pro)); 2.10 – 1.77 (m, 2 CH2(g)(Pro)); 1.70, 1.69, 1.65, 1.63 (4s, 4 Me(Aib)). 13C-NMR
(CD3OD): 175.9, 174.7, 174.2, 171.1 (4s, 4 C¼O); 166.8 (s, ArC¼O); 164.2 (s, 1 arom. C); 130.4 (d, 2 arom.
CH); 126.8 (s, 1 arom. C); 114.8 (d, 2 arom. CH); 63.6, 62.1 (2d, 2 CH(a)(Pro)); 58.2, 57.8 (2s,
2 C(a)(Aib)); 55.9 (q, MeOC6H4); 49.6, 49.1 (2t, 2 CH2(d)(Pro)); 29.6, 29.1 (2t, 2 CH2(b)(Pro)); 26.7,
26.6 (2t, 2 CH2(g)(Pro)); 26.2, 25.4, 24.9, 24.6 (4q, 4 Me(Aib)). ESI-MS: 539 (11, [M þ Na]þ), 517 (16,
[M þ H]þ), 402 (100, [M ꢀ (Pro-OH) þ H]þ), 317 (19, [M ꢀ (Aib-Pro-OH) þ H]þ), 220 (29, [M ꢀ (Pro-
Aib-Pro-OH) þ H]þ), 183 (22, C9H5N2Oþ2 ).
N-(N-[N-(N-{N-[N-(4-Methoxybenzoyl)-2-methylalanyl]prolyl}-2-methylalanyl)prolyl]-2-methyla-
lanyl)proline Methyl Ester (p-MeOBz-(Aib-Pro)3-OMe; 20). According to GP 1, 19 (95 mg, 0.184 mmol)
in dry CH2Cl2 (5 ml) and 1b (40 mg, 0.2024 mmol); stirring for 47 h, CC (CH2Cl2/MeOH from 150 :1 to
10 :1): 115 mg (88%) of 20. M.p. 2938 (dec.). IR: 3272m, 2953w, 2925s, 2854w, 1749m, 1639s, 1605w,
1542m, 1504m, 1467m, 1403m, 1377w, 1363w, 1318w, 1301w, 1255m, 1204w, 1178m, 1115w, 1094w, 1022m,
945w, 892m, 855w, 776w. 1H-NMR: 8.04 (s, NH); 7.90 – 7.87 (m, 2 arom. H); 7.64, 7.57 (2s, 2 NH); 7.01 –
6.98 (m, 2 arom. H); 4.67 – 4.62 (m, CH(a)(Pro)); 4.63 – 4.57 (m, CH(a)(Pro)); 4.55 – 4.42 (m,
CH(a)(Pro)); 3.94 – 3.90 (m, 2 H of 3 CH2(d)(Pro)); 3.87 (s, MeOC6H4); 3.85 – 3.77 (m, 2 H of
3 CH2(d)(Pro)); 3.65 (s, MeO(Pro)); 3.63 – 3.56 (m, 1 H of 3 CH2(d)(Pro)); 3.30 – 3.26 (m, 1 H of
3 CH2(d)(Pro)); 2.22 – 2.01 (m, 3 CH2(b)(Pro)); 1.98 – 1.80 (m, 3 CH2(g)(Pro)); 1.64, 1.63, 1.58, 1.55,
1.50, 1.48 (6s, 6 Me(Aib)). 13C-NMR: 173.4, 172.6, 172.5, 172.0 171.7, 171.6 (6s, 6 C¼O); 166.5 (s,
ArC¼O); 162.7 (s, 1 arom. C); 129.2 (d, 2 arom. CH); 125.1 (s, 1 arom. C); 113.9 (d, 2 arom. CH); 62.4,
62.2, 60.5 (3d, 3 CH(a)(Pro)); 57.1, 56.5, 56.4 (3s, 3 C(a)(Aib)); 55.4 (q, MeOC6H4); 51.7 (q, MeO(Pro));
48.2, 47.9, 47.7 (3t, 3 CH2(d))(Pro)); 28.9, 28.6, 27.9 (3t, 3 CH2(b)(Pro)); 26.2, 25.8, 25.5 (3t,
3 CH2(g)(Pro)); 25.8, 25.7, 25.0, 24.6, 24.5, 24.0 (6q, 6 Me(Aib)). ESI-MS: 751 (9, [M þ K]þ), 735 (25,
[M þ Na]þ), 713 (25, [M þ H]þ) 584 (100, [M ꢀ (Pro-OMe) þ H]þ), 402 (13). Anal. calc. for
C36H52N6O9 (712.85): C 60.66, H 7.35, N 11.79; found: C 60.56, H 7.54, N 11.49.
5. Solvent and Temperature Dependence of the Chemical Shifts of the NH Groups. The peptides 11,
13 – 15, and 18 were dissolved in CDCl3 (ca. 0.2m), and the chemical shifts of the NH groups were
determined at ca. 308. Then, using a syringe, 2, 4, 6, 8, 10, and 12% (v/v) of (D6)DMSO were added, and,
after each addition, the chemical shifts were determined again. For the determination of the temp.
dependence, the NH absorption in CDCl3 soln. (ca. 0.2m), was recorded between 265 and 314 K in
intervals of 7 K.
6. X-Ray Crystal-Structure Determinations of 11, 14, 15, and 18 (see Table 12, and Figs. 2 – 9)3). The
measurements for compounds 11 and 18 were conducted on a Rigaku AFC5R diffractometer using
graphite-monochromated MoKa radiation (l ¼ 0.71073 ꢅ) and a 12-kW rotating anode generator, while
those for compounds 14 and 15 were performed on a Nonius KappaCCD area-detector diffractometer
[20] using graphite-monochromated MoKa radiation (l ¼ 0.71073 ꢅ) and an Oxford Cryosystems
Cryostream 700 cooler. Data reduction for 14 and 15 was accomplished with HKL Denzo and Scalepack
[21]. The intensities were corrected for Lorentz and polarization effects. A numerical absorption
correction [22] was applied in the case of 15, an empirical absorption correction based on azimuthal scans
of several reflections [23] was applied for 11, and an absorption correction based on the multi-scan
method [24] was applied in the case of 14. No absorption correction was applied in the case of 18. In all
cases, equivalent reflections, other than Friedel pairs, were merged. Data collection and refinement
3
)
CCDC-869148 – 869151 contain the supplementary crystallographic data for this article. These data
can be obtained free of charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.

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Helvetica Chimica Acta – Vol. 95 (2012)

Helvetica Chimica Acta – Vol. 95 (2012)
1349
parameters are compiled in Table 12. The structures were solved by direct methods using either SIR92
[25] or SHELXS97 [26].
The crystal lattice of 11 contains highly disordered or diffuse solvent molecules. An analysis of the
residual electron-density peaks suggests that partially occupied sites for CH2Cl2 molecules are present,
but it was not possible to adequately model their contribution to the overall structure in any logical
manner. Therefore, the SQUEEZE routine [27] of the program PLATON [28] was employed. The
procedure gave satisfactory R factors for the refinement and suitable geometric parameters for the
peptide molecule, and there were no significant peaks of residual electron density to be found in the voids
of the structure. The solvent molecules occupy a total volume of 866 ꢅ3 per unit cell, divided into two
symmetry-related regions. The electron count in the solvent region was calculated to be 42 e per unit cell.
This corresponds with the presence of a total of one CH2Cl2 molecule per unit cell, but spread across two
symmetry-related sites. For the purposes of the calculation of the formula weight, density, F(000), and the
linear absorption coefficient, it was assumed that the ratio of peptide 11 to CH2Cl2 in the structure is 4 :1.
The asymmetric unit of 14 contains one peptide and two CH2Cl2 molecules, one of which is highly
disordered. Three partially occupied sets of positions were defined for the Cl-atoms of the disordered
solvent molecule, and the total occupancies of the sets of atoms were restrained to sum to 1.0. Similarity
restraints were applied to the CꢀCl bond lengths, while neighboring atoms within and between each
orientation of the disordered CH2Cl2 molecules were restrained to have similar and pseudo-isotropic
atomic displacement parameters.
In the case of 18, the asymmetric unit contains one peptide and one highly disordered CH2Cl2
molecule plus a site for a H2O molecule which is only one quarter occupied. Two equally occupied
positions were defined for each of the Cl-atoms of the CH2Cl2 molecule, but the large values for their
atomic displacement ellipsoids suggest that this molecule is even more highly disordered within its cavity.
The H2O molecule site is actually very close to that of the CH2Cl2 molecule, and it is possible that either
the H2O molecule is disordered with the CH2Cl2 molecule, or there is no H2O at all, and that the electron
density that has been assigned to the H2O O-atom actually represents another low occupancy disordered
position for an atom of the CH2Cl2 molecule. However, the refined model appears to provide the best
match with the electron density in the solvent region. The central five-membered ring of the peptide
molecule has a disordered envelope conformation in which the envelope flap atom, C(26), is flipped
alternately to both sides of the ring. The major conformation of this ring occurs in 56(2)% of the
molecules. The other five-membered ring also shows slight evidence for similar disorder involving C(31),
but a disordered model could not be refined satisfactorily.
For all structures, the non-H atoms were refined anisotropically. The amide H-atoms of 14 and 18
were located in a difference electron density map, and their positions were allowed to refine together
with individual isotropic displacement parameters. The H-atoms of the CH2Cl2 and H2O solvent
molecules of 18 were not included in the model. All of the remaining H-atoms in each structure were
placed in geometrically calculated positions and refined using a riding model where each H-atom was
assigned a fixed isotropic displacement parameter with a value equal to 1.2 Ueq of its parent C-atom
(1.5Ueq for the Me groups). The refinement of each structure was carried out on F2 using full-matrix least-
squares procedures [26], which minimized the function Sw(F2o ꢀ Fc2 )2. Corrections for secondary
extinction were applied for 11 and 14. Between two and four reflections whose intensities were
considered as outliers were omitted from the final refinement of each structure. Refinement of the
absolute structure parameter [29] confidently confirmed that the refined model represents the true
enantiomorph for 11, 14, and 15 (Table 12), which is consistent with the peptide configurations expected
from the synthesis. The absolute structure for 18 could not be determined owing to the weak anomalous
scattering of the material. The enantiomer used in the refinement was based on the known (S)-
configuration at C(5) and C(11).
Neutral atom scattering factors for non-H-atoms were taken from [30a], and the scattering factors
for H-atoms were taken from [31]. Anomalous dispersion effects were included in Fc [32]; the values for
f’ and f’’ were those of [30b]. The values of the mass attenuation coefficients are those of [30c]. All
calculations were performed using the SHELXL97 [26] program.

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Received March 20, 2012