1310
K. KAI et al.
D-Ile
D-Val
ABA
ABA
O
O
H
H
N
N
HN
HN
O
O
L-Leu
L-Leu
O
O
HN
N
HN
N
O
O
L-Pip
L-Pip
HN
N
HN
N
O
O
L-Ala
L-Ala
O
O
L-MeLeu
L-MeLeu
1
2
Fig. 1. Structures of Compounds 1 and 2.
water; flow rate, 4 ml/min) to yield PF1171C (2, 18 mg). Spectroscopic
data for PF1171A (1): 1H-NMR (400 MHz, CDCl3) ꢁ: 0.86 (3H, d,
J ¼ 6:0 Hz, Leu-ꢁ), 0.89 (3H, d, J ¼ 7:2 Hz, Ile-ꢂ), 0.90 (3H, t,
J ¼ 7:2 Hz, Ile-ꢁ), 0.93 (3H, d, J ¼ 5:2 Hz, Leu-ꢁ0), 0.94, (3H, d,
J ¼ 7:2 Hz, MeLeu-ꢁ0), 0.96 (3H, d, J ¼ 6:8 Hz, MeLeu-ꢁ), 1.26 (3H,
d, J ¼ 6:8 Hz, Ala-ꢃ), 1.37 (2H, m, Ile-ꢂ0), 1.52 (1H, m, Leu-ꢂ), 1.56
(2H, m, Pip-ꢂ), 1.62 (1H, m, MeLeu-ꢂ), 1.75 (2H, m, Leu-ꢃ, Pip-ꢃ),
1.90 (1H, m, MeLeu-ꢃ), 1.97 (1H, m, Leu-ꢃ), 2.03 (3H, m, Pip-ꢃ, Pip-
ꢁ), 2.17 (1H, m, MeLeu-ꢃ), 2.40 (1H, m, Ile-ꢃ), 3.13 (1H, d,
J ¼ 2:0 Hz, Pip-"), 3.17 (3H, s, MeLeu-N-Me), 3.46 (1H, m, MeLeu-
ꢀ), 3.67 (1H, dd, J ¼ 2:4, 10.8 Hz, Pip-ꢀ), 4.11 (1H, d, J ¼ 9:6 Hz,
Pip-"), 4.42 (1H, dd, J ¼ 3:7, 7.2 Hz, Ile-ꢀ), 4.52 (1H, m, Leu-ꢀ), 4.78
(1H, Ala-ꢀ), 7.10 (1H, m, ABA-3), 7.17 (1H, d, J ¼ 8:0 Hz, ABA-5),
7.37 (1H, d, J ¼ 7:6 Hz, Ile-NH), 7.46 (1H, m, ABA-4), 7.60 (1H, d,
J ¼ 9:6 Hz, Ala-NH), 7.98 (1H, d, J ¼ 7:6 Hz, Leu-NH), 8.29 (1H, d,
J ¼ 8:4 Hz, ABA-2), 9.42 (1H, s, ABA-NH). 13C-NMR (100 MHz,
CDCl3) ꢁ: 11.8 (Ile-ꢁ), 14.0 (Ile-ꢂ), 18.5 (Ala-ꢃ), 21.8 (Leu-ꢁ), 22.1
(MeLeu-ꢁ), 23.2 (Leu-ꢁ0), 23.4 (MeLeu-ꢁ0), 24.5 (Leu-ꢂ, Pip-ꢃ), 25.6
(MeLeu-ꢂ), 26.9 (Ile-ꢂ0), 27.4 (Pip-ꢂ), 28.1 (Pip-ꢁ), 36.3 (Leu-ꢃ), 36.5
(Ile-ꢃ), 37.8 (MeLeu-ꢃ), 37.9 (MeLeu-N-Me), 47.9 (Ala-ꢀ), 50.9 (Leu-
ꢀ), 52.6 (Pip-"), 57.3 (Ile-ꢀ), 61.6 (Pip-ꢀ), 65.2 (MeLeu-ꢀ), 122.7
(ABA-1), 123.3 (ABA-3), 123.9 (ABA-2), 127.1 (ABA-5), 131.8
(ABA-4), 137.2 (ABA-6), 168.9 (Pip-CO), 169.3 (MeLeu-CO), 170.3
(ABA-CO), 171.1 (Ile-CO), 174.1 (Leu-CO), 174.3 (Ala-CO). FAB-
D-Ile
L-Ala
(19.4)
(39.3)
A
B
ABA
(25.6)
L-Leu
(33.1)
L-Pip
(27.2)
L-MeLeu
(35.7)
L-Ala
(19.5)
L-Pip
(27.3)
D-Val
(32.1)
L-Leu
(33.2)
L-MeLeu
(35.8)
0
10
20
30
Time (min)
40
60
50
Fig. 2. Marfey’s Analysis of the Hydrolysis Products of 1 (A) and 2
(B).
MS: m=z 655 ½M þ Hꢀþ. ½ꢀꢀD þ25:4ꢁ (c 0.500, MeOH). Spectro-
16
Table 1. HPLC Retention Times of the FDAA Derivatives of Amino
Acids
scopic data for PF1171C (2): 1H-NMR (400 MHz, CDCl3) ꢁ: 0.86 (3H,
d, J ¼ 6:0 Hz, Leu-ꢁ), 0.93 (3H, d, J ¼ 6:8 Hz, Val-ꢂ), 0.95 (6H, d,
J ¼ 6:8 Hz, MeLeu-ꢁ0, Leu-ꢁ), 0.96 (3H, d, J ¼ 6:8 Hz, MeLeu-ꢁ), 1.04
(3H, d, J ¼ 7:2 Hz, Val-ꢂ0), 1.26 (3H, d, J ¼ 6:8 Hz, Ala-ꢃ), 1.54 (3H,
m, Leu-ꢂ, Pip-ꢁ), 1.63 (1H, m, MeLeu-ꢂ), 1.74 (1H, m, Pip-ꢃ), 1.76
(1H, m, Leu-ꢃ), 1.91 (1H, m, MeLeu-ꢃ), 2.00 (1H, m, Leu-ꢃ), 2.04
(1H, m, Pip-ꢃ), 2.08 (2H, m, Pip-ꢂ), 2.17 (1H, m, MeLeu-ꢃ), 2.66 (1H,
m, Val-ꢃ), 3.14 (1H, m, Pip-"), 3.18 (3H, s, MeLeu-N-Me), 3.46 (1H,
m, MeLeu-ꢀ), 3.67 (1H, dd, J ¼ 2:4, 11.2 Hz, Pip-ꢀ), 4.11 (1H, m, Pip-
"), 4.30 (1H, dd, J ¼ 3:2, 7.6 Hz, Val-ꢀ), 4.55 (1H, m, Leu-ꢀ), 4.80
(1H, m, Ala-ꢀ), 7.11 (1H, d, J ¼ 6:8 Hz, ABA-3), 7.17 (1H, d,
J ¼ 8:0 Hz, ABA-5), 7.24 (1H, s, Val-NH), 7.43 (1H, m, ABA-4), 7.61
(1H, d, J ¼ 10:0 Hz, Ala-NH), 7.98 (1H, d, J ¼ 7:6 Hz, Leu-NH), 8.28
(1H, d, J ¼ 8:0 Hz, ABA-2), 9.39 (1H, s, ABA-NH). 13C-NMR
(100 MHz, CDCl3) ꢁ: 16.2 (Val-ꢂ), 18.4 (Ala-ꢃ), 19.7 (Val-ꢂ0), 21.7
(Leu-ꢁ), 22.1 (MeLeu-ꢁ), 23.1 (Leu-ꢁ0), 23.3 (MeLeu-ꢁ0), 24.4 (Leu-ꢂ),
24.5 (Pip-ꢃ), 25.6 (MeLeu-ꢂ), 27.4 (Pip-ꢁ), 28.1 (Pip-ꢂ), 29.9 (Val-ꢃ),
36.2 (Leu-ꢃ), 37.8 (MeLeu-ꢃ), 37.9 (MeLeu-N-Me), 47.8 (Ala-ꢀ), 50.9
(Leu-ꢀ), 52.6 (Pip-"), 59.2 (Val-ꢀ), 61.5 (Pip-ꢀ), 65.1 (MeLeu-ꢀ),
122.7 (ABA-1), 123.3 (ABA-3), 123.9 (ABA-2), 127.1 (ABA-5), 131.7
(ABA-4), 137.1 (ABA-6), 168.8 (Pip-CO), 169.2 (MeLeu-CO), 170.2
(ABA-CO), 170.7 (Val-CO), 174.1 (Leu-CO), 174.3 (Ala-CO). FAB-
Retention time (min)
Amino acid
L
D
Ala
Val
19.4
25.4
33.0
31.6
35.7
27.4
20.0
32.1
40.3
39.2
37.9
24.8
Leu
Ile
MeLeu
Pip
ABA
25.5
although the species could not be determined. A loopful of spores from
a slant culture of the strain was inoculated into 30 g of okara in a Petri
dish of 9 cm in diameter, and cultivation was carried out at 25 ꢁC
for 14 d.
Extraction and isolation. The okara (2.5 kg) that had been
fermented with strain OK-128 was soaked in MeOH. Evaporating
MeOH gave an aqueous concentrate which was extracted with EtOAc.
The EtOAc extract was concentrated and subsequently chromato-
graphed on Wakogel C-200 (Wako Pure Chemical) by eluting with n-
hexane and an increasing ratio of EtOAc to afford 70% and 100%
EtOAc eluates (3.74 g). The active elutes were further chromato-
graphed on Wakogel C-200 by eluting with n-hexane–acetone (75:25).
The active fractions were crystallized from MeOH to afford PF1171A
(1, 1.1 g). The filtrate was subjected to preparative HPLC (column,
Inertsil ODS-3 10 ꢂ 250 mm (GL Sciences); solvent, 75% MeOH in
MS: m=z 641 ½M þ Hꢀþ. ½ꢀꢀD þ40:5ꢁ (c 0.200, MeOH).
16
Marfey’s analysis. The compound (2 mg) was treated at 110 ꢁC for
20 h with 6 M HCl. After cooling to room temperature, the sample was
dried under vacuum. The residue was dissolved in 1 M NaHCO3
(500 ml) and reacted with Marfey’s reagent (120 ml of a 10 mM acetone
solution) and allowed to react at 40 ꢁC for 2 h. After cooling, the
sample was quenched with 2 M HCl and dried under vacuum. The solid