Synthesis of poly-aib oligopeptides and aib-containing peptides via the 'azirine/oxazolone method', and their crystal structures

Article abstract of DOI:10.1002/hlca.201100116

The protected poly-Aib oligopeptides Z-(Aib)_n-N(Me)Ph with n=2-6 were prepared according to the 'azirine/oxazolone method', i.e., by coupling amino or peptide acids with 2,2,N-trimethyl-N-phenyl-2H-azirin-3-amine (1a) as an Aib synthon (Schemea 2). Following the same concept, the segments Z-(Aib)₃-OH (9) and H-L-Pro-(Aib)₃-N(Me)Ph (20) were synthesized, and their subsequent coupling with N,N′- dicyclohexylcarbodiimide (DCC)/ZnCl₂ led to the protected heptapeptide Z-(Aib)₃-L-Pro-(Aib)₃-N(Me)Ph (21; Schemea 3). The crystal structures of the poly-Aib oligopeptide amides were established by X-ray crystallography confirming the 3₁₀-helical conformation of Aib peptides.

Full text of DOI:10.1002/hlca.201100116

Helvetica Chimica Acta – Vol. 94 (2011)
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Synthesis of Poly-Aib Oligopeptides and Aib-Containing Peptides via the
ꢀAzirine/Oxazolone Methodꢁ, and Their Crystal Structures
by Ingeborg Dannecker-Dçrig1), Anthony Linden, and Heinz Heimgartner*
Organisch-Chemisches Institut der Universitꢀt Zꢁrich, Winterthurerstrasse 190, CH-8057 Zꢁrich
(phone: þ 41446354282; fax: þ 41446356812; e-mail: heimgart@oci.uzh.ch)
The protected poly-Aib oligopeptides Z-(Aib)n-N(Me)Ph with n ¼ 2 – 6 were prepared according to
the ꢂazirine/oxazolone methodꢃ, i.e., by coupling amino or peptide acids with 2,2,N-trimethyl-N-phenyl-
2H-azirin-3-amine (1a) as an Aib synthon (Scheme 2). Following the same concept, the segments Z-
(Aib)3-OH (9) and H-l-Pro-(Aib)3-N(Me)Ph (20) were synthesized, and their subsequent coupling with
N,N’-dicyclohexylcarbodiimide (DCC)/ZnCl2 led to the protected heptapeptide Z-(Aib)3-l-Pro-(Aib)3-
N(Me)Ph (21; Scheme 3). The crystal structures of the poly-Aib oligopeptide amides were established by
X-ray crystallography confirming the 310-helical conformation of Aib peptides.
1. Introduction. – The broad interest in Aib-containing oligopeptides is well-
documented [1], and a large number of recent articles confirm that this type of peptides
still attract attention because of their structural (e.g., [2]) and antimicrobial properties
(e.g., [3]). In the last twenty years, we have shown that N,N-disubstituted 2,2-dimethyl-
2H-azirin-3-amines 1 are useful building blocks for 2-aminoisobutyric acid (Aib) in the
syntheses of heterocycles and peptides [4]. The so-called ꢂazirine/oxazolone methodꢃ
proved to be a convenient synthetic approach for the introduction of Aib into peptides
[5] (Scheme 1). After the coupling of an amino acid or peptide acid with 1, the resulting
dipeptide amide 2 was hydrolyzed selectively to give the dipeptide acid 3. Subsequent
coupling with an amino acid ester by using N,N’-dicyclohexylcarbodiimide (DCC)/
ZnCl2 led, via the intermediate 1,3-oxazol-5(4H)-one 4, to the tripeptide 5. This method
has been applied successfully in the syntheses of model peptides and naturally
occurring peptaibols2), e.g., segments of alamethicin [8] and zervamicin II-2 [9],
derivatives of trichovirin I 1B [10] and trichotoxin A-50(G) [11], as well as
hypomurocin A1 [12]. Recently, the ꢂazirine/oxazolone methodꢃ has been adopted
for solid phase synthesis [13].
The repetition of the reaction sequence of azirine coupling and selective hydrolysis
of the C-terminal amide group allows the direct coupling of Aib segments with a
peptide chain. This convenient and efficient method has been used for the preparation
of peptides such as HOꢀCHR1ꢀCOꢀ(NHꢀCMe2ꢀCO)nꢀN(Me)R2 [14] and
ZꢀNHꢀCHR1ꢀCOꢀ(NHꢀCMe2ꢀCO)nꢀN(Me)R2 [15] as precursors of cyclic depsi-
peptides and peptides, respectively.
1
)
)
Part of the Ph.D. thesis of I. D.-D., University of Zꢁrich, 1995.
The term ꢂpeptaibolꢃ is used for amphiphilic, membrane-active Aib-containing peptides with an
acylated N-terminus and a C-terminal amino alcohol, which exhibit antibiotic properties [6][7].
2
ꢄ 2011 Verlag Helvetica Chimica Acta AG, Zꢁrich

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Helvetica Chimica Acta – Vol. 94 (2011)
Scheme 1
The aim of the present study was to demonstrate the usefulness of 2,2-dimethyl-2H-
azirin-3-amines for the preparation of poly-Aib peptides and the determination of the
conformation of these oligopeptides in the crystalline state.
2. Results and Discussion. – 2.1. Synthesis of Poly-Aib Oligopeptides. The synthesis
of the poly-Aib oligopeptides was carried out in analogy to the preparation of the linear
peptides of type XꢀCHR1ꢀCOꢀ(NHꢀCMe2ꢀCO)nꢀN(Me)R2 mentioned above
[14][15]. The (benzyloxy)carbonyl (Z)-protected 2-aminoisobutyric acid (Z-Aib-
OH) [16], which had been prepared in 95% yield by treatment of Aib with benzyl
chloroformate in a mixture of 2n aqueous NaOH and dioxane, was dissolved in Et2O
and reacted with 1.1 equiv. of 2,2,N-trimethyl-N-phenyl-2H-azirin-3-amine (1a) at room
temperature to give Z-Aib-Aib-N(Me)Ph (6a) in quantitative yield (Scheme 2). The
dipeptide amide crystallized directly from the mixture and was isolated by filtration.
Selective hydrolysis of the terminal amide group was achieved in THF/6n HCl 1:1 at
room temperature and led to the dipeptide acid Z-Aib-Aib-OH (7) in 94% yield.
The reaction sequence of azirine coupling and hydrolysis was repeated with 7 and
subsequently three times in addition. A suitable solvent for the coupling step with 7 was
THF; in the cases of the higher homologs 9, 11, and 13, DMF was used. To obtain a clear
solution, the mixtures in DMF were heated to 40 – 708 and then cooled to 08. After
addition of 1a, the solution was allowed to warm to room temperature. The peptide
amides 8a and 12 crystallized directly from the mixture, whereas 10 and 14 were
obtained as yellow oils, but crystallized after treatment with Et2O/petroleum ether and
Et2O, respectively. The pure peptide amides were isolated in 85 – 100% yield. The
selective hydrolysis of 8a and 10 proceeded smoothly at room temperature to give the
peptide acids 9 and 11 in 85 and 98% yield, respectively. In the case of the rather
insoluble 12, the hydrolysis was carried out at 608 and afforded 13 in 98% yield.
2.2. Synthesis of the Heptapeptide Z-(Aib)3-Pro-(Aib)3-N(Me)Ph (21). The
protected heptapeptide amide 21 was synthesized via a combination of the azirine

Helvetica Chimica Acta – Vol. 94 (2011)
995
Scheme 2
coupling/hydrolysis described above and the segment condensation via a 1,3-oxazol-
5(4H)-one (Scheme 3), i.e., by the ꢂazirine/oxazolone methodꢃ. The first segment Z-
(Aib)3-OH (9) was prepared as depicted in Scheme 2, but by using 2,2,N,N-tetramethyl-
2H-azirin-3-amine (1b) as the Aib synthon. The azirine coupling gave 6b and 8b in 87
and 92% yield, respectively, and the selective hydrolysis provided 7 and 9 was almost
quantitative. The second segment, Z-l-Pro-(Aib)3-N(Me)Ph (19), was obtained in a
total yield of 51% by repeated coupling of Z-l-Pro-OH with 1a, followed by the
selective hydrolysis. The reason for the choice of 1a was the faster hydrolysis of the N-
methyl-N-phenyl amides in 6n HCl/THF compared to that of the corresponding N,N-
dimethyl amides. Therefore, the racemization of Pro as well as the acid-catalyzed
cleavage of Pro-Aib [5b][17] was minimized. The hydrolyses of 15 and 17 were
already complete after 3 and 3.5 h, respectively, and afforded the products in 80 and
94% yield.
Finally, the Z group of the tetrapeptide 19 was removed by hydrogenolysis with Pd/
C to yield 20, and the segments 9 and 20 were condensed by using the coupling reagent
DCC/ZnCl2 [5a] to give 21 in 75% yield.
2.3. Crystal Structures of the Poly-Aib Oligopeptide Amides. Nowadays, it is well-
known that the preferred conformation of peptides containing Aib (and some other

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Helvetica Chimica Acta – Vol. 94 (2011)
Scheme 3
a,a-dialkylated glycines) is the 310-helix, which is a sequence of b-turns of type III or
III’, in which the ideal values for all torsion angles f (COꢀNꢀCaꢀCO) and y
(NꢀCaꢀCOꢀN) are ꢁ 608 and ꢁ 308, respectively. The negative values refer to the
right-handed helix (b-turns of type III), and the positive ones to the left-handed helix
(b-turns of type III’). An additional characteristic feature consists of the intramolecular
H-bonds between CO of amino acid i and NH of amino acid i þ 3, which form ten-
membered rings, and thereby stabilize the helical structure.
A rather large series of poly-Aib oligopeptide derivatives have been prepared and
their structures were analyzed (see, e.g., [16][18]). In most cases, peptide esters were
investigated. The crystal structures of the present set of Z-(Aib)n-N(Me)Ph (n ¼ 3 –
6)3), i.e., poly-Aib amides, are in good agreement with the 310-helical structures of
analogous derivatives: the average magnitudes of fi and yi are 55.38 and 31.58, i.e., they
correspond well with the typical values of ꢁ 608 and ꢁ 308 (see, e.g., [16b][18b,e,f]).
Z-(Aib)2-N(Me)Ph (6a). The molecular structure of 6a in the crystal is shown in
Fig. 1. The torsion angles w of the amide groups are in the allowed region for trans-
amide bonds (Table 1). The values of the torsion angle-pair f/y for Aib(2) are
þ 55.2(3)8 and þ 41.9(3)8, i.e., compatible with a left-handed 310-helix, but the
corresponding y-value for Aib(1) is far from the allowed values. Note that, since the
3
)
The dipeptide derivative Z-(Aib)2-N(Me)Ph (6a) is not able to form a b-turn and, therefore, is not
included in this comparison.

Helvetica Chimica Acta – Vol. 94 (2011)
997
Fig. 1. ORTEP Plots [19] of the molecular structures of a) 6a and b) one of the two symmetry-independent
molecules of 8a (50% probability ellipsoids, arbitrary numbering of the atoms)
space group is centrosymmetric, the crystal also contains the equal number of
molecules existing in a right-handed helix.
The molecule 6a does not form intramolecular H-bonds. Each NꢀH group of the
molecule acts as a donor for intermolecular H-bonds: N(2)ꢀH forms an intermolecular

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Helvetica Chimica Acta – Vol. 94 (2011)
Table 1. Torsion Angles w, f, and y of the Backbone of Compounds 6a, 8a, 10, 12, and 14 in the Crystal
(atom numbering refers to Figs. 1 – 3)
Compound
Amino acid
Aib(1)
Atoms
Torsion angles [8]
6a
f1
y1
w1
f2
y2
w2
C(5)ꢀN(3)ꢀC(4)ꢀC(3)
N(3)ꢀC(4)ꢀC(3)ꢀN(2)
C(4)ꢀC(3)ꢀN(2)ꢀC(2)
C(3)ꢀN(2)ꢀC(2)ꢀC(1)
N(2)ꢀC(2)ꢀC(1)ꢀN(1)
C(2)ꢀC(1)ꢀN(1)ꢀC(12)
C(7)ꢀN(4)ꢀC(6)ꢀC(5)
N(4)ꢀC(6)ꢀC(5)ꢀN(3)
C(6)ꢀC(5)ꢀN(3)ꢀC(4)
C(5)ꢀN(3)ꢀC(4)ꢀC(3)
N(3)ꢀC(4)ꢀC(3)ꢀN(2)
C(4)ꢀC(3)ꢀN(2)ꢀC(2)
C(3)ꢀN(2)ꢀC(2)ꢀC(1)
N(2)ꢀC(2)ꢀC(1)ꢀN(1)
C(2)ꢀC(1)ꢀN(1)ꢀC(16)
ꢀ 62.9(3)
165.6(2)
171.1(2)
55.2(3)
41.9(3)
179.0(2)
Aib(2)
8aa)
Aib(1)
Aib(2)
Aib(3)
f1
y1
w1
f2
y2
w2
f3
y3
w3
52.5(3); ꢀ 52.1(3)
37.3(2); ꢀ 37.3(3)
174.4(2); ꢀ 172.9(2)
61.7(3); ꢀ 60.4(3)
29.7(3); ꢀ 30.8(3)
164.5(2); ꢀ 163.9(2)
58.0(3); ꢀ 59.0(3)
58.5(3); ꢀ 56.7(3)
161.7(2); ꢀ 165.4(2)
10
Aib(1)
Aib(2)
Aib(3)
Aib(4)
f1
y1
w1
f2
y2
w2
f3
y3
w3
f4
y4
w4
C(9)ꢀN(5)ꢀC(8)ꢀC(7)
N(5)ꢀC(8)ꢀC(7)ꢀN(4)
C(8)ꢀC(7)ꢀN(4)ꢀC(6)
C(7)ꢀN(4)ꢀC(6)ꢀC(5)
N(4)ꢀC(6)ꢀC(5)ꢀN(3)
C(6)ꢀC(5)ꢀN(3)ꢀC(4)
C(5)ꢀN(3)ꢀC(4)ꢀC(3)
N(3)ꢀC(4)ꢀC(3)ꢀN(2)
C(4)ꢀC(3)ꢀN(2)ꢀC(2)
C(3)ꢀN(2)ꢀC(2)ꢀC(1)
N(2)ꢀC(2)ꢀC(1)ꢀN(1)
C(2)ꢀC(1)ꢀN(1)ꢀC(20)
ꢀ 54.8(3)
ꢀ 29.4(3)
ꢀ 179.9(2)
ꢀ 54.8(3)
ꢀ 28.4(4)
179.6(2)
ꢀ 56.6(4)
ꢀ 29.2(4)
174.7(3)
54.6(4)
53.4(4)
168.7(3)
12
Aib(1)
Aib(2)
Aib(3)
Aib(4)
Aib(5)
f1
y1
w1
f2
y2
w2
f3
y3
w3
f4
y4
w4
f5
y5
w5
C(11)ꢀN(6)ꢀC(10)ꢀC(9)
N(6)ꢀC(10)ꢀC(9)ꢀN(5)
C(10)ꢀC(9)ꢀN(5)ꢀC(8)
C(9)ꢀN(5)ꢀC(8)ꢀC(7)
N(5)ꢀC(8)ꢀC(7)ꢀN(4)
C(8)ꢀC(7)ꢀN(4)ꢀC(6)
C(7)ꢀN(4)ꢀC(6)ꢀC(5)
N(4)ꢀC(6)ꢀC(5)ꢀN(3)
C(6)ꢀC(5)ꢀN(3)ꢀC(4)
C(5)ꢀN(3)ꢀC(4)ꢀC(3)
N(3)ꢀC(4)ꢀC(3)ꢀN(2)
C(4)ꢀC(3)ꢀN(2)ꢀC(2)
C(3)ꢀN(2)ꢀC(2)ꢀC(1)
N(2)ꢀC(2)ꢀC(1)ꢀN(1)
C(2)ꢀC(1)ꢀN(1)ꢀC(24)
55.3(6)
32.5(6)
174.8(4)
55.2(6)
28.9(6)
ꢀ 179.3(5)
54.2(7)
25.9(7)
178.1(4)
60.4(7)
26.5(7)
ꢀ 172.1(5)
ꢀ 47.6(6)
ꢀ 54.1(5)
178.3(4)
14
Aib(1)
Aib(2)
f1
y1
w1
f2
y2
w2
C(13)ꢀN(7)ꢀC(12)ꢀC(11)
N(7)ꢀC(12)ꢀC(11)ꢀN(6)
C(12)ꢀC(11)ꢀN(6)ꢀC(10)
C(11)ꢀN(6)ꢀC(10)ꢀC(9)
N(6)ꢀC(10)ꢀC(9)ꢀN(5)
C(10)ꢀC(9)ꢀN(5)ꢀC(8)
ꢀ 50.8(6)
ꢀ 35.8(5)
ꢀ 178.2(3)
ꢀ 50.4(5)
ꢀ 39.6(5)
ꢀ 172.0(3)

Helvetica Chimica Acta – Vol. 94 (2011)
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Table 1 (cont.)
Compound
Amino acid
Atoms
Torsion angles [8]
Aib(3)
Aib(4)
Aib(5)
Aib(6)
f3
y3
w3
f4
y4
w4
f5
y5
w5
f6
y6
w6
C(9)ꢀN(5)ꢀC(8)ꢀC(7)
N(5)ꢀC(8)ꢀC(7)ꢀN(4)
C(8)ꢀC(7)ꢀN(4)ꢀC(6)
C(7)ꢀN(4)ꢀC(6)ꢀC(5)
N(4)ꢀC(6)ꢀC(5)ꢀN(3)
C(6)ꢀC(5)ꢀN(3)ꢀC(4)
C(5)ꢀN(3)ꢀC(4)ꢀC(3)
N(3)ꢀC(4)ꢀC(3)ꢀN(2)
C(4)ꢀC(3)ꢀN(2)ꢀC(2)
C(3)ꢀN(2)ꢀC(2)ꢀC(1)
N(2)ꢀC(2)ꢀC(1)ꢀN(1)
C(2)ꢀC(1)ꢀN(1)ꢀC(28)
ꢀ 57.8(4)
ꢀ 31.4(4)
ꢀ 175.8(3)
ꢀ 55.5(4)
ꢀ 33.6(4)
ꢀ 176.5(3)
ꢀ 58.3(4)
ꢀ 35.0(4)
174.1(3)
49.3(4)
52.7(4)
ꢀ 179.4(3)
a) Two symmetry-independent molecules.
H-bond with the amide O(1)-atom at the Ph(Me)N end of a neighboring peptide
molecule and thereby links the molecules into extended chains which run parallel to the
[010] direction, and which can be described by a graph set motif [20] of C(5). N(3)ꢀH
forms an intermolecular H-bond with the amide O(2)-atom in the middle of a different
neighboring peptide molecule and thereby links the molecules into centrosymmetric
dimers, in which the interactions can be described by a graph set motif of R22(10). The
combination of these interactions links the molecules into two-dimensional networks
which lie parallel to the (1 0 ꢀ1) plane.
Z-(Aib)3-N(Me)Ph (8a). There are two molecules with almost identical conforma-
tions but with opposite helicity in the asymmetric unit. As the space group is
centrosymmetric, both of the independent molecules exist in the crystal in their left-
and right-handed forms. Aview of molecule A is shown in Fig. 1. Both molecules form a
b-turn, stabilized by an intramolecular H-bond between N(2)ꢀH of Aib(2) and C¼O(4)
of the Z group (Table 2). This is in agreement with the observation of Toniolo et al. that
polypeptides with a Z-protected N-terminal Aib form a b-turn of type III with a 4 ! 1
Table 2. Intramolecular H-Bonds of Compounds 8a, 10, 12, and 14 (atom numbering refers to Figs. 1 – 3)
Compound H-Bond
d(NꢀH) [ꢅ] d(H ··· O) [ꢅ] d(N ··· O) [ꢅ] /(NꢀH ··· O) [8]
8a (A)
8a (B)
10
N(2)ꢀH ··· O(4)
0.79(2)
N(32)ꢀH ··· O(34) 0.83(2)
2.26(2)
2.28(2)
2.11(3)
2.13(3)
2.25(5)
2.04(5)
2.19(5)
2.22(4)
2.30(4)
2.27(4)
2.22(4)
3.027(3)
3.070(3)
2.950(3)
3.001(4)
3.077(5)
2.906(6)
3.087(6)
3.031(4)
3.133(4)
3.094(4)
3.012(4)
162(2)
160(2)
166(2)
168(2)
171(4)
172(4)
168(4)
156(3)
160(3)
156(3)
167(4)
N(2)ꢀH ··· O(4)
N(3)ꢀH ··· O(5)
N(2)ꢀH ··· O(4)
N(3)ꢀH ··· O(5)
N(4)ꢀH ··· O(6)
N(2)ꢀH ··· O(4)
N(3)ꢀH ··· O(5)
N(4)ꢀH ··· O(6)
N(5)ꢀH ··· O(7)
0.85(3)
0.88(3)
0.83(4)
0.87(5)
0.91(4)
0.86(3)
0.87(4)
0.88(4)
0.81(4)
12
14

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Helvetica Chimica Acta – Vol. 94 (2011)
H-bond between NH of the third amino acid and C¼O of the Z group [21]. All torsion
angles w of the amide groups show characteristic values for trans-amide bonds
(Table 1). The average magnitudes of the torsion angle-pairs f1–3/y1–2 are 57.38 and
33.88, typical for a 310-helix. The torsion angles y3A and y3B of the C-terminal Aib differ
significantly from the ideal value of ꢁ 308, an observation that has also been described
earlier [21a].
Each NꢀH group of each independent molecule acts as a donor for H-bonds. In
molecule A, N(2)ꢀH forms an intramolecular H-bond with the urethane O(4)-atom
that is seven atoms along the peptide backbone. This interaction has a graph set motif
[20] of S(10) and serves to maintain a fairly rigid helical conformation of the peptide.
N(3)ꢀH and N(4)ꢀH, which are unable to form an intramolecular interaction because
of their positions in the backbone, form intermolecular H-bonds with the amide O(31’)-
and O(32’)-atoms closest to the Ph(Me)N end of the same neighboring B molecule.
Molecule B exhibits an identical pattern of H-bonds. Each of these specific donors links
molecules A and B alternately into extended chains which run parallel to the [101]
direction, and which can be described by a graph set motif of C22(16). The double-bridge
between adjacent molecules resulting from both the interactions forms a ring with a
graph set motif of R22(12).
Z-(Aib)4-N(Me)Ph (10). The asymmetric unit of the centrosymmetric structure
contains one molecule of the peptide 10 and one molecule of H2O. A view of the
molecule is shown in Fig. 2. The right-handed 310-helical conformation of the reference
molecule is stabilized by two intramolecular 4 ! 1 H-bonds, which form b-turns of type
III’ (N(2)ꢀH ··· O(4), N(3)ꢀH ··· O(5)). As in the previous case, N(3)ꢀH forms a H-
bond with the C¼O(5) group of the urethane. All torsion angles w are in the region of
trans-amide bonds, and the magnitudes of the torsion angles f1–4 and y1–3 correspond
with those of a 310-helical conformation (Table 1; average values ꢀ 55.38 and ꢀ 29.08,
resp.). Again, the value of y4 deviates significantly from ꢁ 308. It is worth mentioning
that f4 and y4 are positive, whereas f1–3 and y1–3 are all negative, i.e., the helicity of the
last Aib is inverted.
Each NꢀH group of the molecule 10 acts as a donor for H-bonds. N(2)ꢀH and
N(3)ꢀH form intramolecular H-bonds with the amide O(4)- and O(5)-atoms that are
seven atoms along the peptide backbone. Each of these interactions has a graph set
motif [20] of S(10). N(5)ꢀ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(1’)-atom at the Ph(Me)N end of a neighboring peptide molecule. These interactions
link the molecules into extended chains which run parallel to the [010] direction, and
which can be described by a graph set motif of C(14). N(4)ꢀH forms an intermolecular
H-bond with the O-atom of the H2O molecule, which, in turn, donates a H-bond to the
amide O(1’)-atom at the Ph(Me)N end of the next peptide molecule in the chain. These
interactions can be described by a binary graph set motif of C22(13).
Z-(Aib)5-N(Me)Ph (12). The molecular structure is shown in Fig. 2. The reference
molecule in the asymmetric unit of the centrosymmetric structure forms a left-handed
310-helix, stabilized by three intramolecular H-bonds of type 4 ! 1, i.e., three b-turns of
type III: N(2)ꢀH ··· O(4), N(3)ꢀH ··· O(5), and N(4)ꢀH ··· O(6), the last one including
the C¼O of the urethane (Table 2). The torsion angles w are typical for trans amide
bonds, and the magnitudes of the torsion angles f1–4 and y1–4 are characteristic for a 310-

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1001
Fig. 2. ORTEP Plots [19] of the molecular structures of a) 10 and b) 12 (50% probability ellipsoids,
arbitrary numbering of the atoms, H2O molecule in 10 omitted)
helical conformation (Table 1; average values þ 56.38 and þ 28.58, resp.). The values
of the last pair, f5 and y5, are quite different and have the opposite sign (ꢀ47.6(6)8 and
ꢀ 54.1(5)8) compared with f1–4 and y1–4
.
Each NꢀH group of the molecule acts as a donor for H-bonds. N(2)ꢀH, N(3)ꢀH,
and N(4)ꢀH form intramolecular H-bonds with the amide O(4)-, O(5)-, and O(6)-
atoms that are seven atoms along the peptide backbone. Each of these interactions has
a graph set motif [20] of S(10). These H-bonds stabilize the peptide in a fairly rigid
helical conformation. N(5)ꢀH and N(6)ꢀH, which are unable to form an intra-
molecular interactions because of their positions in the backbone, form intermolecular
H-bonds with the amide O(1’)- and O(2’)-atoms closest to the Ph(Me)N end of the
same neighboring molecule. Each of these specific donors links the molecules into
extended chains which run parallel to the [100] direction, and which can be described
by a graph set motif of C(14). The double-bridge between adjacent molecules resulting
from both the interactions forms a ring with a graph set motif of R22(12).

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Helvetica Chimica Acta – Vol. 94 (2011)
Z-(Aib)6-N(Me)Ph (14). The molecular structure is shown in Fig. 3. The
asymmetric unit contains one molecule of 14 plus a site, which is ca. 25% occupied
by a H2O molecule. Four intramolecular H-bonds of type 4 ! 1, i.e., four b-turns of
type III’ (N(2)ꢀH ··· O(4), N(3)ꢀH ··· O(5), N(4)ꢀH ··· O(6), and N(5)ꢀH ··· O(7),
Table 2), stabilize a right-handed 310-helix. The compound crystallizes in a chiral space
group, so all molecules exclusively adopt a right-handed helix. As in the cases of 8a, 10,
and 12, the acceptor of the last H-bond is the C¼O group of the urethane. All torsion
angles w are compatible with trans amide bonds, and the values of the torsion angles
f1–5 and y1–5 are characteristic for a 310-helical conformation (Table 1; average values
ꢀ 54.68 and ꢀ 35.18, resp.). Again, the values of the last pair f6 and y6 differ and have
the opposite sign (þ49.3(4)8 and þ 52.7(4)8, resp.).
Fig. 3. ORTEP Plot [19] of the molecular structure of 14 (50% probability ellipsoids, arbitrary
numbering of the atoms, the H2O molecule has been omitted)
Each NꢀH group of the molecule acts as a donor for H-bonds. The four interactions
involving N(2)ꢀH, N(3)ꢀH, N(4)ꢀH, and N(5)ꢀH are intramolecular H-bonds with
the amide O(4)-, O(5)-, O(6)-, and O(7)-atoms that are seven atoms along the peptide
backbone. Each of these interactions has a graph set motif [20] of S(10). This serves to
maintain a fairly rigid helical conformation of the peptide. N(7)ꢀ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-atom at the Ph(Me)N end of a neighboring
peptide molecule. These interactions link the molecules into extended chains which run
parallel to the [001] direction, and which can be described by a graph set motif of C(20).
N(6)ꢀH forms an intermolecular H-bond with the O-atom of the H2O molecule, which,
in turn, donates a H-bond to the first amide O-atom in the next peptide molecule in the
chain. These interactions can be described by a binary graph set motif of C22(19).
3. Conclusions. – With the presented syntheses of poly-Aib oligopeptides and an
Aib-containing heptapeptide, the usefulness of 2,2-dimethyl-2H-azirin-3-amines as
Aib synthons in peptide synthesis was established. Repeated azirine coupling with Z-
protected Aib allowed the smooth and efficient preparation of the Aib oligomers (Z-
(Aib)2–6-N(Me)Ph) with a C-terminal amide function. The X-ray crystal-structure

Helvetica Chimica Acta – Vol. 94 (2011)
1003
analyses of these oligomers confirmed the high preference of b-turns and the 310-helical
conformation. It is worth mentioning that the dipeptide Z-Aib-Aib-N(Me)Ph is not
able to form a b-turn with the corresponding 4 ! 1 H-bond, because it is to short, but
the values of the torsion angles of Aib(2) are close to those of an Aib involved in a b-
turn. As expected, in all oligomers the terminal amide group is not involved in
intramolecular H-bonding and, therefore, does not influence the structure of the
peptide backbone significantly (cf. [18]). Furthermore, the conformation of the Z-
protecting group, which is involved in a 4 ! 1 H-bond stabilizing the first b-turn, is
similar for all investigated oligopeptides and shows the trans,trans-conformation (cf.
[21b]). The torsion angles w of the peptide bonds of the studied Aib oligomers are
typically in the range of 171 – 1808 (trans amide bonds), and only in the two
crystallographically independent molecules of Z-(Aib)3-N(Me)Ph were values of ca.
1648 observed for w2.
With the synthesis of Z-(Aib)3-Pro-(Aib)3-N(Me)Ph, it was demonstrated once
more that the combination of azirine coupling, selective hydrolysis, and coupling of a
peptide segment via a 5(4H)-oxazolone, i.e., the ꢂazirine/oxazolone methodꢃ, is very
suitable for the synthesis of Aib-rich oligopeptides.
We thank the analytical units of our institute for spectra and analyses. Financial support of the work
by the Stipendienfonds der Basler Chemischen Industrie (I. D.-D.), the Swiss National Science
Foundation, and F. Hoffmann-La Roche AG, Basel, is gratefully acknowledged. Dr. B. R. Vincent is
thanked for the crystallographic data collection, and the initial solution and refinement of the structures.
Experimental Part
1. General. Solvents were purified by standard procedures; THF was distilled from Na/
benzophenone, Et2O from Na, and CH2Cl2 from CaCl2; DMF (puriss.) was dried over molecular sieves.
All commercially available chemicals were of anal. grade and were used without purification. The 2H-
azirin-3-amines 1a and 1b were prepared according to the references cited in [4]. TLC: aluminium sheets,
silica gel 60 F254 (Merck). Prep. TLC: glass plates, silica gel 60 F254 (2 mm; Merck). Column
chromatography (CC; flash chromatography [22]): silica gel Merck 60 (0.040 – 0.063 mm). High-
performance liquid chromatography (HPLC): with a Varian 2510 instrument with Varian 2550 UV
detector (254 nm), or Waters 600 E instrument with Waters 484 UV detector (254 nm); Lichrosorb RP 18
or LichroCast RP 18 (reversed phase) columns. M.p.: Mettler-FP-5 apparatus; uncorrected. IR Spectra:
Perkin-Elmer-297 or -781 spectrophotometer; in KBr, in cmꢀ1. 1H- and 13C-NMR Spectra: Varian XL-200
or Bruker-AM-400 instrument (200 and 50.4 MHz, or 400 and 100.7 MHz, resp.); d in ppm, coupling
constants J in Hz; multiplicity of C-atoms from DEPT spectra. MS: Finnigan MAT-90 (EI) or Finnigan
SSQ-700 (CI with NH3) instrument. Elemental analyses were performed at the Institute of Organic
Chemistry of the University of Zꢁrich.
Abbreviations: Aib, 2-aminoisobutyric acid; Bn, benzyl; DCC, N,N’-dicyclohexylcarbodiimide; Z,
(benzyloxy)carbonyl.
General Procedure 1 (GP 1; Azirine Coupling). To a 0.5m soln. of the peptide acid in Et2O, THF, or
DMF at 08 was added azirine 1a or 1b (1.1 equiv.) dropwise, and the mixture was stirred at 08 for 10 min
and at r.t. for several h. The precipitated product was filtered, and washed with Et2O and petroleum
ether. The solvent of the mother liquor was evaporated, and the oily residue was crystallized by treatment
with petroleum ether at 08. The combined crystals were dried in h.v.
General Procedure 2 (GP 2; Selective Hydrolysis). A 0.1m soln. of the peptide amide in THF/6n HCl
1:1 was stirred at r.t. or at 608, and the progress of the reaction was followed by TLC. When the reaction
was complete, the same volume of 2n HCl was added, the product was extracted with Et2O, and the
combined org. phase was dried (Na2SO4). After evaporation of the solvent, the product was dried in h.v.

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Helvetica Chimica Acta – Vol. 94 (2011)
2. Synthesis of Poly-Aib Oligopeptides. 2.1. N-[(Benzyloxy)carbonyl]-2-methylalanine (Z-Aib-OH)
[16]. To a vigorously stirred soln. of Aib (20 g, 194 mmol) in 2n HCl/dioxane 2 :1 (145.5 ml) at 08 were
added simultaneously a soln. of benzyl chloroformate (79.4 g, 466 mmol) in toluene and 4n NaOH
(37 ml) to keep the mixture basic. Then, the mixture was warmed to r.t., and stirring was continued
overnight. After addition of Et2O, the org. phase was separated, and 6n HCl was added to the cooled aq.
phase, which was then extracted with Et2O (3ꢂ). The combined org. phase was evaporated, and the oily
residue was crystallized by addition of petroleum ether at reflux temp. and subsequent cooling to r.t. The
crystalline solid was washed with petroleum ether and dried in h.v. Yield of Z-Aib-OH: 44.5 g (95%).
M.p. 75.58. 1H-NMR (90 MHz, CDCl3): 10.65 – 10.35 (br. s, COOH); 7.35 (s, 5 arom. H); 5.65 – 5.25 (br. s,
NH); 1.60 (s, Me2C).
2.2. N-[(Benzyloxy)carbonyl]-2-methylalanyl-2-methylalanine-N-methyl-N-phenylamide (Z-(Aib)2-
N(Me)Ph; 6a). According to GP 1, to a soln. of Z-Aib-OH (1.30 g, 5.5 mmol) in Et2O (10 ml), 1a (1.0 g,
6.0 mmol) was added. After 5 min, a precipitate formed. The mixture was kept at 48 overnight and then
filtered. Yield: 2.28 g (quant.). Colorless solid. M.p. 152 – 1548. IR (CHCl3): 3430w, 3370w, 2940w, 1725m,
1670m, 1630m, 1595w, 1495s, 1455m, 1390w, 1365w, 1170w, 1120w, 1090w, 1075w, 705w. 1H-NMR
(200 MHz, CDCl3): 7.48 (br. s, NH); 7.42 – 7.30, 7.30 – 7.20 (2m, 10 arom. H); 5.36 (br. s, NH(urethane));
5.07 (s, PhCH2O); 3.28 (s, MeN); 1.47, 1.42 (2s, 2 Me2C). 13C-NMR (50 MHz, CDCl3): 173.6, 172.7 (2s,
2 CO(amide)); 154.9 (s, CO(urethane)); 144.3, 136.5 (2s, 2 arom. C); 129.3, 128.4, 128.3, 128.03, 128.00,
127.9 (6d, 10 arom. CH); 66.4 (t, PhCH2O); 58.4, 57.0 (2s, 2 Me2C); 41.5 (q, MeN); 25.2, 25.1 (2q,
2 Me2C). Anal. calc. for C23H29N3O4 (411.51): C 67.13, H 7.10, N 10.21; found: C 67.25, H 7.03, N 10.30.
Suitable crystals for the X-ray crystal-structure determination were grown from DMSO/H2O by slow
evaporation of the solvent.
2.3. N-[(Benzyloxy)carbonyl]-2-methylalanyl-2-methylalanine (Z-(Aib)2-OH, 7). According to
GP 2, 6a (3.74 g, 9.1 mmol) was hydrolyzed. Evaporation of Et2O gave 7 (2.75 g, 94%). Colorless,
crystalline solid. M.p. 154 – 1588. IR (KBr): 3430m, 3300m, 3040w, 2990w, 2950w, 1725s, 1705s, 1655s,
1535s, 1510m, 1470w, 1460w, 1445w, 1420w, 1380w, 1360w, 1300w, 1255m, 1230m, 1210w, 1185w, 1170w,
1
1080m, 970w, 855w, 785w, 750w, 700w. H-NMR (200 MHz, (D6)DMSO): 12.27 – 12.20 (br. s, COOH);
7.48 (s, NH); 7.37 – 7.26 (m, 5 arom. H, NH); 5.02 (s, PhCH2O); 1.33 (s, 2 Me2C). 13C-NMR (50 MHz,
(D6)DMSO): 175.8 (s, COOH); 173.4 (s, CO(amide)); 154.7 (s, CO(urethane)); 137.2 (s, arom. C); 128.3,
127.7, 127.5 (3d, 5 arom. CH); 65.0 (t, PhCH2O); 56.0, 55.1 (2s, 2 Me2C); 25.0, 24.4 (2q, 2 Me2C). CI-MS:
323 (100, [M þ 1]þ). Anal. calc. for C16H22N2O5 (322.36): C 59.62, H 6.88, N 8.69; found: C 59.54, H 6.82,
N 8.49.
2.4. N-[(Benzyloxy)carbonyl]-2-methylalanyl-2-methylalanyl-2-methylalanine-N-methyl-N-phenyl-
amide (Z-(Aib)3-N(Me)Ph, 8a). According to GP 1, to a soln. of 7 (2.0 g, 6.2 mmol) in THF (14 ml)
was added 1a (1.14 g, 6.5 mmol). After warming to r.t., a precipitate formed. The mixture was stirred at
r.t. for 4 h and kept at 48 overnight: 8a (2.95 g, 95%). Colorless solid. M.p. 177 – 1788. IR (KBr): 3340m,
3290m, 3040w, 2990w, 2940w, 1710m, 1690m, 1660s, 1635s, 1595w, 1540m, 1515m, 1495m, 1465m, 1270m,
1225w, 1205w, 1170w, 1100m, 1090m, 915w, 740w, 710w. 1H-NMR (200 MHz, (D6)DMSO): 7.75, 7.66 (2s,
2 NH); 7.40 – 7.15 (m, 10 arom. H, NH); 5.11 (s, PhCH2O); 3.16 (s, MeN); 1.31, 1.30, 1.29 (3s, 3 Me2C).
13C-NMR (50 MHz, (D6)DMSO): 173.7, 173.1, 172.4 (3s, 3 CO(amide)); 155.5 (s, CO(urethane)); 146.0,
137.2 (2s, 2 arom. C); 128.6, 128.3, 127.6, 126.9, 126.8, 125.9 (6d, 10 arom. CH); 65.1 (t, PhCH2O); 56.2,
56.1, 55.9 (3s, 3 Me2C); 39.1 (q, MeN); 25.4, 24.8, 24.7 (3q, 3 Me2C). Anal. calc. for C27H36N4O5 (496.61):
C 65.30, H 7.31, N 11.28; found: C 65.52, H 7.54, N 11.05.
Suitable crystals for the X-ray crystal-structure determination were grown from DMSO/H2O by slow
evaporation of the solvent.
2.5. N-[(Benzyloxy)carbonyl]-2-methylalanyl-2-methylalanyl-2-methylalanine (Z-(Aib)3-OH, 9).
According to GP 2, 8a (4.95 g, 10.0 mmol) was hydrolyzed. After 2 h, the precipitate was filtered. The
aq. phase was extracted with Et2O, the org. phase was dried (Na2SO4), then Et2O was evaporated, the
combined solid material was washed with Et2O and dried in h.v.: 9 (3.43 g, 85%). Colorless solid. M.p.
195 – 1968. IR (KBr): 3470m, 3410m, 3320m, 3300s, 3080w, 3020w, 2990w, 2930w, 1730s, 1700s, 1660s,
1635s, 1550m, 1535s, 1470w, 1455m, 1390m, 1345m, 1310m, 1275s, 1225w, 1210w, 1180m, 1100s, 1080w,
740m. 1H-NMR (200 MHz, (D6)DMSO): 7.62 (br. s, 2 NH); 7.39 – 7.31 (m, 5 arom. H, NH); 5.07 (s,
PhCH2O); 1.30 (br. s, 3 Me2C). 13C-NMR (50 MHz, (D6)DMSO): 175.5 (s, COOH); 173.4, 173.2 (2s,

Helvetica Chimica Acta – Vol. 94 (2011)
1005
2 CO(amide)); 155.4 (s, CO(urethane)); 137.0 (s, arom. C); 128.3, 127.8, 127.6 (3d, 5 arom. CH); 65.3 (t,
PhCH2O); 56.2, 55.7, 54.8 (3s, 3 Me2C); 24.9, 24.5 (2q, 3 Me2C). CI-MS: 408 (100, [M þ 1]þ). Anal. calc.
for C20H29N3O6 (407.47): C 58.95, H 7.17, N 10.31; found: C 58.87, H 7.33, N 10.55.
2.6. N-[(Benzyloxy)carbonyl]-2-methylalanyl-2-methylalanyl-2-methylalanyl-2-methylalanine-N-
methyl-N-phenylamide (Z-(Aib)4-N(Me)Ph, 10). At ca. 408, 9 (3.20 g, 7.85 mmol) was dissolved in
DMF (17 ml). After cooling to 08, according to GP 1, 1a (1.55 g, 8.2 mmol) was added, and the mixture
was stirred at r.t. for 4 h. The solvent was evaporated, and the yellow oily residue was treated with Et2O.
The formed solid was filtered, dissolved in DMF, precipitated by addition of Et2O (3ꢂ), and dried in h.v.:
10 (4.24 g, 93%). Colorless solid. M.p. 194 – 1958. IR (KBr): 3420w, 3330m, 3300m, 3030w, 2990w, 2940w,
1705s, 1680 – 1650s (br.), 1630m, 1595m, 1530s, 1495m, 1470m, 1455m, 1395m, 1380m, 1365m, 1270m,
1220w, 1170w, 1090m, 710m. 1H-NMR (200 MHz, (D6)DMSO): 8.21, 7.84, 7.55 (3 br. s, 3 NH); 7.38 – 7.16
(m, 10 arom. H, NH); 5.08 (s, PhCH2O); 3.28 (s, MeN); 1.42, 1.34, 1.28, 1.26 (4s, 4 Me2C). 13C-NMR
(50 MHz, (D6)DMSO): 175.1, 173.9, 173.1, 172.7 (4s, 4 CO(amide)); 155.7 (s, CO(urethane)); 146.2, 137.5
(2s, 2 arom. C); 128.6, 128.3, 127.7, 127.2, 126.8, 125.8 (6d, 10 arom. CH); 65.5 (t, PhCH2O); 56.04, 56.0 (2s,
4 Me2C); 39.1 (q, MeN); 25.5, 25.1, 24.6 (3q, 4 Me2C). Anal. calc. for C31H43N5O6 (581.72): C 64.01, H
7.45, N 12.04; found: C 64.15, H 7.48, N 12.02.
Suitable crystals for the X-ray crystal-structure determination were grown from DMSO/H2O by slow
evaporation of the solvent.
2.7. N-[(Benzyloxy)carbonyl]-2-methylalanyl-2-methylalanyl-2-methylalanyl-2-methylalanine (Z-
(Aib)4-OH, 11). According to GP 2, 10 (8.81 g, 15.1 mmol) was hydrolyzed in THF/6n HCl (152 ml).
After 2.5 h, 2n HCl (150 ml) was added, and the precipitate was filtered. The solid material was washed
with CH2Cl2 and dried in h.v.: 11 (7.31 g, 98%). Colorless solid. M.p. 232 – 2338. IR (KBr): 3370w, 3300m,
3070w, 3000w, 1730m, 1710s, 1660s, 1545m, 1530m, 1515m, 1455w, 1400w, 1390w, 1365w, 1300w, 1280w,
1
1260m, 1230w, 1170w, 1080m, 955w, 810w, 790w, 700w. H-NMR (200 MHz, (D6)DMSO): 11.83 (br. s,
COOH); 8.07, 7.78 (2s, 2 NH); 7.41 – 7.25 (m, 5 arom. H, NH); 7.18 (s, NH); 5.09 (s, PhCH2O); 1.33, 1.25,
1.22 (3s, 4 Me2C). 13C-NMR (50 MHz, (D6)DMSO): 175.6 (s, COOH); 174.8, 173.5, 173.1 (3s,
3 CO(amide)); 155.7 (s, CO(urethane)); 137.1 (s, arom. C); 128.3, 127.7, 127.3 (3d, 5 arom. CH); 65.5 (t,
PhCH2O); 56.01, 56.0, 55.5, 54.6 (4s, 4 Me2C); 24.8, 24.6 (2q, 4 Me2C). FAB-MS: 493 (100, [M þ 1]þ).
Anal. calc. for C24H36N4O7 (492.57): C 58.52, H 7.37, N 11.37; found: C 58.56, H 7.10, N 11.56.
2.8. N-[(Benzyloxy)carbonyl]-2-methylalanyl-2-methylalanyl-2-methylalanyl-2-methylalanyl-2-
methylalanine-N-methyl-N-phenylamide (Z-(Aib)5-N(Me)Ph, 12). At ca. 708, 11 (6.50 g, 13.2 mmol)
was dissolved in DMF (30 ml). After cooling to 08, according to GP 1, 1a (2.60 g, 13.7 mmol) was added,
and the mixture was stirred at r.t. overnight. The formed solid was filtered and dried in h.v.: 12 (8.02 g,
91%). Colorless solid. M.p. 235 – 2368. IR (KBr): 3420w, 3300s, 3250m, 3040w, 2990w, 2940w, 1695m,
1675s, 1650s, 1595w, 1555s, 1530s, 1495m, 1465m, 1455m, 1395m, 1385m, 1365m, 1270m, 1225w, 1215w,
1170w, 1090m, 1080m, 770w, 755w, 710m. 1H-NMR (400 MHz, (D6)DMSO, 1008): 7.75, 7.55, 7.42 (3 br. s,
3 NH); 7.38 – 7.16 (m, 10 arom. H, 2 NH); 5.09 (s, PhCH2O); 3.31 (s, MeN); 1.45, 1.42, 1.39, 1.37, 1.34, 1.32,
1.30 (7s, 5 Me2C). 13C-NMR (50 MHz, (D6)DMSO, 1008): 174.4, 174.1, 173.3, 172.8, 172.2 (5s,
5 CO(amide)); 155.3 (s, CO(urethane)); 145.9, 136.5 (2s, 2 arom. C); 127.9, 127.7, 127.2, 126.8, 126.3,
125.2 (6d, 10 arom. CH); 65.2 (t, PhCH2O); 56.0, 55.81, 55.80, 55.7, 55.6 (5s, 5 Me2C); 39.1 (q, MeN); 25.2,
24.8, 24.2, 24.0 (4q, 5 Me2C). FAB-MS: 560 (20, [M þ 1 ꢀ Ph(Me)NH]þ). Anal. calc. for C35H50N6O7
(666.82): C 63.04, H 7.56, N 12.60; found: C 63.08, H 7.48, N 12.44.
Suitable crystals for the X-ray crystal-structure determination were grown from DMSO by slow
evaporation of the solvent.
2.9. N-[(Benzyloxy)carbonyl]-2-methylalanyl-2-methylalanyl-2-methylalanyl-2-methylalanyl-2-
methylalanine (Z-(Aib)5-OH, 13). According to GP 2, a suspension of 12 (200 mg, 0.3 mmol) in THF/
6n HCl (3 ml) was stirred at ca. 608 for 1.5 h. After usual workup, the product was dried in h.v.: 13
(169 mg, 98%). Colorless solid. M.p. 234 – 2358. IR (KBr): 3300m, 3080w, 3030w, 299w, 2940w, 1730m,
1
1710m, 1660s, 1530s, 1455w, 1385w, 1365w, 1300w, 1260m, 1230w, 1170w, 1080m, 750w, 700w. H-NMR
(200 MHz, (D6)DMSO, 508): 11.83 (br. s, COOH); 8.24, 7.85, 7.48 (3s, 3 NH); 7.34 – 7.30 (m, 5 arom. H,
NH); 7.29 (br. s, NH); 5.10 (s, PhCH2O); 1.33, 1.27, 1.22 (3s, 5 Me2C). 13C-NMR (50 MHz, (D6)DMSO,
808): 174.8, 174.4, 174.0, 173.1, 173.0 (5s, COOH, 4 CO(amide)); 155.4 (s, CO(urethane)); 136.7 (s, arom.
C); 127.8, 127.3, 126.9 (3d, 5 arom. CH); 65.3 (t, PhCH2O); 55.8, 55.60, 55.59, 55.5, 54.5 (5s, 5 Me2C); 24.6,

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Helvetica Chimica Acta – Vol. 94 (2011)
24.3, 24.25, 24.1 (4q, 5 Me2C). Anal. calc. for C28H43N5O8 (577.68): C 58.12, H 7.50, N 12.12; found: C
58.35, H 7.57, N 11.95.
2.10. N-[(Benzyloxy)carbonyl]-2-methylalanyl-2-methylalanyl-2-methylalanyl-2-methylalanyl-2-
methylalanyl-2-methylalanine-N-methyl-N-phenylamide (Z-(Aib)6-N(Me)Ph, 14). At ca. 708, 13 (3.0 g,
5.2 mmol) was dissolved in DMF (11 ml). After cooling to 08, according to GP 1, was added 1a (950 mg,
5.45 mmol), and the mixture was stirred at r.t. for 20 h. The obtained oil was treated with Et2O, and the
formed solid was dried in h.v.: 14 (3.58 g, 92%). Colorless solid. M.p. 222 – 2238. IR (KBr): 3320m, 3040w,
2990w, 2940w, 1745w, 1700m, 1670s, 1595w, 1525s, 1455w, 1385w, 1365w, 1270m, 1230w, 1170w, 1090m,
1100w, 740w, 715w, 700w. 1H-NMR (200 MHz, (D6)DMSO þ 5% H2O, 858)4): 7.89, 7.61, 7.50, 7.48 (4 br. s,
4 NH); 7.44 – 7.12 (m, 10 arom. H, 2 NH); 5.10 (s, PhCH2O); 3.31 (s, MeN); 1.45, 1.42, 1.40, 1.37, 1.36, 1.35,
1.32, 1.31, 1.28 (9s, 6 Me2C). 13C-NMR (50 MHz, (D6)DMSO, 708)4): 174.9, 174.7, 174.5, 174.45, 174.1,
173.5, 173.14, 173.1, 172.3 (9s, 6 C¼O(amide)); 155.5, 155.4 (2s, C¼O(urethan)); 146.0, 136.7 (2s, 2 arom.
C); 128.1, 127.9, 127.4, 127.0, 126.5, 125.4 (6d, 10 arom. CH); 65.3 (t, PhCH2O); 56.0, 55.9, 55.8, 55.7, 55.6,
55.5 (6s, 6 Me2C); 38.8 (q, MeN); 25.3, 25.0, 24.5, 24.4, 24.3, 24.1 (6q, 6 Me2C). Anal. calc. for C39H57N7O8
(751.93): C 62.30, H 7.64, N 13.04; found: C 62.08, H 7.62, N 12.82.
Suitable crystals for the X-ray crystal-structure determination were grown from DMSO/H2O by slow
evaporation of the solvent.
3. Synthesis of Z-(Aib)3-l-Pro-(Aib)3-N(Me)Ph (21). 3.1. N-[(Benzyloxy)carbonyl]-2-methylalanyl-
2-methylalanine-N,N-dimethylamide (Z-(Aib)2-NMe2, 6b). According to GP 1, to a soln. of Z-Aib-OH
(4.0 g, 16.9 mmol) in Et2O (36 ml) at 08, azirine 1b (2.1 g, 18.6 mmol) was added, and the mixture was
stirred at r.t. After 1 h, a precipitate formed, and after 24 h, the reaction was complete. The solid was
filtered, washed with Et2O, and dried in h.v.: 6b (5.13 g, 87%). Colorless solid. M.p. 159 – 1618. 1H-NMR
(200 MHz, CDCl3): 7.56 (br. s, NH); 7.40 – 7.30 (m, 5 arom. H); 5.44 (br. s, NH(urethane)); 5.09 (s,
PhCH2O); 3.03 (s, Me2N); 1.58, 1.53 (2s, 2 Me2C). CI-MS: 350 (45, [M þ 1]þ), 305 (100).
3.2. N-[(Benzyloxy)carbonyl]-2-methylalanyl-2-methylalanyl-2-methylalanine-N,N-dimethylamide
(Z-(Aib)3-NMe2, 8b). According to GP 2, 6b (5.0 g, 14.0 mmol) was hydrolyzed to give 7 (4.40 g,
13.65 mmol; see Sect. 2.3). The crude material was dissolved in a mixture of Et2O (28 ml) and THF
(10 ml), and azirine 1b (1.77 g, 15.7 mmol) was added. After 42 h, the precipitate was filtered, and the
product was washed with Et2O, and dried in h.v.: 8b (5.46 g, 92%). Colorless solid. M.p. 185 – 1868.
1H-NMR (200 MHz, CDCl3): 7.43 (br. s, NH); 7.36 – 7.32 (m, 5 arom. H, NH); 6.72 (br. s, NH); 5.13 (s,
PhCH2O); 2.97 (br. s, Me2N); 1.43, 1.42, 1.38 (3s, 3 Me2C). CI-MS: 435 (17, [M þ 1]þ), 390 (100).
3.3. N-[(Benzyloxy)carbonyl]-2-methylalanyl-2-methylalanyl-2-methylalanine (Z-(Aib)3-OH, 9).
The hydrolysis of 8b (5.32 g, 12.2 mmol) according to GP 2 gave 9 (4.10 g, 96%; see Sect. 2.5).
3.4. N-[(Benzyloxy)carbonyl]-l-prolyl-2-methylalanine-N-methyl-N-phenylamide (Z-Pro-Aib-
N(Me)Ph, 15). According to GP 1, to a soln. of Z-Pro-OH (5.0 g, 20.06 mmol) in abs. THF (50 ml) at
08, 1a (3.7 g, 21.06 mmol) was added, and the mixture was stirred under Ar at r.t. for 5 d. The solvent was
evaporated and the residue was crystallized by treatment with Et2O and hexane. The product was washed
with Et2O and dried in h.v.: 15 (7.90 g, 93%). Colorless solid. M.p. 119 – 1208. [a]2D0 ¼ ꢀ51.0 (c ¼ 0.97,
EtOH). IR (KBr): 3420w, 3280m, 3040w, 3000w, 2970w, 2880w, 1710s, 1675s, 1625s, 1590m, 1535m,
1495m, 1470w, 1455w, 1420s, 1390m, 1375w, 1355m, 1250m, 1215w, 1170w, 1120m, 1090m, 1070w, 1030w,
1
1020w, 995w, 960w, 915w, 870w, 775w, 710m, 700w. H-NMR (200 MHz, (D6)DMSO): 8.12 (br. s, NH);
7.40 – 7.10 (m, 10 arom. H); 5.20 – 4.85 (m, PhCH2O); 4.30 – 4.10 (m, CH(2) Pro); 3.45 – 3.30 (m, CH2(5)
Pro); 3.32 (s, MeN); 2.30 – 1.70 (m, CH2(3), CH2(4) Pro); 1.35, 1.31 (2s, Me2C). CI-MS: 424 (43, [M þ
1]þ), 317 (100). Anal. calc. for C24H29N3O4 (423.52): C 68.00, H 6.90, N 9.92; found: C 67.33, H 6.89, N
10.03.
3.5. N-[(Benzyloxy)carbonyl]-l-prolyl-2-methylalanine (Z-Pro-Aib-OH, 16). The hydrolysis of 15
(6.63 g, 15.65 mmol) according to GP 2 for 2.75 h gave 16 (4.19 g, 80%). Colorless foam. 1H-NMR
(90 MHz, CDCl3): 9.30 (br. s, COOH); 7.50 – 7.10 (m, 5 arom. H, NH); 5.10 (br. s, PhCH2O); 4.50 – 4.10
(m, CH(2) Pro); 3.85 – 3.30 (m, CH2(5) Pro); 2.30 – 1.80 (m, CH2(3), CH2(4) Pro); 1.50 (br. s, Me2C).
3.6. N-[(Benzyloxy)carbonyl]-l-prolyl-2-methylalanyl-2-methylalanine-N-methyl-N-phenylamide
(Z-Pro-(Aib)2-N(Me)Ph, 17). According to GP 1, to a soln. of 16 (4.20 g, 12.56 mmol) in Et2O
4
)
Some of the signals were doubled, most likely because of the presence of conformers.

Helvetica Chimica Acta – Vol. 94 (2011)
1007
(130 ml) at 08, 1a (2.53 g, 14.45 mmol) was added, and the mixture was stirred under Ar at 08 overnight.
The precipitate was filtered, washed with Et2O, and dried in h.v.: 17 (5.69 g, 89%). Colorless solid. M.p.
147 – 1488. [a]2D2 ¼ ꢀ23.1 (c ¼ 0.943, EtOH). IR (KBr): 3400m, 3310m, 2990w, 2940w, 2870w, 1695s, 1685s,
1625s, 1595w, 1530w, 1495w, 1440w, 1425m, 1395w, 1365w, 1335m, 1280w, 1240w, 1225w, 1205w, 1110w,
1
1090w, 1000w, 995w, 980w, 770w, 730w, 705w. H-NMR (400 MHz, (D6)DMSO): 8.17, 7.86 (2s, 2 NH);
7.50 – 7.10 (m, 10 arom. H); 5.09 (s, PhCH2O); 4.20 – 4.10 (m, CH(2) Pro); 3.60 – 3.30 (m, CH2(5) Pro);
3.32 (s, MeN); 2.20 – 1.70 (m, CH2(3), CH2(4) Pro); 1.36, 1.35, 1.29, 1.27 (4s, 2 Me2C). CI-MS: 509 (2,
[M þ 1]þ), 402 (100). Anal. calc. for C28H36N4O5 (508.62): C 66.12, H 7.13, N 11.02; found: C 65.89, H 7.22,
N 11.19.
3.7. N-[(Benzyloxy)carbonyl]-l-prolyl-2-methylalanyl-2-methylalanine (Z-Pro-(Aib)2-OH, 18). The
hydrolysis of 17 (5.45 g, 10.72 mmol) according to GP 2 for 3.5 h gave 18 (4.49 g, 94%). Colorless foam.
[a]2D2 ¼ ꢀ36.0 (c ¼ 1.197, EtOH). IR (KBr): 3330m, 3300m, 3030w, 2990w, 2980w, 2940w, 2880w, 1730m,
1685s, 1650s, 1530m, 1500w, 1470m, 1440m, 1395w, 1385w, 1360m, 1315w, 1280w, 1260w, 1240w, 1170m,
1
1130w, 1090w, 1040w, 990w, 975w, 770w, 750w, 700w. H-NMR (400 MHz, (D6)DMSO): 8.21 (s, NH);
7.50 – 7.20 (m, 5 arom. H); 7.18 (s, NH); 5.07 (s, PhCH2O); 4.20 – 4.10 (m, CH(2) Pro); 3.50 – 3.20 (m,
CH2(5) Pro); 2.20 – 1.70 (m, CH2(3), CH2(4) Pro); 1.33, 1.32, 1.31, 1.29 (4s, 2 Me2C). CI-MS: 420 (100,
[M þ 1]þ). Anal. calc. for C21H29N3O6 (419.48): C 60.13, H 6.97, N 10.02; found: C 59.79, H 6.84, N 9.80.
3.8. N-[(Benzyloxy)carbonyl]-l-prolyl-2-methylalanyl-2-methylalanyl-2-methylalanine-N-methyl-N-
phenylamide (Z-Pro-(Aib)3-N(Me)Ph, 19). According to GP 1, to a soln. of 18 (4.0 g, 9.54 mmol) in a
mixture of abs. THF (19 ml) and abs. DMF (10 ml) at 08, 1a (1.94 g, 11.15 mmol) was added slowly, and
the mixture was stirred under Ar at r.t. for 45 h. The precipitate was filtered, washed with Et2O, and dried
in h.v.: 19 (4.64 g, 82%). Colorless solid. [a]2D2 ¼ ꢀ46.0 (c ¼ 1.062, EtOH). IR (KBr): 3400w, 3300w,
3070w, 3040w, 2990w, 2950w, 2880w, 1670s (br.), 1640s (br.), 1595w, 1535s, 1495s, 1470w, 1455s, 1425s,
1395s, 1365s, 1340w, 1290w, 1275w, 1245w, 1215w, 1170w, 1125m, 1100m, 1025w, 985w, 935w, 775w, 740w,
715m, 700w. 1H-NMR (400 MHz, (D6)DMSO): 8.68, 7.43 (2s, 2 NH); 7.40 – 7.10 (m, 10 arom. H); 7.02 (s,
NH); 5.12, 4.98 (AB, JAB ¼ 12.8, PhCH2O); 4.25 – 4.15 (m, CH(2) Pro); 3.55 – 3.35 (m, CH2(5) Pro); 3.30
(s, MeN); 2.20 – 1.75 (m, CH2(3), CH2(4) Pro); 1.36, 1.34 (2s, 3 Me2C). 13C-NMR (50 MHz, CD3OD):
177.0, 176.1, 176.0, 175.4 (4s, 4 C¼O(amide)); 157.0 (s, C¼O(urethane)); 147.4, 138.2 (2s, 2 arom. C);
130.5, 129.9, 129.5, 129.0, 128.5, 128.3 (6d, 10 arom. CH); 68.6 (t, PhCH2O); 62.2 (d, C(2) Pro); 58.7, 58.4,
58.1 (3s, 3 Me2C); 48.2 (t, CH2(5) Pro); 41.3 (q, MeN); 31.4, 26.0 (2t, CH2(3), CH2(4) Pro); 27.5, 26.8,
26.7, 26.3, 24.9, 24.4 (6q, 3 Me2C). CI-MS: 487 (96, [M þ 1 ꢀ Ph(Me)NH]þ), 108 (100). Anal. calc. for
C32H43N5O6 (593.73): C 64.74, H 7.30, N 11.80; found: C 64.53, H 7.20, N 12.01.
3.9. l-Prolyl-2-methylalanyl-2-methylalanyl-2-methylalanine-N-methyl-N-phenylamide (H-Pro-
(Aib)3-N(Me)Ph, 20). To a stirred soln. of 19 (500 mg, 0.84 mmol) in MeOH (5.5 ml) was added in
small portions Pd/C 10% (50 mg) at r.t., and a stream of H2 was bubbled through the mixture for 2 h.
Then, the mixture was filtered through Celite, the filtrate was evaporated, and the residue was crystallized
by treatment with Et2O and dried in h.v.: 20 (345 mg, 89%). Colorless solid. [a]2D2 ¼ ꢀ24.3 (c ¼ 0.420,
EtOH). IR (KBr): 3440m (br.), 3300m (br.), 2980w, 2930w, 1660s (br.), 1595w, 1530m (br.), 1495m,
1
1460w, 1390w, 1365w, 1280w, 1225w, 1170w, 1095w, 770w, 710w. H-NMR (400 MHz, CDCl3): 8.02, 7.05
(2s, 2 NH); 7.40 – 7.20 (m, 5 arom. H, NH); 6.83 (br. s, NH); 3.96 (dd, J ¼ 9.1, 5.3, CH(2) Pro); 3.31 (s,
MeN); 3.10 – 2.85 (m, CH2(5) Pro); 2.20 – 1.65 (m, CH2(3), CH2(4) Pro); 1.49, 1.48, 1.47 (3s, 3 Me2C).
13C-NMR (50 MHz, CDCl3): 175.8, 173.7, 173.2, 173.0 (4s, 4 C¼O(amide)); 145.2 (s, C arom.); 129.1,
127.9, 127.2 (3d, 5 arom. CH); 60.7 (d, C(2) Pro); 57.9, 57.1, 56.7 (3s, 3 Me2C); 47.2 (t, CH2(5) Pro); 40.8
(q, MeN); 30.6, 26.2 (2t, CH2(3), CH2(4) Pro); 25.7, 25.5, 25.4, 25.0, 24.9 (5q, 3 Me2C). FAB-MS: 460 (20,
[M þ 1]þ), 353 (92).
3.10. N-[(Benzyloxy)carbonyl]-2-methylalanyl-2-methylalanyl-2-methylalanyl-l-prolyl-2-methyl-
alanyl-2-methylalanyl-2-methylalanine-N-methyl-N-phenylamide (Z-(Aib)3-Pro-(Aib)3-N(Me)Ph, 21).
To a stirred soln. of 9 (80.6 mg, 0.20 mmol) in DMF (1 ml) at 08, DCC (41 mg, 0.20 mmol) was added.
After 3 min, ZnCl2 (55 mg, 0.40 mol) and then 20 (100 mg, 0.22 mmol) were added in small portions, and
the mixture was stirred under Ar at r.t. for 40 h. Then, aq. Na2CO3 soln. (2.5%) was added, and the
mixture was extracted with CH2Cl2 (3ꢂ). The combined org. phase was washed with 2n HCl (3ꢂ). The
org. phase was dried (Na2SO4), the solvent evaporated, and the residue dried in h.v.: 21 (125 mg, 75%).
Colorless solid. 1H-NMR (400 MHz, CDCl3): 7.77, 7.45, 7.40, 7.23, 6.35, 5.82 (6s, 6 NH); 7.50 – 7.10 (m, 10

1008
Helvetica Chimica Acta – Vol. 94 (2011)
Table 3. Crystallographic Data for Compounds 6a, 8a, 10, 12, and 14
6a
8a
10
12
14
Crystallized from
Empirical formula
Formula weight
DMSO/H2O DMSO/H2O DMSO/H2O
DMSO
DMSO/H2O
C23H29N3O4
411.50
colorless,
prism
C27H36N4O5
496.60
colorless,
prism
C31H43N5O6 · H2O C35H50N6O7 C39H57N7O8 · 0.25 H2O
599.72
colorless,
prism
666.82
colorless,
prism
756.43
colorless,
prism
Crystal color, habit
Temp. [K]
Crystal system
Space group
Z
294(1)
monoclinic
P21/n
294(1)
294(1)
triclinic
P1
294(1)
monoclinic monoclinic
P21/c
4
294(1)
triclinic
ꢀ
ꢀ
P1
P21
2
4
4
2
Reflections for
cell determination
2q Range for cell
determination [8]
Unit cell parameters:
a [ꢅ]
b [ꢅ]
c [ꢅ]
a [8]
b [8]
25
25
25
25
25
20 – 28
20 – 28
24 – 28
20 – 24
20 – 26
13.857(3)
10.796(4)
15.499(4)
90
103.27(2)
90
2259(1)
1.211
0.0835
w
13.171(1)
14.694(3)
15.546(3)
99.42(1)
109.99(1)
91.07(1)
2780.2(8)
1.186
8.660(4)
11.731(5)
16.839(7)
103.25(3)
96.22(3)
90.91(3)
1653(1)
1.204
11.585(4)
18.943(5)
17.718(7)
90
103.67(2)
90
3778(2)
1.172
0.0824
w
9.092(2)
16.052(4)
14.968(4)
90
100.93(2)
90
2144.9(9)
1.171
0.0829
w
g [8]
V [ꢅ3]
Dx [g cmꢀ3
]
m(MoKa) [mmꢀ1
Scan type
]
0.0825
w
0.0858
w
2q(max) [8]
46
46
46
46
52
Total reflections
measured
3952
8238
5013
5831
4749
Symmetry indepen-
dent reflections
Reflections with
I > 2s(I)
2833
2080
7745
5540
4601
3184
5258
2454
4378
3232
Reflections used
Parameters refined;
restraints
2833
285; 0
7745
688; 0
4601
426; 0
5258
464; 2
4378
534; 1
Final R(F) [I > 2s(I)] 0.0431
0.0431
0.1069
0.0514
0.1465
0.0734
0.1883
0.0845; 0
0.943
–
0.0443
0.1137
0.0620; 0.1035
1.020
wR(F2) (all data)
Weights: a; ba)
Goodness-of-fit
0.1092
0.0543; 0.4155 0.0503; 0.3506 0.0776; 0.2280
1.022
1.027
0.0046(5)
1.029
0.008(2)
Secondary extinction 0.008(1)
coefficient
–
Final Dmax/s
0.001
0.001
0.20; ꢀ 0.14
0.001
0.22; ꢀ 0.18
0.002
0.001
D1 (max; min) [e ꢅꢀ3] 0.12; ꢀ 0.13
0.27; ꢀ 0.19 0.13; ꢀ 0.12
a) w ¼ [s2 (Fo2) þ (aP)2 þ bP]ꢀ1 where P ¼ (Fo2 þ 2F2c )/3.
arom. H); 5.19, 5.08 (AB, JAB ¼ 12.6, PhCH2O); 4.22 (t, J ¼ 8.1, CH(2) Pro); 3.75 – 3.55 (m, CH2(5) Pro);
3.45 (s, MeN); 2.40 – 2.25, 2.10 – 1.70 (2m, CH2(3), CH2(4) Pro); 1.61, 1.59, 1.55, 1.51, 1.49, 1.46, 1.45, 1.42,
1.41, 1.34, 1.27, 1.26 (12s, 6 Me2C). 13C-NMR (50 MHz, CDCl3): 174.9, 174.8, 174.5, 174.1, 173.6, 173.2,
173.1 (7s, 7 C¼O(amide)); 156.2 (s, C¼O(urethane)); 146.2, 136.7 (2s, 2 arom. C); 128.9, 128.6, 128.1,

Helvetica Chimica Acta – Vol. 94 (2011)
1009
127.2, 127.1, 126.3 (6d, 10 arom. CH); 66.5 (t, PhCH2O); 64.0 (d, CH(2) Pro); 57.1, 57.0, 56.8, 56.6 (4s,
6 Me2C); 48.4 (t, CH2(5) Pro); 40.1 (q, MeN); 28.8, 26.2 (2t, CH2(3), CH2(4) Pro); 27.8, 27.1, 26.7, 26.1,
25.8, 25.7, 25.5, 24.2, 23.9, 23.4, 23.2 (11q, 6 Me2C). FAB-MS: 871 (15, [M þ Na]þ), 742 (100, [M þ 1 ꢀ
Ph(Me)NH), 390 (100), 305 (100).
4. X-Ray Crystal-Structure Determination of 6a, 8a, 10, 12, and 14 (Table 3 and Figs. 1 – 3)5). All
measurements were performed on a Nicolet R3 diffractometer using graphite-monochromated MoKa
radiation (l 0.71073 ꢅ). The data collection and refinement parameters are given in Table 3, and views of
the molecules are shown in Figs. 1 – 3. The intensities were corrected for Lorentz and polarization effects,
but not for absorption. The structures were solved by direct methods using SHELXS86 [23], which
revealed the positions of all non-H-atoms. In the case of 8a, there are two symmetry-independent
molecules in the asymmetric unit. The atomic coordinates of the two molecules were tested carefully for a
relationship from a higher symmetry-space group using the program PLATON [24], but none could be
found. In the case of 10, the asymmetric unit contains one molecule of the peptide plus one molecule of
H2O. In the case of 14, one significant electron-density peak (ca. 0.5 e/ꢅ3) remained after all other atoms
were accounted for. This peak was assigned as a partially occupied site for a H2O molecule [O(9)], with a
site occupation factor of 0.25. The non-H-atoms were refined anisotropically. The amide H-atoms were
placed in the positions indicated by a difference electron-density map, and their positions were allowed to
refine together with individual isotropic displacement parameters. The H-atoms of the H2O molecule in
14 could not be located. Bond length restraints were applied to the N(5)ꢀH and N(6)ꢀH bonds in
compound 12. All remaining H-atoms were fixed in geometrically calculated positions and refined by
using a rigid 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 (1.5 Ueq for Me groups). The refinements of the structures
were carried out on F2 by using full-matrix least-squares procedures, which minimized the function
Sw(F2o ꢀ F2c )2. A correction for secondary extinction was applied in the cases of 6a, 8a, and 10. Neutral
atom scattering factors for non-H-atoms were taken from [25a], and the scattering factors for H-atoms
were taken from [26]. Anomalous dispersion effects were included in Fc [27]; the values for f’ and f’’ were
those of [25b]. The values of the mass attenuation coefficients are those of [25c]. All calculations were
performed using the SHELXL97 [28] program.
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5
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CCDC-817459 – 817463 contain the supplementary crystallographic data of 6a, 8a, 10, 12, and 14,
respectively, for this article. These data can be obtained free of charge from the Cambridge
Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/data_request/cif.

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Helvetica Chimica Acta – Vol. 94 (2011)
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Received March 17, 2011