Syntheses and CD-spectroscopic investigations of longer-chain β-peptides: Preparation by solid-phase couplings of single amino acids, dipeptides, and tripeptides

Article abstract of DOI:10.1002/hlca.200390133

The synthesis and CD-spectroscopic analysis of eleven water-soluble β-peptides composed of all-β³ or alternating β²- and β³-amino acids is described. Different approaches for the efficient syntheses of longer-chain β-pepti

Full text of DOI:10.1002/hlca.200390133

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Helvetica Chimica Acta Vol. 86 (2003)
Syntheses and CD-Spectroscopic Investigations of Longer-Chain b-Peptides:
Preparation by Solid-Phase Couplings of Single Amino Acids, Dipeptides, and
Tripeptides
by Per I.Arvidsson 1), Jens Frackenpohl2), and Dieter Seebach*
Laboratorium f¸r Organische Chemie der Eidgenˆssischen Technischen Hochschule, ETH Hˆnggerberg,
CH-8093 Z¸rich, Schweiz (Phone: ‡ 4116322990; fax: ‡ 4116321144; e-mail: seebach@org.chem.ethz.ch)
Professor Jack D. Dunitz zum 80. Geburtstag gewidmet
The synthesis and CD-spectroscopic analysis of eleven water-soluble b-peptides composed of all-b3 or
alternating b2- and b3-amino acids is described. Different approaches for the efficient syntheses of longer-chain
b-peptides (> 9 residues) were investigated. They were synthesized on solid phase with Fmoc-protected amino
acids or Fmoc-protected di- or tripeptide fragments (assembled using solution-phase synthesis). The use of
preformed fragments significantly increased the purity of the crude peptides and facilitated purification.
Especially, the use of Fmoc-protected b2/b3-dipeptides for the synthesis of a −mixed× b2/b3-nonapeptide proved to
be remarkably effective, yielding the crude peptide in 95% purity and without detectable epimerization of the
b2-amino acid residues. This is a significant improvement over previously reported procedures for the solid-
phase synthesis of b-peptides, and foreshadows that the field of b-peptide research will now switch from
synthesis to the design and study of complex functional −b-proteins×.
1.Introduction. Solid-phase peptide synthesis (SPPS) has advanced tremendously
since the pioneering work of Merrifield, Sheppard, and others [1]. In fact, milestone
achievements, such as the total synthesis of bradykinin and bovine insuline [2], as well
as more-recent developments in combinatorial chemistry and solid-phase reagents [3]
imply that this is, indeed, a very mature field. Yet, many standard SPPS protocols fail,
mainly due to sequence-specific problems, such as folding and aggregation of the
growing peptide chain [4]. Thus, large efforts are still made on improving the methods
for SPPS, as witnessed from recent developments, such as chaotropic salt [5] and
pseudo-prolines [6], invented to prevent folding during synthesis, and by several
contributions on this topic at international conferences and proceedings.
Considering the obstacles related to a-peptide synthesis, it is not surprising that the
solid-phase synthesis of unnatural oligomers, such as b-peptides, can be particularly
cumbersome. b-Peptides, as well as other oligomeric organic molecules, e.g., oligoureas,
aza-peptides, and oligomers of a-aminoxy acids, have recently received much attention
due to their propensity to form well-defined structures similar to those found in nature
1
)
Postdoctoral fellow at the ETH-Z¸rich (2000/2001), financed by the Swedish Foundation for International
Cooperation in Research and Higher Education (STINT). Present address: Uppsala University, Institute of
Chemistry, Department of Organic Chemistry, Box 599, SE-75124 Uppsala, Sweden.
Postdoctoral fellow at the ETH-Z¸rich (2000/2001), financed by the Deutscher Akademischer
Austauschdienst (Hochschulsonderprogramm III). Present address: Bayer CropScience AG, Geb‰ude
Q18, D-51368 Leverkusen.
2
)

Helvetica Chimica Acta Vol. 86 (2003)
1523
[7]. Hence, b-peptides may be designed to fold into defined secondary structures, like
helices, turns, tubes, and sheets, with as few as six residues [8]. Furthermore, b-peptides
displaying interesting biological properties, e.g., antibacterial [9], antiproliferative [10],
and somatostatin-mimicking [11] activities have been described and proven to be stable
towards proteolytic [12] and metabolic [13] degradation.
While potentially pharmaceutically useful b-peptides are likely to be short and of
low molecular weight [14], the natural expansion of our fundamental research on the
folding and function of b-peptides is to assemble larger sequences capable of forming
tertiary structures, i.e., −b-proteins×. Recently, we paved the way to this goal by reporting
the first native chemical ligation between two unprotected b-peptide strands [15].
Concurrently, Raines and co-workers reported on −hybrid× proteins, where a small b-
dipeptide was introduced into RNase A by means of expressed-protein ligation [16].
Unifying for both these achievements is the need for effective syntheses of medium-
to-long sequences of b-peptides by SPPS. The solid-phase synthesis of b-peptides differs
from that of a-peptides in several aspects: i) increased folding during synthesis (the
high propensity of b-peptides to form stable secondary structures is expected to
influence the coupling and/or deprotection reactions), ii) expensive building blocks
(although many b3-amino acids are now commercially available, the high price or the
labor spent on their preparation mandates that as little material as possible be used in
each coupling), iii) lack of racemization (b3-amino acids cannot racemize during
activation and coupling, while b2-amino acids can; Scheme 1). These differences
suggest that certain variations of the typical SPPS protocol are needed to synthesize
longer-chain b-peptides in an efficient and economic way.
Scheme 1. Possible Racemization Pathway for b-Amino Acids During Coupling. Activated b2-amino acids may
undergo racemization via cyclization to a dihydro-oxazinone, similar to a-amino acids, while b3-amino acids are
configurationally stable.
We have previously established that the standard protocol for a-peptide synthesis,
as implemented on an automated peptide synthesizer, gave the Fmoc-protected
derivative of H-b3-HVal-b3-HGlu-b3-HOrn-b3-HVal-b3-HOrn-b3-HGlu-b3-HVal-OH as
a by-product in the synthesis [8k]. Use of the stronger base DBU (1,8-diazabicy-
clo[5.4.0]undec-7-ene) for Fmoc-deprotection overcomes this limitation, as demon-
strated by the successful SPPS of a 24 amino acid b-peptide via single amino acid

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Helvetica Chimica Acta Vol. 86 (2003)
couplings [17]. In this report, we wish to outline the strategies and methods used to
prepare a large collection of longer-chain b-peptides [18], some of which have been
used in previously reported biological studies.
2.Syntheses. 2.1. Synthesis of b-Amino Acids. The required b3-substituted amino
acids were prepared from the commercially available a-amino acids by the well-
established ArndtÀEistert homologation (1 3 ! 4 6; Scheme 2).
Scheme 2. Synthesis of b3-Amino Acid Derivatives 4 6. The ArndtÀEistert homologation of protected a-amino
acids yields b3-amino acids. All carbamate protecting groups important for peptide chemistry are tolerated, and
products with both protected and free COOH functions may be obtained.
As previously shown by our group, this procedure is compatible with a large variety
of N-protecting groups and allows the preparation of either the free acid or the
corresponding ester-protected derivative [19].
The b2-substituted amino acids 7 and 8 were obtained by diastereoselective
amidomethylation of the titanium enolates formed from acylated oxazolidinone
auxiliaries following a previously reported procedure [20] (Scheme 3).
2.2. Synthesis of a Homologous Series of Amphiphilic b-Peptides by Coupling
Tripeptide Building Blocks in the Solid Phase. b-Peptides capable of forming
amphiphilic 314-helices have been shown to have interesting biological properties.
Our group reported in 1999 that a nine-residue-long b-peptide with the sequence H-(b3-
HAla-b3-HLys-b3-HPhe)3-OH inhibits uptake of small-intestinal cholesterol and lipids
by inhibiting the SR-BI protein [21]. Similar peptides with slightly different aliphatic
side chains and peptide lengths have also been shown to have antibacterial and/or
hemolytic activity [9]3). The need for further biological investigations of this class of
amphiphilic peptides prompted us to synthesize a series of b-peptides with the sequence
H-(b3-HAla-b3-HLys-b3-HPhe)n-OH (n ˆ 3 6). We intended to use these peptides for
the following studies: i) pharmacokinetic investigations of the distribution of b-
3
)
Both the activity and selectivity of such amphiphilic peptides for mammalian vs. bacterial cells strongly
depends upon the nature of the side chains [9d f].

Helvetica Chimica Acta Vol. 86 (2003)
1525
Scheme 3. Synthesis of b2-Amino Acid Derivatives 7 and 8. Diastereoselective amidomethylation of the Ti
enolate formed from an acylated oxazolidinone auxiliary yields Cbz-protected b2-amino acids.
peptides in body tissue4), ii) investigations of antibacterial and hemolytic activities, of
the TFA and HCl salts of the peptides with n ˆ 3 65), and iii) in-vitro and in-vivo
studies of the cholesterol-inhibitory activity of the longer members of this series, i.e.,
n ˆ 4 6. The 15-residue peptide (n ˆ 5), to be used for in-vivo cholesterol-inhibition
studies, had to be prepared on a relatively large scale (ca. 1 g)6), and the nonapeptide
(n ˆ 3) needed for the pharmacokinetic study had to contain one 14C-labeled amino
acid.
These prerequisites, together with the repeating pattern of the sequence, made us
decide to use a combined solution- and solid-phase synthesis7). We, thus, prepared the
tripeptide unit Fmoc-(b3-HAla-b3-HLys(Boc)-b3-HPhe)-OH in solution and then
continued to couple this fragment on the solid support by Fmoc-based SPPS. We
figured that this strategy would simplify the purification of the desired pentadecapep-
tide. Furthermore, the lack of complete coupling and deprotection would not be a
serious obstacle since this would generate the shorter derivatives in the series, i.e., the
nonamer (n ˆ 3) and the dodecamer (n ˆ 4).
2.2.1. Solution-Phase Synthesis of the Protected Tripeptide Fmoc-(b3-HAla-b3-
HLys(Boc)-b3-HPhe)-OH. To follow an orthogonal protecting-group strategy through-
out the synthesis and to be able to use Boc-protection8) of the Lys side chain during
Fmoc SPPS, we chose the (benzyloxy)carbonyl (Cbz) protecting group for the N-
terminus during tripeptide assembly. As outlined in Scheme 4, Cbz-b3-HLys(Boc)-
A pharmacokinetic study on the distribution of the 14C-labelled derivative of the nonapeptide (n ˆ 3) in rat
has been reported recently [13].
4
)
5
6
7
)
)
)
The weak antibacterial and low hemolytic activities of these peptides have been reported elsewhere [9c].
Any peptide chemist would consider this a huge amount of material, especially for a b-peptide.
DeGrado and co-workers reported an alternative method [9a]; in this case the Fmoc-protected tripeptide
building block was prepared by SPPS on a highly acid-labile resin and then cleaved. This peptide was then
used for solid-phase synthesis on Rink amide resin. This procedure is advantageous when smaller
quantities of the peptide are to be prepared.
8
)
For full systematic names and specification of the abbreviations familiar to peptide chemists, see Sect. 1 in
the Exper. Part.

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Helvetica Chimica Acta Vol. 86 (2003)
Scheme 4. Solution-Phase Synthesis of the Fmoc-Protected Tripeptide 16 (overall yield: 60%). For abbrev., see
Sect. 1 in the Exper. Part.
OMe (6a) was deprotected to 9 by means of catalytic hydrogenation, and the latter was
coupled to Cbz-b3-HAla-OH (10) by standard coupling procedures (EDC/HOBt)8).
The dipeptide 11 was obtained in ca. 90% yield after column chromatography.
Saponification of the ester by LiOH in THF afforded the free acid 12 in > 95% yield.
Coupling of the dipeptide acid to H-b3-HPhe-OBn (13) gave the fully protected
tripeptide 14 in ca. 75% yield. Due to the poor solubility of 14, the catalytic
hydrogenation had to be performed in trifluoroethanol; nevertheless, both terminal
protecting groups were smoothly removed in quantitative yield leading to 15. The
Fmoc-group could be introduced with Fmoc-OSu in a dioxane/H2O mixture in up to
93% yield. The total yield of the tripeptide 16 from the corresponding b3-amino acids
was 60% on a 5-g scale.
2.2.2. Solid-Phase Synthesis of H-(b3-HAla-b3-HLys-b3-HPhe)n-OH (n ˆ 2 6). The
Fmoc-protected tripeptide 16 was anchored to Wang resin (initial loading: 0.99 mmol/
g) with N,N-diisopropylcarbodiimide (DIC), HOBt, and a catalytic amount of
DMAP8) (Scheme 5). The degree of anchoring (17), as determined by UV-spectro-

Helvetica Chimica Acta Vol. 86 (2003)
1527
Scheme 5. SPPS of H-(b3-HAla-b3-HLys-b3-HPhe)n-OH (18 22, n ˆ 2 6). For abbrev., see Sect. 1 in the
Exper. Part.
scopic analysis, was determined to be 0.50 mmol/g. This poor loading was probably due
to the relatively low excess (3 equiv.) of the tripeptide (3 equiv.) employed.
The remaining free OH functional groups of the resin were capped with Ac2O and a
catalytic amount of DMAP, and the Fmoc group was removed with 20% piperidine in
NMP and piperidine/DBU/NMP (2 :2 :96). The use of DBU is essential to achieve an
efficient Fmoc deprotection when the growing peptide chain contains more than ca. 6
b-amino acid residues. The use of b3-amino acids eliminates the risk of racemization
and allowed us to develop a special procedure for chain elongation (Scheme 5).
Initially, the resin was treated with 2.5 equiv. of tripeptide 16, 2.4 equiv. of HBTU, 2.5
equiv. of HOBt, and 5 equiv. of DIPEA8). Invariably, when n was > 1, the coupling
reaction was not finished after 48h, as determined by subjecting a small amount of the
beads to the TNBS8) test. To keep the consumption of the tripeptide to a minimum, we
then re-introduced the coupling mixture from the previous step. This solution,
containing the activated tripeptide and DIPEA, was stored at À188 when not used. To
compensate for the probable hydrolysis of some of the activated peptide ester, we also
added additional coupling reagents and DIPEA. This procedure of elongation and

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Helvetica Chimica Acta Vol. 86 (2003)
deprotection was then repeated until the desired chain length (n ˆ 2 6) was reached.
Standard TFA cleavage and precipitation with Et2O afforded the crude peptide as its
TFA salt.
2.2.3. Characterization, HPLC Analysis, and Purification of H-(b3-HAla-b3-HLys-
b3-HPhe)n-OH (n ˆ 2 6). Initially, the synthesis was stopped at the hexapeptide level
(18, n ˆ 2), and part of the beads was removed for subsequent use in the preparation of
the 14C-labeled derivative [13]. A fraction of these beads was subjected to standard
TFA cleavage, and the purity of the crude peptide was determined by HPLC (analytical
reversed-phase C18 column). As expected, the crude hexapeptide was relatively pure
(> 90%). Purification by preparative HPLC yielded 163 mg of pure 18 without major
problems.
Next, the synthesis was stopped at the 15-mer stage, (19, n ˆ 3) and the majority of
the beads were subjected to TFA cleavage. In this case, the analytical HPLC
chromatogram showed three major peaks with several minutes difference in retention
times. Purification of this mixture by preparative HPLC and subsequent analysis by
analytical HPLC revealed the 9-mer (19), 12-mer (20), and 15-mer (21) in a ratio of
1:2 :159). This estimation corresponds well with 34 mg of isolated 9-mer, 58mg of 12-
mer, and 359 mg of 15-mer. Notably, in total, > 800 mg pure pentadecapeptide 21 was
isolated to be used in subsequent in-vivo experiments (this required repetition of the
described solid-phase synthesis and ca. 20 injections on the HPLC system at our
disposal!).
The remaining fraction of beads was subjected to one final coupling to produce the
18-mer 22 (n ˆ 6). Analysis of this peptide by analytical HPLC revealed the presence of
the shorter analogs 19, 20, and 21. Again, the different peptides were easily separated
by preparative HPLC, yielding 91 mg of the pure octadecapeptide 22. To our surprise, a
small fraction with a longer retention time was also seen in the chromatogram, which
was shown by ESI-MS to correspond to the 21-mer. Clearly, the longer reaction times
must have caused partial deprotection of the Fmoc group. This problem was
painstakingly obvious in the synthesis of peptide 19 by the conventional −one-amino-
acid-at-a-time× procedure described below.
2.3. Solid-Phase Synthesis of b3-Peptides by Single-Amino-Acids Couplings. 2.3.1.
Solid-Phase Synthesis of all-(R)-H-(b3-HAla-b3-HLys-b3-HPhe)3-OH. The enantiomer
ent-19, composed solely of (R)-b3-amino acids10), was prepared according to the
improved solid-phase procedure11). In this case, the first amino acid was anchored to
Wang resin (0.99 mmol/g initial substance, 0.63 mmol/g loading), and SPPS was
completed through standard −one-amino-acid-at-a-time× coupling. Positive TNBS tests
indicating incomplete couplings after 3 h were experienced after coupling residues
number five and eight. Prolonged reaction time or surcharge of additional amino acid
and activating agents were then needed in order to complete the reactions. Notably,
crude ent-19 had a purity of only 12%, i.e., much lower than the one produced by
fragment coupling. Furthermore, several peaks had similar retention times, which made
9
)
)
)
The area under each peak was divided by the number of chromophores present in each molecule.
The proteolytic stability of this all-(R)-b3-peptide has previously been reported [12c].
The peptide was assembled under the same conditions as those given in Scheme 4, with the exception that
4 equiv. of the activated amino acid was used.
10
11

Helvetica Chimica Acta Vol. 86 (2003)
1529
the preparative HPLC purification less straightforward than the fragment-coupling
approach. Nevertheless, 15 mg of the nonapeptide ent-19 could be obtained in 95%
purity.
NH2
NH2
NH2
O
O
O
O
O
O
O
O
O
H2N
N
H
N
H
N
H
N
H
N
H
N
H
N
H
N
H
OH
ent-19
The low efficiency of the above synthesis agrees with our general experience that
the main difficulty with solid-phase synthesis of b-peptides is faced when the growing
peptide chain is between six and ten residues long. Despite improvements with Fmoc
deprotection, problems associated with the coupling steps, as judged from TNBS tests,
are frequently encountered at this stage. These problems are probably related to the
pronounced folding tendency of b-peptides, especially those composed of six to ten
residues.
2.3.2. Solid-Phase Synthesis of H-(b3-HAla-b3-HLys-b3-HPhe)3-(b3-HPhe)n-OH
(n ˆ 1 3). Following the synthesis of 21 via the tripeptide-condensation approach
described above, we attempted the synthesis of a larger quantity of the nonapeptide 19
using the standard −one-amino-acid-at-a-time× method. Believing that all hurdles for
successful SPPS of this kind of b-peptide had been overcome, we initiated the synthesis
by anchoring the first amino acid to the Wang resin using 4 equiv. of amino acid, DIC,
and a catalytic amount of DMAP for 24 h. The resin was then carefully washed, shrunk,
and dried in vacuo. The degree of substitution, as determined spectrophotometrically
after Fmoc deprotection, was found to be extraordinarily low (<0.1 mmol/g). Thus, the
loading reaction was repeated once more now with 3 equiv. of amino acid which
resulted in a loading of 0.25 mmol/g. A third attempt was made with 3 equiv. of amino
acid. This time, the degree of substitution was found to be 0.5 mmol/g. Finally, a fourth
loading with only 0.5 equiv. of amino acid was performed before the resin was capped
with Ac2O.
The solid-phase assembly of the peptide was continued despite the low initial
loading. No further complications were observed during the chain elongation, as judged
from TNBS tests after each coupling step. The peptide was cleaved from the resin and
the crude product analyzed by analytical HPLC. However, to our great surprise, a
complex mixture of peptides appeared to be present. Furthermore, none of the signals
had a retention time identical to that of the previously characterized nonapeptide 19.
Three main peaks in a ratio of ca. 1:3 :1 separated by ca. 4 min were observed, each
with a longer retention time than 19. The sample was purified by preparative HPLC,
and three fractions were collected. Analysis by ESI-MS showed that these products
corresponded to H-(b3-HAla-b3-HLys-b3-HPhe)3-(Phe)n-OH (n ˆ 1, 2, and 3), i.e., 23,
24, and 25, respectively.
Obviously, the C-terminal part of these peptides (two to four HPhe residues) was
formed during the repeated loading of the resin. The low degree of substitution must
have been caused by the loss of the Fmoc group during the coupling or washing steps. A
careful analysis of the solvents used revealed no inconsistencies. Thus, the uncontrolled

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Helvetica Chimica Acta Vol. 86 (2003)
NH2
NH2
O
NH2
O
O
O
O
O
O
O
O
O
H2N
N
H
N
H
N
H
N
H
N
H
N
H
N
H
N
H
N
H
OH
23
NH2
NH2
NH2
O
O
O
O
O
O
O
O
O
O
O
N
H
H2N
N
H
N
H
N
H
N
H
N
H
N
H
N
H
N
H
N
H
OH
24
NH2
NH2
NH2
O
O
O
O
O
O
O
O
O
O
O
O
N
H
N
H
OH
H2N
N
H
N
H
N
H
N
H
N
H
N
H
N
H
N
H
N
H
25
Fmoc deprotection must have taken place during the loading steps themselves12). Most
likely, catalytic amounts of DMAP caused this deprotection during the long reaction
times, since it seems unlikely that 1,3-diisoproylcarbodiimide (DIC) or the corre-
sponding urea compound are basic enough in DMF to promote Fmoc deprotec-
tion13 14).
)
2.3.3. Solid-Phase Synthesis of the Hexapeptide H-b3-HCys-(b3-HAla-b3-HLys-b3-
HPhe)5-OH. Part of the beads remaining from the 15-mer synthesis (cf. Sect. 2.2.3)
were used to attach an N-terminal b3-HCys residue, producing 26 (for possible
thioligation [15] or dimerization to the corresponding disulfide-linked peptide
containing 32 b-amino acids).
Hexadecapeptide 26 was obtained by adding Boc-b3-HCys(Trt)-OH15) to the resin
containing bound 19 21. The addition of this single amino acid proceeded smoothly
and provided the crude homocystein-substituted derivatives after cleavage and
12
)
)
)
)
This observation also explains why small quantities of the corresponding 21-mer peptide H-(b3-HAla-b3-
HLys-b3-HPhe)7-OH were isolated in the synthesis described in Sect. 2.2.3.
13
14
15
A 10% solution of DMAP is known to affect Fmoc-deprotection with t1/2 ˆ 85 min [22]; however, in this
case, only a catalytic amount of DMAP was used.
Partial deprotection of the Fmoc group during the normal peptide coupling would not be completely
surprising since DIPEA is known to cause gradual Fmoc deprotection after prolonged reaction times [22].
The decision to use a Boc-protected amino acid was rationalized by the availability of this amino acid
[23a,b] and the fact that this should be the N-terminal amino acid.

Helvetica Chimica Acta Vol. 86 (2003)
1531
deprotection. Although the whole series of peptides could again be obtained after
HPLC purification, only the hexadecamer 26 was collected and characterized.
In summary, a number of hurdles may be encountered when using the conventional
−single-amino-acid-at-a-time× procedure for the preparation of longer-chain b-peptides.
It was assumed that longer reaction times during the loading and coupling steps could
compensate for the high price of b-amino acids, especially since b3-amino acids do not
racemize. However, as evidenced by these observations, care must be taken regarding
the reaction times as well, especially during the loading step (Fmoc deprotection!).
2.4. Synthesis of a Water-Soluble b-Peptide Capable of Forming a 12/10-Helix, by
Solid-Phase Fragment Couplings of b2/b3-Dipeptides. The synthesis of peptides containing
b2-amino acids represents a special challenge, since b2-amino acids may, in contrast to
their b3-analogs, can racemize during activation (via oxazinone formation, cf. Scheme 1).
This racemization may be suppressed by means of less-strongly activating agents and by
keeping the temperature low. However, these factors, especially the temperature, are
difficult to control during SPPS; in fact, partial racemization was reported in our previous
syntheses of b2-amino acid containing peptides [19d].
Bearing these obstacles in mind, we figured that our combined solution- and solid-
phase approach would be especially attractive for the preparation of peptides containing
b2-amino acids. To demonstrate this, we set out to prepare the nonapeptide all-(S)-H-b3-
HPhe-b2-HVal-b3-HPhe-b2-HLeu-b3-HLys-b2-HLeu-b3-HLys-b2-HLeu-b3-HPhe-OH (34)
(Schemes 6 8).
Initially, the dipeptide building blocks Fmoc-b2-HVal-b3-HPhe-OH (28), Fmoc-b2-
HLeu-b3-HPhe-OH (30), and Fmoc-b2-HLeu-b3-HLys(Boc)-OH (33) were prepared
in solution, carbodiimide-mediated couplings (Schemes 6 and 7)16). The couplings
were run at 08 in the presence of HOBt to avoid racemization. Typically, the fully
protected dipeptides 27, 29, and 31 were obtained in > 90% diastereoisomer purity17),
which could be increased by recrystallization to > 98%, as judged from NMR and
HPLC analysis. C-Terminal deprotection and exchange of the N-terminal protection
from Boc- or Cbz groups for Fmoc (27 ! 28, 29 ! 30, and 31 ! 32 ! 33) provided the
desired dipeptide building blocks.
Having the necessary building blocks at hand, we started the solid-phase synthesis.
Prudent by our accumulated experiences, we again looked for possible improvements
in the procedures used. In order to avoid the presence of DMAP during the loading, we
instead turned to MSNT8) [24]. This procedure worked amazingly well. Despite the
fact that only 3 equiv. of dipeptide were employed, the ester formation with the Wang
resin proceeded in 82% yield. This is far more efficient than our previous syntheses on
Wang resins, even when a larger excess of amino acid or peptide was used. All couplings
proceeded smoothly and were finished within 24 h, as judged by TNBS tests. The high
coupling efficiency is remarkable, considering that 2 equiv. of the Fmoc-protected
dipeptide was used at most. In fact, the crude peptide 34 was found to be ca. 95% pure
16
)
The Boc-protected H-b2-HVal-OH (Scheme 6) was prepared from the Cbz-protected derivative
(Scheme 3). The only reason for using the Boc-protected amino acid was the fact that a sample was
available in our laboratory [23c].
The peptide coupling is expected to proceed without notable racemization under these conditions. The
main source of configurational impurity is associated with the lack of complete stereoselectivity in the
preparation of the b2-amino acid.
17
)

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Helvetica Chimica Acta Vol. 86 (2003)
Scheme 6. Solution-Phase Synthesis of the Fmoc-Protected Dipeptide Building Blocks 28 and 30. Carbodiimide
mediated coupling at 08 in the presence of HOBt efficiently suppresses racemization of the b2-amino acids.
Recrystallization of the fully protected dipeptides yields highly diasteroisomerically enriched materials (> 95%
pure) for SPPS. For abbrev., see Sect. 1 in the Exper. Part.
Scheme 7. Solution-Phase Synthesis of the Fmoc-Protected Dipeptide Building Block 33 (in diasteroisomerically
enriched form, >98%). For abbrev., see Sect. 1 in Exper. Part.

Helvetica Chimica Acta Vol. 86 (2003)
1533
Scheme 8. Solid-Phase Synthesis of the −Mixed× b-Peptide 34. The MSNT/MeIm protocol greatly increases the
efficiency of loading of the first dipeptide building block 30 onto the Wang resin. Subsequent chain elongation
with only 2 equiv. of the building blocks proceeded surprisingly well, yielding crude 34 in 95% purity. For
abbrev., see Sect. 1 in the Exper. Part.
after TFA cleavage! This is, so far, the most-effective solid-phase synthesis of a b-
peptide ever conducted in our laboratories.
The reason(s) for the high efficiency of this synthesis is probably related to the
slight modifications of the protocol in Scheme 8 and to the b2/b3-sequence, which might
be less prone to folding during synthesis, and which is known to give rise to better
solubility [8d]18).
The sequence of alternating b2- and b3-amino acids is expected to fold into a 12/10-helix [8d,e,o]. This helix
lacks a net dipole moment, which might affect its folding propensities under the conditions used for
peptide synthesis. b2/b3-Peptides with aliphatic side chains are highly soluble in organic solvents (CHCl3,
AcOEt), in contrast to their b3-counterparts [8d].
18
)

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Helvetica Chimica Acta Vol. 86 (2003)
3.Circular-Dichroism Studies. Circular dichroism (CD) is a tool widely used for
structural investigations of peptides and proteins. In contrast to a-peptides, where an
established correlation between CD spectra and secondary structures exists, such
reliable correlations are only beginning to accumulate for unnatural oligomers [25].
Thus, care should be taken when interpreting the CD spectra of b-peptides, at least until
more structures are unambiguously determined by alternative methods, such as NMR
spectroscopy and X-ray diffraction. Nevertheless, CD spectra are useful for obtaining a
first hint of the conformational preference of b-peptides. The CD spectra of 19 22,
23 26, and 34 are presented in Figs. 1 5.
Fig. 1. CD Spectra of the b-Peptides 19 22 (TFA salts). The spectra were recorded at 20.08 in MeOH at a conc.
of 0.2 mm. The spectrum of 19 resembles that of a −typical× 314-helix (dotted line), while the appearance of the
spectra of 20 22 are different.
The normalized CD spectrum of a b-heptapeptide adopting an (M)-314-helix in
MeOH solution, as confirmed by NMR spectroscopy, is shown in Fig. 1 [8k]. This CD
pattern (maximum near 200 nm, zero crossing between 205 and 210 nm, minimum
between 215 and 220 nm) has been observed in all cases where a 314-helical
conformation was established [8a,h,i,k]. Although recent theoretical studies have
shown that similar CD spectra may result from different conformations [26], our
practical experience suggests the following about the CD spectra of b-peptides
adopting the 314-helical conformation: i) a −stable× 314-helical conformation usually
displays both a positive and a negative band, ii) the intensity of the minimum between
215 and 220 nm correlates with the stability of the 314-helical conformation, and iii) a
low intensity of the maximum near 200 nm is often associated with both a broadening of

Helvetica Chimica Acta Vol. 86 (2003)
1535
the minimum and a hypsochromic shift interpreted as a loss of secondary structure19).
For the peptides 19 22 shown in Fig. 1, these guidelines would suggest that the b-
nonapeptide 19 adopts a 314-helical conformation in MeOH (the overall shape of the
spectrum), while the longer analogs 20 22 adopt alternative conformations to larger
extents when the chain lengths increase (less-intense maxima, broader minima at
shorter wavelength). This suggestion agrees with the fact that there are no indications
(CD, NMR, X-ray), so far, that b-peptides longer than 12 residues and containing
proteinogenic amino acid side chains adopt a 314-helical conformation in the absence of
special additives20).
The CD spectra of the two enantiomeric b-nonapeptides (19 and ent-19) composed
of all-(S)-b3- and all-(R)-b3-amino acids, respectively, are shown in Fig. 2. Clearly, these
b-peptides adopt 314-helices of different handedness, i.e., 19 an (M)-314-helix and ent-19
a (P)-314-helix.
Fig. 2. CD Spectra of the enantiomeric b-peptides 19 and ent-19. The spectra were recorded at 20.08 in MeOH at
a conc. of 0.2 mm. Peptide 19 was present as the HCl salt24), ent-19 as the TFA salt. Both peptides show spectra
characteristic for the 314-helical conformation; the slight difference in intensity is probably a result of the
different salts.
In Fig. 3, the normalized CD spectra of b-decapeptide 23, b-undecapeptide 24, and
b-dodecapeptide 25 are shown together with the spectrum of the −parent× b-peptide 19.
Obviously, the minimum around 215 nm intensifies when the chain length is increased
19
)
Similar changes in the CD spectra were observed for a b-dodecapeptide when the solvent was changed
from MeOH to H2O [27a]. Subsequent NMR spectroscopic analysis established that this b-peptide has a
314-helical conformation in MeOH, but is disordered in H2O [27b]. Salt bridges may be used to stabilize the
314-helix [8k].
CD Measurements in the presence of phospholipid micelles and vesicles indicate formation of an
amphiphilic 314-helix for 21 (data not shown) and similar b-peptides [9a,d].
20
)

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Helvetica Chimica Acta Vol. 86 (2003)
by b3-HPhe residues, (cf. 19, 23, and 24). This trend is not observed for the dodecamer
25 (loss of intensity of the minimum at 215 nm and of the maximum at 200 nm). Our
experience would suggest that the stability of the 314-helical conformation increases up
to the undecapeptide stage, while longer peptides appear to prefer this conformation to
a lesser extent.
Fig. 3. CD Spectra of b-peptides 23 25 compared to 19. All spectra were recorded at 20.08 in MeOH at a conc.
of 0.2 mm. All peptides were measured as their TFA salts. The minimum near 215 nm intensifies with increasing
chain length (addition of b3-HPhe residues; cf. 19, 23, and 24). The dodecamer 25 differs from this trend, and the
overall shape of the spectrum changes, perhaps indicating that the hydrophobic part of this molecule tends to
adopt a 314-helical conformation.
The CD spectrum of the homocysteine-containing b-peptide 26 is shown in Fig. 4,
together with the spectrum of the −parent× compound 21. Again, comparison suggests
that the b-peptide composed of more than twelve residues is less prone to adopt a 314-
helical conformation. In this case, the additional residue leads to a CD spectrum with an
even broader minimum at shorter wavelength. These spectra closely resemble those
obtained for a b-tetracosapeptide in MeOH and H2O [17]21).
The −mixed× b-peptide 34, composed of alternating b2- and b3-amino acid residues, is
expected to adopt the so-called 12/10-helix. High-resolution methods have established
that the CD spectrum of molecules adopting this conformation is characterized by a
very strong maximum near 205 nm [8d,e,o]. As can be seen in Fig. 5, the CD spectrum
of the nonapeptide 34 shows this typical pattern. Therefore, this peptide most likely
adopts the 12/10-helical conformation in MeOH solution22).
21
)
)
In this case, the dispersion of the NH region in 1H-NMR spectra points to a disordered structure [17] (see
also [27]).
A detailed NMR-spectroscopic analysis of 34 is in progress and will be reported in due course.
22

Helvetica Chimica Acta Vol. 86 (2003)
1537
Fig. 4. CD Spectrum of the homocysteine-containing b-hexadecapeptide 26 relative to the pentadecamer 21. Both
spectra were recorded at 20.08 in MeOH at a conc. of 0.2mm. Both peptides were present as their TFA salts. The
minimum observed near 215 nm for 21 is shifted hypsochromically in the case of 26. Similar spectra have been
observed before and correspond, most likely, to less-ordered conformations.
4.Conclusions. In this report, we have tried to impart our experiences on the SPPS
of longer-chain b-peptides. From the various approaches and protocols investigated, we
conclude that the initial loading of the first building block to Wang resin is best
performed using the MSNT/MeIm procedure8), which is efficient even when a small
excess of the amino acid partner is used in the reaction. Alternatively, other resins with
different anchoring groups may be used to increase the loading, which clearly is the
most material-consuming step. Amide-bond formation during chain assembly may be
executed with less than 2 equiv. of the amino acid or peptide to be coupled since the
coupling time may be substantially longer than that commonly used in a-peptide
synthesis. A longer coupling time is allowed when the COOH group of a b3-amino acid
is activated, since such amino acids do not racemize through the oxazinone pathway.
However, coupling times longer than 48h should be avoided since the DIPEA may
cause partial Fmoc deprotection.
The most-critical step during chain elongation is encountered when the growing b-
peptide chain is between seven and ten residues long, as witnessed by incomplete
Fmoc-deprotection and longer coupling times. This may indicate that the optimal
stability of the 314-helical conformation of b-peptides is found with six to twelve all-b3-
amino acid residues (even in solvents like DMF and NMP). For the synthesis of longer-
chain b-peptides, it is, thus, advantageous to decrease the number of couplings in this
range by using preformed, Fmoc-protected di- or tripeptide building blocks. The use of
preformed b2/b3-dipeptides as building blocks for the synthesis of −mixed× b2/b3-peptides
is particularly valuable, since this procedure completely eliminates epimerization at the

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Helvetica Chimica Acta Vol. 86 (2003)
Fig. 5. CD Spectrum of the mixed b2/b3-nonapeptide 34 (TFA salt). The spectrum was recorded at 20.08 in
MeOH at a conc. of 0.2mm. The very strong maximum observed near 205 nm is characteristic for the 12/10-
helical conformation.
b2-amino acid moiety. This approach is, therefore, recommended for the incorporation
of b2-amino acids in solid-phase (peptide) synthesis. The high overall efficiency of the
protocol (purity of crude product > 95%) is surprising and could be explained by the
stability of the 12/10-helical conformation in solvents like DMF and NMP.
Finally, the CD spectroscopic studies, although not as reliable as high-resolution
methods, suggest that b-peptides with a chain length exceeding twelve residues
composed of all-b3-amino acids might have a reduced tendency to adopt the 314-helical
conformation. This implies that special precautions and detailed design are required
before longer elements of b-peptidic secondary (or even tertiary) structures are
prepared. Yet, the lessons learned from the present investigation have taken us several
steps closer to the ultimate goal of production of de novo designed −b-proteins×!
We thank the Swedish Foundation for International Cooperation in Research and Higher Education
(STINT) (P.I.A.) and the Deutscher Akademischer Austauschdienst (Hochschulsonderprogramm III) (J. F.) for
postdoctoral fellowships. Dr. S. Abele, Dr. A. Jacobi, and Dr. A. Bˆhm kindly supplied samples of various amino
acid derivatives. Continuing generous support from Novartis Pharma AG (Basel) is gratefully acknowledged.
Experimental Part
1. General. Abbreviations: Boc ˆ (tert-butoxy)carbonyl, DBU ˆ 1,8-diazabicyclo[5.4.0]undec-7-ene,
DIC ˆ 1,3-diisopropylcarbodiimide, DIPEA ˆ N,N-diisopropyl(ethyl)amine, DMAP ˆ 4-(dimethylamino)pyri-
dine, DMF ˆ dimethylformamide, EDC ˆ 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide hydrochloride,
FC ˆ flash chromatography, Fmoc-OSu ˆ N-{[(9H-fluoren-9-yl-methoxy)carbonyl]oxy}-succinimide, GP ˆ
general procedure, HBTU ˆ N,N,N',N'-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate,

Helvetica Chimica Acta Vol. 86 (2003)
1539
HOBt ˆ 1-hydroxy-1H-benzotriazole hydrate, MeIm ˆ 1-methyl-1H-imidazole, MSNTˆ 1-(mesitylsulfonyl)-3-
nitro-1H-1,2,4-triazole, NMM ˆ N-methylmorpholine, NMP ˆ N-methylpyrrolidin-2-one, PE ˆ petroleum
ether (40/60), TFA ˆ trifluoroacetic acid, TFE ˆ 2,2,2-trifluoroethanol, TIS ˆ triisopropylsilane, TNBS ˆ
2,4,6-trinitrobenzenesulfonic acid; three-letter amino acid abbreviations are used for a-amino acids (Xaa), b-
HXaa ˆ b-homoamino acid [8a][28].
THF was freshly distilled over Na/benzophenone under Ar before use. CHCl3 for optical rotation
measurements (ORD) was filtered over basic Al2O3 (Woelm N, actvity I) to remove EtOH. Solvents for
chromatography and workup procedures were distilled from Sikkon (hexane, AcOEt, MeOH), P2O5 (CH2Cl2,
CHCl3), and KOH/FeSO4 (Et2O). Et3N was distilled from CaH2 and stored over KOH. i-BuOCOCl was
distilled before use and stored at 48 under Ar. a-Amino-acid derivatives were purchased from Bachem, Senn,
Degussa, or Fluka. Fmoc-(R)-b3-HAla-OH (4b) and Fmoc (R)-b3-HPhe-OH (5b) were provided by S. Abele
[19d] and were recrystallized freshly before use. Cbz-(S)-b2-HVal-OH (7) was provided by A. Bˆhm, and Boc-
(R)-b3-HCys(Trt)-OH by A. Jacobi. Wang resin (100 200 mesh) was purchased from Novabiochem. HOBt Was
purchased from Fluka (containing 20% H2O) or from Senn (anh.). Ar was purchased from PanGas. All other
chemicals were used as received from Fluka or Novabiochem. Temps. were monitored with an internal digital
thermometer (Ebro TTX-690). Reactions carried out under the exclusion of light were performed in flasks
completely wrapped in aluminum foil. Caution: The generation and handling of diazomethane requires special
precautions [29]. Agitation in SPPS was achieved by bubbling N2 gas through the mixture. TLC: Merck silica gel
60 F254 plates; UV detection and dipping into solutions of a) ninhydrin (300 mg), AcOH (3 ml), and 1-butanol
(100 ml) or b) anisaldehyde (9.2 ml), AcOH (3.75 ml), conc. H2SO4 (12.5 ml), and EtOH (338ml), followed by
heating with a heat gun. FC: Fluka silica gel 60 (40 63 mm) at r.t. with a pressure of ca. 0.3 bar. Anal. HPLC:
Merck LaChrom HPLC system (L-7150 pump, L-7400 UV detector with variable-wavelength monitor), Merck
Lichrospher-100 column (CRP-18, 100  4.6 mm); unless otherwise stated, binary solvent system (A ˆ MeCN/
TFA 99.9 :0.1, B ˆ H2O/TFA 99.9 :0.1), flow rate 1.2 ml/min, UV detection at 220 nm. Prep. HPLC: Macherey-
Nagel column (Nucleosil 100-7 C18, 250 Â 21 mm), binary solvent system A/B (see anal. HPLC), flow rate 24 ml/
min, UV detection at 220 nm. M.p.: determined in open-end glass capillary tubes on a B¸chi 510 apparatus,
uncorrected. ORD: [a]D determined on a Perkin-Elmer 241 polarimeter (10 cm, 1 ml cell) at r.t.; solvent and the
conc. (in g/100 ml) are indicated. CD Spectra: on a Jasco J-710, from 190 to 250 nm at 25.08, 1-mm rectangular
cell, average of five baseline-corrected, smoothed scans, peptide conc. 0.2 mm or 0.02 mm; molar ellipticity V in
deg ¥ cm2 ¥ dmolÀ1 (l in nm). IR Spectra: on a Perkin-Elmer 782 spectrophotometer, as film, 1 2% CHCl3 soln.,
or KBr-pellets, maxima are classified as s (strong), m (medium), and w (weak); in cmÀ1 1H- and 13C-NMR
.
Spectra: on a Bruker AMX-500 (500/125 MHz), AMX-400 (400/100 MHz), ARX-300 (300/75 MHz), or Varian
Gemini 300 (300/75 MHz). Chemical shifts d in ppm relative to Me4Si (d ˆ 0 ppm) as internal standard, J values
in Hz. MS: ZAB2 SEQ (FAB; 3-nitrobenzylalcohol matrix), on Finnigan MAT TSQ 700 (ESI), or on IonSpec
Ultima 4.7-T FT ion cyclotron resonance (ICR) mass spectrometers; MALDI (matrix-assisted laser-desorption
ionization) in a 2,5-dihydroxybenzoic acid matrix; m/z in % of base peak. Elemental analyses were performed
by the Microanalytical Laboratory of the Laboratorium f¸r Organische Chemie, ETH Z¸rich. All peptides were
characterized by high-resolution (HR) electrospray MS, and their purity was checked by running samples on an
anal. HPLC column using two different gradients.
2. General Procedures (GP) for the Preparation of b-Amino Acids and the b-Tripeptide Building Block 16.
2.1. Synthesis of N-Boc- and N-Cbz-Protected Diazo Ketones (GP 1a). Similarly to the reported procedures
[19a,b,d], the corresponding a-amino acid was dissolved in THF (0.2m) under Ar and cooled to À258. After
addition of Et3N (1.05 equiv.) and i-BuOCOCl (1.05 equiv.), the mixture was stirred at À258 for 30 min. A soln.
of CH2N2 (2.60 equiv.) in Et2O was added to the resulting white suspension, and stirring was continued for 16 h.
Excess CH2N2 was destroyed by addition of AcOH, and the solvent was removed. The yellow residue was
dissolved in AcOEt and washed with sat. aq. NaHCO3, sat. aq. NH4Cl, and sat. aq. NaCl soln. The org. layer was
dried (MgSO4), filtered, and concentrated under reduced pressure. Finally, FC and/or recrystallization afforded
the desired pure diazo ketone.
2.2. Synthesis of N-Fmoc-Protected Diazo Ketones (GP 1b). The pertinent N-Fmoc-protected a-amino
acids were transformed to the corresponding diazo ketones following an optimized procedure [19d] using NMM
(1.05 equiv.) instead of Et3N.
2.3. Rearrangement of a-Diazo Ketones to N-Boc-protected b3-Amino Acids (GP 2a). Similarly to the
reported procedures [19a,b,d], the diazo ketone was dissolved in a 9 :1 mixture (0.25m) of THF and H2O and
then cooled to À258 under Ar and under exclusion of light. After 15 min at À258, a homogeneous soln. of
F3CCO2Ag (0.11 equiv.) in Et3N (2.9 equiv.) was added, and the resulting mixture was allowed to warm to r.t.
within 5 h in the dark. After filtration through Celite, the THF was removed under reduced pressure. The brown

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Helvetica Chimica Acta Vol. 86 (2003)
residue was diluted with sat. aq. NaHCO3 soln. and extracted with Et2O. The aq. layer was adjusted to pH 1
2
with 1n HCl at 08 and then extracted with AcOEt. The org. layer was dried (Na2SO4), filtered, and concentrated
under reduced pressure. Finally, FC and/or recrystallization furnished the pure N-protected b3-amino acids.
2.4. Rearrangement of a-Diazo Ketones to N-Fmoc-Protected b3-Amino Acids (GP 2b). The diazo ketone
was dissolved in THF (0.25m) containing 10% H2O and cooled to À258 under Ar in the dark. A soln. of
CF3CO2Ag (0.11 equiv.) in NMM (2.8equiv.) was added, and the mixture was stirred at r.t. for 4 h under
exclusion of light. Workup as in GP 2a, FC, and recrystallization afforded the pure N-Fmoc-protected b-amino
acids.
2.5. Rearrangement of a-Diazo Ketones to N-Boc-Protected b3-Amino Acid Benzyl Ester Derivatives (GP
2c). Similar to the reported procedure [30], the diazo ketone was dissolved in a mixture of anh. THF and anh.
BnOH (85 :15, 0.25m) and cooled to À258 under Ar and exclusion of light. A homogeneous soln. of silver
benzoate (0.11 equiv.) in Et3N (2.8equiv.) was added, and the resulting mixture was stirred at r.t. for 5 h in the
dark. Subsequent to filtration through Celite, the mixture was concentrated under reduced pressure, and BnOH
was removed by vacuum distillation. The residue was dissolved in AcOEt. After workup with sat. aq. Na2S2O3,
NaHCO3, NH4Cl, and NaCl soln. and drying (Na2SO4), the resulting crude product was purified by FC and
recrystallized to yield the desired b3-amino acid benzyl ester.
2.6. Rearrangement of a-Diazo Ketones to N-Protected b3-Amino Acid Methyl Ester Derivatives (GP 2d).
Similar to the reported procedure [19a,b,d], the diazo ketone was dissolved in anh. MeOH (0.25m) under Ar at
r.t., cooled to À258 under exclusion of light, and treated with a soln. of silver benzoate (0.11 equiv.) in anh. Et3N
(2.8equiv.). The resulting mixture was stirred at r.t. for 5 h in the dark and filtered through Celite. The solvent
was removed under reduced pressure, and the residue was taken up in AcOEt. Workup by washing the org. layer
with sat. aq. Na2S2O3, sat. aq. NaHCO3, sat. aq. NH4Cl, and sat. aq. NaCl soln., drying (Na2SO4), concentration
in vacuo, and FC afforded the desired N-protected b3-amino acid methyl ester.
2.7. Ultrasound-Mediated Rearrangement of a-Diazo Ketones to N-Fmoc-Protected b3-Amino Acids (GP
2e). Following [19e], the diazo ketone was dissolved in a mixture of dioxane and H2O (5 :1, 50 ml/mmol) under
Ar at r.t. Then, CF3CO2Ag (0.11 equiv.) was added, and the mixture was sonicated for 2 h under exclusion of
light. After filtration through Celite, the solvent was removed under reduced pressure, and the residue was taken
up in AcOEt. Workup by washing the org. layer with sat. aq. Na2S2O3, sat. aq. NaHCO3, sat. aq NH4Cl, and sat.
aq. NaCl soln., drying (Na2SO4), concentration in vacuo, FC, and recrystallization afforded the desired Fmoc-
protected b3-amino acid.
2.8. Boc-Deprotection of Amino Acids or Peptides (GP 3). The Boc-protected amino acid or peptide was
dissolved in CH2Cl2 (1m) at r.t. under Ar. After cooling to 08, 1 equiv. of TFA was added, and the resulting
homogeneous mixture was stirred for 2 h at r.t. The solvent was removed under reduced pressure, and the
resulting oily residue was stripped with Et2O (3Â). Finally, the residue was dried in vacuo overnight.
2.9. Cbz-Deprotection of Amino Acids or Peptides (GP 4). The Cbz-protected amino acid or peptide was
dissolved in MeOH or TFE (0.1m), and Pd/C (10%; 0.1 equiv.) was added after careful degassing and saturation
of the solvent with Ar [32]. The flask was evacuated, flushed with H2 several times, and the mixture was stirred
under H2 (1 bar) for 16 h. Subsequent filtration through Celite and concentration under reduced pressure
afforded the desired product, which was used in the following peptide coupling without further purification.
2.10. Peptide Coupling with b3-Amino Acids, EDC, and HOBt (GP 5a). The corresponding free acid and
unprotected amine derivatives were dissolved in anh. CHCl3 (0.5m) at r.t. under Ar. Then, Et3N (1.2 equiv.
without and 5 equiv. with TFA salts present) was added, and the mixture was cooled to 08. After stirring for
15 min at 08, HOBt (1.2 equiv.) was added, and stirring was continued for another 15 min. Then, EDC (1.2
equiv.) was added, and the resulting mixture was stirred at r.t. for 14 h. Dilution with CHCl3 was followed by
thorough washing with 1n HCl, sat. aq. NaHCO3, and sat. aq. NaCl soln. The org. layer was dried (Na2SO4),
filtered, and concentrated under reduced pressure. The resulting pale yellow product was purified by FC or
precipitation.
2.11. Peptide Coupling with b2-Amino Acids, EDC, and HOBt (GP 5b). The free acid and the corresponding
unprotected amine derivative were dissolved in anh. CHCl3 (0.5m) at r.t. After cooling to 08, NMM (1.2 equiv.
without and 3 equiv. with TFA salts present) was added, and the mixture was stirred for 15 min at 08 under Ar.
Then, HOBt (1.2 equiv.) was added, and stirring was continued for 15 min. Finally, EDC (1.2 equiv.) was added,
and the mixture was stirred for 24 h at 08. Subsequent dilution with CHCl3 was followed by thorough washing
with 1n HCl (3Â), sat. aq. NaHCO3 (2Â), and sat. aq. NaCl soln. (1Â). The org. layer was dried (Na2SO4),
filtered, and concentrated under reduced pressure. The resulting pale yellow product was purified by FC and/or
recrystallization.

Helvetica Chimica Acta Vol. 86 (2003)
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2.12. (S)-3-{[(tert-Butoxy)carbonyl]amino}-1-diazobutan-2-one (Boc-(S)-Ala-CHN2; (S)-1). Transforma-
tion of Boc-Ala-OH (18.9 g, 100.00 mmol) according to GP 1a and crystallization (Et2O/pentane) afforded (S)-
1 as yellow crystals (13.75 g, 65%). Spectroscopic data: [19b].
2.13. (S)-3-{[(tert-Butoxy)carbonyl]amino}-1-diazo-4-phenylbutan-2-one (Boc-(S)-Phe-CHN2; (S)-2a).
Transformation of Boc-Phe-OH (26.53 g, 100.00 mmol) according to GP 1a, FC (pentane/AcOEt 3 :1), and
crystallization (Et2O/pentane) afforded (S)-2a as pale yellow crystals (20.30 g, 70%). Spectroscopic data: [19b].
2.14. (S)-1-Diazo-3-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-4-phenylbutan-2-one (Fmoc-(S)-Phe-
CHN2; (S)-2b). Transformation of Fmoc-Phe-OH (8.00 g, 20.65 mmol) according to GP 1b, FC (CH2Cl2/Et2O
6 :1 ! 1:1), and crystallization (Et2O/pentane) afforded diazoketone (S)-2b as pale yellow crystals (5.60 g,
66%). Spectroscopic data: [19c].
2.15. (S)-3-{[(Benzyloxy)carbonyl]amino}-7-{[(tert-butoxy)carbonyl]amino}-1-diazoheptan-2-one (Cbz-
(S)-Lys(Boc)-CHN2; (S)-3a). Transformation of Cbz-Lys(Boc)-OH (38.05 g, 100.00 mmol) according to GP
1a, FC (pentane/AcOEt 5 :1 ! 2 :1), and crystallization (Et2O/pentane) afforded (S)-3a as yellow crystals
(29.70 g, 74%). Spectroscopic data were in accordance with those obtained for Fmoc-(S)-Lys(Boc)-CHN2 [31].
2.16. (R)-7-{[(tert-Butoxy)carbonyl]amino}-1-diazo-3-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)hep-
tan-2-one (Fmoc-(R)-Lys(Boc)-CHN2; (R)-3b). Transformation of Fmoc-Lys(Boc)-OH (5.00 g, 10.67 mmol)
according to GP 1b, FC (CH2Cl2/Et2O 6 :1 ! 1:1), and crystallization (CH2Cl2/pentane) yielded (R)-3b as pale
yellow crystals (4.40 g, 84%). For spectroscopic data of the (S)-enantiomer, see [31].
2.17. (S)-3-{[(tert-Butoxy)carbonyl]amino}butanoic Acid (Boc-(S)-b3-HAla-OH; (S)-4a). Rearrangement
of (S)-1 (13.75, 64.55 mmol) according to GP 2a and recrystallization (AcOEt/pentane) yielded (S)-4a (11.50 g,
56.60 mmol, 88%) as a colorless powder. Spectroscopic data were in agreement with those of ent-4a [19b].
2.18. Benzyl(S)-3-{[(tert-Butoxy)carbonyl]amino}-4-phenylbutanoate (Boc-(S)-b3-HPhe-OBn; (S)-5a).
Rearrangement of (S)-2a (20.30 g, 70.24 mmol) according to GP 2c, FC (AcOEt/pentane 1:6), and
recrystallization (AcOEt/pentane) furnished (S)-5a (21.50 g, 58.19 mmol, 83%) as a colorless powder.
Spectroscopic data: [26a].
2.19. (S)-3-({[(9H-Fluoren-9-yl)methoxy]carbonyl}amino)-4-phenylbutanoic Acid (Fmoc-(S)-b3-HPhe-
OH; (S)-5b). Rearrangement of (S)-2b (1.40 g, 3.41 mmol) according to GP 2e, FC (AcOEt/pentane), and
recrystallization (AcOEt/pentane) furnished (S)-5b (1.31 g, 3.27 mmol, 96%) as a colorless powder. Spectro-
scopic data: [31].
2.20. Methyl (S)-3-{[(Benzyloxy)carbonyl]amino}-7-{[(tert-butoxy)carbonyl]amino}heptanoate (Cbz-(S)-
b3-HLys(Boc)-OMe; (S)-6a). Rearrangement of (S)-3a (29.70 g, 73.52 mmol) according to GP 2d, FC (AcOEt/
pentane 1 :5), and recrystallization (CH2Cl2/pentane) afforded (S)-6a (24.00 g, 58.82 mmol, 80%) as a colorless
powder. M.p. 728. [a]rD:t: ˆ À18.9 (c ˆ 1.095, CHCl3). IR (CHCl3): 3451m, 3008m, 2942m, 1711s, 1509s, 1455m,
1
1438m, 1393w, 1367m, 1169m, 8 64w. H-NMR (400 MHz, CDCl3): 1.35 1.59 (m, 3 CH2); 1.43 (s, t-Bu); 2.53
2.55 (m, CH2CO); 2.95 3.07 (m, CH2N); 3.66 (s, MeO); 3.92 4.03 (m, NCH(R)CH2); 4.56 (br. s, NH); 5.09 (s,
PhCH2); 5.24 (d, J ˆ 8.6, NH); 7.29 7.40 (m, Ph). 13C-NMR (100 MHz, CDCl3): 23.19 (CH2); 28.43 (Me); 29.66
(CH2); 33.99 (CH2); 38.97 (CH2); 40.17 (CH2); 47.97 (CH); 51.69 (Me); 66.68(CH 2); 79.11 (C); 128.11 (CH);
‡
128.51 (CH); 136.55 (C); 156.0 (C); 156.07 (C); 171.95 (C). HR-MALDI-MS: 431.2153 (100, [M ‡ Na] ,
‡
‡
‡
C21H32N2O6 ; calc. 431.2150), 309.1811 (98.50, [M À Boc ‡ 2 H] , C16H25N2O4 ; calc. 309.1815). Anal. calc. for
C21H32N2O6 (408.59): C 61.75, H 7.90, N 6.86; found: C 61.62, H 7.97, N 6.80.
2.21. (R)-7-{[(tert-Butoxy)carbonyl]amino}-3-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)heptanoic
Acid (Fmoc-(R)-b3-HLys(Boc)-OH; 6b). Rearrangement of (R)-3b (4.40 g, 8.93 mmol) according to GP 2b,
FC (AcOEt/pentane/AcOH 10 :10 :1) and recrystallization (CHCl3/pentane) afforded Fmoc-(R)-b3-HLys-
(Boc)-OH (6b) (3.57 g, 7.41 mmol, 83%) as a colorless powder. Spectroscopic data corresponded to those of ent-
6b [19d].
2.22. (S)-2-{[(Benzyloxycarbonyl)amino]methyl}-3-methylbutanoic Acid (Cbz-(S)-b2-HVal-OH; (S)-7).
This material [23c] was prepared according to [19].
2.23. (S)-2-{[(Benzyloxycarbonyl)amino]methyl}-4-methylpentanoic Acid (Cbz-(S)-b2-HLeu-OH; (S)-
(8). This material was prepared according to [19].
2.24. Methyl (S)-3-Amino-7-{[(tert-butoxy)carbonyl]amino}heptanoate ((S)-b3-HLys(Boc)-OMe; (S)-(9).
Compound (S)-6a (24.00 g, 58.82 mmol, 1 equiv.) was deprotected according to GP 4 to afford (S)-9 as a
colorless oil (15.79 g, 57.64 mmol, 98%), which was used in the following peptide coupling without further
purification. [a]rD:t: ˆ ‡6.54 (c ˆ 1.055, CHCl3). IR (CHCl3): 3454w, 3008m, 2936m, 1709s, 1507s, 1438m, 1392w,
1367m, 1168s, 1016w, 8 62w. 1H-NMR (300 MHz, CDCl3): 1.15 1.38( m, 3 CH2); 1.42 (s, t-Bu); 2.10 2.15 (dd,
J ˆ 15.3, 6.3, 1 H, CH2CO); 2.20 2.24 (dd, J ˆ 15.3, 4.4, 1 H, CH2CO); 3.02 3.10 (m, CH2NH2, NCH(R)CH2);

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Helvetica Chimica Acta Vol. 86 (2003)
‡
‡
‡
3.64 (s, MeO); 4.58(br. s, NH). ESI-MS: 571.5 (9, [2M ‡ Na] ), 549.5 (35, [2M ‡ H] ), 297.3 (23, [M ‡ Na] ),
‡
275.3 (100, [M ‡ H] ).
2.25. (S)-3-{[(Benzyloxy)carbonyl]amino}butanoic Acid (Cbz-(S)-b3-HAla-OH; (S)-10). Compound (S)-
4a (11.50 g, 56.60 mmol) was deprotected following GP 3. The crude product was dissolved in 2m NaOH and
cooled to 58. Then, 4m NaOH and benzyl chloroformate (1.2 equiv.) were added simultaneously under vigorous
stirring at 58 over 1 h [33]. The mixture was stirred for 1 h at 58 and was washed with Et2O. The aq. layer was
acidified to pH 1 with conc. HCl, saturated with NaCl, and extracted with AcOEt (3Â). The combined org.
layers were dried (Na2SO4), filtered, and concentrated in vacuo to furnish a pale oil. The crude product was
crystallized from AcOEt/pentane to yield (S)-10 (11.70 g, 49.37 mmol, 87%) as a colorless solid. [a]rD:t: ˆ À15.7
(c ˆ 1.04, CHCl3). IR (CHCl3): 3437w, 3032m, 1713s, 1511s, 1454m, 1342m, 1248m, 1061m, 912w, 600w. 1H-NMR
(400 MHz, CDCl3): 1.22 (d, J ˆ 6.8, Me); 2.53 (d, J ˆ 4.2, CH2CO); 4.11 (br. s, CH); 5.08( s, PhCH2); 5.24 (m,
NH); 7.28 7.36 ( m, Ph), 8.75 (br. s, CO2H). 13C-NMR (100 MHz, CDCl3): 20.36 (Me); 40.43 (CH2); 43.84
(CH); 66.77 (CH2); 128.05 (CH); 128.13 (CH); 128.50 (CH); 136.27 (C); 155.77 (C); 176.37 (C). ESI-MS. 495.1
(4, [2M ‡ Na À 2 H]À), 473.2 (26, [2M À H]À), 236.1 (100, [M À H]À). Anal. calc. for C12H15NO4 (237.25):
C 60.75, H 6.37, N 5.90; found: C 60.79, H 6.30, N 5.71.
2.26. Cbz-(S)-b3-HAla-(S)-b3-HLys(Boc)-OMe (11). Coupling of (S)-10 (10.70 g, 45.15 mmol, 1.0 equiv.)
and (S)-9 (12.37 g, 45.15 mmol, 1.0 equiv.) with Et3N (7.54 ml, 54.18mmol, 1.2 equiv.) according to GP 5a
followed by FC (AcOEt/pentane 60 :40 ! AcOEt) afforded 11 (18.78 g, 38.09 mmol, 84%) as a colorless solid.
On a smaller scale (4.22 mmol), the yield could be increased to 91%. M.p.: 109 1118. [a]rD:t: ˆ À22.7 (c ˆ 1.02,
CHCl3). IR (CHCl3): 3432w, 3007m, 2979m, 1710s, 1505s, 1455m, 1367m, 1170s, 1105m, 1059m, 651w. 1H-NMR
(400 MHz, CDCl3): 1.23 1.25 (d, J ˆ 6.7, Me); 1.26 1.36 (m, CH2); 1.39 1.55 (m, 2 CH2); 1.43 (s, t-Bu); 2.32
(dd, J ˆ 14.5, 5.7, 1 H, CH2CO); 2.47 2.40 (m, 1 H, CH2CO); 2.50 (d, J ˆ 5.5, CH2CO); 2.51 (d, J ˆ 5.3, 1 H,
CH2CO); 3.06 (m, CH2NH); 3.66 (s, MeO); 4.01 4.07 (m, NHCH(R)CH2); 4.19 4.26 (m, NHCH(R)CH2);
4.63 (br. s, NH), 5.08( s, PhCH2), 5.68(br. s, NH); 6.25 (br. s, NH); 7.28 7.35 ( m, Ph). 13C-NMR (100 MHz,
CDCl3): 20.39 (Me); 23.10 (CH2); 28.40 (Me); 29.56 (CH2); 33.51 (CH2); 38.53 (CH2); 40.07 (CH2); 42.28
(CH2); 44.74 (CH); 45.81 (CH); 51.70 (Me); 66.46 (CH2); 79.06 (C); 127.98(CH); 128.44 (CH); 129.27 (CH);
‡
136.58(C); 155.73 (C); 156.09 (C); 170.27 (C); 172.09 (C). HR-MALDI-MS: 516.2685 (36, [ M ‡ Na] ,
‡
‡
‡
C25H39N3O7 ; calc. 516.2681); 416.2162 (35, [M À Boc ‡ H ‡ Na] , C20H31N3O5 ; calc. 416.2169); 394.2345 (100,
‡
‡
[M À Boc ‡ 2 H] , C20H32N3O5 ; calc. 394.2348). Anal. calc. for C25H39N3O7 (493.28): C 60.83, H 7.96, N 8.51;
found: C 60.97, H 8.01, N 8.50.
2.27. Cbz-(S)-b3-HAla-(S)-b3-HLys(Boc)-OH (12). The dipeptide 11 (12.00 g, 24.34 mmol, 1.0 equiv.) was
dissolved in THF (250 ml) and stirred for 15 min at r.t. Then, a 1.0m soln. of LiOH ¥ H2O (1.53 g, 36.51 mmol, 1.5
equiv.) in H2O (36.5 ml) was added. The yellow mixture was stirred for 16 h at r.t., treated with 1n HCl, and
extracted with AcOEt and CHCl3. The combined org. layers were dried (Na2SO4), filtered, and concentrated
under reduced pressure to yield 12 (11.43 g, 23.85 mmol, 98%) as a colorless solid, which was used in the
following coupling without further purification.
2.28. Benzyl-(S)-3-Amino-4-phenylbutanoate (H-(S)-b3-HPhe-OBn; (S)-13). Compound (S)-5a (9.27 g,
25.05 mmol) was deprotected according to GP 3. Concentration under reduced pressure and drying of the
residue in vacuo for 24 h yielded (S)-13 (9.59 g, 25.05 mmol, quant.) as its TFA salt and was used without further
purification.
2.29. Cbz-(S)-b3-HAla-(S)-b3-HLys(Boc)-(S)-b3-Phe-OBn (14). Dipeptide 12 (12.00 g, 25.05 mmol, 1.0
equiv.) and (S)-13 (6.74 g, 25.05 mmol, 1.0 equiv.) were subjected to EDC-mediated coupling with Et3N
(17.43 ml, 125.05 mmol, 5.0 equiv.) according to GP 5a. After workup, the crude product was dissolved in CHCl3/
MeOH 3 :1 and precipitated with MeCN to afford 14 (13.45 g, 18.42 mmol, 74%) as a colorless solid. Rf 0.74
(CH2Cl2/MeOH 9 :1). M.p. 184 1858. [a]rD:t: ˆ À2.62 (c ˆ 0.935, CHCl3/MeOH 3 :1). IR (KBr): 3308s, 3063w,
2974m, 2934m, 2858w, 1734s, 1682s, 1642s, 1538s, 1457m, 1366m, 1312m, 1267s, 1198m, 1170s, 1115m, 1062m,
1
980m, 908w, 8 67w, 750s, 698s. H-NMR (400 MHz, CD3OD): 1.15 1.30 (m, CH2); 1.19 1.21 (d, J ˆ 6.6, Me);
1.35 1.48( m, 2 CH2); 1.43 (s, t-Bu); 2.15 2.20 (dd, J ˆ 14.1, 6.3, 1 H, CH2CO); 2.26 2.33 (m, 3 H, CH2CO);
2.46 2.51 (dd, J ˆ 15.7, 7.1, 1 H, CH2CO); 2.54 2.59 (dd, J ˆ 15.7, 5.6, 1 H, CH2CO); 2.76 2.81 (dd, J ˆ 13.7, 7.5,
1 H, PhCH2); 2.84 2.89 (dd, J ˆ 13.7, 6.9, 1 H, PhCH2); 3.02 (m, CH2NH); 3.95 4.03 (m, 2 NHCH(R)CH2);
4.44 4.46 (m, NHCH(R)CH2); 5.02 (br. s, NH), 5.08( s, PhCH2O); 5.11 (s, PhCH2O); 5.52 (br. s, NH); 6.25 (br.
s, NH); 6.25 (br. s, NH); 7.12 7.40 (m, 3 Ph). 13C-NMR (100 MHz, CDCl3): 20.66 (Me); 23.29 (CH2); 28.51
(Me); 29.54 (CH2); 30.91 (CH2); 33.40 (CH2); 38.19 (CH2); 40.22 (CH2); 41.24 (CH2); 42.82 (CH2); 45.22 (CH);
47.39 (CH); 47.91 (CH); 66.77 (CH2); 66.94 (CH2); 79.45 (C); 126.88 (CH); 128.06 (CH); 128.26 (CH); 128.51
(CH); 128.59 (CH); 128.68 (CH); 128.72 (CH); 128.81 (CH); 129.47 (CH); 135.89 (C); 136.81 (C); 137.79 (C);
‡
156.58(C); 157.12 (C); 171.28(C); 171.36 (C); 171.66 (C). HR-MALDI-MS: 753.3289 (9, [
M ‡ Na] ,

Helvetica Chimica Acta Vol. 86 (2003)
1543
‡
‡
‡
C41H54N4O8 ; calc. 753.3834); 653.3333 (100, [M À Boc ‡ Na] , C36H46N4O6 ; calc. 653.3338); 631.3478 (6, [ M À
‡
‡
Boc ‡ H] , C36H46N4O6 ; calc. 631.3472). Anal. calc. for C41H54N4O8 (730.90): C 67.38, H 7.45, N 7.67; found:
C 67.28, H 7.65, N 7.64.
2.30. H-(S)-b3-HAla-(S)-b3-HLys(Boc)-(S)-b3-Phe-OH (15). Tripeptide 14 (5.80 g, 7.95 mmol) was
dissolved in TFE (320 ml) and deprotected according to GP 4. Filtration through Celite and concentration
under reduced pressure yielded 15 (4.23 g, 8.36 mmol; quant.) as a colorless powder. M.p. 196 1988. Rf 0.05
(CH2Cl2/MeOH 9 :1). [a]rD:t: ˆ À1.84 (c ˆ 0.89, CHCl3). IR (KBr): 3299s, 3007m, 2934s, 2876m, 1711s, 1646s,
1
1540s, 1454m, 1392s, 1366m, 1276m, 1251m, 1170s, 1032m, 8 65w, 747m, 701m. H-NMR (400 MHz, CD3OD):
1.15 1.28( m, CH2); 1.25 (d, J ˆ 6.7, Me); 1.30 1.49 (m, 2 CH2); 1.35 (s, t-Bu); 1.99 2.08( m, 1 H, CH2CO);
2.23 2.36 (m, 3 H, CH2CO); 2.61 2.77 (m, 1 H, CH2CO, PhCH2); 2.91 3.02 (m, CH2NH); 3.47 3.56 (m,
NHCH(R)CH2); 4.19 4.26 (m, NHCH(R)CH2); 4.30 4.40 (m, NHCH(R)CH2); 5.40 (br. s, NH), 7.07 7.19
(m, Ph). 13C-NMR (100 MHz, CDCl3): 18.81 (Me); 23.20 (CH2); 28.50 (Me); 29.61 (CH2); 33.31 (CH2); 39.36
(CH2); 40.33 (CH2); 41.84 (CH2); 42.42 (CH2); 44.74 (CH); 46.92 (CH); 48.34 (CH); 79.39 (C); 126.67 (CH);
128.53 (CH); 129.57 (CH); 138.15 (C); 156.97 (C); 170.14 (C); 171.46 (C); 178.65 (C). ESI-MS: 1013 (4, [2M ‡
‡
‡
‡
‡
H] ); 529 (8, [M ‡ Na] ); 507 (100, [M ‡ H] ); 407 (58, [M À Boc ‡ 2 H] ).
2.31. Fmoc-(S)-b3-HAla-(S)-b3-HLys(Boc)-(S)-b3-Phe-OH (16). Tripeptide 15 (3.81 g, 7.53 mmol, 1
equiv.) was suspended in a 1:1 mixture of dioxane (60.99 ml) and H2O (60.99 ml) [34]. After adding powdered
Na2CO3 (1.59 g, 15.07 mmol, 2 equiv.), the mixture was sonicated for 30 60 min (solubilization). The
homogeneous soln. was cooled to 08, Fmoc-OSu [35] (2.54 g, 7.53 mmol, 1 equiv.) was added, and the resulting
mixture was stirred for 24 h at r.t. The pH was adjusted to 1 2, the soln. was filtered (N4 filter), and the
colorless precipitate was dried in vacuo. The crude product was redissolved in CHCl3/MeOH 3 :1, precipitated
with MeCN, filtered, and dried in vacuo to yield 16 (5.10 g, 7.00 mmol, 93%) as a colorless powder. M.p. 207
2098. Rf 0.52 (CH2Cl2/MeOH 9 :1). Anal. HPLC (20 80% A in 20 min): tR 18.2 min (> 98%). [a]rD:t: ˆ À2.06
(c ˆ 1.02, CHCl3). IR (KBr): 3334s, 3064m, 2970m, 2956m, 1710s, 1684s, 1636s, 1598m, 1540s, 1449m, 1365m,
1259s, 1168m, 1116m, 1060m, 98 3w, 901w, 8 67w, 756m, 737s, 702m, 622m. 1H-NMR (500 MHz, DMSO): 0.95
1.10 (m, CH2); 1.20 (d, J ˆ 6.4, Me); 1.12 1.35 (m, 2 CH2); 1.37 (s, t-Bu); 2.02 2.18( m, 2 CH2CO); 2.24 2.28
(m, 1 H, CH2CO); 2.33 (d, J ˆ 7.9, 1 H, CH2CO); 2.68 2.75 ( m, PhCH2); 2.82 2.84 (m, CH2NH); 3.82 3.86
(m, NHCH(R)CH2); 3.88 3.94 (m, OCH2CH); 4.16 4.23 (m, NHCH(R)CH2); 4.24 4.30 (m,
NHCH(R)CH2, OCH2CH); 6.68 6.73 ( m, NH); 7.16 7.29 (m, 5 arom. H); 7.31 7.34 (m, 2 arom. H); 7.39
7.43 (m, 2 arom. H); 7.67 (d, J ˆ 7.4, 2 arom. H), 7.88 (d, J ˆ 7.5, 2 arom. H); 12.22 (s, CO2H). 13C-NMR
(125 MHz, DMSO): 20.16 (Me); 22.74 (CH2); 28.25 (Me); 29.31 (CH2); 32.98(CH 2); 37.78(CH 2); 39.83 (CH2);
40.26 (CH2); 41.13 (CH2); 42.51 (CH2); 44.22 (CH); 45.89 (CH); 46.74 (CH); 47.19 (CH); 65.13 (CH2); 77.25
(C); 120.08 (CH); 125.12 (CH); 126.07 (CH); 127.02 (CH); 127.57 (CH); 128.09 (CH); 129.07 (CH); 138.59 (C);
140.71 (C); 143.86 (C); 143.93 (C); 155.15 (C); 155.52 (C); 169.16 (C); 169.24 (C); 172.34 (C). HR-MALDI-
‡
‡
‡
‡
MS: 751.3672 (11, [M ‡ Na] , C41H52N4O8 ; calc. 751.3677); 651.3151 (58, [M À Boc ‡ Na] , C36H44N4O6 ; calc.
651.3155); 629.3319 (8, [M À Boc ‡ H] , C36H44N4O6 ; calc. 629.3322). ESI-MS: 1456 (4, [2M À H]À); 727 (100,
[M À H]À); 505 (69, [M À Fmoc]À). Anal. calc. for C41H52N4O8 (728.88): C 67.56, H 7.19, N 7.69; found: C 65.79,
H 7.02, N 7.40.
‡
‡
3. Solid-Phase Synthesis of Long-Chain b-Peptides via Coupling of Tripeptide Building Blocks. 3.1.
Anchoring of Fmoc-(S)-b3-HAla-(S)-b3-HLys(Boc)-(S)-b3-Phe-OH (16) on Wang Resin. Wang resin (2 g, initial
loading 0.99 mmol/g, 1 equiv.) was dried in vacuo for 1 h and swelled in NMP (20 ml) for 45 min. Peptide 16
(4.32 g, 5.94 mmol, 3 equiv.) was dissolved in NMP (30 ml) at 508. The homogeneous soln. was added to the
resin, followed by DIC (0.92 ml, 5.94 mmol, 3 equiv.), HOBt (0.46 g, 5.94 mmol, 3 equiv.), and a cat. amount of
DMAP. The suspension was agitated for 48h. The resin was filtered, washed with NMP (40 ml, 3 Â 5 min) and
CH2Cl2 (40 ml, 5 Â 5 min), and dried in vacuo overnight to afford 17. The peptide loading was determined by
measuring the absorbance of the dibenzofulvene piperidine adduct as follows: a defined amount of resin (ca.
5 mg) was suspended in piperidine/DMF 20 :80 (10.0 ml) in a measuring flask, and the Fmoc-deprotection was
allowed to continue for 1 h. The mixture was transferred to a UV cell, and the absorbance of the resulting soln.
was measured at 290 nm. The concentration (c in [mM]) of the benzofulvene adduct in soln. was determined by
means of a calibration curve23). The loading (Subst, in [mmol/g resin]) was then calculated according to Eqn. 1
[27a]. For 16, the loading (average of two measurements) was 0.50 mmol/g.
23
)
This calibration curve was obtained by plotting the absorbance of a series of benzofulvene piperidine
adducts, prepared by deprotection of Fmoc-Gly, as a function of concentration (0 1 mm).

1544
Helvetica Chimica Acta Vol. 86 (2003)
Substn ˆ cn ¥ Vn / {mrnesin À [cn ¥ Vn ¥ (MW À 18.0) / 1000]}
(1)
3.2. Solid-Phase Syntheses of Peptides 19 22. The loaded resin (17) (0.5 g, 0.25 mmol of 16) was put in a
solid-phase reactor and covered with NMP (5 ml). Unreacted OH groups were capped with Ac2O (0.5 ml,
5.0 mmol, 20 equiv.) and a cat. amount of DMAP (50 mg, 0.40 mmol) for 2.5 h under intensive N2 bubbling. The
resin was filtered and washed with NMP 2 :2 :96 (5 ml, 5 Â 1 min). Solid-phase synthesis was continued by
removing the Fmoc group with 1) 20% piperidine in NMP (5 ml, 2 Â 60 min), 2) DBU/piperidine/NMP (5 ml,
3 Â 60 min), and 3) 20% piperidine in NMP (5 ml, 1 Â 60 min). The resin was washed with NMP (5 ml, 6 Â
3 min), and the elongation sequence was initiated by addition of a homogeneous soln. of 16 (0.455 g,
0.625 mmol, 2.5 equiv.), HBTU (0.228g, 0.600 mmol, 2.4 equiv.), and HOBt (0.101 g, 0.625 mmol, 2.5 equiv.) in
NMP (5 ml), followed by addition of DIPEA (0.214 ml, 1.25 mmol, 5.0 equiv.). The slightly yellow suspension
was agitated by N2 bubbling for 72 h. Monitoring of the coupling reaction was performed with TNBS [36]. In the
case of a positive TNBS test (indicating incomplete coupling), the suspension was allowed to react for another
10 h with 16 (0.5 equiv.) and coupling reagents. The resin was filtered off, the filtrate was collected, dried, and
stored at À208. The dry resin was washed with NMP (5 ml, 6 Â 5 min) prior to Fmoc deprotection using the
following solutions: piperidine/NMP 1:4 (5 ml, 2 Â 60 min), DBU/piperidine/NMP 1:1:48(5 ml, 3 Â 60 min),
and piperidine/NMP 1:4 (5 ml, 1 Â 60 min). After filtration, the resin was washed with NMP (5 ml, 6 Â 3 min),
and SPPS was continued by repeating the following sequence. In each coupling step, a homogeneous soln. of 16
(0.455 g, 0.625 mmol, 2.5 equiv.), HBTU (0.228g, 0.600 mmol, 2.4 equiv.) and HOBt (0.101 g, 0.625 mmol, 2.5
equiv.) in NMP (5 ml) was added. Subsequent to initial mixing by N2 bubbling for 5 min, DIPEA (0.214 ml,
1.25 mmol, 5.0 equiv.) was added, and the resulting suspension was allowed to react for 48h. A soln. of an
additional amount of coupling reagents was added, i.e., HBTU (0.142 g, 1.5 equiv.), HOBt (0.061 g, 1.5 equiv.),
and DIPEA (0.128ml, 3.0 equiv.) when the −re-use× soln. (2 ml) was added. Thereafter, mixing was continued for
48h. When the desired sequence of the peptide was obtained, the resin was filtered off and washed with NMP
(5 ml, 6 Â 3 min). Final Fmoc deprotection was performed with mixtures of piperidine/NMP 1:4 (8ml, 2 Â
60 min), DBU/piperidine/NMP 1:1:48(8ml, 3 Â 60 min), and piperidine/NMP 1:4 (8ml, 1 Â 60 min). The
resin was filtered again and washed extensively with NMP (5 ml, 6 Â 4 min) and CH2Cl2 (8ml, 10 Â 1 min),
shrunk with MeOH (5 ml, 6 Â 3 min) and dried over KOH in vacuo for 24 h. The peptides were cleaved from the
resin by exposure to TFA/H2O 95 :5 (40 ml) for 2h. The resin was removed by filtration, washed with TFA, and
the org. layer was concentrated under reduced pressure. The oily residue was precipitated by addition of cold
Et2O, and the crude product was dried in vacuo. This methodology was used to prepare the 15-mer 21 and the 18-
mer 22. In the case of the 15-mer, 642 mg of crude product were obtained (59%), whereas the 18-mer synthesis
yielded 168mg of crude product (54%). Due to incomplete coupling, both crude products also contained the
lower homologs (9-mer 19, 12-mer 20, and, in the second synthesis, the 15-mer 21.
3.3. Solid-Phase Synthesis of 18. Following the coupling procedure described in Sect. 3.2., 17 (378mg resin,
0.189 mmol) was coupled with 16 (344 mg, 0.47 mmol, 2.5 equiv.) to afford 220 mg of crude product (92%
purity) after TFA-mediated cleavage and precipitation with cold Et2O.
3.4. HPLC Analysis and Purification of b-Peptides 18 22. RP-HPLC Analysis of the crude products were
performed as described in Sect. 1 using a linear gradient (10 100% A in 30 min). Crude products were purified
by prep. RP-HPLC using a gradient of A and B (20 55% A in 40 min, then 55 80% A in 10 min). The purified,
lyophilized peptides 18 22 were analyzed again by anal. RP-HPLC with a linear gradient of A and B (40 60%
A in 20 min).
3.5. H-(S)-b3-HAla-(S)-b3-HLys-(S)-b3-Phe-(S)-b3-HAla-(S)-b3-HLys-(S)-b3-Phe-OH (18). Purification
of the crude product resulting from the SPPS of 18 (220 mg) gave 163 mg of a colorless lyophilizate. Anal.
HPLC (15 50% MeCN in 20 min; 50 80% MeCN in 5 min; 80 98% MeCN in 5 min): tR 6.8min, ( > 98%).
1H-NMR (500 MHz, CD3OD): 1.11 (d, J ˆ 6.7, Me); 1.28 1.47 ( m, 4 CH2); 1.34 (d, J ˆ 6.7, Me); 1.52 1.73 (m, 2
CH2); 2.25 2.61 (m, 6 CH2CO); 2.69 2.90 (m, 2 CH2Ph, 2 CH2NH2); 3.66 3.71 (m, NHCH(R)CH2); 4.17
4.22 (m, NHCH(R)CH2); 4.26 4.35 (m, 2 NHCH(R)CH2); 4.39 4.45 (m, NHCH(R)CH2); 4.50 4.56 (m,
NHCH(R)CH2); 7.17 7.27 (m, 2 Ph); 7.79 (d, J ˆ 8.9, NH); 7.98 (d, J ˆ 8.9, NH); 8.09 (d, J ˆ 8.9, NH).
13C-NMR (125 MHz, CDCl3): 18.84, 20.92 (Me); 23.89, 23.98 (CH2); 28.09, 28.37 (CH2); 35.16, 35.29 (CH2);
39.47, 39.53 (CH2); 40.62, 40.65 (CH2); 41.28, 41.76 (CH2); 42.20, 42.26 (CH2); 43.56 (CH2); 44.22 (CH); 46.57
(CH); 47.35, 47.46 (CH); 49.59, 49.67 (CH); 127.65, 127.68(CH); 129.49, 129.53 (CH); 130.48, 130.54 (CH);
139.46, 139.53 (C); 171.55 (C); 171.83 (C); 172.04 (C); 172.35 (C); 172.80 (C); 175.03 (C). HR-MALDI-MS:
‡
‡
‡
‡
817.4948 (19, [M ‡ Na] , C42H66N8O7 ; calc. 817.4947); 800.4684 (100, [M À OH ‡ Na] , C42H65N8O6 ; calc.
‡
‡
‡
800.4688); 795.5118 (5, [M ‡ H] , C42H67N8O7 ; calc. 795.5116). ESI-MS: 817.6 (2, [M ‡ Na] ); 795.6 (4, [M ‡
H] ); 398.5 (100, [M ‡ 2 H]2‡).
‡

Helvetica Chimica Acta Vol. 86 (2003)
1545
3.6. H-(S)-b3-HAla-(S)-b3-HLys-(S)-b3-Phe-(S)-b3-HAla-(S)-b3-HLys-(S)-b3-Phe-(S)-b3-HAla-(S)-b3-
HLys-(S)-b3-Phe-OH (19). Purification of the crude product resulting from SPPS of 21 by RP-HPLC also
afforded 19 as its TFA salt (34 mg). Anal. HPLC (10 100% A in 30 min): tR 11.5 min (> 95%). 1H-NMR
(500 MHz, D2O): 1.02 1.30 (m, 6 CH2); 1.07 (d, J ˆ 6.6, 3 Me); 1.26 (d, J ˆ 6.7, Me); 1.52 1.73 (m, 3 CH2);
2.12 2.20 (m, CH2CO); 2.21 2.36 (m, 5 CH2CO); 2.38 2.55 ( m, 3 CH2CO); 2.60 2.72 (m, 2 PhCH2); 2.82
2.93 (m, PhCH2, 3 CH2NH2); 3.61 3.66 (m, NHCH(R)CH2); 3.88 3.97 (m, 3 NHCH(R)CH2); 4.07 4.15 (m,
2 NHCH(R)CH2); 4.32 4.46 (m, 3 NHCH(R)CH2); 7.20 7.32 (m, 3 Ph); 7.79 (d, J ˆ 8.9, NH); 7.98 (d, J ˆ 8.7,
NH); 8.09 (d, J ˆ 8.7, NH). 13C-NMR (125 MHz, D2O): 20.11 (Me); 22.09, 22.19, 22.28(CH 2); 24.67, 24.69, 24.73
(CH2); 28.57, 28.85, 28.91 (CH2); 35.02, 35.16 (CH2); 41.55, 41.62, 41.80 (CH2); 42.39 (CH2); 43.57 (CH2); 43.95
(CH2); 44.99, 46.05, 46.07, 47.59, 49.05, 49.19, 50.56, 51.14, 51.24 (CH); 129.43 (CH); 131.24, 131.27 (CH); 131.94,
132.03, 132.23 (CH); 140.51, 140.56, 140.72 (C); 173.65, 174.53, 174.55, 174.63, 174.70, 175.18, 175.36, 175.52,
‡
‡
177.89 (C). ESI-MS: 1207.0 (12, [M ‡ Na] ); 1184.0 (34, [M ‡ H] ); 592.4 (93, [M ‡ 2 H]2‡); 395.5 (100, [M ‡
3 H]3‡).
3.7. H-(S)-b3-HAla-(S)-b3-HLys-(S)-b3-Phe-(S)-b3-HAla-(S)-b3-HLys-(S)-b3-Phe-(S)-b3-HAla-(S)-b3-
HLys-(S)-b3-Phe-(S)-b3-HAla-(S)-b3-HLys-(S)-b3- Phe-OH (20). Purification of the crude product resulting
from the SPPS of 21 by RP-HPLC also afforded 20 as its TFA salt (58mg). Anal. HPLC (10 100% A in
30 min): tR 14.1 min (> 98%). 1H-NMR (500 MHz, CD3OD): 1.13 (d, J ˆ 6.8, Me); 1.14 (d, J ˆ 6.8, 2 Me); 1.30
1.60 (m, 8 CH2); 1.36 (d, J ˆ 6.8, Me); 1.61 1.75 (m, 4 CH2); 2.22 2.50 (m, 6 CH2CO); 2.51 2.72 (m, 6
CH2CO); 2.73 3.05 (m,
4 PhCH2 , 4 NH2CH2); 3.68 3.76 ( m, NHCH(R)CH2); 4.28 4.53 ( m, 8
NHCH(R)CH2); 4.62 4.69 (m, 2 NHCH(R)CH2); 4.72 4.81 (m, NHCH(R)CH2); 6.80 (m, NH); 7.13 7.28
(m, 4 Ph); 7.53 (d, J ˆ 8.5, NH); 7.77 (d, J ˆ 8.5, NH); 7.98 (d, J ˆ 8.6, NH); 8.15 (d, J ˆ 9.6, NH); 8.27 (m, NH);
8.36 (m, NH); 8.46 (d, J ˆ 8.8, NH). 13C-NMR (125 MHz, CD3OD): 18.79, 20.93 (Me); 21.21, 21.40, 23.91, 24.04,
24.41, 24.59, 28.02, 28.12, 28.69, 28.84, 35.89, 35.94, 36.64, 36.74, 39.17, 40.63, 40.69, 41.12, 41.51, 41.55, 41.62,
41.81, 42.19, 43.19, 43.25, 43.32, 43.52, 43.61 (CH2); 43.69, 43.81, 44.47, 46.63, 46.97, 47.17, 47.45, 47.55, 48.66, 49.28,
49.62, 49.78 (CH); 127.59, 127.63, 127.66 (CH); 129.28, 129.44, 129.49, 129.52 (CH); 130.48, 130.59, 130.62, 130.66
(CH); 139.30, 139.50, 139.72, 139.86 (C); 171.23, 171.32, 171.43, 171.59, 171.62, 171.82, 171.89, 171.98, 172.04,
‡
172.13, 173.09, 175.05 (C). ESI-MS: 1573.4 (5, [M ‡ H] ); 786.6 (62, [M ‡ 2 H]2‡); 524.9 (100, [M ‡ 3 H]3‡);
394.1 (40, [M ‡ 4 H]4‡).
3.8. H-(S)-b3-HAla-(S)-b3-HLys-(S)-b3-Phe-(S)-b3-HAla-(S)-b3-HLys-(S)-b3-Phe-(S)-b3-HAla-(S)-b3-
HLys-(S)-b3-Phe-(S)-b3-HAla-(S)-b3-HLys-(S)-b3-Phe-(S)-b3-HAla-(S)-b3-HLys-(S)-b3-Phe-OH (21). Purifi-
cation of the crude product resulting from the SPPS of 21 by RP-HPLC afforded 21 as its TFA salt (359 mg).
Anal. HPLC (10 100% MeCN in 30 min; C18): tR 16.6 min (> 96%). 1H-NMR (500 MHz, CD3OD): 1.12 (m, 3
Me); 1.32 1.78( m, 36 H, CH2 and Me); 2.20 2.49 (m, 8CH 2CO); 2.52 2.75 (m, 7 CH2CO); 2.78 3.02 ( m, 5
PhCH2, 5 NH2CH2); 3.66 3.75 (m, NHCH(R)CH2); 4.25 4.52 (m, 10 NHCH(R)CH2); 4.57 4.78( m, 4
NHCH(R)CH2); 7.08 7.26 ( m, 5 Ph); 7.54 (d, J ˆ 8.6, NH); 7.76 (d, J ˆ 8.5, NH); 7.99 (d, J ˆ 8.7, NH); 8.16 (d,
‡
‡
J ˆ 9.5, NH); 8.23 8.40 (m, NH); 8 .46 (d, J ˆ 8.9, NH). ESI-MS: 1983.7 (8, [M ‡ Na] ); 1961.7 (23, [M ‡ H] );
981.3 (52, [M ‡ 2 H]2‡); 654.2 (48, [M ‡ 3 H]3‡); 491.0 (100, [M ‡ 4 H]4‡); 395.4 (12, [M ‡ 5 H]5‡).
3.9. H-(S)-b3-HAla-(S)-b3-HLys-(S)-b3-Phe-(S)-b3-HAla-(S)-b3-HLys-(S)-b3-Phe-(S)-b3-HAla-(S)-b3-
HLys-(S)-b3-Phe-(S)-b3-HAla-(S)-b3-HLys-(S)-b3-Phe-(S)-b3-HAla-(S)-b3-HLys-(S)-b3-Phe-(S)-b3-HAla-(S)-
b3-HLys-(S)-b3-Phe-OH (22). Purification of the crude product resulting from the SPPS of 22 by RP-HPLC
afforded its TFA salt (91 mg). Anal. HPLC (10 100% A in 30 min): tR 18.8 min (> 98%). ESI-MS: 2349.7 (2,
‡
[M ‡ H] ); 1175.5 (4, [M ‡ 2 H]2‡); 783.9 (47, [M ‡ 3 H]3‡); 588.1 (100, [M ‡ 4 H]4‡); 470.7 (58.6, [M ‡ 5 H]5‡).
3.10. Counter-Ion Replacement of b-Peptides. The multiply charged peptides 18 22 were isolated as the
corresponding TFA salts (one TFA per basic site) after HPLC purification and lyophilization. Replacement of
these counter ions by ClÀ24) was accomplished by dissolving the peptide in 0.1m HCl (>100 equiv.) followed by
re-lyophilization. This step was repeated once. The oily residue was then lyophilized 3 Â from pure H2O or until
a fluffy white lyophilizate was obtained.
4. Solid-Phase Synthesis of b-Peptides by Coupling Single Fmoc-Protected b3-Amino Acids. 4.1. all-(R)-H-
(b3-HAla-b3-HLys-b3-HPhe)3-OH (ent-19). 4.1.1. Anchoring. Wang resin (0.3 g, initial loading 0.99 mmol/g, 1
equiv.) was dried in vacuo for 1 h and swelled in CH2Cl2 (10 ml) for 45 min. Fmoc-(R)-b3-HPhe-OH (R)-5b
(477 mg, 1.19 mmol, 4 equiv.), DIC (0.23 ml, 1.48mmol, 5 equiv.), and DMAP (3.6 mg, 0.03 mmol, 0.1 equiv.)
were dissolved in DMF (3 ml). The homogeneous soln. was added to the resin, and the suspension was mixed for
24
)
Replacement of the counter ions was done in order to avoid possible interference of TFA in antimicrobial
assays [9c].

1546
Helvetica Chimica Acta Vol. 86 (2003)
24 h. The resin was filtered, washed with DMF (5 ml, 3 Â 5 min), CH2Cl2 (5 ml, 6 Â 5 min), and MeOH (5 ml,
3 Â 5 min) and dried in vacuo overnight. The peptide loading (cf. Sect. 3.1) was found to be 0.33 mmol/g. A
second loading was performed with (R)-5b (240 mg, 0.59 mmol, 2 equiv.), DIC (0.12 ml, 0.74 mmol, 2.5 equiv.),
DMAP (3.6 mg, 0.03 mmol, 0.1 equiv.), and HOBt (106 mg, 0.65 mmol, 2.2 equiv.) in DMF (3 ml). After
washing, shrinking, and drying of the resin, the loading was determined to be 0.63 mmol/g.
4.1.2. Solid-Phase Synthesis. The resin with the anchored amino acid (R)-5b (300 mg, 0.63 mmol/g
substitution, 0.19 mmol) was put in a solid-phase reactor and covered with DMF (3 ml). Unreacted OH groups
were capped with Ac2O (0.28ml, 2.7 mmol) and a cat. amount of DMAP (33 mg, 0.27 mmol) overnight under
intensive N2 bubbling. The resin was filtered and washed with DMF (3 ml, 10 Â 1 min). The solid-phase
synthesis was continued by removing the Fmoc group with 20% piperidine in DMF (3 ml, 5 Â 30 min). The resin
was washed with DMF (3 ml, 6 Â 3 min), and the elongation sequence was initiated by addition of a
homogeneous soln. of the Fmoc-protected (R)-amino acid (0.567 mmol, 3 equiv.), HBTU (208mg, 0.548mmol,
2.9 equiv.), and HOBt (92 mg, 0.567 mmol, 3 equiv.) in DMF (3 ml), followed by addition of EtN(i-Pr)2
(0.188 ml, 1.1 mmol, 5.8 equiv.). The resulting slightly yellow suspension was mixed by N2 bubbling, and the
coupling reactions were monitored by withdrawing a small aliquot of the beads and subjecting them to the
TNBS test. Typically, 3 h of coupling were sufficient. A positive TNBS test (incomple coupling) was observed at
residue six (from the C-terminus), i.e., at the hexamer stage. In this case, another 1.5 equiv. of Fmoc-b3-HAla-
OH together with the corresponding quantities of coupling reagents were added, and the coupling was allowed
to proceed for another 1.5 h (still positive TNBS test). The Fmoc-deprotection protocol was then switched to 1)
20% piperidine in DMF (3 ml, 2 Â 30 min), 2) DBU/piperidine/DMF 1:1 :48(3 ml, 3 Â 30 min), and 3) 20%
piperidine in DMF (3 ml, 1 Â 30 min); this protocol was maintained through the rest of the synthesis. Similarly,
coupling of residue eight (Fmoc-b3-HLys(Boc)-OH) required 4 h. However, in this case, no additional amino
acid had to be added. After completing the synthesis and performing the final deprotection, the resin was
washed with DMF (5 ml, 6 Â 4 min) and CH2Cl2 (5 ml, 10 Â 1 min), shrunk with MeOH (5 ml, 6 Â 3 min), and
dried in vacuo for 24 h. Final cleavage was achieved with 40 ml of aq. TFA (95%). The resin was removed by
filtration and washed with TFA. The org. layer was concentrated under reduced pressure. The oily residue was
precipitated with cold Et2O, and the peptide was dried in vacuo to yield 238mg of the crude product as its TFA
salt.
4.1.3. HPLC Analysis and Purification of ent-19. As in Sect. 3.4. Anal. HPLC (10 100% A in 30 min): tR
‡
11.4 min (95%). ESI-MS (pos. mode): 1184.0 (10, [M ‡ H] ); 592.5 (90, [M ‡ 2 H]2‡); 395.5 (100, [M ‡ 3 H]3‡).
ESI-MS (neg. mode): 1182.0 (100, [M À H]À). Other data identical to those reported for 19.
4.2. Solid-Phase Synthesis of H-(b3-HAla-b3-HLys-b3-HPhe)3-(b3-HPhe)n-OH (n ˆ 1 3; 23 25). Wang
resin (500 mg, initial loading 0.99 mmol/g, 1 equiv.) and all glassware used were dried in vacuo overnight. Fmoc-
(S)-b3-HPhe-OH (800 mg, 1.98 mmol, 4 equiv.), DIC (0.39 ml, 2.5 mmol, 5 equiv.), and DMAP (10 mg,
0.08mmol, 0.15 equiv.) were dissolved in DMF (5 ml) and added to the resin. The suspension was allowed to
react for 24 h. The resin was filtered, washed with DMF (10 ml, 3 Â 5min), CH2Cl2 (10 ml, 6 Â 5 min), and
MeOH (10 ml, 3 Â 5 min), and dried in vacuo overnight. The loading was determined as described in Sect. 3.1.
Due to an extraordinarily low loading of < 0.1 mmol/g, two more loadings were performed using Fmoc-(S)-b3-
HPhe-OH (600 mg, 1.5 mmol, 3 equiv.), DIC (0.29 ml, 1.9 mmol, 3.8equiv.), and DMAP (10 mg, 0.08mmol,
0.15 equiv.) in DMF (5 ml) for 24 h. The degree of substitution was raised in this way to 0.25 and 0.50 mmol/g,
resp. A fourth attempt to increase the loading was performed using Fmoc-(S)-b3-HPhe-OH (400 mg, 1.0 mmol,
2 equiv.), DIC (0.20 ml, 1.25 mmol, 2.5 equiv.), and DMAP (5 mg, 0.05 mmol, 0.1 equiv.) in DMF (5 ml)
overnight. The mixture was allowed to further react for 1 h at elevated temp. (gentle heating with a heat-gun)
and 6 h at r.t. The resin was washed with DMF (10 ml, 6 Â 5 min) and capped with Ac2O (0.5 ml, 5.3 mmol) and a
cat. amount of DMAP (64 mg, 0.5 mmol) overnight. A final analysis after washing, shrinking, and drying
indicated a lowered (!) loading of 0.26 mmol/g. Despite these puzzling observations (see discussion), we
decided to continue with the solid-phase synthesis by removing the Fmoc group with DBU/piperidine/DMF
1:1:48(5 ml, 6 Â 30 min) followed by washing with DMF (5 ml, 6 Â 3 min). The elongation sequence was
initiated by addition of a homogeneous soln. of the Fmoc-protected amino acid (0.625 mmol, 2.5 equiv.), HBTU
(230 mg, 0.606 mmol, 2.4 equiv.), and HOBt (101 mg, 0.62 mmol, 2.5 equiv.) in DMF (5 ml), followed by
addition of DIPEA (0.21 ml, 1.25 mmol, 5.0 equiv.). The resulting slightly yellow suspension was mixed by N2
bubbling. The coupling reactions were monitored by withdrawing a small aliquot of the beads and subjecting
them to the TNBS test. Typically, 3 h of coupling were sufficient. No particular difficulties were encountered
during the chain elongation. After completing the synthesis and performing the final Fmoc deprotection, the
resin was washed carefully with DMF (10 ml, 6 Â 4 min) and CH2Cl2 (10 ml, 10 Â 1 min), shrunk with MeOH
(10 ml, 6 Â 3 min), and finally dried in vacuo for 24 h. The dried resin was treated for 2.5 h with TFA/H2O 95 :5

Helvetica Chimica Acta Vol. 86 (2003)
1547
(80 ml). The resin was removed by filtration, washed with TFA, and the org. layer was concentrated under
reduced pressure. The product was precipitated with cold Et2O and dried in vacuo to yield 417 mg of crude
product (TFA salt). The peptides were purified by prep. RP-HPLC (30 70% A in 40 min) and were lyophilized
to yield 18, 58 and 10 mg of 23, 24, and 25, resp., as TFA salts.
Data of H-(S)-b3-HAla-(S)-b3-HLys-(S)-b3-Phe-(S)-b3-HAla-(S)-b3-HLys-(S)-b3-Phe-(S)-b3-HAla-(S)-
b3-HLys-(S)-b3-Phe-(S)-b3-Phe-OH (23). Anal. HPLC (20 80%
A in 20 min): tR 11.3 min (> 98%).
1H-NMR (500 MHz, CD3OD): 1.06 1.57 (m, 6 CH2); 1.09 (d, J ˆ 6.5, CH3); 1.21 (d, J ˆ 6.6, Me); 1.37 (d,
J ˆ 6.6, Me); 1.61 1.82 (m, 3 CH2); 2.25 2.75 (m, 10 CH2CO, 3 PhCH2); 2.82 2.93 (m, PhCH2, 3 NH2CH2);
3.14 3.18( m, NHCH(R)CH2); 3.70 3.78( m, NHCH(R)CH2); 4.30 4.48( m, 6 NHCH(R)CH2); 4.52 4.60
(m, NHCH(R)CH2); 4.63 4.68( m, NHCH(R)CH2); 7.06 7.28( m, 4 Ph); 7.48( d, J ˆ 9.0, NH); 7.78( d, J ˆ 9.3,
NH); 7.91 (d, J ˆ 9.2, NH); 8.16 (d, J ˆ 8.5, NH); 8.23 (d, J ˆ 9.5, NH); 8.26 (m, 3 NH); 8.42 (m, 2 NH).
13C-NMR (125 MHz, CD3OH): 18.99, 21.27, 21.53 (Me); 22.70, 22.84, 23.13 (CH2); 23.59, 23.89, 23.94 (CH2);
24.54 (CH2); 28.22, 28.92, 29.03 (CH2); 35.58, 36.04, 36.09 (CH2); 40.96, 41.00, 41.34 (CH2); 42.57 (CH2); 43.36
(CH2); 43.95 (CH2); 45.94, 46.95, 47.10, 47.55, 48.00, 48.58, 49.45, 49.79, 49.93 (CH); 127.62 (CH); 129.32, 129.49,
129.52, 129.58(CH); 130.51, 130.59, 130.62, 130.64 (CH); 139.35, 139.42, 139.52, 139.64 (C); 171.19, 171.36,
‡
171.56, 171.76, 171.93, 172.10, 172.23, 172.31, 173.26, 175.45 (C). ESI-MS: 1367.0 (11, [M ‡ Na] ); 1345.1 (42,
‡
[M ‡ H] ); 672.8(47, [ M ‡ 2 H]2‡); 449.1 (100, [M ‡ 3 H]3‡).
Data of H-(S)-b3-HAla-(S)-b3-HLys-(S)-b3-Phe-(S)-b3-HAla-(S)-b3-HLys-(S)-b3-Phe-(S)-b3-HAla-(S)-
b3-HLys-(S)-b3-Phe-(S)-b3-Phe-(S)-b3Phe-OH (24). Anal. HPLC (20 80% A in 20 min): tR 14.6 min
(> 98%). 1H-NMR (500 MHz, CD3OD): 1.14 (d, J ˆ 6.6, Me); 1.21 (d, J ˆ 6.6, Me); 1.38( d, J ˆ 6.7, Me);
1.39 1.75 (m, 9 CH2); 2.22 2.77 (m, 11 CH2CO, 3 CH2Ph); 2.79 2.97 (m, 2 CH2Ph, 3 NH2CH2); 3.70 3.75 (m,
2 NHCH(R)CH2); 4.32 4.37 (m, 2 NHCH(R)CH2); 4.38 4.47 ( m, 4 NHCH(R)CH2); 4.52 4.70 (m, 4
NHCH(R)CH2); 4.63 4.68( m, NHCH(R)CH2); 7.08 7.29 ( m, 5 Ph); 7.54 (d, J ˆ 9.6, NH); 7.71 (d, J ˆ 9.3,
NH); 7.74 (m, 2 NH); 7.91 (d, J ˆ 8.9, NH); 8.04 (d, J ˆ 8.8, NH); 8.17 (d, J ˆ 9.5, NH); 8.23 (d, J ˆ 8.9, 2 NH);
8.27 (d, J ˆ 9.0, NH); 8.46 (d, J ˆ 8.9, 2 NH). 13C-NMR (125 MHz, CD3OD): 18.99, 21.29, 21.67 (Me); 23.89,
24.61, 24.64, 28.17, 29.07, 29.09, 36.03, 36.84, 36.97, 39.19, 40.72, 40.95, 40.99, 41.01, 41.36, 41.56, 41.71, 41.89, 42.26,
42.44, 42.85, 43.18, 43.41, 43.50, 43.64 (CH2); 44.00, 44.07, 46.94, 47.04, 47.55, 48.55, 49.45, 49.63, 49.69, 49.79,
49.94 (CH); 127.59, 127.69, 127.69, 127.75 (CH); 129.27, 129.38, 129.50, 129.56 (CH); 130.49, 130.52, 130.57,
130.61, 130.67 (CH); 139.19, 139.40, 139.52, 139.54, 139.61 (C); 171.05, 171.20, 171.54, 171.62, 171.76, 172.09,
‡
‡
172.34, 172.38, 173.28, 175.24 (C). ESI-MS: 1529.2 (20, [M ‡ Na] ); 1507.2 (37, [M ‡ H] ); 753.4 (34, [M ‡
2 H]2‡); 502.8(100, [ M ‡ 3 H]3‡).
Data of H-(S)-b3-HAla-(S)-b3-HLys-(S)-b3-Phe-(S)-b3-HAla-(S)-b3-HLys-(S)-b3-Phe-(S)-b3-HAla-(S)-
b3-HLys-(S)-b3-Phe-(S)-b3-Phe-(S)-b3-Phe-(S)-b3-Phe-OH (25). Anal. HPLC (20 80% A in 20 min.): tR
18.0 min (> 98%). 1H-NMR (500 MHz, CD3OD): 1.13 1.77 (m, 9 CH2); 1.14 (d, J ˆ 6.0, Me); 1.21 (d, J ˆ
6.0, Me); 1.38( d, J ˆ 6.0, Me); 2.22 2.82 (m, 12 CH2CO, 4 PhCH2); 2.83 3.00 (m, 2 PhCH2, 3 NH2CH2); 3.74
(m, NHCH(R)CH2); 4.34 4.50 (m, 6 NHCH(R)CH2); 4.51 4.73 (m, 5 NHCH(R)CH2); 7.06 7.28( m, 6 Ph);
7.47 (d, J ˆ 9.6, NH); 7.71 (d, J ˆ 9.3, NH); 7.92 (d, J ˆ 9.0, NH); 8.05 8.15 (m, 2 NH); 8.19 (d, J ˆ 9.0, NH);
8.24 (m, 3 NH); 8.28 (d, J ˆ 9.0, NH); 8.48 (m, 2 NH). 13C-NMR (125 MHz, CD3OD): 19.00, 21.30, 21.67 (Me);
23.89, 24.65, 24.76, 28.15, 29.09, 30.78, 36.02, 36.87, 37.18, 39.42, 40.96, 41.01, 41.37, 41.69, 42.07, 42.24, 42.68, 43.19,
43.40, 43.64 (CH2); 44.04, 46.94, 47.56, 47.67, 48.37, 48.66, 49.46, 49.63, 49.79 (CH); 127.67 (CH); 129.32, 129.50,
129.54 (CH); 130.51, 130.60 (CH); 139.32, 139.51 (C); 171.29, 171.51, 171.77, 172.12, 172.37, 173.29, 175.31 (C).
ESI-MS: 1690.3 (15, [M ‡ Na] ); 1667.2 (42, [M ‡ H] ); 834.3 (80, [M ‡ 2 H]2‡); 556.4 (100, [M ‡ 3 H]3‡).
4.3. Solid-Phase Synthesis and Purification of the Cys-Containing b-hexadecapeptide 26. A part of the resin
remaining from the SPPS of 21 (cf. Sect. 3) was used for the synthesis of a b3-HCys-containing analog of this
peptide. The resin (160 mg; ca. 0.064 mmol) containing the bound peptides 19 21 was placed in a reactor and
allowed to swell in CH2Cl2 for 1 h. The Fmoc group was removed with DBU/piperidine/DMF 1:1:48(3 ml, 6 Â
10 min). After washing with NMP (3 ml, 9 Â 1 min) and CH2Cl2 (3 ml, 1 Â 1 min), a soln. of Boc-b3-HCys(Trt)-
OH (148mg, 0.32 mmol, 5 equiv.), HBTU (116 mg, 0.31 mmol, 4.8equiv.), and HOBt (52 mg, 0.32 mmol, 5
equiv.) in NMP (3 ml) was added to the resin, followed by DIPEA (0.11 ml, 0.64 mmol, 10 equiv.). The mixture
was allowed to react overnight (negative TNBS test). The resin was washed with NMP (5 ml, 10 Â 1 min) and
CH2Cl2 (5 ml, 10 Â 1 min), shrunk with MeOH (5 ml, 5 Â 1 min), and dried in vacuo. The peptide was cleaved
from the resin and simultaneously deprotected with a mixture of TFA/H2O/EDT/TIS 94.5 :2.5 :2.5 :1 (20 ml) for
3 h to yield after evaporation, precipitation (Et2O), washing (Et2O), and drying 170 mg of crude product.
The crude material was found to contain mainly three components: the b3-HCys derivative of 19 (tR 12.5 min,
8.6%) and 20 (tR 20.0 min, 15%), resp., and the desired peptide 26 (tR 25.5 min, 47%).
‡
‡

1548
Helvetica Chimica Acta Vol. 86 (2003)
Data for H-(S)-b3-Cys-(S)-b3-HAla-(S)-b3-HLys-(S)-b3-Phe-(S)-b3-HAla-(S)-b3-HLys-(S)-b3-Phe-(S)-b3-
HAla-(S)-b3-HLys-(S)-b3-Phe-(S)-b3-HAla-(S)-b3-HLys-(S)-b3-Phe-(S)-b3-HAla-(S)-b3-HLys-(S)-b3-Phe-OH
‡
(26). Anal. HPLC (20 80% A in 20 min): tR 16.5 min (> 98%). ESI-MS: 2076.6 (5, [M ‡ H] ); 1039.8(13,
[M ‡ 2 H]2‡); 693.2 (67, [M ‡ 3 H]3‡); 520.2 (100, [M ‡ 4 H]4‡); 416.6 (17.4, [M ‡ 5 H]5‡).
5. Preparation of b2/b3-Dipeptide Building Blocks 28 33. 5.1. Boc-(S)-b2-HVal-(S)-b3-HPhe-OBn (27).
Boc-(S)-b3-HPhe-OBn ((S)-5a) (320 mg, 0.865 mmol, 1.0 equiv.) was deprotected according to GP 3. The
resulting oily residue was coupled to Boc-(S)-b2-HVal-OH16) (200 mg, 0.86 mmol, 1.0 equiv.) according to GP
5b. FC (AcOEt/pentane 2 :11 ! 1:1) Afforded 27 (408 mg, 0.845 mmol, 98%) as a colorless solid. NMR
Spectroscopic analysis showed that the product was a 85 :15 mixture of diastereoisomers due to the low
enantiomeric purity of the starting Boc-(S)-b2-HVal-OH. Recrystallization from AcOEt/hexane enhanced this
ratio to 94 :6 (70% yield). M.p. 166 1688. Rf 0.33 (AcOEt/pentane 1:2). [a]rD:t: ˆ ‡16.2 (c ˆ 0.5, CHCl3). IR
1
(CHCl3): 3650w, 2900s, 1730s, 1470s, 1380s, 1230s. H-NMR (400 MHz, CDCl3): 0.74 (d, J ˆ 6.6, Me); 0.86 (d,
J ˆ 6.7, Me); 1.40 (s, t-Bu); 1.72 1.81 (m, CH(CHMe2)2); 1.90 1.98( m, CH(R)CO); 2.52 2.57 (m, PhCH2);
2.79 2.84 (dd, J ˆ 13.7, 7.5, CHHCO); 2.87 2.93 (dd, J ˆ 13.6, 7.4, 1 H, CH2CO); 3.11 3.20 (m, 1 H,
BocHNCH2); 3.30 3.38( m, 1 H, BocHNCH2); 4.51 4.58( m, NHCH(R)CH2); 5.03 (br. s, NH); 5.09 5.12 (d,
J ˆ 12.2, 1 H, PhCH2O); 5.09 5.12 (d, J ˆ 12.2, 1 H, PhCH2O); 6.05 (br. s, NH); 7.12 7.15 (m, 2 arom. H);
7.18 7.23 ( m, 1 arom. H); 7.25 7.30 (m, 2 arom. H); 7.34 7.40 (m, 5 arom. H). 13C-NMR (100 MHz, CDCl3):
19.97 (Me); 20.67 (Me); 23.59 (CH); 28.42 (Me); 37.66 (CH2); 40.01(CH2); 40.61 (CH2); 47.35 (CH); 54.07
(CH); 66.64 (CH2); 79.09 (C); 128.80 (CH), 128.46 (CH); 128.50 (CH); 128.65 (CH); 128.68 (CH); 129.58
‡
(CH); 135.55 (C); 137.36 (C); 156.63 (C); 173.69 (C); 174.42 (C). HR-MALDI-MS: 505.2673 (95, [M ‡ Na] ,
‡
C28H38N2O5Na; calc. 505.6016); 405.2 (80, [M À t-Bu ‡ Na] , C24H37N2O7Na; calc. 502.2524); 383.2 (100, [M À t-
‡
Bu] . Anal. calc. for C28H38N2O5 (482.62): C 69.86, H 7.94, N 5.80; found: C 69.83, H 7.95, N 5.98.
5.2. Fmoc-(S)-b2-HVal-(S)-b3-HPhe-OH (28). Boc-(S)-b2-HVal-(S)-b3-HPhe-OBn (27) (0.292 g,
0.60 mmol) was deprotected according to GP 4 and GP 3, which afforded the free dipeptide in quantitative
yield. The residue was directly dissolved in a 1:1 mixture of dioxane/H2O (0.062m) before Na2CO3 (0.136 g,
1.21 mmol, 2 equiv.) was added. Then, Fmoc-OSu [35] (0.204 g, 0.60 mmol, 1 equiv.) was added, the mixture was
sonicated for 60 min and then allowed to react for 24 h at r.t. The pH was adjusted to 1 2, and AcOEt was
added to dissolve the white precipitate formed. The org. layer was washed with H2O (4 Â 20 ml), dried
(Na2SO4), and evaporated to a white solid. The solid was dissolved in a soln. of 5% MeOH in CH2Cl2 (insoluble
materials being removed by filtration), and precipitated by addition of pentane. Filtration and drying in vacuo
afforded 261 mg (82%) of 28 as a colorless powder. This material was pure enough to be used in SPPS. An anal.
sample was obtained by FC (MeOH/CH2Cl2 1:10, 1% AcOH). M.p. 2268. Rf 0.10 (MeOH/CH2Cl2 1:20, 1%
AcOH). Anal. HPLC (20 80% A in 20 min): tR 17.5 min (> 98%). [a]rD:t: ˆ ‡4.2 (c ˆ 0.2, DMF). IR (KBr):
1
3314m, 3066w, 2962s, 1705s, 1646s, 1548s, 1449w, 1264m, 1140w, 738m. H-NMR (400 MHz, CD3OD): 0.59 (d,
J ˆ 6.7, Me); 0.83 (d, J ˆ 6.7, Me); 1.58 1.70 ( m, CHMe2); 2.02 2.08( m, CH(R)CO); 2.48 2.55 ( m, PhCH2);
2.72 2.79 (m, 1 H, CH2CO); 2.88 2.96 (m, 1 H, CH2CO); 3.13 3.20 (m, FmocHNCHH); 3.34 3.40 (m,
FmocHNCH2); 4.19 4.23 (m, CHCH2O); 4.24 4.29 (m, 1 H, CHCH2O); 4.31 4.35 (m, 1 H, CHCH2O);
4.48 4.61 ( m, NHCH(R)CH2); 6.13 (br. s, NH); 6.78( m, NH); 7.12 7.18( m, 1 arom. H); 7.21 7.25 (m, 3 arom.
H); 7.29 7.34 (m, 2 arom. H); 7.36 7.41 (m, 2 arom. H); 7.62 7.66 (m, 2 arom. H); 7.78 7.80 ( d, J ˆ 7.6, 2 arom.
H); 7.93 7.95 (d, J ˆ 8.6, arom. H). 13C-NMR (100 MHz, CD3OD): 20.57 (Me); 21.02 (Me); 29.65 (CH); 40.76
(CH2); 41.20 (CH2); 42.49 (CH2); 48.41 (CH); 48.83 (CH); 55.38 (CH); 68.01 (CH2); 120.94 (CH); 126.27 (CH);
127.51 (CH), 128.21 (CH); 128.82 (CH); 129.44 (CH); 130.39 (CH); 139.74 (C); 142.60 (C); 145.39 (C); 158.81
‡
(C); 175.69 (C); 175.94 (C). HR-MALDI-MS: 537.2366 (37, [M ‡ Na] , C31H34N2O5Na; calc. 537.2369);
‡
‡
515.2545 (21, [M ‡ H] , C31H35N2O5); 497.2440 (30.6, [M À OH] , C31H33N2O4); 273.0401 (100). Anal. calc. for
C31H34N2O5 (482.62): C 69.86, H 7.94, N 5.80; found: C 69.83, H 7.95, N 5.98.
5.3. Cbz-(S)-b2-HLeu-(S)-b3-HPhe-OBn (29). Boc-(S)-b3-HPhe-OBn ((S)-5a) (0.99 g, 2.68mmol, 1.0
equiv.) was deprotected according to GP 3. The resulting oily residue was coupled to Cbz-(S)-b2-HLeu-OH
((S)-(8) (0.75 g, 2.68mmol, 1.0 equiv.) according to GP 5b. FC (AcOEt/pentane 2 :1 ! 1:1) afforded 29
(1.28mg, 2.41 mmol, 90%) as a colorless powder. NMR Spectroscopic analysis showed no presence of the
unwanted diastereoisomer. M.p. 135 1378. Rf 0.30 (AcOEt/pentane 1:2). [a]rD:t: ˆ ‡26.2 (c ˆ 0.5, CHCl3). IR
(CHCl3): 3650w, 3600m, 3000s, 1710s, 1660m, 1510m, 1420m, 1200s, 1140m. 1H-NMR (400 MHz, CDCl3): 0.77
0.83 (m, 2 Me); 1.06 1.15 (m, CH2CHMe2); 1.24 1.42 (m, CH2CHMe2); 2.26 2.34 (m, CH(R)CO); 2.43 2.49
(dd, J ˆ 15.8, 6.2, 1 H, CH2CO); 2.52 2.59 (dd, J ˆ 15.8, 5.0, 1 H, CH2CO); 2.83 (d, J ˆ 7.4, PhCH2); 3.09 3.17
(m, 1 H, BocHNCH2); 3.28 3.36 ( m, 1 H, BocHNCH2); 4.50 4.59 (m, NHCH(R)CH2); 5.02 5.13 (m, 2
PhCH2O); 5.33 (br. s, NH); 5.91 5.94 (br. d, J ˆ 8.7, NH); 7.11 7.14 (d, J ˆ 6.7, 2 arom. H); 7.19 7.40 (m,
13 arom. H). 13C-NMR (100 MHz, CDCl3): 22.23 (Me); 22.94 (Me); 25.61 (CH); 37.97 (CH2); 38.96 (CH2);

Helvetica Chimica Acta Vol. 86 (2003)
1549
40.06 (CH2); 43.36 (CH2); 45.23 (CH); 47.21 (CH); 66.56 (CH2); 66.67 (CH2); 77.02 (C); 126.82 (CH), 128.00
(CH); 128.04 (CH); 128.43 (CH); 128.46 (CH); 128.48 (CH); 128.62 (CH); 128.65 (CH); 129.16 (CH); 135.55
‡
(C); 136.67 (C); 137.21 (C); 156.56 (C); 171.56 (C); 173.93 (C). HR-MALDI-MS: 553.2668(100, [ M ‡ Na] ,
‡
‡
C32H38N2O5Na ; calc. 553.2673); 531.2849 (18, [M ‡ H] , C32H39N2O5; calc. 531.2846); 423.2279 (34.55, [M À
‡
‡
Cbz ‡ Na] , C24H31N2O3Na ; calc. 423.2281). Anal. calc. for C32H38N2O5 (530.66): C 72.43, H 7.22, N 5.28;
found: C 72.61, H 7.41, N 5.15.
5.4. Fmoc-(S)-b2-HLeu-(S)-b3-HPhe-OH (30). Peptide 29 (1.2 g, 2.26 mmol) was deprotected according to
GP 1b. The resulting dried filtrate was dissolved in a 1:1 mixture of dioxane/H2O (0.062m) and Na2CO3 (0.479 g,
4.52 mmol, 2 equiv.). Fmoc-OSu (0.762 g, 2.26 mmol, 1 equiv.) was added, the mixture was sonicated for 60 min
and left to react for 24 h at r.t. The pH was adjusted to 1 2, and AcOEt was added to dissolve the white
precipitate formed. The org. layer was washed with H2O (4 Â 20 ml), dried (Na2SO4), and evaporated to a white
solid. The solid was dissolved in a soln. of 5% MeOH in CH2Cl2 (insoluble materials being removed by
filtration), and precipitated by addition of pentane. Filtration and drying in vacuo afforded 30 (0.95 g, 80%) as a
colorless powder pure enough for SPPS. An anal. sample was obtained by FC (MeOH/CH2Cl2 1:10, 1%
AcOH). M.p. 2188. Rf 0.10 (MeOH/CH2Cl2 1:20, 1% AcOH). Anal. HPLC (20 80% A in 20 min): tR 19.0 min
(> 98%).[a]rD:t: ˆ ‡2.2 (c ˆ 0.5, DMF). IR (CHCl3): 3323s, 3064s, 2954s, 1697s, 1646s, 1541s, 1450m, 1258s,
1152m, 1032m. 1H-NMR (500 MHz, CD3OD): 0.72 0.74 (m, 2 Me); 0.95 1.08( m, CH2CHMe2); 1.32 1.40 (m,
CH2CHMe2); 2.38 2.43 ( m, CH(R)CO); 2.46 2.53 (m, PhCH2); 2.67 2.72 (dd, J ˆ 13.3, 9.5, 1 H, CH2CO);
2.92 2.98( m, 1 H, CH2CO); 3.11 3.14 (m, FmocHNCH2); 4.17 4.23 (m, CHCH2O); 4.25 4.28( m, 1 H,
CHCH2O); 4.32 4.36 (m, 1 H, CHCH2O); 4.51 4.58( m, NHCH(R)CH2); 6.25 (br. s, NH); 6.93 (m, NH);
7.14 7.18( m, 1 arom. H); 7.20 7.26 (m, 3 arom. H); 7.30 7.33 (m, 2 arom. H); 7.37 7.41 (m, 2 arom H); 7.60
7.66 (m, 2 arom. H); 7.78 7.80 ( d, J ˆ 7.5, 2 arom. H); 7.96 7.97 (d, J ˆ 8.7, arom. H). 13C-NMR (125 MHz,
CD3OD): 22.19 (Me); 23.72 (Me); 26.46 (CH); 39.86 (CH2); 40.58(CH 2); 41.02 (CH2); 44.77 (CH2); 46.53
(CH); 48.23 (CH); 48.83 (CH); 67.81 (CH2); 120.78 (CH), 126.14 (CH); 127.29 (CH); 128.04 (CH); 128.65
(CH); 129.22 (CH); 130.19 (CH); 139.49 (C); 142.43 (C); 145.18(C); 158.71 (C); 175.26 (C); 176.07 (C). HR-
‡
‡
‡
‡
MALDI-MS: 551.2516 (100, [M ‡ Na] , C32H36N2O5Na ; calc. 551.2519); 529.2697 (26, [M ‡ H] , C32H37N2O5
;
‡
‡
‡
‡
calc. 529.2690); 511.26 (51, [M À OH] , C32H35N2O4 ); 333.18(32, [ M À C14H11O] , C18H25N2O4 ); 307.20 (53,
‡
[M À Fmoc ‡ H] , C17H27N2O3).
5.5. Cbz-(S)-b2-HLeu-(S)-b3-HLys(Boc)-OMe (31). Cbz-(S)-b3-HLys-OMe ((S)-6a) (3.00 g, 7.35 mmol,
1.0 equiv.) was deprotected according to GP 4. A portion of the resulting oily residue (737 mg, 2.9 mmol, 1.0
equiv.) was coupled to Cbz-(S)-b2-HLeu-OH (8) (750 mg, 2.69 mmol, 1.0 equiv.) according to GP 5b. FC
(AcOEt/pentane 4 :1 ! 1 :1) afforded 31 (869 mg, 1.62 mmol, 61%) as a colorless solid. NMR spectroscopic
analysis did not show the presence of the undesired diastereoisomer. M.p. 68 69 8. Rf 0.46 (AcOEt/pentane
‡
1:2). [a]D ˆ ‡5.32 (c ˆ 0.555, CHCl3). IR (CHCl3): 3453w, 3028w, 3009m, 2956m, 2878m, 1710s, 1671m, 1507m,
1
1439m, 1410w, 1365m, 1229m, 1216s, 1168m, 1089w, 645m. H-NMR (400 MHz, CDCl3): 0.89 (d, J ˆ 5.6, Me);
0.90 (d, J ˆ 5.7, Me); 1.19 1.37 (m, CHMe2, CH2); 1.43 (s, t-Bu); 1.44 1.61 (m, 3 CH2); 2.32 2.47 (m, CH2CO);
2.49 2.55 (m, CHCO); 3.07 (m, CH2NHBoc); 3.13 3.22 (m, 1 H, CbzHNCH2); 3.30 3.41 (m, CbzHNCH2);
3.62 (m, MeO); 4.21 (m, NHCH(R)CH2); 4.56 (br. s, NH); 5.08( s, PhCH2); 5.51 (br. s, NH); 5.98( d, J ˆ 8.7,
NH); 7.30 7.40 (m, Ph). 13C-NMR (100 MHz, CDCl3): 22.31 (Me); 22.95 (Me); 23.23 (CH2); 25.89 (CH); 28.43
(Me); 29.51, 33.85, 38.87, 39.00, 40.23, 43.47 (CH2); 45.38, 46.02 (CH); 51.86 (Me); 66.57 (CH2); 79.14 (C);
128.01, 128.05, 128.48 (CH); 136.67 (C); 156.04, 156.61 (C); 172.30, 174.17 (C). HR-MALDI-MS: 558.3143 (77,
‡
‡
‡
‡
[M ‡ Na] , C28H45N3O7 ; calc. 558.3146); 502.2522 (29, [M À t-Bu ‡ Na] , C24H37N3O7 ; calc. 502.2524);
‡
‡
‡
‡
458.2623 (57, [M À Boc ‡ Na] , C23H37N3O5 ; calc. 458.2627); 436.2803 (100, [M À Boc ‡ H] , C23H37N3O5 ; calc.
436.2805). Anal. calc. for C28H45N3O7 (535.68): C 62.78, H 8.47, N 7.84; found: C 62.74, H 8.33, N 7.66.
5.6. Cbz-(S)-b2-HLeu-(S)-b3-HLys(Boc)-OH (32). Dipeptide 31 (837 mg, 1.56 mmol, 1.0 equiv.) was
dissolved in THF (7.8ml) under Ar and stirred for 15 min at r.t. Then, a 1.0 m soln. of LiOH ¥ H2O (328mg,
7.81 mmol, 5 equiv.) in H2O (7.8ml) was added. The yellow mixture was stirred for 20 h at 0 8, treated with 1n
HCl, and extracted with AcOEt and CHCl3. The combined org. layers were dried (Na2SO4), filtered, and
concentrated under reduced pressure to yield 32 (815 mg, 1.56 mmol; quant) as a colorless solid, which was used
without further purification. IR (CHCl3): 3450w, 3029w, 3008m, 2953s, 2874m, 1711s, 1671m, 1548m, 1501m,
1434m, 1410w, 1363m, 1227m, 1220s, 1168m, 1080w, 8 19m, 645m. 1H-NMR (400 MHz, CDCl3): 0.81 0.83 (m, 2
Me); 1.08 1.17 ( m, CHMe2); 1.20 1.32 (m, CH2); 1.37 (s, t-Bu); 1.38 1.53 ( m, 3 CH2); 2.25 2.38( m, CHCO);
2.41 2.52 (m, CH2CO); 2.98 3.05 ( m, CH2NHBoc); 3.06 3.17 (m, 1 H, CbzHNCH2); 3.20 3.29 (m, 1 H,
CbzHNCH2); 4.17 (m, NHCH(R)CH2); 4.58(br. s, NH); 4.99 5.05 (m, PhCH2); 5.65 (br. s, NH); 6.26 (m,
NH); 7.21 7.32 (m, Ph). 13C-NMR (100 MHz, CDCl3): 22.21 (Me); 23.02 (Me); 23.22 (CH2); 25.61 (CH); 25.92
(CH2); 28.43 (Me); 29.49, 33.76, 38.89, 40.19, 43.51 (CH2); 45.15, 46.26 (CH); 67.97 (CH2); 79.11 (C); 128.04,

1550
Helvetica Chimica Acta Vol. 86 (2003)
‡
128.51 (CH); 136.92 (C); 156.16, 156.87 (C); 172.35, 174.59 (C). HR-MALDI-MS: 544.2999 (39, [M ‡ Na] ,
‡
‡
‡
C27H43N3O7 ; calc. 544.3002); 488.2378 (16, [M À t-Bu ‡ Na] , C23H35N3O7 ; calc. 488.2379); 444.2474 (54, [M À
‡
‡
‡
‡
Boc ‡ Na] , C22H35N3O5 ; calc. 444.2470); 422.2657 (100, [M À Boc ‡ H] , C22H35N3O5 ; calc. 422.2658).
5.7. H-(S)-b2-HLeu-(S)-b3-HLys(Boc)-OH (32b). Peptide 32 (815 mg, 1.56 mmol, 1.0 equiv.) was
deprotected according to GP 4. The resulting viscous oil (696 mg, 1.56 mmol; quant.) was used without further
purification.
5.8. Fmoc-(S)-b2-HLeu-(S)-b3-HLys(Boc)-OH (33). Peptide 32b (696 mg, 1.56 mmol, 1 equiv.) was
suspended in dioxane (13 ml) and H2O (13 ml) [34]. Powdered Na2CO3 (334 mg, 3.13 mmol, 2 equiv.) was
added, and the mixture was sonicated for 45 min until the dipeptide was entirely dissolved. The homogeneous
soln. was cooled to 08, Fmoc-OSu [35] (527 mg, 1.56 mmol, 1 equiv.) was added, and the resulting mixture was
stirred for 24 h at r.t. Then, the pH was adjusted to 1 2, the soln. was filtered (N4 filter), and the colorless
precipitate was dried in vacuo. The crude product was dissolved in a 3 :1 mixture of CHCl3 and MeOH,
precipitated with MeCN, filtered, and dried in vacuo to yield 33 (877 mg, 1.44 mmol; 92%) as a colorless powder.
M.p. 135 1378. Anal. HPLC (20 80% A in 20 min) tR 18.0 min (> 98%). [a]rD:t: ˆ ‡15.51 (c ˆ 0.555, CHCl3). IR
(CHCl3): 3458w, 3022w, 3008m, 2972m, 2954m, 2878m, 1712s, 1671m, 1649s, 1508m, 1431m, 1410w, 1362m,
1220m, 1214s, 1161m, 1080w, 8 70m. 1H-NMR (400 MHz, CD3OD): 0.89 0.93 (m, 2 Me); 1.14 1.19 (m,
CHMe2); 1.28 1.40 ( m, CH2); 1.41 1.63 (m, 3 CH2); 1.42 (s, t-Bu); 2.40 2.58( m, CH2CO, CH(R)CO); 2.98
3.02 (m, CH2NHBoc); 3.20 (m, 1 H, CbzHNCH2); 3.32 (m, 1 H, CbzHNCH2); 4.18 4.30 ( m, NHCH(R)CH2,
OCH2CH); 4.32 4.39 (m, OCH2CH); 7.03 (m, NH); 7.28( m, 2 arom. H); 7.34 (m, 2 arom. H); 7.63 (m, 2 arom.
H); 7.78( d, J ˆ 7.5, 2 arom. Ph). 13C-NMR (100 MHz, CD3OD): 22.38(Me); 23.90 (Me); 24.44 (CH 2); 27.25
(CH); 28.84 (Me); 30.55, 34.90, 40.10, 40.82, 41.29, 44.88 (CH2); 46.75, 47.63, 48.42 (CH); 67.99 (CH2); 79.88 (C);
120.96, 126.24, 128.20, 128.82 (CH); 142.61, 145.37 (C); 158.14, 158.83 (C); 175.15, 176.42 (C). HR-MALDI-
‡
‡
‡
‡
MS: 632.3305 (45, [M ‡ Na] , C34H47N3O7 ; calc. 632.3309); 576.2692 (15, [M À t-Bu ‡ Na] , C30H39N3O7 ; calc.
‡
‡
‡
576.2963); 532.2795 (65, [M À Boc ‡ Na] , C29H39N3O5 ; calc. 532.2794); 510.2963 (100, [M À Boc ‡ H] ,
‡
C29H39N3O5 ; calc. 510.2966).
6. Solid-Phase Synthesis of H-(S)-b3-HPhe-(S)-b2-HVal-(S)-b3-Phe-(S)-b2-HLeu-(S)-b3-HLys-(S)-b2-Leu-
(S)-b3-HLys-(S)-b2-HLeu-(S)-b3-Phe-OH (34) via Partial Fragment Condensation. Wang resin (362 mg, initial
loading 0.90 mmol/g, 1 equiv.), Fmoc-(S)-b2-HLeu-(S)-b3-HPhe-OH (30; 516 mg, 0.977 mmol, 3 equiv.) and all
glassware used were dried in vacuo overnight. The flask containing 30 was capped with a septum and kept under
Ar. A minimum amount of 20% DMF in anh. CH2Cl2 was added to dissolve the dipeptide, and MSNT (289 g,
0.977 mmol, 3 equiv.) and Melm (58ml, 0.733 mmol, 2.25 equiv.) were introduced. After 10 min, this soln. was
transferred to a sealed flask containing the resin, and the mixture was left to react for 2 d under Ar with gentle
agitation. Then, the resin was filtered off, washed with DMF (10 Â ), CH2Cl2 (10 Â ), and shrunk with MeOH
(5Â). After drying in vacuo for 24 h, the degree of loading was found to be 0.74 mmol/g (cf. Sect. 3.1) [26a].
The resin with the anchored dipeptide (531 mg, 0.391 mmol) was put in a solid-phase reactor and swelled
for 30 min by addition of CH2Cl2. Capping was performed twice with Ac2O (0.25 ml, 2.65 mmol, 10 equiv.) and a
cat. amount of DMAP (3.2 mg, 0.02 mmol) in CH2Cl2 for 1 h under intensive N2 bubbling. The resin was filtered
and washed with NMP (5 ml, 10 Â 1 min). The Fmoc group was removed with DBU/piperidine/NMP 2 :2 :96
(5 ml, 6 Â 60 min). The resin was washed with NMP (5 ml, 9 Â 1 min) and CH2Cl2 (5 ml, 1 Â 1 min). The
elongation sequence was initiated by addition of a homogeneous soln. of 33 (477 mg, 0.78mmol, 2.0 equiv.),
HBTU (285 mg, 0.75 mmol, 1.92 equiv.), HOBt (127 mg, 0.78 mmol, 2.0 equiv.), and DIPEA (0.27 ml,
1.56 mmol, 4.0 equiv.) in NMP (5 ml). The slightly yellow suspension was agitated by N2 bubbling for 24 h. This
procedure (deprotection and coupling of 33) was repeated once more to yield the resin-bound hexamer. At this
stage, the resin was shrunk and dried in vacuo. SPPS was continued with one third of the material (ca.
0.130 mmol) by addition of 28 (138mg, 0.26 mmol, 2.0 equiv.), HBTU (95 mg, 0.25 mmol, 1.92 equiv.), HOBt
(42 mg, 0.26 mmol, 2.0 equiv.), and DIPEA (0.09 ml, 0.52 mmol, 4.0 equiv.) in NMP (5 ml), and the reaction was
allowed to proceed for 24 h. The last residue was introduced by reacting with 5b (209 mg, 0.52 mmol, 4.0 equiv.)
in the presence of HBTU (195 mg, 0.51 mmol, 3.96 equiv.), HOBt (84 mg, 0.52 mmol, 4.0 equiv.), and DIPEA
(0.18ml, 1.04 mmol, 8.0 equiv.) in NMP (5 ml) for 24 h. The resin was washed with NMP (5 ml, 10 Â 1 min) and
CH2Cl2 (5 ml, 10 Â 1 min), shrunk with MeOH (5 ml, 5 Â 1 min), and dried in vacuo. The resulting peptide was
cleaved from the resin and deprotected by exposure to TFA/H2O 95 :5 for 3 h to yield after evaporation,
precipitation (Et2O), washing (Et2O), and drying in vacuo crude 34 (100 mg). Purification of the crude
product by RP-HPLC afforded 34 as its TFA salt (78mg, colorless lyophilizate). Anal. HPLC (20 90% A in
30 min): tR 22.3 min (> 98%). 1H-NMR (500 MHz, CD3OD): 0.52 (d, J ˆ 6.7, Me); 0.72 (d, J ˆ 6.6, 2 Me); 0.83
(d, J ˆ 6.7, Me); 0.87 (d, J ˆ 6.0, 2 Me); 0.88 1.02 (m, 2 CH2CHMe2); 0.92 (d, J ˆ 6.6, Me); 0.93 (d, J ˆ 6.5, Me);
1.09 1.14 (m, CH2CHMe2); 1.30 1.71 (m, CHMe2, 3 CH2CHMe2, 6 CH2), 1.94 1.99 (m, CH(R)CO); 2.00

Helvetica Chimica Acta Vol. 86 (2003)
1551
2.05 (t, J ˆ 12.3, CH(R)CO); 2.11 2.16 (t, J ˆ 11.8, CH(R)CO); 2.21 2.26 (t, J ˆ 12.1, CH(R)CO); 2.33 2.42
(m, 1 H, CH2CO); 2.45 2.72 (m, 9 H, CH2CO, PhCH2); 2.75 2.98( m, 3 CH2CO, 2 NH2CH2); 3.04 3.09 (m,
CH2NHR); 3.30 3.32 (m, NH2); 3.37 3.41 (m, 1 H, CH2NHR); 3.43 3.51 (m, 1 H, CH2NHR); 3.58 3.66 ( m,
1 H, CH2NHR); 3.74 3.82 (m, 3 H,
2 CH2NHR); 4.31 4.38( m, NHCH(R)CH2); 4.42 4.50 (m,
NHCH(R)CH2); 4.58 4.67 ( m, 3 NHCH(R)CH2); 7.14 7.31 (m, 13 arom. H); 7.34 7.38( m, 2 arom. H);
7.83 7.86 ( m, NH); 8.17 8.20 (d, J ˆ 9.2, NH); 8.51 8.56 (m, 3 NH); 8.58 8.60 (d, J ˆ 8.9, NH); 8.70 8.72 (d,
J ˆ 9.3, NH); 8.73 8.76 (d, J ˆ 9.2, NH). 13C-NMR (125 MHz, CD3OD): 20.65 (Me); 20.83 (Me); 21.00 (Me);
22.34 (Me); 22.46 (Me); 22.50 (Me); 23.90 (Me); 23.93 (Me); 24.07 (CH2); 24.28(CH 2); 26.68(CH); 27.34
(CH); 28.40 (CH2); 28.49 (CH2); 29.99 (CH); 35.89 (CH2); 35.98(CH 2); 37.24 (CH2); 39.95 (CH2); 39.99 (CH2);
40.02 (CH2); 40.11 (CH2); 40.50 (CH2); 40.59 (CH2); 40.94 (CH2); 41.22 (CH2); 42.34 (CH2); 43.56 (CH2); 43.71
(CH2); 44.21 (CH2); 44.45 (CH2); 37.24 (CH2); 45.18(CH 2); 45.23 (CH); 45.53 (CH); 45.65 (CH); 48.54 (CH);
49.22 (CH); 49.33 (CH2); 49.50 (CH); 49.67 (CH); 51.62 (CH); 51.85 (CH); 55.36 (CH); 127.58 (CH); 127.67
(CH); 128.63 (CH); 129.45 (CH); 129.46 (CH); 130.14 (CH); 130.47 (CH); 130.49 (CH); 130.58 (CH); 136.91
(C); 139.54 (C); 139.65 (C); 171.95 (C); 173.59 (C); 173.74 (C); 173.87 (C); 175.68 (C); 175.82 (C); 176.31 (C);
‡
176.39 (C). ESI-MS (pos. mode): 1281.0 (30, [M ‡ H] ); 640.8(45, [ M ‡ 2 H]2‡); 427.8(100, [ M ‡ 3 H]3‡). ESI-
MS (neg. mode): 1280.0 (100, [M À H]À).
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