Synthesis of cyclopentapeptides with three to five Aib units

Article abstract of DOI:10.1002/hlca.201400359

Four new Aib-containing cyclopentapeptides have been synthesized by cyclization of the corresponding linear pentapeptides using the diethyl phosphorocyanidate (DEPC)/EtN(iPr)₂ method. The linear precursors were prepared via the 'azirine/oxazolo

Full text of DOI:10.1002/hlca.201400359

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Synthesis of Cyclopentapeptides with Three to Five Aib Units
by Franziska S. Arnhold1), Anthony Linden, and Heinz Heimgartner*
Institut fꢀr Chemie der Universitꢁt Zꢀrich, Winterthurerstrasse 190, CH-8057 Zꢀrich
(phone: þ 41446354282; e-mail: heinz.heimgartner@chem.uzh.ch)
Four new Aib-containing cyclopentapeptides have been synthesized by cyclization of the
corresponding linear pentapeptides using the diethyl phosphorocyanidate (DEPC)/EtN(iPr)2 method.
The linear precursors were prepared via the ꢂazirine/oxazolone methodꢃ, i.e., the Aib units were
introduced by the reaction of amino acids or peptide acids with a 2,2-dimethyl-2H-azirin-3-amine,
followed by selective hydrolysis of the terminal amide function. Most remarkably, cyclo[(Aib)5] exists in
CDCl3 solution in a symmetrical conformation, i.e., no intramolecular H-bonds are detectable.
1. Introduction. – Although cyclopeptides have been known for many decades, their
structures, syntheses and biological activities are of continuing interest (see refs. cit. in
[1]). Regarding their structures, cyclopeptides containing a-aminoisobutyric acid (Aib)
are of special relevance because of the conformation-determining properties of a,a-
disubstituted a-amino acids. Besides a few natural Aib-containing cyclopeptides [2],
several have been synthesized, e.g., tetra- [3], penta- [4], and hexapeptides [5], as well
as those with larger rings [6].
Our studies toward the use of 2,2-disubstituted 2H-azirin-3-amines 1 as building
blocks in the synthesis of peptides containing a,a-disubstituted glycines [7] established
that the ꢂazirine/oxazolone methodꢃ is a convenient and efficient approach [8]. Based
on this method, we have also prepared a series of cyclopeptides with Aib or other a,a-
disubstituted glycines in their backbone [1][9]. As expected on the basis of structural
studies of Aib-containing peptides [10], the b-turn motif is also a preferred structure of
cyclopeptides with a,a-disubstituted glycines in their skeleton [1][9][11].
In our recent publication [1], we described the cyclization of several pentapeptides
with 2-methylphenylalanine (Phe(2 Me)), and one or two Aib units, e.g. 2, to give the
corresponding cyclopentapeptides, e.g., 3 (Scheme 1).
In the present study, cyclopentapeptides with three to five Aib units were prepared.
2. Results and Discussion. – The syntheses of the pentapeptides Z-Gly-Aib-Acb-
Aib-Gly-OMe (4a; Acb ¼ 1-aminocyclobutanecarboxylic acid) and Z-Gly-Aib-Pro-
Aib-Aib-N(Me)Ph (4b) have been described in [12]. They were deprotected in the
usual way: saponification of 4a with LiOH · H2O in THF/H2O/MeOH gave the peptide
acid Z-Gly-Aib-Acb-Aib-Gly-OH (5a; 96%), and subsequent hydrogenolysis, either
1
)
In part from the Ph.D. thesis of F. S. A., Universitꢁt Zꢀrich, 1997. Present address: Bachem AG,
Hauptstrasse 144, CH-4416 Bubendorf
ꢄ 2015 Verlag Helvetica Chimica Acta AG, Zꢀrich

Helvetica Chimica Acta – Vol. 98 (2015)
233
Scheme 1
with H2/Pd/C in MeOH or with HCOONH4/Pd/C in MeOH, led to H-Gly-Aib-Acb-
Aib-Gly-OH (6a) in quantitative and 95% yield, respectively. Selective hydrolysis of
4b with 3n HCl in THF/H2O at room temperature yielded Z-Gly-Aib-Pro-Aib-Aib-
OH (5b, 70%) [12]. The latter was deprotected at the N-terminus to furnish H-Gly-
Aib-Pro-Aib-Aib-OH (6b) quantitatively (H2/Pd/C) and in 67% yield (HCOONH4/
Pd/C), respectively.
The cyclization of the two pentapeptides was achieved smoothly by treatment
with diethyl phosphorocyanidate (DEPC)/EtN(iPr)2 (Hꢀnig base) in DMF at room
temperature to give the cyclopentapeptides 7a and 7b in 56 and 89% yield, respectively
(Fig. 1). The structures were elucidated on the basis of the spectroscopic data
compared with those of the previously reported analogs [1]. For example, the dominant
peak in the ESI-MS of 7a appeared at m/z 404 ([M þ Na]þ) besides the [M þ 1]þ peak
at m/z 382. The 1H-NMR spectrum in CDCl3 showed four signals for NH groups at 7.21
(s, 2 NH), 7.08 (t-like, 1 NH), 6.57, and 6.23 ppm (2s, 2 NH), and only two singlets for
the Me groups of two Aib units. In the 13C-NMR spectrum (CDCl3), five signals of
C¼O groups were detected at 174.9, 174.8, 174.0, 171.8, and 171.4 ppm, and the signals
of the Me groups of the two Aib units appeared at 25.2 and 24.7 ppm.
For the attempted synthesis of cyclo(Gly-Aib-Aib-Aib-Aib) (7c), two linear
pentapeptide precursors were prepared as outlined in Schemes 2 and 3. Coupling of
Z-Gly-OH with the known tetrapeptide amide 8 [13] using O-(benzotriazol-1-yl)-
N,N,N’,N’-bis(tetramethylen)uronium hexafluorophosphate (HBPyU)/EtN(iPr)2 gave
Fig. 1. Structures of the cyclopentapeptides 7a and 7b

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Helvetica Chimica Acta – Vol. 98 (2015)
Scheme 2
Scheme 3
4c in 98% yield (Scheme 2). Deprotection of the latter in the usual way yielded 6c
(90%), which was cyclized by treatment with DEPC/EtN(iPr)2 at room temperature
for 17 h to give 7c in 66% yield.
In the first attempt to prepare the alternative precursor 6d, the dipeptide acid 9 [13]
was coupled with H-Gly-OMe to give, after saponification with LiOH · H2O in MeOH,
Z-(Aib)2-Gly-OH (10) in 86% yield (Scheme 3). Subsequent coupling of the latter with
H-(Aib)2-OMe (11a)2), yielded the protected pentapeptide 4d (74%), the structure of
which was established by X-ray crystallography (Fig. 2). Unexpectedly, all attempts to
2
)
Compound 11a was prepared from 9 via esterification with MeOH/BF3 · Et2O, followed by
hydrogenolysis with H2/Pd/C at room temperature, in 86% yield. The product was contaminated
with ca. 6% of 3,3,6,6-tetramethylpiperazine-2,5-dione.

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Fig. 2. ORTEP Plot [14] of the molecular structure of the pentapeptide 4d (50% probability ellipsoids,
arbitrary atom numbering, H-atoms bonded to C-atoms omitted for clarity)
cleave the ester group by treatment with LiOH · H2O to obtain the pentapeptide acid
failed. Therefore, the pentapeptide amide 4e was prepared by condensation of 10 with
H-(Aib)2-N(Me)Ph (11b), which was obtained from Z-(Aib)2-N(Me)Ph [13] by
treatment with H2/Pd/C in MeOH. Selective cleavage of the terminal amide bond by
treatment with 3n HCl in THF/H2O at room temperature and subsequent deprotection
of the N-terminus via transfer hydrogenolysis afforded the desired pentapeptide 6d in
95% yield. The cyclization of 6d under the conditions used for the case of 6c ! 7c, but
for 63 h, gave the same cyclopentapeptide 7c, but in the modest yield of 11%.
In the ESI-MS, characteristic for cyclopentapeptide 7c were the [M þ 1]þ peak at
m/z 398 (100%) together with peaks for [M þ Na]þ (m/z 420) and [M þ K]þ (m/z 436).
The 1H-NMR spectrum in CDCl3 showed a signal for the lactam NH of Gly at 7.31 ppm
(t-like), and four singlets of Aib-NH at 7.05, 6.65, 6.29, and 6.25 ppm. The signals of the
lactam C¼O groups appeared in the 13C-NMR spectrum at 176.5, 174.7, 174.1, 173.9,
and 172.4 ppm. The eight Me groups of the four Aib units resonated as four singlets in
the 1H-NMR spectrum (1.56, 1.55, 1.53, and 1.51 ppm) and gave rise to three signals in
the 13C-NMR spectrum (25.5, 24.9, 24.8 ppm (ca. 1:2 :1)). In addition, four singlets for
C(2) of four Aib and a triplet for CH2 of Gly were detected in the 13C-NMR spectrum.
Suitable crystals of 4d for an X-ray crystal-structure determination were obtained
from AcOEt/hexane by slow evaporation of the solvent. The molecule adopts an
overall helical conformation (Fig. 2). Each NH group of 4d acts as a donor for H-bonds
(Table 1). Three of them are intramolecular ones, forming a regular pattern along the
peptide chain: N(1)ꢀH, N(4)ꢀH, and N(7)ꢀH interact with the amide O-atom that is
seven atoms further along the peptide backbone. Each of these interactions has a graph
set motif [15] of S(10), i.e., three b-turns of type III are formed (Table 2) leading to an
overall 310-helical conformation of the peptide. The remaining N(10)ꢀH and N(13)ꢀH
groups, which are not able to form such intramolecular H-bonds, due to their positions
in the backbone, form intermolecular H-bonds with the ester C¼O and amide O-atoms,
respectively, at the opposite end of the same neighboring molecule. These interactions
link the molecules into extended chains running parallel to the [101] direction; graph
sets C(14) for each interaction.

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Table 1. Intra- and Intermolecular H-Bonds of Pentapeptide 4d (for atom numbering, see Fig. 2)
Donor··· Acceptor
N ··· O [ꢅ]
H ··· O [ꢅ]
NꢀH ··· O [8]
N(1)ꢀH ··· O(8)
N(4)ꢀH ··· O(11)
N(7)ꢀH ··· O(14)
N(10)ꢀH ··· O(24’)
N(13)ꢀH ··· O(2’)
2.952(3)
3.053(3)
2.879(3)
3.053(3)
2.860(3)
2.10(3)
2.21(3)
2.05(3)
2.21(3)
2.03(3)
167(2)
169(2)
158(2)
164(2)
168(2)
Table 2. Torsion Angles f, y, and w [8] of the Backbone of 4d in the Crystal
f(i)
y(i)
ꢀ 54.1(3)
ꢀ 42.1(3)
ꢀ 177.3(2)
ꢀ 55.2(3)
ꢀ 27.5(3)
ꢀ 179.3(2)
ꢀ 58.4(3)
y(iþ2)
w(iþ2)
f(iþ3)
y(iþ3)
w(iþ3)
f(iþ4)
y(iþ4)
ꢀ 23.7(3)
174.9(2)
ꢀ 60.4(3)
ꢀ 20.2(3)
ꢀ 176.9(2)
55.1(3)
w(i)
f(iþ1)
y(iþ1)
w(iþ1)
f(iþ2)
44.5(3)
Finally, the pentapeptide Z-(Aib)5-N(Me)Ph (4f) [13], prepared by repeated
azirine coupling and selective hydrolysis, was deprotected to give 6f in 94% yield
(Scheme 4). As 4f was rather insoluble in THF/H2O, the hydrolysis of the terminal
amide group with 3n HCl had to be performed at 608. The cyclization of 6f proved to be
very difficult. Under the usual conditions, with DEPC/EtN(iPr)2 in DMF at room
temperature as well as at 808, mixtures of products were obtained, among them
formylated pentapeptides, H-(Aib)5-OMe, H-(Aib)10-OMe (MS), etc. Also in CH2Cl2
or in MeCN at reflux, only traces of the desired cyclo[(Aib)5] (7d) could be detected
(MS). Finally, after treatment with DEPC/EtN(iPr)2 in DMF at room temperature for
14 d and repeated chromatographic purification, 7d was obtained in 12% yield.
Scheme 4

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237
The ESI-MS of 7d exhibited a dominant [M þ Na]þ peak (100%) at m/z 448.
1
Surprisingly, the H-NMR spectrum in CDCl3 showed only two singlets at 6.63 (NH)
and 1.54 ppm (Me2C) with an intensity ratio of 1:6. Similarly, only three signals
appeared in the 13C-NMR spectrum (CDCl3): a singlet at 175.2 (C¼O lactam), a singlet
at 58.0 (C(2) of (Aib)), and a quadruplet at 25.0 ppm (Me2C of Aib). From these data,
we concluded that cyclo[(Aib)5] (7d) in CDCl3 solution exists in a perfectly
symmetrical conformation, and no stable intramolecular H-bonds are formed.
3. Conclusions. – Three cyclopentapeptides containing three, four, and five Aib
units, as well as cyclo(Gly-Aib-Acb-Aib-Gly) were prepared by cyclization of the
corresponding linear precursors by treatment with DEPC/EtN(iPr)2 in DMF at room
temperature. The syntheses of the linear pentapeptides were achieved by applying the
ꢂazirine/oxazolone methodꢃ and segment condensation. The results of the cyclization
reactions evidence once more (see Introduction) that the efficiency of the ring closure
strongly depends on steric factors, especially at the N-terminal amino acid, i.e., the
nucleophile in the cyclization. Whereas cyclo[Gly-(Aib)4] was obtained in 66% yield
from H-Gly-(Aib)4-OH, the yield dropped to 11% in the case of H-(Aib)2-Gly-(Aib)2-
OH. Also, the ring closure of penta-Aib to give cyclo[(Aib)5] occurred only sluggishly.
Most remarkable are the NMR spectra of cyclo[(Aib)5], as they indicate a
symmetrical conformation, excluding the presence of stable intramolecular H-bonds.
We thank the analytical sections of our institute for spectra and analyses, and 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-methylalanin); DEPC, diethyl phosphorocyanidate;
EtN(iPr)2, ethyl(diisopropyl)amine (Hꢀnig base); HBPyU, O-(benzotriazol-1-yl)-N,N,N’,N’-bis(tetra-
methylen)uronium hexafluorophosphate; Z, (benzyloxy)carbonyl.
2. General. See [1][12]. Solvents were purified by standard procedures. TLC: Merck glass plates,
silica gel 60 F254. Column chromatography (CC): Uetikon-Chemie, silica gel C-560 (0.04 – 0.063 mm) or
Merck 60, 0.040 – 0.063 mm. M.p.: Mettler-FP-5 apparatus; uncorrected. [a]D Values: Perkin-Elmer-241
polarimeter at 218. IR Spectra: Perkin-Elmer-781 spectrometer, in KBr. 1H- and 13C-NMR spectra:
Bruker AC-300, Bruker ARX-300, or Bruker AMX-600 spectrometer (at 300 or 600 (1H) and 75.5 or
150 MHz, resp.); in CDCl3, CD3OD or (D6)DMSO; multiplicities of 13C signals determined by the
DEPT technique. ESI- and CI-MS: Finnigan TSQ-700 and Finnigan SSQ-700 instrument, respectively; in
m/z (rel. %).
General Procedure 1 (GP 1; Saponification of Peptide Methyl Esters). To a soln. of a peptide methyl
ester (1 mmol) in 10 ml of THF/MeOH/H2O (3 :1:1) at 08 was added LiOH · H2O (2.5 mmol). The
mixture was stirred at 08 for 1 h. Then, it was neutralized by addition of aq. 2n HCl, and the org. solvents
were evaporated (rotavapor). The residue was dissolved in AcOEt, und the mixture was washed with aq.
0.5n HCl. The org. phase was dried (Na2SO4), and the solvent was evaporated.
General Procedure 2 (GP 2; Hydrogenolysis). A mixture of Z-protected peptide in MeOH and ca.
10% Pd/C (10%) at r.t. was stirred under H2 (balloon) overnight. The mixture was filtered through a
Celite pad, and the solvent of the filtrate was evaporated to dryness.
General Procedure 3 (GP 3; Transfer Hydrogenolysis). To a mixture of Z-protected peptide
(1 mmol) and the same amount of Pd/C (10%) in MeOH was added HCOONH4 (5 mmol). The mixture
was heated at reflux for 10 min, and the hot mixture was filtered through a Celite pad and washed with
MeOH. The solvent of the filtrate was evaporated to dryness.

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Helvetica Chimica Acta – Vol. 98 (2015)
General Procedure 4 (GP 4; Hydrolysis of Peptide Amides). A soln. of Z-protected peptide amide
(1 mmol) in 3n HCl (THF/H2O 1:1) was stirred at r.t. for 1 – 4.5 h. Then, 2n HCl was added, and the
mixture was extracted with Et2O. The org. phase was dried (Na2SO4), and the solvent was evaporated.
General Procedure 5 (GP 5; Cyclization with DEPC). To a ca. 1.5 ꢁ 10ꢀ3 m soln. of a deprotected
pentapeptide (0.1 mmol) in DMF (67 ml) at 08 were added dropwise DEPC (0.2 – 0.4 mmol) and
EtN(iPr)2 (1% (v/v)), and the mixture was stirred overnight at r.t. Then, DMF was evaporated, and the
residue was purified chromatographically and crystallized.
General Procedure 6 (GP 6, Segment Condensation). To a mixture of a N-protected peptide
(1 mmol), C-protected amino acid (1.1 mmol), and HBPyU (1 mmol) in CH2Cl2 (1 ml) at r.t. was added
EtN(iPr)2 (2 mmol; 3 mmol in the case of an amino acid chloride), and the mixture was stirred for 1 h.
Then, the solvent was evaporated, and the residue was dissolved in AcOEt (20 ml), washed with aq.
KHSO4 (5%, 3 ꢁ ), aq. NaHCO3 (5%, 3 ꢁ ), and aq. NaCl, and purified by CC.
3. Synthesis of Cyclo(Gly-Aib-Acb-Aib-Gly) (7a). 3.1. Z-Gly-Aib-Acb-Aib-Gly-OH (5a). Hydro-
lysis of Z-Gly-Aib-Acb-Aib-Gly-OMe (4a [12]; 215 mg, 0.339 mmol) was performed with LiOH · H2O
(43 mg, 1.025 mmol) in THF/H2O/MeOH (3 :1:1, 4 ml) according to GP 1: 201 mg (96%) of 5a.
Colorless solid. M.p. 89.6 – 91.48. IR (KBr): 3300s (br), 3060m, 2990m, 2940m, 1755s, 1750s, 1740s, 1730s,
1715s, 1705s, 1695s, 1670s, 1660s, 1650s, 1645s, 1555s, 1540s, 1535s, 1525s, 1505m, 1470m, 1465m, 1455m,
1430m, 1415m, 1390m, 1365m, 1340m, 1310m, 1265m, 1240m, 1185m, 1050m, 1015m, 740m, 695m.
1H-NMR ((D6)DMSO): 8.41, 8.10 (2s, 2 NH); 7.53 (t-like, NH); 7.35 – 7.3 (m, 5 arom. H, NH); 7.25 (s,
NH); 5.02 (s, PhCH2); 3.7 – 3.6 (m, 2 CH2(Gly)); 2.55 – 2.4, 2.1 – 2.0, 1.9 – 1.75 (3m, 3 CH2(Acb)); 1.36,
1.34 (2s, 2 Me2C). 13C-NMR ((D6)DMSO): 174.7, 174.5, 172.5, 170.9, 169.8 (5s, 4 CO(amide), COOH);
156.7 (s, CO(urethane)); 136.8 (s, 1 arom. C); 128.3, 127.7, 127.5 (3d, 5 arom. CH); 65.5 (t, PhCH2); 58.6,
55.9, 55.6 (3s, 2 C(2)(Aib), C(2)(Acb)); 43.7, 40.8 (2t, 2 CH2(Gly)); 30.4 (t, 2 CH2(Acb)); 25.0, 24.7 (2q,
2 Me2C); 15.0 (t, CH2(Acb)). ESI-MS: 556 (100, [M þ Na]þ), 459 (3, [M ꢀ Gly]þ), 374 (10, [M ꢀ Aib-
Gly]þ).
3.2. H-Gly-Aib-Acb-Aib-Gly-OH (6a). a) Hydrogenolysis of 5a (233 mg, 0.437 mmol) in MeOH
(10 ml) with Pd/C (24 mg) according to GP 2; addition of H2O (10 ml), and filtration through Celite:
177 mg (quant.) of 6a.
b) Hydrogenolysis of 5a (114 mg, 0.214 mmol) in MeOH according to GP 3 with Pd/C (114 mg):
1
81 mg (95%) of 6a. Colorless solid. M.p. 239.2 – 240.78. H-NMR (D2O): 3.83, 3.75 (2s, 2 CH2(Gly));
2.65 – 2.5, 2.25 – 2.1, 2.05 – 1.9 (3m, 3 CH2(Acb)); 1.52, 1.50 (2s, 2 Me2C). 13C-NMR (D2O): 179.7, 179.0,
177.9, 169.4 (4s, 4 CO(amide), COOH); 62.1, 59.5, 59.4 (3s, 2 C(2)(Aib), C(2)(Acb)); 46.3, 43.3 (2t,
2 CH2(Gly)); 33.3 (t, 2 CH2(Acb)); 27.1, 26.7 (2q, 2 Me2C); 17.7 (t, CH2(Acb)). ESI-MS: 422 (100, [M þ
Na]þ).
3.3. Cyclo(Gly-Aib-Acb-Aib-Gly) (7a). The cyclization of 6a (27.3 mg, 0.068 mmol) in DMF (45 ml)
was carried out with DEPC (125.5 mg, 0.77 mmol) and EtN(iPr)2 (0.45 ml) according to GP 5. After CC
(CH2Cl2/MeOH/NH3 10 :1:0.1) and crystallization from CHCl3/hexane, 14.5 mg (56%) of 7a were
1
obtained. Colorless solid. M.p. 135.3 – 136.48. H-NMR (CDCl3): 7.21 (s, 2 NH); 7.08 (t-like, NH); 6.57,
6.23 (2s, 2 NH); 3.99 (d, J ¼ 6.3, CH2(Gly)); 3.85 (d, J ¼ 6.4, CH2(Gly)); 2.6 – 2.45, 2.4 – 2.3, 2.15 – 2.0,
2.0 – 1.8 (4m, 2 :2 :1:1, 3 CH2(Acb)); 1.58, 1.55 (2s, 2 Me2C). 13C-NMR (CDCl3): 174.9, 174.8, 174.0,
171.8, 171.4 (5s, 5 CO(amide)); 60.1, 57.7, 57.4 (3s, 2 C(2)(Aib), C(2)(Acb)); 44.7, 44.0 (2t, 2 CH2(Gly));
31.1 (t, 2 CH2(Acb)); 25.2, 24.7 (2q, 2 Me2C); 15.7 (t, CH2(Acb)). ESI-MS: 404 (100, [M þ Na]þ), 382 (7,
[M þ 1]þ).
4. Synthesis of Cyclo(Gly-Aib-Pro-Aib-Aib) (7b). 4.1. H-Gly-Aib-Pro-Aib-Aib-OH (6b). a)
Hydrogenolysis of 5b [12] (86 mg, 0.153 mmol) in MeOH (3 ml) with Pd/C (8.7 mg) was carried out
according to GP 2. After 10 min, the reaction was complete (TLC), and a precipitate formed, which
dissolved again during stirring overnight: 66 mg (quant.) of 6b.
b) Hydrogenolysis of 5b (330 mg, 0.588 mmol) in MeOH (3 ml) according to GP 3 with HCOONH4
(183 mg, 2.902 mmol) and Pd/C (331 mg) afforded 81 mg (95%) of 6b. Colorless solid. M.p. 173.6 –
175.38. IR (KBr): 3320m, 3050m, 2980m, 2930m, 2870m, 1670s, 1620s, 1550s, 1470m, 1450m, 1415m,
1390m, 1360m, 1280m, 1210m, 1190m, 1170m. 1H-NMR (D2O): 4.33 (t-like, CH(2)(Pro)); 3.89 (s,
CH2(Gly)); 3.8 – 3.65, 3.6 – 3.5 (2m, CH2(5)(Pro)); 2.3 – 2.15, 2.1 – 1.8 (2m, 1:3, CH2(3), CH2(4)(Pro));
1.51, 1.48, 1.43, 1.42 (4s, 1:3 :1:1, 3 Me2C). 13C-NMR (D2O): 184.5, 178.2, 176.7, 176.4, 168.5 (5s,

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4 CO(amide), COOH); 65.7 (d, CH(2)(Pro)); 60.5, 59.6 (2s, 1:2, 3 C(2)(Aib)); 51.7 (t, CH2(5)(Pro));
42.8 (t, CH2(Gly)); 31.0, 28.3 (2t, CH2(3), CH2(4)(Pro)); 27.2, 27.04, 26.97, 26.4 (4q, 3 Me2C). ESI-MS:
466 (23, [M þ K]þ, 450 (100, [M þ Na]þ), 428 (52, [M þ 1]þ), 325 (16, [M ꢀ Aib]þ), 286 (20, [Pro-Aib-
Aib]þ).
4.2. Cyclo(Gly-Aib-Pro-Aib-Aib) (7b). The cyclization of 6b (48.2 mg, 0.113 mmol) in DMF (75 ml)
was carried out with DEPC (67.0 mg, 0.411 mmol) and EtN(iPr)2 (0.75 ml) according to GP 5. After CC
(CH2Cl2/MeOH 10 :1) and crystallization from AcOEt/hexane, 41.2 mg (89%) of 7b were obtained.
Colorless solid. M.p. 139.1 – 140.58. [a]2D0 ¼ ꢀ35.6 (c ¼ 0.95, EtOH). IR (KBr): 3300s, 3040m, 2980m,
2940m, 1695s, 1680s, 1670s, 1660s, 1650s, 1645s, 1635s, 1565m, 1555s, 1550s, 1540s, 1520s, 1505s, 1480m,
1470m, 1460m, 1455m, 1445m, 1390s, 1365s, 1270m, 1245m, 1215m, 1195m, 1175m, 1075m, 1050m, 1030m.
1H-NMR ((D6)DMSO): 8.7 (very br. s, NH); 7.83 (br. s, NH); 7.30 (br. s, NH); 6.8 (very br. s, NH); 4.5 –
4.35 (m, CH(2)(Pro)); 4.15 – 3.8 (m, 1 H of CH2(Gly)); 3.55 – 3.3 (m, CH2(5)(Pro), 1 H of CH2(Gly));
2.0 – 1.75 (m, CH2(3), CH2(4)(Pro)); 1.49, 1.44, 1.40, 1.31 (4s, 1:2 :2 :1, 3 Me2C). ESI-MS: 432 (100, [M þ
Na]þ), 410 (20, [M þ 1]þ).
5. Synthesis of Cyclo(Gly-Aib-Aib-Aib-Aib) (7c). 5.1. Via Cyclization of H-Gly-Aib-Aib-Aib-Aib-
OH (6c). 5.1.1. Z-Gly-Aib-Aib-Aib-Aib-N(Me)Ph (4c). According to GP 6, to a mixture of H-Aib-Aib-
Aib-Aib-N(Me)Ph (8 [13], 202.6 mg, 0.453 mmol), Z-Gly-OH (98.2 mg, 0.469 mmol), and HBPyU
(202.7 mg, 0.470 mmol) in CH2Cl2 (4 ml) at r.t. was added EtN(iPr)2 (119.7 mg, 0.926 mmol). After
stirring for 21 h, the precipitate was filtered and washed with a little CH2Cl2 to give 4c (243.7 mg) as a
white powder. The filtrate was evaporated, and CC (CH2Cl2/MeOH) gave an additional 40.7 mg of 4c.
Total yield: 284.4 mg (98%). Colorless powder. M.p. 222.3 – 224.48. IR (KBr): 3310m, 3280m, 2980m,
2940m, 1700m, 1680s, 1670s, 1660s, 1645s, 1635s, 1590m, 1530s, 1495m, 1465m, 1450m, 1395m, 1380m,
1360m, 1270m, 1230m, 1170m, 1090m, 710m. 1H-NMR (CD3OD): 7.4 – 7.2 (m, 10 arom. H); 5.10 (s,
PhCH2); 3.71 (s, CH2(Gly)); 3.38 (s, MeN); 1.55, 1.48, 1.41, 1.38 (4s, 4 Me2C). 13C-NMR (CD3OD): 177.1,
176.7, 176.4, 175.9, 172.1 (5s, 5 CO(amide)); 159.4 (s, CO(urethane)); 147.2, 138.2 (2s, 2 arom. C); 130.3,
129.6, 129.1, 128.8, 128.2, 128.1 (6d, 10 arom. CH); 67.8 (t, PhCH2); 58.4, 58.2, 57.9, 57.6 (4s, 4 C(2)(Aib));
45.2 (t, CH2(Gly)); 41.1 (q, MeN); 26.3, 26.0, 25.34, 25.27 (4q, 4 Me2C). ESI-MS: 661 (16, [M þ Na]þ),
532 (100, [M ꢀ N(Me)Ph]þ), 447 (49, [M ꢀ Aib-N(Me)Ph]þ), 362 (18, [M ꢀ Aib-Aib-N(Me)Ph]þ).
5.1.2. Z-Gly-Aib-Aib-Aib-Aib-OH (5c). According to GP 4, 4c (255.8 mg, 0.400 mmol) was
hydrolyzed in 3n HCl (THF/H2O 1:1, 10 ml, 1 h). Extraction with AcOEt gave 213.4 mg (97%) of 5c.
A sample was recrystallized from boiling AcOEt. Colorless needles. M.p. 201.8 – 203.08. IR (KBr): 3340s,
3310s, 3300s, 3080m, 3070m, 3060m, 3040m, 2980m, 2940m, 2930m, 1730s, 1715s, 1710s, 1695s, 1680s,
1670s, 1660s, 1650s, 1645s, 1550s, 1540s, 1520s, 1510s, 1470m, 1455s, 1385s, 1365m, 1280s, 1240s, 1230s,
1170s, 1160s, 1155s, 1050m, 735m, 700m. 1H-NMR (CD3OD): 7.7 – 7.6 (m, 2 arom. H); 7.4 – 7.3 (m, 3 arom.
H); 5.10 (s, PhCH2); 3.72 (s, CH2(Gly)); 1.49, 1.42, 1.41, 1.39 (4s, 4 Me2C). 13C-NMR (CD3OD): 178.3,
176.9, 176.6, 172.1 (4s, 4 CO(amide), COOH); 159.8 (s, CO(urethane)); 138.2 (s, arom. C); 129.6, 129.1,
128.8 (3d, 5 arom. CH); 67.9 (t, PhCH2); 57.9, 57.7, 57.1 (3s, 4 C(2)(Aib)); 45.3 (t, CH2(Gly)); 25.7, 25.41,
25.36, 25.3 (4q, 4 Me2C). CI-MS: 551 (23), 550 (79, [M þ 1]þ), 533 (26), 532 (93, [M ꢀ OH]þ), 448 (24),
447 (100, [M ꢀ Aib-OH]þ), 442 (32), 424 (10), 416 (18, [M ꢀ BnCOO]þ), 398 (12), 362 (32, [M ꢀ Aib-
Aib-OH]þ), 104 (73, [H-Aib-OH þ 1]þ). Anal. calc. for C24H36N4O7 · 0.5 H2O (558.63): C 55.90, H 7.22, N
12.54; found: C 55.80, H 7.14, N 12.44.
5.1.3. H-Gly-Aib-Aib-Aib-Aib-OH (6c). According to GP 3, 5c (187.7 mg, 0.342 mmol) was
deprotected by treatment with HCOONH4 (109.8 mg, 1.741 mmol) and Pd/C (188.0 mg) in MeOH
(7 ml) to furnish 132.3 mg (93%) of 6c. Colorless solid. 1H-NMR (D2O): 3.84 (s, CH2(Gly)); 1.48, 1.47,
1.43, 1.42 (4s, 4 Me2C). 13C-NMR (D2O): 179.0, 178.8, 178.5, 169.3 (4s, 4 CO(amide), COOH); 60.6, 59.7
(2s, 4 C(2)(Aib)); 43.3 (t, CH2(Gly)); 27.2, 27.1, 26.9, 26.8 (4q, 4 Me2C). CI-MS: 417 (29), 416 (100, [M þ
1]þ), 398 (14, [M ꢀ OH]þ).
5.1.4. Cyclo(Gly-Aib-Aib-Aib-Aib) (7c). According to GP 5, to a soln. of 6c (38.6 mg, 0.093 mmol)
in DMF (63 ml) were added DEPC (46.6 mg, 0.286 mmol) and EtN(iPr)2 (0.6 ml) at r.t. After stirring for
17 h, DMF was evaporated, and the residue was purified by CC (CH2Cl2/MeOH 10 :1): 24.5 mg (66%) of
7c. Colorless solid. M.p. 126.3 – 127.78. IR (KBr): 3330m, 2980w, 2940w, 1690s, 1680s, 1670s, 1660s, 1650s,
1645s, 1635s, 1550s, 1540s, 1530s, 1515s, 1505s, 1470m, 1460m, 1390m, 1375m, 1225m. 1H-NMR (CDCl3):
7.31 (t-like, NH); 7.05, 6.65, 6.29, 6.25 (4s, 4 NH); 3.39 (d, J ¼ 6.1, CH2(Gly)); 1.56, 1.55, 1.53, 1.51 (4s,

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4 Me2C). 13C-NMR (CDCl3): 176.5, 174.7, 174.1, 173.9, 172.4 (5s, 5 CO(amide)); 59.1, 57.6, 57.4, 56.9 (4s,
4 C(2)(Aib)); 45.1 (t, CH2(Gly)); 25.5, 24.9, 24.8 (3q, 4 Me2C). ESI-MS: 436 (12, [M þ K]þ), 420 (19,
[M þ Na]þ), 398 (100, [M þ 1]þ).
5.2. Via Cyclization of H-Aib-Aib-Gly-Aib-Aib-OH (6d). 5.2.1. Z-Aib-Aib-Gly-OMe. According to
GP 6, with Z-Aib-Aib-OH (9 [13], 797.1 mg, 2.473 mmol), H-Gly-OMe · HCl (343.1 mg, 2.733 mmol),
HBPyU (1.070 g, 2.480 mmol), and EtN(iPr)2 (1.3 ml, 7.594 mmol) in CH2Cl2 (7 ml); reaction time 2.5 h.
The precipitate was filtered to give 550.3 mg of tripeptide; the filtrate was evaporated, and CC (AcOEt)
gave an additional portion of tripeptide. Total yield: 906.8 mg (93%). Colorless solid. M.p. 151.5 – 152.28.
IR (KBr): 3350s, 3280s, 3040m, 2980m, 2950m, 1755m, 1700s, 1670s, 1660s, 1535s, 1520s, 1455m, 1435m,
1410m, 1385m, 1370m, 1265m, 1215s, 1210s, 1195s, 1175s, 1090m, 1080s, 985m, 965m, 755m, 700m.
1H-NMR (CDCl3): 7.42 (br. s, NH); 7.4 – 7.3 (m, 5 arom. H); 6.46, 5.32 (2s, 2 NH); 5.10 (s, PhCH2); 3.99
(d, J ¼ 5.6, CH2(Gly)); 3.72 (s, MeO); 1.48 (s, 2 Me2C). 13C-NMR (CDCl3): 174.6, 173.1, 170.5 (3s,
2 CO(amide), COOMe); 155.7 (s, CO(urethane)); 136.0 (s, arom. C); 128.7, 128.5, 128.4 (3d, 5 arom.
CH); 67.1 (t, PhCH2); 57.3, 57.1 (2s, 2 C(2)(Aib)); 51.9 (q, MeO); 41.4 (t, CH2(Gly)); 25.43, 25.38 (2q,
2 Me2C). CI-MS: 412 (6), 411 (30, [M þ NH4]þ), 395 (21), 394 (100, [M þ 1]þ), 331 (14), 306 (22), 305
(8). Anal. calc. for C19H27N3O6 (393.44): C 58.00, H 6.92, N 10.68; found: C 57.88, H 7.04, N 10.62.
5.2.2. Z-Aib-Aib-Gly-OH (10). According to GP 1, Z-Aib-Aib-Gly-OMe (906.8 mg, 2.305 mmol)
was saponified with LiOH · H2O (243.0 mg, 5.791 mmol) in THF/MeOH/H2O 3 :1:1 (7 ml): 809 mg
(93%) of 10. Colorless solid. IR (KBr): 3320s, 3300s, 3085m, 3030m, 3000m, 2940m, 2910m, 2900m,
1765m, 1750m, 1702s, 1665s, 1620s, 1615s, 1565s, 1515s, 1470m, 1450m, 1410m, 1385m, 1370m, 1335m,
1265s, 1220m, 1175s, 1085s, 900m, 740m, 695m. 1H-NMR (CD3OD): 7.90 (br. s, NH); 7.74 (s, NH); 7.4 –
7.25 (m, 5 arom. H); 5.09 (s, PhCH2); 3.89 – 3.87 (m, CH2(Gly)); 1.42, 1.39 (2s, 2 Me2C). 13C-NMR
(CD3OD): 177.6, 176.7, 173.0 (3s, 2 CO(amide), COOH); 158.1 (s, CO(urethane)); 138.2 (s, arom. C);
129.6, 129.24, 129.18 (3d, 5 arom. CH); 67.8 (t, PhCH2); 58.0, 57.8 (2s, 2 C(2)(Aib)); 42.0 (t, CH2(Gly));
25.4 (q, 2 Me2C). CI-MS: 398 (6), 397 (36, [M þ NH4]þ), 381 (19), 380 (100, [M þ 1]þ), 305 (15, [M ꢀ
Gly]þ), 289 (17), 272 (16), 246 (30, [M ꢀ BnOCO]þ.
5.2.3. Z-Aib-Aib-OMe [16]. To a soln. of Z-Aib-Aib-OH [13] (1.070 g, 3.317 mmol) in MeOH
(30 ml) was added BF3 · Et2O (0.42 ml), and the mixture was stirred for 18 h. The solvent was
evaporated, the residue was dissolved in CH2Cl2 (150 ml), and the mixture was extracted with aq. 2n HCl
(2ꢁ), aq. 1n NaOH (2ꢁ), and brine, dried (MgSO4), and the solvent was evaporated. Crystallization
from AcOEt/hexane yielded Z-Aib-Aib-OMe (1.075 g, 97%). Colorless solid. M.p. 107.4 – 108.68. IR
(KBr): 3380m, 3360m, 3320s, 3280s, 3030m, 2980m, 2950m, 1730s, 1715s, 1660s, 1545m, 1535s, 1520s,
1470m, 1465m, 1450m, 1435m, 1385m, 1360m, 1300m, 1255s, 1225m, 1215m, 1190m, 1170m, 1155m,
1085m, 1070s, 960m, 745m, 695m. 1H-NMR (CDCl3): 7.4 – 7.3 (m, 5 arom. H); 6.90 (br. s, 1 NH); 5.31 (br.
s, 1 NH); 5.09 (s, PhCH2); 3.72 (s, MeO); 1.51, 1.50 (2s, 2 Me2C). 13C-NMR (CDCl3): 175.0, 173.5 (2s,
CO(amide), COOMe); 155.1 (s, CO(urethane)); 136.3 (s, arom. C); 128.5, 128.2, 128.1 (3d, 5 arom. CH);
66.7 (t, PhCH2); 57.0, 56.5 (2s, 2 C(2)(Aib)); 52.2 (q, MeO); 25.4, 24.5 (2q, 2 Me2C). CI-MS: 338 (17), 337
(100, [M þ 1]þ), 230 (5), 229 (59). Anal. calc. for C17H24N2O5 (336.39): C 60.70, H 7.19, N 8.33; found: C
60.92, H 7.38, N 8.36.
5.2.4. H-Aib-Aib-OMe [16] (11a). According to GP2, Z-Aib-Aib-OMe (204.8 mg, 0.609 mmol) in
MeOH (3 ml) was deprotected (19.6 mg Pd/C; 30 min): 109.5 mg (89%) of 11a (contaminated with ca.
6% of 3,3,6,6-tetramethylpiperazine-2,5-dione [17]). 1H-NMR ((D6)DMSO): 8.03 (s, NH); 3.57 (s,
MeO); 2.45 – 1.7 (br. signal, NH2); 1.38, 1.15 (2s, 2 Me2C). 13C-NMR ((D6)DMSO): 177.0, 174.4 (2s,
CO(amide), COOMe); 54.7, 54.0 (2s, 2 C(2)(Aib)); 51.7 (q, MeO); 28.3, 24.6 (2q, 2 Me2C). CI-MS: 405
(23, [2 M þ 1]þ), 204 (9), 203 (100, [M þ 1]þ).
5.2.5. H-Aib-Aib-N(Me)Ph (11b). According to GP2, Z-Aib-Aib-N(Me)Ph [13] (613 mg,
1.490 mmol) in MeOH (2 ml) was deprotected (10 mg Pd/C, 30 min): 413 mg (quant.) of 11b. Colorless
oil. 1H-NMR (CDCl3): 7.68 (br. s, NH); 7.45 – 7.25 (m, 5 arom. H); 3.27 (s, MeN); 1.48 (br. s, NH2); 1.49,
1.25 (2s, 2 Me2C). 13C-NMR (CDCl3): 175.9, 173.4 (2s, 2 CO(amide)); 145.2 (s, arom. C); 129.3, 128.0,
127.6 (3d, 5 arom. CH); 57.4, 54.9 (2s, 2 C(2)(Aib)); 41.4 (q, MeN); 28.8, 26.6 (2q, 2 Me2C).
5.2.6. Z-Aib-Aib-Gly-Aib-Aib-OMe (4d). According to GP 6, with 10 (138.8 mg, 0.366 mmol),
freshly prepared 11a (82.3 mg, 0.407 mmol), HBPyU (158.2 mg, 0.367 mmol), and EtN(iPr)2 (131 mg,
1.014 mmol) in CH2Cl2 (3 ml); reaction time 2 h. The precipitate formed during evaporation was

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filtered to give 103.2 mg 4d; the filtrate was evaporated, and CC (AcOEt) gave an additional portion of
4d. Total yield: 151.9 mg (74%). Colorless solid. M.p. 175.3 – 176.18. IR (KBr): 3350s, 3320s, 3300s, 3260s,
3040m, 2980m, 2940m, 1760s, 1700s, 1680s, 1665s, 1660s, 1650s, 1540s, 1515s, 1470m, 1465m, 1455m,
1410m, 1385m, 1360m, 1305m, 1265s, 1230m, 1215m, 1195m, 1160m, 1080s, 700m. 1H-NMR (CDCl3): 7.74
(t-like, NH); 7.33 (br. s, 5 arom. H, NH); 7.08, 6.55, 6.05 (3s, 3 NH); 5.09 (s, PhCH2); 3.70 (d, J ¼ 5.8,
CH2(Gly)); 3.66 (s, MeO); 1.53, 1.51, 1.48, 1.39 (4s, 4 Me2C). 13C-NMR (CDCl3): 175.5, 175.3, 174.5,
174.3, 169.0 (5s, 4 CO(amide), COOMe); 156.5 (s, CO(urethane)); 136.0 (s, arom. C); 128.7, 128.6, 128.1
(3d, 5 arom. CH); 67.5 (t, PhCH2); 57.1, 56.7, 55.8 (3s, 4 C(2)(Aib)); 52.1 (q, MeO); 44.7 (t, CH2(Gly));
25.4, 25.13, 25.08, 25.0 (4q, 4 Me2C). ESI-MS: 586 (100, [M þ Na]þ). Anal. calc. for C27H41N5O8 (563.65):
C 57.54, H 7.43, N 12.42; found: C 57.54, H 7.20, N 12.21.
Crystals suitable for an X-ray crystal-structure determination were grown from AcOEt/hexane by
slow evaporation of the solvent.
5.2.7. Z-Aib-Aib-Gly-Aib-Aib-N(Me)Ph (4e). According to GP 6, with 10 (386.5 mg, 1.019 mmol),
11b (338.2 mg, 1.219 mmol), HBPyU (442 mg, 1.025 mmol), and EtN(iPr)2 (309 mg, 2.391 mmol) in
CH2Cl2 (5 ml); reaction time 16 h. Purification by CC (CH2Cl2/MeOH 20 :1) gave 529.8 mg (81%) of 4e.
A sample was recrystallized from CH2Cl2/hexane. Colorless crystals. M.p. 199.7 – 201.48. IR (KBr): 3310s,
3250m, 2980m, 1690s, 1680s, 1660s, 1645s, 1595m, 1495m, 1470m, 1455m, 1435m, 1395m, 1385m, 1360m,
1270s, 1215m, 1195m, 1175m, 1090m, 1070m, 700m. 1H-NMR (CD3OD): 7.45 – 7.25 (m, 10 arom. H); 5.11
(s, PhCH2); 3.62 (s, CH2(Gly)); 3.36 (s, MeN); 1.53, 1.51, 1.41, 1.35 (4s, 4 Me2C). 13C-NMR (CD3OD):
178.5, 177.4, 176.5, 175.6, 171.8 (5s, 5 CO(amide)); 158.5 (s, CO(urethane)); 147.0, 138.5 (2s, 2 arom. C);
130.3, 129.6, 129.2, 128.9, 128.4 (5d, 10 arom. CH); 68.0 (t, PhCH2); 58.5, 57.2 (2s, 4 C(2)(Aib)); 45.3 (t,
CH2(Gly)); 41.1 (q, MeN); 26.4, 26.0, 25.4, 25.3 (4q, 4 Me2C). ESI-MS: 661 (100, [M þ Na]þ), 532 (6,
[M ꢀ N(Me)Ph]þ).
5.2.8. Z-Aib-Aib-Gly-Aib-Aib-OH. According to GP 4, 4e (448.5 mg, 0.702 mmol) was hydrolyzed in
3n HCl (THF/H2O 1:1; 7 ml): 370.2 mg (96%) of Z-Aib-Aib-Gly-Aib-Aib-OH. Colorless solid. M.p.
215.4 – 216.88. IR (KBr): 3300s, 3060m, 3040m, 2990m, 2940m, 1720s, 1695s, 1670s, 1660s, 1550m, 1540s,
1530s, 1525s, 1470m, 1460m, 1455m, 1385m, 1365m, 1270s, 1220m, 1170m, 1090m, 1080m, 700m. 1H-NMR
(CD3OD): 7.45 – 7.3 (m, 5 arom. H); 5.11 (s, PhCH2); 3.64 (s, CH2(Gly)); 1.49, 1.48, 1.41, 1.36 (4s,
4 Me2C). 13C-NMR (CD3OD): 178.3, 178.0, 177.4, 176.2, 171.8 (5s, 4 CO(amide), COOH); 158.5 (s,
CO(urethane)); 138.5 (s, arom. C); 129.6, 129.2, 128.9 (3d, 5 arom. CH); 68.0 (t, PhCH2); 58.2, 57.8, 57.7,
57.1 (4s, 4 C(2)(Aib)); 45.0 (t, CH2(Gly)); 25.7, 25.4, 25.30, 25.27 (4q, 4 Me2C). ESI-MS: 572 (100, [M þ
Na]þ).
5.2.9. H-Aib-Aib-Gly-Aib-Aib-OH (6d). According to GP 3, Z-Aib-Aib-Gly-Aib-Aib-OH
(281.5 mg, 0.513 mmol) was deprotected in MeOH (9 ml) by treatment with HCOONH4 (163 mg,
1
2.585 mmol) and Pd/C (280.3 mg): 214.0 mg (quant.) of 6d. Colorless solid. H-NMR (D2O): 3.85 (s,
CH2(Gly)); 1.64, 1.51, 1.49, 1.43 (4s, 4 Me2C). 13C-NMR (CD3OD): 184.7, 179.9, 177.8, 175.2, 173.2 (5s,
4 CO(amide), COOH); 60.8, 60.0, 59.3 (3s, 4 C(2)(Aib)); 46.0 (t, CH2(Gly)); 27.1, 26.9, 26.7, 25.9 (4q,
4 Me2C). ESI-MS: 416 (100, [M þ 1]þ).
5.2.10. Cyclo(Gly-Aib-Aib-Aib-Aib) (7c). According to GP 5, to a soln. of 6d (27.2 mg, 0.066 mmol)
in DMF (45 ml) were added DEPC (34.0 mg, 0.21 mmol) and EtN(iPr)2 (0.4 ml) at r.t. After stirring for
63 h, DMF was evaporated, and the residue was purified by CC (CH2Cl2/MeOH 20 :1 ! 10 :1): 2.8 mg
(11%) of 7c.
6. Synthesis of Cyclo(Aib-Aib-Aib-Aib-Aib) (7d). 6.1. Z-Aib-Aib-Aib-Aib-Aib-OH. According to
GP 4, Z-Aib-Aib-Aib-Aib-Aib-N(Me)Ph (4f [13]; 881.1 mg, 1.321 mmol) was hydrolyzed at 608 for 1 h.
The precipitate was filtered and washed with Et2O to give 726.8 mg of Z-Aib-Aib-Aib-Aib-Aib-OH.
Extraction of the aq. phase with Et2O (4ꢁ) gave additional 34.0 mg of the product. Total yield: 760.8 mg
(quant.). Colorless solid. M.p. 226.2 – 227.38. IR (KBr): 3410s, 3320s, 3290s, 3250s, 3040s, 2980s, 2940s,
2920m, 2900m, 1745s, 1730s, 1710s, 1705s, 1695s, 1680s, 1675s, 1665s, 1645s, 1635s, 1580m, 1565m, 1550s,
1530s, 1515s, 1465s, 1455s, 1445s, 1385s, 1365s, 1320s, 1315s, 1280s, 1270s, 1265s, 1225s, 1210s, 1185s, 1180s,
1170s, 1165s, 1090s, 1080s, 955m, 945m, 750m, 700m. 1H-NMR ((D6)DMSO): 11.83 (br. s, COOH); 8.23,
7.85, 7.46 (3s, 3 NH ) ; 7.4 – 7.3 ( m, 5 arom. H, 1 NH); 7.28 (s, NH); 5.09 (s, PhCH2); 1.33, 1.32, 1.21 (3s,
3 :1:1, 5 Me2C). 13C-NMR ((D6)DMSO): 175.5, 174.9, 174.7, 173.42, 173.38 (5s, 4 CO(amide), COOH);
155.7 (s, CO(urethane)); 137.1 (s, arom. C); 128.3, 127.6, 127.1 (3d, 5 arom. CH); 65.4 (t, PhCH2); 55.9,

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55.81, 55.76, 55.6, 54.5 (5s, 5 C(2)(Aib)); 25.0, 24.6, 24.5, 24.3 (4q, 5 Me2C). ESI-MS: 660 (100, [M þ
Na]þ), 578 (80, [M þ 1]þ), 560 (8, [M ꢀ OH]þ), 475 (19, [M ꢀ Aib-OH]þ). Anal. calc. for C28H43N5O8
(577.68): C 58.22, H 7.50, N 12.12; found: C 58.36, H 7.30, N 11.93.
6.2. H-Aib-Aib-Aib-Aib-Aib-OH (6f). According to GP 3, Z-Aib-Aib-Aib-Aib-Aib-OH (233.2 mg,
0.404 mmol) in MeOH (7 ml) was deprotected by treatment with HCOONH4 (129.5 mg, 2.054 mmol)
and Pd/C (240 mg). The crude material was dissolved in MeOH and precipitated with Et2O to give
169.8 mg (95%) of 6f. Colorless powder. 1H-NMR (D2O): 7.54, 7.52 (2s, 2 NH); 1.65, 1.48, 1.43, 1.42 (4s,
1:2 :1:1, 5 Me2C). 13C-NMR (D2O): 178.9, 178.6, 178.4, 175.3 (4s, 4 CO(amide), COOH); 60.1, 59.9,
59.7, 59.6 (4s, 5 C(2)(Aib)); 27.2, 27.1, 26.9, 26.5, 25.9 (5q, 5 Me2C). ESI-MS: 466 (7, [M þ Na]þ), 444 (100,
[M þ 1]þ).
6.3. Cyclo(Aib-Aib-Aib-Aib-Aib) (7d). To a soln. of 6f (171.3 mg, 0.386 mmol) in DMF (260 ml)
were added DEPC (294.8 mg, 1.807 mmol) and EtN(iPr)2 (2.6 ml) at r.t. After stirring at r.t. for 2 weeks,
DMF was removed in high vacuum, and subsequent CC (CH2Cl2/MeOH 15 :1, 3 ꢁ ) yielded 30.7 mg of a
mixture of 7d and an unknown compound. Additional CC (AcOEt/MeOH 20 :1, 2 ꢁ ) gave 19.7 mg
(12%) 7d. Colorless powder. M.p. > 2608. IR (KBr): 3420m, 3320s, 3120m, 2990m, 2980m, 2940m, 1690s,
1670s, 1650s, 1570s, 1520s, 1480m, 1450m, 1385s, 1360s, 1310m, 1280s, 1220s, 1210s, 1185m, 1170m.
1H-NMR (CDCl3): 6.63 (s, 5 NH); 1.54 (s, 5 Me2C). 13C-NMR (CDCl3): 175.2 (s, 5 CO(lactam)); 58.0 (s,
5 C(2)(Aib)); 25.0 (q, 5 Me2C). ESI-MS: 448 (100, [M þ Na]þ).
7. X-Ray Crystal-Structure Determination of 4d (see Table 3 and Fig. 2)3). The measurements were
conducted on a Rigaku AFC5R diffractometer using graphite-monochromated MoKa radiation (l
0.71073 ꢅ) and a 12-kW rotating anode generator. The intensities were corrected for Lorentz and
polarization effects. Azimuthal scans of several reflections indicated no need for an absorption
correction. Equivalent reflections were merged. The data collection and refinement parameters are given
in Table 3, and a view of the molecule is shown in Fig. 2. The structure was solved by direct methods using
SHELXS86 [18], which revealed the positions of all non-H-atoms. The non-H-atoms were refined
anisotropically. The amide H-atoms were placed in the positions indicated by a difference electron-
Table 3. Crystallographic Data for Compound 4d
Crystallized from
Empirical formula
Formula weight
Crystal color, habit
Crystal dimensions [mm]
Temp. [K]
Crystal system
Space group
Z
AcOEt/hexane
C27H41N5O8
563.65
colorless, needle
0.20 ꢁ 0.25 ꢁ 0.45
173(1)
monoclinic
P21/n
4
Dx [gcmꢀ3
]
1.215
0.090
w/2q
55
7404
7068
3881
7068
391
m(MoKa) [mmꢀ1
Scan type
]
2q(max) [8]
Total reflections measured
Symmetry-independent reflections
Reflections with I > 2s(I)
Reflections used in refinement
Parameters refined
Reflections for cell determination
2q Range for cell determination [8] 20 – 37
Unit cell parameters a [ꢅ]
25
Final R(F) [I > 2s (I) reflections] 0.0530
wR(F2) (all data)
Weighting parameter (a)a)
Goodness-of-fit
0.1412
0.0513
1.003
11.674(2)
b [ꢅ]
c [ꢅ]
16.174(2)
16.774(2)
103.29(1)
3082.1(8)
Secondary extinction coefficient
Final Dmax/s
0.0018(5)
0.000
0.25; ꢀ 0.23
b [8]
V [ꢅ3]
D1 (max; min) [eꢅꢀ3
]
a) wꢀ1 ¼ s2(F2o ) þ (aP)2, where P ¼ (Fo2 þ 2F2c )/3
3
)
CCDC-1034190 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.

Helvetica Chimica Acta – Vol. 98 (2015)
243
density map, and their positions were allowed to refine together with individual isotropic displacement
parameters. All remaining 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 C-atom (1.5 Ueq for the Me groups). The refinement of the structure
was carried out on F2 by using full-matrix least-squares procedures, which minimized the function
Sw(F2o – F2c )2. A correction for secondary extinction was applied. Neutral atom scattering factors for non-
H-atoms were taken from [19], and the scattering factors for H-atoms were taken from [20]. Anomalous
dispersion effects were included in Fc [21]; the values for f’ and f’’ were those of [22]. The values of the
mass attenuation coefficients are those of [23]. The SHELXL-2014 program [24] was used for all
calculations.
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Received November 17, 2014