Solution synthesis, conformational analysis, and antimicrobial activity of three alamethicin F50/5 analogs bearing a trifluoroacetyl label

Article abstract of DOI:10.1002/cbdv.201300394

We prepared, by solution-phase methods, and fully characterized three analogs of the membrane-active peptaibiotic alamethicin F50/5, bearing a single trifluoroacetyl (Tfa) label at the N-terminus, at position 9 (central region) or at position 19 (C-termin

Full text of DOI:10.1002/cbdv.201300394

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
1163
Solution Synthesis, Conformational Analysis, and Antimicrobial Activity of
Three Alamethicin F50/5 Analogs Bearing a Trifluoroacetyl Label
a
a
a
b
b
by Marta De Zotti* ), Gema Ballano ), Micha Jost ), Evgeniy S. Salnikov ), Burkhard Bechinger ),
c
a
a
a
Simona Oancea ), Marco Crisma ), Claudio Toniolo ), and Fernando Formaggio )
a) ICB, Padova Unit, CNR, Department of Chemistry, University of Padova, I-35131 Padova
(
e-mail: marta.dezotti@unipd.it)
b) Membrane Biophysics and NMR, Chemistry Institute, UMR 7177, University of Strasbourg/CNRS,
F-67070 Strasbourg
c) Department of Biochemistry and Toxycology, University ꢀLucian Blagaꢁ, RO-550012 Sibiu
We prepared, by solution-phase methods, and fully characterized three analogs of the membrane-
active peptaibiotic alamethicin F50/5, bearing a single trifluoroacetyl (Tfa) label at the N-terminus, at
position 9 (central region) or at position 19 (C-terminus), and with the three Gln at positions 7, 18, and 19
replaced by Glu(OMe) residues. To add the Tfa label at position 9 or 19, a g-trifluoroacetylated a,g-
9
diaminobutyric acid (Dab) residue was incorporated as a replacement for the original Val or
19
Glu(OMe) amino acid. We performed a detailed conformational analysis of the three analogs (using
FT-IR absorption, CD, 2D-NMR, and X-ray diffraction), which clearly showed that Tfa labeling does not
introduce any dramatic backbone modification in the predominantly a-helical structure of the parent
19
peptaibiotic. The results of an initial solid-state F-NMR study on one of the analogs favor the
conclusion that the Tfa group is a very promising reporter for the analysis of peptaibioticꢀmembrane
interactions. Finally, we found that the antimicrobial activities of the three newly synthesized analogs
depend on the position of the Tfa label in the peptide sequence.
Introduction. – In the extremely large family of the naturally occurring antimicro-
bial peptides (AMPs) [1–3], peptaibiotics [4] are members of a peculiar and promising
class, as they possess the following unique features: i) they are active against a variety
of bacteria and viruses [5][6], even drug-resistant strains [7][8]; ii) their a-amino acid
sequences are usually quite short (between 5 and 20 amino acid residues), which is a
good starting point for a potential industrial production; iii) nonetheless, they exhibit
stable and rigid helical conformations, so that it is possible to use them as reliable
scaffolds for drug delivery or other types of applications; iv) they are resistant to the
action of proteolytic enzymes, a key feature for the development of orally
administrable drugs. Moreover, the mechanism of action of peptaibiotics is of great
interest, because, apparently, they do not possess a specific target, such as, for instance,
an enzyme for ꢀconventionalꢁ antibiotics [1–3][6]. Conversely, they exploit their
toxicity by perturbing the permeability of the bacterial membrane, eventually causing
its leakage. This mechanism is highly promising, because it reduces the possibility for
the bacteria to develop resistance. Indeed, numerous studies have been published with
the aim of shedding light on the membrane interaction mechanism of peptaibiotics,
making use of several different spectroscopic techniques applied to tailor-made
synthetic analogs bearing suitable probes [4].
ꢂ 2014 Verlag Helvetica Chimica Acta AG, Zꢃrich

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All of the properties of peptaibiotics mentioned above are due to the presence, in
a,a
their sequences, of a large number of C -dialkyl glycines, the most common of which is
a-aminoisobutyric acid (Aib), which also gives the name to this peptide family
(
peptaibiotics) [4]. Being sterically hindered, Aib can adopt almost exclusively f,y
angles, falling in a limited region of the Ramachandran plot, namely that including the
helical conformation(s) [9–14]. For this reason, Aib is a strong helix inducer, but, for
the same reason, its synthetic insertion in a peptide sequence is rather challenging [15–
17]. Therefore, despite their great potential, single, purified peptaibiotics have been so
far prepared only by specialized researchers who overcame the synthetic difficulties,
which had also precluded for a long time the employment of automatic synthesizers.
The most extensively investigated peptaibiotic is alamethicin, a 19-amino acid
residue-long peptide produced through non-ribosomal synthesis by the fungus
Trichoderma viride as a mixture of numerous components, the most widely present
of which is alamethicin F50/5 (Alm; cf. Table 1) [4][18–32]. Studies on how
alamethicin interacts with membranes have contributed significantly to the advance-
ment of our knowledge on the antimicrobial mechanism of peptaibiotics [33–43] and
membrane pore formation in general [19].
One emerging spectroscopic technique, likely to bring an important contribution to
19
this field is solid-state (ss) F-NMR and, in particular, the center band-only detection
19
of exchange (CODEX) experiment [44]. The employment of F as the heteronucleus
allows collection of reliable information on the number of molecules in the aggregates
[
45]. In this context, our groups have recently studied the quaternary arrangement of
19
the self-aggregated helices of a F-labeled alamethicin component within lipid bilayers
46]. It is then of great importance to find reliable and optimized synthetic strategies to
[
19
insert F labels, suitable for CODEX experiments, in the sequences of peptaibiotics,
without affecting their overall 3D-structure.
Table 1. Amino Acid Sequences of Alamethicin F 50/5 and Its Analogs Discussed in This Article
Name
Sequencea)
Abbreviation
Alamethicin F 50/5
Ac-Aib-Pro-Aib-Ala-Aib-Ala-Gln-Aib-
Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-
Gln-Gln-Phol
Ac-Aib-Pro-Aib-Ala-Aib-Ala-Glu(OMe)-
Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-
Aib-Glu(OMe)-Glu(OMe)-Phol
Tfa-Aib-Pro-Aib-Ala-Aib-Ala-Glu(OMe)-
Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-
Aib-Glu(OMe)-Glu(OMe)-Phol
Alm
[
Glu(OMe)7,18,19]-
Alm’ (1)
Alamethicin F 50/5
(Tfa) ,Glu(OMe)7,18,19]-
0
N TfaAlm’ (2)
a
[
Alamethicin F 50/5
9
[
Dab(Tfa) ,Glu(OMe)7,18,19]-
Ac-Aib-Pro-Aib-Ala-Aib-Ala-Glu(OMe)-
DabTfa9Alm’ (3)
DabTfa19Alm’ (4)
9
Alamethicin F 50/5
Aib-Dab(Tfa) -Aib-Gly-Leu-Aib-Pro-Val-
Aib-Aib-Glu(OMe)-Glu(OMe)-Phol
Glu(OMe)7,18,Dab(Tfa) ]-
19
Ac-Aib-Pro-Aib-Ala-Aib-Ala-Glu(OMe)-
Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-
[
Alamethicin F 50/5
19
Glu(OMe)-Dab(Tfa) -Phol
a) Ac, acetyl; Phol is the 1,2-amino alcohol phenylalaninol; Dab(Tfa), g-trifluoroacetylated a,g-
diaminobutyric acid.

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Recently, the solid-phase peptide syntheses (SPPS) of selected peptaibiotics have
been reported [47–56]. However, the results obtained for the longest members of this
family in terms of yield and purity of the crude are still far from being satisfactory. In
the present work, we exploited a solution-phase synthetic protocol to prepare three
Alm analogs (Table 1), each bearing a single Tfa group i) at the N-terminus, ii) near the
C-terminus (position 19), or iii) in the middle of the sequence (position 9). On the
7,18,19
basis of the synthetic routes already set up for Alm and its Glu(OMe)
analog
[
6][18][19], hereafter termed Alm’ (1), found to be as active as the parent peptaibiotic
[
30], we partially modified the backbone and side-chain protection strategy, thus
improving the final, overall yield. The protocol for the solution-phase synthesis of these
analogs is herein discussed in detail. In addition, the suitability of the Tfa probe in terms
of its influence on the peptaibiotic conformation was assessed by a variety of
spectroscopic techniques (FT-IR absorption, CD, 2D-NMR, and X-ray diffraction).
The results of an antibacterial/antifungal investigation on the three trifluoroacetylated
analogs of Alm’ (1) are also presented. A preliminary account of part of this work was
already reported [57].
Results and Discussion. – Peptide Synthesis. The challenges connected with the
synthesis of peptaibiotics, due to the low reactivity (particularly at the a-amino
a
function) of the C -tetrasubstituted a-amino acids, such as Aib [15][16], especially
when two (or more) of them consecutively occur in the target sequence, and the acid
lability of the extensively present Aib-Pro and Aib-Hyp ((4R)-4-hydroxyproline
(
Hyp)) bonds, are well-documented [15][16]. Nonetheless, in the past few years
attempts were made
by a few research groups at preparing peptaibiotics by using the SPPS approach.
However, even though short peptaibiotics and segments thereof were obtained in
acceptable yield and purity [47][48], the results achieved for the long peptaibiotic
alamethicin are not yet sufficiently good to exploit this methodology for a relatively
large-scale synthesis.
Motivated by these findings, we planned the preparation of the three Tfa-labeled
Alm analogs by solution-phase synthesis, in particular by taking advantage of the
strategies previously described by some of us [29][30] (solution-phase syntheses of Alm
and selected analogs have been already reported by other groups [58–62]). These
strategies imply the use of a combination of step-by-step and segment condensation
approaches (for the latter, by splitting the target sequence into segments at the level of
the Aib-Pro(Hyp) bonds). Inter alia, this protocol offers the following advantages: i)
the formation of the Aib-Pro(Hyp) bonds in the final synthetic steps allows one to
avoid the well-established propensity of this dipeptide at the N-terminus of a sequence
to cyclize to 2,5-dioxopiperazine with the concomitant loss of these two residues from
the growing peptide chain, ii) the presence of an achiral Aib residue at the C-terminus
of the peptide to be activated reduces dramatically the risk of epimerization usually
inherent to the segment condensation approach, iii) the Aib-Pro(Hyp) bonds, if
already synthetically incorporated in the peptide, would force one to avoid the use of
some of the most common protecting groups such as (tert-butoxy)carbonyl (Boc) or
t
tert-butoxy ( BuO), known to be cleaved by acidic treatment. In contrast, this type of
protections may be effectively employed in the step-by-step synthesis of each segment,

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CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
as envisaged in our protocol. In these strategies, however, a still existing problem is the
moderate yield (30%) in the sterically hindered Aib-Pro bond-formation using the
peptide oxazol-5(4H)-one C-component.
In this context, it is worth pointing out that, in our first syntheses of Alm and two
analogs by use of this approach [29], we performed chiral gas-chromatographic
analyses to characterize all final products and found no evidence for the potential
epimerization of the penultimate Leu residue of the C-component. This finding is
corroborated by our HPLC and NMR data on the present synthetic peptaibiotics which
do not suggest formation of any epimer mixture.
In the present work, we maintained the same pattern of segments as described in
[
29][30], but we modified some of the orthogonal protections with the purpose of
accommodating appropriately the different functionalities in our Tfa-labeled target
sequences (Table 1) and improving the reaction yields. For instance, the alcohol
function of the C-terminal 1,2-amino alcohol Phol (phenylalaninol) can be used
without protection, because it is not nucleophilic enough to efficiently compete with
peptide bond-formation under our experimental conditions. However, protecting this
moiety as a benzyl (Bn) ether would increase peptide solubility in organic solvents and
consequently enhance the final overall yield. Therefore, the C-terminal segment
a
employed in the synthesis of two of the analogs discussed in this work, N TfaAlm’ (2;
Scheme 2) and DabTfa9Alm’ (3; Table 1), was obtained using the step-by-step
procedure as outlined in Scheme 1. This segment corresponds to the C-terminal
sequence of Alm’ (1). The overall yield for the synthesis of this segment increased from
Scheme 1. Synthetic Strategy Adopted for the Alm’ (1) C-terminal Segment Boc-Pro-Val-(Aib)2-
[
Glu(OMe)] -Phol
2
i) Boc-Phol/NaH/BnBr 1:1:1, anh. THF. ii) 10 equiv. of CF COOH (TFA), anh. CH Cl . iii) 1-Ethyl-3-
3
2
2
[
3-(dimethylamino)propyl]carbodiimide (EDC)/1-hydroxy-7-azabenzotriazole (HOAt), N-methylmor-
pholine (NMM; to keep pH 8). iv) H /Pd, MeOH, 4 d.
2

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18% (with the unprotected Phol) [29] to 24% (with the present strategy involving Phol
protection).
Furthermore, we synthesized the C-terminal segment needed to obtain the
DabTfa19Alm’ (4) analog following either of the two synthetic pathways, namely with
or without the Bn protection of the C-terminal Phol. It turned out that the protection of
the Phol alcoholic moiety is indeed strongly advisable, as it allowed us to improve the
yield of this C-terminal segment from 5% to more than 16%. Moreover, we found that
we could exploit the same building block Z-Dab(Tfa)-OH (Z, (benzyloxy)carbonyl)
for the synthesis of both target analogs DabTfa9Alm’ (3; Scheme 3) and DabT-
fa19Alm’ (4; Scheme 4), i.e., in the latter case, also in the presence of Phol-Bn, making
the overall schemes simpler and the related syntheses faster. Indeed, we could
selectively and quantitatively remove the N-protecting group Z in the presence of the
C-terminal Bn ether thanks to the difference between the kinetics of their removal
reactions, the former requiring less than 1 h, while the latter needs several days of
catalytic hydrogenation [63]. Unfortunately, no mechanistic explanation for this
interesting finding was provided by the authors.
The versatile strategies employed to prepare the N-terminal (residues 1–5) and
central (residues 6–13) segments are illustrated in Schemes 2–4. The total syntheses of
the three target analogs were carried out with moderate overall yields (10–12%, more
than 100 mg each) and high purities (> 95%). The protocol in [29] indicated a 6%
overall yield for Alm’ (1). The HPLC elution profiles (which replace the data on the
TLC runs reported for all intermediate compounds) of the three fluorinated analogs of
Alm, depicted in Fig. 1, highlight the purities of these final products. The analog with
Fig. 1. Analytical RP-HPLC elution profiles (retention times (t )) of the three Tfa-containing Alm’ (1)
R
analogs. The profile of Alm’ (1; t , 12.21 min) is shown for comparison.
R

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1171
Tfa in the central position of the sequence (DabTfa9Alm’ (3)) appears to be markedly
less hydrophobic than its two congeners and the parent peptide (Alm’). Additional
1
characterizations of the final peptaibiotics include solid-state FT-IR absorption, H-
13
and C-NMR, and ESI-TOF-MS data.
IR Absorption. The conformational preferences of the three Tfa-labeled peptides
were initially investigated by FT-IR spectroscopy in CDCl solution. In Fig. 2, the
3
amide A (a) and amide I/II (b) regions, recorded for the peptides at 1 mm
concentration, are displayed along with those of Alm’ (1) [29], shown for comparison.
ꢀ
1
In Fig. 2,b, the spectral intensities were normalized on the amide II band at 1538 cm .
The amide A regions of all peptides analyzed were dominated by a strong band at ca.
ꢀ
1
3
320 cm , that we assigned to the NꢀH stretching mode of H-bonded ꢀCOꢀNHꢀ
a
groups [64][65]. A slightly different behavior was exhibited by N TfaAlm’ (2), the
corresponding intense band of which was shifted to 3330 cm . Also, the weak
absorption generally observed at ca. 3430 cm , associated with free (solvated) amide
ꢀ
1
ꢀ
1
ꢀ
1
ꢀ1
Fig. 2. FT-IR Absorption spectra in a) 3500–3200-cm region and b) 1750–1500-cm region in CDCl3
a
solution (peptide concentration, 1 mm) of Alm’ (1), N TfaAlm’ (2), DabTfa9Alm’ (3), and
DabTfa19Alm’ (4). Effect of peptide concentration in CDCl for c) N TfaAlm’ (2) and d) DabTfa9Alm’
a
3
(
3): 1 mm (full line) and 0.1 mm (dashed line).

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a
groups, was completely absent in the spectrum of N TfaAlm’ (2). The dilution effect on
the amide A band, illustrated in Fig. 2,c and d, for two of the peptides, highlights the
presence of a highly folded conformation extensively stabilized by a large number of
intramolecular H-bonds at the lowest concentration (0.1 mm) examined. Nonetheless, a
contribution, albeit modest, of intermolecular NꢀH· · ·O¼C interactions was also
apparent.
ꢀ
1
The position of the intense C¼O stretching mode (amide I) near 1655 cm
Fig. 2,b) in all of the analyzed spectra strongly supports the view that the con-
formation preferentially adopted by all of the peptides investigated in CDCl was of the
(
3
310-/a-helical type [66].
ꢀ
1
Fig. 3. FT-IR Absorption spectra in 3500–3200-cm
region in CDCl3 solution for three sets of
representative short sequences. a) Boc-Ala-Aib-OBn (1); Boc-Aib-Ala-Aib-OBn (2); Boc-Pro-Aib-Ala-
t
t
Aib-OBn (3); Ac-Aib-Pro-Aib-Ala-Aib-OBn (5; 4). b) Z-Leu-Aib-O Bu (1); Z-Gly-Leu-Aib-O Bu (2);
t
t
Z-Aib-Gly-Leu-Aib-O Bu (3); Z-Dab(Tfa)-Aib-Gly-Leu-Aib-O Bu (4); Z-Aib-Dab(Tfa)-Aib-Gly-
Leu-Aib-O Bu (6; 5); Z-Glu(OMe)-Aib-Dab(Tfa)-Aib-Gly-Leu-Aib-O Bu (6); Z-Ala-Glu(OMe)-
Aib-Dab(Tfa)-Aib-Gly-Leu-Aib-O Bu (7). c) Boc-Phol (1); Boc-Phol(Bn) (2); Boc-Glu(OMe)-
t
t
t
Phol(Bn) (3); Boc-[Glu(OMe)] -Phol(Bn) (4); Boc-Aib-[Glu(OMe)] -Phol(Bn) (5); Boc-(Aib) -
2
2
2
[
[
Glu(OMe)] -Phol(Bn) (6); Boc-Val-(Aib) -[Glu(OMe)] -Phol(Bn) (7); Boc-Pro-Val-(Aib)2-
2
2
2
Glu(OMe)] -Phol(Bn) (8); Boc-Pro-Val-(Aib) -[Glu(OMe)] -Phol (9). Peptide concentration, 1 mm.
2
2
2

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To follow the onset of the folded conformation as a function of increasing peptide
main-chain length, we recorded the FT-IR absorption spectra of three sets of
representative short sequences in CDCl solution. Fig. 3 (amide A region) shows that
3
the intramolecularly H-bonded, folded conformation was already present at a level as
low as that of the pentapeptides and further developed in the longer sequences, with a
much more significantly growing structuration closely related to the percentage of the
helix-inducing Aib residues.
Circular Dichroism. The far-UV/CD spectra of the three final Tfa-containing
peptides in MeOH and 100 mm sodium dodecylsulfate (SDS) in H O (a membrane-
2
mimicking environment) are shown in Fig. 4,a and b, respectively, together with those
of Alm’ (1). Acquisition of the near-UV absorption spectra in the same solvents
allowed us to normalize the peptide concentrations, even though only the modest
aromatic chromophore of Phol is present in the sequences [67]. Under each of the two
experimental conditions examined, all analogs exhibited similar spectral shapes. We
have already reported [29] that Alm and Alm’ (1) show almost the same CD curves in
MeOH. All these CD spectra are indicative of a right-handed, predominantly a-helical
structure [68] as the R ratio [69–71] between the intensities of the two negative bands
at 222 and 208 nm is 0.80ꢁ0.05. The population of 3 -helix segments was definitely
10
lower [10][72–74]. In the presence of SDS micelles, the intensity of the negative
maximum at 222 nm increased, becoming higher than that at 208 nm (R value 1.03ꢁ
a
Fig. 4. Far-UV/CD Spectra for Alm’ (1), N TfaAlm’ (2), DabTfa9Alm’ (3), and DabTfa19Alm’ (4) in
a) MeOH and b) 100 mm SDS solution. Peptide concentration, 0.1 mm.

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0.02). This observation strongly suggests the onset of an extremely well-developed a-
helical structure in this membrane-mimicking environment for all of the peptides
investigated. We conclude that neither the presence of a non-proteinogenic a-amino
acid (Dab) bearing a side-chain Tfa probe nor the replacement of the N-terminal
capping group (Ac) with Tfa affects dramatically the overall peptide conformation in
the two environments of different polarity investigated.
NMR Spectroscopy. We extended our conformational analysis of the Tfa-labeled
analogs of Alm’ (1) in CD OH solution by using the 2D-NMR technique. Despite the
3
length of the peptide sequence and the occurrence of many amino acids of the same
type (e.g., eight Aib residues) and two Pro (lacking the NH H-atom) the assignment of
all of the H-atom resonances was successfully achieved by exploiting the standard
Wꢀthrich procedure [75], supported by the 2D HMBC spectrum [76]. Several NOESY
spectra were acquired at different mixing times. Eventually, the mixing time of 250 ms
was chosen, as it provided the best signal-to-noise ratio without the presence of spin-
diffusion effects.
As a typical example, two portions of the NOESY spectrum of DabTfa9Alm’ (3)
are shown in Figs. 5 and 6. The presence of all NH -NH cross-peaks (Fig. 5), except
i
iþ1
a
a
those covered by the diagonal peaks, along with a number of C H !NH and C H !
i
iþ1
i
Fig. 5. Section (amide NH H-atom region) of the NOESY spectrum of DabTfa9Alm’ (3) in CD OH
3
solution (600 MHz, 298 K). Peptide concentration, 1.64 mm.

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
1175
Fig. 6. Fingerprint region of the NOESY spectrum of DabTfa9Alm’ (3) in CD OH solution (600 MHz,
3
a
2
98 K). Peptide concentration, 1.64 mm. The medium-range connectivities C H !NH (full line) and
i
iþ3
a
C H !NH (dashed line) are underlined.
i
iþ4
NH
connectivities (Fig. 6), highlights the occurrence of a well-defined, helical
iþ3
structure throughout the amino acid sequence. Furthermore, the detection of several
a
C H !NH medium-length connectivities, well spread all over the sequence (Fig. 6),
i
iþ4
allowed us to conclude that DabTfa9Alm’ (3) is folded in a largely a-helical

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CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
conformation, closely resembling that of the parent peptide [32]. The corresponding
NOESY spectra for the other two trifluoroacetylated analogs (deposited) indicated
that the overall prevaling a-helical conformation was largely mantained.
X-Ray Diffraction. A variety of attempts to grow single crystals appropriate for an
X-ray diffraction analysis allowed us to solve the 3D structures of the terminally
protected pentapeptide ester Ac-Aib-Pro-Aib-Ala-Aib-OBn (5), hexapeptide ester Z-
t
Aib-Dab(Tfa)-Aib-Gly-Leu-Aib-O Bu (6), and the tetrapeptide amide Boc-(Aib) -
2
Glu(OMe)-Dab(Tfa)-Phol(Bn) (7; Fig. 7). These compounds represent the N-
terminal, central (residues 8–13), and C-terminal sequences, respectively, of the
Alm analogs synthesized in this work. The most relevant conformational and
intramolecular H-bond parameters for the three oligopeptides are compiled in Tables 2
and 3, respectively.
Bond lengths and bond angles (deposited) are in general agreement with previously
reported values for the geometry of the Boc-NHꢀ [77] and Z-NHꢀ [78] urethane
moieties, the peptide unit [79][80], the methyl/benzyl ester group [81], and the Aib
[
82][83] residue.
In the four molecules of the three peptides, all amide/peptide bonds (w torsion
angles) are in the common trans disposition [79][80]. The conformation of the peptide
backbone (f, y torsion angles) of the pentapeptide ester 5 is helical, stabilized by two
strong and one weak intramolecular C¼O· · ·HꢀN H-bonds [84–86]. From the N-
3
terminus of the main chain, two strong H-bonds are formed by the Aib NH and
4
1
Ala NH groups as donors, and the Ac and Aib C¼O groups as acceptors, respectively.
1
Rather unusually, the Aib C¼O group is the acceptor of a further, weak H-bond, from
5
the Aib NH group as the donor. Collectively, the intramolecular H-bonding scheme is
characterized by two consecutive C10 (b-turn [87–89]) forms (incipient 3 -helix
10
[
72][73]), the second of which is embraced by a larger C (a-turn [88][90]) form. This
13
4
latter conformation is generated by the wide f torsion angle, observed for the Ala
residue. Also the C-terminal Aib residue, partially external to the helix structure, is
helical, but its screw sense is opposite to that of the preceding residues. This is a
common observation for 3 -helical peptide esters with a C-terminal C -dialkyl glycine
[
[
5
a,a
10
4
4
91]. The pyrrolidine ring of the Pro residue has a symmetry between twist T and E
3
92][93] with ring-puckering parameters [94] q ¼0.380(7) ꢄ and f ¼ ꢀ81.6(7)8.
2
2
Two helical, but conformationally distinct molecules 6A and 6B (Fig. 7) occur in the
asymmetric unit of the hexapeptide ester 6. Not only some of the c torsion angles of the
2
5
long side chains of Dab(Tfa) and Leu residues (deposited) differ remarkably, but a
4
5
few main-chain f, y torsion angles as well. Notably, the f values for Gly and Leu ,
5
both in molecule 6A, and for Leu in molecule 6B are considerably expanded, although
4
to a different extent, from the regular helical values. Moreover, the y values for Gly in
2
molecules 6A and 6B, and particularly for Dab(Tfa) in 6B are very compressed. As a
result, the four intramolecular C¼O· · ·HꢀN H-bonds in molecule 6B are those
expected for a 3 -helix, whereas those in molecule 6A are not. Indeed, the N6ꢀH· · ·
10
O3 H-bond (C form), which should have characterized the 3 -helix of molecule 6B, is
10
10
missing, and it is replaced by a N6ꢀH· · ·O2 H-bond (C form). Here too, as already
13
highlighted above for 5, near the C-terminus, a C¼O O-atom (the O2 atom in this case)
is a double acceptor of intramolecular H-bonds. Again, in both molecules 6A and 6B,
the screw sense of the C-terminal helical Aib residue is opposite to that of the preceding

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
1177

1
178
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
Table 2. Backbone Torsion Angles for Peptides 5–7
Residue
5
Residue
7
f [8]
y [8]
w [8]
f [8]
y [8]
w [8]
179.5(4)
1
1
Aib
Pro
ꢀ52.8(6) ꢀ37.4(6) ꢀ178.3(4) Aib
ꢀ54.5(6) ꢀ30.7(6)
2
2
ꢀ58.3(5) ꢀ29.5(6)
179.4(4) Aib
ꢀ45.2(6) ꢀ39.9(6) ꢀ171.2(4)
3
3
Aib
ꢀ55.4(6) ꢀ38.6(5) ꢀ178.8(4) Glu(OMe)
ꢀ57.8(6) ꢀ30.7(6) ꢀ177.1(4)
4
4
Ala
ꢀ78.2(5) ꢀ34.1(6)
173.5(5) Dab(Tfa)
173.7(6)
ꢀ87.8(5)
ꢀ4.7(7) ꢀ175.2(4)
5
Aib
52.2(8)
48.7(7)
Residue
Molecule 6A
Molecule 6B
f [8]
f [8]
y [8]
w [8]
y [8]
w [8]
Aib1
Dab(Tfa)
ꢀ60.5(6) ꢀ31.5(6) ꢀ178.2(4)
ꢀ52.2(6) ꢀ34.6(5) ꢀ174.7(4)
ꢀ52.1(6) ꢀ39.2(6) ꢀ174.1(5)
2
ꢀ67.2(6)
ꢀ8.4(7)
167.9(5)
179.7(6)
175.0(5)
3
Aib
Gly
ꢀ60.2(6) ꢀ26.4(6)
179.7(4)
ꢀ51.4(8) ꢀ29.6(8)
ꢀ61.6(8) ꢀ19.4(8)
4
ꢀ82.9(6) ꢀ17.0(7) ꢀ176.8(5)
5
Leu
Aib
ꢀ99.7(6) ꢀ54.8(6)
54.2(7) 46.5(6)
176.4(5)
174.7(5)
ꢀ68.6(6) ꢀ25.1(6) ꢀ177.2(5)
5
45.7(7)
46.2(7) ꢀ176.4(5)
Table 3. Intramolecular H-Bond Parameters for Peptides 5–7
Donor
DꢀH
Acceptor
A
Distance [ꢄ]
D· · ·A
Distance [ꢄ]
H· · ·A
Angle [8]
DꢀH· · ·A
5
N3ꢀH
N4ꢀH
N5ꢀH
O0
O1
O1
3.041(6)
2.984(5)
3.337(5)
2.28
2.29
2.51
147
138
163
6
N3ꢀH
N4ꢀH
N5ꢀH
N6ꢀH
N13ꢀH
N14ꢀH
N15ꢀH
N16ꢀH
O0A
O1
O2
3.070(5)
2.966(5)
3.054(5)
3.038(5)
3.018(7)
3.012(6)
2.907(6)
3.020(6)
2.25
2.18
2.26
2.22
2.18
2.16
2.07
2.22
159
152
153
159
163
169
164
155
O2
O0B
O11
O12
O13
7
N3ꢀH
N4ꢀH
N5ꢀH
N4DꢀH
O0
O1
O2
O3E1
2.982(5)
3.026(5)
3.029(5)
2.920(6)
2.14
2.24
2.20
2.11
165
153
163
156
residues. Finally, in both molecules, the Dab side-chain trifluoroacetamide NH group is
intermolecularly H-bonded to a corresponding peptide C¼O O-atom of a molecule
related by the same symmetry operation.
The backbone of 7 (Fig. 7) is folded in a (partially irregular) 3 -helical structure
10
with three strong intramolecular C¼O· · ·HꢀN H-bonds. The donors of the C10 forms

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
1179
3
4
are the main-chain Glu(OMe) , Dab(Tfa) , and Phol C-terminal amide NH groups, and
1
2
the acceptors are the Boc urethane and the peptide C¼O groups of the Aib and Aib
residues. The C-terminal b-turn is type I [87–89], produced by the non-helical f, y
4
values of the Dab(Tfa) residue. A surprising, previously unreported in crystalline
peptides, property of this 3D structure is the occurrence of an additional intramolecular
C¼O· · ·HꢀN H-bond which links the two long, functionalized side chains of the
3
4
consecutive Glu(OMe) and Dab(Tfa) residues. The former affords the methyl ester
C¼O acceptor, and the latter acts as the trifluoroacetamido NꢀH donor. The crystal
packing modes and intermolecular H-bondings for peptides 5–7 are presented and
discussed in the Supplementary Information (available upon request from the authors).
To summarize our results in the crystalline state, the three short peptides are
extensively folded in helical structures. This 3D-structural disposition is associated with
the remarkable amount (ꢂ 50%) of Aib residues in the sequences [10][72][73]. Not
surprisingly, in view of the limited length (<seven residues) of the peptides
[
10][72][73], the helix adopted is primarily of the 3 -type, although with minor
10
irregularities near the C-terminus. A comparison among the 3D structures reported
above, and the conformations of the corresponding 1–5-, 8–13-, and 16–19-Phol
peptide segments in the published full-length X-ray structures of Alm (three
7,18,19
16
crystallographically independent molecules) [20] and its (Glu(OMe)
, TOAC )
(
TOAC¼4-amino-2,2,6,6-tetramethyl-1-oxylpiperidine-4-carboxylic acid) analog (two
independent molecules) [38] indicates an overall conformational similarity, in that
these segments are also helical in the full-length molecules. Differences, however, are
found in the details of the H-bonding patterns (C10 vs. C13 forms, C10 forms
encompassed within C13 forms). The most notable difference is found for the 8–13
segment (Fig. 7), which is mainly (molecule 6A) or fully (molecule 6B) 3 -helical in
10
the structure of 6 reported in this work, whereas it shows an almost exclusive a-helical
7
,18,19
16
character when part of Alm or its (Glu(OMe)
helical structure was also observed for Alm when reconstituted into a supported POPC
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) phospholipid bilayer by using
, TOAC ) analog. A mixed a-/3 -
10
(
15
solid-state N-NMR spectroscopy [95].
Solid-State 19F-NMR Spectroscopy. As a result of an initial MAS (magic angle
spinning) solid-state F-NMR analysis, the spectrum of a sample of N TfaAlm’ (2)
reconstituted in a POPC bilayer and recorded in the frozen state is shown in Fig. 8,a.
The chemical-shift anisotropy of 40 ppm allows for the magnetization exchange
19
a
between magnetically inequivalent CF groups as they occur for chemically equivalent
3
peptide molecules in an oligomeric aggregate (Fig. 8,b). The normalized magnet-
ization exchange curve (S/S in Fig. 8,b) is indicative of the presence of oligomers.
0
Antimicrobial Activity. The antimicrobial activities of the three trifluoroacetylated
analogs of Alm’ (1) were assessed on Gram-positive (Staphylococcus aureus and
Enterococcus faecalis) and Gram-negative (Escherichia coli, Yersinia enterocolitica,
and Shigella flexneri) bacteria, and on the fungus Candida albicans, and were compared
to those of the parent compound Alm’ (1; Table 4). The most interesting results are
those of DabTfa19Alm’ (4) which exhibits a broad spectrum of action including against
both Gram-positive and Gram-negative bacteria. This finding is at variance with those
a
obtained for N TfaAlm’ (2), DabTfa9Alm’ (3), and Alm’ (1) (the latter appreciably
active only on the thick cellular wall of the Gram-positive E. faecalis strain), all of

1
180
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
Fig. 8. a) Experimental solid-state 19F-NMR spectrum of 11 mol-% N TfaAlm’ (2) in POPC (¼1-
palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) at ꢀ258. The MAS (magic angle spinning) frequency
is 8 kHz. The resulting spinning side bands reveal the chemical-shift anisotropy. b) Experimental
CODEX curve obtained at a MAS frequency of 15 kHz. The data points are indicated by black squares
a
(
uncertaintyꢁ0.2).
a
Table 4. Antimicrobial Activities of Peptides Alm’ (1), N TfaAlm’ (2), DabTfa9Alm’ (3), and
a
DabTfa19Alm’ (4) (at 100 mg/disk) )
a
Bacterial strain
Alm’ (1) N TfaAlm’ (2) DabTfa9Alm’ (3) DabTfa19Alm’ (4)
Staphylococcus aureus ATCC 25923 6
6
7
6
6
6
6
6
7
6
6
6
6
6
8
8
10
7
6
Enterococcus faecalis ATCC 29212
Escherichia coli ATCC 25922
Yersinia enterocolitica ATCC 27739
Shigella flexneri clinical isolate
Candida albicans ATCC 1028
9
6
6
6
6
a) Diameter of inhibition zone ([mm]).
which appear to be unable to penetrate the highly selective external membrane of the
Gram-negative bacterial cells. For a comparison with the related antimicrobial
activities of the Alm natural mixture, the reader is referred to [26]. Since these Tfa-
labeled peptaibiotics are potentially toxic compounds, it is not superfluous to remind
19
that their syntheses were exclusively aimed towards a solid-state F-NMR inves-
tigation.
Conclusions. – Peptaibiotics bind to lipid membranes and self-associate into
19
oligomers. The solid-state F-NMR technique provides in-depth information about the
D structure, dynamics, and topology of peptides incorporated into membranes. We are
currently investigating this biophysical phenomenon in F-labeled analogs of the long
3

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
1181
(19 amino acid residues), highly helical peptaibiotic Alm which forms channels in the
phospholipid membranes [46][57].
In this contribution, we described the solution-phase synthesis and chemical
characterization of three such peptaibiotics, each containing a single Tfa probe either at
the N-terminus, in the central region, or near the C-terminus, respectively. To this end,
we partially modified our previously published synthetic schemes of Alm and some of
its analogs [29][30] with beneficial effects. We demonstrated by a number of
spectroscopic techniques (FT-IR absorption, CD, and NMR) in SDS micelles, in
organic solvents of different polarity (CDCl and MeOH), and in the crystalline state
3
(
by X-ray diffraction analyses of three segments spanning almost entirely the Alm’ (1)
amino acid sequence) as well that the typical helical properties of this peptaibiotic
1
9
[
29][30][32] are essentially preserved, i.e., that the herein described Alm’ (1) F-
labeling strategy is conformationally harmless. Our initial solid-state 9F-NMR
investigation on the N TfaAlm’ (2) analog unambiguously indicated the formation of
1
a
oligomeric aggregates when this peptide is immersed into a phospholipid membrane.
All three trifluoroacetylated analogs of Alm’ (1) are antimicrobially active, although
with partially different specific properties.
This work was supported by the Italian Ministry of Education, University, and Research, MIUR
(
grant PRIN 2008).
Experimental Part
General. Amino acids and their derivatives were purchased from IRIS Biotech. N-Ethyl-N’-[3-
(
dimethylamino)propyl]carbodiimide (EDC) and 1-hydroxy-7-aza-1,2,3-benzotriazole (HOAt) [96]
were GL Biochem products. All chiral amino acids employed are of the l-configuration. All other
chemicals were obtained from SigmaꢀAldrich. The final products were analyzed by anal. RP-HPLC on a
Jupiter Phenomenex C column (4.6ꢃ250 mm, 5 mm, 300 ꢄ) using an Agilent 1200 HPLC pump. The
4
2 2
binary elution system used was: A, H O/MeCN 9 :1; B, MeCN/H O 9 :1; gradient 60–80% B in 20 min
(
flow rate 1 ml/min); spectrophotometric detection at l 226 nm. Anal. TLC and prep. column
chromatography (CC): Kieselgel F254 and Kieselgel 60 (0.040–0.063 mm; Merck), resp. The retention
factor (R ) values were determined using four solvent mixtures as eluants: Rf1: CHCl / EtOH 9 :1; Rf2
BuOH/AcOH/H O 3 :1:1; R : toluene/EtOH 7:1; R : CH Cl / MeOH 98 :2. M.p.: Cap. tube immersed
f
3
:
2
f3
f4
2
2
in an oil bath (Tottoli apparatus, Bꢀchi); uncorrected. ESI-TOF-MS: PerSeptive Biosystem Mariner
instrument; in m/z.
CD Experiments. CD Recordings were carried out on a JASCO J-715 spectropolarimeter. The CD
spectra were acquired and processed using the J-700 program for Windows. All spectra were recorded at
r.t., using Hellma quartz cells with Suprasilꢅ windows and optical path length of 0.1 cm. The signal-to-
noise ratio was improved by accumulating eight scans. The values are expressed in terms of total molar
2
ꢀ1
ellipticity, [q] [degree·cm ·dmol ]. Spectrograde MeOH (99.9%) and 100 mm aq. SDS soln. were used
T
as solvents.
FT-IR Absorption Experiments. The FT-IR absorption spectra were recorded with a PerkinElmer
1
2
720X spectrophotometer, N -flushed, equipped with a sample-shuttle device, at a nominal resolution of
cm , averaging 100 scans; ˜n in cm . Solvent (baseline) spectra were obtained under the same
2
ꢀ
1
ꢀ1
conditions. Cells with path lengths of 1.0 and 10 mm (with CaF windows) were used. Spectrograde
2
CDCl (99.8%, D) was purchased from Merck.
3
NMR Experiments. 1D H- and 13C-NMR spectroscopy was employed to characterize the synthesized
1
compounds. CDCl and (D )DMSO were used as solvents. All spectra were acquired on a Bruker Avance
3
6
DMX-600 or DRX-400 operating at 600 or 400 MHz, resp., using the TOPSPIN software package; d in
ppm rel. to Me Si as internal standard, J in Hz. The 2D-NMR experiments were carried out on
4

1
182
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
DabTfa9Alm’ (3; 2 mg of the peptide dissolved in 0.55 ml of CD OH; final peptide concentration;
3
1.6 mm). All experiments were acquired at 298 K on a Bruker Avance DMX-600 instrument. Suppression
of the OH solvent signal was obtained applying a WATERGATE gradient program. The standard
Wꢀthrich procedure [75] was employed to obtain the assignment of the spin systems of the trisubstituted
residues. The CLEAN-TOCSY [97][98] spectrum (spin lock pulse, 70 ms) was acquired by conducting
4
00 experiments of 40 scans each, whilst for the NOESY spectrum 400 experiments, each one consisting
of 96 scans, were carried out. The sequential assignment was performed by means of NOESY (mixing
time, 250 ms) and HMBC [76] (160 t increments of 200 scans each and 2K points; spectral width,
1
2
00 ppm, centered at 95 ppm in F1) experiments.
19
Solid-State F-NMR Experiments. A homogeneous mixture of POPC (¼ 1-palmitoyl-2-oleoyl-sn-
a
glycero-3-phosphocholine; Avanti Polar Lipids) and N TfaAlm’ (2) at a molar ratio of 1:8 was obtained
in CHCl . The soln. was dried under a flow of N and then left in vacuo overnight. The mixture was
3
2
dispersed in 10 mm Tris buffer (pH 7.5) by vortex mixing. Thereafter, the hydrated lipid bilayers were
subjected to five rapid freeze thaw cycles, centrifuged, and concentrated by pelleting in a benchtop
centrifuge. After removal of the excess buffer, a dense suspension of multilamellar vesicles in H O (ca.
2
19
5
0% w/w H O content) was obtained. Solid-state F-NMR spectra were acquired at 470.4 MHz on a
2
Bruker Avance widebore 500 NMR spectrometer equipped with a triple resonance MAS 3.2 mm probe
1
19
that allows simultaneous tuning of the H and F frequencies on a single channel through a combiner/
splitter assembly. For the CODEX experiments [45], the spinning speed was set to 15 kHz. Experiments
19
1
were conducted at ꢀ258. Typical radio frequency (rf) field strengths were 70 and 50 kHz for F and H,
1
9
19
resp. The recycle delay was 1 s. F Chemical shifts were referenced to the Teflon F signal at ꢀ122 ppm.
Boc-Phol-Bn. Yield: 90%. M.p. 54–558. R : 0.95, R : 0.95, R : 0.80, R : 0.85. IR (KBr): 3375, 1683,
517. H-NMR (400 MHz, CDCl ): 7.39–7.14 (m, 10 arom. H, Bn, Phol); 4.89 (m, BocNH); 4.49 (m, CH ,
3 2
Bn); 3.96 (m, a-CH, Phol); 3.43–3.37 (m, CH O, Phol); 2.94–2.76 (m, b-CH ); 1.43 (s, Boc). C-NMR
f1
f2
f3
f4
1
1
13
2
2
(
150 MHz, CDCl ): 155.38; 138.27; 129.48; 128.45; 128.39; 127.78; 126.30; 73.25; 70.13; 51.68; 37.91; 28.42.
3
þ
ESI-TOF-MS: 342.20 ([MþH] ; calc. 342.27).
Boc-Glu(OMe)-Phol-Bn. Yield: 80%. M.p. 81–828. R 0.95, Rf2 0.95, Rf3 0.95, Rf4 0.50. IR (KBr):
f1
1
3
350, 3314, 1732, 1683, 1657, 1522. H-NMR (400 MHz, CDCl ): 7.37–7.15 (m, 10 arom. H, Bn, Phol); 6.48
3
(
d, 1 H); 5.18 (m, 1 H); 4.50 (m, CH , Bn); 4.27 (m, 1 H); 4.11 (m, 1 H); 3.68 (s, Glu(OMe)); 3.40 (m,
2
CH O, Phol); 2.88 (m, b-CH , Phol); 2.36 (m, b-CH , Glu(OMe)); 2.05 (m, 1 H of g-CH , Glu(OMe));
2
2
2
2
13
1
.88 (m, 1 H of g-CH , Glu(OMe)); 1.43 (s, Boc). C-NMR (150 MHz, CDCl ): 173.78; 170.85; 155.63;
2 3
1
37.85; 129.38; 128.46; 127.82; 126.48; 73.27; 69.62; 53.85; 51.81; 50.49; 45.79; 37.48; 30.20; 28.31; 28.02.
Boc-Glu(OMe)-Glu(OMe)-Phol-Bn. Yield: 81%. M.p. 123–1248. R 0.95, R 0.95, R 0.20, R 0.95.
f1
f2
f3
f4
1
IR (KBr): 3326, 3292, 1733, 1685, 1646, 1524. H-NMR (400 MHz, CDCl ): 7.34–7.16 (m, 10 arom. H, Bn,
3
Phol); 7.02 (d, NH); 6.60 (d, NH); 5.25 (d, NH); 4.49 (m, CH , Bn); 4.38 (m, a-CH); 4.29 (m, a-CH);
2
4
.08 (m, a-CH); 3.68 (s, Glu(OMe)); 3.67 (s, Glu(OMe)); 3.41 (m, CH O Phol); 2.89 (m, b-CH , Phol);
2 2
2
.38 (m, 2 b-CH , Glu(OMe)); 2.06 (m, 2 H, 2 g-CH , Glu(OMe)); 1.90 (m, 2 H, 2 g-CH , Glu(OMe));
2
2
2
13
1.45 (s, Boc). C-NMR (150 MHz, CDCl ): 173.94; 173.76; 171.58; 170.06; 165.92; 165.64; 165.14; 164.75;
3
1
3
63.94; 155.71; 137.91; 129.34; 128.42; 127.78; 127.73; 126.44; 73.20; 69.82; 54.06; 52.70; 51.90; 50.57; 37.41;
þ
0.09; 28.31; 27.65; 27.58. ESI-TOF-MS: 628.30 ([MþH] ; calc. 628.36).
Boc-Aib-Glu(OMe)-Glu(OMe)-Phol-Bn. Yield: 81%. M.p. 91–928. R 0.95, Rf2 0.95, Rf3 0.10, Rf4
f1
1
0
.95. IR (KBr): 3309, 1738, 1657, 1526. H-NMR (400 MHz, CDCl
3
): 7.89 (m, NH); 7.62 (d, NH); 7.37–
7
.12 (m, 10 arom. H, Bn, Phol); 6.97 (d, NH); 5.00 (s, NH, Aib); 4.54 (m, CH , Bn); 4.39 (m, 2 a-CH);
2
4
.20 (m, a-CH); 3.72 (s, Glu(OMe)); 3.64 (s, Glu(OMe)); 3.51 (m, CH
2
2
O, Phol); 3.07 (m, 1 H, b-CH ,
Phol); 2.79 (m, 1 H, b-CH , Phol); 2.57–2.42 (m, 2 H); 2.41–2.06 (m, 5 H); 1.94 (m, 1 H of g-CH ,
2
2
13
Glu(OMe)); 1.48 (s, 1 Me, Aib); 1.44 (s, Boc); 1.41 (s, 1 Me, Aib). C-NMR (150 MHz, CDCl
3
): 175.61;
1
7
7
75.44; 173.15; 171.07; 170.57; 155.52; 138.67; 129.44; 128.23; 128.18; 127.62; 127.32; 126.07; 81.04; 73.03;
0.92; 56.76; 55.38; 53.26; 52.22; 51.51; 50.59; 37.61; 30.74; 28.25; 27.00; 26.55; 25.39; 24.00. ESI-TOF-MS:
þ
13.34 ([MþH] ; calc. 713.32).
Boc-Aib-Aib-Glu(OMe)-Glu(OMe)-Phol-Bn. Yield: 85%. M.p. 124–1258. R 0.95, R 0.95, R 0.95,
f1
f2
f3
1
R 0.95. IR (KBr): 3370, 3293, 1740, 1680, 1653, 1532. H-NMR (400 MHz, CDCl ): 7.64 (d, NH); 7.62 (d,
f4
3
NH); 7.36–7.13 (m, 10 arom. H, Bn, Phol); 6.90 (d, NH); 6.62 (s, NH, Aib); 4.99 (s, NH, Aib); 4.54 (m,
CH , Bn); 4.35 (m, 2 a-CH); 4.17 (m, a-CH); 3.65 (s, Glu(OMe)); 3.64 (s, Glu(OMe)); 3.50 (m, CH O,
2
2

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
1183
Phol); 3.06 (m, 1 H); 2.78 (m, 1 H); 2.57–2.42 (m, 2 H); 2.40–2.20 (m, 4 H); 2.15–1.91 (m, 2 H); 1.49 (s,
1
3
1
1
7
2
Me, Aib); 1.45 (s, Boc); 1.43 (s, 1 Me, Aib); 1.41 (s, 2 Me, Aib). C-NMR (150 MHz, CDCl ): 175.73;
3
75.09; 173.25; 171.82; 171.13; 155.83; 138.49; 129.43; 128.20; 128.08; 127.68; 127.33; 126.01; 81.10; 73.07;
1.22; 56.71; 56.63; 54.93; 53.37; 51.56; 51.43; 50.48; 37.74; 30.99; 30.80; 28.18; 27.60; 26.64; 26.49; 26.02;
þ
3.16. ESI-TOF-MS: 798.38 ([MþH] ; calc. 798.42).
Boc-Val-Aib-Aib-Glu(OMe)-Glu(OMe)-Phol-Bn. Yield: 86%. M.p. 139–1408. R 0.95, Rf2 0.95, Rf3
.95, R 0.95. IR (KBr): 3288, 1740, 1651, 1534. H-NMR (400 MHz, CDCl ): 7.66 (d, NH); 7.58 (d, NH);
f1
1
0
f4
3
7.41–7.18 (m, 10 arom. H, Bn, Phol); 7.14 (d, NH); 6.99 (d, NH); 6.51 (s, NH, Aib); 4.97 (s, NH, Aib); 4.56
(
m, CH , Bn); 4.37 (m, 2 a-CH); 4.13 (m, a-CH); 3.71 (m, a-CH); 3.65 (s, Glu(OMe)); 3.63 (s,
2
Glu(OMe)); 3.51 (m, CH O, Phol); 3.10 (m, 1 H); 2.79 (m, 1 H); 2.58–2.48 (m, 2 H); 2.44–2.14 (m,
2
5
1
1
6
2
H); 2.12–1.95 (m, 2 H); 1.49 (m, Boc, 1 Me of Aib); 1.47 (s, 1 Me, Aib); 1.46 (s, 1 Me, Aib); 1.40 (s,
13
Me, Aib); 1.01 (2d, 2 g-Me, Val). C-NMR (150 MHz, CDCl ): 176.25; 174.64; 173.28; 173.02; 172.28;
3
72.15; 171.19; 156.92; 150.19; 138.65; 129.46; 128.16; 128.04; 127.61; 127.23; 125.92; 81.25; 73.04; 71.17;
2.31; 57.00; 56.63; 55.03; 53.36; 51.53; 51.39; 50.58; 37.73; 31.03; 30.89; 29.62; 28.21; 27.45; 27.23; 26.52;
þ
6.08; 23.26; 23.03; 18.94; 18.59. ESI-TOF-MS: 897.45 ([MþH] ; calc. 897.52).
Boc-Pro-Val-Aib-Aib-Glu(OMe)-Glu(OMe)-Phol-Bn. Yield: 87%. M.p. 68–698. R 0.95, Rf2 0.95,
f1
1
Rf3 0.95, Rf4 0.95. IR (KBr): 3301, 1739, 1653, 1533. H-NMR (400 MHz, CDCl ): 7.71 (s, NH, Aib); 7.66
3
(d, NH); 7.59 (d, NH); 7.29–7.04 (m, 10 arom. H, Bn, Phol); 7.14 (d, NH); 6.95 (d, NH); 6.31 (d, NH);
4
.48 (m, CH , Bn); 4.30 (m, 2 a-CH); 4.13 (m, a-CH); 4.05 (m, a-CH); 3.86 (m, a-CH); 3.57 (s,
2
Glu(OMe)); 3.55 (s, Glu(OMe)); 3.51–3.41 (m, CH O of Phol, d-CH of Pro); 3.03 (m, 1 H, b-CH ,
2
2
2
Phol); 2.71 (m, 1 H, b-CH , Phol); 2.51–2.36 (m, 2 H); 2.35–2.12 (m, 7 H); 2.00–1.84 (m, 4 H); 1.44 (m,
2
13
Boc); 1.42 (s, 2 Me, Aib); 1.37 (s, 2 Me, Aib); 0.92 (d, g-Me, Val); 0.86 (d, g-Me, Val). C-NMR
150 MHz, CDCl ): 175.28; 174.59; 173.33; 172.92; 171.54; 171.00; 155.71; 138.70; 129.51; 128.22; 128.13;
(
3
1
3
27.66; 127.27; 126.01; 81.61; 73.05; 70.95; 56.94; 56.70; 54.91; 53.22; 51.62; 51.46; 50.60; 37.66; 31.09;
þ
0.99; 28.21; 27.75; 26.56; 26.06; 23.33. ESI-TOF-MS: 994.48 ([MþH] ; calc. 994.51).
Boc-Pro-Val-Aib-Aib-Glu(OMe)-Glu(OMe)-Phol. Yield: 99%. M.p. 86–878. R 0.95, Rf2 0.95, Rf3
f1
1
0
.95, Rf4 0.95. IR (KBr): 3303, 1740, 1656, 1535. H-NMR (400 MHz, CDCl ): 7.94 (d, NH); 7.82 (s, NH,
3
Aib); 7.68 (d, NH); 7.32–7.22 (m, 5 arom. H, Phol); 7.15 (d, NH); 7.04 (d, NH); 6.40 (d, NH); 4.31 (m, a-
CH); 4.20 (m, a-CH); 4.10 (m, a-CH); 3.93 (m, a-CH); 3.72 (m, a-CH); 3.65 (s, Glu(OMe)); 3.64 (s,
Glu(OMe)); 3.59–3.49 (m, 3 H); 2.96 (m, 2 H); 2.60–2.46 (m, 2 H); 2.42–2.10 (m, 7 H); 2.10–1.91 (m,
13
4
H); 1.60 (s, 6 H); 1.51 (m, 12 H); 1.46 (s, 6 H); 0.99 (d, g-Me, Val); 0.94 (d, g-Me, Val). C-NMR
(
150 MHz, CDCl ): 177.42; 175.32; 173.73; 173.22; 172.95; 172.51; 171.56; 171.31; 156.04; 138.89; 129.64;
3
1
3
1
28.15; 125.98; 94.99; 81.92; 63.43; 61.84; 60.48; 57.04; 56.95; 55.59; 53.96; 53.32; 51.58; 51.43; 47.53;
6.82; 31.13; 31.11; 30.06; 28.86; 28.38; 28.31; 27.83; 27.49; 26.96; 26.53; 26.02; 24.87; 23.08; 22.94; 17.07;
þ
7.67. ESI-TOF-MS: 904.45 ([MþH] ; calc. 904.51).
Z-Dab(Tfa)-Phol-Bn. Yield: 69%. M.p. 135–1368. R 0.95, Rf2 0.95, Rf3 0.50, Rf4 0.75. IR (KBr):
f1
1
3
304, 1698, 1645, 1532. H-NMR (400 MHz, (D )DMSO): 9.31 (m, e-NH, Dab); 7.87 (d, J¼8.4, NH,
6
Dab); 7.41 (d, J¼8.0, NH, Phol); 7.39–7.06 (m, 15 arom. H, Z, Bn, Phol); 5.01 (m, CH , Z); 4.44 (m, CH ,
2
2
Bn); 4.12–4.05 (m, a-CH); 4.04–3.96 (m, a-CH); 3.38–3.32 (m, 2 H); 3.21–3.09 (m, CH O, Phol); 2.84–
2
2
.77 (m, 1 H); 2.75–2.64 (m, 1 H); 1.85–1.72 (m, 1 H); 1.70–1.57 (m, 1 H). 13C-NMR (150 MHz,
CDCl ): 170.14; 157.74; 157.50; 156.73; 137.65; 137.53; 135.83; 129.31; 128.64; 128.53; 128.44; 128.13;
3
1
27.99; 126.63; 73.33; 69.39; 67.49; 52.28; 50.92; 37.41; 35.93; 32.94; 22.86. ESI-TOF-MS: 572.21 ([Mþ
þ
H] ; calc. 572.23).
Boc-Glu(OMe)-Dab(Tfa)-Phol-Bn. Yield: 70%. M.p. 146–1478. R 0.95, Rf2 0.95, Rf3 0.40, Rf4 0.35.
f1
1
IR (KBr): 3304, 1701, 1638, 1552. H-NMR (400 MHz, CDCl ): 7.82 (m, e-NH, Dab); 7.35–7.05 (m, 10
3
arom. H, Phol, Bn); 6.84 (d, J¼7.6, NH, Phol); 6.21 (d, J¼8.4, NH, Dab); 5.11 (br. d, J¼4.8, NH, Glu);
4
.46–4.39 (m, CH , Bn); 4.29–4.14 (m, 2 a-CH); 4.03–3.95 (m, a-CH); 3.63 (s, Glu(OMe)); 3.68–3.56
2
(
m, 1 H); 3.37–3.28 (m, CH O, Phol); 3.03–2.90 (m, 1 H); 2.86–2.78 (m, 2 H); 2.45–2.25 (m, 2 H);
2
2
1
1
.05–1.94 (m, 1 H); 1.92–1.79 (m, 2 H); 1.71–1.59 (m, 1 H); 1.37 (s, Boc). 13C-NMR (150 MHz, CDCl ):
3
73.60; 172.70; 169.98; 157.75; 157.52; 155.87; 137.70; 129.28; 128.49; 127.92; 126.56; 118.84; 116.93;
15.02; 113.12; 80.47; 73.25; 69.52; 54.33; 51.94; 50.96; 50.80; 37.33; 35.88; 32.59; 30.28; 28.29; 28.24; 27.33.
þ
ESI-TOF-MS: 681.29 ([MþH] ; calc. 681.39).

1
184
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
Boc-Aib-Glu(OMe)-Dab(Tfa)-Phol-Bn. Yield: 85%. M.p. 106–1078. R 0.95, Rf2 0.95, Rf3 0.35, Rf4
f1
1
0
(
(
.30. IR (KBr): 3304, 1720, 1661, 1639, 1527. H-NMR (400 MHz, CDCl ): 8.01 (br. d, J¼4.0, NH); 7.87
3
m, e-NH, Dab); 7.76 (d, J¼8.0, NH); 7.38–7.10 (m, 10 arom. H, Phol, Bn, 2 NH); 5.07 (s, NH, Aib); 4.56
m, CH , Bn); 4.42–4.36 (m, a-CH); 4.35–4.28 (m, a-CH); 4.20–4.13 (m, a-CH); 3.73 (s, Glu(OMe));
2
3
.58–3.49 (m, CH O, Phol); 3.39–3.29 (m, 1 H); 3.12–3.07 (m, 1 H); 3.05–2.98 (m, 1 H); 2.76–2.66 (m,
2
1
H); 2.58–2.42 (m, 2 H); 2.20–2.09 (m, 2 H); 1.92–1.84 (m, 2 H); 1.53 (s, b-Me, Aib); 1.45 (s, Boc); 1.41
13
(s, b-Me, Aib). C-NMR (150 MHz, CDCl ): 175.88; 171.10; 171.08; 157.42; 157.17; 155.74; 138.56;
3
1
7
3
38.51; 129.59; 129.51; 129.28; 128.55; 128.52; 128.28; 128.20; 128.14; 128.02; 127.64; 127.43; 126.21; 81.25;
3.35; 73.09; 71.10; 69.58; 63.47; 56.72; 55.54; 52.30; 51.87; 50.76; 37.88; 37.47; 36.59; 36.01; 32.92; 30.75;
0.51; 30.36; 29.41; 29.02; 28.29; 28.22; 27.11; 25.12; 25.00; 23.82; 23.39. ESI-TOF-MS: 766.32 ([MþH] ;
þ
calc. 766.33).
Boc-Aib-Aib-Glu(OMe)-Dab(Tfa)-Phol-Bn (7). Yield: 65%. M.p. 128–1298. R 0.95, Rf2 0.95, Rf3
f1
1
0
.30, Rf4 0.20. IR (KBr): 3301, 1724, 1661, 1533. H-NMR (400 MHz, CDCl ): 8.04 (m, e-NH, Dab); 7.72
3
(
d, J¼6.0, NH); 7.68 (d, J¼8.0, NH); 7.38–7.10 (m, 10 arom. H, Phol, Bn); 7.03 (d, J¼8.8, NH); 6.72 (s,
NH, Aib); 5.13 (s, NH, Aib); 4.58–4.51 (m, CH , Bn); 4.43–4.28 (m, 2 a-CH); 4.21–4.10 (m, a-CH);
2
3
1
1
.64 (s, Glu(OMe)); 3.53–3.49 (m, 1 H); 3.36–3.23 (m, CH O, Phol); 3.05–2.98 (m, 1 H); 3.09–3.01 (m,
2
H); 2.79–2.69 (m, 1 H); 2.58–2.42 (m, 2 H); 2.39–2.27 (m, 1 H); 2.14–1.98 (m, 2 H); 1.96–1.88 (m,
H); 1.51 (s, b-Me, Aib); 1.44 (s, b-Me Aib, Boc); 1.41 (s, b-Me, Aib); 1.26 (s, b-Me, Aib). 1 C-NMR
3
(
150 MHz, CDCl ): 175.51; 175.04; 171.72; 171.41; 157.38; 157.14; 155.78; 138.41; 138.38; 129.44; 128.22;
3
1
3
28.10; 127.65; 127.38; 126.15; 81.52; 73.07; 70.94; 56.84; 56.68; 54.80; 51.64; 50.77; 37.76; 36.54; 30.94;
þ
0.24; 28.17; 27.64; 26.74; 25.78; 23.21. ESI-TOF-MS: 851.40 ([MþH] ; calc. 851.41).
Boc-Val-Aib-Aib-Glu(OMe)-Dab(Tfa)-Phol-Bn. Yield: 87%. M.p. 158–1598. R 0.95, Rf2 0.95, Rf3
f1
1
0
.30, R 0.25. IR (KBr): 3294, 1725, 1652, 1535. H-NMR (400 MHz, CDCl ): 7.99 (m, e-NH, Dab); 7.70–
f4
3
7
.62 (m, 2 NH); 7.52 (s, NH, Aib); 7.38–7.13 (m, 10 arom. H, Phol, Bn); 7.07 (d, J¼8.8, NH); 6.54 (s, NH,
Aib); 4.98 (br. d, J¼2.6, NH); 4.59–4.52 (m, CH , Bn); 4.46–4.25 (m, 2 a-CH); 4.17–4.07 (m, a-CH);
2
3
.75–3.68 (m, a-CH); 3.65 (s, Glu(OMe)); 3.56–3.46 (m, CH O, Phol); 3.36–3.21 (m, 2 H); 3.13–3.03
2
(
(
(
m, 1 H); 2.79–2.69 (m, 1 H); 2.57–2.48 (m, 2 H); 2.42–2.29 (m, 1 H); 2.22–2.13 (m, 1 H); 2.12–1.88
m, 3 H); 1.52 (s, b-Me, Aib); 1.50–1.47 (br. s, Boc); 1.46 (s, b-Me, Aib); 1.41 (s, b-Me, Aib); 1.03–0.97
13
m, 2 g-Me, Val). C-NMR (150 MHz, CDCl ): 176.14; 174.63; 173.08; 171.95; 171.54; 157.14; 156.86
3
1
6
2
38.57; 129.51; 129.27; 128.46; 128.22; 128.09; 127.94; 127.63; 127.32; 126.10; 81.71; 73.29; 73.07; 70.97;
9.55; 62.17; 57.12; 56.62; 54.83; 51.72; 51.62; 50.85; 37.76; 36.67; 31.02; 30.30; 29.66; 28.23; 27.48; 25.88;
þ
3.33; 23.25; 19.02; 18.39. ESI-TOF-MS: 950.43 ([MþH] ; calc. 950.41).
Boc-Pro-Val-Aib-Aib-Glu(OMe)-Dab(Tfa)-Phol-Bn. Yield: 71%. M.p. 84–858. R 0.95, R 0.95, R
f3
f1
f2
1
0
.25, Rf4 0.25. IR (KBr): 3303, 1723, 1657, 1533. H-NMR (400 MHz, CDCl ): 8.03 (m, e-NH, Dab); 7.81
3
(
8
4
s, NH, Aib); 7.76–7.68 (m, 2 NH); 7.35–7.16 (m, 10 arom. H, Phol, Bn); 7.14 (d, J¼7.2, NH); 7.10 (d, J¼
.8, NH); 6.38 (d, J¼5.2, NH); 4.59–4.52 (m, CH , Bn); 4.41–4.31 (m, 2 a-CH); 4.24–4.18 (m, a-CH);
2
2 2
.17–4.08 (m, a-CH); 3.96–3.89 (m, a-CH); 3.64 (s, Glu(OMe)); 3.60–3.46 (m, CH O, Phol, d-CH ,
Pro); 3.55–3.20 (m, 2 H); 3.15–3.03 (m, 1 H); 2.79–2.69 (m, 1 H); 2.56–2.45 (m, 2 H); 2.43–2.31 (m,
H); 2.27–2.20 (m, 1 H); 2.08–1.88 (m, 6 H); 1.57 (s, 2 b-Me, Aib); 1.52 (s, Boc); 1.45 (s, b-Me, Aib);
2
1
3
1
.43 (s, b-Me, Aib); 0.99 (d, J¼6.8, g-Me, Val); 0.93 (d, J¼6.8, g-Me, Val). C-NMR (150 MHz, CDCl ):
3
1
1
3
76.38; 175.32; 173.69; 173.10; 172.15; 171.48; 171.39; 157.33; 157.08; 156.01; 138.67; 129.55; 128.20;
28.05; 127.64; 127.28; 126.03; 81.92; 73.05; 71.04; 61.81; 60.40; 57.00; 54.81; 51.81; 51.56; 50.72; 47.51;
7.82; 36.64; 31.10; 30.36; 30.05; 28.83; 28.29; 27.55; 26.94; 25.90; 24.84; 23.20; 22.97; 19.08; 17.61. ESI-
þ
TOF-MS: 1047.51 ([MþH] ; calc. 1047.58).
Boc-Pro-Val-Aib-Aib-Glu(OMe)-Dab(Tfa)-Phol. Yield: 99%. M.p. 95–968. R 0.95, Rf2 0.95, Rf3
f1
1
0
.20, Rf4 0.10. IR (KBr): 3302, 1724, 1656, 1537. H-NMR (400 MHz, CDCl ): 7.88–7.81 (m, 2 NH); 7.77
3
(
s, NH, Aib); 7.65 (d, J¼7.6, NH); 7.26–7.13 (m, 5 arom. H, Phol); 7.09 (d, J¼7.2, NH); 7.04 (d, J¼8.8,
NH); 6.32 (br. d, J¼4.4, NH); 4.26–4.18 (m, a-CH); 4.17–4.06 (m, 2 a-CH); 4.04–3.95 (m, a-CH);
.89–3.82 (m, a-CH); 3.71–3.66 (m, 1 H of d-CH Pro); 3.58 (s, Glu(OMe)); 3.55–3.44 (m, CH OH,
3
2
2
Phol, 1 H of d-CH Pro); 3.39–3.29 (m, 1 H); 3.17–3.04 (m, 1 H); 2.89–2.82 (m, 1 H of b-CH Phol);
2
2
2
.80–2.73 (m, 1 H of b-CH Phol); 2.52–2.40 (m, 2 H); 2.36–2.21 (m, 2 H); 2.20–2.40 (m, 2 H); 1.98–
2
1
.83 (m, 5 H); 1.55 (s, 2 b-Me, Aib); 1.45 (s, Boc); 1.40 (s, b-Me, Aib); 1.39 (s, b-Me, Aib); 0.93 (d, J¼6.8,
13
g-Me, Val); 0.88 (d, J¼6.8, g-Me, Val). C-NMR (150 MHz, CDCl ): 177.46; 175.64; 173.83; 173.07;
3

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
1185
1
6
2
72.56; 171.79; 171.69; 157.44; 157.19; 156.07; 138.68; 129.65; 128.14; 126.14; 116.92; 115.01; 81.96; 63.76;
1.85; 60.58; 57.00; 56.97; 55.56; 53.29; 52.26; 51.66; 47.54; 36.88; 36.71; 30.98; 30.27; 30.08; 28.88; 28.32;
þ
7.44; 26.95; 25.82; 24.86; 23.25; 22.89; 19.06; 17.74. ESI-TOF-MS: 957.45 ([MþH] ; calc. 957.51).
Tfa-Aib-Pro-Aib-Ala-Aib-OBn. Yield: 99%. M.p. 221–2228. R 0.60, R 0.95, R 0.25. IR (film on
f1
f2
f3
1
KBr): 3334, 1739, 1707, 1664, 1627, 1528. H-NMR (400 MHz, CDCl ): 8.89 (s, NH); 7.51–7.49 (d, NH);
3
7
.44 (s, NH); 7.33 (m, 5 arom. H, Bn); 7.14 (s, NH); 5.11–5.10 (m, CH , Bn); 4.28 (m, a-CH); 4.20 (m, a-
2
CH); 3.93 (m, 1 H); 3.22–3.20 (m, 1 H); 2.38 (m, 1 H); 1.91–1.89 (m, 1 H); 1.73 (m, 2 H); 1.56 (s, 3 H);
1
.54 (s, 6 H); 1.53 (s, 3 H); 1.52 (s, 3 H); 1.45 (s, 3 H); 1.40–1.68 (d, 3 H). 13C-NMR (150 MHz, CDCl ):
3
1
1
2
74.81; 174.20; 172.98; 172.30; 172.06; 157.40; 157.14; 136.13; 128.31; 127.70; 121.07; 118.52; 116.61;
14.70; 112.80; 66.42; 64.74; 57.79; 57.00; 55.91; 49.72; 48.82; 28.68; 27.25; 26.33; 25.47; 25.14; 24.33; 23.72;
þ
3.28; 17.05. ESI-TOF-MS: 628.26 ([MþH] ; calc. 628.29).
t
Z-Dab(Tfa)-Aib-Gly-Leu-Aib-O Bu. Yield: 82%. M.p. 91–928. R 0.50, R 0.90, R 0.25. IR (KBr):
f1
f2
f3
1
3
374, 1716, 1660, 1538. H-NMR (400 MHz, CDCl ): 7.90 (m, NH); 7.47 (m, NH); 7.39–7.28 (m, 5 arom.
3
H, Z); 6.98 (m, NH); 7.06 (m, NH); 6.91 (s, NH); 6.44 (d, NH); 5.10 (2d, CH , Z); 4.35 (m, a-CH, Leu);
2
4
1
.08–3.93 (m, a-CH Gly, a-CH of Dab); 3.71–3.63 (m, a-CH of Gly); 3.62–3.50 (m, 1 H); 3.41–3.28 (m,
H); 2.23–2.11 (m, 1 H); 2.01–1.89 (m, 1 H); 1.75–1.59 (m, 3 H); 1.64 (s, b-Me, Aib); 1.45 (s, b-Me,
t
Aib); 1.42 (s, BuO); 1.38 (s, b-Me, Aib); 0.91 (d, J¼5.6, 1 d-Me, Leu); 0.86 (d, J¼5.6, 1 d-Me, Leu).
13
C-NMR (150 MHz, CDCl ): 175.18; 173.47; 172.46; 172.42; 172.28; 170.69; 158.02; 157.73; 157.12;
3
1
3
36.17; 128.60; 128.34; 127.77; 116.89; 114.99; 81.33; 67.32; 57.00; 56.70; 53.33; 52.85; 43.45; 39.88; 36.60;
þ
0.08; 27.78; 24.84; 24.77; 24.65; 23.01; 21.19. ESI-TOF-MS: 745.43 ([MþH] ; calc. 745.34).
t
Z-Aib-Dab(Tfa)-Aib-Gly-Leu-Aib-O Bu (6). Yield: 86%. M.p. 200–2018. R 0.50, R 0.80, R 0.25.
f1
f2
f3
1
IR (KBr): 3365, 3309, 1705, 1650, 1538. H-NMR (400 MHz, CDCl ): 7.97 (m, NH); 7.63 (d, J¼7.4, NH);
3
7
.58 (s, NH); 7.46 (d, J¼7.9, NH); 7.39–7.28 (m, 5 arom. H, Z); 7.16 (m, NH); 6.79 (s, NH); 5.30 (s, NH);
5
.06 (m, CH , Z); 4.42–4.36 (m, a-CH, Leu); 4.26–4.14 (m, a-CH of Gly, a-CH of Dab); 3.70–3.64 (m,
2
a-CH Gly); 3.49–3.40 (m, 1 H); 3.38–3.30 (m, 1 H); 2.17–2.01 (m, 2 H); 1.82–1.63 (m, 3 H); 1.59 (s, b-
Me, Aib); 1.52 (s, b-Me, Aib); 1.50 (s, b-Me, Aib); 1.48 (s, b-Me, Aib); 1.46 (s, b-Me, Aib); 1.44 (s, b-Me,
t
13
Aib); 1.43 (s, BuO); 0.93 (d, J¼6.3, 1 d-Me, Leu); 0.90 (d, J¼6.3, 1 d-Me, Leu). C-NMR (150 MHz,
CDCl ): 175.53; 174.60; 172.00; 171.87; 170.47; 157.26; 155.84; 135.74; 134.08; 128.64; 128.46; 127.91;
3
9
2
7.05; 94.98; 81.30; 67.37; 57.26; 57.08; 52.05; 51.84; 43.21; 39.72; 37.00; 29.07; 27.79; 27.75; 26.10; 25.63;
þ
5.28; 24.79; 24.61; 24.45; 23.91; 23.03; 21.65. ESI-TOF-MS: 830.43 ([MþH] ; calc. 830.40).
t
Z-Glu(OMe)-Aib-Dab(Tfa)-Aib-Gly-Leu-Aib-O Bu. Yield: 80%. M.p. 88–898. R 0.45, R 0.85, R
f1
f2
f3
1
0
.20. IR (KBr): 3396, 1722, 1658, 1533. H-NMR (400 MHz, CDCl ): 8.07 (m, NH); 7.57 (d, J¼7.0, NH);
3
7.55 (s, NH); 7.49 (d, J¼7.6, NH); 7.41–7.31 (m, 5 arom. H, Z); 6.98 (s, NH); 6.68 (s, NH); 6.26 (d, NH);
5
.09 (m, CH , Z); 4.42–4.31 (m, a-CH, Leu); 4.09–4.03 (m, a-CH, Gly); 3.99–3.89 (m, a-CH of Dab, a-
2
CH of Glu(OMe)); 3.72 (s, Glu(OMe)); 3.69–3.61 (m, a-CH, Gly); 3.54–3.37 (m, 2 H); 2.63–2.55 (m,
1
H); 2.52–2.43 (m, 1 H); 2.20–1.95 (m, 4 H); 1.80–1.67 (m, 3 H); 1.59 (s, b-Me, Aib); 1.62 (s, b-Me,
t
Aib); 1.47 (s, 2 b-Me, Aib); 1.45 (s, b-Me, Aib); 1.41 (s, BuO); 1.39 (s, b-Me, Aib); 0.92 (d, J¼6.3, 1 d-Me,
13
Leu); 0.89 (d, J¼6.3, 1 d-Me, Leu). C-NMR (150 MHz, CDCl ): 175.60; 175.45; 173.95; 173.36; 172.53;
3
1
5
2
72.40; 172.04; 170.51; 157.78; 157.53; 157.47; 135.81; 128.69; 128.46; 127.65; 116.83; 114.92; 81.15; 67.53;
7.24; 56.93; 56.73; 56.42; 52.97; 52.33; 52.12; 43.48; 39.46; 36.97; 30.39; 29.80; 27.79; 27.77; 27.74; 25.88;
þ
5.16; 24.87; 24.81; 24.52; 24.43; 24.36; 23.00; 21.43. ESI-TOF-MS: 973.51 ([MþH] ; calc. 973.51).
t
Z-Ala-Glu(OMe)-Aib-Dab(Tfa)-Aib-Gly-Leu-Aib-O Bu. Yield: 77%. M.p. 94–958. R 0.45, R
f1
f2
0
.85, Rf3 0.15. HPLC: tR 10.68 min (flow rate, 1 ml/min; gradient, 40–90% B in 25 min; C Vydac
4
1
column). IR (KBr): 3370, 1722, 1659, 1533. H-NMR (400 MHz, CDCl ): 8.02 (m, NH, Glu(OMe)); 7.67
3
(
m, e-NH, Dab); 7.64 (s, NH, Aib); 7.52 (d, J¼7.7, NH, Leu); 7.46 (d, J¼6.6, a-NH, Dab); 7.40 (s, NH,
Aib); 7.37–7.29 (m, 5 arom. H, Z); 7.06 (s, NH, Aib); 5.69 (d, NH, Ala); 5.14 (m, CH , Z); 4.40–4.30 (m,
2
a-CH, Leu); 4.27–4.17 (m, a-CH, Dab); 4.08–3.91 (m, a-CH of Ala, a-CH of Glu(OMe), 1 H of a-CH2,
Gly); 3.78–3.72 (m, 1 H of a-CH , Gly); 3.69 (s, Glu(OMe)); 3.52–3.37 (m, b-CH , Dab); 2.59–2.45 (m,
2
2
b-CH , Glu); 2.20–2.10 (m, 1 H of g-CH , Dab); 2.09–1.98 (m, 1 H of g-CH of Dab: g-CH2 of
2
2
2
Glu(OMe)); 1.82–1.63 (m, b-CH and g-CH, Leu); 1.51 (s, b-Me, Aib); 1.50 (s, b-Me, Aib); 1.49 (s, b-Me,
2
t
Aib); 1.48 (m, b-Me, Aib); 1.43 (m, 2 b-Me of Aib, b-Me of Ala); 1.39 (s, BuO); 0.91 (d, J¼5.6, 1 d-Me,
1
3
Leu); 0.88 (d, J¼7.0, 1 d-Me, Leu). C-NMR (150 MHz, CDCl ): 175.63; 175.55; 175.04; 173.50; 172.70;
3
1
72.00; 171.87; 171.77; 170.13; 157.26; 157.08; 135.94; 128.63; 128.44; 127.79; 80.85; 67.53; 57.21; 57.14;

1
186
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
5
2
1
6.55; 56.29; 56.02; 52.89; 52.80; 52.70; 52.33; 52.17; 43.63; 39.35; 36.83; 30.57; 30.16; 27.82; 27.72; 26.74;
6.69; 25.10; 24.92; 24.59; 24.34; 23.35; 23.29; 23.08; 22.89; 21.46; 20.89; 17.37; 17.32. ESI-TOF-MS:
þ
044.52 ([MþH] ; calc. 1044.56).
Z-Ala-Glu(OMe)-Aib-Dab(Tfa)-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Glu(OMe)-Glu(OMe)-Phol.
1
Yield: 30%. IR (film on KBr): 3321, 1737, 1656, 1533. H-NMR (600 MHz, CDCl ): 8.33 (m, 1 H); 7.94
3
(
7
(
m, 1 H); 7.92–7.91 (m, 2 H); 7.87–7.85 (m, 2 H); 7.80 (m, 1 H); 7.73 (m, 1 H); 7.69–7.67 (m, 2 H); 7.60–
.56 (m, 4 H); 7.49–7.47 (m, 2 H); 7.40 (m, 2 H); 7.36–7.34 (m, 7 H); 7.23 (s, 1 H); 7.22 (s, 1 H); 7.19–7.14
m, 2 H); 5.14 (m, 2 H); 4.43–4.16 (m, 5 H); 4.09–4.01 (m, 4 H); 3.89–3.88 (m, 1 H); 3.76–3.74 (m,
1 H); 3.72 (m, 1 H); 3.69–3.67 (m, 4 H); 3.66 (m, 1 H); 3.64 (m, 2 H); 3.634 (m, 1 H); 3.627 (m, 3 H);
3.60 (m, 3 H); 3.56–3.51 (m, 3 H); 2.95–2.77 (m, 3 H); 2.75–2.65 (m, 1 H); 2.51 (m, 4 H); 2.38–2.36 (m,
2 H); 2.35–2.26 (m, 4 H); 2.25–2.15 (m, 6 H); 2.12–1.98 (m, 9 H); 1.95–1.66 (m, 21 H); 1.64 (m, 1 H);
1.61 (s, 3 H); 1.57 (s, 3 H); 1.55 (s, 6 H); 1.53 (s, 6 H); 1.52 (s, 3 H); 1.50 (s, 3 H); 1.49 (s, 3 H); 1.48 (s,
6
H); 1.45 (s, 6 H); 1.44 (s, 3 H); 1.41 (s, 3 H); 1.03 (m, 3 H); 0.97–0.92 (m, 6 H); 0.90–0.89 (m, 3 H).
1
3
C-NMR (150 MHz, CDCl ): 177.60; 177.39; 176.35; 175.81; 174.58; 174.11; 174.06; 166.78; 159.85;
3
1
6
2
37.15; 136.81; 135.80; 135.42; 129.62; 128.70; 128.59; 128.20; 123.23; 122.89; 112.98; 68.10; 63.51; 63.41;
1.27; 56.40; 51.72; 49.43; 49.17; 44.57; 44.32; 43.11; 33.84; 33.16; 30.98; 30.91; 30.85; 30.19; 29.66; 29.59;
9.28; 29.25; 27.03; 26.34; 26.31; 26.26; 25.65; 18.11; 17.98; 17.76; 17.43; 17.29. ESI-TOF-MS: 1774.81
þ
2þ
(
[MþH] ); 887.40 ([Mþ2 H] ; calc. 887.40).
Z-Ala-Glu(OMe)-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Glu(OMe)-Dab(Tfa)-Phol. Yield:
1
3
0%. IR (film on KBr): 3321, 1727, 1655, 1533. H-NMR (600 MHz, CDCl ): 8.33 (m, 1 H); 7.93–7.91
3
(
m, 5 H); 7.82–7.80 (m, 1 H); 7.76 (t, 1 H); 7.72 (s, 1 H); 7.58 (s, 1 H); 7.56–7.55 (d, 1 H); 7.52–7.51 (d,
1
2
2
1
H); 7.42–7.40 (m, 2 H); 7.36–7.34 (m, 7 H); 7.22–7.19 (m, 5 H); 7.16–7.15 (m, 2 H); 5.15–5.13 (m,
H); 4.41–4.37 (m, 1 H); 4.31–4.26 (m, 3 H); 4.06–3.96 (m, 6 H); 3.78–3.76 (m, 2 H); 3.72–3.70 (m,
H); 3.68–3.65 (m, 7 H); 3.64–3.59 (m, 6 H); 3.55–3.54 (m, 1 H); 3.39–3.37 (m, 1 H); 3.21–3.16 (m,
H); 2.82–2.79 (m, 2 H); 2.68–2.64 (m, 1 H); 2.50 (m, 5 H); 2.35–2.17 (m, 21 H); 2.11–2.06 (m, 5 H);
1
6
.97–1.93 (m, 6 H); 1.80–1.79 (m, 2 H); 1.61 (s, 3 H); 1.56 (s, 3 H); 1.54 (s, 6 H); 1.53 (s, 3 H); 1.50 (s,
H); 1.49 (s, 3 H); 1.48 (s, 3 H); 1.47 (s, 6 H); 1.45 (s, 6 H); 1.44 (s, 3 H); 1.42 (s, 3 H); 1.39 (m, 5 H);
13
1.07–0.88 (m, 12 H). C-NMR (150 MHz, CDCl ): 177.53; 177.41; 176.34; 176.18; 174.69; 173.49; 173.17;
3
1
1
3
2
8
72.50; 170.42; 157.27; 136.28; 129.60; 128.75; 128.67; 128.45; 128.34; 128.16; 128.11; 127.71; 127.60;
26.24; 67.32; 64.14; 63.50; 56.73; 56.42; 56.21; 53.46; 53.19; 52.93; 52.43; 51.60; 49.24; 44.46; 40.13; 39.82;
6.95; 36.49; 30.72; 30.62; 29.29; 29.09; 27.83; 27.03; 26.99; 26.21; 25.56; 25.16; 24.62; 24.40; 23.38; 23.12;
þ
3.03; 22.62; 22.55; 21.11; 20.11; 19.58; 19.12; 18.97; 17.35; 17.24. ESI-TOF-MS: 1730.95 ([MþH] );
2
þ
65.93 ([Mþ2 H] ; calc. 866.01).
Tfa-Aib-Pro-Aib-Ala-Aib-Ala-Glu(OMe)-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Glu(OMe)-
a
Glu(OMe)-Phol (N TfaAlm’; 2). Yield: 51%. HPLC: t 22.58 min (flow rate: 1 ml/min; gradient: 60–
R
1
1
00% B in 30 min; C18 Agilent Zorbax column). IR (film on KBr): 3328, 1740, 1666, 1535. H-NMR
(
600 MHz, CDCl
3
): 9.92 (s, 1 H); 8.25 (s, 1 H); 8.08 (m, 1 H); 7.98 (s, 1 H); 7.93 (m, 1 H); 7.88–7.85 (m,
3
H); 7.79 (s, 1 H); 7.65–7.50 (m, 5 H); 7.39–7.38 (d, 1 H); 7.30–7.28 (m, 2 H); 7.24–7.21 (m, 2 H); 7.16–
7.14 (m, 1 H); 7.045 (s, 1 H); 4.41 (m, 1 H); 4.30–4.28 (m, 1 H); 4.19 (m, 2 H); 4.14–4.13 (m, 1 H); 4.07–
3.97 (m, 5 H); 3.88 (m, 1 H); 3.81 (m, 2 H); 3.66 (m, 2 H); 3.64 (s, 3 H); 3.63 (s, 3 H); 3.61 (s, 3 H); 3.55–
3.49 (m, 2 H); 3.29 (m, 1 H); 2.82–2.81 (d, 2 H); 2.73–2.66 (m, 2 H); 2.56–2.52 (m, 2 H); 2.40 (m, 10 H);
2.32–2.06 (m, 12 H); 1.96–1.89 (m, 4 H); 1.80–1.72 (m, 3 H); 1.62–1.60 (m, 10 H); 1.55 (m, 15 H); 1.53
(
1
m, 9 H); 1.51 (s, 3 H); 1.50 (m, 6 H); 1.49 (s, 3 H); 1.57 (m, 6 H); 1.12–1.11 (d, 3 H); 1.08–1.07 (d, 3 H);
.00–0.99 (d, 3 H); 0.94–0.91 (m, 9 H). 13C-NMR (150 MHz, CDCl ): 177.53; 177.35; 176.60; 176.51;
3
1
1
5
3
2
1
76.23; 176.18; 175.31; 174.82; 174.08; 173.97; 173.87; 173.69; 173.31; 173.27; 173.11; 173.08; 172.89;
70.94; 157.88; 157.63; 138.54; 129.52; 128.10; 126.07; 64.71; 63.97; 63.53; 63.39; 57.87; 56.75; 56.72; 56.41;
6.35; 56.12; 56.02; 55.08; 53.18; 52.82; 52.68; 52.44; 51.56; 51.47; 51.31; 49.36; 49.30; 44.52; 40.41; 37.21;
0.78; 30.74; 30.72; 29.67; 29.22; 29.09; 28.99; 28.92; 26.97; 26.94; 26.87; 26.47; 26.38; 26.21; 26.01; 25.92;
5.57; 25.24; 24.53; 23.50; 23.30; 22.99; 22.84; 22.67; 22.58; 22.47; 21.21; 20.55; 20.14; 19.26; 18.80; 16.76;
þ
2þ
6.60. ESI-TOF-MS: 2062.12 ([MþH] ; calc. 2062.10); 1031.54 ([Mþ2 H] ).
Ac-Aib-Pro-Aib-Ala-Aib-Ala-Glu(OMe)-Aib-Dab(Tfa)-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-
Glu(OMe)-Glu(OMe)-Phol (DabTfa9Alm’; 3). Yield: 52%. HPLC: tR 16.52 min (flow rate, 1 ml/min;
1
gradient, 40–90% B in 30 min; C Vydac column). IR (film on KBr): 3317, 1738, 1655, 1535. H-NMR
4

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
1187
8
10
11
13
(
600 MHz, CD OH): 8.30–8.27 (m, 3 NH, Aib , Aib , Gly ); 8.18 (s, NH, Aib ); 8.09–8.08 (d, NH,
3
12
7
6
5
Leu ); 8.05–8.04 (d, NH, Glu(OMe) ); 7.98–7.97 (d, NH, Ala ); 7.96 (s, NH, Aib ); 7.90–7.87 (m, 3 NH,
Aib , Glu(OMe) , Glu(OMe) ); 7.80 (s, NH, Aib ); 7.78–7.77 (d, NH, Dab(Tfa) ); 7.62 (s, NH, Aib );
.59–7.58 (m, 2 NH, Val , Ala ); 7.57 (s, NH, Aib ); 7.34–7.31 (m, NH, H Phol); 7.25–7.22 (t, 2 H
Phol); 7.17–7.15 (t, 2 H Phol); 4.48–4.40 (m, a-CH Leu ); 4.40–4.38 (m, a-CH Pro ); 4.28–4.25 (m, a-
CH Pro ); 4.21–4.14 (m, 2 a-CH Glu(OMe) , Phol); 4.10–4.09 (m, a-CH Ala ); 4.06–4.03 (m, 2 a-CH
Glu(OMe) , Ala ); 3.99–3.95 (m, 1 H of a-CH Gly , 2 a-CH Dab(Tfa) , Glu(OMe) , 1 H of d-CH
Pro ); 3.92–3.89 (m, 1 H of d-CH Pro ); 3.77–3.76 (m, 1 H of a-CH Gly , a-CH Val , 1 H of d-CH
Pro ); 3.67–3.66 (m, CH OH Phol, 2 Glu(OMe)); 3.63 (s, Glu(OMe)); 3.53–3.47 (m, 1 H of d-CH Pro ,
1
18
19
17
9
3
15
4
16
7
p
m
12
14
o
2
19
6
18
4
11
9
7
2
2
2
14
11
15
2
2
2
14
2
2
2
9
g-CH Dab(Tfa) ); 2.98–2.94 (dd, 1 H of b-CH Phol); 2.77–2.69 (m, 1 H of b-CH Phol, 1 H of g-CH
2
2
2
2
7,18,19
18
7,19
Glu(OMe)
2
); 2.65–2.63 (m, 1 H of g-CH (OMe) ); 2.53–2.50 (m, 1 H of g-CH (OMe) ); 2.37–
.27 (m, 7 H, 1 b-CH Pro , Pro , Val , Glu(OMe)
Glu(OMe) , Dab(Tfa) ); 2.10 (m, 2 H, 1 H of g-CH Pro , Pro ); 2.08 (s, Ac); 2.05–2.04 (m, 3 H, 1 H
of b-CH Glu(OMe) , 1 H of g-CH Pro , Pro ); 1.92 (m, g-CH, 1 H of b-CH Leu ); 1.84–1.79 (m, 2 H,
H of b-CH Pro , Pro ); 1.64–1.49 (m, 55 H, 16 Me of 8 Aib, 2 Me of 2 Ala, 1 H of b-CH , Leu ), 1.09–
2 2
.08 (d, 1 g-Me, Val ); 0.98–0.92 (m, 1 g-Me of Val , 2 d-Me of Leu ). C-NMR (150 MHz, CDCl ):
2
2
2
14
15
7,18,19
9
, Dab(Tfa) ); 2.21–2.15 (m, 3 H, 1 b-CH
7,18
9
2
14
2
19
2
14
12
2
2
2
2
14
12
1
1
1
1
1
(
6
5
15
15
12
13
3
11
86.55; 179.03; 177.60 (C¼O, Gly ); 177.10 (C¼O, Aib); 177.00; 176.90 (C¼O, Aib); 176.30 (C¼O, Aib);
4 18 14 12 2
75.90 (2 C¼O, Ala , Glu(OMe) ); 175.00 (C¼O, Pro ) 174.40 (2 C¼O, Leu , Pro ); 174.25 (C¼O, Aib);
15
13
19
73.9 (C¼O, Val ); 173.6 (C¼O, Aib ); 173.1 (C¼O, Glu(OMe) ); 172.38; 171.30 (C¼O, Ac); 138.53
C(1), Phol); 129.18 (C(4), Phol); 127.78 (C(2), Phol); 125.78 (C(3), Phol); 118.61; 107.00; 106.91; 91.39;
a
2
a
14
a
15
a
13
a
7
4.39 ( C, Pro ); 63.13 ( C, Pro ); 62.83 ( C, Val ); 56.85 ( C, Aib ); 56.75; 56.62 ( C, Glu(OMe) );
a 17 a a a a
5
3
18
6.48 ( C, Aib ); 56.36; 56.23 ( C, Aib ); 56.17; 56.10 ( C, Aib ); 55.69 ( C, Glu(OMe) ); 54.42 ( C,
19
a
a
12
b
d
14
d
Glu(OMe) ); 53.14 ( C, Phol); 52.23 ( C, Leu ); 50.56 ( CH OH Phol); 49.22 (1 C, Pro ); 48.86 (1 C,
Pro ); 43.55 ( C, Gly ); 43.06; 40.16 ( C, Leu ); 36.76 (1 C, Phol); 36.42 ( C, Dab(Tfa) ); 35.63; 30.18
2
2
a
11
b
12
b
b
9
b
7,19
b
15
b
7
g
14
g
2
(
(
C, Glu(OMe) ); 29.22 ( C, Val ); 29.02 (1 C, Glu(OMe) ); 28.83 ( C, Pro ); 28.48 ( C, Pro ); 28.02
g
9
b b b b b
C, Dab(Tfa) ); 26.11 (1 C, Aib); 26.08 (2 C, Aib); 25.78 (1 C, Aib); 25.73 (1 C, Aib); 25.73 (1 C,
b
b
b
b
b
Aib); 25.46 (1 C, Aib); 25.41 (1 C, Aib); 25.35 (1 C, Aib); 25.31 (1 C, Aib); 22.51 (2 C, Aib); 22.14 (1
C, Aib); 22.00 (2 C, Aib); 21.87 (1 C, Aib); 21.83 (1 C, Aib); 21.75 (1 C of Aib, 1 C of Ala ); 21.71 (1
C, Ala ); 21.02 ( C, Ac); 20.04 (1 C, Leu ); 18.96 (1 C, Val ); 18.10 (1 C, Val ). ESI-TOF-MS:
b
b
b
b
b
b
4
b
6
a
d
12
g
15
g
15
þ
2þ
2
105.50 ([MþH] ; calc. 2105.11); 1053.10 ([Mþ2 H] ).
Ac-Aib-Pro-Aib-Ala-Aib-Ala-Glu(OMe)-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Glu(OMe)-
Dab(Tfa)-Phol (DabTfa19Alm’; 4). Yield: 40%. HPLC: t 23.48 min (flow rate, 1 ml/min; gradient, 40–
R
1
9
0% B in 30 min; C Vydac column). IR (film on KBr): 3320, 1724, 1658, 1534. H-NMR (600 MHz,
4
CDCl ): 8.06–8.04 (t, 1 H); 7.99–7.92 (m, 7 H); 7.82–7.80 (m, 4 H); 7.64–7.41 (m, 9 H); 7.30–7.29 (m,
3
3 H); 7.23–7.21 (m, 3 H); 7.17–7.16 (m, 2 H); 4.49–4.34 (m, 2 H); 4.31–4.28 (m, 1 H); 4.23–4.20 (m,
3 H); 4.05–3.96 (m, 6 H); 3.90–3.87 (m, 2 H); 3.81–3.806 (m, 1 H); 3.75–3.72 (m, 3 H); 3.67–3.65 (m,
3 H); 3.635 (s, 3 H); 3.363 (s, 3 H); 3.57–3.49 (m, 3 H); 3.42–3.39 (m, 2 H); 3.27–3.24 (m, 1 H); 2.98–
2.81 (m, 2 H); 2.71–2.64 (m, 2 H); 2.85–2.52 (m, 2 H); 2.43–2.17 (m, 14 H); 2.10–2.07 (m, 6 H); 1.98–
1
0
1
1
7
4
2
2
.91 (m, 10 H); 1.61–1.44 (m, 81 H); 1.30–1.12 (d, 3 H); 1.08–1.07 (d, 3 H); 1.01–1.00 (d, 3 H); 0.95–
13
.92 (m, 9 H). C-NMR (150 MHz, CDCl ): 191.26; 189.92; 186.20; 182.09; 182.00; 181.86; 181.79;
3
76.01; 175.30; 174.14; 173.98; 173.91; 173.71; 173.47; 173.41; 173.30; 173.05; 173.01; 170.63; 147.37;
44.55; 144.04; 138.83; 138.51; 136.12; 135.19; 134.16; 129.64; 128.15; 128.10; 126.18; 106.25; 82.49;
3.69; 73.62; 64.63; 63.55; 56.77; 56.70; 56.57; 56.46; 53.08; 52.85; 51.57; 49.44; 49.40; 49.21; 48.69; 47.65;
6.40; 45.86; 45.29; 33.07; 30.76; 29.99; 29.89; 27.18; 26.97; 26.47; 26.30; 25.50; 23.35; 23.09; 22.74; 22.61;
þ
2.54; 21.23; 19.29; 18.95; 16.80; 16.59. ESI-TOF-MS: 2061.30 ([MþH] ; calc. 2061.12); 1031.12 ([Mþ
2
þ
H] ).
X-Ray Diffraction Analysis. Colorless crystals of Ac-Aib-Pro-Aib-Ala-Aib-OBn (5), Z-Aib-
t
Dab(Tfa)-Aib-Gly-Leu-Aib-O Bu (6), and Boc-(Aib) -Glu(OMe)-Dab(Tfa)-Phol(Bn) (7) were grown
2
i
by slow evaporation from CH Cl , a MeOH/Et O mixture, and an PrOH/MeOH mixture, resp. Data
2
2
2
collection was performed on a Philips PW1100 four-circle diffractometer in the q–2q scan mode using
graphite-monochromated CuK radiation. The structures were solved by direct methods of the SIR 2002
a
2
program [99], and refined by full-matrix block least-squares procedures on F , using all data, by
application of the SHELXL 97 program [100], with anisotropic displacement parameters for all non-H-

1
188
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
atoms, and allowing the positional parameters and the anisotropic displacement parameters to refine at
alternate cycles. Evaluation and modelling of disordered parts were performed by exploitation of three-
dimensional difference electron-density maps available through the SHELXLe program [101]. Ph Rings,
whenever occurring (Phol side chain, Z and Bn protecting groups), were constrained to the idealized
geometry. All H-atoms were calculated at idealized positions and refined using a riding model.
The asymmetric unit of 5 is composed of one peptide molecule and one co-crystallized CH Cl
2
2
molecule. Two independent peptide molecules characterize the asymmetric unit of 6. The Dab side chain
g
d
in molecule 6B (Fig. 7) is disordered. Its C and N atoms, and the C¼O O-atom of the Tfa protecting
group as well, were refined on two sets of positions, each with a population parameter of 0.50. In each of
the two independent molecules, the rotationally disorder CF moiety of the Tfa group was refined on two
3
sets of positions, each with a population parameter of 0.50. Restraints were applied to the bond distances
and bond angles involving atoms of the disordered parts, as well as to their anisotropic displacement
parameters, the latter to approach an isotropic behavior. In 7, the CF moiety of the Tfa group showed
3
rotational disorder. It was refined on two sets of positions, with population parameters of 0.60 and 0.40,
resp. Restraints were applied to the bond distances and bond angles involving atoms of the disordered
parts.
Relevant crystallographic data and structure refinement parameters are avaiable as Supporting
Material. CCDC-942778–942780 contain the crystallographic data for this article. These data can be
obtained from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
Antimicrobial Activity Assays. Peptide antibacterial activities were tested against Gram-positive and
Gram-negative bacteria by the standardized disk-diffusion method of Bauer et al. [102] according to the
National Committee for Clinical Laboratory Standards (NCCLS)-recommended MuellerꢀHilton agar
culture medium (pH 7.2–7.4) [103] and 5-mm diameter paper disks (Macherey-Nagel 615, Germany).
Disks were prepared by using blank paper disks previously autoclaved and saturated with the peptide
soln. The peptide samples were dissolved in DMSO such as to give a 10 mg/ml soln. The peptide-
impregnated disks were placed aseptically onto inoculated plates and incubated at 378. The results were
collected after 24 h incubation by measuring the inhibition zones (in mm). The antifungal activity was
tested against Candida albicans (ATCC 1028, where ATCC stands for American Type Culture Collection)
strain grown on the Sabouraud dextrose agar culture medium [104]. The antibacterial activities were
assayed against the reference bacterial strains and clinical isolates (cf. Table 4).
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