Design, Synthesis and Structural Investigations of a β-Peptide Forming a 314-Helix Stabilized by Electrostatic Interactions

Article abstract of DOI:10.1002/chem.200305571

Two different strategies have been employed for the synthesis of Fmoc-protected β³-homoarginine; the Arndt-Eistert homologation of α-arginine and the guanidinylation of β³-homoornithine. Solid-phase β-peptide synthesis was used for t

Full text of DOI:10.1002/chem.200305571

D. Seebachet al.
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Chem. Eur. J. 2004, 10, 1607 1615
DOI: 10.1002/chem.200305571
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

FULL PAPER
Design, Synthesis and Structural Investigations of a b-Peptide
Forming a 314-Helix Stabilized by Electrostatic Interactions
Magnus Rueping, Yogesh R. Mahajan, Bernhard Jaun, and Dieter Seebach*[a]
Abstract: Two different strategies have
been employed for the synthesis of
Fmoc-protected b3-homoarginine; the
Arndt Eistert homologation of a-argi-
nine and the guanidinylation of b3-ho-
moornithine. Solid-phase b-peptide
synthesis was used for the preparation
of b-heptapeptide 1, which was de-
signed to form a helix stabilized by
electrostatic interactions through posi-
tively (b3hArg) and negatively charged
(b3hGlu) amino acid residues. CD
measurements and corresponding
NMR investigations in MeOH and
aqueous solutions do indeed show that
the b-peptidic 314-helix can be stabi-
lized by salt-bridge formation.
Keywords: b-peptides ¥ electrostat-
ic interactions ¥ helical structures ¥
NMR spectroscopy ¥ salt bridges
Introduction
tous a-peptides; they can be designed to fold into secondary
structures, suchas ehlices,
[4,5]
turns[6] and sheets;[4,6a] and
Electrostatic interactions are important in many biological
processes, suchas enzyme catalysis, protein protein and pro-
tein nucleic acid binding, protein folding, flexibility and sta-
bility. Close range electrostatic interactions, that is, salt -
bridges and their networks have been shown to contribute
to peptide and protein stability.[1] Salt bridges are formed by
amino acid residues withopposite charges (Asp or Glu with
Arg, Orn, Lys or His), which are often near each other in
the amino acid sequence. The strength of a salt bridge is de-
termined by the geometry and distance of interaction,
degree of exposure to solvent, and effects of neighboring
charged residues. Many charged residues that form salt -
bridges are found in a-helical conformations where the op-
positely charged side chains occur one turn apart on the
same face of the helix in i and i+3,4 positions.
Various synthetic oligomers with conformations similar to
those in natural peptides and proteins have recently been
studied to increase our understanding of protein folding and
stability.[2] Especially, b- and g-peptides (peptides consisting
of chiral b- and g-amino acid residues) have received consid-
erable attention.[2,3] These peptides have several attractive
characteristics: they are structurally related to the ubiqui-
they are resistant to proteolytic degradation by common
proteases and peptidases,[7] suggesting that they might be
useful peptidomimetics.
Short b-peptides consisting solely of b3-amino acids, that
are derived from natural l-amino acids via Arndt Eistert
homologation, have been shown to fold into left-handed 314-
helical structures with the side chains of residues i and i+3
in juxtapositions at a distance of approximately 5 ä.[3] The
role of specific side-chain interactions of residues i and i+3
in stabilizing the helical structure of b-peptide 314-helices
has already been addressed. Previously, we showed, that in-
troduction of a conformational constraint by covalently link-
ing b3hCys side chains in i and i+3 positions stabilizes the
314-helix.[8] In addition, we and others were able to demon-
strate that polar side chains, as well as properly chosen
charged side chains of b-amino acids can lead to helical con-
formations in aqueous solution.[9] However, these results
e]
relied mainly on CD-spectroscopic measurements,[9c which
are not always incisive.[10]
Now, we describe a combined and detailed CD- and
NMR-spectroscopic structural analysis of a b-heptapeptide 1
designed to be stabilized by electrostatic interactions
(Figure 1).
[a] Dr. M. Rueping, Dr. Y. R. Mahajan, Prof. Dr. B. Jaun,
Prof. Dr. D. Seebach
We selected b-peptide 1, an all-b3-peptide, which is ex-
pected to form a 314-helix, and introduced negatively and
positively charged residues in i and i+3 positions to allow
electrostatic interactions. The potential salt bridges are es-
tablished by b3hGlu (residues 2 and 6) and b3hArg (resi-
dues 3 and 5). b3hArg was specifically chosen, rather than
b3hOrn or b3hLys, as the guanidinium group is known to
play an important role in many organic and biological proc-
Laboratorium f¸r Organische Chemie
der Eidgenˆssischen Technischen Hochschule Z¸rich
ETH-Hˆnggerberg, Wolfgang-Pauli-Strasse 10
8093 Z¸rich(Switzerland)
Fax : (+41)1-632-1144
E-mail: seebach@org.chem.ethz.ch
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
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¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/chem.200305571
Chem. Eur. J. 2004, 10, 1607 1615

1607 1615
acids starting from the corre-
sponding a-amino acids[19,20] in-
cluding Na-Boc- and Na-Z-pro-
tected arginine derivatives.[21]
The second approach relies on
the guanidinylation[22] of a suit-
ably protected ornithine deriva-
tive, a strategy that has long
been used in a-peptide chemis-
try to synthesize arginine-con-
taining peptides from the ap-
propriate ornithine-containing
precursors.[23]
In accordance witheth
Arndt Eistert
homologation
procedure of Na-Fmoc-protect-
ed a-amino acids developed in
our group,[20] the mixed anhy-
drides of commercially availa-
ble Na-Fmoc-protected a-argi-
nine, witheither a Pmc or Pbf
protecting group on the Nw-po-
sition, were converted to the
corresponding Fmoc-protected
Figure 1. Schematic representation of a 314-helical structure of b3-heptapeptide 1 from the side and top. Color
code: black=hydrophobic residues (b3hVal), red=negatively charged residues (b3hVal) and blue=positively
charged residues (b3hArg).
esses involving the binding of negatively charged substrates
for example, synthetic molecular receptors,[11] protein nucle-
ic acid interactions,[12] DNA/RNA recognition and cellular
uptake.[13,14] Furthermore, the guanidinium group is basic
(pKa ~12, that is, the positive charge is maintained over a
wide pH range), planar, and demonstrates directionality in
hydrogen-bonding interactions. The remaining three b3-
amino acid residues of heptapeptide 1 were provided by
bhVal residues to facilitate helix formation by hydrophobic
interactions. In addition the C-terminus was amidated to
prevent interactions between the otherwise free carboxylic
acid and the bhArg residues.
diazoketones which were subsequently homologated using a
modification of the base-free, silver(i)-catalysed, ultrasound-
promoted Wolff rearrangement protocol of Sewald and co-
workers.[24] In a typical homologation procedure used, the
diazoketones were ultrasonicated in the presence of catalytic
amounts of PhCOOAg and BnOH to provide the benzyl
esters 2a and 2b in moderate yields (57 and 46%, respec-
tively over two steps). Finally, debenzylation (H2, Pd/C) of
the fully protected b-amino acids 2 furnished the desired
compounds, Fmoc-b3hArgw(Pbf)-OH 3a and Fmoc-
b3hArgw(Pmc)-OH[25] 3b in 91 and 90%, respectively
(Scheme 1).
Results and Discussion
Preparation of b3-homoarginine derivatives: The trifunction-
al guanidine group displays a strong nucleophilic character,
and thus if it is improperly protected, side reactions occur
suchas intramolecular cyclisation to d-lactam derivatives,
acylation followed by decomposition to ornithine, and intra-
molecular cyclisation to 4-carboxy-2-imino-1,3-diazacyclo-
heptane derivatives.[15,16] Many different protecting groups
based on nitro, urethane, arylsulphonyl or aryl derivatives
have been proposed, but so far none of them satisfies the
basic requirements for an ideal protecting group, that is,
Scheme 1. Synthesis of b3-homoarginine derivatives via Arndt Eistert ho-
mologation of the corresponding a-amino acids.
robust protection to prevent undesired side reactions, and
[16]
clean and smoothremoval under mild conditions.
Never-
theless, acid-labile protections, such as Nw,Nw’-bis(Boc), Nw-
Pmc[17] or Nw-Pbf[18] are reported to be amenable to solid-
phase peptide synthesis using the Fmoc strategy.
Fmoc-b3hOrn(Boc)-OH, which was required for the orni-
thine-to-arginine transformation, was obtained by homolo-
gation of Fmoc-(S)-Orn(Boc)-OH using the Arndt Eistert
reaction[20] in combination withteh ultrasound-promoted
Wolff rearrangement. Fmoc-b3hOrn(Boc)-OH was Boc-de-
protected (TFA) and subsequently treated with N,N’-
bis(Boc)-1-amidinopyrazole[26] (4) under basic conditions
For the synthesis of b3-homoarginine, two different ap-
proaches were chosen. The first approach involves the use
of the Arndt Eistert homologation method which has been
successfully applied to the synthesis of various b3-amino
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D. Seebachet al.
FULL PAPER
(Et3N) in formamide/dioxane to afford Fmoc-b3hArgw,w’
(Boc)2-OH 5 in 70% yield over two steps (Scheme 2).
-
Scheme 2. Guanidinylation of Fmoc-b3hOrn(TFA)-OH using N,N’-
bis(Boc)-1-amidinopyrazole 4.
Synthesis of b3-heptapeptide 1: b-Peptide 1 was envisaged to
be synthesized by coupling Fmoc-protected-amino acids on
a Rink amide resin.[27] The Fmoc-protected amino groups on
the Rink amide resin were liberated upon treatment with
20% piperidine in DMF. Anchoring of the first amino acid
was achieved by reacting the resin with Fmoc-b3-amino
acids (3 5 equiv withrespect to the resin loading) activated
by HBTU/HOBt/(iPr)2NEt at RT in DMF for 2 4 h. Com-
pletion of the coupling was confirmed by a TNBS test.[28]
For elongating the peptide chain, the Fmoc-protecting group
of the anchored amino acid was removed using a combina-
tion of 20% piperidine in DMF and DBU/piperidine/DMF
1:1:48. The amino acids were coupled under conditions simi-
lar to those used for anchoring, namely HBTU/HOBt/
(iPr)2NEt. The deprotection and coupling cycles were re-
peated six times in total to furnishthe Fmoc-protected b-
heptapeptides on the resin. The Fmoc-protecting group was
comfortably removed from the resin-bound b-peptide 1
using the standard DBU/piperidine protocol. Subsequently,
the resin-bound peptide was cleaved off the resin with 10%
TFA in CH2Cl2, and the side-chain-protected peptide thus
obtained was further subjected to acidolysis in the presence
of a scavenger (TFA/(iPr)3SiH/H2O 95:2.5:2.5) to afford the
crude b-peptide 1. After preparative HPLC, the b-peptide 1
(33% yield) was isolated in >98% purity (Scheme 3).
Scheme 3. Solid-phase synthesis of b-heptapeptide 1 on a Rink amide
resin.
corresponding NMR investigations have established that b3-
peptides forming a 314-helical structure exhibit a characteris-
tic CD pattern (a negative Cotton effect near 215 nm and
zero crossing at ca. 207 nm).[4,8b,9a,b,29] As anticipated, the
CD spectra of b3-heptapeptide 1 in MeOH and aqueous sol-
ution display a pattern characteristic of a 314-helix (Fig-
ure 2a, b). However, the Cotton effect at 215 nm gradually
decreases withincreasing amount of water, indicating a loss
of secondary structure (Figure 2a).
Remarkably, when one compares the mean molar elliptic-
ity of all b3-heptapeptides synthesized in our laboratory,
heptapeptide 1 exhibits the strongest ellipticity in MeOH.
Additionally, the pH dependence of the CD spectrum in
aqueous solution supports our view that electrostatic inter-
Structural Analysis
CD-Spectroscopic
measure-
ments: Circular dichroism spec-
troscopy is a frequently used
method for analyzing a-peptidic
structures. Although, for b-pep-
tides, the correlation between
CD pattern and secondary
structure is not yet fully estab-
lished, for certain b-peptides,
CD spectra have been correlat-
ed withsecondary structures
when used in combination with
other spectroscopic techniques.
Figure 2. CD Spectra of 1 a) in MeOH and H2O; b) in H2O at different pH values. The spectra were recorded
Thus, CD measurements and at a concentration of 0.2 mm at 208C and are not normalized to the number of residues.
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b-Peptide 314-Helix
1607 1615
actions strongly stabilize the helical conformation of peptide
1 (Figure 2b). The ellipticity decreases at pH values above
the pKa values expected for the basic b3hArg side chains
and the terminal amino group, and below the pKa values of
the acidic b3hGlu side chains. The strongest molar ellipticity
is observed at pH 3.5, the value where the arginine residues
are anticipated to be protonated and the glutamic acid resi-
dues to be deprotonated (Figure 2b). These observations
clearly indicate that electrostatic interactions can be used to
stabilize the b-peptidic 314-helix, while non salt-bridge-form-
ing b-peptides without any conformational constraints do
not show the characteristic CD pattern in aqueous solutions.[29]
NMR Spectroscopy: In order to obtain more detailed infor-
mation on the secondary structure and its stability towards
unfolding, a detailed NMR-spectroscopic investigation of b3-
heptapeptide 1 was carried out. First, we examined peptide
1 in MeOH.[30] The presence of a regular secondary struc-
ture was indicated by a large dispersion of the chemical
shifts, as well as by the observation of large and small values
Figure 3. NMR Structure of b-heptapeptide 1 in MeOH. The peptide
forms a well-defined 314-helical structure. Side view without side chains
(left) and top view with side chains (right).
3
for the vicinal coupling constants J(H-C(b),Hax-C(a)) and
3J(H-C(b),Hla-C(a)), respectively. The amino acid spin sys-
tems were assigned using DQF-COSY and TOCSY techni-
ques, whereas the sequence assignment was derived from
by the CD measurements.[29] In order to obtain information
about the transition from a stable and well-defined 314-helix
in MeOH to either a more extended or multiconformational
state in aqueous solution, we carried out an NMR titration
experiment starting withpure MeOH and adding increasing
amounts of water (Figure 4). To our surprise, up to a con-
centration of ~25% H2O (v/v) little change of the amide
chemical shifts was observed and the dispersion of the
amide and side chain NH chemical shifts actually increased,
causing us to wonder whether the 314-helical structure may
be maintained or even further stabilized by the presence of
up to 25% (v/v) of water?
Therefore, a detailed NMR structural investigation of
peptide 1 in a MeOH/water (3:1) mixture was carried out.
The obtained spectra were analyzed along the same lines as
described for the MeOH solution.[30] Again, NOEs typical of
the 314-helix are found in the ROESY spectra, but some of
these NOEs are considerable weaker than those observed in
MeOH. A simulated annealing calculation was carried out
to test whether the NMR derived restraints were still consis-
tent witha single conformation. Teh resulting structures
withthe lowest restraint violation formed a bundle of struc-
tures (Figure 5) that has the shape of a 314-helical conforma-
tion. However, due to the lower number of restraints
(NOEs) and lower intensity of the cross-peaks, the 314-helix
is now less well-defined than in MeOH. This indicates that,
although the 314-helix is still present, other conformations
become increasingly populated when water is added.
d
aN(i,i+1) and dN,N(i,i+1) sequential NOEs and HSQC/
3
ꢀ
HMBC correlations. Large J(NH,H C(b)) coupling con-
stants (~9 Hz) established that the NH and H-C(b) protons
are in an antiperiplanar arrangement. The diastereotopic
H-C(a) protons were assigned assuming that the axial pro-
tons exhibit a large, and the lateral protons a small coupling
withH-C( b). This is in agreement with stronger NOEs
being observed from H-C(b) to the lateral Hla-C(a) protons,
compared withthe axial H ax-C(a) protons, and withstronger
NOEs from NHi+1 to the axial Hax-C(a)i protons.
To determine the three-dimensional structure, ROESY
spectra at different mixing times (150, 300 ms) were record-
ed, the NOE values were extracted, calibrated and classified
according to their volume into strong, medium and weak
distance categories. Examination of the NOEs and compari-
son withformer b3-peptides showed that all the characteris-
tic cross-peaks[8a] for a 314-helical conformation were present
for peptide 1 in MeOH. In fact, they were either compara-
ble or even stronger than the corresponding NOEs earlier
observed for other b3-peptides[5e] suggesting that b3-hepta-
peptide 1 forms the most stable 314-helix we have observed
to date. The NOE derived distances as well as the dihedral
angles derived from coupling constants and NOEs were
used in a restrained molecular dynamics simulated annealing
protocol.[30] The calculation yielded 20 structures that could
be clustered into a well-defined left-handed 314-helix with
side chains of b3hGlu and b3hArg on top of each other, in
keeping with the presence of salt-bridges between these
charged groups (Figure 3).
Conclusion
Subsequently, b3-heptapeptide 1 was examined in an
aqueous solution. Unfortunately, poor dispersion of the
chemical shifts and overlapping resonance signals hampered
complete assignment by NMR and at the same time indicat-
ed that peptide 1 may adopt a more extended or partially
unwound conformation in aqueous solution as also indicated
In summary, we have synthesized b3-homoarginine via two
different strategies–method A: Arndt Eistert homologa-
tion of a-arginine and method B: guanidinylation of an ap-
propriately protected b3-homoornithine derivative–suitable
for solid-phase peptide synthesis. Solid-phase b-peptide syn-
thesis was used to prepare b3-heptapeptide 1. Th e Fmoc
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D. Seebachet al.
FULL PAPER
of secondary structure as the water content increases in the
medium. This observation suggests that the transition from
the folded to the unfolded structures proceeds progressively,
which would be in agreement with the proposed non-coop-
erative folding of b-peptides.[31] Furthermore, it confirms our
previous findings that hydrogen-bonding is more important
in stabilizing b-peptidic helices than the b-amino acid resi-
dues backbone.[32]
The surprisingly increased dispersion of the NH chemical
shifts at a water concentration of 25% (v/v) led us to the
first NMR structural analysis of a b-peptide 314-helical struc-
ture in a mixture of methanol and water. This result, in con-
junction withthe pH-dependent CD measurements, clearly
demonstrates that electrostatic interactions can be utilized
to stabilize secondary structures, while similar to a-peptides,
non salt-bridge-forming peptides without any conformation-
al constraints remain unstructured under these condi-
tions.[29b]
Thus, we believe that these findings are of great impor-
tance withrespect to our goals of designing b-peptidic terti-
ary structures, and of investigating the influences of b-pepti-
dic secondary structures on the folding, stability, activity of
mixed a/b-peptides and proteins.
Figure 4. 1H NMR Spectroscopic titration study of a MeOH solution of 1
withwater. Up to a water concentration of ~25% (v/v) no considerable
change of the amide chemical shifts can be observed; but the dispersion
of the amide and the side-chain NH protons even increases. From 50 to
75% water, the amide protons move together and at 100% water their
assignment becomes difficult.
Experimental Section
General methods: Starting materials and reagents: THF was distilled
from K under an Ar atmosphere prior to use. Solvents for chromatogra-
phy and workup procedures were distilled over anhydrous CaSO4, P2O5
or KOH/FeSO4 (Et2O). Et3N and (iPr)2NEt were distilled from CaH2.
Amino acid derivatives were purchased from Novabiochem. All other re-
agents were used as received from Fluka or Aldrich.
Caution: The generation and handling of CH2N2 requires special precau-
tions.[33]
Reactions carried out with the exclusion of light were performed in
flasks completely wrapped in aluminium foil. Acronyms: Pbf=2,2,4,6,7-
pentamethyl-dihydrobenzofurane-5-sulfonyl, Pmc=2,2,5,7,8-pentameth-
yl-chromane-6-sulfonyl, HBTU=2-(1H-benzotriazol-1-yl)-1,1,3,3-tetra-
methyluronium hexafluorophosphate, HOBt=1-hydroxybenzotriazole,
NMM=N-methyl morpholine, TNBS=2,4,6-trinitro-benzenesulfonic
acid, DBU=1,8-diazabicyclo[5.4.0]undec-7-ene, TFA=trifluoroacetic
acid, TIS=triisopropylsilane.
Equipment: Thin-layer chromatography (TLC): silica gel 60 F254 plates
(Merck); detection withUV and dipping into a solution of ™Mo-stain∫
(25 g phosphomolybdic acid, 10 g Ce(SO4)2¥H2O, 60 mL conc. H2SO4 and
940 mL H2O) followed by heating. Flash column chromatography (FC):
silica gel 60 (40 63 mm, Fluka) at 0.2 0.3 bar. Analytical HPLC: Knauer
HPLC system (pump type WellChrom K-1000 Maxi-Star, degasser, UV
detector (variable-wavelengthmonitor)), column: Nucleosil 100 5 C
8
(250î4 mm, Macherey Nagel). Preparative HPLC: Merck/Hitachi
HPLC system (pump type L-6250, UV detector L-4000) column: Nucleo-
sil 100-7C8 (250î21 mm, Macherey Nagel). Optical rotations: Perkin
Elmer 241 polarimeter (10 cm, 1 mL cell) at RT. CD Spectra: Jasco J-710
spectropolarimeter recording from 190 to 250 nm at 208C; 1 mm cell;
average of 5 scans; peptide concentration 0.2 mm; smoothing was done
by Jasco software. Solvents: MeOH (HPLC grade), aq. buffers: pH 1.7,
3.5: 0.1m AcOK/AcOH, pH 5.7, pH 7.0 and 7.9: 0.1m KH2PO4/
K2HPO4;[34] pH 9.6 and 11.0: 0.05m NaHCO3/NaOH.[35] IR Spectra: Per-
kin Elmer-782 spectrophotometer. CHCl3 (Fluka) was filtered over Alu-
mina N, Akt. I (ICN Biomedicals GmbH, Germany) before use. NMR
Spectra: Bruker AMX 500 (1H: 500 MHz, 13C: 125 MHz) or Varian
Gemini 300 or Varian Mercury 300 (1H: 300 MHz, 13C: 75 MHz); chemi-
cal shifts d in ppm downfield from internal Me4Si (d=0). MS: IonSpec
Ultima (MALDI FT-MS, high resolution MS (HRMS), in a 2,5-dihydroxy-
Figure 5. NMR Structure of b-heptapeptide 1 in 3:1 MeOH/H2O. Side
view without side chains (left) and top view with side chains (right).
strategy and the acid labile Rink amide resin allowed a mild
cleavage of the final b3-heptapeptide amide 1. Based on the
strong ellipticity in the CD spectrum and the corresponding
NMR-structural investigations we conclude that b3-hepta-
peptide adopts a stable 314-helical conformation in MeOH.
The CD- and NMR-titration studies indicate a gradual loss
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b-Peptide 314-Helix
1607 1615
benzoic acid (DHB) matrix). Elemental analyses were performed by the
Microanalytical Laboratory, Laboratorium f¸r Organische Chemie, ETH-
Z¸rich.
stroyed by vigorous stirring. The mixture was then diluted with Et2O and
washed with sat. aq. NaHCO3, 1n HCl, and brine. The organic phase was
dried (MgSO4) and concentrated under reduced pressure. FC (EtOAc/
hexane 8:2!9:1) afforded Fmoc-(S)-Arg(Pbf)-CHN2 (6.73 g, 63%) as a
yellow foam. Rf =0.3 (EtOAc/hexane 9:1); 1H NMR (300 MHz, CDCl3):
d=1.42 (s, 6H; Me), 1.47 1.62 (m, 3H; CH2), 1.80 1.85 (m, 1H; CH2),
2.06 (s, 3H; Me), 2.49 (s, 3H; Me), 2.57 (s, 3H; Me), 2.90 (s, 2H; CH2),
3.22 (brm, 2H; CH2N), 4.11 4.16 (m, 2H; CHCH2O, CHN), 4.30 4.42
(m, 2H; CHCH2O), 5.53 (s, 1H; CHN2), 5.99 (d, J(H,H)=8.1 Hz, 1H;
NHFmoc), 6.23 (brs, 3H; NH), 7.25 7.33 (m, 2H; arom.), 7.36 (t,
J(H,H)=7.5 Hz, 2H; arom.), 7.55 (d, J(H,H)=7.2 Hz, 2H; arom.), 7.72
(d, J(H,H)=7.5 Hz, 2H; arom.); 13C NMR (75 MHz, CDCl3): d=12.6,
18.1, 19.4, 25.3, 28.7, 29.9, 40.8, 43.3, 47.2, 66.9, 86.4, 117.5, 119.9, 124.6,
124.9, 126.9, 127.6, 132.2, 132.5, 138.2, 141.2, 143.5, 156.1, 156.3, 158.7; IR
(CHCl3): n˜ = 3432 (w), 3347 (w), 3007 (w), 2977 (w), 2111 (s), 1719 (s),
1624 (s), 1556 (s), 1508 (m), 1451 (m), 1370 (s), 1150 (m), 1105 (s), 1035
(w), 852 (w), 658 cmꢀ1 (w); MS (MALDI): m/z (%): 822 (11), 821 (25),
695 (9) [M+Na]+, 686 (15), 685 (35), 683 (15), 670 (12), 669 (33), 668
(35), 667 (90), 663 (11), 646 (16), 645 (38), 548 (13), 547 (40), 529 (22),
467 (10), 457 (11), 421 (18), 411 (33), 395 (22), 394 (14), 393 (59), 389
(16), 374 (22), 373 (100), 277 (17), 273 (16), 199 (15); HRMS: calcd for
[C35H40N6O6SNa]+ : 695.2622; found: 695.2621.
Reversed-phase (RP) HPLC analysis and purification: RP-HPLC Analy-
sis was performed on a Nucleosil 100-5C8 column (250î4 mm, Macher-
ey Nagel) witha linear gradient of A (0.1% TFA in H 2O) and B
(MeCN) at a flow rate of 1 mLminꢀ1 (Knauer HPLC system); UV detec-
tion at 220 nm; tR in min. RP-HPLC purification was performed on a Nu-
cleosil 100-7C8 column (250î21 mm, Macherey Nagel) with a linear gra-
dient of A and B at a flow rate of 20 mLminꢀ1 (Merck/Hitachi system),
UV detection at 215 nm.
Heptapeptide 1: The Rink amide resin[27] (410 mg, 0.25 mmol; loading
0.61 mmolgꢀ1) was swelled in DMF (6 mL) for 30 min and Fmoc depro-
tected using 20% piperidine in DMF (6 mL, 3î20 min) under Ar bub-
bling. A solution of Fmoc-b3hVal-OH (4.0 equiv), HBTU (3.8 equiv) and
HOBt (4.0 equiv) in DMF (4 mL) and (iPr)2NEt (7.8 equiv) were added
successively to the resin and the suspension was mixed for 1 2 h by Ar
bubbling. The coupling was monitored with TNBS test.[28] The resin was
then filtered and washed with DMF (6 mL, 6î1 min) prior to the follow-
ing Fmoc deprotection step. By measuring the absorbance of the benzo-
fulvene/piperidine adduct the loading was determined to be 82%. The
Fmoc group was removed using 20% piperidine in DMF (6 mL, 2î
10 min), DBU/piperidine/DMF 1:1:48 (6 mL, 3î10 min), 20% piperidine
in DMF (6 mL, 10 min) under Ar bubbling. The resin was filtered and
washed with DMF (50 mLmmolꢀ1, 6î3 min). For eachcoupling step, a
solution of the Fmoc-b3-amino acid (4/5 equiv), HBTU (3.8/4.8 equiv)
and HOBt (4/5 equiv) in DMF (4 mL) and (iPr)2EtN (7.8/9.6 equiv) were
added successively to the resin and the suspension was mixed by Ar bub-
bling for 1 2 h. In case of a positive TNBS test (indicating incomplete
coupling), the suspension was allowed to react further for 1 2 h. The
resin was then filtered and washed with DMF (6 mL, 6î1 min). After
coupling the last amino acid, the Fmoc group was cleaved and the resin
washed with DMF (6 mL, 6î1 min), DCM (6 mL, 6î1 min) and MeOH
(6 mL, 3î1 min). Drying overnight under hn afforded the Fmoc-depro-
tected peptide-resin (568 mg).
PhCO2Ag (0.12 g, 0.5 mmol) was added to
a solution of Fmoc-(S)-
Arg(Pbf)-CHN2 (3.36 g, 5.0 mmol) in THF/BnOH (8.5:1.5, 8.0 mL). The
resulting mixture was ultrasonicated for 4 hin the dark at RT. After re-
moving the bulk of THF under reduced pressure, the residue was dis-
solved in EtOAc and washed with sat. aq. Na2S2O3 (2î), sat. aq.
NaHCO3 (2î), sat. aq. NH4Cl solutions and brine. The organic phase
was dried (MgSO4) and concentrated under reduced pressure. FC
(EtOAc/pentane 1:1 ! 7:3) afforded 2a (3.44 g, 91%) as white foam.
Rf =0.22 (EtOAc/pentane 3:1); [a]RDT =ꢀ12.4 (c=1.00 in CHCl3);
1H NMR (300 MHz, CDCl3): d=1.42 (s, 6H; 2Me), 1.42 1.63 (m, 3H),
1.66 (brm, 1H), 2.06 (s, 3H; Me), 2.49 (s, 3H; Me), 2.49 2.57 (m, 2H;
CH2COOBn), 2.57 (s, 3H; Me), 2.89 (s, 3H; Me), 3.19 (m, 2H; CH2N),
3.96 (brs, 1H; CHNHFmoc), 4.13 (t, J(H,H)=7.2 Hz, 1H; CHCH2O),
4.34 (d, J(H,H)=6.2 Hz, 2H; CHCH2O), 5.09 (s, 2H; CH2Ph), 5.48 (d,
J(H,H)=9.0 Hz, 1H; NHFmoc), 6.05 (brs, 3H; 3NH), 7.23 7.39 (m,
9H), 7.55 (d, J(H,H)=7.5 Hz, 2H), 7.73 (d, J(H,H)=7.5 Hz, 2H);
13C NMR (75 MHz, CDCl3): d=12.6, 18.0, 19.4, 25.6, 28.7, 32.1, 39.6,
40.9, 43.3, 47.3, 47.5, 66.6, 66.8, 86.3, 117.4, 119.9, 124.5, 126.9, 127.6,
128.2, 128.3, 128.5, 132.2, 132.9, 135.4, 138.2, 141.2, 143.6, 143.7, 155.9,
156.3, 158.6, 171.0; IR (CHCl3): n˜ =3621 (w), 3430 (w), 2976 (m), 1723
(m), 1621 (m), 1558 (m), 1514 (m), 1451 (m), 1390 (w), 1248 (m), 1106
(m), 1046 (m), 877 (w), 658 cmꢀ1 (w); MS (MALDI): m/z (%): 776 (15),
775 (29) [M+Na]+, 502 (33), 501 (100) [MꢀPbf+2H]+, 305 (40); elemen-
tal analysis calcd (%) for C42H48N4O7S (752.9): C 67.00, H 6.43, N 7.44;
found: 66.75, H 6.49, N 7.33.
The dry Fmoc-deprotected peptide-resin (160 mg) was treated with a
mixture of CH2Cl2/TFA/TIS 90:9:1 (5î3 mL), allowing the solvent to
pass through the resin bed slowly. Excess TFA/CH2Cl2 was evaporated
and the side-chain protecting groups removed by stirring the oily residue
in TFA/TIS/H2O 95:2.5:2.5 for 3 h. The solvent was evaporated, co-
evaporated withCH 2Cl2, to yield an oily residue. The precipitate formed
upon addition of cold Et2O to the oily residue was separated by decant-
ing the solvent. The precipitate was dissolved in H2O/dioxane solution
and lyophilized to yield 94 mg of the crude peptide. Purification of the
crude peptide by RP-HPLC (10 20% B in 40 min) afforded the TFA salt
of 1 (19 mg, 33%) as a white fluffy solid. Analytical RP-HPLC (17 27%
B in 40 min) tR 38.7 min, purity >98%. 1H NMR (500 MHz, D2O/H2O
1:9): d=0.87 (d, J(H,H)=6.7 Hz, 6H; Me), 0.88 (d, J(H,H)=6.5, 6H;
Me), 0.99 (d, J(H,H)=6.8, 3H; Me), 1.00 (d, J(H,H)=6.9 Hz, 3H; Me),
1.44 1.66 (m, 9H), 1.68 1.79 (m, 4H), 1.83 1.88 (m, 2H), 1.91 1.99 (m,
1H), 2.26 2.58 (m, 17H; 8CH2CO, CHHCO), 2.69 (dd, J(H,H)=16.2,
4.5 Hz, 1H; CHHCO), 3.17 3.18 (m, 4H; CH2N), 3.44 3.48 (m, 1H),
3.53 3.54 (m, 1H; CHN), 4.03 4.09 (m, 2H; CHN), 4.16 4.22 (m, 4H;
CHN), 6.83 (s, 1H; NH), 6.64 (brs, 7H), 7.15 (brs, 2H, 2NH), 7.52 (s,
1H; NH), 7.19 7.96 (m, 3H; 4NH), 7.96 (d, J(H,H)=10 Hz, 1H; NH),
8.12 (d, J(H,H)=9.7 Hz, 1H; NH), 8.14 (d, J(H,H)=9.6 Hz, 1H; NH);
13C NMR (125 MHz, D2O/H2O 1:9): d=19.9, 20.0, 20.9, 21.1, 27.3, 27.9,
28.1, 31.9, 32.7, 33.6, 33.7, 34.6, 37.3, 40.4, 41.1, 49.3, 49.4, 49.5, 55.1, 55.2,
57.1, 61.2, 72.7, 159.5, 159.6, 165.5, 165.8, 174.6, 174.9, 175.1, 175.8, 179.7,
180.8; IR (KBr): n˜ =3307 (m), 2969 (w), 1654 (s), 1560 (w), 1438 (w),
1207 (s), 1136 (s), 982 (w), 841 (w), 801 (m), 723 (m), 602 (w), 518 (w),
418 cmꢀ1 (w); MS (MALDI): m/z (%): 1006.6 (10), 1005.6 (19) [M+Na]+
, 985.6 (16), 984.6 (54), 983.6 (100) [M+H]+, 967.6 (10), 966.6 (20), 941.6
(10); HRMS: calcd for [C44H83N14O11]+ : 983.6360; found 983.6349.
Compound 2b: Fmoc-(S)-Arg(Pmc)-CHN2 was synthesized from Fmoc-
(S)-Arg(Pmc)-OH (9.94 g, 15.0 mmol) in a procedure analogous to that
of Fmoc-(S)-Arg(Pbf)-CHN2. FC (EtOAc/hexane 8:2!9:1) afforded
Fmoc-(S)-Arg(Pmc)-CHN2 (5.70 g, 55%) as
a yellow foam. Rf =0.3
(AcOEt/hexane 90:10); 1H NMR (300 MHz, CDCl3): d=1.26 (s, 6H;
Me), 1.58 (brm, 3H; CH2), 1.72 1.80 (m, 1H; CH2), 1.74 (t, J(H,H)=
6.7 Hz, 2H; CH2), 2.08 (s, 3H; Me), 2.54 (s, 3H; Me), 2.57 (s, 3H; Me),
3.22 (brm, 2H; CH2N), 4.09 4.16 (m, 2H; CHCH2O, CHN), 4.30 4.37
(m, 2H; CHCH2O), 5.51 (s, 1H; CHN2), 5.99 (d, J(H,H)=7.8 Hz, NH),
6.13 (brs, NH), 6.22 (brs, 2H; NH), 7.24 7.27 (m, 2H; arom.), 7.35 (t,
J(H,H)=7.3 Hz, 2H; arom.), 7.54 (d, J(H,H)=7.5 Hz, 2H; arom.), 7.72
(d, J(H,H)=7.5 Hz, 2H; arom.); 13C NMR (75 MHz, CDCl3): d=12.3,
17.6, 18.7, 21.5, 25.3, 26.8, 29.9, 32.8, 40.8, 47.2, 66.9, 73.7, 117.9, 119.9,
124.1, 125.0, 126.9, 127.6, 132.9, 134.8, 135.4, 141.2, 143.6, 153.6, 156.1,
156.3; IR (CHCl3): n˜ = 3345 (w), 2879 (w), 2944 (w), 2111 (m), 1720
(m), 1624 (s), 1552 (s), 1509 (m), 1450 (m), 1370 (m), 1299 (m), 1166 (w),
1110 (s), 1046 (w), 834 (w), 657 cmꢀ1 (w); MS (ESI, pos.): m/z: 725.0 (20)
[M+K]+, 710.0 (24), 709.0 (52) [M+Na]+, 687.0 (100); HRMS: calcd for
[C32H46N4O6SNa]+ : 681.2723; found: 681.2726.
Compound 2a: Fmoc-(S)-Arg(Pbf)-OH (9.73 g, 15.00 mmol) was dis-
solved in THF (43 mL) under Ar and cooled to ꢀ208C. After addition of
ClCO2iBu (2.04 mL, 15.75 mmol) and NMM (1.74 mL, 15.75 mmol), the
mixture was stirred at ꢀ208C for 30 min. The resulting white suspension
was allowed to warm up to ꢀ58C and a solution of CH2N2 in Et2O was
added until the rich yellow colour persisted. Stirring was continued for
4 has the mixture was allowed to warm to RT. Excess CH 2N2 was de-
Compound 2b was synthesized from Fmoc-(S)-Arg(Pmc)-CHN2 (3.36 g,
5.0 mmol) in a procedure analogous to that of 2a. FC (EtOAc/pentane
3:2!5:1) afforded 2b (6.33 g, 83%) as white foam. Rf =0.32 (EtOAc/
pentane 3:1); [a]RDT =ꢀ12.7 (c=0.82 in CHCl3); 1H NMR (300 MHz,
CDCl3): d=1.27 (s, 6H; Me), 1.31 1.61 (m, 4H; 2CH2), 1.75 (t, J(H,H)=
1613
Chem. Eur. J. 2004, 10, 1607 1615
www.chemeurj.org
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

D. Seebachet al.
FULL PAPER
6.7, 2H; CH2), 2.08 (s, 3H; Me), 2.50 2.60 (m, 2H; CH2COOBn), 2.55 (s,
3H; Me), 2.57 (s, 3H; Me), 3.96 (brm, 1H; CHNHFmoc), 4.14 (t,
J(H,H)=6.7, 1H; CHCH2O), 4.34 4.37 (m, 2H; CHCH2O), 5.09 (s, 2H;
CH2Ph), 5.43 (d, J(H,H)=9.3 Hz, 1H; NHFmoc), 5.96 (brs, 3H; NH),
7.24 7.39 (m, 9H), 7.54 (d, J(H,H)=7.5 Hz, 2H), 7.74 (d, J(H,H)=
7.5 Hz, 2H); 13C NMR (75 MHz, CDCl3): d=12.2, 17.6, 18.6, 25.4, 26.8,
32.1, 32.8, 39.5, 40.9, 47.2, 66.6, 66.7, 73.6, 117.8, 119.9, 123.9, 124.9, 126.9,
127.2, 128.2, 128.3, 128.5, 133.2, 134.8, 135.3, 135.4, 141.1, 143.5, 143.6,
153.4, 155.7, 156.3, 171.0; IR (CHCl3): n˜ =3430 (w), 3352 (w), 3008 (w),
2946 (w), 1722 (m), 1621 (m), 1553 (s), 1513 (m), 1451 (m), 1385 (w),
1353 (w), 1299 (m), 1262 (m), 1167 (m), 1111 (s), 1013 (w), 657 cmꢀ1 (w);
MS (MALDI): m/z (%): 805 (8) [M+K]+, 791 (16), 790 (49), 789 (100)
[M+Na]+, 502 (33), 501 (98) [MꢀPmc+2H]+, 481 (11), 305 (59); elemen-
tal analysis calcd (%) for C43H50N4O7S (766.9): C 67.34, H 6.57, N 7.31
found C 67.37, H 6.73, N 7.28.
ing rotamers); 1H NMR (75 MHz, CDCl3): d=1.48 (s, 9H; tBu), 1.49 (s,
9H; tBu), 1.62 1.69 (br, 4H; CH2), 2.35, 2.59 2.68 (m, 2H; CH2CO),
3.25 3.40, 3.63 (br, 2H; CH2N), 4.00 (brs, 1H; CHN), 4.20 (t, J(H,H)=
6.7, 1H; CHCH2O), 4.39, 4.55 (d, J(H,H)=9.3, 2H; CHCH2O), 5.70 (d,
J(H,H)=9.3, 1H; NH), 7.27 7.41 (m, 5H; Ph), 7.59, 7.60 (d, J(H,H)=
7.2, 7.2 Hz, 2H; arom.), 7.75 (d, J(H,H)=7.5, 2H; arom.), 8.38 (br, 1H;
NH), 11.46 (br, 1H; NH); 13C NMR (75 MHz, CDCl3): d = 26.2, 28.2,
28.4, 31.1, 39.1, 40.5, 47.4, 48.1, 66.6, 79.5, 83.3, 119.9, 125.0, 126.9, 127.6,
141.2, 143.7, 153.1, 156.1, 163.1, 174.3; IR (CHCl3): n˜ =3325 (w), 2984
(w), 1720 (s), 1616 (s), 1511 (w), 1450 (w), 1416 (m), 1333 (m), 1248 (m),
1135 (s), 1054 (w), 1028 (w), 622 cmꢀ1 (w); MS (MALDI): m/z (%): 633.3
(3) [M+Na]+, 412.2 (23), 411.2 (100); elemental analysis calcd (%) for
C32H42N4O8 (610.7): C62.94, H 6.93, N 9.17; found: C 62.92, H 6.95, N
9.13.
Compound 3a: A few drops of AcOH and 10% Pd/C (140 mg) under Ar
were added to a solution of 2a (1.51 g, 2.0 mmol) in methanol (20 mL).
The apparatus was evacuated, flushed three times with H2 and the mix-
ture was stirred under an atmosphere of H2 for 2 hand 30 min. The mix-
ture was diluted withMeOH, filtered throughCelite and concentrated
under reduced pressure. FC (CH2Cl2/MeOH/AcOH 95:5:1!90:10:1) af-
forded 3a (1.21 g, 91%) as a white foam. Rf =0.14 (CH2Cl2/MeOH/
AcOH 95:5:1); [a]RDT =ꢀ6.86 (c=1.02, CHCl3); 1H NMR (300 MHz,
CDCl3, signals of rotamers are given in italics): d=1.39 (s, 6H; Me), 1.56
(br, 4H; CH2), 2.04 (s, 3H; Me), 2.25 2.31 (m, 2H; CH2CO), 2.47 (s, 3H;
Me), 2.54 (s, 3H; Me), 2.86 (s, 3H; Me), 3.17 (brs, 2H; CH2CMe2), 3.59,
3.94 (brs, 1H; CHN), 4.10 4.16 (t, J(H,H)=6.9 Hz, 1H; CHCH2O), 4.30,
4.47 (br, 2H; CHCH2O), 5.81 (d, J(H,H)=8.4 Hz, 1H; NH), 6.36 (br,
3H; NH), 7.20 7.34 (m, 4H; arom.), 7.51 (d, J(H,H)=7.2 Hz, 2H;
arom.), 7.68 (d, J(H,H)=7.5 Hz, 2H; arom.); 13C NMR (75 MHz,
CDCl3): d=12.6, 18.0, 19.4, 25.5, 28.6, 31.4, 39.6, 40.8, 43.2, 47.8, 66.8,
86.4, 117.6, 119.8, 124.7, 124.9, 126.9, 127.5, 132.3, 138.3, 141.0, 143.5,
143.6, 156.2, 156.3, 158.8, 174.9; IR (CHCl3): n˜ =3436 (w), 3348 (w), 3008
(w), 2976 (w), 1713 (s), 1622 (m), 1555 (s), 1451 (m), 1408 (m), 1371 (w),
1107 (s), 909 (w), 658 (w), 621 (w) cmꢀ1; MS (MALDI): m/z (%): 686
(32), 685 (79) [M+Na]+, 412 (22), 411 (100), 394 (18), 393 (79), 215 (18).
Acknowledgment
We thank Mrs. B. Brandenberg for recording NMR spectra, and grateful-
ly acknowledge Novartis Pharma AG (Basel) for ongoing financial sup-
port.
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Compound 3b: Compound 3b was synthesized starting from 2b (5.50 g,
7.3 mmol) in a procedure analogous to that of 3a. FC (CH2Cl2/MeOH/
AcOH 95:5:1) afforded 3b (4.45 g, 90%) as a white foam. Rf =(CH2Cl2/
MeOH/AcOH 95:5:1); [a]RDT =ꢀ5.2 (c=1.12 in CHCl3); 1H NMR
(300 MHz, CDCl3, signals of rotamers are given in italics): d = 1.26 (s,
6H; Me), 1.38 1.46 (m, 1H; CH2), 1.57 (br, 3H; CH2), 1.73 (t, J(H,H)=
6.5 Hz, 2H; CH2), 2.07 (s, 3H; Me), 2.30, 2.52 2.54 (m, 2H; CH2CO),
2.52 (s, 3H; Me), 2.55 (s, 3H; Me), 3.17 (br, 1H; CH2N), 3.57, 3.93 (br,
1H; CHN), 4.09 4.16 (m, 1H; CHCH2O), 4.31 4.33, 4.49 (br, 2H;
CHCH2O), 5.75 (d, J(H,H)=9.0 Hz, 1H; NH), 6.27 (br, 3H; NH), 7.20
7.26 (m, 2H; arom.), 7.33 (t, J(H,H)=7.5 Hz, 2H; arom.), 7.52 (d,
J(H,H)=7.5 Hz, 2H; arom.), 7.69 (d, J(H,H)=7.5 Hz, 2H; arom.);
13C NMR (75 MHz, CDCl3): d=12.24, 17.6, 18.6, 21.5, 25.5, 26.8, 29.8,
31.4, 32.7, 39.51, 40.8, 47.2, 66.7, 73.7, 117.9, 119.8, 124.1, 125.0, 126.9,
127.5, 132.7, 134.8, 135.4, 141.1, 143.5, 143.7, 153.6, 156.2, 156.3, 174.7; IR
(CHCl3): n˜ =3348 (w), 2944 (w), 1713 (m), 1622 (m), 1651 (s), 1450 (m),
1385 (w), 1299 (m), 1248 (w), 1166 (w), 1110 (s), 1014 (w), 658 cmꢀ1 (w);
MS (MALDI): m/z (%): 715 (5) [M+K]+, 7.1 (12), 700 (43), 699 (100)
[M+Na]+, 412 (20), 411 (86) [MꢀPmc+2H]+, 393 (17), 393 (77)
[MꢀPmcꢀNH2]+, 215 (19); elemental analysis calcd (%) for
C36H44N4O7S (676.3): C 63.89, H 6.55, N 8.28; found C 63.77, H 6.68, N
8.12.
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Compound 5: TFA (12 mL) was added at 08C to a solution of Fmoc-(S)-
b3hOrn(Boc)-OH (2.92 g, 6.23 mmol) in CH2Cl2 (12 mL), and the mixture
allowed to warm to RT while stirring. After 4 h the solvent was removed
under reduced pressure, coevaporated withCH 2Cl2 and the residue dried
under high vacuum to yield the TFA salt. The TFA salt was used as is.
(iPr)2NEt (3.4 mL, 20.14 mmol) was added to a suspension of the TFA
salt in formamide followed by a solution of 4 (2.9 g, 9.34 mmol) in diox-
ane (9 mL). After stirring the mixture for 48 h, 1n HCl (40 mL) was
added and extracted with EtOAc (3î100 mL). The organic phase was
washed with brine and concentrated under reduced pressure. FC
(EtOAc/pentane/AcOH 4:6:0.2) afforded 5 (2.67 g, 70%) as a white
foam. Rf = 0.43 (EtOAc/pentane/AcOH 5:5:0.2); [a]RDT =+1.88 (c=0.96
1
in CHCl3); H NMR (300 MHz, CDCl3, a mixture of slowly interconvert-
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b-Peptide 314-Helix
1607 1615
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Received: September 25, 2003 [F5571]
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¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim