Total synthesis of the peptaibols hypomurocin A3 and hypomurocin A5, and their conformation analysis

Article abstract of DOI:10.1002/cbdv.201200285

The total syntheses of hypomurocin A3 and hypomuricin A5 (HM A3 and HM A5, resp.) in solution phase are described. These syntheses have been successfully achieved by applying the 'azirine/oxazolone method' to introduce the two Aib-Pro units into the backbone of these undecapeptaibols in one step with methyl 2,2-dimethyl-2H-azirine-3-prolinate as the 'Aib-Pro synthon'. The coupling of Z-protected (Z=(benzyloxy)carbonyl) amino acids or peptide acids with amino acid tert-butyl esters and of peptide segments was carried out according to the TBTU (=O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate) and HOBt (=1-hydroxybenzotriazole) protocol. Purification by reversed-phase HPLC gave the peptides in pure form. The products were characterized by optical rotation, NMR and IR spectroscopy, mass spectrometry, and elemental analysis. The crystal structures of HM A3 and of an octapeptide fragment of HM A5 could be obtained. An NMR analysis was also carried out with HM A3 and HM A5 to determine their conformations in solution. A global structural comparison between the three sequences of HM A1, HM A3, and HM A5 was performed, as well as the HPLC correlation of the natural HM A family and the synthetic samples.

Full text of DOI:10.1002/cbdv.201200285

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Total Synthesis of the Peptaibols Hypomurocin A3 and Hypomurocin A5, and
Their Conformation Analysis
by Nicolas Pradeille1), Manuel Tzouros, Kerstin Mçhle, Anthony Linden, and Heinz Heimgartner*
Organisch-chemisches Institut der Universitꢀt Zꢁrich, Winterthurerstrasse 190, CH-8057 Zꢁrich
(phone: þ41-44-6354282; fax: þ41-44-6356812; e-mail: heimgart@oci.uzh.ch)
Dedicated to Professor Dieter Seebach on the occasion of his 75th birthday
The total syntheses of hypomurocin A3 and hypomuricin A5 (HM A3 and HM A5, resp.) in solution
phase are described. These syntheses have been successfully achieved by applying the ꢂazirine/oxazolone
methodꢃ to introduce the two Aib-Pro units into the backbone of these undecapeptaibols in one step with
methyl 2,2-dimethyl-2H-azirine-3-prolinate as the ꢂAib-Pro synthonꢃ. The coupling of Z-protected (Z¼
(benzyloxy)carbonyl) amino acids or peptide acids with amino acid tert-butyl esters and of peptide
segments was carried out according to the TBTU (¼O-(benzotriazol-1-yl)-N,N,N’,N’-tetramethyluro-
nium tetrafluoroborate) and HOBt (¼1-hydroxybenzotriazole) protocol. Purification by reversed-phase
HPLC gave the peptides in pure form. The products were characterized by optical rotation, NMR and IR
spectroscopy, mass spectrometry, and elemental analysis. The crystal structures of HM A3 and of an
octapeptide fragment of HM A5 could be obtained. An NMR analysis was also carried out with HM A3
and HM A5 to determine their conformations in solution. A global structural comparison between the
three sequences of HM A1, HM A3, and HM A5 was performed, as well as the HPLC correlation of the
natural HM A family and the synthetic samples.
1. Introduction. – In the last few decades, a remarkable interest in a special class of
oligopeptides called ꢀpeptaibolsꢁ was documented (cf. [1] and refs. cit. therein). The
term comprises antibiotic peptides containing a-aminoisobutyric acid (Aib) and, in
some cases, a-ethylalanine (¼ isovaline, Iva), an acetylate N-terminus, and a C-
terminal amino alcohol [2]. These peptides are of natural origin, mainly produced by
fungi of the genera Trichoderma, Gliocladium, and Emericillopsis [3]. The biological
interest in these peptides is based on their membrane-active properties [4], i.e., their
ability to form ion channels through biological membranes [5]. These properties are the
result of relatively rigid helical conformations of peptaibols because of the presence of
a,a-disubstituted a-amino acids such as Aib [6] (for crystal structures, see [7]). Very
recently, peptaibols were also investigated as potential suppressors of tumor cells [8].
With the development of the so-called ꢀazirine/oxazolone methodꢁ [9], a new
approach to Aib-containing peptides was established. The key steps are i) the coupling
of a 2,2,N,N-tetrasubstituted 2H-azirin-3-amine of type 1 with amino or peptide acids to
give 2, ii) the selective hydrolysis of the terminal amide group of the extended peptide
leading to the peptide acid 3, and iii) coupling with the next amino acid via an
intermediate 1,3-oxazol-5(4H)-one to yield the peptide 4 [10] (Scheme 1).
1
)
Part of the Ph.D. thesis of N. P., Universitꢀt Zꢁrich, 2006.
ꢄ 2012 Verlag Helvetica Chimica Acta AG, Zꢁrich

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2529
Scheme 1
By this method, several peptaibols or segments of peptaibols have been prepared
[11]. For example, the synthesis of hypomurocin A1 has been described, as well as its
3D-NMR structure in solution and in deuterated micelles [12]. The used concept of the
synthesis of HM A1 was not justified, but it was chosen as optimal also for the synthesis
of HM A3 and HM A5. In this article, the strategy of the synthesis of HM A1, HM A3,
and HM A5 will be detailed, and the synthesis of HM A3 and HM A5 described.
Furthermore, 2D-NMR structural studies in solution have been performed, thus
allowing a comparison of the structures of the three molecules. Moreover, the
correlation of the synthetic molecules and the corresponding natural products was
carried out by HPLC.
2. Results and Discussion. – As the entire project comprises the synthesis of three
hypomurocins, HM A1, A3, and A5, with the aim of comparing their structures, the first
requirement was an efficient preparative strategy to minimize the number of reaction
steps required for all three syntheses. The choice of the three major segments in the
synthesis of HM A1 [12] was based on the fact that some segments are common to HM
A3 and HM A5 (Fig. 1) and, therefore, can be used in all three syntheses.
Fig. 1. Common sequences of HM A1, HM A3, and HM A5
The N-termini of the three selected hypomurocins HM A1, HM A3, and HM A5 are
identical, i.e., Ac-Aib-Gln. Therefore, the first idea was to prepare the dipeptide Ac-
Aib-Gln-OH by coupling Ac-Aib-OH and H-Gln-OtBu and deprotecting the C-
terminus of the resulting dipeptide. The subsequent coupling step with H-Val-OtBu (for
HM A1 and A3) and H-Ile-OtBu (for HM A5) might result in a substantial loss of
material, as Gln bears a rather bulky side chain. As commercial Ac-Aib-OH is very
expensive, and it has to be prepared by acetylating H-Aib-OH, a reaction which can be

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
performed in low yield only2), we decided to introduce Aib in position 1 for the
tripeptides Ac-Aib-Gln-Val-OH (6) and Ac-Aib-Gln-Ile-OH (7) in the last step,
because the commercial starting materials for Gln, Val, and Ile are rather inexpensive.
The tripeptide 6 can be used for the synthesis of both HM A1 and HM A3, 7 is needed
for the preparation of HM A5. Thus, Z-Gln-OH was coupled first with H-Val-OtBu and
t
H-Ile-OtBu, respectively; the Bu protecting group was chosen to allow a smooth
deprotection by treatment with CF3COOH (TFA) and to avoid the cleavage of the Ac
group on Aib. The obtained products Z-Gln-Val-OtBu (8) and Z-Gln-Ile-OtBu (9)
were deprotected via hydrogenolysis (H2, Pd/C, MeOH) to give H-Gln-Val-OtBu (10)
and H-Gln-Ile-OtBu (11), respectively, and then coupled with Ac-Aib-OH [12] to lead
to the two tripeptides 12 and 13. Deprotection of the latter by treatment with TFA
yielded 6 and 7 (Scheme 2).
Scheme 2
The comparison of the rest of the three peptaibols (Fig. 2) reveals that two Aib-Pro
sequences in positions 5 and 6, and 9 and 10 are in common, which should be introduced
via azirine coupling at an amino acid by using methyl N-(2,2-dimethyl-2H-azirin-3-yl)-
l-prolinate (14) [12][13] as the building block. So, the tripeptides Val-Aib-Pro, Leu-
Aib-Pro, and Ile-Aib-Pro were prepared. Whereas Val-Aib-Pro is a segment of only
HM A1, Leu-Aib-Pro is a common sequence in both HM A1 (residues 8–10) and HM
A3 (residues 4–6), which allows this segment to be used in both syntheses. Moreover,
HM A1 and HM A5 contain the same C-terminal pentapeptide Leu-Leu-Aib-Pro-
Leuol (residues 7–11). This again reduces the total number of reaction steps. Finally,
HM A3 and HM A5 contain the common tripeptide sequence Ile-Aib-Pro (residues 8–
10 for HM A3 and 4–6 for HM A5). For the coupling of two peptide chains and/or
2
)
Alternatively, Ac-Aib-OH can be prepared via azirine coupling of 2,2,N-trimethyl-N-phenyl-2H-
azirin-3-amine with AcOH to give Ac-Aib-N(Me)Ph (5) and subsequent selective hydrolysis of the
amide group. The coupling occurred smoothly and the structure of 5 was established by X-ray
crystallography (Table 2).

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2531
Fig. 2. Overview of the syntheses of HM A1 [12], HM A3, and HM A5

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
amino acids, the TBTU (¼O-(benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium tetra-
fluoroborate)/HOBt (¼1-hydroxybenzotriazole) strategy was used successfully. When
needed, the N-termini of peptides/amino acids were Z-protected (Z¼(benzyloxy)car-
bonyl), and its deprotection was performed under H2 with Pd/C (5%). The hydrolysis
of the peptide methyl esters was achieved with 3 equiv. of LiOH in THF/MeOH/H2O
3 :1:1 (v/v), and the tert-butyl ester was hydrolyzed by treatment with TFA.
The complete strategy for the syntheses is outlined in Fig. 2. The common parts of
A1, A3, and A5 are highlighted.
2.1. Synthesis of HM A3. The synthesis of HM A3 started by preparing the
unprotected segment 7–11, i.e., H-Leu-Ile-Aib-Pro-Leuol (15; Scheme 3). The reaction
of Z-Ile-OH with the 2H-azirin-3-amine 14 gave the protected tripeptide methyl ester
Z-Ile-Aib-Pro-OMe (16) in 58% yield3). The latter was hydrolyzed with LiOH to give
Z-Ile-Aib-Pro-OH (17; 80% yield4), which then was coupled with H-Leuol to give the
tetrapeptide Z-Ile-Aib-Pro-Leuol (18) in 63% yield.
Scheme 3
Surprisingly, 18 crystallized from the crude mixture after the evaporation of the
solvent and gave crystals of good quality, suitable for X-ray crystallography (Fig. 3,a).
The OH group and the adjacent amide group, N(19)ꢀH, of Leuol form intramolecular
H-bonds with the C¼O group of Aib and Ile, respectively, that are seven atoms back
along the peptide backbone. Each of these interactions has the graph set motif [15] of
S(10). This serves to maintain the helical conformation of the tetrapeptide. N(10)ꢀH
(Ile) and N(13)ꢀH (Aib), which are unable to form an intramolecular interaction
because of their position in the backbone, form intermolecular H-bonds with the same
C¼O group of Pro adjacent to the OH end of a neighboring peptide molecule, although
the latter interaction is very weak. These intermolecular interactions link the molecules
3
)
)
This low yield is unusual for a coupling with a 2H-azirin-3-amine and may be explained by the use
of a partially decomposed 14, which was kept for more than one year before use.
Half of the amount of 17 was set apart for the synthesis of HM A5.
4

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2533
Fig. 3. ORTEP Plots [14] of the molecular structure of a) 18 and b) one of the symmetry-independent
molecules of 19 (50% probability ellipsoids; arbitrary numbering of the atoms; H-atoms bonded to C-
atoms have been omitted for clarity)

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
into extended chains which run parallel to the [001] direction with graph set motifs
C(11) and C(8), respectively.
The purification of the tetrapeptide 18 by flash column chromatography afforded a
second product 19 in very small amounts, which was also crystallized, and its structure
was established by X-ray-analysis (Fig. 3, b). The molecular structure of 19 proved to
be almost identical with that of 18 except for the configuration of the Pro residue, which
was found to be (R). Although peptide couplings with HOBt additives are known to
proceed without epimerization, we assume that the introduction of H-Leuol at this
position is a weak point in the synthesis, as a similar result was obtained in the synthesis
of HM A1 [12]. The yields for this coupling reaction are 63% for 18, and additional 1–
2% of 19 were isolated, i.e., the epimerization occurs only to a small extent.
The molecular structure of 19 is shown in Fig. 3,b5). The space group permitted the
compound in the crystal to be enantiomerically pure, but the absolute configuration of
the molecule has not been determined. The enantiomer used in the refinement was
based on the known (S)-configuration of Leu, Ile, and Leuol, but the configuration of
Pro is (R). There are two symmetry-independent molecules in the asymmetric unit,
which have the same configuration. One of the molecules is disordered at three
positions, the benzyl group, and the Ile and Leu side chains. The crystal was also
twinned with the major twin fraction being ca. 81%.
Each OH and amide NH group of each symmetry-independent molecule acts as a
donor for H-bonds. The OH group in each molecule forms an intramolecular H-bond
with the amide O-atom of Ile. This interaction has a graph set motif of S(13). The NH
group of Leuol in each molecule also forms an intramolecular H-bond with the same
amide O-atom of Ile. This interaction has a graph set motif of S(10). The amide H-atom
of Ile (N(1)ꢀH) in molecule A forms an intermolecular H-bond with the OH group of
molecule B. In turn, the corresponding amide group of molecule B interacts in the same
way with the original molecule A. These interactions form a ring with a graph set motif
of R22(28). The amide H-atom of Aib (N(4)ꢀH) in molecule A forms an intermolecular
H-bond with the Aib amide O-atom from an adjacent molecule A, thereby linking the
A molecules into extended chains, which run parallel to the [010] direction with a graph
set motif of C(5). The identical interaction links the B molecules into extended chains,
which run antiparallel to the chains of A molecules. The combination of the
intermolecular interactions leads to a two-dimensional network of A and B molecules
that lies parallel to the (100) plane.
The deprotection of the amino group of 18 with Pd/C under H2 gave the
tetrapeptide H-Ile-Aib-Pro-Leuol (20) quantitatively (Scheme 3). From this com-
pound, suitable crystals for an X-ray structure determination could also be obtained
(Fig. 4).
The space group permits 20 in the crystal to be enantiomerically pure, but the
absolute configuration of the molecule has not been determined. The enantiomer used
in the refinement was based on the known (S)-configuration of Ile in the molecule. All
stereogenic centers in the molecule have the (S)-configuration. The asymmetric unit
contains two molecules of 20, both of which are the same stereoisomer, plus a site for a
5
)
The X-ray crystal-structure analysis of 19 does not allow conclusions concerning bond lengths or
angles that might appear to be unusual because of the poor overall quality of the crystals.

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2535
Fig. 4. ORTEP Plot [14] of the molecular structure of one of the symmetry-independent molecules of 20
(50% probability ellipsoids; arbitrary numbering of the atoms; H-atoms bonded to C-atoms have been
omitted for clarity)
H2O molecule that is both partially occupied and disordered about a C2-axis. Thus, the
peptide/H2O ratio in the crystal is 8 :1. The Ile group in one of the independent peptide
molecules (molecule B) is disordered over two significantly different conformations.
The conformations of molecules A and B also differ significantly in the orientations of
the Leuol end of the peptide chain.
In each of the symmetry-independent peptide molecules 20, the amide NꢀH of
Leuol forms an intramolecular H-bond with the Aib amide O-atom that is seven atoms
back along the peptide backbone. Each of these interactions has the graph set motif of
S(10). In molecule A, the terminal NH2 group forms intermolecular H-bonds with
amide O-atoms of a neighboring molecule A (Aib), forming chains of A molecules
which run parallel to the [010] direction and have a graph set motif of C(8), and with a
neighboring molecule B (Pro; graph set motif D). The terminal NH2 group of molecule
B only forms one H-bond, that being with the Pro amide O-atom of another molecule
B, linking the B molecules into chains which also run parallel to the [010] direction with
a graph set motif of C(11). The amide H-atom of Aib of molecule A forms an
intermolecular H-bond with the OH O-atom of the same neighboring molecule A that
interacts with the terminal NH2 group. This interaction within the chains of A
molecules has a graph set motif of C(11). The amide H-atom of Aib of molecule B
forms an intermolecular H-bond with the Pro amide O-atom of a neighboring molecule
A (graph set motif D). Further, the OH group of molecule A forms an intermolecular
H-bond with the terminal amine N(1)-atom of an adjacent molecule A in the same
chains described previously (graph set motif C(14)), while the OH group of molecule B
forms an intermolecular H-bond with the Pro O(9)-atom of a neighboring molecule A

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
(graph set motif D). Together, the intermolecular interactions link the molecules into
two-dimensional networks, which lie parallel to the (100) plane6).
Then, 20 was coupled with Z-Leu-OH to give the pentapeptide Z-Leu-Ile-Aib-Pro-
Leuol (21; 97% yield), which was deprotected to give quantitatively 15, i.e., the
segment 7–11 of HM A3 (Scheme 3). The coupling of 15 with Z-Leu-Aib-Pro-OH
(22), which was set apart in the synthesis of HM A1 [12], was performed as usual with
TBTU/HOBt leading to the protected octapeptide Z-Leu-Aib-Pro-Leu-Ile-Aib-Pro-
Leuol (23) in 97% yield, which is again a surprisingly high yield for such a coupling
reaction. The latter was deprotected to furnish H-Leu-Aib-Pro-Leu-Ile-Aib-Pro-Leuol
(24) in 98% yield. Finally, HM A3 was obtained by the coupling of 24 with Ac-Aib-Gln-
Val-OH (6) in 68% yield (Scheme 4).
Scheme 4
Suitable crystals of HM A3 for an X-ray-analysis could be obtained (Fig. 5,a)7).
The space group permits the compound in the crystal to be enantiomerically pure, but
the absolute configuration of the molecule has not been determined. The enantiomer
used in the refinement was based on the known (S)-configuration of the amino acids in
the molecule. The structure determination confirmed that there were no unexpectedly
inverted configurations present. The asymmetric unit contains one peptide molecule
and one disordered H2O molecule. The peptide molecule displays disorder in several
side chains, i.e., the terminal two Me groups of Leu4 and the terminal Me groups of
Leuol, as well as three of the ring C-atoms of Pro10.
Most of the amide groups are involved in intramolecular H-bonds, but a few
intermolecular interactions do occur, linking the peptide and H2O molecules into two-
dimensional networks (Fig. 5,b). The O(33)ꢀH group forms an intermolecular H-bond
with the amide O(18)-atom of the central Pro6 of a neighboring molecule. This
interaction links the peptide molecules into extended chains which run parallel to the
6
)
Although the H-atoms of the H2O molecule could not be located, the H2O molecule is close to the
OH O-atoms of peptide molecules A and B, and can be assumed to be involved in H-bonding
interactions with these groups.
The crystals were quite small needles of poor quality and were very weakly diffracting. As a result,
there was a paucity of data, and the overall quality of the structure was significantly lower than
desirable. The overall molecular conformation was clearly defined, but the large standard
uncertainties on the geometric parameters mean that no far-reaching conclusions should be drawn
from a detailed analysis of individual bond lengths, bond angles, or torsion angles.
7
)

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2537
Fig. 5. a) ORTEP Plot [14] of the molecular structure of hypomurocin A3 (HM A3) (50% probability
ellipsoids; arbitrary numbering of the atoms; H-atoms bonded to C-atoms have been omitted for
clarity). b) Molecular packing of hypomurocin A3 (HM A3) projected down the b-axis showing the H-
bonding scheme (equivalent isotropic spheres for atoms; uninvolved H-atoms omitted for clarity; color
code: C-atoms: black, O-atoms: red, N-atoms: blue, H-atoms: green, and H-bonds: red).

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
[010] direction with a graph set motif of C(19). The primary amide group of the Gln2
side chain forms intermolecular H-bonds with two different neighboring peptide
molecules, with the acceptor amide O(30)- and O(27)-atoms, respectively, being in the
Pro10 and Aib9 moieties. One of these interactions links the peptide molecules into
extended chains which run parallel to the [011] direction and can be described by the
graph set motif of C(32), while the other interaction links the peptide molecules into
extended chains which run parallel to the [010] direction with the graph set motif of
C(29). The amide H-atom of Aib1 (N(1)ꢀH) forms an intermolecular H-bond with the
O-atom of the disordered H2O molecule. Although the H-atoms of the H2O molecule
could not be located, suitable O· · ·O distances indicate that the H2O molecule donates
two H-bonds. One interaction is with the OH O-atom in the peptide molecule from
which the amide N(1)ꢀH (Aib1) interaction originates, thereby linking pairs of peptide
and H2O molecules into loops with a binary graph set motif of R22(37). The second
interaction is with the amide O-atom of the Gln2 side chain of a different peptide
molecule, thereby linking pairs of peptide and H2O molecules into chains which run
parallel to the [011] direction and can be described by the graph set motif of C22(12). All
of the intermolecular H-bonding interactions combine to link the peptide and H2O
molecules into two-dimensional networks, which lie parallel to the (100) plane. With
the exception of N(25)ꢀH of Aib9, which is not involved in any H-bonding interaction,
all amide groups not mentioned above form intramolecular H-bonds with amide O-
atoms. The amide H-atoms N(7)ꢀH (Val3), N(19)ꢀH (Leu7), N(22)ꢀH (Ile8), and
N(31)ꢀH (Leuol11) form intramolecular H-bonds with the amide O-atoms that are
seven atoms back along the peptide backbone. Each of these interactions has the graph
set motif of S(10). The amide H-atom of Gln2 (N(4)ꢀH) forms an intramolecular H-
bond with the amide O-atom of the Gln2 side chain, thereby creating a loop with a
graph set motif of S(7). The amide H-atoms N(10)ꢀH of Leu4 and N(13)ꢀH of Aib5 do
not interact as expected with the amide O-atoms O(3) of Aib1 and O(6) of Gln2,
respectively, but interact instead with O(34) of Ac and O(3) of Aib1, respectively, to
give loops with a graph set motif of S(13). Therefore, HM A3 contains elements of two
types of helical structures, i.e., a 310-helix (graph set motif S(10)) and an a-helix (graph
set motif S(13)).
2.2. Synthesis of HM A5. Having prepared HM A1 and HM A3, the synthesis of HM
A5 was rather short. It can be resumed at the synthesis of the segment 1–3 (Ac-Aib-
Gln-Ile-OH; 7; Scheme 2), which has to be coupled with the two segments 4–6 (Z-Ile-
Aib-Pro-OH; 17; Scheme 3) and 7–11 (H-Leu-Leu-Aib-Pro-Leuol; 25) from the HM
A1 synthesis [12] (Scheme 5). The Z-protected octapeptide 26 was obtained by the
condensation of 17 and 25 with TBTU/HOBt in 64% yield. The deprotection of 27
under the usual conditions gave 26 quantitatively, which was then coupled with the
tripeptide 7 to give HM A5 in 31% yield.
Suitable crystals of 26 for an X-ray crystal-structure determination were obtained
from Et2O/MeOH/AcOEt/hexane/cyclohexane (Fig. 6). The space group permits the
compound in the crystal to be enantiomerically pure, but the absolute configuration of
the molecule has not been determined. The enantiomer used in the refinement was
based on the expected (S)-configuration of the chiral amino acids used. The structure
determination confirms that all peptide units do indeed have the (S)-configuration.
There are two symmetry-independent molecules in the asymmetric unit. Both have the

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2539
Scheme 5
same configuration, but their conformations are slightly different (Fig. 6, b). Some of
the side chains in each molecule are disordered over two conformations, i.e., the iPr side
chain of Leuol and the entire side chain of Leu5 of molecules A and B, respectively, as
i
well as the Pr side chain of Leu4 in molecule A and the entire side chain of Ile1 in
molecule B. In addition, Pro3 of each molecule has two envelope conformations with
the C-atom of the envelope flap being ꢂupꢃ or ꢂdownꢃ. The asymmetric unit includes two
molecules of the peptide plus two disordered solvent molecules, which, from the
arrangement of residual electron-density peaks, appeared to be AcOEt.
The OH group in each molecule forms an intramolecular H-bond with the amide O-
atom of Pro7, thereby forming a loop with a graph set motif of S(10). The amide H-atom
of Ile1 in molecule A forms an intermolecular H-bond with the OH O-atom of a
neighboring molecule A. This interaction links the A molecules into extended chains,
which run parallel to the [010] direction and can be described by the graph set motif of
C(26) (Fig. 7). The corresponding interaction also links the B molecules into extended
chains, which run parallel to the [010] direction. The amide H-atom of Aib2 (N(4)ꢀH)
in molecule A forms an intermolecular H-bond with the amide O-atom of Pro7 of the
same neighboring molecule A that undergoes the N(1)ꢀH· · ·O interaction with the
amide H-atom of Ile1, so that this interaction is also a link in the chains described above
(graph set motif C(20)). Molecule B behaves in an identical fashion. The amide H-
atoms of Leu4 (N(10)ꢀH), Leu5 (N(13)ꢀH), Aib6 (N(16)ꢀH), and Leuol8 (N(22)ꢀH)
form intramolecular H-bonds with the amide O-atoms that are seven atoms back along
the peptide backbone. Each of these interactions has the graph set motif of S(10), and
they serve to maintain a fairly rigid helical conformation of the peptide. The same
pattern of intramolecular interactions occurs in molecule B with one exception. The
amide H-atom of Aib6 (N(76)ꢀH) in molecule B does not interact as expected with
O(69) of Pro3, but interacts instead with O(66) of Aib2 in molecule B to give a loop with
a graph set motif of C(13). The N· · ·O and H· · ·O distances for N(76)ꢀH· · ·O(69) are
3.641(6) and 3.00 ꢅ, respectively, and are well beyond the distance considered as an H-
bonding interaction. Presumably, this difference results from a small difference in the
turn structure of molecule B compared with that in molecule A. Superposition of the
two molecules shows that, although the conformations at the Z-end are similar, small
deviations develop after the Pro3 unit (Fig. 6, b).
2.3. Correlation of the Synthetic Samples with the Natural Peptaibols. The synthetic
peptaibols HM A1 [12], HM A3, and HM A5 were analyzed by reversed-phase HPLC/

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Fig. 6. a) ORTEP Plot [14] of the molecular structure of one of the symmetry-independent molecules of 26
(conformation A, 50% probability ellipsoids; arbitrary numbering of the atoms; H-atoms bonded to C-
atoms have been omitted for clarity). b) Superposition of the two symmetry-independent molecules of 26.
ESI-MS individually or as defined mixtures with the fraction containing the set of
hypomurocins A (HM A) of the extract of the fungus H. muroiana. According to
Brꢂckner and co-workers [16], the HM A fraction is a mixture composed of six
structurally similar peptaibols possessing three different quasi-molecular ions ([Mþ
H]þ ). In Fig. 8, the base-peak chromatograms (BPC) of the natural fungus extract, and
of mixtures of the natural extract with the three synthetic materials are shown. In our
experiment, the natural peptaibol HM A1 ([MþH]þ , m/z 1161) was detected at tR

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2541
Fig. 7. Molecular packing of 26 projected down the a-axis showing the H-bonding scheme (equivalent
isotropic spheres for atoms; uninvolved H-atoms omitted for clarity; color code: C-atoms: black, O-
atoms: red, N-atoms: blue, H-atoms: green, and H-bonds: red)
28.5 min, the three compounds HM A2, A3, and A4 ([MþH]þ , m/z 1175 each) all co-
eluted at tR 29.5 min, and the isomeric HM A5 and A5a ([MþH]þ , m/z 1189 each)
were recorded at tR 30.7 min (Fig. 8, d). It is easily recognized from the chromatograms
that the addition of synthetic HM A1 to the natural mixture induces an increase of the
peak at tR 28.9 min (Fig. 8,c). The same experiment performed with synthetic HM A3
leads to an increase of the peak at tR 29.7 min (Fig. 8,b), and the addition of synthetic
HM A5 results in an increase of the peak at tR 31.2 min (Fig. 8,a).
The synthetic peptaibols were further analyzed by MS/MS (see also [17]) to confirm
their amino acid sequences (see Exper. Part). Due to the presence of two proline
residues in the HM A peptaibols, abundant fragment ions of the b- and y-type
(nomenclature of the peptide fragment ions according to Roepstorff and Fohlman [18],
and Biemann [19]), deriving from cleavages of the amide bond adjacent to the proline
N-terminus, are observed in the mass spectra. This gas-phase behavior of proline-rich
peptides has already been reported [20]. As a consequence, the intensities of the quasi-
molecular ions [MþH]þ observed for the HM A derivatives were low (<1% rel.
intensity). The MS/MS data obtained clearly revealed the amino acid sequence of the
three synthetic compounds, and were identical with those obtained for the natural
products, thus confirming the structures reported [16].
2.4. Structural Analysis in Solution by NMR. The conformations of HM A3 and HM
A5 were investigated by NMR spectroscopy in (D6)DMSO solutions at 300 K.

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Fig. 8. HPLC-ESI-MS Base-peak chromatograms (BPC) representing the hypomurocin A (HM A)
fraction of H. muroiana spiked with synthetic HM A5 (a), HM A3 (b), and HM A1 (c), as well as the BPC
of the natural HM A sample (d)

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2543
Chemical-shift assignments were achieved by standard methods. The experimental
methods were detailed in [12].
The conformation of HM A1 in solution was characterized previously by NMR
spectroscopy [12]. It could be shown that HM A1 adopts well-defined helical structures
in (D6)DMSO solution as well as in the presence of micelles. For comparison with HM
A1, the NMR structure determinations were performed in the present study with the
analogs HM A3 and HM A5.
Both compounds show a single set of 1H-NMR resonances similar to the spectrum
of HM A1. In the case of HM A5, selective broadening of some backbone 1H
resonances were observed indicating peptide association or conformational exchange
processes. Nevertheless, the resonance assignment was completed for both compounds.
The observed ROE connectivities indicate the trans-orientation of all peptide bonds.
Even prior to the structure calculation, the analysis of the chemical shifts, and
sequential and medium-range ROEs revealed the formation of helical structures in
solution. Especially, the difference between the observed aH chemical-shift values and
the corresponding random-coil values (Dd¼dobserved ꢀdrandom) are widely used as an
indicator of secondary-structure formation [21]. Negative values of Dd less than
ꢀ0.1 ppm, reflecting helical regions, could be observed for residues 2, 3, 6, 7, 10, and 11
in all three compounds (Fig. 9).
Fig. 9. Deviation of aH chemical shifts relative to the random-coil values of residues (except Aibs) in HM
A1, HM A3, and HM A5
Similar to HM A1, numerous characteristic ROEs were observed with HM A3 and
HM A5, which clearly indicate predominantly helical and/or turn conformations. In
particular, the presence of sequential HN,HN ROEs, as well as medium-range aN(i,i þ
2), aN(i,iþ3), aN(i,iþ4), and ab(i,iþ3) ROEs are evident for helical folded peptides
(in the case of Pro: HN is replaced by d(H); in the case of Aib: aH is replaced by bH).

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
The average solution structures of HM A3 and HM A5 were determined using 113 and
122 ROE connectivities, respectively. The NMR structures converge to a narrow range
of backbone conformations with RMSD values of 0.56 ꢅ for HM A3 and 0.25 ꢅ for
HM A5. The calculated structures of HM A3 (Fig. 10) and HM A5 (Fig. 11) represent
well-defined helical structures. The helical conformation of HM A3 and HM A5 seem
to be slightly distorted, indicated by the non-ideal helical backbone torsion angles
(Table 1). Therefore, the structure can be described as helical with mixed secondary-
structure elements of a-helices, 310-helices, and type I b-turns.
Fig. 10. a) Superposition of the final 20 NMR structures of HM A3. The backbone conformations are
shown by a ribbon representation. b) A typical NMR structure in ball-and-stick representation. The
branched-aliphatic residues at position 3, 4, 7, 8, and 11 are indicated in green.
Finally, it could be shown that the solution structures of HM A3 and HM A5
correlate very well with the structure of HM A1 determined previously. Therefore, it
can be concluded that the sequence variations at positions 3, 4, and 8 have only minor
impact on the general fold of these peptides in solution. An important feature of the
structures is the orientation of all hydrophobic side chains on one side of the helix. This
amphiphilic structure is a condition for the ability to form ion channels [5].
3. Conclusions. – The two hypomurocins A3 and A5 were successfully synthesized.
We have shown that the introduction of the two Aib-Pro units via the ꢂazirine/

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2545
Fig. 11. a) Superposition of the final 20 NMR structures of HM A5. The backbone conformations are
shown by a ribbon representation. b) A typical NMR structure in ball-and-stick representation. The
branched-aliphatic residues at position 3, 4, 7, 8 and 11 are indicated in green.
Table 1. Calculated f and y Torsion Angles of the NMR Structures of HM A3 and HM A5
Residue
HM A3
HM A5
f [8]
y [8]
f [8]
y [8]
Aib1
61.6ꢁ99.3
7.4ꢁ95.3
172.4ꢁ8.4
ꢀ83.0ꢁ63.9
ꢀ22.0ꢁ11.1
ꢀ4.1ꢁ29.6
ꢀ32.2ꢁ7.5
ꢀ61.9ꢁ1.2
ꢀ22.0ꢁ0.1
ꢀ9.0ꢁ2.5
Gln2
ꢀ89.7ꢁ26.4
ꢀ81.3ꢁ15.2
ꢀ94.2ꢁ11.6
ꢀ53.1ꢁ9.2
ꢀ63.8ꢁ0.1
ꢀ93.6ꢁ4.8
ꢀ82.7ꢁ4.7
ꢀ42.9ꢁ3.0
ꢀ64.1ꢁ0.3
ꢀ126.6ꢁ15.3
ꢀ6.7ꢁ14.0
ꢀ41.2ꢁ0.2
2.4ꢁ20.4
ꢀ98.2ꢁ72.7
ꢀ89.6ꢁ12.6
ꢀ102.4ꢁ31.4
ꢀ38.1ꢁ1.4
ꢀ70.6ꢁ0.0
ꢀ76.0ꢁ1.0
ꢀ83.7ꢁ4.3
ꢀ39.1ꢁ0.7
ꢀ70.6ꢁ0.0
ꢀ63.1ꢁ10.9
Val3/Ile3
Leu4/Ile4
Aib5
ꢀ65.4ꢁ1.3
ꢀ0.1ꢁ0.1
ꢀ35.7ꢁ8.0
ꢀ21.3ꢁ3.4
ꢀ65.4ꢁ2.3
ꢀ6.8ꢁ3.0
ꢀ32.1ꢁ14.8
Pro6
Leu7
Ile8/Leu8
Aib9
ꢀ18.2ꢁ2.7
ꢀ61.7ꢁ1.0
ꢀ17.4ꢁ0.6
162.8ꢁ17.5
Pro10
Leu11
oxazolone methodꢃ proceeds with almost quantitative yield [12]. The hydrolysis of
methyl esters with LiOH and the deprotection of tert-butyl esters with TFA were also

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
performed in very high yield, as well as the hydrogenolytic deprotection of the Z
protecting group and the coupling of small peptide segments. As expected, the sensitive
points in the synthesis were the coupling reactions of the segments: in the case of HM
A3, the yields were excellent, being almost quantitative for five steps. Even the last
coupling leading to HM A3 was performed with high yield (68%). For HM A5, yields
are also good, except for the final coupling to give HM A5 in only 31% yield, as well as
for the introduction of Aib1 in only 66% yield.
Concerning the epimerization, analogous to the synthesis of HM A1 [12], the
problem also appeared with the coupling of H-Leuol to Pro. This was the only partial
epimerization observed in the three HM A molecules targeted, and the bond-formation
between Pro10 and Leuol with TBTU/HOBt is the only weak point of the chosen
strategy for the synthesis of hypomurocins. Here again, although the epimer could be
separated and isolated, its amount was negligible in term of yields. These total
syntheses illustrate once more the advantages of the ꢂazirine/oxazolone methodꢃ in the
preparation of peptaibols.
Several intermediates or final products were obtained as crystals suitable for X-ray
structure determination, which confirmed the helical structure of Aib-containing
oligopeptides. Furthermore, NMR studies revealed that all three synthesized hypo-
murocins HM A1, HM A3, and HM A5 adopt helical conformations in solution with
amphiphilic properties. The final HPLC correlation between synthetic and natural
hypomurocins A confirmed the original structure determinations of the hypomurocins
by Brꢂckner and co-workers [16].
We thank Prof. Dr. Hans Brꢂckner for providing the sample of natural hypomurocin A. We also
thank the analytical sections of our institute for spectra and analyses, especially Nadja Bross, Dr. Gudrun
Hopp, and Simon Jurt for numerous NMR studies, and the Swiss National Foundation and F. Hoffmann-
La Roche AG, Basel, for financial support.
Experimental Part
General. See [12].
RP-HPLC Systems, Correlation of Synthetic and Natural Hypomurocins. HPLC-MS: Hewlett-
Packard HP 1100 HPLC system (Hewlett-Packard, Palo Alto, CA, USA) equipped with a HTS PAL
autosampler (CTC Analytics, CH-Zwingen), connected to a Bruker ESQUIRE-LC quadrupole ion-trap
instrument (Bruker Daltonik GmbH, D-Bremen) equipped with a Hewlett-Packard electrospray (ESI)
ion source. Solvents and reagents: MeCN (HPLC grade, Scharlau, E-Barcelona); HCOOH puriss. p.a.
(Fluka, CH-Buchs). H2O was purified with a MILLI-QRG apparatus (Millipore, Milford, MA, USA).
Samples preparation: synthetic material (40 mg) was dissolved in H2O/MeCN 9 :1 (500 ml); the fraction
containing the natural HM A peptaibols was dissolved in H2O/MeCN 9 :1 (70 ml). Samples of 1 ml of the
synthetic peptaibol stock soln., of 1 ml of the natural HM A peptaibols stock soln., or of a 1:1 mixture of
synthetic/natural stock soln. were injected for the individual runs and were analyzed by HPLC/ESI-MS.
Column and chromatographic conditions: Interchrom Uptisphere C18 HDO m column (UP3 HDOA5,
3 mm, 0.25ꢂ50 mm, Interchim, F-MontluÅon); the eluent from the HPLC was split using an Acurate high-
pressure pre-column flow splitter (LC Packings, NL-Amsterdam), and the output flow was set at a rate of
3 ml minꢀ1. Mobile phase: gradient over 50 min from 10 to 90% of solvent B (solvent A: 0.1% HCOOH in
H2O; solvent B: 0.1% HCOOH in MeCN); tR in min. MS Conditions: Nebulizer gas (N2), 13 psi; dry gas
(N2), 8 l minꢀ1; dry temp., 2808; cap. voltage, 4500 V; end plate, ꢀ500 V; cap. exit, 70 V; and skimmer 1,
19 V. The MS acquisitions were performed at normal resolution (0.6 m full width at half maximum),
under ion-charge control (ICC) conditions (10000) in the mass range from m/z 50 to 1300, and an m/z

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2547
50.2 trap-drive value. The fragmentation experiments were performed in the auto MS/MS mode by
selecting two precursor ions, the isolation width was 4 m, the fragmentation cut-off set by ꢂfast calcꢃ, and
the fragmentation amplitude set at 0.7 V in the ꢂSmartFragꢃ mode.
Abbreviations. See [12].
General Procedure 1 (GP 1; Peptide Coupling). To a soln. of an N-protected amino acid or N-
protected peptide and 1 equiv. of a C-protected amino acid or C-protected peptide in MeCN were added
1 equiv. of TBTU and HOBt. When the mixture was completely dissolved, 3 equiv. of Et3N were added.
The mixture was stirred overnight at r.t. (N2 atmosphere) and checked by TLC. After completion of the
reaction, the mixture was washed with brine (3ꢂ ). The org. layer was evaporated, and the product was
purified by flash chromatography (FC) or prep. layer chromatography (PLC).
General Procedure 2 (GP 2; Coupling with 2H-Azirin-3-amines). To a soln. of 1.2 equiv. of the
corresponding 2H-azirin-3-amine in THF or CH2Cl2, 1 equiv. of an N-protected amino acid or N-
protected peptide was added, and the mixture was stirred at r.t. overnight (N2 atmosphere). After
completion of the reaction, the solvent was evaporated, and the product was purified by FC or PLC.
General Procedure 3 (GP 3; Saponification of Peptide Methyl Esters). To a soln. of the peptide ester
in THF/MeOH/H2O 3 :1:1, 3 equiv. of LiOH·H2O were added, and the mixture was stirred at r.t.
overnight (N2 atmosphere). After completion of the reaction (TLC), the mixture was acidified with 1n
HCl to reach pH 1, and then extracted with CH2Cl2 (3ꢂ ), the org. layers were combined, dried
(Na2SO4), and evaporated. The product was purified by FC or PLC.
General Procedure 4 (GP 4; Hydrogenolytic Deprotection). The N-protected peptide was dissolved
in MeOH, and 10% Pd/C (1/3 of the weight of the peptide) was added to the soln. The mixture was stirred
overnight under H2 at r.t. After completion of the reaction (TLC), the soln. was filtered through a Celite
pad, and the solvent was evaporated. The product was dried in high vacuum and used for the next
reaction step without any further purification.
RP-HPLC Correlation of Synthetic and Natural Hypomurocins. According to [16], the fragments
Pro-Leu, Pro-Leu-Leu, Pro-Leu-Leu-Aib, Pro-Leu-Ile, and Pro-Leu-Ile-Aib were labeled PL, PLL,
PLLU, PLI and PLIU, resp.
Data for HM A1. HPLC/MS: tR 28.9 min; 539.2 (100, bþ5 ), 623.5 (91, yþ6 ), 947.6 (8, b9þ), 1161.5 (<1,
[MþH]þ ); MS/MS (539.2): 256.1 (14, bþ2 ), 327.2 (4, bþ3 – CO), 355.2 (46, b3þ), 454.2 (100, b4þ); MS/MS
(623.5): 183.2 (11, [PLþHꢀCO]þ ), 211.1 (30, [PLþH]þ ), 215.2 (6, yþ2 ), 279.2 (6), 296.2 (3, [PLIþHꢀ
CO]þ ), 324.2 (97, [PLIþH]þ ), 381.2 (6, [PLIUþHꢀCO]þ ), 409.3 (100, [PLIUþH]þ ).
Data for HM A3: HPLC/MS: tR 29.7 min; 215.2 (5, yþ2 ), 553.3 (100, b5þ), 623.4 (95, y6þ), 962.5 (9, bþ9 ),
1175.6 (<1, [MþH]þ ). MS/MS (553.3): 256.1 (18, b2þ), 327.2 (4, bþ3 ꢀCO), 355.2 (51, bþ3 ), 440.3 (<1,
bþ4 ꢀCO), 468.2 (100, b4þ); MS/MS (623.4): 183.2 (12, [PLþHꢀCO]þ ), 211.1 (32, [PLþH]þ ), 215.2 (6,
yþ2 ), 279.2 (3), 296.2 (2, [PLIþHꢀCO]þ ), 324.2 (71, [PLIþH]þ ), 381.3 (3, [PLIUþHꢀCO]þ ), 409.3
(100, [PLIUþH]þ ).
Data for HM A5. HPLC/MS: tR 31.2 min; 567.4 (77, bþ5 ), 623.4 (100, yþ6 ), 975.5 (9, b9þ), 1189.6 (<1,
[MþH]þ ). MS/MS (567.4): 256.1 (25, bþ2 ), 341.2 (4, bþ3 ꢀCO), 369.2 (61, b3þ), 482.3 (100, b4þ); MS/MS
(623.4): 183.1 (14, [PLþHꢀCO]þ ), 211.1 (33, [PLþH]þ ), 215.1 (5, yþ2 ), 279.2 (8), 324.2 (98, [PLIþ
H]þ ), 381.3 (6, [PLIUþHꢀCO]þ ), 409.3 (100, [PLIUþH]þ ).
Z-Ile-Aib-Pro-OMe (16). According to GP 2, 14 (1782.4 mg, 7.266 mmol) was dissolved in CH2Cl2
(80 ml), Z-Ile-OH (1752.5 mg, 6.605 mmol) was added, and the mixture was stirred overnight. Then, the
solvent was evaporated, and the crude product was purified by FC to give 16 (1559 mg, 51%). White
foam. Rf (AcOEt) 0.65. M.p. 115–1178. [a]D20 ¼ ꢀ69.0 (c ¼ 0.10, MeOH). RP-HPLC (II): tR 21.35, l
219 nm. IR (KBr): 3279vs, 3065s, 3036m, 2966vs, 2928s, 2877m, 1752vs, 1718vs, 1666vs, 1622vs, 1540vs,
1454vs, 1416vs, 1384s, 1364vs, 1271vs, 1241vs, 1205vs, 1162vs, 1125s, 1095m, 1046s, 1027s, 777m, 754m,
1
739s, 697s, 646m, 616m. H-NMR (300 MHz, CD3OD): 0.86–0.97 (m, Me(g)(Ile), Me(d)(Ile)); 1.13–
1.30 (m, CH2a(g)(Ile)); 1.43, 1.45 (2s, 2 Me(b)(Aib)); 1.47–1.57 (m, CH2b(g)(Ile)); 1.58–1.68 (m,
CH2a(g)(Pro)); 1.69–1.80 (m, CH2b(b)(Pro), CH(b)(Ile)); 1.80–1.95 (m, CH2b(g)(Pro), CH2a(b)(Pro));
3.46–3.57 (m, CH2a(d)(Pro)); 3.57–3.65 (m, CH2b(d)(Pro)); 3.67 (s, MeO); 3.93–4.00 (m, CH(a)(Ile));
4.38 (dd, J¼8.4, 3.7, CH(a)(Pro)); 5.02, 5.14 (AB, J ¼ 12.6, PhCH2); 7.07 (br. d, J ¼ 8.8, NH(Ile)); 7.25–
7.38 (m, 5 arom. CH); 8.49 (br. s, NH(Aib)). 13C-NMR (75.5 MHz, CD3OD): 11.1 (q, Me(d)(Ile)); 15.9
(q, Me(g)(Ile)); 24.5, 25.7 (2q, 2 Me(b)(Aib)); 25.9 (t, CH2(g)(Ile)); 26.7 (t, CH2(g)(Pro)); 28.8 (t,

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
CH2(b)(Pro)); 38.2 (d, CH(b)(Ile)); 49.1 (t, CH2(d)(Pro)); 52.5 (q, MeO); 57.6 (s, C(a)(Aib)); 60.5 (d,
CH(a)(Ile)); 62.0 (d, CH(a)(Pro)); 67.5 (t, PhCH2); 128.7, 129.0, 129.5 (3d, 5 arom. CH); 138.4 (s, arom.
C); 158.4 (s, OꢀC¼O); 173.5, 173.9, 174.8 (3s, 3 C¼O). ESI-MS (MeOHþNaI): 484.2 (100, [M þ Na]þ ).
Anal. calc. for C24H35N3O6 ·0.5 H2O (470.51): C 61.27, H 7.71, N 8.93; found: C 61.33, H 7.89, N 8.87.
Z-Ile-Aib-Pro-OH (17). According to GP 3, 16 (1.71 g, 3.70 mmol) was dissolved in THF/MeOH/
H2O (126 ml), and LiOH·H2O (466 mg, 11.11 mmol) was added. After the workup, the solvent was
evaporated to give 17 (1.32 g, 80%). White foam. Rf (AcOEt) 0.09. M.p. 90–928. [a]2D0 ¼ ꢀ48.6 (c ¼ 1.00,
MeOH). RP-HPLC (II): tR 29.22, l 218 nm. IR (KBr): 3417vs, 3036s, 2965vs, 2933vs, 2877s, 1713vs,
1611vs, 1535vs, 1455vs, 1403vs, 1251vs, 1175s, 1130s, 1089s, 1042vs, 1027s, 983m, 776m, 737s, 697s, 661m,
1
619m. H-NMR (300 MHz, CD3OD): 0.86–0.95 (m, Me(g)(Ile), Me(d)(Ile)); 1.06–1.23 (m, CH2a(g)-
(Ile)); 1.44, 1.46 (2s, 2 Me(b)(Aib)); 1.50–1.59 (m, CH2b(g)(Ile)); 1.59–1.68 (m, CH2a(g)(Pro)); 1.69–
1.95 (m, CH2b(g)(Pro), CH2ab(b)(Pro), CH(b)(Ile)); 3.47–3.66 (m, CH2ab(d)(Pro)); 3.97 (t, J¼8.3,
CH(a)(Ile)); 4.38 (dd, J¼8.4, 3.5, CH(a)(Pro)); 5.02, 5.15 (AB, J ¼ 12.5, PhCH2); 7.06 (br. d, J ¼ 8.8,
NH(Ile)); 7.26–7.37 (m, 5 arom. CH); 8.49 (br. s, NH(Aib)). 13C-NMR (75.5 MHz, CD3OD): 11.2 (q,
Me(d)(Ile)); 15.9 (q, Me(g)(Ile)); 24.6, 25.7 (2q, 2 Me(b)(Aib)); 26.0 (t, CH2(g)(Ile)); 26.7 (t,
CH2(g)(Pro)); 29.1 (t, CH2(b)(Pro)); 38.2 (d, CH(b)(Ile)); 49.1 (t, CH2(d)(Pro)); 57.6 (s, C(a)(Aib));
60.5 (d, CH(a)(Ile)); 62.0 (d, CH(a)(Pro)); 67.5 (t, PhCH2); 128.7, 129.0, 129.5 (3d, 5 arom. CH); 138.4
(s, arom. C); 158.5 (s, OꢀC¼O); 173.5, 173.9, 176.1 (3s, 3 C¼O). ESI-MS (MeOHþNaI): 470.1 (100,
[M þ Na]þ ).
Z-Ile-Aib-Pro-Leuol (18). According to GP 1, a mixture of 17 (1.237 g, 2.76 mmol), H-Leuol·HCl
(424.7 mg, 2.76 mmol), TBTU (887.5 mg, 2.76 mmol), and HOBt (373 mg, 2.76 mmol) was dissolved in
MeCN (160 ml), and Et3N (1166 ml, 8.29 mmol) was added. After completion of the reaction, the mixture
was purified by FC to give 18 (946.5 mg, 63%). White powder. Rf (AcOEt/MeOH 6 :1) 0.57. M.p. 192–
1958. [a]2D0 ¼ ꢀ28.7 (c ¼ 1.00, MeOH). RP-HPLC (II): tR 49.53, l 219 nm. IR (KBr): 3466vs, 3392s,
3294vs, 3005s, 2972vs, 2877s, 1720vs, 1630vs, 1555vs, 1514vs, 1469s, 1413vs, 1381s, 1363m, 1327s, 1310m,
1286s, 1245vs, 1198s, 1171s, 1150m, 1087s, 1067s, 1037s, 1009m, 992m, 758s, 721m, 701m, 651w, 625m.
1H-NMR (300 MHz, CD3OD): 0.86–0.96 (m, 2 Me(d)(Leuol), Me(g)(Ile), Me(d)(Ile)); 1.10–1.38 (m,
CH2a(b)(Leuol), CH2b(g)(Ile)); 1.41, 1.47 (2 br. s, 2 Me(b)(Aib)); 1.50–1.85 (m, CH2b(b)(Leuol),
CH2b(g)(Ile), CH(g)(Leuol), CH2(g)(Pro), CH2a(b)(Pro), CH(b)(Ile)); 2.10–2.20 (m, CH2b(b)(Pro));
3.33–3.43 (m, CH2a(d)(Pro)); 3.46 (d, J¼5.3, CH2OH(Leuol)); 3.63–3.71 (m, CH2b(d)(Pro)); 3.90–4.00
(m, CH(a)(Leuol)); 4.01 (d, J¼8.1, CH(a)(Ile)); 4.40 (dd, J¼8.5, 5.3, CH(a)(Pro)); 5.03, 5.13 (AB, J ¼
12.7, PhCH2); 7.26–7.38 (m, 5 arom. CH). 13C-NMR (75.5 MHz, CD3OD): 11.6 (q, Me(d)(Ile)); 16.3 (q,
Me(g)(Ile)); 22.2, 23.9 (2q, 2 Me(d)(Leuol)); 24.3, 26.0 (2q, 2 Me(b)(Aib)); 26.1 (t, CH2(g)(Pro)); 26.1
(d, CH(g)(Leuol)); 26.6 (t, CH2(g)(Ile)); 30.0 (t, CH2(b)(Pro)); 38.0 (d, CH(b)(Ile)); 40.7 (t,
CH2(b)(Leuol)); 49.6 (t, CH2(d)(Pro)); 51.2 (d, CH(a)(Leuol)); 57.9 (s, C(a)(Aib)); 61.4 (d,
CH(a)(Ile)); 64.2 (d, CH(a)(Pro)); 65.8 (t, CH2OH(Leuol)); 67.7 (t, PhCH2); 128.8, 129.0, 129.5 (3d,
5 arom. CH); 138.3 (s, arom. C); 158.4 (s, OꢀC¼O); 174.1, 174.5, 174.6 (3s, 3 C¼O). ESI-MS (MeOHþ
NaI): 569.3 (100, [M þ Na]þ ). Anal. calc. for C29H46N4O6 ·H2O (564.72): C 61.68, H 8.57, N 9.92; found:
C 61.98, H 8.21, N 9.99.
Crystallization from MeCN by evaporation of the solvent gave colorless crystals suitable for X-ray
crystallography.
Z-Ile-Aib-(R)-Pro-Leuol (19). After the purification of 18 by FC, a small fraction (ca. 10 mg, ca.
1%) of colorless crystals was isolated and identified as 19. The obtained crystals were suitable for X-ray
crystallography.
H-Ile-Aib-Pro-Leuol (20). According to GP 4, to a soln. of 18 (946.5 mg, 1.731 mmol) in MeOH
(67 ml), Pd/C was added, and the mixture was stirred under H2 overnight. After completion of the
reaction and filtration through Celite, the solvent was evaporated to give 20 (738 mg, quant.). White
foam. Rf (AcOEt/MeOH 6 :1) 0.13. M.p. 140–1418. [a]2D0 ¼ ꢀ5.2 (c ¼ 0.50, MeOH). RP-HPLC (II): tR
25.02, l 218 nm. IR (KBr): 3406vs, 3360s, 3278vs, 3071w, 2958s, 2933s, 2875m, 1639vs, 1548s, 1504m, 1465s,
1453m, 1406s, 1380m, 1361m, 1305w, 1279w, 1236w, 1195w, 1173m, 1149m, 1093w, 1073w, 1048w, 1033w,
708w, 618w. 1H-NMR (300 MHz, CD3OD): 0.88–0.96 (m, 2 Me(d)(Leuol), Me(d)(Ile)); 1.05 (d, J¼6.8,
Me(g)(Ile)); 1.14–1.28 (m, CH2a(g)(Ile)); 1.29–1.40 (m, CH2a(b)(Leuol)); 1.48, 1.49 (2s, 2 Me-
(b)(Aib)); 1.45–1.57 (m, CH2b(b)(Leuol), CH2b(g)(Ile), CH(g)(Leuol)); 1.76–1.97 (m, CH2(g)(Pro),

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2549
CH(b)(Ile), CH2a(b)(Pro)); 2.18–2.31 (m, CH2b(b)(Pro)); 3.38 (d, J¼4.2, CH(a)(Ile)); 3.46–3.56 (m,
CH2OH(Leuol), CH2a(d)(Pro)); 3.72–3.82 (m, CH2b(d)(Pro)); 3.94–4.04 (m, CH(a)(Leuol)); 4.43 (dd,
J¼8.2, 5.8, CH(a)(Pro)). 13C-NMR (75.5 MHz, CD3OD): 12.1 (q, Me(d)(Ile)); 16.3 (q, Me(g)(Ile));
22.2, 23.8 (2q, 2 Me(d)(Leuol)); 24.3 (q, Me(b)(Aib)); 25.1 (t, CH2(g)(Pro)); 26.1 (d, CH(g)(Leuol));
26.3 (q, Me(b)(Aib)); 26.7 (t, CH2(g)(Ile)); 30.1 (t, CH2(b)(Pro)); 39.8 (d, CH(b)(Ile)); 40.7 (t,
CH2(b)(Leuol)); 49.6 (t, CH2(d)(Pro)); 50.1 (d, CH(a)(Leuol)); 57.8 (s, C(a)(Aib)); 60.3 (d,
CH(a)(Ile)); 64.3 (d, CH(a)(Pro)); 65.8 (t, CH2OH(Leuol)); 174.4, 174.7, 175.4 (3s, 3 C¼O). ESI-MS
(MeOHþNaI): 435.3 (100, [M þ Na]þ ). Anal. calc. for C21H40N4O4 ·0.5 H2O (421.58): C 59.83, H 9.80, N
13.29; found: C 60.13, H 9.96, N 13.17.
Crystallization from MeOH/AcOEt/hexane/CH2Cl2/petroleum ether by slow evaporation of the
solvent gave colorless crystals suitable for X-ray crystallography.
Z-Leu-Ile-Aib-Pro-Leuol (21). According to GP 1, a mixture of 20 (700 mg, 1.70 mmol), Z-Leu-OH
(450.1 mg, 1.70 mmol), TBTU (544.8 mg, 1.70 mmol), and HOBt (260.5 mg, 1.70 mmol) was dissolved in
MeCN (70 ml), and Et3N (715 ml, 5.09 mmol) was added. As 20 was spairingly soluble in MeCN, 20 ml of
DMF were added. After completion of the reaction, the mixture was purified by FC to give 21 (1086 mg,
97%). White foam. Rf (AcOEt/MeOH 6 :1) 0.70. M.p. 88–908. [a]2D0 ¼ ꢀ56.9 (c ¼ 1.00, MeOH). RP-
HPLC (II): tR 29.18, l 218 nm. IR (KBr): 3308vs, 3035m, 2960vs, 2874s, 1704vs, 1648s, 1544vs, 1468s,
1454s, 1410s, 1384s, 1366s, 1342m, 1263s, 1172m, 1120m, 1043s, 697m, 616m. 1H-NMR (300 MHz,
CD3OD): 0.86–0.98 (m, 2 Me(d)(Leu), 2 Me(d)(Leuol), Me(d)(Ile), Me(g)(Ile)); 1.10–1.26 (m,
CH2a(g)(Ile)); 1.28–1.39 (m, CH2a(b)(Leuol)); 1.40, 1.43 (2s, 2 Me(b)(Aib)); 1.44–1.64 (m,
CH2(b)(Leu), CH2b(b)(Leuol), CH2b(g)(Ile), CH(g)(Leuol)); 1.64–1.77 (m, CH(g)(Leu)); 1.77–1.85
(m, CH2a(b)(Pro)); 1.80–1.97 (m, CH2(g)(Pro), CH(b)(Ile)); 2.14–2.25 (m, CH2b(b)(Pro)); 3.51 (t, J¼
5.6, CH2OH(Leuol)); 3.56–3.64 (m, CH2(d)(Pro)); 3.94–4.04 (m, CH(a)(Leuol)); 4.12–4.18 (m,
CH(a)(Leu)); 4.23 (d, J¼7.2, CH(a)(Ile)); 4.39–4.45 (m, CH(a)(Pro)); 5.09, 5.16 (AB, J ¼ 12.7,
PhCH2); 7.26–7.38 (m, 5 arom. CH). 13C-NMR (75.5 MHz, CD3OD): 11.5 (q, Me(d)(Ile)); 16.3 (q,
Me(g)(Ile)); 21.9 (q, Me(d)(Leu)); 22.2 (q, Me(d)(Leuol)); 23.4 (q, Me(d)(Leu)); 23.8 (q, Me(d)-
(Leuol)); 24.2, 25.8 (2q, 2 Me(b)(Aib)); 25.9 (t, CH2(g)(Pro)); 26.0 (d, CH(g)(Leuol)); 26.1 (d,
CH(g)(Leu)); 26.7 (t, CH2(g)(Ile)); 30.1 (t, CH2(b)(Pro)); 37.5 (d, CH(b)(Ile)); 40.7 (t,
CH2(b)(Leuol)); 41.9 (t, CH2(b)(Leu)); 49.3 (t, CH2(d)(Pro)); 51.0 (d, CH(a)(Leuol)); 55.4 (d,
CH(a)(Ile)); 58.0 (s, C(a)(Aib)); 59.0 (d, CH(a)(Leu)); 64.2 (d, CH(a)(Pro)); 65.8 (t, CH2OH(Leuol));
67.7 (t, PhCH2); 128.7, 129.1, 129.6 (3d, 5 arom. CH); 138.3 (s, arom. C); 158.6 (s, OꢀC¼O); 173.1, 174.4,
174.6, 175.4 (4s, 4 C¼O). ESI-MS (MeOHþNaI): 682.3 (44, [M þ Na]þ ), 435.3 (100). Anal. calc. for
C35H57N5O7 ·0.5 H2O (668.41): C 62.89, H 8.75, N 10.48; found: C 62.62, H 8.91, N 10.54.
H-Leu-Ile-Aib-Pro-Leuol (15). According to GP 4, to a soln. of 21 (1033 mg, 1.565 mmol) in MeOH
(60 ml), Pd/C was added and the mixture was stirred under H2 overnight. After completion of the
reaction and filtration through Celite, the solvent was evaporated to give 15 (821 mg, quant.). White
foam. Rf (AcOEt/MeOH 6 :1) 0.15. M.p. 76–778. [a]2D0 ¼ ꢀ46.7 (c ¼ 1.00, MeOH). RP-HPLC (II): tR
30.15, l 220 nm. IR (KBr): 3311vs, 2959vs, 2874s, 1648vs, 1547vs, 1469s, 1408s, 1385s, 1366s, 1307m, 1243w,
1198m, 1172m, 1103w, 1068w, 1038w, 618w. 1H-NMR (300 MHz, CD3OD): 0.86–1.00 (m, 2 Me(d)(Leu),
2 Me(d)(Leuol), Me(d)(Ile), Me(g)(Ile)); 1.15–1.33 (m, CH2a(g)(Ile)); 1.31–1.40 (m, CH2a(b)(Leu),
CH2a(b)(Leuol)); 1.46 (br. s, 2 Me(b)(Aib)); 1.47–1.65 (m, CH2b(b)(Leu), CH2b(b)(Leuol),
CH2b(g)(Ile), CH(g)(Leuol)); 1.66–1.78 (m, CH(g)(Leu)); 1.79–2.00 (m, CH2a(b)(Pro), CH2(g)(Pro),
CH(b)(Ile)); 2.16–2.27 (m, CH2b(b)(Pro)); 3.50–3.64 (m, CH(a)(Leu), CH2OH(Leuol)); 3.64–3.74
(m, CH2(d)(Pro)); 3.97–4.07 (m, CH(a)(Leuol)); 4.28 (d, J¼7.3, CH(a)(Ile)); 4.43 (dd, J¼9.0, 5.3,
CH(a)(Pro)). 13C-NMR (75.5 MHz, CD3OD): 11.5 (q, Me(d)(Ile)); 16.3 (q, Me(g)(Ile)); 22.2 (q,
Me(d)(Leuol)); 22.2 (q, Me(d)(Leu)); 23.5 (q, Me(d)(Leuol)); 23.8 (q, Me(d)(Leu)); 24.0, 25.5 (2q,
2 Me(b)(Aib)); 25.6 (t, CH2(g)(Pro)); 25.9 (d, CH(g)(Leuol)); 26.1 (d, CH(g)(Leuol)); 26.5 (t,
CH2(g)(Ile)); 29.9 (t, CH2(b)(Pro)); 37.6 (d, CH(b)(Ile)); 40.5 (t, CH2(b)(Leuol)); 45.2 (t,
CH2(b)(Leu)); 49.5 (t, CH2(d)(Pro)); 50.9 (d, CH(a)(Leuol)); 54.2 (d, CH(a)(Ile)); 57.9 (s, C(a)(Aib));
58.9 (d, CH(a)(Leu)); 64.1 (d, CH(a)(Pro)); 65.8 (t, CH2OH(Leuol)); 173.3, 174.2, 174.4, 177.7 (4s,
4 C¼O). ESI-MS (MeOHþNaI): 548.3 (100, [M þ Na]þ ). Anal. calc. for C27H51N5O5 ·1.5 H2O (552.73):
C 58.67, H 9.85, N 12.67; found: C 58.84, H 9.73, N 12.48.

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Z-Leu-Aib-Pro-Leu-Ile-Aib-Pro-Leuol (23). According to GP 1, a mixture of 15 (781.2 mg,
1.49 mmol), Z-Leu-Aib-Pro-OH (22) [12] (665 mg, 1.49 mmol), TBTU (477.2 mg, 1.49 mmol), and
HOBt (228.2 mg, 1.49 mmol) was dissolved in MeCN (110 ml), and Et3N (627 ml, 4.46 mmol) was added.
After completion of the reaction, the mixture was purified by FC to give 23 (1381 mg, 97%). White foam.
Rf (AcOEt/MeOH 6 :1) 0.22. Rf (AcOEt/MeOH 3 :1) 0.35. M.p. 116–1178. [a]2D0 ¼ ꢀ5.5 (c ¼ 1.00,
MeOH). RP-HPLC (II): tR 51.5, l 218 nm. IR (KBr): 3442vs, 3296vs, 3036m, 2958vs, 2926s, 2874s, 1722s,
1648vs, 1539vs, 1469s, 1454s, 1415s, 1383s, 1365s, 1342m, 1304m, 1260s, 1220m, 1202m, 1172s, 1126w,
1047m, 700m, 620m. 1H-NMR (600 MHz, CD3OD): 0.82 (t, J¼7.2, Me(d)(Ile)); 0.86–1.01 (m,
4 Me(d)(Leu), 2 Me(d)(Leuol), Me(g)(Ile)); 1.09–1.21 (m, CH2a(g)(Ile)); 1.24–1.40 (m, CH2a-
(b)(Leuol)); 1.42–1.46 (m, CH2b(g)(Ile)); 1.46, 1.47 (2 br. s, 4 Me(b)(Aib)); 1.50–1.68 (m,
CH2(b)(Leu), CH2b(b)(Leu), CH2b(b)(Leuol), CH(g)(Leu), CH(g)(Leu), CH(g)(Leuol)); 1.68–
1.80 (m, 2 CH2b(b)(Pro)); 1.79–1.88 (m, 2 CH2a(g)(Pro)); 1.88–2.00 (m, CH(b)(Ile), 2 CH2b(g)(Pro),
CH2a(b)(Leu)); 2.20–2.33 (m, 2 CH2a(b)(Pro)); 3.23–3.31 (m, CH2b(d)(Pro)); 3.47–3.59 (m, CH2ab(d)-
(Pro), CH2OH(Leuol)); 3.77–3.86 (m, CH2a(d)(Pro)); 3.92–4.05 (m, CH(a)(Leuol)); 4.12 (dd, J¼8.7,
5.7, CH(a)(Leu)); 4.16–4.25 (m, CH(a)(Leu)); 4.25–4.36 (m, CH(a)(Ile), CH(a)(Pro)); 4.43 (t, J¼7.7,
CH(a)(Pro)); 5.08, 5.19 (AB, J ¼ 12.6, PhCH2); 7.27–7.43 (m, 5 arom. CH). 13C-NMR (150.9 MHz,
CD3OD): 11.7 (q, Me(d)(Ile)); 16.3 (q, Me(g)(Ile)); 21.0, 21.2 (2q, 2 Me(d)(Leu)); 22.1 (q,
Me(b)(Aib)); 23.4 (q, Me(d)(Leu)); 23.5 (q, Me(b)(Aib)); 23.7 (q, Me(d)(Leu)); 24.1, 24.3 (2q,
2 Me(d)(Leuol)); 25.8 (q, Me(b)(Aib)); 26.0 (d, CH(g)(Leuol)); 26.0 (d, CH(g)(Leu)); 26.1 (q,
Me(b)(Aib)); 26.2 (d, CH(g)(Leu)); 26.7 (t, CH2(g)(Ile)); 26.8, 27.0 (2t, 2 CH2(g)(Pro)); 29.8, 30.2 (2t,
2 CH2(b)(Pro)); 38.0 (d, CH(b)(Ile)); 40.4 (t, CH2(b)(Leu)); 40.4 (t, CH2(b)(Leuol)); 41.3 (t,
CH2(b)(Leu)); 49.9, 50.3 (2t, 2 CH2(d)(Pro)); 51.2 (d, CH(a)(Leuol)); 53.1, 55.1 (2d, 2 CH(a)(Leu));
57.9 (s, 2 C(a)(Aib)); 62.0 (d, CH(a)(Ile)); 64.3, 65.1 (2d, 2 CH(a)(Pro)); 65.8 (t, CH2OH(Leuol)); 67.9
(t, PhCH2); 128.9, 129.2, 129.6 (3d, 5 arom. CH); 138.3 (s, arom. C); 158.4 (s, OꢀC¼O); 174.3, 174.4,
174.5, 174.7, 175.0, 175.8 (6s, 7 C¼O). ESI-MS (MeOHþNaI): 977.5 (100, [M þ Na]þ ). Anal. calc. for
C50H82N8O10 ·H2O (973.22): C 61.71, H 8.70, N 11.51; found: C 61.72, H 8.73, N 11.48.
H-Leu-Aib-Pro-Leu-Ile-Aib-Pro-Leuol (24). According to GP 4, to a soln. of 23 (155 mg,
0.162 mmol) in MeOH (30 ml), Pd/C was added, and the mixture was stirred under H2 overnight.
After completion of the reaction and filtration through Celite, the solvent was evaporated to give 24
(130.4 mg, 98%). White foam. Rf (AcOEt/MeOH 3 :1) 0.22. M.p. 95–968. [a]2D0 ¼ ꢀ1.4 (c ¼ 1.00,
MeOH). RP-HPLC (II): tR 85, l 217 nm. IR (KBr): 3312vs, 2959vs, 2873s, 1647vs, 1536vs, 1469vs, 1412vs,
1
1384s, 1365s, 1340m, 1286s, 1245m, 1196s, 1172s, 1147m, 1101w, 1069m, 1039w, 673w, 618m. H-NMR
(300 MHz, CD3OD): 0.84 (t, J¼7.4, Me(d)(Ile)); 0.87–1.03 (m, 4 Me(d)(Leu), 2 Me(d)(Leuol),
Me(g)(Ile)); 1.28–1.44 (m, CH2b(b)(Leu), CH2b(g)(Ile)); 1.44–1.69 (m, CH2a(g)(Ile), CH2b(b)(Leu),
CH2(b)(Leuol), CH2a(b)(Leu), CH(g)(Leu)); 1.47, 1.48, 1.50, 1.53 (4 br. s, 4 Me(b)(Aib)); 1.69–1.85 (m,
2 CH2b(b)(Pro), CH(g)(Leu), CH(g)(Leuol)); 1.85–2.04 (m, CH2a(b)(Leu), 2 CH2(g)(Pro),
CH(b)(Ile)); 2.21–2.43 (m, 2 CH2a(b)(Pro)); 3.24–3.32 (m, CH2b(d)(Pro)); 3.39–3.45 (m, CH(a)-
(Leu)); 3.46–3.60 (m, CH2b(d)(Pro), CH2OH(Leuol)); 3.78–4.02 (m, 2 CH2a(d)(Pro), CH(a)(Leuol));
4.21 (dd, J¼11.3, 3.8, CH(a)(Leu)); 4.28–4.40 (m, CH(a)(Ile), CH(a)(Pro)); 4.44 (t, J¼7.5,
CH(a)(Pro)). 13C-NMR (75.5 MHz, CD3OD): 10.8 (q, Me(d)(Ile)); 16.1 (q, Me(g)(Ile)); 21.2, 22.1
(2q, 2 Me(d)(Leu)); 22.2 (q, Me(b)(Aib)); 23.6 (q, Me(d)(Leu)); 23.8 (q, Me(b)(Aib)); 24.0 (q,
Me(d)(Leu)); 24.0, 24.3 (2q, 2 Me(d)(Leuol)); 25.8 (q, Me(b)(Aib)); 26.0 (d, CH(g)(Leuol)); 26.3 (d,
CH(g)(Leu)); 26.4 (q, Me(b)(Aib)); 26.4 (d, CH(g)(Leu)); 26.7 (t, CH2(g)(Ile)); 26.9, 27.1 (2t,
2 CH2(g)(Pro)); 30.1, 30.2 (2t, 2 CH2(b)(Pro)); 37.8 (d, CH(b)(Ile)); 40.5 (t, CH2(b)(Leuol)); 40.7, 45.4
(2t, 2 CH2(b)(Leu)); 50.1, 50.3 (2t, 2 CH2(d)(Pro)); 51.2 (d, CH(a)(Leuol)); 54.6, 55.0 (2d, 2 CH(a)-
(Leu)); 57.4, 58.1 (2s, 2 C(a)(Aib)); 59.3 (d, CH(a)(Ile)); 64.5, 65.0 (2d, 2 CH(a)(Pro)); 65.8 (t,
CH2OH(Leuol)); 173.2, 174.4, 174.6, 175.1, 175.4, 175.8, 178.6 (7s, 7 C¼O). ESI-MS (MeOHþNaI):
843.5 (100, [M þ Na]þ ). Anal. calc. for C42H76N8O8 ·0.5 H2O (831.11): C 60.77, H 9.35, N 13.50; found: C
60.75, H 9.35, N 13.50.
Ac-Aib-Gln-Val-Leu-Aib-Pro-Leu-Ile-Aib-Pro-Leuol (HM A3). According to GP 1, a mixture of 24
(130.4 mg, 0.16 mmol), Ac-Aib-Gln-Val-OH (6 [12], 59.1 mg, 0.16 mmol), TBTU (51 mg, 0.16 mmol),
and HOBt (24.4 mg, 0.16 mmol) was dissolved in MeCN (15 ml), and Et3N (67 ml, 0.48 mmol) was added.
After completion of the reaction, the mixture was purified by FC to give HM A3 (126 mg, 68%). White

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2551
foam. Rf (AcOEt/MeOH 3 :1) 0.26. M.p. 239–2408. [a]2D0 ¼ ꢀ9.0 (c ¼ 1.00, MeOH). RP-HPLC (II): tR
117, l 217 nm. IR (KBr): 3608s, 3394vs, 3059w, 2959vs, 2928vs, 2874s, 1647vs, 1539vs, 1469vs, 1440s, 1415s,
1384s, 1364s, 1290s, 1265s, 1219s, 1193s, 1172s, 1126m, 1094m, 1050m, 1030m, 688w, 618s, 591m, 523w.
1H-NMR (600 MHz, CD3OD): 0.84 (t, J¼7.3, Me(d)(Ile)); 0.87 (d, J¼6.4, Me(d)(Leu)); 0.90–0.94 (m,
2 Me(d)(Leu), Me(d)(Leuol)); 0.95–0.98 (m, Me(g)(Ile), Me(d)(Leuol)); 1.00–1.03 (m, Me(g)(Val),
Me(d)(Leu)); 1.06 (d, J¼6.9, Me(g)(Val)); 1.28–1.38 (m, CH2b(g)(Ile), CH2b(b)(Leuol)); 1.47, 1.48,
1.49 (3 br. s, 6 Me(b)(Aib)); 1.49–1.55 (m, CH2a(g)(Ile)); 1.57–1.64 (m, CH2a(b)(Leuol),
2 CH2b(b)(Leu)); 1.64–1.69 (m, CH(g)(Leuol)); 1.71–1.81 (m, CH(g)(Leu), 2 CH2b(b)(Pro), CH2a-
(b)(Leu)); 1.81–1.86 (m, CH(g)(Leu)); 1.86–1.91 (m, CH2b(g)(Pro)); 1.91–2.02 (m, CH2a(b)(Leu),
CH(b)(Ile), CH2a(g)(Pro), CH2b(g)(Pro), Me(Ac)); 2.05–2.08 (m, CH2a(g)(Pro)); 2.08–2.15 (m,
CH2(b)(Gln)); 2.24–2.31 (m, CH2a(b)(Pro), CH(b)(Val)); 2.31–2.38 (m, CH2a(b)(Pro)); 2.38–2.45 (m,
CH2b(g)(Gln)); 2.45–2.51 (m, CH2a(g)(Gln)); 3.25–3.30 (m, CH2b(d)(Pro)); 3.50, 3.56 (ABX, J¼11.3,
5.7, CH2OH(Leuol)); 3.58–3.63 (m, CH2b(d)(Pro)); 3.80–3.86 (m, CH2a(d)(Pro), CH2a(d)(Pro)); 3.94
(d, J¼8.3, CH(a)(Val)); 3.94–3.99 (m, CH(a)(Leuol)); 4.06 (dd, J¼7.7, 4.7, CH(a)(Gln)); 4.20 (dd, J¼
11.7, 3.7, CH(a)(Leu)); 4.28 (d, J¼7.9, CH(a)(Ile)); 4.33–4.37 (m, CH(a)(Pro), CH(a)(Leu)); 4.44 (t,
J¼7.7, CH(a)(Pro)). 13C-NMR (150.9 MHz, CD3OD): 10.9 (q, Me(d)(Ile)); 16.0 (q, Me(g)(Ile)); 19.7,
20.0 (2q, 2 Me(g)(Val)); 21.1 (q, Me(d)(Leu)); 21.2 (q, Me(d)(Leuol)); 22.1 (q, Me(d)(Leu)); 23.2 (q,
Me(Ac)); 23.5, 23.9 (2q, 2 Me(d)(Leu)); 24.1 (q, Me(d)(Leuol)); 24.1, 24.2 (2q, 2 Me(b)(Aib)); 25.7 (d,
CH(g)(Leu)); 26.0 (d, CH(g)(Leuol)); 26.2 (d, CH(g)(Leu)); 26.3 (t, CH2(g)(Ile)); 26.4 (t,
CH2(b)(Gln)); 26.4 (q, 2 Me(b)(Aib)); 26.7 (t, CH2(g)(Pro)); 26.7 (q, Me(b)(Aib)); 26.9 (t,
CH2(g)(Pro)); 27.0 (q, Me(b)(Aib)); 30.2 (t, 2 CH2(b)(Pro)); 30.5 (d, CH(b)(Val)); 32.2 (t,
CH2(g)(Gln)); 37.8 (d, CH(b)(Ile)); 40.4 (t, CH2(b)(Leuol)); 40.7, 41.2 (2t, 2 CH2(b)(Leu)); 50.3, 50.6
(2t, 2 CH2(d)(Pro)); 51.1 (d, CH(a)(Leuol)); 53.9, 55.0 (2d, 2 CH(a)(Leu)); 57.4, 57.8 (2s, 2 C(a)(Aib));
57.8 (d, CH(a)(Gln)); 58.0 (s, C(a)(Aib)); 59.4 (d, CH(a)(Ile)); 63.0 (d, CH(a)(Val)); 64.4, 64.9 (2d,
2 CH(a)(Pro)); 65.8 (t, CH2OH(Leuol)); 173.2, 173.7, 174.3, 174.6, 175.0, 175.5, 175.9, 175.8, 178.0, 178.5
(10s, 12 C¼O). ESI-MS (MeOHþNaI): 1197.6 (100, [M þ Na]þ ). Anal. calc. for C58H102N12O13 ·H2O
(1193.52): C 58.37, H 8.78, N 14.08; found: C 58.26, H 9.00, N 13.93.
Crystallization from H2O/MeCN gave colorless crystals suitable for X-ray crystallography.
Z-Gln-Ile-OtBu (9). According to GP 1, a mixture of Z-Gln-OH (62.6 mg, 0.223 mmol), H-Ile-
OtBu·HCl (50 mg, 0.223 mmol), TBTU (71.7 mg, 0.223 mmol), and HOBt (34.3 mg, 0.223 mmol) was
dissolved in MeCN (14 ml), and Et3N (94 ml, 0.67 mmol) was added. After completion of the reaction,
the mixture was purified by FC to give 9 (91.9 mg, 92%). White powder. Rf (AcOEt/MeOH 6 :1) 0.80.
M.p. 1468. [a]2D0 ¼ ꢀ19.1 (c ¼ 1.56, MeOH). RP-HPLC (II): tR 23.05, l 213 nm. IR (KBr): 3411s, 3316s,
2969m, 2920m, 2855w, 1724s, 1689s, 1651vs, 1543s, 1455m, 1400w, 1369m, 1329w, 1289m, 1263m, 1168m,
1139m, 1088w, 1061m, 976w, 743w, 695w, 665w, 624w. 1H-NMR (300 MHz, CD3OD): 0.89–0.96 (m,
Me(g)(Ile), Me(d)(Ile)); 1.19–1.32 (m, CH2a(g)(Ile)); 1.42–1.54 (m, CH2b(g)(Ile)); 1.46 (s, Me3CO);
1.81–1.95 (m, CH2b(b)(Gln); CH(b)(Ile)); 2.01–2.14 (m, CH2a(b)(Gln)); 2.34 (t, J ¼ 7.6, CH2(g)(Gln));
4.18–4.27 (m, CH(a)(Gln), CH(a)(Ile)); 5.09 (br. s, PhCH2); 7.25–7.38 (m, 5 arom. H). 13C-NMR
(75.5 MHz, CD3OD): 11.9 (q, Me(d)(Ile)); 16.0 (q, Me(g)(Ile)); 26.4 (t, CH2(g)(Ile)); 28.4 (q, Me3CO);
29.3 (t, CH2(g)(Gln)); 32.6 (t, CH2(b)(Gln)); 38.5 (d, CH(b)(Ile)); 55.7 (d, CH(a)(Gln)); 59.0 (d,
CH(a)(Ile)); 67.8 (t, PhCH2); 83.0 (s, Me3CO); 128.9, 129.0, 129.5 (3d, 5 arom. CH); 172.1, 177.9
(2s, 3 C¼O). The OꢀC¼O and arom. C could not be identified. ESI-MS (MeOHþNaI): 472.2 (100,
[M þ Na]þ ). Anal. calc. for C23H35N3O6 (449.54): C 61.45, H 7.85, N 9.35; found: C 61.35, H 7.90,
N 9.34.
H-Gln-Ile-OtBu (11). According to GP 4, to a soln. of 9 (638.7 mg, 1.421 mmol) in MeOH (42 ml),
Pd/C was added, and the mixture was stirred under H2 overnight. After completion of the reaction (TLC)
and filtration through Celite, the solvent was evaporated to give 11 (448 mg, quant.). White foam. Rf
(AcOEt/MeOH 6 :1) 0.09. RP-HPLC (II): tR 19.27, l 203 nm. 1H-NMR (300 MHz, CD3OD): 0.92–0.99
(m, Me(g)(Ile), Me(d)(Ile)); 1.20–1.36 (m, CH2a(g)(Ile)); 1.42–1.56 (m, CH2b(g)(Ile)); 1.47 (s,
Me3CO); 1.84–1.98 (m, CH2b(b)(Gln), CH(b)(Ile)); 1.98–2.11 (m, CH2a(b)(Gln)); 2.42 (t, J ¼ 7.8,
CH2(g)(Gln)); 3.68 (t, J¼6.4, CH(a)(Gln)); 4.27 (d, J¼5.4, CH(a)(Ile)). 13C-NMR (75.5 MHz,
CD3OD): 11.9 (q, Me(d)(Ile)); 16.0 (q, Me(g)(Ile)); 26.4 (t, CH2(g)(Ile)); 28.3 (q, Me3CO); 31.0 (t,

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
CH2(g)(Gln)); 32.3 (t, CH2(b)(Gln)); 38.4 (d, CH(b)(Ile)); 54.7 (d, CH(a)(Gln)); 58.9 (d, CH(a)(Ile));
83.1 (s, Me3CO); 172.1, 174.5, 178.0 (3s, 3 C¼O).
Ac-Aib-Gln-Ile-OtBu (13). According to GP 1, a mixture of 11 (338 mg, 1.072 mmol), Ac-Aib-OH
(155.6 mg, 1.072 mmol), TBTU (344.2 mg, 1.072 mmol), and HOBt (164.6 mg, 1.072 mmol) was
dissolved in MeCN (65 ml), and TEA (452 ml, 3.215 mmol) was added. After completion of the
reaction, the mixture was purified by FC to give 13 (311.2 mg, 66%). White foam. Rf (AcOEt/MeOH
6 :1) 0.30. M.p. 80–858. [a]2D0 ¼ ꢀ26.4 (c ¼ 2.97, MeOH). RP-HPLC (II): tR 18.09, l 203 nm. IR (KBr):
3417vs, 3064w, 2973s, 2935s, 2878w, 1731vs, 1660vs, 1536vs, 1454s, 1383s, 1368s, 1300s, 1257m, 1221s,
1147vs, 1038w, 987w, 847w, 589m. 1H-NMR (300 MHz, CD3OD): 0.89–0.97 (m, Me(g)(Ile), Me(d)-
(Ile)); 1.16–1.32 (m, CH2a(g)(Ile)); 1.43–1.57 (m, CH2b(g)(Ile)); 1.43, 1.45 (2s, 2 Me(Aib)); 1.46 (s,
Me3CO); 1.85–2.02 (m, CH2b(b)(Gln), CH(b)(Ile)); 1.97 (s, Me(Ac)); 2.08–2.20 (m, CH2a(b)(Gln));
2.31 (t, J ¼ 7.1, CH2(g)(Gln)); 4.16 (d, J¼6.3, CH(a)(Ile)); 4.36 (dd, J¼9.6, 4.5, CH(a)(Gln)). 13C-NMR
(75.5 MHz, CD3OD): 11.8 (q, Me(d)(Ile)); 15.9 (q, Me(g)(Ile)); 23.2, 25.6 (2q, 2 Me(b)(Aib)); 25.7 (q,
Me(Ac)); 26.6 (t, CH2(g)(Ile)); 28.4 (q, Me3CO); 32.5 (t, CH2(b)(Gln), CH2(g)(Gln)); 38.0 (d,
CH(b)(Ile)); 54.4 (d, CH(a)(Gln)); 57.7 (s, C(a)(Aib)); 59.2 (d, CH(a)(Ile)); 82.8 (s, Me3CO); 172.0,
173.2, 173.9, 177.0, 178.1 (5s, 5 C¼O). ESI-MS (MeOHþNaI): 465.2 (100, [M þ Na]þ ). Anal. calc. for
C21H38N4O6 · 0.5 H2O (451.56): C 55.86, H 8.71, N 12.41; found: C 55.94, H 8.54, N 12.41.
Ac-Aib-Gln-Ile-OH (7). At 08, 13 (275 mg, 0.621 mmol) was dissolved in TFA (15 ml) under N2.
After 2.5 h, TFA was evaporated. The residue was dissolved in CH2Cl2/Et2O 1:1 (v/v) and evaporated
(3ꢂ ) to give 7 (238 mg, quant.). White powder. Rf (AcOEt/MeOH 6 :1) 0.15, Rf (AcOEt/MeOH 3 :1)
0.26. RP-HPLC (II): tR 30.46, l 205 nm. IR (KBr): 3437vs, 2926s, 2853m, 1659vs, 1541vs, 1455s, 1204vs,
1139s, 835m, 802m, 723m, 673m, 619m. 1H-NMR (300 MHz, CD3OD): 0.89–0.98 (m, Me(g)(Ile),
Me(d)(Ile)); 1.18–1.33 (m, CH2a(g)(Ile)); 1.45–1.58 (m, CH2b(g)(Ile)); 1.44, 1.45 (2s, 2 Me(b)(Aib));
1.88–2.01 (m, CH2b(b)(Gln), CH(b)(Ile)); 1.96 (s, Me(Ac)); 2.07–2.19 (m, CH2a(b)(Gln)); 2.31 (t, J ¼
7.1, CH2(g)(Gln)); 4.31 (d, J¼6.0, CH(a)(Ile)); 4.37 (dd, J¼9.2, 4.4, CH(a)(Gln)). 13C-NMR
(75.5 MHz, CD3OD): 11.8 (q, Me(d)(Ile)); 16.0 (q, Me(g)(Ile)); 23.1, 25.3 (2q, 2 Me(b)(Aib)); 25.9
(q, Me(Ac)); 26.4 (t, CH2(g)(Ile)); 28.4 (t, CH2(b)(Gln)); 32.5 (t, CH2(g)(Gln)); 38.1 (d, CH(b)(Ile));
54.4 (d, CH(a)(Gln)); 57.8 (s, C(a)(Aib)); 58.5 (d, CH(a)(Ile)); 82.8 (s, Me3CO); 173.2, 173.9, 174.4,
177.1, 178.2 (5s, 5 C¼O). ESI-MS (MeOHþNaI): 409.3 (100, [M þ Na]þ ).
Z-Ile-Aib-Pro-Leu-Leu-Aib-Pro-Leuol (26). According to GP 1, a mixture of 17 (546 mg,
1.22 mmol), H-Leu-Leu-Aib-Pro-Leuol (25 [12]; 641 mg, 1.22 mmol), TBTU (392 mg, 1.22 mmol),
and HOBt (187.3 mg, 1.22 mmol) was dissolved in MeCN (70 ml), and Et3N (514 ml, 3.66 mmol) was
added. After completion of the reaction, the mixture was purified by FC to give 26 (739 mg, 63%). White
foam. Rf (AcOEt/MeOH 6 :1) 0.50. M.p. 1448. [a]2D0 ¼ ꢀ4.5 (c ¼ 2.40, MeOH). RP-HPLC (II): tR 58.21, l
205 nm. IR (KBr): 3441vs, 2960s, 2872m, 1726s, 1624vs, 1529vs, 1468s, 1412s, 1385s, 1365m, 1339m, 1253s,
1
1218s, 1171s, 1126m, 1100m, 1039m, 750w, 699w, 616m. H-NMR (300 MHz, CD3OD): 0.82–0.99 (m,
Me(g)(Ile), Me(d)(Ile), 2 Me(d)(Leuol), 4 Me(d)(Leu)); 1.21–1.31 (m, CH2a(g)(Ile)); 1.32–1.40 (m,
CH2a(b)(Leuol)); 1.45, 1.48 (2 br. s, 4 Me(b)(Aib)); 1.51–1.57 (m, CH2b(g)(Ile)); 1.57–1.61 (m,
CH2b(b)(Leu), CH2b(b)(Leuol)); 1.62–1.71 (m, CH2(b)(Leuol), CH(g)(Leu), CH(g)(Leuol)); 1.71–
1.79 (m, 2 CH2b(b)(Pro), CH(g)(Leu)); 1.79–1.88 (m, CH2(g)(Pro), CH2b(g)(Pro)); 1.88–2.00 (m,
CH2a(b)(Leu), CH(b)(Ile), CH2a(g)(Pro)); 2.21–2.32 (m, CH2a(b)(Pro), CH2a(b)(Pro)); 3.28–3.37 (m,
CH2b(d)(Pro)); 3.46–3.64 (m, CH2OH(Leuol), CH2ab(d)(Pro)); 3.79–3.86 (m, CH2a(d)(Pro)); 3.91–
4.03 (m, CH(a)(Ile), CH(a)(Leuol)); 4.18 (dd, J ¼ 11.5, 4.0, CH(a)(Leu)); 4.28–4.35 (m, CH(a)(Pro));
4.40–4.49 (m, CH(a)(Pro), CH(a)(Leu)); 5.09, 5.20 (AB, J ¼ 12.6, PhCH2); 7.28–7.50 (m, 5 arom. H).
13C-NMR (75.5 MHz, CD3OD): 11.7 (q, Me(d)(Ile)); 16.3 (q, Me(g)(Ile)); 21.0, 21.2 (2q, 2 Me(d)-
(Leu)); 22.1, 23.5 (2q, 2 Me(d)(Leuol)); 23.6, 23.7 (2q, 2 Me(d)(Leu)); 24.1, 24.3 (2q, 2 Me(b)(Aib));
25.8 (d, CH(g)(Leu)); 26.0 (d, CH(g)(Leuol)); 26.1 (q, Me(b)(Aib)); 26.2 (d, CH(g)(Leu)); 26.7 (t,
CH2(g)(Ile), CH2(g)(Pro)); 26.8 (q, Me(b)(Aib)); 27.0 (t, CH2(g)(Pro)); 29.9, 30.2 (2t, 2 CH2(b)(Pro));
38.0 (d, CH(b)(Ile)); 40.4 (t, CH2(b)(Leu), CH2(b)(Leuol)); 41.3 (t, CH2(b)(Leu)); 49.5, 50.3 (2t,
2 CH2(d)(Pro)); 51.3 (d, CH2(a)(Leuol)); 53.2, 55.1 (2d, 2 CH2(a)(Leu)); 57.9 (s, 2 C(a)(Aib)); 62.1 (d,
CH(a)(Ile)); 64.4, 65.1 (2d, 2 CH2(a)(Pro)); 65.8 (t, CH2OH(Leuol)); 67.9 (t, PhCH2); 129.0, 129.2, 129.6
(3d, 5 arom. CH); 138.3 (s, arom. C); 158.4 (s, OꢀC¼O); 174.3, 174.4, 174.5, 174.7, 175.0, 175.9, 176.0 (7s,

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2553
7 C¼O). ESI-MS (MeOHþNaI): 977.6 (100, [M þ Na]þ ). Anal. calc. for C50H82N8O10 ·2 H2O (991.27):
C 60.58, H 8.75, N 11.30; found: C 60.83, H 8.42, N 11.28.
Crystallization from Et2O/MeOH/AcOEt/hexane/cyclohexane gave colorless crystals suitable for
X-ray crystallography.
H-Ile-Aib-Pro-Leu-Leu-Aib-Pro-Leuol (27). According to GP 4, to a soln. of 26 (14 mg,
0.015 mmol) in MeOH (10 ml), Pd/C was added, and the mixture was stirred under H2 overnight.
After completion of the reaction and filtration through Celite, the solvent was evaporated to give 27
(12 mg, quant.). White foam. Rf (AcOEt/MeOH 6 :1) 0.08. M.p. 100–1058. [a]2D0 ¼ ꢀ5.4 (c ¼ 1.20,
MeOH). RP-HPLC (II): tR 92.86, l 207 nm. IR (KBr): 3311vs, 2959vs, 2873s, 1644vs, 1536vs, 1470s,
1412vs, 1385s, 1365s, 1340m, 1283m, 1196m, 1172s, 1101w, 1069w, 1038w, 618m. 1H-NMR (300 MHz,
CD3OD): 0.82–1.02 (m, Me(g)(Ile), Me(d)(Ile), 2 Me(d)(Leuol), 4 Me(d)(Leu)); 1.11–1.42 (m,
CH2a(g)(Ile), CH2a(b)(Leuol)); 1.44, 1.48, 1.51, 1.54 (4 br. s, 4 Me(b)(Aib)); 1.54–1.83 (m, CH2b(g)(Ile),
CH2b(b)(Leuol), CH2b(b)(Leu), CH(g)(Leu), 2 CH2b(b)(Pro), CH(b)(Ile), CH(g)(Leuol),
CH2b(b)(Leu), CH(g)(Leu)); 1.83–2.05 (m, CH2a(b)(Leu), 2 CH2(g)(Pro), CH2a(b)(Leu)); 2.20–
2.32, 2.32–2.42 (2m, 2 CH2a(b)(Pro)); 3.29–3.38 (m, CH2b(d)(Pro), CH(a)(Ile)); 3.43–3.59 (m,
CH2OH(Leuol), CH2b(d)(Pro)); 3.77–4.02 (m, 2 CH2a(d)(Pro), CH(a)(Leuol)); 4.18 (dd, J ¼ 11.4, 4.2,
CH(a)(Leu)); 4.31 (t, J ¼ 8.0, CH(a)(Pro)); 4.40–4.48 (m, CH(a)(Pro), CH(a)(Leu)). 13C-NMR
(75.5 MHz, CD3OD): 12.4 (q, Me(d)(Ile)); 16.4 (q, Me(g)(Ile)); 21.1, 21.3 (2q, 2 Me(d)(Leu)); 22.2, 23.5
(2q, 2 Me(d)(Leuol)); 23.6, 23.7 (2q, 2 Me(d)(Leu)); 24.2, 24.4 (2q, 2 Me(b)(Aib)); 25.2 (d,
CH(g)(Leu)); 25.8 (d, CH(g)(Leuol)); 26.0 (q, Me(b)(Aib)); 26.2 (d, CH(g)(Leu)); 26.2 (t,
CH2(g)(Ile)); 26.7 (t, CH2(g)(Pro)); 26.8 (q, Me(b)(Aib)); 26.8 (t, CH2(g)(Pro)); 30.0, 30.1 (2t,
2 CH2(b)(Pro)); 39.9 (d, CH(b)(Ile)); 40.3 (t, CH2(b)(Leu); CH2(b)(Leuol)); 41.2 (t, CH2(b)(Leu));
49.9, 50.2 (2t, 2 CH2(d)(Pro)); 51.2 (d, CH(a)(Leuol)); 53.1, 55.0 (2d, 2 CH(a)(Leu)); 57.5, 57.8 (2s, 2
C(a)(Aib)); 60.8 (d, CH(a)(Ile)); 64.3, 65.1 (2d, 2 CH(a)(Pro)); 65.8 (t, CH2OH(Leuol)); 174.2, 174.4,
174.6, 175.3, 175.8, 177.5 (6s, 7 C¼O). ESI-MS (MeOHþNaI): 843.6 (100, [M þ Na]þ ). Anal. calc. for
C42H76N8O8 ·H2O (839.12): C 60.12, H 9.37, N 13.35; found: C 59.96, H 9.13, N 13.15.
Ac-Aib-Gln-Ile-Ile-Aib-Pro-Leu-Leu-Aib-Pro-Leuol (HM A5). According to GP 1, a mixture of 7
(207 mg, 1.22 mmol), 27 (440 mg, 0.536 mmol), TBTU (172 mg, 0.536 mmol), and HOBt (187.3 mg,
0.536 mmol) was dissolved in MeCN (30 ml), and Et3N (226 ml, 1.608 mmol) was added. After
completion of the reaction, the mixture was purified by FC to give HM A5 (195.3 mg, 31%). White foam.
Rf (AcOEt/MeOH 3 :1) 0.28. M.p. 156–1588. [a]2D0 ¼ ꢀ9.0 (c ¼ 0.67, MeOH). RP-HPLC (II): tR 49.51, l
203 nm. IR (KBr): 3436vs, 2960s, 2875m, 1641vs, 1539s, 1469m, 1415m, 1385m, 1365m, 1341m, 1292m,
1171m, 1101m, 1068m, 614m. 1H-NMR (600 MHz, CD3OD): 0.83–0.99 (m, 2 Me(d)(Leu), Me(d)(Ile),
2 Me(d)(Leuol), Me(d)(Leu), Me(d)(Ile), 2 Me(g)(Ile)); 1.01 (d, J¼7.0, Me(d)(Leu)); 1.27–1.39 (m,
CH2b(b)(Leuol), 2 CH2b(g)(Ile)); 1.46, 1.49, 1.51, 1.55 (4 br. s, 6 Me(b)(Aib)); 1.54–1.68 (m,
CH(g)(Leuol), CH2a(b)(Leuol), 2 CH2a(g)(Ile), 2 CH2b(b)(Leu)); 1.68–1.82 (m, CH2a(b)(Leu),
2 CH2b(b)(Pro), CH(g)(Leu)); 1.83–1.91 (m, CH2b(g)(Pro), CH(g)(Leu)); 1.93–2.01 (m,
CH2b(g)(Pro), CH2a(g)(Pro), CH(b)(Ile), Me(Ac)); 2.01–2.15 (m, CH2(b)(Gln), CH2a(g)(Pro),
CH(b)(Ile), CH2a(b)(Leu)); 2.25–2.31, 2.31–2.37 (2m, 2 CH2a(b)(Pro)); 2.38–2.43 (m, CH2b(g)(Gln));
2.45–2.51 (m, CH2a(g)(Gln)); 3.31–3.36 (m, CH2b(d)(Pro)); 3.48–3.53 (m, CH2aOH(Leuol)); 3.53–3.56
(m, CH2bOH(Leuol), CH2b(d)(Pro)); 3.78–3.83, 3.83–3.87 (2m, 2 CH2a(d)(Pro)); 3.94–3.99 (m,
CH(a)(Leuol)); 4.00 (d, J¼8.8, CH(a)(Ile)); 4.05 (dd, J¼7.8, 4.4, CH(a)(Gln)); 4.15–4.20 (m,
CH(a)(Leu)); 4.18 (d, J¼8.5, CH(a)(Ile)); 4.33 (t, J¼7.8, CH(a)(Pro)); 4.43–4.48 (m, CH(a)(Pro),
CH(a)(Leu)). 13C-NMR (150.9 MHz, CD3OD): 10.8, 11.1 (2q, 2 Me(d)(Ile)); 15.9, 16.3 (2q, 2 Me(g)-
(Ile)); 21.0, 21.1 (2q, 2 Me(d)(Leu)); 22.1 (q, Me(d)(Leuol)); 23.2 (q, Me(Ac)); 23.5, 23.7 (2q,
2 Me(d)(Leu)); 23.8 (q, Me(d)(Leuol)); 24.1, 24.2, 24.3 (3q, 3 Me(b)(Aib)); 25.8, 26.00 (2d,
2 CH(g)(Leu)); 26.00 (q, Me(b)(Aib)); 26.1 (d, CH(g)(Leuol)); 26.5 (t, CH2(g)(Pro)); 26.6 (t,
CH2(b)(Gln)); 26.7 (t, CH2(g)(Ile)); 26.7, 27.0 (2q, 2 Me(b)(Aib)); 27.1 (t, CH2(g)(Pro)); 27.2 (t,
CH2(g)(Ile)); 30.0, 30.2 (2t, 2 CH2(b)(Pro)); 32.3 (t, CH2(g)(Gln)); 36.7, 37.4 (2d, 2 CH(b)(Ile)); 40.4 (t,
CH2(b)(Leu)); 41.3 (t, CH2(b)(Leuol)); 50.3, 50.6 (2t, 2 CH2(d)(Pro)); 51.1 (d, CH(a)(Leuol)); 53.1,
55.1 (2d, 2 CH(a)(Leu)); 57.4 (s, C(a)(Aib)); 57.5 (d, CH(a)(Gln)); 57.8, 58.0 (2s, 2 C(a)(Aib)); 60.1,
61.4 (2d, 2 CH(a)(Ile)); 64.3, 65.2 (2d, 2 CH(a)(Pro)); 65.8 (t, CH2OH(Leuol)). ESI-MS (MeOHþ

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
NaI): 1212.5 (100, [M þ Na]þ ). Anal. calc. for C59H104N12O13 ·2 H2O (1225.37): C 57.82, H 8.88, N 13.71;
found: C 57.75, H 8.85, N 13.81.
X-Ray Crystal-Structure Determination of 5, 18, 19, 20, 26, and HM A3 (Table 2 and Figs. 3–7)8). All
measurements were conducted on a Nonius KappaCCD area-detector diffractometer [22] using
graphite-monochromated MoKa radiation (l 0.71073 ꢅ) and an Oxford Cryosystems Cryostream 700
cooler. The data collection and refinement parameters are compiled in Table 2, and views of the
molecules are shown in Figs. 3–7. Data reduction was performed with HKL Denzo and Scalepack [23].
The intensities were corrected for Lorentz and polarization effects, but not for absorption. Equivalent
reflections were merged.
The structures were solved by direct methods, by using SIR92 [24] for 5, 18, 20, and HM A3,
SHELXS97 [25] for 19, and the Shake-N-Bake program [26] for 26, which, in each case, revealed the
positions of all non-H-atoms. For each structure, the non-H-atoms were refined anisotropically. The H-
atoms were generally 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 atom (1.5 Ueq for the Me groups, the OH group of 19, and the NH2 and OH groups of 20). In the
case of 19, the OꢀH vector was rotated so as to correspond with the direction that would bring the H-
atom closest to the nearest H-bond acceptor. The amide H-atom for 5, and the amide and OH H-atoms
for 18, 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.
In the case of 19, 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 [27], but none could be found. The benzyl (Bn), Ile, and Leu groups
in one molecule are disordered. Two sets of overlapping positions were defined for all atoms of the Bn
group, for the entire Ile group, and for the end iPr atoms of the Leu group. The site occupation factors of
the major conformations of these groups refined to 0.66(3), 0.63(8), and 0.52(6), resp. Similarity
restraints were applied to the chemically equivalent bond lengths and angles involving all disordered C-
atoms, while neighboring atoms within and between each conformation of the disordered groups were
restrained to have similar atomic displacement parameters. Pseudo-isotropic restraints were also applied
to the atomic displacement parameters of the disordered C-atoms of the Ile group. The crystal was found
to be twinned by a two-fold rotation about the [100] direction. The twin law was included in the
refinement, which led to a value of 0.814(4) for the major twin component.
The asymmetric unit of 20 contains two molecules of the peptide, plus a site for a H2O molecule that
is both partially occupied and disordered about a C2 axis. The best results were obtained when the site
occupation factor of the H2O molecule was set to 0.25. Thus, the 20/H2O ratio in the crystal is 8 :1. The
two independent peptide molecules have significantly different conformations, thus obviating the
likelihood of overlooked symmetry. The Ile group in one of the independent peptide molecules
(molecule B) is disordered over two conformations. Two sets of positions were defined for the Me and Et
atoms of the Ile group, and the site occupation factor of the major conformation refined to 0.706(7).
Similarity restraints were applied to the chemically equivalent bond lengths and angles involving all
disordered C-atoms, while neighboring atoms within and between each conformation of the disordered
group were restrained to have similar atomic displacement parameters.
In the case of 26, the asymmetric unit includes two molecules of the peptide plus two disordered
solvent molecules, which, from the arrangement of residual electron-density peaks, appeared to be
AcOEt. The atomic coordinates were tested carefully for a relationship from a higher symmetry space
group using the program PLATON [27], but none could be found. The disorder of the solvent molecules
could not be modelled adequately, so the SQUEEZE routine [28] of the program PLATON was
employed. This procedure, which allows the disordered solvent molecules to be omitted entirely from the
subsequent refinement model, gave better refinement results, and there were no significant peaks of
residual electron density to be found in the voids of the structure. Omission of the solvent molecules
8
)
CCDC-890150–890155 contain the supplementary crystallographic data for this paper. 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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leaves a total void volume of 725 ꢅ3 per unit cell distributed mainly across four localities, with two
cavities having a volume of 201 ꢅ3 and two of 118 ꢅ3. The number of electrons contributing to the voids
in the structure was calculated by the SQUEEZE routine to be ca. 256 e per unit cell. Allowing for four
AcOEt molecules per unit cell yields 192 e, and this was used in the subsequent calculation of the
empirical formula, formula weight, density, linear absorption coefficient, and F(000). It is possible that
additional H2O molecules are present in the larger voids, which might serve to increase the overall
electron count to a value closer to that determined experimentally, but these were not included in the
calculations, and no obvious peaks of residual electron density were present to support this hypothesis.
Some of the side groups in each molecule are disordered over two conformations. Two positions were
i
defined for the terminal Pr atoms and the entire side chain of the Leu group at the Leu-ol end of
molecules A and B, resp. The site occupation factors of the major conformations of these groups refined
to 0.59(3) and 0.74(2), resp. Two positions were defined for the terminal iPr atoms of the first Leu group
(Z-end) in molecule A and the entire side chain of the Ile group in molecule B. The site occupation
factors of the major conformations of these groups were refined to 0.62(2) and 0.61(3), resp. The first
proline group at the Z-end of each molecule has two envelope conformations with two positions being
defined for the C-atom of the envelope flap. The site occupation factors of the major conformations of
these groups refined to 0.59(3) and 0.68(2), for molecules A and B, resp. Similarity restraints were
applied to the chemically equivalent bond lengths and angles involving all disordered C-atom, while
neighboring atoms within and between each disordered conformation were restrained to have similar
atomic displacement parameters.
The asymmetric unit of HM A3 contains one peptide molecule and one H2O molecule, which is
disordered over two closely positioned sites. The peptide molecule displays disorder in several side
groups. Two positions were defined for the terminal Me groups of the Leu group closest to the Ac end of
the molecule and the terminal Me groups of the Leuol group, as well as three of the ring C-atoms of the
Pro group next to the Leuol end of the molecule. The site occupation factors of the major conformations
were refined to 0.53(5), 0.89(2), and 0.80(3), resp. Similarity restraints were applied to the chemically
equivalent bond lengths and angles involving all disordered atoms, while neighboring atoms within and
between each conformation of the disordered groups were restrained to have similar atomic
displacement parameters. The atomic displacement parameters of the disordered Leu groups were
further restrained to be pseudo-isotropic.
The refinement of each structure was carried out on F2 using full-matrix least-squares procedures,
which minimized the function Sw(F2o ꢀFc2 )2. A correction for secondary extinction was applied in the
cases of 5, 18, 19, 20, and HM A3. In the case of 18, 20, 26, and HM A3, two, five, nine, and seven, resp.,
reflections, whose intensities were considered to be extreme outliers, were omitted from the final
refinement. Neutral atom-scattering factors for non-H-atoms were taken from [29], and the scattering
factors for H-atoms were taken from [30]. Anomalous dispersion effects were included in Fc [31]; the
values for f’ and f ’’ were those of [32]. The values of the mass attenuation coefficients are those of [33].
All calculations were performed using the SHELXL97 [25] program.
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