K. Okimura et al.
Bull. Chem. Soc. Jpn. Vol. 80, No. 3 (2007)
551
25
1 mol Lꢁ1 HCl (50 mL). Yield: 9.51 mg (36.1%). ½ꢀꢄD ꢁ46:9ꢃ (c
pound 21 (2.3 mg, 1.6 mmol) was treated with DMF:H2O:piperi-
dine (5:6:2), and the product was purified by HPLC in the same
0.2, 12% AcOH), FAB-MS calcd for C57H101N14O12 ½M þ Hꢄþ
23
1
2
1173, found 1173. HP-TLC: Rf 0.49, Rf 0.49.
manner as that described for 7. Yield: 0.9 mg (46.9%). ½ꢀꢄD
Colistin A and B (15 and 16). Colistin sulfate (Wako, 600
mg) was purified by RP-HPLC using YMC Pack D-ODS-5ST
(20 ꢂ 150 mm) with 23% CH3CN in 0.1% TFA at a flow rate of
6 mL minꢁ1. The peak products corresponding to colistin A (tR:
39.6 min) and colistin B (tR: 21.7 min) were obtained and lyophi-
ꢁ50:7ꢃ (c 0.5, 12% AcOH), FAB-MS calcd for C48H91N14O12
½M þ Hꢄþ 1055, found 1055. HP-TLC: Rf 0.48, Rf 0.46.
1
2
ꢀ
Boc–Dab(Fmoc)–Tetrakis(N -trifluoroacetyl)–Colistin (2–
10) (23). Compound 19 (13.5 mg, 10 mmol) was coupled with
Boc–Dab(Fmoc)–OH (17.6 mg, 40 mmol) and HATU (15.2 mg,
40 mmol), and the product was isolated in the same manner as that
25
lized. Colistin A (15), yield: 195.5 mg (33.6%). ½ꢀꢄD ꢁ59:2 (c
0.5, 12% AcOH), FAB-MS: 1169 ½M þ Hꢄþ, (calcd. for C53H101
-
N16O13; 1169), HP-TLC: Rf 0.11, Rf 0.41. Colistin B (16),
described for 8. Yield: 17.3 mg (99.7%). ½ꢀꢄD ꢁ26:1ꢃ (c 0.5,
27
DMF), FAB-MS calcd for C72H99F12N16O20 ½M þ Hꢄþ 1735,
1
2
25
1
2
yield: 219.8 mg (38.2%). ½ꢀꢄD ꢁ61:6ꢃ (c 0.5, 12% AcOH), FAB-
found 1735. HP-TLC: Rf 0.92, Rf 0.85.
ꢀ
MS calcd for C52H99N16O13 ½M þ Hꢄþ 1155, found 1155. HP-
Octanoyl–Dab(Fmoc)–Tetrakis(N -trifluoroacetyl)–Colistin
(2–10) (24). Compound 23 (13.0 mg, 7.5 mmol) was treated with
TFA, and the resulting H–Dab(Fmoc)–tetrakis(N -trifluoroace-
1
2
TLC: Rf 0.11, Rf 0.41.
ꢀ
ꢁ
Pentakis(N -trifluoroacetyl)–Colistin A (17). Purified coli-
stin A (15) (164 mg, 0.12 mmol) was trifluoroacetylated with S-
ethyl trifluorothioacetate (380 mg, 2.4 mmol), and the product 17
was precipitated in the same manner as that described for 2. Yield:
tyl)–colistin (2–10) was coupled with octanoic acid in the same
manner as that described for 9. The product was purified on a
Toyopearl HW-40 column in the same manner as that described
27
27
183 mg (95.6%). ½ꢀꢄD ꢁ41:3ꢃ (c 0.5, DMF), FAB-MS calcd for
for 9. Yield: 12.0 mg (90.9%). ½ꢀꢄD ꢁ23:8ꢃ (c 0.5, DMF), FAB-
C63H96F15N16O18 ½M þ Hꢄþ 1649, found 1649. HP-TLC: Rf
MS calcd for C75H105F12N16O19 ½M þ Hꢄþ 1761, found 1761. HP-
1
2
1
2
0.92, Rf 0.84.
Pentakis(N -trifluoroacetyl)–Colistin (18). Colistin sulfate
TLC: Rf 0.90, Rf 0.85.
ꢀ
Octanoyl–Dab–Colistin (2–10) Hydrochloride (Octanoyl–
Colistin, 25). Compound 24 (7.9 mg, 4.5 mmol) was treated with
DMF:H2O:piperidine (5:6:2) in the same manner as that described
for 5, and the product was purified by HPLC under conditions
(Wako, 1.5 g, 1.1 mmol, calculated as 15) was trifluoroacetylated
with S-ethyl trifluorothioacetate (3.4 g, 22 mmol) and the product
18 was precipitated in the same manner as that described for 3.
Yield: 1.69 g (96.6%, calculated as 17). A small amount of the
product 18 dissolved in DMSO was subjected to RP-HPLC on a
YMC Pack D-ODS-5ST column (150 ꢂ 20 mm) with linear gradi-
ent elution (30 min) from 51.3 to 55.1% CH3CN in 0.1% TFA as
the eluent at a flow rate of 5 mL minꢁ1. Two main peaks were
observed, and one of the peaks was characterized as 17 based
on the FAB-MS, HPLC, and HP-TLC analytical data. The other
25
similar to those used for 10. Yield: 2.5 mg (41.7%). ½ꢀꢄD ꢁ51:5ꢃ
(c 0.5, 12% AcOH), FAB-MS calcd for C52H99N16O13 ½M þ Hꢄþ
1
2
1155, found 1155. HP-TLC: Rf 0.10, Rf 0.42.
ꢀ
Acetyl–Tetrakis(N -trifluoroacetyl)–Colistin (2–10) (26).
Compound 19 (26.97 mg, 20 mmol) in 90% DMF (500 mL) was
treated with pyridine 2.25 mL (28 mmol) and acetic anhydride
2.65 mL (28 mmol), and the product was isolated in the same man-
25
ꢁ
peak corresponded to pentakis(N -trifluoroacetyl)–colistin B:
27
ner as that described for 11. Yield: 19.68 mg (70.7%). ½ꢀꢄD
½ꢀꢄD ꢁ41:1ꢃ (c 0.5, DMF), FAB-MS calcd for C62H94F15N16O18
ꢁ41:3ꢃ (c 0.5, DMF), FAB-MS calcd for C50H75F12N14O16 ½M þ
½M þ Hꢄþ 1635, found 1635. HP-TLC: Rf 0.92, Rf 0.84.
Hꢄþ 1355, found 1355. HP-TLC: Rf 0.80, Rf 0.79.
1
2
1
2
ꢀ
Tetrakis(N -trifluoroacetyl)–Colistin (2–10) Hydrochloride
Acetyl–Colistin (2–10) Tetrahydrochloride (27). Compound
26 (27.08 mg, 20 mmol) was treated with DMF:H2O:piperidine
(5:6:2), and the product was purified by HPLC in the same manner
(19). Route A: Purified 17 (150 mg, 91 mmol) was treated with
MSA (90 mL), H2O (45 mL), and dioxane (45 mL), and the prod-
uct was isolated in the same manner as that described for 4. Yield:
25
as that described for 12. Yield: 9.01 mg (40.36%). ½ꢀꢄD ꢁ47:3ꢃ (c
25
7.6 mg (6.2%). ½ꢀꢄD ꢁ42:4ꢃ (c 0.5, 12% AcOH), FAB-MS calcd
0.5, 12% AcOH), FAB-MS calcd for C42H79N14O12 ½M þ Hꢄþ
for C48H73F12N14O15 ½M þ Hꢄþ 1313, found 1313. HP-TLC: Rf
971, found 971. HP-TLC: Rf 0.07, Rf 0.33.
ꢀ
1
1
2
2
0.71, Rf 0.73.
Route B: Pentakis(N -trifluoroacetyl)–colistin (18) (490 mg)
Myristoyl–Tetrakis(N -trifluoroacetyl)–Colistin (2–10) (28).
ꢁ
Compound 19 (53.94 mg, 40 mmol) was coupled with myristoic
acid (11.52 mg, 80 mmol) and HATU (30.4 mg, 80 mmol), and
the product was isolated in the same manner as that described
was treated with MSA (300 mL), H2O (150 mL), and dioxane
(150 mL), and the product was isolated in the same manner as that
25
25
described for 4. Yield: 34.1 mg (8.5%). ½ꢀꢄD ꢁ40:4ꢃ (c 0.5, 12%
for 13. Yield: 46.58 mg (76.5%). ½ꢀꢄD ꢁ37:3ꢃ (c 0.5, DMF),
AcOH), FAB-MS calcd for C48H73F12N14O15 ½M þ Hꢄþ 1313,
FAB-MS calcd for C62H99F12N14O16 ½M þ Hꢄþ 1523, found1523.
1
2
1
2
found 1313. HP-TLC: Rf 0.70, Rf 0.73.
HP-TLC: Rf 0.91, Rf 0.85.
Colistin (2–10) Pentahydrochloride (20). Compound 19 (2.0
mg, 1.5 mmol) was treated with a mixture of DMF:H2O:piperidine
(5:6:2), and the product was isolated in the same manner as that
Myristoyl–Colistin (2–10) Tetrahydrochloride (29). Com-
pound 28 (30.44 mg, 20 mmol) was treated with DMF:H2O:piperi-
dine (5:6:2), and the product was purified by HPLC in the same
25
25
described for 5. Yield: 1.0 mg (60.7%). ½ꢀꢄD ꢁ45:6ꢃ (c 0.5, 12%
manner as that described for 14. Yield: 6.47 mg (25.2%). ½ꢀꢄD
AcOH), FAB-MS calcd for C40H77N14O11 ½M þ Hꢄþ 929, found
ꢁ46:4ꢃ (c 0.5, 12% AcOH), FAB-MS calcd for C54H103N14O12
929. HP-TLC: Rf 0.04, Rf 0.16.
ꢀ
½M þ Hꢄþ 1139.788, found: 1139.8. HP-TLC: Rf 0.48, Rf 0.48.
Antimicrobial Activities of Synthetic Polymyxin B and
Colistin Analogs. The antimicrobial activities of synthetic pep-
tides were estimated by the standard micro plate dilution method
as reported previously.21
1
2
1
2
Octanoyl–Tetrakis(N -trifluoroacetyl)–Colistin (2–10) (21).
Compound 19 (13.5 mg, 10 mmol) was coupled with octanoic acid
(5.76 mg, 40 mmol) and HATU (15.2 mg, 40 mmol), and the prod-
uct was isolated in the same manner as that described for 6. Yield:
27
14.0 mg (97.3%). ½ꢀꢄD ꢁ26:3ꢃ (c 0.5, DMF), FAB-MS calcd for
LPS Binding Activity. LPS binding activity was examined
according to the method reported by Moore.31 A solution of [Dab-
(Dansyl–Gly)1]–polymyxin B3 (4 nmol in 4 mL) and 30 mg of LPS
(E. coli, serotype005:B5, Sigma Chemical Co.) in 5 mM HEPES
C56H87F12N14O16 ½M þ Hꢄþ 1439, found1439. HP-TLC: Rf 1 0.89,
2
Rf 0.84.
Octanoyl–Colistin (2–10) Tetrahydrochloride (22). Com-