Attempts toward the synthesis of the peptaibol antiamoebin by using the 'azirine/oxazolone method'

Article abstract of DOI:10.1002/cbdv.201200386

The two segments, 1-9 and 10-16, of the peptaibol antibiotic antiamoebin I, i.e., the nonapeptide Ac-Phe-Aib-Aib-Aib-D,L-Iva-Gly-Leu-Aib-Aib-OH (15) and the heptapeptide Z-Hyp-Gln-D,L-Iva-Hyp-Aib-Pro-Pheol (34), have been prepared as mixtures of the epimers containing D,L-Iva. All α,α-disubstituted α-amino acids were introduced by the 'azirine/oxazolone method', in which amino or peptide acids are coupled with the corresponding 2H-azirin-3-amines, followed by selective hydrolysis of the terminal amide bond. The amino acids Hyp and Gln were introduced as Z-protected⁴) (2S,4R)-4-(tert-butoxy) proline (19) and methyl N-[bis(4-methoxyphenyl)methyl]glutamine (26). Coupling of peptide segments was achieved via the 'mixed anhydride' method, the DCC/HOBt or TBTU/HOBt strategy. The crystal structure of the segment 6-9 was determined by X-ray crystallography and displayed the presence of a β-turn conformation. Copyright

Full text of DOI:10.1002/cbdv.201200386

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Attempts toward the Synthesis of the Peptaibol Antiamoebin I by Using the
ꢀAzirine/Oxazolone Methodꢁ1)
by Pia Blaser2), Werner Altherr3)†, Anthony Linden, and Heinz Heimgartner*
Organisch-chemisches Institut der Universitꢀt Zꢁrich, Winterthurerstrasse 190, CH-8057 Zꢁrich
(phone: þ41-44-6354282; fax: þ41-44-6356836; e-mail: heimgart@oci.uzh.ch)
The two segments, 1–9 and 10–16, of the peptaibol antibiotic antiamoebin I, i.e., the nonapeptide
Ac-Phe-Aib-Aib-Aib-d,l-Iva-Gly-Leu-Aib-Aib-OH (15) and the heptapeptide Z-Hyp-Gln-d,l-Iva-
Hyp-Aib-Pro-Pheol (34), have been prepared as mixtures of the epimers containing d,l-Iva. All a,a-
disubstituted a-amino acids were introduced by the ꢂazirine/oxazolone methodꢃ, in which amino or
peptide acids are coupled with the corresponding 2H-azirin-3-amines, followed by selective hydrolysis of
the terminal amide bond. The amino acids Hyp and Gln were introduced as Z-protected4) (2S,4R)-4-
(tert-butoxy)proline (19) and methyl N-[bis(4-methoxyphenyl)methyl]glutamine (26). Coupling of
peptide segments was achieved via the ꢂmixed anhydrideꢃ method, the DCC/HOBt or TBTU/HOBt
strategy. The crystal structure of the segment 6–9 was determined by X-ray crystallography and
displayed the presence of a b-turn conformation.
1. Introduction. – The reaction of 2H-azirine-3-amines of type 1 with N-protected a-
amino acids or peptides proved to be useful for the synthesis of peptides containing a,a-
disubstituted a-amino acids, e.g., a-aminoisobutyric acid (Aib) [2]. It has been shown
that the so-called ꢂazirine/oxazolone methodꢃ [3] is an attractive tool for the
preparation of peptaibol segments [4] and peptaibols [5]. Furthermore, a solid-phase
protocol of the ꢂazirine/oxazolone methodꢃ has been developed [6].
Peptaibols are naturally occurring linear, bioactive peptides, produced by
filamentous fungi such as Trichoderma and Gliocladium (with teleomorphs in
Hypocrea), Emericillopsis, Stilbella, and Acremonium. According to definition, these
N-acylated peptides contain Aib, frequently together with other a,a-disubstituted a-
amino acids, as well as a C-terminal b-amino alcohol such as Pheol, Valol, or Leuol [7–
9]. They attracted increasing interest because of their biological activities as antifungal,
antibacterial, and anticancer compounds [10]. Recently, it has been shown that they are
synthesized by non-ribosomal peptide synthetases [11]. These oligopeptides are well-
known to occur in rather stable helical conformations, i.e., a-helix, 310-helix, and b-bend
ribbon spiral, as a result of the high content of Aib residues [12] (for some exceptions,
see [13]). Based on these structural properties, peptaibols are ꢂmembrane-activeꢃ
1
)
)
)
Presented partly at the ꢂ22nd European Peptide Symposiumꢃ, Interlaken, Switzerland, 1992 [1].
2
3
In part from the Diploma thesis of P. B., Universitꢀt Zꢁrich, 1990.
†
Part of the Ph.D. thesis of W. A., Universitꢀt Zꢁrich, 1994. Dr. Werner Altherr passed away on
October 4, 2012.
For all abbreviations, cf. the Exper. Part.
4
)
ꢄ 2013 Verlag Helvetica Chimica Acta AG, Zꢁrich

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
921
polypeptide antibiotics, which, after aggregation to bundles, form ꢂion-channelsꢃ
through biological membranes [7a][14][15].
The natural peptaibol Antiamoebin was reported initially as a mixture of two
compounds (antiamoebins I and II (AAM I and AAM II, resp.); Fig. 1) in a ratio of ca.
98 :2. The antibiotic activity of this peptaibol was described for the first time by
Thirumalachar [16], and the primary structure was established stepwise by Vaidya
et al., Pandey et al., and Brꢀckner et al. [17]. It was shown that antiamoebin consists of
mixtures of up to 16 microheterogeneous 16-mer peptaibols with varying compositions
depending on the source of the natural material [17e]. The crystal structure of AAM I
has been solved by X-ray crystallography independently by Karle et al. and Snook et al.
[18], establishing the presence of a right-handed helix. On the other hand, it was
reported that the N-terminus of AAM I in solution (DMSO or MeOH) forms a left-
handed helical structure [14b][19]. A recent study of the conformation in MeOH
solution showed a rapid exchange between the right-handed and left-handed 310-helical
conformation of the N-terminal segment Ac-Phe-Aib-Aib-Aib-Iva-Gly [20]. Further-
more, several studies on the ability of antiamoebin to form ion channels have been
published [19][21].
Fig. 1. Sequences of antiamoebins I and II [18]
The aim of the present study was the elaboration of a practical synthesis of the
segments 1–9 and 10–16 of AAM I by applying the ꢂazirine/oxazolone methodꢃ.
2. Results and Discussion. – 2.1. Synthesis of Ac-Phe-Aib-Aib-Aib-d,l-Iva-Gly-Leu-
Aib-Aib-OH (1). – Based on our experience, the ꢂazirine couplingꢃ should be ideally
suited for the synthesis of the segment 1–5 (i.e., Ac-Phe-Aib3-d,l-Iva-OH, 2)) with
four consecutive a,a-disubstituted a-amino acids. We have shown that similar peptide
segments could be prepared conveniently by repeating the sequence ꢂazirine couplingꢃ
and selective hydrolysis [3c][5b][22]. Therefore, N-acetylated l-phenylalanine, Ac-
Phe-OH, in THF was reacted with 2,2,N-trimethyl-N-phenyl-2H-azirin-3-amine (1a) at
20–358, leading to dipeptide amide 3 in quantitative yield (Scheme 1). Subsequent
hydrolysis under standard conditions (3n HCl, THF/H2O 1:1, r.t.) gave the
corresponding peptide acid 4 in 90% yield. These two reaction steps were repeated
to give tripeptide 6 and tetrapeptide 85). The reaction of the latter with the racemic
isovaline (Iva) synthon 1b [5b] was carried out at 20–508 to give pentapeptide amide 9
as a ca. 1:1 mixture of the epimers in 96% yield6). Selective hydrolysis then yielded 2 as
a mixture of epimers (92%).
5
)
)
This tetrapeptide has been prepared previously by conventional coupling methods within the
synthesis of emerimicins III and IV [23].
An attempted separation of the epimers by means of preparative HPLC (LiChrosorb RP18,
MeOH/H2O gradient) gave the epimers in enriched form (8:1 and 1:7, resp.). In the mean time, we
have elaborated a preparative method for the synthesis of enantiomerically pure Iva synthons
[4b][24].
6

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Scheme 1
In a similar manner, the protected segment 6–9, 10, was prepared, starting with Z-
Gly-Leu-OH, by azirine coupling with 1a to give 11, hydrolysis to 12, and again
coupling with the Aib-synthon 1a to yield 10 (Scheme 2). Finally, the N-terminus was
deprotected by hydrogenolysis to give tetrapeptide amide 13.
For the coupling of the two segments 2 and 13, different conditions were evaluated,
i.e., via the ꢂmixed anhydrideꢃ [25] or via a 1,3-oxazol-5(4H)-one by treatment with
DCC4) or CME-CDI [26] in the presence of CSA or ZnCl2 [3b] (see Exper. Part). The
best result was obtained when a solution of 2 in THF/DMF 1:1 was treated
subsequently with equimolar amounts of N-methylmorpholine (NMM), isobutyl
chloroformate, and 13, to give 14 in 69% yield (Scheme 2). Hydrolysis of the latter with
3n HCl (THF/H2O 1:1) at room temperature afforded 15 in 67% yield as a mixture of
epimers.
In addition to these reactions, hydrolysis of 10 was performed with 3n HCl under
standard conditions to yield Z-Gly-Leu-Aib-Aib-OH (16; 82%), and subsequent
deprotection of the NH2 group (H2, Pd/C) gave tetrapeptide H-Gly-Leu-Aib-Aib-OH
(17) in almost quantitative yield (Scheme 3).
Single crystals of 16, of modest quality for an X-ray crystal-structure analysis, were
obtained from CH2Cl2. Although the results have low precision, the overall
composition of the molecule has been determined unambiguously. The crystal
structure of 16 is shown in Fig. 2. The space group of 16 permits the compound in
the crystal to be enantiomerically pure, but the absolute configuration has not been

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
923
Scheme 2
Scheme 3
determined. The enantiomer used in the refinement was based on the known (S)-
configuration of Leu. Both ends of the molecule are disordered over two approximately
equally occupied conformations. The N(1)ꢀH group of Aib4 forms an intramolecular
H-bond with O(4) of Gly1 thus creating a loop that can be described by the graph set
motif [28] of S(10) (b-turn). The N(4)ꢀH and N(3)ꢀH groups form intermolecular H-
bonds with an amide O-atom (N(4)ꢀH· · ·O(2’)) and the C¼O group of the acid
function (N(3)ꢀH· · ·O(1’)), respectively, of the same neighboring molecule. These
interactions link the molecules into extended chains running parallel to the [110]
direction, and each interaction can be described by the C(11) graph set motif [28]. The
chains also include an intermolecular H-bond between the OH group and an adjacent
amide O-atom (O(11)ꢀH· · ·O(3^), graph set motif C(10)), but the direction of
propagation of this interaction is opposite to that of the other two interactions. The
three-fold screw axis parallel to [001] results in two additional sets of symmetry-related
chains running parallel to [100] and [010]. The interaction of N(2)ꢀH with an amide O-
atom in a different neighboring molecule (N(2)ꢀH· · ·O(5’’)) serves at the local level to

924
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Fig. 2. ORTEP Plot [27] of the molecular structure of one of the disordered conformations of 16 (50%
probability ellipsoids; arbitrary numbering of atoms; H-atoms bonded to C-atoms have been omitted for
clarity)
link pairs of molecules into dimers in which the R22(20) ring motif is evident. At the
extended level, these latter interactions cross-link the above-mentioned chains to yield
overall a three-dimensional H-bonded network of peptide molecules.
2.2. Synthesis of Z-Hyp-Gln-d,l-Iva-Hyp-Aib-Pro-Pheol (34). The synthesis of
segment 10–16 of AAM I started with the preparation of the C-terminal tetrapeptide
18 (Scheme 4). Although the coupling of Z-Hyp-OH with the Aib synthon 1a and the
subsequent hydrolysis furnished Z-Hyp-Aib-OH in good yield [29], the attempted
coupling with H-Pro-Pheol via the mixed anhydride [25] failed. Therefore, the O-
protected Hyp derivative 19 was used, and the reaction with 1a in CH2Cl2 at room
temperature gave dipeptide amide 20 in 96% yield, and subsequent hydrolysis afforded
the acid 21 (86%). Hydrogenolytic deprotection of 20 to give 22 was achieved in 90%
yield. The segment H-Pro-Pheol (24) was obtained from Z-Pro-OH and H-Pheol via
the mixed anhydride and deprotection of 23. The coupling of 21 with 24 by using DCC/
HOBt in the presence of CSA yielded 25 (78%). Hydrogenolytic deprotection of the
amino group led to the desired 18.
For the preparation of segment 10–12, Gln derivative 26 with the Dod-protected
[30] amide function was chosen [5b]. Treatment of 19 and 26 with DCC/HOBt/CSA in
DMF at 08 led to the fully protected dipeptide 27, which was saponified to give the
dipeptide acid 28 in 88% yield (Scheme 5). Reaction of the latter with the Iva synthon

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
925
Scheme 4
1b gave tetrapeptide 29 as a mixture of epimers in 80% yield7), and subsequent
selective hydrolysis led to the tripeptide acid 30 (95%).
Unfortunately, the attempted coupling of the segments 30 and 18 by treatment with
DCC/HOBt/CSA in DMF failed. It is worth mentioning that, under the same
conditions, the related tripeptide Z-Hyp(tBu)-Gln(Dod)-Aib-OH reacted with 18 to
give Z-Hyp(tBu)-Gln(Dod)-Aib-Hyp(tBu)-Aib-Pro-Pheol [29], i.e., the protected C-
terminal heptapeptide of zervamicin IA [32], albeit in only 11% yield.
Finally, the desired segment 10–16 of AAM I was prepared by the 3þ2þ2
condensation depicted in Scheme 6. The coupling of the tripeptide 30 with the dipeptide
7
)
Attempts to separate the diastereoisomers by prep. HPLC (Nucleosil 100-7) were not successful.
Therefore, the analogous reaction of 28 with the Iva synthons 1c [29] and 1d [31] bearing a chiral
auxiliary group were carried out, leading to the corresponding tripeptides of type 29 in 75 and 76%
yield, respectively. Again, in each case two epimers were formed in almost equal amounts, which
could not be separated [29].

926
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Scheme 5
22 by using TBTU/HOBt/EtNiPr2 was successful and gave the pentapeptide 31 in a
yield of 60%8). Subsequent selective hydrolysis of the terminal amide group was
accomplished under the usual conditions (3n HCl, H2O/THF 1:1, r.t.), and the peptide
acid 32 was obtained in 94% yield. The latter was coupled with the earlier prepared C-
terminal dipeptide 24, with DCC/HOBt/CSA as the coupling reagents, to yield the
protected terminal heptapeptide 33 (60%). All side-chain functional groups, i.e., the
tBuO groups of two Hyp and the Dod group of Gln, were deprotected simultaneously
by treating 33 with CF3COOH in the presence of a small amount of anisole. The
heptapeptide Z-Hyp-Gln-d,l-Iva-Hyp-Aib-Pro-Pheol (34) was obtained in 93% yield
as a crystalline material, which was purified by preparative HPLC without separation of
the two epimers.
3. Conclusions. – The presented results show that the ꢂazirine/oxazolone methodꢃ
offers a convenient approach to the synthesis of the peptaibol antibiotic antiamoebin
AAM I. Epimer mixtures of the two main segments 1–9 and 10–16 containing d,l-
isovaline were obtained in reasonable yields. Whereas the introduction of the a,a-
disubstituted a-amino acids Aib and d,l-Iva via coupling with the corresponding 2H-
azirin-3-amines occurred smoothly and with high yields, the coupling of the segments
was much more demanding. The success of this reaction strongly depended on the
8
)
The attempted coupling with DCC/HOBt/CSA gave the desired 31 in only 15% yield.

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
927
Scheme 6
chosen strategy, i.e., the disconnection of the target molecule into segments. The
separation of epimeric segments containing d,l-Iva seems to be possible by HPLC but
proved to be rather difficult. As enantiomerically pure 2H-azirin-3-amines as d- and l-
Iva synthons are now available [4b][24], the synthesis of the natural AAM I should be
possible according to the presented method.
We thank the analytical sections of our institute for spectra and analyses, and the Swiss National
Science Foundation, the Stipendienfonds der Basler Chemischen Industrie, and F. Hoffmann-La Roche
AG, Basel, for financial support.
Experimental Part
1. Abbreviations. Aib, 2-Aminoisobutyric acid (2-methylalanine); CME-CDI, N-cyclohexyl-N’-[2-(4-
methylmorpholin-4-ylium)ethyl]carbodiimide 4-toluenesulfonate; CSA, camphor-10-sulfonic acid;
DCC, N,N’-dicyclohexylcarbodiimide; Dod, ꢂ4,4’-dimethoxyditylꢃ (¼4,4’-dimethoxybenzhydryl
(Mbh)); HOBt, 1-hydroxybenzotriazole; Hyp, trans-4-hydroxyproline; Iva, ꢂisovalineꢃ (¼2-ethylala-
nine); NMM, 4-methylmorpholine; TBTU, O-(1H-benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium

928
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
tetrafluoroborate; TFA, trifluoroacetic acid; Pheol, l-phenylalaninol (¼(S)-2-amino-3-phenylpropan-1-
ol); Z, (benzyloxy)carbonyl.
2. General. See [5b]. Amino acids, Ac-Phe-OH, and Pheol were purchased from Novabiochem and
Bachem, and are all l-configured; other reagents and solvents from Aldrich, Bachem, Fluka, and Merck,
resp. Solvents were dried according to known protocols. The syntheses of 2,2,N-trimethyl-N-phenyl-2H-
azirin-3-amine (1a) and rac-2-ethyl-2,N-dimethyl-N-phenyl-2H-azirin-3-amine (1b) have been described
previously [5b][33]. M.p.: Mettler-FP-5 apparatus, uncorrected. Optical rotations ([a]D): Zeiss-LEP-A2
or Perkin-Elmer-241 polarimeter at 21–238. IR Spectra: Perkin-Elmer 781 spectrometer, in KBr; ˜n in
cmꢀ1. 1H- and 13C-NMR spectra: Bruker AC-300, Bruker AM-400, and Bruker AMX-600 spectrometer at
300, 400, and 600 (1H), and 75.5, 100.8, and 151.2 MHz (13C), resp., in CD3OD or (D6)DMSO, if not
otherwise stated; d in ppm rel. to Me4Si as internal standard, J in Hz. ESI-MS: Finnigan TSQ-700
instrument; in m/z (rel. %). FAB-MS: Finnigan MAT-90; in m/z (rel. %). CI-MS: Finnigan MAT-90 or
SSQ-700 instrument with NH3 or isobutane; in m/z (rel. %).
General Procedure 1 (GP 1): Coupling with 2H-Azirin-3-amines. To a soln. of an N-protected amino
acid or N-protected peptide in THFor CH2Cl2 at 08, ca. 1.2 equiv. of the corresponding 2H-azirin-3-amine
in THF or CH2Cl2 were added, and the mixture was stirred at r.t. for several hours under Ar. After
completion of the reaction, the solvent was evaporated, and the product was purified by column
chromatography (CC), prep. layer chromatography (PLC), or crystallization.
General Procedure 2 (GP 2): Selective Hydrolysis of Peptide N-Methyl-N-phenylamides. A soln. of
the peptide amide in 3n HCl (THF/H2O 1:1) was stirred at r.t. for 1–50 h. Then, aq. 2n HCl was added,
and the mixture was extracted with CH2Cl2 (3ꢁ ). The org. layers were combined, dried (Na2SO4), and
evaporated. The product was purified by crystallization.
General Procedure 3 (GP 3): Hydrogenolytic Deprotection. The N-protected peptide (Z-peptide)
was dissolved in MeOH, and 10% Pd/C was added to the soln. The mixture was stirred at r.t. under H2
overnight. After completion of the reaction (TLC), the soln. was filtered through a pad of Celite, and the
solvent was evaporated. The product was dried in high vacuum (i.v.).
2. Preparation of the Pentapeptide Ac-Phe-Aib-Aib-Aib-d,l-Iva-OH (2). 2.1. Ac-Phe-Aib-N(Me)Ph
(3). According to GP 1, Ac-Phe-OH (3.04 g, 14.7 mmol) in THF (80 ml) at 08 was treated with 1a (2.82 g,
16.2 mmol); stirring at r.t. for 49 h and at 358 for 3 h. The solvent was evaporated, the residue was
dissolved in a small amount of AcOEt, and the product precipitated by addition of hexane: 5.54 g (99%)
of 3. Colorless crystals. M.p. 140.8–142.28. [a]2D2 ¼ þ7.1 (c¼0.91, EtOH). IR (KBr): 3375s, 3060s, 3030m,
2990m, 2930m, 1650s, 1595s, 1565s, 1550s, 1535s, 1495s, 1390s, 1360s, 1290s, 1250s, 1200s, 1170m, 1115m,
1090s, 1075m, 1030m, 770m, 745s, 700s. 1H-NMR ((D6)DMSO): 7.95 (d, J¼8.9, NH); 7.94 (s, NH); 7.38–
7.12 (m, 10 arom. H); 4.40 (td, J¼8.8, 5.5, CH(2) (Phe)); 3.12 (s, MeN); 2.91, 2.71 (AB of ABM, JAB
¼
13.7, JAM ¼8.9, JBM ¼5.5, PhCH2); 1.78 (s, MeCO); 1.32, 1.28 (2s, Me2C). 13C-NMR ((D6)acetone): 173.0,
170.9, 170.2 (3s, 3 CO (amide)); 146.3, 138.5 (2s, 2 arom. C); 130.3, 129.9, 128.9, 128.5, 127.8, 127.1 (6d,
10 arom. CH); 57.9 (s, Me2C); 54.9 (d, C(2) (Phe)); 40.8 (q, MeN); 38.6 (t, PhCH2); 26.84, 26.75
(2q, Me2C); 22.8 (q, MeCO). CI-MS: 382 (21, [Mþ1]þ ), 276 (17, [MꢀPh(Me)Nþ1]þ ), 275 (100,
[MꢀPh(Me)N]þ ), 178 (23), 175 (35), 108 (20, [Ph(Me)Nþ2]þ ), 107 (14, [Ph(Me)Nþ1]þ ), 89 (18).
Anal. calc. for C22H27N3O3 (381.47): C 69.27, H 7.13, N 11.02; found: C 69.23, H 7.14, N 11.21.
2.2. Ac-Phe-Aib-OH (4) [23b]. According to GP 2, a soln. of 3 (7.30 g, 19.0 mmol) in 3n HCl
(192 ml) was stirred at r.t. for 21 h. The solvent was evaporated, and the precipitate was filtered and
washed with Et2O: 5.04 g (90%) of 4. Colorless crystals. M.p. 197.3–202.88. [a]2D2 ¼ þ2.0 (c¼0.75,
EtOH). IR (KBr): 3290s, 3060m, 3030m, 2990m, 2930m, 1720s, 1660m, 1640s, 1565s, 1500w, 1440m,
1380m, 1356w, 1310w, 1290w, 1265w, 1220w, 1190w, 1170w, 1120w, 1040w, 1020w, 995w, 745w, 700w.
1H-NMR ((D6)DMSO): 8.17 (s, NH); 8.03 (d, J¼8.7, NH); 7.41–7.15 (m, 5 arom. H); 4.51 (td, J¼9.1, 4.6,
CH(2) (Phe)); 2.94, 2.68 (AB of ABM, JAB ¼13.7, JAM ¼9.8, JBM ¼4.6, PhCH2); 1.73 (s, MeCO); 1.33, 1.30
(2s, Me2C). 13C-NMR ((D6)acetone): 177.6 (s, COOH); 172.9, 172.7 (2s, 2 CO (amide)); 138.4 (s, 1 arom.
C); 130.4, 129.3, 127.7 (3d, 5 arom. CH); 57.0 (s, Me2C); 55.8 (d, C(2) (Phe)); 39.1 (t, PhCH2); 25.2, 25.1
(2q, Me2C); 22.4 (q, MeCO). CI-MS: 294 (17), 293 (100, [Mþ1]þ ), 275 (15, [MꢀH2Oþ1]þ ), 190 (15,
[AcPhe]þ ), 104 (11). Anal. calc. for C15H20N2O4 (292.33): C 61.63, H 6.90, N 9.58; found: C 61.76, H 6.98,
N 9.53.

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
929
2.3. Ac-Phe-Aib-Aib-N(Me)Ph (5). According to GP 1, to a soln. of 4 (385 mg, 132 mmol) in THF
(9 ml) at 08 was added 1a (253 mg, 1.45 mmol); stirring at r.t. for 65 h. The solvent was evaporated, and
the crude 5 was used for the hydrolysis. IR (KBr): 3300s, 3060m, 2980m, 2940m, 1650s, 1590m, 1560m,
1550s, 1535s, 1515s, 1495s, 1470m, 1455m, 1390m, 1365m, 1280m, 1220m, 1090m, 1025w, 970w, 745m, 705s.
1H-NMR ((D6)DMSO): 8.25 (d, J¼6.4, NH); 8.08 (s, NH); 7.36–7.16 (m, 10 arom. H, NH); 4.33 (dt, J¼
8.9, 6.3, CH(2) (Phe)); 3.21 (s, MeN); 2.91, 2.77 (AB of ABM, JAB ¼13.7, JAM ¼8.9, JBM ¼6.2, PhCH2);
1.80 (s, MeCO); 1.36, 1.31, 1.27. 1.22 (4s, 2 Me2C). 13C-NMR (CD3OD): 176.1, 175.5, 173.5, 173.4 (4s, 4 CO
(amide)); 146.8, 137.9 (2s, 2 arom. C); 130.5, 130.3, 129.5, 128.4, 128.3, 127.9 (6d, 10 arom. CH); 58.6, 58.0
(2s, 2 Me2C); 57.3 (d, C(2) (Phe)); 41.1 (q, MeN); 32.2 (t, PhCH2); 26.8, 26.4, 26.2, 23.9 (4q, 2 Me2C); 22.3
(q, MeCO). CI-MS: 361 (11, [MꢀPh(Me)Nþ1]þ ), 360 (53, [MꢀPh(Me)N]þ ), 108 (100, [Ph(Me)Nþ
2]þ ), 107 (25, [Ph(Me)Nþ1]þ ).
2.4. Ac-Phe-Aib-Aib-OH (6). According to GP 2, a soln. of 5 (350 mg, 0.75 mmol) in 3n HCl (7.5 ml)
was stirred at r.t. for 23 h. Then, 2n HCl (3.5 ml) was added, the mixture was extracted with CH2Cl2
(3ꢁ ), and the org. phase was dried (Na2SO4). The solvent was evaporated, Et2O was added, and the
precipitate was filtered: 216 mg (87%, two steps) of 6. Colorless crystals. M.p. 197.0–198.28. [a]D22
¼
þ39.3 (c¼1.13, EtOH). IR (KBr): 3390s, 3330s, 3290s, 3060m, 2980m, 2930m, 1745s, 1735m, 1680s, 1645s,
1625s, 1560m, 1550s, 1500m, 1465m, 1450m, 1380m, 1370m, 1290m, 1245m, 1210m, 1175m, 1160s, 1120w,
1050w, 1030w, 1005w, 960w, 730m, 695m. 1H-NMR ((D6)DMSO): 12.04 (s, COOH); 8.24 (d, J¼6.7, NH);
8.14 (s, NH); 7.27–7.15 (m, 5 arom. H, NH); 4.33 (dt, J¼9.1, 6.4, CH(2) (Phe)); 2.91, 2.75 (AB of ABM,
JAB ¼13.7, JAM ¼9.1, JBM ¼5.9, PhCH2); 1.78 (s, MeCO); 1.32, 1.27, 1.25, 1.23 (4s, 2 Me2C). 13C-NMR
(CD3OD): 178.3 (s, COOH); 176.2, 176.1, 173.7 (3s, 3 CO (amide)); 138.2 (s, 1 arom. C); 130.8, 129.8,
128.2 (3d, 5 arom. CH); 58.1, 57.3 (2s, 2 Me2C); 57.8 (d, C(2) (Phe)); 38.6 (t, PhCH2); 26.8, 25.9, 24.9, 24.2
(4q, 2 Me2C); 22.7 (q, MeCO). CI-MS: 379 (22), 378 (100, [Mþ1]þ ), 361 (6, [MꢀH2Oþ1]þ ), 360 (31,
[MꢀH2O]þ ), 275 (18). Anal. calc. for C19H27N3O5 (377.44): C 60.46, H 7.21, N 11.13; found: C 60.32, H
7.21, N 11.06.
2.5. Ac-Phe-Aib-Aib-Aib-N(Me)Ph (7). According to GP 1, to a soln. of 6 (419 mg, 1.11 mmol) in
THF (6 ml) at 08 was added 1a (206 mg, 1.18 mmol); stirring at r.t. for 51 h. The solvent was evaporated,
the residue was dissolved in AcOEt, Et2O was added, and the precipitate was filtered: 590 mg (96%) of 7.
Colorless crystals. M.p. 181.5–184.58. [a]2D2 ¼ þ24.7 (c¼0.55, EtOH). IR (KBr): 3440m, 3350s, 3290s,
3060m, 2990m, 2935w, 1660s, 1635s, 1595m, 1535s, 1510m, 1490m, 1470m, 1455m, 1395m 1380m, 1365m,
1280w, 1230w, 1090m, 1070w, 1025w, 980w, 745w, 705m. 1H-NMR ((D6)DMSO): 8.55 (s, NH); 8.34 (d, J¼
5.5, NH); 8.08 (s, NH); 7.37–7.18 (m, 10 arom. H, NH); 7.13 (s, NH); 4.35–4.28 (m, CH(2) (Phe)); 3.27
(s, MeN); 2.93, 2.84 (AB of ABM, JAB ¼13.7, JAM ¼8.8, JBM ¼6.5, PhCH2); 1.81 (s, MeCO); 1.41, 1.37, 1.31,
1.22. 1.19 (5s, 3 Me2C). 13C-NMR (CD3OD): 176.8, 175.8, 175.7, 174.1, 173.4 (5s, 5 CO (amide)); 147.0,
137.9 (2s, 2 arom. C); 130.5, 130.2, 129.5, 128.3, 128.1, 127.9 (6d, 10 arom. CH); 58.5, 58.2, 57.7 (3s, 3
Me2C); 57.2 (d, C(2) (Phe)); 41.0 (q, MeN); 38.0 (t, PhCH2); 27.5, 26.58, 26.57, 26.2, 24.2, 23.7 (6q, 3
Me2C); 22.4 (q, MeCO). CI-MS: 446 (28, [MꢀPh(Me)Nþ1]þ ), 445 (100, [MꢀPh(Me)N]þ ), 361 (8),
360 (41), 108 (44, [Ph(Me)Nþ2]þ ), 107 (11, [Ph(Me)Nþ1]þ ). Anal. calc. for C30H41N5O5 (551.68): C
65.32, H 7.49, N 12.69; found: C 65.11, H 7.35, N 12.78.
2.6. Ac-Phe-Aib-Aib-Aib-OH (8). According to GP 2, a soln. of 7 (368 mg, 0.67 mmol) in 3n HCl
(7.0 ml) was stirred at r.t. for 39 h. Then, 2n HCl (3.5 ml) was added, the mixture was extracted with
CH2Cl2 (3ꢁ ), and the org. phase was dried (Na2SO4). The solvent was evaporated, Et2O was added, and
the precipitate was filtered: 259 mg (84%) of 8. Colorless crystals. M.p. 216.0–217.58. [a]2D2 ¼ þ31.9 (c¼
0.86, EtOH). IR (KBr): 3440s, 3300s, 3065m, 3030m, 3000s, 2980s, 2940m, 2880m, 1710s, 1680s, 1675s,
1640s, 1560s, 1550s, 1495m, 1470m, 1455m, 1450s, 1435m, 1415m, 1370s, 1365s, 1310s, 1275m, 1245s,
1220m, 1180s, 1125w, 1085w, 1060w, 1005w, 985w, 750m, 700m. 1H-NMR ((D6)DMSO): 8.52 (s, NH); 8.31
(d, J¼5.8, NH); 7.45–7.18 (m, 5 arom. H, NH); 7.10 (s, NH); 4.34–4.27 (m, CH(2) (Phe)); 2.90, 2.81 (AB
of ABM, JAB ¼13.7, JAM ¼8.7, JBM ¼6.5, PhCH2); 1.82 (s, MeCO); 1.35, 1.33, 1.32, 1.26, 1.21, 1.18 (6s, 3
Me2C). 13C-NMR (CD3OD): 178.4 (s, COOH); 176.7, 176.2, 174.5, 174.0 (4s, 4 CO (amide)); 138.1 (s, 1
arom. C); 130.8, 129.8, 128.2 (3d, 5 arom. CH); 58.1, 58.0, 57.5 (3s, 3 Me2C); 57.3 (d, C(2) (Phe)); 38.3 (t,
PhCH2); 27.4, 26.9, 26.4, 24.8, 24.4, 24.0 (6q, 2 Me2C); 22.6 (q, MeCO). CI-MS: 446 (21), 445 (72, [Mꢀ
H2Oþ1]þ ), 361 (22), 360 (100, [445ꢀAib]þ ). Anal. calc. for C23H34N4O6 (462.54): C 59.73, H 7.41, N
12.11; found: C 59.48, H 7.50, N 12.27.

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
2.7. Ac-Phe-Aib-Aib-Aib-d,l-Iva-N(Me)Ph (9). According to GP 1, to a soln. of 8 (185 mg,
0.40 mmol) in THF (6 ml) and abs. DMF (1 ml) at 08 was added 1b (83 mg, 0.44 mmol); stirring at r.t. for
24 h and at 508 for 94 h. Then, Et2O was added, the precipitate was filtered and washed with Et2O:
251 mg (96%) of 9 as a ca. 1:1 mixture of diastereoisomers. Colorless crystals. M.p. 247.0–252.08. [a]D22
¼
þ11.4 (c¼0.54, EtOH). IR (KBr): 3440m, 3310s, 3024w, 2990w, 2940m, 1665s, 1650s, 1595m, 1530s,
1495m, 1470m, 1455m, 1415m, 1380m, 1360m, 1310w, 1290w, 1225w, 1175w, 1110w, 1090w, 1070w, 1030w,
1010w, 765w, 705m. 1H-NMR ((D6)DMSO): 8.57 (d, J¼8.1, NH); 8.26 (s, NH); 7.48–7.16 (m, 10 arom. H,
3 NH); 4.37–4.24 (m, CH(2) (Phe)); 3.29 (s, MeN); 2.94–2.76 (m, PhCH2); 1.95–1.73 (m, MeCH2
(Iva)); 1.81 (s, MeCO); 1.41, 1.40, 1.35, 1.34, 1.29, 1.27, 1.26, 1.25, 1.23, 1.21 (10s, 3 Me2C, Me (Iva)); 0.82 (t,
J¼7.4, MeCH2 (Iva)). 13C-NMR ((D6)DMSO): 174.83, 174.75, 173.7, 173.6, 173.5, 173.4, 172.63, 172.56,
172.2, 172.1, 170.5, 170.4 (12s, 6 CO (amide)); 146.5, 137.50, 137.46 (3s, 2 arom. C); 129.3, 128.8, 128.2,
127.0, 126.5, 126.0 (6d, 10 arom. CH); 59.5, 59.4 (2s, C(2) (Iva)); 56.3, 56.2, 56.1, 56.0, 55.9 (5s, 3 Me2C);
55.3 (d, C(2) (Phe)); 39.3 (q, MeN); 36.4 (t, PhCH2); 29.3 (t, MeCH2 (Iva)); 26.9, 26.4, 26.0, 25.9, 25.7,
24.9, 24.5, 24.0, 23.7, 23.5, 23.3, 22.3, 22.2 (12q, 3 Me2C, Me (Iva), MeCO); 8.04, 7.99 (2q, MeCH2 (Iva)).
CI-MS: 546 (24), 545 (73), 544 (100, [MꢀPh(Me)N]þ ), 446 (13), 445 (32, [544–Iva]þ ), 380 (12), 360
(16, [445ꢀAib]þ ), 285 (13), 284 (50), 282 (10), 256 (38), 190 (19, [AcPhe]þ ), 181 (17), 109 (13), 108
(90, [Ph(Me)Nþ2]þ ), 107 (34, [Ph(Me)Nþ1]þ ), 91 (27). Anal. calc. for C35H50N6O5 (650.82): C 64.59,
H 7.74, N 12.91; found: C 64.40, H 8.00, N 12.86.
The epimers of 9 were separated by prep. HPLC (LiChrosorb RP18; gradient MeOH/H2O). Epimer
9a (ca. 8 :1 mixture). [a]2D2 ¼ þ8.6 (c¼0.95, EtOH). 1H-NMR (CD3OD): 7.83 (s, NH); 7.58 (s, NH); 7.39–
7.20 (m, 10 arom. H, 3 NH); 4.37 (t-like, J¼8.0, 7.8, CH(2) (Phe)); 3.41 (s, MeN); 3.02, 2.94 (AB of ABM,
JAB ¼13.6, JAM,BM ¼8.0, 7.8, PhCH2); 2.12–1.95 (m, MeCH2 (Iva)); 1.94 (s, MeCO); 1.56, 1.54, 1.48, 1.38,
1.36, 1.29, 1.25 (7s, 3 Me2C, Me (Iva)); 0.95 (t, J¼7.5, MeCH2 (Iva)).
Epimer 9b (ca. 1:7 mixture): [a]2D2 ¼ þ13.0 (c¼0.90, EtOH). 1H-NMR (CD3OD): 7.72 (br. s, NH);
8.62 (s, NH); 7.54 (s, NH); 7.42–7.26 (m, 10 arom. H, 2 NH); 4.35 (t-like, J¼7.9, 7.8, CH(2) (Phe)); 3.40 (s,
MeN); 3.04–2.95 (m, PhCH2); 2.11–1.95 (m, MeCH2 (Iva)); 1.95 (s, MeCO); 1.55, 1.54, 1.48, 1.39, 1.35,
1.28, 1.20 (7s, 3 Me2C, Me (Iva)); 0.95 (t, J¼7.4, MeCH2 (Iva)). 13C-NMR ((D6)DMSO): 174.7, 173.6,
173.3, 172.5, 172.0, 170.4 (6s, 6 CO (amide)); 146.3, 137.4 (2s, 2 arom. C); 129.2, 128.6, 128.0, 126.9, 126.4,
125.9 (6d, 10 arom. CH); 59.4 (s, C(2) (Iva)); 56.2, 56.1, 55.8 (3s, 3 Me2C); 55.2 (d, C(2) (Phe)); 39.2 (q,
MeN); 36.3 (t, PhCH2); 29.2 (t, MeCH2 (Iva)); 26.8, 25.9, 25.8, 24.4, 23.6, 23.2, 22.2, 22.0 (8q, 3 Me2C, Me
(Iva), MeCO); 7.93 (q, MeCH2 (Iva)).
2.8. Ac-Phe-Aib-Aib-Aib-d,l-Iva-OH (2). According to GP 2, a soln. of 9 (277 mg, 0.43 mmol) in 3n
HCl (4.5 ml) was stirred at r.t. for 25 h. Then, 2n HCl (2.2 ml) was added, the mixture was extracted with
CH2Cl2 (4ꢁ ), and the org. phase was dried (Na2SO4). The solvent was evaporated, and the residue was
crystallized from AcOEt/Et2O: 222 mg (92%) of 2. Colorless crystals. M.p. 234.8–236.08. [a]2D2 ¼ þ16.6
(c¼0.61, EtOH). IR (KBr): 3370m, 3320s, 3060w, 3030w, 2980s, 2935m, 1750m, 1740m, 1660s, 1535s,
1455m, 1445m, 1385m, 1365m, 1230m, 1170w, 750w, 700w. 1H-NMR ((D6)DMSO): 11.93 (br. s, COOH);
8.54 (s, NH); 8.28 (s, NH); 7.37 (d, J¼4.8, NH (Phe)); 7.29–7.19 (m, 5 arom. H); 7.16 (s, NH); 7.13 (s,
NH); 4.31–4.29 (m, CH(2) (Phe)); 2.93, 2.82 (AB of ABM, JAB ¼13.6, JAM ¼8.9, JBM ¼6.2, PhCH2); 2.05–
1.60 (m, MeCH2 (Iva)); 1.81 (s, MeCO); 1.31, 1.28, 1.26, 1.24, 1.21 (5s, 3 Me2C, Me (Iva)); 0.77 (m,
MeCH2 (Iva)). 13C-NMR (CD3OD): 177.7, 177.3, 176.5, 176.4, 176.3, 174.02, 173.97, 173.6 (8s, COOH, 5
CO (amide)); 137.8 (s, 1 arom. C); 130.4, 129.5, 127.9 (3d, 5 arom. CH); 60.83, 60.76 (2s, C(2) (Iva));
57.94, 57.90, 57.1 (3s, 3 Me2C); 57.6 (d, C(2) (Phe)); 37.9 (t, PhCH2); 31.3 (t, MeCH2 (Iva)); 27.0, 26.71,
26.68, 26.46, 26.43, 26.3, 25.0, 24.8, 24.3, 24.0, 23.8, 22.6, 22.4, 22.2 (14q, 3 Me2C, Me (Iva), MeCO); 8.68,
8.38 (2q, MeCH2 (Iva)). CI-MS: 562 (18, [Mþ1]þ ), 545 (33, [MꢀH2Oþ1]þ ), 544 (100, [MꢀH2O]þ ),
463 (17), 446 (20, [544–Ivaþ1]þ ), 445 (20, [544–Iva]þ ), 360 (38, [445ꢀAib]þ ). Anal. calc. for
C28H43N5O7 (561.68): C 59.88, H 7.72, N 12.47; found: C 59.93, H 7.75, N 12.65.
3. Preparation of the Tetrapeptide H-Gly-Leu-Aib-Aib-N(Me)Ph (13). 3.1. Z-Gly-Leu-Aib-
N(Me)Ph (11). According to GP 1, Z-Gly-Leu-OH (3.51 g, 10.7 mmol) in THF (70 ml) at 08 was
treated with 1a (2.08 g, 11.9 mmol); stirring at r.t. for 3 d. The solvent was evaporated, the residue was
dissolved in a small amount of AcOEt, and the product precipitated by addition of petroleum ether:
5.25 g (98%) of 11. Colorless crystals. M.p. 150.0–151.08. [a]2D2 ¼ ꢀ20.4 (c¼0.53, EtOH). IR (KBr):
3290s, 3080m, 2960m, 1740s, 1685s, 1660s, 1620s, 1595s, 1565s, 1550s, 1540s, 1495s, 1470m, 1455m, 1395m,

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
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1365m, 1260s, 1235s, 1210m, 1170w, 1120w, 1090w, 1075w, 1050m, 775m. 1H-NMR ((D6)DMSO): 7.99 (br.
s, NH); 7.89 (d, J¼8.5, NH); 7.52 (t, J¼6.0, NH); 7.34–7.15 (m, 10 arom. H); 5.02 (s, PhCH2O); 4.25–
4.16 (m, CH(2) (Leu)); 3.65 (d, J¼6.0, CH2 (Gly)); 3.14 (s, MeN); 1.56–1.23 (m, CH(4) (Leu), CH2(3)
(Leu)); 1.34 (s, Me2C); 0.84 (t-like, J¼8.2, 6.7, Me2CH). 13C-NMR ((D6)acetone): 173.1, 171.9, 169.9 (3s,
3 CO (amide)); 157.7 (s, CO (urethane)); 146.4, 138.0 (2s, 2 arom. C); 129.9, 129.2, 128.6, 128.51, 128.45,
127.8 (6d, 10 arom. CH); 66.9 (t, PhCH2O); 57.9 (s, Me2C); 51.9 (d, C(2) (Leu)); 45.1 (t, C(2) (Gly)); 42.0
(t, C(3) (Leu)); 40.9 (q, MeN); 26.8, 25.2 (d, C(4) (Leu) and q, Me2C); 23.6, 22.0 (2q, Me2CH). CI-MS:
391 (14), 390 (67, [MꢀPhCH2Oþ1]þ ), 282 (18), 256 (15), 245 (9), 200 (13), 108 (100, [PhCH2Oþ1]þ or
[Ph(Me)Nþ2]þ ), 107 (42, [Ph(Me)Nþ1]þ or [PhCH2O]þ ), 91 (26). Anal. calc. for C27H36N4O5
(496.61): C 65.30, H 7.31, N 11.28; found: C 65.28, H 7.42, N 11.26.
3.2. Z-Gly-Leu-Aib-OH (12). According to GP 2, a soln. of 11 (4.45 g, 8.95 mmol) in 3n HCl (90 ml)
was stirred at r.t. for 12 h. Then, 2n HCl (45 ml) was added and the mixture was extracted with CH2Cl2
(5ꢁ ). The combined org. phases were dried (Na2SO4), the solvent was evaporated, the residue was
dissolved in a small amount of acetone, and hexane was added. The precipitate was filtered: 3.07 g (84%)
of 12. Colorless crystals. M.p. 151.9–153.98. [a]2D2 ¼ ꢀ37.5 (c¼0.75, EtOH). IR (KBr): 3435m, 3280s,
3080m, 2960m, 1720s, 1670s, 1630s, 1565s, 1550s, 1510m, 1470m, 1455m, 1420w, 1385w, 1370m, 1295m,
1275s, 1245s, 1170m, 1155m, 1100w, 1085w, 1050w, 1030w, 735m, 695m. 1H-NMR ((D6)DMSO): 12.13 (br.
s, COOH); 8.07 (s, NH); 7.87 (d, J¼8.6, NH); 7.46 (t, J¼6.0, NH); 7.35–7.33 (m, 5 arom. H); 5.03 (s,
PhCH2O); 4.39–4.31 (m, CH(2) (Leu)); 3.63 (d, J¼6.0, CH2 (Gly)); 1.60–1.34 (m, CH(4) (Leu),
CH2(3) (Leu)); 1.34, 1.31 (2s, Me2C); 0.85 (t-like, J¼8.4, 6.7, Me2CH). 13C-NMR (CD3OD): 177.6 (s,
COOH); 173.8, 172.0 (2s, 2 CO (amide)); 159.1 (s, CO (urethane)); 138.1 (s, arom. C); 129.5, 129.0, 128.8
(3d, 5 arom. CH); 67.8 (t, PhCH2O); 57.0 (s, Me2C); 52.8 (d, C(2) (Leu)); 45.0 (t, C(2) (Gly)); 41.9 (t,
C(3) (Leu)); 25.7, 25.3 (d, C(4) (Leu); q, Me2C); 23.4, 22.0 (2q, Me2CH). CI-MS: 409 (23), 408 (100,
[Mþ1]þ ), 391 (14), 390 (61, [MꢀH2Oþ1]þ ), 305 (17, [390 – Aib]þ ), 277 (9), 104 (12), 91 (11). Anal.
calc. for C20H19N3O6 (407.47): C 58.95, H 7.17, N 10.31; found: C 59.00, H 7.08, N 10.41.
3.3. Z-Gly-Leu-Aib-Aib-N(Me)Ph (10). According to GP 1, 12 (1.18 g, 2.9 mmol) in THF (15 ml) at
08 was treated with 1a (0.56 g, 3.19 mmol); stirring at r.t. for 50 h. The solvent was evaporated, and the
crude product was obtained as a foam: 1.68 g (quant.) of 10. IR (KBr): 3310s, 3060m, 3030m, 2950m,
2770m, 1710s, 1660s, 1590m, 1545s, 1530s, 1495s, 1470m, 1455m, 1390m, 1365m, 1250s, 1170m, 1115w,
1090m, 1070w, 1050m, 770w, 740w, 705w. 1H-NMR (CDCl3): 7.60 (s, NH); 7.40–7.17 (m, 10 arom. H); 6.83
(s, NH); 6.56 (d, J¼8.2, NH); 6.04 (br. s, NH); 5.13, 5.11 (AB, JAB ¼12.2, PhCH2O); 4.39 (dt, J¼8.3, 6.2,
CH(2) (Leu)); 4.09, 3.79 (AB of ABX, JAB ¼17.8, JAX ¼7.1, JBX ¼4.7, CH2 (Gly)); 3.23 (s, MeN); 1.73–1.42
(m, CH(4) (Leu), CH2(3) (Leu)); 1.51, 1.46, 1.41, 1.38 (4s, 2 Me2C); 0.91 (m, Me2CH). 13C-NMR
(CD3OD): 176.0, 175.6, 174.4, 172.6 (4s, 4 CO (amide)); 159.0 (s, CO (urethane)); 146.8, 138.0 (2s, 2
arom. C); 130.3, 129.5, 129.1, 128.8, 128.4, 128.3 (6d, 10 arom. CH); 67.8 (t, PhCH2O); 58.6, 58.2 (2s, 2
Me2C); 54.3 (d, C(2) (Leu)); 44.7 (t, C(2) (Gly)); 41.1 (t, C(3) (Leu); q, MeN); 26.4, 26.3, 26.2, 25.8 (4q, 2
Me2C); 24.8 (d, C(4) (Leu)); 23.3, 22.2 (2q, Me2CH). CI-MS: 476 (18), 475 (59, [MꢀPhCH2Oþ1]þ or
[MꢀPh(Me)N]þ ), 390 (9, [475ꢀAib]þ ), 360 (42), 108 (100, [PhCH2Oþ1]þ or [Ph(Me)Nþ2]þ ), 107
(36, [Ph(Me)Nþ1]þ or [PhCH2O]þ ).
3.4. H-Gly-Leu-Aib-Aib-N(Me)Ph (13). According to GP 3, a mixture of 10 (2.03 g, 3.5 mmol) in
MeOH (28 ml) and Pd/C (206 mg) under H2 was stirred at r.t. for 15 h. The crude product 13 was
obtained as an oil: 1.57 g (quant.), and the pure product was obtained after crystallization from CH2Cl2/
Et2O. Colorless crystals. M.p. 64.5–68.58. [a]D22 ¼ þ2.3 (c¼0.48, EtOH). IR (KBr): 3300s, 3060m, 2950m,
2870w, 1655s, 1595s, 1560m, 1550s, 1530s, 1495s, 1470m, 1390m, 1365m, 1285m, 1255m, 1220m, 1200m,
1170m, 1110m, 1090m, 1070w, 1025w, 770w, 740w, 710w. 1H-NMR (CDCl3): 7.57 (d, J¼7.9, NH); 7.52 (s,
NH); 7.45–7.17 (m, 5 arom. H); 6.93 (s, NH); 4.32 (dt, J¼8.2, 5.9, CH(2) (Leu)); 3.40, 3.34 (AB, JAB ¼7.2,
CH2 (Gly)); 3.27 (s, MeN); 1.89 (br. s, NH2); 1.78–1.52 (m, CH(4) (Leu), CH2(3) (Leu)); 1.50, 1.45, 1.44,
1.41 (4s, 2 Me2C); 0.94 (d, J¼8.0, Me (Leu)); 0.92 (d, J¼7.8, Me (Leu)). 13C-NMR (CDCl3): 173.8, 173.6,
172.7, 171.2 (4s, 4 CO (amide)); 144.4 (s, arom. C); 129.3, 128.2, 127.8 (3d, 5 arom. CH); 58.2, 57.4 (2s, 2
Me2C); 51.9 (d, C(2) (Leu)); 44.7 (t, C(2) (Gly)); 41.2 (q, MeN); 40.0 (t, C(3) (Leu)); 25.7, 24.91, 24.85,
24.78, 24.3 (4q, 2 Me2C; d, C(4) (Leu)); 22.3, 22.0 (2q, Me2CH). CI-MS: 342 (19, [MꢀPh(Me)Nþ1]þ ),
341 (100, [MꢀPh(Me)N]þ ), 178 (37), 177 (56), 151 (15), 108 (13, [Ph(Me)Nþ2]þ ), 107 (7,

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
[Ph(Me)Nþ1]þ ), 89 (28). Anal. calc. for C23H37N5O4 (447.58): C 61.72, H 8.33, N 15.65; found: C 61.68,
H 8.44, N 15.45.
3.5. Z-Gly-Leu-Aib-Aib-OH (16). According to GP 2, a soln. of 10 (332 mg, 0.57 mmol) in 3n HCl
(7 ml) was stirred at r.t. for 25 h. Then, 2n HCl (3.5 ml) was added, and the mixture was extracted with
CH2Cl2 (4ꢁ ). The combined org. phases were dried (Na2SO4), the solvent was evaporated, and the oily
residue dissolved in a small amount of CH2Cl2 was left standing at r.t. The formed precipitate was filtered:
211 mg (82%) of 16. Colorless crystals. M.p. 159.5–162.08. [a]2D2 ¼ ꢀ20.9 (c¼0.89, EtOH). IR (KBr):
3320s, 3060m, 3030m, 2990m, 2930m, 2870m, 1710s, 1660s, 1650s, 1550s, 1530s, 1470m, 1450m, 1405m,
1385m, 1365w, 1335w, 1300m, 1265s, 1220m, 1160s, 1125w, 1015w, 735m, 695m. 1H-NMR ((D6)DMSO):
12.05 (s, COOH); 8.07 (d, J¼6.5, NH); 8.03 (s, NH); 7.44 (t, J¼6.0, NH); 7.39–7.26 (m, 5 arom. H); 7.21
(s, NH); 5.01 (s, PhCH2O); 4.12 (q-like, Jꢂ7.1, CH(2) (Leu)); 3.68, 3.60 (AB of ABX, JAB ¼16.9, JAX
¼
6.3, JBX ¼6.0, CH2 (Gly)); 1.64–1.50 (m, CH(4) (Leu)); 1.48–1.41 (m, CH2(3) (Leu)); 1.32, 1.30, 1.27 (3s,
2 Me2C); 0.89, 0.84 (2d, J¼6.5, Me2CH). 13C-NMR (CD3OD): 178.3 (s, COOH); 176.2, 174.7, 172.9 (3s,
3 CO (amide)); 159.3 (s, CO (urethane)); 138.3 (s, arom. C); 129.7, 129.3, 129.1 (3d, 5 arom. CH); 68.2 (t,
PhCH2O); 58.2, 57.5 (2s, 2 Me2C); 54.5 (d, C(2) (Leu)); 45.1 (t, C(2) (Gly)); 41.5 (t, C(3) (Leu)); 26.1,
25.6, 25.2 (d, C(4) (Leu); 2q, 2 Me2C); 23.6, 22.4 (2q, Me2CH). CI-MS: 493 (10, [Mþ1]þ ), 476 (13), 475
(53, [MꢀH2Oþ1]þ ), 408 (26), 391 (23, [MꢀH2OꢀAibþ2]þ ), 390 (100, [Z-Gly-Leu-Aibþ1]þ ), 305
(8, [Z-Gly-Leuþ1]þ ), 277 (9), 104 (60). Anal. calc. for C24H36N4O7 (492.57): C 58.52, H 7.37, N 11.37;
found: C 58.38, H 7.22, N 11.47.
Suitable crystals of 16 for an X-ray crystal-structure determination were obtained from MeOH/Et2O
by slow evaporation of the solvent.
3.6. H-Gly-Leu-Aib-Aib-OH (17). According to GP 3, a mixture of 16 (270 mg, 0.55 mmol) in
MeOH (4 ml) and Pd/C (30 mg) under H2 was stirred at r.t. for 5.5 h. The crude product 17 was obtained
as a colorless solid: 204 mg (quant.). 1H-NMR ((D6)DMSO): 8.50 (d, J¼9.0, NH); 8.37, 7.80 (2s, 2 NH);
4.48 (q-like, Jꢂ7.8, CH(2) (Leu)); 3.92 (br. s, NHþ3 ); 3.40, 3.30 (AB, JAB ¼7.0, CH2 (Gly)); 1.52–1.37 (m,
CH(4) (Leu), CH2(3) (Leu)); 1.32, 1.28, 1.25 (3s, 2 Me2C); 0.90, 0.85 (2d, J¼6.0, Me2CH). 13C-NMR
(CD3OD): 181.8 (s, COOꢀ ); 175.0, 172.7, 167.7 (3s, 3 CO (amide)); 58.9, 58.3 (2s, 2 Me2C); 53.1 (d, C(2)
(Leu)); 42.0 (t, C(2) (Gly)); 41.1 (t, C(3) (Leu)); 27.2, 26.0, 24.3, 24.2, 24.0 (4q, 2 Me2C; d, C(4) (Leu));
22.0, 22.6 (2q, Me2CH). CI-MS: 360 (20), 359 (100, [Mþ1]þ ), 341 (14, [MꢀH2Oþ1]þ ), 256 (12, [341ꢀ
Aib]þ ), 189 (6), 171 (6).
4. Coupling of 2 and 13 to Give Ac-Phe-Aib-Aib-Aib-d,l-Iva-Gly-Leu-Aib-Aib-N(Me)Ph (14). 4.1.
Via the Mixed Anhydride. To a stirred soln. of 2 (265 mg, 0.47 mmol) in a mixture of THF (0.5 ml) and
DMF (0.5 ml) at ꢀ158, NMM (48 mg, 0.47 mmol) and isobutyl chloroformate (64 mg, 0.47 mmol) were
added. After stirring for 4 min at ꢀ158, 13 (213 mg, 0.47 mmol) in THF (1 ml) was added, and the
mixture was stirred for 3 h in an ice-bath and for 50 h at r.t. To the suspension was added CH2Cl2, and the
mixture was extracted with an aq. soln. of citric acid (5%), 1n NaOH, and sat. aq. NaCl soln. The org.
phase was dried (Na2SO4), the solvent evaporated, and the product precipitated by addition of Et2O:
321 mg (69%) of 14.
4.2. With DCC and Catalytic Amounts of CSA. To a stirred soln. of 2 (175 mg, 0.31 mmol) in DMF
(1.4 ml) at 08 was added DCC (65 mg, 0.32 mmol). After stirring for 10 min at 08, HOBt (47 mg,
0.35 mmol) and CSA (20 mg) were added, and, after 2 min, 13 (162 mg, 0.36 mmol), and the mixture was
stirred for 50 h at r.t. Then, the mixture was filtered through a pad of Celite and washed with DMF. The
filtrate was dissolved in CH2Cl2, and the soln. was extracted with an aq. soln. of citric acid (5%), 1n
NaOH, and sat. aq. NaCl soln. The org. phase was dried (Na2SO4), and the solvent was evaporated:
284 mg (92%) of crude 14, which still contained dicyclohexylurea (1H-NMR). Part of the crude material
(78 mg) was purified by column chromatography (CC; SiO2, CH2Cl2/MeOH 19 :1) to give 46 mg of pure
14.
4.3. With DCC and Catalytic Amounts of ZnCl2. To a stirred soln. of 2 (63 mg, 0.11 mmol) in abs.
DMF (10.5 ml) at 08 was added DCC (25 mg, 0.12 mmol). After stirring for 3 min at 08, ZnCl2 (31 mg,
0.23 mmol), and a soln. of 13 (57 mg, 0.13 mmol) and Et3N (20 mg, 0.20 mmol) in abs. DMF (0.5 ml)
were added, and the mixture was stirred for 77.5 h at r.t. Then, the mixture was filtered through a pad of
Celite and washed with DMF. The filtrate was dissolved in CH2Cl2 and the soln. was extracted with 2n
HCl, 1n NaOH, and sat. aq. NaCl soln. The org. phase was dried (Na2SO4), and the solvent was

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
933
evaporated. The residue was dissolved in a small amount of CH2Cl2, and the product was crystallized by
addition of hexane: 25 mg (23%) of 14.
4.4. With CME-CDI and Catalytic Amounts of CSA. To a stirred soln. of 2 (293 mg, 0.52 mmol) in
DMF (1.5 ml) at 08 was added CME-CDI (221 mg, 0.52 mmol). After stirring for 5 min at 08, HOBt
(72 mg, 0.53 mmol) and CSA (30 mg) were added, and, after another 5 min, 13 (238 mg, 0.53 mmol) was
added. The mixture was stirred for 5 h at r.t., CH2Cl2 (0.5 ml) was added, and stirring at r.t. was continued
for 98 h. Then, the mixture was diluted with CH2Cl2, and the soln. was extracted with an aq. soln. of citric
acid (5%), 1n NaOH, and sat. aq. NaCl soln. The org. phase was dried (Na2SO4), and the solvent was
evaporated: 132 mg (26%) of 14.
Data of 14. Colorless crystals. M.p. 230.0–232.58. [a]2D2 ¼ þ7.3 (c¼0.48, EtOH). IR (KBr): 3460m,
3300s, 3060w, 2980m, 2940m, 1660s, 1595m, 1545s, 1535s, 1495m, 1465m, 1440m, 1385m, 1365m, 1280w,
1220w, 1170w, 1090w, 770w, 745w, 705m. 1H-NMR ((D6)DMSO): 8.61, 8.60 (2s, NH (Aib2)); 8.30 (d, J¼
5.8, NH (Phe)); 7.91 (t-like, J¼5.6, NH (Gly)); 7.70, 7.69 (2s, NH (Aib4)); 7.62–7.59 (m, NH (Aib3), NH
(Aib9), NH (Leu)); 7.49, 7.47 (2s, NH (Iva)); 7.34–7.17 (m, 10 arom. H, NH (Aib8)); 4.35–4.29 (m, CH(2)
(Phe)); 4.04–4.02 (m, CH(2) (Leu)); 3.75–3.56 (m, CH2 (Gly)); 3.23 (s, MeN); 2.96, 2.82 (AB of ABMX,
JAB ¼13.7, JAM ¼8.9, JBM ¼6.0, JBX ¼2.5, PhCH2); 2.04–1.91 (m, 1 H of MeCH2 (Iva)); 1.81 (s, MeCO);
1.76–1.44 (m, 1 H of MeCH2 (Iva), CH(4) (Leu), CH2(3) (Leu)); 1.37, 1.35, 1.32, 1.300, 1.296, 1.27, 1.26,
1.25 (8s, 5 Me2C, Me (Iva)); 0.88 (d, J¼5.9, (Me (Leu)); 0.84 (d, J¼5.5, Me (Leu)); 0.80–0.74 (m,
MeCH2 (Iva)). 13C-NMR ((D6)DMSO): 175.8, 175.7, 175.5, 175.21, 175.18, 175.0, 173.5, 172.63, 172.58,
171.7, 170.5, 170.1 (12s, 10 CO (amide)); 146.2, 137.6 (2s, 2 arom. C); 129.4, 128.8, 128.2, 127.2, 126.5, 126.2
(6d, 10 arom. CH); 59.3, 59.2 (2s, C(2) (Iva)); 56.5, 56.4, 56.1, 56.0 (4s, 5 Me2C); 55.4 (d, C(2) (Phe)); 52.5
(d, C(2) (Leu)); 43.4 (t, C(2) (Gly)); 39.7 (q, MeN); 37.4 (t, C(3) (Leu)); 36.5 (t, C(3) (Phe)); 29.2, 28.8
(2t, MeCH2 (Iva)); 25.83, 25.75, 25.6, 25.4, 25.2, 24.9, 24.7, 24.2, 23.9, 23.7, 23.0, 22.5, 21.6, 21.4, 21.3
(d, C(4) (Leu), 14q, 5 Me2C, Me2CH, Me (Iva), MeCO); 7.77, 7.67 (2q, MeCH2 (Iva)). FAB-MS: 1013
(5, [MþNa]þ ), 886 (14), 885 (36, [MꢀPh(Me)Nþ1]þ ), 884 (65, [MꢀPh(Me)N]þ ), 800 (11, [884ꢀ
Aibþ1]þ ), 799 (18, [884ꢀAib]þ ), 714 (10, [799ꢀAibþ1]þ ), 601 (18, [714ꢀLeu]þ ), 545 (25, [601–
Glyþ1]þ ), 544 (65, [601–Gly]þ ), 446 (27, [544–Ivaþ1]þ ), 445 (100, [544–Iva]þ ). Anal. calc. for
C51H78N10O10 (991.24): C 61.80, H 7.93, N 12.13; found: C 61.60, H 8.22, N 13.93.
4.5. Hydrolysis of 14 to Give Ac-Phe-Aib-Aib-Aib-d,l-Iva-Gly-Leu-Aib-Aib-OH (15). According to
GP 2, a soln. of 14 (170 mg, 0.17 mmol) in 3n HCl (2 ml) was stirred at r.t. for 10 h. Then, 2n HCl (1 ml)
was added, and the mixture was extracted with CH2Cl2 (4ꢁ ). The combined org. phases were dried
(Na2SO4), the solvent was evaporated, and the product precipitated by addition of Et2O: 103 mg (67%)
of 15. Colorless crystals. M.p. 208.0–210.58. [a]2D2 ¼ þ7.3 (c¼0.30, EtOH). 1H-NMR ((D6)DMSO): 12.05
(br. s, COOH); 8.63, 8.62 (2s, NH (Aib2)); 8.31 (d, J¼5.9, NH (Phe)); 7.93 (dd-like, J¼5.9, 5.4, NH
(Gly)); 7.71 (s, NH (Aib4)); 7.61, 7.59 (2s, NH (Aib3), NH (Aib9), NH (Leu)); 7.51, 7.49 (2s, NH (Iva));
7.30–7.20 (m, 5 arom. H); 7.04 (s, NH (Aib8)); 4.34 (M of ABMX, JMX ¼6.0, JMA ¼8.6, JMB ¼6.1, CH(2)
(Phe)); 4.10–4.03 (m, CH(2) (Leu)); 3.76–3.58 (m, CH2 (Gly)); 2.98, 2.83 (AB of ABMX, JAB ¼13.7,
JAM ¼9.0, JBM ¼6.0, JBX ¼3.0, PhCH2); 2.04–1.96 (m, 1 H of MeCH2 (Iva)); 1.83 (s, MeCO); 1.75–1.47
(m, 1 H of MeCH2 (Iva), CH(4) (Leu), CH2(3) (Leu)); 1.39, 1.37, 1.35, 1.33, 1.31, 1.30, 1.29, 1.27 (8s, 5
Me2C, Me (Iva)); 0.89 (d, J¼5.7, Me (Leu)); 0.83 (d, J¼6.1, Me (Leu)); 0.80–0.75 (m, MeCH2 (Iva)).
13C-NMR ((D6)DMSO): 175.8, 175.7, 175.6, 175.31, 175.27, 175.1, 173.2, 172.6, 171.7, 170.7, 170.0 (11s,
COOH, 9 CO (amide)); 137.6 (s, arom. C); 129.4, 128.3, 126.6 (3d, 5 arom. CH); 59.4, 59.3 (2s, C(2)
(Iva)); 56.2, 56.1, 55.0 (3s, 5 Me2C); 55.4 (d, C(2) (Phe)); 52.4 (d, C(2) (Leu)); 43.5 (t, C(2) (Gly)); 38.5
(t, C(3) (Leu)); 36.6 (t, C(3) (Phe)); 29.3, 28.9 (2t, MeCH2 (Iva)); 25.6, 25.5, 25.2, 24.9, 24.7, 24.3, 23.9,
23.7, 23.1, 22.5, 21.6, 21.4, 21.3 (d, C(4) (Leu), 12q, 5 Me2C, Me2CH, Me (Iva), MeCO); 7.85, 7.69
(2q, MeCH2 (Iva)). FAB-MS: 903 (7), 902 (15, [Mþ1]þ ), 886 (12), 800 (12), 799 (25, [MꢀH2OꢀAibþ
1]þ ), 714 (11, [798ꢀAibþ1]þ ), 601 (14, [714ꢀLeu]þ ), 545 (17, [601–Glyþ1]þ ), 544 (50, [601–Gly]þ ),
446 (27, [544–Ivaþ1]þ ), 445 (58, [544–Iva]þ ), 361 (21, [445ꢀAibþ1]þ ), 360 (81, [445ꢀAib]þ ), 276
(11, [360ꢀAibþ1]þ ), 275 (49, [360ꢀAib]þ ), 190 (12, [275ꢀAib]þ ), 171 (16), 169 (14), 162 (27), 155
(14), 141 (12), 128 (12), 127 (18), 126 (12), 120 (62), 112 (15). Anal. calc. for C44H71N9O11 (902.10): C
58.58, H 7.93, N 13.97; found: C 58.29, H 8.21, N 13.88.
5. Synthesis of the Heptapeptide Z-Hyp-Gln-d,l-Iva-Hyp-Aib-Pro-Pheol (34). 5.1. Z-Hyp(tBu)-Aib-
N(Me)Ph (20). According to GP 1, Z-Hyp(tBu)-OH (19, 2.51 g, 7.8 mmol) in CH2Cl2 (30 ml) at 08 was

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
treated with 1a (1.70 g, 9.75 mmol); stirring at r.t. for 17.5 h. The solvent was evaporated, and the residue
was purified by CC (AcOEt/hexane 3 :2!4 :2): 3.73 g (96%) of 11. Colorless foam. M.p. 53.5–54.28.
[a]2D1 ¼ ꢀ22.1 (c¼1.09, EtOH). IR (KBr): 3440w, 2990w, 2970w, 1685s, 1635m, 1590m, 1530w, 1490m,
1450m, 1410m, 1385m, 1355m, 1290w, 1240w, 1180m, 1120m, 1065m, 1020w, 900w, 700w. 1H-NMR
(CD3OD; two conformers): 7.43–7.12 (m, 10 arom. H); 5.17–5.00 (m, PhCH2O); 4.43–4.18 (m, CH(2)
(Hyp), CH(4) (Hyp)); 3.72–3.60, 3.43–3.23 (2m, CH2(5) (Hyp)); 3.15 (s, MeN); 2.20–1.98 (m, CH2(3)
(Hyp)); 1.46, 1.43, 1.35 (3s, Me2C); 1.20, 1.18 (2s, tBu). 13C-NMR (CD3OD; two conformers): 175.0, 173.7,
173.6 (3s, 2 CO (amide)); 156.8, 156.4 (2s, CO (urethane)); 146.4, 146.3, 138.1, 137.7 (4s, 2 arom. C);
130.3, 129.5, 129.0, 128.7 (4d, 10 arom. CH); 75.3 (s, Me3C); 70.7, 70.0 (2d, C(4) (Hyp)); 68.3, 68.1 (2t,
PhCH2O); 60.2, 60.0 (2d, C(2) (Hyp)); 58.5, 58.3 (2s, Me2C); 55.7, 55.1 (2t, C(5) (Hyp)); 41.2, 41.0 (2q,
MeN); 40.4, 39.1 (2t, C(3) (Hyp)); 28.5 (q, Me3C); 26.8, 26.6, 26.4, 26.3 (4q, Me2CH). ESI-MS: 534 (9,
[MþK]þ ), 518 (100, [MþNa]þ ). Anal. calc. for C28H37N3O5 (495.62): C 67.86, H 7.53, N 8.48; found: C
67.95, H 7.62, N 8.32.
5.2. Z-Hyp(tBu)-Aib-OH (21). According to GP 2, a soln. of 20 (2.95 g, 5.95 mmol) in 3n HCl
(60 ml) was stirred at r.t. for 23 h. Then, 2n HCl (3.5 ml) was added, and the mixture was extracted with
CH2Cl2 (4ꢁ ). The combined org. phases were dried (Na2SO4), the solvent was evaporated, and the
residue was recrystallized from CH2Cl2/hexane: 2.10 g (86%) of 21. Colorless crystals. M.p. 144.1–145.28.
[a]2D2 ¼ ꢀ32.5 (c¼1.01, EtOH). IR (KBr): 3430w, 3390w, 3220w, 2980m, 2880w, 1690s, 1545w, 1515w,
1455m, 1420m, 1390w, 1355m, 1310w, 1260w, 1170m, 1130m, 1100m, 1070w, 998w, 915w, 900w, 695w.
1H-NMR (CDCl3 ; two conformers): 7.35 (br. s, 5 arom. H); 5.30–5.02 (m, PhCH2O); 4.55–4.22 (m,
CH(2) (Hyp), CH(4) (Hyp)); 3.72–3.55, 3.55–3.35, 3.35–3.20 (3m, CH2(5) (Hyp)); 2.50–2.32, 2.28–
t
2.02, 2.02–1.83 (3m, CH2(3) (Hyp)); 1.54, 1.52, 1.50 (3s, Me2C); 1.18 (s, Bu). 13C-NMR (CD3OD; two
conformers): 177.6, 177.5, 174.1, 173.9 (4s, COOH, CO (amide)); 156.7, 156.3 (s, CO (urethane)); 137.9,
137.8 (2s, arom. C); 129.5, 129.4, 128.9 (3d, 5 arom. CH); 75.3 (s, Me3C); 70.6, 70.0 (2d, C(4) (Hyp)); 68.5,
68.2 (2t, PhCH2O); 60.3, 60.2 (2d, C(2) (Hyp)); 57.0, 56.9 (2s, Me2C); 55.4, 54.0 (2t, C(5) (Hyp)); 40.0,
38.9 (2t, C(3) (Hyp)); 28.5 (q, Me3C); 25.8, 24.7 (2q, Me2CH). CI-MS: 407 (100, [Mþ1]þ ). Anal. calc.
for C21H30N2O6 (406.48): C 62.05, H 7.44, N 6.89; found: C 62.14, H 7.18, N 7.09.
5.3. H-Hyp(tBu)-Aib-N(Me)Ph (22). According to GP 3, a mixture of 20 (700 mg, 1.41 mmol) in
MeOH (15 ml) and Pd/C (91 mg) under H2 was stirred at r.t. for 23 h. The crude product was purified by
CC (AcOEt/hexane 95 :5) to give 22 (463 mg, 90%). Yellowish resin. [a]2D1 ¼ ꢀ30.2 (c¼1.24, EtOH). IR
(KBr): 3320w, 2960m, 2920w, 2860w, 1660s, 1635s, 1590m, 1510m, 1490m, 1455m, 1415w, 1385m, 1360m,
1290w, 1240w, 1185m, 1115m, 1090m, 1070m, 1020m, 995m, 900w, 700w. 1H-NMR (CDCl3): 7.50–7.20 (m,
5 arom. H); 4.19 (br. s, CH(2) (Hyp)); 3.52–3.35 CH(4) (Hyp)); 3.23 (s, MeN); 3.00 (dd, J¼11.3, 4.8, 1 H
of CH2(5) (Hyp)); 2.68 (dd, J¼11.3, 3.0, 1 H of CH2(5) (Hyp)); 2.05–1.85, 1.85–1.60 (2m, CH2(3)
t
(Hyp)); 1.47, 1.46 (2s, Me2C); 1.17 (s, Bu). 13C-NMR (CD3OD; two conformers): 175.5, 175.3, 174.8,
174.7 (4s, 2 CO (amide)); 146.7 (s, arom. C); 130.6, 130.5, 128.8, 128.4 (4d, 5 arom. CH); 74.7, 74.6 (2s,
Me3C); 70.9, 70.8 (2d, C(4) (Hyp)); 60.7, 60.6 (2d, C(2) (Hyp)); 58.6, 58.0 (2s, Me2C); 55.5 (t, C(5)
(Hyp)); 41.8, 41.6 (2 br. q, MeN); 40.4, 39.7 (2t, C(3) (Hyp)); 28.7 (q, Me3C); 27.4, 27.1, 26.8, 26.5 (4q,
Me2CH). ESI-MS: 384 (15, [MþNa]þ ), 362 (100, [Mþ1]þ ); FAB-MS: 362 (100, [Mþ1]þ ). Anal. calc.
for C20H31N3O3 (361.49): C 66.45, H 8.64, N 11.62; found: C 66.17, H 8.45, N 11.39.
5.4. Z-Pro-Pheol (23). To a stirred soln. of Z-Pro-OH (4.00 g, 16.06 mmol) in THF (80 ml) at ꢀ98,
NMM (1.77 ml, 16.06 mmol) and isobutyl chloroformate (2.09 ml, 16.00 mmol) were added. After 6 min
stirring at ꢀ58, H-PheOH (2.57 mg, 16.99 mmol) was added. The mixture was stirred for 10 min at ꢀ58
and for 20 h at r.t. Then, the solvent was evaporated, the residue was dissolved in CH2Cl2, and the soln.
extracted with 2n HCl (2ꢁ ) and 1n NaOH (2ꢁ ), dried (Na2SO4), and evaporated: 5.86 g (95%) of 23.
Colorless crystals. M.p. 135.1–135.48. [a]2D1 ¼ ꢀ67.0 (c¼1.02, EtOH). IR (KBr): 3420w, 3000w, 2960w,
2890w, 1680s, 1515m, 1500w, 1450w, 1420m, 1355m, 1180w, 1115m, 1090w, 1040w, 985w, 920w, 700m.
1H-NMR (CD3OD): 7.45–7.05 (m, 10 arom. H); 5.16, 5.10 (AB, J¼12.6, PhCH2O); 5.01 (s, PhCH2O);
4.28–4.02 (m, CH(2) (Pro), CH(2) (Pheol)); 3.58–3.35 (m, CH2(5) (Pro), CH2OH); 3.00–2.55 (m,
CH2(3) (Pheol)); 2.25–1.92, 1.92–1.60 (2m, 1 H and 3 H, resp., CH2(3) (Pro), CH2(4) (Pro)). 13C-NMR
(CD3OD; two conformers): 174.9, 174.6 (2s, CO (amide)); 156.5 (s, CO (urethane)); 139.7, 137.9 (2s, 2
arom. C); 130.3, 129.3, 129.0, 127.3 (4d, 10 arom. CH); 68.2, 68.1 (2t, PhCH2O); 63.9 (t, CH2OH); 62.1,
61.6 (2d, C(2) (Pro)); 54.2, 53.9 (2d, C(2) (Pheol)); 48.6, 48.2 (2t, C(5) (Pro)); 37.7 (t, C(3) (Pheol)); 32.6,

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
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31.3, 25.1, 24.2 (4t, C(3) (Pro), C(4) (Pro)). CI-MS: 401 (21), 400 (100, [Mþ1þNH3]þ ), 384 (15), 383
(71, [Mþ1]þ ). Anal. calc. for C22H26N2O4 (382.46): C 69.09, H 6.85, N 7.32; found: C 69.03, H 6.83, N
7.16.
5.5. H-Pro-Pheol (24). According to GP 3, a mixture of 23 (3.00 g, 7.85 mmol) in MeOH (70 ml) and
Pd/C (412 mg) under H2 was stirred at r.t. for 21 h. The crude product was crystallized from CH2Cl2/
hexane to give 24 (1.79 g, 91%). Colorless crystals. [a]2D2 ¼ ꢀ54.1 (c¼0.99, EtOH). IR (KBr): 3350m,
3260m, 3060w, 3020w, 2960w, 2940m, 2860m, 1650s, 1600w, 1520m, 1480w, 1455m, 1435w, 1300w, 1240w,
1220w, 1100w, 1080w, 1055m, 1000w, 920w, 740m, 700m. 1H-NMR (CD3OD): 7.32–7.10 (m, 5 arom. H);
4.18–4.05 (m, CH(2) (Pheol)); 3.62–3.45 (m, CH(2) (Pro), CH2OH); 3.01–2.65 (m, CH2(3) (Pheol),
CH2(5) (Pro)); 2.10–1.90, 1.71–1.41 (2m, CH2(3) (Pro), CH2(4) (Pro)). 13C-NMR (CDCl3): 176.1 (s, CO
(amide)); 137.7 (s, arom. C); 129.1, 128.4, 126.4 (3d, 5 arom. CH); 65.1 (t, CH2OH); 60.3 (d, C(2) (Pro));
52.7 (d, C(2) (Pheol)); 47.0 (t, C(5) (Pro)); 37.0 (t, C(3) (Pheol)); 30.5 (t, C(3) (Pro)); 25.8 (t, C(4)
(Pro)). CI-MS: 249 (100, [Mþ1]þ ). Anal. calc. for C14H20N2O2 (248.33): C 67.72, H 8.12, N 11.28; found:
C 67.58, H 7.95, N 10.99.
5.6. Z-Hyp(tBu)-Aib-Pro-Pheol (25). To a soln. of 21 (1.75 g, 4.31 mmol) in DMF (7 ml) at 08 was
added DCC (889 mg, 4.31 mmol), and the mixture was stirred for 6 min. Then, HOBt (643 mg,
7.76 mmol), CSA (60 mg), and 24 (1.23 g, 4.95 mmol) were added, and the mixture was stirred at r.t. for
18.5 h. Usual workup (see 5.4) and CC (AcOEt/MeOH 95 :5) afforded 25 (2.16 g, 78%). Colorless foam.
M.p. 73.1–74.08. [a]2D1 ¼ ꢀ37.4 (c¼1.03, EtOH). IR (KBr): 3450w, 3400w, 3320w, 3050w, 2980w, 2870w,
1675s, 1655s, 1540m, 1530m, 1495w, 1465m, 1450m, 1410m, 1360m, 1350m, 1305w, 1240w, 1170m, 1125m,
1100m, 1065m, 970w, 945w, 910w, 695m. 1H-NMR (CDCl3 ; two conformers): 8.03, 7.65 (2s, 2 NH); 7.47 (d,
J¼8.6, NH); 7.45–7.15 (m, 10 arom. H); 5.23, 5.17 (AB, J¼12.5, PhCH2O); 4.63–4.43, 4.43–4.18 (2m,
CH(2) (Hyp), CH(4) (Hyp), CH(2) (Pro), CH(2) (Pheol)); 3.95–3.66, 3.66–3.46, 3.46–3.18 (3m,
CH2(3) (Pheol), CH2OH, CH2(5) (Hyp)); 3.02–2.79 (m, CH2(5) (Pro)); 2.53–2.24, 2.08–1.92, 1.92–
1.72, 1.72–1.52 (4m, CH2(3) (Hyp), CH2(3) (Pro), CH2(4) (Pro)); 1.41, 1.37 (2s, Me2C); 1.18, 1.17 (2s,
tBu). 13C-NMR (CD3OD; two conformers): 174.6, 174.4, 174.3, 174.2, 174.1, 174.0 (6s, 3 CO (amide));
157.0, 156.4 (2s, CO (urethane)); 139.9, 138.1, 137.8 (3s, 2 arom. C); 130.3, 130.2, 129.5, 129.3, 129.1, 128.7,
127.3 (7d, 10 arom. CH); 75.4, 75.3 (2s, Me3C); 70.9, 70.2 (2d, C(4) (Hyp)); 68.3, 68.2 (2t, PhCH2O); 64.3,
64.2 (2t, CH2OH); 64.1 (d, C(2) (Pro)); 60.8, 60.0 (2d, C(2) (Hyp)); 57.9 (s, Me2C); 56.1, 55.5 (2t, C(5)
(Hyp)); 54.1, 53.9 (2d, C(2) (Pheol)); 49.6 (t, C(5) (Pro)); 40.8, 39.4 (2t, C(3) (Hyp)); 37.6, 37.5 (2t, C(3)
(Pheol)); 29.8, 26.5 (2t, C(3) (Pro), C(4) (Pro)); 28.6 (q, Me3C); 26.3, 26.1, 24.2, 24.0 (4q, Me2C). ESI-
MS: 675 (11, [MþK]þ ), 659 (100, [MþNa]þ ). Anal. calc. for C35H48N4O7 (636.79): C 66.02, H 7.60, N
8.80; found: C 65.79, H 7.74, N 8.83.
5.7. H-Hyp(tBu)-Aib-Pro-Pheol (18). According to GP 3, a mixture of 25 (802 mg, 1.26 mmol) in abs.
MeOH (10 ml) and Pd/C (104 mg) under H2 was stirred at r.t. for 25 h. After filtration, the solvent was
evaporated, and the product 18 was dried i.v. (633 mg, quant.). Colorless foam: M.p. 52.3–53.18. IR
(KBr): 3440w, 3380w, 3050w, 3060w, 3005m, 2980m, 2870w, 1655s, 1535m, 1510m, 1470w, 1455w, 1405m,
1365m, 1340w, 1305w, 1240w, 1190m, 1170w, 1150w, 1095w, 1070w, 1040w, 1000w, 945w, 900w, 865w, 700w.
1H-NMR (CDCl3 ; two conformers): 7.42 (s, NH); 7.30–7.05 (m, 5 arom. H); 4.85–4.70, 4.70–4.55, 4.55–
4.28, 4.28–4.05 (4m, CH(2) (Hyp), CH(4) (Hyp), CH(2) (Pro), CH(2) (Pheol)); 3.95–3.40, 3.40–3.15
(2m, CH2(3) (Pheol), CH2OH, CH2(5) (Hyp)); 2.97–2.75 (m, CH2(5) (Pro)); 2.45–1.90, 1.90–1.35 (2m,
t
CH2(3) (Hyp), CH2(3) (Pro), CH2(4) (Pro), Me2C); 1.18, 1.16 (2s, Bu). 13C-NMR (CD3OD; two
conformers): 176.2, 175.9, 174.4, 174.0 (4s, 3 CO (amide)); 140.0 (s, arom. C); 130.3, 129.2, 127.3 (3d, 5
arom. CH); 74.9 (s, Me3C); 73.6 (d, C(4) (Hyp)); 64.6, 64.4 (2t, CH2OH); 64.1, 64.0 (2d, C(2) (Pro)); 60.8
(d, C(2) (Hyp)); 57.8, 57.5 (2s, Me2C); 55.9 (t, C(5) (Hyp)); 53.9, 53.8 (2d, C(2) (Pheol)); 49.7 (t, C(5)
(Pro)); 40.7, 40.5 (2t, C(3) (Hyp)); 37.5 (t, C(3) (Pheol)); 29.7, 26.5 (2t, C(3) (Pro), C(4) (Pro)); 28.8,
28.7 (2q, Me3C); 26.4, 24.2 (2q, Me2C). ESI-MS: 541 (15, [MþK]þ ), 525 (100, [MþNa]þ ).
5.8. Z-Hyp(tBu)-Gln(Dod)-OMe (27). A mixture of Z-Hyp(tBu)-OH (19, 204 mg, 0.64 mmol) and
DCC (130 mg, 0.63 mmol) in DMF (3 ml) was stirred at 08 for 6 min. Then, HOBt (96 mg, 0.71 mmol),
CSA (10 mg), and a soln. of H-Gln(Dod)-OMe·HCl (26, 304 mg, 0.72 mmol) and Et3N in DMF (1 ml)
were added. The mixture was stirred for 19 h at r.t. The solvent was evaporated, the residue was dissolved
in CH2Cl2, the soln. was extracted with 2n HCl (2ꢁ ) and 1n NaOH (2ꢁ ), dried (Na2SO4), and purified
by CC (AcOEt/hexane 4 :1): 385 mg (88%) of 27. Colorless foam. M.p. 62.8–63.78. [a]2D1 ¼ ꢀ20.0 (c¼

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
1.15, EtOH). IR (KBr): 3430w, 3340w, 3000w, 2980w, 2840w, 1740m, 1680s, 1610w, 1585w, 1510s, 1465w,
1455w, 1420m, 1390w, 1355m, 1305m, 1250m, 1175m, 1120m, 1085m, 1065w, 1035m, 830w, 700w. 1H-NMR
(CDCl3; two conformers): 7.36–7.04, 6.85–6.68 (2m, 14 H); 6.14 (d, J¼8.1, 1 H); 5.15–5.00, 4.82, 4.61
(m, AB, J¼12.6, PhCH2O); 4.43–4.27 (m, 1 H); 4.24 (dd, J¼8.1, 4.8, 1 H); 3.82–3.58 (m, 10 H); 3.25
t
(dd, J¼10.7, 5.1, 1 H); 2.47–1.83 (m, CH2(3) (Hyp), CH2(4) (Gln), CH2(3) (Gln)); 1.17 (s, Bu).
13C-NMR (CDCl3): 172.0, 171.9, 171.0 (3s, 2 CO (amide), 1 CO (ester)); 158.6, 158.5 (2s, 2 arom. C
(Dod)); 155.3 (s, CO (urethane)); 136.1, 134.2, 134.1 (3s, 3 arom. C); 128.5, 128.4, 128.3, 127.9, 126.6 (5d,
9 arom. CH); 113.7 (d, 4 arom. H (Dod)); 73.9 (s, Me3C); 69.3 (d, C(4) (Hyp)); 67.0 (t, PhCH2O); 59.3 (d,
C(2) (Hyp)); 55.5 (d, CH (Dod)); 55.1 (q, 2 MeO (Dod)); 53.5 (t, C(5) (Hyp)); 52.3 (q, MeO (Gln));
51.7 (d, C(2) (Gln)); 37.3 (t, C(3) (Hyp)); 32.0, 28.4 (2t, C(4) (Gln), C(3) (Gln)); 28.1 (q, Me3C). CI-MS:
690 (6, [Mþ1]þ ), 227 (100). Anal. calc. for C38H47N3O9 (689.81): C 66.17, H 6.87, N 6.09; found: C 66.11,
H 6.95, N 5.96.
5.9. Z-Hyp(tBu)-Gln(Dod)-OH (28). To a soln. of 27 (401 mg, 0.58 mmol) in dioxane (4.5 ml), was
added 1n NaOH (4.5 ml) and the mixture was stirred at r.t. for 4.5 h. Then, excess 2n HCl was added, and
the mixture was extracted with CH2Cl2 (3ꢁ ). The org. phase was dried (Na2SO4), the solvent was
evaporated, and the residue was dried in high vacuum: 392 mg (quant.) of 28. Colorless foam. M.p. 72.6–
73.68. IR (KBr): 3420w, 3310w, 3060w, 3030w, 3000m, 2980m, 2940w, 2840w, 1680s (br.), 1660m, 1590w,
1510s, 1465m, 1455m, 1440m, 1425m, 1390w, 1355m, 1305w, 1250m, 1175m, 1120w, 1085w, 1065w, 1035m,
915w, 830w, 695w. 1H-NMR (CDCl3; two conformers): 7.79 (d, J¼8.4, NH); 7.38–7.16, 7.16–7.03, 6.88–
6.68 (3m, 14 H); 6.18 (d, J¼8.5, 1 H); 4.63–4.47, 4.37–4.17 (2m, PhCH2O, CH(2) (Gln), CH(2) (Hyp),
CH(4) (Hyp)); 3.72, 3.64 (2s, 2 MeO); 3.80–3.57 (m, 1 H of CH2(5) (Hyp)); 3.25 (dd, J¼10.9, 3.9, 1 H of
CH2(5) (Hyp)); 2.72–2.54, 2.54–2.32, 2.32–2.00 (3m, CH2(3) (Hyp), CH2(4) (Gln), CH2(3) (Gln)); 1.15
(s, tBu). 13C-NMR (CD3OD; two conformers): 175.2, 174.9, 174.6, 174.4, 173.9, 173.6 (6s, 2 CO (amide), 1
CO (ester)); 160.2 (s, 2 arom. C (Dod)); 156.6, 156.4 (2s, CO (urethane)); 137.7, 135.3, 135.2 (3s, 3 arom.
C); 129.7, 129.5, 129.1, 128.8 (4d, 9 arom. CH); 114.8 (d, 4 arom. CH (Dod)); 75.3 (s, Me3C); 70.8, 70.0
(2d, C(4) (Hyp)); 68.2 (t, PhCH2O); 60.4, 60.1 (2d, C(2) (Hyp)); 57.1, 57.0 (2d, CH (Dod)); 55.7 (q,
2 MeO); 55.5 (t, C(5) (Hyp)); 53.4, 52.9 (2d, C(2) (Gln)); 40.2, 39.2 (2t, C(3) (Hyp)); 33.4, 33.1 (2t, C(4)
(Gln)); 28.9 (t, C(3) (Gln)); 28.5 (q, Me3C). ESI-MS: 714 (7, [MþK]þ ), 698 (100, [MþNa]þ ).
5.10. Z-Hyp(tBu)-Gln(Dod)-d,l-Iva-N(Me)Ph (29). According to GP 1, 28 (1.20 g, 1.76 mmol) in
CH2Cl2 (15 ml) at 08 was treated with 1b (468 mg, 2.49 mmol); stirring at r.t. for 21 h. The solvent was
evaporated, and the residue was purified by CC (AcOEt/hexane 95 :5!100 :0): 1.23 g (80%) of 29.
Colorless foam. M.p. 95.7–96.58. IR (KBr): 3420w, 3330w, 3060w, 3000m, 2970m, 2930w, 2880w, 2840w,
1665s (br.), 1610m, 1595m, 1510s, 1465m, 1455m, 1420m, 1390w, 1360m, 1305w, 1245m, 1175m, 1120w,
1075w, 1030w, 970w, 900w, 830w, 810w, 700w. 1H-NMR (CD3OD; two conformers): 7.43–7.07 (m, 14
arom. H); 6.90–6.79 (m, 5 H); 6.12–6.02 (m, 1 H); 5.10–4.86 (m, PhCH2O); 4.50–4.25 (m, 2 H); 4.20–
4.03 (m, 1 H); 3.75, 3.74, 3.72, 3.70, 3.69 (5s, 2 MeO); 3.80–3.56, 3.42–3.30 (2m, CH2(5) (Hyp)); 3.22,
3.21 (2s, MeN); 2.65–2.30, 2.30–1.96, 1.96–1.58 (3m, CH2(3) (Hyp), MeCH2 (Iva), CH2(4) (Gln),
CH2(3) (Gln)); 1.41, 1.39, 1.33 (3s, MeC(2) (Iva)); 1.16 (s, tBu); 0.92–0.76 (m, MeCH2 (Iva)). 13C-NMR
(CD3OD; two conformers): 174.8, 174.7, 174.6, 174.1, 172.3, 172.2 (6s, 4 CO (amide)); 160.2 (s, 2 arom. C
(Dod)); 156.9, 156.3 (2s, CO (urethane)); 146.0 (br. s, 1 arom. C); 137.8, 137.7, 135.4, 135.2 (4s, 3 arom. C);
130.6, 130.5, 129.8, 129.0, 128.6 (5d, 14 arom. CH); 114.9 (d, 4 arom. CH (Dod)); 75.3 (s, Me3C); 70.8, 70.4
(2d, C(4) (Hyp)); 68.4 (t, PhCH2O); 62.0, 61.7 (2s, C(2) (Iva)); 60.8, 60.0 (2d, C(2) (Hyp)); 57.1, 57.0 (2d,
CH (Dod)); 55.7 (q, 2 MeO); 55.7 (t, C(5) (Hyp)); 53.8, 53.7 (2d, C(2) (Gln)); 41.5 (br. q, MeN); 40.4,
39.5 (2t, C(3) (Hyp)); 32.8, 31.4 (2t, C(4) (Gln)); 30.7, 29.3, 29.2, 28.8 (4t, C(3) (Gln), MeCH2 (Iva)); 28.6
(q, Me3C); 23.4, 23.3 (2q, MeC(2) (Iva)); 8.6, 8.4 (2q, MeCH2 (Iva)). ESI-MS: 902 (8, [MþK]þ ), 866
(100, [MþNa]þ ). Anal. calc. for C49H61N5O9 (864.06): C 68.11, H 7.12, N 8.11; found: C 68.10, H 7.22, N
7.97.
5.11. Z-Hyp(tBu)-Gln(Dod)-d,l-Iva-OH (30). According to GP 2, a soln. of 29 (1.10 g, 1.27 mmol)
in 3n HCl (20 ml) was stirred at r.t. for 24 h. Then, 2n HCl (3.5 ml) was added, and the mixture was
extracted with CH2Cl2 (4ꢁ ). The combined org. phases were dried (Na2SO4), the solvent was
evaporated, and the residue was recrystallized from CH2Cl2/hexane: 939 mg (95%) of 30. Colorless
crystals. M.p. 103.4–104.38. IR (KBr): 3320w, 3060w, 3020w, 3000m, 2970m, 2930w, 2830w, 1680s, 1660s,
1610m, 1585m, 1510s, 1465m, 1455m, 1420m, 1390w, 1355m, 1305w, 1245m, 1175m, 1130w, 1085w, 1065w,

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
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1
1035w, 870w, 830w, 810w, 695w. H-NMR (CDCl3; two conformers): 8.34 (br. s, NH); 7.85 (d, J¼8.9,
NH); 7.38–7.00, 6.91–6.68 (2m, 14 arom. H); 6.07 (d, J¼6.0, 1 H); 5.13–4.72, 4.72–4.18 (2m, PhCH2O),
CH(2) (Gln), CH(2) (Hyp), CH(4) (Hyp)); 3.88–3.61 (m, 1 H of CH2(5) (Hyp), 2 MeO)); 3.41–3.24
(m, 1 H of CH2(5) (Hyp)); 2.68–2.36, 2.36–1.67, 1.55–1.20 (3m, CH2(3) (Hyp), MeCH2 (Iva), CH2(4)
t
(Gln), CH2(3) (Gln), MeC(2) (Iva)); 1.15, 1.14 (2s, Bu); 0.98–0.64 (m, MeCH2 (Iva)). 13C-NMR
(CD3OD; two conformers): 176.9, 176.8, 174.7, 174.1, 173.6, 172.3 (6s, 4 CO (amide)); 160.0 (s, 2 arom. C
(Dod)); 156.7 (s, CO (urethane)); 137.5, 137.4, 135.2, 134.9 (4s, 3 arom. C); 129.6, 129.2, 129.1, 128.9,
128.6, 128.4 (6d, 9 arom. CH); 114.2 (d, 4 arom. CH (Dod)); 75.2 (s, Me3C); 70.6, 69.8 (2d, C(4) (Hyp));
68.2, 68.1 (2t, PhCH2O); 61.1, 60.9 (2s, C(2) (Iva)); 60.6, 59.8 (2d, C(2) (Hyp)); 56.9 (d, CH (Dod)); 55.6
(q, 2 MeO); 55.2 (t, C(5) (Hyp)); 54.4, 54.0 (2d, C(2) (Gln)); 40.0, 39.2 (2t, C(3) (Hyp)); 33.0, 32.8 (2t,
C(4) (Gln)); 30.8, 30.5, 30.2, 28.7 (4t, C(3) (Gln), MeCH2 (Iva)); 28.5 (q, Me3C); 22.5, 22.3 (2q, MeC(2)
(Iva)); 8.6, 8.5 (2q, MeCH2 (Iva)). ESI-MS: 813 (12, [MþK]þ ), 797 (100, [MþNa]þ ), 741 (22). Anal.
calc. for C42H54N4O10 (774.92): C 65.10, H 7.02, N 7.23; found: C 64.89, H 7.31, N 7.18.
5.12. Z-Hyp(tBu)-Gln(Dod)-d,l-Iva-Hyp(tBu)-Aib-N(Me)Ph (31). To a soln. of 30 (100 mg,
0.13 mmol) in CH2Cl2 (2 ml) at 08 was added EtNiPr2 (Hꢀnigꢃs base; 34 mg, 0.26 mmol). After stirring for
3 min, TBTU (43 mg, 0.13 mmol), HOBt (21 mg, 0.16 mmol), and 22 (55 mg, 0.15 mmol) were added,
and the mixture was stirred at r.t. for 48 h. Then, the solvent was evaporated, the residue was dissolved in
AcOEt, the soln. was extracted with 2n HCl (2ꢁ ) and 1n NaOH (2ꢁ ), dried (Na2SO4), and the product
was purified by PLC (CH2Cl2/MeOH 9 :1): 87 mg (60%) of 31. Pale-yellow solid. M.p. 103.6–104.68.
1H-NMR (CD3OD; two conformers): 7.42–7.02 (m, 14 arom. H); 6.92–6.78 (m, 4 arom. H); 6.12, 6.10 (2s,
1 H); 5.14–4.88 (m, PhCH2O); 4.60–4.46, 4.46–4.20, 4.20–4.03 (3m, CH(2) (Gln), 2 CH(2) (Hyp),
2 CH(4) (Hyp)); 3.75, 3.74, 3.721, 3.718 (4s, 2 MeO); 3.70–3.53, 3.46–3.34 (2m, 2 CH2(5) (Hyp)); 3.32 (s,
MeN); 2.57–2.40, 2.27–1.70 (2m, 2 CH2(3) (Hyp), MeCH2 (Iva), CH2(4) (Gln), CH2(3) (Gln)); 1.51,
1.50 (2s, Me2C (Aib)); 1.40, 1.25 (2s, MeC(2) (Iva)); 1.18, 1.16 (2s, 2 tBu); 0.82, 0.76 (2t, J¼7.5, MeCH2
(Iva)). ESI-MS: 1157 (24, [Mþ1þK]þ ), 1141 (100, [Mþ1þNa]þ ); FAB-MS: 1011 (100, [Mꢀ
Ph(Me)N]þ ).
5.13. Z-Hyp(tBu)-Gln(Dod)-d,l-Iva-Hyp(tBu)-Aib-OH (32). According to GP 2, a soln. of 31
(315 mg, 0.28 mmol) in 3n HCl (10 ml) was stirred at r.t. for 20 h. Usual workup and crystallization from
CH2Cl2/hexane gave 274 mg (94%) of 32. Colorless crystals. M.p. 111.3–112.28. 1H-NMR (CD3OD; two
conformers): 7.36–7.20, 7.20–7.05, 6.92–6.78 (3m, 13 arom. H); 6.11, 6.08, 6.07 (3s, 1 H); 5.06, 4.92 (AB,
J¼12.6, PhCH2O); 5.05 (s, PhCH2O); 4.55–4.43, 4.43–4.20, 4.20–4.08 (3m, CH(2) (Gln), 2 CH(2)
(Hyp), 2 CH(4) (Hyp)); 3.76, 3.75, 3.74, 3.72 (4s, 2 MeO); 3.72–3.33 (m, 2 CH2(5) (Hyp)); 2.57–2.38,
2.27–1.70 (2m, 2 CH2(3) (Hyp), MeCH2 (Iva), CH2(4) (Gln), CH2(3) (Gln)); 1.49, 1.48, 1.47, 1.41, 1.27
(5s, Me2C (Aib), MeC(2) (Iva)); 1.18, 1.17, 1.16 (3s, 2 tBu); 0.95–0.73 (m, MeCH2 (Iva)). ESI-MS: 1007
(24, [MþK]þ ), 1051 (100, [MþNa]þ ), 995 (14).
5.14. Z-Hyp(tBu)-Gln(Dod)-d,l-Iva-Hyp(tBu)-Aib-Pro-Pheol (33). To a soln. of 32 (219 mg,
0.21 mmol) in DMF (3 ml) at 08 was added DCC (45 mg, 0.22 mmol). After stirring for 6 min, HOBt
(33 mg, 0.24 mmol), CSA (8 mg), and 24 (62 mg, 0.25 mmol) were added, and the mixture was stirred at
r.t. for 43 h. Workup as described in Sect. 5.12 and purification by CC (AcOEt/MeOH 95 :5!9 :1) gave
163 mg (60%) of 33. Colorless foam. M.p. 122.8–123.38. 1H-NMR (CD3OD; two conformers): 7.36–7.20,
7.20–7.05, 6.92–6.80 (3m, 20 H); 6.12, 6.10 (2s, 1 H); 5.09, 4.96 (AB, J¼12.6, PhCH2O); 5.08 (s,
PhCH2O); 4.73–4.62, 4.44–4.20, 4.20–4.07 (3m, CH(2) (Gln), 2 CH(2) (Hyp), 2 CH(4) (Hyp), CH(2)
(Pheol), CH(2) (Pro)); 3.75, 3.74, 3.73, 3.72 (4s, 2 MeO); 3.70–3.52, 3.52–3.34 (2m, 2 CH2(5) (Hyp),
CH2(5) (Pro), CH2OH); 3.03–2.93, 2.80–2.68 (2m, CH2(3) (Pheol)); 2.58–2.42, 2.28–1.95, 1.95–1.75,
1.75–1.52 (4m, 2 CH2(3) (Hyp), MeCH2 (Iva), CH2(4) (Gln), CH2(3) (Gln), CH2(3) (Pro), CH2(4)
(Pro)); 1.48, 1.47, 1.45, 1.42, 1.41 (5s, Me2C (Aib)); 1.31, 1.28 (2s, MeC(2) (Iva)); 1.19, 1.17, 1.158, 1.156 (4s,
t
2 Bu); 0.92–0.82 (m, MeCH2 (Iva)); 0.79 (t, J¼7.5, MeCH2 (Iva)). ESI-MS: 1299 (12, [Mþ1þK]þ ),
1282 (100, [Mþ1þNa]þ ), 652 (66, [MþNa]2þ ).
5.15. Z-Hyp-Gln-d,l-Iva-Hyp-Aib-Pro-Pheol (34). To a soln. of 33 (63 mg, 0.05 mmol) in TFA
(1.2 ml) at 08 was added anisol (0.1 ml), and the soln. was stirred at r.t. for 4 h. Then, TFA was
evaporated, the residue was dissolved in CH2Cl2, and the solvent was evaporated again. This procedure
was repeated several times. The crude product was purified by prep. HPLC (LiChrosorb RP18, 7 mm,
MeCN/H2O 100 :0!97:3!9 :1, 8 ml/min): 43 mg (93%) of 34. Colorless crystals. M.p. 130.7–131.78.

938
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
1H-NMR (CD3OD; two conformers): 8.10–7.96 (m, NH); 7.92–7.82 (m, NH); 7.42–7.08 (m, 10 arom. H);
5.28–5.07 (m, PhCH2O); 4.78–4.66, 4.54–4.25, 4.18–4.07 (3m, CH(2) (Gln), 2 CH(2) (Hyp), 2 CH(4)
(Hyp), CH(2) (Pro), CH(2) (Pheol)); 3.95–3.72, 3.72–3.53, 3.53–3.43 (3m, 2 CH2(5) (Hyp), CH2(5)
(Pro), CH2OH); 3.05–2.82 (m, CH2(3) (Pheol)); 2.44–2.24, 2.24–2.08, 2.08–1.92, 1.92–1.75, 1.75–1.57
(5m, 2 CH2(3) (Hyp), MeCH2 (Iva), CH2(4) (Gln), CH2(3) (Gln), CH2(3) (Pro), CH2(4) (Pro)); 1.50,
1.49, 1.48, 1.42, 1.36, 1.28 (6s, Me2C (Aib), MeC(2) (Iva)); 1.00–0.75 (m, MeCH2 (Iva)). ESI-MS: 943
(100, [MþNa]þ ), 921 (21, [Mþ1]þ ), 483 (36, [MþNa]2þ ).
6. X-Ray Crystal-Structure Determination of 16 (see the Table and Fig. 2)9). All measurements were
carried out on a Nicolet diffractometer using graphite-monochromated MoKa radiation (l¼0.71073 ꢅ).
The intensities were corrected for Lorentz and polarization effects, but not for absorption. Equivalent
reflections were merged. The data collection and refinement parameters are given in the Table, and a
view of the molecule is shown in Fig. 2. The structure was solved by direct methods using SHELXS86
[34], which revealed the positions of all non-H-atoms. The atoms of the terminal acid function, from the
CH2 group onwards, and the atoms of the benzoyl function, from the amide group onwards, are
Table 1. Crystallographic Data for Compound 16
Crystallized from
Empirical formula
Formula weight
Crystal color, habit
Crystal dimensions [mm]
Temp. [K]
MeOH/Et2O
C24H36N4O7
492.57
colorless, prism
0.36ꢁ0.37ꢁ0.75
295(1)
Crystal system
Space group
trigonal
P3121
Z
6
Reflections for cell determination
2q Range for cell determination [8]
Unit cell parameters:
a [ꢅ]
25
48–52
13.144(15)
c [ꢅ]
29.41(4)
4401(13)
1.115
0.0823
V [ꢅ3]
Dx [g cmꢀ3
m(MoKa) [mmꢀ1
Scan type
]
]
w
2q(max) [8]
50
Total reflections measured
Symmetry-independent reflections
Reflections with I>2s(I)
Reflections used in refinement
Parameters refined; restraints
Final R(F) [I>2s(I) reflections]
wR(F2) (all data)
16229
5190
2892
5190
478; 505
0.0894
0.2929
Weights
Goodness-of-fit
w¼[s2(F2o )þ(0.1830P)2]ꢀ1 where P¼(Fo2 þ 2F2c )/3
1.057
Final Dmax/s
D1(max; min) [e ꢅꢀ3
0.001
0.50; ꢀ0.31
]
9
)
CCDC-910784 contains the supplementary crystallographic data for this article. These data can be
obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif.

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
939
disordered. Two sets of overlapping positions were defined for the atoms of each disordered fragment.
The site occupation factors of the major conformations of these fragments refined to similar values and
were then refined as a common value yielding a final value of 0.53(1). 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 fragments were restained to have similar
atomic displacement parameters. The non-H-atoms were refined anisotropically. All of the H-atoms were
placed in geometrically calculated positions and refined by 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 and OH groups). The refinement of the structure was carried out on F2 by using full-matrix
least-squares procedures, which minimized the function Sw(F2o ꢀFc2 )2. A correction for secondary
extinction was not applied. Neutral atom-scattering factors for non-H-atoms were taken from [35], and
the scattering factors for H-atoms were taken from [36]. Anomalous dispersion effects were included in
Fc [37]; the values for f ’ and f ’’ were those of [38]. The values of the mass-attenuation coefficients are
those of [39]. The SHELXL97 program was used for all calculations [40].
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Received November 15, 2012