Synthesis of low-hemolytic antimicrobial dehydropeptides based on gramicidin S

Article abstract of DOI:10.1021/jm061051v

The synthesis and biological activity of a novel cyclic β-sheet-type antimicrobial dehydropeptide based on gramicidin S (GS) is described. The GS analogue, containing two (Z)-(β-3-pyridyl)-α,β-dehydroalanine (ΔZ3Pal) residues at the 4 and 4′ positions (2), was synthesized by solution-phase methodologies using Boc-Leu-ΔZ3Pal azlactone. Analogue 2 exhibited high antimicrobial activity against Gram-positive bacteria and had much lower hemolytic activity than wild-type GS and the corresponding (Z)-α,β-dehydrophenylalanine (ΔZ-Phe) analogue (1).

Full text of DOI:10.1021/jm061051v

7592
J. Med. Chem. 2006, 49, 7592-7595
[D-Dap4,4′]GS9 and γ-amino-L-proline-modified GS13,16 did not
induce hemolysis and can highly permeabilize through outer-
membrane of Gram-negative bacteria. These peptides, however,
are less active against Gram-positive bacteria than wild-type
GS, suggesting that polycationic analogues of GS preferentially
interact with the outer membrane of Gram-negative bacteria.
Synthesis of Low-Hemolytic Antimicrobial
Dehydropeptides Based on Gramicidin S
Keiichi Yamada,† Shun-suke Shinoda,† Hiroyuki Oku,†
Keiko Komagoe,‡ Takashi Katsu,‡ and Ryoichi Katakai*,†
Department of Chemistry, Gunma UniVersity, Tenjin-cho, Kiryu,
Gunma 376-8515, Japan, and Graduate School of Medicine,
Dentistry and Pharmaceutical Science, Okayama UniVersity,
Tsushima, Okayama 700-8530, Japan
Biological activities of GS mutants, with D-Phe residues at
the 4 and 4′ positions replaced by other D-amino acids, depend
on their conformations. For example, a water-soluble D-Tyr4,4′
analogue, [D-Tyr4,4′]GS, exhibits a weaker hemolytic activity
than wild-type GS by maintaining moderate antimicrobial
activity while mutation by D-Asn, D-His, or D-Ser results in loss
of both activities because these D-amino acids did not induce
â-turn conformation.5 Very recently, Grotenbreg et al. have
reported that aryl substituents in the turn regions of GS and
analogues are indispensable for bactericidal action.20 R,â-
Dehydroamino acids (∆AAs) are hitherto used for â-turn
inducers of de novo peptides.18 Shimohigashi et al. reported
that replacement of D-Phe residues in GS with (Z)-R,â-
dehydrophenylalanine (∆ZPhe, Figure 1a) residues stabilizes its
â-sheet conformation with maintaining strong antimicrobial
activity.22,23 Recently, we designed a novel ∆AA, (Z)-(â-3-
pyridyl)-R,â-dehydroalanine (∆Z3Pal, Figure 1a).24 In the present
study, we found that replacement of ∆ZPhe residues in [∆Z-
Phe4,4′]GS (1, Figure 1b) with ∆Z3Pal drastically reduces
cytotoxicity to human erythrocyte without loss of antimicrobial
activity.
ReceiVed September 4, 2006
Abstract: The synthesis and biological activity of a novel cyclic
â-sheet-type antimicrobial dehydropeptide based on gramicidin S (GS)
is described. The GS analogue, containing two (Z)-(â-3-pyridyl)-R,â-
dehydroalanine (∆Z3Pal) residues at the 4 and 4′ positions (2), was
synthesized by solution-phase methodologies using Boc-Leu-∆Z3Pal
azlactone. Analogue 2 exhibited high antimicrobial activity against
Gram-positive bacteria and had much lower hemolytic activity than
wild-type GS and the corresponding (Z)-R,â-dehydrophenylalanine (∆Z-
Phe) analogue (1).
The global spread of multidrug-resistant bacteria is a growing
threat to human health.1 Cationic antimicrobial peptides (CAPsa)
that attack bacterial membranes are promising agents for
combating bacterial pathogens;2 consequently, the modes of
action of CAPs, and in particular R-helical CAPs, have been
extensively studied by various synthetic and spectroscopic
techniques.3
∆Z3Pal-containing analogue of GS, [∆Z3Pal4,4′]GS (2), was
synthesized by solution-phase method as shown in Scheme 1.
Boc-Leu-∆Z3Pal azlactone (9) was initially synthesized from
â-3-pyridyl-DL-serine (7) as previously reported.24 Treatment
of 9 with H-Pro-OMe (1.05 equiv) in the presence of a catalytic
amount of DMAP (0.05 equiv)25 afforded Boc-Leu-∆Z3Pal-Pro-
OMe (10) in 96% yield. Addition of excess amounts of H-Pro-
OMe (1.5 equiv) gave a mixture of 10 and Boc-D-Leu-∆Z3Pal-
Pro-OMe (L/D ) 51/49 estimated by 1H NMR). Stepwise
elongation of Orn(For) and Val residues afforded protected
pentapeptide derivative (12). Protected decapeptide 15 was
derived from 12 by segment condensation. Saponification of
15 followed by acidic deprotection of Boc group gave a linear
decapeptide 17. Cyclization of 17 under a high-dilution condition
in DMF afforded [Orn(For)2,2′, ∆Z3Pal4,4′]GS (4), whose struc-
ture was confirmed by 1H NMR and ESI-MS. At first, yield of
the cyclic product 4 was extremely low in spite of using potential
condensation reagents such as BOP-Cl (trace) or PyBOP (15%).
The use of HATU improved the yield up to 85%. Finally,
deprotection of formyl groups afforded 2 in good yield. [∆Z-
Phe4,4′]GS (1), [∆ZPhe,4 ∆Z3Pal4′]GS (3), and a tetrahydro
analogue of 2 ([D-3Pal4,4′]GS, 6) were also synthesized in
comparison (see Supporting Information). These pyridine-
containing analogues were highly soluble in water, while wild-
Gramicidin S (GS, cyclo(Val-Orn-Leu-D-Phe-Pro)2) is a
membrane-lytic cyclic peptide antibiotic that acts against both
Gram-positive and Gram-negative bacteria.4,5 NMR6 and X-ray
crystallographic studies7 established that the main-chain con-
formation of GS is a stable antiparallel â-sheet conformation.
Type II′ â-turn moieties that connect two short â-strands are
essential for the bioactive conformation of GS.5-8 Although the
mode of action of GS toward biomembranes is not completely
understood, GS is generally believed to perturb lipid packing,
resulting in the destruction of the cytoplasmic membrane’s
integrity and enhancement of its permeability.8 Unfortunately,
the use of GS for therapeutic purposes has been limited to topical
application because of its high toxicity to human red blood cells.
Therefore, structure-activity relationships of GS and related
cyclic peptides have been studied to dissociate its antimicrobial
and hemolytic activities and to elucidate the mode of action.9-18
The selectivity of CAPs including GS toward biomembrane
is governed by a net positive charge of peptides and their
amphiphilicity.19 Polycationic decapeptide analogues of GS,
* To whom correspondence should be addressed. Phone: +81-277-30-
1340. Fax: +81-277-30-1343. E-mail: katakai@chem.gunma-u.ac.jp.
† Gunma University
‡ Okayama University
a Abbreviations: Boc, tert-butoxycarbonyl; BOP-Cl, bis(2-oxo-3-oxazo-
lidinyl)phosphinic chloride; CAPs, cationic antimicrobial peptides; CD,
circular dichroism; D-Dap, D-R,γ-diaminopropionic acid; Dap(Z), Nγ-
benzyloxycarbonyl-D-R,γ-diaminopropionic acid; DIEA, N,N-diisopropyl-
ethylamine; DMAP, 4-dimethylaminopyridine; EDC‚HCl, 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride; ESI-MS, electrospray
ionization mass spectroscopy; For, formyl; HATU, 1-[bis(dimethylanimo)-
methylene]-1H-1,2,3-triazolo(4,5-b)pyridinium 3-oxide hexafluorophos-
phate; HFIP, 1,1,1,3,3,3-hexafluoro-2-propanol; HOBt, N-hydroxybenzo-
triazole; NMM, N-methylmorpholine; NOESY, nuclear Overhauser and
exchange spectroscopy; D-3Pal, (â-3-pyridyl)-D-alanine; PyBOP, benzo-
triazole-1-yloxypyrrolidinophosphonium hexafluorophosphate; TFA, tri-
fluoroacetic acid; Z, benzyloxycarbonyl.
1
type GS and 1 were hardly soluble. H NMR and ESI-MS
measurements revealed that 2 contains small amount of ∆E-
3Pal4,4′ isomer (E/Z ) 9/91 estimated by HPLC).
As shown in Figure 2, NOESY spectral analysis indicated
that the configuration of the â-substituent in ∆3Pal residues is
the (Z)-form because strong NOE between â-CH(∆Z3Pal4,4′) and
δ-CH2(Pro5,5′) was observed. Steric proximity between δ-NH3
-
+
(Orn2,2′) and R-CH(Pro5,5′) suggests the stabilization of â-sheet
10.1021/jm061051v CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/08/2006

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26 7593
Figure 1. (a) ∆Z3Pal, ∆ZPhe, and D-3Pal and (b) GS analogues in this
study.
Scheme 1. Solution-Phase Total Synthesis of 2a
Figure 2. Selective NOESY spectrum of 2 (20 °C, DMSO-d6, 8.95
mM, τm ) 1000 ms).
Table 1. Amide Proton Temperature Shift Coefficients (∆δ/∆T, ppb/K)
and Coupling Constants (3JNH-CH, Hz) of GS and Its Analogues in
a
DMSO-d6
peptides
Val1,1
′
Orn2,2
′
Leu3,3
′
Xaa4,4
′
GS
1
-1.8 (-c)
-1.1 (-c)
-0.9 (10.0)
c
-4.8 (9.1)
-5.6 (8.9)
-5.6 (9.5)
-4.8 (9.1)
-4.8 (9.1)
-4.2 (9.5)
-2.8 (8.8)
-2.6 (8.6)
-2.6 (8.5)
-2.6 (9.5)
-2.8 (9.0)
-2.7 (8.5)
-7.2 (3.0)
-5.3 (-d)
-4.6 (-d)
-5.3 (-d)
-4.6 (-d)
-5.7 (4.0)
2
3b
c
6
-2.3 (-c)
a 3
J
values are in the parentheses and are measured at 20 °C.
NH-CH
b Upper: Val1-∆ZPhe4. Lower: Val1′-∆Z3Pal4′. c Overlapped with aromatic
protons. d Singlet.
a Reagent and conditions: (a) Boc-Leu-OSu, 10% aqueous NaHCO3/
1,2-dimethoxyethane ) 1:1 (v/v), 0 °C f room temp, 24 h; (b) Ac2O,
AcONa, 3 h; (c)HCl‚H-Pro-OMe, DMAP (5 mol%), NMM, CHCl3, 0 °C
f room temp, 24 h; (d) 4 M HCl in dioxane, 0 °C, 2 h; (e) Boc-Orn(For)-
OH, EDC‚HCl, HOBt, NMM, CHCl3, 0 °C f room temp, 24 h; (f) TFA,
0 °C, 2 h; (g) Boc-Val-OH EDC‚HCl, HOBt, NMM, CHCl3, 0 °C f room
temp, 24 h; (h) 1 M aqueous NaOH, 50% aqueous MeOH, 0 °C f room
temp, 2 h; (i) EDC‚HCl, HOBt, NMM, CHCl3, 0 °C f room temp, 24 h;
(j) HATU, DIEA, DMF (final concentration, 1 mM), 0 °C f room temp,
2 days; (k) 10% HCl in MeOH, room temp, 2 days.
Figure 3. CD spectra of GS, 1, 2, and 6 in MeOH (20 °C).
+
conformation by hydrogen-bonding interaction between δ-NH3
(Orn2,2′) and CdO(∆Z3Pal4,4′) like wild-type GS.26
-
with a shoulder at 217 nm. The difference is mainly due to the
R,â-double bond chromophore of ∆ZPhe, and the band at 280
nm is characterized as a ∆ZPhe-Pro â-turn structure.23 In the
case of ∆Z3Pal4,4′ analogue 2, the spectrum is essentially similar
to that of 1, suggesting that 2 adopts a cyclic â-sheet conforma-
tion similar to that of 1 and GS. D-3Pal4,4′ analogue 6 exhibited
a CD spectrum similar to that of wild-type GS but with much
weaker intensity. In an earlier study by Kopple et al., cyclic
hexapeptide related to GS, cyclo(Orn-D-Phe-Pro)2, exhibits two
negative bands centered at 222 and 200 nm in HFIP, which is
characteristic of type II′ â-turn conformation.27 In the case of
6, the decrement of the ellipticity suggests that the main-chain
conformation of 6 was distorted or destabilized compared with
the wild-type GS, which is consistent with the result of 1H NMR
analysis.
Shimohigashi et al. have previously reported that ∆ZPhe4,4′
analogue 1 adopts a stable antiparallel â-sheet conformation like
1
1
parent GS by H NMR analysis.22,23 Variable-temperature H
NMR experiments revealed that four NRHs of Val1,1′ and Leu3,3′
in ∆Z3Pal-containing analogues 2 and 3 were hydrogen-bonded.
3JNH-CH values of Val, Orn, and Leu residues (ranging from
8.6 to 9.5 Hz) correspond to the â-sheet conformation (Table
1). In contrast, the temperature shift coefficient (∆δ/∆T) and
3
the JNH-CH value of R-NH(D-3Pal) in 6 slightly differ from
that of R-NH(D-Phe) in wild-type GS, suggesting that the main-
chain conformation of 6 is slightly distorted.
CD is also useful for studying conformational changes of GS
analogues from parent GS. Figure 3 shows CD spectra of GS
and analogues in MeOH. 1 has three negative bands at 210,
236, and 280 nm, while GS has a negative band at 206 nm
Biological activities of GS analogues were evaluated by a
similar method reported previously.16 Table 2 shows the

7594 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26
Letters
Table 2. Antimicrobial Activity of GS Analogues
MIC (µg/mL)
Staphylococcus
aureus 209P
Escherichia coli
K12 strain W3110
peptidesa
GS
1
2
3
5
4
4
8
4
4
16
32
32
32
32
64
6
64
a Cells were cultured at 37 °C for 20 h in a Mueller-Hinton broth.
Figure 5. Correlation among biological activities of GS-based cyclic
decapeptides and their retention time in HPLC analysis. Closed circles
and opened triangles indicate MIC values toward S. aureus 209P and
ED50 values toward human erythrocyte. The following conditions were
applied for RP-HPLC analysis: column, YMC-pack ODS R&D (6.0
mm i.d. × 250 mm); flow rate, 1 mL/min; eluent, 40-85% aqueous
CH3CN (containing 0.1%TFA); detection, 270 nm (1-3), 220 nm
(others).
Therefore, it is considered that ∆Z3Pal analogue 2 possessing
similar aromatic ∆AA residues in the â-turn moieties could
interact with bacterial membranes like 1 and [Ala2,2′,∆ZPhe4,4′]-
GS. At present, however, the mechanisms of action of 2 are
not fully understood.
Figure 4. Dose-dependence curves of hemolysis induced by GS
analogues.
In conclusion, we have discovered a novel antimicrobial
cyclic peptide 2 that exhibits high solubility in water and low
hemolytic activity. Our findings should be helpful in obtaining
new insights to the rational design of novel â-sheet-type CAPs
with low hemolytic activity.
antibacterial activity determined by the liquid-broth method and
that ∆Z3Pal4,4′ analogue 2 exhibited potent antimicrobial activity
against Gram-positive Staphylococcus aureus, which was
comparable to those of wild-type GS and ∆ZPhe4,4′ analogue 1.
The MIC value of the analogue containing ∆Z3Pal and ∆ZPhe
residues (3) was comparable to that of [D-Tyr4,4′]GS (5). It is
noteworthy that hemolytic activity of 2 was drastically reduced
in comparison with those of other analogues (Figure 4). These
observations suggest that incorporation of ∆Z3Pal residues leads
to diminished cytotoxicity to human erythrocyte without dis-
rupting the bioactive conformation of GS. To clarify the
structural importance of ∆Z3Pal residues in antibacterial action,
biological activities of [D-3Pal4,4′]GS (6) were evaluated. As
expected, water-soluble 6 did not induce hemolysis (Figure 4),
but it was inactive against S. aureus and E. coli (Table 2).
Relative hydrophobicity and hydrophilicity of peptides in-
cluding GS-related peptides could be evaluated by HPLC
analysis.28 Figure 5 shows the correlation among biological
activities of GS-based peptides and their retention time. ∆Z-
3Pal analogue 2 had a lower retention time compared with all
hemolytic analogues and higher retention time compared with
6, indicating that 6 is slightly more hydrophilic than 2. At
present, we speculate that the lost of antimicrobial activity of 6
is related to its higher hydrophilicity and conformational
distortion mentioned above. Increased hemolytic activity was
accompanied by a retardation of elution time. A remarkable
difference between MIC and ED50 was observed only in the
case of two ∆Z3Pal-containing 2. As a result, introduction of
two ∆Z3Pal residues into the GS framework provides moderate
hydrophilicity without interfering with permeation of 2 across
bacterial membranes. Interestingly, Katsu et al. have reported
that antimicrobial [Ala2,2′,∆ZPhe4,4′]GS, an analogue of 1 lacking
cationic Orn residues, enhances the K+ efflux from human
erythrocyte, resulting in changes in its morphology. On the other
hand, inactive [Ala2,2′]GS scarcely induces the change.29 The
difference implies that the aromatic and conjugated planes in
∆ZPhe residues play a crucial role in membrane-peptide
interaction and/or its permeation through a phospholipid bilayer.
Acknowledgment. The authors are grateful to Prof. Masao
Kawai (Nagoya Institute of Technology, Japan) for his helpful
comments.
Supporting Information Available: Experimental details,
spectroscopic data, and HPLC of GS analogues. This material is
available free of charge via the Internet at http://pubs.acs.org.
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