Synthesis and antibacterial activity of tripropeptin C

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Synthesis and antibacterial activity of tripropeptin C derivatives
modified at the carboxyl groups
Sehei Hirosawa1, Yoshiaki Takahashi1,*, Hideki Hashizume2, Toshiaki Miyake1 and
Yuzuru Akamatsu2
1
Institute of Microbial Chemistry (BIKAKEN), Hiyoshi, Japan
2
Institute of Microbial Chemistry (BIKAKEN), Tokyo, Japan
* Corresponding author: Dr Y Takahashi, Institute of Microbial Chemistry (BIKAKEN),
Hiyoshi, 3-34-17, Ida, Nakahara-ku, Kawasaki-shi, Kanagawa 211-0035, Japan.
E-mail: [email protected]
Supplementary material
Index:
1.
2.
3.
4.
General information
Synthesis of compounds 1-11
Biological assays
Mass analysis of complex formation with UPP
S1
S2
S2
S6
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1. General information
1
H NMR spectra were recorded on an AVANCE 500 spectrometer (500 MHz) or
AVANCEIII400 HD spectrometer (400 MHz, Bruker Biospin, Rheinstetten, Germany)
at 300K. The chemical shifts () were measured downfield from tetramethylsilane,
which was used as an internal standard, and were confirmed, when necessary, by
shift-correlated 2D spectra. The mass spectra were recorded on a LTQ XL electrospray
ionization (ESI) spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) and/or a
JMS-T100LC (ESI) spectrometer (JEOL JMS-T100LC, Tokyo, Japan). TLC was
performed on a Kieselgel 60 F254 (Merck, Darmstadt, Germany), and column
chromatography was carried out on Wakogel C-200 (Wako Pure Chemical Industries,
Ltd., Osaka, Japan).
2. Synthesis of compounds 1–11
Tripropeptin C 16-methyl ester (1) and tripropeptin C 4-methyl ester (2).
To a solution of tripropeptin C (92 mg, 0.080 mmol) in methanol (8 ml) was added
trimethylsilyldiazomethane (13.7 mg, 0.12 mmol) in hexane (0.2 ml) and the solution
was kept for 1 h at room temperature. A further charge of trimethylsilyldiazomethane
(13.7 mg, 0.12 mmol) in hexane (0.2 ml) was then added and the solution kept likewise
for further 1 h. TLC (CHCl3-MeOH-H2O, 10:5:1) of the solution showed four spots at
RF=0.55 (1), 0.35, 0.25 and 0.15 (2) (cf. TPPC: RF= 0.10). Concentration gave a residue,
which was purified by chromatography (CHCl3-MeOH-H2O, 10:5:1) to give 1 (14.1 mg,
15%) and 2 (26.3 mg, 28%) as colorless solids.
1: 1H NMR (500 MHz, DMSO-d6):  0.84 (6H, d, J = 6.5 Hz), 3.68 (3H, s, OCH3), 3.95
(1H, m), 4.00 (1H, br s), 4.40 (1H, d, J = 6 Hz), 4.50 (1H, d, J = 6.5 Hz), 4.81 (1H, dd, J
= 3.5 and 10 Hz), 4.88 (1H, br s, OH), 4.95 (1H, d, J = 10 Hz, OH), 6.61 (1H, d, J = 6
Hz), 7.37 (1H, d, J = 5.5 Hz, NH), 7.55 (1H, d, J = 9.5 Hz, NH), 7.95 (1H, d, J = 10 Hz,
NH), 8.22 (1H, d, J = 9.5 Hz, NH), 8.92 (1H, d, J = 9 Hz, NH), 8.99 (1H, br s, NH).
ESI-MS: m/z calcd. for C52H85N11O19 1167.6; found [M+H]+ 1168.9.
2: 1H NMR (500 MHz, DMSO-d6):  0.84 (6H, d, J = 6.5 Hz), 0.98 (3H, d, J = 6 Hz),
2.34 (1H, d, J = 13 Hz), 2.60 (1H, t, J = 13 Hz), 3.53 (3H, s, OCH3), 4.23 (1H, d, J = 8
Hz), 4.38 (1H, br s, OH), 4.80 (1H, d, J = 7.5 Hz), 4.95 (1H, t, J = 5.5 Hz, OH), 5.66
(1H, d, J = 7.5 Hz, OH), 6.71 (1H, d, J = 7 Hz, NH), 7.18 (1H, d, J = 6.5 Hz, NH), 7.99
(1H, d, J = 10 Hz, NH), 8.21 (1H, d, J = 9.5 Hz, NH), 8.55 (1H, d, J = 8.5 Hz, NH),
8.80 (1H, br s, NH). ESI-MS: m/z calcd. for C52H85N11O19 1167.6; found [M+H]+
1168.9.
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Tripropeptin C 16-diphenylmethyl ester (3) and tripropeptin C 4-diphenymethyl
ester (4).
To a solution of tripropeptin C (115 mg, 0.10 mmol) in methanol (5 ml) was added
diphenyldiazomethane (60 mg, 0.31 mmol) and the solution was kept for 2 h at room
temperature. Concentration gave a residue, which was washed with diethyl ether and
chromatographed (CHCl3-MeOH-H2O, 60:20:3) to give 3 (32.9 mg, 25%) and 4 (23.6
mg, 18%) as colorless solids, together with recovered tripropeptin C (58 mg).
3: 1H NMR (500 MHz, DMSO-d6):  0.84 (6H, d, J = 6.5 Hz), 0.97 (3H, d, J = 6 Hz),
2.37 (1H, slightly br d, J = ~13 Hz), 2.84 (1H, dd, J = 11.5 and 15 Hz), 3.17 (1H, t, J =
9 Hz), 4.24 (1H, dt, J = 4, 10 and 10 Hz), 4.56 (1H, dd, J = 4.5 and 6 Hz), 4.85 (1H, dd,
J = 4 and 9 Hz), 4.90 (1H, m), 5.12 (1H, d, J = 4 Hz, OH), 6.24 (1H, d, J = 6 Hz, OH),
6.71 (1H, s, Ph2CH), 7.68 (1H, t, J = ~5 Hz, NH), 7.90 (1H, d, J = 9 Hz, NH), 8.10
(1H, d, J = 9 Hz, NH), 8.15 (1H, d, J = 9 Hz, NH), 8. 61(1H, d, J = 8.5 Hz, NH), 10.89
(1H, d, J = 9 Hz, NH). ESI-MS: m/z calcd. for C64H93N11O19 1319.7; found [M+Na]+
1342.7.
4: 1H NMR (500 MHz, DMSO-d6):  0.84 (6H, d, J = 6.5 Hz), 0.99 (3H, d, J = 6 Hz),
2.29 (1H, d, J = 13.5 Hz), 2.54 (1H, t, J = 13.5 Hz), 4.95 (1H, d, J = ~2 Hz, OH), 5.11
(1H, m), 6.21 (1H, d, J = 7 Hz, OH), 6.90 (1H, s, Ph2CH), 7.07 (1H, d, J = 7.5 Hz,
NH), 7.75 (1H, d, J = 9 Hz, NH), 8.05 (1H, d, J = 9 Hz, NH), 8.26 (1H, d, J = 8.5 Hz,
NH), 8.55 (1H, br s, NH). ESI-MS: m/z calcd. for C64H93N11O19 1319.7; found
[M+Na]+ 1342.7.
Tripropeptin C 4, 16-bis-methyl ester (5).
To a solution of tripropeptin C (115 mg, 0.10 mmol) in methanol (2.5 ml) was added
p-toluenesulfonic acid (43.3 mg, 0.25 mmol) and the solution was kept for 0.5 h at room
temperature. The reaction solution was concentrated to a small volume and diethyl ether
was added. The resulting precipitate was washed thoroughly with diethyl ether to give a
solid of tripropeptin C bis-p-toluenesulfonate. To a solution of this solid in methanol (6
ml) was added trimethylsilyldiazomethane (171 mg, 1.50 mmol) in hexane (2.5 ml) and
the resulting solution was kept for 0.5 h at room temperature. Concentration gave a
residue, which was washed with diethyl ether and chromatographed
(CHCl3-MeOH-H2O, 60:20:3) to give a colorless solid of 5 (110 mg, 82%) as the
p-toluenesulfonic acid salt.
5 p-toluenesulfonate: 1H NMR (500 MHz, DMSO-d6):  0.84 (6H, d, J = 6.5 Hz), 0.98
(3H, d, J = 6 Hz), 2.29 (3H, s, CH3C6H4SO2), 3.58 (3H, s, OCH3), 3.60 (3H, s, OCH3),
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4.15 (1H, s), 4.52 (1H, d, J =5 Hz, OH), 4.92 (1H, t, J =6 Hz, OH), 5.13 (1H, d, J =3 Hz,
OH), 5.70 (1H, d, J =7.5 Hz, OH), 6.07 (1H, d, J =5.5 Hz, OH), 7.11 & 7.47 (each 2H, d,
J =8 Hz, CH3C6H4SO2), 7.84 (1H, d, J =9.5 Hz, NH), 8.03 (1H, d, J =9 Hz, NH), 8.83
(1H, d, J =9 Hz, NH). ESI-MS: m/z calcd. for C53H87N11O19 1181.6; found
[M+p-TsOH+Na]+ 1353.64.
Tripropeptin C 4, 16-bis-diphenylmethyl ester (6).
To a solution of tripropeptin C (115 mg, 0.10 mmol) in methanol (3 ml) was added
p-toluenesulfonic acid (43.0 mg, 0.25 mmol) and the solution was kept for 0.5 h at room
temperature. The reaction solution was concentrated to a small volume and diethyl ether
was added. The resulting precipitate was thoroughly washed with diethyl ether to give a
solid of tripropeptin C bis-p-toluenesulfonate. To a solution of this solid in methanol (3
ml) was added diphenyldiazomethane (348 mg, 1.79 mmol) and the solution was kept
for 1 h at room temperature. Concentration gave a residue, which was washed with
diethyl ether and chromatographed (CHCl3-MeOH-H2O, 60:20:3) to give a colorless
solid of 6 (149 mg, 90%) as the p-toluenesulfonic acid salt.
6 p-toluenesulfonate: 1H NMR (500 MHz, DMSO-d6):  0.84 (6H, d, J = 6.5 Hz), 1.02
(3H, d, J = 6 Hz), 2.29 (3H, s, CH3C6H4SO2), 6.01 (1H, d, J = 7 Hz, OH), 6.07 (1H, d, J
= 6 Hz, OH), 6.73 (1H, s, Ph2CH), 6.82 (1H, s, Ph2CH). ESI-MS: m/z calcd. for
C77H103N11O19 1485.7; found [M+p-TsOH+Na]+ 1680.8.
Tripropeptin C 4-(2-carboxy)ethyl amide (7).
To a solution of 3 (158 mg, 0.12 mmol) in methanol (4 ml) was added p-toluenesulfonic
acid (20.7 mg, 0.12 mmol) and the solution was stirred for 1 h at room temperature.
Concentration gave a residue, which was washed with diethyl ether to give 3
p-toluenesulfonate. A mixture of 3 p-toluenesulfonate, -alanine diphenylmethylester
p-toluenesulfonate (205 mg, 0.48 mmol), N-methylmorpholine (36.4 mg, 0.36 mmol),
1-hydroxybenzotriazole (24.3 mg, 0.18 mmol) and N, N-dicyclohexylcarbodiimide
(37.1 mg, 0.18 mmol) in N, N-dimethylformamide (3.6 ml) was stirred overnight at
room temperature. Concentration gave a residue, which was washed with diethyl ether
and chromatographed on a Sepadex LH 20 column (MeOH) to give a solid of 4-amide
of 3. To a solution of the solid in chloroform (2.5 ml) was added trifluoroacetic acid (1.2
ml) and anisole (0.6 ml) and the solution was kept for 1 h at room temperature.
Concentration gave a residue, which was chromatographed (CHCl3-MeOH-H2O,
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10:5:1) to give a colorless solid of 7 (93.6 mg, 56%) as the p-toluenesulfonic acid salt.
H NMR (400 MHz, DMSO-d6):  0.84 (6H, d, J = 6.5 Hz), 2.29 (3H, s, CH3C6H4SO2),
4.79 (1H, d, J = 8 Hz), 5.08 (1H, br s), 7.82 (1H, d, J =9 Hz), 8.04 (1H, d, J =8 Hz),
1
8.27 (1H, d, J =9 Hz). ESI-MS: m/z calcd. for C54H88N12O20 1224.6; found [M–H]–
1223.7.
Tripropeptin C 16-(2-carboxy)ethyl amide (8).
This compound was prepared from 4 following the procedure in preparation of 7 in 58%
yield. 1H NMR (500 MHz, DMSO-d6):  0.85 (6H, d, J = 6.5 Hz), 0.97 (3H, d, J = 6
Hz), 2.38 (1H, d, J =13 Hz), 2.84 (1H, t, J =13 Hz), 4.72 (1H, dd, J = 5.5 and 9 Hz),
4.84 (1H, d, J = 8 Hz), 4.90 (1H, br s), 5.08 (1H, d, J =3 Hz), 6.20 (1H, d, J =6 Hz),
7.72 (1H, t, J =5 Hz), 8.13 (1H, d, J = 9 Hz), 8.51 (1H, d, J =8 Hz). ESI-MS: m/z calcd.
for C54H88N12O20 1224.6; found [M–H]– 1223.6.
Tripropeptin C 4-[(S)-4-carboxy-3-hydroxy]butyl amide (9).
This compound was prepared from 3 and (S)-5-amino-2-hydroxypentanoic acid
diphenylmethylester p-toluenesulfonate following the procedure in preparation of 7 in
61% yield. 1H NMR (500 MHz, DMSO-d6):  0.84 (6H, d, J = 6.5 Hz), 0.99 (3H, d, J =
6 Hz). ESI-MS: m/z calcd. for C56H92N12O21 1268.7; found [M–H]– 1267.6.
Tripropeptin C 16-[(S)-4-carboxy-3-hydroxy]butyl amide (10).
This compound was prepared from 4 and (S)-5-amino-2-hydroxypentanoic acid
diphenylmethylester p-toluenesulfonate following the procedure in preparation of 7 in
71% yield. 1H NMR (500 MHz, DMSO-d6):  0.85 (6H, d, J = 6.5 Hz), 0.97 (3H, d, J =
6 Hz), 2.37 (1H, d, J =13 Hz), 2.82 (1H, t, J =13 Hz), 4.85 (1H, d, J = 8 Hz), 4.91 (1H,
br s), 5.09 (1H, s), 6.09 (1H, br s), 8.10 (1H, d, J = 8 Hz), 8.48 (1H, br s). ESI-MS: m/z
calcd. for C56H92N12O21 1268.7; found [M–H]– 1267.6.
Tripropeptin C 4-(4-amino)butyl amide (11).
This compound was prepared from 3 and t-butoxycarbonylaminobutylamine following
the procedure in preparation of 7 in 52% yield. 1H NMR (500 MHz, DMSO-d6):  0.84
(6H, d, J = 6.5 Hz), 5.03 (1H, br s), 5.15 (1H, br s) 8.85 (1H, br s). ESI-MS: m/z calcd.
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for C55H93N13O18 1223.7; found [M+H]+ 1224.5.
3. Biological assays
The minimum inhibitory concentrations (MICs) were determined by the serial agar
dilution method using Mueller-Hinton agar (Difco) for Staphylococcus aureus.1
Ref.1 Japan Society of Chemotherapy. Method of MIC determination. Chemotherapy
38, 102-105 (1990) (in Japanese).
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4. Mass analysis of complex formation with UPP
TPPC or bis-methyl ester 5 was mixed with undecaprenyl pyrophosphate (UPP) in 50%
aqueous MeOH, and the resulting mixture was subjected to mass spectroscopic analysis.
The mass spectra were recorded using a JMS-T100LC (ESI) spectrometer (JEOL
JMS-T100LC, Tokyo, Japan). For the TPPC, complex formation with UPP was detected
at 2120 Da, whereas the complex formed between the bis-methyl ester 5 and UPP was
not observed [refer to (A)-(D) of Figure S1].
Abbreviations: UP, undecaprenyl monophosphate; UPP, undecaprenyl pyrophosphate.
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