Semi-synthesis of polymyxin B (2-10) and colistin (2-10) analogs employing the trichloroethoxycarbonyl (Troc) group for side chain protection of α, γ-diaminobutyric acid residues

Article abstract of DOI:10.1248/cpb.55.1724

Improved strategies for the chemical conversion of natural polymyxin B and colistin to their N-terminal analogs are reported. First, the protection of the side chains of five L-α,γ-diaminobutyric acid (Dab) residues in natural polymyxin B and colistin was achieved with trichloroethoxycarbonyl (Troc), then the resulting pentakis(N^γ-Troc)-polymyxin B and pentakis(N^γ-Troc)-colistin were treated with trifluoroacetic acid (TFA): methanesulfonic acid (MSA): dimethylformamide (DMF) :H₂O (10:30:55:5) at 40 ° C in order to remove N^αalkanoyl- Dab(Troc)-OH selectively. The new key compounds, tetrakis(N^γ- Troc)-polymyxin B (2-10) and tetrakis(N^γ-Troc)-colistin (2-10), were obtained in 19% and 15% yields, respectively, which is higher than previous reports using trifluoroacetyl (Tfa) for tetrakis(N^γ- Tfa)-polymyxin B (2-10) and tetrakis(N^γ-Tfa)-colistin (2-10), respectively.²⁾ Acylation of tetrakis(N^γ-Troc)- polymyxin B (2-10) and tetrakis(N^γ-Troc)-colistin (2-10) with various hydrophobic acids bearing aliphatic or aromatic ring structures, followed by the deprotection of Troc by Zn in AcOH, produced polymyxin B (2-10) and colistin (2-10) analogs which were used for structure-activity relationship studies. It was found that cyclohexylbutanoyl-, 4-biphenylacetyl-, and 1-adamantaneacetyl-polymyxin B (2-10) showed potent antimicrobial activity equal to that of polymyxin B against three Gram-negative bacterial strains. The lipopolysacharide (LPS) binding activity of cyclohexylbutanoyl-, 4-biphenylacetyl-, and cyclododecanecarbonyl-polymyxin B (2-10) increased greatly in comparison with that of polymyxin B (2-10). The various N ^α-acylated polymyxin B (2-10) analogs showed slightly higher antimicrobial and LPS binding activities than the corresponding N ^α-acylated colistin (2-10) analogs.

Full text of DOI:10.1248/cpb.55.1724

1724
Chem. Pharm. Bull. 55(12) 1724—1730 (2007)
Vol. 55, No. 12
Semi-synthesis of Polymyxin B (2-10) and Colistin (2-10) Analogs
Employing the Trichloroethoxycarbonyl (Troc) Group for Side Chain
1)
a g
Protection of , -Diaminobutyric Acid Residues
Keiko OKIMURA, Kazuhiro OHKI, Yuki SATO, Kuniharu OHNISHI, and Naoki SAKURA*
Faculty of Pharmaceutical Sciences, Hokuriku University; Kanagawa-machi, Kanazawa 920–1181, Japan.
Received August 9, 2007; accepted September 22, 2007
Improved strategies for the chemical conversion of natural polymyxin B and colistin to their N-terminal
analogs are reported. First, the protection of the side chains of five L-a,g-diaminobutyric acid (Dab) residues
in natural polymyxin B and colistin was achieved with trichloroethoxycarbonyl (Troc), then the resulting
pentakis(Ng-Troc)-polymyxin B and pentakis(Ng-Troc)-colistin were treated with trifluoroacetic acid (TFA) :
methanesulfonic acid (MSA) : dimethylformamide (DMF) : H2O (10 : 30 : 55 : 5) at 40 °C in order to remove Na-
alkanoyl-Dab(Troc)-OH selectively. The new key compounds, tetrakis(Ng-Troc)-polymyxin B (2-10) and
tetrakis(Ng-Troc)-colistin (2-10), were obtained in 19% and 15% yields, respectively, which is higher than previ-
ous reports using trifluoroacetyl (Tfa) for tetrakis(Ng-Tfa)-polymyxin B (2-10) and tetrakis(Ng-Tfa)-colistin (2-
10), respectively.2) Acylation of tetrakis(Ng-Troc)-polymyxin B (2-10) and tetrakis(Ng-Troc)-colistin (2-10) with
various hydrophobic acids bearing aliphatic or aromatic ring structures, followed by the deprotection of Troc by
Zn in AcOH, produced polymyxin B (2-10) and colistin (2-10) analogs which were used for structure–activity re-
lationship studies. It was found that cyclohexylbutanoyl-, 4-biphenylacetyl-, and 1-adamantaneacetyl-polymyxin
B (2-10) showed potent antimicrobial activity equal to that of polymyxin B against three Gram-negative bacterial
strains. The lipopolysacharide (LPS) binding activity of cyclohexylbutanoyl-, 4-biphenylacetyl-, and cyclodode-
canecarbonyl-polymyxin B (2-10) increased greatly in comparison with that of polymyxin B (2-10). The various
Na-acylated polymyxin B (2-10) analogs showed slightly higher antimicrobial and LPS binding activities than the
corresponding Na-acylated colistin (2-10) analogs.
Key words polymyxin
trichloroethoxycarbonyl group; antimicrobial activity
B
(2-10) analog; tetrakis(Ng-Troc)-colistin nonapeptide; semi-synthesis; methanesulfonic acid;
Polymyxin
B
and colistin, produced by Bacillus function of Dab1 in polymyxin B1, the main component of
polymyxa3,4) and Bacillus colistinus,5) are cationic cyclic de- polymyxin B, is acylated by (S)-6-methyloctanoic acid, and
capeptide antibiotics active against Gram-negative bacteria most of the other minor components are replaced by various
that bind to the lipid A portion of lipopolysaccharides fatty acids.7) Colistin A (polymyxin E1) and colistin B
(LPS).6) Both peptides contain six L-a,g-diaminobutyric acid (polymyxin E2) are the main components of colistin. Colistin
(Dab) residues. The Ng-amino function of Dab at position 4 A is acylated by (S)-6-methyloctanoic acid at the N-terminus,
(Dab4) is acylated by C-terminal Thr10 to form a 23-member and colistin B is acylated by 6-methylheptanoic acid.9—11)
lactam ring (Fig. 1). The peptides have identical amino acid
Therapeutic applications of polymyxin B and colistin have
sequences except for position 6, which is occupied by D-Phe been limited to topical use because of their toxicity to hu-
in polymyxin B and by D-Leu in colistin.7,8) The Na-amino mans.12) Most of the toxic activity of these peptide antibiotics
Fig. 1. Synthesis of Tetrakis(Ng-Troc)-polymyxin B (2-10) (3) or Tetrakis(Ng-Troc)-colistin (2-10) (4) from Polymyxin B1 or Colistin A
∗ To whom correspondence should be addressed. e-mail: n-sakura@hokuriku-u.ac.jp
© 2007 Pharmaceutical Society of Japan

December 2007
1725
are due to the N-terminal fatty acyl-Dab1. Polymyxin B (2- istin. In the present study, Troc was examined as an acid-re-
10) and colistin (2-10) are cyclic nonapeptides obtained re- sistant protecting group for the Dab side chains of polymyxin
spectively from polymyxin B and colistin by enzymic re- B and colistin. Pentakis(Ng-Troc)-polymyxin B (1) and pen-
moval of fatty acyl-Dab-OH with ficin13) or papain.14) The takis(Ng-Troc)-colistin (2) were prepared almost quantita-
toxicity of the nonapeptides is known to be markedly low in tively by treating commercially available polymyxin B or col-
comparisons with the parent compounds, polymyxin B and istin with Troc-Cl and triethylamine (TEA) in 90% dimethyl-
colistin; however, both nonapeptides are very weak bacterici- formamide (DMF). To avoid derivatizing Thr side chains at
dals despite the fact that they retain considerable binding ac- positions 2 and 10 with Troc, aqueous DMF was used as the
tivity to LPS.15) Therefore, the development of nonapeptide solvent. The resulting pentakis(Ng-Troc)-polymyxin B (1)
analogs with increased antimicrobial activity and/or higher and pentakis(Ng-Troc)-colistin (2) were dissolved in trifluo-
LPS binding activity is of interest.
roacetic acid (TFA) : MSA : DMF : H2O (10 : 30 : 55 : 5), and
Previously, we reported a strategy for chemically convert- the solution was allowed to react at 40 °C for 48 h to remove
ing natural polymyxin B and colistin to their N-terminal fatty acyl-Dab(Troc)-OH. After cleavage, the product was
analogs.2) The side chains of Dab residues in the natural pep- treated with diluted ammonia to reverse the N–O migration
tides were first protected by trifluoroacetyl (Tfa) groups; the products, as reported previously.2) Following purification by
key step of the conversion strategy was the treatment of the RP-HPLC, the new key compounds, tetrakis(Ng-Troc)-
Ng-protected natural peptides with aqueous methanesulfonic polymyxin B (2-10) (3) and tetrakis(Ng-Troc)-colistin (2-10)
acid (MSA)16—20) in order to cleave Na-alkanoyl-Dab(Tfa)- (4), were obtained in 18.9% and 15.2% yield, respectively
OH. Tetrakis(Ng-Tfa)-polymyxin B (2-10) and tetrakis(Ng- (Fig. 1). Higher yields of these key compounds compared to
Tfa)-colistin (2-10), which have Na-free and Ng-protected that of their Tfa derivatives may be attributable not only to
amino functions, were useful starting materials for the semi- changing the protecting group from Tfa to Troc, but also to
synthesis of their N-terminal derivatives. However, the yields changes in the reaction conditions, since using a very low
of tetrakis(Ng-Tfa)-polymyxin B (2-10) and tetrakis(Ng-Tfa)- content of H2O in the MSA–TFA–DMF mixture should re-
colistin (2-10) were only 8—11%. In this study, we investi- sult in higher selectivity during the hydrolysis reaction as re-
gated the trichloroethoxycarbonyl (Troc) group as an alterna- ported in the selective chemical removal of acyl amino acids
tive acid-resistant Ng-protecting group for the Dab residues from the N-terminally acylated peptides such as pGlu-pep-
of polymyxin B and colistin, and sixteen Na-acylated non- tides and Myr-Gly-peptides.2,16—19) The removal of fatty acyl-
apeptide analogs (5—20) were synthesized by an improved Dab(Troc)-OH from 1 and 2 by the cleavage of Dab(Troc)-
semi-synthetic route in order to develop nonapeptide analogs Thr bond at the positions 1–2 was considered to be facilitated
with potent antimicrobial and LPS binding activities.
through N–O migration,2) however, the cleavage of another
bond at positions 9–10 seemed to be unavoidable, resulting
in the still unsatisfactory yields of 3 and 4.
Results and Discussion
Semi-synthesis As reported previously,2) the preparation
The N-terminal acylation of tetrakis(Ng-Troc)-polymyxin
of Na-free and Ng-protected nonapeptides from Ng-protected B (2-10) (3) and tetrakis(Ng-Troc)-colistin (2-10) (4) with
cyclic decapeptides was the key step for the semi-synthesis fatty acids was achieved using HATU as the coupling
of N-terminal nonapeptide analogs of polymyxin B and col- reagent. Eight fatty acids bearing aliphatic or aromatic ring
Fig. 2. Synthesis of Polymyxin B (2-10) and Colistin (2-10) Derivatives

1726
Vol. 55, No. 12
tures to the nonapeptides polymyxin B (2-10) and colistin (2-
10) greatly increased their bactericidal activity. Three
polymyxin B (2-10) analogs, acylated with 1-adaman-
taneacetyl- (5), 4-biphenylacetyl- (7), and cyclohexylbu-
tanoyl- (8), showed potent antimicrobial activities equal to
that of polymyxin B. 2-Anthracenecarbonyl- (6), 1-pyrenebu-
tanoyl- (9), and cyclododecanecarbonyl- (11) analogs exhib-
ited slightly reduced antimicrobial activity, equal to that of
octanoyl-polymyxin
B
(2-10)24) and octanoyl-colistin
(2-10),2) which were recently reported to have MIC values
of 1.0—4.0 nmol/ml against E. coli and Salmonella Ty-
phimurium (S. Typhimurium), respectively. trans-4-Propylcy-
clohexanecarbonyl-polymyxin B (2-10) (10) and 2-cyclo-
pentylphenylacetyl-polymyxin B (2-10) (12) showed reduced
Fig. 3. HPLC Profile of Cyclohexylbutanoyl-polymyxin B (2-10) (8) Pre-
pared by Chemical Conversion
Column, Tosoh TSK-GEL ODS-120T (4.6ꢀ250 mm); linear 40 min gradient elution
from 19.0 to 57.0% CH3CN in 0.1% TFA; flow, 1 ml/min; detection, 210 nm.
structures were examined in this study (Fig. 2). The resulting activities, with MIC values of 4 and 8 nmol/ml, respectively
Na-fatty acylated tetrakis(Ng-Troc)-polymyxin B (2-10) (Table 1), similar to that reported by Tsubery for Fmoc-
(5a—12a) and Na-fatty acylated tetrakis(Ng-Troc)-colistin polymyxin B (2-10), which has a MIC value of 8 mg/ml.25) It
(2-10) (13a—20a) were treated with Zn in AcOH to remove seems likely that the antimicrobial activities of polymyxin B
the Troc groups. The products were purified first by RP- (2-10) analogs bearing various fatty acyl groups are slightly
HPLC and then by gel-filtration, using 5 mmol/l hydrochloric higher against E. coli and S. Typhimurium than those of the
acid as the eluent, to yield fatty acyl-nonapeptides as their corresponding colistin (2-10) analogs. The activities of vari-
tetrahydrochlorides. The structures of the synthetic poly- ous colistin (2-10) analogs varied in an approximately similar
myxin B (2-10) analogs (5—12) and colistin (2-10) analogs manner as the corresponding polymyxin B (2-10) analogs.
(13—20), shown in Fig. 2, were confirmed by FAB-MS. The These results suggest that, for antimicrobial activity, D-Phe at
purity of these synthetic peptides was demonstrated by ana- position 6 is superior to D-Leu in this family of peptides.
lytical HPLC as representatively shown in Fig. 3 for analog 8
It is well known that the nonapeptides polymyxin B (2-10)
(the purity 97%), as well as HP-TLC. Thus, starting from the and colistin (2-10) have almost no antimicrobial activ-
new key compounds 3 and 4 highly pure fatty acyl-nonapep- ity.13,14,24) However, introduction of octanoyl to the Na-amino
tides were obtained using a single coupling reaction, fol- function of nonapeptides greatly increases their activity.2,24)
lowed by deprotection. Recently, a strategy for the synthesis In this study it was found that cyclohexylbutanoyl-polymyxin
of tetra-tert-butyloxycarbonyl (Boc)-protected polymyxin B B (2-10) (8) showed two-fold higher antimicrobial activity
nonapeptide was reported by O’Dowd et al.21) employing against E. coli compared with polymyxin B, and its activity
non-protected polymyxin B (2-10) as the starting material, against S. Typhimurium and Pseudomonas aeruginosa (P.
and the selective tetra-Boc-protection of the Dab side chain aeruginosa) was equal to that of polymyxin B. Analog 8 is
amino groups to produce Na-free tetrakis(Ng-Boc)- the most potent synthetic polymyxin B nonapeptide analog
polymyxin (2-10) was described.
reported to date. The cyclohexyl ring structure located at the
Concerning the bactericidal activity of synthetic poly- terminus (the 4-position of butanoyl) in 8 seems to cause fa-
myxin peptides, there were the discrepancies among pub- vorable interactions with the bacterial cell membrane. How-
lished reports. Octanoyl-colistin (2-10) synthesized in our pre- ever, a cyclohexyl ring located at the carbonyl of the acyl
vious study showed the potent activity against Escherichia group seems to be unfavorable, since trans-4-propylcyclo-
coli (E. coli) with the MIC value of 1 nmol/l,2) however, the hexylcarbonyl-polymyxin B (2-10) (10) shows low activity.
same compound reported by Chihara was 10—20 times less In this regard, biphenylacetyl-analogs (7, 15) show high ac-
active, which was prepared by the reaction of octanoyl chlo- tivity, while 2-cyclopentylphenylacetyl-analogs (12, 20) with
ride directly with colistin nonapeptide bearing five free a branched bulky structure close to the N-terminus of Thr2
amino functions.22) This product was contaminated with 20% show low activity. The sensitivity of E. coli and S. Ty-
of Ng-octanoyl-colistin nonapeptide(s), which were estimated phimurium to polymyxin B (2-10) and colistin (2-10) analogs
by quantitative analysis of Na-DNP-Thr and Ng-DNP-Dab in varied according to the N-terminal fatty acyl group on the
the dinitrophenylated octanoyl-colistin nonapeptide prepara- nonapeptide, but the sensitivity of P. aeruginosa did not. Pre-
tion.22) The activity of our synthetic myristoyl-colistin (2-10) viously we reported that acetyl-polymyxin B (2-10) and
also differed from that reported 30 years ago.23) Thus, we acetyl-colistin (2-10) retained the potent bactericidal activity
demonstrated in the previous paper2) that the N-terminal only against P. aeruginosa, but not against E. coli and S. Ty-
structure–activity relationship of polymyxin B and colistin phimurium.2) Taking into account the previous results, it is
should be re-examined using highly pure synthetic peptides possible to say that the peptide portions of polymyxin B and
as these descrepancies are believed to be due to differences colistin contributes greater to destroy the cell membrane of P.
in the purities of the synthetic peptides. In the present study, aeruginosa than fatty acyl portion, and it seems likely that
the chemical conversion route described above was applied the constitution of the P. aeruginosa cell membrane differs
to search for potent bioactive acyl-nonapeptide analogs bear- from that of other Gram-negative bacteria.
ing aliphatic or aromatic ring structure in the acyl group (Fig.
2).
LPS Binding Activity The lipopolysaccharide (LPS, de-
rived from E. coli) binding activities of the synthetic analogs
Antimicrobial Activity of Synthetic Peptides Introduc- were examined according to the method reported previ-
tion of fatty acids bearing aliphatic or aromatic ring struc- ously24) (Figs. 4, 5). The activities of 2-anthracenecarbonyl-

December 2007
1727
Fig. 4. Displacement Assay of Synthetic Polymyxin Derivatives
Fig. 5. Displacement Assay of Synthetic Colistin Derivatives
Table 1. Antimicrobial Activity of Synthetic Polymyxin Analogs
MIC (nmol/ml)
Peptide
Salmonella
Typhimurium
Pseudomonas
aeruginosa
Escherichia coli
Polymyxin B (Sigma)
1.0
1.0
4.0
0.5
0.5
2.0
4.0
2.0
8.0
2.0
4.0
1.0
2.0
2.0
8.0
4.0
16.0
0.5
0.5
2.0
1.0
0.5
2.0
4.0
2.0
8.0
1.0
2.0
2.0
1.0
2.0
8.0
2.0
16.0
1.0
1.0
2.0
1.0
1.0
2.0
2.0
2.0
2.0
1.0
2.0
1.0
1.0
1.0
2.0
2.0
2.0
Ada-Ac-polymyxin B (2-10) (5)
2-Anthracene-CO-polymyxin B (2-10) (6)
4-Biphenyl-Ac-polymyxin B (2-10) (7)
cHex-Bu-polymyxin B (2-10) (8)
1-Pyrene-Bu-polymyxin B (2-10) (9)
Propyl-cHex-CO-polymyxin B (2-10) (10)
Cyclododecane-CO-polymyxin B (2-10) (11)
cPenPh-Ac-polymyxin B (2-10) (12)
Ada-Ac-colistin (2-10) (13)
2-Anthracene-CO-colistin (2-10) (14)
4-Biphenyl-Ac-colistin (2-10) (15)
cHex-Bu-colistin (2-10) (16)
1-Pyrene-Bu-colistin (2-10) (17)
Propyl-cHex-CO-colistin (2-10) (18)
Cyclododecane-CO-colistin (2-10) (19)
cPenPh-Ac-colistin (2-10) (20)
analogs (6, 14) and 1-pyrenebutanoyl-analogs (9, 17) were (2-10) (8), and cyclododecanecarbonyl-polymyxin B (2-10)
not examined because these compounds disturbed fluores- (11) showed LPS binding activities close to that observed
cence measurements of the dansyl group. Although none of for octanoyl-polymyxin B (2-10).24) 1-Adamantaneacetyl-
the synthetic nonapeptides examined showed LPS binding polymyxin B (2-10) (5) and propylcyclohexanecarbonyl-
activity comperable to polymyxin B, 4-biphenylacetyl- polymyxin B (2-10) (10) showed low LPS binding activity.
polymyxin B (2-10) (7), cyclohexylbutanoyl-polymyxin B Analog 5, as well as 7 and 8, were potent bactericidals, com-

1728
Vol. 55, No. 12
Pentakis(Ng-trichloroethoxycarbonyl)-colistin (2) Colistin sulfate
(Wako, 847.89 mg, 0.60 mmol, calculated as Colistin A sulfate) was sus-
pended in 95% DMF (80 ml) containing TEA (834 ml, 6.0 mmol), and Troc-
Cl (822 mg, 6.0 mmol) was added while the mixture was cooled with ice.
The mixture was allowed to react for 24 h at room temperature while the pH
was maintained at 7.8 with TEA. The product was solidified by addition of
cold H2O (320 ml), collected by filtration, dried and then reprecipitated from
MeOH–AcOEt–ether–petr. ether. Yield: 1025.91 mg (83.5%, calculated as
pentakis(Ng-trichloroethoxycarbonyl)-colistin A). The crude product was
used for the next reaction without further purification. Small amounts of ma-
terial from the two main peaks of this product were isolated by RP-HPLC on
a CAPCELL PAK UG80 column (20ꢀ250 mm). Purified products were
lyophilized and characterized. Pentakis(Ng-trichloroethoxycarbonyl)-colistin
A: [a]D27 ꢁ28.3° (cꢂ0.5, DMF), FAB-MS m/z: 2047.3 (Calcd for
C68H106Cl15N16O23: 2047.46). HP-TLC: Rf 1 0.90, Rf 2 0.88. Pentakis(Ng-
trichloroethoxycarbonyl)-colistin B: [a]D27 ꢁ28.3° (cꢂ0.5, DMF), FAB-MS
m/z: 2033.3 (Calcd for C67H104Cl15N16O23: 2033.43). HP-TLC: Rf 1 0.90, Rf 2
perable to polymyxin B (Table 1). Thus, the antimicrobial
and LPS binding activities of these compounds are not paral-
lel. These results suggest that 1-adamantaneacetyl-polymyxin
B (2-10) (5) is a potent bactericidal agent that exhibits mod-
erate activity for neutralizing the endotoxin. Previously we
reported that myristoyl-polymyxin B (2-10) and myristoyl-
colistin (2-10) show potent LPS binding activity but have
lower antimicrobial activity than polymyxin B, with a MIC
value of 4.0 nmol/ml against E. coli.2) Thus, myristoyl-
polymyxin B (2-10) may have potent neutralizing activity
against endotoxin while retaining moderate antimicrobial ac-
tivity. 2-Cyclopentylphenylacetyl-polymyxin B (2-10) (12)
and cyclopentylphenylacetyl-colistin (2-10) (20), which have
a bulky structure close to the N-terminus of Thr2, showed
very low LPS binding activity and 8—16 fold lower antimi- 0.88.
crobial activity. Colistin (2-10) analogs less effectively dis-
Tetrakis(Ng-trichloroethoxycarbonyl)-polymyxin B (2-10) Hydrochlo-
ride (3) Compound 1 (93.58 mg, 45 mmol) was dissolved in trifluoroacetic
acid (TFA) (3 ml) cooled in ice, then a mixture of methanesulfonic acid
(MSA) : dimethylformamide (DMF) : water (30 : 55 : 5) (27 ml) was added
and the reaction mix was stirred at 40 °C for 48 h. The mixture was added
directly to a Wakogel 50C18 column (5ꢀ6 cm), which was washed with
H2O (400 ml) to remove MSA. Reaction products were eluted with 75%
aqueous dioxane (350 ml). The eluates were combined, cooled in an ice bath,
and a dilute ammonia solution (0.5 mol/l, 35 ml) was then added. The mix-
ture was stirred for 30 min while cooled with ice, then the solvent was re-
moved under vacuum. The residue was re-dissolved in 90% aqueous dioxane
(50 ml), and then lyophilized. The obtained powder was dissolved in 90%
dioxane, and then purified by RP-HPLC on a YMC Pack D-ODS-5ST col-
umn (150ꢀ20 mm) using a linear gradient (30 min) from 45.6 to 56.05%
CH3CN in 0.1% TFA at a flow rate of 5 ml/min. The eluate was evaporated
and lyophilized, then the obtained product was re-lyophilized from 75%
dioxane (50 ml) containing 1 mol/l HCl (0.1 ml). The yield of 3 was
14.49 mg (18.9%). [a]D27 ꢁ49.7° (cꢂ0.2, 50% AcOH), FAB-MS m/z: 1665.2
(Calcd for C55H79Cl12N14O19: 1665.73). HP-TLC: Rf 1 0.71, Rf 2 0.78.
placed dansylated polymyxin B3 bound to LPS than
polymyxin B (2-10) analogs, suggesting that D-Phe at posi-
tion 6 is superior to D-Leu for LPS binding activity in this
family of peptides.
In conclusion, the synthetic strategy for converting natural
polymyxin B and colistin to their N-terminal analogs was im-
proved twice in the present study with regard to the yields of
the key intermediates, tetrakis(Troc)-polymyxin B (2-10) and
tetrakis(Troc)-colistin (2-10). Acylation of these key inter-
mediates with various fatty acids, followed by deprotection
with Zn–AcOH to remove the Troc protecting group, pro-
vided various nonapeptide analogs in good yields. The pres-
ent study demonstrates that fatty acylated polymyxin B (2-
10) analogs bearing aliphatic or aromatic ring structures are
synthetic antibiotics whose characteristics depend on the spe-
cific fatty acyl group in the compound. Among sixteen syn-
thetic analogs, cyclohexylbutanoyl-polymyxin B (2-10) (8)
Tetrakis(Ng-trichloroethoxycarbonyl)-colistin (2-10) Hydrochloride
(4) Pentakis(Ng-trichloroethoxycarbonyl)-colistin (2) (92.05 mg, 45 mmol)
was dissolved in trifluoroacetic acid (TFA) (3 ml) cooled with ice, a solu-
was found to be as potent a bactericidal agent as polymyxin tion of methanesulfonic acid (MSA) : dimethylformamide (DMF) : water
(30 : 55 : 5) (27 ml) was added, then the mixture was stirred at 40 °C for 48 h.
B against E. coli, S. Typhimurium and P. aeruginosa, but its
binding activity was slightly lower than polymyxin B or
myristoyl-polymyxin B (2-10). 1-Adamantaneacetyl-poly-
myxin B (2-10) (5) showed lower LPS binding activity than
polymyxin B, but the analog 5 was a potent bactericidal.
The product was isolated in the same manner as described for 3. Yield:
11.37 mg (15.2%). [a]D27 ꢁ34.3° (cꢂ0.2, 50% AcOH), FAB-MS m/z: 1631.2
(Calcd for C52H81Cl12N14O19: 1631.71). HP-TLC: Rf1 0.70, Rf2 0.81.
1-Adamantaneacetyl-polymyxin B (2-10) Tetrahydrochloride (5) To
a solution of 3 (25.5 mg, 15 mmol) and N-methylmorpholine (NMM)
(3.06 ml, 30 mmol) in DMF (600 ml), a mixture of 1-adamantaneacetic acid
(11.66 mg, 60 mmol) and HATU (22.8 mg, 60 mmol) in DMF (300 ml) was
Experimental
General Fast-atom bombardment mass spectra (FAB-MS) were ob- added while the mixture was cooled with ice. The mixture was stirred at
tained on a JMS-DX300 mass spectrometer (JEOL Ltd., Japan). The optical
room temperature for 3 h and its pH was maintained at 7.8 with NMM. The
rotations of the peptides were measured using a DIP-370 digital polarimeter mixture was applied to a Toyopearl HW-40 column (100ꢀ2 cm) and eluted
(JASCO Co., Ltd., Japan). HPLC was performed on a system comprised of a
PX-8010 gradient controller (Tosoh, Japan), two CCPD pumps (Tosoh), a
Rheodyne 7125 injector (Rheodyne Inc., U.S.A.) or an AS-8020 automatic
with DMF : water (9 : 1). The eluate fractions containing the main product
were combined and evaporated in vacuo. The residue was lyophilized from
dioxane (3 ml) to give 1-adamantaneacetyl-tetrakis(Ng-trichloroethoxycar-
sample injector (Tosoh), a UV 8000 detector (Tosoh), and an 805 data sta- bonyl)-polymyxin B (2-10) (5a). Yield: 30.39 mg (84.2%). [a]D27 ꢁ25.2°
tion (Waters). HP-TLC was performed on precoated silica-gel plates (cꢂ0.5, DMF), FAB-MS m/z: 1841.3 (Calcd for C67H95Cl12N14O20:
(Kieselgel 60, E. Merck, Germany). Rf 1: BuOH : AcOH : AcOEt : H2O 1841.99). HP-TLC: Rf 1 0.88, Rf 2 0.85.
(1 : 1 : 1 : 1), Rf 2: BuOH : pyridine : AcOH : H2O (30 : 20 : 6 : 24).
Compound 5a (27.6 mg, 15 mmol) was treated with Zn (6 mmol,
Pentakis(Ng-trichloroethoxycarbonyl)-polymyxin B (1) Polymyxin B 392.28 mg)–AcOH (5 ml) at 40 °C overnight. After filtration, the product
sulfate (Wako, Japan; 868 mg, 0.60 mmol, calculated as Polymyxin B1 sul- was purified by RP-HPLC on
YMC Pack D-ODS-5ST column
fate) was suspended in 95% DMF (80 ml) containing TEA (834 ml,
(20ꢀ150 mm) using linear gradient elution (30 min) from 25.65 to 30.4%
a
6.0 mmol), and Troc-Cl (822 mg, 6.0 mmol) was added while the mixture CH3CN in 0.1% TFA at a flow rate of 5 ml/min. The eluate fractions contain-
was cooled with ice. The mixture was allowed to react for 24 h at room tem- ing the main peak product were collected, evaporated and lyophilized. The
perature while the pH was maintained at 7.8 with TEA. The product was so- product was applied to a Toyopearl HW-40 column (100ꢀ2 cm) and eluted
lidified by addition of cold H2O (320 ml), collected by filtration, dried, and
with 25% CH3CN in 5 mM HCl. The collected eluate fractions containing the
then reprecipitated from MeOH–AcOEt–ether–petr. ether. Yield: 1019 mg main product were combined, evaporated and lyophilized. Purified 5 was re-
(81.7%, calculated as pentakis(Ng-trichloroethoxycarbonyl)-polymyxin B1). lyophilized from 75% dioxane. Yield (5): 10.46 mg (54.3%). [a]D27 ꢁ62.8°
The crude product was used for the next reaction without further purifica-
(cꢂ0.5, 12% AcOH), FAB-MS m/z: 1139 (Calcd for C55H91N14O12: 1139).
tion. A small amount of material from the main peak of this product was iso- HP-TLC: Rf 1 0.38, Rf 2 0.44.
lated by RP-HPLC on a YMC Pack D-ODS-5ST column (150ꢀ20 mm).
2-Anthracenecarbonyl-polymyxin B (2-10) Tetrahydrochloride (6)
The purified product was lyophilized and characterized. [a]D27 ꢁ27.2° To a solution of 3 (25.5 mg, 15 mmol) and NMM (3.06 ml, 30 mmol) in DMF
(cꢂ0.5, DMF), FAB-MS m/z: 2081.3 (Calcd for C71H104Cl15N16O23: (600 ml), a mixture of 2-anthracenecarboxylic acid (13.33 mg, 60 mmol) and
2081.47). HP-TLC: Rf 1 0.89, Rf 2 0.87.
HATU (22.8 mg, 60 mmol) in DMF (300 ml) was added. The mixture was

December 2007
1729
stirred at room temperature for 3 h and treated in the same manner as de-
decanecarbonyl-tetrakis(Ng-trichloroethoxycarbonyl)-polymyxin
B
(2-10)
scribed for 5a to give 2-anthracenecarbonyl-tetrakis(Ng-trichloroethoxycar- (11a). Yield: 23.30 mg (83.60%). [a]D27 ꢁ27.0° (cꢂ0.4, DMF), FAB-MS
bonyl)-polymyxin B (2-10) (6a). Yield: 27.01 mg (96.5%). [a]D27 ꢁ11.2° m/z: 1859.4 (Calcd for C68H101Cl12N14O20: 1860.05). HP-TLC: Rf 1 0.88, Rf 2
(cꢂ0.5, DMF), FAB-MS m/z: 1869.2 (Calcd for C70H87Cl12N14O20: 0.87.
1869.96). HP-TLC: Rf 1 0.88, Rf 2 0.85.
Compound 11a (18.58 mg, 10 mmol) was treated with Zn (4 mmol,
Compound 6a (18.68 mg, 10 mmol) was treated with Zn (4 mmol, 261.52 mg)–AcOH (2.5 ml) at 40 °C overnight. After filtration, the product
261.52 mg)–AcOH (2.5 ml) at 40 °C for 18 h. After filtration, the product
was purified in the same manner as described for 5. Yield (6): 3.72 mg
(28.4%). [a]D27 ꢁ42.5° (cꢂ0.5, 12% AcOH), FAB-MS m/z: 1167 (Calcd for
C58H83N14O12: 1167). HP-TLC: Rf 1 0.37, Rf 2 0.43.
was purified in the same manner as described for 5. Yield (11): 6.84 mg
(52.53%). [a]D27 ꢁ66.8° (cꢂ0.5, 12% AcOH), FAB-MS m/z: 1157 (Calcd
for C56H97N14O12: 1157). HP-TLC: Rf 1 0.38, Rf 2 0.46.
Cyclopentylphenylacetyl-polymyxin B (2-10) Tetrahydrochloride (12)
4-Biphenylacetyl-polymyxin B (2-10) Tetrahydrochloride (7) To a so- To a solution of 3 (25.5 mg, 15 mmol) and NMM (3.06 ml, 30 mmol) in DMF
lution of 3 (25.5 mg, 15 mmol) and NMM (3.06 ml, 30 mmol) in DMF (600 ml), a mixture of cyclopentylphenylacetic acid (12.26 mg, 60 mmol) and
(600 ml), a mixture of 4-biphenylacetic acid (12.73 mg, 60 mmol) and HATU HATU (22.8 mg, 60 mmol) in DMF (300 ml) was added while the mixture
(22.8 mg, 60 mmol) in DMF (300 ml) was added while the reaction mix was was cooled with ice. The mixture was stirred at room temperature for
cooled with ice. The mixture was stirred at room temperature for 5 h and
treated in the same manner as described for 5a to give 4-biphenylacetyl- clopentylphenylacetyl-tetrakis(Ng-trichloroethoxycarbonyl)-polymyxin B (2-
tetrakis(Ng-trichloroethoxycarbonyl)-polymyxin (2-10) (7a). Yield: 10) (12a). Yield: 23.45 mg (84.5%). [a]D27 ꢁ27.6° (cꢂ0.5, DMF), FAB-MS
1 h and treated in the same manner as described for 5a to give cy-
B
27.85 mg (100.0%). [a]D27 ꢁ21.2° (cꢂ0.5, DMF), FAB-MS m/z: 1859.3 m/z: 1851.3 (Calcd for C98H93Cl12N14O20: 1851.98). HP-TLC: Rf 1 0.88, Rf 2
(Calcd for C69H89Cl12N14O20: 1859.96). HP-TLC: Rf 1 0.88, Rf 2 0.86.
Compound 7a (18.58 mg, 10 mmol) was treated with Zn (4 mmol,
261.52 mg)–AcOH (2.5 ml) at 40 °C for 3 h. After filtration, the product was
purified in the same manner as described for 5. Yield (7): 6.74 mg (51.8%).
[a]D27 ꢁ61.6° (cꢂ0.5, 12% AcOH), FAB-MS m/z: 1157 (Calcd for
C57H85N14O12: 1157). HP-TLC: Rf 1 0.37, Rf 2 0.42.
0.86.
Compound 12a (18.50 mg, 10 mmol) was treated with Zn (4 mmol,
261.52 mg)–AcOH (2.5 ml) at 40 °C for 5 h. After filtration, the product was
purified in the same manner as described for 5. Yield (12): 5.61 mg
(43.35%). [a]D27 ꢁ51.7° (cꢂ0.4, 12% AcOH), FAB-MS m/z: 1149 (Calcd
for C56H89N14O12: 1149). HP-TLC: Rf 1 0.38, Rf 2 0.46.
Cyclohexylbutanoyl-polymyxin B (2-10) Tetrahydrochloride (8) To a
1-Adamantaneacetyl-colistin (2-10) Tetrahydrochloride (13) To the
solution of 3 (25.5 mg, 15 mmol) and NMM (3.06 ml, 30 mmol) in DMF solution of 4 (25.0 mg, 15 mmol) and NMM (3.06 ml, 30 mmol) in DMF
(600 ml), a mixture of cyclohexylbutyric acid (10.22 mg, 60 mmol) and (600 ml), a mixture of 1-adamantaneacetic acid (11.66 mg, 60 mmol) and
HATU (22.8 mg, 60 mmol) in DMF (300 ml) was added while the reaction HATU (22.8 mg, 60 mmol) in DMF (300 ml) was added. The mixture was
mix was cooled with ice. The mixture was stirred at room temperature for stirred at room temperature for 1 h and treated in the same manner as de-
2 h and treated in the same manner as described for 5a to give cyclohexylbu-
scribed for 5a to give 1-adamantaneacetyl-tetrakis(Ng-trichloroethoxycar-
tanoyl-tetrakis(Ng-trichloroethoxycarbonyl)-polymyxin B (2-10) (8a). Yield: bonyl)-colistin (2-10) (13a). Yield: 22.83 mg (84.3%). [a]D27 ꢁ28.4° (cꢂ0.5,
23.10 mg (84.8%). [a]D27 ꢁ25.2° (cꢂ0.5, DMF), FAB-MS m/z: 1817.3
(Calcd for C65H95Cl12N14O20: 1817.97). HP-TLC: Rf 1 0.88, Rf 2 0.86.
Compound 8a (18.16 mg, 10 mmol) was treated with Zn (4 mmol,
261.52 mg)–AcOH (2.5 ml) at 40 °C for 3 h. After filtration, the product was
purified in the same manner as described for 5. Yield (8): 6.94 mg (55.1%).
[a]D27 ꢁ67.3° (cꢂ0.5, 12% AcOH), FAB-MS m/z: 1115 (Calcd for
C53H91N14O12: 1115). HP-TLC: Rf 1 0.37, Rf 2 0.43.
DMF), FAB-MS m/z: 1807.3 (Calcd for C64H97Cl12N14O20: 1807.97). HP-
TLC: Rf 1 0.88, Rf 2 0.87.
Compound 13a (18.06 mg, 10 mmol) was treated with Zn (4 mmol,
261.52 mg)–AcOH (2.5 ml), and the product was isolated in the same man-
ner as described for 5. Yield (13): 7.44 mg (59.52%). [a]D27 ꢁ54.9° (cꢂ0.5,
12% AcOH), FAB-MS m/z: 1105 (Calcd for C52H93N14O12: 1105). HP-TLC:
Rf 1 0.37, Rf 2 0.47.
1-Pyrenebutanoyl-polymyxin B (2-10) Tetrahydrochloride (9) To a
2-Anthracenecarbonyl-colistin (2-10) Tetrahydrochloride (14) To the
solution of 3 (25.5 mg, 15 mmol) and NMM (3.06 ml, 30 mmol) in DMF solution of 4 (25.0 mg, 15 mmol) and NMM (3.06 ml, 30 mmol) in DMF
(600 ml),
a mixture of 1-pyrenebutyric acid (17.3 mg, 60 mmol) and (600 ml), a mixture of 2-anthracenecarboxylic acid (13.33 mg, 60 mmol) and
O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate HATU (22.8 mg, 60 mmol) in DMF (300 ml) was added while the mixture
(HATU) (22.8 mg, 60 mmol) in DMF (300 ml) was added while the mixture was cooled with ice. The mixture was stirred at room temperature for 1 h
was cooled with ice. The mixture was stirred at room temperature for 2 h
and treated in the same manner as described for 5a to give 2-anthracenecar-
and treated in the same manner as described for 5a to give 1-pyrenebu-
bonyl-tetrakis(Ng-trichloroethoxycarbonyl)-colistin (2-10) (14a). Yield:
tanoyl-tetrakis(Ng-trichloroethoxycarbonyl)-polymyxin B (2-10) (9a). Yield: 27.78 mg (100.8%). [a]D27 ꢁ9.6° (cꢂ0.5, DMF), FAB-MS m/z: 1835.3
27.36 mg (94.3%). [a]D27 ꢁ22.4° (cꢂ0.5, DMF), FAB-MS m/z: 1935.3 (Calcd for C67H89Cl12N14O20: 1835.94). HP-TLC: Rf 1 0.89, Rf 2 0.87.
(Calcd for C75H93Cl12N14O20: 1936.06). HP-TLC: Rf 1 0.88, Rf 2 0.86.
Compound 14a (18.34 mg, 10 mmol) was treated with Zn (4 mmol,
Compound 9a (19.34 mg, 10 mmol) was treated with Zn (4 mmol, 261.52 mg)–AcOH (2.5 ml), and the product was isolated in the same man-
261.52 mg)–AcOH (2.5 ml) at 40 °C for 20 h. After filtration, the product
was purified in the same manner as described for 5. Yield (9): 6.87 mg
(49.9%). [a]D27 ꢁ59.2° (cꢂ0.4, 12% AcOH), FAB-MS m/z: 1233 (Calcd for
C63H89N14O12: 1233). HP-TLC: Rf 1 0.37, Rf 2 0.43.
ner as described for 5. Yield (14): 4.26 mg (33.33%). [a]D27 ꢁ31.2° (cꢂ0.5,
12% AcOH), FAB-MS m/z: 1133 (Calcd for C55H85N14O12: 1133). HP-TLC:
Rf 1 0.37, Rf 2 0.45.
4-Biphenylacetyl-colistin (2-10) Tetrahydrochloride (15) To the solu-
trans-4-Propylcyclohexanecarbonyl-polymyxin B (2-10) Tetrahydro- tion of 4 (25.0 mg, 15 mmol) and NMM (3.06 ml, 30 mmol) in DMF (600 ml),
chloride (10) To a solution of 3 (25.5 mg, 15 mmol) and NMM (3.06
mixture of 4-biphenylacetic acid (12.73 mg, 60 mmol) and HATU
ml, 30 mmol) in DMF (600 ml), a mixture of trans-4-Propylcyclohexane- (22.8 mg, 60 mmol) in DMF (300 ml) was added while the mixture was
carboxylic acid (17.3 mg, 60 mmol) and HATU (22.8 mg, 60 mmol) in DMF
cooled with ice. The mixture was stirred at room temperature for 2 h and
(300 ml) was added while the reaction mix was cooled with ice. The mixture treated in the same manner as described for 5a to give 4-biphenylacetyl-
was stirred at room temperature for 1 h and treated in the same manner as
tetrakis(Ng-trichloroethoxycarbonyl)-colistin (2-10) (15a). Yield: 23.67 mg
described for 5a to give trans-4-propylcyclohexanecarbonyl-tetrakis(Ng- (86.5%). [a]2D7 ꢁ22.3° (cꢂ0.2, DMF), FAB-MS m/z: 1825.3 (Calcd for
a
trichloroethoxycarbonyl)-polymyxin
B
(2-10) (10a). Yield: 23.03 mg C66H91Cl12N14O20: 1825.95). HP-TLC: Rf 1 0.89, Rf 2 0.88.
(84.54%). [a]D27 ꢁ23.7° (cꢂ0.2, DMF), FAB-MS m/z: 1817.3 (Calcd for
C65H95Cl12N14O20: 1817.97). HP-TLC: Rf 1 0.88, Rf 2 0.86.
Compound 15a (18.24 mg, 10 mmol) was treated with Zn (4 mmol,
261.52 mg)–AcOH (2.5 ml), and the product was isolated in the same man-
Compound 10a (18.16 mg, 10 mmol) was treated with Zn (4 mmol, ner as described for 5. Yield (15): 9.17 mg (72.3%). [a]D27 ꢁ51.5° (cꢂ0.5,
261.52 mg)–AcOH (2.5 ml) at 40 °C for 5 h. After filtration, the product was
purified in the same manner as described for 5. Yield (10): 8.41 mg (66.8%).
[a]D27 ꢁ61.5° (cꢂ0.5, 12% AcOH), FAB-MS m/z: 1115 (Calcd for
C53H91N14O12: 1115). HP-TLC: Rf 1 0.37, Rf 2 0.44.
12% AcOH), FAB-MS m/z: 1123 (Calcd for C54H87N14O12: 1123). HP-TLC:
Rf 1 0.37, Rf 2 0.44.
Cyclohexylbutanoyl-colistin (2-10) Tetrahydrochloride (16) To the
solution of 4 (25.0 mg, 15 mmol) and NMM (3.06 ml, 30 mmol) in DMF
Cyclododecanecarbonyl-polymyxin B (2-10) Tetrahydrochloride (11) (600 ml), a mixture of cyclohexylbutyric acid (10.22 mg, 60 mmol) and
To a solution of 3 (25.5 mg, 15 mmol) and NMM (3.06 ml, 30 mmol) in DMF HATU (22.8 mg, 60 mmol) in DMF (300 ml) was added while the mixture
(600 ml), a mixture of cyclododecanecarboxylic acid (12.74 mg, 60 mmol)
was cooled with ice. The mixture was stirred at room temperature for 1 h
and HATU (22.8 mg, 60 mmol) in DMF (300 ml) was added while the mix- and treated in the same manner as described for 5a to give cyclohexyl-
ture was cooled with ice. The mixture was stirred at room temperature for butanoyl-tetrakis(Ng-trichloroethoxycarbonyl)-colistin (2-10) (16a). Yield:
1 h and treated in the same manner as described for 5a to give cyclodo- 25.32 mg (94.7%). [a]2D7 ꢁ23.3° (cꢂ0.2, DMF), FAB-MS m/z: 1783.3

1730
Vol. 55, No. 12
(Calcd for C62H97Cl12N14O20: 1783.95). HP-TLC: Rf 1 0.88, Rf 2 0.88.
Compound 16a (17.82 mg, 10 mmol) was treated with Zn (4 mmol,
261.52 mg)–AcOH (2.5 ml), and the product was isolated in the same man-
ner as described for 5. Yield (16): 9.30 mg (75.9%). [a]D27 ꢁ48.3° (cꢂ0.5,
12% AcOH), FAB-MS m/z: 1081 (Calcd for C50H93N14O12: 1081). HP-TLC:
Rf 1 0.37, Rf 2 0.45.
polymyxin B3 (4 nmol) and 30 mg of LPS (E. coli, serotype005:B5, Sigma
Chemical Co.) in 5 mM N-2-hydroxyethylpiperazine-Nꢃ-2-ethanesulfonic
acid (HEPES) buffer (pH 7.2, 1 ml) was incubated in a quartz cuvette at
30 °C for 1 h, then a peptide solution (1 mm/ml) (4 ml each) was added in the
same manner as reported previously.24) The fluorescence spectra were meas-
ured using a fluorescence spectrophotometer F-4500 (Hitachi Instrument
1-Pyrenebutanoyl-colistin (2-10) Tetrahydrochloride (17) To the so- Co., Tokyo, Japan) at an excitation wavelength of 330 nm and an emission
lution of 4 (25.0 mg, 15 mmol) and NMM (3.06 ml, 30 mmol) in DMF wavelength of 490 nm.
(600 ml), a mixture of 1-pyrenebutyric acid (17.3 mg, 60 mmol) and HATU
(22.8 mg, 60 mmol) in DMF (300 ml) was added while the mixture was
cooled with ice. The mixture was stirred at room temperature for 1 h and
treated in the same manner as described for 5a to give 1-pyrenebutanoyl-
Acknowledgements We are grateful to Ms. Mayumi Niwa of the Cen-
tral Analytical Center of Hokuriku University for performing the FAB-MS
measurements. This work was supported in part by a special grant from
tetrakis(Ng-trichloroethoxycarbonyl)-colistin (2-10) (17a). Yield: 26.09 mg Hokuriku University.
(91.5%). [a]D27 ꢁ23.2° (cꢂ0.5, DMF), FAB-MS m/z: 1901.3 (Calcd for
C72H95Cl12N14O20: 1902.04). HP-TLC: Rf 1 0.88, Rf 2 0.87.
References and Notes
Compound 17a (20.9 mg, 11 mmol) was treated with Zn (4.4 mmol,
284.67 mg)–AcOH (2.75 ml) at 40 °C for 20 h, and the product was isolated
in the same manner as described for 5. Yield (17): 5.82 mg (39.4%). [a]2D7
ꢁ50.0° (cꢂ0.5, 12% AcOH), FAB-MS m/z: 1199 (Calcd for C60H91N14O12:
1199). HP-TLC: Rf 1 0.37, Rf 2 0.44.
trans-4-Propylcyclohexanecarbonyl-colistin (2-10) Tetrahydrochloride
(18) To the solution of 4 (25.0 mg, 15 mmol) and NMM (3.06 ml, 30 mmol)
in DMF (600 ml), a mixture of trans-4-propylcyclohexanecarboxylic acid
(17.3 mg, 60 mmol) and HATU (22.8 mg, 60 mmol) in DMF (300 ml) was
added while the mixture was cooled with ice. The mixture was stirred at
room temperature for 1 h and treated in the same manner as described for 5a
to give trans-4-propylcyclohexanecarbonyl-tetrakis(Ng-trichloroethoxycar-
bonyl)-colistin (2-10) (18a). Yield: 21.6 mg (81.0%). [a]D27 ꢁ24.8° (cꢂ0.5,
DMF), FAB-MS m/z: 1783.3 (Calcd for C62H97Cl12N14O20: 1787.95). HP-
TLC: Rf 1 0.89, Rf 2 0.88.
1) A part of this work was reported previously as a preliminary commu-
nication; Okimura K., Ohki K., Sato Y., Ohnishi K., Sakura N., “Pep-
tide Science 2006,” ed. by Ishida H., Mihara H., The Japanese Peptide
Society, Japan, 2006, p. 190.
2) Okimura K., Ohki K., Sato Y., Ohnishi K., Uchida Y., Sakura N., Bull.
Chem. Soc. Jpn., 80, 543—552 (2007).
3) Ainsworth G. C., Brown A. M., Brownlee G., Nature (London), 160,
263 (1947).
4) Benedict R. G., Langlykke A. F., J. Bacteriol., 54, 24—25 (1947).
5) Koyama Y., Kurosawa A., Tsuchiya A., Takakuta K., J. Antibiot., 3,
457—458 (1950).
6) Morrison D. C., Jacobs D. M., Immunochemistry, 13, 813—818
(1976).
7) Storm D. R., Rosenthal K. S., Swanson P. E., Ann. Rev. Biochem., 46,
723—763 (1977).
Compound 18a (17.82 mg, 10 mmol) was treated with Zn (4 mmol,
261.52 mg)–AcOH (2.5 ml), and the product was isolated in the same man-
ner as described for 5. Yield (18): 9.70 mg (79.1%). [a]D27 ꢁ48.3° (cꢂ0.5,
12% AcOH), FAB-MS m/z: 1081 (Calcd for C50H93N14O12: 1081). HP-TLC:
Rf 1 0.37, Rf 2 0.46.
Cyclododecanecarbonyl-colistin (2-10) Tetrahydrochloride (19) To
the solution of 4 (25.0 mg, 15 mmol) and NMM (3.06 ml, 30 mmol) in DMF
(600 ml), a mixture of cyclododecanecarboxylic acid (12.74 mg, 60 mmol)
8) Orwa J. A., Govaerts C., Busson R., Roets E., Van Schepdael A.,
Hoogmartens J., J. Antibiot., 54, 595—599 (2001).
9) Suzuki T., Inouye H., Fujikawa K., Suketa Y., J. Biochem. (Tokyo), 54,
25—33 (1963).
10) Suzuki T., Inouye H., Fujikawa K., Nagasawa S., J. Biochem. (Tokyo),
54, 173—180 (1963).
11) Suzuki T., Hayashi K., Fujikawa K., J. Biochem. (Tokyo), 54, 412—
418 (1963).
and HATU (22.8 mg, 60 mmol) in DMF (300 ml) was added while the mix- 12) Hermsen E. D., Sullivan C. J., Rotschafer J. C., Infect. Dis. Clin. N.
ture was cooled with ice. The mixture was stirred at room temperature for
Am., 17, 545—562 (2003).
1 h and treated in the same manner as described for 5a to give cyclodode- 13) Chihara S., Tobita T., Yahata M., Ito A., Koyama Y., Agric. Biol.
canecarbonyl-tetrakis(Ng-trichloroethoxycarbonyl)-colistin (2-10) (19a).
Chem., 37, 2455—2463 (1973).
Yield: 23.0 mg (84.0%). [a]D27 ꢁ25.5° (cꢂ0.5, DMF), FAB-MS m/z: 1825.4 14) Vaara M., Vaara T., Antimicrob. Agents Chemother., 24, 107—113
(Calcd for C65H103Cl12N14O20: 1826.03). HP-TLC: Rf 1 0.87, Rf 2 0.87.
(1983).
Compound 19a (18.24 mg, 10 mmol) was treated with Zn (4 mmol, 15) Warren H. S., Kania S. A., Siber G. R., Antimicrob. Agents
261.52 mg)–AcOH (2.5 ml), and the product was isolated in the same man-
ner as described for 5. Yield (19): 9.55 mg (75.3%). [a]D27 ꢁ53.1° (cꢂ0.5,
12% AcOH), FAB-MS m/z: 1123 (Calcd for C53H99N14O12: 1123). HP-TLC:
Rf 1 0.38, Rf 2 0.47.
Cyclopentylphenylacetyl-colistin (2-10) Tetrahydrochloride (20) To
the solution of 4 (25.0 mg, 15 mmol) and NMM (3.06 ml, 30 mmol) in DMF
(600 ml), a mixture of cyclopentylphenylacetic acid (12.26 mg, 60 mmol) and
Chemother., 28, 107—112 (1985).
16) Hashimoto T., Ohki K., Sakura N., Chem. Pharm. Bull., 44, 2033—
2036 (1996).
17) Ohki K., Sakura N., Hashimoto T., Chem. Pharm. Bull., 45, 194—197
(1997).
18) Kobayashi J., Ohki K., Okimura K., Hashimoto T., Sakura N., Chem.
Pharm. Bull., 54, 827—831 (2006).
HATU (22.8 mg, 60 mmol) in DMF (300 ml) was added while the mixture 19) Okimura K., Sugimoto K., Ohki K., Sakura N., Hashimoto T., Chem.
was cooled with ice. The mixture was stirred at room temperature for
Pharm. Bull., 47, 1249—1255 (1999).
4 h and treated in the same manner as described for 5a to give cy- 20) Okimura K., Ohki K., Nagai S., Sakura N., Biol. Pharm. Bull., 26,
clopentylphenylacetyl-tetrakis(Ng-trichloroethoxycarbonyl)-colistin (2-10)
1166—1169 (2003).
(20a). Yield: 24.1 mg (88.4%). [a]D27 ꢁ19.2° (cꢂ0.5, DMF), FAB-MS m/z: 21) O’Dowd H., Kim B., Margolis P., Wang W., Wu C., Lopez S. L., Blais
1817.3 (Calcd for C65H95Cl12N14O20: 1817.97). HP-TLC: Rf 1 0.88, Rf 2 0.87.
J., Tetrahedron Lett., 48, 2003—2005 (2007).
Compound 20a (18.16 mg, 10 mmol) was treated with Zn (4 mmol, 22) Chihara S., Ito A., Yahata M., Tobita T., Koyama Y., Agric. Biol.
261.52 mg)–AcOH (2.5 ml), and the product was isolated in the same man-
ner as described for 5. Yield (20): 5.1 mg (40.48%). [a]D27 ꢁ38.0° (cꢂ0.2,
12% AcOH), FAB-MS m/z: 1115 (Calcd for C53H91N14O12: 1115). HP-TLC:
Rf 1 0.38, Rf 2 0.46.
Chem., 37, 2709—2717 (1973).
23) Chihara S., Ito A., Yahata M., Tobita T., Koyama Y., Agric. Biol.
Chem., 38, 521—529 (1974).
24) Sakura N., Itoh T., Uchida Y., Ohki K., Okimura K., Chiba K., Sato Y.,
Sawanishi H., Bull. Chem. Soc. Jpn., 77, 1915—1924 (2004).
Antimicrobial Activities of Synthetic Polymyxin
B and Colistin
Analogs The antimicrobial activities of the synthetic peptides were esti- 25) Tsubery H., Ofek I., Cohen S., Fridkin M., Peptides, 22, 1675—1681
mated using the standard micro plate dilution method reported previously.24)
(2001).
LPS Binding Activity LPS binding activity was examined according to 26) Moore R. A., Bates N. C., Hancock R. E. W., Amtimicrob. Agents
the method reported by Moore.26) A solution of [Dab(Dansyl-Gly)1]-
Chemother., 29, 496—500 (1986).