How to stabilize or break β-peptidic helices by disulfide bridges: Synthesis and CD investigation of β-peptides with cysteine and homocysteine side chains

Article abstract of DOI:10.1002/(SICI)1522-2675(19990804)82:8<1150::AID-HLCA1150>3.0.CO;2-O

all-L-β³-Penta-, hexa-, and heptapeptides with the proteinogenic side chains of valine, leucine, serine, cysteine, and methionine have been prepared by previously described procedures (12, 13, 14, 15; Schemes 2-5). Thioether cleavage with Na/NH

Full text of DOI:10.1002/(SICI)1522-2675(19990804)82:8<1150::AID-HLCA1150>3.0.CO;2-O

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Helvetica Chimica Acta ± Vol. 82 (1999)
How To Stabilize or Break b-Peptidic Helices by Disulfide Bridges:
Synthesis and CD Investigation of b-Peptides with Cysteine
and Homocysteine Side Chains
1
by Albrecht Jacobi ) and Dieter Seebach*
Laboratorium für Organische Chemie der Eidgenössischen Technischen Hochschule, ETH-Zentrum,
Universitätstrasse 16, CH-8092 Zürich
all-l-b3-Penta-, hexa-, and heptapeptides with the proteinogenic side chains of valine, leucine, serine,
cysteine, and methionine have been prepared by previously described procedures (12, 13, 14, 15; Schemes 2 ± 5).
Thioether cleavage with Na/NH3 in b-HMet residues has also provided a b3-hexapeptide with homocysteine
(CH2CH2S) side chains (13e). The HS (CH2)n groups were positioned on the b-peptidic backbone in such a
way that, upon disulfide-bridge formation, the corresponding b-peptide was expected to maintain either a 31-
helical secondary structure (1, 2) (Fig. 1) or to be forced to adopt another conformation (3, 4). The 13-, 17-, 19-,
and 21-membered-ring macrocyclic disulfide derivatives and their open-chain precursors, as well as all synthetic
intermediates, were purified (crystallization, flash or preparative HPL chromatography; Fig. 5) and fully
characterized (m.p., [a]D, CD, IR, NMR, FAB or ESI mass spectroscopy, and elemental analysis, whenever
possible; Fig. 2 and Exper. Part). The structures in MeOH and H2O of the new b-peptides were studied by CD
spectroscopy (Figs. 3 and 4), where the characteristic 215-nm-trough/200-nm-peak pattern was used as an
indicator for the presence or absence of (M)-31-helical conformations. A CH2 S2 CH2 and, somewhat less so, a
(CH2)2 S2 (CH2)2 bracket between residues i and i ‡ 3 (1 vs. 12d, and 2 vs. 13e in Fig. 3) give rise to CD spectra
which are compatible with the presence of 31-helical structures, while CH2 S2 CH2 brackets between residues
i and i ‡ 2 (3 vs. 14c) or i and i ‡ 4 (4 vs. 15c in Fig. 4) do not.
1. Introduction. ± b-Peptides consisting of as few as six residues and carrying
proteinogenic side chains have been shown to form stable secondary structures in
MeOH solution. Thus, the NMR structure of a simple b3-hexapeptide2) H-(b3-HVal-b3-
HAla-b3-HLeu)2-OH, built from homologated a-amino acids, is a left-handed 31 or 314
helix2) [1], that of the isomer H-(b2-HVal-b3-HAla-b2-HLeu-b3-HVal-b2-HAla-b3-
HLeu)-OH is a right-handed 12/10/12 helix [3], and that of H-(u-b2,3-HAla(a-Me)-u-
b2,3-HVal(a-Me)-b2-HVal-b3-HLys-u-b2,3-HAla(a-Me)-u-b2,3-HLeu(a-Me))-OH is
a
hairpin with an antiparallel pleated sheet [4]3). Two schematic representations of the
b3-peptidic 31 helix are shown in Fig. 1, a1 (side view) and a2 (top view), with the lateral
substituents indicated as simple spheres. The helical-wheel-type top view reveals the
juxtaposition (in 5-Š distance) of the side chain of amino acids i and i ‡ 3. A proper
covalent link between two such neighboring positions should stabilize the helix and a
short clamp between i and i ‡ 2, or i and i ‡ 4 positions will enforce new backbone
1
)
)
Postdoctoral Fellow 1998/99, partially funded by the Deutsche Forschungsgemeinschaft (DFG).
In contrast to our original report (Fig. 4, a, in [2]) and in agreement with a recently described effect of
protecting groups on the stability of the 314 helix [3], the fully protected derivative Boc-(b-HVal-b-HAla-b-
HLeu)2-OMe does not form the 31 helix in MeOH, according to CD measurements by J. V. Schreiber
(hitherto unpublished results, ETH-Zürich, 1998).
2
3
)
For reviews on b-peptides, see [5 ± 7].

Helvetica Chimica Acta ± Vol. 82 (1999)
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Fig. 1. Schematical representation of some possible disulfide-bridging modes in short b-oligopeptides. a1) Side
view of a b-peptidic left-handed or (M)-31-helix; top: N-terminus, bottom: C-terminus (colored arrows indicate
sulfur side chain positions resulting in varying disulfide-ring sizes as shown in b) ± d)). a2) Top view of a b-
peptidic left-handed or (M)-31-helix. b) ± d) Cross-sectional helical wheel-type representations of the top views
of b3-peptide with bridging disulfide bonds.
conformations of the b-peptide. We have now synthesized b3-peptides with cysteine
(CH2SH) and homocysteine (CH2CH2SH) side chains to be able to test the structural
modifications caused by disulfide-bond formation4). Thus, b3-hexapeptides with
(CH2)nSH groups in the 2- and 5-position should be able to form 17- and 19-membered
disulfide rings that prevent the helix from unwinding (Fig. 1, b and c; see the Formulae
1 and 2). On the other hand, disulfide bridges formed by oxidative coupling of b3-HCys
side chains in the 2- and 4-positions of a b3-pentapeptide (Fig. 1, d) or in the 2- and 6-
4
)
Various strategies for the introduction of conformational constraint by covalent linkages have been
employed in the case of a-peptidic helices (e.g., side-chain lactamization [8], disulfide bridging [9], and
ring-closing metathesis [10]).

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Helvetica Chimica Acta ± Vol. 82 (1999)
positions of a b3-heptapeptide (Fig. 1, e), with 13- and 21-membered rings, are not
compatible with the 31 helical structure (see the Formulae 3 and 4). We chose CD
spectroscopy as tool for structural analysis in solution. The CD spectra of numerous b-
peptides in CF3CH2OH, MeOH [11] and in H2O [12] have led to assignment of a 216-
nm trough followed by a 198-nm peak as pattern characteristic of the 31 helix of (M)-
configuration. With the serine side chain in the 4-position of b-heptapeptide 4, we
expected to be able to test the helix-breaking effect of the disulfide bridge in H2O (cf.
[12a]).
Besides the possibility of disulfide-bond formation, the sulfanyl group in cysteine is
of great interest with respect to its capacity as a metal-ligating functional group [13]5).
Furthermore, it is intriguing to note that the a-amino-acid sequence Cys
X X Cys
occurs frequently at the active sites of various metal-containing [15] and metal-free
proteins6).
-
5
)
SH-Group-mediated fixation of b-peptides to a gold surface is of particular interest, as it should thus be
possible to obtain structural data about b-peptide monolayers from STM methods [14].
6
)
Examples for the occurrence of the Cys
X X Cys sequence in proteins are the zinc-finger motif [16],
iron-sulfur clusters [17], oxidoreductases like thioredoxins and glutaredoxins [18], and cytochrome c,
where the cysteine residues form a thioether linkage between the apoprotein and the prosthetic group [19].

Helvetica Chimica Acta ± Vol. 82 (1999)
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2. Synthesis of the b-Peptides 1 ± 4. ± We employed solution synthesis with N-
terminal Boc and C-terminal MeO protection7). Starting from the commercial
N-Boc-protected a-amino acids S-benzylcysteine and methionine8), and by the Arndt-
Eistert homologation procedure as described previously [1][20] for the other b-amino-
acid building blocks, we prepared Boc-(R)-b3-HCys(Bn)-OH (5) and Boc-(R)-b3-
HMet-OH (6)9) and coupled them to give the b-di-, tri-, and tetrapeptide derivatives
7 ± 11 in good yields (79 ± 86%; see Scheme 1). C-Terminal deprotection of the b-
tripeptides 9a and 10a by methyl-ester hydrolysis gave 9b (91%) and 10b (92%),
respectively.
The fully protected hexapeptide 12a was obtained by fragment coupling of Boc-
deprotected 9a with 9b (yield: 71%) (Scheme 2). Due to poor solubility in organic
7
)
This particular set of protecting groups was chosen for being orthogonal to alkyl protection of the sulfanyl
group. Protecting groups susceptible to removal by hydrogenolysis could not be employed because of the
catalyst-poisoning effect of thioether and thiol functions.
8
9
)
We were confident that the methionine side chain in b-peptides would be amenable to demethylation by
Na/NH3, a reaction which had been performed successfully on a-methionine [21].
As pointed out before for the homologative conversion of (S)- or l-valine to (R)- or l-b3-homovaline [1],
the retentive homologation of (S)- or l-methionine takes place with change of the chirality sense, caused
by reversal of the CIP priority sequence.
)

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Scheme 1. Preparation of (R)-b3-Amino-Acid Derivatives 5 and 6 by Arndt-Eistert Homologation and
Preparation of Peptide Derivatives 7 ± 11. Xaa ˆ b-Amino acid with various substitution patterns. For other
abbreviations, see General in Exper. Part.
solvents (as encountered before with other b3-hexa- and b3-heptapeptides [1][2]), flash
chromatography (FC) was not possible with 12a, which was instead purified by
recrystallization from MeOH. The conditions employed for the methyl-ester hydrolysis
of hexapeptide derivative 12a were more drastic than for the smaller peptides; the yield
of 12b was nevertheless high (94%) and no side products were observed.
The elaboration of the subsequent deprotection and cyclization protocol yielding
the cyclic disulfide-bridged peptide 1 required considerable experimental effort. While
the cleavage of the S-benzyl groups in 12b was found to proceed smoothly with fivefold
molar excess of Na in liquid NH3, the resulting crude linear dithiol peptide was found to
be very sensitive to air oxidation. Reversed-phase HP liquid chromatography (RP-
HPLC) of Boc-deprotected samples revealed the formation of several less and more

Helvetica Chimica Acta ± Vol. 82 (1999)
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Scheme 2
polar species only minutes after exposure to air10). Most of these species (not
identified, probably disulfide oligomers of different molecular weight and connectivity)
were, in turn, susceptible to reductive cleavage. Treatment of the mixture with excess
dithiothreitol resulted in only two major products (by RP-HPLC analysis). As a
consequence of these findings, the S-benzyl deprotection and cyclization of 12b was
conducted in a one-pot procedure: upon completion of the reductive cleavage, the Na/
NH3 solution was quenched and immediately diluted with MeOH to an 80 mm peptide
concentration, in order to prevent aggregation and intermolecular side reactions (see
GP 4 in Exper. Part). Air oxidation yielded a single cyclic peptide derivative, which was
Boc-deprotected to give 1 in a rather good yield (53% after purification by prep.
RP-HLPC)11).
The hexapeptide derivative 13b with methionine side-chains was also prepared in
two steps starting from Boc-deprotected 10a and 10b (Scheme 3). Cleavage of the
sulfanylmethyl groups in 13b required forcing Birch conditions (large excess of Na,
prolonged reaction times of up to 24 h) but proceeded without considerable side
reactions (e.g., degradation of the main chain, desulfuration12)) to yield the dithiol 13d.
Boc-Deprotection followed by preparative RP-HPLC purification gave the free dithiol
10
)
Addition of catalytic amounts dithiothreitol (0.1 equiv.) to these mixtures of fully deprotected peptide
species in MeOH (degassed, 1% Et3N) and stirring for 5 days under Ar did not result in a reduced number
of compounds (as judged by the RP-HPLC analysis) (cf. [22]).
For comparison of thiol oxidation methods generating cyclic disulfides in peptides, see [23].
A side reaction, which had been observed during S-debenzylation of the A chain of porcine insuline under
Birch conditions [24].
11
12
)
)

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Helvetica Chimica Acta ± Vol. 82 (1999)
Scheme 3
peptide 13e (positive Ellmanꢀs test, see Exper. Part), which could be cyclized in
MeOH/NH3 to give the macrocycle 213).
The fully protected pentapeptide 14a was prepared by coupling of Boc-deprotected
7 with 9b (Scheme 4). Deprotection and cyclization steps were performed as described
above, while this particular cyclization to a 13-membered ring in 3 proceeded in slightly
lower yield (38% after prep. RP-HPLC purification) than with the larger cyclic
disulfides.
The fully protected heptapeptide 15a was prepared in the same way, from the Boc-
deprotected tetrapeptide 11 and the tripeptide acid 9b (Scheme 5). Compound 15a is
well soluble in organic solvents (CHCl3, MeOH) and was purified by FC (yield: 42%);
subsequent methyl-ester saponification gave the N-Boc-peptide acid 15b (yield: 96%).
13
)
Compound 2 could also be prepared from 13b by the one-pot deprotection/cyclization procedure
(described for 12b), followed by prep. RP-HPLC purification (yield: 51%). Earlier attempts to cyclize 13d
or 13e by oxidation with H2O2 [23] had failed. DMSO-Mediated oxidation of 13e [25] had given 2 in low
and unreproducible yields, and additional difficulties were encountered in the removal of DMSO from the
product.

Helvetica Chimica Acta ± Vol. 82 (1999)
1157
Scheme 4
Scheme 5
Simultaneous debenzylation of the ether and thioether moieties in the acid 15b under
Birch conditions (see above) provided no difficulties, and the macrocycle 4 was
obtained after oxidative cyclization and prep. RP-HPLC purification in 65% overall
yield.

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While the purity of the cyclic disulfide-bridged peptides 1 ± 4 was established by
analytical RP-HLPC (see Fig. 5 in Exper. Part)14), electrospray-ionization mass
spectroscopy (ESI-MS) proved to be the method of choice to further corroborate the
structures of these compounds (see Fig. 2). The cyclic compound 2 and its reduction
product 13e could easily be distinguished by a mass difference of two Da in the positive-
as well as the negative-ion mode. Correct ESI mass spectra were also obtained for
peptides 1, 3, and 4 (see Exper. Part).
3. CD Spectroscopy. ± As concluded from numerous CD measurements and
corresponding NMR structural investigations of b-peptides [1][2][8][9], the CD
pattern of a trough at ca. 216 and a peak at ca. 198 nm is indicative of a 31 helix of (M)-
configuration. While the chromophoric groups in the linear peptides studied here are
essentially the same as in the earlier reports, the macrocyclic peptides 1 ± 4 have an
additional disulfide chromophore [26]. Considering a possible CD contribution of this
unit (which results from axial chirality of the S S bond), we recorded CD spectra of
the disulfide-bridged peptides 1 ± 4 in the range from 190 to 350 nm15). None of them
shows any Cotton effect above 230 nm16).
The CD spectra of cysteine-containing b3-hexapeptide derivatives 1, 12c, and 12d
are shown in Fig. 3, a. Compound 1 exhibits a pattern typical for a 31-helical structure in
MeOH, but with a small blue shift. A stronger shift in this direction, accompanied by a
decreased intensity of the Cotton effect, is detected for 1 in H2O. Nevertheless, a
considerable amount of helical structure must still be present for 1 in aqueous solution,
as judged from its CD spectrum17).
Reductive opening of the 17-membered ring of 1 mediated by excess dithiothreitol
(see GP 6 in Exper. Part) yields 12d, the CD spectrum of which shows a strong 200-nm
maximum followed by a 216-nm minimum indicating an undisturbed 31 helix (see
above). Interestingly, the S-benzyl side chains in 12c appear to break the secondary
structure: a complete loss of the 216-nm minimum is observed.
The oxidative cyclization of 13e yields peptide 2 with a 19-membered disulfide-
bonded ring, and thus with a ring size increased by two atoms as compared to 1. Apart
from decreased intensities, the CD spectra of 2 and 13e are similar (Fig. 3,b). If at all,
the helical conformation of 2 is only marginally maintained in aqueous solution. This
means that, while the 17-membered ring in 1 allows for preservation of certain features
of the helical secondary structure in H2O, the 19-membered ring in 2 is not able to
14
)
)
Ellmanꢀs tests, which were performed on samples of 1 ± 4 after prep. RP-HPLC purification, showed that
no free thiol groups were present.
According to calculations, the Cotton effect of a disulfide group in the region of the amide-backbone
transitions (below 230 nm) should be negligible [27]. For further theoretical work on disulfide
stereochemistry and chiroptical properties, see [28].
A possible explanation for these findings is that the energy difference between the two diastereoisomeric
species resulting from (P)- or (M)-configuration of the S S unit is very small and, as the barrier for the
rotation about the S S bond is low, their respective Cotton effects cancel each other [27][28].
One must keep in mind that linking, and thus conformationally locking, the central residues of a helix as
done in peptide 1 (cf. Fig. 1,b) cannot prevent the helix from unwinding at its termini, which could account
for the decreased CD intensities observed upon switching from a less protic (MeOH) to a more
competitive solvent such as H2O.
15
16
17
)
)

Helvetica Chimica Acta ± Vol. 82 (1999)
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Fig. 3. Overlay of CD spectra of linear and cyclic b-peptides 1, 2, 12c, 12d, 13a ± c, 13e, and 13f in MeOH and
H2O. a) b-Peptides 1, 12c, and 12d. b) b-Peptides 2 and 13e. c) b-Peptides 13a ± c and 13f. Molar ellipticity [V] in
10 deg ´ cm2 ´ mol 1. All N-deprotected b-peptides were measured as their TFA salts (see Exper. Part).

Helvetica Chimica Acta ± Vol. 82 (1999)
1161
enforce the helical structure to the extent required for survival of the competitive H-
bonding in aqueous media.
The effect of the main-chain protecting groups on the formation and stability of the
31 helix2) was investigated with the linear methionine peptide derivatives 13a ± c and
13f in MeOH (Fig. 3,c): whereas the fully protected hexapeptide 13a shows rather no
pattern at all, N-Boc-peptide acid 13b exhibits a ꢁdistortedꢀ 31 helical pattern, which is
nervertheless clearly present, with a pronounced maximum at 200 nm but only weak
minimum at 222 nm. Boc-Deprotected methyl ester 13c gives the familiar pattern,
which is almost as strong as for the fully deprotected peptide 13f (molar ellipticity:
‡ 1.15 ´ 105 (201 nm); 3.2 ´ 104 (218 nm)18). While the influence of protecting groups
on the stability of the 31 helix can not be rationalized simply in terms of intramolecular
H-bonding19), the interaction between the charged terminal groups and the helix
macrodipole has been proposed to cause this effect20). Thus, the positive charge at the
‡
H3N terminus (negative end of the macrodipole) should have a stabilizing effect on
the b-peptidic 31 helix, and the same is true for the negative charge at the CO2 terminus
(positive end of the macrodipole)21). Clearly, a careful investigation of pH and solvent
dependence, as well as of salt effects [30][31], will be necessary for better under-
standing.
The 13-membered cyclic b-pentapeptide 3, which can not possibly form a 31 helix
(Fig. 4, a), gives rise to a CD curve with a single minimum below 200 nm. Upon
reductive cleavage of the disulfide bond in 3 with excess dithiothreitol, the pattern is
almost reversed (14c: molar ellipticity ‡ 1.05 ´ 105 at 198 nm; 9.0 ´ 103 at 220 nm), and
a gross pattern results similar to that associated with the 31 helix. That a pronounced
trough is missing supports the proposed minimum number of six residues necessary to
form a stable b-peptidic 31 helix [1]. In contrast to all other compounds described
herein, the CD spectrum of 3 in H2O does not differ much from its spectrum in MeOH.
This weak solvent dependence could be due to a comparatively rigid backbone induced
by the 13-membered ring in 3.
Finally, the b-heptapeptide 4 contains a 21-membered ring, which should not fit
with a 31-helical structure (cf. Fig. 1). Indeed, 4 shows a novel CD pattern in MeOH
with a 200 nm trough, which collapses in aqueous solution22). Upon reductive ring
cleavage in 4, the characteristic 31-helical CD pattern is fully restored (see the spectrum
of 15c).
A particular feature of the CD pattern exhibited by the helical b-peptides described
herein is a variation of the relative peak and trough intensities (cf. Figs. 3 and 4). The
18
)
)
The protecting-group effect was found to be independent of the peptide concentration in the range from
0.02 to 0.4 mm in MeOH.
While in the case of the a-peptidic a and 310 helices [29], and also in the b-peptidic 12/10/12 helix [3] an N-
terminal Boc group can account for an additional H-bond with a main-chain amide group, the b-peptidic 31
helix lacks this possibility for additional stabilization, as all NH groups are pointing towards the C-
terminus (cf. Fig. 1, a1).
19
20
21
22
)
)
)
As proposed in the helix dipole model for a-peptides [30], charges on side chains which are opposite to the
resulting helix macrodipole charge should also stabilize the helix structure of b-peptides.
As pointed out before, the a-peptidic a helix and the b-peptidic 31 helix have opposite polarity with
respect to their C- and N-termini [5].
The H2O solubility of 4 (0.7 mm), containing one serine side chain, is moderately increased in comparison
to 1 (0.3 mm).

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Fig. 4. Overlay of CD spectra of linear and cyclic b-peptides 3, 4, 14c, and 15c in MeOH and H2O. a) b-Peptides 3
and 14c. b) b-Peptides 4 and 15c. Molar ellipticity [V] in 10 deg ´ cm2 ´ mol 1. All N-deprotected b-peptides were
measured as their TFA salts (see Exper. Part).
intensity ratios R ˆ [V]217/[V]200 in MeOH of the two extremes associated with the
31-helical peptide structure range from R ˆ 0.1 in 13e to R ˆ 0.8 in H-(b3-HVal-b3-
HAla-b3-HLeu)2-OH [1]. It is possible that these changes of R correspond to only small
structural changes within the 31 helix23).
Structural determinations by NMR spectroscopy are currently under way in order
to establish a better correlation between ꢁknownꢀ CD and structural pattern; in
addition, the NMR structures of compounds exhibiting a novel CD pattern remain to
be determined.
23
)
The ratio R ˆ [V]222/[V]207 (corresponding to the n ! p* and p ! p* backbone amide transitions,
respectively) was taken as an indicator to distinguish a-helical from 310 helical conformations in a-peptides
[32]. CD Spectra of poly(b-amino acid) helices were recently calculated and compared with experimental
data [33].

Helvetica Chimica Acta ± Vol. 82 (1999)
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4. Conclusion. ± We have prepared and studied b3-peptides with cysteine and
homocysteine side chains in order to demonstrate the general feasibility of disulfide
bridging in b-peptide chemistry. Thus, the cyclic peptides 1 ± 4 were obtained in
reasonable yields by air oxidation from their corresponding open-chain precursors.
Structural features of the new peptides were elucidated by CD spectroscopy:
Whereas the cyclic peptides 1 and 2 show weaker Cotton effects than their respective
linear analogues in MeOH (which could mean that the geometry imposed on the 31
helix by cyclization is not optimal in either case), a net stabilization was found for 1 in
H2O: The Cotton effect detected for 1 was stronger than for the linear 31-helical
peptides with proteinogenic side chains known to date [1][12].
On the other hand, the helix-disrupting effect of disulfide linkages attached to
positons that do not stack above each other in the 31 helix was clearly demonstrated
with the cyclic peptides 3 and 4.
In summary, it may be said that disulfide linkages in b-peptides are ± as they are in
a-peptides ± a valuable tool for introducing conformational restrictions. This tool can
be used advantageously in the design of novel secondary and possibly tertiary
structures, and thus will allow us to rationally proceed with investigations of b-peptides.
We gratefully acknowledge the financial support of the Deutsche Forschungsgemeinschaft (DFG)
(fellowship No. JA 918/1-1 to A. J.). D. Rigo is thanked for the preparation and characterization of Boc-(R)-
b3-HCys(Bn)-OH, K. Gademann for a donation of Boc-(R)-b3-HSer(Bn)-OH, which was used for the
preparation of the b3-heptapeptide 4. We gratefully acknowledge the assistance by the staff in our analytical
division: B. Brandenberg and P. Zumbrunnen (NMR), Dr. W. Amrein, O. Greter, and R. Häfliger (MS) and D.
Manser (elemental analysis). Continuing support of Novartis Pharma AG, Basel, is greatly appreciated.
Experimental Part
1. General. Abbreviations: Boc2O: di(tert-butyl) dicarbonate, DTT: dithiothreitol, EDC: 1-[3-(dimethyl-
amino)propyl]-3-ethylcarbodiimide hydrochloride, FC: flash chromatography, HOBt: 1-hydroxy-1H-benzo-
triazole, h.v.: high vacuum, 0.01 ± 0.1 Torr, b-HXaa: b-homoamino acid. Solvents for chromatography and
workup procedures were distilled from Sikkon (anh. CaSO4; Fluka). Et3N was distilled from CaH2 and stored
over KOH. ClCO2Et was distilled and stored at ‡ 48 (refrigerator) under Ar. All indicated temp. were
monitored with an internal thermometer (Ebro-TTX-690 digital thermometer). Amino-acid derivatives were
purchased from Bachem, Senn, or Degussa. All other reagents were used as received from Fluka. The diazo
ketones Boc-(S)-Cys(Bn)-CHN2 and Boc-(S)-Met-CHN2 [34], as well as the b-amino-acid derivatives Boc-
(R)-b3-HVal-OH [1], Boc-(R)-b3-HSer(Bn)-OH [35], Boc-(R)-b3-HMet-OH (6) [35] and, Boc-(S)-b3-HLeu-
OMe [1], were prepared according to literature procedures. Caution: The generation and handling of CH2N2
requires special precautions [36]. Reactions carried out with the exclusion of light were performed in flasks
completely wrapped in aluminum foil. TLC: Merck silica gel 60 F254 plates; detection with UV, and anisaldehyde
or I2. FC: Fluka silica gel 60 (40 ± 63 mm); at ca. 0.2 bar. Anal. HPLC: Knauer HPLC System K 1000,
EuroChrom 2000 Integration Package, degasser, UV detector K 2000 (variable-wavelength monitor),
Macherey-Nagel C18 column: Nucleosil 100-5 C18 (250 Â 4 mm). Prep. HPLC: Knauer HPLC system: pump
type 64, programmer 50, UV detector (variable-wavelength monitor); Macherey-Nagel C18 column: Nucleosil
100-7 C18 (250 Â 21 mm). M.p.: Büchi-510 apparatus; uncorrected. Optical rotations: Perkin-Elmer 241
polarimeter (10 cm, 1 ml cell) at r.t. Circular dichroism (CD) spectra: Jasco J-710 recording from 190 to
250 nm at r.t.; 1-mm rectangular cell; average of five scans, corrected for the baseline; peptide concentration
1
0.2 mm in MeOH or H2O; molar ellipticity V in deg ´ cm2 ´ dmol (l in nm); smoothing by Jasco software. IR
Spectra: Perkin-Elmer-782 spectrophotometer. NMR Spectra: Bruker AMX 500 (1H: 500 MHz, 13C: 125 MHz),
AMX 400 (1H: 400 MHz, 13C: 100 MHz), ARX 300 (1H: 300 MHz), Varian Gemini 300 (1H: 300 MHz, 13C:
75 MHz); chemical shifts d in ppm downfield from internal Me4Si (ˆ 0 ppm); J values in Hz. MS: VG Tribrid
(EJ), VG ZAB2-SEQ (FAB, in a 3-nitrobenzyl-alcohol matrix), and Finnigan TSQ 7000 (ESI, sprayed from a
10 5 m methanolic solution; volumetric flow 20 ml/min) spectrometer; in m/z (% of basis peak). Elemental

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Helvetica Chimica Acta ± Vol. 82 (1999)
analyses were performed by the Microanalytical Laboratory of the Laboratorium für Organische Chemie, ETH-
Zürich.
2. Boc Deprotection: General Procedure 1 (GP 1). Similarly to the reported procedure [1][2], the Boc-
protected amino acid was dissolved in CH2Cl2 (0.5m) and cooled to 08. An equal volume of CF3CO2H was
added, and the mixture was allowed to slowly warm to r.t. and then stirred for further 1.5 h. Concentration under
reduced pressure and drying of the residue under h.v. (12 h) yielded the crude CF3CO2H salt, which was
identified by NMR and used without further purification.
3. Methyl-Ester Hydrolysis: General Procedure 2a (GP 2a). The fully protected oligopeptide was dissolved
in MeOH (0.1m) and treated with 1n NaOH (1.2 equiv.). After 24 h, the mixture was diluted with H2O, and the
pH was adjusted to 2 ± 3 with 1n HCl. The soln. was extracted with AcOEt, and the combined org. phases were
washed successively with sat. aq. NaCl soln. and H2O. The org. phase was evaporated and dried under h.v.
General Procedure 2b (GP 2b). The fully protected oligopeptide was dissolved in CF3CH2OH (0.1m),
treated with 5n NaOH (100 equiv.), and heated to 458. After completion of the reaction (TLC), the mixture was
diluted with H2O, and the pH was adjusted to 2 ± 3 with 5n HCl (08). The soln. was extracted with AcOEt, and
the combined org. phases were washed successively with sat. NaCl soln. and H2O. The org. phase was evaporated
and dried under h.v.
4. Peptide Coupling with EDC: General Procedure 3a (GP 3a). The appropriate CF3CO2H salt was
dissolved in CHCl3 (0.5m) and cooled to 08. The soln. was treated successively with Et3N (5 equiv.), HOBt
(1.2 equiv.), a soln. of the Boc-protected fragment (1 equiv.) in CHCl3 (0.5m), and EDC (1.2 equiv.). The
mixture was allowed to warm to r.t. and then stirred for 18 h. Subsequent dilution with CHCl3 was followed by
thorough washing with 1n HCl, sat. aq. NaHCO3 soln., and sat. aq. NaCl soln. The org. phase was dried (MgSO4)
and then concentrated under reduced pressure. FC yielded the pure peptide.
General Procedure 3b (GP 3b). The appropriate CF3CO2H salt was dissolved in CHCl3 (0.5m) and cooled
to 08. The soln. was treated successively with Et3N (5 equiv.), HOBt (1.2 equiv.), a soln. of the Boc-protected
fragment (1 equiv.) in DMF (0.1m), and EDC (1.2 equiv.). The mixture was allowed to warm to r.t. and then
stirred for 18 h. The solvents were removed under reduced pressure, and the residue was dispersed in CHCl3
(0.02m). The resulting suspension was washed with 1m HCl, sat. aq. NaHCO3 soln., sat. aq. NaCl soln., and H2O.
The org. phase was concentrated under reduced pressure. FC or recrystallization yielded the pure peptide.
24
5. Cleavage of S-Benzyl Groups/O-Benzyl Groups ) and Cyclization to the Disulfide. General Procedure 4
(GP 4). The Boc-protected oligopeptide was dissolved in NH3 (2 mm) at 338; 5 equiv. of Na per Bn-group was
added. The dark blue soln. was stirred for 1 h and subsequently quenched by addition of MeOH. The resulting
methanolic solution of NH3 was immediately diluted to a peptide concentration of 0.08 mm. Air oxidation was
allowed to proceed while air was bubbled through the soln. with slow stirring. Aliquots were removed at
different times in order to monitor reaction progress by RP-HPLC (after workup and Boc-deprotection of these
samples). When oxidation was judged to be complete (Ellmanꢀs test [38] was negative), MeOH was removed
under reduced pressure and the resulting crude cyclic peptide was Boc-deprotected according to GP 1.
6. Reversed Phase (RP) HPLC Analysis and Purification of b-Peptides: General Procedure 5 (GP 5). RP-
HPLC Analysis was performed on a Macherey-Nagel C18 column/Nucleosil 100-5 C18 (250 Â 4 mm) by using a
linear gradient of A (0.1% CF3CO2H in H2O) and B (MeCN) at a flow rate of 1 ml/min with UV detection at
220 nm. tR in min. Crude products were purified by prep. RP-HPLC (Macherey-Nagel C18 column/Nucleosil 100-
7 C18 (250 Â 21 mm) using a gradient of A and B at a flow rate of 20 ml/min with UV detection at 220 nm) and
then lyophilized.
7. S S Reduction of Cyclic Disulfide-Bridged Oligo-b-peptides: General Procedure 6 (GP 6). A 0.2 mm
methanolic solution of the cyclic peptide was purged with Ar and treated with 50 equiv. KOH and 25 equiv. DTT
under Ar. The reaction was monitored by RP-HPLC (Fig. 5). After 1 h, the soln. was acidified by addition of
CF3CO2H and stored under Ar.
Boc-(R)-b3-HCys(Bn)-OH (5). According to [1], a soln. of Boc-(S)-Cys(Bn)-CHN2 (6.71 g, 20 mmol) in
THF (50 ml) containing 10% H2O at 258 (bath temp.) was treated with a soln. of CF3COOAg (486 mg,
2.2 mmol) in Et3N (5.86 g, 58 mmol) under Ar with the exclusion of light. The mixture was allowed to warm to
r.t. within 4 h in the dark. After 12 h, the soln. was reduced to half of its initial volume, diluted with Et2O (35 ml)
and extracted with sat. aq. NaHCO3 soln. The aq. phase was carefully adjusted to pH 2 ± 3 at 08 with 5n HCl and
extracted with Et2O. The org. phase was dried (MgSO4) and then concentrated under reduced pressure.
24
)
For a detailed description of a Birch reduction (experimental setup and procedure), see [37].

Helvetica Chimica Acta ± Vol. 82 (1999)
1165
Fig. 5. HPLC Profiles of purified b-peptides 1 ± 4, 12c, and 13e (RP-C18 column, linear gradient of A (0.1%
CF3COOH in H2O and B (MeCN); see GP 7 in Exper. Part)
Recrystallization from Et2O/pentane yielded 5 (3.97 g, 61%). White solid. M.p. 112 ± 1138. Rf (CH2Cl2/MeOH
15 : 1) 0.45. [a]rD:t: ˆ‡ 4.3 (c ˆ 1.0, CHCl3). IR (KBr): 3365m, 2979m, 1682s, 1517s, 1365m, 1299m, 1250m, 1165s,
1047w, 1018w, 768w, 695m. 1H-NMR (300 MHz, CDCl3): 1.45 (s, t-Bu); 2.57 ± 2.71 (m, CH2CO, CH2S); 3.73
(s, SCH2Ph); 4.05 ± 4.16 (m, CHN); 5.10 (br., NH); 7.21 ± 7.34 (m, 5 arom. H). 13C-NMR (75 MHz, CDCl3):
28.4 (Me); 35.2, 36.3, 37.3 (CH2); 46.6 (CH); 79.9 (C); 127.2, 128.6, 129.0 (arom. C); 155.3, 176.6 (C). FAB-MS:
‡
‡
326 (44, [M ‡ 1] ), 307 (100), 289 (46), 270 (48), 226 (16, [M Boc ‡ 1] ). Anal. calc. for C16H23NO4S
(325.42): C 59.05, H 7.12, N 4.30; found: C 59.10, H 7.07, N 4.23.
Boc-(R)-b3-HMet-OH (6). Treatment of a soln. of Boc-(S)-Met-CHN2 (10.90 g, 40 mmol) according to [1]
followed by recrystallization of the crude product from Et2O/pentane yielded 6 (7.57 g, 73%). White solid. M.p.
83 ± 848. Rf (Et2O/pentane 2 :1) 0.15. Spectroscopical data in agreement with [35]. [a]Dr:t:
ˆ
20.2 (c ˆ 1.0,
CHCl3). ([35]: [a]Dr:t:
ˆ
19.9 (c ˆ 1.0, CHCl3)).

1166
Helvetica Chimica Acta ± Vol. 82 (1999)
Boc-(R)-b3-HCys(Bn)-(S)-b3-HLeu-OMe (7). Boc-(S)-b3-HLeu-OMe (3.89 g, 15 mmol) was deprotected
according to GP 1, dissolved in CHCl3 (30 ml), and treated with Et3N (7.58 g, 75 mmol), HOBt (2.43 g,
18 mmol), 5 (5.03 g, 15 mmol) in CHCl3 (30 ml), and EDC (3.44 g, 18 mmol) according to GP 3a. FC (CH2Cl2/
MeOH 20 :1) yielded 7 (5.87 g, 83%). White solid. M.p. 105 ± 1068. Rf (CH2Cl2/MeOH 20 :1) 0.6. [a]Dr:t:
ˆ
8.4
(c ˆ 1.0, CHCl3). IR (KBr): 3340s, 2959s, 1738s, 1685s, 1643s, 1521s, 1436m, 1367m, 1303m, 1277m, 1251m,
1161s, 1050m, 1023w, 764w, 704m. 1H-NMR (400 MHz, CDCl3): 0.90 (d, J ˆ 6.6, 2 Me); 1.24 ± 1.31 (m, CH); 1.44
(s, t-Bu); 1.38 ± 1.50 (m, 1 H, CH2); 1.52 ± 1.62 (m, 1 H, CH2); 2.41 ± 2.75 (m, 2 CH2CO, CH2S); 3.67 (s, MeO);
3.74 (s, SCH2Ph); 3.98 ± 4.05 (m, CHN); 4.26 ± 4.35 (m, CHN); 5.63 (br., NH); 6.05 (br., NH); 7.21 ± 7.35
(m, 5 arom. H). 13C-NMR (100 MHz, CDCl3): 22.1, 22.9 (Me); 25.0 (CH); 28.4 (Me); 35.3, 36.4, 38.6, 38.9, 43.1
(CH2); 44.2, 47.5 (CH); 51.7 (Me); 79.4 (C); 127.1, 128.5, 129.0 (arom. C); 138.2, 155.3, 170.0, 172.1 (C). FAB-
‡
‡
‡
MS: 489 (16, [M ‡ Na] ), 467 (52, [M ‡ 1] ), 367 (100, [M Boc ‡ 1] ). Anal. calc. for C24H38N2O5S (466.64):
C 61.77, H 8.21, N 6.00, S 6.87; found: C 61.88, H 8.15, N 5.98, S 6.87.
Boc-(R)-b3-HVal-(R)-b3-HCys(Bn)-(S)-b3-HLeu-OMe (9a). Compound
7 (4.87 g, 10.5 mmol) was
deprotected according to GP 1, dissolved in CHCl3 (21 ml), and treated with Et3N (5.35 g, 53 mmol), HOBt
(1.70 g, 12.6 mmol), Boc-(R)-b3-HVal-OH (2.42 g, 10.5 mmol) in CHCl3 (21 ml), and EDC (2.41 g, 12.6 mmol)
according to GP 3a. FC (CH2Cl2/MeOH 15 :1) yielded 9a (4.76 g, 79%). White solid. M.p. 154 ± 1558. Rf
(CH2Cl2/MeOH 15 :1) 0.6. [a]Dr:t:
ˆ
19.5 (c ˆ 1.0, CHCl3). IR (KBr): 3304s, 2960s, 1741s, 1687s, 1647s, 1540s,
1437m, 1367m, 1310m, 1248m, 1174s, 1047m, 1021w, 770w, 705m. 1H-NMR (400 MHz, CDCl3): 0.89 ± 0.92
(m, 4 Me); 1.24 ± 1.32 (m, CH); 1.42 (s, t-Bu); 1.40 ± 1.50 (m, 1 H, CH2); 1.54 ± 1.63 (m, 1 H, CH2); 1.77 ± 1.82
(m, CH); 2.24 ± 2.81 (m, 3 CH2CO, CH2S); 3.67 (s, MeO); 3.67 ± 3.74 (m, CHN); 3.74 (s, SCH2Ph); 4.22 ± 4.83
(m, 2 CHN); 5.15 (br., NH); 6.33 (br., NH); 6.96 (br., NH); 7.21 ± 7.35 (m, 5 arom. H). 13C-NMR (100 MHz,
CDCl3): 18.5, 19.5, 22.1, 22.9 (Me); 25.0 (CH); 28.4 (Me); 32.2 (CH); 34.7, 36.3, 38.4, 39.2, 39.3, 43.2 (CH2);
44.5, 46.5 (CH); 51.8 (Me); 53.5 (CH); 79.1 (C); 127.1, 128.6, 129.0 (arom. C); 138.2, 156.0, 170.3, 170.8, 172.2
‡
‡
‡
(C). FAB-MS: 602 (28, [M ‡ Na] ), 580 (79, [M ‡ 1] ), 480 (100, [M Boc ‡ 1] ). Anal. calc. for C30H49N3O6S
(579.79): C 62.15, H 8.52, N 7.25, S 5.53; found: C 62.00, H 8.42, N 7.18, S 5.52.
Boc-(R)-b3-HVal-(R)-b3-HCys(Bn)-(S)-b3-HLeu-OH (9b). Compound 5a (1.16 g, 2 mmol) was dissolved
in CF3CH2OH (20 ml) and treated with 40 ml of 5n NaOH (200 mmol), according to GP 2b (reaction time:
12 h) yielding 9b (1.03 g, 91%). White solid. M.p. 171 ± 1728. Rf (CH2Cl2/MeOH 15 :1) 0.36. [a]Dr:t:
ˆ
8.6 (c ˆ
0.5, MeOH). IR (KBr): 3335s, 2960s, 1740s, 1685s, 1646s, 1540s, 1367m, 1308m, 1248m, 1172s, 1017w, 775w,
698m. 1H-NMR (400 MHz, CD3OD): 0.88 ± 0.92 (m, 4 Me); 1.26 ± 1.33 (m, CH); 1.40 (s, t-Bu); 1.40 ± 1.50
(m, 1 H, CH2); 1.53-1.64 (m, 1 H, CH2); 1.73 ± 1.81 (m, CH); 2.19 ± 2.58 (m, 3 CH2CO, CH2S); 3.70 ± 3.78
(m, CHN); 3.74 (s, SCH2Ph); 4.26 ± 4.35 (m, 2 CHN); 6.35 (br., NH); 7.18 ± 7.34 (m, 5 arom. H); 7.79
(br., NH); 7.87 (br., NH). 13C-NMR (100 MHz, CD3OD): 18.3, 19.8, 22.2, 23.7 (Me); 26.1 (CH); 28.9 (Me);
33.5 (CH); 36.2, 37.0, 40.2, 41.0, 44.5 (CH2); 46.0, 47.8, 54.8 (CH); 80.0 (C); 128.0, 129.5, 130.2 (arom. C); 139.9,
‡
‡
‡
158.1, 172.2, 173.5, 174.9 (C). FAB-MS: 588 (71, [M ‡ Na] ), 566 (60, [M ‡ 1] ), 466 (100, [M Boc ‡ 1] ).
Boc-(R)-b3-HVal-(R)-b3-HCys(Bn)-(S)-b3-HLeu-(R)-b3-HVal-(R)-b3-HCys(Bn)-(S)-b3-HLeu-OMe
(12a). Compound 9a (495 mg, 0.85 mmol) was deprotected according to GP 1, dissolved in CHCl3 (1.7 ml), and
treated with Et3N (431 mg, 4.3 mmol), HOBt (136 mg, 1 mmol), 9b (483 mg, 0.85 mmol) in DMF (8.5 ml), and
EDC (196 mg, 1 mmol) according to GP 3b. Recrystallization from MeOH yielded 12a (623 mg, 71%). White
solid. M.p. 225 ± 2278. Rf (CH2Cl2/MeOH 10 :1) 0.56. [a]Dr:t:
ˆ
0.6 (c ˆ 0.5, MeOH). IR (KBr): 3302s, 2958s,
1739s, 1686s, 1648s, 1540s, 1437m, 1367m, 1311m, 1248m, 1173s, 1028w, 772w, 706m. 1H-NMR (400 MHz, CDCl3/
CD3OD 3 : 1): 0.89 ± 0.93 (m, 8 Me); 1.24 ± 1.31 (m, 2 CH); 1.42 (s, t-Bu); 1.40 ± 1.47 (m, CH2); 1.54 ± 1.62
(m, CH2); 1.79 ± 1.83 (m, 2 CH); 2.23 ± 2.68 (m, 6 CH2CO, 2 CH2S); 3.67 (s, MeO); 3.72, 3.73 (2s, 2 SCH2Ph);
3.74 ± 3.85 (m, CHN); 4.05 ± 4.11 (m, CHN); 4.25 ± 4.40 (m, 4 CHN); 5.83 (br., NH); 7.20 ± 7.36 (m,
10 arom. H). 13C-NMR (100 MHz, CDCl3/CD3OD 3 : 1): 18.3, 18.6, 19.1, 19.3, 22.2, 23.1, 23.2 (Me); 25.1
(CH); 28.5 (Me); 32.0, 32.4 (CH); 35.5, 35.7, 36.4, 36.6, 38.4, 38.9, 39.5, 39.7, 39.8, 41.7, 43.8, 43.9 (CH2); 44.6,
45.5, 46.5, 46.6 (CH); 52.0 (Me); 52.2, 53.7 (CH); 79.4 (C); 127.2, 127.3, 128.7, 128.7, 129.1, 129.2 (arom. C);
‡
138.3, 138.5, 156.8, 170.7, 170.9, 171.4, 171.5, 172.1, 172.6 (C). FAB-MS: 1050 (100, [M ‡ Na] ).
Boc-(R)-b3-HVal-(R)-b3-HCys(Bn)-(S)-b3-HLeu-(R)-b3-HVal-(R)-b3-HCys(Bn)-(S)-b3-HLeu-OH (12b).
Compound 12a (425 mg, 0.41 mmol) was dissolved in CF3CH2OH (4.1 ml) and treated with 8.2 ml of 5n
NaOH (41 mmol), according to GP 2b (reaction time: 48 h) yielding 12b (394 mg, 94%). White solid. M.p.
233 ± 2358 (dec.). Rf (CH2Cl2/MeOH 10 :1) 0.37. [a]rD:t: ˆ‡ 1.3 (c ˆ 0.5, MeOH). IR (KBr): 3299s, 2958s, 1687s,
1647s, 1541s, 1453m, 1367m, 1311m, 1247m, 1173s, 1029w, 773w, 707m. 1H-NMR (400 MHz, CDCl3/CD3OD
3 : 1): 0.89 ± 0.92 (m, 8 Me); 1.21 ± 1.35 (m, 2 CH); 1.42 (s, t-Bu); 1.42 ± 1.50 (m, CH2); 1.52 ± 1.63 (m, CH2);
1.80 ± 1.94 (m, 2 CH); 2.28 ± 2.67 (m, 6 CH2CO, 2 CH2S); 3.71, 3.73 (2s, 2 SCH2Ph); 3.73 ± 3.80 (m, CHN);
4.02 ± 4.07 (m, CHN); 4.22 ± 4.41 (m, 4 CHN); 7.20-7.38 (m, 10 arom. H). 13C-NMR (100 MHz, CDCl3/CD3OD

Helvetica Chimica Acta ± Vol. 82 (1999)
1167
3 : 1): 18.2, 18.6, 19.1, 19.3, 22.3, 23.1, 23.2 (Me); 25.1 (CH); 28.5 (Me); 32.0, 32.4 (CH); 35.8, 35.9, 36.5, 36.8,
38.2, 38.8, 39.6, 39.9, 40.1, 41.6, 44.0, 44.2 (CH2); 44.9, 45.3, 46.4, 46.6 (CH); 52.1, 53.6 (CH); 79.4 (C); 127.2,
127.3, 128.7, 128.7, 129.1, 129.2 (arom. C); 138.3, 138.5, 156.8, 170.5, 171.0, 171.4, 171.6, 172.3, 174.1 (C). FAB-
‡
MS: 1036 (100, [M ‡ Na] ).
H-(R)-b3-HVal-(R)-b3-HCys(Bn)-(S)-b3-HLeu-(R)-b3-HVal-(R)-b3-HCys(Bn)-(S)-b3-HLeu-OH ´ CF3 -
CO2H (12c). Compound 12b (33 mg, 0.033 mmol) was deprotected according to GP 1. The crude peptide was
purified by prep. RP-HPLC (50 ! 90% B in 25 min) according to GP 5 yielding 12c (19 mg, 58%). White solid.
RP-HPLC (40 ! 90% B in 25 min) tR 20.6 min, purity > 98%. M.p. 198 ± 1998 (dec.). CD (0.2 mm in MeOH):
‡ 1.48 ´ 105 (200 nm). IR (KBr): 3289m, 3084m, 2961m, 1654s, 1550s, 1420w, 1387w, 1308w, 1201m, 1138m, 800w,
701w. 1H-NMR (400 MHz, CD3OD): 0.89 ± 0.96 (m, 6 Me); 1.08 (d, J ˆ 6.9, 2 Me); 1.22 ± 1.33 (m, 2 CH); 1.37 ±
1.46 (m, CH2); 1.51 ± 1.64 (m, CH2); 1.67 ± 1.75 (m, CH); 2.02 ± 2.10 (m, CH); 2.18 ± 2.85 (m, 6 CH2CO,
2 CH2S); 3.50 ± 3.55 (m, CHN); 3.65, 3.70 (2d, J ˆ 13.1, SCH2Ph); 3.77 (s, SCH2Ph); 3.73 ± 3.80 (m, CHN);
4.14 ± 4.20 (m, CHN); 4.35 ± 4.44 (m, 2 CHN); 4.48 ± 4.55 (m, CHN); 4.65 ± 4.72 (m, CHN); 7.16 ± 7.34
(m, 10 arom. H); 7.48 (br., NH); 7.74 (br., NH); 7.77 (br., NH); 8.19 (br., NH). 13C-NMR (100 MHz,
CD3OD): 18.2, 19.2, 19.5, 19.5, 22.9, 23.0, 23.3, 23.8 (Me); 26.0, 26.1, 32.1, 34.0 (CH); 36.1, 36.9, 37.1, 37.7, 38.1,
38.9, 40.6, 40.7, 40.9, 41.7 (CH2); 45.4, 45.8 (CH); 46.6 (CH2); 47.1, 52.7, 56.2 (CH); 128.0, 128.1, 129.5, 129.5,
130.1, 130.2 (arom. C); 139.7, 139.8, 171.0, 172.0, 172.1, 172.5, 173.1, 175.0 (C). ESI-MS (pos.): 958 (14, [M ‡
‡
‡
‡
2 Na H] ), 936 (45, [M ‡ Na] ), 914 (100, [M ‡ 1] ). ESI-MS (neg.): 912 (100, [M 1] ).
cyclo-H-(R)-b3-HVal-(R)-b3-HCys-(S)-b3-HLeu-(R)-b3-HVal-(R)-b3-HCys-(S)-b3-HLeu-OH ´ CF3CO2H
(1). Compound 12b (80 mg, 0.079 mmol) was deprotected and cyclized according to GP 4. The crude peptide
was purified by prep. RP-HPLC (20 ! 50% B in 30 min) according to GP 5 yielding 1 (35 mg, 53%), which gave
a negative Ellmanꢀs test [38]. White solid. RP-HPLC (20 ! 50% B in 20 min, then 50 ! 100% B in 5 min)
tR 22.3 min, purity >95%. M.p. 145 ± 1478. CD (0.2 mm in MeOH): ‡ 1.3 ´ 104 (200 nm); 4.5 ´ 104 (213 nm).
CD (0.2 mm in H2O): 2.6 ´ 104 (209 nm). IR (KBr): 3284m, 3081m, 2961m, 1654s, 1544s, 1466w, 1388w, 1309w,
1203s, 1138m. 1H-NMR (500 MHz, CD3OD): 0.88 ± 0.96 (m, 6 Me); 1.08 (2d, J ˆ 6.9, 2 Me); 1.24 ± 1.71
(m, 2 CH2, 3 CH); 2.03 ± 2.10 (m, CH); 2.31 ± 3.25 (m, 6 CH2CO, 2 CH2S); 3.49 ± 3.53 (m, CHN); 4.12 ± 4.16
(m, CHN); 4.35 ± 4.40 (m, CHN); 4.47 ± 4.53 (m, CHN); 4.57 ± 4.64 (m, CHN). 13C-NMR (125 MHz, CD3OD):
18.1, 18.8, 19.0, 19.6, 22.4, 23.0, 23.3, 23.6 (Me); 26.1, 26.1, 32.1, 34.0 (CH); 36.1, 37.5, 37.6, 39.5, 40.6, 40.9, 41.8,
44.9 (CH2); 45.8, 46.0 (CH); 46.5 (CH2); 47.3, 47.6 (CH); 48.1 (CH2); 52.2, 56.5 (CH); 172.1, 172.4, 172.4, 172.5,
‡
‡
‡
173.3, 175.0 (C). ESI-MS (pos.): 776 (12, [M ‡ 2 Na H] ), 754 (38, [M ‡ Na] ), 732 (100, [M ‡ 1] ). ESI-MS
(neg.): 730 (100, [M 1] ).
H-(R)-b3-HVal-(R)-b3-HCys-(S)-b3-HLeu-(R)-b3-HVal-(R)-b3-HCys-(S)-b3-HLeu-OH ´ CF3CO2H (12d).
Compound 12d was prepared from 1 according to GP 6. CD (0.2 mm in MeOH): ‡ 1.2 ´ 105 (200 nm); 4.8 ´
104 (216 nm).
Boc-(R)-b3-HMet-(S)-b3-HLeu-OMe (8). Boc-(S)-b3-HLeu-OMe (1.297 g, 5 mmol) was deprotected
according to GP 1, dissolved in CHCl3 (10 ml), and treated with Et3N (2.525 g, 25 mmol), HOBt (0.811 g,
6 mmol), 6 (1.317 g, 5 mmol) in CHCl3 (10 ml), and EDC (1.150 g, 6 mmol) according to GP 3a. FC (CH2Cl2/
MeOH 15 :1) yielded 8 (1.738 g, 86%). White solid. M.p. 112 ± 1138. Rf (CH2Cl2/MeOH 15 :1) 0.45. [a]Dr:t:
ˆ
38.2 (c ˆ 1.0, CHCl3). IR (KBr): 3302s, 2955s, 1742s, 1687s, 1656s, 1537s, 1441m, 1365m, 1274m, 1172s,
1
1053m, 1026w. H-NMR (300 MHz, CDCl3): 0.90 (d, J ˆ 6.8, Me); 0.90 (d, J ˆ 6.5, 2 Me); 1.24 ± 1.33 (m, CH);
1.42 (s, t-Bu); 1.42 ± 1.51 (m, 1 H, CH2); 1.54 ± 1.61 (m, 1 H, CH2); 1.75 ± 1.85 (m, CH2); 2.08 (s, MeS); 2.32 ± 2.59
(m, 2 CH2CO, CH2S); 3.68 (s, MeO); 3.91 ± 3.95 (m, CHN); 4.29 ± 4.34 (m, CHN); 5.40 (br., NH); 6.12
(br., NH). 13C-NMR (75 MHz, CDCl3): 15.5, 22.1, 22.8 (Me); 25.0 (CH); 28.4 (Me); 30.9, 34.0, 38.9, 40.7, 43.1
‡
(CH2); 44.2, 47.7 (CH); 51.7 (Me); 79.2, 155.6, 170.1, 172.2 (C). FAB-MS: 427 (6, [M ‡ Na] ), 405 (100, [M ‡
‡
1] ). Anal. calc. for C19H36N2O5S (404.57): C 56.41, H 8.97, N 6.92, S 7.93; found: C 56.60, H 8.76, N 6.94, S 7.73.
Boc-(R)-b3-HVal-(R)-b3-HMet-(S)-b3-HLeu-OMe (10a). Compound 8 (4.04 g, 10 mmol) was deprotected
according to GP 1, dissolved in CHCl3 (20 ml), and treated with Et3N (5.05 g, 50 mmol), HOBt (1.62 g,
12 mmol), Boc-(R)-b3-HVal-OH (2.31 g, 10 mmol) in CHCl3 (20 ml), and EDC (2.30 g, 12 mmol) according to
GP 3a. FC (CH2Cl2/MeOH 15 :1) yielded 10a (4.23 g, 82%). White solid. M.p. 176 ± 1778. Rf (CH2Cl2/MeOH
15 :1) 0.30. [a]Dr:t:
ˆ
31.1 (c ˆ 1.0, CHCl3). IR (KBr): 3304s, 2961m, 1741m, 1687s, 1645s, 1540s, 1437w, 1367w,
1
1249w, 1174m, 1050w. H-NMR (300 MHz, CDCl3): 0.89 ± 0.92 (m, 4 Me); 1.25 ± 1.34 (m, CH); 1.43 (s, t-Bu);
1.43 ± 1.62 (m, CH2); 1.74 ± 1.85 (m, CH2); 1.97 ± 2.02 (m, CH); 2.08 (s, MeS); 2.23 ± 2.60 (m, 3 CH2CO, CH2S);
3.69 (s, MeO); 3.61 ± 3.74 (m, CHN); 4.06 ± 4.19 (m, CHN); 4.25 ± 4.37 (m, CHN); 5.11 (br., NH); 6.50
(br., NH); 6.90 (br., NH). 13C-NMR (75 MHz, CDCl3): 15.5, 18.4, 19.4, 22.1, 22.9 (Me); 25.0 (CH); 28.4 (Me);
30.9 (CH2); 32.3 (CH); 33.2, 39.3, 39.5, 40.4, 43.2 (CH2); 44.6, 47.2 (CH); 51.8 (Me); 53.6 (CH); 79.2, 156.1,

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Helvetica Chimica Acta ± Vol. 82 (1999)
‡
‡
‡
170.5, 171.0, 172.4 (C). FAB-MS: 540 (38, [M ‡ Na] ), 518 (54, [M ‡ 1] ), 418 (54, [M Boc ‡ 1] ). Anal.
calc. for C25H47N3O6S (517.72): C 58.00, H 9.15, N 8.12, S 6.19; found: C 57.87, H 9.36, N 8.27, S 6.10.
Boc-(R)-b3-HVal-(R)-b3-HMet-(S)-b3-HLeu-OH (10b). Compound 10a (2.07 g, 4 mmol) was dissolved in
MeOH (10 ml) and treated with 10 ml of 1n NaOH (10 mmol), according to GP 2a. Recrystallization from
MeOH yielded 10b (1.85 g, 92%). White solid. M.p. 198 ± 1998. Rf (CH2Cl2/MeOH 15 :1) 0.3. [a]Dr:t:
ˆ
11.0 (c ˆ
0.5, MeOH). IR (KBr): 3348s, 3315s, 2956m, 1728m, 1682s, 1646s, 1593m, 1549s, 1442w, 1365w, 1228w, 1172m,
1057w. 1H-NMR (400 MHz, CD3OD): 0.85 ± 0.88 (m, 4 Me); 1.21 ± 1.32 (m, CH); 1.38 (s, t-Bu); 1.40 ± 1.48
(m, 1 H, CH2); 1.51 ± 1.61 (m, 1 H, CH2); 1.69 ± 1.81 (m, CH2, CH); 2.04 (s, MeS); 2.15 ± 2.50 (m, 3 CH2CO,
CH2S); 3.63 ± 3.67 (m, CHN); 4.06 ± 4.11 (m, CHN); 4.21 ± 4.27 (m, CHN). 13C-NMR (100 MHz, CD3OD):
15.3, 18.1, 19.2, 22.0, 23.2 (Me); 25.2 (CH); 28.5 (Me); 30.8 (CH2); 32.7 (CH); 33.4, 39.3, 40.1, 41.4, 43.5 (CH2);
‡
45.0, 47.3, 54.0 (CH); 79.6, 156.8, 171.3, 172.6, 174.1 (C). FAB-MS: 504 (99, [M ‡ 1] ), 404 (100, [M
‡
Boc ‡ 1] ).
Boc-(R)-b3-HVal-(R)-b3-HMet-(S)-b3-HLeu-(R)-b3-HVal-(R)-b3-HMet-(S)-b3-HLeu-OMe (13a). Com-
pound 10a (518 mg, 1 mmol) was deprotected according to GP 1, dissolved in CHCl3 (2 ml), and treated with
Et3N (505 mg, 5 mmol), HOBt (162 mg, 1.2 mmol), 10b (503 mg, 1 mmol) in DMF (10 ml), and EDC (230 mg,
1.2 mmol) according to GP 3b. Recrystallization from MeOH yielded 13a (687 mg, 76%). White solid. M.p. 2558
(dec.). Rf (CH2Cl2/MeOH 15 :1) 0.45. [a]Dr:t:
ˆ
3.6 (c ˆ 1.0, CF3CH2OH). IR (KBr): 3298m, 3079w, 2959m,
1
1740m, 1686m, 1647s, 1541s, 1438m, 1367m, 1248w, 1174m, 1051w. H-NMR (400 MHz, CDCl3/CD3OD 3 : 1):
0.90 ± 0.94 (m, 8 Me); 1.26 ± 1.33 (m, 2 CH); 1.44 (s, t-Bu); 1.45 ± 1.50 (m, CH2); 1.57 ± 1.65 (m, CH2); 1.77 ± 1.84
(m, 6 H, 2 CH2, 2 CH); 2.09, 2.10 (2s, 2 MeS); 2.24 ± 2.56 (m, 6 CH2CO, 2 CH2S); 3.70 (s, MeO); 3.70 ± 3.74
(m, CHN); 4.04 ± 4.12 (m, CHN); 4.18 ± 4.28 (m, 3 CHN); 4.29 ± 4.35 (m, CHN); 5.96 (br., NH); 7.49 (br.,
NH); 7.59 (br., NH); 7.64 (br., NH). 13C-NMR (100 MHz, CDCl3/CD3OD 3 : 1): 15.4, 15.5, 18.3, 18.5, 19.2, 19.3,
22.1, 23.2, 23.3 (Me); 25.3 (CH); 28.6 (Me); 30.9 (CH2); 32.2, 32.9 (CH); 33.9, 34.2, 38.8, 39.2, 40.2, 41.2, 41.6,
42.1, 43.9, 44.0 (CH2); 45.0, 45.8, 47.0, 47.1 (CH); 52.1 (Me); 52.5, 54.0 (CH); 79.6, 157.0, 171.3, 171.4, 171.8,
‡
‡
‡
172.1, 172.6, 172.7 (C). FAB-MS: 926 (65, [M ‡ Na] ), 904 (28, [M ‡ 1] ), 804 (76, [M Boc ‡ 1] ), 559 (100).
Boc-(R)-b3-HVal-(R)-b3-HMet-(S)-b3-HLeu-(R)-b3-HVal-(R)-b3-HMet-(S)-b3-HLeu-OH (13b). Com-
pound 13a (226 mg, 0.20 mmol) was dissolved in CF3CH2OH (2 ml) and treated with 4 ml of 5n NaOH
(20 mmol) according to GP 2b (reaction time: 24 h) yielding 13b (213 mg, 96%). White solid. M.p. 245 ± 2468
(dec.). Rf (CH2Cl2/MeOH 10 :1) 0.18. CD (0.2 mm in MeOH): ‡ 6.9 ´ 104 (200 nm); 6.0 ´ 103 (222 nm). IR
(KBr): 3296m, 3079w, 2960m, 1689m, 1648s, 1542s, 1438m, 1367m, 1248w, 1173m, 1048w. 1H-NMR (400 MHz,
CDCl3/CD3OD 3 : 1): 0.89 ± 0.94 (m, 8 Me); 1.22 ± 1.34 (m, 2 CH); 1.39 ± 1.45 (m, CH2); 1.45 (s, t-Bu); 1.56 ± 1.66
(m, CH2); 1.75 ± 1.81 (m, 2 CH2, 2 CH); 2.09, 2.10 (2s, 2 MeS); 2.26 ± 2.57 (m, 6 CH2CO, 2 CH2S); 3.76 ± 3.85
(m, CHN); 4.08 ± 4.15 (m, CHN); 4.30 ± 4.44 (m, 4 CHN). 13C-NMR (100 MHz, CDCl3/CD3OD 3 : 1): 15.5,
15.6, 18.1, 19.0, 19.1, 19.2, 22.5, 22.6, 23.3 (Me); 25.4, 25.4 (CH); 28.6 (Me); 30.9, 31.0 (CH2); 32.8 (CH); 35.2,
38.0, 39.0, 40.9, 41.5, 41.8, 42.7 (CH2); 45.1, 46.0, 46.2, 46.4 (CH); 52.0, 53.6 (CH); 79.6, 157.4, 170.5, 171.4, 171.7,
‡
‡
172.1, 173.2, 176.5 (C). FAB-MS: 912 (100, [M ‡ Na] ), 790 (22, [M Boc ‡ 1] ).
H-(R)-b3-HVal-(R)-b3-HMet-(S)-b3-HLeu-(R)-b3-HVal-(R)-b3-HMet-(S)-b3-HLeu-OMe´ CF3CO2H (13c).
Compound 13a (50 mg, 0.055 mmol) was deprotected according to GP 1, yielding 13c (49 mg, 98%). White
solid. M.p. 218 ± 2198 (dec.). CD (0.2 mm in MeOH): ‡ 8.3 ´ 104 (198 nm); 2.3 ´ 104 (218 nm). IR (KBr):
3294m, 3105w, 2962m, 1646s, 1545s, 1439m, 1370w, 1311w, 1262w, 1204m, 1133m. 1H-NMR (400 MHz, CD3OD):
0.88 ± 0.96 (m, 6 Me); 1.09 (d, J ˆ 6.9, 2 Me); 1.22 ± 1.45 (m, 2 CH, CH2); 1.54 ± 2.10 (m, 3 CH2, 2 CH); 2.08, 2.08
(2s, 2 MeS); 2.18 ± 2.92 (m, 6 CH2CO, 2 CH2S); 3.53 ± 3.57 (m, CHN); 3.71 (s, MeO); 4.21 ± 4.33 (m, CHN);
4.35 ± 4.60 (m, 4 CHN); 7.43 (br., NH); 7.71 (br., NH); 7.73 (br., NH); 8.23 (br., NH). ESI-MS (pos.): 826 (11,
‡
‡
[M ‡ Na] ), 804 (100, [M ‡ 1] ).
Boc-(R)-b3-HVal-(R)-b3-HHcy-(S)-b3-HLeu-(R)-b3-HVal-(R)-b3-HHcy-(S)-b3-HLeu-OH (13d). Com-
pound 13b (400 mg, 0.45 mmol) was dissolved in 135 ml of NH3 at 338 [37]. Na (1.04 g, 45 mmol) was
added. The dark-blue soln. was stirred for 24 h and subsequently quenched by adding 3 g (56 mmol) of NH4Cl.
NH3 was allowed to evaporate completely, and the residue was taken up in 100 ml of H2O. The pH was adjusted
to 2 ± 3 with 1n HCl, resulting in the precipitation of 13d (337 mg, 87%), which was not further purified. White
solid. M.p. 212 ± 2138 (dec.). Rf (CH2Cl2/MeOH 10 : 1) 0.18. [a]Dr:t:
ˆ
0.1 (c ˆ 0.25, MeOH). IR (KBr): 3276m,
1
3081w, 2960m, 1689m, 1641s, 1544s, 1444m, 1368m, 1250w, 1171m. H-NMR (400 MHz, CDCl3/CD3OD 3 : 1):
0.90 ± 0.94 (m, 8 Me); 1.24 ± 1.36 (m, 2 CH); 1.45 (s, t-Bu); 1.45 ± 1.49 (m, CH2); 1.57 ± 1.63 (m, CH2); 1.77 ± 1.89
(m, 2 CH2, 2 CH); 2.27 ± 2.55 (m, 6 CH2CO, 2 CH2S); 3.74 ± 3.81 (m, CHN); 4.06 ± 4.14 (m, CHN); 4.27 ± 4.42
(m, 4 CHN). 13C-NMR (100 MHz, CDCl3/CD3OD 3 : 1): 18.2, 18.7, 19.1, 19.2 (Me); 21.3, 21.3 (CH2); 22.4, 23.3
(Me); 25.3 (CH); 28.6 (Me); 32.5, 33.0 (CH); 38.4, 39.0, 39.3, 39.6, 40.5, 41.1, 41.5, 41.9, 44.4, 44.6 (CH2); 44.9,

Helvetica Chimica Acta ± Vol. 82 (1999)
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45.1, 45.5, 46.0, 46.1 (CH); 52.3, 53.9 (CH); 79.6, 157.2, 170.9, 171.3, 171.6, 172.0, 173.0, 174.4 (C). ESI-MS
(neg.): 860 (100, [M 1] ).
H-(R)-b3-HVal-(R)-b3-HHcy-(S)-b3-HLeu-(R)-b3-HVal-(R)-b3-HHcy-(S)-b3-HLeu-OH´ CF3CO2H (13e).
Compound 13d (51 mg, 0.059 mmol) was deprotected according to GP 1. The crude peptide was purified by
prep. RP-HPLC (45 ! 65% B in 30 min) according to GP 5 yielding 13e (36 mg, 71%). White solid. RP-HPLC
(45 ! 65% B in 20 min) tR 15.5 min, purity >98%. M.p. 202 ± 2038 (dec.). CD (0.2 mm in MeOH): ‡ 1.92 ´ 105
(197 nm); 2.3 ´ 104 (218 nm). IR (KBr): 3281m, 3081w, 2961m, 1654s, 1547s, 1466w, 1438w, 1387w, 1309w,
1
1202m, 1137m, 1045w. H-NMR (400 MHz, CD3OD): 0.88 ± 0.96 (m, 6 Me); 1.09 (d, J ˆ 6.9, 2 Me); 1.21 ± 1.33
(m, 2 CH); 1.37 ± 1.46 (m, CH2); 1.51 ± 2.11 (m, 3 CH2, 2 CH); 2.20 ± 2.89 (m, 6 CH2CO, 2 CH2S); 3.51 ± 3.56
(m, CHN); 4.17 ± 4.23 (m, CHN); 4.34 ± 4.44 (m, 2 CHN); 4.46 ± 4.54 (m, CHN); 4.59 ± 4.66 (m, CHN); 7.50
(br., NH); 7.64 (br., NH); 7.72 (br., NH); 8.22 (br., NH). 13C-NMR (100 MHz, CD3OD): 18.3, 19.2, 19.3, 19.5,
19.5 (Me); 21.7, 21.9 (CH2); 23.0, 23.3, 23.8 (Me); 26.1, 32.1, 34.0 (CH); 36.1, 38.8, 40.8, 41.2, 41.4, 41.6, 41.7, 41.8
(CH2); 45.3, 45.7 (CH); 45.8, 46.2, 46.6, 46.7 (CH2); 52.7, 56.3 (CH); 171.1, 172.3, 172.4, 172.5, 173.1, 175.0 (C).
‡
‡
ESI-MS (pos.): 784 (17, [M ‡ Na] ), 762 (100, [M ‡ 1] ). ESI-MS (neg.): 760 (100, [M 1] ].
H-(R)-b3-HVal-(R)-b3-HMet-(S)-b3-HLeu-(R)-b3-HVal-(R)-b3-HMet-(S)-b3-HLeu-OH ´ CF3CO2H (13f).
Compound 13b (59 mg, 0.066 mmol) was deprotected according to GP 1. The crude peptide was purified by
prep. RP-HPLC (35 ! 70% B in 35 min) according to GP 5, yielding 13f (35 mg, 59%). White solid. RP-HPLC
(35 ! 70% B in 20 min) tR 17.1 min, purity >95%. M.p. 214 ± 2158 (dec.). CD (0.2 mm in MeOH): ‡ 1.15 ´ 105
(201 nm); 3.2 ´ 104 (218 nm). IR (KBr): 3308m, 3083w, 2964m, 1684s, 1644s, 1558m, 1446m, 1210s, 1142s,
844m, 803m, 725m. 1H-NMR (400 MHz, CD3OD): 0.89 ± 0.96 (m, 6 Me); 1.09 (d, J ˆ 6.9, 2 Me); 1.21 ± 1.34
(m, 2 CH); 1.37 ± 1.46 (m, CH2); 1.51 ± 2.11 (m, 3 CH2, 2 CH); 2.08, 2.09 (2s, 2 MeS); 2.26 ± 2.87 (m, 6 CH2CO,
2 CH2S); 3.51 ± 3.56 (m, CHN); 4.16 ± 4.23 (m, CHN); 4.38 ± 4.49 (m, 3 CHN); 4.52 ± 4.58 (m, CHN); 7.52 (br.,
NH); 7.69 (br., NH); 7.74 (br., NH); 8.22 (br., NH). 13C-NMR (100 MHz, CD3OD): 15.6, 18.3, 19.2, 19.5, 19.5,
23.0, 23.0, 23.3, 23.8 (Me); 26.0, 26.1 (CH); 31.5, 31.8 (CH2); 32.1, 34.0 (CH); 36.2, 36.5, 36.7, 38.9, 40.8, 41.5,
41.6, 41.8 (CH2); 45.3, 45.7 (CH); 45.8, 46.6 (CH2); 46.7, 47.4, 52.8, 56.3 (CH); 171.2, 172.2, 172.6, 173.2, 174.9
‡
‡
‡
‡
(C). ESI-MS (pos.): 834 (15, [M ‡ 2 Na H] ), 828 (14, [M ‡ K] ), 812 (71, [M ‡ Na] ), 790 (100, [M ‡ 1] ).
ESI-MS (neg.): 788 (100, [M 1] ).
cyclo-H-(R)-b3-HVal-(R)-b3-HHcy-(S)-b3-HLeu-(R)-b3-HVal-(R)-b3-HHcy-(S)-b3-HLeu-OH ´ CF3CO2H
(2). Compound 13b (35 mg, 0.041 mmol) was cyclized and deprotected according to GP 4. The crude peptide
was purified by prep. RP-HPLC (20 ! 40% B in 30 min) according to GP 5, yielding 2 (18 mg, 51%), which
gave a negative Ellmanꢀs test [38]. White solid. RP-HPLC (20 ! 50% in 20 min) tR 14.5 min, purity >95%. M.p.
128 ± 1308 (dec.). CD (0.2 mm in MeOH): ‡ 7.7 ´ 104 (200 nm); 2.4 ´ 104 (218 nm). CD (0.2 mm in H2O):
‡ 2.2 ´ 104 (203 nm); 0.6 ´ 104 (226 nm). IR (KBr): 3293m, 3098m, 2962m, 1654s, 1550s, 1472w, 1399m, 1203s,
1137m. 1H-NMR (500 MHz, CD3OD): 0.89 ± 0.96 (m, 6 Me); 1.08 (d, J ˆ 6.9, 2 Me); 1.24 ± 2.21 (m, 4 CH2,
4 CH); 2.24 ± 3.17 (m, 6 CH2CO, 2 CH2S); 3.52 ± 3.56 (m, CHN); 4.14 ± 4.24 (m, CHN); 4.38 ± 4.45 (m, 2 CHN);
4.59 ± 4.64 (m, CHN); 4.71 ± 4.78 (m, CHN); 7.62 (br., NH); 7.71 (br., NH); 7.74 (br., NH); 8.22 (br., NH).
13C-NMR (100 MHz, CDCl3): 18.1, 19.2, 19.6, 19.6, 23.0, 23.2, 23.3, 23.7 (Me); 26.1, 32.1 (CH); 33.7 (CH2); 34.0
(CH); 35.6, 36.0, 36.4, 36.6, 38.8, 40.0, 41.2, 41.9 (CH2); 45.5, 45.7 (CH); 45.7 (CH2); 46.0 (CH); 46.4, 46.5 (CH2);
‡
‡
52.9, 56.5 (CH); 171.3, 171.8, 172.7, 173.0, 175.3 (C). ESI-MS (pos.): 782 (10, [M ‡ Na] ), 760 (100, [M ‡ 1] ).
ESI-MS (neg.): 758 (100, [M 1] ).
Boc-(R)-b3-HVal-(R)-b3-HCys(Bn)-(S)-b3-HLeu-(R)-b3-HCys(Bn)-(S)-b3-HLeu-OMe (14a). Com-
pound 7 (128 mg, 0.275 mmol) was deprotected according to GP 1, dissolved in CHCl3 (0.6 ml), and treated
with Et3N (140 mg, 1.4 mmol), HOBt (45 mg, 0.35 mmol), 9b (156 mg, 0.275 mmol) in DMF (2.8 ml), and EDC
(64 mg, 0.35 mmol) according to GP 3b. FC (CH2Cl2/MeOH 13 :1) yielded 14a (225 mg, 89%). White solid. M.p.
201 ± 2028. Rf (CH2Cl2/MeOH 13 :1) 0.33. [a]Dr:t:
ˆ
1.4 (c ˆ 0.5, MeOH). IR (KBr): 3302s, 2957s, 1684s, 1636s,
1541w, 1451s, 1366m, 1310w, 1260m, 1173m, 1019m, 804w, 699m. 1H-NMR (400 MHz, CDCl3/CD3OD 3 : 1):
0.89 ± 0.94 (m, 6 Me); 1.24 ± 1.33 (m, 2 CH); 1.41 (s, t-Bu); 1.41 ± 1.47 (m, CH2); 1.53 ± 1.61 (m, CH2); 1.80 ± 1.84
(m, CH); 2.23 ± 2.69 (m, 5 CH2CO, 2 CH2S); 3.66 (s, MeO); 3.66 ± 3.70 (m, CHN); 3.74, 3.74 (2s, 2 SCH2Ph);
4.20 ± 4.31 (m, 4 CHN); 7.11 ± 7.34 (m, 10 arom. H). 13C-NMR (100 MHz, CDCl3/CD3OD 3 : 1): 18.3, 19.4, 22.0,
23.1, 23.4 (Me); 25.1 (CH); 28.5 (Me); 32.4 (CH); 35.2, 35.4, 36.5, 39.1, 39.5, 39.6, 39.8, 42.0, 43.4, 43.5 (CH2);
44.8, 45.4, 46.8 (CH); 51.9 (Me); 53.8 (CH); 79.5 (C), 127.3, 127.3, 128.8, 129.2, 129.2 (arom. C); 138.4, 138.4,
‡
‡
156.7, 170.8, 170.9, 171.0, 171.4, 172.1, 172.5 (C). FAB-MS: 937 (7, [M ‡ Na] ), 915 (17, [M ‡ 1] ), 815 (100,
‡
[M Boc ‡ 1] ).
Boc-(R)-b3-HVal-(R)-b3-HCys(Bn)-(S)-b3-HLeu-(R)-b3-HCys(Bn)-(S)-b3-HLeu-OH (14b). Compound
14a (197 mg, 0.22 mmol) was dissolved in CF3CH2OH (2.2 ml) and treated with 4.4 ml of 5n NaOH (22 mmol),
according to GP 2b (reaction time: 12 h) yielding 14b (185 mg, 94%). White solid. M.p. 211 ± 2128. Rf (CH2Cl2/

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Helvetica Chimica Acta ± Vol. 82 (1999)
MeOH 15 :1) 0.13. [a]Dr:t:
ˆ
0.7 (c ˆ 0.25, MeOH). IR (KBr): 3297s, 2957s, 1686s, 1647s, 1541s, 1451s, 1366m,
1313w, 1248m, 1172m, 1029m, 770w, 699m. 1H-NMR (400 MHz, CDCl3/CD3OD 3 : 1): 0.87 ± 0.96 (m, 6 Me);
1.21 ± 1.33 (m, 2 CH); 1.39 (s, t-Bu); 1.39 ± 1.62 (m, 2 CH2); 1.73 ± 1.81 (m, CH); 2.25 ± 2.80 (m, 5 CH2CO,
2 CH2S); 3.68 ± 3.80 (m, CHN); 3.74 (br. s, 2 SCH2Ph); 4.39 ± 4.56 (m, 4 CHN); 7.23 ± 7.38 (m, 10 arom. H).
13C-NMR (100 MHz, CDCl3/CD3OD 3 : 1): 18.0, 19.6, 22.1, 23.1, 23.4 (Me); 25.0, 25.0 (CH); 28.5 (Me); 31.8
(CH); 35.7, 36.2, 36.5, 40.1, 40.6, 41.6, 43.6, 44.4 (CH2); 44.9, 45.9, 46.6, 53.4 (CH); 80.1 (C); 127.4, 127.8, 128.8,
‡
129.0, 129.1 (arom. C); 138.0, 138.1, 157.2, 172.5, 173.8 (C). FAB-MS: 923 (100, [M ‡ Na] ), 800 (12, [M
‡
Boc ‡ 1] ).
cyclo-H-(R)-b3-HVal-(R)-b3-HCys-(S)-b3-HLeu-(R)-b3-HCys-(S)-b3-HLeu-OH ´ CF3CO2H (3). Com-
pound 14b (60 mg, 0.067 mmol) was deprotected and cyclized according to GP 4. The crude peptide was
purified by prep. RP-HPLC (20 ! 40% B in 30 min) according to GP 5, yielding 3 (21 mg, 38%), which gave a
negative Ellmanꢀs test [38]. White solid. RP-HPLC (20 ! 50% B in 20 min) tR 14.5 min, purity >95%. M.p.
170 ± 1728 (dec.). CD (0.2 mm in MeOH): 8.3 ´ 104 (198 nm). CD (0.2 mm in H2O): 6.1 ´ 104 (198 nm). IR
(KBr): 3314m, 3112m, 2962m, 1671s, 1558s, 1400m, 1307w, 1204s, 1136s. 1H-NMR (500 MHz, CD3OD): 0.90 ±
0.95 (m, 4 Me); 1.01 ± 1.04 (m, 2 Me); 1.28 ± 1.62 (m, 2 CH2, 2 CH); 1.93 ± 2.01 (m, CH); 2.19 ± 3.25
(m, 5 CH2CO, 2 CH2S); 3.44 ± 3.50 (m, CHN); 3.83 ± 3.94 (m, CHN); 4.25 ± 4.30 (m, CHN); 4.34 ± 4.39
(m, 2 CHN); 7.89 (br., NH); 8.03 (br., NH). 13C-NMR (125 MHz, CD3OD): 18.2, 18.6, 22.2, 22.6, 23.4, 23.6
(Me); 26.1, 26.2, 31.7 (CH); 35.3, 40.2, 41.0, 41.3, 41.7, 42.1, 44.5, 44.6, 45.3 (CH2); 46.1, 46.1, 55.6 (CH); 172.0,
‡
‡
172.0, 172.2, 173.1, 174.9 (C). ESI-MS (pos.): 663 (8, [M ‡ 2 Na H] ), 656 (10, [M ‡ K] ), 640 (45, [M ‡
‡
‡
Na] ), 618 (100, [M ‡ 1] ). ESI-MS (neg.): 616 (100, [M 1] ).
H-(R)-b3-HVal-(R)-b3-HCys-(S)-b3-HLeu-(R)-b3-HCys-(S)-b3-HLeu-OH ´ CF3CO2H (14c). Compound
14c was prepared from 3 according to GP 6. CD (0.2 mm in MeOH): ‡ 1.05 ´ 105 (198 nm); 9.0 ´ 103 (220 nm).
Boc-(R)-b3-HSer(Bn)-(R)-b3-HVal-(R)-b3-HCys(Bn)-(S)-b3-HLeu-OMe (11). Compound 9a (232 mg,
0.4 mmol) was deprotected according to GP 1, dissolved in CHCl3 (0.8 ml), and treated with Et3N (202 mg,
2 mmol), HOBt (65 mg, 0.48 mmol), Boc-(R)-b3-HSer(Bn)-OH (124 mg, 0.4 mmol) in CHCl3 (0.8 ml), and
EDC (95 mg, 0.48 mmol) according to GP 3a. FC (CH2Cl2/MeOH 13 :1) yielded 11 (257 mg, 83%). White solid.
M.p. 159 ± 1608. Rf (CH2Cl2/MeOH 15 :1) 0.48. [a]Dr:t:
ˆ
6.1 (c ˆ 1.0, CHCl3). 1H-NMR (400 MHz, CDCl3):
0.86 ± 0.92 (m, 3 Me); 1.24 ± 1.31 (m, CH); 1.42 (s, t-Bu); 1.42 ± 1.49 (m, 1 H, CH2); 1.51 ± 1.62 (m, 1 H, CH2);
1.74 ± 1.83 (m, CH); 2.25 ± 2.77 (m, 4 CH2CO, CH2S); 3.47 ± 3.58 (m, CH2O); 3.66 (s, MeO); 3.71 (s, SCH2Ph);
3.94 ± 4.02 (m, CHN); 4.07 ± 4.17 (m, CHN); 4.18 ± 4.26 (m, CHN); 4.26 ± 4.36 (m, CHN); 4.51 (s, CH2O); 5.51
(br., NH); 6.35 (br., NH); 6.69 (br., NH); 7.01 (br., NH); 7.20 ± 7.36 (m, 10 arom. H). 13C-NMR (100 MHz,
CDCl3): 19.0, 19.6, 22.1, 22.9 (Me); 25.0 (CH); 28.4 (Me); 31.5 (CH); 34.8, 36.2, 38.3, 38.5, 38.7, 39.1, 43.2
(CH2); 44.5, 46.5, 47.9 (CH); 51.8 (Me); 52.1 (CH); 71.5, 73.3 (CH2); 79.1 (C); 127.1, 127.7, 127.8, 128.4, 128.6,
‡
129.0 (arom. C); 138.0, 138.1, 155.6, 170.2, 170.5, 170.8, 172.2 (C). FAB-MS: 793 (14, [M ‡ Na] ), 771 (47,
‡
‡
[M ‡ 1] ), 671 (100, [M Boc ‡ 1] ).
Boc-(R)-b3-HVal-(R)-b3-HCys(Bn)-(S)-b3-HLeu-(R)-b3-HSer(Bn)-(R)-b3-HVal-(R)-b3-HCys(Bn)-(S)-b3-
HLeu-OMe (15a). Compound 11 (212 mg, 0.275 mmol) was deprotected according to GP 1, dissolved in CHCl3
(0.6 ml), and treated with Et3N (140 mg, 1.4 mmol), HOBt (45 mg, 0.35 mmol), 9b (156 mg, 0.275 mmol) in
DMF (2.8 ml), and EDC (64 mg, 0.35 mmol) according to GP 3b. FC (CH2Cl2/MeOH 7:1) yielded 15a (140 mg,
42%). White solid. M.p. 220 ± 2228. Rf (CH2Cl2/MeOH 16 :1) 0.29. [a]Dr:t:
ˆ
2.6 (c ˆ 1.0, CHCl3). IR (KBr):
3291s, 3063m, 2958s, 1648s, 1542s, 1453s, 1367m, 1311w, 1248m, 1174m, 1028m, 743w, 699m. 1H-NMR (400 MHz,
CDCl3): 0.85 ± 0.92 (m, 8 Me); 1.15 ± 1.70 (m,4 CH, 2 CH2, t-Bu); 2.38 ± 3.31 (m, 7 CH2CO, 2 CH2S); 3.49 ± 3.79
(m, 2 SCH2Ph, MeO); 4.05 ± 4.16 (m, 2 CHN); 4.27 ± 4.38 (m, CHN); 4.41 ± 4.55 (m, 3 CHN, 2 CH2O); 4.74 ±
4.86 (m, CHN); 5.49 (br., NH); 7.19 ± 7.34 (m, 15 arom. H); 7.51 (br., NH); 7.61 (br., NH); 7.76 (br., NH); 8.31
(br., NH); 8.57 (br., NH); 8.83 (br., NH); 8.93 (br., NH). 13C-NMR (100 MHz, CDCl3): 19.1, 19.3, 19.4, 22.8,
23.1 (Me); 24.7, 25.0 (CH); 28.5 (Me); 32.5, 32.8 (CH); 35.5, 36.8, 37.7, 37.9, 39.6, 39.8, 40.1, 41.4 (CH2); 44.0
(CH); 44.8 (CH2); 45.0 (CH); 45.1 (CH2); 46.7, 47.2 (CH); 52.2 (Me); 52.4 (CH); 71.6, 73.0 (CH2); 80.2 (C);
111.1, 118.4, 123.7, 124.7, 126.9, 127.1, 127.3, 127.8, 128.1, 128.4, 128.4, 128.6, 128.9 (arom. C); 137.7, 138.1, 157.5,
‡
170.6, 170.8, 170.90, 171.4, 171.6, 173.0, 174.5 (C). FAB-MS: 1119 (100, [M Boc ‡ 1] ).
Boc-(R)-b3-HVal-(R)-b3-HCys(Bn)-(S)-b3-HLeu-(R)-b3-HSer(Bn)-(R)-b3-HVal-(R)-b3-HCys(Bn)-(S)-
b3-HLeu-OH (15b). Compound 15a (91 mg, 0.075 mmol) was dissolved in CF3CH2OH (1 ml) and treated with
1.5 ml of 5n NaOH (7.5 mmol), according to GP 2b (reaction time: 48 h) yielding 15b (87 mg, 96%). White
solid. M.p. 2418 (dec.). Rf (CH2Cl2/MeOH 10 :1) 0.22. [a]Dr:t:
ˆ
0.4 (c ˆ 1.0, CF3CH2OH). IR (KBr): 3308m,
2961m, 1637s, 1542w, 1451s, 1366m, 1261m, 1172m, 1098m, 1028w, 802w, 698w. 1H-NMR (400 MHz, CD3OD):
0.85 ± 0.92 (m, 8 Me); 1.14 ± 1.83 (m, 4 CH, 2 CH2, t-Bu); 2.33 ± 2.78 (m, 7 CH2CO, 2 CH2S); 3.31 (m, 1 H);
3.48 ± 3.53 (m, CHN); 3.71 (d, J ˆ 4.4, SCH2Ph); 3.76 (d, J ˆ 2.4, SCH2Ph); 3.84 ± 3.88 (m, CHN); 4.27 ± 4.35

Helvetica Chimica Acta ± Vol. 82 (1999)
1171
(m, 3 CHN); 4.43 ± 4.64 (m, 2 CHN, 2 CH2O); 7.17 ± 7.34 (m, 15 arom. H); 7.49 (br., NH); 7.82 (br., NH); 8.30
(br., NH). 13C-NMR (100 MHz, CD3OD): 19.0, 19.1, 19.3, 22.3, 22.8, 23.2, 23.3 (Me); 25.0, 25.2 (CH); 28.5
(Me), 32.5, 32.7 (CH); 36.5, 36.6, 36.7, 37.5, 37.7, 37.9, 38.7, 39.7, 40.3, 41.8 (CH2); 44.7 (CH); 44.9 (CH2); 45.5
(CH); 45.7 (CH2); 46.2, 47.5, 51.8, 53.2 (CH); 71.9, 73.3 (CH2); 79.2 (C); 127.2, 127.3, 127.8, 127.9, 128.0, 128.6,
128.7, 129.1, 129.2, 131.3 (arom. C); 138.3, 138.3, 138.4, 157.4, 170.0, 170.7, 171.1, 171.4, 172.7, 173.9 (C). FAB-
‡
MS: 1233 (100, [M ‡ 29] ).
cyclo-H-(R)-b3-HVal(R)-b3-HCys-(S)-b3-HLeu-(R)-b3-HSer-(R)-b3-HVal-(R)-b3-HCys-(S)-b3-HLeu-OH ´
CF3CO2H (4). Compound 15b (60 mg, 0.050 mmol) was deprotected and cyclized according to GP 4. The crude
peptide was purified by prep. RP-HPLC (20 ! 40% B in 30 min) according to GP 5, yielding 4 (27 mg, 65%),
which gave a negative Ellmanꢀs test [38]. White solid. RP-HPLC (20 ! 50% B in 20 min) tR 18.1 min, purity
>98%. M.p. 190 ± 1918 (dec.). CD (0.2 mm in MeOH): 5.7 ´ 104 (200 nm). CD (0.2 mm in H2O): 1.5 ´ 104
(203 nm). IR (KBr): 3295m, 3110m, 2975m, 1676s, 1545s, 1437m, 1400m, 1315w, 1207s, 1136s, 1052w. 1H-NMR
(500 MHz, CD3OD): 0.91 ± 0.98 (m, 6 Me); 1.03 ± 1.06 (m, 2 Me); 1.24 ± 1.84 (m, 2 CH2, 3 CH); 1.97 ± 2.03
(m, CH); 2.24 ± 3.06 (m, 7 CH2CO, 2 CH2S); 3.41 ± 3.44 (m, CHN); 3.46 ± 3.57 (m, CH2CO); 4.05 ± 4.09
(m, CHN); 4.31 ± 4.38 (m, 4 CHN); 4.42 ± 4.48 (m, CHN); 7.89 (br., NH). 13C-NMR (125 MHz, CD3OD):
18.2, 18.8, 19.0, 19.3, 22.4, 22.4, 23.6, 23.7 (Me); 26.1, 26.1, 32.0, 34.0 (CH); 36.2, 38.5, 38.7, 40.0, 40.1, 41.4, 43.2,
44.7, 44.9, 45.2, 45.7 (CH2); 46.1, 46.6, 48.2, 53.4, 56.1 (CH); 64.5 (CH2); 171.7, 172.0, 172.1, 172.5, 173.2, 173.5,
‡
‡
‡
175.4 (C). ESI-MS (pos.): 877 (20, [M ‡ 2 Na H] ), 855 (24, [M ‡ Na] ), 833 (100, [M ‡ 1] ). ESI-MS
(neg.): 831 (100, [M 1] ).
H-(R)-b3-HVal-(R)-b3-HCys-(S)-b3-HLeu-(R)-b3-HSer-(R)-b3-HVal-(R)-b3-HCys-(S)-b3-HLeu-OH ´
CF3CO2H (15c). Compound 15c was prepared from 4 according to GP 6. CD (0.2 mm in MeOH): ‡ 6.6 ´ 104
(204 nm); 3.3 ´ 104 (216 nm).
REFERENCES
[1] D. Seebach, M. Overhand, F. N. M. Kühnle, B. Martinoni, L. Oberer, U. Hommel, H. Widmer, Helv. Chim.
Acta 1996, 79, 913.
[2] D. Seebach, P. E. Ciceri, M. Overhand, B. Jaun, D. Rigo, L. Oberer, U. Hommel, R. Amstutz, H. Widmer,
Helv. Chim. Acta 1996, 79, 2043.
[3] D. Seebach, S. Abele, K. Gademann, G. Guichard, T. Hintermann, B. Jaun, J. L. Matthews, J. V. Schreiber,
L. Oberer, U. Hommel, H. Widmer, Helv. Chim. Acta 1998, 81, 932.
[4] D. Seebach, S. Abele, K. Gademann, B. Jaun, Angew. Chem. 1999, submitted.
[5] D. Seebach, J. L. Matthews, J. Chem. Soc., Chem. Commun., 1997, 21, 2015.
[6] S. H. Gellman, Acc. Chem. Res. 1998, 31, 173.
[7] K. Gademann, T. Hintermann, J. V. Schreiber, Curr. Med. Chem. 1999, 111, 1700; ibid., Int. Ed. 1999, 38,
1595.
[8] D. Y. Jackson, D. S. King, J. Chmielewski, S. Singh, P. G. Schultz, J. Am. Chem. Soc. 1991, 113, 9391.
[9] M. Chorev, E. Roubini, R. L. McKee, S. W. Gibbons, M. E. Goldman, M. P. Caulfield, M. Rosenblatt,
Biochemistry 1991, 30, 5968.
[10] H. E. Blackwell, R. H. Grubbs, Angew. Chem. 1998, 110, 3469; ibid. Int. Ed. 1998, 37, 3281.
[11] a) T. Hintermann, D. Seebach, Synlett 1997, 437; b) G. Guichard, D. Seebach, Chimia 1997, 51, 315; c) G.
Guichard, S. Abele, D. Seebach, Helv. Chim. Acta 1998, 52, 187.
[12] a) S. Abele, G. Guichard, D. Seebach, Helv. Chim. Acta 1998, 81, 2141; b) B. W. Gung, D. Zou, A. M.
Stalcup, C. E. Cottrell, J. Org. Chem. 1999, 64, 2176; c) D. A. Appella, J. J. Barchi, S. R. Durell, S. H.
Gellman, J. Am. Chem. Soc. 1999, 121, 2309.
[13] E. Block, J. Zubieta, in ꢁAdvances in Sulfur Chemistryꢀ, Ed. E. Block, Jai Press Inc., Greenwich, CT, 1994,
p. 133.
[14] E. P. Fries, J. E. T. Andersen, L. L. Madsen, P. Moller, R. J. Nichols, K. G. Olesen, J. Ulstrup, Electro-
chimica Acta 1998, 43, 2889.
[15] T. V. Oꢀ Halloran, Science 1993, 261, 715.
[16] S. J. Lippard, J. M. Berg, ꢁPrinciples of Bioinorganic Chemistryꢀ, University Science Books, Mill Valley, CA,
1994, p. 178.
[17] K. T. Yasunobu, M. Tanaka, in ꢁIron Sulfur Proteinsꢀ. Ed. E. Lovenberg, Academic Press Inc., London
1973, Vol. 2, p. 27.
[18] H. J. Dyson, Method. Enzymol. 1995, 252, 293.

1172
Helvetica Chimica Acta ± Vol. 82 (1999)
[19] H. Tuppy, in ꢁSulfur in Proteinsꢀ Eds. R. Benesch, R. E. Benesch, P. D. Boyer, I. M. Klotz, W. R.
Middlebrook, A. G. Uzent-Györgi, Academic Press Inc., London 1959, p. 141.
[20] C. Guibourdenche, J. Podlech, D. Seebach, Liebigs Ann. Chem. 1996, 1121.
[21] G. Grue-Sùrensen, E. Kelstrup, A. Kjñr, J. égaard Madsen, J. Chem. Soc., Perkin Trans. I 1984, 1091.
[22] T. D. Clark, K. Kobayashi, M. R. Ghadiri, Chem. Eur. J. 1999, 5, 782.
Â
[23] a) D. Andreu, F. Albericio, N. A. Sole, M. C. Munson, M. Ferrer, G. Barany in ꢁPeptide Synthesis
Protocolsꢀ, Eds. M. W. Pennington, B. M. Dunn, Humana Press Inc., Totowa, NJ, 1994, Vol. 35, p. 91; b) J.
Eichler, R. A. Houghten, Protein Pep. Lett. 1997, 4, 157; c) E. V. Kudryavtseva, M. V. Sidorova, R. P.
Evstigneeva, Russ. Chem. Rev. 1998, 67, 545.
[24] Y. Shimonishi, H. Zahn, W. Puls, Z. Naturforschg. 1969, 24b, 422.
[25] J. P. Tam, C.-R. Wu, W. Liu, J.-W. Zhang, J. Am. Chem. Soc. 1991, 113, 6657.
[26] R. W. Woody, A. K. Dunker in ꢁCircular Dichroism and the Conformational Analysis of Biomoleculesꢀ,
Ed. G. D. Fasman, Plenum Press, New York, NY, 1996, p. 109.
[27] A. Rauk, J. Am. Chem. Soc. 1984, 106, 6517.
[28] G. Siligardi, M. M. Campbell, W. A. Gibbons, A. F. Drake, Eur. J. Biochem. 1992, 207, 25.
[29] a) I. L. Karle, J. L. Flippen-Anderson, K. Uma, P. Balaram, Proteins: Struct. Funct. Genet. 1990, 7, 62;
b) I. L. Karle, J. L. Flippen-Anderson, K. Uma, P. Balaram, Biopolymers 1993, 33, 827; c) C. Toniolo, E.
Benedetti, Trends Biochem. Sci. 1991, 16, 350.
[30] K. R. Shoemaker, P. S. Kim, E. J. York, J. M. Stewart, R. L. Baldwin, Nature 1987, 326, 563.
[31] D. J. Lockhart, P. S. Kim, Science 1993, 260, 198.
[32] C. Toniolo, A. Polese, F. Formaggio, M. Crisma, J. Kamphuis, J. Am. Chem. Soc. 1996, 118, 2744.
[33] K. A. Bode, J. Applequist, Macromolecules 1997, 30, 2144.
[34] J. Podlech, D. Seebach, Liebigs Ann. Chem. 1995, 1217.
[35] K. Gademann, hitherto unpublished results, ETH-Zürich 1998.
[36] P. Lombardi, Chem. Ind. (London) 1990, 708; S. Moss, ibid. 1994, 122.
[37] M. Bodanszky, A. Bodanszky, ꢁPractice of Peptide Synthesisꢀ, Springer Verlag, Berlin, Heidelberg, 1994,
p. 135.
[38] G. L. Ellman, Arch. Biochem. Biophys. 1959, 82, 70.
Received May 3, 1999