Synthesis, CD Spectra, and Enzymatic Stability β 2-Oligoazapeptides Prepared from (S)-2-Hydrazino Carboxylic Acids Carrying the Side Chains of Val, Ala, and Leu

Article abstract of DOI:10.1002/hlca.200390342

β-Peptides offer the unique possibility to incorporate additional heteroatoms into the peptidic backbone (Figs. 1 and 2). We report here the synthesis and spectroscopic investigations of β²-peptide analogs consisting of (S)-3-aza-β-amino acids

Full text of DOI:10.1002/hlca.200390342

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Synthesis, CD Spectra, and Enzymatic Stability of b2-Oligoazapeptides
Prepared from (S)-2-Hydrazino Carboxylic Acids Carrying the Side Chains of
Val, Ala, and Leu
by Ge¬rald Lelais1) and Dieter Seebach*
Laboratorium f¸r Organische Chemie der Eidgenˆssischen Technischen Hochschule, ETH Hˆnggerberg, HCI,
Wolfgang-Pauli-Strasse 10, CH-8093 Z¸rich
(phone: ‡4116322990; fax: ‡4116321144; e-mail: seebach@org.chem.ethz.ch)
Dedicato al Professore Duilio Arigoni in occasione del suo 758 compleanno
b-Peptides offer the unique possibility to incorporate additional heteroatoms into the peptidic backbone
(Figs. 1 and 2). We report here the synthesis and spectroscopic investigations of b2-peptide analogs consisting of
(S)-3-aza-b-amino acids carrying the side chains of Val, Ala, and Leu. The hydrazino carboxylic acids were
prepared by a known method: Boc amidation of the corresponding N-benzyl-l-a-amino acids with an
oxaziridine (Scheme 1). Couplings and fragment coupling of the 3-benzylaza-b2-amino acids and
a
corresponding tripeptide (N-Boc/C-OMe strategy) with common peptide-coupling reagents in solution led to
b2-di, b2-tri-, and b2-hexaazapeptide derivatives, which could be N-debenzylated (4 9; Schemes 2 4). The new
compounds were identified by optical rotation, and IR, 1H-and 13C-NMR, and CD spectroscopy (Figs. 4 and 5)
and high-resolution mass spectrometry, and, in one case, by X-ray crystallography (Fig. 3). In spite of extensive
measurements under various conditions (temperatures, solvents), it was not possible to determine the secondary
structure of the b2-azapeptides by NMR spectroscopy (overlapping and broad signals, fast exchange between the
two types of NH protons!). The CD spectra of the N-Boc and C-OMe terminally protected hexapeptide analog
9 in MeOH and in H2O (at different pH) might arise from a (P)-314-helical structure. The N-Boc-b2-tri and N-
Boc-b2-hexaazapeptide esters, 7 and 9, were shown to be stable for 48 h against the following peptidases:
pronase, proteinase K, chymotrypsin, trypsin, carboxypeptidase A, and 20S proteasome.
1. Introduction.
The synthesis of non-natural peptide mimics, so called
peptidomimetics, has received increasing attention in recent years. Their rapid
development is driven by the fact that they will eventually replace both a-peptide
substrates of enzymes and a-peptide ligands of protein receptors, due to the enhanced
resistance toward proteases, increased bioavailability, and reduced immunogenicity
they may have, as compared to a-peptide analogs. In addition, many of these oligomers
adopt defined secondary structures, a necessary feature for promising candidates in
drug design.
Among all synthetic strategies, the modification of the peptide backbone seems to
be the most relevant for the construction of secondary structures [1]; this may involve
isosteric or isoelectronic exchange of units (e.g., peptoids, azapeptides, azatides, ethane-
1,2-amines, oxazolidin-2-ones, and pyrrolidinones) or the introduction of additional
fragments (e.g., b-peptides, a-aminoxy peptides, hydrazino peptides, g-peptides,
1
)
Part of the projected Ph.D. thesis of G. L., ETH-Z¸rich 2004.

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oligocarbamates, oligoureas, vinylogous sulfonopeptides, and many more other
compounds) (Fig. 1)2).
b-Peptides are probably the most thoroughly investigated peptidomimetic oligo-
mers. They are stable to rat metabolism [2], exhibit slow microbial degradation [3], and
are inherently stable to a range of proteases and peptidases [4 8]. Additionally, as few
as six amino acids are necessary to allow the peptides to fold into well-ordered
secondary structures consisting of helices, turns, and sheets [9 13]. To expand the
prolific field of b-peptides towards the construction of possible new secondary
structures, the replacement of the Ca and Cb atoms of the b-amino acids with
heteroatoms could be an attractive modification. In fact, oligomers of a-aminoxy acids,
i.e., b-amino acids where the Cb-atom is replaced by O, display a defined helical
structure in solution, characterized by 1.8 residues per turn and H-bonds between the
CO of residue i and NH of residue i ‡ 2. The so-formed eight-membered H-bonded
rings (consecutive NÀO turns) differ from the helices found in −normal× b-peptides
[14 17]. Another feasible modification would be the replacement of the Cb-atom of b-
peptides by NR, leading to b2-azapeptides, also called hydrazino peptides. These novel
peptides are of great interest, owing to the possibility for additional H-bonds via the
sp3-N-atom. Quantum-chemical calculations carried out on 3-aza-b-homoglycine
oligomers by G¸nther and Hofmann [18] show the presence of a wide variety of
secondary-structure elements. Whereas some characteristics correspond to those
structural types found in b-peptides, others are new. Initial experimental studies on this
class of peptides were attempted by Cheguillaume etal. [19] in synthesizing Na-
substituted hydrazino acetic acid oligomers (R ˆ Me, iPr, iBu). These achiral analogs of
b3-peptides were studied by NMR techniques and expected to fold to a helical structure
containing eight-membered H-bonded rings, where both N-atoms are part of the
ring3). This feature has effectively been proved previously by Aubry and co-workers
[20 27] while studying structural and conformational aspects of a-hydrazino acids and
1
mixed dipeptide derivatives by combined use of H-NMR and IR spectroscopy, X-ray
diffraction, and molecular dynamics. The following features should be noted: i) When
both N-atoms are acylated, the two amide groups are in a rigid orthogonal disposition
in order to minimize steric hindrance. ii) When only the Nb-atom is acylated, the Na-
atom has sp3 character (bond angles are clearly smaller than 1208), and adopts either
the (R)-or ( S)-configuration according to the possible interactions in which the NÀHa
bond could be involved. In fact, such proton-accepting and -donating properties result
in a variety of inter-and intramolecular contacts. iii) The conformational arrangement
of the NaÀNb bond in the hydrazide group is much more flexible than in the diacylated
compound. Instead of the single orthogonal arrangement, the COÀNbÀNaÀCa
torsional angle effectively assumes values between 808 and 1308 (absolute values),
with a preference for 1208. iv) The hydrazide group can participate in a complex H-
bond network in which the COÀNHb amide moiety resembles a standard peptide
group, with a proton-donating site the NÀHb bond and a proton-accepting site the
2
)
A detailed discussion with extensive references will be given in a forthcoming review article by D.
Seebach, A. K. Beck, and D. Bierbaum in Angew. Chem.
It should be noted that the presence of pyramidal N-atoms, i.e., stereogenic centers with −free×
configuration, renders any folded structure chiral.
3
)

Fig. 1. Structural lineage of peptidomimetics backbones. The family tree of peptidomimetic backbones is organized horizontally from left to right by the number of
atoms separating peptide (or peptide-like) units and vertically by the functional group classes. For clarity, each peptidomimetic is represented by the residue formula
of only one configuration.

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carbonyl O-atom. The sp3-Na-atom is both a proton-accepting site, and a proton donor
when protonated; the lone pair is able to participate in the stabilization of a particular
turn4) containing a double intramolecular interaction and eight atoms in the H-bonded
ring.
Due to the fact that these peptides have not received much attention from the
scientific community, and that the folding propensity of this type of peptides still needs
to be investigated experimentally, we present in this paper the synthesis of chiral
hydrazino oligomers with Val, Ala, and Leu side chains by coupling the 3-aza-b2-amino
acid derivatives in solution, and their comparison with analogous b-peptides (Fig. 2). In
addition, spectroscopic and enzymatic-cleavage investigations are described.
Fig. 2. Structural comparison of b2-aza- and b2-peptides
2. Preparation of 3-Aza-b2-Amino Acids and Peptides. Several methods for the
synthesis of 3-aza-b2-homoamino acids are described in the literature. Typically, they
can be synthesized by reduction of N-nitrosoamino acids [29 32], asymmetric
synthesis [33 39], N-amidation [26][40 43], the Shestakov urea rearrangement
[44 46], or Mitsunobu reaction [47][48]. Thus, most hydrazino analogs of the coded a-
amino acids have been prepared before.
To attempt the synthesis of the b-amino acid derivatives needed for the hydrazino
peptide coupling in solution, we decided to use an established method patented by
Colletetal. [43]. This approach involves the N-amidation of secondary amines (in our
case the N-benzyl-l-a-amino acids 1a 1c) with a Boc-protected oxaziridine as
electrophile [49]. The use of enantiomerically pure a-amino acids as starting materials
for the preparation of the Boc-protected 3-benzylaza-b2-homoamino acids is very
convenient due to their ready availability. Compounds 1a 1c were prepared in
acceptable-to-good yields by formation of the imine from the corresponding a-amino
acids (H-Ala-OH, H-Val-OH, and H-Leu-OH) and benzaldehyde, and subsequent
NaBH4 reduction [50][51] (Scheme 1). Isolation problems were encountered only with
the alanine derivative 1a, due to its solubility in H2O at any pH. Therefore, 1a could be
4
)
With reference to the so called g-turn in a-peptides, in which an intramolecular i ‡ 1 ! i À 1 H-bond closes
a seven-membered ring [28], the a-hydrazino acid residue in position i exhibits a similar folded
conformation, characterized by an intramolecular bifurcated H-bond in which the NÀHi‡1 bond interacts
both with the COiÀ1 (to form an eight-membered cycle) and with the lone pair of the Nai -atom. This has
been referred to as an expanded g-like turn. The involvement of the NÀHi‡1 proton in a double
interaction is possibly responsible for the stability of this folded structure.

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isolated in acceptable yield only after purification by ion-exchange chromatography.
Further reaction of these intermediates with N-[(tert-butoxy)carbonyl]-3-(4-cyano-
phenyl)oxaziridine in CH2Cl2 at 08 [43] afforded 2a 2c in good yields. Ester protection
of the leucine derivative 2c (for the C-terminal residue), with MeI in the presence of
NaHCO3 in DMF at room temperature, afforded 3 in nearly quantitative yield after
purification (Scheme 1).
Scheme 1. Preparation of the Boc-Protected 3-Benzylaza-b2-homoamino Acids 2a 2c and of the Ester 3 from
Natural l-a-Amino Acids
Slow cooling and evaporation of a CH2Cl2/iPr2O mixture from a solution of the a-
hydrazino acid 2a led to the formation of crystals suitable for structure determination
by single-crystal X-ray diffraction (Fig. 3). The crystal structure5) was determined with
SHELXS-97 [52] and displays some interesting features: the pyramidal sp3 character of
the Na-atom is clearly demonstrated by the magnitude of its three bond angles, the sum
of which is ca. 3358. This N-atom is able to participate in the stabilization of the eight-
membered H-bonded ring formed by the carbazate CO and the COOH H-atom
(bifurcated H-bond) and, therefore, assumes a (S)-configuration (Fig. 3,a). On the
other hand, the NHb H-atom is involved in an intermolecular H-bond with a COOH
CO group of another molecule. This conformation probably experiences additional
stabilization by an intramolecular CH-p contact6) between the Bn ring and the MeCH
of the Boc group. The crystal packing displays an additional intermolecular CH-p
contact between the Me group of one molecule and the Bn ring of another (Fig. 3,b
and c).
With all the required aza-amino acids in hand, we envisaged the synthesis of various
oligomers under the same coupling conditions as for b-peptides. Consequently, the
ester 3 was Boc-deprotected by treatment with CF3COOH (TFA) in CH2Cl2 and
then submitted to 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride
(EDC)/1-hydroxy-1H-benzotriazole (HOBt) coupling conditions with the Boc-
protected amino acid 2a (Scheme 2). The resulting dipeptide 4 could be isolated in
5
)
)
We thank Dr. B. Schweizer for the determination of the X-ray crystal structure.
For reviews on CH-p interactions, see [53][54].
6

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Fig. 3. X-Ray crystal structure of 2a. a) The Na-atom has a pyramidal conformation with (S)-configuration and
participates in the stabilization of the eight-membered H-bonded ring formed by the carbazate CO and the
COOH H-atom (bifurcated H-bond). b) Side-view (along the a,b-plane), and c) top-view (along the c axis) of
the unit cell. The molecules are interconnected by H-bonds between the NHb H-atom of one molecule and a
COOH O-atom of another. The packing is additionally stabilized by an intramolecular CH-p contact between
the Bn ring and the MeCH of the Boc group, and an intermolecular CH-p contact between the Me group and
the Bn ring of two different molecules.
good yield, even though the reaction was not clean. Following Boc deprotection of 4
and reaction with 2b, coupling with EDC and HOBt gave 5 in disappointing yield, the
main product isolated being the valine active ester derivative as well as the unreacted
Boc-deprotected dipeptide (data not shown).
Better results were obtained with O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethy-
luronium hexafluorophosphate (HATU) as coupling reagent. It is known that its
presence accelerates the coupling reaction of amino acids, probably through intra-
molecular base catalysis [55][56]. Indeed, the reaction of the Boc-deprotected

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Scheme 2. Preparation of the b2-Azapeptide Derivatives 4
6
dipeptide with 2b, HATU, and N-methylmorpholine (NMM) in DMF gave, after
purification, the tripeptide 5 in 88% yield (Scheme 2). As the use of HATU avoided
competitive side reactions, the same coupling conditions were also used for the
synthesis of the dipeptide 4. Boc Deprotection of 3, followed by the reaction with 2a,
HATU, and NMM in CH2Cl2, led to the isolation of dipeptide 4 in 87% yield
(Scheme 2). For fragment coupling, the methyl ester group of 5 was saponified by
treatment with LiOH in MeOH/H2O [57]. The resulting Boc-protected tripeptide
derivative 6 was obtained in quantitative yield and with no loss of stereochemical
integrity. Catalytic hydrogenolysis of the N-Bn groups of 5 with H2 in EtOH in the
presence of 10% Pd/C at room temperature gave the debenzylated Boc-tripeptide ester
7 in 84% yield (Scheme 3), without any NÀN bond cleavage.
Unfortunately, attempts at subsequent cleavage of the Boc and methyl ester groups
to prepare the fully deprotected tripeptide were in vain. Neither saponification of the
ester group in 7, followed by Boc deprotection, nor Boc deprotection of the acid 6,
followed by catalytic hydrogenation, led to the desired product.
The last step required for the synthesis of the fully protected hexapeptide 8 was the
fragment coupling, which involved the reaction of the peptide acid 6 with the TFA salt
derived from 5. The use of HATU and NMM for this coupling reaction resulted in an
inseparable mixture of products, none of which seemed to be the desired compound.
Further attempts with other reagents were, therefore, investigated: the use of EDC/
HOBt resulted in the isolation of the active ester of the acid 6 as major product; the
reaction with EDC and DMAP (4-(dimethylamino)pyridine) resulted, again, in an
inseparable mixture of products, and the use of PyBroP (bromotris(pyrrolidine)phos-
phonium hexafluorophosphate) afforded the peptide 8 in only 19% yield. Step-by-step
coupling was also undertaken and shown to be inefficient upon scale-up, due to the
formation of inseparable by-products at each step. The problem could be partially

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Scheme 3. Multiple N-Debenzylation of the Fully Protected Tripeptide Analog 5
solved when BOPCl (bis(2-oxooxazolidin-3-yl)phosphinic chloride) and NMM were
used for the coupling reaction. In fact, the peptide 8 could, thus, be isolated after
column chromatography in reasonable yield and purity (Scheme 4). The hydro-
genolysis of the NÀBn bonds in 8 with H2 in CF3CH2OH/DMF 1:1, with ca. 30% Pd/C,
afforded, after preparative HPLC and without any NÀN bond cleavage, the peptide 9
in 44% yield. For the NMR measurements (vide infra), part of the compound had to be
washed with aqueous bicarbonate solution to remove TFA.
3. Spectroscopic Investigations towards Secondary-Structure Elucidation. Circu-
lar dichroism (CD) is extensively used to obtain information on the secondary structure
of peptides and proteins in solution. For a-peptides and proteins, characteristic CD
patterns are assigned to b-sheet, a-helix, and random-coil structures. When many
secondary-structure motifs are present at the same time, only the population of each is
extracted from the CD data. In the b-peptide world, the correlation is somehow more
difficult. Even though certain patterns of the CD spectra have been correlated with
secondary structures, such as 314-, 2.512-, and 12/10-helices, or hairpin turns [58 60],
there are limitations. It seems that only the 314-helix can be derived unambiguously
from the CD spectra: it has been established, by numerous CD measurements and
corresponding NMR investigations, that b-peptides consisting of l-b3-amino acids
exhibit a characteristic CD pattern with a negative Cotton effect near 215 nm, a zero-
crossing between 205 and 210 nm, and a peak near 200 nm; this pattern is assigned to a
left-handed (M)-314-helical structure [59][60]. The 12/10-helix [12][61][62] and the
hairpin turn [59] both show a maximum at around 200 nm and are, therefore, similar to
that of a random-coil structure (in H2O) [63].
b2-Azapeptides are new in the field of peptidomimetics and should be compared
with the b2-peptide analogs only with due care7). As a first step in probing the existence
7
)
Experience in our group has led us to wait for NMR solution-structure determinations before we try to
correlate structures with CD data [64].

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Scheme 4. Preparation of the b2-Hexaazapeptide Derivative 8 by FragmentCoupling of 5 with 6 and Subsequent
Debenzylation to the Terminally Protected b2-Hexaazapeptide 9
of a secondary structure, CD spectra (0.2m solutions) of the fully protected (5 and 8)
and of the N-debenzylated peptides (7 and 9) were recorded. As indicated by the CD
spectra (normalized), none of the Bn-protected peptides show a significant Cotton
effect. In fact, they show hardly any difference in comparison with the simple amino
acid derivative 3. In addition, going from 5 (three aza-amino acid residues) to 8 (six
residues), the intensity of the CD curve actually decreases (Fig. 4, left)! On the other
hand, the debenzylated peptides 7 and 9 (Fig. 4, right) give rise to a totally different
effect: the CD spectra in MeOH of both peptides show, with different intensities, a
maximum at ca. 225 nm, a zero-crossing at ca. 216 nm, and a more-intense minimum at
ca. 198 nm.
Additional CD measurements with the peptide 9 in H2O display a pattern similar to
that obtained in MeOH and show a moderate pH dependence: in acidic media the
curve is superimposable to that obtained in MeOH, and, at higher pH, it becomes
somewhat less intense. It should be noted that the b-peptide A8), which was assigned
[62][65] a (P)-314-helical structure, shows a similar CD pattern, with the peak being
8
)
The b-peptide A represents the terminally deprotected analog of 9.

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Fig. 4. CD Spectra of the 3-benzylaza-b2-amino acid ester 3 and the azapeptide derivatives 5, and 7 9. Left: The
normalized CD spectra (0.2 mm in MeOH) of the fully protected amino acid 3, and of the fully protected
peptides 5 and 8 do not show any representative Cotton effects. Right: The CD curves (normalized) of the Bn-
deprotected peptides 7 and 9 show, with different intensities, a peak at ca. 225 nm, a zero-crossing at ca. 216 nm,
and a trough at ca. 198 nm. CD Curves of 9 are similar in MeOH and in H2O; at acidic pH (blue and green) the
curves are almost superimposable with the pattern in MeOH (red), a decrease in intensity is observed at basic
pH. All CD measurements were performed at 228 on a JASCO J-710 spectropolarimeter in a 1-mm cell, with
0.2 mm solutions.
blue-shifted by ca. 10 nm (Fig. 5; not normalized). Thus, from the CD spectra shown in
Fig. 4 we would have to conclude that our −azapeptide× also forms a right-handed helical
secondary structure.
To obtain more reliable7) information about the secondary structure of the new
peptide analogs, NMR investigations of the hexapeptide 9 have also been undertaken,
Fig. 5. CD Spectrum and formula of the b2-hexapeptide A in MeOH. The pattern consists of a positive Cotton
effect at ca. 216 nm, a zero-crossing at ca. 210 nm, and a negative peak at ca. 198 nm, and it is believed to build to
some extend a (P)-314-helix. The CD spectrum was measured at À208, resulting in a 40% increase of the molar
ellipticity of the 216-nm Cotton effect in comparison with the data collected at 228 [62]. Concentration 0.2 mm,
molar ellipticity V in 10 deg cm2 molÀ1
.

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unfortunately without success. A strong overlapping of the NHb (peptidic) signals and
the absence of signals from the NHa (hydrazino) H-atoms characterized the 1H-NMR
spectrum of the peptide 9 (as TFA salt) in CDCl3. Measurements in CD2Cl2 at various
temperatures (between À708 and room temperature) did not solve the problem: the
signals of the peptidic H-atoms were overlapping, and the hydrazino NHa signals were
still absent; in addition, all other signals were degenerate at low temperatures. On the
other hand, the 1H-NMR spectrum of the TFA-free hexapeptide 9 in CD2Cl2 displayed
much better dispersion of the peptidic H-atoms, but the signals from the NHa H-atoms
were broad and overlapping. Attempts to improve the resolution in other deuterated
solvents were not successful. In all cases, the NH H-atoms appeared as broad singlets,
probably due to fast exchange between both types of NH H-atoms, and structural
assignment was impossible.
4. Enzymatic Stability of the Tri- and Hexapeptide Analogs 7 and 9. Despite the
fact that no secondary structure could be assigned by NMR spectroscopy, we consider
these peptides to be interesting candidates as peptidomimetics. In the early seventies,
two natural peptides containing a a-hydrazino acid residue were isolated: linatine from
linseed [30][66] and negamycin [67][68], a mushroom antibiotic, containing the
hydrazino analogues of proline and sarcosine. Moreover, a-hydrazino acids are also
known to be potent inhibitors of pyridoxal phosphate-dependent enzymes (trans-
aminases and decarboxylases) through the probable formation of a hydrazone with the
pyridoxal phosphate aldehyde group [36][69 71]. For example, Carbidopa (a-
hydrazino-a-(3,4-dihydroxybenzyl)propanoic acid) is used for the treatment of
Parkinson×s disease as a dopa-decarboxylase inhibitor [72]. Since proteolytic stability
is a prerequisite for possible pharmacological applications, we wondered whether these
hydrazino peptides would be as stable as the b-peptides [4 8]. We have, therefore,
tested compounds 7 and 9 in vitro with several peptidases: pronase, proteinase K,
chymotrypsin, trypsin, carboxypeptidase A, and 20S proteasome (human, yeast, arch.
flug.)9). We were pleased to observe that both compounds are stable for at least 48 h
under conditions where a-peptides are degraded within minutes!
5. Conclusions. We have shown that chiral a-hydrazino carboxylic acid derivatives
can be coupled in solution for the preparation of b2-azapeptides. The a-hydrazino
derivative 2a displays, in the solid state, the characteristic bifurcated H-bond, which
might be responsible for the formation of helical conformations in corresponding
peptides. The CD spectra of the fully protected tri-and hexapeptides, 5 and 8,
respectively, do not show any significant Cotton effect, indicating lack of folding into
any secondary structure. On the other hand, the N-debenzylated peptides 7 and 9
display patterns similar to those of the corresponding b2-hexapeptide A, albeit with
lower intensities and a red-shifted maximum. Unfortunately, no secondary-structure
determination was possible by NMR techniques: there is fast H-exchange between
both types of NH H-atoms and insufficient dispersion of their signals. Interestingly,
both hydrazino peptides 7 and 9 are stable against peptidases and make this class of
peptides interesting candidates for drug design.
9
)
We gratefully acknowledge the help of Dr. J. Frackenpohl for conducting these experiments.

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Experimental Part
1. General. Abbreviations: BOPCl: Bis(2-oxooxazolidin-3-yl)phosphinic chloride, DMAP: 4-(dimethyla-
mino)pyridine, EDC: 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride, FC: (flash chromatog-
raphy), HATU: O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, HOBt: 1-hy-
droxy-1H-benzotriazole, h.v.: high vacuum, 0.01 0.1 Torr, IEC: ion-exchange chromatography (Amberlite IR-
120), NMM: N-methylmorpholine. Solvents for chromatography and workup procedures were distilled from
Sikkon (anh. CaSO4; Fluka) and from KOH (Et2O), resp. Et3N was distilled from CaH2 and stored over 4-ä
molecular sieves. MeI was filtered over Alox before use. Amino acids were purchased from Fluka or Senn. All
other reagents were used as received from Fluka. TLC: Merck silica gel 60 F254 plates; detection with UV,
anisaldehyde soln. (9.2 ml of anisaldehyde, 12.5 ml of conc. H2SO4, 3.75 ml of AcOH, 340 ml of EtOH), or −Mo-
stain× soln. (25 g of phosphormolybdic acid, 10 g of Ce(SO4)2 ¥ H2O, 60 ml of conc. H2SO4, 940 ml of H2O), FC:
Fluka silica gel 60 (40 63 mm); at ca. 0.2 bar. Anal. HPLC: Knauer HPLC system (pump type WellChrom K-
1000 Maxy-Star, degasser, UV detector (variable-wavelength monitor) or Merck HPLC system (LaChrom,
pump type L-7150, UV detector L-7400, interface D-7000, HPLC manager D-7000), Macherey-Nagel C8 column
(Nucleosil 100-5 C8 (250 Â 4 mm) or Macherey-Nagel C18 column (Nucleosil 100-5 C18 (250 Â 4 mm). Prep.
HPLC: Merck HPLC system (LaChrom, pump type L-7150, UV detector L-7400, interface D-7000, HPLC
manager D-7000). Macherey-Nagel C8 column (Nucleosil 100-7 C8 (250 Â 21 mm); TFA for prep. HPLC was
used as UV-grade quality (> 99% GC). Lyophilization: Heterosicc cooling condenser with h.v. pump. M.p.:
B¸chi-510 apparatus; uncorrected. CD Spectra: Jasco J-710 recording from 190 to 250 nm at r.t.; 1-mm
rectangular cell; average of five scans, corrected for the baseline; peptide concentration 0.2 mm; molar ellipticity
V in deg ¥ cm2 ¥ dmolÀ1 (l in nm); smoothing by Jasco software. IR Spectra: Perkin-Elmer 1600 FT-IR
spectrophotometer. NMR Spectra: Bruker AMX II 500 (1H: 500 MHz, 13C: 125 MHz), Bruker AMX-400 (1H:
400 MHz, 13C: 100 MHz), Bruker AMX-300 (1H: 300 MHz, 13C: 75 MHz), Varian Mercury XL 300 (1H:
300 MHz, 13C: 75 MHz); chemical shifts d in ppm downfield from internal Me4Si (ˆ0 ppm); J values in Hz. MS:
IonSpec Ultima 4.7 T FT Ion Cyclotron Resonance (ICR, HR-MALDI, in 2,5-dihydroxybenzoic acid matrix)
spectrometer; in m/z (% of basis peak). Elemental analyses were performed by the Microanalytical Laboratory
of the Laboratorium f¸r Organische Chemie, ETH-Z¸rich.
2. Synthesis of N-Bn-Protected a-Amino Acids: General Procedure 1 (GP 1). Similar to the procedures
reported in [50][51], the a-amino acid (1 equiv.) was dissolved in 2n NaOH (1 equiv.), PhCHO (1 equiv.) was
added, and the mixture was stirred at r.t. for 15 min. After cooling to 08, NaBH4 (0.33 equiv.) was added in one
portion (Caution: strong development of gas!). One h later, PhCHO and NaBH4 were added again according to
the same procedure. After stirring 1 h more, the mixture was washed with Et2O (2Â), the aq. layer was saturated
with solid NaCl and acidified to pH 7 in an ice-bath with 2n HCl. The white solid formed was collected, and the
crude product was purified by washing with cold H2O or by IEC.
3. Synthesis of Boc-(S)-b2-3-aza(Bn)-Homoamino Acids: General Procedure 2 (GP 2). Similar to the
procedure reported in [43], 1.5m methanolic soln. of Et4NOH (1 equiv.) was added to N-benzyl-a-amino acid
(1 equiv.). The suspension was stirred until the soln. became clear. The MeOH was evaporated, and the
yellow oily residue was dissolved in CH2Cl2 (0.33m). The soln. was cooled to 08, and N-[(tert-butoxy)carbonyl]-
3-(4-cyanophenyl)oxaziridine (1.05 equiv., synthesised according to [49]), dissolved in CH2Cl2 (0.5m), was
added dropwise. The flask was hermetically sealed and placed in a refrigerator for 12 24 h. The solvent
was then evaporated, and the residue was taken up in an aq. NaOH soln. (12 mg of NaOH/50 ml of H2O for
1 mmol). After stirring for 30 min, the precipitated 4-formylbenzonitrile was separated by filtration. The
aq. phase was slightly salinized, washed with Et2O (7Â), and then acidified to pH 3 with solid KHSO4. The
resulting milky soln. was extracted with Et2O (3Â), the combined org. layers dried (MgSO4) and concentrated
under reduced pressure. The resulting crude product was either recrystallized or used without further
purification.
4. Boc Deprotection: General Procedure 3 (GP 3). Similar to the procedure reported in [4], the Boc-
protected amino acid/peptide was dissolved in CH2Cl2 (0.5m) and cooled to 08. An equal volume of TFA was
added, and the mixture was stirred at 08 for 4 h (TLC control). The solvent was evaporated; the residue was
taken up in dried toluene or CHCl3, and evaporated twice. The resulting trifluoroacetate salt was dried under
h.v., identified by NMR, and used in a next step without further purification.
5. Peptide Coupling: General Procedures 4 (GP 4). a) The appropriate trifluoroacetate salt was dissolved in
CH2Cl2 (0.5m) and cooled to 08. This soln. was treated successively with NMM (3 5.3 equiv.), a soln. of the Boc-
protected fragment (1 equiv.) in CH2Cl2 (0.25m), and HATU or BOPCl (1 equiv.). The mixture was allowed to
warm to r.t. and then stirred until TLC indicated complete reaction. Subsequent dilution with AcOEt was

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Helvetica Chimica Acta Vol. 86 (2003)
followed by washing with 5% aq. KHSO4 soln. (1Â), 5% aq. NaHCO3 soln. (1Â) and sat. aq. NaCl-soln. (1Â).
The org. layer was dried (MgSO4) and concentrated under reduced pressure. FC yielded the pure peptide.
b) The appropriate trifluoroacetate salt was dissolved in DMF (0.5m) and cooled to 08. This soln. was
treated successively with NMM (4 equiv.), a soln. of the Boc-protected fragment (1 equiv.) in DMF (0.25m), and
HATU (1 equiv.). The mixture was allowed to warm to r.t. and then stirred until TLC indicated complete
reaction. Sat. aq. NaHCO3 soln. was added, and the mixture was stirred for another 10 min. The mixture was
diluted with H2O and extracted with Et2O (3Â). The combined org. layers were washed with sat. aq. NaCl soln.
(1Â), dried (MgSO4), and concentrated under reduced pressure. FC afforded the pure peptide.
6. Bn Deprotection of the Hydrazino Group: General Procedure 5 (GP 5). The fully protected peptide was
dissolved in EtOH or CF3CH2OH/DMF 1 :1 (0.07 0.2m). Some drops of AcOH and ca. 10 30% (w/w) Pd/C
(10%) were added. The apparatus was evacuated and flushed with H2 (3Â), and the mixture was stirred under
H2 atmosphere (balloon) for 4 6 d. Subsequent filtration through Celite and concentration under reduced
pressure yielded the Bn-free peptide, which was further purified by FC or HPLC.
(S)-2-(Benzylamino)propanoic Acid (Bn-Ala-OH; 1a). Alanine (6.24 g, 70.0 mmol) was transformed
according to GP 1 and purified by IEC according to the following procedure: Amberlite IR-120 (250 g) was
mixed with H2O and packed in a chromatography column. The column was first washed with 2n HCl (8 l) (flow
rate: 100 1000 ml/h) and then with H2O (3 l) until the pH was neutral. Thereafter, the product was put on the
column as a soln. in H2O (250 ml), and washing was continued with additional H2O (8.5 l). Finally, washing with
1m NH4OH (1 l) eluted the product, which was concentrated under reduced pressure and lyophilized to yield 1a
(7.34 g, 58%). White powder. M.p. 248 2498 (dec.) ([73]: m.p. 251). [a]rD:t: ˆ ‡2.5 (c ˆ 1.02, 6n HCl) ([73]:
[a]Dr:t: ˆ ‡4.3 (c ˆ 1, 6n HCl)). 1H-NMR (300 MHz, D2O): 1.47 (d, J ˆ 7.1, Me); 3.67 (q, J ˆ 7.1, CHN); 4.17
(d, J ˆ 13.0, 1 H, PhCH2); 4.23 (d, J ˆ 13.0, 1 H, PhCH2); 7.46 (s, 5 arom. H). The NMR data of the
corresponding HCl salt are reported in [74] and are in agreement with the data of 1a.
(S)-2-(Benzylamino)-3-methylbutanoic Acid (Bn-Val-OH; 1b). Transformation of valine (3.52 g,
30.0 mmol) according to GP 1 and washing with a large amount of cold H2O yielded 1b (5.35 g, 86%). The
product was used in a next step without further purification. White solid. M.p. 256 2578 ([73]: m.p. 2558).
[a]rD:t: ˆ ‡10.5 (c ˆ 1.02, 6n HCl) ([73]: [a]rD:t: ˆ ‡14.8 (c ˆ 1, 6n HCl)). 1H-NMR (200 MHz, D2O): 0.92 (d, J ˆ
7.1, Me); 0.98 (d, J ˆ 7.1, Me); 2.00 2.20 (m, Me2CH); 3.32 (d, J ˆ 4.6, CHN); 4.07 (d, J ˆ 13.3, 1 H, PhCH2);
4.22 (d, J ˆ 13.3, 1 H, PhCH2); 7.46 (s, 5 arom. H). The NMR data of the corresponding HCl salt were reported
in [74] and are in agreement with the data of 1b.
(S)-2-(Benzylamino)-4-methylpentanoic Acid (Bn-Leu-OH; 1c). Transformation of leucine (3.69 g,
30.0 mmol) according to GP 1 and washing with a large amount of cold H2O yielded 1c (3.96 g, 64%). The
product was used in a next step without further purification. White solid. M.p. 222 2238 ([73]: m.p. 242).
[a]rD:t: ˆ ‡14.6 (c ˆ 1.00, 6n HCl) ([73]: [a]rD:t: ˆ ‡14.2 (c ˆ 1, 6n HCl)). 1H-NMR (300 MHz, D2O): 0.84 (d, J ˆ
5.6, Me); 0.89 (d, J ˆ 5.6, Me); 1.59 1.76 (m, Me2CH); 3.56 (m, CHN); 4.15 (d, J ˆ 13.1, 1 H, PhCH2); 4.26
(d, J ˆ 13.1, 1 H, PhCH2); 7.48 (s, 5 arom. H). The physical data are in agreement with the values reported in
[73].
(S)-2-{1-Benzyl-2-[(tert-butoxy)carbonyl]hydrazino}propanoic Acid ((S)-Boc-b2-3-aza(Bn)-hAla-OH;
2a). Compound 1a (500 mg, 2.80 mmol) was first treated with methanolic Et4NOH (4.00 ml, 2.80 mmol) and
then with N-[(tert-butoxy)carbonyl]-3-(4-cyanophenyl)oxaziridine (720 mg, 2.92 mmol) according to GP 2.
Purification of the crude product by recrystallization (Et2O/hexane) yielded 2a (787 mg, 91%). White solid.
1H-NMR (300 MHz, CDCl3): 1.34 (s, t-Bu); 1.41 (d, J ˆ 7.2, Me); 3.63 3.73 (m, CHN); 3.99 (s, PhCH2); 6.15
(br. s, 0.8 H, NH); 7.05 (br. s, 0.2 H, NH); 7.25 7.40 (m, 5 arom. H); 9.62 (br. s, OH). The physical data are in
agreement with the values reported in [43].
(S)-2-{1-Benzyl-2-[(tert-butoxy)carbonyl]hydrazino}-3-methylbutanoic Acid ((S)-Boc-b2-3-aza(Bn)-hVal-
OH; 2b). Compound 1b (2.07 g, 10.0 mmol) was first treated with methanolic Et4NOH (6.67 ml, 10.0 mmol) and
then with N-[(tert-butoxy)carbonyl]-3-(4-cyanophenyl)oxaziridine (2.59 g, 10.5 mmol) according to GP 2.
Purification of the crude product by recrystallization (Et2O/hexane) yielded 2b (2.09 g, 65%). White crystals.
1H-NMR (300 MHz, CDCl3): 0.98 (d, J ˆ 6.5, Me); 1.10 (d, J ˆ 6.5, Me); 1.38 (s, t-Bu); 2.05 2.17 (m, Me2CH);
3.00 3.20 (br. m, CHN); 3.90 4.10 (br. m, PhCH2); 6.55 (br. s, 0.5 H, NH); 7.13 (br. s, 0.5 H, NH); 7.24 7.30
(m, 3 arom. H); 7.31 7.42 (m, 2 arom. H); 8.06 (br. s, OH). The physical data are in agreement with the values
reported in [43].
(S)-2-{1-Benzyl-2-[(tert-butoxy)carbon yl]hydrazino}-4-methylpentanoic Acid ((S)-Boc-b2-3-aza(Bn)-
hLeu-OH; 2c). Treatment of 1c (2.14 g, 9.67 mmol) with methanolic Et4NOH (6.5 ml, 9.75 mmol) and then
with N-[(tert-butoxy)carbonyl]-3-(4-cyanophenyl)oxaziridine (2.50 g, 10.2 mmol) according to GP 2 yielded 2c
1
(2.87 g, 88%). The crude product was used in a next step without further purification. White foam. H-NMR

Helvetica Chimica Acta Vol. 86 (2003)
4165
(300 MHz, CDCl3): 0.77 (br. s, Me); 0.91 (d, J ˆ 6.2, Me); 1.36 (s, t-Bu); 1.36 1.49 (m, 1 H, CH2); 1.67 1.77
(m, 1 H, CH2); 2.07 (br. s, Me2CH); 3.46 3.54 (br. m, CHN); 3.92 4.18 (br. m, PhCH2); 6.51 (br. s, NH);
7.24 7.39 (m, 5 arom. H); 9.56 (br. s, OH).
Methyl (S)-2-{1-Benzyl-2-[(tert-butoxy)carbonyl]hydrazino}-4-methylpentanoate ((S)-Boc-b2-3-aza(Bn)-
hLeu-OMe; 3). Similar to the procedure reported in [75], compound 2c (2.66 g, 7.91 mmol) was dissolved in
DMF (0.61m), and solid NaHCO3 (1.33 g, 15.8 mmol) was added, followed by MeI (0.79 ml, 12.7 mmol). After
stirring at r.t. for 25 h, H2O was added, and the mixture was extracted with AcOEt (4Â). The combined org.
layers were successively washed with 5% aq. Na2SO3-soln. (1Â) and sat. aq. NaCl-soln. (1Â), and dried
(MgSO4). Removal of the solvent and FC (Et2O/pentane 1:6) yielded 3 (2.69 g, 97%). Colorless oil. Rf (Et2O/
Pentan 1 :6) 0.20. [a]rD:t: ˆ ‡25.4 (c ˆ 0.99, CHCl3). IR (CHCl3): 3364w, 3008w, 2958m, 2868w, 1730s, 1696s,
1483m, 1455m, 1435m, 1392m, 1368m, 1159s, 1077w, 1050w, 1028w, 993w. 1H-NMR (300 MHz, DMSO, 908): 0.80
(d, J ˆ 6.5, Me); 0.87 (d, J ˆ 6.5, Me); 1.31 (s, t-Bu); 1.40 (ddd, J ˆ 14.0, 8.1, 5.6, 1 H, Me2CHCH2); 1.62 (ddd, J ˆ
14.0, 8.7, 5.3, 1 H, Me2CHCH2); 1.85 1.97 (m, Me2CH); 3.52 (dd, J ˆ 8.7, 5.6, CHN); 3.69 (s, MeO); 3.95
(s, PhCH2); 7.05 (br. s, NH); 7.20 7.32 (m, 3 arom. H); 7.35 7.39 (m, 2 arom. H). 13C-NMR (100 MHz, CDCl3):
21.10, 23.29 (Me); 23.98 (CH); 28.20 (Me); 39.19 (CH2); 51.48 (Me); 61.31 (CH2); 62.25 (CH); 79.49 (C);
127.49, 128.18, 129.45 (CH); 136.99, 155.06, 174.81 (C). MALDI-MS: 373.2 (7.20), 317.1 (12.72), 295.0 (3.77),
251.2 (17.65), 191.1 (14.95), 91.0 (8.15). Anal. calc. for C19H30N2O4 (350.46): C 65.12, H 8.63, N 7.99; found: C
64.94, H 8.58, N 8.18.
Methyl (S)-2-[1-Benzyl-2-((S)-2-{1-benzyl-2-[(tert-butoxy)carbonyl]hydrazino}propionyl)hydrazino]-4-
methylpentanoate (Boc-(S)-b2-3-aza(Bn)-hAla-(S)-b2-3-aza(Bn)-hLeu-OMe; 4). Compound
3 (766 mg,
2.18 mmol) was Boc-deprotected according to GP 3. The resulting trifluoroacetate salt was coupled with 2a
(644 mg, 2.18 mmol), NMM (0.73 ml, 6.63 mmol), and HATU (831 mg, 2.18 mmol) according to GP 4a for 24 h.
FC (CH2Cl2/Et2O 10 :1) yielded 4 (1.00 g, 87%). White foam. Rf (CH2Cl2/Et2O 10 :1) 0.20. [a]rD:t: ˆ ‡30.8 (c ˆ
1.00, CHCl3). IR (CHCl3): 3340w, 3008m, 2960m, 1727s, 1687m, 1494m, 1368m, 1160s, 996w. 1H-NMR
(400 MHz, CDCl3): 0.78 (d, J ˆ 6.5, Me); 0.90 (d, J ˆ 6.7, Me); 1.20 (d, J ˆ 7.0, Me); 1.33 (s, t-Bu); 1.29 1.46
(m, 1 H, Me2CHCH2); 1.66 1.73 (m, 1 H, Me2CHCH2); 1.99 2.09 (br. m, Me2CH); 3.32 3.35 (m, CHN);
3.54 3.58 (m, CHN); 3.78 (s, MeO); 3.81 (s, PhCH2); 3.98 (s, PhCH2); 6.29 (br. s, NH); 7.20 7.32 (m, 8 ar-
om. H); 7.40 (d, J ˆ 6.6, 2 arom. H); 8.36 (br. s, NH). 13C-NMR (100 MHz, CDCl3): 14.87, 21.29, 23.20 (Me);
24.17 (CH); 28.24 (Me); 39.28 (CH2); 51.65 (Me); 60.06 (CH2); 61.16, 62.50 (CH); 79.71 (C); 127.51, 127.62,
128.24, 128.27, 129.24, 129.25 (CH); 136.59, 155.34, 171.83, 174.38 (C). MALDI-MS: 549.3 (3.41), 449.2 (100.00),
427.3 (38.93), 358.2 (9.04), 329.2 (16.17), 305.2 (59.85), 267.1 (24.87), 256.1 (41.70), 249.1 (10.47), 245.2 (21.36),
238.1 (15.30), 171.1 (12.31), 149.1 (23.27). Anal. calc. for C29H42N4O5 (526.67): C 66.14, H 8.04, N 10.64; found:
C 66.25, H 8.18, N 10.67.
Methyl (S)-2-(1-Benzyl-2-{(S)-2-[1-benzyl-2-((S)-2-{1-benzyl-2-[(tert-butoxy)carbonyl]hydrazino}-3-
methylbutanoyl)hydrazino]propanoyl}hydrazino)-4-methylpentanoate (Boc-(S)-b2-3-aza(Bn)-hVal-(S)-b2-3-
aza(Bn)-hAla-(S)-b2-3-aza(Bn)-hLeu-OMe; 5). Compound 4 (1.76 g, 3.34 mmol) was Boc-deprotected
according to GP 3. The resulting trifluoroacetate salt was coupled with 2b (1.08 g, 3.34 mmol) according to
GP 4b for 46 h. FC (CH2Cl2/Et2O 3 :1) yielded 5 (2.15 g, 88%). White foam. CD (0.2 mm in MeOH) ‡4.67 ¥ 104
(195 nm). Rf (CH2Cl2/Et2O 3 :1) 0.21. [a]Dr:t: ˆ ‡50.3 (c ˆ 0.98, CHCl3). IR (CHCl3): 3335w, 3007m, 2962m,
1
1725s, 1679s, 1479m, 1368m, 1166m, 992w. H-NMR (400 MHz, CDCl3): 0.59 (d, J ˆ 6.4, Me); 0.86 (d, J ˆ 6.5,
Me); 0.93 (d, J ˆ 6.7, Me); 1.00 (d, J ˆ 6.5, Me); 1.24 (d, J ˆ 6.8, Me); 1.34 (s, t-Bu); 1.43 1.51 (m, 1 H,
Me2CHCH2); 1.69 1.77 (m, 1 H, Me2CHCH2); 1.96 2.04 (br. m, Me2CH); 2.04 2.29 (br. m, Me2CH); 2.45
2.51 (br. d, CHN); 3.28 3.33 (br. q, CHN); 3.56 3.60 (br. d, CHN); 3.62 (d, J ˆ 5.2, 1 H, PhCH2); 3.64 (d, J ˆ
5.2, 1 H, PhCH2); 3.71 (d, J ˆ 13.2, 1 H, PhCH2); 3.79 (s, MeO); 3.89 (d, J ˆ 13.2, 1 H, PhCH2); 3.99 (s, PhCH2);
6.99 7.21 (br. s, NH); 7.23 7.40 (m, 15 arom. H); 7.80 8.00 (br. s, NH); 8.00 8.20 (br. s, NH). 13C-NMR
(100 MHz, CDCl3): 15.90, 19.26, 20.23, 21.27, 23.22 (Me); 24.27 (CH); 28.26 (Me); 39.43 (CH2); 51.79 (Me);
59.42 (CH); 59.74, 61.26 (CH2); 62.90, 70.02 (CH); 79.28 (C); 127.37, 127.79, 127.82, 128.18, 128.32, 128.40,
128.50, 128.76, 128.91, 129.20, 129.30 (CH); 136.53, 137.15, 154.97, 171.30, 172.15, 174.57 (C). MALDI-MS: 753.4
(9.56), 653.4 (100.00), 631.4 (12.63), 519.3 (18.91), 471.3 (19.35), 434.2 (91.21), 410.2 (48.96), 375.2 (12.58),
342.2 (10.40), 305.2 (21.03), 258.1 (12.94), 245.2 (17.20), 217.8 (10.82), 199.1 (10.07), 177.1 (17.75), 175.1 (13.14),
166.1 (10.30). Anal. calc. for C41H58N6O6 (730.95): C 67.37, H 8.00, N 11.50; found: C 67.24, H 8.00, N 11.37.
(S)-2-(1-Benzyl-2-{(S)-2-[1-benzyl-2-((S)-2-{1-benzyl-2-[(tert-butoxy)carbonyl]hydrazino}-3-methylbu-
tyryl)hydrazino]propionyl}hydrazino)-4-methylpentanoic Acid (Boc-(S)-b2-3-aza(Bn)-hVal- (S)-b2-3-aza(Bn)-
hAla-(S)-b2-3-aza(Bn)-hLeu-OH; 6). A soln. of the fully protected tripeptide 5 (250 mg, 0.34 mmol) in MeOH
(0.2m) was cooled to 08, treated with 2.7m LiOH soln. (0.76 ml, 2.05 mmol), and stirred at r.t. overnight. The
mixture was diluted with H2O, acidified to pH 2 3 with 1n HCl, and extracted with AcOEt (3Â). The combined

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Helvetica Chimica Acta Vol. 86 (2003)
org. layers were dried (MgSO4) and concentrated under reduced pressure to yield 6 (245 mg, quant.). The acid
was used in a next step without further purification.
Methyl (S)-2-(2-{(S)-2-[2-((S)-2-{2-[(tert-Butoxy)carbonyl]hydrazino}-3-methyl-butanoyl)hydrazino]-
propionyl}hydrazino)-4-methylpentanoate (Boc-(S)-b2-3-aza-hVal-(S)-b2-3-aza-hAla-(S)-b2-3-aza-hLeu-OMe;
7). Compound 5 (151 mg, 0.21 mmol) was debenzylated in EtOH (0.21m) with 10% Pd/C (10%) according to
GP 5 for 4 d. Purification by FC (CH2Cl2/MeOH 15 :1) yielded 7 (79.4 mg, 84%). Colorless oil. CD (0.2 mm in
MeOH): À2.58 ¥ 103 (198 nm); ‡820 (225 nm). Rf (CH2Cl2/MeOH 15 :1) 0.27. IR (CHCl3): 3407m, 2965s,
2873m, 1714s, 1673s, 1468s, 1392w, 1369m, 1158s. 1H-NMR (300 MHz, CDCl3): 0.94 (d, J ˆ 6.5, Me); 0.96 (t, J ˆ
6.5, 2 Me); 1.03 (d, J ˆ 7.1, Me); 1.33 (d, J ˆ 6.8, Me); 1.42 (s, t-Bu); 1.50 1.65 (m, Me2CHCH2); 1.68 1.79
(m, Me2CH); 2.00 2.10 (m, Me2CH); 3.36 (d, J ˆ 5.0, CHN); 3.55 (q, J ˆ 6.8, CHN); 3.62 (t, J ˆ 7.1, CHN); 3.73
(s, MeO); 4.00 (br. s, NHCH); 4.57 (br. s, NHCH); 4.96 (br. s, NHCH); 6.76 (s, NHCO); 8.43 (s, NHCO); 8.52
(s, NHCO). 13C-NMR (75 MHz, CDCl3): 17.21, 18.44, 19.20, 22.39, 22.81 (Me); 24.96 (CH); 28.36 (Me); 30.40
(CH); 39.02 (CH2); 52.42 (Me); 60.34, 61.28, 69.83 (CH); 80.80, 156.68, 171.53, 171.94, 173.55 (C). MALDI-MS:
411.3 (16.62), 383.2 (77.23), 337.2 (29.22), 280.2 (10.38), 254.1 (100.00), 195.1 (91.15), 168.1 (18.53), 163.1
‡
(11.54), 149.1 (13.73). HR-MALDI-MS: 483.2907 ([C20H40N6NaO6] ; calc. 483.2907).
Boc-(S)-b2-3-aza(Bn)-hVal-(S)-b2-3-aza(Bn)-hAla-(S)-b2-3-aza(Bn)-hLeu-(S)-b2-3-aza(Bn)-hVal-(S)-b2-
3-aza(Bn)-hAla-(S)-b2-3-aza(Bn)-hLeu-OMe (8). Compound 5 (250 mg, 0.34 mmol) was Boc-deprotected
according to GP 3. The resulting trifluoroacetate salt was coupled with 6 (245 mg, 0.34 mmol), NMM (0.20 ml,
1.82 mmol), and BOPCl (87.0 mg, 0.34 mmol) according to GP 4a for 29 h. Purification of the crude product by
FC (CH2Cl2/Et2O 3 :1) yielded 8 (182 mg, 40%). White glass. CD (0.2 mm in MeOH): ‡2.98 ¥ 104 (199 nm). Rf
(CH2Cl2/Et2O 3 :1) 0.26. IR (CHCl3): 3326m, 3007m, 2961m, 1954w, 1877w, 1815w, 1724s, 1674s, 1477s, 1368m,
1160m. 1H-NMR (300 MHz, CDCl3): 0.54 (br. d, Me); 0.65 (d, J ˆ 6.8, Me); 0.81 (br. d, Me); 0.86 (d, J ˆ 6.5,
Me); 0.89 (d, J ˆ 6.5, Me); 0.94 (d, J ˆ 6.5, Me); 0.97 (d, J ˆ 6.5, Me); 1.11 (d, J ˆ 6.5, Me); 1.22 (d, J ˆ 7.5,
2 Me); 1.34 (s, t-Bu); 1.36 1.80 (m, 2 Me2CHCH2); 1.80 2.16 (br. m, 4 Me2CH); 2.36 2.63 (br. m, 2 CHN);
3.17 3.32 (m, 2 CHN); 3.61 3.68 (m, 2 CHN); 3.72 3.92 (m, 4 PhCH2); 3.78 (s, MeO); 3.83 (s, PhCH2); 3.97
(s, PhCH2); 6.99 7.01 (br. d, NH); 7.00 7.45 (m, 30 arom. H); 7.60 7.94 (br. m, 2 NH); 8.30 8.80
(br. m, 3 NH). 13C-NMR (75 MHz, CDCl3): 16.98, 19.32, 20.41, 21.36, 21.93, 23.21, 23.36 (Me); 24.39 (CH);
28.36 (Me); 39.59 (CH2); 51.91 (Me); 59.69 (CH); 60.10, 60.17, 60.31, 60.40, 60.76, 60.95, 61.52, 61.72 (CH2);
62.90, 70.02 (CH); 79.24 (C); 127.23, 127.68, 128.06, 128.22, 128.37, 128.70, 128.92, 129.16, 129.32 (CH); 136.20,
137.09, 154.90, 155.64, 171.29, 171.79, 172.28, 174.45 (C). MALDI-MS: 1351.8 (10.66), 1251.7 (33.51), 1032.6
(19.05), 797.5 (40.52), 693.4 (12.53), 638.4 (27.96), 619.3 (12.47), 593.4 (100.00), 578.3 (42.01), 487.3 (11.66),
434.2 (40.31), 410.2 (17.16), 374.2 (10.44), 342.2 (15.79), 304.1 (11.04), 245.2 (17.23). HR-MALDI-MS:
‡
1351.7937 ([C76H104N12NaO9] ; calc. 1351.7947).
Boc-(S)-b2-3-aza-hVal-(S)-b2-3-aza-hAla-(S)-b2-3-aza-hLeu-(S)-b2-3-aza-hVal-(S)-b2-3-aza-hAla-(S)-b2-
3-aza-hLeu-OMe (9). Compound 8 (76.2 mg, 0.06 mmol) was debenzylated in CF3CH2OH/DMF 1 :1 (0.8 ml)
with 30% Pd/C (10%) according to GP 5 for 6 d. Purification by RP-HPLC (column: Macherey-Nagel, Nucleosil
100-7 C8, 250 Â 21 mm; linear gradient: MeCN/0.1% TFA in H2O 30 :70 ! 60 :40 in 30 min; detection: UV
215 nm; tR 11.6 min) and lyophilization yielded 9 (19.8 mg, 44%). White powder. For anal. reasons, a small
amount of the debenzylated peptide (10 mg) was dissolved in CH2Cl2, washed with semi-sat. aq. NaHCO3 soln.
(1Â) and with sat. aq. NaCl soln. (1Â), dried (MgSO4), and concentrated under reduced pressure to afford the
TFA-free 9 (7 mg). Colorless solid. CD (0.2 mm in MeOH): À9.0 ¥ 103 (198 nm); ‡1.88 ¥ 103 (226 nm). 1H-NMR
(500 MHz, C5D5N): 0.86 (d, J ˆ 6.7, Me); 0.87 (d, J ˆ 6.6, Me); 0.89 (d, J ˆ 6.6, Me); 0.92 (d, J ˆ 6.6, Me); 1.13
(d, J ˆ 6.8, Me); 1.14 (d, J ˆ 6.8, Me); 1.17 (d, J ˆ 6.7, 2 Me); 1.23 1.31 (m, Me2CHCH2); 1.48 (s, t-Bu); 1.51
(d, J ˆ 6.9, Me); 1.52 (d, J ˆ 6.9, Me); 1.70 1.76 (m, Me2CHCH2); 1.87 1.95 (br. m, 2 Me2CH); 2.28 2.53
(br. m, 2 Me2CH); 3.72 (s, MeO); 3.77 3.80 (m, 2 CHN); 3.95 4.03 (m, 3 CHN); 4.14 4.15 (m, CHN); 5.31
(br. s, NH); 5.62 (br. s, NH); 5.73 (br. s, NH); 5.80 (br. s, NH); 9.06 (br. s, NH). 13C-NMR (125 MHz, C5D5N):
17.47, 17.55, 18.92, 19.09, 19.52, 19.70, 22.41, 22.56, 22.81, 23.09 (Me); 25.20 (CH); 28.48 (Me); 30.72, 30.85 (CH);
40.26, 40.99 (CH2); 51.84 (Me); 60.00, 62.35, 63.08, 70.37 (CH); 79.42, 157.89, 172.91, 173.14, 173.42, 173.52,
173.69, 174.38 (C). MALDI-MS: 811.5 (26.73), 737.4 (23.49), 711.5 (100.00), 529.4 (11.59), 475.3 (14.75), 329.2
‡
(10.26). HR-MALDI-MS: 811.5096 ([C34H60N12NaO9] ; calc. 811.5130).

Helvetica Chimica Acta Vol. 86 (2003)
4167
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Received September 11, 2003