Design, synthesis and structural analysis of mixed α/β-peptides that adopt stable cyclic hairpin-like conformations

Article abstract of DOI:10.1016/j.tet.2012.01.015

The strategic replacement of four α-amino acid residues of a cyclo-(ααααα)₂ peptide by β-, β²- or β³-amino acids residues provided a series of novel 2:1 α/β-mixed peptides that were designed to adopt cyclic hairpin-like st

Full text of DOI:10.1016/j.tet.2012.01.015

Tetrahedron 68 (2012) 2391e2400
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Design, synthesis and structural analysis of mixed
cyclic hairpin-like conformations
a/b-peptides that adopt stable
a
b
c
d
e
ꢀ
Matthijs van der Knaap , Jose M. Otero , Antonio Llamas-Saiz , Mark J. van Raaij , Lianne I. Lageveen ,
Henk J. Busscher e, Gijsbert M. Grotenbreg f, Gijsbert A. van der Marel a, Herman S. Overkleeft a,
Mark Overhand a
,
*
a Bio-Organic Synthesis, Leiden Institute Of Chemistry, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands
b Departamento de Bioquímica y Biología Molecular, Universidad de Santiago, Santiago de Compostela, Spain
c Unidad de Rayos X (RIAIDT), Edificio CACTUS, Universidad de Santiago de Compostela, Santiago de Compostela, Spain
d Laboratorio M-4, Dpto de Estructura de Macromoleculas, Centro Nacional de Biotecnologia, c/Darwin 3, Campus Cantoblanco, E-28049 Madrid, Spain
e Department of Biomedical Engineering, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
f Department of Microbiology, National University of Singapore, CeLs, 28 Medical Drive, Singapore 117456, Singapore
a r t i c l e i n f o
a b s t r a c t
b -
2- or b3
Article history:
The strategic replacement of four
amino acids residues provided a series of novel 2:1 a/b
a
-amino acid residues of a cyclo-(aaaaa
)
peptide by
b
-,
-mixed peptides that were designed to adopt
cyclic hairpin-like structures. It was shown that conformationally stable cyclo-(ababa isomers can be
2
Received 8 September 2011
Received in revised form 23 December 2011
Accepted 9 January 2012
)
2
obtained using both enantiomers of the central two basic
turn sequence and several combinations of facing -amino acid residues with no side chain or a hy-
a-amino acid residues, a known a-amino acid
Available online 18 January 2012
b
drophobic side chain having specific regio- and stereochemistry. The X-ray analysis of two derivatives
provides molecular details of the intra-molecular hydrogen bonding interaction, dihedral angles of the
backbone and side chain positioning of the novel cyclic hairpin-like structures. One of these isomers
forms an unprecedented hexagon-shaped nano-channel assembly in the crystal structure. Well-defined
cyclic hairpin-like structures as described here and derivatives that can be readily designed based on this
research can be used as scaffolds onto which functional groups can be grafted in a spatially controlled
Keywords:
Cyclic mixed a/b-peptides
H-bonding pattern
Hairpin-like structure
Nano-channels
manner and as
b-hairpin mimics with specific biological properties.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
the position in these sequences, as well as the regio- and stereo-
chemistry of the side chains of the built-in -amino acid residues
b
Enormous progress has been made in the design of linear and
cyclic oligomers with predetermined structures by making use of
strongly influence the stability of the secondary structures. Guided
by these results and our own structural work on modified cyclic
peptides26e32 we realized that related rigid hairpin structures
the properties of b- and g
-amino acids.1e9 In particular hydrogen
bond formation between specific homologue residues govern the
formation of turn-, helix-, and sheet-like structures, as well as
molecular assemblies. In recent years, it has been shown that even
greater structural diversity can be obtained by combining different
classes of amino acids homologues with each other.10e15 Though
containing mixed
backbone.
To attain well-defined hairpin-like structures a cyclic seam-
less peptide backbone must comply with specific geometrical
requirements to allow a regular pattern of intra-molecular hy-
drogen bonding interactions. The requirements for the forma-
a-/b-amino acids can be designed having a cyclic
many mixed
variants of hairpin structures were obtained as well.16e25 For in-
stance, the peptide sequences bbaabb 23 aabaabaa 24
aabaaaa
baa 22 and aabaaaaaabaa 16
all form hairpin-like structures, each
with a distinct intra-molecular hydrogen bonding pattern. Besides
the turn-forming propensity of the central two -amino acids, both
a/b
-peptides adopt helical structures,13,14 several
tion of conformationally stable cyclic hairpin structures for
a-
,
,
peptides are well established,33e37 as exemplified by the natural
product gramicidin S (GS, cyclo-(Pro-Val-Orn-Leu-(R)-Phe)2). GS
is a C2 symmetric cyclic decapeptide (Fig. 1a) containing four
intra-molecular hydrogen bonding interactions (blue arrows
Fig. 1a),38e41 resulting in a central 14-membered H-bonding ring,
flanked by two 10-membered H-bonding rings (Fig. 1b). In ad-
dition to the alignment of the interstrand hydrogen bond
forming amides, the stereochemistry of the amino acid side
,
,
a
* Corresponding author. Fax: þ31 71 5274307; e-mail addresses: overhand@
chem.leidenuniv.nl, m.overhand@chem.leidenuniv.nl (M. Overhand).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2012.01.015

2392
M. van der Knaap et al. / Tetrahedron 68 (2012) 2391e2400
a
b
c
Fig. 1. (a) The structure of the antibiotic peptide gramicidin S (GS). Blue arrows indicate intra-molecular hydrogen bonds, in green are the two (R)-
Schematic representation of the cyclo-(aaaaa 2 motif in which the central 14-membered H-bonding ring segment is flanked by two 10-membered H-bonded ring segments and two
two-residue turns. (c) Schematic representation of the designed cyclo-(ababa motif, indicating is the alternative hydrogen bonding pattern.
a-amino acid residues. (b)
)
)
2
chains is of importance for the formation of a stable secondary
structure. The presence of an (R)- -amino acid residue (corre-
sponding to a
-amino acid in the Fisher notation)42 in each turn
2. Results and discussion
2.1. Synthesis
a
D
region stabilizes the cyclic hairpin structure in solution. The side
chains of the strand residues in the cyclic hairpin structure
adopt well-defined positions with the hydrophobic side chains
(i-Pr and i-Bu) on one face of the molecule and the hydrophilic
groups (aminopropyl) of the central residues on the opposing
face.38e41 GS does not readily aggregate and is soluble in aque-
ous media.
The linear precursors of the cyclo-(ababa
) peptides 1e11 were
2
prepared using suitably protected
blocks following a solid-phase Fmoc peptide chemistry strategy. The
required (S)- or (R)-configured Fmoc- -amino acids of which the Orn
residues have their side chains protected with a Boc-group,
Fmoce
AlaeOH and both enantiomers of the hydrophobic b3
amino acids are commercially available. Both enantiomers of
Fmoce
2hVal were synthesized following adaptation of the litera-
ture procedures.49e51 The hyper-acid labile HMPB-MBHA resin52 was
functionalized with eitherFmoce AlaeOH or Fmoce(R)-PheeOH, in
a- and b-amino acid building
a
b
-
Using GS as a basis we set out to replace
dues in the strand regions by -amino acids, in order to obtain
mixed -sequences with hairpin characteristics. It was decided
to retain the two turn -amino acid residues as well as the two
central residues in the strand regions. Thus, the two -amino
a-amino acid resi-
b
b
a/b
a
b
a
the presence of the coupling reagent DIC and catalytic DMAP and
elongated using standard conditions. The assembled decameric
partially protected peptides were cleaved from the resin by repetitive
mild acid treatment and cyclization of the C/N-terminus realized in
DMF under high dilution (0.01 M) under the agency of PyBOP/HOBt/
DiPEA. Removal of reagents was affected by LH-20 gel filtration, the
Boc-groups of the Orn residues of the cyclic peptides were removed
using TFA and the fully deprotected peptides were purified using
preparative HPLC purification (20e45% overall yield).
acid residues neighboring the central ornithine residue in each
strand of the regular cyclic hairpin structure of GS were replaced
by unsubstituted
b-amino acids (1). An alternative pattern of
stabilizing intra-molecular hydrogen bonding16e25 interactions
would ensue to accommodate the four additional methylene
groups in the peptide backbone, namely a central 10-membered
H-bonding ring, flanked by two 14-membered H-bonding rings
(Fig. 1c). iso-Propyl and iso-butyl side chains were introduced,
analogously to the stereochemistry and the relative position of
the side chains in the strand regions of GS, via the dual in-
2.2. Crystallographic analysis
corporation of (R)-
Fig. 2). To probe the stability of the secondary structure the
stereochemistry of the side chains of the -amino acid residues
b b
2hVal and (S)- 3hLeu residues (peptides 2e4,
The cyclo-(ababa
) peptides 1e11 were subjected to controlled
2
b
evaporation conditions using a homemade set of crystallization
were varied (peptides 5e7) and a second series of compounds
were prepared (8e11, Fig. 2) containing two (R)-Orn residues in
buffers and solvents. cyclo-(ab2ab3a 6 and cyclo-(ab3ab2a
)
)
2
11
2
provided single crystals that were suitable for X-ray diffraction and
their high resolution structures are compared with those of GS38 in
Fig. 3. Based on the orientation and distances of the amides of the
opposing strands (Table 1), the crystal structures of compounds 6
and 11 have four well-defined intra-molecular hydrogen bonding
interactions. These results confirm the anticipated four stabilizing
which the
positions in the sequence. Here we present the synthesis of
a series of symmetric and asymmetric cyclo-(ababa peptides
b b
2- and 3-amino acid residues have swapped their
)
2
(1e11) and the study of their structural properties using spec-
troscopic and diffraction techniques. In addition, as it is known
and well documented that
b
-amino acid containing peptides may
intra-molecular hydrogen bonding pattern of cyclo-(ababa) pep-
2
display antibacterial activity (for examples of antibacterial
peptides see Refs. 43e45, for examples of antibacterial mixed a/
b
-
tides. The distances and angles of the two H-bonds of the turn re-
gions and the dihedral angles of the (R)-Phe-Pro sequences of all
three peptides have comparable values (Table 2). The turn regions
of GS and peptides 6 and 11 are thus highly similar.
b
-peptides see Refs. 46e48), we also report the antibacterial
activity of peptides 1e11.
The side chains of the two facing strands of all three peptides
position themselves perpendicular with respect to the hydrogen

M. van der Knaap et al. / Tetrahedron 68 (2012) 2391e2400
2393
Fig. 2. The structures of the symmetric and asymmetric cyclo-(ababa
) peptides 1e11.
2
Table 2
Dihedral angles of the amino acid residues of GS, 6 and 11 based on the X-ray data. In
brackets the deviation of the dihedral angles of the turn residues from an ideal type
II0 b-turn are indicated (angleobservedꢁangleideal
)
Peptide
GS
Residue
4(
D
4) [ꢀ]
q
[ꢀ]
j(
Dj) [ꢀ]
Pro(1)
Val(2)
Orn(3)
Leu(4)
(R)-Phe(5)
Pro(6)
Val(7)
Orn(8)
Leu(9)
ꢁ84(ꢁ4)
ꢁ87
d
d
d
d
d
d
d
d
d
d
ꢁ2(ꢁ2)
139
134
ꢁ135
ꢁ137
107
52(ꢁ8)
ꢁ80(0)
ꢁ108
ꢁ126(ꢁ6)
5(5)
126
135
103
ꢁ129(ꢁ9)
ꢁ113
ꢁ141
(R)-Phe(10)
54(ꢁ6)
6
Pro(1)
ꢁ76(4)
ꢁ127
ꢁ96
d
ꢁ10(ꢁ10)
120
162
(R)-
Orn(3)
(R)-
3hLeu(4)
b
2hVal(2)
178
d
b
96
ꢁ162
d
ꢁ154
(R)-Phe(5)
53(ꢁ7)
ꢁ92(ꢁ12)
ꢁ126
ꢁ128
78
ꢁ130(ꢁ10)
15 (15)
149
Pro(6)
d
(R)-
Orn(8)
(R)-
3hLeu(9)
b
2hVal(7)
177
d
Fig. 3. X-ray crystal structures of GS (top) and the peptides 6 (middle) and 11 (bottom),
with their intra-molecular hydrogen bonding patterns (left, side chains of the aaa and
the bab-strand regions are omitted for clarity) and their side chain positions (right).
124
b
ꢁ161
d
ꢁ125
(R)-Phe(10)
55(ꢁ5)
ꢁ127(ꢁ7)
11
Pro(1)
ꢁ81(9)
ꢁ79
d
ꢁ7(ꢁ7)
116
(S)-
(R)-Orn(3)
(R)-
2hVal(4)
b
3hLeu(2)
160
d
Table 1
144
ꢁ145
Intra-molecular hydrogen bonding distances and angles of facing strand residues of
b
ꢁ147
51(ꢁ9)
ꢁ71(19)
ꢁ85
177
d
109
the X-ray structures of GS,6a 6 and 11
(R)-Phe(5)
ꢁ133(ꢁ13)
ꢁ15(ꢁ15)
130
Pro(6)
d
D/O]C [ꢀ]
ꢁ
ꢁ
Peptide
GS
Donor
Acceptor
d(D/A) [A]
d(H/A) [A]
(S)-
(R)-Orn(8)
(R)-
2hVal(9)
(R)-Phe(10)
b
3hLeu(7)
156
d
N(2)
N(4)
N(7)
N(9)
O(9)
O(7)
O(4)
O(2)
2.94
2.91
3.04
2.90
2.11
2.06
2.19
2.03
134
159
134
150
142
ꢁ144
b
ꢁ145
175
d
118
56(ꢁ4)
ꢁ130(ꢁ10)
6
N(2)
N(3)
N(7)
N(8)
O(9)
O(8)
O(4)
O(3)
3.03
2.92
2.92
2.84
2.19
2.09
2.08
1.98
132
155
143
145
bond pattern. As a result of the regio- and stereochemistry of the
facing bab strands of peptide 6, that is (R)- -(R)-
2-(S)- 3, both iso-
b
a
b
propyl groups are oriented on the concave face of the molecule
11
N(2)
N(3)
N(7)
N(8)
O(9)
O(8)
O(4)
O(3)
3.00
2.98
3.07
2.94
2.16
2.11
2.25
2.07
132
164
134
171
while the iso-butyl and the aminopropyl groups are located on the
convex face (Fig. 3 right middle). The (S)-
stereochemistry of the facing bab strands of peptide 11 provides
b a-(R)-b
3-(R)- 2 regio- and

2394
M. van der Knaap et al. / Tetrahedron 68 (2012) 2391e2400
a cyclic hairpin-like structure in which both hydrophobic iso-butyl
and iso-propyl side chains are located on the concave face with the
two hydrophilic aminopropyl side chains on the opposite face
(Fig. 3 right bottom). This side chain positioning introduces am-
phiphilic characteristics to the molecule and in this respect, peptide
11 resembles GS. Peptides 6 and 11 adopt a bent hairpin-like
structure, rather than a pleated sheet, and the observed values of
of 11 interact with two other subunits in the lateral direction via
hydrophobic and -stacking interactions. Six lateral subunits come
p
full circle, forming a hexagon-shaped nano-channel structure with
ꢁ
a cationic interior diameter of w26 A. The hydrophobic exterior
ꢁ
(w45 A) of the hexagon-shaped molecular assembly interacts with
its neighbors with each of its six molecular walls.
the dihedral angles53 4,
q and j are listed in Table 2.
2.3. NMR analysis
The hydrogen bonding network between the peptide subunits
as observed in the crystal packing of GS and peptides 6 and 11 is
depicted in Fig. 4. In our previously published crystal structure of
GS38 not all amides in the strand regions are involved in inter-
subunit hydrogen bonding interactions. The GS monomers in the
crystal structure form closely packed double-helical tubes with
a hydrophobic exterior and a hydrophilic interior (Fig. 4, top).38
Peptide 6 forms a hydrogen bonding network with no discrete
channels in its crystal (Fig. 4, middle). Apart from the four intra-
The structures of the peptides 6 and 11 in solution were evalu-
ated using NMR and compared with the well studied cyclic
b-
hairpin structure of GS using this technique.41,54e61 Compounds 6
and 11 dissolve readily in polar solvents and as judged by the dis-
persion of the amide proton signals of their NMR spectra there are
no multiple conformations in CD3OH (Fig. 5, left). Using a combi-
nation of COSYand TOCSY spectra the NMR signals of 6 and 11 could
3
be assigned. The JNHeH coupling constant values of the Orn and
a
(R)-Orn residues are >7 Hz, while the 3JNHeHa values of the (R)-Phe
molecular hydrogen bonds present per monomer 11, each bab
-
strand interacts with a neighboring molecule via inter-subunit
hydrogen bonds (Fig. 4, bottom). The turn regions of each subunit
residues are <4 Hz (Fig.
5
middle). The chemical shift
perturbation26,30,62e64 of the Orn and (R)-Orn residues have a DdHa
Fig. 4. Molecular assembly of GS, peptide 6 and 11 in their crystals. (Top): GS subunits assemble in a helical fashion with inter-subunit hydrogen bonds (blue dashed lines) in the
pattern (3,3,2,3,3)n forming a double-helical channel (14 A).6a (Middle): Part of the hydrogen bonding network observed in the crystal X-ray structure of peptide 6, each monomers
ꢁ
has four hydrogen bonds with four other subunits (blue dashed lines), no channels are present. (Bottom): Each monomer 11 forms inter-subunit hydrogen bonding interactions
(blue dashed lines) with its neighbors, the turn regions interact via
channels (26 A).
p-stacking interactions with one lateral subunit, six subunits come full circle forming a hexagon-shaped nano-
ꢁ

M. van der Knaap et al. / Tetrahedron 68 (2012) 2391e2400
2395
Fig. 5. The amide regions of the 600 MHz 1D NMR spectra in CD3OH of GS (blue) and peptides 6 (green) and 11 (red) (left). 3JNHeHa coupling constant values of the Orn and (R)-Orn
and (R)-Phe residues (middle). The chemical shift perturbation of the
a-protons of the
a-amino acid residues of GS, 6, and 11 (right).
>0.1 ppm and the Pro and (R)-Phe residues a DdHa <0 ppm (Fig. 5
right). Both the magnitude of the 3JNHeHa coupling constant values
Orn residues are part of an extended conformation of a strand re-
gion and that the (R)-Phe, Pro residues are part of a turn region.65,66
The interpretation of the coupling constants of peptides 6 and 11
and the chemical shift perturbation of the
a-protons, of the a-
amino acid residues of GS, 6 and 11 concord that the Orn and (R)-
corresponding to the b
-amino acid residues,53 although not directly

2396
M. van der Knaap et al. / Tetrahedron 68 (2012) 2391e2400
comparable with GS, is of interest. For instance the amide signal of
peptides 6 and 11. More structural data is required, however, to
the (R)-
b
2hVal residues of peptide 11 appears as a doublet with
determine the exact conformation of these peptides. In contrast,
3
a large coupling constant JNHeH
of 10 Hz. Using the Karplus
the NMR spectra of peptides 5 and 7, containing (S)-
b
2hVal and (S)-
ba/b
equation for 4 dihedral angles of amino acid residues,67,68 this large
coupling constant corresponds to 4 wꢁ120ꢀ. The value of this di-
hedral angle is in agreement with the measured dihedral angle 4 as
determined from the X-ray crystal structure (Table 2).
b
3hLeu residues and (S/R)- 2hVal and (S)- 3hLeu, respectively, ap-
b
b
pear as broad signals. As peptides 5 and 7 readily dissolve, the
broad NMR signals are most likely due to the absence of a well-
defined secondary structure in solution, though it is possible that
these peptides are actually not monomeric in methanol solution.
The NOESY spectra of 6 and 11 displayed several signals char-
acteristic of a hairpin-like structure. Indicative of an extended
conformation of an amino acid residue as part of a strand region is
the presence of a strong sequential NOE interaction between Ha in
the i position and the NH of the amino acid in the iþ1 position,
together with a weak or absence NHeNH NOE between these
residues.65,66 As can be seen in Fig. 6 in both peptide 6 and 11 the
2.4. Biological activity
Finally peptides 1e11 were evaluated for their biological activity
(Table 3). As can be seen, none of the tested peptides, with the
exception of 8 displayed any antibacterial activity comparable to
strong NOE signal between the H
with the NH of the sequential
interaction is absent. Furthermore, both peptides 6 and 11 have
a characteristic sequential NHeNH interaction of the -amino acid
a
of the (S)-Orn/(R)-Orn residues
b-amino acid, while the NHeNH
the all-a-peptide starting point, gramicidin S.
b
residue at the iꢁ1 position with the NH of the Orn (6) or (R)-Orn
with the amino acid in the iþ1 position (11) (Fig. 6). This data
strongly indicates the presence of a bab-strand. Peptide 11 also
displays a long range NOE interaction between Ha of (R)-Phe and
3. Conclusion
The strategic replacement of four
a
-amino acid residues of
-amino acid
counterparts provided a series of novel 2:1 a/b-mixed peptides
a cyclo-(aaaaa
)
2
peptide by their hydrophobic b
the NH (R)-
addition to a NOE signal between Ha (R)-Phe and NH (R)-
peptide 6 also shows a weak interstrand NOE between NH (R)-
2hVal and H 2hLeu, also in agreement with the crystal
(R)-
b
3hLeu, which supports the presence of a
b
-turn. In
b
2hVal,
(1e11) that were designed to adopt cyclic hairpin-like structures. It
was shown that conformationally stable cyclo-(ababa)2 isomers can
b
b
b
be obtained using several stereochemical combinations of
a, b, -
b2
structure. More details concerning the NMR data can be found in
and
b
3-amino acids in the facing bab strand regions while keeping
the experimental section (Tables S1e3).
the two turn regions constant. Two of the here reported thirteen
peptides, that is, 6 and 11, provided X-ray crystal structures. The X-
ray data showed the anticipated stabilizing intra-molecular hy-
drogen bonding pattern, specific side chains positioning and an
unaltered structure of the two turn regions as compared with the
parent compound (GS). The regio- and stereochemistry of the
amino acids of the two facing bab strands determine the side chain
positioning. Peptides 6 and 11 adopt a bent conformation, rather
than a pleated sheet structure. The bent conformation is intrinsic to
the cyclo-(ababa
)
2
structure and is probably enhanced by the
presence of the four unsubstituted sp3-hybridized carbon atoms in
the macrocyclic backbone. By using NMR techniques it was shown
that peptides 1e4 and 8e11 adopt similar stable cyclic hairpin-like
structures in solution. However, not every
nation provided a structurally stable cyclo-(ababa
a
/
b
-amino acid combi-
structure, as
)
2
peptides 5 and 7 appear not to adopt a well-defined conformation
in solution. At this moment we do not have a plausible explanation
for this latter finding. Deeper understanding of stabilizing/desta-
bilizing factors of this type of cyclic hairpin-like peptides may be
obtained by synthesizing derivatives containing disubstituted b2,3
-
amino acids,1e9,69,70 turn modified analogues,19 or the design of
related cyclic hairpin-like structures combining -, -, and -amino
a
b
g
acids with a predetermined intra-molecular hydrogen bonding
pattern.
The here described cyclo-(ababa)2 molecules may be applicable
in various ways. Well-defined cyclic hairpin-like structures can be
used as scaffolds onto which functional groups can be grafted in
a spatially controlled manner71,72 and as
b-hairpin mimics with
specific biological properties.73e78 With respect to the latter, we
aim for the development of membrane disrupting amphiphilic
cationic peptides antibiotics that kill antibiotic resistant strains. The
hexagon-shaped cationic nano-channel assemblies as observed in
the crystal packing of peptide 11 are related yet distinct from our
Fig. 6. Enlargement of the NOESY spectra of peptide 6 (top) and 11 (bottom), dis-
playing the NHeHa interactions (NHeHb for the b-amino acids).
In conclusion, the NMR data is in full agreement with the
structures of peptides 6 and 11 as determined by X-ray. Based on
earlier reported hexameric
a GS turn modified cyclo-(aaaadaaaa
b
-barrel-like pore X-ray structure of
3
the magnitude of the JNHeH coupling constant values and the
)
derivative31 and self-
a
chemical shift perturbation of the
a
-protons of the
a
-amino acid
assembling peptide nano-tubes with remarkable material and bi-
residues of peptides 1e4, and 8e11 (Fig. 7), as well as the dispersion
of the amide signals (Table S1) it is concluded that they all adopt
distinct, but generally similar, cyclic hairpin-like structures as
ological properties.11,79e81 Currently, we are preparing a series of
cyclo-(ab3ab2a
) derivatives of 11 to explore the possibility of in-
2
ducing their self-assembly under chemically controlled conditions.

M. van der Knaap et al. / Tetrahedron 68 (2012) 2391e2400
2397
3
Fig. 7. JNHeHa coupling constant values of the (S)-Orn, (R)-Orn and (R)-Phe residues of peptides 1e4 and 8e10 (left), and the chemical shift perturbation of the
amino acid residues of GS (1e4 and 8e10) (right).
a-protons of the a-
Table 3
A: H2O, B: MeCN, and C: 1.0% aq TFA. HPLC purifications were
performed with Gilson GX-281 automated HPLC system,
equipped with a preparative Gemini C18 column (150ꢃ21.20 mm,
). The applied buffers were: A: 0.2% aq TFA, B: MeCN. The
analysis of coupling constants, temperature coefficients, and
chemical shift perturbations were performed with GraphPad
Prism version 5.01 for Windows, GraphPad Software, San Diego
California USA, www.graphpad.com.
Antibacterial activity of the cyclic mixed
a
/
b
-peptides 1e11 in
m
g/mL (MIC)
10
32 >64 >64 >64 >64 >64 32 >64 >64
a
Strain
GS
1
2
3
4
5
6
8
11
5
m
S. aureus 7323
S. aureus 7388
CNS 5277
CNS 5115
CNS 7368
8
8
4
32 >64 >64
16 >64 >64
32 >64 >64
64 >64 >64
16 >64 >64
32 >64 64
32 >64 >64
32
64
64
64
64
64
64
16 >64
8
8
8
>64 >64
>64 >64
>64 >64
8
8
32
E. faecalis 1131
16 >64 >64 >64
64 >64
E. coli ATCC 25922 32 >64 >64 >64 >64 >64 >64
P. aeruginosa AK1 16 >64 >64 >64 >64 >64 >64 >64 >64 >64
64 >64 >64
4.2. General peptide synthesis
P. aeruginosa
ATCC19582
S. mitis BMS
S. mitis ATCC
33399
8
>64 >64 >64 >64 >64 >64 >64 >64 >64
Loading of the HMPB-MBHA-resin: The HMPB-MBHA-resin
(theoretical loading¼1.2 mmol/g, 2 mmol, 1.67 g) was suspended
in 1,2-dichloroethane (10 mL) and concentrated thrice. Then a so-
lution of the first amino acid (5 equiv, 10 mmol), DIC (5 equiv,
8
4
>64 >64 >64
>64 >64 >64
16 >64 >64
16 64 >64
32 >64 >64
64 >64 >64
10 mmol, 1.54 mL), and DMAP (0.01 equiv, 20 mmol, 3 mg) in dry
4. Experimental
4.1. General
DCM/DMF (50 mL,10:1 v/v) was added. The mixture was shaken for
3 h and then drained, washed subsequently with DCM, NMP, DCM,
and Et2O. The resin was dried before determination of the loading.
The loading procedure was repeated when the loading of the resin
was found to be too low.
(S)-Fmoceb b
3hLeueOH and (R)-Fmoce 3hLeueOH were ob-
Stepwise elongation: FmoceDPheeHMPB-MBHA-resin (loading
tained from Senn Chemicals AG. Solvents and chemicals were
used as received from their supplier. Solvents were stored over
of the resin was 0.50 mmol/g, 100
nine cycles of Fmoc solid-phase synthesis with the appropriate
commercial amino acid building blocks, or Fmoce
2hValeOH. The
mmol, 200 mg) was submitted to
ꢁ
4 A molecular sieves. Solvents for column chromatography and
extractions were of technical grade and distilled prior to use. THF
was distilled over LiAlH4, CH2Cl2 over CaH2 and MeOH over
NaBH4 prior to use. All reactions were performed at room tem-
perature under inert atmosphere. Flash chromatography was
performed on Screening Devices silica gel 60 (0.04e0.063 mm).
TLC analysis was conducted on DC-alufolien (Merck, Kieselgel 60,
F254) with detection by UV-absorption at 254 nm or by spraying
with a solution of ninhydrin (3 g/L) in EtOH/AcOH (20:1 v/v), or
KMnO4 solution (20 g in 1% aq K2CO3), followed by charring at
ꢂ150 ꢀC. 1H and 13C NMR spectra were recorded with a Bruker
DMX-600 spectrometer (600/150 MHz) or with a Bruker AV-
b
side chain of ornithine is protected with a Boc-group. Fmoc removal
was effected by treatment with 20% piperidine in NMP for
2ꢃ10 min. The resin was subsequently washed with NMP, DCM,
MeOH, and finally NMP. FmoceAAeOH (2.5 equiv, 250 mmol), HCTU
(2.5 equiv, 250
mmol, 103 mg) in NMP was pre-activated for 1 min
after the addition of DiPEA (3 equiv, 300
m
mol, 53 L) and then
m
added to the resin. The suspension was shaken for 1.5 h. The resin
was washed with NMP, DCM, MeOH, and NMP.
Cleavage from the resin: After the final Fmoc deprotection the
resin was washed with NMP and DCM and treated with 5 mL 1% TFA
in DCM (6ꢃ10 min). The filtrates were collected, diluted with tol-
uene (15 mL), and concentrated under reduced pressure. The resi-
due was coevaporated with toluene (2ꢃ50 mL).
400liq spectrometer (400/100 MHz). Chemical shifts
in parts per million relative to TMS (0 ppm) or CD3OH (3.31 ppm)
as internal standard. High resolution mass spectra were recorded
d are given
by direct injection (2 mL of a 2 mM solution in water/acetonitrile;
Cyclization: In DMF (80 mL) were dissolved PyBOP (5 equiv,
50:50 v/v and 0.1% formic acid) on a mass spectrometer (Thermo
Finnigan LTQ Orbitrap) equipped with an electro spray ion source
in positive mode (source voltage 3.5 kV, sheath gas flow 10,
capillary temperature 250 ꢀC) with resolution R¼60,000 at m/z
400 (mass range m/z¼150e2000) and dioctylphthalate (m/
z¼391.28428) as a ‘lock mass’. The high resolution mass spec-
trometer was calibrated prior to measurements with a calibration
mixture (Thermo Finnigan). LC/MS analyses were performed on
an LCQ Adventage Max (Thermo Finnigan) equipped with
a Gemini C18 column (Phenomenex). The applied buffers were
500
m
mol, 260 mg), HOBt (5 equiv, 500
mmol, 77 mg), and DiPEA
(15 equiv, 1.5 mmol, 262
mL). The linear decapeptide was dissolved
in DMF (5 mL) and added dropwise over 1 h to the reaction mix-
ture. After addition the mixture was stirred for 16 h. The reaction
mixture was concentrated in vacuo and the crude mixture was
subjected to LH-20 size exclusion chromatography.
Boc deprotection: The peptide was dissolved in DCM (2 mL) and
TFA (2 mL) was added. The mixture was stirred for 2 h, concen-
trated, and coevaporated with toluene (2ꢃ10 mL). The obtained
crude product was applied to preparative HPLC purification. Using

2398
M. van der Knaap et al. / Tetrahedron 68 (2012) 2391e2400
gradients of aqueous TFA and acetonitrile the cyclic peptides 1e13
were obtained in the yield range 20e45%.
173.0, 172.7, 172.5, 172.5, 137.4, 130.3, 129.6, 128.2, 74.8, 68.8, 64.0,
61.6, 58.4, 30.2, 25.9, 23.7, 22.6; LC/MS: tR¼6.61 min (10/90%
MeCN, 15 min run); ESI-MS: m/z 1197.40 [MþH]þ; HRMS: calcu-
lated for [C64H101N12O10]þ: m/z 1197.77581; found: m/z 1197.77691.
4.2.1. cyclo-(Pro-
CD3OH)
b b
Ala1-Orn- Ala2-(R)-Phe)2 (1). 1H NMR (600 MHz,
d
8.47 (NH Phe, d, J¼3.7 Hz, 2H), 8.37 (NH
b
Ala2, t, J¼6.0 Hz,
2H), 8.23 (NH Orn, d, J¼8.2 Hz, 2H), 7.85 (H 3 Orn, 4H), 7.83 (NH
4.2.5. cyclo-(Pro-(S)-
b
2hVal-Orn-(S)-
b
3hLeu-(R)-Phe)
(5). LC/MS:
b
Ala1, dd, J¼5.7, 6.4 Hz, 2H), 7.34e7.26 (Ar. Phe, 10H), 4.66 (Ha Orn,
tR¼6.10 min (10/90% MeCN, 15 min run); ESI-MS: m/z 1197.67
[MþH]þ; HRMS: calculated for [C64H101N12O10]þ: m/z 1197.77581;
found: m/z 1197.77705.
2H), 4.40 (Ha Phe, 2H), 4.31 (Ha Pro, 2H), 3.67 (Hd Pro, 2H), 3.62 (Hb
b
Ala1, 2H), 3.51 (Hb
b
Ala2, 2H), 3.31 (Hb
b
Ala1, 2H), 3.19 (Hb
b
Ala2,
2H), 3.00 (Hb Phe, 2H), 2.95 (Hd Orn, 4H), 2.94 (Hb Phe, 2H), 2.51 (Ha
b
Ala2, 2H), 2.47 (Hd Pro, 2H), 2.41 (Ha
b
Ala1, 4H), 2.35 (Ha Ala2, 2H),
b
4.2.6. cyclo-(Pro-(R)-
(600 MHz, CD3OH)
b b
2hVal-Orn-(R)- 3hLeu-(R)-Phe)2 (6). 1H NMR
2.00 (Hg Orn, 2H), 1.97 (Hb Pro, 2H), 1.87 (Hb Orn, 2H), 1.76 (Hg Pro,
d
8.78 (NH Phe, d, J¼2.5 Hz, 2H), 8.58 (NH Orn, d,
2H), 1.66 (Hb Orn, 2H), 1.66 (Hb Pro, 2H), 1.51 (Hg Pro, 2H); 13C
J¼9.3 Hz, 2H), 8.26 (NH
b
3hLeu, d, J¼9.3 Hz, 2H), 7.87 (H 3 Orn, 4H),
(150 MHz, CD3OH)
d
174.9, 173.8, 173.9, 173.5, 173.2, 137.0, 130.5,
7.45 (NH
b
2hVal, d, J¼10.0 Hz, 2H), 7.32e7.24 (Ar. Phe, 10H), 4.60
129.6, 128.4, 61.7, 55.7, 53.6, 49.9, 47.9, 40.4, 37.9, 37.2, 37.1, 36.6,
35.0, 30.1, 24.9, 24.5; LC/MS: tR¼4.76 min (10/90% MeCN, 15 min
run); ESI-MS: m/z 1001.67 [MþH]þ; HRMS: calculated for
[C50H73N12O10]þ: m/z 1001.55671; found: m/z 1001.55734.
(Ha Orn, 2H), 4.40 (Ha Phe, 2H), 4.44 (Hb 2hVal, 2H), 4.34 (Hb
b
b
3hLeu, 2H), 4.30 (Ha Pro, 2H), 3.74 (Hd Pro, 2H), 3.05 (Hb Phe, 2H),
3.02 (Hg Orn, 2H), 2.92 (Hg Orn, 2H), 2.94 (Hb Phe, 2H), 2.86 (Ha
2hVal, 2H), 2.77 (Ha 3hLeu, 2H), 02.57 (Ha 3hLeu, 2H), 2.54 (Hd
Pro, 2H), 2.05 (Hb Pro, 4H), 1.87 (Hb 2hVal, 2H), 1.77 (Hg Orn, 4H),
1.74 (Hg Pro, 2H), 1.68 (Hd 3hLeu, 2H), 1.67 (Hb Orn, 4H), 1.64 (Hg
3hLeu, 2H), 1.58 (Hg Pro, 2H), 1.22 (Hg0 3hLeu, 2H), 0.94 (H 3 b3hLeu,
3H), 0.85 (H 3 b3hLeu, 3H), 0.83 (Hg 2hVal, 6H); 13C (150 MHz,
CD3OH) 174.4, 173.4, 173.3, 173.2, 172.6, 136.9, 130.4, 129.6, 128.3,
b
b
b
b
4.2.2. cyclo-(Pro-(S)-
(600 MHz, CD3OH)
b
3hLeu-Orn-
b
Ala-(R)-Phe)2
(2). 1H
NMR
b
d
8.63 (NH Phe, d, J¼4.1 Hz, 2H), 8.46 (NH Orn, d,
b
b
J¼8.3 Hz, 2H), 8.17 (NH
b
3hLeu, d, J¼9.0 Hz, 2H), 7.74 (NH
b
Ala, t,
b
J¼5.1 Hz, 2H), 7.31e7.23 (Ar. Phe, 10H), 4.48 (Ha Phe, 2H), 4.42 (Ha,
d
Orn, 2H), 4.32 (Ha Pro, 2H), 4.29 (Hb
b
3hLeu, 2H), 3.56 (Hd Pro, 2H),
61.7, 55.7, 52.6, 51.3, 49.9, 47.9, 46.4, 42.9, 42.3, 40.3, 39.4, 37.5, 31.6,
30.3, 27.6, 26.0, 25.0, 24.4, 23.8, 22.9, 21.4; LC/MS: tR¼6.32 min
(10/90% MeCN, 15 min run); ESI-MS: m/z 1197.67 [MþH]þ; HRMS:
calculated for [C64H101N12O10]þ: m/z 1197.77581; found: m/z
1197.77698.
3.51 (Ha
Orn, 4H), 2.62 (Ha
2.28 (Ha 3hLeu, 2H), 1.94 (Hb Pro, 4H), 1.84 (Hg Orn, 4H), 1.70 (Hg
Pro, 2H), 1.69 (Hb Orn, 4H), 1.55 (Hd 3hLeu, 2H), 1.52 (Hg Pro, 2H),
1.44 (Hg 3hLeu, 4H), 0.91 (H 3 b3hLeu, 6H), 0.82 (H 3 b3hLeu, 6H); 13
(150 MHz, CD3OH) 173.8, 173.5, 173.1, 172.9, 172.2, 137.3, 130.4,
b
Ala, 2H), 3.30 (Ha
b
Ala, 2H), 2.99 (Hb Phe, 4H), 2.95 (Hd
b
Ala, 4H), 2.56 (Hd Pro, 2H), 2.42 (Ha
b
3hLeu, 2H),
b
b
b
C
d
4.2.7. cyclo-(Pro-(S)-b b
2hVal-Orn-(S)- 3hLeu-(R)-Phe-Pro-(R)-
129.6, 128.2, 61.6, 55.5, 53.6, 47.9, 47.8, 46.7, 44.1, 42.7, 40.4, 37.9,
37.6, 36.2, 31.1, 30.3, 25.9, 24.9, 24.6, 23.6; LC/MS: tR¼5.55 min
(10/90% MeCN, 15 min run); ESI-MS: m/z 1113.67 [MþH]þ; HRMS:
calculated for [C58H89N12O10]þ: m/z 1113.68191; found: m/z
1113.68273.
b
2hVal-Orn-(S)-
b
3hLeu-(R)-Phe) (7). LC/MS: tR¼6.28 min (10/90%
MeCN, 15 min run); ESI-MS: m/z 1197.60 [MþH]þ; HRMS: calcu-
lated for [C64H101N12O10]þ: m/z 1197.77581; found: m/z 1197.77714.
4.2.8. cyclo-(Pro-
b
Ala1-(R)-Orn-
b
Ala2-(R)-Phe)2
(8). 1H
NMR
(600 MHz, CD3OH)
J¼7.6 Hz, 2H), 8.24 (NH
d
8.64 (NH Phe, d, J¼3.8 Hz, 2H), 8.30 (NH Orn, d,
4.2.3. cyclo-(Pro-(R)-
b
2hVal-Orn-
b
Ala-(R)-Phe)2
(3). 1H
NMR
Ala,
b
Ala2, t, J¼5.4 Hz, 2H), 7.90 (H 3 Orn, 4H),
(600 MHz, CD3OH)
d
8.64 (NH Phe, d, J¼3.8 Hz, 2H), 8.35 (NH
b
7.69 (NH
b
Ala1, t, J¼5.1 Hz, 2H), 7.31e7.17 (Ar Phe, 10H), 4.50 (Ha
t, J¼5.4 Hz, 2H), 8.25 (NH Orn, d, J¼8.3 Hz, 2H), 7.88 (H 3 Orn, 4H),
Phe, 2H), 4.39 (Ha Orn, 2H), 4.26 (Ha Pro, 2H), 3.61 (Hd Pro, 2H), 3.59
7.50 (NH
b
2hVal, t, J¼5.4 Hz, 2H), 7.32e7.19 (Ar. Phe, 10H), 4.50 (Ha
(Hb
Ala2, 2H), 3.03 (Hd Orn, 2H), 3.02 (Hb Phe, 2H), 2.96 (Hd Orn, 2H),
2.96 (Hb Phe, 2H), 2.56 (Hd Pro, 2H), 2.53 (Ha Ala1, 4H), 2.51 (Ha
b b b
Ala2, 2H), 3.46 (Hb Ala1, 2H), 3.38 (Hb Ala1, 2H), 3.35 (Hb
Phe, 2H), 4.50 (Ha Orn, 2H), 4.30 (Ha Pro, 2H), 3.85 (Hb
b
Ala, 2H),
b
3.60 (Hd Pro, 2H), 3.54 (Hb
b
2hVal, 2H), 3.40 (Hb
b
Ala, 2H), 3.26 (Hb
b
b
2hVal, 2H), 3.04 (Hb Phe, 2H), 3.01 (Hd Orn 4H), 2.93 (Hb Phe, 2H),
b b
Ala2, 2H), 2.43 (Ha Ala2, 2H), 1.89 (Hb Pro, 2H), 1.85 (Hb Orn, 2H),
2.57 (Ha
b
Ala, 4H), 2.57 (Hd Pro, 2H), 2.50 (Ha
b
2hVal, 2H), 1.98 (Hg
1.73 (Hb Pro, 2H), 1.72 (Hg Orn, 2H), 1.71 (Hb Orn, 2H), 1.68 (Hg Pro,
0
Pro, 4H), 1.84 (Hb
b
2hVal, 2H), 1.73 (Hg Orn, 04H), 1.66 (Hb Orn, 4H),
2H), 1.65 (Hg Orn, 2H), 1.51 (Hg Pro, 2H); 13C NMR (150 MHz,
0
1.66 (Hb Pro, 2H), 1.52 (Hb Pro, 2H), 0.93 (Hg
b
2hVal, 6H), 0.89 (Hg
CD3OH) d 173.8, 173.7, 173.4, 173.4, 173.1, 137.1, 130.3, 129.5, 128.3,
b
2hVal, 6H); 13C (150 MHz, CD3OH)
d
175.5, 173.8, 173.7, 173.4, 173.1,
61.8, 55.5, 53.8, 48.0, 40.8, 40.4, 37.9, 37.3, 36.8, 36.2, 35.5, 30.4,
24.9, 24.6; LC/MS: tR¼4.84 min (10/90 min MeCN, 15 min run);
ESI-MS: m/z 1001.60 [MþH]þ; HRMS: calculated for
[C50H72N12O10]þ: m/z 1001.55671; found: m/z 1001.55734.
137.1, 130.4, 129.6, 128.3, 61.7, 55.5, 53.3, 52.8, 48.6, 47.9, 41.0, 40.5,
38.0, 36.9, 35.8, 30.8, 30.1, 29.6, 24.6, 21.1, 20.1; LC/MS: tR¼5.28 min
(10/90% MeCN, 15 min run); ESI-MS: m/z 1085.40 [MþH]þ;
HRMS: calculated for [C56H85N12O10]þ: m/z 1085.65061; found: m/z
1085.65151.
4.2.9. cyclo-(Pro-(S)-b b
3hLeu-(R)-Orn- Ala-(R)-Phe)2 (9). 1H NMR
(600 MHz, CD3OH)
d
8.64 (NH Phe, d, J¼2.9 Hz, 2H), 8.44 (NH
bAla,
4.2.4. cyclo-(Pro-(R)-
(600 MHz, CD3OH)
J¼7.2 Hz, 2H), 8.11 (NH
7.65 (NH
b
2hVal-Orn-(S)-
b
3hLeu-(R)-Phe)2 (4). 1H NMR
t, J¼2.9 Hz, 2H), 8.42 (NH Orn, J¼8.6 Hz, 2H), 7.92 (H 3 Orn, 4H), 7.37
d
8.51 (NH Phe, d, J¼3.5 Hz, 2H), 8.42 (NH Orn, d,
(NH
b
3hLeu, d, J¼9.5 Hz, 2H), 7.30e7.23 (Ar Phe, 10H), 4.52 (Ha Orn,
b
3hLeu, d, J¼8.9 Hz, 2H), 7.89 (H 3 Orn, 4H),
2H), 4.44 (Ha Phe, 2H), 4.43 (Hb 3hLeu, 2H), 4.30 (Ha Pro, 2H), 3.79
b
b
2hVal, br s, 2H), 7.31e7.23 (Ar. Phe, 10H), 4.44 (Ha Phe,
(Hb
2H), 3.02 (Hd Orn, 4H), 2.92 (Hb Phe, 2H), 2.56 (Ha
(Ha Ala, 2H), 2.45 (Hd Pro, 2H), 2.43 (Ha 3hLeu, 4H), 1.94 (Hb Pro,
2H), 1.86 (Hb Orn, 2H), 1.73 (Hb Orn, 2H), 1.71 (Hg Orn, 2H), 1.68 (Hb
Pro, 2H), 1.67 (Hg Orn, 2H), 1.62 (Hg Pro, 2H), 1.60 (Hg 3hLeu, 2H),
1.53 (Hg Pro, 2H), 1.52 (Hd 3hLeu, 2H), 1.19 (Hg 3hLeu, 2H), 0.87 (H 3
3hLeu, 6H), 0.83 (H 3 b3hLeu, 6H); 13C NMR (150 MHz, CD3OH)
173.7, 173.3, 173.1, 173.0, 172.7, 137.0, 130.4, 129.6, 128.3, 61.9, 55.7,
b
Ala, 2H), 3.60 (Hd Pro, 2H), 3.23 (Hb
b
Ala, 2H), 3.05 (Hb Phe,
2H), 4.41 (Ha Orn, 2H), 4.31 (Ha Pro, 2H), 4.29 (Hb
b
3hLeu, 2H), 3.65
b
Ala, 2H), 2.46
(Hb 2hVal, 2H), 3.52 (Hd Pro, 2H), 3.15 (Hb 2hVal, 2H), 3.04 (Hd Orn,
b
b
b
b
2H), 2.98 (Hb Phe, 4H), 2.98 (Hd Orn, 2H), 2.74 (Hd Pro, 2H), 2.65 (Ha
b
2hVal, 2H), 2.44 (Ha
b
3hLeu, 2H), 1.93 (Ha
b
3hLeu, 2H), 1.91 (Hb Pro,
b
’
2H), 1.85 (Hb
b
2hVal, 2H), 1.81 (Hb Orn, 2H), 1.73 (Hb Pro, 2H), 1.69
b
b
(Hg
b
b
3hLeu, 2H), 1.69 (Hg Pro, 2H), 1.68 (Hg Orn, 4H), 1.60 (Hg
b
d
3hLeu, 2H), 1.59 (Hg Pro, 2H), 1.47 (Hb Orn, 2H), 1.32 (Hd0 b3hLeu,
0
2H), 0.99 (Hg
b
2hVal, 6H), 0.99 (H 3 b3hLeu, 6H), 0.93 (Hg
b
d
2hVal,
173.3,
53.0, 47.8, 46.2, 44.3, 43.0, 40.5, 37.7, 37.0, 35.4, 31.1, 31.1, 30.4, 26.2,
24.7, 24.5, 23.8; LC/MS: tR¼6.82 min (10/90 min MeCN, 15 min
6H), 0.93 (H 3 b3hLeu, 6H); 13C NMR (150 MHz, CD3OH)

M. van der Knaap et al. / Tetrahedron 68 (2012) 2391e2400
2399
run); ESI-MS: m/z 1113.47 [MþH]þ; HRMS: calculated for
[C58H89N12O10]þ: m/z 1113.68191; found: m/z 1113.68281.
performed at beamline BM30A and high and low resolution in-
tensity data were collected at beamline ID14-1 at the ESRF (Gre-
noble, France) by performing one 4 scan (1ꢀ oscillation per image).
2hVal-(R)-Phe)2 (10). 1H NMR
Images were collected with DNA software84 and processed to 1.10 A
ꢁ
4.2.10. cyclo-(Pro-
b
Ala-(R)-Orn-(R)-
b
(600 MHz, CD3OH)
J¼7.7 Hz, 2H), 7.92 (NH
d
8.49 (NH Phe, d, J¼5.3 Hz, 2H), 8.30 (NH Orn, d,
with MOSFLM.85 Low and high resolution datasets were scaled and
merged with POINTLESS and SCALA.86
b
Ala, t, J¼5.2 Hz, 2H), 7.86 (H 3 Orn, 4H), 7.81
(NH
b
2hVal, t, J¼5.4 Hz, 2H), 7.33e7.25 (Ar. Phe, 10H), 4.72 (Ha Phe,
2H), 4.36 (Ha Orn, 2H), 4.25 (Ha Pro, 2H), 3.73 (Hd Pro, 2H), 3.43 (Hb
4.3.3. Structure determination of compounds 6 and 11. Initial phases
for solving the structure of compound 11 were obtained with
ACORN,87 using a mixed molecular replacement/ab initio pro-
b
2hVal, 2H), 3.42 (Hb Ala, 2H), 3.33 (Hb 2hVal, 2H), 3.33 (Hb
b b bAla,
2H), 2.98 (Hb Phe, 4H), 2.98 (Hd Orn, 2H), 2.94 (Hd Orn, 2H), 2.78 (Hd
Pro, 2H), 2.61 (Ha
1.85 (Ha
b
Ala, 2H), 2.46 (Ha
b
Ala, 2H), 1.86 (Hb Pro, 2H),
cedure with the Leu-DPhe-Pro-Val -turn of native GS38 used as
b
0
b
2hVal, 2H), 1.85 (Hb Orn, 2H), 1.80 (Hb Pro, 2H), 1.76 (Hb
a starting search fragment. ACORN produced suitable electronic
density maps and by means of COOT88 the complete structure of
compound 11 could be traced. The structure of compound 6 was
solved by direct methods using the program SHELXD.89 Both
structures were refined with no intensity cutoff using the full-
matrix least-squares methods on F2 refinement implemented in
SHELXL89 included in the WinGX package.90 Throughout the re-
finement, bond-length, bond-angle, and planarity restraints were
imposed. All non-H atoms were refined anisotropically with suit-
able rigid-bond and similarity restraints. All hydrogen positions
were calculated and refined using a riding atom model. Both
structures have one crystallographically independent molecule per
asymmetric unit with several disordered side chains. Selected
crystallographic data is reported in Tables S4 and S5. Final figures
were created using PyMOL (Delano Scientific, Palo Alto CA, USA).
CCDC 773292 and CCDC 805623 contain the supplementary crys-
tallographic data for structures 6 and 11, respectively. These data
can be obtained free of charge from The Cambridge Crystallo-
graphic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
b
2hVal, 2H), 1.75 (Hg Pro, 2H), 1.72 (Hb Orn, 2H), 1.65 (Hg Orn, 4H),
0
0
1.59 (Hg Pro, 2H), 0.94 (Hg
NMR (150 MHz, CD3OH)
b
2hVal, 6H), 0.85 (Hg
b
2hVal, 6H); 13C
d
176.5, 174.1, 173.8, 173.5, 172.6, 137.3,
130.4, 129.5, 128.2, 61.6, 54.6, 54.0, 53.4, 48.2, 40.6, 40.5, 38.5, 37.3,
36.1, 30.5, 30.3, 30.0, 24.9, 24.9, 21.0, 20.3; LC/MS: tR¼5.84 min
(10/90 min MeCN, 15 min run); ESI-MS: m/z 1085.33 [MþH]þ;
HRMS: calculated for [C56H85N12O10]þ: m/z 1085.65061; found: m/z
1085.65166.
4.2.11. cyclo-(Pro-(S)-
b
3hLeu-(R)-Orn-(R)-
b
2hVal-(R)-Phe)2 (11). 1H
NMR (600 MHz, CD3OH)
d
8.54 (NH Orn, d, J¼7.7 Hz, 2H), 8.47 (NH
Phe, s, 2H), 8.12 (NH b
2hVal, s, 2H), 7.94 (H 3 Orn, 4H), 7.57 (NH
b
3hLeu, d, J¼8.8 Hz, 2H), 7.32e7.24 (Ar. Phe, 10H), 4.50 (Ha Orn, 2H),
4.41 (Ha Phe, 2H), 4.34 (Ha Pro, 2H), 4.30 (Hb 3hLeu, 2H), 3.76 (Hb
b
b
2hVal, 2H), 3.58 (Hd Pro, 2H), 3.15 (Hb
2H), 3.01 (Hd Orn, 4H), 2.91 (Hb Phe, 2H), 2.51 (Ha
(Hd Pro, 2H), 2.40 (Ha 2hVal, 2H), 2.34 (Ha 3hLeu, 2H), 2.00 (Hb
Pro, 2H), 1.94 (Hb Orn, 2H), 1.87 (Hb’ b2hVal, 2H), 1.75 (Hb Orn, 2H),
b
2hVal, 2H), 3.08 (Hb Phe,
b
3hLeu, 2H), 2.50
b
b
1.75 (Hg Orn, 2H), 1.73 (Hg 3hLeu, 2H), 1.68 (Hg Orn, 2H), 1.61 (Hg
b
Pro, 2H), 1.60 (Hb Pro, 2H), 1.57 (Hd
b
3hLeu, 2H), 1.530 (Hg Pro, 2H),
2hVal, 6H),
Acknowledgements
0
1.22 (Hg
b
3hLeu, 2H), 1.00 (Hg
b
2hVal, 6H), 0.96 (Hg
b
0.88 (H 3 b3hLeu, 6H), 0.82 (H 3 b3hLeu, 6H); 13C NMR (150 MHz,
CD3OH) 176.3, 173.3, 173.0, 172.9, 172.8, 136.9, 130.4, 129.6, 128.3,
We thank STW for financing this research, Spanish Ministry of
d
ꢀ
Science and Innovation for grant BFU2008-01588 and for a Jose
61.7, 55.6, 53.3, 52.6, 47.9, 46.8, 43.7, 42.9, 40.7, 40.6, 38.0, 31.3, 30.7,
30.3, 26.1, 24.6, 24.4, 23.8, 21.4, 20.5, 20.3; LC/MS: tR¼7.83 min
(10/90 min MeCN, 15 min run); ESI-MS: m/z 1097.53 [MþH]þ;
HRMS: calculated for [C64H101N12O10]þ: m/z 1197.77581; found: m/
z 1197.77631.
Castillejo fellowship to J.M.O. and the Xunta de Galicia for an
~
Angeles Alvarino fellowship to J.M.O. We thank the ESRF for the
provision of beam time on beamlines ID14-1 and BM30A. We thank
Kees Erkelens, Fons Lefeber, and Hans van den Elst for their tech-
nical assistance with the analysis and purification of the peptides.
4.3. Crystallographic data
Supplementary data
4.3.1. Crystallization and crystallographic data collection of com-
pound 6. Colorless plate-shaped crystals were obtained after slow
Supplementary data related to this article can be found online at
doi:10.1016/j.tet.2012.01.015.
evaporation of 2
mL droplets of 16 mg/mL peptide in 50% solution of
MeOH in H2O plus 2
mL of 1.0 M NaOH in MeOH under paraffin oil in
a Terasaki plate. A plate-shaped crystal was mounted in air and
then rapidly placed under a 100 K dry nitrogen stream. Intensity
data were collected using a BrukereNonius FR591 Kappa CCD2000
References and notes
1. Seebach, D.; Beck, A. K.; Bierbaum, D. J. Chem. Biodivers. 2004, 1, 1111e1239.
2. Cheng, R. P.; Gellman, S. H.; DeGrado, W. F. Chem. Rev. 2001, 101, 3219e3232.
3. Hill, D. J.; Mio, M. J.; Prince, R. B.; Hughes, T. S.; Moore, J. S. Chem. Rev. 2001, 101,
3893e4011.
4. Gademann, K.; Hintermann, T.; Schreiber, J. V. Curr. Med. Chem. 1999, 6,
905e925.
5. DeGrado, W. F.; Schneider, J. P.; Hamuro, Y. J. Pept. Res. 1999, 54, 206e217.
6. Gellman, S. H. Acc. Chem. Res. 1998, 31, 173e180.
7. Seebach, D.; Matthews, J. L. Chem. Commun. 1997, 2015e2022.
8. Appella, D. H.; Christianson, L. A.; Karle, I. L.; Powell, D. R.; Gellman, S. H. J. Am.
Chem. Soc. 1996, 118, 13071e13072.
X-ray diffractometer with Cu K
optics by performing one 4 and five
image) at different and 2 angle settings. The exposure time used
a
radiation and multilayer confocal
u
scans (2ꢀ oscillations per
k
q
was 300 s per degree at generator settings (45 kV, 90 mA). Raw
images were integrated using DENZO.83 The resulting intensities
were scaled, corrected for absorption effects and the crystal unit-
cell parameters calculated by global refinement using
SCALEPACK.83
9. Seebach, D.; Overhand, M.; Kuhnle, F. N. M.; Martinoni, B.; Oberer, L.; Hommel,
U.; Widmer, H. Helv. Chim. Acta 1996, 79, 913e941.
10. Vasudev, P. G.; Chatterjee, S.; Shamala, N.; Balaram, P. Chem. Rev. 2011, 111,
657e687.
11. Brea, R. J.; Reiriz, C.; Granja, J. R. Chem. Soc. Rev. 2010, 39, 1448e1456.
12. Vasudev, P. G.; Chatterjee, S.; Shamala, N.; Balaram, P. Acc. Chem. Res. 2009, 42,
1628e1639.
4.3.2. Crystallization and crystallographic data collection of com-
pound 11. Colorless crystals were obtained by slow evaporation of
2
mL droplets of 17 mg/mL peptide in 50% (vol/vol) solution of
MeOH in H2O plus 2 L of 20% (vol/vol) of glycerol and 0.1 M Na-
m
13. Horne, W. S.; Gellman, S. H. Acc. Chem. Res. 2008, 41, 1399e1408.
14. Chatterjee, S.; Roy, R. S.; Balaram, P. J. R. Soc. Interface 2007, 4, 587e606.
cacodylate in MeOH under paraffin oil in a Terasaki plate. A
prism-shaped crystal was mounted in a cryoloop and directly flash-
frozen in liquid nitrogen. Preliminary diffraction tests were
€
15. Seebach, D.; Hook, D. F.; Glattli, A. Biopolymers 2006, 84, 23e37.
16. Lengyel, G. A.; Frank, R. C.; Horne, W. S. J. Am. Chem. Soc. 2011, 133, 4246e4249.

2400
M. van der Knaap et al. / Tetrahedron 68 (2012) 2391e2400
17. Chatterjee, S.; Vasudev, P. G.; Raghothama, S.; Ramakrishnan, C.; Shamala, N.;
Balaram, P. J. Am. Chem. Soc. 2009, 131, 5956e5965.
45. Arvidsson, P. I.; Ryder, N. S.; Weiss, H. M.; Gross, G.; Kretz, O.; Woessner, R.;
Seebach, D. ChemBioChem 2003, 4, 1345e1347.
18. Seebach, D.; Jaun, B.; Sebesta, R.; Mathad, R. I.; Flogel, O.; Limbach, M.; Sellner,
46. Ahmed, S.; Kaur, K. Chem. Biol. Drug Des. 2009, 73, 545e552.
47. Schmitt, M. A.; Weisblum, B.; Gellman, S. H. J. Am. Chem. Soc. 2004, 126,
6848e6849.
48. Arvidsson, P. I.; Ryder, N. S.; Weiss, H. M.; Hook, D. F.; Escalante, J.; Seebach, D.
Chem. Biodivers. 2005, 2, 401e420.
H.; Cottens, S. Helv. Chim. Acta 2006, 89, 1801e1825.
19. Rai, R.; Vasudev, P. G.; Ananda, K.; Raghothama, S.; Shamala, N.; Karle, I. L.;
Balaram, P. Chem.dEur. J. 2007, 13, 5917e5926.
20. Roy, R. S.; Gopi, H. N.; Raghothama, S.; Karle, I. L.; Balaram, P. Chem.dEur. J.
2006, 12, 3295e3302.
49. Seebach, D.; Beck, A. K.; Capone, S.; Deniau, G.; Groselj, U.; Zass, E. Synthesis
21. Roy, R. S.; Gopi, H. N.; Raghothama, S.; Gilardi, R. D.; Karle, I. L.; Balaram, P.
Biopolymers 2005, 80, 787e799.
22. Gopi, H. N.; Roy, R. S.; Raghothama, S. R.; Karle, I. L.; Balaram, P. Helv. Chim. Acta
2002, 85, 3313e3330.
23. Karle, I. L.; Gopi, H. N.; Balaram, P. Proc. Natl. Acad. Sci. U.S.A. 2002, 99,
5160e5164.
2009, 1e32.
50. Lee, H.-S.; Park, J.-S.; Kim, B. M.; Gellman, S. H. J. Org. Chem. 2003, 68,
1575e1578.
51. Hintermann, T.; Seebach, D. Synlett 1997, 437e438.
€
52. Riniker, B.; Florsheimer, A.; Fretz, H.; Sieber, P.; Kamber, B. Tetrahedron 1993, 49,
9307e9320.
24. Karle, I. L.; Gopi, H. N.; Balaram, P. Proc. Natl. Acad. Sci. U.S.A. 2001, 98,
3716e3719.
53. Roy, R. S.; Balaram, P. J. Pept. Res. 2004, 63, 279e289.
54. Bach, A. C.; Markwalder, J. A.; Ripka, W. C. Int. J. Pept. Protein Res. 1991, 38,
314e323.
55. Marchettini, N.; Donati, A.; Rossi, C. Chem. Phys. Lett. 1991, 180, 19e22.
56. Liquori, A.; De Santis, P. Int. J. Biol. Macromol. 1980, 2, 112e115.
57. Rachovsky, S.; Scheraga, H. A. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 6965e6967.
58. Hawkes, G. E.; Randall, E. W.; Hull, W. E.; Convert, O. Biopolymers 1980, 19,
1815e1826.
59. Kricheldorf, H. R. Org. Magn. Reson. 1981, 15, 162e177.
60. Rae, I. D.; Scheraga, H. A. Biochem. Biophys. Res. Commun. 1978, 81, 481e485.
61. Komoroski, R. A.; Peat, I. R.; Levy, G. C. Biochem. Biophys. Res. Commun. 1975, 65,
272e279.
62. Griffiths-Jones, S. R.; Maynard, A. J.; Searle, M. S. J. Mol. Biol. 1999, 292,
1051e1069.
25. Liskamp, R. M. J. Recl. Trav. Chim. Pays-Bas 1994, 113, 1e19.
26. Knijnenburg, A. D.; Tuin, A. W.; Spalburg, E.; De Neeling, A. J.; Mars-Groe-
nendijk, R. H.; Noort, D.; Otero, J. M.; Llamas-Saiz, A. L.; Van Raaij, M. J.; Van
der Marel, G. A.; Overkleeft, H. S.; Overhand, M. Chem.dEur. J. 2011, 17,
3995e4004.
27. Kapoerchan, V. V.; Knijnenburg, A.; Niamat, M.; Spalburg, E.; De Neeling, A. J.;
Nibbering, P.; Mars-Groenendijk, R. H.; Noort, D.; Otero, J. M.; Llamas-Saiz, A. L.;
Van Raaij, M. J.; Van der Marel, G. A.; Overkleeft, H. S.; Overhand, M. Chem.dEur. J.
2010, 16, 12174e12181.
28. Kapoerchan, V. V.; Spalburg, E.; De Neeling, A. J.; Mars-Groenendijk, R. H.;
Noort, D.; Otero, J. M.; Ferraces-Casais, P.; Llamas-Saiz, A. L.; Van Raaij, M. J.;
Van Doorn, J.; Van der Marel, G. A.; Overkleeft, H. S.; Overhand, M. Chem.dEur.
J. 2010, 16, 4259e4265.
29. Grotenbreg, G. M.; Buizert, A. E. M.; Llamas-Saiz, A. L.; Spalburg, E.; Van Hooft,
P. A. V.; De Neeling, A. J.; Noort, D.; Van Raaij, M. J.; Van der Marel, G. A.;
Overkleeft, H. S.; Overhand, M. J. Am. Chem. Soc. 2006, 128, 7559e7565.
30. Grotenbreg, G. M.; Kronemeijer, M.; Timmer, M. S. M.; El Oualid, F.; Van Well, R.
M.; Verdoes, M.; Spalburg, E.; Van Hooft, P. A. V.; De Neeling, A. J.; Noort, D.;
Van Boom, J. H.; Van der Marel, G. A.; Overkleeft, H. S.; Overhand, M. J. Org.
Chem. 2004, 69, 7851e7859.
63. Maynard, A. J.; Sharman, G. J.; Searle, M. S. J. Am. Chem. Soc. 1998, 120,
1996e2007.
64. The
dHa of the amino acid residues in GS are not significantly affected when
using methanol instead of water as solvent system. For the determination of
the DdHa Orn, the random coil value of the
65. Wuthrich, K. NMR of Proteins and Nucleic Acids; John Wiley & Sons (Wiley-In-
d
Ha of Lys is used.
€
terscience): New York, NY, 1986.
€
66. Pardi, A.; Billeter, M.; Wuthrich, K. J. Mol. Biol. 1984, 180, 741e751.
31. Grotenbreg, G. M.; Timmer, M. S. M.; Llamas-Saiz, A. L.; Verdoes, M.; Van der
Marel, G. A.; Van Raaij, M. J.; Overkleeft, H. S.; Overhand, M. J. Am. Chem. Soc.
2004, 126, 3444e3446.
32. Roy, S.; Lombart, H.-G.; Hancock, R. E. W.; Farmer, S. W.; Lubell, W. D. J. Pept. Res.
2002, 60, 198e214.
67. Karplus, M. J. Chem. Phys. 1959, 30, 11e15.
68. Karplus, M. J. Am. Chem. Soc. 1963, 85, 2870e2871.
69. Krauthauser, S.; Christianson, L. A.; Powell, D. R.; Gellman, S. H. J. Am. Chem. Soc.
1997, 119, 11719e11720.
70. Seebach, D.; Abele, S.; Gademann, K.; Jaun, B. Angew. Chem., Int. Ed. 1999, 38,
33. Gibbs, A. C.; Kondejewski, L. H.; Gronwald, W.; Nip, A. M.; Hodges, R. S.; Sykes,
1595e1597.
B. D.; Wishart, D. S. Nat. Struct. Biol. 1998, 5, 284e288.
34. Kondejewski, L. H.; Farmer, S. W.; Wishart, D. S.; Kay, C. M.; Hancock, R. E. W.;
Hodges, R. S. J. Biol. Chem. 1996, 271, 25261e25268.
35. Schwyzer, R.; Tunkyi, A.; Carrion, J. P.; Gorup, B.; Nolting, H. Helv. Chim. Acta
1964, 47, 441e464.
36. Schwyzer, R. Chimia 1958, 12, 53e68.
37. Solanas, C.; De la Torre, B. G.; Fernandez-Reyes, M.; Santiveri, C. M.; Angeles
Jimenez, M.; Rivas, L.; Jimenez, A. I.; Andreu, D.; Cativiela, C. J. Med. Chem. 2010,
53, 4119e4129.
38. Llamas-Saiz, A. L.; Grotenbreg, G. M.; Overhand, M.; van Raaij, M. J. Acta Crys-
tallogr. 2007, D63, 401e407.
71. Renaudet, O.; Garcia, J.; Boturyn, D.; Spinelli, N.; Defrancq, E.; Labbe, P.; Dumy, P.
Int. J. Nanotechnol. 2010, 7, 738e752.
72. Mutter, M.; Vuilleumier, S. Angew. Chem., Int. Ed. Engl. 1989, 28, 535e554.
73. Freire, F.; Gellman, S. H. J. Am. Chem. Soc. 2009, 131, 7970e7971.
74. Khakshoor, O.; Nowick, J. S. Curr. Opin. Chem. Biol. 2008, 12, 722e729.
75. Robinson, J. A. Acc. Chem. Res. 2008, 41, 1278e1288.
76. Moriuchi, T.; Hirao, T. Chem. Soc. Rev. 2004, 33, 294e301.
77. Kessler, H. Angew. Chem., Int. Ed. Engl. 1982, 21, 512e523.
78. Nowick, J. S.; Smith, E. M.; Pairish, M. Chem. Soc. Rev. 1996, 25, 401e415.
79. Bong, D. T.; Clark, T. D.; Granja, J. R.; Ghadiri, M. R. Angew. Chem., Int. Ed. 2001,
40, 988e1011.
39. Tishchenko, G. N.; Andrianov, V. I.; Vainstein, B. K.; Woolfson, M. M.; Dodson, E.
80. Ghadiri, M. R.; Granja, J. R.; Milligan, R. A.; McRee, D. E.; Khazanovich, N. Nature
1993, 366, 324e327.
Acta Crystallogr. 1997, D53, 151e159.
40. Hull, S. E.; Karlsson, R.; Main, P.; Woolfson, M. M.; Dodson, E. J. Nature 1978,
275, 206e207.
41. Stern, A.; Gibbons, W. A.; Craig, L. C. Proc. Natl. Acad. Sci. U.S.A. 1968, 61, 734e741.
81. Tomasic, L.; Lorenzi, G. P. Helv. Chim. Acta 1987, 70, 1012e1016.
83. Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 276, 307e326.
84. Leslie, A. G. W.; Powell, H. R.; Winter, G.; Svensson, O.; Spruce, D.; McSweeney,
S.; Love, D.; Kinder, S.; Duke, E.; Nave, C. Acta Crystallogr. 2002, D58, 1924e1928.
85. Leslie, A. G. W. Acta Crystallogr. 2006, D62, 48e57.
86. Evans, P. Acta Crystallogr. 2006, D62, 72e82.
42.
a
-Amino acids, with the exception of cysteine, have the (S)-configuration,
corresponding to the configuration according to the Fisher notation: Prelog,
V.; Helmchen, G. Angew. Chem., Int. Ed. Engl. 1982, 21, 567e583 For clarity
L
reasons, we always indicate the absolute stereochemistry of the mentioned
amino acids.
b
-
87. Jia-xing, Y.; Woolfson, M. M.; Wilson, K. S.; Dodson, E. J. Acta Crystallogr. 2005,
D61, 1465e1475.
43. Porter, E.A.;Wang,X.;Lee,H.-S.;Weisblum,B.;Gellman, S.H. Nature 2000, 404, 565.
44. Hamuro, Y.; Schneider, J. P.; DeGrado, W. F. J. Am. Chem. Soc. 1999, 121,
12200e12201.
88. Emsley, P.; Cowtan, K. Acta Crystallogr. 2004, D60, 2126e2132.
89. Sheldrick, G. M. Acta Crystallogr. 2008, D64, 112e122.
90. Farrugia, L. J. J. Appl. Crystallogr. 1999, 32, 837e838.