Structure-activity relationships of bacterial outer-membrane permeabilizers based on polymyxin B heptapeptides

Article abstract of DOI:10.1016/j.bmcl.2010.01.040

A series of cationic cyclic heptapeptides based on polymyxin B have been synthesized for use as permeabilizers of the outer membrane of Gram-negative bacteria. Only analogs with the Dab²-d-Phe³-Leu⁴-Xxx⁵ sequence (Xxx = Dab or Orn) showed a synergistic bactericidal effect when combined with conventional antibiotics, indicating that the Dab² residue plays a critical role in permeation of the outer membrane of Gram-negative bacteria.

Full text of DOI:10.1016/j.bmcl.2010.01.040

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1771–1775
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationships of bacterial outer-membrane
permeabilizers based on polymyxin B heptapeptides
b
c
a
Hirotoshi Urakawa a, Keiichi Yamada a, , Keiko Komagoe , Setsuko Ando , Hiroyuki Oku ,
*
Takashi Katsu b, Ichiro Matsuo a
a Department of Chemistry and Chemical Biology, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan
b Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700-8530, Japan
c Department of Chemistry, Faculty of Sciences, Fukuoka University, Fukuoka 814-0180, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of cationic cyclic heptapeptides based on polymyxin B have been synthesized for use as permea-
bilizers of the outer membrane of Gram-negative bacteria. Only analogs with the Dab2- -Phe3-Leu4-Xxx5
D
Received 31 October 2009
Revised 4 January 2010
Accepted 6 January 2010
Available online 20 January 2010
sequence (Xxx = Dab or Orn) showed a synergistic bactericidal effect when combined with conventional
antibiotics, indicating that the Dab2 residue plays a critical role in permeation of the outer membrane of
Gram-negative bacteria.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Polymyxin B
Antimicrobial peptide
Gram-negative outer-membrane
Solid-phase peptide synthesis
The global spread of multidrug-resistant (MDR) bacteria is a
growing threat to human health. Antimicrobial and host defense
peptides are promising agents for new anti-infective chemother-
apy. Polymyxins and collistins are cyclic peptides which exhibit
strong antimicrobial activity against Gram-negative bacteria.1
They can bind to the outer membrane of such bacteria, namely
lipopolysaccharide (LPS) or Gram-negative endotoxin. Although
polymyxins have fallen out of favor since their use in the 1960s
due to nephrotoxicity and neurotoxicity, they have re-emerged
as a promising antibiotic to combat against MDR pathogens despite
the toxicity. In fact, polymyxin B (PMB, Fig. 1) in combination with
other antimicrobials is considered a reasonable and safe treatment
option of MDR Gram-negative pathogens2 and endotoxin shock.3
Two N-terminal truncated forms of PMB, namely PMB nonapep-
tide (PMBN, Fig. 1) and PMB heptapeptide (PMBH, 1) (Fig. 2), are
not bactericidal to Gram-negative bacteria but still can bind to
LPS and increase the permeability of their outer membrane to
hydrophobic antibiotics.4 These features make them clinically
important leads for the development of outer-membrane permea-
bilizers5 and endotoxin-neutralizing agents.6
association between PMB and LPS. Replacement of the Dab resi-
dues with Lys in the cyclic heptapeptide moiety of PMBN has been
reported to drastically reduce its outer-membrane permeability.6
To further clarify this functional loss, we synthesized a series of
PMBH analogs possessing various basic amino acid residues
through systematic alternations of the Dab residues at the 2-, 5-
and 6-positions for determining the essential Dab residues in
PMBH for high outer-membrane permeability (Fig. 2).
Compound 1 was previously prepared by enzymatic hydrolysis
of PMB4 or a solution-phase method.7 As illustrated in Scheme 1,
we synthesized PMBH analogs using solid-phase synthesis and
cyclization-cleavage (SPS–CC) using Kaiser–Oxime resin,8 which
is known to be efficient for cyclic peptide synthesis.
Based on a previous report for Fmoc-solid-phase peptide syn-
thesis (SPPS) of PMB and PMBN, with cycling between
c-NH2(Dab)
and CO2H(Thr),9 we cyclized the linear heptapeptides between
a
-
NH2(Dab5) and CO2H(Leu4), as Thr(Bzl) is not a suitable C-terminal
residue for segment condensation.10 Boc-Leu-OH (substitution:
0.3–0.5 mmol/g resin) was attached to the resin using dicyclohex-
ylcarbodiimide in CH2Cl2. The protected Dab derivatives,
Boc-Dab(Z)-OH, and Z-Dab(Boc)-OH, were synthesized as previ-
ously reported.7 The Boc group was removed by treatment with
25% trifluoroacetic acid (TFA)/dichloromethane at room tempera-
ture for 30 min. Stepwise elongation of the protected amino
acid residues using 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetrameth-
ylaminium hexafluorophosphate (HBTU)/diisopropylethylamine
(DIEA) gave the protected linear peptidyl resin. The protected
The L-2,4-diaminobutyric acid (Dab) residues in PMB are known
for their important role in binding to LPS, which increases the out-
er-membrane permeability of the antibiotic. To date, many PMB
analogs have been designed and synthesized to investigate the
* Corresponding author. Tel.: +81 277 30 1345; fax: +81 277 30 1343.
E-mail address: kyamada@chem-bio.gunma-u.ac.jp (K. Yamada).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.040

1772
H. Urakawa et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1771–1775
Figure 1. Structure of polymyxin B1 and polymyxin B nonapeptide (PMBN).
Figure 2. Primary structure of PMBH and analogs in this study.
Scheme 1. Solid-phase synthesis of PMBH. Reagents and conditions: (a) Boc-Leu-OH (3 equiv), DCC (3 equiv) in DCM 24 h; (b) Boc-amino acid (3 equiv), HBTU (3 equiv),
HOBt (3 equiv), DIEA (5 equiv) in DMF 45 min, deprotection: 25% TFA/CH2Cl2, 30 min; (c) DIEA (3 equiv), AcOH (3 equiv) in DMF, 24 h; and (d) H2, 10% Pd/C, MeOH–H2O–TFA
(40:10:2 (v/v/v)), 5 h.
peptides underwent cleavage from the resin via on-resin macro-
cyclization8 and then catalytic hydrogenation in TFA/water/meth-
anol (0.2:1:4, v/v/v) to give the desired PMBH analogs in fair
yield; their structures were confirmed by 1H NMR and electrospray
ionization-MS. 1H NMR chemical shifts and temperature coeffi-
cients of the amide protons of 1–6 in DMSO-d6 were summarized
in Table 1.
The ability of cationic peptides to destabilize the outer mem-
brane of Gram-positive bacteria can be evaluated using conven-
tional antibiotics that are only active against these microbes;11 in
the present study, novobiocin and erythromycin were used as
the probe antibiotics. The MIC values of these antibiotics in the
presence of 1 and its analogs are shown in Table 2. PMBN was used
as a positive control. As expected, the antibiotics inhibited the
growth of Escherichia coli upon the addition of 1 and PMBN. As
the concentration of 1 was increased, the antibiotics exhibited
higher activity, confirming that PMBH can destabilize the outer
membrane of E. coli.
Next, we synthesized analogs in which all Dab residues except
the 1-position in PMBH were replaced by Orn ([Orn2,5,6]PMBH, 2)
or L-2,3-diaminopropionic acid (Dap) ([Dap2,5,6]PMBH, 3) to evalu-
ate their membrane permeability. Interestingly, both analogs were
less active than 1. Furthermore, the PMB3 analog that was derived
from 2 ([Orn5,8,9]PMB3, Fig. 3) exhibited weaker antimicrobial
activity against E. coli and was inactive against Gram-positive
Staphylococcus aureus (Table 3). These results indicate that replace-
ment of the basic amino acid residues in PMBH reduces outer-
membrane permeability.
Thus, we synthesized three additional PMBH analogs containing
only one Orn residue, namely [Orn2]PMBH (4), [Orn5]PMBH (5),
and [Orn6]PMBH (6), to further evaluate membrane permeability.
Interestingly, only 4 did not induce membrane permeability of

H. Urakawa et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1771–1775
1773
Table 1
1H NMR chemical shifts (d [ppm] at 20 °C) and temperature coefficients (
Dd/DT [ppb/K]) of the amide protons of 1–6 in DMSO-d6
Residue
1
2
3
4
5
6
X=Dab
Y=Dab
Z=Dab
X=Orn
Y=Orn
Z=Orn
X=Dap
Y=Dap
Z=Dap
X=Orn
Y=Dab
Z=Dab
X=Dab
Y=Orn
Z=Dab
X=Dab
Y=Dab
Z=Orn
Dab1
X2
d(
c
-NH)
7.47
ꢀ0.5
8.94
ꢀ3.8
8.79
ꢀ4.8
8.37
ꢀ3.2
7.78
ꢀ1.2
8.51
ꢀ3.4
7.53
ꢀ1.6
7.67
—
8.53
ꢀ5.0
9.06
ꢀ3.3
8.62
ꢀ3.6
7.93
ꢀ1.4
9.36
ꢀ7.0
8.32
ꢀ0.9
7.54
ꢀ0.3
7.36
ꢀ0.8
8.83
ꢀ5.6
8.80
ꢀ3.7
8.44
ꢀ3.4
7.81
ꢀ1.2
8.68
ꢀ3.7
7.57
ꢀ1.6
7.40
ꢀ0.6
7.31
—
a
b
D
d/
D
T
d( -NH)
d/
d( -NH)
d/
d( -NH)
d/
d( -NH)
d/
d( -NH)
d/
d( -NH)
d/
a
8.27
ꢀ3.6
8.66
ꢀ4.7
8.27
ꢀ3.5
7.35
ꢀ1.6
8.66
ꢀ4.7
6.99
0.02
8.74
8.92
ꢀ3.2
8.78
ꢀ4.7
8.41
ꢀ3.1
7.73
ꢀ1.3
8.68
ꢀ3.2
7.34
ꢀ0.6
c
D
D
T
—
-Phe3
a
8.77
D
d
D
D
T
—
Leu4
Y5
a
8.45
ꢀ2.9
7.53
ꢀ0.6
D
DT
a
D
DT
Z6
a
8.79
e
D
D
T
—
Thr7
a
7.31
ꢀ0.4
D
DT
a
b
c
Overlaped with the signal of
Overlaped with the signal of arom. (
Overlaped with the signal of
Overlaped with the signal of
Overlaped with the signal of
c
-NH(Dab1).
D
-Phe3).
a
a
a
-NH(D
-Phe3 and Dab6).
d
e
-NH(Dab2,6).
-NH(Dab2 and -Phe3).
D
Table 2
Minimum Inhibitory Concentrations (MIC,
l
g/ml) of antibiotics against E. coli K12 W3110 in the presence of PMB analogsa
Polymyxin B analogb
Antibioticd
MIC of antibiotic in the presence of the indicated concentration (lg/ml) of polymyxin B analog
0.5
1
2
4
8
16 32
PMBNc
Novobiocin
Erythromycin
Novobiocin
Erythromycin
Novobiocin
Erythromycin
Novobiocin
Erythromycin
Novobiocin
Erythromycin
Novobiocin
Erythromycin
Novobiocin
Erythromycin
16
32
64
64
8
16
32
32
4
8
4
8
8
2
8
8
8
32
64
16
16
32
64
8
2
8
8
1
4
4
PMBH (1)
16
16
64
64
32
32
64
64
16
8
16
64
64
32
16
64
64
16
8
8
4
[Orn2,5,6]PMBH (2)
[Dap2,5,6]PMBH (3)
[Orn2]PMBH (4)
[Orn5]PMBH (5)
[Orn6]PMBH (6)
—
128
64
64
32
64
64
32
16
32
32
32
64
16
16
32
32
8
4
8
8
16
32
8
16
32
32
4
4
8
8
e
64
64
64
64
64
32
32
32
32
8
8
8
32
16
16
16
a
b
c
Measured in Mueller–Hinton broth (see Supplementary data).
MICs of PMBH and analogs alone were >128
PMBN was obtained from Sigma.
lg/ml in all cases.
d
e
MICs of novobiocin and erythromycin alone were 128 and 64
Not determined.
l
g/ml, respectively.
Table 3
MICs (
g/ml) of polymyxin B3 (PMB3) and [Orn5,8,9]PMB3
a
l
Peptides
MIC (lg/ml)
S. aureus 209P
E. coli K12 W3110
PMBb
64
>128
>128
0.5
0.5
16
PMB3
[Orn5,8,9]PMB3
a
Measured in Mueller–Hinton broth (see Supplementary data).
PMB was obtained from Sigma.
b
Figure 3. Structure of polymyxin B3 (n = 2) and [Orn5,8,9]PMB3 (n = 3).
CD spectra of 1–6 were measured to obtain information about
the antibiotics. (Table 1) These results indicated that the basic ami-
no acid residue at the 2-position in PMBH is important for high
outer-membrane permeability.
conformational behavior of PMBH analogs in water and membrane-
mimetic environment (Fig. 4). 1,1,1,3,3,3-Hexafluoroisopropanol
(HFIP) was used as a membrane-mimetic solvent.12 Native PMBH

1774
H. Urakawa et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1771–1775
Figure 4. CD spectra of 1–6 in water and HFIP. (20 °C, [peptide] = 1 mg/mL) Dotted lines and solid lines indicate the spectra in water and HFIP, respectively. (a) 1, (b) 2, (c) 3,
(d) 4, (e) 5, and (f) 6.
(1) has two minimum at 199 and 217 nm in water (Fig. 4a, dotted
line). In HFIP, the spectrum was slightly red-shifted to 201 and
220 nm (Fig. 4a, solid line). According to the literature, cyclic hep-
creases both antimicrobial and LPS-binding activities.15 The Dab
residues in the heptapeptide provide the critical electrostatic con-
tacts with phosphate groups of lipid A.16,17 In our case, replace-
tapeptide moiety of PMBN adopts type II0 b-turn around
D
-Phe-Leu
-turn around Thr residue in water.13 In addition, Kopple and
co-workers have reported that CD spectrum of cyclo(Orn- -Phe-
ment of the Dab residue at position 2 in PMBH by Orn
and
c
significantly reduced outer-membrane permeability despite
remaining cationic side chain. CD spectral comparison among na-
tive PMBH (1) and the inactive analogs (2 and 4) suggested that
the conformation of 2 and 4 are somewhat different from that of
1 in membrane-mimicking environment. We consider that the
conformational differences could weaken the electrostatic interac-
tion between the cationic side chain of Orn2 and a phosphate group
of Lipid A but the differences still remain unclear. To address this
issue, detailed NMR studies are in progress.
D
Pro)2 has two negative bands centered at 200 and 222 nm in HFIP,
which is characteristic of type II0 b-turn conformation.14 These
facts suggests that 1 adopts type II0 b-turn conformation in water
and HFIP. In the case of 2, red-shift of the two negative bands
was not observed by changing solvent from water to HFIP but
the intensity of the spectrum was significantly increased (Fig. 4b,
solid line), suggesting that 2 adopts an ordered conformation in
membrane-mimicking environment but the conformation is not
preferable for outer-membrane permeation. CD spectra of 3 in
water and HFIP were also similar to those of 1 but with weaker
intensity, indicating that the main-chain conformation of 3 is
slightly distorted (Fig. 4c). We consider that this conformational
destabilization reduces the outer-membrane permeability.
In summary, we have found that the replacement of the Dab2
residue in PMBH by a basic amino acid reduces outer-membrane
permeability of Gram-negative bacteria. Our results should con-
tribute to designing outer-membrane permeabilizers or LPS-neu-
tralizing agents using PMB and related peptides.
Among the PMBH analogs containing only one Orn residue, out-
er-membrane permeable analog 5 showed similar CD spectral
change to native PMBH 1 (Fig. 4e). On the other hand, CD spectra
of the inactive analog 4 in water and HFIP were resemble to those
of Orn2,5,6 analog 2. CD spectrum of the analog 6 in HFIP showed
two negative bands at 199 and 216 nm with a shoulder at
221 nm (Fig. 4f). Although the CD spectrum of 6 has characteristics
of both native PMBH 1 and Orn2 analog 4 in HFIP, the outer-mem-
brane permeability of 6 was comparable to that of 1.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.01.040.
References and notes
1. Li, J.; Nation, R. L.; Milne, R. W.; Turnidge, J. D.; Coulthard, K. Int. J. Antimicrob.
Agents 2005, 25, 11.
2. Zavascki, A. P.; Goldani, L. Z.; Li, J.; Nation, R. L. J. Antimicrob. Chemother. 2007,
60, 1206.
Recently, Sakura and co-workers have also reported that substi-
tution of the Dab residue at the 5-position in PMB3 by Ala de-

H. Urakawa et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1771–1775
1775
3. Bhor, V. M.; Thomas, C. J.; Surolia, N.; Surolia, A. Mol. Biosyst. 2005, 1, 213.
4. Kimura, Y.; Matsunaga, H.; Vaara, M. J. Antibiot. 1992, 45, 742.
5. Li, C.; Budge, L. P.; Driscoll, C. D.; Willardson, B. M.; Allman, G. W.; Savage, P. B.
J. Am. Chem. Soc. 1999, 121, 931.
6. Tsubery, H.; Ofek, I.; Cohen, S.; Fridkin, M. J. Med. Chem. 2000, 43, 3085.
7. Yamada, K.; Urakawa, H.; Oku, H.; Katakai, R. J. Pept. Res. 2004, 64, 43.
8. (a) Ösapay, G.; Profit, A.; Taylor, J. W. Tetrahedron Lett. 1990, 31, 6121; (b)
Mihara, H.; Yamabe, S.; Niidome, T.; Aoyagi, H.; Kumagai, H. Tetrahedron Lett.
1995, 36, 4837.
9. (a) Sharma, S. K.; Wu, A. D.; Chandramouli, N.; Fotsch, C.; Kardash, G.; Bair, K.
W. J. Pept. Res. 1999, 53, 501; (b) Kline, T.; Hokub, D.; Therrien, J.; Ryckman, D. J.
Pept. Res. 2001, 57, 175; (c) Tsubery, H.; Ofek, I.; Cohen, S.; Fridkin, M. Peptides
2001, 22, 1675; (d) Tsubery, H.; Ofek, I.; Cohen, S.; Eisenstein, M.; Fridkin, M.
Mol. Pharmacol. 2002, 62, 1036; (e) Okimura, K.; Ohki, K.; Ohnishi, K.; Sakura, N.
Chem. Pharm. Bull. 2007, 55, 1724.
10. Sakakibara, S. Biopolymers (Peptide Science) 1999, 51, 279.
11. Vaara, M.; Vaara, T. Antimicrob. Agents Chemother. 1983, 24, 107.
12. Xue, R.; Wang, S.; Wang, C.; Zhu, T.; Fei Li, F.; Sun, H. Biopolymers 2006, 84, 329.
13. Bhattacharjya, S.; David, S. A.; Mathan, V. L.; Balaram, P. Biopolymers 1996, 41,
251.
14. Bush, C. A.; Sarkar, S. K.; Kopple, K. D. Biochemistry 1978, 17, 4951.
15. Kanazawa, K.; Sato, Y.; Ohki, K.; Okimura, K.; Uchida, Y.; Shindo, M.; Sakura, N.
Chem. Pharm. Bull. 2009, 57, 240.
ˇ
16. Pristovšek, P.; Kidric, J. J. Med. Chem. 1999, 42, 4604.
17. Bruch, M. D.; Cajal, Y.; Koh, J. T.; Jain, M. K. J. Am. Chem. Soc. 1999, 121, 11993.