Synthesis and evaluation of strand and turn modified ring-extended gramicidin S derivatives

Article abstract of DOI:10.1016/j.bmc.2011.04.031

In this paper, we describe the crystal structure of previously reported ring-extended gramicidin S (GS) derivative 2 (GS14K4), containing a d-amino acid residue in one of the β-strand regions. This structure is in agreement with a previously reported mode

Full text of DOI:10.1016/j.bmc.2011.04.031

Bioorganic & Medicinal Chemistry 19 (2011) 3402–3409
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of strand and turn modified ring-extended
gramicidin S derivatives
Annemiek D. Knijnenburg a, Varsha V. Kapoerchan a, Gijsbert M. Grotenbreg a, , Emile Spalburg b,
Albert J. de Neeling b, Roos H. Mars-Groenendijk c, Daan Noort c, José M. Otero d,e, Antonio L. Llamas-Saiz f,
Mark J. van Raaij d,g, Bep Ravensbergen h, Peter H. Nibbering h, Gijs A. van der Marel a, Herman S. Overkleeft a,
Mark Overhand a,
⇑
a Leiden Institute of Chemistry, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands
b National Institute of Public Health and the Environment, Laboratory for Infectious Diseases, PO Box 1, 3720 BA Bilthoven, The Netherlands
c TNO, Prins Maurits Laboratory, PO Box 45, 2280 AA Rijswijk, The Netherlands
d Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Campus Vida, Universidad de Santiago, 15782 Santiago de Compostela, Spain
e Laboratoire des Proteines Membranaires, Institut de Biologie Structurale J. P. Ebel, 41 Rue Jules Horowitz, F-38027 Grenoble, France
f Unidad de Rayos X (RIAIDT), Laboratorio Integral de Dinámica y Estructura de Biomoléculas ‘José R. Carracido’, Edificio CACTUS, Campus Vida, Universidad de Santiago,
15782 Santiago de Compostela, Spain
g Laboratorio M-4, Departamento de Estructura de Macromoleculas, Centro Nacional de Biotecnologia, c/Darwin 3, Campus Cantoblanco, E-28049 Madrid, Spain
h Center for Infectious Diseases, Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 February 2011
Revised 5 April 2011
Accepted 13 April 2011
Available online 22 April 2011
In this paper, we describe the crystal structure of previously reported ring-extended gramicidin S (GS)
derivative 2 (GS14K4), containing a
is in agreement with a previously reported modeling study of the same molecule. The polar side chain
of the additional -amino acid residue is positioned at the same face of the molecule as the hydrophobic
side chains, and we believe that because of this compound 2 is considerably less hydrophobic than
extended GS derivatives in which the strand regions are exclusively composed of -amino acids. Using
D-amino acid residue in one of the b-strand regions. This structure
D
L
Keywords:
this backbone structure as our benchmark we prepared a small series of ring-extended GS analogues fea-
turing sugar amino acid dipeptide isosteres of varied hydrophobicity at the turn region. We show that via
this approach hydrophobicity of extended GS analogues can be tuned without affecting the secondary
structure (as observed from NMR and CD spectra). Biological evaluation reveals that hydrophobicity cor-
relates to cell toxicity, but still bacteriolysis is induced with GS analogues that are too hydrophilic to effi-
ciently lyse human red blood cells.
Cationic antimicrobial peptides
Gramicidin S, GS14K4
Antibiotics
Secondary structure
Amphipathic peptides
Dipeptide isostere
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
two type II’ b-turns. The amino acid sequence and its secondary
structure render the molecule amphipathic with the cationic side
The increasing number of pathogenic bacteria that are resistant
to clinically applied antibiotics has led to a search for potent new
antibiotics with alternative targets.1 Cationic antimicrobial pep-
tides (CAPs) comprise a broad class of molecules that target the li-
pid bilayer of bacteria. Bacteria do not easily build up resistance
against such peptides,2 and therefore CAPs are considered as prom-
ising leads for the development of new antibiotics. A prominent
member of the CAP family is the cyclo-decapeptide gramicidin S
(GS) isolated from Aneurinibacillus migulans.3 Its secondary struc-
ture consists of a short antiparallel b-sheet interconnected by
chains oriented on one face of the b-sheet and the hydrophobic
side chains on the opposing face.4 The cationic amphipathic char-
acteristics of the peptide are responsible for its membrane disrupt-
ing properties, and GS is active against both Gram-positive and to a
lesser extent against Gram-negative bacteria.5 In combination with
the antibiotic agent framycetin, which targets Gram-negative bac-
teria, GS has proven to be an effective antibiotic against external
ear infections.6 The major drawback of GS, however, is that it is
hemolytic and therefore it is only used to treat topical infections.
Over the years many GS derivatives have been designed with the
aim to improve the therapeutic index, that is, decreasing hemolytic
activity while retaining potent antimicrobial activity.7 One promis-
ing strategy is modification of the b-turn region of the peptide with
unnatural amino acids8 or dipeptide isosteres.9 In this context,
sugar amino acids (SAAs) featuring a carboxylate and an amine10
⇑
Corresponding author. Tel.: +31 715274483; fax: +31 715274307.
E-mail address: overhand@chem.leidenuniv.nl (M. Overhand).
Present address: Department of Microbiology, National University of Singapore
CeLs, 28 Medical Drive, Singapore 117456, Singapore.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.04.031

A. D. Knijnenburg et al. / Bioorg. Med. Chem. 19 (2011) 3402–3409
3403
can be used to influence conformational behavior, and at the same
2. Results and discussion
2.1. Synthesis
time allow the introduction of additional functionalities and thus
modulation of the overall physical properties. An alternative strat-
egy to improve the therapeutic index of GS is to adjust the amphi-
pathic nature by modifying the b-strand regions.11 In this context,
the design and evaluation of tetradecameric GS analogues have
been reported, including derivatives containing six apolar and four
cationic residues. These appeared to behave as ring-extended GS
analogues in that they adopt stable cyclic b-hairpin conformations
in solution with altered amphipathicity.12 Kondejewski and co-
workers reported the substitution of several amino acids in both
the b-turn and b-strand regions without significantly changing sec-
The precursors of peptides 213a and 4 were assembled on the
highly acid sensitive HMPB–BHA-resin using a standard step-wise
solid phase Fmoc-based peptide synthesis protocol.15 After mild
acidic cleavage from the solid support, the linear peptides having
the basic side chains protected with Boc-groups were cyclized un-
der high dilution and purified using gel filtration. Boc deprotection
under strong acidic conditions and purification by preparative
HPLC afforded peptides 2 and 4. Peptides 8–10 were synthesized
according to a previous described procedure14 using the appropri-
ately functionalized SAA building blocks 1116 and 12 (Scheme 1).
SAA 12 was prepared by treating known SAA 11 with excess so-
dium hydride and 1.2 equiv of benzyl bromide (Scheme 1). SAAs
11 and 12 were coupled to preloaded HMPB-BHA-resin (13) yield-
ing peptide 14 and 15, respectively. After reducing the azide func-
tionalities using aqueous trimethylphosphine the sequences were
elongated by solid phase peptide synthesis after which the ensuing
linear peptides 16 and 17 were cleaved from resin and converted to
the cyclic peptides 18 and 19. The benzyl group in Boc-protected
peptide 19 were removed using 10% palladium hydroxide on char-
coal in a hydrogen gas atmosphere yielding Boc protected cyclic
peptide 20. Removal of the Boc protective groups of peptides 18–
20 was accomplished using trifluoroacetic acid in the presence of
triethylsilane and the target peptides were purified using prepara-
tive HPLC. Compounds 2, 4 and 8–10 were obtained in 25–40%
overall yield. Peptides 1, 3 and 5–7 were prepared as reported
previously.14
ondary structure characteristics.13 Single
D-amino acid substitution
of the fourth position in the b-strand of tetradecameric GS ana-
logues such as in 2 (GS14K413a, Figure 1), led to a compound with
a reported improved therapeutic index compared to the
acid substituted 1 (GS14, Fig. 1). Molecular modeling studies on
2 indicate that the inserted -amino acid in the sequence has no
L-amino
D
major influence on the cyclic b-hairpin secondary structure.
Recently, we reported the incorporation of furanoid SAAs as b-
turn mimetic in tetradecameric GS derivatives (3 and 5–7,
Fig. 1).14 NMR- and CD spectroscopy showed that these peptides
adopt cyclic b-hairpin structures. Variation of the substitution pat-
tern on the furanoid core provided derivatives that displayed an in-
creased antibacterial activity and reduced hemolytic activity
compared to 3. Compared to GS and 3, these analogues display en-
hanced antimicrobial activity against Gram-negative bacterial
strains. Because of these promising results we decided to synthe-
size tetradecameric GS parent compound 4 that contains an addi-
tional D-amino acid in the sequence and the furanoid SAAs b-turn
modified derivatives 8–10 (Fig. 1). The new peptides were sub-
jected to a detailed study of their structural features and these
were compared with those of 1–3 and 5–7. The entire series 1–
10 was evaluated for their bactericidal properties against an ex-
tended panel of bacterial strains, as well as their hemolytic
properties.
2.2. X-ray crystallography
Thus far no crystal structure of any cyclic tetradecameric GS
analogue has been reported. We were therefore pleased to see that
compound 2 gave crystals, which was used to determine the atom-
ic structure by high resolution X-ray crystallography. The side
chain of the
D
-lysine residue situated at the 4-position cleary re-
H
N
O
O
O
R2
H
N
H
N
H
N
N
H
N
H
N
H
N
O
OR3
O
O
O
R1
R1
R1
R1
O
OR4
O
O
O
H
N
H
N
H
N
N
O
N
N
H
N
H
O
H
R2
O
O
O
5
1, GS14: R1 = (CH2)4NH2, R2 = OH
, R1 = (CH2)3NH2, R2 = H, R3 = R4 = Bn
3
6, R1 = (CH2)3NH2, R2 = R4 = H, R3 = Bn
7, R1 = (CH2)3NH2, R2 = R3 = R4 = H
, R1 = (CH2)3NH2, R2 = H
O
O
H
O
H
N
R2
H
N
H
N
N
N
H
N
H
N
H
N
O
OR3
O
O
O
R1
R1
R1
R1
O
O
O
O
OR4
H
N
H
N
H
N
N
O
N
N
H
N
H
O
H
R2
O
O
O
2,
8
, R1 = (CH2)3NH2, R2 = H, R3 = R4 = Bn
9, R1 = (CH2)3NH2, R2 = R4 = H, R3 = Bn
10, R1 = (CH2)3NH2, R2 = R3 = R4 = H
GS14K4: R1 = (CH2)4NH2, R2 = OH
4, R1 = (CH2)3NH2, R2 = H
Figure 1. Structures of the tetradecameric Gramicidin S derivatives 1–4 and analogues with a SAA modified b-turn (5–10). Numbering of amino acids N ? C: cyclo-(Val1-
Orn2-Leu3- -Orn4-Val5- -Phe6-Pro7/SAA-Leu8-Orn9-Val10-Orn11-Leu12- -Phe13-Pro14).
D
D
D

3404
A. D. Knijnenburg et al. / Bioorg. Med. Chem. 19 (2011) 3402–3409
O
O
O
O
O
O
b
N
N
OH
N
OH
N
L O V O L
3
a
Fmoc-L-O-V-O-L
3
3
13
BnO
O
OH
BnO
O
OBn
R O
3
OR
2
11
12
14
15
R
R
= H, R = Bn
3
2
2
= R = Bn
3
O
O
H
N
H
c, d
N
N
N
N
HN
O
H
H
H
O
O
R
R
R
1
O
1
1
OR
OR
e, f
3
2
D
D
R
1
O
H N
F
P
V
O
L
O V
N
H
L O V O L
O
O
O
2
H
N
H
N
H
N
O
N
H
N
N
R O
3
OR
2
H
H
O
O
O
16 R = H, R = Bn
2
2
3
17 R = R = Bn
3
8
18, R = Boc-NH(CH ) , R = Bn, R = Bn
, R = (CH ) NH .TFA
1 2 3 2
1
1
2 3
2 3
2
2
3
h
19, R = Boc-NH(CH ) , R = H, R = Bn
9, R = (CH ) NH .TFA
1 2 3 2
3
g
20, R = Boc-NH(CH ) , R = H, R = H
10, R = (CH ) NH .TFA
1 2 3 2
1
2 3
2
3
Scheme 1. Reagents and conditions: (a) NaH (2.5 equiv), BnBr (1.2 equiv), DMF, 0 °C, 5 h, 71%.; (b) (i) 20% piperidine in NMP. (ii) using SAA 11 and 12 (1.5 equiv), HBTU
(3 equiv), DiPEA (6 equiv), DMF, 16 h; (c) PMe3 (1 M in 9:1 THF/H2O, 25 equiv), 16 h; (d) standard sequential Fmoc SPPS; (e) 1% TFA in DCM; (f) pyBOP (5 equiv), HOBt
(5 equiv), DiPEA (15 equiv), DMF; (g) H2, 10% Pd(OH)2/C in MeOH, 5 h; (h) 95/2.5/2.5 TFA/Tis/H2O.
sides on the same face as the hydrophobic side chains of the valine
and leucine residues (Fig. 2A). Because of this it is likely that com-
pound 2 possesses amphipathic characteristics rather different
from those of the lead compound 1. A small distortion is seen in
the pleated sheet structure of the molecule at the position of the
2.3. NMR and CD analysis
Compounds 4 and 8–10 give well-resolved 1H NMR spectra in
CD3OH, which allowed the assessment of their secondary structure
in solution. To enable comparison between L- and D-ornithine
D
-lysine substitution and the b-sheet is slightly curved (Fig. 2A).
substituted analogues and the influence of the furanoid SAA in
the type II’ b-turn, all amide coupling constants and chemical shift
perturbations of peptides 1–10 were determined (Table S1 in Sup-
plementary data). In all cases the peptides appear to possess the
typical b-sheet/b-hairpin character as observed from their amide
The presence of the -amino acid residue however does not pre-
D
clude the formation of the cyclic b-hairpin conformation in which
the distances between specific amides and carbonyl functionalities
of opposing strands clearly indicate the presence of six intramolec-
ular hydrogen bonding interactions (Fig. 2B). Notable is the great
similarity with the reported molecular modeling structure of 217
in which the same arrangement of the side chain functionalities
was observed. In the crystal structure the molecules are packed
in a roof-tile shape with the hydrophilic residues at the concave
face and the hydrophobic residues at the convex face (Fig. 2C).
coupling constants and chemical shift perturbations (3JHN : 7–
a
9 Hz for Val, Leu and Orn residues; 4 Hz for
D-Phe and DdH :
a
>0.1 for Val, Leu, and Orn; <0 for
D
-Phe and Pro).19 In general, pep-
tides 8–10 exhibit lower values in chemical shift perturbation and
amide coupling constants than the parent peptide 4. The same
trend was observed for the L-ornithine substituted analogues 5–7
One of each
D
-Tyr-Pro sequences of alternate molecules are placed
compared to 3. This indicates that the presence of the SAA turn mi-
metic results in a less pronounced secondary structure. Interest-
ingly, no major differences in coupling constants and chemical
shift perturbation were observed between peptides 5–7 and 8–
such that they close the concave face like a zipper forming an
asymmetric channel with a hydrophilic core (Fig. 2D and E). This
crystal packing differs from the crystal lattice reported for GS,
which is reported to line up into double stranded helical
channels.18
10. This finding suggests that the incorporation of
D-ornithine
Figure 2. (A) Side view of the crystal structure of 2; (B) top view of (side chains are omitted for clarity); (C) top view of roof-tile shaped crystal lattice; (D) Side view of the
‘zipper’ forming an asymmetric channel; (E) cross-cut through the asymmetric channel.

A. D. Knijnenburg et al. / Bioorg. Med. Chem. 19 (2011) 3402–3409
3405
has little influence on the stability of the cyclic b-hairpin structure
in CD3OH.
the most potent bactericidals of the series presented here and
are at least as active as the parent compound, GS. From a therapeu-
tic point of view however, compound 8 might be the most interest-
ing of the series. Although slightly less potent than GS against most
of the bacterial strains assayed, this compound is considerably less
CD was used to study the conformational behavior of the sec-
ondary structures of the peptides in solvent systems of different
polarity. The CD spectra measured in methanol are depicted in Fig-
ure 3A. Peptides 3 and 4 show the typical GS curve with negative
ellipticities at 220 and 205 nm confirming the b-sheet/b-hairpin
character.20 CD corroborates the similarities in secondary structure
in the series 5–7 and 8–10 (Fig. 3A) observed in the NMR measure-
ments. The molar ellipticity of compounds 5–7 in TFE/H2O (Fig-
ure 3B) is slightly enhanced compared to 8–10 at 220 nm which
suggests that these former peptides (5–7) have a more pronounced
b-sheet/b-hairpin structure in this solvent system.21,22 In buffered
aqueous environments somewhat more disordered curves were
observed for several analogues. For instance, peptide 3 exhibits
b-sheet/b-hairpin character although less pronounced than in
TFE/H2O. Peptide 4 has a high molar ellipticity at 220 nm, but also
shows a wavelength shift (towards 200 nm), which can indicate a
different backbone conformation.13 The CD curves of 5–10 in water
exhibit less pronounced negative molar ellipticities at 220 nm
combined with a high negative molar ellipticity at 195 nm, sug-
gesting that these peptides have a more disordered structure with
less b-sheet character.
hemolytic, and complete hemolysis is achieved only at 500
lM
(compare the value for GS, being 62.5 M). To a lesser extend the
l
same trend is observed for compound 9. This in contrast to the
hemolytic properties of the most potent bactericidal compounds
of the series, namely 5–7, which with values between 15 and
30 lM appear to be at least tenfold more toxic (Fig. 4).
2.5. Correlation of hydrophobicity and biological activity
With the aim to get insight into the hydrophobicity of the pep-
tides we established their HPLC retention times under controlled
conditions (Table 1).23,24 The analogues containing a
D
-ornithine
residue (2, 4, 8–10) are significantly less hydrophobic than their
-ornithine counterparts (1, 3, 5–7). This result matches the trend
L
previously observed for 1 and 2.13 As expected, the GS derivatives
featuring benzylated SAA turn regions are more hydrophobic than
their non-benzylated counterparts. The most hydrophilic com-
pound of the series, compound 10, appeared to be inactive in both
biological assays, that is, it is neither antibacterial nor hemolytic.
At the opposite site of the hydrophobicity spectrum is compound
5, one of the most active compounds (both bactericidal and hemo-
lytic) that has the longest retention time on the reverse phase
HPLC. Interestingly, compounds 8 and 9, which we identified as
the most promising compounds that combine bactericidal activity
2.4. Biological evaluation
The peptides 1–10 and GS were screened for their hemolytic
activity and antibacterial activity against a panel of Gram-positive
and Gram-negative bacteria (Table 1, Fig. 4). Compounds 5–7 are
Figure 3. (A) CD spectra measured in CH3OH; (B) CD spectra measured in 50% TFE/0.01 M aq NaOAc (pH 5.3); (C) CD spectra measured in aq NaOAc (pH 5.3). All peptides
were measured at 0.1 mM concentration.
Table 1
Hemolytic (mg/L), antimicrobial activity (MIC, mg/L) and HPLC retention times of GS and ring-extended derivatives 1–10
Peptide Hemolysisa S. aureusb S. epider midisb E. faecalisb B. cereusb P. aeruginosac E. colic K. pneumoniaec E. cloacaec P. mirabilisc HPLC retention timed
GS
1
2
3
4
5
6
7
8
62.5
3.9
500
3.9
125
15.6
15.6
31.3
500
500
>500
4
4
64
8
8
8
4
4
4
8
8
4
32
16
16
16
2
4
4
8
8
64
>64
>64
64
8
16
16
16
64
64
>64
32
>64
64
32
64
16
16
8
64
>64
64
64
32
32
16
16
32
32
>64
64
>64
64
64
32
32
16
16
32
32
64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
8.38
7.26
5.95
8.00
6.48
6.32
6.28
5.70
5.67
5.33
4.98
>64
64
16
8
4
4
8
8
16
>64
32
64
8
16
8
8
8
16
32
>64
32
32
64
9
10
8
16
64
MW GS: 1369,49; MW 1 (GS14), 2 (GS14K4): 2126.23; MW 3,4: 2038.12; MW 5,8: 2133,22; MW 6,9: 2043,09; MW 7,10: 1952,97.
a
Hemolytic activity (lM) in which 100% of the erythrocytes are lysed, hemolytic curves are shown in supporting information.
Gram-positive bacteria, MIC in mg/L.
Gram-negative bacteria, MIC mg/L.
HPLC retention time in minutes. For conditions see experimental section.
b
c
d

3406
A. D. Knijnenburg et al. / Bioorg. Med. Chem. 19 (2011) 3402–3409
Figure 4. (A) Hemolytic activity of analogue 1–5 and GS; (B) hemolytic activity of analogue 6–10 and GS.
with limited hemolytic properties, appear to be in the middle of
the hydrophobic range of the series. This observation underscores
previous findings from our and other laboratories8,9,11–14 with
related molecules and suggests that there is an optimum in hydro-
phobicity in a series of amphiphilic peptides with otherwise unal-
tered secondary structure with respect to an optimal bactericidal/
hemolytic ratio.
were recorded using the presaturation solvent suppression. All
spectra were recorded in phase-sensitive mode using either the
TPPI or states-TPPI for quadrature detection in the indirect
dimension.
4.2. CD spectroscopy
CD spectra were recorded at 298 K on a Jasco J-815 spectropo-
larimeter using 0.1 cm path length quartz cells. The CD spectra
are an average of four scans, collected at 0.1 nm intervals between
190 and 250 nm with scanning speed 50 nm/min. The peptides
were prepared at concentrations 0.1 mM in MeOH, 0.1 mM in
50% TFE/0.01 M NaOAc (pH 5.3) or 0.1 mM in 0.01 M NaOAc (pH
5.3). Ellipticity is reported as mean residue ellipticity [h], with
approximate errors of 10% at 220 nm.
3. Conclusions
In conclusion, we have for the first time obtained a crystal
structure of an extended gramicidin S analogue, the known com-
pound 2 (GS14K4). This structure unambiguously demonstrates
that insertion of an additional D-amino acid in a cyclic b-hairpin
structure does not necessarily lead to a drastically altered second-
ary structure, and compound 2 appears to closely resemble the b-
sheet/b-hairpin structure of 1 (GS14). As a result, the basic side
4.3. Crystallization and crystal structure determination of 2
chain of this D-amino acid residue points towards the same direc-
Colorless plate-shaped crystals were obtained after slow evapo-
tion as the aliphatic side chains of the Leu/Val residues and thus
whereas the secondary structure of the two compounds is much
the same, the amphiphilic characteristics are rather more different.
Building on these results and based on our previous findings,14 we
proceeded to generate analogues in which one of the turn regions
contains a furanoid SAA of increasing hydrophobicity. NMR- and
CD spectroscopy indicate that these analogues are all capable of
adopting a cyclic b-hairpin structure. Notable is the observation
in CD that compounds 5–10 are less structured under physiological
conditions (aqueous buffer). Although it is difficult to link confor-
mational properties directly to biological activity, compound 8–
10 are less hemolytic.
ration of 2
in H2O plus 2
l
L droplets of 9 mg/mL peptide in 80% solution of MeOH
L of 0.2 M HCl in MeOH under paraffin oil in Tera-
l
saki plates. A 0.20 Â 0.05 Â 0.05 mm crystal was mounted in air
and directly flash-frozen in liquid nitrogen. Synchrotron data were
collected at beamline BM14 at the ESRF (Grenoble, France). Images
were collected with DNA software,26 processed with MOSFLM27
and scaled with POINTLESS and SCALA28 to 1.00 Å resolution. The
structure was solved by direct methods using the program ilMili-
one29 and refined with no intensity cutoff using the full-matrix
least-squares methods on F2 refinement implemented in SHELXH30
included in the WinGX package.31 Throughout the refinement,
bond-length, bond-angle and planarity restraints were imposed.
All non-H atoms were refined anisotropically with suitable rigid-
bond and similarity restraints. All hydrogen positions were calcu-
lated and refined using a riding atom model. The asymmetric unit
contains two crystallographically independent molecules of 2 with
several disordered amino acids, plus one molecule of trifluoroace-
tate and twelve point five molecules of water. Selected crystallo-
graphic data is reported in Table S2 in Supplementary data. Final
figures were created using PyMOL (Delano Scientific, Palo Alto
CA, USA). CCDC-814883 contains the supplementary crystallo-
graphic data for this paper. These data can be obtained free of
charge from The Cambridge Crystallographic Data Centre via
http://www.ccdc.cam.ac.uk/data_request/cif.
The hydrophobic character of extended GS can be tuned by
introduction at the appropriate site of a D-amino acid residue, by
varying the nature of one of the b-turn regions, or a combination
thereof. We have evaluated the consequences of these modifica-
tions on biological activity and have strong indications (compound
8, to a lesser extend compound 9) that there may be an optimal
hydrophobicity in a series of closely related compounds (especially
with respect to the secondary structure) with cell disrupting prop-
erties, in that bacteriolytic activity might be separated from hemo-
lytic properties.
4. Experimental section
4.1. NMR spectroscopy
4.4. Antimicrobial assay
The peptides were recorded on a Bruker DMX 600 equipped
with a pulsed field gradient accessory and a cryo-probe. For the
The following bacterial strains were used: Staphylococcus aureus
(ATCC 29213), Staphylococcus epidermidis (ATCC 12228), Enterococ-
cus faecalis (ATCC 29212), Bacillus cereus (ATCC 11778), Escherichia
coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Klebsiella
pneumoniae 1190901525, Enterobacter cloacae 1190900252, Pro-
2D cROESY25 spectra (
smix = 200 msec) the peptides were dissolved
in CD3OH. Standard DGF-COSY (512c  2084c) and TOCSY
(400c  2048c) spectra were recorded using presaturation for sol-
vent suppression. cROESY spectra (400c  2048c,
smix = 180 ms)

A. D. Knijnenburg et al. / Bioorg. Med. Chem. 19 (2011) 3402–3409
3407
teus mirabilis 1190901236. Bacteria were stored at À70 °C and
grown at 30 °C on Columbia Agar with sheep blood (Oxoid, Wesel,
Germany) suspended in physiological saline until an optical den-
sity of 0.1 AU (at 595 nm, 1 cm cuvette). The suspension was di-
pling with the resin and shaken for 4 h; (d) washing with NMP
(2 Â 50 mL) and DCM (2 Â 50 mL); mini cleavage: LCMS 14: Rf
5.69 min, linear gradient 10 ? 90% B in 13.5 min.; m/z = 847.0
[M+H]+. LCMS 15: Rf 7.05 min, linear gradient 10 ? 90% B in
13.5 min.; m/z = 937.4 [M+H]+.
Azide reduction: Resin 14 and 15, respectively were washed with
1,4-dioxane (3 Â 10 mL), and taken up in 1,4-dioxane (10 mL) to
which trimethylphosphine (16 equiv, 1 M in THF) pre-mixed with
H2O (0.6 equiv) was added. The resin was shaken for 24 h; the
reduction of the azide functionality was monitored with the Kaiser
test.
luted (10Â) with physiological saline and 2
lL of this inoculum
were added to 100 L of growth medium, [Nutrient Broth from Dif-
l
co (ref. nr. 234000, lot nr. 6194895) with yeastextract (Oxoid LP
0021, lot nr. 900711, 2 g/400 mL of broth)] in microtiter plates
(96 wells). The peptides (1–10 and GS) were dissolved in ethanol
(4 g/L) and further diluted in distilled water (1 g/L), and two-fold
diluted in the broth (64, 32, 16, 8, 4 and 1 mg/L). The plates were
incubated at 30 °C (24–96 h) and the MIC was determined as the
lowest concentration inhibiting bacterial growth at 24 h. The
experiments were conducted once, the experimental error is one
MIC interval (a factor two).
Automated SPPS elongation: Azide reduced resins 14 (0.2 mmol)
and 15 (0.1 mmol), respectively were subjected to 7 cycles of SPPS
with the use of commercially available building blocks in the fol-
lowing order: Fmoc-Val-OH, Fmoc-D-Orn(Boc)-OH, Fmoc-Leu-OH,
Fmoc-Orn(Boc)-OH, Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-d-Phe-OH
and subsequent Fmoc deprotection. Minicleavage LCMS 16: Rf
4.84 min, linear gradient 10 ? 90% B in 13.5 min.; m/z = 1607.2
[M+H]+. LCMS 17: Rf 5.18 min, linear gradient 10 ? 90% B in
13.5 min.; m/z = 1695.5 [M+H]+.
Cleavage from resin: The peptides were released from the resin
by mild acidic cleavage (4 Â 10 min, 10 mL 1% TFA in DCM). The
fractions were collected and coevaporated with toluene
(3 Â 50 mL) to give the crude linear peptides which were immedi-
ately cyclized without further purification.
4.5. Hemolytic assays
Freshly drawn heparinized blood was centrifuged for 10 min at
1000 g at 10 °C. Subsequently, the erythrocyte pellet was washed
three times with 0.85% saline solution and diluted with saline to
a 1/25 packed volume of red blood cells. The peptides (1–10 and
GS) were dissolved in a 30% DMSO/0.5 mM saline to a stock of
1.5 mM. If required (suspension formed) the stock solution was
sonicated for a few seconds. prior to use. As a possitive control a
1% Triton-X100 solution was prepared. For the assay 100
ine were dispensed in columns 1–11 of a microtiter plate and
l
L of sal-
Cyclization: To a solution of HOBt (5 equiv), pyBOP (5 equiv) and
DIPEA (15 equiv) in DMF (160 mL) were dropwise added the crude
peptides in DMF (20 mL) over a periode of 16 h using the syringe-
pump. The solvent was removed under reduced pressure and the
resulting mixture was applied to a SephadexÒ size exclusion col-
umn (50.0 mmD Â 1500 mmL) and eluted with MeOH. LCMS 18
(deprotected in vial): Rf 5.12 min, linear gradient 10 ? 90% B in
13.5 min.; m/z = 1588.2 [M+H]+; HRMS (ESI) m/z 1987.21414
[M+H]+, Calcd 1987.21296 for C100H164N17O24. LCMS 19 (deprotec-
ted in vial): Rf 5.46 min, linear gradient 10 ? 90% B in 13.5 min.;
m/z = 1678.6 [M+H]+; HRMS (ESI) m/z 1039.13508 [½ M+H]+, Calcd
1039.13360 for C107H170N17O24
100
100
100
l
l
l
L of 1% Triton solution in column 12. To wells A1-C1,
L of the peptide were added and mixed properly. Next,
L of wells A1–C1 were dispensed into wells A2–C2. This pro-
cess was repeated until wells A10–C10, followed by discarding
100 L from wells A10–C10. These steps were repeated for the
other peptides. Subsequently, 50 L of the red blood cell solution
l
l
were added to the wells and the plates were incubated at 37 °C
for 4 h. After incubation, the plates were centrifuged at 1000 g at
RT for 5 min. In a new microtitre plate, 50 lL of the supernatant
of each well were dispensed into a corresponding well. The absor-
bance at 405 nm was measured and the percentage of hemolysis
was determined. The experiment was conducted once and carried
out in triplicate. A maximum of 10% experimental error was found.
Hydrogenation: Protected peptide 19 (121 mg, 60.9 lmol) was
hydrogenated under a hydrogen atmosphere in MeOH (30 mL)
with 10% Pd(OH)2/C (40 mg). The mixture was filtered over celite
yielding the crude peptide 20 (99 mg, 52.1 lmol); LC/MS 20
4.6. Synthesis peptides 2,4,8–10
(deprotected in vial): Rf 4.79 min, linear gradient 10 ? 90% B in
13.5 min.; m/z = 1497.1 [M+H]+; HRMS (ESI) m/z 1898.17050
[M+H]+, Calcd 1898.16937 for C93H159N17O24
4.6.1. General procedure GS analogues 8–10
Stepwise elongation: Preloaded resin with Fmoc-Leucine (2.8 g,
0.53 mmol/g, 1.5 mmol) was submitted to 4 cycles of Fmoc solid-
phase synthesis with use of commercially available building blocks
in the order: Fmoc-Orn(Boc)-OH, Fmoc-Val-OH, Fmoc-Orn(Boc)-
OH, Fmoc-Val-OH. (a) deprotection with 20% piperidine in NMP
(3 Â 50 mL, 15 min); (b) washing with NMP (2 Â 50 mL) and
DCM (2 Â 50 mL). (c) pre-activation of the up-following building
block (3 equiv) in 50 mL of NMP, 0.2 M HBTU in NMP and DIPEA
(6 equiv) and subsequent coupling with the resin and shaken for
4 h. (d) washing with NMP (2 Â 50 mL) and DCM (2 Â 50 mL). Cou-
plings were monitored for completion by the Kaiser test and LC/
MS. (e) The resin was capped with 0.45 M Ac2O in NMP (5 equiv)
and DIPEA (1 equiv). (f) The resin was washed with MeOH
(2 Â 50 mL), NMP (2 Â 50 mL), DCM (2 Â 50 mL). Dry resin 13:
3.73 g, 0.4 mmol/g. LC/MS: Rf 5.61 min, linear gradient 10 ? 90%
B in 13.5 min.; m/z = 794.7 [M+H]+.
Deprotection: The Boc-protection groups were removed by addi-
tion of TFA/Tis/H2O mixture (10 mL, 95/2.5/2.5) and subsequently
the peptide was purified by preparative RP-HPLC. Yield of peptides
8–10 after HPLC is based on purified protected state.
4.6.2. cyclo-[Leu-Lys-Val-Lys-Leu-D-Tyr-Pro-Val- Lys-Leu-D-Lys-
Val- -Tyr-Pro] (2)
D
HMPB-BHA-Fmoc-Orn(Boc) preloaded resin was subjected to 13
cycles of SPPS; cleaved from resin; cyclized; washed; deprotected;
purified by semi-preparative RP-HPLC (linear gradient of 29–38%, 3
CV) and lipophilization of the combined fractions furnished the
peptide (71.7 mg, 33.7 lmol, 34%); LC/MS Rf 5.95 min, linear gradi-
ent 10 ? 90% B in 13.5 min.; m/z = 1669.9 [M+H]+; HRMS (ESI) m/z
835.54176 [½ M+H]+, Calcd 835.54201 for C85H141N18O16; IR
3268.1, 2958.1, 1668.0, 1621.9, 1538.5, 1455.8, 1200.6, 1132.7,
839.1, 800.0, 722.4, 599.7; 1H NMR (600 MHz, CD3OH) d 8.92 (d,
Incorporation of SAA building blocks: (a) The resin 13 (498 mg)
was washed with MeOH (2 Â 50 mL), NMP (2 Â 50 mL), DCM
(2 Â 50 mL); (b) deprotection with 20% piperidine in NMP
(3 Â 10 mL); (c) pre-activation of 11 (1.75 equiv, 0.7 mmol) and
12 (1.5 equiv, 0.3 mmol), respectively with HBTU (3 equiv, 0.2 M
HBTU in NMP), DIPEA (6 eq) in 10 mL of NMP and subsequent cou-
J = 2.3, 1H, NH DTyr6), 8.81 (d, J = 3.9, 1H, NH DTyr13), 8.81 (d,
J = 7.8, 1H, NH Val10), 8.76 (d, J = 9.1, 1H, NH Leu12), 8.65(d,
J = 9.3, 1H, NH Leu3), 8.59 (d, J = 8.6, 1H, NH Lys11), 8.40 (d,
J = 9.7, 1H, NH Lys4), 8.38 (d, J = 10.5, 1H, NH Lys2), 7.99 (d,
J = 8.4, 1H, NH Val5), 7.97 (d, J = 9.4, 1H, NH Leu8), 7.81 (br s, NH2
Lys), 7.64 (d, J = 9.2, 1H, NH Val1), 7.55 (d, J = 9.3, 1H, NH Lys9),

3408
A. D. Knijnenburg et al. / Bioorg. Med. Chem. 19 (2011) 3402–3409
7.04 (dd, J = 2.0, 8.4, 4H, H2,6
D
Tyr), 6.72 (t, J = 7.8, 4H, H3,5
D
Tyr),
gradient 10 ? 90% B in 13.5 min.; m/z = 1677.6 [M+H]+; HRMS
(ESI) m/z 1677.04996 [M+H]+, Calcd 1677.05020 for
5.46 (m, 1H, H Lys4), 5.19 (m, 1H, H Lys9), 5.14 (m, 1H, H
a
a
a
Lys2), 4.97 (m, 1H, H Lys11), 4.80 (m, 1H, H Leu3), 4.61 (q,
C87H137N17O16; IR 3281.9, 2962.4, 1667.9, 1633.9, 1538.2, 1455.7,
a
a
J = 7.6, 1H, H Leu12), 4.52–4.34 (m, 7H, H Leu8, Val10,5
,
D ,
Tyr13,6
1201.0, 1130.0, 835.3, 798.8, 721.5, 608.1; 1H NMR (600 MHz,
a
a
D
Pro7,14), 4.31 (t, J = 8.4, 1H, H
(m, 1H, Hdd Pro7), 3.04-2.77 (m, 12H, H
a
Val1), 3.71 (m, 1H, Hdd Pro14), 3.62
Lys, Hbd,u Tyr), 2.53
CD3OH) d 8.84 (d, J = 3.2, 1H, NH Phe13), 8.57 (d, J = 7.5, 1H, NH
D
Orn2), 8.52 (d, J = 8.1, 1H, NH Orn9), 8.51 (m, 1H, NH SAA12), 8.50
(d, J = 9.3, 1H, NH Leu12), 8.48 (d, J = 8.6, 1H, NH Orn4), 8.31 (d,
J = 7.0, 1H, NH Orn11), 8.28 (d, J = 6.7, 1H, NH Leu3), 8.27 (d,
J = 6.9, 1H, NH Leu8), 8.20 (d, J = 6.9, 1H, NH Val5), 8.06 (d, J = 8.0,
1H, NH Val10), 7.90 (br s, NH2 Orn), 7.79 (d, J = 8.4, 1H, NH Val1),
e
d,u
(m, 1H, Hdu Pro14), 2.49 (m, 1H, Hdu Pro7), 2.23 (dd, J = 6.8, 13.6,
1H, Hb Val1), 2.13–1.89 (m, 8H, Hb Val10,5, Hbd Pro, Hbd Lys), 1.86
(m, 3H, Hbd Leu), 1.80–1.30 (m, 30H, Hbu, Leu, Hbu, Lys,
cd
cd,udd,u
Hbu,
Pro), 1.28 (m, 1H, H Lys2), 1.10 (m, 1H, H Lys9) 1.07–
cu cu
c
d,u
0.80 (m, 36H, CH3 Val, CH3 Leu); 13C NMR (151
D
MHz, CD3OH) d
7.45–7.12 (m, 15H, CH Ar), 4.77 (m, 1H, H Orn4), 4.71–4.52 (m,
a
D
173.99, 173.86, 173.78, 173.64, 173.47, 173.42, 173.35, 173.17,
173.15, 172.76, 172.61, 172.37, 172.31, 163.02, 162.79, 158.14,
158.08, 131.45, 131.37, 127.37, 126.95, 117.13, 116.37, 116.33,
61.81, 61.71, 59.79, 58.67, 58.38, 56.09, 55.87, 53.82, 53.57,
53.20, 52.70, 52.14, 52.01, 51.76, 47.89, 47.59, 44.76, 42.10,
41.00, 40.91, 40.68, 40.54, 39.32, 36.77, 36.58, 35.77, 35.46,
33.73, 33.56, 33.37, 33.10, 32.59, 31.05, 30.77, 30.35, 28.73,
28.47, 28.33, 25.84, 25.63, 24.53, 24.39, 24.30, 24.04, 23.76,
23.63, 23.30, 23.20, 23.17, 23.00, 22.39, 22.03, 19.95, 19.51,
19.06, 18.86, 18.76, 18.36.
6H, H Orn2,9,11, Leu3,9,12
,
Phe13, H1 SAA12), 4.45 (m, 10H, CH2
a
Bn) 4.37 (m, 1H, H Pro14), 4.21 (d, J = 3.6, 1H, H2 SAA12), 4.12
a
(m, 3H, H4 SAA12, H Val1,5), 4.04 (m, 1H, H5d SAA12), 3.88 (s, 1H,
a
D
H3 SAA12), 3.69 (m, 1H, Hdd Pro14), 3.06 (m, 1H, Hbd,u Phe13), 2.95
(m, 7H, Hdd,u Orn), 2.79 (m, 1H, Hdu Orn9), 2.58 (m, 1H, Hdu Pro14),
2.23 (dd, J = 6.9, 14.7, 1H, Hb Val1), 2.17 (dd, J = 6.7, 13.3, 1H, Hb
Val5), 1.99 (m, 1H, Hb Val10, Hbd Pro14), 1.90 (m, 2H, Hbd, Orn4,11),
1.83–1.48 (m, 23H, Hbd, Leu, Hbu,
Orn), 1.39 (m, 1H, H
c
c
d,u
c
Leu12), 1.05–0.82 (m, 36H, CH3 Val, CH3 Leu); 13C NMR (151 MHz,
CD3OH) d 175.12, 174.64, 173.85, 173.80, 173.67, 173.41, 173.31,
173.22, 173.16, 172.99, 172.68, 171.36, 138.71, 138.68, 137.02,
130.39, 129.63, 129.60, 129.46, 129.16, 129.05, 128.93, 128.76,
128.40, 87.19, 84.05, 83.82, 82.96, 73.41, 72.59, 61.81, 60.68,
59.77, 55.64, 54.01, 53.60, 53.32, 53.23, 52.46, 52.03, 49.57,
49.42, 49.28, 49.14, 49.08, 49.07, 49.00, 48.86, 48.80, 48.78,
48.72, 48.57, 47.85, 43.41, 42.47, 42.17, 40.74, 40.50, 40.40,
40.12, 37.45, 32.40, 32.18, 31.78, 31.38, 30.72, 30.55, 29.92,
25.98, 25.76, 25.71, 25.20, 24.71, 24.66, 24.55, 23.72, 23.26,
22.91, 22.86, 22.00, 21.81, 19.78, 19.71, 19.39, 18.67, 18.56.
4.6.3. cyclo-[Leu-Orn-Val-Orn-Leu-D-Phe-Pro-Val-Orn-Leu-D-
Orn-Val- -Phe-Pro] (4)
D
HMPB-BHA-Fmoc-Orn(Boc) preloaded resin was subjected to 13
cycles of SPPS; cleaved from resin; cyclized; washed; deprotected;
purified by semi-preparative RP–HPLC (linear gradient of 29–38%,
3 CV) and lipophilization of the combined fractions furnished the
peptide (53.3 mg, 26.1
ent 10 ? 90% B in 13.5 min; m/z = 1582.9 [M+H]+; HRMS (ESI) m/z
1582.02418 [M+H]+, Calcd 1582.02432 for C81H132N18O14
IR
lmol, 26%); LCMS Rf 6.48 min, linear gradi-
;
3273.4, 2920.1, 1683.6, 1633.9, 1538.6, 1455.7, 1202.5, 1131.5,
4.6.5. cyclo-[(2R,3S,4R,5R)-5-aminomethyl-4-benzyloxy-3-
1008.7, 837.1, 800.1, 721.4, 612.0; 1H NMR (600 MHz, CD3OH) d
hydroxytetrahydrofuran-2-carboxyl-Leu-Orn-Val-Orn-Leu-
Phe-Pro-Val-Orn-Leu- -Orn-Val] (9)
Peptide 19 (125 mg, 62.9 mol), as prepared described above,
D-
D
9.06 (d, J = 2.1, 1H, NH Phe6), 8.98 (d, J = 2.9, 1H, NH dPhe13),
D
8.67 (d, J = 8.6, 3H, NH Val10, Orn11, Leu12), 8.58 (d, J = 8.9, 2H, NH
Leu3, Orn4), 8.48 (d, J = 9.5, 1H, NH Orn2), 8.02 (d, J = 7.8, 1H, NH
Val5), 7.88 (d, J = 9.1, 1H, NH Leu8), 7.69 (d, J = 8.9, 1H, NH Val1),
7.58 (d, J = 9.7, 1H, NH Orn9), 7.33–7.25 (m, 10H, CH Ar), 5.45 (m,
l
was deprotected using TFA/Tis/H2O (95/2.5/2.5) and subsequently
the peptide was purified by preparative RP-HPLC (linear gradient
of 33-42%, 3CV and yielded 31.7 mg, 15.5 lmol, 25%; LCMS Rf
1H, H Orn4), 5.34 (m, 1H, H Orn9), 5.12 (m, 1H, H Orn2), 5.02–
5.33 min, linear gradient 10 ? 90% B in 13.5 min.; m/z = 1587.2
[M+H]+; HRMS (ESI) m/z 1587.00388 [M+H]+, Calcd 1587.00325
for C80H131N17O16; IR 3269.8, 2962.4, 1667.9, 1634.1, 1538.9,
1455.8, 1201.5, 1132.0, 837.3, 799.8, 722.1, 616.0; 1H NMR
a
a
a
D
4.68 (m, 3H, H Orn11, Leu12,3), 4.57–4.50 (m, 2H, H Leu8, Phe13),
a
a
D
4.46 (m, 2H, H Val10, Phe6), 4.38 (m, 2H, H Val5, Pro7), 4.36 (m,
a
a
1H, H Pro14), 4.27 (t, J = 8.2, 1H, H Val1), 3.73 (m, 1H, Hdd Pro7),
a
a
D
D
3.62 (m, 1H, Hdd Pro14), 3.10 (m, 2H, Hbd Phe), 3.04 (m, 1H, Hdd
Orn11), 2.92 (m, 5H, Hbu Phe, Hdd,u Orn2,4,11), 2.80 (m, 1H, Hdd
(600 MHz, CD3OH) d 8.72 (d, J = 3.8, 1H, NH Phe13), 8.50 (t,
D
J = 5.2, 1H, NH SAA11), 8.49 (d, J = 7.9, 1H, NH Orn2), 8.43 (d,
J = 5.7, 1H, NH Orn9), 8.40 (d, J = 8.2, 1H, NH Orn4), 8.38 (d,
J = 7.6, 1H, NH Leu12), 8.26 (d, J = 6.8, 2H, NH Orn11, Leu8), 8.07
(d, J = 7.5, 1H, NH Leu3, Val5), 7.98 (d, J = 6.3, 1H, NH Val10), 7.88
(br s, 1H, NH2 Orn), 7.83 (d, J = 7.9, 1H, NH Val1), 7.73 (br s, 1H,
Orn9), 2.71 (m, 1H, Hdu Orn9), 2.47 (m, 1H, Hdu Pro7,14), 2.28 (dd,
J = 6.9, 13.7, 1H, Hb Val1), 2.12–1.84 (m, 11H, Hbd Pro, Hbd Orn,
Hbd Leu, Hb Val5,10), 1.85–1.46 (m, 27H, Hbu,
Leu, Hbu,
Orn,
c
d,u
c
d,u
Hbu,
Pro), 1.44 (m, H Leu8), 1.35 (m, 1H, H Leu12), 1.03–
cu cu
c
d,u
0.80 (m, 36H, CH3 Val, CH3 Leu); 13C NMR (151 MHz, CD3OH) d
174.46, 173.90, 173.72, 173.61, 173.58, 173.47, 173.22, 173.07,
172.97, 172.83, 172.70, 172.48, 172.40, 162.96, 162.72, 136.86,
136.33, 130.43, 130.36, 129.65, 128.57, 128.46, 101.28, 61.89,
60.08, 58.83, 58.34, 55.92, 55.61, 53.05, 52.78, 52.65, 52.16,
51.97, 51.83, 48.05, 47.73, 44.73, 42.08, 41.05, 40.76, 40.57,
40.51, 38.99, 37.56, 37.26, 33.82, 33.39, 33.29, 33.07, 32.28,
31.05, 30.94, 30.59, 30.45, 30.32, 25.90, 25.75, 25.65, 25.07,
24.96, 24.69, 24.54, 24.45, 24.35, 24.29, 23.41, 23.22, 22.91,
22.20, 21.90, 20.70, 19.93, 19.53, 19.19, 19.07, 18.72, 18.32.
NH2 Orn), 7.39–7.20 (m, 10H, CH Ar), 4.76 (m, 1H, H Orn11),
a
D
4.65–4.59 (m, 5H, H Orn2,4, Leu3, Phe13, CH2 Bn), 4.58 (d, J = 3.6,
a
1H, H1 SAA11), 4.56 (m, 1H, H Orn9), 4.51 (m, 2H, H Leu8,12, H2
a
a
SAA11), 4.37 (d, J = 5.9, 1H, H Pro14), 4.25 (t, J = 7.0, 1H, H
a
a
a
Val10), 4.15 (m, 2H, H Val5, H4 SAA11), 4.06 (t, J = 8.1, 1H, H
a
Val1), 4.01 (m, 1H, H5d SAA11), 3.84 (s, 1H, H3 SAA11), 3.71 (m,
D
1H, Hdd Pro14), 3.12–3.02 (m, 3H, H5u SAA11, Hbd,u Phe13), 3.02–
2.85 (m, 9H, Hdd,u Orn, H5u SAA11), 2.69 (m, 1H, Hdu Pro14), 2.21
(dd, J = 7.3, 14.3, 1H, Hb Val1), 2.16 (dd, J = 6.7, 13.5, 1H, Hb Val5),
2.08 (dd, J = 6.8, 13.6, 1H, Hb Val10), 2.00 (m, 1H, Hbd Pro14), 1.90
(m, 3H, Hbd Orn4,9,11), 1.84–1.46 (m, 23H, Hbd, Leu, Hbu, Orn),
cd,u
c
4.6.4. cyclo-[(2R,3S,4R,5R)-5-aminomethyl-3,4-dibenzyloxy-
1.40 (m, 1H, H Leu12) 1.11–0.82 (m, 36H, CH3 Val, CH3 Leu); 13C
c
tetrahydrofuran-2-carboxyl-Leu-Orn-Val-Orn-Leu-
Val-Orn-Leu- -Orn-Val] (8)
Peptide 18 (116 mg, 55.8
was deprotected using TFA/Tis/H2O (95/2.5/2.5) and subsequently
purified by semi-preparative RP–HPLC (linear gradient of 37–46, 3
D
-Phe-Pro-
NMR (151 MHz, CD3OH) d 174.55, 174.40, 173.97, 173.95, 173.66,
173.42, 173.31, 173.00, 172.97, 172.35, 138.89, 137.16, 130.41,
129.62, 129.45, 128.90, 128.79, 128.34, 86.86, 86.78, 84.44, 76.72,
72.59, 61.81, 60.91, 60.42, 55.49, 54.27, 53.88, 53.56, 53.31,
52.49, 52.38, 47.99, 43.52, 43.25, 42.14, 41.18, 40.53, 40.43,
40.33, 37.67, 32.27, 32.16, 31.49, 31.41, 30.64, 30.37, 29.86,
D
l
mol), as prepared described above,
CV and yielded 49.4 mg, 23.1 lmol, 42%; LCMS Rf 5.67 min, linear

A. D. Knijnenburg et al. / Bioorg. Med. Chem. 19 (2011) 3402–3409
3409
29.11, 25.95, 25.84, 25.72, 25.26, 24.82, 24.68, 23.78, 23.30, 23.06,
22.60, 21.97, 21.90, 19.91, 19.86, 19.76, 19.44, 18.89, 18.54.
3. (a) Berditsch, M.; Afonin, S.; Ulrich, A. S. Appl. Environ. Microbiol. 2007, 73,
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4.6.6. cyclo-[(2R,3S,4R,5R)-5-aminomethyl-3,4-
dihydroxytetrahydrofuran-2-carboxyl-Leu-Orn-Val-Orn-Leu-
Phe-Pro-Val-Orn-Leu- -Orn-Val] (10)
Peptide 19 (99 mg, 52.1 mol) was hydrogenated as described
D-
D
l
above. Subsequently 20 was deprotected using TFA/Tis/H2O (95/
2.5/2.5) and purified by preparative RP–HPLC (linear gradient of
31–40%, 3 CV and yielded 38.9 mg, 19.9 lmol, 38%; LCMS Rf
7. (a)Synthetic Aspects of Biologically Active Cyclic Peptides: Gramicidin
S and
Tyrocidines; Izumiya, N., Kato, T., Aoyaga, H., Waki, M., Kondo, M., Eds.; Halsted
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P. J. Am. Chem. Soc. 2005, 127, 5742.
4.98 min, linear gradient 10 ? 90% B in 13.5 min.; m/z = 1497.1
[M+H]+; HRMS (ESI) m/z 1496.95527 [M+H]+, Calcd 1496.95630 for
C
73H125N17O16; IR 3278.2, 2962.0, 1668.1, 1627.8, 1531.9, 1455.8,
1201.0, 1132.8, 836.6, 799.4, 721.8, 620.1; 1H NMR (600 MHz,
D
CD3OD) d 8.70 (d, J = 3.4, 1H, NH Phe13), 8.51 (t, J = 5.9, 1H, NH
SAA), 8.49 (d, J = 8.4, 1H, NH Orn2), 8.39 (d, J = 7.8, 2H, NH Orn4,9),
8.37 (d, J = 8.9, 1H, NH Leu12), 8.31 (d, J = 6.8, 1H, NH Leu8), 8.24 (d,
J = 6.8, 1H, NH Orn11), 8.08 (d, J = 7.3, 2H, NH Leu3 Val5), 7.98 (d,
J = 7.2, 1H, NH Val10), 7.88 (br s, 2H, NH2 Orn), 7.84 (d, J = 7.8, 1H,
NH Val1), 7.73 (br s, 2H, NH2 Orn), 7.28 (m, 10H, CH Ar), 4.74 (m,
8. (a) Solanas, C.; de la Torre, B. G.; Fernandez-Reyes, M.; Santiveri, C. M.; Jimenez,
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14. Knijnenburg, A. D.; Spalburg, E.; de Neeling, A. J.; Mars-Groenendijk, R. H.;
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1H, H Orn11), 4.65, (m, 1H, H Orn4), 4.63 (m, 1H, H Orn2), 4.62
a
a
a
D
(m, 1H, H Leu3), 4.62 (d, J = 3.4, 1H, H1 SAA), 4.60 (m, 1H, H PHe13),
a
a
4.55 (m, 1H, H Orn9), 4.51 (m, 1H, H Leu12), 4.50 (m, 1H, H Leu8),
a
a
a
4.37 (d, J = 5.9, 1H, H Pro14), 4.30 (d, J = 3.2, 1H, H2 SAA), 4.23 (t,
a
J = 7.4, 1H, H Val10), 4.13 (t, J = 6.7, 1H, H Val5), 4.06 (t, J = 8.0, 1H,
a
a
H
Val1), 4.06 (m, 1H, H4 SAA), 4.01 (m, 2H, H3, H5d SAA), 3.71 (m,
a
D
1H, Hdd Pro14), 3.06 (m, 3H, H5u SAA, Hbd,bu Phe13), 3.03–2.89 (m,
8H, Hdd,du Orn), 2.70 (m, 1H, Hdu Pro14), 2.21 (dd, J = 7.1, 14.3, 1H,
Hb Val1), 2.16 (dd, J = 6.8, 13.4, 1H, Hb Val5), 2.09 (dd, J = 6.8, 13.6,
1H,Hb Val5), 2.01 (m, 1H, Hbd Pro) 1.91 (m, 4H Hbd Orn), 1.83–1.44
(m, 23H, Hbd,u, Leu, Hbu,, d,u Orn), 1.41 (m, 1H, H Leu12), 1.13–0.77
c
c
c
(m, 36H, CH3 Val, CH3 Leu); 13C NMR (151 MHz, CD3OD) d 174.58,
174.37, 173.92, 173.61, 173.49, 173.40, 172.93, 172.77, 172.73,
172.56, 162.92, 162.69, 138.38, 136.93, 130.38, 129.64, 129.52,
129.11, 128.78, 128.42, 86.16, 83.02, 77.26, 72.21, 61.87, 60.94,
60.77, 59.42, 55.73, 54.05, 53.63, 53.45, 53.19, 52.76, 51.79, 47.90,
43.42, 42.15, 42.07, 41.47, 40.75, 40.71, 40.60, 40.43, 37.47, 33.56,
31.47, 30.71, 30.64, 30.51, 30.40, 30.26, 29.82, 25.78, 25.75, 25.63,
25.16, 25.00, 24.68, 24.55, 23.90, 23.31, 23.02, 22.88, 22.29, 21.62,
19.88, 19.83, 19.68, 19.54, 19.37, 18.95.
Acknowledgements
This work was supported by the Leiden Institute of Chemistry
and grant BFU2008-01588 from the Spanish Ministry of Science
and Innovation. Assistance in performing NMR measurements by
C. Erkelens, A. W. M. Lefeber and K. Babu is kindly acknowledged.
We thank Hans van den Elst for recording HRMS data and main-
taining LC/MS systems. We thank ESRF-Grenoble for provision of
beamtime on beamline BM14. J.M.O. thanks the Xunta de Galicia
and Spanish Ministry of Science and Innovation for ‘Ángeles Alvar-
iño’ and ‘José Castillejo’ fellowships, respectively.
17. Jelokhani-Niaraki, M.; Prenner, E. J.; Kondejewski, L. H.; Kay, C. M.; McElhaney,
R. N.; Hodges, R. S. J. Pep. Res. 2001, 58, 293.
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K. NMR of Proteins and Nucleic Acids; John Wiley & Sons: New York, 1986.
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Sykes, B. D. In Techniques in Protein Chemistry VI; Crabb, J. W., Ed.; Academic
Press: Orlando, 1995; pp 451–458.
Supplementary data
21. Blanco, F. J.; Jimenez, M. A.; Pineda, A.; Rico, M.; Santoro, J.; Nieto, J. L.
Biochemistry 1994, 33, 6004.
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1986, 359, 499.
Supplementary data (Table S1 (Amide coupling constants and
chemical shift pertubations) and table S2 (selected crystallographic
data of 2)can be found in the supporting information. In addition 1H
spectra of 2,4, 8–10 are given.) associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.2011.04.031.
25. Griesinger, C.; Ernst, R. R. J. Magn. Reson. 1987, 75, 261.
26. 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, 1924.
27. Leslie, A. G. W. Acta Crystallogr. 2006, D62, 48.
28. Evans, P. Acta Crystallogr. 2006, D62, 72.
References and notes
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