The cyclo-β-tetrapeptide (β-HPhe-β-HThr-β-HLys-β-HTrp): Synthesis, NMR structure in methanol solution, and affinity for human somatostatin receptors

Article abstract of DOI:10.1002/(SICI)1522-2675(20000119)83:1<16::AID-HLCA16>3.0.CO;2-3

The known solid-state structure (Fig. 1, top) of cyclo(β-HAla)₄ was used to model the structure of the title compound 1 as a prospective somatostatin mimic (Fig. 1, bottom). The synthesis started with the N-protected natural amino acids Boc-Phe-OH, Boc-Trp-OH, Boc-Lys(2-Cl-Z)-OH, and Boc-Thr(OBn)-OH, which were homologated to the corresponding β-amino-acid derivatives (Scheme 1) and coupled to the β-tetrapeptide Boc-β-HTrp-β-HPhe-β-HThr(OBn)-β-HLys(2-Cl-Z)-OMe (16); the (N-Me)-β-HThr-(N-Me)-β-HPhe analog 17 was also prepared. C- and N-terminal deprotection and cyclization through the pentafluorophenyl ester gave the insoluble β-tetrapeptide with protected Thr and Lys side chains (18). Solubilization and debenzylation could only be effected in LiCl-containing THF (ca. 10% yield; with ca. 55% recovery). HPLC Purification provided a sample of the title compound 1, the structure of which, as determined by NMR-spectroscopy (Fig. 2, left) was drastically different from the 'theoretical' model (Fig. 1). There is a transannular H-bond dividing the macrocyclic 16-membered ring, thus forming a ten- and a twelve-membered H-bonded ring, the former mimicking, or actually being superimposable on, an α-peptidic so-called β-turn. Still, the four side chains occupy equatorial positions on the ring, as planned, albeit with somewhat different geometry as compared to the 'original'. The cyclo-β-tetrapeptide has micromolar affinities to the human somatostatin receptors (hsst 1-5). Thus, we have demonstrated for the first time that it is possible to mimic a natural peptide hormone with a small β-peptide. Furthermore, we have discovered a simple way to construct the ubiquitous β-turn motif with β-peptides (which are known to be stable to mammalian peptidases).

Full text of DOI:10.1002/(SICI)1522-2675(20000119)83:1<16::AID-HLCA16>3.0.CO;2-3

16
Helvetica Chimica Acta ± Vol. 83 (2000)
The Cyclo-b-Tetrapeptide (b-HPhe-b-HThr-b-HLys-b-HTrp):
Synthesis, NMR Structure in Methanol Solution, and Affinity for Human
Somatostatin Receptors1)
2
3
by Karl Gademann ), Martin Ernst ), and Dieter Seebach*
Laboratorium für Organische Chemie der Eidgenössischen Technischen Hochschule Zürich,
Universitätstrasse 16, ETH-Zentrum, CH-8092 Zürich
and Daniel Hoyer
Nervous System Research, S-386-745, Novartis Pharma AG, CH-4002 Basel
The known solid-state structure (Fig. 1, top) of cyclo(b-HAla)4 was used to model the structure of the title
compound 1 as a prospective somatostatin mimic (Fig. 1, bottom). The synthesis started with the N-protected
natural amino acids Boc-Phe-OH, Boc-Trp-OH, Boc-Lys(2-Cl-Z)-OH, and Boc-Thr(OBn)-OH, which were
homologated to the corresponding b-amino-acid derivatives (Scheme 1) and coupled to the b-tetrapeptide Boc-
b-HTrp-b-HPhe-b-HThr(OBn)-b-HLys(2-Cl-Z)-OMe (16); the (N-Me)-b-HThr-(N-Me)-b-HPhe analog 17
was also prepared. C- and N-terminal deprotection and cyclization through the pentafluorophenyl ester gave the
insoluble b-tetrapeptide with protected Thr and Lys side chains (18). Solubilization and debenzylation could
only be effected in LiCl-containing THF (ca. 10% yield; with ca. 55% recovery). HPLC Purification provided a
sample of the title compound 1, the structure of which, as determined by NMR-spectroscopy (Fig. 2, left) was
drastically different from the ꢀtheoreticalꢁ model (Fig. 1). There is a transannular H-bond dividing the
macrocyclic 16-membered ring, thus forming a ten- and a twelve-membered H-bonded ring, the former
mimicking, or actually being superimposable on, an a-peptidic so-called b-turn. Still, the four side chains occupy
equatorial positions on the ring, as planned, albeit with somewhat different geometry as compared to the
ꢀoriginalꢁ. The cyclo-b-tetrapeptide has micromolar affinities to the human somatostatin receptors (hsst 1 ± 5).
Thus, we have demonstrated for the first time that it is possible to mimic a natural peptide hormone with a small
b-peptide. Furthermore, we have discovered a simple way to construct the ubiquitous b-turn motif with b-
peptides (which are known to be stable to mammalian peptidases).
Introduction. ± Oligomers of b-amino acids (b-peptides) have received much
attention in the realm of artificial compounds that fold into compact and well-defined
secondary structures. This is due to their ability to display a large variety of different
secondary structures with just one type of building block (see reviews: [2 ± 4]). After
the initial discovery of the 314 helix without [5] and with conformational restraints [6],
two other types of helices have been disclosed [7 ± 9]. Two different turns [7][8],
parallel [5] and antiparallel [10 ± 12] sheets, as well as meander-type secondary
structures [13][14] have been found in solution and in the solid state.
Cyclic b-peptides, however, have received less attention. Several cyclic b-peptides
have been prepared [5][15 ± 18], but structure determination was hampered by the
poor solubility of these compounds. It was powder X-ray diffraction that led to the
1
)
)
)
Partially published in a preliminary communication [1].
Part of the projected dissertation of K. G., ETH-Zürich.
Part of the Diplomarbeit (master thesis) of M. E., ETH-Zürich 1998.
2
3

Helvetica Chimica Acta ± Vol. 83 (2000)
17
assignment of the solid-state conformation of three cyclo-b-tetrapeptides [19]4) (see the
structure of the (all-S)-cyclo(b-HAla)4 (A) in Fig. 1, top). They are characterized by a
tubular stacking with an infinite network of pleated-sheet-type H-bonds (so called
peptide nanotubes). This nanotube formation has been proposed to promote single-
channel ion transport through phospholipid bilayers [18]. Only very recently, the first
NMR structure in water of the cyclo-b-tripeptide ((b-HGlu)3) with carboxylate side chains
has been reported, being characterized by an all-up arrangement of the CˆO groups [17].
b-Peptides are resistant to degradation by mammalian proteolytic enzymes [20][21],
and their building blocks, the b-amino acids, have been shown to be non-mutagenic in
Fig. 1. Top: Powder X-ray structure of (all-S)-cyclo(b-HAla)4. Bottom: Top view of the superposition of the
solution structure of octreotide [41] (C-atoms in gray) and a model of 1 derived from the powder X-ray structure
of cyclo-b-tetrapeptide A (C-atoms in green). The overlap of the side chains indicates good mimicry of the
somatostatin pharmacophore by 1.
4
)
The rings of the three diastereoisomers of (S,S,S,S), and (R,S,R,S) and (R,R,S,S) configurations have
different shapes and different relative geometries of the equatorial side chains, with all four CˆO groups
being involved in intermolecular H-bonding.

18
Helvetica Chimica Acta ± Vol. 83 (2000)
the Ames test [20]. b3-Amino acids (two synthetic steps from the corresponding amino
acids [22]), as well as the b-peptides (obtainable by solution- or solid-phase coupling
methods [23]) are readily available.
All these features make b-peptides promising candidates for the use as peptidomi-
metics in medicinal chemistry 5). However, before our preliminary communication [1],
nothing had been known in the literature about the potential of b-peptides as
biologically and pharmacologically active compounds. In this paper, we describe in full
detail the design, synthesis, and biological evaluation of a b-peptide mimicking a
natural a-peptide. Furthermore, we disclose the first solution structure of a cyclo-b-
tetrapeptide, which turns out to be different from the known solid- state structures.
Results and Discussion. ± Target and Design. There are few a-peptide hormones
that have been studied as intensively as the tetradecapeptide somatostatin (SRIF:
Somatotropin Release Inhibiting Factor). After its isolation in 1973 from 500,000
hypothalami [24], it has been characterized as a potent inhibitor of growth hormone
(GH) from the anterior pituitary [24]. In addition, it also inhibits the pancreatic
secretion of glucagon and insulin [25], as well as the secretion of gastrin from the gut
[26]. Furthermore, SRIF acts as a neurotransmitter in the central nervous system and
peripheral tissue, where it modulates several processes, such as smooth muscle motor
activity [27] and the release of other neurotransmitters [28][29]. Five different soma-
tostatin receptors (sst 1±5) have been identified and characterized as G-protein-coupled
transmembrane receptors [30]. So far, the physiological function of only two, sst2 (media-
tion of the release of GH) and sst5 (inhibition of the release of insulin), are known.
All these physiological functions render SRIF very important for the treatment of
several diseases. Many analogues have been synthesized and some are in clinical use,
e.g., octreotide (Sandostatinꢂ) by Novartis [31][32]. It is applied in the treatment of
acromegaly and of certain gastro-entero-pancreatic tumors. Its elimination half-life
from the blood serum (90 min) is still rather short. It is, therefore, of great interest to
find non-peptide analogues that mimick SRIF or octreotide6). For these reasons, we
chose somatostatin as the target to demonstrate the potential of b-peptides as mimetics
of natural a-peptide hormones.
The structure of octreotide has been studied both in the solid state [37] and in
solution [38 ± 41] (Fig. 1). All structures are characterized by a type II' b-turn spanning
Trp4 and Lys5. Furthermore, it is known that Phe3 and Thr6 are important for
biological activity7). Computer-aided modelling revealed similarities between the
powder X-ray structure of cyclo-b-tetrapeptides (Fig. 1, top) and type II' b-turns of a-
peptides. Thus, we have attached the side chains necessary for biological activity to the
b-peptide backbone of the solid-state structure that led to the cyclo-b-tetrapeptide 1,
the side chains of which in the ꢀtheoreticalꢁ model overlapped well with the solution
structure of octreotide (Fig. 1, bottom). In addition, we intended to synthesize an N-
5
)
There are many b-amino acids that are part of natural products, especially of marine origin. Furthermore,
they have been introduced in pharmaceutically active compounds for a long time to increase stability
against proteolytic degradation or to induce certain structural elements. For references, see [2].
Recently, several groups reported non-peptidic compounds that mimick SRIF [33 ± 36].
Most a-peptide SRIF analogues with biological activity contain the amino-acid sequence Phe-Trp-Lys-Thr
[32].
6
7
)
)

Helvetica Chimica Acta ± Vol. 83 (2000)
19
NH2
O
O
H
HN
S
N
N
H
S
O
O
NH
NH2
O
HN
O
H
H
O
N
N
H
NH
H
HO
HO
CH2OH
Octreotide, Sandostatin®
H
N
H–Ala–Gly–Cys–Lys–Asn–Phe–Phe–Trp
NH
O
O
O
HN
O
HO–Cys–Ser–Thr–Phe–Thr–Lys
Somatostatin, SRIF
H
HN
H
N
NH2
OH
1
methylated derivative in order to circumvent possible solubility problems, often
encountered in peptide chemistry (especially for cyclic b-peptides [15][16]).
Synthesis. We chose the Boc/benzyl (Bn) protecting-group strategy for the synthesis
of 1 in solution. The Boc-protected b3-amino acids 4±9 were prepared by the Arndt-Eistert
method [22][42], via the corresponding diazo ketones 3a± d (Scheme 1). To our surprise,
and in contrast to earlier observations [22], Trp derivatives 3c and 6 (containing an
unprotected indole heterocycle) were easily isolated in good yields. In addition, we
prepared the N-methylated building blocks 10 and 11, following a protocol developed
for a-amino acids [43][44] that has been recently adapted for b-amino acids [15][45].
Boc Deprotection of 4 and subsequent conventional peptide coupling to 6 with N-
[3-(dimethylamino)propyl]-N'-ethylcarbodiimide hydrochloride (EDC)/1-hydroxy-
1H-benzotriazole (HOBt) furnished the b-dipeptide 12 (85%). Similarly, 8 was
deprotected and coupled with 9 and 11 to give the b-dipeptide 13 and the N-methylated
derivative 14. Removal of the N-terminal protecting group of 14 and coupling with 10
proceeded smoothly in 79% yield to give the b-tripeptide 15 8). Fragment coupling of
C-terminally deprotected 12 with the N-terminally deprotected 13 gave b-tetrapeptide
8
)
The N-methylation appeared to be no obstacle to peptide-bond formation.

20
Helvetica Chimica Acta ± Vol. 83 (2000)
Scheme 1. Preparation of b-Amino-Acid Building Blocks 4 ± 11.
R1
R1
H
R1
O
a)
b)
Boc
OH
Boc
Boc
OR2
N
N
N2
N
H
H
H
O
O
2
3
4 R1 = Bn, R2 = Me
5 R1 = Bn, R2 = H
6 R1 = (1H-indol-3-yl)methyl, R2 = H
7 R1 = (CH2)4NH(2-Cl-Z), R2 = H
8 R1 = (CH2)4NH(2-Cl-Z), R2 = Me
9 R1 = (R)-1-(Benzyloxy)ethyl, R2 = H
2, 3
R1
a
b
c
d
Bn
(CH2)4NH(2-Cl-Z)
(1H-Indol-3-yl)methyl
(R)-1-(Benzyloxy)ethyl
R1
O
R1
O
c)
Boc
Boc
N
OH
N
OH
H
Me
5 R1 = Bn
9 R1 = (R)-1-(Benzyloxy)ethyl
10 R1 = Bn
11 R1 = (R)-1-(Benzyloxy)ethyl
a) ClCO2Et, Et3N, THF, then CH2N2, Et2O; 3a: 86%, 3b: 70%, 3c: 63%, 3d: 55%. b) CF3CO2Ag (cat.), Et3N,
THF, R2OH; 4: 98%, 5: 44%, 6: 84%, 7: 91%, 8: 81%, 9: 88%. c) NaH, MeI, THF; 10: 92%, 11: 83%.
16 in 93% yield. However, the analogous coupling of 15 to the doubly N-methylated b-
tetrapeptide 17 proceeded with a yield of only 66%. The N-Me groups increase the
solubility in organic solvents tremendously, making the isolation and purification of 17
much more facile than that of 16.
b-Tetrapeptide 16 was deprotected at the C-terminus and allowed to react with penta-
fluorophenol and EDC, the resulting active ester was, after Boc deprotection, cyclized
(Hünigꢁs base in MeCN at 708) in dilute solution to furnish the fully protected cyclo-b-
tetrapeptide 18 (38%; see Scheme 2). This compound is essentially insoluble in all
solvents commonly used in peptide chemistry. Being aware of our previous work on the
solubilization by Li-salts of peptides in organic solvents [17][46], we dissolved 18 in
THF with 6 equiv. of LiCl and carried out the hydrogenolysis (Pd on charcoal/H2). After
purification by RP-HPLC, the deprotected cyclo-b-tetrapeptide 1 was obtained in only
poor yield (9%), however, more than 50% of the starting material were recovered.
In contrast to the non-N-methylated b-peptide, the much more soluble N-
methylated b-peptide 17 could not be cyclized via deprotection and pentafluorophen-
yl-ester formation. Also, other cyclization methods common in peptide chemistry, such
as the use of diphenylphosphoryl azide (DPPA) in DMF 9) or the application of HATU/
HOAt10), were not successful. This observation is in agreement with the problems
9
)
)
See, for example, [47].
10
HATU: O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, HOAt: 1-hy-
droxy-7-azabenzotriazole; see, for example, [18].

Helvetica Chimica Acta ± Vol. 83 (2000)
21
NH–(2-Cl-Z)
O
HN
Me
OBn
Ph
O
O
O
Boc
Boc
N
R
N
OMe
N
N
OMe
H
H
H
H
12 (85%)
13 R = H (96%)
14 R = Me (89%)
NH–(2-Cl-Z)
O
NH–(2-Cl-Z)
O
HN
Ph
Ph Me
O
OBn
Me
OBn
O
O
O
O
Boc
Boc
N
N
N
R
N
OMe
N
N
N
OMe
H
H
H
H
H
R
Me
Me
16 R = H (93%)
17 R = Me (66%)
15 (79%)
Scheme 2. Synthesis of Cyclo-b-tetrapeptide 1.
HN
O
HN
H
H
N
N
a)
b)
NH
O
O
O
O
NH
O
O
O
16
H
H
NH
H
N
NH
H
N
HO
BnO
NH2
NH-(2-Cl-Z)
18
1
a) i) 50 equiv. NaOH, H2O, CF3CH2OH, ii) EDC, C6F5OH, DMF, iii) CF3CO2H, HSCH2CH2SH, CH2Cl2, iv)
EtN(i-Pr)2, MeCN, 708, syringe pump (38% from 16). b) Ca. 6 equiv. LiCl, THF, Pd/C, H2; 9% (53% starting
material recovered).
encountered during attempted synthesis of cyclic N-methylated b3-HAla peptides [15].
Steric effects do not play a role, since the nucleophilic amino group of b3-HTrp is not
methylated. A possible reason might be the (helical) preorganization of non-N-
methylated b-peptides that facilitates cyclization. Of course, substituents on the amide

22
Helvetica Chimica Acta ± Vol. 83 (2000)
N-atom have an impact on the conformation of any peptide11), and, thus, influence its
reactivity.
Biological Investigations. The affinity of the cyclo-b-tetrapeptide 1 for the five
different human somatostatin receptors (hsst 1 ± 5, expressed in CHO or CCL-39 cell
lines)12) was measured by radioligand-binding assays (Table 1). Displacement experi-
ments for the specific binding of [125I]LTT-SRIF28 to these receptors were carried out as
previously described, and the affinities are reported as pKd Æ SEM values. The data in
Table 1 clearly show that 1 has affinity, although the concentrations are in the
micromolar and not in the nanomolar range as for octreotide13).
Table 1. Comparison of the Affinitiy Values of 1 and Octreotide by Radioligand Binding Assays (displacement
experiments of the specific binding of [125I]-LTT-SRIF28) with the Five Different Human Somatostatin Receptors
(hsst 1 ± 5) expressed in CCL-39 (Chinese Hamster Lung Fibroblast) or CHO (Chinese Hamster Ovary) Cell
Lines. The values are reported as pKd Æ SEM of 4 independent determinations, the values for octreotide are
taken from [69]. The affinities of 1 are in the micromolar range, at least an order of magnitude lower than those
of octreotide.
Receptor
b-Peptide 1
Octreotide
hsst1 (CCL-39)
hsst2 (CCL-39)
hsst3 (CHO)
hsst4 (CCL-39)
hsst5 (CCL-39)
4.85 Æ 0.04
4.44 Æ 0.09
5.48 Æ 0.01
5.00 Æ 0.05
3.73 Æ 0.15
6.65
9.19
7.88
6.40
7.17
Structure Determination by NMR Spectroscopy. Having the first soluble cyclo-b-
tetrapeptide 1 in hand, we thought that an NMR structure determination might provide
insights into its hitherto unknown conformation and, thus, explain the reduced affinity
1
as compared to octreotide. All resonances in the H-NMR spectrum obtained at
500 MHz in CD3OH at 298 K were assigned by DQF-COSY and TOCSY experiments
(Table 2), and it was possible to extract the J values of all backbone H-atoms directly
from the 1D-NMR spectrum.
From the spectrum, two important facts became immediately evident: a)
Investigations on octreotide demonstrated that the aromaticity of the indole ring leads
to a shift of the resonances of the lysine side-chain protons to lower frequencies (higher
fields), since both side-chains are in close proximity [38 ± 41]. This phenomenon was
not observed in the 1H-NMR spectrum of 1, indicating a lack of closeness of the Trp and
Lys side-chains. b) The J(NH, H C(b)) values show that the solution conformation
differs drastically from that of the ꢀtheoreticalꢁ model (see Fig. 1). According to this
model, four equal J(NH, H C(b)) values would have been expected, however, two
large (b3-HPhe, b3-HThr), one medium (b3-HTrp), and one small (b3-HLys) were
measured. In addition, the backbone conformation around the central C(a) C(b)
bond for the lysine residue must be different from those of the other three b-amino
acids, as can be judged from the J(H C(a),H C(b)) values.
11
)
For ꢀnormalꢁ peptides, a reversed situation is often encountered: while regular a-peptides cyclize in poor
yields, their N-methylated counterparts give higher cyclization yields (see, for example, [48]).
For abbreviations: see the legend of Table 1 and Exper. Part.
The molecular mass of 1 is only 60% of octreotide: 1 (618.77), octreotide (1019.24), and somatostatin
(1637.88)
12
13
)
)

Helvetica Chimica Acta ± Vol. 83 (2000)
23
Table 2. Assignment of 1H-NMR Resonances (500 MHz, CD3OH, 298 K) by a Combination of COSY and
TOCSY Experiments. The J values were extracted from the 1D spectrum.
Thr
7.22
4.01
2.42
2.18
3.73
±
1.10
±
±
±
±
Lys
Phe
7.66
4.36
2.35
2.24
2.71
2.65
±
±
±
±
±
Trp
8.35
4.36
2.39
2.31
2.87
3.07
±
±
±
±
±
NH
H
H
8.22
3.42
2.49
2.14
1.65
1.53
±
1.51
1.51
1.62
1.62
2.95
2.89
5.2
C(b)
C(a)
H' C(a)
C(g)
H
H' C(g)
Me
H
C(d)
H' C(d)
C(e)
H' C(e)
C(f)
H
H
±
±
9.5
12.9
3.3
±
±
8.6
11.2
2.3
±
±
7.0
11.7
2.3
H' C(f)
J(NH, H C(b))
J(H C(b), H C(a))
J(H C(b), H C(a))
5.7
3.3
Additional information was obtained from a ROESY spectrum. The NOEs were
extracted and classified into three intensity categories based on their relative crosspeak
volumes (Table 3). These NOEs and all 3J values for the backbone protons have been
used as restraints in molecular-dynamics calculations by the QUANTA package. The
resulting conformation, in agreement with all experimental values, is shown in Fig. 2.
The conformation of the cyclo-b-tetrapeptide 1 is characterized by an intra-
molecular H-bond that divides the 16-atom backbone into a ten-membered and a
twelve-membered H-bonded ring. The ten-membered ring mimicks a natural a-peptide
b-turn and is very similar to the ring observed in the 12/10/12-helix [7][8], in a hairpin
[12], and in a b2,2-peptide [13]. However, there is no reason obvious to us why the H-
bond should form from NH of b3-HTrp to the CˆO of b3-HPhe. In addition, the other
Table 3. NOEs Extracted from the 150-ms ROESY Spectrum (500 MHz, CD3OH, 298 K). Intraresidual NOEs
from H C(b) to NH were not classified due to COSY artefacts.
Atom
Residue
Atom 2
Residue
Intensity
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
b3-HTrp
b3-HTrp
b3-HTrp
b3-HTrp
b3-HTrp
b3-HLys
b3-HLys
b3-HLys
b3-HLys
b3-HPhe
b3-HPhe
b3-HPhe
b3-HThr
b3-HThr
b3-HThr
H
H
C(b)
C(a)
b3-HLys
b3-HLys
b3-HLys
b3-HTrp
b3-HTrp
b3-HLys
b3-HThr
b3-HThr
b3-HThr
b3-HPhe
b3-HTrp
b3-HTrp
b3-HThr
b3-HPhe
b3-HPhe
s
s
H' C(a)
C(a)
H' C(a)
w
s
m
m
s
s
m
s
s
s
m
s
s
H
H
H
H
C(a)
C(b)
C(a)
H' C(a)
H' C(a)
H
H' C(a)
H' C(a)
H
C(a)
C(a)
H' C(a)

24
Helvetica Chimica Acta ± Vol. 83 (2000)

Helvetica Chimica Acta ± Vol. 83 (2000)
25
two amide groups are pointing up and down, thus further reducing the net dipole
moment of the molecule. All side chains occupy lateral positions on the macrocyclic
ring, which results in a flat ring shape.
The superposition with octreotide (Fig. 2, right) shows that the overlap of the side
chains is reduced as compared to the model (Fig. 1). This might explain the weaker
binding of compound 1, compared to octreotide, although the receptor-bound conforma-
tion of both compounds might be different [49]. The ꢀfitꢁ of the side chains might have
been increased if the intramolecular H-bond had been formed between NH of b3-HLys
and CˆO of b3-HTrp, mimicking a b-turn with the side chains of Lys and Trp.
This is the first example of a solution structure of a cyclo-b-tetrapeptide. Of course,
we should not conclude that, in general, all cyclo-b-tetrapeptides exhibit such H-
bonding patterns. However, if one compares this solution structure to the solid state
conformation of cyclo(b3-HAla)4 (A; Fig. 1, top), there is only the fundamental
difference of the H-bonding pattern, while the overall shape of the molecules is flat,
and side-chain orientation is comparable. Cyclic a-peptides (starting with pentapep-
tides, which have a comparable ring size) often exhibit intramolecular H-bonds14).
CD Spectroscopy. CD-Spectroscopy is a valuable tool for analysis of secondary
structure in a straightforward manner, once a correlation between the observed pattern
and the structure in solution is established15). Although CD spectroscopy never proves
structure, it is complementary to NMR spectroscopy. The CD spectrum of 1 in MeOH
(Fig. 3) displays a single maximum at ca. 200 nm, and a zero crossing at 193 nm. This
pattern, which is very similar to that observed for the 12/10/12 helix [7][8] and also for
the b-peptide hairpin [12][53], has been assigned to ten- and/or twelve-membered H-
bonded rings. This spectrum thus further corroborates the structure derived from NMR
spectroscopy.
However, this observed CD pattern differs from that observed for A (in
trifluoroethanol), which displays a minimum at 215 nm, a zero crossing at 207 nm
and a maximum at 196 nm [15]. This suggests that different cyclo-b-tetrapeptides fold
into different conformations in solution.
Conclusion. ± We have shown for the first time that it is possible to mimic a natural
a-peptide hormone with a small b-peptide. This was not at all obvious, since in b-
peptides not only the amide bonds but also the side chains are shifted compared to their
natural analogs. While some proteins (e.g., proteases) do not recognize b-peptides as
substrates, the somatostatin receptors do! It is now in the hands of medicinal chemists
to exploit these specific properties of b-peptides, and to explore their scope and
limitations as peptidomimetics. This search is facilitated by the ease of the synthesis of
the building blocks as well as of b-peptides. In addition, there are various possibilities
for optimization of receptor binding by simple variation of the substitution pattern of
the side chains on the b-amino-acid backbone [54]. All these features make b-peptides
promising candidates for peptidomimetics.
We thank B. Brandenberg for recording NMR spectra and Prof. Dr. B. Jaun for valuable discussions. We
thank A. K. Beck for reading the experimental part, and G. Lelais and S. Odermatt for carrying out some of the
experiments described herein, as part of the requirements for the advanced laboratory course in organic
14
)
)
For an early review by Kessler, see [49].
For work on b-peptides, see [2][8][50 ± 53].
15

26
Helvetica Chimica Acta ± Vol. 83 (2000)
Fig. 3. CD Spectrum of b-peptide 1 in MeOH (c ˆ 0.323 mm, molar ellipticity in 10 deg cm2 mol 1).
chemistry. Generous supply of TFA by Solvay and of Phe and Trp by Degussa are greatly appreciated, as is the
ongoing financial support from Novartis Pharma and Agro, Basel. M. E. thanks the DAAD for a grant and the
Studienstiftung des Deutschen Volkes for generous support.
Experimental Part
1. General. Abbreviations: Boc: (tert-butoxy)carbonyl, EDC: N-[3-(Dimethylamino)propyl]-N'-ethyl-
carbodiimide hydrochloride, FC: flash chromatography, GP: General Procedure, HOBt: 1-hydroxy-1H-
benzotriazole, h.v.: high vacuum, 0.01 ± 0.1 Torr, b-HXxx: b-homoamino acid, NMM: N-methylmorpholine,
SEM: standard error, TFA: trifluoroacetic acid, Z: benzyloxycarbonyl, 2-Cl-Z: (2-chlorobenzyloxy)carbonyl.
THF was freshly distilled over K under Ar before use. Et3N was distilled over CaH2. Solvents for
chromatography and workup were distilled over Sikkon (Fluka). Boc-Amino acids were purchased from Senn
or were prepared according to the literature [55]. All other reagents were used as received from Fluka. All
reactions were carried out under Ar. All given temp. refer to the bath temp. TLC: Merck silica gel 60 F254 plates;
detection with UVor dipping into a soln. of anisaldehyde (9.2 ml), AcOH (3.75 ml), conc. H2SO4 (12.5 ml), and
EtOH (338 ml), followed by heating. FC: Fluka silica gel 60 (40 ± 63 mm); at ca. 0.3 bar. Anal. HPLC: Knauer
HPLC system (pump type 64, EuroChrom 2000 integration package, degaser, UV detector (variable-
wavelength monitor)), Macherey-Nagel C18 column (Nucleosil 100-7 C18 (250 Â 4 mm)), solvent A: 1% TFA in
H2O, solvent B: MeCN. 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. CD: Jobin-
Yvon-Mark III, 1-mm cell length. 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),

Helvetica Chimica Acta ± Vol. 83 (2000)
27
Varian Gemini 300 (1H: 300 MHz, 13C: 75 MHz), or Gemini 200 (1H: 200 MHz, 13C: 50 MHz); chemical shifts
(d) in ppm downfield from SiMe4 (ˆ0 ppm); J values in Hz. Hitachi Perkin-Elmer RHU-6M (FAB, in a 3-
nitrobenzyl-alcohol matrix) spectrometer; in m/z (% of basis peak). Elemental analyses were performed by the
Microanalytical Laboratory of the Laboratorium für Organische Chemie, ETH-Zürich.
2. Preparation of Diazo Ketones. General Procedure 1 (GP 1). Caution: The generation and handling of
CH2N2 requires special precautions [56][57]. The N-Boc-protected a-amino acid was dissolved in THF (0.2m)
under Ar. The soln. was cooled to 158, and Et3N (1 equiv.) and ClC(O)OEt (1 equiv.) were added. After
15 min, the suspension was allowed to warm to 08, and a soln. of CH2N2 in Et2O was added until a strong yellow
color persisted. The suspension was allowed to warm to r.t. and stirred for 3 h. The excess of CH2N2 was
destroyed by the addition of few drops of AcOH. The mixture was diluted with H2O and the bulk of THF
evaporated. After addition of Et2O, the mixture was washed with sat. NaHCO3, NH4Cl, and NaCl solns. The org.
layer was dried (MgSO4) and evaporated. FC or recrystallization gave the pure diazo ketones.
3. Homologation of the Diazo Ketones to the Corresponding b-Amino Acids. General Procedure 2 (GP 2).
The diazo ketone was dissolved in THF/H2O 9 :1 (v/v) (0.25m). Under the exclusion of light, the soln. was
cooled to ca. 258, and a soln. of CF3CO2Ag (0.11 equiv.) in Et3N (2.3 equiv.) was added. The mixture was
allowed to warm up to r.t. and stirred for 3 h in the dark. THF was evaporated, and the residue was dissolved in
Et2O. The b-amino acid was extracted with sat. NaHCO3 soln., and the H2O-layer was carefully acidified at 08 to
pH 2 ± 3 and extracted with Et2O. The combined org. layers were dried (MgSO4) and evaporated. The resulting
b-amino acids were used without further purification.
4. Homologation of the Diazo Ketones to the Corresponding b-Amino Acid Methyl Esters. General
Procedure 3 (GP 3). The diazo ketone was dissolved in MeOH (0.25m). Under the exclusion of light, the soln.
was cooled to ca. 258, and a soln. of CF3CO2Ag (0.11 equiv.) in Et3N (2.3 equiv.) was added. The mixture was
allowed to warm up to r.t. and stirred for 3 h in the dark. The solvent was evaporated, and the residue was
dissolved in Et2O. The org. layer was washed with sat. NaHCO3, NH4Cl, and NaCl solns., dried (MgSO4), and
evaporated.
5. N-Methylation of Boc-b-Amino Acids. General Procedure 4 (GP 4). The b-amino acid was dissolved in
THF (0.1m), MeI (8 equiv.) was added, the soln. was cooled to 08, and NaH (3 equiv.) was added in portions. The
mixture was allowed to warm to r.t. and stirred for 22 h, then cooled to 108 and excess NaH was hydrolyzed
with ice. The solvent was evaporated, and the residue was dissolved in H2O. The aq. phase was washed with Et2O
(the pH was adjusted to ca. 2 with sat. aq. KHSO4 soln.) and extracted with Et2O. The org. phase was washed
with 0.5m HCl soln. and dried (MgSO4). The solvent was removed under reduced pressure to yield the Boc-
protected N-methyl-b-amino acid which was used without further purification.
6. Boc Deprotection with TFA. General Procedure 5 (GP 5). The Boc-protected compound was dissolved in
CH2Cl2 (0.25m) and cooled to 08. The same amount (v/v) of TFA was added, and the mixture was stirred for 1 h
at 08 and for 1 h at r.t. The solvent was evaporated, the residue was twice taken up in CHCl3 and evaporated. The
resulting trifluoroacetate was dried under h.v. and used without further purification.
7. Peptide Coupling with EDC and HOBt. General Procedure 6 (GP 6). The trifluoroacetate of the amino
fragment was dissolved in CHCl3 (0.5m) and cooled to 08. Et3N (4 equiv.), HOBt (1.2 equiv.), a soln. of the Boc-
protected fragment (1 equiv.) in CHCl3 (0.25m), and EDC (1.2 equiv.) were added, and the resulting mixture
was stirred for 30 min at 08. The mixture was then allowed to warm up and stirred for additional 12 h (TLC
control). The mixture was diluted with CHCl3 and was washed with 1m HCl, sat. NaHCO3, and sat. NaCl solns.
The org. layer was dried (MgSO4) and evaporated, and the resulting peptides were either purified or used
without further purification.
8. Synthesis of Cyclo-b-tetrapeptide 1. tert-Butyl N-[(1S)-1-benzyl-3-diazo-2-oxopropyl]carbamate (3a)
[22]: 15.9 g (60 mmol) of Boc-Phe-OH (2a), 5.75 ml (1 equiv.) of ClC(O)OEt, and 8.4 ml (1 equiv.) of Et3N
were transformed according to GP 1. Recrystallization (AcOEt/pentane) gave 3a (15.0 g, 86%). Yellow solid. Rf
(Et2O/pentane 1 :1) 0.4. M.p. 95 ± 968 ([22]: M.p. 69 ± 708). [a]D
ˆ
11.8 (c ˆ 1.00, CHCl3). 1H-NMR (300 MHz,
CDCl3): 1.41 (s, t-Bu); 3.02 (d, J ˆ 6.8, CH2); 4.42 (br., CHN); 5.11 (br., NHBoc); 5.22 (br., CHN2); 7.18 ± 7.33
(m, 5 arom. H).
2-Chlorobenzyl N-((5S)-5-{[(tert-Butoxy)carbonyl]amino}-7-diazo-6-oxoheptyl)carbamate (3b): 25
g
(60.3 mmol) of Boc-Lys(Ne-2-Cl-Z)-OH (2b), 7.9 ml (1 equiv.) of ClC(O)OiBu, and 8.0 ml (1 equiv.) of Et3N
were transformed according to GP 1. FC (Et2O/pentane 3 :1) gave 3b (18.5 g, 70%). Yellow solid. Rf (AcOEt/
pentane 4 :9) 0.34. M.p. 72 ± 748. [a]D
ˆ
18.0 (c ˆ 1.03, CHCl3). IR (CHCl3): 3446w, 3008w, 2960w, 2940w,
2110s, 1800w, 1711s, 1642m, 1500m, 1446w, 1368m, 1248w, 1163w, 1042w, 859w, 630w. 1H-NMR (300 MHz,
CDCl3): 1.36 ± 1.83 (m, 3 CH2, t-Bu); 3.17 ± 3.23 (m, NCH2CH2); 4.14 (br., CHN); 4.91 (br., NHBoc); 5.21 (m,
NH, PhCH2); 5.45 (s, CHN2); 7.23 ± 7.29 (m, 2 arom. H); 7.35 ± 7.43 (m, 2 arom. H). 13C-NMR (75 MHz, CDCl3):

28
Helvetica Chimica Acta ± Vol. 83 (2000)
22.33; 28.36; 29.55; 32.10; 40.56; 53.97; 57.34; 64.05; 80.20; 127.11; 129.51; 129.61; 129.77; 130.01; 133.83; 134.57;
‡
‡
‡
155.86; 193.48. FAB-MS: 877.2 (1.7, [2M ‡ H] ), 439.1 (23.1, [M ‡ H] ), 339.0 (24.7, [M Boc ‡ 2 H] ), 311.1
‡
(27.8 [M N2 Boc ‡ 2 H] ). Anal. calc. for C20H27N4O5Cl (438.91): C 54.73, H 6.20, N 12.76; found: C 54.65,
H 6.17, N 12.50.
tert-Butyl N-{(1S)-3-Diazo-1-[(1H-3-indolyl)methyl]-2-oxopropyl}carbamate (3c): 20 g (65.7 mmol) of
Boc-Trp-OH (2c), 8.6 ml (1 equiv.) of ClC(O)OiBu, and 9.0 ml (1 equiv.) of Et3N were transformed according
to GP 1. FC (Et2O/pentane 2 :1) gave 3c (13.6 g, 63%). Yellow solid. Rf (Et2O/pentane 1:1) 0.43. M.p. 53 ± 548.
[a]D ˆ ‡6.0 (c ˆ 1.00, CHCl3). IR (CDCl3): 3689w, 3477m, 3428w, 3008w, 2980w, 2110s, 1706s, 1636m, 1495m,
1456w, 1368s, 1165m, 1092w, 1044w, 1011w, 857w. 1H-NMR (300 MHz, CDCl3): 1.43 (s, t-Bu); 3.19 ± 3.21 (m,
CH2 C(1)); 4.52 (br., CHN); 5.19 (br., NHBoc, CHN2); 7.02 ± 7.37 (m, 4 arom. H); 7.62 (d, J ˆ 7.5, 1 arom. H);
8.28 (br, NH of indole). 13C-NMR (75 MHz, CDCl3): 28.33; 54.32; 57.92; 80.07; 110.35; 111.26; 118.83; 119.77;
‡
‡
122.29; 123.10; 127.52; 136.20; 155.32; 194.29. FAB-MS: 329.5 (60.7, [M ‡ H] ), 328.5 (37.7, M ), 154.2 (100.0).
Anal. calc. for C17H20N4O3 (328.37): C 62.18, H 6.14, N 17.06; found: C 61.97, H 6.37, N 16.93.
tert-Butyl N-{(1S)-3-Diazo-1-[(1R)-1-(benzyloxy)ethyl]-2-oxopropyl}carbamate (3d): 30.4 g (98 mmol) of
Boc-Thr(OBn)-OH (2d), 10.3 ml (98 mmol) of ClC(O)OEt, and 10.5 ml (93.5 mmol) of Et3N were transformed
according to GP 1. Recrystallization (AcOEt/pentane) gave 3d (17.9 g, 55%). Rf (AcOEt/pentane 1:2) 0.82.
M.p. 104 ± 1058. [a]D
ˆ
39.0 (c ˆ 0.80, CHCl3). IR (CHCl3): 3435w, 3008w, 2981w, 2111s, 1710m, 1635m, 1494m,
1454w, 1368m, 1162m, 1072w, 867w, 624w. 1H-NMR (300 MHz, CDCl3): 1.21 (d, J ˆ 6.2, Me); 1.46 (s, t-Bu);
4.15 ± 4.23 (m, CHN, CHOBn); nA ˆ 4.47, nB ˆ 4.57 (AB, JAB ˆ 11.5, OCH2Ph); 5.44 (d, J ˆ 7.8, NHBoc); 5.58 (s,
CHN2); 7.28 ± 7.38 (m, 5 arom. H). 13C-NMR (75 MHz, CDCl3): 16.25; 28.33; 54.22; 62.03; 71.50; 74.55; 80.15;
‡
‡
127.82; 128.41; 137.98; 155.81; 193.26. FAB-MS: 334.2 (55.3, [M ‡ H] ), 90.8 (100.0, [C7H7] ). Anal. calc. for
C17H23N3O4 (333.39): C 61.25, H 6.95, N 12.60; found: C 61.27, H 7.02, N 12.55.
Methyl (3S)-3-{[(tert-Butoxy)carbonyl]amino}-4-phenylbutanoate (Boc-b3-HPhe-OMe, 4) [22]: 3.0 g
(10.4 mmol) of 3a, 0.25 g (1.14 mmol) of CF3CO2Ag, and 3.4 ml (24 mmol) of Et3N were reacted according
to GP 3. FC (Et2O/pentane 3 :5) gave 4 (3.0 g, 98%). Colorless wax. Rf (AcOEt/pentane 1:1) 0.77. [a]D
ˆ
9.7
(c ˆ 1.00, CHCl3) ([22]: [a]D
ˆ
10.0 (c ˆ 0.99, CHCl3)).
(3S)-3-{[(tert-Butoxy)carbonyl]amino}-4-phenylbutanoic Acid (Boc-b3-HPhe-OH, 5) [58]: 20.9 g
(72 mmol) of 3a were reacted according to GP 2. Recrystallization (AcOEt/pentane) gave 5 (8.9 g, 44%).
Colorless solid. M.p. 108 ± 1098 ([58]: 100 ± 1058).
(3S)-3-{[(tert-Butoxy)carbonyl]amino}-4-(1H-indol-3-yl)butanoic Acid (Boc-b3-HTrp-OH, 6): 9.0 g
(27 mmol) of 3c, 0.65 g (2.9 mmol) of CF3CO2Ag, and 8.8 ml (63 mmol) of Et3N were reacted according to
GP 2. Drying under h.v. gave 6 (7.2 g, 84%). Colorless foam. M.p. 144 ± 1458. [a]D
ˆ
12.1 (c ˆ 1.00, CHCl3). IR
(CHCl3): 3674w, 3476m, 3007w, 2981w, 1706s, 1504m, 1456m, 1418w, 1393w, 1368m, 1166m, 1090w, 1047w, 869w,
1
622w. H-NMR (300 MHz, CDCl3): 1.42 (s, t-Bu); 2.48 ± 2.64 (m, 2 H C(2)); 3.00 ± 3.13 (m, 2 H C(4)); 4.31
(br., CHN); 5.01 (br., NHBoc); 7.04 ± 7.22 (m, 3 arom. H); 7.36 (d, J ˆ 7.8, 1 arom. H); 7.65 (d, J ˆ 7.5, 1 arom.
H); 8.14 (br., NH of indole). 13C-NMR (75 MHz, CDCl3): 28.35; 30.01; 37.99; 47.99; 79.62; 111.16; 111.61;
‡
‡
118.94; 119.61; 122.15; 123.02; 127.65; 136.27; 155.58; 176.06. FAB-MS: 319.2 (48.3, [M ‡ H] ), 318.2 (55.1, M ),
307.1 (100.0). Anal. calc. for C17H22N2O4 (318.37): C 64.13, H 6.96, N 8.80; found: C 64.05, H 6.92, N 8.71.
(3S)-3-{[(tert-Butoxy)carbonyl]amino}-7-({[(2-chlorobenzyl)oxy]carbonyl}amino)heptanoic Acid (Boc-
b3-HLys(Ne-2-Cl-Z)-OH, 7), [59]. 2.0 g (4.3 mmol) 3b, 0.11 g (0.5 mmol) CF3CO2Ag and 1.5 ml (11 mmol)
Et3N were transformed according to GP 2. Drying under h.v. and precipitation with pentane gave 7 (1.8 g, 91%).
Colorless powder. [a]D
ˆ
12.9 (c ˆ 0.98, CHCl3). ([59]: ˆ 11.4 (c ˆ 1.19, CHCl3)). 1H-NMR (200 MHz,
CDCl3): 1.32 ± 1.62 (s, 3 CH2, t-Bu); 2.57 (d, J ˆ 5.4, CH2C(O)); 3.23 (q, J ˆ 6.4, NCH2CH2); 3.91 (br., CHN);
4.96 (br., 2 NH); 5.23 (s, CH2(2-Cl-Ph)); 7.26 ± 7.36 (m, 2 arom. H); 7.38-7.47 (m, 2 arom. H).
Methyl
(3S)-3-{[(tert-Butoxy)carbonyl]amino}-7-({[(2-chlorobenzyl)oxy]carbonyl}amino)heptanoate
(Boc-b3-HLys(Ne-2-Cl-Z)-OMe, 8) [60]: 2 g (4.6 mmol) of 3b were reacted according to GP 3. FC (Et2O/
pentane) gave 8 (1.63 g, 81%). White solid. Rf (Et2O) 0.67. M.p.: 77 ± 798 ([60]: 77.5 ± 798). [a]D
ˆ
16.7 (c ˆ
1.03, CHCl3) ([60]: [a]D
ˆ
15.5 (c ˆ 1.00, CHCl3)). 1H-NMR (300 MHz, CDCl3): 1.34 ± 1.63 (m, 3 CH2, t-Bu);
2.51 (d, J ˆ 4.4, 2 H C(2)); 3.20 (q, J ˆ 6.5, NCHCH2); 3.67 (s, MeO); 3.90 (br., CHN); 4.85 ± 5.0 (br., 2 NH);
5.21 (s, ClC6H4CH2); 7.24 ± 7.28 (m, 2 arom. H); 7.35 ± 7.43 (m, 2 arom. H).
(3R,4R)-4-(Benzyloxy)-3-{[(tert-butoxy)carbonyl]amino}pentanoic Acid (Boc-b3-HThr(OBn)-OH, 9):
15.4 g (42 mmol) of 3d, 1.12 g (4.62 mmol) of CF3CO2Ag, and 15.0 ml (107 mmol) of Et3N were reacted
according to GP 2. Drying under h.v. gave 9 (13.1 g, 88%). Colorless glass. [a]D
ˆ
1.0 (c ˆ 1.30, CHCl3). IR
(CHCl3): 3439w, 2980m, 1710s, 1500s, 1455w, 1393w, 1368m, 1166m, 1098w, 908w, 863w, 617w. 1H-NMR
(300 MHz, CDCl3): 1.22 (d, J ˆ 6.23, Me); 1.45 (s, t-Bu); 2.52 ± 2.69 (m, 2 H C(2)); 3.65 ± 3.73 (m, H C(4));
4.05 ± 4.07 (m, CHN); nA ˆ 4.39, nB ˆ 4.59 (AB, JAB ˆ 11.5, PhCH2); 5.11 (d, J ˆ 9.6, NHBoc); 7.26 ± 7.37 (m, 5

Helvetica Chimica Acta ± Vol. 83 (2000)
29
arom. H); 10.45 (br., OH). 13C-NMR (75 MHz, CDCl3): 17.03; 29.24; 38.00; 52.58; 71.95; 75.91; 80.46; 128.41;
‡
‡
128.60; 128.73; 129.26; 139.02; 156.79; 177.31. FAB-MS: 669.5 (4.1, [2M ‡ Na] ), 647.5 (5.1, [2M ‡ H] ), 346.2
(29.6, [M ‡ Na] ), 324.2 (53.9, [M ‡ H] ), 268.1 (73.8, [M ‡ H tBu] ), 224.2 (100, [M Boc ‡ 2 H] ). A small
amount of 9 was treated with excess cyclohexylamine and recrystallized from MeOH. Anal. calc. for
C17H25NO3 ´ C6H13N (422.56): C 65.38, H 9.06, N 6.63; found: C 65.21, H 9.17, N 6.61.
‡
‡
‡
‡
(3S)-3-{[(tert-butoxy)carbonyl](methyl)amino}-4-phenylbutanoic Acid (Boc-N-Me-b3-HPhe-OH (10):
1.00 g (3.6 mmol) of 5 was reacted according to GP 4 to yield 10 (968 mg, 92%). White powder. M.p. 78 ±
818. 1H-NMR (200 MHz, CDCl3, Rotamers!): 1.28 ± 1.43 (br. s, t-Bu); 2.51 ± 2.96 (m, 2 H C(2), PhCH2, MeN);
4.55 (br. s, NCH); 7.18 ± 7.33 (m, 5 arom. H).
(3R,4R)-4-(Benzyloxy)-3-{[(tert-butoxy)carbonyl](methyl)amino}pentanoic Acid (Boc-N-Me-b3-HThr-
(OBn)-OH (11): 5.36 g (16.6 mmol) 9 was reacted according to GP 4 to yield 11 (4.64 g, 83%). Yellow oil.
1H-NMR (200 MHz, CDCl3, Rotamers!): 1.19 (d, MeCH); 1.45 (s, t-Bu); 2.50 ± 2.87 (m, 2 H C(2), MeN);
3.64 ± 3.83 (br., H C(4)); 4.40 ± 4.50 (br., CHN); nA ˆ 4.34, nB ˆ 4.62 (AB, JAB ˆ 11.6, PhCH2); 7.26 ± 7.38 (m, 5
arom. H).
Boc-b3-HTrp-b3-HPhe-OMe (12): 2.16 g (7.4 mmol) of 4 was reacted according to GP 5. The resulting
trifluoroacetate was coupled, according to GP 6, with 2.34 g (7.4 mmol) of 6. FC (AcOEt/pentane 2 :1 to 4 :1)
gave 12 (3.1 g, 85%). Brownish solid. Rf (AcOEt/pentane 2 :1) 0.46. M.p. 178 ± 1798. [a]D
ˆ
22.4 (c ˆ 0.82,
CHCl3). IR (CHCl3): 3478m, 3428m, 3008m, 1700s, 1496s, 1456m, 1438m, 1367m, 1166s, 1090w, 1046w, 930w,
650w. 1H-NMR (500 MHz, CDCl3): 1.42 (s, t-Bu); nA ˆ 2.26, nB ˆ 2.34 (ABM, JAB ˆ 14.9, JBM ˆ 5.8, JAM ˆ 5.3,
CH2C(O)OMe); nA ˆ 2.46, nB ˆ 2.50 (ABM, JAB ˆ 16.1, JBM ˆ 5.3, JAM ˆ 5.4, CH2C(O)N); 2.77 ± 2.81 (m, 1 H,
CH2-Aryl); 2.88 ± 2.94 (m, CH2-Aryl); 3.03 ± 3.10 (br., CH2-Aryl); 3.67 (s, MeO); 4.14 ± 4.21 (m, CHN); 4.43 ±
4.55 (m, CHN); 5.43 (br., NHBoc), 6.15 (d, J ˆ 8.6, NHCO); 6.97 (d, J ˆ 1.6, 1 arom. H); 7.08 ± 7.34 (m, 8 arom.
H); 7.65 (d, J ˆ 7.8, 1 H, indole), 8.19 (s, NH of indole). 13C-NMR (125 MHz, CDCl3): 28.43; 29.92; 37.10; 39.34;
39.92; 47.31; 48.78; 51.79; 79.21; 111.09; 112.14; 119.11; 122.05; 122.87; 126.77; 127.69; 128.63; 129.25; 136.25;
‡
‡
137.44; 155.58; 170.56; 172.11. FAB-MS: 1009.7 (8.62, [2M ‡ Na] ), 516.3, (58.4, [M ‡ Na] ), 494.3 (47.6, [M ‡
‡
‡
‡
H] ), 416.2 (8.2, [M Boc ‡ Na] ), 394.2 (66.2, [M Boc ‡ 2 H] ), 376.1 (100.0). Anal. calc. for C28H35N3O5
(493.6): C 68.13, H 7.15, N 8.51; found: C 67.85, H 7.42, N 8.41.
Boc-b3-HThr(OBn)-b3-HLys(Ne-2-Cl-Z)-OMe (13): 3.3 g (7.5 mmol) 8 was reacted according to GP 5 and
coupled, according to GP 6, with 2.44 g (7.5 mmol) of 9. Drying under h.v. gave 13 (4.636 g, 96%), which was
used without further purification. For anal. purposes, a small amount of 13 was recrystallized from MeOH. Rf
(AcOEt) 0.63. M.p. 121 ± 1228. [a]D
ˆ
4.3 (c ˆ 2.20, CHCl3). IR (CHCl3): 3677w, 3436m, 3007m, 2979m,
1
2997m, 1711s, 1506s, 1439m, 1368m, 1248m, 1168m, 1061m, 864w, 653w. H-NMR (400 MHz, CDCl3): 1.18 (d,
J ˆ 6.1, Me); 1.26 ± 1.14 (m, CH2CH2NH); 1.42 (s, t-Bu); 1.46 ± 1.58 (m, 2 CH2 (Lys)); 2.32 ± 2.49 (m, 2
CH2C(O)); 3.1 ± 3.23 (m, NCH2CH2 (Lys)); 3.67 (s, MeO); 3.91 ± 3.93 (m, CHN); 4.21 ± 4.23 (m, CHN); nA
ˆ
4.39, nB ˆ 4.60 (AB, JAB ˆ 11.5 PhCH2); 5.12 ± 5.19 (br., CHOBn, NHBoc); 5.19 (s, CH2(2-Cl-C6H4)); 6.42 (d, J ˆ
7.6, NH-(2-Cl-Z)); 7.22 ± 7.44 (m, 9 arom. H). 13C-NMR (100 MHz, CDCl3): 16.13; 23.06; 28.37; 29.34; 33.57;
38.69; 39.91; 40.66; 45.77, 51.71; 52.47; 63.77; 71.09; 75.98; 79.52; 126.85; 127.76; 127.80; 128.23; 129.25; 129.46;
‡
129.06; 133.43; 134.44; 138.28; 156.17; 156.28; 170.31; 172.03. FAB-MS: 1295.8 (18.2, [2M] ), 670.38 (65.1, [M ‡
‡
‡
‡
Na] ), 648.4 (86.5, [M ‡ H] ), 548.3 (100.0, [M Boc ‡ 2 H] ). Anal. calc. for C33H46N3O8Cl (648.19): C 61.15,
H 7.15, N 6.48; found: C 61.10, H 7.24, N 6.46.
Boc-N-Me-b3-HThr(OBn)-b3-HLys(Ne-2-Cl-Z)-OMe (14): 4.19 g (9.5 mmol) of 8 was reacted according to
GP 5 and coupled, according to GP 6, with 3.19 g (9.5 mmol) of 11. FC (CH2Cl2/MeOH 20 :1) gave 14 (5.6 g,
89%). Colorless Oil. Rf (CH2Cl2/MeOH 20 :1) 0.30. IR (CHCl3): 3449w, 3008m, 2977m, 1724s, 1675s, 1514s,
1440m, 1367m. 1H-NMR (200 MHz, CDCl3, Rotamers!): 1.17 (d, J ˆ 5.8, MeCH); 1.21 ± 1.48 (m, 3 CH2, t-Bu);
2.37 ± 2.87 (m, 2 CH2C(O), MeN); 3.12 ± 3.33 (br. m, NHCH2); 3.69 (br. s, MeO, CHOBn); 4.18 ± 4.30 (br. m,
2 CHN); nA ˆ 4.33, nB ˆ 4.62 (AB, JAB ˆ 11.6, PhCH2); 5.22 (br., NH); 5.22 (s, CH2-Aryl); 6.22, 6.42 (br. d,
‡
‡
rotamers, NH); 7.24 ± 7.46 (m, 9 arom. H). FAB-MS: 684.32 (4.1, [M ‡ Na] ), 662.33 (28.0, [M ‡ H] ), 562.27
‡
(100.0, [M Boc ‡ 2 H] ).
Boc-N-Me-b3-HPhe-N-Me-b3-HThr(OBn)-b3-HLys(Ne-2-Cl-Z)-OMe (15): 2.04 g (3.1 mmol) of 14 was
reacted according to GP 5 and coupled, according to GP 6, with 861 mg (3.1 mmol) of 10. FC (80 g, AcOEt/
CH2Cl2 1:1) gave 15 (2.01 g, 79%). Colorless foam. Rf (AcOEt/CH2Cl2 1:1) 0.29. 1H-NMR (300 MHz, CDCl3,
Rotamers!): 1.12 (d, MeCH); 1.13 ± 1.58 (br. m, 3 CH2, t-Bu); 2.30 ± 3.00 (br. m, 3 CH2C(O), 2 MeN, PhCH2);
3.05 ± 3.22 (br. m, NHCH2); 3.64 (br. s, MeO, CHOBn); 4.05 ± 4.40 (br., 2 CHN); 4.50 ± 4.60 (br. m, CHN); nA
ˆ
4.29, nB ˆ 4.57 (AB, JAB ˆ 11.5, PhCH2); 5.17 ± 5.19 (m, NH(2-Cl-Z)); 5.19 (s, CH2-Aryl); 5.57, 6.30, 6.58, 6.79 (br.
‡
‡
m, rotamers, NH); 7.10 ± 7.41 (m, 14 arom. H). FAB-MS: 859.1 (6.0, [M ‡ Na] ), 837.2 (19.5, [M ‡ H] ), 737.2
‡
(100.0, [M Boc ‡ 2 H] ).

30
Helvetica Chimica Acta ± Vol. 83 (2000)
Boc-b3-HTrp-b3-HPhe-b3-HThr(OBn)-b3-HLys(Ne-2-Cl-Z)-OMe (16): 1.6 g (3.25 mmol) of 12 and 0.65 g
(16 mmol) of NaOH were dissolved in 25 ml of MeOH/3 ml of H2O, and the reaction mixture was stirred for 24 h
at r.t. Additional 0.6 g (16 mmol) of NaOH were added, and the mixture was stirred overnight at 408 (TLC
control). The peptide was precipitated by the addition of 1m HCl and collected by filtration. Drying under h.v.
over P2O5 gave the peptide acid Boc-b3-HTrp-b3-HPhe-OH (1.54 g, 98%), which was used without further
purification. Peptide ester 13 (2.094 g, 3.2 mmol) was deprotected according to GP 6 and coupled with 1.54 g
(3.28 mmol) of Boc-b3-HTrp-b3-HPhe-OH according GP 7. Precipitation gave 16 (3.01 g, 93%). Colorless solid.
Rf (AcOEt) 0.30. Anal. RP-HPLC (50 ± 100% B in 10 min, then 100% B): tR 12.4 min. M.p. 195 ± 1968 (dec.).
[a]D
ˆ
7.3 (c ˆ 0.55, AcOH). IR (KBr): 3650w, 3303s, 3059m, 3028m, 2931m, 2857m, 1683s, 1646s, 1534s,
1440m, 1366m, 1342m, 1248s, 1163s, 1099m, 1020m, 854w, 806w, 741s, 699m, 668m, 591m, 508w, 431w. 1H-NMR
(500 MHz, CD3OD/CDCl3 1:4): 1.12 (d, J ˆ 6.2, Me); 1.25 ± 1.52 (m, 3 CH2 (Lys), t-Bu); 2.21 ± 2.46 (m,
4 CH2C(O)); nA ˆ 2.77, nB ˆ 2.85 (ABM, JAB ˆ 13.9, JAM ˆ 6.9, JBM ˆ 6.7, CH2-Aryl); 2.89 ± 2.90 (m, 1 H, CH2-
Aryl); 2.97 (br., 1 H, CH2-Aryl); 3.12 (t, J ˆ 6.7, 2 H); 3.61 (s, MeO); 3.67 ± 3.86 (m, CHOBn); 4.11 ± 4.13 (br.,
2 CHN); 4.23 (br., CHN); 4.36 ± 4.38 (m, CHN); nA ˆ 4.40, nB ˆ 4.57 (AB, JAB ˆ 11.2 PhCH2); 5.17 (s, CH2(2-Cl-
Ph)); 5.21 (br., NH-(2-Cl-Z)); 7.01 ± 7.41 (m, 18 arom. H); 7.60 ± 7.62 (m, 1 arom. H (indole)). 13C-NMR
(125 MHz, CD3OD/CDCl3 1 :4): 16.06; 18.01; 23.24; 28.47; 29.45; 30.28; 33.91; 38.74; 39.16; 39.70; 40.27; 40.74,
46.40; 47.77; 48.60; 51.40; 51.85; 63.93; 71.48; 75.56; 79.53; 111.27; 111.41; 118.95; 119.14; 121.72; 123.59; 126.77;
126.88; 127.07; 127.90; 128.10; 128.25; 128.65; 128.72; 129.45; 129.50; 129.59; 133.43; 134.61; 136.58; 137.93;
‡
138.22 ;156.19; 157.09; 171.24; 171.61 (2 C); 172.41. FAB-MS: 1031.9 (51.1, [M ‡ Na] ), 1009.9 (100.0, [M ‡
‡
‡
H] ), 909.9 (59.5, [M Boc ‡ 2 H] ), 307.2 (100.0).
Boc-b3-HTrp-N-Me-b3-HPhe-N-Me-b3-HThr(OBn)-b3-HLys(Ne-2-Cl-Z)-OMe (17): 3.47 g (4.1 mmol) of
15 was reacted according to GP 5 and coupled, according to GP 6, with 1.32 g (4.1 mmol) of 6. FC (AcOEt)
gave 17 (2.85 g, 66%). Colorless foam. Rf (AcOEt) 0.3. [a]D
ˆ
5.55 (c ˆ 1.035, MeOH). 1H-NMR (300 MHz,
CDCl3, Rotamers!): 1.13 (d, MeCH); 1.13 ± 1.48 (br. m, 3 CH2, t-Bu); 2.30 ± 3.00 (br. m, 4 C(O)CH2, 2 MeN,
PhCH2, CH2-indole); 3.02 ± 3.17 (br. m, NHCH2); 3.64 (br. s, MeO, CHOBn); 4.04 ± 4.38 (br. m, 4 CHN); nA ˆ
4.27, nB ˆ 4.59 (AB, JAB ˆ 11.5, PhCH2); 5.19 (m, NH(2-Cl-Z)); 5.19 (s, CH2-Aryl); 5.55, 6.39, 6.59, 6.93 ± 7.00 (br.
‡
m, rotamers, NH); 7.04 ± 7.40 (m, 18 arom. H); 7.58 ± 7.64 (m, 1 arom. H). FAB-MS: 1060.0 (11.5, [M ‡ Na] ),
‡
‡
1037.8 (58.2, [M ‡ H] ), 937.6 (100.0, [M Boc ‡ 2 H] ). Anal. calc. for C57H73N6O10Cl (1037.02): C 65.98,
H 7.09, N 8.10; found: C 65.81, H 7.26, N 7.90.
Cyclo(b3-HTrp-b3-HPhe-b3-HThr(OBn)-b3-HLys(Ne-2-Cl-Z)) (18): 2.5 g (2.49 mmol) of 16 and 6.0 g
(150 mmol) of NaOH were suspended in 20 ml of H2O and 25 ml of TFE, and the suspension was stirred for 2 d
at r.t. and for another 24 h at 458. The peptide acid was precipitated with 1m HCl soln., washed with H2O and
MeOH, and dried under h.v. The resulting b-peptide acid was dissolved in 100 ml of DMF and 50 ml of CHCl3,
and was treated at r.t. with 2.0 g (10 mmol) of EDC and 1.0 g (5.4 mmol) of C6F5OH. Molecular sieves (4 Š)
were added to the mixture. After 24 h, additional EDC (1.0 g, 5 mmol) and C6F5OH (0.5 g, 2.7 mmol) were
added. The solvent was evaporated after 48 h (TLC control), and the residue was triturated with MeOH,
centrifuged and washed with H2O. The resulting b-peptide acid ester (2.85 g) was treated with a mixture of TFA/
ethane-1,2-dithiol/CH2Cl2 (5 :4 :2) under Ar at 08 (0.5m). The mixture was stirred for 10 min at 08, and the
solvent was removed under h.v. The oily residue was co-evaporated twice with CHCl3 and dried for 3 h under h.v.
The residue was subsequently dissolved in MeCN (0.025m) and was slowly added (syringe pump) to a soln. of
0.43 ml (2.4 equiv.) of EtN(i-Pr)2 in MeCN (3.3 mm) at 708 during 16 h. A colorless precipitate was observed
after 2 h. After completion of the addition, the mixture was stirred for additional 2 h and cooled to r.t. under
stirring. The precipitate was collected by filtration and washed with MeCN and MeOH. Drying under h.v. gave
18 (0.84 g, 38%). Colorless powder. M.p. 255 ± 2608 (dec.). CD: (c ˆ 0.2 mmol/ml, CF3CH2OH): no extrema
observed. IR (KBr): 3752w, 3300s, 3060m, 2929m, 2858m, 1656s, 1547s, 1455s, 1440s, 1377m, 1368m, 1261s,
1141m, 1090m, 1060m, 1026m, 813w, 746s, 699s, 599m, 427w. 1H-NMR (500 MHz, 24 mg 18 ‡ 5 mg LiCl, 0.75 ml
of (D8)THF): 1.00 (d, J ˆ 6.4, Me); 1.37 ± 1.62 (m, 3 CH2); nA ˆ 2.08, nB ˆ 2.39 (AB, JAB ˆ 12.5, CH2); nA ˆ 2.25,
nB ˆ 2.30 (AB, JAB ˆ 10.8, CH2); 2.52 (t, J ˆ 12.6, CH2); 2.61 ± 2.72 (m, CH2); 2.86 (dd, J ˆ 9.2, 13.8, 1 H, CH2);
3.00 ± 3.19 (m, 3 CH2); 3.68 ± 3.71 (m, CHO); nA ˆ 4.54, nB ˆ 4.88 (AB, JAB ˆ 12.0, CH2OBn); 4.53 (m, CHN);
4.72 (t, J ˆ 8.3, CHN); 4.96 (br., CHN); 5.09 (br., CHN); 5.17 (s, CH2(2-Cl-C6H4)); 6.68 (t, J ˆ 5.7, NH-(2-Cl-Z));
6.92 (t, J ˆ 7.2, 1 arom. H); 6.98 (t, J ˆ 7.2, 1 arom. H); 7.06 (t, J ˆ 7.3, 1 arom. H); 7.13 ± 7.28 (m, 12 arom. H); 7.36
(d, J ˆ 7.6, 1 arom. H); 7.42 (d, J ˆ 7.3, 2 arom. H); 7.49 (d, J ˆ 7.2, 1 arom. H); 7.65 (d, J ˆ 7.9, 1 arom. H); 8.90 (d,
J ˆ 6.9, NH); 8.97 (d, J ˆ 8.1, 2 NH); 9.17 (d, J ˆ 9.0, NH); 10.18 (br., NH). 13C-NMR (75 MHz, 24 mg 18 ‡ 5 mg
LiCl, 0.75 ml (D8)THF): 11.65; 20.67; 26.88; 28.59; 31.71; 32.14; 37.10; 37.89; 38.62; 39.17; 42.89; 44.71; 46.53;
47.28; 60.24; 63.94; 67.76; 71.19; 108.32; 108.72; 115.81; 116.37; 118.28; 120.76; 123.56; 124.30; 124.43; 125.32 (2 C);

Helvetica Chimica Acta ± Vol. 83 (2000)
31
125.53; 125.61; 126.48; 126.57; 126.99; 130.45; 132.99; 134.36; 136.45; 137.24; 153.66; 170.04 (2 C); 170.41; 170.99.
‡
‡
MALDI-TOF-MS: 899.8 ([M ‡ Na] ), 877.6 ([M ‡ H] ).
Cyclo(b3-HTrp-b3-HPhe-b3-HThr-b3-HLys) (1): 200 mg (0.23 mmol) of 18 were dissolved in 13 ml of THF
by the addition of 60 mg (1.2 mmol) of LiCl; 250 mg of Pd/C (10%) were added under Ar. The apparatus was
evacuated and flushed with H2 three times. The mixture was stirred for 24 h, few drops of MeOH and ca. 100 mg
LiCl (2.5 mmol) were added, and the mixture was stirred for another 24 h. The residue was filtered and washed
with a soln. of LiCl in THF. The combined org. layers were evaporated and dissolved in 6 ml of MeOH.
Purification by prep. RP-HPLC (C18; 10 min 0% B; 20 min 0 ± 35% B; at 30% B isocratic until product
completely eluted; then 9.5 min 35 ± 60% B; 5 min 60 ± 98% B; 10 min 98% B): 106 mg (53%) of 18 and 12 mg
(9%) of 1. Anal. RP-HPLC (C18; gradient 5 min 20 ± 30% B; 10 min 30 ± 45% B; 5 min 45 ± 100% B; 5 min 100%
B): tR 12.8 min. [a]D ˆ ‡96 (c ˆ 0.25, MeOH). 1H-NMR (500 MHz, CD3OH): See Table 2. 13C-NMR (125 MHz,
CD3OH): 20.38; 22.88; 27.07; 31.40; 34.77; 40.72; 40.89; 41.07; 42.38; 45.25; 51.29; 55.40; 69.74; 112.04; 112.33;
119.72 (2 C); 122.37; 124.62; 127.62; 129.34; 129.42 (2 C); 129.45; 130.58 (2 C); 130.62; 138.30; 139.27; 172.83;
‡
‡
173.30; 175.19; 175.20. FAB-MS: 641.5 (43.8, [M ‡ Na] ), 619.5 (100.0, [M ‡ H] ).
9. NMR Analysis of Cyclo-b-tetrapeptide 1. Sample: 3 mg of 1 dissolved in 0.6 ml of CD3OH. 1D-NMR
(AMX 500): 1H-NMR (500 MHz): suppression of the CD3OH signal by presat.; 90 K data points, 128 scans, 5.6-s
acquisition time. 2D-NMR: Solvent suppression by presat. DQF.COSY (500 MHz, CD3OH) with pulsed field
gradients (PFG) for coherence pathway selection [61]: acquisition: 4 K(t2) Â 512(t1) data points. 8 scans per t1
increment, 0.33-s acquisition time in t2; relaxation delay 2.0 s. TPPI Quadrature detection in w1. Processing:
Zero filling and FT to 1 K Â 1 K real/real data points after multiplication with sin2 filter shifted by p/3 in w2 and
p/2 in w1. TOCSY (DIPSI-2 SL; 10 kHz) [62] (500 MHz, CD3OH): acquisition: 2 K(t2) Â 1 K(t1) data points. 32
scans per t1 increment, mixing time 125 ms, TPPI quadrature detection. Processing: Zero filling and FT to 1 K Â
1 K real/real data points after multiplication with sin2 filter shifted by p/3 in w2 and p/2 in w1. ROESY [63]
(500 MHz, CD3OH): acquisition: a ROESY spectrum with a mixing time of 150 ms was acquired. Solvent
suppression by presat., CW-spin lock (3.8 kHz) between trim pulses, 2 K(t2) Â 480(t1) data points, 96 scans per t1
increment. 0.16-s acquisition time in t2, other parameters identical to DQF.COSY. Processing: Zero filling and
FT to 2 K Â 2 K real/real data points after multiplication by cos2 filter in w2 and in w1. Baseline correction with
3rd degree polynomial in both dimensions.
10. Structure Determination with Restrained Molecular Dynamics (RMD). Based on the powder X-ray
structure of cyclo(b3-HAla)4 [19], a model of 1 was generated using the QUANTA program package, with the
Lys side chain NH2 bearing no charge. Fifteen NOEs (cf. Table 3) were classified in three distance categories
according to their relative cross-peak volume (determined by integration): s (strong), m (medium), and w
(weak). Upper boundary distance values of 3, 3.5 and 4.5 Š, and lower boundary distance values of 1.9 Š were
assigned to these three categories. The H2C(a) of Thr, Trp, and Phe were stereospecifically assigned by their
distinct coupling constants to H C(b) after inspection of a model of the structure. The H' C(a) (see Table 2)
of Thr, Trp, and Phe correspond to the HRe protons, which show large J-values to their H C(b). These 15
distance restraints were used together with 8 dihedral restraints for all NH C(b)H and C(b)H C(a)H angles,
obtained via Karplus-type equations [64][65], in RMD with the QUANTA program package and the
CHARMm force field. The resulting model, which is in agreement with all experimentally determined values, is
depicted in Fig. 2.
11. Biological Evaluation of Cyclo-b-tetrapeptide 1. 11.1. Human Recombinant Receptors Expressed in
CCL39 or CHO Cells. CCL39 Cells (established line of Chinese hamster lung fibroblasts; American Type
Culture Collection) or CHO (hsst3 receptors in Chinese hamster oocytes, American Type Culture Collection)
were cultured as described in [66][67], and used for stable expression of the human sst1 ± 5 receptor genes.
Transfection method and G418 selection have been described in detail in [66][67]. Receptor expression of single
cell-derived colonies was tested by radioligand binding as described in [66][67]. For crude membrane
preparations, cells were harvested by washing with 10 mm HEPES, pH 7.5, scraping off the culture plates with
4 ml of the same buffer, and centrifugation at 48 for 5 min at 2500 Â g. The cell pellet was either stored at 808
or directly used. The cells were resuspended in binding assay buffer (10 mm HEPES, pH 7.5, 0.5% (w/v)) bovine
serum albumin by homogenization with the Polytron at 50 Hz for 20 s. In competition experiments, cell
homogenates (hsst1 and hsst2: ca. 1.5 Â 105; hsst3 and hsst5: 0.75 Â 105; hsst4: 4.5 Â 105 cells, depending on the
expression level of each receptor) were used in assay buffer (10 mm HEPES, pH 7.5, 0.5% (w/v) bovine serum
albumin, 5 mm MgCl2, 5 mg/ml bacitracin). Cell homogenate (150 ml) was incubated with 50 ml of 25 ± 50 pm
[
125I]LTT-SRIF28, and 50 ml of assay buffer in the absence (total binding) or presence competing ligands or 1 mm
SRIF14 (non-specific binding). Reactions were terminated by vacuum filtration and bound radioactivity was
measured in a Packard TopCount.

32
Helvetica Chimica Acta ± Vol. 83 (2000)
11.2. Binding. Competition and saturation curves from experiments performed in triplicate determination
were analyzed as described in [66][67]. Data were analyzed by non-linear regression curve fitting with the
computer program SCTFIT [68]. The data are reported as pKd values ( log mol/l). Protein concentrations were
determined by the method of Bradford with bovine serum albumin as a standard.
11.2. Ligands. SRIF14 (Ala-Gly-cyclo(Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys)-OH) was pur-
chased from Bachem AG (Bubendorf, Switzerland); octreotide (SMS 201-995; d-Phe-cyclo(Cys-Phe-d-Trp-Lys-
Thr-Cys)-Thr-OH) was from Novartis Pharma, Basel; ([125I]LTT-SRIF28; Ser-Ala-Asn-Ser-Asn-Pro-Ala-Leu-
Ala-Pro-Arg-Glu-Arg-Lys-Ala-Gly-cyclo(Cys-Lys-Asn-Phe-Phe-d-Trp-Lys-Thr-(125I-Tyr)-Thr-Ser-Cys)-OH),
the radioligand was custom labelled by ANAWA AG (Wangen, Switzerland).
REFERENCES
[1] K. Gademann, M. Ernst, D. Hoyer, D. Seebach, Angew. Chem. 1999, 111, 1302; Angew. Chem., Int. Ed.
1999, 38, 1223.
[2] K. Gademann, T. Hintermann, J. V. Schreiber, Curr. Med. Chem. 1999, 6, 905.
[3] S. H. Gellman, Acc. Chem. Res. 1998, 31, 173.
[4] D. Seebach, J. L. Matthews, Chem. Commun. 1997, 2015.
[5] D. Seebach, M. Overhand, F. N. M. Kühnle, B. Martinoni, L. Oberer, U. Hommel, H. Widmer, Helv. Chim.
Acta 1996, 79, 913.
[6] D. H. Apella, L. A. Christianson, I. L. Karle, D. R. Powell, S. H. Gellman, J. Am. Chem. Soc. 1996, 118,
13071.
[7] D. Seebach, K. Gademann, J. V. Schreiber, J. L. Matthews, T. Hintermann, B. Jaun, L. Oberer, U. Hommel,
H. Widmer, Helv. Chim. Acta 1997, 80, 2033.
[8] 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.
[9] D. H. Apella, L. A. Christianson, D. A. Klein, D. R. Powell, S. Huang, J. J. Barchi, S. H. Gellman, Nature
(London) 1997, 387, 381.
[10] S. Krauthäuser, L. A. Christianson, D. R. Powell, S. H. Gellman, J. Am. Chem. Soc. 1997, 119, 11719.
[11] Y. J. Chung, L. A. Christianson, H. E. Stanger, D. R. Powell, S. H. Gellman, J. Am. Chem. Soc. 1998, 120,
10555.
[12] D. Seebach, S. Abele, K. Gademann, B. Jaun, Angew. Chem. 1999, 111, 1700; Angew. Chem., Int. Ed. 1999,
38, 1595.
[13] D. Seebach, S. Abele, T. Sifferlen, M. Hänggi, S. Gruner, P. Seiler, Helv. Chim. Acta 1998, 81, 2218.
[14] S. Abele, P. Seiler, D. Seebach, Helv. Chim. Acta 1999, 82, 1559.
[15] J. L. Matthews, M. Overhand, F. N. M. Kühnle, P. E. Ciceri, D. Seebach, Liebigs Ann. Chem. 1997, 1371.
[16] J. L. Matthews, K. Gademann, B. Jaun, D. Seebach, J. Chem. Soc., Perkin Trans. 1 1998, 3331.
[17] K. Gademann, D. Seebach, Helv. Chim. Acta 1999, 82, 957.
[18] T. D. Clark, L. K. Buehler, M. R. Ghadiri, J. Am. Chem. Soc. 1998, 120, 651.
[19] D. Seebach, J. L. Matthews, A. Meden, T. Wessels, C. Baerlocher, L. B. McCusker, Helv. Chim. Acta 1997,
80, 173.
[20] T. Hintermann, D. Seebach, Chimia 1997, 51, 244.
[21] D. Seebach, S. Abele, J. V. Schreiber, B. Martinoni, A. K. Nussbaum, H. Schild, H. Schulz, H. Hennecke, R.
Woessner, F. Bitsch, Chimia 1998, 52, 734.
[22] J. Podlech, D. Seebach, Liebigs Ann. Chem. 1995, 1217.
[23] G. Guichard, S. Abele, D. Seebach, Helv. Chim. Acta 1998, 81, 187.
[24] P. Brazeau, W. Vale, R. Burgus, N. Ling, M. Butcher, J. Rivier, R. Guillemin, Science 1973, 179, 77.
[25] L. Mandarino, D. Stenner, W. Blanchard, S. Nissen, J. Gerich, N. Ling, P. Brazeau, P. Bohlen, F. Esch, R.
Guillemin, Nature 1981, 291, 76.
[26] S. Bloom, C. Mortimer, M. Thorner, G. Besser, R. Hall, A. Gomez-Pan, U. Roy, R. Russel, D. Coy, A.
Kastin, A. Schally, Lancet 1974, 2, 1106.
[27] V. Havlicek, M. Rezek, H. Freisen, Pharmacol. Biochem. Behav. 1976, 4, 455.
[28] M. F. Chesselet, T. Reisine, J. Neurosci. 1983, 3, 232.
[29] M. Gothert, Nature 1980, 273, 674.
[30] T. Reisine, G. I. Bell, Neuroscience 1995, 67, 777.
[31] W. Bauer, U. Breiner, W. Doepfner, R. Haller, R. Huguenin, P. Marbach, T. J. Petcher, J. Pless, Life Sci.
1982, 31, 1133.

Helvetica Chimica Acta ± Vol. 83 (2000)
33
[32] ꢀProgress in Basic and Clinical Pharmacologyꢁ, Eds. P. Lomax, C. Scarpignato, and E. S. Vesell, Vol. 10,
Karger, Basel, Freiburg, Paris, London, New York, New Delhi, Bangkok, Singapore, Tokyo, Sidney, 1996.
[33] R. Hirschmann, K. C. Nicolaou, S. Petranico, J. Salvino, E. M. Leahy, P. A. Sprengeler, G. Furst, C. D.
Strader, M. A. Cascieri, M. R. Candelore, C. Donalson, W. Vale, L. Maechler, J. Am. Chem. Soc. 1992, 114,
9217.
[34] S. P. Rohrer, E. T. Birzin, R. T. Mosley, S. C. Berk, S. M. Hurchins, D.-M. Shen, Y. Xiong, E. C. Hayes,
R. M. Parmar, F. Foor, S. W. Mitra, S. J. Degrado, M. Shu, J. M. Klopp, S.-J. Cai, A. Blake, W. W. S. Chan, A.
Pasternak, L. Yang, A. A. Patchett, R. G. Smith, K. T. Chapman, J. M. Schaeffer, Science 1998, 282, 737.
[35] A. J. Souers, A. A. Virgillo, W. Feniuk, J. A. Ellman, J. Am. Chem. Soc. 1999, 121, 1817.
[36] R. Hirschmann, J. J. Hynes, M. A. Cichy-Knight, R. D. v. Rijn, P. A. Sprengeler, P. G. Spoors, W. C.
Shakespeare, S. Pietranico-Cole, J. Barbarosa, J. Liu, W. Yao, S. Rohrer, I. A. B. Smith, J. Med. Chem. 1998,
41, 1382.
[37] E. Pohl, A. Heine, G. M. Sheldrick, Z. Dauter, K. S. Wilson, J. Kallen, W. Huber, P. J. Pfäffli, Acta
Crystallogr., Sect. D 1995, 51, 48.
[38] C. Wynants, G. v. Binst, H. R. Loosli, Int. J. Pept. Protein Res. 1985, 25, 608; ibid. 615.
[39] C. Wynants, D. Tourwe, W. Kazmierski, V. J. Hruby, G. v. Binst, Eur. J. Biochem. 1989, 185, 371.
[40] H. Widmer, A. Widmer, W. Braun, J. Biomol. NMR 1993, 3, 307.
[41] G. Melacini, Q. Zhu, M. Goodman, Biochemistry 1997, 36, 1233.
[42] J. Podlech, D. Seebach, Angew. Chem. 1995, 107, 507; ibid., Int. Ed. 1999, 34, 471.
[43] J. R. McDermott, N. L. Benoiton, Can. J. Chem. 1972, 51, 1915.
[44] S. T. Cheung, N. L. Benoiton, Can. J. Chem. 1977, 55, 916.
[45] T. Hintermann, C. Mathes, D. Seebach, Eur. J. Org. Chem. 1998, 2379.
[46] D. Seebach, A. K. Beck, A. Studer, in ꢀModern Synthetic Methods 1995ꢁ, Eds. B. Ernst, C. Leumann,
VHCA, Basel, and VCH, Weinheim, 1995.
[47] R. Hirschmann, W. Yao, B. Arison, L. Maechler, A. Rosegay, P. A. Sprengeler, I. A. B. Smith, Tetrahedron
1998, 54, 7179.
[48] D. Seebach, O. Bezençon, B. Jaun, T. Pietzonka, J. L. Matthews, F. N. M. Kühnle, W. B. Schweizer, Helv.
Chim. Acta 1996, 79, 588.
[49] H. Kessler, Angew. Chem. 1982, 94, 509; ibid., Int. Ed. 1982, 21, 512.
[50] K. Gademann, B. Jaun, D. Seebach, R. Perozzo, L. Scapozza, G. Folkers, Helv. Chim. Acta 1999, 82, 1.
[51] J. Applequist, K. A. Bode, D. H. Appella, L. A. Christianson, S. H. Gellman, J. Am. Chem. Soc. 1998, 120,
4891.
[52] K. A. Bode, J. Applequist, Macromolecules 1997, 30, 2144.
[53] D. Seebach, J. V. Schreiber, S. Abele, X. Daura, W. F. van Gunsteren, Helv. Chim. Acta 2000, 83, 34.
[54] K. Gademann, A. Haene, T. Kimmerlin, D. Hoyer, D. Seebach, unpublished results, ETH-Zürich and
Novartis Pharma, 1999.
[55] O. Keller, W. E. Keller, G. V. Look, Org. Synth. 1985, 63, 160.
[56] P. Lombardi, Chem. Ind. (London) 1990, 708.
[57] S. Moss, Chem. Ind. (London) 1994, 122.
[58] M. Rodriguez, A. Aumelas, J. Martinez, Tetrahedron Lett. 1990, 31, 5153.
[59] S. Abele, G. Guichard, D. Seebach, Helv. Chim. Acta 1998, 81, 2141.
[60] S. Abele, Dissertation Nr. 13203, ETH-Zürich 1999.
[61] A. L. Davis, E. D. Laue, J. Keeler, D. Moskau, J. Lohman, J. Magn. Reson. 1991, 94, 637.
[62] S. P. Rucker, A. J. Shaka, Mol. Phys. 1989, 68, 509.
[63] C. Griesinger, R. R. Ernst, J. Magn. Reson. 1987, 75, 261.
[64] A. d. Marco, M. Llinas, K. Wüthrich, Biopolymers 1978, 17, 617.
[65] A. Pardi, M. Billeter, K. Wüthrich, J. Mol. Biol. 1984, 180, 741.
[66] S. Siehler, K. Seuwen, D. Hoyer, Eur. J. Pharmacol. 1998, 348, 311.
[67] S. Siehler, K. Seuwen, D. Hoyer, Naunyn-Schmiedebergꢀs Arch. Pharmacol. 1998, 357, 483.
[68] De Lean, SCIFIT: A Computer Program for Simultaneous Analysis of Saturation and Competition Curves,
Howard Hughes Medical Institute, Duke Univ. Medical Center, Durham, North Carolina.
[69] S. Siehler, K. Seuwen, D. Hoyer, Naunyn-Schmiedebergꢀs Arch. Pharmacol. 1999, 360, 488.
Received October 27, 1999