Small cyclic disulfide peptides: Synthesis in preparative amounts and characterization by means of NMR and FT-IR spectroscopy

Article abstract of DOI:10.1002/ejoc.200400174

Two cyclic disulfide-bridged tetrapeptides [cyclo(Boc-Cys-Pro-Aib-Cys-OMe) and cyclo(Boc-Cys-Pro-Phe-Cys-OMe)] were prepared in high yield and purity. The use of double-walled reaction flasks, which were attached to an external cryostat, gave perfect temperature control of the reaction. The acetamidomethyl and trityl group were used for the protection of the thiol groups of Cys. Disulfide bond formation was obtained by cleavage of the protection groups and subsequent oxidation of the free thiols with iodine under high dilution conditions in one step. The obtained cyclic peptides were checked for purity by analytical HPLC, and identified by NMR spectroscopy and a variety of standard analytical methods (IR, m. p., FAB-MS, ELA, TLC). Conformational properties of the peptides were derived from one- and two-dimensional NMR experiments. Temperature-dependent NMR and FT-IR experiments allowed for the determination of the degree of inter- and intramolecular hydrogen bonding of the cyclic tetrapeptides. The results from the temperature-dependent NMR experiments are in good agreement with the observed dynamics of the peptides in the amide I and II region, which were determined by means of temperature-dependent FT-IR spectroscopy using the ATR technique. Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004.

Full text of DOI:10.1002/ejoc.200400174

FULL PAPER
Small Cyclic Disulfide Peptides: Synthesis in Preparative Amounts and
Characterization by Means of NMR and FT-IR Spectroscopy
Christoph Kolano,[a] Klaus Gomann,[a] and Wolfram Sander*[a]
Keywords: Peptides / Disulfide bond formation / High dilution / Hydrogen bonds / ATR technique / Temperature-
dependent IR spectroscopy
Two cyclic disulfide-bridged tetrapeptides [cyclo(Boc−Cys−
Pro−Aib−Cys−OMe) and cyclo(Boc−Cys−Pro−Phe−Cys−
OMe)] were prepared in high yield and purity. The use of
double-walled reaction flasks, which were attached to an ex-
ternal cryostat, gave perfect temperature control of the reac-
tion. The acetamidomethyl and trityl group were used for the
ical methods (IR, m. p., FAB-MS, ELA, TLC). Conformational
properties of the peptides were derived from one- and two-
dimensional NMR experiments. Temperature-dependent
NMR and FT-IR experiments allowed for the determination
of the degree of inter- and intramolecular hydrogen bonding
of the cyclic tetrapeptides. The results from the temperature-
protection of the thiol groups of Cys. Disulfide bond forma- dependent NMR experiments are in good agreement with
tion was obtained by cleavage of the protection groups and
subsequent oxidation of the free thiols with iodine under
the observed dynamics of the peptides in the amide I and II
region, which were determined by means of temperature-
high dilution conditions in one step. The obtained cyclic pep- dependent FT-IR spectroscopy using the ATR technique.
tides were checked for purity by analytical HPLC, and identi- ( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
fied by NMR spectroscopy and a variety of standard analyt- Germany, 2004)
Introduction
gering event (that perturbs the protein/peptide from a ‘‘star-
ting’’ conformation) is required to initiate the folding or
unfolding process. In time-resolved spectroscopy, in general,
the triggering event is a short laser pulse. Cyclic tetrapep-
tides containing a disulfide bridge are well-suited, since the
disulfide bridge is a weak covalent bond (bond dissociation
energy, 64.5 kcal molϪ1) and thus provides a predetermined
breaking point. Thus, a disulfide bridge can easily and irre-
versibly be cleaved by UV light.
The dynamic behavior of BR (bacteriorhodopsin), which
undergoes a reversible photocycle on irradiation,[1Ϫ3] is best
known among all biomolecules. This protein has been ex-
tensively investigated by time-resolved infrared spec-
troscopy using the STEP/SCAN technique.[4] Up to now,
no dynamic investigations have been carried out on much
smaller biomolecules, which play an important role in the
human organism. As many primary processes of small bi-
omolecules take place on a time-scale faster than microse-
conds, real time dynamic investigations in the nanosecond
time region provide an insight to the primary processes of
protein folding in detail. Therefore, such results are well-
suited to optimize algorithms of molecular modeling pro-
gram packages. The hormone peptides oxytoxin and vasop-
ressin are examples of small bioactive peptides. Such cyclic
tetrapeptides containing a disulfide bridge and a folding
motif have been of interest for a long time. A variety of
peptides were synthesized and investigated by a variety of
techniques to determine structural and conformational
properties, binding affinity, catalytic properties and biologi-
cal activity. To study protein- or peptide folding, a trig-
For the investigation of cyclic tetrapeptides containing a
disulfide bridge with regard to the dynamic behavior (fold-
ing processes, formation of a motif or aggregation) using
the STEP/SCAN technique, a few aspects have to be taken
into consideration. Water is best suited for biological sys-
tems, but, due to the strong absorptions of water in the IR
region, unsuitable for infrared spectroscopy. The problem
can be avoided by a minimization of the spacer size of the
spectroscopic cell or the use of the ATR technique. A good
alternative is to keep a suitable protection group (photost-
able towards UV irradiation) at the N-terminus and the C-
terminus of the peptides to make the compound soluble in
solvents like tetrachloromethane, acetonitrile or cyclohex-
ane, which are more suitable for IR spectroscopy. The inves-
tigation of folding processes by time-resolved IR spec-
troscopy, thus requires (i) that the peptides are available on
a large scale (several grams); (ii) that the peptides are appro-
priately modified to ensure good solubility in the solvents
used for the time-resolved investigations (C- and N-ter-
minus); (iii) that no further photoactive or labile groups are
present in the peptide.
[a]
Lehrstuhl für Organische Chemie II,
Ruhr-Universität Bochum,
Universitätstrasse 150, 44801 Bochum, Germany
Fax (internat.): ϩ49-234-3214353
E-mail: Wolfram.Sander@rub.de
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
Eur. J. Org. Chem. 2004, 4167Ϫ4176
DOI: 10.1002/ejoc.200400174
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4167

C. Kolano, K. Gomann, W. Sander
FULL PAPER
SPPS (solid-phase peptide synthesis) is a well-established dation of the linear form 2 with iodine in methanol under
technique and offers numerous advantages over the classical high dilution conditions.
method in solution, but, due to the limited loading capacity
The cyclic tetrapeptide cyclo(BocϪCysϪProϪPheϪ
of the resin, it is less suitable for the synthesis of peptides CysϪOMe) (6) was prepared according to Scheme 2. Lin-
on a large scale (several grams) in a research laboratory. ear tetrapeptide 7 was synthesized by a stepwise elongation
A well-designed and optimized classical synthetic strategy using the mixed anhydride method with isobutyl chloro-
under slightly modified and optimized reaction conditions formate. Deprotection of the Boc-intermediates 8 and 9 was
leads more easily to the desired amount of peptides. In this once again performed with the use of TFA. The cyclization
paper we demonstrate the advantage of the classical solu- that yields 6 was performed as described before.
tion method in combination with specially designed
Discussion
doubled-walled reaction flasks attached to an external cryo-
stat to give perfect temperature control for the large scale
synthesis of peptides. We focus on the synthesis of two cyc-
lic tetrapeptides cyclo(BocϪCysϪProϪAibϪCysϪOMe)
(1) and cyclo(BocϪCysϪProϪPheϪCysϪOMe) (6). Fur-
ther, we characterized the structural properties of these pep-
tides by NMR and temperature-dependent IR spectroscopy
in view of time-resolved investigations, which are currently
in progress.
Synthesis of Peptides
The strategy for the synthesis of peptide 1 is based on
the fragment condensation of smaller peptides. In our case,
dipeptide 4 was chosen as the starting material, as a good
synthetic protocol for this compound was published by
Jung et al.[5] several years ago. We increased the yield of
this reaction by 5Ϫ10% with the use of double-walled reac-
tion flasks in combination with an external cryostat, which
gave perfect temperature control. This technique allowed
the optimization of reaction temperatures and times for the
coupling reactions. The coupling between cysteine deriva-
Chemistry
The cyclic tetrapeptide cyclo(BocϪCysϪProϪAibϪ tive 10 and dipeptide 4 proved to be the most difficult step.
CysϪOMe) (1) was prepared according to Scheme 1. The The esterification of HCl*HϪCys(Acm)ϪOH was de-
key intermediate, dipeptide BocϪProϪAibϪOH (4), was scribed to yield a colorless, hygroscopic foam,[6] which can
synthesized in a slightly modified way according to the be crystallized from isopropyl alcohol.[7] We obtained the
method published by Jung et al.[5] Tripeptide 3 was pre- resulting ester HCl*HϪCys(Acm)ϪOMe (10) as a colorless
pared by the elongation of the dipeptide 4 with CDMT as crystalline compound. After careful removal of the solvent
a suitable coupling reagent. Elongation to the linear tetra- and remaining thionyl chloride, the product from a large-
peptide 2 was done with the use of DCCI/HOBt. Deprotec- scale preparation showed no tendency of forming a foam.
tion of the Boc-intermediate 3 was performed by treatment Smaller quantities can be crystallized successively from dry
with TFA. The cyclic tetrapeptide 1 was obtained by oxi- ether and pentane.
Scheme 1
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Eur. J. Org. Chem. 2004, 4167Ϫ4176

Synthesis and Characterization of Small Cyclic Disulfide Peptides
FULL PAPER
Scheme 2
Various coupling reagents and temperatures have been Cys(Trt)ϪProϪPheϪCys(Acm)ϪOMe (7) was synthesized
checked for the coupling reaction involving the a, a-dialkyl- by a stepwise elongation using the mixed anhydride method
ated amino acid Aib (aminoisobutyric acid) in the case of with isobutyl chloroformate, again in combination with
peptide 3. Although an excellent coupling method for Aib temperature-controlled double-walled reaction flasks. De-
has been published by Carpino[8a] (using TFFH), we found protection of the Boc protected intermediates was per-
two methods to be most suitable for attaching the cysteine formed by treatment with TFA, as described before. Cycli-
derivative 10 to the dipeptide BocϪProϪAibϪOH. The use zation used I2/methanol under high dilution conditions at
of DCCI/HOBt as a coupling reagent gives the desired tri- ambient temperature. The yield of this oxidation reaction is
peptide 3 in fairly high yields; however, column chromatog- much lower than in the case of cyclic tetrapeptide 1. Col-
raphy is necessary to obtain the peptide in good purity. The umn chromatography yields the desired cyclic tetrapeptide
second method uses CDMT[8b] as a coupling reagent giving 6 in moderate yields (40Ϫ45%). The Aib/Phe exchange in-
the tripeptide in lower yields as in the method described fluences the conformation of the backbone. In the linear
before, but no further purification by column chromatogra- tetrapeptide 2, the nonchiral amino acid Aib is used instead
phy is necessary. The slightly basic properties of the triazine of the chiral amino acid Phe. This leads to a conformation
allows extraction under acidic conditions. We performed of 2 that is less flexible than the conformation of 6, resulting
coupling reactions using the MA method for attaching Aib in a much closer arrangement of the Cys groups (see also
to the dipeptide 4. However, an oily product was obtained, NMR and IR investigations.), and thus higher yields of the
which gave only very low amounts of 3 after purification. cyclized product 1 relative to 6.
Methods like preactivation or the use of very powerful
coupling reagents like HATU, TBTU and HBTU showed
NMR Studies
no significant improvement over the coupling reaction. De-
Well-resolved NMR spectra were recorded for the linear
protection was performed with TFA, which was diluted peptides 2 and 7, and the cyclic peptides 1 and 6 in
with hexane. The pure deprotected peptides were obtained [D6]DMSO at ambient temperature. 1H NMR spectra for 1
after coevaporation with hexane and precipitation with dry and 2 are shown in Figure 1 (for data for 6 and 7, see Sup-
ether in good yield. The deprotected peptides are stable at porting Information). The assignments are based on
room temperature for about 3 days if stored in a desiccator TOCSY, HMBC, HMQC and ROESY experiments.
under argon. Good coupling results can be obtained by TOCSY and HMQC spectra for 1 and 2 are shown in the
using of a large excess of base, and dissolving the mixture Supplementary Information.
by ultrasonication. The standard method DCCI/HOBt was
The 1H NMR spectra of the cyclic tetrapeptide
used for coupling to the linear tetrapeptide 2. The use of cyclo[BocϪCysϪProϪPheϪCysϪOMe] (6) in [D6]DMSO
two different thiol protection groups in combination with a shows a variety of complex signals, while one- and two-
high dilution condition minimizes the amount of oligomers dimensional NMR investigations of 6 in CDCl3 show two
and polymers in the cyclization reaction. The yield of the well-resolved sets of signals, which belong to two confor-
desired cyclic tetrapeptide 1 after column chromatography mations. A similar influence of the solvent on peptide con-
is 60Ϫ65%.
formation has been observed by Balaram et al.[9] for the
The cyclic tetrapeptide 6 was obtained from cyclization derivative cyclo[BocϪCysϪProϪPheϪCysϪNHMe] (12).
of the key intermediate 7. The linear tetrapeptide BocϪ The complete assignment of the signals of 6 in CDCl3 is
Eur. J. Org. Chem. 2004, 4167Ϫ4176
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4169

C. Kolano, K. Gomann, W. Sander
FULL PAPER
Figure 3. Schematic representation of the cis/trans conformers of a
XxxϪPro peptide bond
based on HMQC, HMBC, TOCSY and ROESY experi-
ments. The two conformers were identified as the trans and
cis species of 6 with respect to the XxxϪPro amide bond.
A distinction between the two conformers is based on the
significant shifts of the β- and γ-carbon atom of the proline
ring.[10,11] This information is easily extracted (13C NMR,
HMQC and HMBC). 13C spectra of 1, 2 and 7 show
characteristic signals of Pro near δ ϭ 29.5 ppm (Cβ) and
24.2 ppm (Cγ) indicating a trans XxxϪPro peptide bond
(Table 1).[10,11] As expected, 1, 2 and 7 exist in the energeti-
cally favored trans conformation. In contrast, 6 shows a
trans and a cis XxxϪPro peptide bond in the major (M)
and minor (m) forms, respectively. The ratio of the confor-
mations (M:m) was estimated to approximately 2:1 by inte-
1
gration of the signals of the H NMR spectra. This result
is in agreement with the well-known activation barrier of
16.7Ϫ21.5 kcal molϪ1 for the cis/trans isomerization along
the XxxϪPro peptide bond and the small energy difference
of 0.5Ϫ1.4 kcal·molϪ1 between cis XxxϪPro and trans
XxxϪPro.[12Ϫ15] The complete data from the NMR experi-
ments is summarized in Table 2
1
1
Figure 1. Top: H spectra of 2; bottom: H spectra of 1; both 600-
MHz in [D6]DMSO; signals marked with an asterisk belong to
ethyl acetate (eluate)
1
Figure 2. 600-MHz H NMR spectrum of 6 in CDCl3; (M) refers to the major, (m) to the minor conformation; signals marked with an
asterisk or circle belong to the eluate; inset: top: magnification of the 4.2Ϫ5.5 ppm region; bottom: magnification of the 2.0Ϫ3.6 ppm
region
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Eur. J. Org. Chem. 2004, 4167Ϫ4176

Synthesis and Characterization of Small Cyclic Disulfide Peptides
FULL PAPER
Table 1. Characteristic shifts for Cβ and Cγ indicating a trans or
cis XxxϪPro peptide bond
Table 2. Chemical shifts of NH and CαH protons of 1, 2, 6, and 7
relative to cyclo[BocϪCysϪProϪPheϪCysϪNHMe] (12)[9]
Compound
Cβ [ppm]
Cγ [ppm]
∆δ(CβϪCγ) [ppm]
Exp. δ [ppm]
Cys(1) Pro Aib Phe Cys(4) NHMe
2
1
7
6
28.52
28.40
28.48
28.20 (M)
32.00 (m)
29.50[10]
24.64
24.85
24.06
3.88
3.55
4.42
2
1
7
6
δNH[([D6]DMSO)]
δCαH[([D6]DMSO)] 3.99
δNH[([D6]DMSO)] 7.22
δCαH[([D6]DMSO)] 4.41
δNH[([D6]DMSO)] 7.05
δCαH[([D6]DMSO)] 4.47
δNH[(CDCl3)] (M) 5.19
δCαH[(CDCl3)] (M) 4.48
7.02
Ϫ
4.16
Ϫ
4.31
Ϫ
4.20
Ϫ
4.43
Ϫ
4.42
7.85 Ϫ
7.61
4.34
7.40
4.14
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
24.80 (M)
21.60 (m)
24.20[10]
3.40 (M)
8.47 Ϫ
10.40 (m)
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
trans
Ϫ
Ϫ
8.30 7.63
4.46 4.51
6.13 6.85
4.94 4.85
6.10 7.02 6.50
4.99 4.67
25.0 Ϯ 1.0[11]
25.1 Ϯ 0.5[11]
22.50[10]
1.3 to 6.0[11]
Ϫ
cis
31.30[10]
22.4 Ϯ 0.8[11]
23.4 Ϯ 0.3[11]
Ϫ
12 δNH[(CDCl3)] (M)[9] 5.19
δCαH[(CDCl3)] (M)[9] 4.51
8.3 to 10.0[11]
Ϫ
Information of relevance to conformational analysis can tween NH Cys(4) and CO Cys(1) (β-turn), which plays an
easily be taken from the 1H NMR spectra. For example, the important role in fixing this structure, 7 and 6 show no
temperature-dependence of the chemical shift of the NHЈs uniform conformation. Neither 6 nor 7 exhibit intramolecu-
indicate hydrogen bonding, while the vicinal [3J(NH,HϪ lar hydrogen bonds. Rather, the characteristic ∆δ values of
C(α)] coupling constants give information on the dihedral 6 and 7 indicate solvent exposed NH groups, which cannot
angles ϕ in the peptide part (Tables 3 and 4).
strengthen and fix the backbone. As the polarity of the sol-
The data obtained by the temperature-dependent NMR vents plays an important role, intramolecular hydrogen
experiments underline the influence of the sequence on the bonds in CDCl3 are much stronger than in [D6]DMSO, and
conformation. While 2 and 1 exist in only one rigid confor- it is not astonishing that 7 exists in a variety of confor-
mation and exhibit one intramolecular hydrogen bond be- mations in [D6]DMSO, but only in two in CDCl3.
Figure 4. Linear (2 and 7) and cyclic tetrapeptides (1 and 6) investigated by temperature-dependent NMR and FTIR spectroscopy;
dashed lines indicate inter- or intramolecular hydrogen bonding
Eur. J. Org. Chem. 2004, 4167Ϫ4176
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C. Kolano, K. Gomann, W. Sander
FULL PAPER
3
This effect is in agreement with the results from the
NMR experiments. If a hydrogen bond gets weaker, a new
band at higher wavenumbers is expected to appear. More-
over, the band that is involved in the intramolecular hydro-
gen bond should show a less rapid decrease in intensity.
However, none of these features has been observed for 2. In
contrast, cyclic tetrapeptide 1 showed a completely different
behavior in the amide I and II regions (Figure 6).
Table 3. Vicinal J[NH,HϪC(α)] coupling constants
Compound
Amino acid
3JHNCαH [Hz]
2 ([D6]DMSO)
1 ([D6]DMSO)
7 ([D6]DMSO)
Cys(1)
Cys(4)
Cys(1)
Cys(4)
Cys(1)
Cys(4)
8.53
7.02
8.03
6.52
6.99
8.12
7.75
8.50
7.36
9.44
8.88
Phe
6[a] (CDCl3)
Cys(1) (M)
Cys(4) (M)
Phe (M)
Phe (m)
[a]
Cys(m): 8.68 and 5.67 Hz. Values may contain errors due to
signal overlapping.
Table 4. T-gradients of the NHЈs given in ∆δ/T*10Ϫ3 ppm/K indi-
cating intra- or intermolecular hydrogen bonding
Compound
Acm NH Aib NH Cys NH(1) Cys NH(2) Phe NH
2 ([D6]DMSO)
1 ([D6]DMSO)
7 ([D6]DMSO)
6 (CDCl3)
5.5
Ϫ
5.5
Ϫ
5.8
6.0
Ϫ
9.2
10.3
2.4
1.7
Ϫ
Ϫ
9.5
5.2
6.3
[a]
[a]
[a]
Ϫ
[a]
No values determined due to signal overlapping.
Figure 6. Temperature-dependent FTIR spectrum of 1 in CHCl3;
the spectral resolution is 4 cmϪ1
Temperature-Dependent FTIR Studies
To support the results from the temperature-dependent
NMR experiments, we carried out FTIR measurements
with compounds 2 and 1 in trichloromethane in the tem-
perature range from Ϫ10.0 to 50.0 °C with a spectral reso-
lution of 4 cmϪ1 using an ATR unit and a thermojacket.
All characteristic bands of the linear tetrapeptide 2 in the
amide I and II regions showed a decrease in intensity on
annealing (Figure 5).
Three bands (1749, 1675 and 1618 cmϪ1) of the amide I
region exhibit a decrease in intensity or the appearance of
new bands on warming the solution. The decrease in inten-
sity of the signal at 1749 cmϪ1 is very slow relative to the
bands at 1675 and 1618 cmϪ1. Both bands rapidly lose
intensity on annealing and simultaneously form two new
signals (shoulders) at 1695 and 1633 cmϪ1. These obser-
vations are in good agreement with the results from the
NMR experiments. This leads us to the conclusion that the
band at 1749 cmϪ1 is assigned to the CϭO stretching vi-
bration of Cys(1) which, according to the NMR spectro-
scopic data, is involved in the formation of an intramolecu-
lar hydrogen bond with NH Cys(4) and the resulting β-
turn. The other two bands can be assigned to the remaining
CϭO stretching vibrations of 1, which are involved in inter-
molecular hydrogen bonds with the solvent or further mol-
ecules. On annealing, these intermolecular hydrogen bonds
are rapidly cleaved. As this causes an increase in the bond
order of the CϭO stretching vibration, the new signals are
observed at higher wavenumbers. On annealing, the bands
of the amide II region react in a similar way (loss of inten-
sity and simultaneous formation of new bands). However,
a complete assignment of each band to a single amino acid
is not possible without selective isotopic labeling or site-
directed mutagenesis.
Figure 5. Temperature-dependent FTIR spectrum of 2 in CHCl3;
the spectral resolution is 4 cmϪ1
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Synthesis and Characterization of Small Cyclic Disulfide Peptides
FULL PAPER
bodiimide, DCM ϭ dichloromethane, DMF ϭ dimethylformam-
ide, HOBt ϭ hydroxybenzotriazole, NMM ϭ N-methylmorpholine,
MA ϭ mixed-anhydride method, TFA ϭ trifluoroacetic acid, Trt ϭ
trityl, Cys(1) refers to the Boc-protected cysteine, Cys(4) refers to
the cysteine carrying the methyl ester.
Conclusion
The selective use of temperature control in the classical
peptide synthesis has been demonstrated to allow the large-
scale synthesis of two cyclic tetrapeptides. These peptides
are derivatives of the bioactive hormone peptides Oxytocine
and Vassopressine. Double-walled reaction flasks attached
to an external cryostat allowed perfect temperature control,
and resulted in high yields of the two cyclic tetrapeptides
and their precursors. We obtained information on the con-
formation of the peptides from one- and two-dimensional
NMR experiments. Further, we investigated the dynamics
of the peptides in the amide I and II region, by tempera-
ture-dependent FT-IR spectroscopy using the ATR tech-
nique. The results are in good agreement with the data ob-
tained by temperature-dependent NMR spectroscopy.
General Deprotection Procedure: The Boc-protected peptide or am-
ino acid (10 mmol) was stirred in TFA (41 mL) in a flask sealed
with a drying tube for 100 minutes at ambient temperature. Hexane
(345 mL) was added, and the solution was stirred for a further 30
minutes. The solvent was evaporated, and the residue treated with
a small portion of dry ether. Precipitated, deprotected peptide/am-
ino acid was obtained as a colorless powder, which was checked for
complete deprotection by 1H NMR spectroscopy (removal of the
Boc signal at about 1.35 ppm). The deprotected compound can be
stored for about 3 days if placed in a desiccator under argon.
Yield 95Ϫ98%.
Cyclization: BocϪCys(Trt)ϪProϪXϪCys(Acm)ϪOMe (2 mmol)
was dissolved in freshly distilled methanol (400 mL). A solution of
iodine (1.55 g, 6 mmol) in freshly distilled methanol (60 mL) was
added over 10 minutes with vigorous mechanical stirring. Stirring
was continued at ambient temperature for another 90 minutes. The
reaction was quenched by the dropwise addition of a Na2S2O3 solu-
tion (0.1 ) until the color of the methanolic solution had com-
pletely disappeared. The solution was evaporated to dryness, the
residue taken up in water (50 mL) and extracted three times each
with trichloromethane (150 mL). Drying of the combined organic
phases, and removal of the solvent yielded an oil, which was treated
with dry ether (few mL). This yielded a colorless precipitate, which
was filtered off. The remaining solution was evaporated to dryness,
and the foam obtained thus was purified by column chromatogra-
phy. Elution with DCM/EtOAc (9:1 and 8:2) and ethyl acetate af-
forded pure colorless cyclic tetrapeptide. Yield 40Ϫ65%.
Experimental Section
General: Melting points: Dr. Tottoli apparatus, uncorrected. IR
spectra (KBr pellet): PerkinϪElmer 983G or 841 spectrometer.
FAB mass: Varian MAT CH5, meta-nitrobenzyl alcohol matrix.
Elementary analyses: Vario EL; averaged value of two measure-
ments of the same sample. TLC: MachereyϪNagel SIL G/UV254
silica gel plates. Column chromatography: MATREXTM silica Si
chromatography medium, Amicon Europe, CH-1001 Lausanne,
pore diam. 60 A, part size 020Ϫ45 my. 1H NMR: Bruker DPX
˚
200 (200.13 MHz), DRX 400 (400.13 MHz) and Bruker DRX 600
(600.13 MHz) instruments. 13C NMR: Bruker DPX 200
(50.3 MHz), DRX 400 (100.4 MHz) and Bruker DRX 600
(150.9 MHz) instruments, δ rel. to Me4Si. 2D NMR experiments
(TOCSY, HMQC, HMBC, ROESY) on a Bruker DRX 400 or
DRX 600 instrument. Drying agent: Na2SO4, if not mentioned
otherwise. Boc protected amino acids were purchased from Calbi-
ochem-Novabiochem. Double-walled reaction flasks (Supporting
Information, see also the footnote on the first page of this article)
were made at the glassblowing facility of our department. The tem-
peratures given refer to the temperatures inside the reaction flask
of the double-walled flasks. Temperature control was achieved by
connecting an external cryostat with temperature control to the
flasks. The cryostats can operate in a cooling or a heating mode
(Haake Modell Fison CH or Lauda RC 20 CS).
General Peptide Coupling Procedure, Elongation by Mixed Anhy-
dride Method: BocϪXϪOH (0.033 mol) and NMM (7.3 mL, 0.066
mol) were dissolved, whilst stirring, in DCM (170 mL) and cooled
for 15 minutes at Ϫ15 °C. Isobutyl chloroformate (4.34 mL, 0.033
mol) was slowly added dropwise with continued stirring. The mix-
ture was kept at Ϫ15 °C for 10 to 15 minutes. Thereafter, a well-
dissolved solution of amino acid methyl ester hydrochloride or de-
protected peptide (0.033 mol), NMM (7.3 mL, 0.066 mol), and
DMF (15 mL) in DCM (120 mL) was added dropwise over 15
minutes to the stirred mixture at Ϫ15 °C. The mixture was stirred
at Ϫ15 °C for another 15 minutes and then at room temperature
overnight. The mixture was then taken up in EtOAc (800 mL). The
organic layers were successively washed with 5% KHSO4 (250 mL),
water (250 mL) with sat. NaHCO3 (260 mL), sat. NaHCO3 (200
Temperature-dependent FTIR measurements: For temperature-de-
pendent FTIR measurements, a Bruker IFS66v/S (Bruker Optics,
Ettlingen) was used in combination with an ATR unit (horizontal mL), and sat. NaCl (300 mL). The collected organic layers were
multireflection unit ‘‘Gateway’’, Specac, UK). The ATR unit was
placed in the sample compartment of the FTIR spectrometer and
equipped with a continuous-flow sample cell with a thermojacket
(45°-ZnSe-crystal, 550 µL sample volume, ZnSe-crystal 6 reflec-
tions, temperature range Ϫ10 to 90 °C, Specac, UK). Temperature
control was achieved by connecting the thermojacket to a cryostat
(Haake Modell Fison CH or Lauda RC 20 CS). The temperature
was also checked near the ZnSe-crystal by an electronic tempera-
ture sensor (Voltcraft 300 K Digital thermometer, Conrad Elec-
tronics, Germany). All measurements were carried out with 4 cmϪ1
dried, and the solvent was evaporated.
HCl*Aib؊OMe (11): Aminoisobutyric acid (10.5 g, 0.1018 mol)
was suspended in methanol (100 mL) and cooled by an ice bath.
Whilst stirring, SOCl2 (7.8 mL, 0.107 mol) was added dropwise to
the solution. After the complete addition of thionyl chloride, the
mixture was heated for four hours at 55 °C. The mixture was al-
lowed to stand at room temperature whilst stirring overnight. The
solvent was coevaporated five times each with methanol (50 mL).
After complete removal of the solvent, the residue was recrys-
tallized from methanol/ether (50 mL, 1:1) and placed in a desic-
cator with NaOH overnight. This yielded 15.36 g (98.3%) of a col-
orless compound. M.p. 180.0 °C (ref. 183Ϫ184 °C[16]). Rf ϭ 0.59
resolution in the spectral range between 400 and 3000 cmϪ1
.
The abbreviations used are as follows: Acm ϭ acetamidomethyl,
Aib ϭ aminoisobutyric acid, Boc ϭ tert-butoxycarbonyl, CDMT ϭ
2-chloro-4,6-dimethoxy-1,3,5-triazine, DCCI ϭ dicyclohexylcar-
1
in BuOH/glacial acetic acid/water/pyridine (15:3:12:10). H NMR
(400 MHz, CDCl3): δ ϭ 8.95 (s, br., NH3ϩ), 3.79 (s, OCH3), 1.71
Eur. J. Org. Chem. 2004, 4167Ϫ4176
www.eurjoc.org
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4173

C. Kolano, K. Gomann, W. Sander
FULL PAPER
(s, CH3) ppm. 13C NMR (100 MHz, CDCl3): δ ϭ 171.59 (CO),
57.48 (Cα), 53.33 (OCH3), 23.93 (Cβ) ppm. IR: ν˜ ϭ 2960, 2658,
at once. Stirring was continued for another 30 minutes at Ϫ5 °C.
The temperature was then raised to 45 °C. The clear solution was
2585, 2036, 1748, 1596, 1523, 1469, 1319, 1237, 1196, 1086, 977, stirred for 4 hours at 45 °C, after which the solvent was removed
877, 770 cmϪ1. FAB-MS m/z: 118.1 (100) [M Ϫ HCl ϩ H]ϩ. carefully. During this process, the solution became oily, and then
C5H12ClNO2 (153.6): calcd. C 39.1, H 7.8, N 9.1; found C 38.46,
H 7.54, N 8.82.
formed a colorless dry foam, which could be cracked by a spatula.
The crude was placed overnight in a desiccator over KOH under
dynamic vacuum of a membrane pump. This yielded 7.88 g (98.4%)
of a colorless crystalline compound.
Boc-Pro-Aib؊OMe (5): Dipeptide 5 was synthesized according to
the procedure published by Jung et al.[5] Changes have been im-
plemented that involve the temperatures, as the reaction was carried
out in a double-walled flask under complete temperature control
by an external cryostat.
In the case of a smaller reaction scale, the foam sometimes became
oily or assumed the consistency of chewing gum if exposed to air.
If this occurred, one could purify the compound in the following
manner (for example, 10 mmol scale): dry ether (20 mL) are added
to the compound. The flask is then placed in a super sonic bath
for 30 minutes. After removal of the solvent and repetition of this
procedure with pentane (20 mL), the solvent is removed completely,
yielding a colorless crystalline compound. M.p. 130.0Ϫ132.0 °C
(ref. 132.0Ϫ134.0 °C[7]). Rf ϭ 0.65 in methanol. 1H NMR
(400 MHz, (D6)DMSO): δ ϭ 8.81 (br., Acm NH), 4.27 (d, Acm β),
4.25 (Cys α), 4.12 (br., NH3ϩ), 3.73 (s, OCH3), 3.33 (Cys β), 3.11
(Cys β), 1.84 (s, Acm CH3) ppm. 13C NMR (100 MHz,
(D6)DMSO): δ ϭ 169.84 (CO), 168.69 (CO), 53.05 (OCH3), 52.21
(Cα, Cys), 40.70 (Cβ, Acm), 30.34 (Cβ, Cys), 22.70 (Acm CH3)
ppm. IR: ν˜ ϭ 2933, 1751, 1654, 1504, 1441, 1376, 1326, 1244, 1090,
1005, 851 cmϪ1. FAB-MS m/z: 207.1 (100) [M Ϫ HCl ϩ H]ϩ.
C7H15ClN2O3S (242.7): calcd. C 34.6, H 6.2, N 11.5, S 13.2; found
C 34.64, H 6.13, N 11.59, S 13.11.
The starting solution of BocϪPro-OH in DCM/DMF was stirred
for 30 minutes at Ϫ15 °C. Isobutyl chloroformate was added drop-
wise over 15Ϫ20 minutes without raising the temperature of the
reaction mixture. The solution was then stirred for a further 10
minutes before adding a completely dissolved and precooled (Ϫ5
to 0 °C) solution of HCl*Aib-OMe (11) in NMM and DCM/DMF
at Ϫ18 °C over a period of 15 minutes. The mixture was stirred for
an additional hour at Ϫ15 °C. The temperature was then increased
to Ϫ5 °C. After 90 minutes at Ϫ5 °C, the temperature was raised
to room temperature and stirring was continued for an additional
90 minutes.
Another change was made during the workup procedure. The crude
oil, which was obtained after drying and removal of the solvent,
was crystallized from pentane/ethyl acetate (1:1) to yield an off-
white compound (>82% yield). M.p. 79.5 °C (ref. 80 °C[5]). Rf ϭ
0.83 in BuOH/acetic acid/water (3:1:1). 1H NMR (200 MHz,
[(D6]DMSO): δ ϭ 8.16 (s, NH Aib), 4.05 (Pro α), 3.53 (s, OCH3),
3.30 (br., Pro δ), 2.07Ϫ1.75 (m, br., Pro β,γ), 1.37Ϫ1.30 (m, Boc
CH3, Aib CH3) ppm. 13C NMR (50 MHz, CDCl3): δ ϭ 174.76
(CO), 171.62 (CO), 156.69 (CO), 80.42 (qC Boc), 61.14 (Cα, Pro),
56.14 (Cα, Aib), 52.47 (OCH3), 46.99 (Cδ, Pro), 30.78 (Cβ, Pro),
28.33 (Boc CH3), 24.99 (Cβ, Aib), 24.66 (Cγ, Pro) ppm. IR: ν˜ ϭ
Boc؊Pro؊Aib؊Cys(Acm)؊OMe (3). Procedure I: Compound 4
(2.48 g, 0.00825 mol) and DCCI (1.87 g, 0.00908 mol) were dis-
solved whilst stirring in DMF (20 mL) and cooled at Ϫ2 °C for 15
minutes. A precooled (Ϫ5 to 0 °C) solution of 10 (2.0 g, 0.00825
mol) and triethylamine (2.30 mL, 0.0165 mol) in DMF (15 mL)
were added dropwise. The mixture was stirred for 2 hours at Ϫ2
°C and for a further 18 hours at 20 °C, after which it was stirred
for 18 hours at room temperature. Precipitated dicyclohexylurea
was filtered off, and the solvent was removed in vacuo. The residue
was dissolved in chloroform (15 mL) and successively washed with
HCl (0.1 , 60 mL), NaHCO3 (0.5 , 60 mL), and water (60 mL).
The organic layer was dried and the solvent removed. The crude
product was purified by column chromatography. Elution with
DCM/Et2O (7:3 and 1:1) and ethyl acetate afforded pure colorless
tripeptide 3 (2.26 g, 56.6%).
3319, 2990, 1741, 1672, 1533, 1407, 1288, 1153, 987, 775, 616 cmϪ1
.
FAB-MS m/z: 337.2 (54) [M ϩ Na]ϩ, 315.2 (96) [M ϩ H]ϩ, 259.1
(47), 241.1 (7), 215.1 (100). C15H26N2O5 (314.4): calcd. C 57.3, H
8.3, N 8.9; found C 56.94, H 8.13, N 8.80.
Boc؊Pro؊Aib؊OH (4): Saponification of dipeptide 5 was per-
formed according to the literature procedure of Jung et al.[5] The
reaction was performed in a double-walled reaction flask. The tem-
perature inside the reaction flask was kept at 37.5 °C, controlled
by an external cryostat. The reaction yielded 78.0% of an off white
compound. Completeness of saponification was checked by NMR
spectroscopy. No further purification was necessary. M.p. 159.0 °C
(ref. 161 °C[5]). Rf ϭ 0.78 in BuOH/acetic acid/water (3:1:1). 1H
NMR (200 MHz, [D6]DMSO): δ ϭ 12.15 (s, br., COOH), 7.96 (s,
NH Aib), 4.02 (Pro α), 3.31 (br., Pro δ), 2.05Ϫ1.75 (k, br., Pro
β,γ), 1.36Ϫ1.30 (m, Boc CH3, Aib CH3) ppm. 13C NMR (50 MHz,
CDCl3): δ ϭ 176.68 (CO), 173.14 (CO), 146.00 (CO), 81.00 (qC
Boc), 61.36 (Cα, Pro), 56.80 (Cα, Aib), 47.10 (Cδ, Pro), 33.24 (Cβ,
Pro), 28.31 (Boc CH3), 25.74/25.07 (Cβ, Aib), 24.62 (Cγ, Pro) ppm.
IR: ν˜ ϭ 3316, 2985, 2879, 1713, 1680, 1529, 1408, 1302, 1245, 1166,
1127, 943, 778, 615 cmϪ1. FAB-MS m/z: 323.0 (100) [M ϩ Na]ϩ,
Procedure II: CDMT (3.86 g, 0.022 mmol) and 4 (6.79 g, 0.02244
mol) were dissolved, whilst stirring, in DCM (31 mL), and cooled
at Ϫ5 °C for 15 minutes. NMM (4.95 mL, 0.04488 mol) was slowly
added dropwise to the solution in such a manner that the reaction
temperature did not rise above 0 °C. The solution was stirred for a
further 4 hours at Ϫ1 °C until nearly all CDMT was consumed. A
mixture of 10 (5.34 g, 0.022 mol) and NMM (4.85 mL, 0.044 mol)
in DCM/THF/CH3CN (15:15:25 mL) was added dropwise to the
solution whilst stirring at Ϫ5 °C. Stirring was continued for 2.5
hours at 0 °C. The temperature was then raised to room tempera-
ture, and the mixture was stirred overnight. The solvent was evapo-
rated, and the residue was taken up in ethyl acetate (90 mL) and
successively washed with water (24 mL), 10% citric acid (26 mL),
water (26 mL), saturated NaHCO3 solution (24 mL), and water (24
mL). The organic layer was dried, and the solvent evaporated. This
yielded 5.62 g (52.3%) of pure tripeptide. No further purification
301.1 (47) [M
ϩ
H]ϩ, 245.0 (36), 223.0 (16), 201.0 (83).
C14H24N2O5 (300.4): calcd. C 55.9, H 8.1, N 9.3; found C 55.39,
H 8.79, N 9.05.
1
HCl*Cys(Acm)؊OMe (10): Methanol (40 mL) was placed in the
reaction flask at Ϫ15 °C. Whilst stirring, thionyl chloride (2.65 mL,
was necessary. M.p. 120.0 °C (dec.). H NMR (400 MHz, CDCl3):
δ ϭ 7.31 (br., Acm NH), 7.22 (br., NH Cys), 6.71 (br., NH Aib),
0.036 mol) was added dropwise to the methanol in a manner that 4.67 (Cys α), 4.44/4.19 (Acm β), 4.19 (Pro α), 3.71 (s, OCH3), 3.43
the reaction temperature did not increase beyond Ϫ5 °C. HCl*
Cys(Acm)ϪOH (7.5 g, 0.033 mol) was then added to the solution
(Pro δ), 2.96 (Cys β), 2.19Ϫ1.86 (m, br., Pro β, γ), 1.99 (s, Acm
CH3), 1.50 (Aib CH3), 1.44 (s, Boc CH3) ppm. 13C NMR
4174
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2004, 4167Ϫ4176

Synthesis and Characterization of Small Cyclic Disulfide Peptides
(100 MHz, CDCl3): 173.97 (CO), 172.22 (CO), 170.96 (CO), 170.69 441.0 (4), 419.0 (52). C21H34N4O7S2 (518.64): calcd. C 48.6, H 6.6,
FULL PAPER
(CO), 156.85 (CO), 80.83 (qC Boc), 60.43 (Cα, Pro), 57.10 (Cα,
Aib), 52.93 (Cα, Cys), 52.61 (OCH3), 47.20 (Cδ, Pro), 41.53 (Cβ,
Acm), 33.91 (Cβ, Pro), 32.98 (Cβ, Cys), 28.34 (Boc CH3), 26.16/
25.60 (Cβ, Aib), 24.92 (Cγ, Pro), 23.00 (Acm CH3) ppm. IR: ν˜ ϭ
3315, 2982, 2937, 1748, 1672, 1539, 1389, 1252, 1167, 1125, 1030,
755 cmϪ1. FAB-MS m/z: 511.3 (100) [M ϩ Na]ϩ, 489.3 (41) [M ϩ
H]ϩ, 389.2 (40). C21H36N4O7S (488.6): calcd. C 51.6, H 7.4, N 11.5,
S 6.6; found C 51.29, H 7.20, N 11.31, S 6.28.
N 10.8, S 12.4; found C 48.21, H 7.11, N 10.03, S 12.50.
Boc؊Phe؊Cys(Acm)؊OMe (9): Dipeptide 9 was synthesized as
described above (MA method). No further purification was neces-
sary. The reaction yielded a colorless powder (12.37 g, 82.2%). M.p.
99.0Ϫ100.0 °C (ref. 102.0Ϫ103.0 °C[17]). 1H NMR (200 MHz,
CDCl3): δ ϭ 7.32Ϫ7.19 (m, Phe), 7.04 (d, NH Cys), 6.75 (br., Acm
NH), 5.06 (d, NH Phe), 4.74 (Cys α), 4.43 (Phe α), 4.30 (Acm β),
3.72 (s, OCH3), 3.19Ϫ2.81 (Cys β, Phe β), 2.01 (s, Acm CH3), 1.38
(Boc CH3) ppm. 13C NMR (100 MHz, CDCl3): δ ϭ 171.73 (CO),
170.55 (CO), 170.41 (CO), 152.98 (CO), 136.43 (Phe), 129.33 (Phe),
128.68 (Phe), 126.99 (Phe), 80.38 (qC Boc), 55.96 (Cα, Phe), 53.01
(Cα, Cys), 52.73 (OCH3), 42.28 (Cβ, Acm), 38.04 (Cβ, Phe), 33.88
(Cβ, Cys), 28.24 (Boc CH3), 23.18 (Acm CH3) ppm. IR: ν˜ ϭ 3334,
3060, 2975, 1729, 1691, 1648, 1527, 1437, 1415, 1249, 1167, 1049,
953, 859, 756, 712, 698, 634 cmϪ1. FAB-MS m/z: 476.1 (52) [M ϩ
Na]ϩ, 454.2 (31) [M ϩ H]ϩ. C21H31N3O6S (453.5): calcd. C 55.6,
H 6.8, N 9.3, S 7.1; found C 55.72, H 7.36, N 9.14, S 6.91.
Boc؊Cys(Trt)؊Pro؊Aib؊Cys(Acm)؊OMe (2): BocϪCys(Trt)Ϫ
OH (5.0 g, 0.0108 mol) , DCCI (2.92 g, 0.014 mol), and HOBt
(1.90 g, 0.014 mol) were dissolved, whilst stirring, in DMF (52 mL)
and cooled for 30 minutes at Ϫ20 °C. A solution of equivalent
amounts of deprotected 3 (0.0108 mol) [see general procedure for
deprotection] and NMM (4.84 mL, 0.046 mol) in DMF (82 mL)
was precooled for 15 minutes in an ice bath. After 30 minutes the
precooled solution was added. The mixture was stirred for 2 hours
at Ϫ20 °C. The temperature was then raised to 23 °C and stirring
was continued at this temperature for a further 20 hours. The mix-
ture was allowed to stand at room temperature for 1 hour. The
precipitated dicyclohexylurea was filtered off, and the solvent was
removed in vacuo. The residue was taken up in EtOAc/5% KHCO3
(165 mL, 1:1). Again the solution was filtered to remove further
precipitated dicyclohexylurea. The filtrate was successively washed
three times each with 5% KHCO3 (82 mL), three times each with
5% KHSO4 (82 mL), and once with saturated NaCl solution (82
mL). Drying of the combined organic layers, and removal of the
solvent yielded a colorless powder, which was further purified by
column chromatography. Elution with DCM/Et2O (1:1), DCM/
EtOAc (4:6), and ethyl acetate afforded pure colorless linear tetra-
peptide 2 (5.2 g, 57.8%). M.p. 102.0 °C (dec.). 1H NMR (600 MHz,
(D6)DMSO): δ ϭ 8.42 (t, Acm NH), 7.85 (s, NH Aib), 7.61 [d, NH
Cys(4)], 7.32Ϫ7.21 (m, Trt), 7.02 [d, NH Cys(1)], 4.34 [Cys α(4)],
4.25/4.15 (Acm β), 4.16 (Pro α), 3.99 [Cys α(1)], 3.59 (s, OCH3),
3.20/2.78 (Pro δ), 2.97/2.82 [Cys β(4)], 2.52/2.34 [Cys β(1)],
1.94Ϫ1.71 (m, br., Pro β, γ), 1.84 (s, Acm CH3), 1.34Ϫ1.28 (m, Boc
CH3, Aib CH3) ppm. 13C NMR (100 MHz, [D6]DMSO): δ ϭ
174.07 (CO), 171.06 (CO), 170.71 (CO), 169.60 (CO), 169.08 (CO),
155.23 (CO), 144.47 (Trt), 129.34 (Trt), 128.14 (Trt), 126.88 (Trt),
78.33 (qC Boc), 66.58 (qC Trt), 60.04 (Cα, Pro), 56.02 (Cα, Aib),
52.62 [Cα, Cys(4)], 52.47 [Cα, Cys(1)], 52.00 (OCH3), 46.64 (Cδ,
Pro), 40.63 (Cβ, Acm), 32.52 [Cβ, Cys(1)], 31.77 [Cβ, Cys(4)], 28.52
(Cβ, Pro), 28.23 (Boc CH3), 25.50/24.53 (Cβ, Aib), 24.64 (Cγ, Pro),
22.67 (Acm CH3) ppm. IR: ν˜ ϭ 3314, 3062, 2982, 1743, 1684, 1659,
1522, 1444, 1369, 1248, 1169, 1045, 938, 863, 745, 702, 676, 619
cmϪ1. FAB-MS m/z: 856.1 (92) [M ϩ Na]ϩ, 834.1 (12) [M ϩ H]ϩ.
C43H55N5O8S2 (834.07): calcd. C 61.9, H 6.6, N 8.4, S 7.6; found
C 60.62, H 6.30, N 7.90, S 7.90.
Boc؊Pro؊Phe؊Cys(Acm)؊OMe (8): Tripeptide 8 was synthesized
as described above (MA method). The residue was taken up in dry
ether (approximately 250 mL) and refluxed for 20 minutes. This
afforded a colorless powder, which was removed by suction fil-
tration yielding 13.78 g (75.9%) of pure tripeptide. M.p. 113.0 °C
(dec.). 1H NMR (400 MHz, [D6]DMSO): δ ϭ 8.53 (d, Cys NH),
8.48 (t, Acm NH), 7.80 (d, Phe NH), 7.19 (m, Phe), 4.66 (Phe α),
4.50 (Cys α), 4.22 (Acm β), 4.04 (Pro α), 3.62 (s, OCH3), 3.25 (Pro
δ), 3.03Ϫ2.77 (Cys β, Phe β), 2.01Ϫ1.64 (m, br., Pro β, γ), 1.84
(s, Acm CH3), 1.38Ϫ1.16 (Boc CH3) ppm. 13C NMR (100 MHz,
[D6]DMSO): δ ϭ 172.26 (CO), 171.43 (CO), 170.90 (CO), 169.48
(CO), 153.38 (CO), 137.67 (Phe), 129.13 (Phe), 127.98 (Phe), 126.25
(Phe), 78.41 (qC Boc), 59.55 (Cα, Pro), 53.56 (Cα, Phe), 52.41 (Cα,
Cys), 52.06 (OCH3), 46.45 (Cδ, Pro), 40.51 (Cβ, Acm), 37.57 (Cβ,
Cys), 30.77 (Cβ, Phe), 29.40 (Cβ, Pro), 27.85 (Boc CH3), 22.88 (Cγ,
Pro), 22.55 (Acm CH3) ppm. IR: ν˜ ϭ 3296, 3064, 2974, 1742, 1655,
1540, 1395, 1306, 1256, 1162, 1123, 1029, 845, 749, 702 cmϪ1. FAB-
MS m/z: 573.2 (59) [M
ϩ ϩ
Na]ϩ, 551.2 (30) [M H]ϩ.
C26H38N4O7S (550.68): calcd. C 56.7, H 6.9, N 10.2, S 5.8; found
C 56.41, H 6.54, N 10.00, S 5.43.
Boc؊Cys(Trt)؊Pro؊Phe؊Cys(Acm)؊OMe (7): Linear tetrapep-
tide 7 was synthesized as described above (MA method). The resi-
due was purified by column chromatography. Elution with DCM/
Et2O (1:1) and DCM/EtOAc (4:6) afforded pure colorless linear
tetrapeptide 7 (20.70 g, 70.0%). M.p. 115.0 °C (dec.). 1H NMR
(600 MHz, [D6]DMSO): δ ϭ 8.42 (t, Acm NH), 8.30 (d, Phe NH),
7.63 [d, Cys NH(4)], 7.33Ϫ7.15 (m, Phe, Trt), 7.05 [d, Cys NH(1)],
4.51 [Cys α(4)], 4.47 [Cys α(1)], 4.46 (Phe α), 4.22 (Acm β), 4.20
(Pro α), 3.62 (s, OCH3), 3.19/2.73 (Pro δ), 2.97/2.74 [Cys β(4)], 2.83
(Phe β), 2.52/2.34 [Cys β(1)], 1.93Ϫ1.59 (m, br., Pro β, γ), 1.83 (s,
Acm CH3), 1.35 (Boc CH3) ppm. 13C NMR (150 MHz,
Cyclo[Boc؊Cys؊Pro؊Aib؊Cys؊OMe] (1): See general procedure
for cyclization. M.p. 113.0Ϫ115.0 °C. 1H NMR (600 MHz,
[D6]DMSO): δ ϭ 8.47 (s, NH Aib), 7.40 [d, NH Cys(4)], 7.22 [d, [D6]DMSO): δ ϭ 170.72 (CO), 170.70 (CO), 170.20 (CO), 169.40
NH Cys(1)], 4.41 [Cys α(1)], 4.31 (Pro α), 4.14 [Cys α(4)], 3.64 (CO), 168.87 (CO), 155.05 (CO), 144.34 (Trt), 137.37 (Phe), 129.17
(s, OCH3), 3.55 (Pro δ), 3.46/2.58 [Cys β(1)], 3.18/3.10 [Cys β(4)], (Trt), 129.09 (Phe), 127.94 (Trt), 127.89 (Phe), 126.67 (Trt), 126.17
2.10Ϫ1.83 (m, br., Pro β, γ), 1.39Ϫ1.29 (m, Boc CH3, Aib CH3) (Phe), 78.19 (qC Boc), 66.40 (qC Trt), 59.65 (Cα, Pro), 59.38 [Cα,
ppm. 13C NMR (150 MHz, [D6]DMSO): δ ϭ 174.42 (CO), 171.38 Cys(4)], 53.49 (Cα, Phe), 52.36 [Cα, Cys(1)], 51.93 (OCH3), 46.26
(CO), 170.51 (CO), 168.75 (CO), 154.85 (CO), 78.90 (qC Boc),
(Cδ, Pro), 40.56 (Cβ, Acm), 37.41 [Cβ, Cys(4)], 32.76 [Cβ, Cys(1)],
60.15 (Cα, Pro), 55.98 (Cα, Aib), 53.54 [Cα, Cys(1)], 52.23 (OCH3), 31.60 (Cβ, Phe), 28.48 (Cβ, Pro), 28.10 (Boc CH3), 24.06 (Cγ, Pro),
51.95 [Cα, Cys(4)], 46.92 (Cδ, Pro), 37.23 [Cβ, Cys(4)], 34.70 [Cβ, 22.49 (Acm CH3) ppm. IR: ν˜ ϭ 3303, 3058, 2975, 1656, 1522, 1442,
Cys(1)], 28.40 (Cβ, Pro), 28.15 (Boc CH3), 24.85 (Cγ, Pro), 22.89 1367, 1247, 1168, 1044, 856, 744, 702, 620 cmϪ1. FAB-MS m/z:
(Cβ, Aib) ppm. IR: ν˜ ϭ 3323, 2983, 2882, 1752, 1684, 1641, 1519,
918.3 (55) [M ϩ Na]ϩ, 243.1 (100) [Trt ϩ H]ϩ. C48H57N5O8S2
1444, 1367, 1305, 1244, 1168, 1044, 1016, 933, 858, 773 cmϪ1. FAB- (896.15): calcd. C 64.3, H 6.4, N 7.8, S 7.2; found C 63.65, H 6.75,
MS m/z: 541.0 (22) [M ϩ Na]ϩ, 519.1 (42) [M ϩ H]ϩ, 463.0 (14), N 7.34, S 6.73.
Eur. J. Org. Chem. 2004, 4167Ϫ4176
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 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4175

C. Kolano, K. Gomann, W. Sander
FULL PAPER
Cyclo[Boc؊Cys؊Pro؊Phe؊Cys؊OMe] (6): See general procedure
for cyclization. M.p. 85.0 °C (dec.). H NMR (600 MHz, CDCl3):
1
[1]
[2]
[3]
[4]
K. Gerwert, Ber. Bunsen-Ges. 1988, 92, 978Ϫ982.
δ ϭ ca. 7.20 (m, Phe), 6.85 [d, Cys NH(4)], 6.13 (d, Phe NH), 5.19
[d, Cys NH(1)], 4.94 (Phe α), 4.85 [Cys α(4)], 4.48 [Cys α(1)], 4.43
(Pro α), 3.71 (s, OCH3), 3.67/3.39 (Pro δ), 3.45/3.06 [Cys β(1)], 3.28/
3.19 (Phe β), 3.16/3.02 [Cys β(4)] 2.19Ϫ1.94 (m, br., Pro β, γ), 1.40
(Boc CH3) ppm. 13C NMR (150 MHz, CDCl3): δ ϭ 172.33 (CO),
170.10 (CO), 169.91 (CO), 169.73 (CO), 154.62 (CO), 137.89 (Phe),
129.66 (Phe), 128.69 (Phe), 127.29 (Phe), 80.93 (qC Boc), 61.96
(Cα, Pro), 55.66 [Cα, Cys(4)], 52.73 (OCH3), 52.53 (Cα, Phe), 51.11
[Cα, Cys(1)], 47.77 (Cδ, Pro), about 40.00 [Cβ, Cys(1), Cys(4)],
36.17 (Cβ, Phe), 29.32 (Cβ, Pro), 28.26 (Boc CH3), 24.86 (Cγ, Pro)
ppm. IR: ν˜ ϭ 3325, 3067, 3034, 2982, 1747, 1676, 1520, 1439, 1369,
1304, 1248, 1167, 1046, 1024, 919, 866, 749, 702, 618 cmϪ1. FAB-
K. Gerwert, B. Hess, Mikrochim. Acta 1988, 1, 255Ϫ258.
K. Gerwert, B. Hess, Prog. Clin. Biol. Res. 1988, 273, 321Ϫ326.
J. R. Burie, W. Leibl, E. Nabedryk, J. Breton, Appl. Spectrosc.
1993, 47, 1401Ϫ1404.
H. Schmitt, G. Jung, Liebigs Ann. Chem. 1985, 321Ϫ344.
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Rittel, Helv. Chim. Acta 1976, 59, 1489Ϫ1497.
[5]
[6]
[7]
[8] [8a]
L. A. Carpino, A. El-Faham, J. Am. Chem. Soc. 1995, 117,
[8b]
5401Ϫ5402.
Z. J. Kaminski, Synthesis 1987, 917Ϫ920.
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27, 2462Ϫ2471.
H. Kessler, Angew. Chem. 1982, 94, 509Ϫ520.
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[10]
[11]
MS m/z: 603.2 (55) [M
ϩ ϩ
Na]ϩ, 581.2 (30) [M H]ϩ.
C26H36N4O7S2 (580.73): calcd. C 53.8, H 6.3, N 9.7, S 11.0; found
C 51.46, H 6.27, N 8.46, S 11.11.
[12]
C. Schiene, G. Fischer, Curr. Opin. Struct. Biol. 2000, 10,
40Ϫ45.
[13]
[14]
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[17]
G. Fischer, T. Tradler, T. Zarnt, FEBS Lett. 1998, 426, 17Ϫ20.
G. Fischer, Angew. Chem. Int. Ed. Engl. 1994, 33, 1415Ϫ1436.
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E. Klieger, H. Gibian, Ann. 1961, 649, 183Ϫ202.
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ello, S. Spisani, Arch. Pharm. 1993, 326, 955Ϫ958.
Received March 16, 2004
Acknowledgments
The authors thank Prof. M. Feigel for his valuable cooperation
and fruitful discussions. This work was financially supported by
the Deutsche Forschungsgemeinschaft (SFB 452) and the Fonds
der Chemischen Industrie.
4176
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2004, 4167Ϫ4176