Structural and biological evaluation of some loloatin C analogues

Article abstract of DOI:10.1016/j.bmc.2009.07.049

Loloatin C is a cyclic cationic antimicrobial peptide which is active against Gram positive as well as certain Gram negative bacteria. Unfortunately, it is equally potent against human erythrocytes. To probe the structure-activity relationship of this promising antibiotic peptide, amino acid substitution and/or incorporation of a constraint sugar amino acid dipeptide isoster has been applied. Six new derivatives have been synthesized using SPPS and their solution structure investigated using NMR studies. Finally, the antimicrobial and the hemolytic activities have been determined.

Full text of DOI:10.1016/j.bmc.2009.07.049

Bioorganic & Medicinal Chemistry 17 (2009) 6233–6240
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structural and biological evaluation of some loloatin C analogues
Adriaan W. Tuin a, Gijsbert M. Grotenbreg a, Emile Spalburg b, Albert J. de Neeling b,
Roos H. Mars-Groenendijk c, Gijsbert A. van der Marel a, Daan Noort c, Herman S. Overkleeft a,
,
*
Mark Overhand a,
*
a Bioorganic Synthesis, Leiden Institute of Chemistry, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands
b Institute of Public Health and the Environment, Research Laboratory for Infectious Diseases, PO Box 1, 3720 BA Bilthoven, The Netherlands
c TNO Defense, Security and Safety, Busisness unit Biological and Chemical Protection, PO Box, 45, 2280 AA Rijswijk, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
Loloatin C is a cyclic cationic antimicrobial peptide which is active against Gram positive as well as cer-
tain Gram negative bacteria. Unfortunately, it is equally potent against human erythrocytes. To probe the
structure–activity relationship of this promising antibiotic peptide, amino acid substitution and/or incor-
poration of a constraint sugar amino acid dipeptide isoster has been applied. Six new derivatives have
been synthesized using SPPS and their solution structure investigated using NMR studies. Finally, the
antimicrobial and the hemolytic activities have been determined.
Received 15 June 2009
Accepted 22 July 2009
Available online 25 July 2009
Keywords:
Loloatin C
Sugar amino acid
Ó 2009 Elsevier Ltd. All rights reserved.
Structure–activity relationship
Cationic antimicrobial peptide
1. Introduction
amphiphilic orientation of the individual side chains, with the
hydrophobic Val and Leu residues on one side of the molecule and
The loloatins are cationic antimicrobial peptides (CAPs) isolated
from laboratory cultures of bacteria collected from the reefs of Lol-
oata Island, Papua New Guinea.1 The loloatins comprise four cyclic
peptides that are named loloatin A, B, C and D (Fig. 1). Loloatins A–
D are active against Gram positive bacteria with MIC values be-
the hydrophilic Orn side chains on the other. The solution structure
of loloatin C is much less well defined,4 and is found to be solvent
dependent. In DMSO, a hydrogen bond accepting solvent, the sec-
ondary structure of loloatin C was analyzed by means of chemical
shift perturbation and NOESY analysis. A b-strand composed of
-helical5 Trp- Phe-Asn strand are
D
tween 1 and 8
l
g/mL against a panel of eight bacterial strains
Leu-Orn-Val and an opposing
a
tested.2 Besides being the most potent of the four family members,
loloatin C is also the only one active against the Gram negative
strain Escherichia coli, making it an interesting lead compound for
further development towards broad spectrum antibiotics.
interconnected via two turn motifs composed of Pro- Tyr and
Asp-Trp. The lack of chemical shift reference data in water/trifluoro-
ethanol (TFE, a hydrogen bond donating solvent known to stabilize
secondary structural elements)6 defies conclusions about the con-
formation of loloatin C. In a 30% TFE/H2O mixture, NOESY analysis
D
Loloatin C has a remarkable structural resemblance to Gramici-
din S (GS). Both are cyclic decapeptides, and share 4 out of the 10
amino acids in their primary sequence. GS is composed of two iden-
tical pentapeptides, each consisting of a b-strand (Val-Orn-Leu) con-
revealed a less defined structure with only one secondary structural
D
element, namely, an inverse
c
-turn around the Tyr-Pro-Trp se-
quence. Increasing the TFE content in the solvent mixture to 70% in-
duced a remarkable structural change. A dumbell-like conformation
D
nected to a type II’ b-turn motif ( Phe-Pro). Loloatin C has one closely
D
D
D
related pentapeptide motif (with a Tyr instead of a Phe) connected
was identified centred around the Orn and Phe residues. Interest-
D
to a Trp- Phe-Asn-Asp-Trp sequence. Whereas GS is likely to carry
ingly, this conformation orientates the hydrophilic sidechains
(Orn, Asn and Asp) to one side of the molecule and the remaining
hydrophobic side chains to the other, resulting in an amphiphilic
overall structure.
two positive charges in biological surroundings, loloatin C has a
zwitterionic character. The solution structure of GS has been exten-
sively studied in a variety of solvents.3 In each of these solvents GS
adopts a rigid cyclic b-hairpin conformation that is stabilized by four
interstrand hydrogen bonds. This structure is characterized by an
We have previously reported on GS analogue 7 that contains a
sugar amino acid (SAA) dipeptide isoster7 derived from 6 as
8
D
replacement of one of the two Pro- Phe b-turns. We found that
the secondary structure of this derivative differs slightly from that
of GS, but that the biological activities of the two compounds were
closely related. We here report on the synthesis and evaluation of a
* Corresponding authors. Tel.: +31 71 527 4483; fax: +31 71 527 4307 (M.O.).
E-mail addresses: h.s.overkleeft@lic.leidenuniv.nl (H.S. Overkleeft), overhand@
chem.leidenuniv.nl (M. Overhand).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.07.049

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A. W. Tuin et al. / Bioorg. Med. Chem. 17 (2009) 6233–6240
Figure 1. Structures of loloatins A–D, GS and sugar amino acid 6 containing GS derivative 7.
set of loloatin C analogues modified in both strand and turn re-
gions, and that include derivatives containing SAA 6 as a replace-
ment of the turn region.
2. Results and discussion
The target molecules are depicted in Figure 2. In compound 8,
D
SAA 6 replaces the Pro- Tyr motif (Fig. 2). In compound 9 the
Figure 2. Loloatin C derivatives 8–13 that are the subject of the research described here.

A. W. Tuin et al. / Bioorg. Med. Chem. 17 (2009) 6233–6240
6235
D
Asn- Phe sequence is replaced (Fig. 2). Replacing the Asp-Trp motif
measured chemical shift of the
a-proton of a certain amino acid
with SAA 6 leads to compound 10, whereas analogue 11 (Fig. 2)
and the standard value of that same amino acid in a random coil
configuration (
dH = observed dH À random coil dH ). This is
based on the observation that
an -helix experience an upfield shift and residues within a
D
L
SAA 6 replaces the Asp-Trp motif and the Phe is replaced by Phe.
D
a
a
a
L
D
The influence of using Phe instead of Phe is further studied in 12.
a-protons of amino acids within
L
D
Compound 13 is analogous to 12, but with Asp replaced by Asp
(Fig. 2).
a
b-sheet experience a downfield shift compared to the chemical
The synthesis of the loloatin C analogues is exemplified in
Scheme 1 for the construction of compound 8. Standard Fmoc
based solid-phase peptide synthesis (SPPS) starting from commer-
cially available HMPB-BHA resin preloaded with Fmoc-Leu is
deprotected using 20% piperidine in NMP and subjected to seven
coupling cycles under the agency of HCTU en DIPEA affording lin-
ear octapeptide 14. Sugar amino acid 6 was coupled to deprotected
14 using 1.5 equiv of 6, with prolonged coupling time. Staudinger
reduction of the terminal azide functionality in 15 using PMe3 in
wet THF followed by acidic cleavage from the resin afforded 16
which was subsequently cyclized upon PyBOP/HOBt/DIPEA treat-
ment in DMF under dilute conditions, deprotected using 50%TFA/
DCM. HPLC purification yielded the target compound 8 in 7% yield.
Compounds 9–13 were prepared following the same strategy in
yields ranging from 3% to 37% after purification.
shift of that residue in a random coil configuration.
The observed values for the vicinal coupling constants JNH-H
for the loloatin C (3) and analogues are depicted in Figure 3A and
3
a
the chemical shift perturbations are depicted in Figure 3B. The sec-
D
ondary structure of all derivatives contain a turn motif around Tyr
(3JNH-H <4 Hz), flanked by two strand regions (8.5 < 3JNH-
a
< 9.5 Hz). The Leu-Orn-Val sequence forms a b-strand in all ana-
Ha
logues. Interpretation of the chemical shift perturbation underlines
/
L
D
these findings. The residues in the Trp6- Phe-Asn motif show both
positive and negative values for the perturbation among the differ-
ent compounds and no general trend is observed. In compound 10,
D
D
the Tyr, Trp6 and Phe residues appear to form a helical region/
turn, as evidenced by their negative chemical shift perturbation
values. The position of residues 9 and 10 in these analogues are
(
or )
L
D
connected either by SAA 6 (in 11) or by
Asp-Trp motif (in
12 and 13, respectively). On the basis of these 1D NMR analyses
the structures of compounds 11 and 12 seem remarkably similar
3. NMR studies of loloatin C (3) and derivatives 8–13
except for the regions round the Phe residues. The 1H NMR spec-
L
tra of
9 and 13 show severe peak broadening indicating
Next, attention was focused on the structural analysis of lol-
oatin C (3) and the newly designed analogues 8–13. The NMR
spectra of our previously reported7 GS derivatives were mea-
sured using CD3OH as solvent. Therefore, the structure of each
conformational flexibility, preventing complete assignment of
their spectra.
To obtain more details concerning the structural properties of
loloatin C and derivatives NOESY spectra were recorded. For this
purpose NOESY spectra of loloatin C (3) in CD3OH and in TFE/
H2O 7/3 were compared. At 500 MHz the amide region of com-
pound 3 in TFE/H2O showed only poor resolution at ambient tem-
perature hampering the assignment of the 2D spectra. Increasing
the temperature resulted in an improved resolution with an opti-
mum at 305 K (Fig. 4A). Despite the elevated temperature four
NOE signals could be identified in the NH–NH region (Fig. 4B). In
methanol, well resolved spectra at ambient temperature were ob-
tained (Fig. 4C). Two important long range NOE’s are observed in
of the loloatine analogues was assessed in methanol by the anal-
3
ysis of the vicinal JNH-H coupling constant9 and the chemical
a
shift perturbation.10 The vicinal JNH-H coupling constant corre-
3
a
lates with the dihedral angle within an amino acid residue. It
can be used in a qualitative manner to identify secondary struc-
tural elements within a peptide. Consecutive large values (8.5–
9.5 Hz) correspond to dihedral angles commonly found in b-
3
sheets, small values for JNH-H (3.5–4.5 Hz) correspond to those
a
found in an
a-helix/turns. Isolated occurrences of small values
3
for JNH-H (<4 Hz) indicate the presence of a turn within the
a
D
both solvents, with the NH–NH NOE between Phe and Val (B in
Fig 4B and C) being the most prominent. Besides minor differences
in these two solvent systems, loloatin C is folded back onto itself in
peptide chain. Another qualitative method to distinguish
between secondary structural elements is the chemical shift
perturbation. This method relies of the comparison of the
Scheme 1. Synthesis of lolatin C derivatives using SPPS. Reagents and conditions: 7 cycles of (a) 20% pip/NMP, 3 Â 5 min; (b) Fmoc-AA-OH (5 equiv), HCTU (5 equiv), DIPEA
(10 equiv) 90 min; (ii) 6 (1.5 equiv), HCTU (1.5 equiv), DIPEA (3 equiv), 16 h; (iii) (a) PMe3 (1 M in 9:1 THF/H2O, 25 equiv), 16 h; (b) 1% TFA/DCM; (iv) (a) PyBOP (5 equiv),
HOBt (5 equiv), DIPEA (10 equiv), 16 h; (b) 50% TFA/DCM, 1 h.

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A. W. Tuin et al. / Bioorg. Med. Chem. 17 (2009) 6233–6240
3
Figure 3. Values of the vicinal JNH-H (A) and of the chemical shift perturbation (B) for loloatin C (3) and derivatives 8–13 in CD3OH. For some residues, amide protons
a
appeared as broad singlets, a-protons were buried under other peaks or residues were replaced by 6 resulting in their omission.
agreement with adopting a dumbbell-like structure as described in
the literature.4
gram negative strains used in this assay. In addition, loloatin C
is also hemolytically active.11 As for the analogues 8, 9, 10 and
13 both the antimicrobial and hemolytical12 activities are highly
reduced compared to loloatin C. Only analogues 11 and 12 are
marginally active in both assays.
Because of the ease of recording NMR spectra at ambient
temperature the NOESY spectra of compounds 8, 10, 11 and 12
were for comparison also recorded in methanol. In Figure 4D,
E and F parts of the NH–NH regions of compounds 8, 11 and
12 are depicted. Compound 8 has a distorted turn region, pre-
sumably due to the presences of the sugar amino acid moiety
5. Conclusion and discussion
D
and the Phe residue (Fig. 4D). The NOE’s of compound 11 and
Six novel analogues of loloatin C were synthesized and analyzed
by means of NMR spectroscopy. The unexpectedly low yield for
some of the derivatives can be explained by a difficult macrocycli-
zation and a laborious HPLC purification. The secondary structures
of these analogous were analyzed using 1D- and 2D-NMR spectros-
copy. Analogues 3, 8, 10, 11 and 12 adopt a secondary structure in
solution (methanol), compounds 9 and 13 proved to have multiple
conformations in the same solvent. The peak broadening of certain
NH signals observed in the NMR spectrum of loloatin C (3) indicate
partial conformational flexibility, however, a dumbbell-like confor-
mation4 is apparent as judged by the observed long range NOE’s.
The secondary structures of compounds 8, 10, 11 and 12 as judged
by the NMR experiments differs substantially from the loloatin C
structure.
12 indicate beta-hairpin structures similar to gramicidine
S
(Fig. 4E and F). Compound 10 does not have long range NH–
NH NOE’s. Sharp NH signals in the proton spectrum, however to-
gether with several other NOE correlations are indicative of the
presence of a secondary structure.
4. Biological evaluation
The antimicrobial and hemolytical potencies of the novel lol-
oatin
C derivatives were determined (Table 1 and Fig. 5).
Whereas the native loloatin C (3) shows some antimicrobial po-
tency against gram positive strains, it is inactive against the

A. W. Tuin et al. / Bioorg. Med. Chem. 17 (2009) 6233–6240
6237
Figure 4. (A) The amide region of the 1D proton spectrum (500 MHz) of Loloatin C in TFE/H2O 7/3 at different temperatures. (B) A close-up of the NH–NH region of Loloatin C
in TFE/H2O 7/3 at 305 K and assigned correlations. (C–F) Close-ups of the NH–NH region of the NOE spectra (600 MHz) in CD3OH of Loloatin C (C), 8 (D), 11 (E) and 12 (F) and
the assigned correlations. Dashed arrows and lowercase signals indicate sequential NOEs, solid lines and uppercase are long range NOEs.

6238
A. W. Tuin et al. / Bioorg. Med. Chem. 17 (2009) 6233–6240
Table 1
MIC values for compounds 3 and 8–13
Gram pos. Staph. aureus
Gram pos. Staph. epidermis
Gram pos. Enterococ faecalis
Gram. neg. E. coli
Gram neg. P. auriginosa
Gram pos. Bacillus cereus
3
8
9
10
11
12
13
8
8
8
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
8
>64
>64
>64
>64
64
>64
>64
>64
32
32
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
64
32
>64
>64
6.1. General procedure for peptide synthesis
(a) Stepwise elongation: Fmoc-Leu-HMPB-BHA resin (196 mg,
0.51 mmol/g, 0.1 mmol) was submitted to seven cycles of Fmoc
solid-phase synthesis with the appropriate commercially available
amino acid building blocks Fmoc-Orn(Boc)-OH, Fmoc-Val-OH,
D
Fmoc-Tyr(tBu)-OH, Fmoc-Pro-OH, Fmoc- Phe-OH, Fmoc-Phe-OH,
Fmoc-Asp(tBu)-OH, Fmoc-Asn(Trt)-OH and Fmoc-Trp(Boc)-OH as
follows: (a) deprotection with piperidine/NMP (1/4, v/v, 5 mL,
15 min); (b) wash with NMP (5 mL, 3Â, 3 min); (c) coupling of
the appropriate Fmoc amino acid (5 equiv, 0.5 mmol) in the pres-
ence of HCTU (5 equiv, 0.5 mmol, 206 mg) and DIPEA (10 equiv,
1 mmol, 162 lL) which was preactivated for 2 min in NMP
(5 mL) and shaken for 90 min; (d) wash with NMP (5 mL, 3Â,
3 min). Couplings were monitored for completion by the Kaiser
test.14 Finally, the N-terminal amine was liberated by Fmoc-depro-
tection with piperidine/NMP (1/4, v/v, 5 mL, 15 min) followed by
washing with NMP (5 mL, 3Â, 3 min). Coupling of SAA 6 was per-
formed as follows: To the resin-bound peptide, a preactivated solu-
tion of SAA 6 (1.5 equiv, 44 mg, 0.150 mmol), HCTU (1.5 equiv,
Figure 5. Hemolytic activity of loloatin C and derivatives 8–13.
The biological activities of all analogues were rather low as
compared with loloatin C with only marginal activities of ana-
logues 11 and 12 in both the antimicrobial and hemolytical assay.
The reduced activity of analogues 8–13 are likely caused by
their inability to adopt a similar amphiphilic biologically active
conformation as the parent compound loloatin C. Thus, the strat-
egy of replacing particular dipeptide sequences with the constraint
sugar amino acid 6, as successfully applied in the case of the cat-
ionic cyclic decapeptide GS, does not work in the case of the related
zwitterionic peptide loloatin C.
62 mg, 0.150 mmol) and DIPEA (3.0 equiv, 74 lL, 0.45 mmol) in
NMP (3 mL) was added and the resulting suspension was shaken
for 16 h. The resin was finally washed with NMP (5 mL, 3Â,
3 min) to give the cyclized peptide.
(b) On-resin Staudinger reduction: The appropriate resin-bound
azide was treated with a pre-mixed cocktail of H2O (0.5 mL) and
PMe3 (3.5 mL, 1 M in THF) and shaken for 16 h. The resin was
washed with methanol (4 mL, 3Â, 3 min) and DMF (4 mL, 3Â,
3 min).
(c) Cleavage from the resin: Resin-bound nonapeptide was
repeatedly treated with 1% TFA/DCM (4 mL). The filtrate was
coevaporated with toluene (3Â) and the residue was used as such
in the cyclization step.
(d) Cyclization: The linear nonapeptide was taken up in DMF
(5 mL) and added dropwise over the course of an hour to a solution
of benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexa-
fluorophosphate (PyBOP) (5 equiv, 270 mg, 0.5 mmol), HOBt
6. Experimental section
For LC/MS analysis, an JASCO HPLC system (detection simulta-
neously at 214 and 254 nm) equipped with an analytical C18 col-
umn (4.6 mmD Â 250 mmL, 5
l particle size) in combination with
buffers A: H2O, B: MeCN and C: 0.5% aq TFA and coupled to a mass
instrument with a custom-made Electronspray Interface (ESI) was
used. For reversed-phase HPLC purification of the final compounds,
an automated HPLC system supplied with a semi-preparative C18
(5 equiv, 67 mg, 0.5 mmol) and DIPEA (15 equiv, 254 lL, 1.5 mmol)
in DMF (70 mL) and allowed to stir for 16 h. The solvent was re-
moved in vacuo and the resulting mixture was used without fur-
ther purification in the deprotection step.
(e) Deprotection: The crude cyclized peptide was treated with
50% TFA/DCM (10 mL) for 1 h, before it was concentrated and puri-
fied by HPLC purification.
column (10.0 mmD Â 250 mmL, 5
plied buffers were A: H2O, B: MeCN and C: 1.0% aq TFA. High res-
olution mass spectra were recorded by direct injection (2 L of a
M solution in water/acetonitrile; 50/50; v/v and 0.1% formic
l particle size) was used. The ap-
l
2
l
acid) on a mass spectrometer (Thermo Finnigan LTQ Orbitrap)
equipped with an electrospray ion source in positive mode (source
voltage 3.5 kV, sheath gas flow 10, capillary temperature 250 °C)
with resolution R = 60,000 at m/z 400 (mass range m/z = 150–
2000) and dioctylpthalate (m/z = 391.28428) as a ‘lock mass’.13
The high resolution mass spectrometer was calibrated prior to
measurements with a calibration mixture (Thermo Finnigan). 1H
and 13C NMR spectra were measured on a Bruker AV-500 (500/
125 MHz) or a Bruker DMX-600 (600/125 MHz). Chemical shifts
are given in ppm (d) relative to TMS (0 ppm) or MeOD
(3.30 ppm) and coupling constants are given in Hz.
6.2. Antibacterial assays
The following bacterial strains were used: Staphylococcus aureus
(ATCC 29213), Staphylococcus epidermidis (ATCC 12228), Enterococ-
cus faecalis (ATCC 29212), Bacillus cereus (ATCC 11778),E. coli (ATCC
25922) and Pseudomonas aeruginosa (ATCC 27853). 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 density of 0.1 AU (at 595 nm, 1 cm cuvette). The

A. W. Tuin et al. / Bioorg. Med. Chem. 17 (2009) 6233–6240
6239
suspension was diluted (10Â) with physiological saline, and 2
l
L of
(m, 2H), 1.85–1.76 (m, 1H), 1.72–1.57 (m, 4H), 1.57–1.46 (m,
2H), 1.36–1.34 (m, 1H), 1.32–1.25 (m, 2H), 1.25–1.16 (m, 2H),
1.01 (d, J = 6.7, 3H), 0.97 (d, J = 6.6, 3H), 0.85 (d, J = 5.9, 3H), 0.79
(d, J = 5.9, 3H).
this inoculum was added to 100 L growth medium, Nutrient
l
Broth from Difco (ref. nr. 234000, lot nr. 6194895) with yeast ex-
tract (Oxoid LP 0021, lot nr. 900711, 2 g/400 mL broth) in microti-
ter plates (96 wells). The peptides were dissolved in ethanol (4 g/L)
and diluted in distilled water (1000 mg/L), and twofold diluted in
the broth (64, 32, 16, 8, 4 and 1 mg/L). Incubation at 30 °C (24–
96 h) and the MIC was determined as the lowest concentration
inhibiting bacterial growth.
D
6.3.3. cyclo-[Val-Orn-Leu- Tyr-Pro-Trp-SAA-Asp-Trp] 9
Prepared according to the general procedure. Yield: 29.8 mg,
22.5 lmol, 23%. LC–MS: tR 6.04 min (linear gradient 10?90% B
in 13.5 min), m/z = 1324.0 [M+H]+, 662.8 [2M+H]+. 1H NMR
(600 MHz, MeOD) d 10.67 (s, 1H), 10.65 (s, 1H), 10.32 (s, 1H),
8.99 (s, 1H), 8.38 (d, J = 9.5, 1H), 7.85 (br s, 1H), 7.82 (br s, 1H),
7.74 (d, J = 7.7, 2H), 7.57 (d, J = 8.1, 1H), 7.55 (br s, 1H), 7.49 (d,
J = 7.8, 1H), 7.45 (s, 1H), 7.38 (d, J = 7.6, 3H), 7.32 (t, J = 7.5, 2H),
7.27 (t, J = 7.9, 2H), 7.22–7.14 (m, 3H), 7.08 (s, 1H), 7.03 (dd,
J = 7.2, 12.4, 2H), 6.96 (t, J = 7.4, 1H), 6.92 (d, J = 8.3, 2H), 6.83–
6.73 (m, 1H), 6.65 (d, J = 8.4, 2H), 4.65–4.59 (m, 2H), 4.59–4.50
(m, 3H), 4.33 (d, J = 14.4, 1H), 4.29 (d, J = 13.2, 2H), 4.19 (t, J = 5.0,
1H), 4.15 (br s, 1H), 4.11–4.06 (m, 2H), 4.06–4.00 (m, 1H), 3.90
(d, J = 8.1, 1H), 3.88 (br s, 1H), 3.72 (s, 1H), 3.52–3.45 (m, 2H),
3.45–3.35 (m, 1H), 3.34 (s, 1H), 3.28–3.16 (m, 4H), 2.90 (t,
J = 12.6, 1H), 2.77 (dt, J = 5.4, 17.6, 1H), 2.77 (br s, 1H), 2.61 (br s,
1H), 2.19–2.06 (m, 2H), 2.01–1.87 (m, 2H), 1.78–1.69 (m, 1H),
1.63 (dd, J = 11.0, 22.2, 2H), 1.60–1.53 (m, 2H), 1.47–1.38 (m,
2H), 1.38–1.25 (m, 4H), 1.25–1.17 (m, 7H), 1.15–1.06 (m, 2H),
1.06–0.98 (m, 5H), 0.98–0.81 (m, 6H), 0.74–0.62 (m, 1H), 0.61–
0.47 (m, 1H), 0.44–0.29 (m, 1H).
6.3. Hemolytic assays
Freshly drawn heparinized blood was centrifuged for 10 min at
1000g 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 to be evalu-
ated were dissolved in a 30% DMSO/0.5 mM saline solution to give
a 1.5 mM solution of peptide. If a suspension was formed, the sus-
pension was sonicated for a few seconds. A 1% Triton-X solution
was prepared. Subsequently, 100
pensed in columns 1–11 of a microtiter plate, and 100
ton solution was dispensed in column 12. To wells A1-C1, 100
l
L of saline solution was dis-
L of 1% Tri-
L of
l
l
the peptide was added and mixed properly. Hundred microlitres of
wells A1-C1 were dispensed into wells A2-C2. This process was re-
peated until wells A10-C10, followed by discarding 100
A10-C10. These steps were repeated for the other peptides. Subse-
quently, 50 L of the red blood cell solution was added to the wells
lL of wells
l
and the plates were incubated at 37 °C for 4 h. After incubation, the
plates were centrifuged at 1000g at 10 °C for 4 min. In a new
microtitre plate, 50 lL of the supernatant of each well was dis-
pensed into a corresponding well. The absorbance at 405 nm was
measured and the percentage of hemolysis was determined.
D
D
6.3.4. cyclo-[Val-Orn-Leu- Tyr-Pro-Trp- Phe-Asn-SAA] 10
Prepared according to the general procedure. Yield: 4.0 mg,
3.0 lmol, 3%. LC–MS: tR 6.14 min (linear gradient 10?90% B in
13.5 min), m/z = 1336.0 [M+H]+, 668.7 [2M+H]+. 1H NMR
(600 MHz, CD3OH) d 10.30 (s, 2H), 8.95 (s, 1H), 8.79 (d, J = 8.4,
1H), 8.59 (d, J = 8.1, 1H), 8.55 (d, J = 7.7, 1H), 8.49 (d, J = 8.7, 1H),
8.18 (br s, 1H), 8.14 (d, J = 7.7, 1H), 8.02 (d, J = 9.0, 1H), 7.78 (d,
J = 9.2, 2H), 7.73 (d, J = 7.9, 1H), 7.55 (d, J = 7.9, 1H), 7.32 (d,
J = 8.1, 1H), 7.27 (d, J = 8.2, 1H), 7.21–7.14 (m, 4H), 7.14–7.09 (m,
4H), 7.09–6.96 (m, 10H), 6.94 (t, J = 7.4, 2H), 6.66 (d, J = 8.4, 2H),
5.41–5.31 (m, 1H), 4.82–4.75 (m, 1H), 4.69 (dd, J = 7.9, 15.5, 1H),
4.60–4.54 (m, 1H), 4.46 (t, J = 8.0, 1H), 4.39–4.33 (m, 1H), 4.16
(dd, J = 2.4, 7.5, 1H), 4.12–4.07 (m, 1H), 3.37 (d, J = 7.6, 3H), 3.26
(d, J = 9.3, 1H), 3.18 (dd, J = 4.4, 14.4, 1H), 3.07 (dd, J = 6.8, 13.8,
1H), 3.05–2.98 (m, 2H), 2.96 (dd, J = 4.7, 12.7, 2H), 2.91–2.74 (m,
5H), 2.72–2.55 (m, 3H), 2.27 (dd, J = 9.5, 17.2, 1H), 2.20 (dd,
J = 9.3, 17.1, 1H), 2.13 (dd, J = 6.8, 13.7, 1H), 1.96–1.88 (m, 1H),
1.79–1.72 (m, 1H), 1.72–1.61 (m, 6H), 1.61–1.54 (m, 2H), 1.44–
1.33 (m, 3H), 1.31–1.24 (m, 1H), 1.02–0.91 (m, 18H), 0.43–0.32
(m, 1H).
D
D
6.3.1. cyclo-[Val-Orn-Leu- Tyr-Pro-Trp- Phe-Asn-Asp-Trp] 3
Prepared according to the general procedure. Yield: 19.7 mg,
13.5 lmol, 14%. LC–MS: tR 7.26 min (linear gradient 10?90% B
in 13.5 min), m/z = 1335.6 [M+H]+, 668.5 [2M+H]+. 1H NMR
(500 MHz, CD3OH) d 10.25 (s, 1H), 10.22 (s, 1H), 9.43 (bs, 1H),
9.34 (br s, 1H), 9.23 (br s, 1H), 8.93 (br s, 1H), 8.80 (d, J = 9.3,
1H), 8.68 (br s, 1H), 8.28 (d, J = 2.6, 1H), 8.14 (s, 1H), 8.00 (d,
J = 8.6, 1H), 7.65 (d, J = 7.7, 1H), 7.55 (dd, J = 13.3, 29.2, 4H), 7.43–
7.32 (m, 3H), 7.28–7.13 (m, 5H), 7.09–6.89 (m, 6H), 6.84 (s, 1H),
6.72 (br s, 1H), 6.63 (d, J = 8.1, 2H), 5.94–5.84 (m, 1H), 5.52 (d,
J = 5.9, 1H), 4.78–4.68 (m, 1H), 4.68–4.60 (m, 2H), 4.32 (br s, 2H),
4.06 (d, J = 7.1, 1H), 3.65–3.63 (m, 1H), 3.45–3.35 (m, 2H), 3.22–
3.00 (m, 4H), 3.00–2.79 (m, 4H), 2.64 (t, J = 13.5, 1H), 2.40–2.14
(m, 5H), 2.14–2.05 (m, 1H), 2.03–1.93 (m, 2H), 1.93–1.84 (m,
2H), 1.83–1.75 (m, 4H), 1.74–1.65 (m, 2H), 1.61–1.48 (m, 2H),
1.39–1.23 (m, 3H), 1.20 (s, 3H), 1.16 (m, 6H), 1.12–1.04 (m, 6H),
0.94–0.82 (m, 1H), 0.19 (s, 1H).
D
6.3.5. cyclo-[Val-Orn-Leu- Tyr-Pro-Trp-Phe-Asn-SAA] 11
Prepared according to the general procedure. Yield: 46.6 mg,
D
36.6 lmol, 37%. LC–MS: tR 6.34 min (linear gradient 10?90% B
6.3.2. cyclo-[Val-Orn-Leu-SAA-Trp- Phe-Asn-Asp-Trp] 8
in 13.5 min), m/z = 1283.9 [M+H]+, 642.8 [2M+H]+. 1H NMR
(600 MHz, MeOD) d 10.41 (br s, 1H), 8.99 (d, J = 3.1, 1H), 8.82 (d,
J = 8.4, 1H), 8.78 (d, J = 9.1, 2H), 8.72 (d, J = 8.4, 2H), 8.64 (d,
J = 7.6, 2H), 7.94 (d, J = 8.4, 1H), 7.85–7.79 (m, 5H), 7.70 (d,
J = 8.6, 2H), 7.69–7.65 (m, 2H), 7.63–7.60 (m, 1H), 7.57–7.53 (m,
1H), 7.51–7.48 (m, J = 12.5, 1H), 7.40–7.37 (m, 5H), 7.37–7.30 (m,
7H), 7.29–7.22 (m, 7H), 7.18–7.14 (m, 1H), 7.12 (t, J = 7.3, 2H),
7.07–6.99 (m, 5H), 5.18 (q, J = 7.6, 1H), 5.02 (s,1H), 4.94–4.85 (m,
1H), 4.82–4.76 (m, 1H), 4.76–4.71 (m, 1H), 4.71–4.67 (m, 2H),
4.62 (d, J = 11.7, 1H), 4.57–4.47 (m, 5H), 4.43–4.36 (m, 2H), 4.28–
4.25 (m, 1H), 4.24 (d, J = 3.0, 1H), 4.17 (dd, J = 1.7, 8.2, 1H), 4.12
(t, J = 10.6, 1H), 3.92 (d, J = 2.8, 2H), 3.81 (d, J = 16.7, 1H), 3.76–
3.70 (m, 1H), 3.65 (s, 1H), 3.31 (dd, J = 9.1, 14.7, 18H), 3.23–3.18
(m, 25H), 3.03–2.96 (m, 3H), 2.89–2.81 (m, 3H), 2.80–2.73 (m,
Prepared according to the general procedure. Yield: 9.73 mg,
6.76 lmol, 7%. LC–MS: tR 5.60 min (linear gradient 10?90% B in
13.5 min), m/z = 1324.7 [M+H]+, 663.1 [2M+H]+. 1H NMR
(500 MHz, CD3OH) d 10.39 (s, 1H), 10.33 (s, 1H), 8.88 (d, J = 7.1,
1H), 8.53 (d, J = 8.3, 1H), 8.39 (d, J = 8.9, 1H), 8.34 (d, J = 3.4, 1H),
8.25 (d, J = 8.2, 1H), 8.19 (d, J = 9.3, 1H), 8.08–8.03 (m, 1H), 7.98–
7.93 (m, 1H), 7.75 (d, J = 8.9, 1H), 7.73 (s, 1H), 7.66 (d, J = 7.8,
1H), 7.52 (d, J = 7.9, 1H), 7.38 (br s, 1H), 7.34–6.94 (m, 20H), 5.48
(d, J = 7.7, 1H), 4.82 (dd, J = 8.1, 13.8, 1H), 4.71 (dt, J = 4.8, 10.2,
1H), 4.67–4.62 (m, 1H), 4.56 (d, J = 11.7, 1H), 4.52 (d, J = 11.6,
1H), 4.46–4.39 (m, 1H), 4.38–4.30 (m, 3H), 4.21 (d, J = 10.0, 2H),
3.84 (s, 1H), 3.76–3.68 (m, 1H), 3.34 (s, 1H), 3.24–3.15 (m, 3H),
3.11–3.04 (m, 3H), 2.99–2.92 (m, 1H), 2.91–2.79 (m, 3H), 2.73 (br
s, 1H), 2.69 (dd, J = 3.9, 17.4, 2H), 2.30–2.22 (m, 1H), 2.06–1.94

6240
A. W. Tuin et al. / Bioorg. Med. Chem. 17 (2009) 6233–6240
1H), 2.70 (dd, J = 5.7, 15.5, 1H), 2.55–2.47 (m, 1H), 2.31–2.20 (m,
3H), 2.09–2.02 (m, 11H), 2.01–1.93 (m, 2H), 1.93–1.87 (m, 21H),
1.84–1.76 (m, 2H), 1.76–1.67 (m, 2H), 1.67–1.52 (m, 7H), 1.52–
1.38 (m, 4H), 1.38–1.30 (m, 2H), 1.28 (s, 1H), 1.11–1.03 (m, 20H),
1.03–0.93 (m, 5H), 0.42–0.30 (m, 1H).
Acknowledgements
We are very grateful for generous financial support by TNO De-
fense, Security and Safety, Rijswijk, the Netherlands. We thank
Hans van der Elst for assistance with the LC–MS purifications
and Kees Erkelens and Fons Lefeber for assistance with the record-
ing of NMR spectra.
D
6.3.6. cyclo-[Val-Orn-Leu- Tyr-Pro-Trp-Phe-Asn-Asp-Trp] 12
Prepared according to the general procedure. Yield: 39 mg,
30 lmol, 30%. LC–MS: tR 6.40 min (linear gradient 10?90% B
Supplementary data
in 13.5 min), m/z = 1283.9 [M+H]+, 642.7 [2M+H]+. 1H NMR
(600 MHz, MeOD) d 10.32 (s, 1H), 8.91 (d, J = 2.9, 1H), 8.75 (d,
J = 7.9, 1H), 8.71 (d, J = 9.0, 1H), 8.64 (d, J = 8.4, 1H), 8.57 (d,
J = 7.5, 1H), 7.86 (d, J = 8.4, 1H), 7.78–7.71 (m, 3H), 7.62 (d,
J = 8.6, 1H), 7.60 (br s, 1H), 7.55–7.50 (m, 1H), 7.47 (t, J = 7.6,
1H), 7.42 (s, 1H), 7.33–7.22 (m, 9H), 7.21–7.14 (m, 5H), 7.04 (t,
J = 7.5, 1H), 6.98–6.93 (m, 3H), 6.67 (s, 1H), 6.64 (d, J = 8.4,
2H), 5.10 (d, J = 5.7, 1H), 4.74–4.69 (m, 1H), 4.66 (dd, J = 7.8,
16.2, 1H), 4.61 (d, J = 11.7, 1H), 4.54 (d, J = 11.7, 1H), 4.47 (d,
J = 3.9, 1H), 4.45–4.40 (m, 2H), 4.34–4.29 (m, 1H), 4.16 (d,
J = 2.9, 1H), 4.09 (d, J = 8.3, 1H), 3.85 (d, J = 2.6, 1H), 3.73 (d,
J = 14.3, 1H), 3.62 (d, J = 11.1, 1H), 3.58–3.54 (m, 1H), 3.29–3.17
(m, 8H), 3.17–3.11 (m, 2H), 2.95–2.88 (m, 2H), 2.81–2.73 (m,
2H), 2.73–2.66 (m, 1H), 2.62 (dd, J = 5.6, 15.4, 1H), 2.47–2.38
(m, 1H), 2.23–2.13 (m, 2H), 2.01–1.93 (m, 4H), 1.92–1.80 (m,
1H), 1.77–1.68 (m, 1H), 1.68–1.59 (m, 1H), 1.59–1.45 (m, 4H),
1.45–1.31 (m, 2H), 1.30–1.24 (m, 1H), 1.02–0.95 (m, 14H),
0.94–0.85 (m, 2H), 0.33–0.24 (m, 1H).
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.07.049.
References and notes
1. Gerard, J. M.; Haden, P.; Kelly, M. T.; Anderson, R. J. J. Nat. Prod. 1999, 62, 80.
2. Scherkenbeck, J.; Chen, H.; Haynes, R. K. Eur. J. Org. Chem. 2002, 2350.
3. Selected examples include: (a) Stern, A.; Gibbons, W. A.; Graig, L. C. Proc. Natl.
Acad. Sci. U.S.A. 1968, 61, 734; (b) Gibbons, W. A.; Némethy, G.; Stern, A.; Graig,
L. C. Proc. Natl. Acad. Sci. U.S.A. 1970, 67, 239; (c) Jones, C. R.; Kuo, M.; Gibbons,
W. A. J. Biol. Chem. 1979, 254, 10307; (d) Krauss, E. M.; Chan, S. I. J. Am. Chem.
Soc. 1982, 104, 6953; (e) Mihailescu, D.; Smith, J. C. J. Phys. Chem. B 1999, 103,
1586.
4. (a) Chen, H.; Haynes, R. K.; Scherkenbeck, J.; Sze, K. H.; Zhu, G. Eur. J. Org. Chem.
2004, 31; (b) Chen, H.; Guo, X.-K. Chin. J. Struct. Chem. 2005, 24, 273.
5. Although the values of the chemical shift perturbation indicate an
a-helical
conformation, geometric constraints in this small decapeptide will most likely
D
enforce the Trp- Phe-Asn sequence in
a a-helical turn. See: Rose, G. D.;
Gierasch, L. M.; Smith, J. A. Adv. Prot. Chem. 1985, 37, 1–109.
6. Reviewed in: Povey, J. F.; Mark Smales, C.; Hassard, S. J.; Howard, M. J. J. Struct.
Biol. 2007, 157, 329–338.
7. (a) Stöckle, M.; Voll, G.; Günther, R.; Lohof, E.; Locardi, E.; Gruner, S.; Kessler, H.
Org. Lett. 2002, 4, 2501–2504; (b) van Well, R. M.; Marinelli, L.; Altona, C.;
Erkelens, K.; Siegal, G.; van Raaij, M.; Llamas-Saiz, A. L.; Kessler, H.; Novellino,
E.; Lavecchia, A.; van Boom, J. H.; Overhand, M. J. Am. Chem. Soc. 2003, 125,
10822–10829.
8. Grotenbreg, G. M.; Buizert, A. E. M.; Llamas-Saiz, A. L.; Spalburg, E.; van Hooft,
P. A.; 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, 7559–7565.
9. Wüthrich, K. NMR of Proteins and Nucleic Acids; John Wiley & Sons: New York, 1986.
10. Wishart, D. S.; Sykes, B. D.; Richards, F. M. Biochemistry 1992, 31, 1647–1651.
11. Ding, Y.; Qin, C.; Guo, Z.; Niu, W.; Zhang, R.; Li, Y. Chem. Biodivers. 2007, 4, 2827–2834.
12. Compounds 8, 10, 11 and 12 did not fully dissolve for the hemolytical assay in
up to 30% DMSO. Since their antimicrobial activities were also low, the
solubility issue was not further pursued.
D
6.3.7. cyclo-Val-Orn-Leu- Tyr-Pro-Trp-Phe-Asn-DAsp-Trp] 13
Prepared according to the general procedure. Yield: 7.2 mg
5.4 lmol, 5%. LC–MS: tR 6.16 min (linear gradient 10?90% B in
13.5 min), m/z = 1335.9 [M+H]+, 668.8 [2M+H]+. 1H NMR
(600 MHz, CD3OH) d 10.32 (s, 2H), 10.30 (s, 2H), 9.05 (br s, 2H),
8.72 (br s, 2H), 8.61 (d, J = 8.6, 2H), 8.53 (d, J = 6.6, 2H), 8.45 (br s,
3H), 7.95 (d, J = 10.2, 2H), 7.78 (br s, 2H), 7.71 (d, J = 7.9, 3H),
7.66 (d, J = 8.8, 2H), 7.47 (br s, 2H), 7.28 (d, J = 8.0, 7H), 7.25–7.11
(m, 18H), 7.10–6.96 (m, 20H), 6.93 (t, J = 7.5, 3H), 6.69 (d, J = 8.2,
5H), 5.45 (s, 1H), 4.84–4.57 (m, 2H), 4.49–4.24 (m, 3H), 4.19 (s,
2H), 3.45–3.32 (m, 6H), 3.27–3.22 (m, 4H), 3.10 (s, 9H), 3.03–2.83
(m, 6H), 2.74 (dd, J = 4.2, 17.3, 4H), 2.67 (dd, J = 5.2, 16.7, 5H),
2.30–2.04 (m, 6H), 1.88–1.52 (m, 21H), 1.42 (s, 7H), 1.35–1.21
(m, 4H), 1.13–0.74 (m, 43H), 0.50 (s, 2H).
13. Olsen, J. V.; de Godoy, L. M. F.; Li, G. Q.; Macek, B.; Mortensen, P.; Pesch, R.;
Makarov, A.; Lange, O.; Horning, S.; Mann, M. Mol. Cell. Proteomics 2005, 4,
2010–2021.
14. Kaiser, E.; Colescott, R. L.; Bossering, C. D.; Cook, P. I. Anal. Biochem. 1970, 34, 595.