Synthesis and biological evaluation of gramicidin S-inspired cyclic mixed α/β-peptides

Article abstract of DOI:10.1002/cbdv.201200277

Via a Mannich reaction involving a dibenzyliminium species and the titanium enolates of Evans' chiral acylated oxazolidinones the β²-amino acids (R)- and (S)-Fmoc-β²homovaline and (R)-Fmoc- β²homoleucine are synthesized. T

Full text of DOI:10.1002/cbdv.201200277

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Synthesis and Biological Evaluation of Gramicidin S-Inspired Cyclic Mixed
a/b-Peptides
by Matthijs van der Knaapa), Fatih Basalana), Henny C. van de Meib), Henk J. Busscherb),
Gijsbert A. van der Marela), Herman S. Overkleefta), and Mark Overhand*a)
a) Bio-Organic Synthesis, Leiden Institute of Chemistry, Leiden University, Einsteinweg 55,
2333 CC Leiden, The Netherlands (fax: þ31-71-5274307; e-mail: m.overhand@chem.leidenuniv.nl)
b) UMCG: Biomaterials, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands
Dedicated to Professor Dieter Seebach on the occasion of his 75th birthday
Via a Mannich reaction involving a dibenzyliminium species and the titanium enolates of Evansꢀ
chiral acylated oxazolidinones the b2-amino acids (R)- and (S)-Fmoc-b2homovaline and (R)-Fmoc-
b2homoleucine are synthesized. These building blocks were used, in combination with commercially
available a- and b3-amino acids, for the synthesis of the cyclo-(ab3ab2a)2 peptide 2 and the cyclo-
(ab2ab3a)2 peptides 3–5. The peptides 2–5 were screened for their ability to inhibit a small panel of
Gram-negative and Gram-positive bacterial strains.
Introduction. – The discovery of the structural and functional properties of
oligomers entirely composed of b- or g-amino acids has revolutionized the field of
peptide chemistry [1]. By combining amino acid homologs with each other, the
availability of peptide-like molecules with diverse predetermined structures is
extended even further. For instance, linear mixed a/b-peptides can be designed that
are able to adopt helical [2–4] or b-hairpin-like structures [5–8]. We recently reported
the design, synthesis, and structural analysis of a series of cyclic mixed a/b-peptides [9].
A representative member out of this series is the cyclo-(ab3ab2a)2 peptide 1, a
derivative of the antibiotic gramicidin S (GS; Fig. 1). Compound 1 adopts a rigid cyclic
hairpin-like structure with an alternative intramolecular H-bonding pattern of the
strand regions in comparison with GS (Fig. 1). The iBu and iPr side chains of the b3- and
b2-amino acid residues, and the 3-aminopropyl side chains of the central two a-amino
acids form an apolar and polar molecular face, structurally resembling the hydrophobic
and hydrophilic side-chain positioning of GS (Fig. 1). X-Ray analysis of the crystal
structures show that GS adopts a pleated-sheet structure, while peptide 1 is slightly
bent. Peptide 1 is not able to inhibit the growth of bacterial strains.
Here, we present a synthetic approach involving dibenzyliminium [10] and the
titanium enolate of Evansꢀ chiral oxazolidinone [11], towards (R)- and (S)-Fmoc-
b2homovaline and (R)-Fmoc-b2homoleucine (Fmoc¼(9H-flouren-9-yl)methoxycar-
bonyl) [12][13]. These b2-amino acid building blocks were used to extend the scope of
cyclic mixed a/b-peptides, i.e., to allow the synthesis of peptide 2, and several of its
regioisomers and diastereoisomers 3–5 (Fig. 2). Peptide 2 contains two (R)-
ꢁ 2012 Verlag Helvetica Chimica Acta AG, Zꢂrich

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Fig. 2. The structures of the target peptides cyclo(ab3ab2a)2 2, cyclo(ab2ab3a)2 3–5, and the reference
compound 6
b3homovaline and two (R)-b2homoleucine residues, as opposed to the two (S)-
b3homoleucine and two (R)-b2homovaline residues of peptide 1. Peptide 3 is the
regioisomer of 2, containing two (S)-b2homovaline and two (R)-b3homoleucine
residues. Peptide 4 is the diastereoisomer of 3 in which all the b-amino acid residues
have the opposite configuration, while peptide 5 is the diastereoisomer of 3 in which the
ornithine residues have opposite configuration. The cyclo-(ab3ab2a)2 peptide 2 and the
cyclo-(ab2ab3a)2 peptides 3–5 were screened for their antimicrobial activities against a
panel of Gram-positive and Gram-negative bacterial strains, and compared with the
activities of the natural product GS, 1, and reference compound 6, a diastereoisomer of
GS (Fig. 2).

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Results and Discussion. – Suitably protected (R)- and (S)-b3-amino acids, are
commercially available or readily accessible through various well-established methods
[14–16]. b2-Amino acids are more difficult to obtain [15][17–19]. A fruitful approach
proceeds via the Mannich reaction and makes use of a chiral auxiliary [19]. In this
reaction, the protective groups of the iminium component [17][20] are of importance
with respect to their ease of removal in a later stage of the synthesis. Dibenzyliminium
ion 8 (Scheme) was selected as an easily accessible aminomethylating agent. It is
generated from methylenebis(dibenzylamine), a shelf-stable compound, which, in turn,
can be synthesized from Bn2NH and formalin in high yields [10].
For the synthesis of the required suitably protected b2-amino acids, the acylated
oxazolidinones 7a–7c (Scheme) were prepared according to literature procedures
[21][22]. Titanium enolates [13] of 7a–7c were reacted with freshly prepared
dibenzyliminium trifluoroacetate 8 (Scheme). The dibenzyl-protected Mannich
products 9a–9c were obtained in 80–99% yield with high diastereoselectivity, with
the minor diastereoisomer readily separable by silica-gel column chromatography.
Hydrogenolysis of 9a (or 9b) in the presence of catalytic amounts of Pd on charcoal
provided the heterocycle 10a (or 10b), presumably via intramolecular attack of the
partially deprotected amine 11 on the endocyclic C¼O group [23][24]. Protonation of
the Bn2NH, to render it non-nucleophilic, resulted in elimination products 12. Removal
of the chiral auxiliary, prior to debenzylation, was hampered by the attack of commonly
employed nucleophiles, including lithium peroxide, magnesium-, or lithium alcoholates,
on the endocyclic C¼O group of 9a (or 9b) leading to undesired products. The
application of lithium ethylthiolate [25][26] to remove the chiral auxiliaries of 9a (or
9b), provided the thioesters 14a (or 14b) in good yields. The success of this procedure
can be rationalized by regeneration of the starting material in case the ethylthiolate
attacks the endocyclic C¼O group to give intermediate 13 (Scheme). Mercury
trifluoroacetate-mediated hydrolysis of the thioesters 14a (or 14b) led smoothly to the
dibenzyl-protected amino acid 15a (or 15b). The two Bn groups of compounds 15a (or
15b) were cleanly removed using Pd/C and H2 in MeOH/H2O/AcOH, followed by
Fmoc protection under standard SchottenꢀBaumann conditions to provide (R)-Fmoc-
b2homovaline (17a) and (R)-Fmoc-b2homoleucine (17b) in good yields. Accordingly,
compound 9c was converted into (S)-Fmoc-b2homovaline (17c) (Scheme).
The cyclic mixed a/b-peptides 2–5 and peptide 6 were prepared by assembly of
their linear precursors on solid support, followed by cyclization in solution under high
dilution. For this purpose, (R)-Fmoc-phenylalanine was immobilized under standard
conditions on the hyper-acid-labile HMPB (¼4-(hydroxymethyl)-3-(methoxyphenoxy)-
butanoic acid) linker [27][28] grafted on the MBHA (¼4-methylbenzhydrilamine)
resin. The target immobilized oligopeptides were assembled by stepwise elongation via
a Fmoc synthesis protocol with the appropriate amino acid using HCTU (¼2-(6-
chloro-1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate)/Et-
NiPr2. The full-length peptide sequences having unprotected N-termini were cleaved
from the solid support by repeated treatment with 1% CF3COOH (TFA) in CH2Cl2.
This provided the decapeptides with the Ne-Boc protecting groups on the ornithine side
chains, allowing their C!N cyclization using PyBOP (¼(Benzotriazol-1-yloxy)tri-
pyrrolidinophosphonium hexafluorophosphate)/HOBt (¼1-hydroxybenzotriazole)/
EtNiPr2 in DMF at low concentration. The products were fully deprotected using

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Scheme
a) TiCl4, Et3N, CH2Cl2, 08, then 4, ꢀ708; 9a: 84%, 9b: 100%, 9c: 95%. b) H2, Pd/C, MeOH. c) Hþ. d)
BuLi, EtSH, THF, 08, then 9a–9c; 14a: 92%, 14b: 40%, 14c: 77%. e) Hg(TFA)2, H2O/THF; 15a–15c:
100%. f) H2, Pd/C, MeOH/H2O/AcOH. g) [(9H-Fluoren-9-yl)methoxy]carbonyloxy succinimide
(Fmoc-OSu), NaHCO3, dioxane/H2O; over two steps: 17a: 87%, 17b: 82%, 17c: 88%.

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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strong acidic conditions and subjected to HPLC purification to give the compounds 2–
6 in yields of 20–45%. All peptides were analyzed by high-resolution mass
spectrometry and several NMR techniques. The peptides, with the exception of
1
peptide 4 and 6, gave well-resolved H spectra in CD3OH, with sharp amide signals
between 7 and 9 ppm. Previously [9], we determined that the solution structure of
peptide 1 based on its NMR data agrees well with the X-ray structure (Fig. 1).
Comparing the NMR data of peptides 1 and 2, and 3 and 5, it is concluded that the
isomers 1 and 2 have comparable structural properties, and that compounds 3 and 5
adopt distinct cyclic hairpin-like structures (see Exper. Part).
The peptides 2–5 were screened for their antimicrobial activities against a panel of
Gram-positive and Gram-negative bacterial strains and compared with the activities of
the natural product GS, 1, and the GS diastereoisomer 6 (Table 1). The natural product
GS has the best antibacterial activity, and peptide 3 is second best. Peptides 1 and 2 are
largely inactive. Peptides 5 and 6 are inactive.
Table 1. Antibacterial Activity of the Cyclic Mixed a/b-Peptides 1–5 (MIC in mg/ml), Compared to GS
and Peptide 6
Strain
GS
1
2
3
4
5
6
S. aureus 7323
S. aureus 7388
CNS 5277
CNS 5115
8
8
4
16
8
8
32
16
8
8
4
>64
16
32
64
16
>64
>64
>64
>64
16
32
16
32
64
16
64
64
>64
>64
32
16
8
8
>8
16
32
32
>64
>64
8
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
CNS 7368
E. faecalis 1131
E. coli ATCC 25922
P. aeruginosa AK1
P.aeruginosa ATCC19582
S. mitis BMS
S. mitis ATCC 33399
16
64
32
In conclusion, a synthetic route towards the synthesis of (R)- and (S)-Fmoc-
b2homovaline and (R)-Fmoc-b2homoleucine is described following a Mannich reaction
using titanium enolates of Evansꢀ acyloxazolidinones and dibenzyliminium trifluoro-
acetate as the aminomethylating agent. An appropriate deprotection procedure was
developed allowing the synthesis of suitably functionalized b2-homoamino acid
building blocks in good yields. We presume that this procedure is especially suitable
for the synthesis of b2-amino acids with sterically demanding side chains. The prepared
Fmoc-b2-homoamino acids were used for the synthesis of cyclic mixed a/b-decapep-
tides having both hydrophilic and hydrophobic functionalities. The cyclic amphiphilic
a/b-decapeptide 3, containing two (S)-b2homovaline and two (R)-b3homoleucine
residues, possesses modest antibacterial activity. Interestingly, the regioisomer of 3, i.e.,
peptide 2, containing two (R)-b3homovaline and two (R)-b2homoleucine residues, is
largely inactive. Both peptides 2 and 3 adopt cyclic hairpin-like structures based on
i
i
their NMR data. The Pr and Bu moieties of peptide 2 are separated by CH2 groups
from the central ornithine moiety, while peptide 3 and GS have these hydrophobic
groups directly adjacent to the ornithine moiety (Fig. 3). This structural difference has

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
an effect on the biological activity. More potent derivatives of 3 may be obtained by
fine-tuning of the hydrophobic characteristics and increasing the positive charge of the
strand regions, such as including adamantane glycine residues [29] flanked by b2/b3-
amino acids with ornithine side chains. In addition, incorporation of appropriately
functionalized anti-b2,3-amino acids [1], leading to even more rigid conformational
properties of the cyclic mixed a/b peptides, may have a beneficial effect on the
biological activity.
Fig. 3. Comparison of the positioning of the central amino acid side chains of the symmetric strands of
peptide 2, GS, and peptide 3
We thank STW for grant to support this research. We thank Fons Lefeber and Kees Erkelens for
NMR assistance, and Hans van der Elst and Nico Meeuwenoord for their help with LC/MS, HR-MS, and
analytical and preparative HPLC.
Experimental Part
General. Solvents and chemicals were used as received from their supplier. Solvents were stored over
4-ꢃ molecular sieves (MeOH on 3 ꢃ). Solvents for column chromatography (CC) and extractions were
of technical grade and distilled prior to use. THF was distilled over LiAlH4, CH2Cl2 over CaH2, and
MeOH over NaBH4 prior to use. (S)-Fmoc-b3hLeu-OH and (R)-Fmoc-b3hLeu-OH were obtained from
Senn Chemicals AG. All reactions were performed at r.t. under inert atmosphere. Flash chromatography
(FC) was performed on Screening Devices silica gel 60 (0.04–0.063 mm). TLC Analysis was conducted
on DC-Alufolien (Merck, Kieselgel 60, F254) with detection by UVabsorption at l 254 nm or by spraying
with a soln. of ninhydrin (3 g/l) in EtOH/AcOH 20 :1 (v/v), or KMnO4 soln. (20 g in 1% aq. K2CO3),
followed by charring at ꢁ1508. LC/MS Analyses were performed on a LCQ Adventage Max (Thermo
Finnigan) equipped with a Gemini C18 column (Phenomenex). The applied buffers were A: H2O, B:
MeCN, and C: 1.0% aq. TFA. HPLC Purifications were performed with a Gilson GX-281 automated
HPLC system, equipped with a prep. Gemini C18 column (150ꢂ21.20 mm, 5 m). The applied buffers
were: A: 0.2% aq. TFA, B: MeCN. Optical rotations were measured on a Propol automatic polarimeter
(sodium D line, l 589 nm) at r.t. IR Spectra were recorded on a Perkin-Elmer Paragon 1000; ˜n in cmꢀ1
.
1H- and 13C-NMR spectra were recorded with a Bruker AV-400liq spectrometer (400 and 100 MHz, resp.)
or a Bruker DMX-600 spectrometer (600 and 150 MHz, resp.); chemical shifts d are given in ppm rel. to
Me4Si (0 ppm), or CD3OD/CD3OH (3.31 ppm) as internal standard. The analysis of coupling constants
(J in Hz), temp. coefficients and chemical-shift perturbations were performed with GraphPad Prism
version 5.01 for Windows, GraphPad Software, San Diego, California, USA, www.graphpad.com. HR
Mass spectra were recorded by direct injection (2 ml of a 2 mm soln. in H2O/MeCN 50 :50 (v/v) and 0.1%
HCOOH) with a mass spectrometer (Thermo Finnigan LTQ Orbitrap) equipped with an electro-spray
ion source in positive-ion mode (source voltage, 3.5 kV; sheath gas flow, 10; cap. temp., 2508) with
resolution R¼60000 at m/z 400 (mass range m/z 150–2000) and dioctylphthalate (m/z 391.28428) as a
ꢄlock massꢀ. The HR mass spectrometer was calibrated prior to measurements with a calibration mixture
(Thermo Finnigan).

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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Antibacterial Screening. Bacteria were stored at ꢀ708 and grown at 308 on Columbia Agar with
sheep blood (Oxoid, D-Wesel) suspended in physiological saline until an optical density of 0.1 AU (at
595 nm, 1-cm cuvette). The suspension was diluted (10ꢂ) with physiological saline, and 2 ml of this
inoculum was added to 100 ml growth medium, Nutrient Broth from Difco (ref. No. 234000, lot No.
6194895) with yeast extract (Oxoid LP 0021, lot No. 900711, 2 g/400 ml broth) in Microtiter plates (96
wells). The peptides GS and 1–6 were dissolved in EtOH (4 g/l) and diluted in distilled H2O (1000 mg/l),
and two-fold diluted in the broth (64, 32, 16, 8, 4 and 1 mg/l). After incubation at 308 (24–96 h), the MIC
value was determined as the lowest concentration inhibiting bacterial growth.
General Procedure for the Aminomethylation (GP 1). Acylated oxazolidinone 7a, 7b, or 7c was
dissolved in CH2Cl2 (0.1m) and cooled in an ice/water bath. TiCl4 (1 equiv., 1m soln. in CH2Cl2) was added
slowly, upon which the soln. turned bright yellow. The mixture was stirred for 5 min, then Et3N (1 equiv.)
was added dropwise, turning the soln. dark red. After stirring for 15 min, the mixture was cooled to ꢀ808,
and subsequently a soln. of 8 in CH2Cl2 (0.85 equiv.) was added. The mixture was left stirring for 16 h at
ꢀ808. Then, the reaction was quenched at ꢀ808 by addition of sat. NH4Cl soln. After warming to r.t., the
pH was adjusted to 2, and the mixture was extracted with AcOEt. The org. layer was washed with
NaHCO3 soln. and brine, dried (MgSO4), filtered, and evaporated. Purification was carried out by FC.
(4S)-4-Benzyl-3-{(2R)-2-[(dibenzylamino)methyl]-3-methylbutanoyl}-1,3-oxazolidin-2-one (9a).
Starting from 4.62 g (17.7 mmol) of 7a, according to GP 1, FC (10% Et2O/petroleum ether (PE)).
Yield: 7.0 g (15.1 mmol, 84%). Colorless glass. [a]D ¼ þ14.4 (c¼1.0, CHCl3). IR (neat): 2961.1, 1772.5,
1697.5, 1382.9, 1348.5, 1209.4, 1099.9, 743.9, 699.7. 1H-NMR (400 MHz, CDCl3): 7.41–7.16 (m, 15 H); 4.67
(sept., J¼3.6, 1 H); 4.30 (br. s, 1 H); 4.15 (m, 2 H); 3.82 (d, J¼13.7, 2 H); 3.47 (dd, J¼13.2, 2.9, 1 H); 3.42
(d, J¼13.7, 2 H); 3.09 (dd, J¼10.8, 12.0, 1 H); 2.72 (dd, J¼13.1, 10.2, 1 H); 2.59 (dd, J¼12.5, 3.8, 1 H);
1.84 (dq, J¼13.4, 6.7, 1 H); 0.92 (d, J¼6.8, 3 H); 0.88 (d, J¼6.8, 3 H). 13C-NMR (100 MHz, CDCl3):
175.5; 153.4; 138.9; 135.7; 129.4; 129.0; 128.9; 128.0; 127.2; 126.8; 65.7; 58.3; 55.7; 54.2; 46.4; 38.3; 30.3;
20.3; 20.0. HR-MS: 471.26382 (C30H35N2Oþ3 ; calc. 471.26422).
(4S)-4-Benzyl-3-{(2R)-2-[(dibenzylamino)methyl]-4-methylpentanoyl}-1,3-oxazolidin-2-one (9b).
Starting from 3.16 g (11.4 mmol) of 7b, according to GP 1, FC (10% Et2O/toluene). Yield: 3.5 g
(11.4 mmol, 100%). Slightly yellow oil. [a]D ¼ þ12.8 (c¼1.0, CHCl3). IR (neat): 2958.8, 2886.2, 1691.6,
1
1548.8, 1448.5, 1298.1, 1263.4, 1251.8, 1161.2, 1039.0, 1045.4, 756.1, 729.1. H-NMR (400 MHz, CDCl3):
7.35–7.14 (m, 15 H); 4.59 (ddt, J¼10.3, 6.6, 3.1, 1 H); 4.43 (ddd, J¼13.8, 8.4, 5.2, 1 H); 4.03 (m, 2 H); 3.63
(m, 4 H); 3.36 (dd, J¼2.8, 13.2, 1 H); 2.90 (dd, J¼12.3, 8.2, 1 H); 2.68 (dd, J¼13.2, 10.1, 1 H); 2.49 (dd,
J¼12.3, 6.0, 1 H); 1.60–1.34 (m, 3 H); 0.88 (dd, J¼6.2, 2.5, 6 H). 13C-NMR (101 MHz, CDCl3): 176.1;
153.1; 139.0; 135.6; 129.3; 128.9; 128.9; 128.1; 127.2; 126.8; 65.8; 60.2; 58.4; 57.0; 55.6; 40.0; 39.3; 38.1;
26.3; 23.1; 22.6. HR-MS: 485.27928 (C31H37N2Oþ3 ; calc. 485.27987).
(4R)-4-Benzyl-3-{(2S)-2-[(dibenzylamino)methyl]-3-methylbutanoyl}-1,3-oxazolidin-2-one (9c).
Starting from 2.9 g (11.1 mmol) of 7c, according to GP 1, FC (10% Et2O/PE). Yield: 4.5 g (9.52 mmol,
95%). Colorless glass. [a]D ¼ ꢀ14.0 (c¼1.0, CHCl3). IR (neat): 2961.1, 1772.5, 1697.5, 1382.9, 1348.5,
1209.4, 1099.9, 743.9, 699.7. 1H-NMR (400 MHz, CDCl3): 7.41–7.16 (m, 15 H); 4.67 (sept., J¼3.6, 1 H);
4.30 (br. s, 1 H); 4.15 (m, 2 H); 3.82 (d, J¼13.7, 2 H); 3.47 (dd, J¼13.2, 2.9, 1 H); 3.42 (d, J¼13.7, 2 H);
3.09 (dd, J¼10.8, J¼12.0, 1 H); 2.72 (dd, J¼13.1, 10.2, 1 H); 2.59 (dd, J¼12.5, 3.8, 1 H); 1.84 (dq, J¼
13.4, 6.7, 1 H); 0.92 (d, J¼6.8, 3 H); 0.88 (d, J¼6.8, 3 H). 13C-NMR (100 MHz, CDCl3): 175.5; 153.4;
138.9; 135.7; 129.4; 129.0; 128.9; 128.0; 127.2; 126.8; 65.7; 58.3; 55.7; 54.2; 46.4; 38.3; 30.3; 20.3; 20.0. HR-
MS: 471.26382 (C30H35N2Oþ3 ; 471.26422).
General Procedure for the Thiolytic Removal of the Evansꢀ Template (GP 2). BuLi (1.1 equiv., 1.6m in
hexanes) was added dropwise to a stirred soln. of EtSH in THF (3 equiv., 0.3m) at 08, and the soln. was
stirred for 15 min. Then, a soln. of 9a, 9b, or 9c in THF (1m) was added slowly to the thiolate soln. When
TLC indicated completion, the reaction was quenched with sat. NH4Cl, and the mixture was warmed to
r.t. The product was extracted with Et2O and washed with NaHCO3 soln. The Et2O layer was dried
(MgSO4), filtered, and concentrated in vacuo. The crude product was purified by FC.
S-Ethyl (R)-2-[(Dibenzylamino)methyl]-3-methylbutanethioate (14a). Starting from 7.1 g
t
(15.1 mmol) of 9a, according to GP 2, FC (5% BuOMe in PE). Yield: 4.95 g (13.9 mmol, 92%). Oil.
[a]D ¼ þ12.2 (c¼1.0, CHCl3). IR (neat): 2960.5, 1681.8, 1452.3, 918.1, 734.8, 696.3. 1H-NMR (400 MHz,
CDCl3): 7.33–7.20 (m, 10 H); 3.66 (d, J¼13.6, 2 H); 3.40 (d, J¼13.6, 2 H); 2.95–2.86 (m, 3 H); 2.64–2.58

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
(m, 1 H); 2.53 (dd, J¼4.2, J¼12.6, 1 H); 1.80 (sext., J¼6.8, 1 H); 1.29 (t, J¼7.4, 3 H); 0.86 (d, J¼6.8,
3 H); 0.82 (d, J¼6.8, 3 H). 13C-NMR (100 MHz, CDCl3): 201.9; 139.1; 129.0; 128.0; 126.9; 59.3; 58.5;
54.6; 29.8; 23.3; 20.7; 20.3; 14.8. HR-MS: 356.20419 (C22H30NOSþ ; 356.30426).
S-Ethyl (2R)-2-[(Dibenzylamino)methyl]-4-methylpentanethioate (14b). Starting from 3.5 g
t
(11.4 mmol) of 9b, according to GP 2, FC (0!5% BuOMe/PE). Yield: 2.2 g (4.53 mmol, 40%).
Slightly yellow oil. [a]D ¼ ꢀ5.0 (c¼1.0, CHCl3). IR (neat): 3026.31, 2954.95, 1681.93, 1450.47, 1367.53,
1070.49. 1H-NMR (400 MHz, CDCl3): 7.40–7.23 (m, 8 H); 7.23–7.16 (m, 2 H); 3.62 (d, J¼13.6, 2 H); 3.44
(d, J¼13.6, 2 H); 2.97–2.77 (m, 4 H); 2.38 (dd, J¼5.0, 12.4, 1 H); 1.58–1.40 (m, 2 H); 1.25 (t, J¼7.4,
3 H); 1.18 (ddd, J¼4.7, 8.3, 13.4, 1 H); 0.84 (d, J¼6.4, 3 H); 0.80 (d, J¼6.4, 3 H). 13C-NMR (100 MHz,
CDCl3): 202.3; 128.8; 128.0; 126.8; 58.6; 57.3; 51.0; 40.1; 25.6; 23.1; 23.1; 22.0; 14.7. HR-MS: 370.21994
(C23H32NOSþ ; calc. 370.21991).
S-Ethyl (2S)-2-[(Dibenzylamino)methyl]-3-methylbutanethioate (14c). Starting from 4.5 g
t
(9.5 mmol) of 9c, according to GP 1, FC (5% BuOMe in PE). Yield: 2.6 g (7.3 mmol, 77%). Oil.
[a]D ¼ þ11.9 (c¼1.0, CHCl3). IR (neat): 2960.5, 1681.8, 1452.3, 918.1, 734.8, 696.3. 1H-NMR (400 MHz,
CDCl3): 7.33–7.20 (m, 10 H); 3.66 (d, J¼13.6, 2 H); 3.40 (d, J¼13.6, 2 H); 2.95–2.86 (m, 3 H); 2.64–2.58
(m, 1 H); 2.53 (dd, J¼4.2, 12.6, 1 H); 1.80 (sext., J¼6.8, 1 H); 1.29 (t, J¼7.4, 3 H); 0.86 (d, J¼6.8, 3 H);
0.82 (d, J¼6.8, 3 H). 13C-NMR (100 MHz, CDCl3): 201.9; 139.1; 129.0; 128.0; 126.9; 59.3; 58.5; 54.6; 29.8;
23.3; 20.7; 20.3; 14.8. HR-MS: 356.20419 (C22H30NOSþ ; calc. 356.30426).
General Procedure for the Thioester Hydrolysis (GP 3). Thioester 14a, 14b, or 14c was dissolved in
THF/H2O 5 :1 (0.2m, v/v) and Hg(TFA)2 (1.5 equiv.) was added, upon which the soln. turned yellow,
which subsided after some minutes. When TLC analysis indicated completion, brine was added, and the
suspension was filtered over a double Whatman filter. The layers were separated, and the organics were
evaporated. The residue was redissolved in AcOEt, washed with brine (5ꢂ ), H2O, dried (MgSO4),
filtered, and evaporated. FC was applied when necessary to obtain the product.
(2R)-2-[(Dibenzylamino)methyl]-3-methylbutanoic Acid (15a). Starting from 4.79 g (13.5 mmol) of
14a, according GP 3. Yield: 4.2 g (13.5 mmol, quant.). [a]D ¼ ꢀ38.8 (c¼1.0, CHCl3). IR (neat): 2963.8,
1717.9, 1457.9, 1197.7, 752.0, 700.2. 1H-NMR (400 MHz, CDCl3): 11.71 (br. s, 1 H); 7.47–7.28 (m, 10 H);
4.17 (d, J¼13.2, 2 H); 4.03 (d, J¼13.2, 2 H); 3.25 (t, J¼12.2, 1 H); 2.87 (dd, J¼3.0, 13.0, 1 H); 2.76–2.73
(m, 1 H); 2.14 (sext., J¼6.8, 1 H); 0.83 (d, J¼6.8, 3 H); 0.79 (d, J¼6.8, 3 H). 13C-NMR (100 MHz,
CDCl3): 175.9; 131.5; 130.5; 129.1; 129.0; 57.4; 50.8; 46.1; 28.7; 19.7; 18.7. HR-MS: 312.19595 (C20H26NOþ2 ;
calc. 312.19581).
(2R)-2-[(Dibenzylamino)methyl]-4-methylpentanoic Acid (15b). Starting from 2.2 g (4.53 mmol)
14b, according to GP 3, FC (5% MeOH/CH2Cl2). Yield: 2.0 g (4.53 mmol, 100%). [a]D ¼ ꢀ76.6 (c¼1.0,
1
CHCl3). IR (neat): 2954.95, 1705.07, 1600.92, 1452.40, 1369.46, 1240.23. H-NMR (400 MHz, CDCl3):
13.87 (s, 1 H); 7.43–7.23 (m, 10 H); 4.01 (d, J¼13.3, 2 H); 3.66 (d, J¼13.2, 2 H); 2.97–2.83 (m, 1 H); 2.68
(dd, J¼4.7, 12.9, 1 H); 2.63–2.54 (m, 1 H); 1.79–1.68 (m, 1 H); 1.62 (dt, J¼6.7, 13.0, 1 H); 1.09 (dt, J¼
6.7, 13.5, 1 H); 0.84 (dd, J¼6.5, 8.3, 6 H). 13C-NMR (100 MHz, CDCl3): 177.2; 134.3; 129.8; 128.8; 128.4;
57.7; 54.9; 38.5; 38.4; 25.6; 22.6; 22.2. HR-MS: 326.21135 (C21H28NOþ2 ; calc. 326.21146).
(2S)-2-[(Dibenzylamino)methyl]-3-methylbutanoic Acid (15c). Starting from 2.26 g (7.3 mmol) of
14c, according to GP 3. Yield: 2.3 g (7.3 mmol, quant.). [a]D ¼ ꢀ38.8 (c¼1.0, CHCl3). IR (neat): 2963.8,
1717.9, 1457.9, 1197.7, 752.0, 700.2. 1H-NMR (400 MHz, CDCl3): 11.71 (br. s, 1 H); 7.47–7.28 (m, 10 H);
4.17 (d, J¼13.2, 2 H); 4.03 (d, J¼13.2, 2 H); 3.25 (t, J¼12.2, 1 H); 2.87 (dd, J¼3.0, 13.0, 1 H); 2.76–2.73
(m, 1 H); 2.14 (sext., J¼6.8, 1 H); 0.83 (d, J¼6.8, 3 H); 0.79 (d, J¼6.8, 3 H). 13C-NMR (100 MHz,
CDCl3): 175.9; 131.5; 130.5; 129.0; 129.0; 57.4; 50.8; 46.1; 28.7; 19.7; 18.7. HR-MS: 312.19595
(C20H26NOþ2 ; calc. 312.19581).
General Procedure for Benzyl Removal and Fmoc-Protection (GP 4). The carboxylic acid 15a, 15b or
15c was dissolved in MeOH/H2O/AcOH 80 :10 :10 (v/v/v), purged with Ar, and a cat. amount of 10% Pd/
C was added. A H2 atmosphere was applied (1 bar), and the suspension was stirred for 16 h; then, the
mixture was filtered over Whatman pads and concentrated. The residue was redissolved in dioxane, and
Fmoc-OSu (1.1 equiv.) and sat. NaHCO3 soln. were added, and the mixture was stirred for 8 h. The
product was extracted with AcOEt, and the organics were washed with H2O and brine, dried (MgSO4),
filtered and evaporated. The crude was purified by FC.

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2503
(2R)-2-({[(9H-Fluoren-9-ylmethoxy)carbonyl]amino}methyl)-3-methylbutanoic Acid (17a). Start-
ing from 0.46 g (1.49 mmol) of 15a, according to GP 4, FC (0!3% MeOH/CH2Cl2). Yield: 0.41 g
(1.15 mmol, 78%; in two steps). 1H-NMR (400 MHz, CDCl3): 7.77–7.26 (m, 9 H); 5.23 (t, J¼11.2, 1 H);
4.54 (d, J¼6, 1 H); 4.37 (sext., J¼44.8, 1 H); 4.22 (q, J¼18.8, 1 H); 3.35 (m, 2 H); 2.95 (s, 1 H); 2.21
(quint., J¼21.2, 1 H); 2.03 (q, J¼26.8, 1 H); 1.03 (t, J¼11.2, 3 H); 0.86 (d, J¼6.4, 3 H). 13C-NMR
(100 MHz, CDCl3): 129.0; 127.6; 127.0; 125.2; 124.8; 124.3; 120.3; 119.9; 66.6; 47.2; 45.2; 40.4; 28.6; 19.6;
NMR and further analysis in agreement with previously reported data [30].
(2R)-2-({[(9H-Fluoren-9-ylmethoxy)carbonyl]amino}methyl)-4-methylpentanoic Acid (17b). Start-
ing from 2.0 g (4.5 mmol) of 15b, according to GP 4, FC (0!3% MeOH/CH2Cl2). Yield: 0.42 g
(2.87 mmol, 82%; in two steps). 1H-NMR (400 MHz, CDCl3): 12.12 (s, 1 H); 7.89–7.30 (m, 8 H); 4.28 (d,
J¼6.8, 2 H); 4.21 (d, J¼6.4, 1 H); 3.25 (s, 2 H); 2.54 (s, 1 H); 1.57 (t, J¼12.8, 1 H); 1.41 (q, J¼14, 1 H);
1.24 (s, 1 H); 0.86 (q, J¼10, 6 H). 13C-NMR (100 MHz, CDCl3): 175.5; 143.7; 140.6; 125.5; 123.5; 123.3;
122.9; 121.3; 65.5; 45.4; 43.1; 42.6; 38.4; 26.2; 20.0; 17.8; NMR and further analysis in agreement with
previously reported data [30].
(2S)-2-({[(9H-Fluoren-9-ylmethoxy)carbonyl]amino}methyl)-3-methylbutanoic Acid (17c). Starting
from 0.96 g (2.97 mmol) of 15c, according to GP 4, FC (0!3% MeOH/CH2Cl2). Yield: 1.06 g
1
(2.87 mmol, 68%; in two steps. H-NMR (400 MHz, CDCl3): 7.77–7.26 (m, 9 H); 5.23 (t, J¼11.2, 1 H);
4.54 (d, J¼6, 1 H); 4.37 (sext., J¼44.8, 1 H); 4.22 (q, J¼18.8, 1 H); 3.35 (m, 2 H); 2.95 (s, 1 H); 2.21
(quint., J¼21.2, 1 H); 2.03 (q, J¼26.8, 1 H); 1.03 (t, J¼11.2, 3 H); 0.86 (d, J¼6.4, 3 H). 13C-NMR
(100 MHz, CDCl3): 129.0; 127.6; 127.0; 125.2; 124.8; 124.3; 120.3; 119.9; 66.6; 47.2; 45.2; 40.4; 28.6; 19.9;
NMR and further analysis in agreement with previously reported data [30].
General Peptide Synthesis. Loading of the HMPB-MBHA Resin. MBHA Resin (theoretical loading,
1.2 mmol/g, 2 mmol, 1.67 g) was suspended in ClCH2CH2Cl and concentrated thrice. Then, a soln. of (R)-
Fmoc-Phe-OH (5 equiv., 10 mmol, 3.9 g), N,N’-diisopropylcarbodiimide (DIC; 5 equiv., 10 mmol,
1.54 ml) and 4-(dimethylamino)pyridine (DMAP; 0.01 equiv., 20 mmol, 3 mg) in dry CH2Cl2/DMF 10 :1
(50 ml, v/v) was added. The mixture was shaken for 3 h and then drained, washed subsequently with
CH2Cl2, N-methylpyrrolidin-2-one (NMP), CH2Cl2, and Et2O. The resin was dried before determination
of the loading. The procedure was repeated when necessary.
Stepwise Elongation. Fmoc-DPhe-HMPB-MBHA resin (loading of the resin was 0.50 mmol/g,
100 mmol, 200 mg) was submitted to nine cycles of Fmoc solid-phase peptide synthesis with the
appropriate amino acid building blocks. The amino group on the side chain of ornithine was protected
with a Boc group. Fmoc removal was effected by treatment with 20% piperidine in NMP for 2ꢂ10 min.
The resin was subsequently washed with NMP, CH2Cl2, MeOH, and finally NMP. The Fmoc-AA-OH
(2.5 equiv., 250 mmol) and HCTU (2.5 equiv., 250 mmol, 103 mg) in NMP were pre-activated for 1 min
after the addition of EtNiPr2 (3 equiv., 300 mmol, 53 ml) and then added to the resin. The suspension was
shaken for 1.5 h. The resin was washed with NMP, CH2Cl2, MeOH, and NMP.
Cleavage from the Resin. After the final Fmoc deprotection the resin was washed with NMP and
CH2Cl2 and treated with 5 ml 1% TFA in CH2Cl2 (6ꢂ10 min). The filtrates were collected and
coevaporated with toluene (3ꢂ50 ml).
Cyclization. In DMF (80 ml) were dissolved PyBOP (5 equiv., 500 mmol, 260 mg), HOBt (5 equiv.,
500 mmol, 77 mg), and EtNiPr2 (15 equiv., 1.5 mmol, 262 ml). The linear decapeptide was dissolved in
DMF (5 ml) and added dropwise over 1 h to the coupling cocktail. After addition, the mixture was stirred
for 16 h. The mixture was concentrated in vacuo, and the crude mixture was subjected to LH-20 size-
exclusion chromatography.
Boc Deprotection. The peptide was dissolved in CH2Cl2 (2 ml), and TFA (2 ml) was added. The
mixture was stirred for 2 h, concentrated, and co-evaporated with toluene (2ꢂ10 ml). The obtained
crude product was applied to prep. HPLC purification. Using gradients of aq. TFA and MeCN, the cyclic
peptides 2–6 were obtained in the yield range of 20–45%.
cyclo(Pro-(R)-b3hVal-(R)-Orn-(R)-b2hLeu-(R)-Phe)2 (2). LC/MS: tR 7.58 min (10!90 min
1
MeCN, 15 min run). H-NMR (600 MHz, CD3OH): 8.52 (d, J¼4.14, 2 H, NH, Phe); 8.10 (d, J¼7.20,
2 H, NH, Orn); 7.95 (4 H, He, Orn); 7.79 (s, 2 H, NH, b2hLeu); 7.63 (d, J¼9.36), 7.33–7.25 (10 H, NH,
b3hVal; Ar, Phe); 4.58 (2 H, Ha, Orn); 4.55 (2 H, Ha, Phe); 4.34 (2 H, Ha, Pro); 4.16 (2 H, Hb, b3hVal);
3.63 (2 H, Hd, Pro); 3.23 (4 H, Hb, b2hLeu); 3.03 (2 H, Hb, Phe); 2.96 (4 H, Hd, Orn); 2.95 (2 H, Hb, Phe);

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2.70 (2 H, Ha, b2hLeu); 2.62 (2 H, Hd, Pro); 2.49 (4 H, Ha, b3hVal); 2.02 (2 H, Hb, Orn); 2.00 (2 H, Hb,
Pro); 1.79 (2 H, Hg, b3hVal); 1.77 (2 H, Hg, Orn); 1.75 (2 H, Hb, Orn); 1.73 (2 H, Hb, Pro); 1.71 (2 H, Hg,
Pro); 1.65 (2 H, Hg, Orn); 1.53 (2 H, Hg, Pro); 1.52 (2 H, Hb’, b2hLeu); 1.50 (2 H, Hg’, b2hLeu); 1.14 (2 H,
Hb’, b2hLeu); 0.89 (6 H, Hd, b3hVal); 0.87 (12 H, Hd’, b2hLeu); 0.84 (6 H, Hd, b3hVal). For comparison of
3J(NH,Ha)/3J(NH,Hb) values, NOE data, and chemical-shift perturbations of the Ha proton, see
Tables 2–4, resp. 13C-NMR (150 MHz, CD3OH): 176.6; 174.1; 173.8; 173.3; 172.8; 137.2; 130.4; 129.6;
129.4; 128.3; 61.9; 55.3; 53.3; 52.8; 47.9; 44.5; 43.0; 40.2; 39.7; 39.2; 38.0; 33.9; 31.1; 30.3; 27.1; 24.7; 23.5;
22.4; 19.7; 17.7. ESI-MS: 1197.80 ([MþH]þ ). HR-MS: 1197.77714 (C64H101N12O1þ0 ; calc. 1197.77581).
Table 2. 3J(NH,Ha)/3J(NH,Hb) Values [Hz] for Peptides 2–5
Peptide
b-Amino acid (II)
Orn (III)
b-Amino acid (IV)
(R)-Phe
2
3
4
5
9.36
7.20
8.87
–c)
–a)
9.51
–c)
4.14
2.25
–c)
3.73/8.56b)
–c)
5.13a)
8.92
7.93
–c)
a) Broad singlet. b) Double doublet. c) Impossible to assign signals.
Table 3. Selected NOE Data for Peptides 2, 3, and 5. NOE Spectra were recorded at 600 MHz in CD3OH;
mixing time, 200 ms (s¼strong (ca. 1.5 – 3 ꢃ), m ¼medium (ca. 3–3.5 ꢃ), w¼weak (ca. 3.5–5 ꢃ)).
2
3
5
NH Orn
Ha b3hVal (s)
Ha b2hLeu (m)
Ha Orn (s)
Ha Pro (m),
Hd Pro (m)
Hd Pro (m)
NH Phe1
Ha b3hLeu1 (m)
Ha b3hLeu2 (m)
Ha b2hVal1 (s)
Ha Orn1 (m)
NH Orn
Ha b2hVal (s)
Ha b3hLeu (m)
Ha Orn (s)
Ha Pro (m),
Hd Pro (m)
Hd Pro (m)
NH Phe
NH b2hLeu
NH b3hVal
NH Phe2
NH Phe
NH b3hLeu
NH b2hVal
NH Orn1
NH b3hLeu1
Ha Phe
NH b2hVal1
NH b2hVal2
Ha Pro1 (s),
Hd Pro2 (m)
Ha Pro2 (s),
Hd Pro2 (m)
Hd Pro2 (m)
Ha b3hLeu1 (m)
Ha Phe
Ha Phe1
Ha Orn1
Table 4. Chemical-Shift Perturbations [ppm] of the Protons Ha of Peptides 2, 3, and 5, Recorded in
CD3OH [31]
Peptide
Pro
Orn
(R)-Phe
2
3
5
ꢀ0.10
ꢀ0.12
ꢀ0.11
0.22
0.81
0.22
ꢀ0.11
ꢀ0.45
ꢀ0.25
cyclo(Pro-(S)-b2hVal-Orn-(R)-b3hLeu-(R)-Phe)2 (3). LC/MS: tR 4.86 min (10!90% MeCN,
1
15 min run). H-NMR (600 MHz, CD3OH): 8.45 (d, J¼9.51, 2 H, NH, b3hLeu); 8.26 (d, J¼2.25, 2 H,
NH, Phe); 8.02 (d, J¼8.87, 2 H, NH, Orn); 7.57 (dd, J¼3.73, J¼8.56, 2 H, NH, b2hVal); 7.36–7.27 (10 H,
Ar, Phe); 5.17 (2 H, Ha, Orn); 4.32 (2 H, Ha, Pro); 4.21 (2 H, Ha, Phe); 4.16 (2 H, Hb, b3hLeu); 3.88 (2 H,
Hb, b2hVal); 3.76 (2 H, Hd, Pro); 3.00 (2 H, Hb, b2hVal); 2.97 (4 H, Hb, Phe); 2.87 (2 H, Hd, Orn); 2.81
(2 H, Hd, Orn); 2.44 (4 H, Ha, b3hLeu); 2.32 (2 H, Hd, Pro); 2.03 (2 H, Ha, b2hVal); 2.01 (2 H, Hb, Pro);
1.85 (2 H, Hg, Pro); 1.76 (2 H, Hb’, b2hVal); 1.74 (2 H, Hg, Orn); 1.72 (2 H, Hb, Orn); 1.65 (2 H, Hd,

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2505
b3hLeu); 1.65 (2 H, Hg, Orn); 1.65 (2 H, Hb, Orn); 1.61 (2 H, Hb, Pro); 1.52 (2 H, Hg, b3hLeu); 1.48 (2 H,
Hg, Pro); 1.23 (2 H, Hg, b3hLeu); 1.01 (3 H, Hg’, b2hVal); 0.95 (3 H, Hg’, b2hVal); 0.91 (3 H, He, b3hLeu);
0.85 (3 H, He, b3hLeu). For comparison of 3J(NH,Ha)/3J(NH,Hb) values, NOE data, and chemical-shift
perturbations of the Ha proton, see Tables 2–4, resp. 13C-NMR (151 MHz, CD3OH): 175.5; 173.8; 173.3;
173.3; 173.2; 136.9; 130.5; 129.7; 128.5; 61.6; 55.7; 55.5; 53.4; 44.6; 41.8; 40.8; 40.5; 39.3; 37.9; 32.0; 30.7;
30.3; 28.8; 26.2; 25.1; 24.5; 23.3; 22.0; 21.1; 20.6. ESI-MS: 1198.0 ([MþH]þ ). HR-MS: 1197.77619
(C64H101N12Oþ10 ; calc. 1197.77581).
cyclo(Pro-(R)-b2hVal-(R)-Orn-(S)-b3hLeu-(R)-Phe)2 (4). LC/MS: tR 7.93 min (10!90 min MeCN,
15 min run). 1H-NMR (600 MHz, CD3OH): broad signals. ESI-MS: 1198.0 ([MþH]þ ). HR-MS:
1197.77691 (C64H101N12Oþ10 : 1197.77581).
cyclo(Pro-(S)-b2hVal-(S)-Orn-(R)-b3hLeu-(R)-Phe)2 (5). LC/MS: tR 6.31 min (10!90 min MeCN,
15 min run). 1H-NMR (600 MHz, CD3OH): 8.76 (d, J¼8.92, 2 H, NH, Orn); 8.55 (s, 2 H, NH, Phe); 8.06
(d, J¼7.93, 2 H, NH, b3hLeu); 7.51 (t, J¼5.13, 2 H, NH, b2hVal); 7.32–7.22 (10 H, Ar, Phe); 4.58 (2 H, Ha,
Orn); 4.45 (2 H, Hb, b3hLeu); 4.41 (2 H, Ha, Phe); 4.33 (2 H, Ha, Pro); 3.74 (2 H, Hb, b2hVal); 3.60 (2 H,
Hd, Pro); 3.17 (2 H, Hb, b2hVal); 3.08 (2 H, Hd, Orn); 3.06 (2 H, Hb, Phe); 2.93 (2 H, Hb, Phe); 2.91 (2 H,
Hd, Orn); 2.88 (2 H, Ha, b2hVal); 2.46 (2 H, Ha, b3hLeu); 2.45 (2 H, Hd, Pro); 2.26 (2 H, Ha, b3hLeu); 2.02
(2 H, Hb, Pro); 1.99 (2 H, Hb’, b2hVal); 1.87 (2 H, Hb, Orn); 1.74 (2 H, Hg, Orn); 1.69 (2 H, Hb, Orn); 1.67
(2 H, Hg, Orn); 1.59 (2 H, Hg, Pro); 1.56 (2 H, Hb, Pro); 1.49 (2 H, Hg, Pro); 1.48 (2 H, Hg, b3hLeu); 1.43
(2 H, Hg, b3hLeu); 1.21 (2 H, Hd, b3hLeu); 0.91 (12 H, Hg’, b2hVal); 0.87 (12 H, He, b3hLeu). For
comparison of 3J(NH,Ha)/3J(NH,Hb) values, NOE data, and chemical-shift perturbations of the Ha
proton, see Tables 2–4, resp. 13C-NMR (150 MHz, CD3OH): 175.2; 173.2; 172.9; 172.2; 136.6; 130.1;
129.3; 128.1; 47.5; 47.4; 46.1; 43.9; 42.3; 40.3; 40.2; 37.5; 31.3; 30.5; 30.4; 30.2; 30.2; 30.0; 25.6; 24.4; 23.9;
23.6; 21.7; 20.3; 19.3. ESI-MS: 1198.0 ([MþH]þ ). HR-MS: 1197.77698 (C64H101N12O1þ0 ; calc. 1197.77581).
1
cyclo(Pro-Val-dOrn-Leu-dPhe)2 (6). LC/MS: tR 5.34 min (10!90% MeCN, 15 min run). H-NMR
(600 MHz, CD3OH): broad signals. 13C-NMR (150 MHz, CD3OH): 174.3; 173.5; 173.4; 173.1; 171.9;
137.1; 130.5; 129.6; 128.4; 101.1; 62.5; 60.8; 55.1; 54.7; 52.7; 52.4; 48.2; 44.0; 40.6; 40.5; 30.6; 30.6; 30.4;
26.2; 25.1; 24.6; 24.5; 24.1; 23.8; 22.6; 20.1; 19.3. ESI-MS: 1142.2 ([MþH]þ ). HR-MS: 1141.71454
(C60H93N12Oþ10 ; calc. 1141.71321).
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Received July 31, 2012