Journal of Natural Products
ARTICLE
were performed by MIDI Laboratories, Inc. (Newark, DE, USA), and
the D2 variable region of the large subunit (LSU) rRNA was sequenced
and compared to their database. This analysis suggested that this fungus
was related to Trichothecium sp. (order Hypocreales); these data were
deposited in Genbank (accession no. JF930284). The culture was stored
on a malt extract slant and was transferred periodically. A fresh culture
was grown on a similar slant, and a piece was transferred to a medium
containing 2% soy peptone, 2% dextrose, and 1% yeast extract (YESD
media). Following incubation (7 d) at 22 ꢀC with agitation, the culture
was used to inoculate 50 mL of a rice medium, prepared using rice to
which was added a vitamin solution and twice the volume of rice with
H-4), 4.20 (d, J = 5, H-2), 3.95 (br s., H-8), 3.92 (d, J = 12, Ha-13), 3.83
(br s., H-7), 3.63 (d, J = 7, H-11), 3.61 (d, J = 12, Hb-13), 2.39 (dd, J =
15.5, 8.6, Ha-3), 2.12 (m, Hb-3), 2.11 (dd, J = 7.5, 2, H-4 ), 1.84 (s,
0
1
3
H
3
-16), 1.03 (s, H
3
-15), 0.92 (s, H
3
-14); C NMR (125 MHz, CDCl
3
)
0
0
0
δ 165.9 (C-1 ), 146.7 (C-3 ), 136.8 (C-9), 121.4 (C-10), 120.2 (C-2 ),
82.6 (C-2), 77.0 (C-7), 74.8 C-4), 74.6 (C-12), 72.5 (C-8), 69.7 (C-11),
66.7 (C-13), 50.1 (C-5), 38.9 (C-6), 37.3 (C-3), 20.7 (C-16), 15.8
(C-15), 15.7 (C-4 ), 6.7 (C-14); HMBC data, H-2 f C-5, C-11, C-12;
-3 f C-2, C-4; H-4 f C-1 , C-2, C-5, C-6, C-12; H-7 f C-5, C-8,
0
0
H
2
C-7; H-8 f C-6, C-7, C-9, C-10, C-16; H-10 f C-6, C-8, C-11, C-16;
H-11 f C-2, C-9, C-10; H -13 f C-2, C-5, C-12; H -14 f C-4, C-5,
2
3
0
0
0
C-6; H
3
-15 f C-5, C-6; H
3
-16 f C-8, C-9, C-10; H-2 f C-1 , C-4 ;
H O, in a 250 mL Erlenmeyer flask. This was incubated at 22 ꢀC until
2
0
0
0
0
0
0
H-3 f C-1 , C-4 ; H -4 f C-1 , C-2 ; HRESIMS m/z 373.1620 [M +
the culture showed good growth (approximately 14 d). The scale-up
culture was grown in a 2.8 L Fernbach flask containing 150 g of rice and
3
+
Na] , calcd for C19
26 6
H O Na, 373.1622.
2
5
1
13
Guangomide A (5): [α]
D
3
+6.5 (c 0.31, CHCl ); H and C NMR
2
300 mL of H O and was inoculated using a seed culture grown in YESD
3
data matched the literature; HRMALDITOFMS m/z 641.3163 [M +
medium. This was incubated at 22 ꢀC for 14 d.
Extraction and Isolation. To the large-scale solid fermentation
+
Na] , calcd for C H N O Na, 641.3157.
3
1 46 4 9
1
3ꢀ15
Amino Acid Analysis by Chiral HPLC.
Independently, an
was added 500 mL of 1:1 MeOHꢀCHCl
4 h, then filtered, and the solvent was evaporated (waxy yellow solid).
This crude extract was partitioned between CHCl
ꢀMeOHꢀH
4:1:5), and the organic soluble material was dried and further parti-
tioned between hexanes and CH CN (1:1). The CH CN partition (4 g)
3
. The mixture was shaken for
aliquot of compounds 1 (2 mg) and 2 (2 mg) were subjected to
hydrolysis with 6 N HCl (1 mL) in a sealed tube at 100 ꢀC for 24 h. Upon
cooling, the solutions were evaporated to dryness. Each residue was
2
3
2
O
(
dissolved in H O and extracted with diethyl ether (2 ꢁ 2 mL). Both
2
3
3
organic and aqueous solutions were subjected to chiral HPLC analysis at
a flow rate of 1.0 mL/min using four different solvent systems: (1) 5%
was fractionated via flash chromatography using silica gel via a hexanesꢀ
CHCl ꢀMeOH gradient to afford four fractions. Fraction 2 (1.5 g),
3
4 R R
2-propanol in 2 mM CuSO : L-Hiv (t = 15 min), D-Hiv (t = 27 min),
3
which eluted with 0 to 10% MeOH in CHCl over 14 min, was separated
L-Hic (t = 49 min), D-Hic (t = 68 min), L-NMeLeu (t = 12 min),
R
R
R
further by reversed-phase HPLC (20ꢀ100% CH
3
CNꢀH
2
O over 100 min)
D-NMeLeu (t
R
= 17 min); (2) 5% MeOH in 2 mM CuSO
= 5.3 min); (3) 30% MeOH in 2 mM CuSO
t = 25 min), D-Phe (t = 35 min), L-Leu (t = 15 min), D-Leu (t =
4
: L-Ser (t
R
=
to obtain 18 fractions. Fraction 13 yielded guangomide A (4; 91 mg).
4.9 min), D-Ser (t
R
4
: L-Phe
Fraction 14 (11.4 mg) was purified by semipreparative reversed-phase
(
R
R
R
R
HPLC (20ꢀ100% CH
on A (3, 3 mg). Fraction 16 (9.4 mg) was separated further by semipre-
O over 15 min)
3
CNꢀH
2
O over 15 min) to yield trichothospor-
1
4
8 min); (4) 100% 1 mM CuSO : L-Val (t = 26 min), D-Val (t =
7 min). Dilutions were adjusted ineach case to approximate the response
4 R R
parative reversed-phase HPLC (20ꢀ100% CH
3
CNꢀH
2
of the UV detector (λ = 214 nm) with those of hydrolyzed samples.
to obtain trichodepsipeptide B (2; 4 mg), while similar separation con-
ditions were utilized on fraction 17 (18 mg) to yield trichodepsipeptide
A (1; 7 mg). An initial sample of this same fungus was processed in the
2
3
Amino Acid Analysis of Marfey’s Derivatives by UPLC. A
sample of compound 5 (2.3 mg) was subjected to acid hydrolysis in a
manner similar to the above. The dried aqueous solution was resus-
same fashion to yield a CH CN partition (672 mg), which was subjectedto
3
pended in H O (100 μL), and 1% Marfey’s reagent in acetone (200 μL)
2
flash chromatography using silica gel via a hexanesꢀCHCl
gradient to afford seven fractions. Fraction 5 (490 mg) was separated fur-
therby preparative reversed-phase HPLC (20ꢀ100% CH CNꢀH O over
00 min) to yield a new trichothecene analogue (4; 17 mg), trichothecin
3
ꢀMeOH
and 1 M NaHCO (40 μL) were added. The mixture was heated at 40 ꢀC
3
for 1 h in a shaking water bath, after which it was removed and cooled.
The mixture was quenched with 2 M HCl (20 μL), dried, and dissolved
in MeOH for UPLC analysis. Amino acid standards (1 mg each) were
derivatized with Marfey’s reagent in a manner similar to the above. The
UPLC analysis was performed using two solvent systems: (1) 10ꢀ50%
CH CNꢀ0.1% TFA in H O over 10 min at a flow rate of 0.5 mL/min:
3
2
1
(
(
6 mg), crotocin (7 mg), a mixture of roseotoxin S and trichothecinol B
16 mg), guangomide A (5; 22 mg), and trichothosporon A (3; 22 mg).
2
5
Trichodepsipeptide A (1): white powder; [α]
ꢀ45.6 (c 0.3,
D
1
13
3
2
CHCl ); UV (MeOH) λ
(log ε) 206 (2.97); H, C NMR, and
3
max
+
L-NMePhe (t = 8.334 min), D-NMePhe (t = 8.447 min), L-Ala (t =
R
R
R
HMBC data, see Table 1; HRESIMS m/z 711.3923 [M + Na] calcd for
Na, 711.3945.
Trichodepsipeptide B (2): white powder; [α]
5
.281 min), D-Ala (t
R
= 6.186 min); (2) 10ꢀ70% MeOHꢀ0.1% TFA
36 56 4 9
C H N O
2
5
in H O over 10 min at a flow rate of 0.4 mL/min: L-NMeAla (t =
2
R
ꢀ79.0 (c 0.1,
D
1
13
R R
7.730 min), D-NMeAla (t = 7.682 min), L-Ala (t = 6.836 min), D-Ala
CHCl
3
); UV (MeOH) λmax (log ε) 203 (2.67); H, C NMR and
+
R
(t = 8.067 min).
HMBC data, see Table 1; HRESIMS m/z 697.3770 [M + Na] , calcd for
2
4
Cytotoxicity Assay. The cytotoxicity measurements against MCF-7
human breast carcinoma (Barbara A. Karmanos Cancer Center,
C
35
H
54
N
4
O
9
Na, 697.3788.
2
5
Trichothosporon A (3): white powder; [α] ꢀ8.8 (c 0.1, CHCl );
D
3
2
5
1
Detroit, MI, USA), NCI-H460 human large-cell lung carcinoma
(HTB-177, American Type Culture Collection, Manassas, VA, USA),
3
H NMR (500 MHz, CDCl ) δ 5.23 (s, H-1), 3.82 (d, J = 3, H-4), 4.63
0 0
d, J = 3, H-5), 3.74 (s, H-7), 5.19 (d, J = 9, H-1 ), 5.28 (d, J = 9, H-2 ),
(
6
2
2
6
0 0 0
.11 (d, J = 15, H-4 ), 6.47 (dd, J = 15, 11, H-5 ), 5.85 (d, J = 11, H-6 ),
.08 (m, H-8 ), 2.08 (m, H-9 ), 5.06 (m, H-10 ), 1.58 (s, H-12 ), 1.66 (s,
and SF-268 human astrocytoma (NCI Developmental Therapeutics
Program, Frederick, MD, USA) cell lines were performed as described
0
0
0
0
2
2
27
0
0
0
13
previously. Compounds 1 and 2 were also tested against the HT-29
human colorectal adenocarcinoma (HTB-38, American Type Cul-
H-13 ), 1.92 (d, J = 1, 3 -CH
CDCl ) δ 99.3 (C-1), 193.6 (C-2), 61.5 (C-3), 56.1 (C-4), 65.9 (C-5),
70.8 (C-6), 56.8 (C-7), 64.4 (C-1 ), 125.7 (C-2 ), 140.1 (C-3 ), 134.2
), 1.77 (s, 7 -CH ); C NMR (125 MHz,
3 3
3
2
8
0
0
0
ture Collection) and MDA-MB-435 human melanoma (HTB-129,
American Type Culture Collection) cell lines as described previously.
1
29
0 0 0 0 0
0
(
(
(
C-4 ), 126.4 (C-5 ), 125.1 (C-6 ), 140.6 (C-7 ), 40.3 (C-8 ), 26.8
C-9 ), 124.0 (C-10 ), 132.0 (C-11 ), 17.9 (C-12 ), 25.9 (C-13 ), 13.6
3 -CH
0
0
0
0
0
0
+
3
), 17.1 (7 -CH
3
); HRESIMS m/z 447.1981 [M + Na] , calcd for
’ ASSOCIATED CONTENT
C H O Na, 447.1995.
2
2 32 8
2
5
1
13
S
Supporting Information. H and C NMR spectra of
Trichothecene analogue (4): colorless film; [α]
D
ꢀ11.0 (c 0.1,
b
1
0
CHCl
3
); H NMR (500 MHz, CDCl
3
) δ 6.35 (dq, J = 11.5, 7.5, H-3 ),
compounds 1ꢀ4. These materials are available free of charge via
0
5.77 (dq, J = 11.5, 2, H-2 ), 5.58 (d, J = 6, H-10), 5.51 (dd, J = 8, 3.4,
the Internet at http://pubs.acs.org.
2
141
dx.doi.org/10.1021/np2004243 |J. Nat. Prod. 2011, 74, 2137–2142