(S)-β3-homolysine- and (S)-β3-homoserine-containing β-peptides: CD spectra in aqueous solution

Article abstract of DOI:10.1002/(SICI)1522-2675(19981216)81:12<2141::AID-HLCA2141>3.0.CO;2-5

For further structural studies and for physiological investigations of β-peptides, it is necessary to have H₂O-soluble derivatives. Thus, we have prepared β-hexa-, β-hepta-, and β-nonapeptides (1-6) with two, three, and seven side chains of lys

Full text of DOI:10.1002/(SICI)1522-2675(19981216)81:12<2141::AID-HLCA2141>3.0.CO;2-5

Helvetica Chimica Acta ± Vol. 81 (1998)
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(S)-b3-Homolysine- and (S)-b3-Homoserine-Containing b-Peptides:
CD Spectra in Aqueous Solution
1
2
by Stefan Abele ), Gilles Guichard ), and Dieter Seebach*
Laboratorium für Organische Chemie der Eidgenössischen Technischen Hochschule, ETH-Zentrum,
Universitätstrasse 16, CH-8092 Zürich
For further structural studies and for physiological investigations of b-peptides, it is necessary to have H2O-
soluble derivatives. Thus, we have prepared b-hexa-, b-hepta-, and b-nonapeptides (1 ± 6) with two, three, and
seven side chains of lysine and serine. To detect possible p-p interactions, we also included the b-amino acid b2-
HHop, resulting from homologation of so-called homophenylalanine (Hop) (5 and 6). The Fmoc-b2- and b3-
amino-acid derivatives (11 ± 14 and 19), and the corresponding b-peptides were prepared by methods previously
described (solid-phase peptide coupling; HPLC-pure samples, Fig. 1). Circular-dichroism spectra (Fig. 2)
indicate the presence of less pronounced secondary structures (especially of the lysine analogues with multiple
positive charge) in H2O as compared to MeOH. The b3-heptapeptide (3) with two serine side chains is well
soluble in H2O and exhibits the CD pattern typical of the 31-helical structure.
1. Introduction. ± Short-chain w-peptides [1] that can form stable folded structures
have recently joined the rapidly growing family of structurally defined non-natural
oligomers [2]. We and others have demonstrated that peptides consisting exclusively of
b- [3 ± 7] or g- [8] [9] rather than a-amino acids, can form stable and novel helical
structures in MeOH, pyridine, or in the solid state. More recently, further structural
diversity has been achieved with the successful design and synthesis of parallel [4]
[10] and antiparallel [11] pleated-sheet-type structures based on b3- and b2,3-amino
acids.
With respect to potential applications of b-peptides in biological systems, the
current challenge is to design, based on secondary structural elements, b-peptides with
a given secondary structure and with specific biological functions. For example, cyclo-
b3-tetrapeptides that have been found to form nanotubes in the solid state [12], have
recently been used as transmembrane ion channels [13]. The elucidation of structural
propensities of linear b-peptides in aqueous environment represents a further
important issue that still needs to be addressed for future developments in bioorganic
chemistry. Here, we report the synthesis and CD measurements in organic solvents and
aqueous solution of b-hepta- and b-nonapeptides 1 ± 6 containing two or more polar
hydrophilic side chains of Lys or Ser, with b3-amino-acid residues (1 ± 5) or with mixed
sequences of b2- and b3-amino-acid residues (6).
From our previous studies [4] [5] [14], b-peptides 1 ± 6 are expected to adopt the
(M)-31-helical structure. Compound 1 can be considered as a section of the b-peptide
1
)
)
Part of the projected Ph. D. thesis of S.A., ETH-Zürich.
Grant from the Association pour la Recherche sur le Cancer (ARC), France. Present address: UPR 9021
2
Â
CNRS, Institut de Biologie Moleculaire et Cellulaire, F-67084 Strasbourg.

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analogue of poly-a-lysine, a cationic polymer that has been widely used to study the
transition between a-helical and pleated-sheet structures by CD spectroscopy [15 ± 17],
and to explore its complexation with anionic partners, including DNA [18] or bilirubin
[19]. Peptides 2 and 3 were prepared in order to examine the effect of lysine and
serine side chains on the helix stability. A comparison of the b-heptapeptide 2 with
the homologous b-nonapeptide 4 should allow us to assess the stabilizing effect of
growing chain length3). Analysis of the 31-helix model suggests that the incorpo-
ration of an additional CH2 group in the side chain of b2- and b3-HPhe might favor the
stacking of the Ph rings. In addition to electrostatic, H-bonding, and hydrophobic
interactions, aromatic interactions may also contribute to the stabilization of helical
secondary structures. For example, in the case of oligo-N-substituted-glycines
(ꢀpeptoidsꢁ) with aromatic side chains, p-stacking and dipole-dipole repulsion between
side chains and the CˆO groups of the backbone have been suggested to account for
the stabilization of helical structures in H2O [21]. Hence, compound 5 and 6, containing
the b-amino-acid analogue of so-called homophenylalanine (Hop), were thought to be
suitable for evaluating the influence of aromatic side chains on folding propensities of
b-peptides.
2. Preparation of the b2- and b3-Amino-Acid Building Blocks. ± For rendering b-
peptides H2O-soluble, incorporation of polar side-chains is a prerequisite. For this
purpose, we synthesized several new b-amino acids with the appropriate orthogonally
protected functional groups. Diazo ketones 7 ± 10 were prepared by procedures used in
our laboratory [4] [22] [23], starting from the corresponding commercially available
enantiomerically pure a-amino acids. The b3-amino acids 11 ± 14 were obtained in good
yields (40 ± 50% from the a-amino acids) by Arndt-Eistert homologation. Whereas the
Boc-protected b3-amino acids 11a and 12 were prepared for coupling in solution, Fmoc-
protected acids 11b, 13, and 14 are the appropriate building blocks for solid-phase
synthesis4). The N,N'-diphthalyl-[25a], N,N'-dibenzyloxy-, and N,N'-bis(tert-butyl-
oxy)carbonyl [25b] derivatives of 13 have already been synthesized in order to confirm
the structure of ꢀisolysineꢁ5), a hydrolysis product of several antibiotics. However, since
orthogonality of the amino protecting groups in these compounds is not secured, we
prepared the b-Fmoc-/e-Boc-protected amino acid 13 that can be employed in Fmoc-
coupling reactions.
Evansꢁ enolate chemistry was used for the synthesis of b2-amino-acid derivatives
(Scheme). Unlike in our previous syntheses [6] [26], we used benzyl N-(methoxy-
methyl) carbamate [27] for the amidomethylation of (R)-4-benzyl-3-(4-phenylbuta-
noyl)-1,3-oxazolidin-2-one (15), through the TiCl4-enolate to give the unlike-product
166). The somewhat lower yield (44%), as compared to the previously used
electrophile (benzoylamino)methyl chloride, is more than compensated for by the
3
)
)
)
)
b-Peptides 2 and 4 are also promising candidates for the inhibition of cholesterol uptake through brush
border membrane [20].
Like its a-analogues [24], the b3-homotyrosine derivative 14 has a low solubility in common organic
solvents (CH2Cl2, CHCl3, AcOEt).
4
5
6
3,6-Diaminohexanoic acid, often called b-lysine, is a constitutional isomer of lysine. According to the
nomenclature used for b-amino acids [4] [6] [14], this compound is b3-HOrn
1
The configurational purity of the chromatographed product was >97:3 (by H-NMR).

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milder conditions of N-deprotection. Hydrolytic removal of the auxiliary group with
LiOH/H2O2 led to the Z-protected acid 17 which was N-deprotected by hydrogenolysis
(H2, Pd/C) to yield the free b2-amino acid 18 which was converted to the desired Fmoc-
protected b2-amino acid 19 (33% starting from 16).
3. Preparation of b-Peptides. ± b3-Heptapeptides 1 ± 3 and 5, 6, and b3-nonapeptide
4 were synthesized on ortho-chlorotrityl chloride [28] resin by conventional solid-phase
Scheme. Preparation of (S)-b2-Amino Acid Derivatives 17 ± 19 by Aminomethylation of Acyl-oxazolidinones

Helvetica Chimica Acta ± Vol. 81 (1998)
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peptide-synthesis procedures [29] [30] (BOP/HOBt/(i-Pr)2EtN)7). The yields of the
crude peptides, obtained after cleavage from the resin and precipitation with Et2O,
were between 45 and 99%8). The crude products had a purity of 48 ± 80%, as
determined by reversed-phase (RP) HPLC (see Exper. Part). The HPLC profiles of
purified b-peptides 1 ± 3, 5 and 6 are shown in Fig. 1. FAB-MS Analysis of the major
impurities isolated by preparative RP-HPLC revealed that poor purities were due to
incomplete Fmoc removal9). It seems that this complication may arise during the
synthesis of b3-HLys-containing b-peptides exceeding seven residues. An elemental
analysis was performed with b3-heptahomolysine 1 in order to determine the amount of
CF3COOH present after purification and lyophilization. The values found correspond
to the heptapeptide with eight molecules of CF3COOH. This result is of importance for
other b-peptides containing free amino groups10). It shows that all free amino groups
form a CF3COOH salt. All the b-peptides were white powders except the polycationic
b-peptide 1 which was obtained as a colorless highly viscous oil.
4. CD Spectroscopy. ± CD Spectra of b-peptides 1 ± 6 (Fig. 2) were measured in
MeOH, in CF3CH2OH (TFE), and in buffered aqueous solutions at different pH in the
range of 195 ± 250 nm11) (0.2 mm concentration, see Exper. Part). In MeOH, all b-
peptides showed the typical CD pattern (positive Cotton effect at 200 nm, negative
Cotton effect at 215 ± 220 nm) that we previously assigned to the (M)-31 helix [6]
[14]12 13). The weakest negative Cotton effect (ca. 1.2 ´ 104 at 219 nm) was measured
)
for b3-hepta(homolysine) 1 suggesting that the large number of neighboring cationic
side chains destabilizes the secondary structure (Fig. 2,a). It is noteworthy that the b3-
HSer-containing peptide 3 displays a more intensive negative Cotton effect (ca. 6.1 ´
104 at 219 nm) than its b3-HLys-containing analogue 2 (ca. 3.7 ´ 104 at 219 nm)
(Fig. 2,b and c). Obviously, the OH groups in 3 do not destabilize the 31 helix, while the
‡
NH3 groups in 2 do. A comparison of the mean residue molar ellipticity of b-
heptapeptide 2 and b-nonapeptide 4 ( 5.2 ´ 103 and 5.3 ´ 103, resp.) shows that there is
no stabilization of the 31 helix due to longer chain length, in this case (Fig. 2,b and d).
We then tested the effect of aromatic side-chain interaction on the Cotton effect (see 2
vs. 5 and 6): CD curves of b-peptides 2 and 5 in MeOH do not differ substantially
(
3.7 ´ 104 vs. 4.2 ´ 104 at 220 nm resp.) (Fig. 2,b and e). This indicates that the presence
of an additional CH2 group in the aromatic side chains (vide supra) (b-peptides 5 and
6), although it could favor p-stacking, does not stabilize the 31 helix in MeOH.
7
)
)
)
Abbreviations are defined in the Exper. Part.
% Mass recovered based on polymer loading.
8
9
The corresponding Fmoc-protected octa- and hexapeptides were identified by mass spectroscopy as
impurities in 4 and 5, respectively, indicating that incomplete Fmoc deprotection had taken place in the
penultimate coupling step. Use of the stronger base 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) instead of
20% piperidine in DMF [29] did not substantially improve the purity of crude products 4 and 5.
The molar ellipticity F can only be determined accurately from the CD spectra if the exact mass of the
sample is known.
Because of inherent absorption of buffer solutions, data were recorded for l> 195 nm. Changes of the CD
spectra in the range 195 ± 200 nm are not discussed.
When measured in TFE, the CD spectra exhibit a stronger negative Cotton effect (Fig. 2).
The high content of aromatic chromophors (up to 45% of the side chains) did not alter the magnitude of
the Cotton effect, compared with previously reported 31-helical b-peptides [4] [6] [14] [29].
10
11
)
)
12
13
)
)

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Fig. 1. HPLC Profiles of purified b-heptapeptides 1 ± 3, 5, and 6 (RP-C18 column, linear gradient of 0.1%
CF3COOH in H2O and MeCN; see GP 7 in Exper. Part)
b-Peptide 6 has a novel sequence pattern ((b2-HXaa-b3HXaa-b3HXaa)n) that is
compatible with a 31-helical structure, and this is confirmed by its CD spectrum
(Fig. 2,f ).
Next, we compared the CD spectra of the new b-peptides in aqueous solution. The
trough at ca. 220 nm is only observed with compounds 3 and 6, albeit with much lower
intensities ( 2.4 ´ 104 and 1.1 ´ 104 at pH 5.6) than in MeOH. In the case of
compounds 3 and 6, the intensity of the Cotton effect increases at high pH values
(
3.0 ´ 104 and 2.1 ´ 104, at pH 11). Taken together, these data and those obtained in
MeOH suggest that b-HSer has a smaller helix-disrupting effect in H2O than b-HLys. In
the case of b-peptides 1, 2, 4, and 5, the molar ellipticity at 215 nm is only slightly

Helvetica Chimica Acta ± Vol. 81 (1998)
2147
Fig. 2. Overlay of CD spectra of b-peptides 1 ± 6 under various solvent conditions. a) b-Peptide 1. b) b-Peptide 2.
c) b-Peptide 3. d) b-Peptide 4. e) b-Peptide 5. f) b-Peptide 6. Molar ellipticity [V] in 10 deg ´ cm2 ´ mol 1. The
curves specified by pH values all refer to aqueous solutions.

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negative or even positive14). A common feature of peptides 2, 4, and 6 is the occurrence
of a positive shoulder at 205 ± 210 nm, that might reflect either a single new
conformation or the co-existence of several conformational isomers. Surprisingly, the
CD spectrum of the highly basic b-peptide 1 did not show any significant pH
dependence.
5. Conclusion. ± We have investigated by CD spectroscopy the conformational
features of short-chain b-peptides 1 ± 6 in aqueous environment. In contrast to CD
spectra measured in MeOH, spectra of b3-HLys-containing b-peptides 1 and 2, and 4
and 5 recorded in H2O and in buffered solutions do not display the typical pattern
assigned to the 31-helical structure. This may be due to a destabilization of the helix
caused by partial or total disruption of the H-bond network15). In contrast, CD spectra
of both b3-heptapeptide 3 (with side chains of Ala, Phe, and Ser) and mixed b2, b3-
heptapeptide 6 (with side chains of Ala, Hop, and Lys) show, although with a moderate
intensity, the helix-specific trough at 215 ± 220 nm. In this peptide series, the apparent
order of stabilization of the 31 helix in H2O would then be b3-HSer> b3-HLys for polar
residues in positions 3 and 6, and b2-HHop > b3-HHop ˆ b3-HPhe for aromatic residues
in 1-, 4-, and 7-position. A more systematic approach based on combinatorial libraries
would be of interest to determine the 31-helix-disrupting and -generating propensities
of all b2- and b3-amino acids with proteinaceous side chains in H2O. This would be in
analogy to the existing scales describing the effect of an a-amino-acid side chain on a-
helix formation [31].
Alternative strategies that could be used to achieve 31-helix stabilization in aqueous
solution would include: i) The creation of ion pairs or salt bridges between side chains
of two b-amino-acid residues i and (i ‡ 3) (i.e., side chains of Glu and Lys). ii) Since the
31 helix has a resulting dipole moment with the positive pole near the C- and the
negative near the N-terminus [6], the ideal situation would be to insert the negatively
charged side chains near the C- and the positively charged ones near the N-terminus.
iii) The design of covalent macrocycles between i and (i ‡ 3) side chains. iv) The design
of H2O-soluble b-peptides built completely from b2,3-amino acids (b2,3-peptides have
very slow NH/ND exchange kinetics, probably due to the diminished solvent accessibility
of the NH protons [6]). v) The design of H2O-soluble long-chain b-peptides [6] [29b].
We would like to thank Karl Gademann for the characterization of compound 8. We gratefully
acknowledge the assistance of B. Brandenberg and P. Zumbrunnen (NMR), Dr. W. Amrein, R. Häfliger, and O.
Greter (MS), and D. Manser (elemental analysis).
Experimental Part
1. General. Abbreviations: BOP: benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophos-
phate, DMAP: 4-(dimethylamino)pyridine. FC: flash chromatography, GP: general procedure, HOBt: 1-
hydroxy-1H-benzotriazole, h.v.: high vacuum, 0.01 ± 0.1 Torr, NMM: N-methylmorpholin, TFA: CF3COOH,
TIS: triisopropylsilane, b-HXaa: b-homoamino acid. THF was freshly distilled over K under Ar before use.
Solvents for chromatography and workup procedures were distilled from Sikkon (anh. CaSO4; Fluka). Et3N was
14
)
)
A first demonstration of the disruptive effect of H2O on b-peptide secondary structures was described in
one of our previous studies on b-peptides [29b].
This effect is well-known for a-helices which are more stable in MeOH and TFE than in H2O.
15

Helvetica Chimica Acta ± Vol. 81 (1998)
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distilled from CaH2 and stored over KOH. i-BuOCOCl was distilled and stored at ‡48 under Ar. All indicated
temp. were monitored with an internal thermometer (Ebro TTX 690 digital thermometer). Amino-acid
derivatives were purchased from Bachem, Senn, or Degussa. ortho-Chlorotrityl chloride and Wang resins were
purchased from Novabiochem. All other reagents were used as received from Fluka. Caution: The generation
and the handling of CH2N2 requires special precautions [32]. TLC was performed on Merck silica gel 60 F254
plates; detection with UV and ninhydrine. FC: Fluka silica gel 60 (40 ± 63 mm) at ca. 0.2 bar. Anal. HPLC:
Knauer HPLC system (pump type 64, EuroChrom 2000 Integration Package, degaser, UV detector (variable-
wavelength monitor)). Prep. HPLC: Knauer HPLC system (pump typ 64, programmer 50, UV detector
(variable-wavelength monitor)). M.p.: Büchi 510 apparatus; uncorrected. Optical rotations: Perkin-Elmer 241
polarimeter (10 cm, 1 ml cell) at r.t. CD Spectra: Jasco J-710 between 190 and 250 nm in a 1 mm rectangular cell
at r.t. All spectra were the average of five scans and were corrected for the baseline. (Peptide concentration
0.2 mm). The molar ellipticity (V) is reported in deg ´ cm2 ´ dmol 1. Smoothing was performed using the software
provided by Jasco. IR Spectra: Perkin-Elmer-782 spectrophotometer. NMR Spectra: Bruker AMX 500 (1H:
500 MHz, 13C: 125 MHz), AMX 400 (1H: 400 MHz, 13C: 100 MHz), Varian Gemini 300 (1H: 300 MHz, 13C:
75 MHz), or Varian Gemini 200 (1H: 200 MHz, 13C: 50 MHz); chemical shifts d in ppm downfield from internal
Me4Si (ˆ 0 ppm); J values in Hz; some compounds show the presence of rotamers which are indicated. MS: VG
Tribrid (EI), Hitachi Perkin-Elmer RHU-6M (FAB, in a nitrobenzyl-alcohol matrix), LDI 1700 (MALDI), or
Finnigan MAT TSQ 7000 (ESI) spectrometer. Elemental analyses were performed by the Microanalytical
Laboratory of the Laboratorium für Organische Chemie, ETH-Zürich.
2. Diazo Ketones 7 ± 10: General Procedure 1 (GP 1). Similarly to the procedure reported in [23], the N-
protected amino acid was dissolved in THF (0.35m) under Ar and cooled to 208. After addition of i-BuOCOCl
(1.05 equiv.) and NMM (1.05 equiv.), the mixture was stirred at 208 for 20 min. The resulting white suspension
was allowed to warm up to 58, and a soln. of CH2N2 in Et2O was added until the rich yellow color persisted.
Stirring was continued for 4 h as the mixture was allowed to warm to r.t. Excess CH2N2 was destroyed by
vigorous stirring and by the addition of a few drops of AcOH. The mixture was then diluted with Et2O and
washed with sat. NaHCO3 soln., 1n HCl, and sat. NaCl soln. The org. phase was dried (MgSO4) and
concentrated under reduced pressure. FC and/or recrystallization afforded the pure diazo ketone.
3. Homologated Carboxylic Acids 11 ± 14: General Procedures 2 (GP 2). GP 2a. Similar to the procedures
reported in [4] [23], the diazo ketone was dissolved in THF (0.25m) containing 10% H2O and then cooled to
258 under Ar with the exclusion of light. A soln. of CF3COOAg (0.11 equiv.) in Et3N (2.8 equiv.) was added,
and the resulting mixture was allowed to warm to r.t. in 4 ± 5 h in the dark. After removing the bulk of THF
under reduced pressure, the mixture was diluted with aq. sat. NaHCO3 soln. and extracted with Et2O. The aq.
phase was then carefully adjusted to pH 2 ± 3 at 08 with 1n HCl and extracted with AcOEt. The org. layer was
dried (MgSO4) and concentrated under reduced pressure. FC and/or recrystallization afforded the pure N-
protected b3-amino acids.
GP 2b. The diazo ketone was dissolved in THF (0.25m) containing 10% H2O and then cooled to 08 under Ar
with the exclusion of light. A soln. of CF3COOAg (0.11 equiv.) in NMM (2.5 equiv.) was added and the resulting
mixture was allowed to warm to r.t. in 4 h in the dark. Workup as in GP 2a. FC and/or recrystallization afforded
the pure N-protected b3-amino acids.
4. N-Fmoc Protection: General Procedure 3 (GP 3). A stirred soln. of the b-amino acid in 0.15m aq Na2CO3
(2 ± 3 equiv.) was treated with a soln. of Fmoc-OSu (1.2 equiv.) in acetone (0.1m). After 2 h, the mixture was
concentrated in vacuo, diluted with H2O and extracted with Et2O. The aq. phase was carefully adjusted to pH
2 ± 3 at 08 with 1n HCl and extracted with AcOEt. The org. layer was dried (MgSO4) and evaporated. FC and/or
recrystallization afforded the pure N-Fmoc-protected b-amino acid.
5. Anchoring of N-Fmoc-Protected b-Amino Acids on the Resin: General Procedure 4 (GP 4). ortho-
Chlorotrityl-chloride resin was esterified according to [28]. The resin (initial loading: 1.00 or 1.30 mmol Cl/g)
was dried under h.v. for 30 min and swelled in CH2Cl2 (10 ml/mmol) for 10 min. A soln. of the b-amino acid
(0.8 ± 1.0 equiv.) in CH2Cl2 (10 ml/mmol) and (i-Pr)2EtN (3.2 ± 4.0 equiv.) were then added successively, and the
suspension was mixed under Ar for 4 h. Subsequently, the resin was filtered, washed (10 ml/mmol) with CH2Cl2/
MeOH/(i-Pr)2EtN 17:2:1 (3 Â 3 min), CH2Cl2 (3 Â 3 min), DMF (2 Â 3 min), CH2Cl2 (3 Â 3 min), MeOH (2 Â
3 min) and finally dried over KOH under h.v. for 12 h. The loading of the resin was then determined on a
3 ± 5 mg sample (after treatment with 20% piperidine in DMF for 20 min), by measuring the absorbance of the
dibenzofulvene-piperidine adduct (formed during deprotection) at 300, 289, and 266 nm (e ˆ 7800, 5800, and
17500, resp.), taking the average absorbance from the three measurements.
6. b-Peptide on Solid Support: General Procedures 5 (GP 5). GP 5a (cf. Scheme). The Fmoc group of the
first amino acid attached to the resin was removed using 20% piperidine in DMF (30 ml/mmol, 2 Â 20 min)

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Helvetica Chimica Acta ± Vol. 81 (1998)
under Ar bubbling. The resin was then filtered and washed with DMF (30 ml/mmol, 6 Â 3 min). For each
coupling step, a soln. of the Fmoc-b-amino acid (2 ± 3 equiv.), BOP (2 ± 3 equiv.), and HOBt (2 ± 3 equiv.) in
DMF (2 ml) and (i-Pr)2EtN (6 ± 9 equiv.) were added successively to the resin, and the suspension was mixed
for 15 ± 60 min under Ar. Monitoring of the coupling reaction was performed with 2,4,6-trinitrobenzenesulfonic
acid (TNBS). In case of a positive TNBS test (indicating incomplete coupling), the suspension was allowed to
react further for 15 ± 60 min. The resin was then filtered and washed (12 ml/mmol) with DMF (3 Â 3 min) prior
to the following Fmoc deprotection step. After the removal of the last Fmoc protecting group, the resin was
washed (12 ml/mmol) with DMF (6 Â 3 min), CH2Cl2 (3 Â 3 min), Et2O (5 Â 1 min), and dried under h.v. over
KOH for 4 h.
GP 5b. As in GP 5a, except that the Fmoc group is removed using 30 ml/mmol DBU/piperidine/DMF
(1:1:48, 1 Â 3 min and 1 Â 8 min) under Ar bubbling.
7. Resin Cleavage and Final Deprotection: General Procedures 6 (GP 6). GP 6a. The dry Fmoc-deprotected
peptide-resin was treated with CF3COOH/H2O 95:5 (20 ml/mmol, 5 Â 15 min) under Ar bubbling. The resin
was removed by filtration, washed with CF3COOH and the org. phase containing the peptide was concentrated
under reduced pressure. The precipitate, which formed upon addition of cold Et2O to the oily residue, was
collected by filtration or centrifugation. The solid was then dissolved in H2O or H2O/AcOH 9:1 and lyophilized
to afford a crude product which was analyzed and purified by RP-HPLC.
8. Reversed Phase (RP) HPLC Analysis and Purification of b-Peptides: General Procedure 7 (GP 7). RP-
HPLC Analysis was performed on a Macherey-Nagel C8 column/Nucleosil 100-5 C8 (250 Â 4 mm) or Macherey-
Nagel C18 column/Nucleosil 100-5 C8 (250 Â 4 mm) by using a linear gradient of A (0.1% CF3COOH in H2O)
and B (MeCN) at a flow rate of 1 ml/min with UV detection at 220 nm. tR in min. Crude products were purified
by prep. RP-HPLC (Macherey-Nagel C18 column/Nucleosil 100-7 C18 (250 Â 21 mm)) using gradient of A and B
at a flow rate of 4 ml/min with UV detection at 214 nm and then lyophilized.
9. Diazo Ketones 7 ± 10. (S)-3-{[(tert-Butoxy)carbonyl]amino}-1-diazo-5-phenylpentan-2-one (7). (S)-2-
{[(tert-Butoxy)carbonyl]amino}-4-phenylbutyric acid (4.8 g, 17.18 mmol) was transformed according to GP 1.
Recrystallization from Et2O/hexane yielded 7 (3.6 g, 69%). Yellowish solid. M.p. 83 ± 848. [a]rD:t: ˆ ‡ 12.6 (c ˆ
0.97, CHCl3). IR (CHCl3): 3438w, 3008w, 2111s, 1708s, 1643m, 1496s, 1455w, 1369s, 1241w, 1164m, 1046w, 1021w.
1H-NMR (300 MHz, CDCl3): 1.45 (s, t-Bu); 1.79 ± 1.91 (m, 1 H, CHCH2); 2.06 ± 2.18 (m, 1 H, CHCH2);
2.68 (t, J ˆ 7.5, PhCH2); 4.21 (br., CHN); 5.15 (d, J ˆ 6.53, NH); 5.40 (s, CHN2); 7.17 ± 7.32 (m, 5 arom. H).
13C-NMR (50 MHz, CDCl3): 25.9 (Me); 29.2, 31.7 (CH2); 51.6, 54.5 (CH); 123.9, 126.0, 126.2 (CH); 138.5, 153.1,
‡
191.5 (C). FAB-MS: 304 (7, [M ‡ 1] ). Anal. calc. for C16H21N3O3 (303.36): C 63.35, H 6.98, N 13.85; found:
C 63.50, H 6.89, N 13.58.
(S)-3-{[(tert-Butoxy)carbonyl]amino}-7-{[(2-chlorobenzyloxy)carbonyl]amino}-1-diazoheptan-2-one (Boc-
(S)-Lys(2-Cl-Z)-CHN2; 8). Boc-(S)-Lys(2-Cl-Z)-OH (30.0 g, 72.0 mmol) was transformed according to GP 1.
FC (AcOEt/pentane 2:3) yielded 8 (28.09 g, 89%). Yellow solid. M.p. 72 ± 748. Rf 0.29 (AcOEt/pentane 2:3). IR
(CHCl3): 3446w, 3008w, 2960w, 2940w, 2110s, 1800w, 1711s, 1642m, 1500m, 1446w, 1368m, 1248w, 1163w, 1042w,
859w, 630w. 1H-NMR (300 MHz, CDCl3): 1.36 ± 1.69 (m, t-Bu, 5 CH); 1.74 ± 1.83 (m, CH); 3.17 ± 3.23 (m, CH2);
4.14 (br., CHN), 4.91 (br., NH); 5.21 (br. s, CH2, NH); 5.45 (br. s, CHN2); 7.23 ± 7.29 (m, 2 arom. H); 7.35 ±
7.43 (m, 2 arom. H). 13C-NMR (76 MHz, CDCl3): 22.33, 28.36, 29.55, 32.10, 40.56, 53.97, 57.34, 64.05, 80.21,
‡
127.11, 129.51, 129.61, 129.77, 130.01, 133.83, 134.57, 155.86, 156.62, 193.48. FAB-MS: 877 (1.7, 2M ), 439 (23.1,
‡
[M ‡ 1] ), 355(100), 339(24.7), 311(27.8), 252(50.2), 154.1 (23.8), 127(33.8). Anal. calc. for C20H27ClN4O5
(438.91): C 54.73, H 6.20, N 12.76; found: C 54.65, H 6.17, N 12.50.
(S)-6-{[(tert-Butoxy)carbonyl]amino}-1-diazo-3-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)hexan-2-
one (Fmoc-(S)-Orn(Boc)-CHN2; 9). Fmoc-(S)-Orn(Boc)-OH (5.0 g, 11.0 mmol) was transformed according to
GP 1. FC (Et2O/CH2Cl2 1:8) and recrystallization (AcOEt/hexane) yielded 9 (3.18 g, 60%). Bright-yellow
powder. M.p. 1158 (dec., sintering at 868). Rf 0.09 (Et2O/CH2Cl2 1:8). [a]Dr:t:
ˆ
24.0 (c ˆ 1.0, CHCl3). IR
(CHCl3): 3453w, 3007w, 2111m, 1712s, 1641m, 1507s, 1450m, 1391m, 1367s, 1166m, 1044w, 867w. 1H-NMR
(400 MHz, CDCl3): 1.44 (s, t-Bu); 1.47 ± 1.60 (m, 3 CH); 1.81 ± 1.87 (m, 1 CH); 3.13 ± 3.16 (m, CH2N);
4.21 (t, J ˆ 7.0, CHO); 4.27 (br. s, NH); 4.39 ± 4.49 (m, CH2O); 4.60 (br. s, NH); 5.43 (br. s, CHN2); 5.62 (br.
d, J ˆ 7.0, NH); 7.30 ± 7.34 (m, 2 arom. H); 7.38 ± 7.43 (m, 2 arom. H); 7.59 ± 7.62 (m, 2 arom. H); 7.77 (d, J ˆ
7.5, 2 arom. H). 13C-NMR (100 MHz, CDCl3): 26.25 (CH2); 28.42 (Me); 29.56, 39.74 (CH2); 47.29, 54.02, 57.35
(CH); 66.77 (CH2); 79.36 (C); 120.0, 124.71, 125.06, 125.13, 127.10, 127.73 (CH); 141.37, 143.76, 156.05, 156.20,
‡
193.37 (C). FAB-MS: 479 (3.2, [M ‡ 1] ), 178(78.0), 165(34.3), 132(100). Anal. calc. for C26H30N4O5 (478.55):
C 65.26, H 6.32, N 11.71; found: C 65.20, H 6.42, N 11.52.
(S)-4-[(4-tert-Butoxy)phenyl]-1-diazo-3-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)butan-2-one (Fmoc-
(S)-Tyr(t-Bu)-CHN2; 10). Fmoc-(S)-Tyr(t-Bu)-OH (20.0 g, 43.5 mmol) was transformed according to GP 1. FC

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2151
(AcOEt/pentane 1:3) and recrystallization (CH2Cl2/hexane) yielded 10 (14.4 g, 69%). Yellow powder. M.p.
120.5 ± 121.58. Rf 0.18 (AcOEt/pentane 1:3). [a]rD:t: ˆ ‡3.5 (c ˆ 1.0, CHCl3). IR (CHCl3): 3426w, 3005w, 2985m,
2113s, 1718s, 1641m, 1503s, 1451m, 1390m, 1364s, 1318m, 1159m, 1108w, 1082w, 1041w, 918w, 897m, 851w, 826w.
1H-NMR (400 MHz, CD3COCD3, signals of rotamers in italics): 1.25 (s, t-Bu); 2.86 (dd, J ˆ 14.0, 9.6, H C(4));
3.14 (dd, J ˆ 14.0, 4.9, H C(4)); 4.16 (t, J ˆ 6.9, CHO); 4.31 (d, J ˆ 7.0, CH2O); 3.32 ± 4.44 (m, CHN); 5.86,
6.16 (s, CHN2); 6.80 (d, J ˆ 8.4, NH); 6.88 (d, J ˆ 8.4, 2 arom. H); 7.09, 7.19 (d, J ˆ 8.3, 2 arom. H); 7.30 ±
7.34 (m, 2 arom. H); 7.39 ± 7.43 (m, 2 arom. H); 7.65 (d, J ˆ 7.3, 2 arom. H); 7.85 (d, J ˆ 8.5, 2 arom. H).
13C-NMR (100 MHz, CD3COCD3, signals of rotamers in italics): 29.11 (Me); 37.31, 48.07 (CH2); 53.71, 60.81,
67.01 (CH); 78.41 (C); 120.79, 124.66, 126.11, 126.14, 127.93, 128.51, 130.61 (CH); 133.12, 142.15, 142.16, 144.92,
‡
‡
145.07, 155.23, 156.74, 194.59 (C). FAB-MS: 967 (8.6, 2M ), 484 (40.6, [M ‡ 1] ), 456(41.4), 399(13.5),
222(83.5), 179(100), 165(14.7), 154(53.1), 136(34.4). Anal. calc. for C29H29N3O4 (483.57): C 72.03, H 6.04,
N 8.69; found: C 72.11, H 6.17, N 8.58.
10. b3-Amino Acids 11 ± 14. (S)-3-{[(tert-Butoxy)carbonyl]amino}-5-phenylpentanoic Acid (Boc-(S)-b3-
HHop-OH; 11a) [33]. Diazo ketone 7 (1.4 g, 4.6 mmol) was transformed according to GP 2a. FC (CHCl3/
MeOH 10:1) and recrystallization from AcOEt/hexane yielded 11a (890 mg, 66%). White solid. M.p. 81 ± 828.
[a]rD:t:
ˆ
4.0 (c ˆ 0.75, CHCl3). IR (CHCl3): 3439w, 3018s, 2978w, 2400m, 1708m, 1510m, 1422m, 1210m, 1047w,
928w. 1H-NMR (200 MHz, CDCl3, signals of rotamers in italics): 1.46 (s, t-Bu); 1.71 ± 2.02 (m, CHCH2); 2.59 ±
2.81 (m, PhCH2, COCH2); 3.96 (br., CHN); 4.98, 5.84 (br., NH); 7.17 ± 7.32 (m, 5 arom. H). 13C-NMR
(50 MHz, CDCl3): 28.4 (Me); 32.6, 36.4, 39.3 (CH2); 47.4 (CH); 79.7 (C); 126.0, 128.4, 128.5 (CH); 141.4,
‡
155.6, 176.6 (C). FAB-MS: 304 (7, [M ‡ 1] ). Anal. calc. for C16H23NO4 (293.36): C 65.51, H 7.90, N 4.77;
found: C 65.62, H 7.85, N 4.83.
(S)-3-({[(9H-Fluoren-9-yl)methoxy]carbonyl}amino)-5-phenylpentanoic Acid (Fmoc-(S)-b3-HHop-OH;
11b). Diazoketone 11a (541 mg, 1.84 mmol) was dissolved in CF3COOH (0.25m) at 08. After stirring at r.t. for
1 h and concentration under reduced pressure, the crude trifluoroacetate salt which precipitated upon addition
of Et2O, was collected by filtration, dried under h.v., and used without further purification. The trifluoroacetate
salt (440 mg, 1.43 mmol) was transformed according to GP 3. FC (AcOEt/hexane/AcOH 4:6:0.3) and
recrystallization from CHCl3/hexane yielded 11b (490 mg, 64%). White solid. M.p. 177.5 ± 1788. Rf 0.47 (AcOEt/
hexane/AcOH 4:6:0.3). [a]Dr:t:
ˆ
7.62 (c ˆ 0.79, CHCl3). IR (CHCl3): 3434w, 3008w, 2944w, 1717s, 1510m,
1451w, 1405w, 1339w, 1248m, 1127w, 1082w, 1051w. 1H-NMR (400 MHz, CD3COCD3): 1.88 ± 1.91 (m, CHCH2);
2.53± 2.79 (m, PhCH2, COCH2); 4.01 ± 4.09 (m, CHN); 4.24 (t, J ˆ 7.0, CHCH2O); 4.32 ± 4.41 (m, CHCH2O);
6.51 (d, J ˆ 8.8, NH); 7.14 ± 7.42 (m, 4 arom. H); 7.70 (d, J ˆ 7.5, 2 arom. H); 7.85 (d, J ˆ 7.6, 2 arom. H).
13C-NMR (100 MHz, CD3COCD3): 33.1, 37.4, 40.1 (CH2); 48.2, 49.0 (CH); 66.7 (CH2); 120.8, 126.1, 126.5, 128.1,
‡
‡
128.5, 129.2 (CH); 142.1, 143.0, 145.3, 156.7, 172.7 (C). FAB-MS: 416.2 (100, [M ‡ 1] ), 415.1 (14, M ). Anal.
calc. for C26H25NO4 (415.49): C 75.16, H 6.06, N 3.37; found: C 75.04, H 5.98, N 3.41.
(S)-3-{[(tert-Butoxy)carbonyl]amino}-7-{[(2-chlorobenzyloxy)carbonyl]amino}heptanoic Acid (Boc-(S)-
b3-HLys(2-Cl-Z)-OH; 12). Diazo ketone 8 (6.85 g, 15.61 mmol) was transformed according to GP 2a.
Recrystallization (CH2Cl2/pentane) yielded 12 (5.05 g, 75%). White powder. M.p. 72 ± 788. Rf 0.3 (CH2Cl2/
MeOH 12:1). [a]Dr:t:
ˆ
11.43 (c ˆ 1.19, CHCl3). IR (CHCl3): 3448w, 3326w, 2981m, 2941m, 2859w, 1709s, 1597w,
1506s, 1445m, 1393m, 1368m, 1167s, 1039m, 867w. 1H-NMR (400 MHz, CD3COCD3): 1.39 (s, t-Bu);
2.45 (dd, J ˆ 15.4, 6.7, H C(2)); 2.54 (dd, J ˆ 15.4, 6.0, H C(2)); 3.14 ± 3.19 (m, CH2N); 3.91 (br., CHN);
5.16 (s, PhCH2); 5.86 (br. s, NH); 6.43 (br. s, NH); 7.32 ± 7.37 (m, 2 arom. H); 7.40 ± 7.44 (m, 1 arom. H); 7.48 ±
7.50 (m, 1 arom. H); 10.74 (br., COOH). 13C-NMR (100 MHz, CD3COCD3): 23.92 (CH2); 28.64 (Me); 30.42,
35.00, 40.13, 41.45 (CH2); 48.50 (CH); 63.63 (CH2); 78.60 (C); 127.99, 130.13, 130.20, 130.33 (CH); 133.57,
‡
‡
‡
136.05, 156.22, 156.91, 172.94 (C). FAB-MS: 857 (5.2, 2M ), 451 (10.4, [M ‡ Na] ), 429 (20.8, M ), 329 (100),
125 (60.0). Anal. calc. for C20H29ClN2O6 (428.91): C 56.01, H 6.81, N 6.53; found: C 56.02, H 6.79, N 6.36.
(S)-6-{[(tert-Butoxy)carbonyl]amino}-3-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)hexanoic Acid (Fmoc-
(S)-b3-Orn(Boc)-OH; 13). Diazo ketone 9 (2.65 g, 5.54 mmol) was transformed according to GP 2b.
Recrystallization (CHCl3/hexane) yielded 13 (1.63 g, 63%). White powder. M.p. 1048 (dec.). Rf 0.27 (MeOH/
CH2Cl2 1:9). [a]Dr:t:
ˆ
8.0 (c ˆ 1.0, CHCl3). IR (CHCl3): 3436w, 3008w, 2981w, 1710s, 1511m, 1450m, 1406w,
1367w, 1169m, 1107w, 1082w, 1046w, 872w. 1H-NMR (400 MHz, CD3COCD3; signals of rotamers in italics):
1.40 (s, t-Bu); 1.52 ± 1.66 (m, 2 CH2); 2.51 (dd, J ˆ 15.6, 6.5, H C(2)); 2.58 (dd, J ˆ 15.6, 6.6, H C(2)); 3.09 ±
3.18 (m, CH2N); 3.95 ± 4.05 (m, CHN); 4.22 (t, J ˆ 7.1, CHO); 4.29 ± 4.40 (m, CH2O); 5.58, 5.95 (br., NH);
6.46 (d, J ˆ 8.7, NH); 7.30 ± 7.34 (m, 2 arom. H); 7.39 ± 7.43 (m, 2 arom. H); 7.60 ± 7.70 (m, 2 arom. H);
7.86 (d, J ˆ 7.5, 2 arom. H). 13C-NMR (100 MHz, CD3COCD3; signals of rotamers in italics): 14.34, 28.67 (Me);
23.28, 27.55, 32.58, 40.17, 40.90 (CH2); 48.16, 49.10 (CH); 66.72 (CH2); 78.38 (C); 120.79, 126.13, 126.16, 127.93,
‡
128.48 (CH); 142.11, 145.12, 145.23, 156.65, 156.71, 172.79 (C). FAB-MS: 938 (6.2, [2 M ‡ 1] ), 491 (4.9, [M ‡

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‡
‡
Na] ), 469 (30.3, [M ‡ 1] ), 369(50.9), 307(47.0), 289(24.8), 178(100). Anal. calc. for C26H32N2O6 (468.55):
C 66.65, H 6.88, N 5.98; found: C 66.53, H 6.79, N 5.84.
(S)-3-({[(9H-Fluoren-9-yl)methoxy]carbonyl}amino)-4-[4-(tert-butoxy)phenyl]butanoic Acid (Fmoc-(S)-
b3-HTyr(t-Bu)-OH; 14). Diazo ketone 10 (8.0 g, 16.5 mmol) was transformed according to GP 2b. FC (CH2Cl2/
MeOH 15:1 ! 6:1) and recrystallization (AcOEt/pentane) yielded 14 (5.81 g, 74%). White powder. M.p. 190 ±
1918 (dec.). Rf 0.44 (CH2Cl2/MeOH 10:1). [a]Dr:t:
ˆ
20.8 (c ˆ 0.5, DMF). IR (KBr): 3500 ± 2700 (br.), 1695s,
1656m, 1606w, 1562m, 1534m, 1506s, 1451m, 1367m, 1262m, 1234s, 1162m, 1106m, 1084m, 1045m, 901m, 851w,
1
756m, 734m, 623w, 573w. H-NMR (400 MHz, CDCl3, signals of rotamers in italics): 1.25, 1.30 (s, t-Bu); 2.10 ±
2.95 (m, 2 H C(2), PhCH2); 3.90, 4.16 ± 4.20 (m, CHO, CHN); 4.30 ± 4.51 (m, CH2O); 5.25, 5.79 (br., NH);
6.89 (d, J ˆ 7.5, 2 arom. H); 7.00 ± 7.10 (m, 2 arom. H); 7.28 ± 7.31 (m, 2 arom. H); 7.36 ± 7.40 (m, 2 arom. H);
7.55 (d, J ˆ 7.4, 2 arom. H); 7.74 (d, J ˆ 7.5, 2 arom. H). 13C-NMR (100 MHz, CDCl3, signals of rotamers
in italics): 28.82, 29.70 (Me); 37.37, 39.51 (CH2); 47.25, 49.25 (CH); 66.72 (CH2); 78.42, 119.98, 124.26,
125.05, 127.07, 127.70, 129.77, 132.03 (CH); 141.33, 143.87, 154.17, 155.74, 176.10 (C). FAB-MS: 1497 (41.4,
‡
‡
‡
‡
‡
[3M 1 ‡ 2 K] ), 985 (8.8, [2M ‡ K] ), 512 (17.9, [M ‡ K] ), 496 (43.8, [M ‡ Na] ), 474 (78.3, [M ‡ 1] ),
307(22.3), 179(100). Anal. calc. for C29H31NO5 ´ 0.75 H2O (487.09): C 71.51, H 6.73, N 2.88; found: C 71.54,
H 6.81, N 2.92.
11. b2-Amino Acid 19. (R)-4-Benzyl-3-(4-phenyl-1-oxobutyl)oxazolidin-2-one (15). Similarly to the
procedure reported in [34], 4-phenylbutyric acid (4.08 g, 24.87 mmol) and Et3N (3.6 ml, 26.07 mmol) were
dissolved in THF (50 ml) under Ar. After cooling to 788 (internal temp.), pivaloyl chloride (3.2 ml,
26.1 mmol) was slowly added, and the white suspension was stirred at 08 for 1 h. In a separate flask, (R)-4-
benzyloxazolidin-2-one (4.2 g, 23.7 mmol) was dissolved in THF (100 ml) at r.t. and treated with a cat. amount
of DMAP (144 mg, 1.19 mmol) and Et3N (3.3 ml, 23.7 mmol). The resulting soln. was added to the mixed pivalic
anhydride at 788 over 5 min. After stirring for 12 h at r.t., the mixture was diluted with 1m NaOH and extracted
with CH2Cl2. The org. layer was washed with sat. NaCl soln., H2O, dried (Na2SO4), and concentrated in vacuo.
FC (AcOEt/hexane 3:7) yielded (R)-4-benzyloxazolidin-2-one (1.0 g, 24%) and 15 (5.0 g, 65%). White solid.
M.p. 108 ± 1098. [a]Dr:t:
ˆ
48.5 (c ˆ 1.04, CHCl3). IR (CHCl3): 3011w, 2924w, 1781s, 1698m, 1497w, 1454w,
1
1385m, 1352m, 1291w, 1108w, 1080w, 1051w, 1014w. H-NMR (200 MHz, CDCl3): 1.99 ± 2.14 (m, CH2); 2.71 ±
2.84 (m, CH2CO, 1 H, PhCH2); 2.92 ± 3.10 (m, PhCH2); 3.31 (dd, J ˆ 13.3, 3.3, 1 H, PhCH2); 4.16 ± 4.21
(m, CH2O); 4.62 ± 4.74 (m, CHN); 7.18 ± 7.41 (m, 5 arom. H). 13C-NMR (50 MHz, CDCl3): 23.39, 32.53,
32.69, 35.49 (CH2); 52.72 (CH); 63.77 (CH2); 123.66, 125.03, 126.08, 126.20, 126.65, 127.09 (CH); 133.00, 139.22,
‡
‡
151.15, 170.77 (C). FAB-MS: 324.1 (100, [M ‡ 1] ), 423.1 (7, M ). Anal. calc. for C20H21NO3 (323.39): C 74.28,
H 6.54, N 4.33; found: C 74.31, H 6.59, N 4.48.
(4S)-4-Benzyl-3-[(2R)-2-({[(benzyloxy)carbonyl]amino}methyl)-1-oxo-4-phenylbutyl]oxazolidin-2-one
(16). Compound 15 (4.8 g, 14.86 mmol) was dissolved in CH2Cl2 under Ar and cooled to 58 (internal temp.).
TiCl4 (1.64 ml, 15 mmol) was slowly added (internal temp. <18) and the resulting orange soln. stirred for 10 min.
Et3N (2.09 ml, 15 mmol) was then added and the resulting dark red soln. stirred for 45 min at 58. Benzyl N-
(methoxymethyl) carbamate (4.35 g, 22.29 mmol) [27] was added, and the mixture was stirred at 08 for 3 h. The
mixture was quenched with sat. NH4Cl soln. and the org. phase washed twice with 1m HCl and sat. NaCl soln.,
dried (MgSO4), and evaporated. FC (Et2O/pentane 1:1) yielded 15 (960 mg, 20%) and 16 (3.19 g, 44%).
Colorless oil. [a]Dr:t:
ˆ
41.1 (c ˆ 1.3, CHCl3). IR (CHCl3): 3450w, 3010s, 2977w, 1779m, 1720m, 1515m, 1422w,
1389w, 1351w, 1215w, 1046w, 928w. 1H-NMR (300 MHz, CDCl3): 1.76 ± 1.88 (m, 1 H, PhCH2CH2); 2.06 ±
2.12 (m, 1 H, PhCH2CH2); 2.60 ± 2.70 (m, 3 H, PhCH2CH2, PhCH2CHN); 3.21 (dd, J ˆ 13.4, 3.4, 1 H,
PhCH2CHN); 3.52 ± 3.60 (m, CH2N); 3.96 ± 3.98 (m, CHCO); 4.04 ± 4.12 (m, CH2O); 4.46 (m, CHN);
5.10 (s, PhCH2); 5.15 (br., NH); 7.14 ± 7.61 (m, 15 arom. H). 13C-NMR (100 MHz, CDCl3): 28.7, 31.1, 35.4,
40.0 (CH2); 40.1, 53.0 (CH); 63.8, 64.3 (CH); 123.7, 124.9, 125.7, 125.9, 126.1, 126.2, 126.5, 127.0 (CH); 132.9,
‡
‡
134.1, 138.7, 150.7, 153.9, 172.3 (C). FAB-MS: 509 (100, [M ‡ Na] ), 487 (54, [M ‡ 1] ).
(S)-2-({[(Benzyloxy)carbonyl]amino}methyl)-4-phenylbutanoic Acid (Z-(S)-b2-HHop-OH; 17). Com-
pound 16 (3.0 g, 6.16 mmol) was dissolved in THF/H2O 4:1 (0.2m) and cooled (ice-bath). H2O2 (30% aq. soln.,
2.5 ml, 25 mmol) followed by LiOH ´ H2O (414 mg, 9.86 mmol.), was added, and the soln. was stirred for 90 min
at 08. The mixture was treated with 1m Na2SO3 soln. (20 ml), concentrated and extracted with CH2Cl2 (3Â). The
chiral auxiliary was recovered from the CH2Cl2 phase (590 mg, 60%). The aq. phase was acidified (pH 1 ± 2)
with 6m HCl and extracted with AcOEt (3Â). The combined AcOEt phases were dried (MgSO4) and
evaporated. Recrystallization from CHCl3/hexane yielded 17 (1.40 g, 70%). Colorless needles. M.p. 103 ± 103.58.
[a]rD:t:
ˆ
4.7 (c ˆ 0.96, CHCl3). IR (CHCl3): 3446w, 3011w, 2948w, 1717s, 1515m, 1455w, 1410w, 1328w, 1261w,
1143w, 1082w, 1046w. 1H-NMR (400 MHz, CD3OD): 1.74 ± 1.83 (m, 1 H, PhCH2CH2); 1.84 ± 1.93 (m, 1 H,
PhCH2CH2); 2.55 ± 2.71 (m, 3 H, PhCH2CH2, CHCO); 3.27 ± 3.41 (m, CH2N); 4.84 (s, PhCH2); 7.04 (br., NH);

Helvetica Chimica Acta ± Vol. 81 (1998)
2153
7.12 ± 7.34 (m, 10 arom. H). 13C-NMR (100 MHz, CD3OD): 32.5, 34.4, 43.4 (CH2); 46.6 (CH); 67.5 (CH2);
‡
127.1, 129.1, 129.2, 129.5 (CH); 138.4, 143.0, 158.9, 177.9 (C). FAB-MS: 655.3 (2, [2M ‡ 1] ), 350.1 (13, [M ‡
‡
‡
‡
Na] ), 328.1 (100, [M ‡ 1] ), 327.1 (7, M ). Anal. calc. for C19H21NO4 (327.38): C 69.71, H 6.47, N 4.28; found:
C 69.75, H 6.61, N 4.31.
(S)-2-(Aminomethyl)-4-phenylbutanoic Acid (H-(S)-b2-HHop-OH; 18). Compound 17 (600 mg,
1.83 mmol) was dissolved in EtOH (0.1m), and a cat. amount of 10% Pd/C was added. The apparatus was
evacuated, flushed three times with H2, and the mixture was stirred under an atmosphere of H2, for 3 h.
Filtration, concentration under reduced pressure, and recrystallization from EtOH/H2O yielded 18 (222 mg,
63%). Colorless needles. M.p. 228 ± 2308. [a]Dr:t:
ˆ
26.4 (c ˆ 0.6, 1m HCl). IR (KBr): 2948s, 1633s, 1506s, 1261w.
1H-NMR (200 MHz, D2O): 1.71 ± 2.00 (m, PhCH2CH2); 2.47 ± 2.72 (m, 3 H, PhCH2CH2, CHCO); 3.00 ±
3.20 (m, CH2N); 7.22 ± 7.40 (m, 5 arom. H). 13C-NMR (50 MHz, D2O): 31.9, 32.6, 41.1 (CH2); 45.0 (CH);
‡
‡
67.5 (CH2); 126.5, 128.7, 128.9 (CH); 142.1, 180.6 (C). ESI-MS: 232.0 (10, [M ‡ K] ), 216.0 (28, [M ‡ Na] ),
‡
194.0 (100, [M ‡ 1] ). Anal. calc. for C11H15NO2 (193.25): C 68.37, H 7.82, N 7.25; found: C 68.17, H 7.72,
N 7.21.
(S)-2-[({[(9H-Fluoren-9-yl)methoxy]carbonyl}amino)methyl]-4-phenylbutanoic Acid (Fmoc-(S)-b2-HHop-
OH; 19). The free b2-amino acid 18 was transformed according to GP 3. Recrystallization from CHCl3/hexane
yielded 17 (310 mg, 75%). White solid. M.p. 182 ± 1848. Rf 0.41 (AcOEt/hexane/AcOH 4:6:0.3). [a]Dr:t:
ˆ
9.7
(c ˆ 1.2, CH3COCH3). IR (CHCl3): 3451w, 3019s, 2400m, 1522w, 1477w, 1423w, 1410w, 1328w, 1215s, 1046w,
929w. 1H-NMR (400 MHz, CD3COCD3): 1.81 ± 1.98 (m, PhCH2CH2); 2.62 ± 2.77 (m, 3 H, PhCH2CH2, CHCO);
3.36 ± 3.48 (m, CH2N); 4.22 (t, J ˆ 7.0, CHCH2O); 4.33 (d, J ˆ 6.9, CHCH2O); 6.59 (br., NH); 7.14 ± 7.42 (m, 9
arom. H); 7.69 (d, J ˆ 7.5, 2 arom. H); 7.85 (d, J ˆ 7.5, 2 arom. H). 13C-NMR (100 MHz, CD3COCD3): 32.3,
34.0, 43.0 (CH2); 45.9, 48.1 (CH); 67.0 (CH2); 120.8, 126.1, 126.6, 127.7, 128.5, 129.3 (CH); 142.1, 142.4, 145.2,
‡
‡
157.3, 175.7 (C). FAB-MS: 832.5 (1, [2M ‡ 1] ), 416.2 (100, [M ‡ 1] ). Anal. calc. for C26H25NO4 (415.49):
C 75.16, H 6.06, N 3.37; found: C 75.08, H 5.95, N 3.39.
12. b-Peptides. H-(S)-b3-HLys-(S)-b3-HLys-(S)-b3-HLys-(S)-b3-HLys-(S)-b3-HLys-(S)-b3-HLys-(S)-b3-
HLys-OH ´ 8 CF3COOH (1). According to GP 4 the ortho-chlorotrityl-chloride resin (189 mg, 1.00 mmol Cl/
g) was esterified with Fmoc-(S)-b3-HLys(Boc)-OH [29] (64.0 mg, 0.168 mmol). Loading 0.338 mmol/g (57%)
corresponding to 64 mmol of anchored Fmoc-(S)-b3-HLys(Boc)-OH. Synthesis according to GP 5b and
cleavage from the resin according to GP 6 afforded crude 1 as CF3COOH salt (112 mg, 91%), purity 79% (RP-
HPLC). The peptide was purified by prep. RP-HPLC (2 ± 30% B in 20 min) according to GP 7: CF3COOH salt
of 1 (67.8 mg, 55%). Colorless high viscous liquid. RP-HPLC (0 ± 25% B in 20 min) tR 10.0 min, purity >99%.
M.p. 2358 (dec., sintering at 2008). CD (0.2 mm in MeOH): 1.2 ´ 104 (219 nm). IR (KBr): 3600 ± 3000 (br.),
2956m, 2867m, 2362s, 2340s, 1850 ± 1350br, 1206m, 1172m, 1128m, 839w, 800w, 722w, 666m, 606w, 522w.
1H-NMR (400 MHz, D2O): 1.29 ± 1.70 (m, 21 CH2); 2.28 ± 2.46 (m, 11 CHCO); 2.50 ± 2.66 (m, 3 CHCO); 3.55 ±
3.59 (m, CHN); 4.07 ± 4.17 (m, 6 CHN). 13C-NMR (100 MHz, D2O): 24.34, 24.89, 29.07, 29.13, 29.17, 34.35,
35.69, 35.81, 39.68, 41.77, 41.88, 41.97, 43.73, 43.79, 43.93 (CH2); 49.02, 49.70, 49.75, 51.43 (CH); 119.07 (q, J ˆ
583.5, 291.6, CF3); 165.61 (q, J ˆ 70.7, 35.1, CF3CO); 173.85, 175.14, 175.20, 175.27, 178.05 (C). FAB-MS: 1052
‡
‡
‡
(4.9, [M ‡ K] ), 1036 (14.9, [M ‡ Na] ), 1015 (100, [M ‡ 1] ), 307(19), 137(63.5), 84(62.5). Anal. calc. for
C49H100N14O8 ´ 8 CF3COOH (1925.61): C 40.54, H 5.65, N 10.18; found: C 39.42, H 5.39, N 9.82.
H-(S)-b3-HPhe-(S)-b3-HAla-(S)-b3-HLys-(S)-b3-HPhe-(S)-b3-HAla-(S)-b3-HLys-(S)-b3-HPhe-OH (2).
According to GP 4 the ortho-chlorotrityl-chloride resin (200 mg, 1.30 mmol Cl/g) was esterified with Fmoc-
b3-(S)-HPhe-OH (104.0 mg, 0.260 mmol). Loading 0.42 mmol/g (48%). Synthesis (62 mmol) according to GP 5b
and cleavage from the resin according to GP 6 afforded crude 2 as CF3COOH salt (58.1 mg, 72%), purity 81%
(RP-HPLC). The peptide was purified by prep. RP-HPLC (10 ± 60% B in 10 min) according to GP 7:
CF3COOH salt of 2 (46 mg, 57%). White solid. RP-HPLC (30 ± 90% B in 20 min) tR 12.0 min, purity >96%.
M.p. 225.2 ± 236.48. CD (0.2 mm in MeOH): 3.7 ´ 104 (219 nm). IR (KBr): 3600 ± 3000 (br.), 2960m, 1650s,
1
1550s, 1205m, 1133m, 800m, 722m. H-NMR (400 MHz, CD3OD): 1.12 (d, J ˆ 6.7, Me); 1.13 (d, J ˆ 6.7, Me);
1.35 ± 1.50 (m, CHCH2CH2CH2); 1.56 ± 1.75 (m, CHCH2CH2CH2); 2.25 ± 3.07 (m, 24 H, CH2CO, CH2NH2,
PhCH2); 3.75 ± 3.82 (m, CHN); 4.16 ± 4.18 (m, CHN); 4.29 ± 4.35 (m, CHN); 4.36 ± 4.53 (m, 2 CHN); 4.57 ±
4.64 (m, 2 CHN); 7.16 ± 7.36 (m, 15 arom. H); 7.62 (d, J ˆ 8.6, NH); 7.77 (d, J ˆ 9.0, NH); 7.97 (d, J ˆ 9.1,
NH); 8.21 (d, J ˆ 9.2, NH); 8.25 (d, J ˆ 8.8, NH). 13C-NMR (100 MHz, D2O): 17.1, 17.5 (Me); 20.1, 24.3, 30.5,
30.6, 34.8, 36.1, 37.2, 37.3, 37.9, 38.9, 39.0, 40.0, 40.1, (CH2); 41.3, 41.4, 44.5, 44.6, 46.0, 46.7, 48.1 (CH); 124.8,
‡
125.8, 126.7, 127.1, 127.5 (CH); 133.1, 136.1, 168.5, 170.1, 170.2, 170.6, 173.6 (C). FAB-MS: 958 (57, [M ‡ 1] ),
‡
957 (100, M ).
H-(S)-b3-HPhe-(S)-b3-HAla-(S)-b3-HSer-(S)-b3-HPhe-(S)-b3-HAla-(S)-b3-HSer-(S)-b3-HPhe-OH (3). Ac-
cording to GP 4 the ortho-chlorotrityl-chloride resin (250 mg, 1.00 mmol Cl/g) was esterified with Fmoc-b3-(S)-

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Helvetica Chimica Acta ± Vol. 81 (1998)
HPhe-OH (90.0 mg, 0.225 mmol). Loading 0.68 mmol/g (92%). Synthesis (62 mmol) according to GP 5a and
cleavage from the resin according to GP 6 afforded crude 3 as CF3COOH salt (35.4 mg, 58%), purity 70% (RP-
HPLC). The peptide was purified by prep. RP-HPLC (10 ± 60% B in 120 min) according to GP 7: CF3COOH
salt of 3 (15 mg, 25%). White solid. RP-HPLC (30 ± 90% B in 20 min) tR 9.9 min, purity >96%. M.p. 115.2 ±
149.48. CD (0.2 mm in MeOH): 6.1 ´ 104 (219 nm). IR (KBr): 3600 ± 3000 (br.), 3286s, 3086m, 2929m, 1653s,
1546s, 1455m, 1378m, 1260w, 1205m, 1134m, 1055w, 968w, 834w, 800w, 750w, 721w, 700w. 1H-NMR (400 MHz,
CD3OD): 1.13 (d, J ˆ 6.2, Me); 2.25 ± 3.07 (m, 20 H, CH2CO, PhCH2); 3.42 ± 3.52 (m, CH2O); 3.75 ±
3.82 (m, CHN); 4.16 ± 4.22 (m, CHN); 4.44 ± 4.55 (m, 3 CHN); 4.63 ± 4.71 (m, 2 CHN); 7.16 ± 7.38 (m, 15 arom.
H); 7.45 (d, J ˆ 8.5, NH); 7.60 (d, J ˆ 9.0, NH); 7.97 (d, J ˆ 9.0, NH); 8.44 (d, J ˆ 9.7, NH); 8.50 (d, J ˆ 8.9,
NH). 13C-NMR (100 MHz, D2O): 20.9, 21.3 (Me); 38.2, 38.3, 38.9, 39.2, 40.2, 41.4, 42.3, 43.2, 43.3, 43.4 (CH2);
43.6, 43.8, 52.0 (CH); 64.9 (CH2); 127.7, 128.6, 129.6, 130.1, 130.6 (CH); 136.9, 139.3, 139.8, 171.2, 171.5, 171.6;
‡
‡
‡
171.9, 172.3, 173.7, 175.2 (C). FAB-MS: 912 (4.7, [M ‡ K] ), 896 (26.5, [M ‡ Na] ), 875 (93.6, [M ‡ 1] ), 874
‡
(100, M ).
H-(S)-b3-HAla-(S)-b3-HLys-(S)-b3-HPhe-(S)-b3-HAla-(S)-b3-HLys-(S)-b3-HPhe-(S)-b3-HAla-(S)-b3-HLys-
(S)-b3-HPhe-OH (4). According to GP 4 , the ortho-chlorotrityl-chloride resin (158.6 mg, 1.00 mmol Cl/g) was
esterified with Fmoc-b3-(S)-HPhe-OH (48 mg, 0.12 mmol). Loading 0.38 mmol/g (65%) corresponding to 60 mmol
of anchored Fmoc-b3-(S)-HPhe-OH. Synthesis according to GP 5b and cleavage from the resin according to GP 6
afforded crude 4 as CF3COOH salt (99 mg, quant.), purity 49% (RP-HPLC). The peptide was purified by RP-
HPLC (10 ± 35% B in 20 min, then 35±45% B in 15 min; C8) according to GP 7: CF3COOH salt of 4 (25.7 mg,
26%). White solid. HPLC (15 ± 65% B in 20 min; C8) tR 11.7 min, purity >95%. M.p. 1378 (dec.). CD (0.2 mm in
MeOH): 4.8 ´104 (219 nm). IR (KBr): 3293 (br.), 3096m, 2974m, 2947m, 1676s, 1654s, 1560m, 1541m, 1437w,
1265w, 1205s, 1136m, 1031w, 838w, 800w, 723m, 701w. 1H-NMR (400 MHz, CD3OD): 1.12 (d, J ˆ 6.7, Me);
1.14 (d, J ˆ 6.6, Me); 1.29± 1.76 (m, Me, 9 CH2); 2.25± 2.70 (m, 9 CH2CO); 2.74± 2.96 (m, 3 PhCH2, 3 CH2N);
3.69± 3.75 (m, CHN); 4.38 ± 4.68 (m, 8 CHN); 7.16± 7.28 (m, 15 arom. H); 7.77 (d, J ˆ 9.0, NH); 8.16 (d, J ˆ 9.8,
NH); 8.23 (d, Jˆ 8.8, NH); 8.41± 8.44 (m, NH). 13C-NMR (100 MHz, CD3OD): 18.84, 20.93, 21.56 (Me); 23.90,
24.09, 24.45, 28.03, 28.73, 28.82, 35.89, 36.54, 40.64, 40.67, 40.70, 41.11, 41.57, 41.65, 42.25, 43.23, 43.37 (CH); 43.49,
43.64, 43.81, 46.67, 46.99, 47.26, 47.41, 127.70, 129.50, 129.57, 130.52, 130.62, 130.65 (CH); 139.40, 139.52, 139.66,
‡
171.43, 171.46, 171.64, 171.75, 171.91, 172.13, 172.21, 173.11. FAB-MS: 1222 (18.0, [M ‡ K] ), 1206 (39.8, [M ‡
‡
‡
Na] ), 1184 (100, [M ‡ 1] ), 1042 (13.4).
H-(S)-b3-HHop-(S)-b3-HAla-(S)-b3-HLys-(S)-b3-HHop-(S)-b3-HAla-(S)-b3-HLys-(S)-b3-HHop-OH (5).
According to GP 4 the ortho-chlorotrityl-chloride resin (150 mg, 1.00 mmol Cl/g) was esterified with 11b
(56 mg, 0.135 mmol). Loading 0.61 mmol/g (91%) corresponding to 0.123 mmol of anchored 11b. Synthesis
according to GP 5b and cleavage from the resin according to GP 6 afforded crude 5 as AcOH salt (60.4 mg,
45%), purity 48% (RP-HPLC). The peptide was purified by prep. RP-HPLC (20 ± 80% B in 20 min, C8)
according to GP 7: CF3COOH salt of 5 (15 mg, 25%). White solid. RP-HPLC (20 ± 80% B in 20 min): tR
12.94 min, purity >98%. M.p. 101.5 ± 108.48. CD (0.2 mm in MeOH): 4.2 ´ 104 (220 nm). IR (KBr): 3600 ±
3000 (br.), 3283s, 3066s, 2955s, 1650s, 1555s, 1433m, 1383m, 1261w, 1206s, 1133s, 978w, 833w, 800m, 750w, 722m,
700m. 1H-NMR (500 MHz, CD3OD): 1.18 (d, J ˆ 6.7, Me); 1.21 (d, J ˆ 6.7, Me); 1.37 ± 1.83 (m, 16 H,
PhCH2CH2, CHCH2CH2CH2); 1.95 ± 2.12 (m, PhCH2CH2); 2.28 ± 2.95 (m, 24 H, PhCH2, CH2N, CH2CO);
3.64 ± 3.69 (m, CHN); 4.29 ± 4.57 (m, 6 CHN); 7.16 ± 7.38 (m, 15 arom. H); 7.63 (d, J ˆ 9.0, NH); 7.77 (d, J ˆ
9.2, NH); 7.92 (d, J ˆ 9.2, NH); 8.35 (d, J ˆ 9.3, NH); 8.37 (d, J ˆ 9.1, NH). 13C-NMR (125 MHz, CD3OD):
21.0, 21.2 (Me); 24.0, 24.2, 28.3, 28.6, 32.4, 33.7, 33.9, 35.9, 36.2, 36.3, 38.4, 39.0, 39.1, 39.9, 40.6, 40.7, 42.0, 43.3
(CH2); 43.6 (CH); 43.7(CH2); 44.0, 47.1, 47.3, 47.4, 48.2, 50.7 (CH); 127.0, 127.5, 129.3, 129.4, 129.5, 129.8 (CH);
‡
141.8, 142.8, 142.9, 171.2, 171.7, 171.9, 172.0, 172.6, 173.4, 175.1 (C). FAB-MS: 1021 (5.0, [M ‡ Na] ), 999 (70.3,
‡
‡
[M ‡ 2] ), 998 (100, [M ‡ 1] ).
H-(S)-b2-HHop-(S)-b3-HAla-(S)-b3-HLys-(S)-b2-HHop-(S)-b3-HAla-(S)-b3-HLys-(S)-b2-HHop-OH (6).
According to GP 4 the ortho-chlorotrityl-chloride resin (150 mg, 1.00 mmol Cl/g) was esterified with 19 (56 mg,
0.135 mmol). Loading 0.66 mmol/g (99%) corresponding to 0.133 mmol of anchored 19. Synthesis according to
GP 5a and cleavage from the resin according to GP 6 afforded crude 6 as AcOH salt (72 mg, 46%), purity 80%
(RP-HPLC). The peptide was purified by prep. RP-HPLC (20 ± 80% B in 20 min, C18) according to GP 7:
CF3COOH salt of 6 (59 mg, 33%). White solid. RP-HPLC (20 ± 80% B in 20 min): tR 12.6 min, purity >97%.
M.p. 76.5 ± 106.78. CD (0.2 mm in MeOH): 4.8 ´ 104 (219.8 nm). IR (KBr): 3600 ± 3000 (br.), 3277s, 3067s,
1
2966s, 1650s, 1555s, 1456m, 1394m, 1261m, 1200s, 1133s, 833m, 800m, 750w, 722m, 700m. H-NMR (500 MHz,
CD3OD): 1.22 (d, J ˆ 6.7, Me); 1.23 (d, J ˆ 6.6, Me); 1.36 ± 1.96 (m, 18 H, PhCH2CH2, CHCH2CH2CH2); 2.30 ±
2.73 (m, 16 H, PhCH2, CH2CO, CHCO); 2.87 ± 2.97 (m, 5 H, CH2CH2N, CHCO); 3.09 ± 3.29 (m, 4 H, CH2N);
3.55 (dd, J ˆ 13.5, 9.0, 1 H, CH2N); 3.62 (dd, J ˆ 13.2, 10.8, 1 H, CH2N); 4.25 ± 4.36 (m, 2 CHN); 4.43 ± 4.53

Helvetica Chimica Acta ± Vol. 81 (1998)
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(m, 2 CHN); 7.05 ± 7.26 (m, 15 arom. H). 13C-NMR (125 MHz, CD3OD): 21.2, 21.4 (Me); 24.0, 24.1, 28.3, 28.5,
33.5, 33.6, 34.0, 34.4, 34.5, 34.8, 35.5, 36.0, 40.7, 40.8, 41.8, 41.9, 42.0, 42.3, 42.9, 43.4, 43.4, 43.5 (CH2); 44.1, 44.4,
45.6, 46.2, 47.3, 47.4, 47.7 (CH); 127.1, 127.2, 127.3, 129.3, 129.5, 129.6 (CH); 142.4, 142.7, 172.4, 172.8, 173.4,
‡
‡
‡
173.8, 174.8, 177.9 (C). FAB-MS: 1021 (9.6, [M ‡ Na] ), 999 (58.6, [M ‡ 2] ), 998 (100, [M ‡ 1] ).
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Received September 22, 1998