Journal of Natural Products
Article
tR 17.4 min), indicating that they were major components of the
Table 2. Minimum Inhibitory Concentration Assay Results
for 1
extract.
1
The H NMR spectrum of the HPLC fraction 14 (0.6 mg, tR 42.2
compound (MIC; μg/mL)
min) showed complex signals characteristic of a mixture. Therefore, a
larger portion of the MeCN fraction (339 mg) was subjected to silica
gel CC using gradient elution with hexanes/EtOAc−MeOH to yield
11 fractions. Fraction 4 (45.7 mg), eluted with 75% EtOAc in
hexanes, was further purified by semipreparative RP-HPLC as above
to afford 1 (5.0 mg, tR 42.2 min) and 2 (0.5 mg, tR 45.4 min). Fraction
3 (104 mg), eluted with 50% EtOAc in hexanes, was further separated
by semipreparative RP-HPLC (Agilent 1260 Infinity-C18 column; 5
μm; 9.4 × 250 mm; gradient elution 40−100% MeCN in H2O over
60 min; 2 mL/min) to afford 6 (1.5 mg, tR 9.1 min), and fraction 5
(43 mg), eluted with 100% EtOAc in hexanes, was further separated
by semipreparative RP-HPLC (Agilent 1260 Infinity-C18 column; 5
μm; 9.4 × 250 mm; gradient elution 40−100% MeCN in H2O over
60 min; 2 mL/min) to afford 3 (0.5 mg, tR 39.0 min). Known
compounds 5 and 7 were identified by comparison of their NMR and
specific rotation data with literature values.13,17
a
organism
1
control
Staphylococcus aureus ATCC 43300
Candida albicans ATCC 10231
Cryptococcus neoformans H99 (23 °C)
Cryptococcus neoformans H99 (37 °C)
128
64
128
8
0.16
1.70
0.85
0.85
a
Chlortetracycline + streptomycin was the control for S. aureus.
Amphotericin B was the control for fungal strains.
with an MIC value of 8 μg/mL at 37 °C, while showing lesser
inhibitory effects against C. albicans and S. aureus with MIC
values of 64 and 128 μg/mL, respectively (Figure S20).
EXPERIMENTAL SECTION
Broomeanamide A (1): white solid; [α]20 −6 (c 0.05, MeOH);
D
■
1H and 13C NMR and HMBC data, see Table 1; positive ion
HRESIMS m/z 909.6197 [M + H]+ (calcd for C49H80N8O8 + H,
909.6177).
General Experimental Procedures. Specific rotations were
measured on an AUTOPOL III automatic polarimeter. H and 13C
1
NMR spectra were recorded using Bruker AVANCE-600 or
AVANCE-500 spectrometers. Chemical shift values were referenced
to residual solvent signals for CDCl3 (δH/δC, 7.26/77.2). HSQC,
HMBC, TOCSY, and ROESY data were recorded using the Bruker
AVANCE-600 instrument. HRESIMS and HRESIMSMS data were
obtained using a Waters Q-Tof Premier mass spectrometer.
Semipreparative HPLC separations were carried out using an Agilent
1260 Infinity LC system instrument with a diode array detector
equipped with a semipreparative Apollo C18 column (Grace, 1.0 × 25
cm, 5 μm) with UV detection at 210, 254, and 350 nm or an Agilent
1220 Infinity LC system instrument equipped with the same column
type under UV detection at 254 nm. Conditions for analytical
separation of Marfey derivatives are described below. Single-crystal X-
ray data were collected using a Bruker D8-Venture Duo diffractometer
equipped with a Photon III detector, Mo-target and Cu-target
microfocus X-ray tubes, and an Oxford Cryostreams 800 series N2 gas
stream sample cooler/heater.
Fungal Material and Fermentation. The culture of S.
broomeana TFC201724 was isolated from mycelium effused on an
old basidioma of Heterobasidion cf. linzhiense (H. insulare group)
growing on a decaying stump. The material was collected by one of
the authors (K.P.) in a cedar forest 4 km west of Dhanaulti, Dehradun
district, Uttarakhand, India, on October 9, 2012, and deposited at the
fungarium of the University of Tartu (TUF119036). It included only
an anamorph with abundant conidia that were isolated, and colonies
were grown on 1.5% malt extract agar (MEA, Oxoid, Cambridge, UK)
in the dark at 24 °C. The culture has been deposited at the Tartu
Fungal Culture Collection (TFC) of the University of Tartu, and the
TEF (MH795104) and ITS-LSU rDNA (MH795096) sequences are
available in GenBank.3 Maximum likelihood analyses of the combined
rDNA and TEF sequences of the S. broomeana group3 in RAxML,18
applying the default settings, revealed TFC201724 to form the sister
group of isolates of S. broomeana originating from Europe (Figure S2).
A subculture on PDA was used to inoculate 10 1-L Erlenmeyer flasks,
each containing 100 g of autoclaved rice in 100 mL of distilled
water.19 Two 0.25 cm2 agar plugs from the subculture were aseptically
transferred into each flask, and the cultures were incubated statically
at room temperature for 30 days.
Broomeanamide B (2): white solid; [α]20 −18 (c 0.03, MeOH);
D
1H and 13C NMR and HMBC data, see Table S1; positive ion
HRESIMS m/z 895.6032 [M + H]+ (calcd for C48H78N8O8 + H,
895.6021).
Broomeanamide C (3): white solid; [α]20 −42 (c 0.03, MeOH);
D
1H and 13C NMR and HMBC data, see Table S2; negative ion
HRESIMS m/z 893.5887 [M − H]− (calcd for C48H78N8O8 − H,
893.5864).
1
Compound 4: white solid; H NMR (CDCl3, 500 MHz) δ 7.74
(1H, br s, NH-8), 7.43 (2H, br t, J = 7.3 Hz, H-3′, 5′), δ 7.37 (2H, br
d, J = 7.6 Hz, H-2′, 6′), 7.34 (1H, br t, J = 7.3 Hz, H-4′), 6.99 (1H, s,
H-10), 4.31 (1H, dd, J = 10.2, 6.5 Hz, H-6), 3.82 (1H, m, H-3a), 3.65
(1H, m, H-3b), 2.47 (1H, m, H-5a), 2.10 (1H, m, H-5b), 2.03 (1H,
m, H-4a), 1.96 (1H, m, H-4b); 13C NMR (CDCl3, 150 MHz) 166.0
(C, C-7), 158.0 (C, C-1), 133.3 (C, C-9), 129.5 (CH, C-2′, 6′), 128.8
(C, C-1′), 128.6 (CH, C-3′, 5′), 127.5 (CH, C-4′), 116.0 (CH, C-
10), 59.3 (CH, C-6), 45.8 (CH2, C-3), 29.1 (CH2, C-5), 22.0 (CH2,
C-4), negative ion HRESIMS m/z 241.0977 [M − H]−) (calcd for
C14H14N2O2 − H, 241.0978).
1
Compound 6: white solid; H NMR (CDCl3, 500 MHz) δ 7.74
(1H, br s, NH-8), 7.43 (2H, br t, J = 7.6 Hz, H-3′, 5′), δ 7.39 (2H, br
d, J = 7.3 Hz, H-2′, 6′), 7.35 (1H, br t, J = 7.3 Hz, H-4′), 7.06 (1H, s,
H-10), 3.89 (1H, m, H-3a), 3.77 (1H, m, H-3b), 3.19 (1H, s, OH-6),
2.38 (1H, m, H-5a), 2.29−2.21 (2H, m, H-5b, H-4a), 2.08 (1H, m,
H-4b); 13C NMR (CDCl3, 150 MHz) 165.1 (C, C-7), 158.3 (C, C-
1), 133.0 (C, C-9), 129.6 (CH, C-2′, 6′), 129.1 (C, C-1′), 128.7 (CH,
C-3′, 5′), 126.6 (CH, C-4′), 117.7 (CH, C-10), 87.2 (C−OH, C-6),
45.8 (CH2, C-3), 36.8 (CH2, C-5), 19.5 (CH2, C-4), negative ion
HRESIMS m/z 257.0921 [M − H]− (calcd for C14H14N2O3 − H,
257.0926).
Marfey’s Analysis of Broomeanamide A. Determination of the
absolute configuration of the amino acid units in 1 was accomplished
using Marfey’s method in conjunction with both C3 and C18
chromatographic separation.9−11 For the derivatization reaction, 0.2
mg of 1 was transferred to a 2 mL glass tube, to which 500 μL of 6 M
HCl was added and kept at 110 °C for 16 h. After hydrolysis, the
liquid was evaporated under a stream of air, and 50 μL of H2O, 20 μL
of 1 M NaHCO3, and 100 μL of 1% Marfey’s reagent (1-fluoro-2,4-
dinitrophenyl-5-L-alanine amide, L-FDAA) in acetone were added.
The tube was sealed and kept at 40 °C with occasional agitation. The
reaction was quenched with addition of 40 μL of 2 M HCl and dried
under a stream of air. The reaction product was dissolved in 200 μL of
MeOH, filtered with a 0.22 μm hydrophilic PTFE filter, and
submitted to C3 and C18 analysis using an Agilent 1260 HPLC
coupled to an Agilent 6120 single quadrupole MS, collecting positive
and negative ESIMS at m/z 160−1500. Mobile phases consisted of
0.1% formic acid in MeCN (A), 0.1% aqueous formic acid (B), and
Extraction and Isolation. Each fermented flask was extracted
with EtOAc (0.2 L × 2) and filtered, and the combined filtrate was
air-dried to yield a crude extract (1.5 g). The extract was partitioned
between hexanes (10 mL) and MeCN (10 mL) to obtain hexanes and
MeCN fractions. Upon evaporation of solvent, these samples were
found to contain 0.4 and 1.1 g, respectively. A small portion of the
MeCN fraction (14.4 mg) was subjected directly to semipreparative
RP-HPLC (Agilent 1220 Infinity-C18 column; 5 μm; 9.4 × 250 mm;
gradient elution 40−100% MeCN in H2O over 50 min; 2 mL/min) to
afford 7 (2.0 mg, tR 9.2 min), 4 (1.1 mg, tR 11.3 min), and 5 (0.8 mg,
2032
J. Nat. Prod. 2021, 84, 2028−2034