The contribution of the N-terminal structure of polymyxin B peptides to antimicrobial and lipopolysaccharide binding activity

Article abstract of DOI:10.1246/bcsj.77.1915

To elucidate the N-terminal structure-activity relationships of polymyxin B peptides, seven polymyxin B component peptides, the structures of which having been elucidated, and seven N-terminal fatty acid and/or amino acid deletion analogs were synthesized, and their antimicrobial activities determined. The lipopolysaccharide (LPS) binding activities of synthetic peptides were evaluated using [Dab(Dansyl-Gly)¹]-polymyxin B₃ (Dab; L-α,γ-diaminobutyric acid) as a fluorescent probe. The results indicated that the fatty acyl moiety was not indispensable for LPS binding, but the C₉ fatty acyl groups of polymyxin B peptides contributed to the binding affinity to a slightly greater extent than C₈ or C ₇. The fatty acyl moieties of polymyxin B contributed greatly to the antimicrobial activity, while the distinct N-terminal structures of polymyxin B₁-B₆, bearing normal-, iso-, or anteiso-fatty acids, or 3-hydroxy-fatty acid with chain lengths between C₇ and C₉, did not affect bactericidal potency.

Full text of DOI:10.1246/bcsj.77.1915

ꢀ 2004 The Chemical Society of Japan
Bull. Chem. Soc. Jpn., 77, 1915–1924 (2004) 1915
The Contribution of the N-Terminal Structure of Polymyxin B Peptides
to Antimicrobial and Lipopolysaccharide Binding Activity
ꢀ
Naoki Sakura, Tatsuya Itoh, Yoshiki Uchida,1 Kazuhiro Ohki, Keiko Okimura,
Kenzo Chiba, Yuki Sato, and Hiroyuki Sawanishi
Faculty of Pharmaceutical Sciences, Hokuriku University, Kanagawa-machi, Kanazawa 920-1181
1Department of Food Science and Nutrition, Osaka Shoin Women’s University, Hishiyanishi, Higashi-Osaka 577-8550
Received April 22, 2004; E-mail: n-sakura@hokuriku-u.ac.jp
To elucidate the N-terminal structure–activity relationships of polymyxin B peptides, seven polymyxin B compo-
nent peptides, the structures of which having been elucidated, and seven N-terminal fatty acid and/or amino acid dele-
tion analogs were synthesized, and their antimicrobial activities determined. The lipopolysaccharide (LPS) binding ac-
tivities of synthetic peptides were evaluated using [Dab(Dansyl-Gly)1]-polymyxin B3 (Dab; L-ꢀ,ꢁ-diaminobutyric acid)
as a fluorescent probe. The results indicated that the fatty acyl moiety was not indispensable for LPS binding, but the C9
fatty acyl groups of polymyxin B peptides contributed to the binding affinity to a slightly greater extent than C8 or C7.
The fatty acyl moieties of polymyxin B contributed greatly to the antimicrobial activity, while the distinct N-terminal
structures of polymyxin B1–B6, bearing normal-, iso-, or anteiso-fatty acids, or 3-hydroxy-fatty acid with chain lengths
between C7 and C9, did not affect bactericidal potency.
Polymyxin B family peptides, isolated from Bacillus poly-
myxa,1,2 have potent antimicrobial activity against Gram-neg-
ative bacteria and a high binding ability to the lipid A portion
of lipopolysaccharides (LPS), which is the active principle of
endotoxins. One of the characteristics of these peptide antibi-
otics is that they contain six L-ꢀ; ꢁ-diaminobutyric acid
(Dab) units, among which the ꢁ-amino group of the Dab at po-
sition 4 (Dab4) is acylated by a C-terminal Thr10 to form a 23-
member lactam ring.3,4 Commercially available polymyxin B
is a mixture composed of numerous closely related peptides,
the N-terminals of which are acylated with various fatty acids
(Fig. 1). Polymyxin B1 is the main component in peptide mix-
tures bearing (6S)-6-methyloctanoic acid as the fatty acyl moi-
ety. The minor components, i.e., polymyxin B2, B3, and B4,
have been shown to have 6-methylheptanoyl, octanoyl, and
heptanoyl fatty acyl groups, respectively.3,5 [Ile7]-polymyxin
B1 might be included in this peptide family, because a minor
component was reported6 to contain L-Ile instead of L-Leu,
and Leu at position 7 of polymyxin B1 was deduced to be re-
placed by Ile, as determined structurally in the case of colistin
peptides.7–9 Polymyxin B5 and B6 were recently reported to
have the same structure as polymyxin B1, except that the fatty
acyl moieties were nonanoyl and 3-hydroxy-6-methyloctanoyl,
respectively, though the configuration of 3-hydroxy-6-methyl-
octanoic acid has not been determined.10
The chemical syntheses of polymyxin B peptides by classi-
cal methods11,12 and the application of solid-phase synthe-
ses13–15 have been performed to confirm the structure or clarify
1
2
3
4
5
6
7
Fatty acyl -Dab-Thr-Dab-Dab-Dab- D-Phe- X
Thr Dab
Dab
8
10
9
Polymyxin B (PMB) peptides
Fatty acyl
X
Polymyxin B
Polymyxin B
Polymyxin B
Polymyxin B
Polymyxin B
Ia
Ib
Ic
Id
Ie
If
(6S)-6-methyloctanoyl
6-methylheptanoyl
octanoyl
Leu
Leu
Leu
Leu
Leu
Leu
Ile
1
2
3
4
5
6
heptanoyl
nonanoyl
Polymyxin B
(3RS,6S)-3-hydroxy-6-methyloctanoyl
(6S)-6-methyloctanoyl
7
[Ile ]-Polymyxin B
Ig
1
Fig. 1. Structures of polymyxin B peptides.
Published on the web October 9, 2004; DOI 10.1246/bcsj.77.1915

1916 Bull. Chem. Soc. Jpn., 77, No. 10 (2004)
Activity of Polymyxin B N-Terminal Analogs
ꢂ
and alter the biological activities of polymyxin B. The present
investigation was undertaken to determine the antimicrobial
activities of each polymyxin B component peptide (Fig. 1)
and the N-terminally deleted-analogs (Fig. 4) employing syn-
thetic peptides. We report here the contributions of the N-ter-
minal structure of polymyxin B to the LPS binding activity by
LPS binding assay using mono-dansylated polymyxin B3 as a
fluorescent probe.
Thr(Bzl)10 and ꢁ-NH2 of Dab4 at 4 C in minimal amounts
of DMSO–DMF to dissolve the starting materials. Interesting-
ly, in the lactam formation reaction, the intramolecular reac-
tion mediated by DPPA proceeded quantitatively in the pres-
ence of a high concentration of a linear partially protected pep-
tide (III). Although the reason for this cannot be explained, no
polymer formation was observed at peptide concentrations as
high as 0.01 mmol/mL. Neither polymer production nor ꢂ-
elimination20,21 was observed as reported previously.14 Gel
filtration on a Toyopearl HW-40 column was carried out for
purification of protected peptides (III and IV) using DMF–
H2O (9:1).
For the LPS binding assay, a mono-dansylated polymyxin
B3 analog bearing a dansyl fluorescent probe on the side chain
of Dab at position 1 through Gly as a spacer was designed.
According to the synthetic route shown in Fig. 3, Fmoc-Dab-
(ivDde)-OH (ivDde; 1-(4,4-dimethyl-2,6-dioxocyclohexyli-
dene)-3-methylbutyl) was introduced at position 1 of the pro-
tected peptide resin, and then octanoyl was substituted for
Results and Discussion
Synthesis of Dab Derivatives and Fatty Acids. Fmoc-
ꢀ
ꢁ
Dab(2-ClZ)-OH (N -(9-flluorenylmethyloxycarbonyl)-N -(2-
chlorobenzyloxycarbonyl)-L-ꢀ; ꢁ-diaminobutyric acid) and
Fmoc-Dab(Boc)-OH were prepared from Fmoc-Dab-OH,
which was obtained by a Hofmann rearrangement16 of
Fmoc-Gln-OH. Among the N-terminal fatty acyl moieties, S-
(þ)-6-methyloctanoic acid for polymyxin B1 and 6-methyl-
heptanoic acid for polymyxin B2, were synthesized starting
with S-(ꢁ)-2-methylbutanol and 4-methylpentanol, respective-
ly, according to the methods described previously.17,18 As the
configuration of 3-hydroxy-6-methyloctanoic acid for poly-
myxin B6 was not determined, (3RS; 6S)-3-hydroxy-6-methyl-
octanoic acid was synthesized as shown in Fig. 2 and used in
this study.
ꢀ
the N -Fmoc to yield Octanoyl-Dab(ivDde)-Thr(Bzl)-Dab-
(2-ClZ)-Dab(Boc)-Dab(2-ClZ)-D-Phe-Leu-Dab(2-ClZ)-Dab(2-
ClZ)-Thr(Bzl)-O-HMP-resin (IIh). The ivDde protecting
ꢁ
group on N was then removed selectively by hydrazine treat-
ment,22 and Fmoc-Gly-OH was coupled, followed by dansyl
chloride to give Octanoyl-Dab(Dansyl-Gly)-Thr(Bzl)-Dab-
(2-ClZ)-Dab(Boc)-Dab(2-ClZ)-D-Phe-Leu-Dab(2-ClZ)-Dab(2-
ClZ)-Thr(Bzl)-O-HMP-resin (IIh0). TFA treatment of IIh0 fol-
lowed by a cyclization reaction with DPPA yielded Octanoyl-
Peptide Synthesis. The synthesis of polymyxin B compo-
nent peptides was carried out as shown in Fig. 3. Starting from
a hydroxymethylphenoxymethyl resin (HMP-resin) anchored
with Fmoc-Thr(Bzl), the peptide chain was elongated by sol-
id-phase synthesis using Fmoc-amino acids bearing benzyl-
type protecting groups on the side chain functional groups, ex-
cept Dab at position 4, which was protected by Boc. After in-
troduction of all of the amino acids and the N-terminal fatty
acid, cleavage of the HMP-resin support was achieved by
TFA (trifluoroacetic acid), and the Boc protecting group of
Dab4 was removed simultaneously to give a linear partially
protected peptide (III).
ꢀ
Dab(Dansyl-Gly)-Thr(Bzl)-Dab(2-ClZ)-cyclic-[Dab -Dab(2-
ꢀ
ꢀ
ClZ)-D-Phe-Leu-Dab(2-ClZ)-Dab(2-ClZ)-Thr(Bzl) ] ( amide
bond between the ꢁ-NH2 of Dab4 and ꢀ-COOH of Thr10)
(IVh).
The final removal of the benzyl-type protecting groups of
the protected cyclic polymyxin B peptide (IV) was performed
by HF treatment in the presence of anisole. The product was
purified by RP-HPLC and highly purified polymyxin B peptide
(I) was obtained as a hydrochloride form after gel filtration
using a 5 mmol/L HCl solution containing 25% acetonitrile,
The cyclization reaction of III was carried out using DPPA
(diphenyl phosphorazidate) reagent19 between ꢀ-COOH of
COCH3
a
b
OH
Cl
CO2Et
O
O
O
6
7
c
d
e
CO2Et
CO2Et
CO2H
OH
O
OH
10
9
8
a: thionyl chloride,
d: NaBH4
b: ethyl acetoacetate,
e: KOH
c: NaOH,
Fig. 2. Synthesis of (3RS; 6S)-3-hydroxy-6-methyloctanoic acid.

N. Sakura et al.
Bull. Chem. Soc. Jpn., 77, No. 10 (2004) 1917
Fmoc-Thr(Bzl)-O-HMP-Resin
1)
2) Fmoc-AA-OH/HATU
20% piperidine/NMP
Fatty acyl -Dab(2-ClZ)-Thr(Bzl)-Dab(2-ClZ)-Dab(Boc)-
Dab(2-ClZ)-D-Phe-Leu-Dab(2-ClZ)-Dab(2-ClZ)-Thr(Bzl)-O-HMP-Resin ( II )
TFA
Fatty acyl -Dab(2-ClZ)-Thr(Bzl)-Dab(2-ClZ)-Dab-
( III )
Dab(2-ClZ)-D-Phe-Leu-Dab(2-ClZ)-Dab(2-ClZ)-Thr(Bzl)-OH
DPPA in DMF in an ice bath
Fatty acyl -Dab(2-ClZ)-Thr(Bzl)-Dab(2-ClZ)-Dab-Dab(2-ClZ)-D-Phe
( IV )
Leu
Thr(Bzl)-Dab(2-ClZ)-Dab(2-ClZ)
HF
Fatty acyl -Dab-Thr-Dab-Dab-Dab-D-Phe
( I )
Leu
Dab
Thr Dab
Fig. 3. Synthesis of polymyxin B peptides.
Table 1. Characteristics of Synthetic Polymyxin B (PMB) Peptides
29
Peptide
[ꢀ]D
FAB-MS
[M + H]þ [M + Na]þ
HPLCaÞ
HP-TLCbÞ
1
2
(c ¼ 0:5, 12% AcOH)
Formula
tR/min
Rf
Rf
PMB1 (Ia)
PMB2 (Ib)
PMB3 (Ic)
PMB4 (Id)
PMB5 (Ie)
PMB6 (If)
ꢁ68:2ꢂ
ꢁ68:2ꢂ
ꢁ72:5ꢂ
ꢁ77:4ꢂ
ꢁ73:5ꢂ
ꢁ71:3ꢂ
C56H98N16O13
C55H96N16O13
C55H96N16O13
C54H94N16O13
C56H98N16O13
C56H98N16O14
1203
1189
1189
1175
1203
1219
1225
1211
1211
1197
1225
1241
29.01 0.37
25.90 0.42
26.66 0.46
23.11 0.43
30.08 0.48
24.67 0.45
0.52
0.50
0.52
0.41
0.47
0.43
and 24.83
½Ile7]-PMB1 (Ig)
ꢁ63:6ꢂ
ꢁ54:4ꢂ
ꢁ59:5ꢂ
ꢁ46:1ꢂ
ꢁ49:2ꢂ
ꢁ49:7ꢂ
ꢁ66:1ꢂ
ꢁ74:2ꢂ
ꢁ66:5ꢂ
C56H98N16O13
C69H110N18O16S1
C47H82N16O12
C43H74N14O11
C39H67N13O9
C35H59N11O8
C51H88N14O12
C47H81N13O10
C43H73N11O9
1203
1479
1063
963
1225
1501
1085
985
27.81 0.48
38.68 0.46
12.93 0.09
12.79 0.28
13.32 0.15
12.41 0.46
31.14 0.59
27.16 0.61
28.22 0.66
0.42
0.64
0.03
0.05
0.06
0.13
0.43
0.44
0.48
½Dab(Dansyl-Gly)1]-PMB3 (Ih)
Des-FA-PMB (1–10) (Ii)
Des-FA-PMB (2–10) (Ij)
Des-FA-PMB (3–10) (Ik)
Des-FA-PMB (4–10) (Il)
Octanoyl-PMB (2–10) (Im)
Octanoyl-PMB (3–10) (In)
Octanoyl-PMB (4–10) (Io)
862
762
884
784
1089
988
888
1111
1010
910
a) HPLC conditions: Column, Capcell Pak C18 ACR (4:6 ꢃ 250 mm); Elution, a linear gradient from 16 to 32% CH3CN in 0.1% TFA
1
(30 min) followed by maintaining final condition; Flow rate, 1 mL/min; Detection, 210 nm. b) TLC solvent systems: Rf ;
2
BuOH:Pyridine:AcOH:H2O (30:20:6:24). Rf ; BuOH:AcOH:AcOEt:H2O (1:1:1:1).
followed by lyophilization. The structure and purity of the syn-
thetic polymyxin B component peptides (Ia–Ig) were con-
firmed by FAB-MS analysis, amino acid analysis of acid hy-
drolysates, high-performance thin-layer chromatography (HP-
TLC), and analytical HPLC (Table 1). Individual synthetic
polymyxin B peptides appeared as single peaks on analytical
RP-HPLC, except for polymyxin B6 (If). As If contained
(3RS; 6S)-3-hydroxy-6-methyloctanoyl as the fatty acyl group,
the diastereomeric mixture appeared as split peaks on analyti-
cal HPLC.
For the fatty acyl deletion analogs (Ii–Il), N-terminally
Fmoc-protected cyclic peptides (IVi–IVl) were prepared.
The Fmoc was removed by piperidine prior to the final depro-
tection with HF. Thus, the Fmoc strategy in combination with
the benzyl-type protection for side chain functional groups was
adopted because it was a versatile synthetic method for struc-
turally complicated peptides, such as polymyxin B, composed
of six diamino acids, Dab, and a lactam ring. In this study, not
only the polymyxin B component peptides, but also [Dab(Dan-
syl-Gly)1]-polymyxin B3 (Ih) were successfully synthesized

1918 Bull. Chem. Soc. Jpn., 77, No. 10 (2004)
Activity of Polymyxin B N-Terminal Analogs
1
2
3
4
5
6
7
8
9
10
Des-FA-PMB (1-10)
Des-FA-PMB (2-10)
Des-FA-PMB (3-10)
Des-FA-PMB (4-10)
Octanoyl-PMB (2-10)
Octanoyl-PMB (3-10)
Octanoyl-PMB (4-10)
Ii
H-Dab-Thr-Dab-Dab*-Dab-D-Phe-Leu-Dab-Dab-Thr*-
H-Thr-Dab-Dab*-Dab-D-Phe-Leu-Dab-Dab-Thr*-
H-Dab-Dab*-Dab-D-Phe-Leu-Dab-Dab-Thr*-
Ij
Ik
Il
H-Dab*-Dab-D-Phe-Leu-Dab-Dab-Thr*-
Im
In
Io
C H CO-Thr-Dab-Dab*-Dab-D-Phe-Leu-Dab-Dab-Thr*-
7
15
C H CO-Dab-Dab*-Dab-D-Phe-Leu-Dab-Dab-Thr*-
7
15
C H CO-Dab*-Dab-D-Phe-Leu-Dab-Dab-Thr*-
7
15
4
10
* : amide linkage between γ-NH of Dab and α-COOH of Thr
2
Fig. 4. Structure of N-terminal deletion polymyxin B-related peptides.
Table 2. Antimicrobial and LPS Binding Activities of Synthetic Polymyxin B (PMB) Peptides
Peptide
Minimum inhibitory concentration (MIC)/nmol mLꢁ1
LPS binding
aÞ
Escherichia coli
Salmonella
typhimurium
Pseudomonas
aeruginosa
IC50
/nmol mLꢁ1
PMB (Sigma)
PMB1 (Ia)
PMB2 (Ib)
PMB3 (Ic)
PMB4 (Id)
PMB5 (Ie)
PMB6 (If)
½Ile7]-PMB1 (Ig)
1.0
1.0
1.0
0.5
0.5
1.0
0.5
0.5
0.5
1.0
1.0
0.5
0.5
1.0
0.5
0.5
1.0
1.0
1.0
1.0
0.5
1.0
0.5
0.5
1.0
4.0
128
64
4
4
9
8
10
6
7
5
½Dab(Dansyl-Gly)1]-PMB3 (Ih)
Des-FA-PMB (1–10) (Ii)
Des-FA-PMB (2–10) (Ij)
Des-FA-PMB (3–10) (Ik)
Des-FA-PMB (4–10) (Il)
Octanoyl-PMB (2–10) (Im)
Octanoyl-PMB (3–10) (In)
Octanoyl-PMB (4–10) (Io)
1.0
8.0
1.0
16
—
13
19
21
>32
12
14
30
>256
128
>256
2.0
16
128
>256
256
>256
2.0
16
128
128
2.0
8.0
128
a) The concentrations required for 50% inhibition of Ih–LPS binding were derived from the quenching curves of syn-
thetic peptides in Fig. 6.
using the four different amino protecting groups, Fmoc, ivDde,
Boc, and ClZ, followed by their selective removal during the
synthetic procedure.
did not affect the antimicrobial potency. The contribution of
the various fatty acyl groups of colistin (polymyxin E) deriva-
tives was examined by Chihara,23,24 however, the synthetic
peptides, prepared directly from colistin nonapeptide, were
heterogeneous, making it difficult to evaluate the results. It
was shown that substitution of Ile for Leu7 in polymyxin B1
did not have any significance with respect to biological func-
tion, and a similar result was recently reported regarding
[Ile7]-polymyxin E1.25 The results outlined above indicated
that all polymyxin B molecules isolated and structurally eluci-
dated to date retain similar antimicrobial activity. The results
indicated that any polymyxin B molecule could be selected
for further study of the structure–activity relationships, with
deletion, replacement, or modification of the amino acid resi-
dues. Therefore, polymyxin B3 (Ic), with octanoyl at the N-ter-
minal, was selected as a lead compound for the following ad-
vanced study using synthetic peptides. [Dab(Dansyl-Gly)1]-
polymyxin B3 (Ih), a key compound for the LPS binding
assay, retained potent bactericidal activity with MIC of
1 nmol/mL.
Antimicrobial Activities of Synthetic Polymyxin B
Peptides. The antimicrobial activities of fifteen synthetic
peptides (Ia–Io) were estimated by the standard micro plate di-
lution method. The minimum inhibitory concentration (MIC)
values of synthetic polymyxin B component peptides (Ia–Ig)
were 0.5–1.0 mol/mL toward Escherichia coli, Salmonella
typhimurium, and Pseudomonas aeruginosa (Table 2). No sig-
nificant differences in antimicrobial potency were observed
among the five component peptides (Ia–Ie) despite their dis-
tinct fatty acyl structures. Synthetic polymyxin B6 (If), bearing
(3RS; 6S)-3-hydroxy-6-methyloctanoic acid as the N-terminal
fatty acid, was also as active as the other component peptides.
Based on the above results, it was difficult to determine wheth-
er one of the diastereomeric mixtures of If would be active,
while the counterpart would be inactive. Thus, the structural
differences between normal-, iso-, and anteiso-fatty acids, or
3-hydroxy fatty acid with chain lengths of C7, C8, and C9

N. Sakura et al.
Bull. Chem. Soc. Jpn., 77, No. 10 (2004) 1919
Octanoyl-polymyxin B (4–10) (Io), devoid of the N-termi-
nal tripeptide Dab-Thr-Dab in polymyxin B3 and instead com-
505 nm
490 nm
10
9
ꢀ
posed of a cyclic heptapeptide and an octanoyl group at N of
Dab4, retained some antimicrobial activity, although it was
less than 1/100 of that of polymyxin B3 (Ic). Elongation of
10
µ L
8
7
6
5
4
8
µ L
ꢀ
the peptide chain from the N of Dab4 increased the potency
in a stepwise manner, and octanoyl-polymyxin B (2–10)
(Im) had almost full activity with an MIC of 2.0 nmol/mL.
However, deletion of the octanoyl group from Im resulted in
des-fatty acyl-polymyxin B (2–10) (Ij), which showed very lit-
tle activity. These results were in agreement with the lack of
antimicrobial activity of polymyxin B nonapeptide, prepared
by enzymatic treatment of native polymyxin B with papain
or ficin.26,27 It was shown in this study that a shorter peptide,
des-fatty acyl-polymyxin B (3–10) (Ik), had greater bacterici-
dal activity than that of Ij, although its activity was very low.
In a recent synthetic study of polymyxin B nonapeptide an-
alogs, it was reported that elongation from polymyxin B non-
apeptide with tri-alanine (Ala3-polymyxin B nonapeptide) or
hexa-alanine (Ala6-polymyxin B nonapeptide) resulted in a
complete loss of antimicrobial activity.28 However, des-fatty
acyl-polymyxin B (1–10) (Ii) showed considerable activity
with MIC values of 8.0, 16, and 4.0 nmol/mL against E. coli,
S. typhimurium, and P. aeruginosa, respectively. Thus, it was
of interest that the elongation of des-fatty acyl-polymyxin B
(2–10) (Ij) with a hydrophilic and basic amino acid, Dab, re-
gained the antimicrobial activity. However, the N-terminal fat-
ty acyl group was demonstrated to make a greater contribution
to the bactericidal activity, as the antibacterial potency of Ii
was still low similar to that of octanoyl-polymyxin B (3–10)
(In).
LPS Binding Activity. Dansyl-polymyxin B is usually
prepared from native commercially available polymyxin B
by coupling with dansyl chloride.29 The reaction yields heter-
ogeneous products because of the existence of five free amino
functions of Dab residues at positions 1, 3, 5, 8, and 9 in the
molecule. In this study, a structurally well-defined mono-dan-
sylated fluorescent probe, [Dab(Dansyl-Gly)1]-polymyxin B3
(Ih), was prepared and used to establish an LPS binding assay
system. As Ih showed almost full antimicrobial activity, it
may bind to LPS of the living bacterial membrane in the same
way as polymyxin B itself. Furthermore, [Ala1]-polymyxin B3
retained full antimicrobial activity with an MIC value of 1
nmol/mL,30 suggesting that a basic amino acid residue at po-
sition 1 is not a structural requirement for bactericidal activity.
That is, modification of the side chain amino function of Dab1
was assumed not to disturb the interaction of polymyxin B
with LPS. The maximum fluorescence spectrum of Ih was
shifted from 560 to 505 nm, and the intensity was greatly
enhanced at 505 nm by titration with LPS (Fig. 5).
6
4
µ L
µ
L
2
3
2
µ
L
0
µ
L
1
0
560 nm
550
450
500
600
Wavelength / nm
Fig. 5. Fluorescence emission spectra of [Dab(Dansyl-
Gly)1]-polymyxin B3 (Ih). A solution of Ih (10 nmol/
mL) in HEPES buffer (5 mmol/L, pH 7.2) was titrated
with a solution of E. coli LPS (3 mg/mL) (2 mL each)
in a quartz cuvette. Excitation was at 330 nm. The arrows
labeled 505 nm and 490 nm indicate the maximum wave-
length of fluorescent enhancement and the wavelength
used for LPS binding assay of various synthetic peptides,
respectively.
myxin B1, B5, B6, and [Ile7]-polymyxin B1 (Ia, Ie, If, and
Ig) bearing a C9 fatty acyl showed almost as potent LPS bind-
ing activity as native polymyxin B, while polymyxin B2, B3,
and B4 (Ib, Ic, and Id) bearing a C8 and C7 fatty acyl showed
slightly but significantly lower binding activity. Among the N-
terminal deletion analogs, des-fatty acyl-polymyxin B (1–10)
(Ii), octanoyl-polymyxin B (2–10) (Im), and octanoyl-poly-
myxin B (3–10) (In) showed high binding activities, but their
activities were lower than those of polymyxin B component
peptides. The IC50 values of des-fatty acyl-polymyxin B (2–
10) (Ij) and des-fatty acyl-polymyxin B (3–10) (Ik) were 2–
5 fold higher than those of polymyxin B component peptides.
Octanoyl-polymyxin B (4–10) (Io) had a further reduced bind-
ing activity to LPS, and des-fatty acyl-polymyxin B (4–10) (Il)
had a greatly reduced binding activity. Introduction of an oc-
tanoyl group into these des-fatty acyl-polymyxin B peptides
caused increases in the LPS binding activity to the same de-
gree as introduction of one amino acid, i.e., the IC50 value
of octanoyl-polymyxin B (2–10) (Im) was similar to that of
des-fatty acyl-polymyxin B (1–10) (Ii), and that of octanoyl-
polymyxin B (3–10) (In) was similar to des-fatty acyl-poly-
myxin B (2–10) (Ij). However, although octanoyl-polymyxin
B (3–10) (In) and des-fatty acyl-polymyxin B (2–10) (Ij)
showed similar binding activities, this was not reflected in their
antimicrobial activities, as LPS binding activity did not paral-
lel antimicrobial activity.
LPS binding activity was examined according to the method
reported by Moore.31 The fluorescence of the LPS–Ih com-
plex, measured at 490 nm, was quenched by the addition of
synthetic peptides in a concentration-dependent manner. The
apparent binding affinities of various polymyxin B component
peptides to LPS and of N-terminal deletion analogs were esti-
mated from their quenching curves (Figs. 6A and 6B), from
which the peptide concentration to inhibit 50% binding of Ih
to LPS (IC50 value) was obtained (Table 2). Synthetic poly-
Conclusion
Seven polymyxin B component peptides and seven N-termi-
nal fatty acid and/or amino acid deletion analogs were synthe-

1920 Bull. Chem. Soc. Jpn., 77, No. 10 (2004)
Activity of Polymyxin B N-Terminal Analogs
A
B
125
125
100
75
Des- FA- PMB (1-10) (Ii)
Des- FA- PMB (2-10) (Ij)
Des- FA- PMB (3-10) (Ik)
Des- FA- PMB (4-10) (Il)
Octanoyl- PMB (2-10) (Im)
Octanoyl- PMB (3-10) (In)
Octanoyl- PMB (4-10) (Io)
PMB (Sigma)
PMB1 (Ia)
100
PMB2 (Ib)
PMB3 (Ic)
75
50
PMB4 (Id)
PMB5 (Ie)
PMB6 (If)
[Ile7]-PMB1 (Ig)
50
25
0
25
0
0
5
10
15
20
25
-1
30
35
0
5
10
15
20
25
-1
30
35
Peptide / nmol mL
Peptide / nmol mL
Fig. 6. Quenching curves of [Dab(Dansyl-Gly)1]-polymyxin B3 (Ih) caused by addition of various polymyxin B (A) and N-terminal
deletion polymyxin B peptides (B).
Fmoc-Dab-OH (1). Fmoc-Gln-OH (36.8 g, 0.10 mol) was
subjected to a Hofmann rearrangement with I,I-bis(trifluoroace-
toxy)iodobenzene16 (47.3 g, 0.11 mol) in DMSO (225 mL)–H2O
(25 mL). After 20 h, the reaction mixture was diluted with cold
H2O (300 mL) and washed with ether. The pH was then adjusted
to 6.5 with 4 mol/L NaOH to yield a precipitate, which was col-
lected and washed with boiling ethanol (200 mL). Yield 30.22 g
(88.9%). mp 169.5–171.7 C, [ꢀ]D ꢁ4:0ꢂ (c 1.0, 50% AcOH),
high-resolution FAB-MS (JMS-DX300 mass spectrometer, JEOL
Ltd.): 341.1502 [M + H]þ, (calcd for C19H21N2O4; 341.1501).
sized, and their antimicrobial activities were determined. The
LPS binding activities of the synthetic peptides were evaluated
using [Dab(Dansyl-Gly)1]-polymyxin B3 as a fluorescent
probe. The results revealed that the fatty acyl moiety was
not indispensable in terms of LPS binding, but the C9 fatty ac-
yl groups of the polymyxin B peptides did contribute to the
binding affinity to a slightly greater extent than the C8 or C7.
The fatty acyl moieties of polymyxin B contributed greatly
to the antimicrobial activity, while the distinct N-terminal
structures of polymyxin B1–B6, bearing normal-, iso-, and an-
teiso-fatty acids, or a 3-hydroxy-fatty acid with chain lengths
between C7 and C9, did not change the bactericidal potency.
The introduction of an octanoyl group into the amino termini
of shorter polymyxin B peptides demonstrated the contribution
of the fatty acyl group to the antimicrobial activity, as well as
to the increases in the LPS binding activity.
ꢂ
29
Found: C, 65.64; H, 6.14; N, 8.02%. Calcd for C19H20N2O4
1/2H2O: C, 65.30; H, 6.14; N, 7.71%.
ꢄ
Fmoc-Dab(2-ClZ)-OH (2). To a solution of 1 (17.02 g, 0.050
mol) and TEA (triethylamine) (7.0 mL, 0.05 mol) in H2O (100
mL)–DMSO (30 mL) was added a solution of Z(2-Cl)-OSu
(15.60 g, 0.055 mol) in DMSO (70 mL). The mixture was stirred
for 3 h at room temperature while keeping the pH at 8 with TEA.
The mixture was diluted with H2O (150 mL)–TEA (7.0 mL),
washed three times with ether (200 mL each), and then acidified
with 6 mol/L HCl. The product was extracted three times with
AcOEt (150 mL) and washed three times with 0.5 mol/L HCl
and saturated NaCl. The combined organic phases were dried over
Na2SO4 and evaporated to give an oil, which was solidified with
isopropyl alcohol (150 mL). The product was collected and wash-
ed with boiling AcOEt (50 mL). Yield 21.80 g (85.7%). mp
Experimental
General. HPLC was performed on an apparatus equipped
with a 590 and 510 pump (Waters Corp., Milford, MA, USA), a
U6K injector (Waters), an S310 model II UV detector (Soma Op-
tics Ltd., Tokyo, Japan), a 680 Automated Gradient Controller
(Waters), and a chromatocorder 21 (System Instruments Co.,
Ltd., Tokyo, Japan). Gel column chromatography was carried
out with a Toyopearl HW-40S (Tosoh Corporation, Tokyo,
Japan). Amino acid analysis of the acid hydrolysate was conduct-
ed on a model 7300 amino acid analyzer system (Beckman Instru-
ments Ltd., Fullerton, CA USA). HF cleavage reactions were car-
ried out in a Teflon HF apparatus (Peptide Institute Inc., Osaka,
Japan). Fast-atom bombardment mass spectra (FAB-MS) were ob-
tained on a JMS-DX300 mass spectrometer (JEOL Ltd., Tokyo,
Japan). The 1H NMR spectra were recorded in CDCl3 as a solvent
using TMS as an internal standard on a JEOL-PMX 60-SI spec-
trometer. The optical rotations of peptides were measured with a
DIP-370 digital polarimeter (Nippon Bunko Co., Ltd., Tokyo,
Japan). HP-TLC was performed on precoated silica gel plates
(Kieselgel 60; Merck, Darmstadt, Germany). All reagents, sol-
vents, and Fmoc-amino acids were obtained as reagent grade
products from Watanabe Chem. Ind. Ltd. (Hiroshima, Japan) or
Wako Pure Chem. Ind. Ltd. (Tokyo, Japan). Octanoic acid, hepta-
noic acid, and nonanoic acid were from Aldrich Chemical Compa-
ny Inc. (Milwaukee, WI, USA).
29
134.6–136.2 ꢂC, [ꢀ]D ꢁ13:0ꢂ (c 0.5, methanol), high-resolution
FAB-MS: 509.1477 [M + H]þ, (calcd for C27H26ClN2O6;
509.1476). Found: C, 63.66; H, 5.04; N, 5.59%. Calcd for
C27H25ClN2O6: C, 63.72; H, 4.95; N, 5.50%.
Fmoc-Dab(Boc)-OH (3). To a solution of 1 (10.21 g, 0.030
mol) and TEA (4.20 mL, 0.03 mol) in H2O (60 mL)–DMSO
(30 mL) was added a solution of Boc2O (8.51 g, 0.039 mol) in
DMSO (30 mL). The mixture was treated in the same manner
as described above for 2. The product was purified with AcOEt
(25 mL)-MeOH (5 mL)–petr. ether (60 mL). Yield 9.80 g
29
(74.2%). mp 102.0–103.7 ꢂC, [ꢀ]D ꢁ13:4ꢂ (c 0.5, methanol),
high-resolution FAB-MS: 441.2026 [M + H]þ, (calcd for
C24H29N2O6; 441.2026). Found: C, 65.49; H, 6.42; N, 6.33%.
Calcd for C24H28N2O6: C, 65.44; H, 6.41; N, 6.36%.
(6S)-6-Methyloctanoic Acid (4) and 6-Methylheptanoic Acid
(5). The fatty acids used for the preparation of polymyxin B1 and
B2 were synthesized by methods reported previously,17,18 starting
with (2S)-2-methylbutanol and 4-methylpentanol, respectively. 4:

N. Sakura et al.
Bull. Chem. Soc. Jpn., 77, No. 10 (2004) 1921
High-resolution FAB-MS: 159.1387 [M + H]þ, (calcd for
C9H19O2; 159.1385). 5: MS (m=z): 145 [M + H]þ.
d, J ¼ 6:0 Hz, one of CH(OH)CH2COOH), 4.03 (1H, m,
CH(OH)), 6.87 (2H, br s, OH, COOH, exchangeable by D2O),
IR (liquid) cmꢁ1: 3423, 1712. High-resolution FAB-MS (m=z):
175.1334 [M + H]þ, (Calcd for C9H19O3: 175.1331). Total yield
of 10 was 13.0% from (4S)-4-methylhexanoic acid.
Protected Polymyxin B1 Peptide Resin [(6S)-6-Methylocta-
noyl-Dab(2-ClZ)-Thr(Bzl)-Dab(2-ClZ)-Dab(Boc)-Dab(2-ClZ)-
D-Phe-Leu-Dab(2-ClZ)-Dab(2-ClZ)-Thr(Bzl)-OMP-Resin]
(IIa) and Related Compounds (IIb–IIg). General Procedure 1:
The protected amino acids used were Fmoc-Dab(2-ClZ)-OH,
Fmoc-Dab(Boc)-OH, Fmoc-Thr(Bzl)-OH, Fmoc-D-Phe-OH, and
Fmoc-Leu-OH. Fmoc-Thr(Bzl)-O-HMP-resin was prepared by
the coupling of Fmoc-Thr(Bzl)-OH (8 mol equiv.) to 4-hydoxy-
methylphenoxymethyl-resin (HMP-resin or Wang-resin, 0.74
(4S)-4-Methylhexanoyl Chloride (6). A mixture of (4S)-4-
methylhexanoic acid (33.7 g, 0.26 mol) and thionyl chloride
(83.3 g, 0.70 mol) was refluxed for 5 h. The reaction mixture
was distilled to give 6 (22.9 g, 59.1%; bp 94 ꢂC/63 mm). 1H NMR
(CDCl3) ꢃ 0.78–1.00 (6H, m, CH3 ꢃ 2), 0.98–1.90 (5H, m,
CH3CH2(CH3)CHCH2), 2.87 (2H, t, J ¼ 8:0 Hz, CH2CO). IR
(liquid) cmꢁ1: 1803. FAB-MS (m=z): 149 [M + H]þ.
(6S)-Ethyl 2-Acetyl-6-methyl-3-oxooctanoate (7). Ethyl ace-
toacetate (40.6 g, 0.31 mol) was added in a dropwise manner to a
solution of clean sodium (3.6 g, 0.15 g atom) in dry diethyl ether
(470 mL) at room temperature, followed by stirring for 12 h. To
the above solution was added 6 (22.9 g, 0.155 mol) in a dropwise
manner at room temperature. The mixture was then refluxed for 10
h. Water was added to the reaction mixture. The ether layer was
extracted and washed with brine, dried, and concentrated. The res-
idue was distilled to give 7 (22.15 g, 59.0%; bp 106–109 ꢂC/4
mm). 1H NMR (CDCl3) ꢃ 0.78–1.00 (6H, m, CH3 ꢃ 2), 0.98–
1.90 (5H, m, CH3CH2(CH3)CHCH2), 1.30 (3H, t, J ¼ 8:0 Hz,
CH3CH2), 2.30 (3H, s, COCH3), 2.63 (2H, t, J ¼ 8:0 Hz,
CH2CO), 4.00 (1H, s, Ac(CO)CHCO2Et), 4.23 (2H, d, J ¼ 8:0
Hz, CH3CH2). IR (liquid) cmꢁ1: 1762, 1716. High-resolution
MS (m=z): 242.1521 (Calcd for C13H22O4: 242.1518).
mmol/g, Novabiochem, Laufelfingen, Switzerl) with DCC (4
¨
mol equiv.) in the presence of 4-dimethylaminopyridine (DMAP,
0.1 mol equiv.). Starting from Fmoc-Thr(Bzl)-O-HMP-resin (344
mg, 0.2 mmol), the peptide chain was elongated through a solid-
phase methodology using a peptide synthesizer (ABI 433A; Ap-
plied Biosystems, Foster City, CA, USA). Deprotection of the
Fmoc group was performed by treatment with 20% piperidine in
N-methylpyrolidone (NMP). Fmoc-amino acids (0.5 mmol),
HATU (0.5 mmol), and N,N-diisopropylethylamine (1.0 mmol)
were stirred with the resin for 1 h in NMP for each coupling reac-
tion. After introduction of (6S)-6-methyloctanoic acid (4) to N-ter-
minal of Dab1, the peptide resin was washed consecutively three
times with DMF, dichloromethane, MeOH, and ether, and then
dried in vacuo to yield IIa (686 mg).
(6S)-Ethyl 6-Methyl-3-oxooctanoate (8). To a solution of so-
dium hydroxide (1.79 g, 0.045 mol) in water (142 mL) was added
7 (0.045 mol). The solution was heated at 100 ꢂC for 45 min. After
cooling rapidly in an ice bath, the solution was extracted twice
with ether. The ether solution wꢂas dried and distilled in vacuo
The other protected peptide resins (IIb–IIg) were prepared in
the same manner as described in General procedure 1 using 6-
methylheptanoic acid (5), octanoic acid, heptanoic acid, nonanoic
acid (Wako Pure Chem. Ind. Ltd., Osaka, Japan), or (3RS; 6S)-3-
hydroxy-6-methyloctanoic acid (10) as fatty acids.
1
to give 8 (4.72 g, 52.4%; 92–94 C/4 mm). H NMR (CDCl3) ꢃ
0.78–l.00 (6H, m, CH3 ꢃ 2), 0.98–1.90 (5H, m, CH3CH2CH-
(CH3)CH2CH2), l.25 (3H, t, J ¼ 8:0 Hz, CH3CH2), 2.50 (2H, t,
J ¼ 8:0 Hz, CH2CO), 3.40 (2H, s, COCH2COEt), 4.20 (2H, q,
J ¼ 8:0 Hz, OCH2CH3). IR (liquid) cmꢁ1: 1747, 1718. High-re-
solution MS (m=z): 200.1414 (Calcd for C11H20O3: 200.1413).
(3RS; 6S)-Ethyl 3-Hydroxy-6-methyloctanoate (9). To a so-
lution of 8 (4.72 g, 24 mmol) in ethanol (100 mL) and water (2
mL) was added sodium tetrahydroborate (445 mg, 12 mmol) at
room temperature. The mixture was then stirred for 2 h. After re-
moval of the solvent under reduced pressure, the residue was ex-
tracted with ether. The ether layer was washed with brine, dried,
and evaporated. The oily residue was purified by column chroma-
tography [SiO2, CH2Cl2] to give 9 (viscous oil: 3.66 g, 75.1%).
1H NMR (CDCl3) ꢃ 0.78–1.00 (6H, m, CH3 ꢃ 2), 0.98–1.90
(5H, m, CH3CH2(CH3)CHCH2), 1.23 (3H, t, J ¼ 8:0 Hz, OCH2-
CH3), 2.42 (1H, d, J ¼ 8:0 Hz, one of CH(OH)CH2COOEt), 2.46
(1H, d, J ¼ 6:0 Hz, one of CH(OH)CH2COOEt), 3.10 (1H, br s,
OH, exchangeable by D2O), 4.0 (1H, m, CH(OH)), 4.13 (2H, q,
J ¼ 8:0 Hz, OCH2CH3). IR (liquid) cmꢁ1: 3452, 1735. High-
resolution FAB-MS (m=z): 203.1644 [M + H]þ, (Calcd for
C11H23O3: 203.1647).
(3RS; 6S)-3-Hydroxy-6-methyloctanoic Acid (10). A mix-
ture of 9 (1.65 g, 8.2 mmol), potassium hydroxide (0.82 g, 15
mmol), water (2 mL), and ethanol (9 mL) was refluxed for 90
min. After ethanol was removed by distillation, water (3 mL)
was added to the residue, neutralized with hydrochloric acid,
and then extracted with dichloromethane. The dichloromethane
layer was washed with brine, dried, and concentrated. The residue
was purified by column chromatography [SiO2, CH2Cl2] to give
10 (viscous oil: 1.31 g, 91.2%). 1H NMR (CDCl3) ꢃ 0.70–l.10
(6H, m, CH3 ꢃ 2), 0.98–l.90 (5H, m, CH3CH2(CH3)CHCH2).
2.50 (lH, d, J ¼ 8:0 Hz, one of CH(OH)CH2COOH), 2.53 (1H,
Linear Partially Protected Polymyxin B1 [(6S)-6-Methyloc-
tanoyl-Dab(2-ClZ)-Thr(Bzl)-Dab(2-ClZ)-Dab-Dab(2-ClZ)-D-
Phe-Leu-Dab(2-ClZ)-Dab(2-ClZ)-Thr(Bzl)-OH] (IIIa) and
Related Compounds (IIIb–IIIg). General Procedure 2: TFA–
H2O (95:5) (6 mL) was added to IIa (630 mg) on ice and then stir-
red for 90 min at room temperature. After filtration to remove the
resin, the TFA solution was evaporated in vacuo, and the residue
was lyophilized from dioxane. The crude product (240 mg) was
dissolved in DMSO (1 mL)–DMF (1 mL) and applied to a column
(1:6 ꢃ 95 cm) of Toyopearl HW-40S (Tosoh Co., Tokyo, Japan)
using DMF:H2O (9:1) as the eluent. Fractions containing the main
product were combined, evaporated, and lyophilized from dioxane
to give IIIa 175 mg (39.0% from Fmoc-Thr(Bzl)-OHMP-resin).
28
[ꢀ]D ꢁ20:8ꢂ (c 0.5, DMF), FAB-MS; Found (for the most
abundant isotopic variant); 2244.85 [M + H]þ, Calcd for
C
110H137Cl5N16O24, HPLC: tR 34.3 min [Column, YMC-Pack
A-803 C4 (4:6 ꢃ 250 mm); Elution, a linear gradient (30 min)
from 38 to 76% MeCN in 0.1% TFA; Flow rate, 1 mL/min;
Detection, 210 nm].
The other linear partially protected polymyxin B peptides
(IIIb–IIIg) were prepared in the same manner as described in
General procedure 2.
28
Linear Partially Protected Polymyxin B2 (IIIb). [ꢀ]D
ꢁ22:7ꢂ (c 0.5, DMF), FAB-MS; Found; 2230.83 [M + H]þ, Calcd
for C109H135Cl5N16O24, HPLC: tR 33.5 min.
Linear Partially Protected Polymyxin B3 (IIIc). [ꢀ]D
28
ꢁ33:7ꢂ (c 0.5, DMF), FAB-MS; Found; 2230.82 [M + H]þ, Calcd
for C109H135Cl5N16O24, HPLC: tR 33.5 min.
Linear Partially Protected Polymyxin B4 (IIId). [ꢀ]D
28

1922 Bull. Chem. Soc. Jpn., 77, No. 10 (2004)
Activity of Polymyxin B N-Terminal Analogs
ꢁ45:8ꢂ (c 0.5, DMF), FAB-MS; Found; 2217.81 [M + H]þ, Calcd
for C108H133Cl5N16O24, HPLC: tR 33.1 min.
hydrolysate: Thr 2.12 (2), Dab 5.94 (6), Phe 0.96 (1), Leu 0.97 (1).
The other polymyxin B peptides Ib–Ig were prepared from
IVb–IVg in the same manner as described in General procedure
4, and the characteristics of the synthetic polymyxin B peptides
are shown in Table 1.
28
Linear Partially Protected Polymyxin B5 (IIIe). [ꢀ]D
ꢁ15:5ꢂ (c 0.5, DMF), FAB-MS; Found; 2244.85 [M + H]þ, Calcd
for C110H137Cl5N16O24, HPLC: tR 34.8 min.
Linear Partially Protected Polymyxin B6 (IIIf).
28
[ꢀ]D
[Dab(Dansyl-Gly)1]-polymyxin B3 [Octanoyl-Dab(Dansyl-
ꢁ7:8ꢂ (c 0.5, DMF), FAB-MS; Found; 2261.86 [M + H]þ, Calcd
Gly)-Thr-Dab-cyclic-(Dab -Dab-D-Phe-Leu-Dab-Dab-Thr )
(
Ã
Ã
Ã
for C110H137Cl5N16O25, HPLC: tR 32.7 and 33.2 min.
Linear Partially Protected [Ile7]-Polymyxin B1 (IIIg).
Amide Bond between the ꢀ-NH2 of Dab4 and ꢁ-COOH of
Thr10)] (Ih). Octanoyl-Dab(ivDde)-Thr(Bzl)-Dab(2-ClZ)-Dab-
(Boc)-Dab(2-ClZ)-D-Phe-Leu-Dab(2-ClZ)-Dab(2-ClZ)-Thr(Bzl)-
O-HMP-resin (IIh) (0.1 mmol) was prepared in the same manner
as described in General procedure 1, except that Fmoc-Dab-
(ivDde)-OH (Watanbe Chem. Ind. Ltd., Hiroshima, Japan) was in-
troduced to position 1. After the incorporation of octanoic acid,
IIh was stirred for 10 min in 2% hydrazine in DMF. Hydrazine
treatment was repeated three times to remove the ivDde protecting
group.22 The amino function of the side chain of Dab1 was then
acylated with Fmoc-Gly-OH, followed by treatment with 20% pi-
peridine in DMF and coupling with dansyl chloride to give Oc-
tanoyl-Dab(Dansyl-Gly)-Thr(Bzl)-Dab(2-ClZ)-Dab(Boc)-Dab(2-
ClZ)-D-Phe-Leu-Dab(2-ClZ)-Dab(2-ClZ)-Thr(Bzl)-O-HMP-resin
(IIh0). According to General procedure 2, IIh0 was treated with
95% TFA to yield a linear partially protected peptide, Octanoyl-
Dab(Dansyl-Gly)-Thr(Bzl)-Dab(2-ClZ)-Dab-Dab(2-ClZ)-D-Phe-
Leu-Dab(2-ClZ)-Dab(2-ClZ)-Thr(Bzl)-OH (IIIh). FAB-MS;
Found (for the most abundant isotopic variant); 2352.9 [M +
H]þ, Calcd. for C115H144Cl4N18O25S. According to General pro-
cedure 3, IIIh was reacted with DPPA to yield protected cyclic
[Dab(Dansyl-Gly)1]-polymyxin B3 (IVh). FAB-MS; Found (for
the most abundant isotopic variant); 2334.9 [M + H]þ, Calcd
for C115H142Cl4N18O24S. According to General procedure 4,
IVh was treated with HF to yield Ih. Yield: 14 mg. The character-
istics of Ih are shown in Table 1.
[ꢀ]D ꢁ15:5ꢂ (c 0.5, DMF), FAB-MS; Found; 2244.84 [M +
28
H]þ, Calcd for C110H137Cl5N16O24, HPLC: tR 34.0 min.
Cyclic Protected Polymyxin B1 [(6S)-6-Methyloctanoyl-
Ã
Dab(2-ClZ)-Thr(Bzl)-Dab(2-ClZ)-cyclic-[Dab -Dab(2-ClZ)-D-
Ã
Ã
Phe-Leu-Dab(2-ClZ)-Dab(2-ClZ)-Thr(Bzl) ] ( Amide Bond
between the ꢀ-NH2 of Dab4 and ꢁ-COOH of Thr10)] (IVa).
General Procedure 3: To an ice-cold solution of IIIa (0.03
mmol, 67 mg) in DMSO (1 mL)–DMF (2 mL) was added diphen-
yl phosphorazidate (DPPA)19 (0.12 mmol, 33 mg) and 1-methyl-
morpholine (0.30 mmol, 30 mL). The mixture was stirred at 4
ꢂC for 24 h and evaporated to half the original volume. The solu-
tion was applied to a Toyopearl HW-40S column in the same man-
ner as described in General procedure 2. The fractions containing
the product were combined, evaporated, and lyophilized from di-
oxane to give IVa: 63.5 mg (95.1%). [ꢀ]D ꢁ18:2ꢂ (c 0.5, DMF),
28
FAB-MS; Found (for the most abundant isotopic variant); 2227.85
[M + H]þ, Calcd for C110H135Cl5N16O23. HPLC; tR 37.7 min
(under the same conditions as described above for IIIa).
The other cyclic protected polymyxin B peptides (IVb–IVg)
were prepared in the same manner as described in General proce-
dure 3. The yields of IVb–IVg were almost quantitative.
28
Cyclic Protected Polymyxin B2 (IVb). [ꢀ]D ꢁ18:6ꢂ (c 0.5,
DMF), FAB-MS; Found; 2213.83 [M
C
+
H]þ, Calcd for
109H133Cl5N16O23, HPLC: tR 36.9 min.
Cyclic Protected Polymyxin B3 (IVc). [ꢀ]D ꢁ16:2ꢂ (c 0.5,
28
Des-Fatty Acyl-Polymyxin B (1–10), [H-Dab-Thr-Dab-cy-
Ã
Ã
Ã
DMF), FAB-MS; Found; 2213.83 [M
109H133Cl5N16O23, HPLC: tR 36.9 min.
+
H]þ, Calcd for
clic-(Dab -Dab-D-Phe-Leu-Dab-Dab-Thr ) ( Amide Bond
between the ꢀ-NH2 of Dab4 and ꢁ-COOH of Thr10)] (Ii) and
Related Compounds (Ij–Il). General Procedure 5: Fmoc-
Dab(2-ClZ)-Thr(Bzl)-Dab(2-ClZ)-Dab(Boc)-Dab(2-ClZ)-D-Phe-
Leu-Dab(2-ClZ)-Dab(2-ClZ)-Thr(Bzl)-O-HMP-resin (IIi), ob-
tained as an intermediate in General procedure 1, was treated with
95% TFA according to General procedure 2 to yield a linear par-
tially protected peptide, Fmoc-Dab(2-ClZ)-Thr(Bzl)-Dab(2-ClZ)-
Dab-Dab(2-ClZ)-D-Phe-Leu-Dab(2-ClZ)-Dab(2-ClZ)-Thr(Bzl)-
OH (IIIi). FAB-MS; Found (for the most abundant isotopic var-
iant); 2327.8 [M + H]þ, Calcd for C116H131Cl5N16O25. The DPPA
cyclization of IIIi according to General procedure 3 yielded
C
Cyclic Protected Polymyxin B4 (IVd). [ꢀ]D ꢁ19:4ꢂ (c 0.5,
28
DMF), FAB-MS; Found; 2199.81 [M
108H131Cl5N16O23, HPLC: tR 36.1 min.
+
H]þ, Calcd for
C
Cyclic Protected Polymyxin B5 (IVe). [ꢀ]D ꢁ20:0ꢂ (c 0.5,
28
DMF), FAB-MS; Found; 2227.83 [M
110H135Cl5N16O23, HPLC: tR 37.7 min.
+
H]þ, Calcd for
C
28
Cyclic Protected Polymyxin B6 (IVf). [ꢀ]D ꢁ12:7ꢂ (c 0.5,
DMF), FAB-MS; Found; 2243.84 [M
110H135Cl5N16O24, HPLC: tR 35.6 and 36.0 min.
Cyclic Protected [Ile7]-Polymyxin B1 (IVg). [ꢀ]D ꢁ12:7ꢂ
(c 0.5, DMF), FAB-MS; Found; 2227.84 [M + H]þ, Calcd for
+
H]þ, Calcd for
C
28
ꢀ
Fmoc-Dab(2-ClZ)-Thr(Bzl)-Dab(2-ClZ)-cyclic-[Dab -Dab(2-ClZ)-
ꢀ
ꢀ
C
110H135Cl5N16O23, HPLC: tR 37.5 min.
Polymyxin B1 [(6S)-6-Methyloctanoyl-Dab-Thr-Dab-cyclic-
D-Phe-Leu-Dab(2-ClZ)-Dab(2-ClZ)-Thr(Bzl) ] ( amide bond
between the ꢁ-NH2 of Dab4 and ꢀ-COOH of Thr10) (IVi).
FAB-MS; Found (for the most abundant isotopic variant);
2309.8 [M + H]þ, Calcd for C116H129Cl5N16O24. IVi was treated
with 20% piperidine in DMF for 30 min to remove the Fmoc
group. The solution was evaporated in vacuo, and the residue
was lyophilized from dioxane. The product was treated with HF
and purified according to General procedure 4 to yield Ii. The
characteristics of Ii are shown in Table 1.
Ã
Ã
Ã
(Dab -Dab-D-Phe-Leu-Dab-Dab-Thr ) ( Amide Bond be-
tween the ꢀ-NH2 of Dab4 and ꢁ-COOH of Thr10)] (Ia) and Re-
lated Compounds (Ib–Ig). General Procedure 4: Cyclic pro-
tected polymyxin B1 (IVa) (44.5 mg, 0.02 mmol) was treated with
anhydrous HF (2 mL)–anisole (0.2 mL) for 1 h in an ice bath. The
excess HF was then removed in vacuo. The residue was dissolved
in H2O (15 mL), washed three times with ether, and lyophilized.
The crude product was purified by HPLC employing a Capcell
Pak C18 (1 ꢃ 25 cm). The eluate containing the main product
was lyophilized. The product was passed through a Toyopearl
HW-40 column (1:5 ꢃ 57 cm) using 25% CH3CN in 5 mmol/L
HCl as the eluent to give purified polymyxin B1 as a hydrochlo-
ride salt (Ia): 14.5 mg (52.3%). Amino acid analysis of the acid
Des-Fatty Acyl-Polymyxin B (2–10) (Ij), Des-Fatty Acyl-Pol-
ymyxin B (3–10) (Ik), and Des-Fatty Acyl-Polymyxin B (4–10)
(Il). In the same manner as described in General procedure 5, Ij,
Ik, and Il were prepared. Their characteristics are shown in
Table 1.
Octanoyl-Polymyxin B (2–10) [Octanoyl-Thr-Dab-cyclic-

N. Sakura et al.
Bull. Chem. Soc. Jpn., 77, No. 10 (2004) 1923
Ã
Ã
Ã
(Dab -Dab-D-Phe-Leu-Dab-Dab-Thr ) ( Amide Bond be-
tween the ꢀ-NH2 of Dab4 and ꢁ-COOH of Thr10)] (Im). Gen-
fer (5 mM, pH 7.2) (1 mL), followed by a solution of LPS in H2O
(3 mg/mL) (10 mL, 30 mg). The solutions were kept at 30 ꢂC for 1
h until the fluorescence intensity reached a plateau. The intensity
of the dansyl fluorescence of Ih was measured at an excitation
wavelength of 330 nm and an emission wavelength of 490 nm.
A solution of each polymyxin B component peptide (Ia–Ig) or
N-terminal deletion analog (Ii–Io) (1 mmol/mL) (4 mL each)
was added cumulatively to the quartz cuvettes at 5-min intervals
to obtain 8 points (4–32 nmol). The changes in fluorescence inten-
sity were measured after each addition. The initial intensity of
fluorescence was taken as 100%. The percent fluorescence inten-
sity was plotted as a function of the peptide concentration. The
concentrations required for 50% quenching of Ih bound to LPS
(IC50) were derived from the quenching curves of synthetic pep-
tides (Table 2 and Fig. 6). The binding experiments were repeated
at least three times for each peptide to obtain reproducible results.
Comparisons among the groups of the IC50 values of polymyxin B
component peptides were made using ANOVA. The IC50 values
of Ib, Ic, and Id were significantly higher than that of Ia
(p < 0:01), and those of Ia, Ie, If, and Ig were not significantly
different. No significant differences among the IC50 values of
Ib, Ic, Id, and If were observed.
eral Procedure 6:
The coupling of octanoic acid with
H-Thr(Bzl)-Dab(2-ClZ)-Dab(Boc)-Dab(2-ClZ)-D-Phe-Leu-Dab(2-
ClZ)-Dab(2-ClZ)-Thr(Bzl)-O-HMP-resin, obtained as an inter-
mediate in the General procedure 1, yielded Octanoyl-Thr(Bzl)-
Dab(2-ClZ)-Dab(Boc)-Dab(2-ClZ)-D-Phe-Leu-Dab(2-ClZ)-Dab-
(2-ClZ)-Thr(Bzl)-O-HMP-resin (IIm). Treatment of IIm with
95% TFA according to General procedure 2 yielded Octanoyl-
Thr(Bzl)-Dab(2-ClZ)-Dab-Dab(2-ClZ)-D-Phe-Leu-Dab(2-ClZ)-
Dab(2-ClZ)-Thr(Bzl)-OH (IIIm). FAB-MS; Found (for the most
abundant isotopic variant); 1961.9 [M + H]þ, Calcd for
C97H122Cl4N14O21. The DPPA cyclization reaction of IIIm ac-
cording to General procedure 3 yielded Octanoyl-Thr(Bzl)-Dab-
ꢀ
(2-ClZ)-Dab -Dab(2-ClZ)-D-Phe-Leu-Dab(2-ClZ)-Dab(2-ClZ)-
Thr(Bzl)
ꢀ
ꢀ
(
amide bond between the ꢁ-NH2 of Dab4 and ꢀ-
COOH of Thr10) (IVm). FAB-MS; Found (for the most abundant
isotopic variant); 1943.9 [M + H]þ, Calcd for C97H120Cl4N14O20.
Treatment of IVm with HF followed by purification of the product
according to General procedure 4 yielded Im. The characteristics
of Im are shown in Table 1.
Octanoyl-Polymyxin B (3–10) (In) and Octanoyl-Polymyxin B
(4–10) (Io) were prepared in the same manner as described in
General procedure 6 and the characteristics are shown in Table 1.
We thank Dr. C. Yanaihara, Osaka College of Pharmacy, for
her critical reading of the manuscript. This work was partially
supported by a special grant from Hokuriku University.
Bacteria and Susceptibility Test.
Escherichia coli IFO
12734, Salmonella typhimurium IFO 12529, and Pseudomonas
aeruginosa IFO 3080 were purchased from the Institute for Fer-
mentation, Osaka (IFO), Japan. These bacteria were grown over-
ꢂ
References
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