Microbial transformation of curvularin

Article abstract of DOI:10.1021/np0580309

The microbiological transformation of curvularin (1) with Beauveria bassiana ATCC 7159 afforded three new metabolites identified as curvularin-7-O-β-D-glucopyranoside (2), curvularin-4′-O-methyl-7-O- β-D-glucopyranoside (3), and 6-hydroxycurvularin-4′-O-methyl-6-O- β-D-glucopyranoside (4) resulting from hydroxylation, glucosidation, and methylglucosidation of the substrate. Isolation of 6-methyl-2,4-dihydroxyphenyl- 4′-O-methyl-1-O-β-D-glucopyranoside (5) from the fermentation broth of B. bassiana ATCC 7159 without any added substrate suggested that hydroxylase, glucosyl transferase, and methylase are constitutive enzymes of this organism.

Full text of DOI:10.1021/np0580309

J. Nat. Prod. 2005, 68, 1271-1273
1271
Microbial Transformation of Curvularin
Jixun Zhan and A. A. Leslie Gunatilaka*
Southwest Center for Natural Products Research and Commercialization, Office of Arid Lands Studies, College of Agriculture
and Life Sciences, The University of Arizona, 250 East Valencia Road, Tucson, Arizona 85706-6800
Received March 1, 2005
The microbiological transformation of curvularin (1) with Beauveria bassiana ATCC 7159 afforded three
new metabolites identified as curvularin-7-O-â-D-glucopyranoside (2), curvularin-4′-O-methyl-7-O-â-D-
glucopyranoside (3), and 6-hydroxycurvularin-4′-O-methyl-6-O-â-D-glucopyranoside (4) resulting from
hydroxylation, glucosidation, and methylglucosidation of the substrate. Isolation of 6-methyl-2,4-
dihydroxyphenyl-4′-O-methyl-1-O-â-D-glucopyranoside (5) from the fermentation broth of B. bassiana
ATCC 7159 without any added substrate suggested that hydroxylase, glucosyl transferase, and methylase
are constitutive enzymes of this organism.
Curvularins are macrocyclic lactones produced by a
number of fungi of the genera Curvularia,1 Penicillium,2
and Alternaria3 and have been reported to possess a variety
of biological activities, including phytotoxicity,3 cytotoxicity
toward sea urchin embryogenesis,2a inhibition of cell
division,2a inhibition of expression of human inducible nitric
oxide synthase,4 and growth-promoting activity in farm
animals.5 They have also been reported to cause hepatic
necrosis.6 Our recent isolation of several curvularins with
moderate cytotoxicity from two Sonoran desert microorgan-
isms,7 together with interesting biological activities associ-
ated with these macrolides, prompted us to obtain further
curvularin analogues for structure-activity relationship
studies. Although microbial biotransformation of the re-
lated zearalenone group of macrolides is known,8 this is
2, indicating that the newly introduced glucose moiety
must be attached to one of the phenolic hydroxyls of 1. The
correlation of the anomeric 1′-H signal at δH 4.94 to C-7
signal at δC 157.4 in the HMBC spectrum of 2 revealed
that the glucose moiety was located at C-7. The above data
together with the detailed analysis of its 2D NMR spectra
including the HMBC spectrum (Figure 1) confirmed its
structure as curvularin-7-O-â-D-glucopyranoside (2). The
HRFABMS of the second product (3) revealed the molecular
formula C23H32O10, suggesting that it contains an ad-
the first report of the biotransformation of curvularin-type
microbial macrolides.
Of the eight fungal strains screened, all except Cunning-
hamella elegans ATCC 9245 were able to completely
deplete and transform curvularin (1). However, Beauveria
bassiana ATCC 7159 was selected because the TLC analy-
sis indicated that it was able to biotransform 1 into three
products compared with six other strains (Absidia coerulia
MR 27, Cunninghamella blakesleeana ATCC 8688a, Cun-
ninghamella echinulata ATCC 9244, C. echinulata NRRL
3655, Mucor mucedo UI 4605, and Streptomyces griseus
ATCC 13273), which resulted in the formation of only one
biotransformation product. The preparative-scale incuba-
tion of 1 with B. bassiana for 7 days afforded three polar
products, 2-4, identified as due to glucosidation, methyl-
glucosidation, and hydroxylation of 1. B. bassiana (Clavi-
cipitaceae), also known as sugar icing fungus, has previ-
ously been used in the biotransformation of more than 300
different substrates,9 resulting in hydroxylation,10 hydroly-
sis,11 methylglucosidation,12 and oxidation.13
1
ditional CH2 compared with that of 2. H and 13C NMR
spectra of 3 were similar to those of 2 except for the signals
due to the sugar moiety. Only two aliphatic OH signals
(at δH 4.64 and 4.49) were observed in its 1H NMR
spectrum compared with three OH signals for compound
2. The NMR signals at δH 3.55 (3H) and δC 60.5 suggested
the presence of an OMe group in 3, and strong HMBC
correlation of the signal at δH 3.55 to the signal at δC 80.1
(C-4′) indicated that this OMe is attached to C-4′ of the
sugar. Therefore, this compound was identified as curvu-
larin-4′-O-methyl-7-O-â-D-glucopyranoside (3).
The molecular formula, C22H30O10, of the most polar
product (2) determined by its HRFABMS, together with
the presence of three OH signals at δH 4.60, 4.42, and 4.33
The HRFABMS of the third biotransformation product
(4) was consistent with the molecular formula C23H32O11,
1
in its H NMR spectrum, and five CH signals at δc 102.3,
78.2, 77.8, 74.6, and 71.3 and one CH2 signal at δ 62.6 in
its DEPT spectrum, suggested it to be an O-glucoside
derivative of curvularin. Compared with 1, only one low
field D2O-exchangeable signal (δH 8.96) was observed for
* To whom correspondence should be addressed. Tel: (520) 741-1691.
Fax: (520) 741-1468. E-mail: leslieg@ag.arizona.edu.
Figure 1. Selected HMBC correlations of 2 and 4.
10.1021/np0580309 CCC: $30.25
© 2005 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/13/2005

1272 Journal of Natural Products, 2005, Vol. 68, No. 8
Notes
conditions for an additional 7 days. Culture control consisted
of fermentation broth of B. bassiana ATCC 7159 without the
substrate but with the same volume of acetone, and the
substrate control consisted of sterile PDB medium with the
same amount of a solution of 1 in acetone. Both controls were
incubated under the same conditions. A preparative scale
experiment was carried out in 30 × 250 mL flasks, each
containing 100 mL of PDB under the same circumstances and
conditions as the small scale fermentation. A total of 40 mg of
1 was used (1.33 mg/flask). For isolation of 5, B. bassiana
ATCC 7159 was cultivated under the same conditions, but
without the substrate in 10 × 250 mL flasks, each containing
100 mL of PDB.
Extraction, Isolation, and Identification of the Me-
tabolites. The cultures were filtered, and the combined filtrate
(1860 mL) was neutralized with aqueous NaOH and extracted
with EtOAc (3 × 1200 mL). Evaporation of EtOAc under
reduced pressure yielded a dark brown solid (129.8 mg), which
was subjected to gel filtration on Sephadex LH-20 (3.0 g) and
eluted with hexane-acetone (10:1) (160 mL), hexane-acetone
(2:1) (120 mL), hexane-acetone (1:1) (160 mL), acetone (80
mL), and MeOH (20 mL). Twenty-seven fractions (20 mL each)
were collected and combined on the basis of their TLC profiles,
yielding combined fractions A (14.7 mg), B (20.6 mg), and C
(75.7 mg). Further separation of fraction C (75.0 mg) by
preparative TLC (12% MeOH-dichloromethane) furnished 2
(6.7 mg, 16.8%), 3 (9.1 mg, 22.8%), and 4 (2.7 mg, 6.8%).
Compound 5 (8.3 mg, 16.6%) was isolated by preparative TLC
(12% MeOH in CH2Cl2) of the EtOAc extract (50.1 mg) of a
culture (1000 mL) of B. bassiana ATCC 7159.
indicating the presence of an additional oxygen compared
with 3, which suggested that a hydroxyl group and a
glucose molecule were introduced to curvularin (1). Com-
parison of 1H and 13C NMR data with those of 3 suggested
that compound 4 contained a 4′-O-methylglucose moiety.
1
In its H NMR spectrum, 4 had only one aromatic proton
signal (at δ 6.44) compared with two aromatic proton
signals for both 2 and 3. This signal at δH 6.44 of 4 had a
strong HMBC correlation to the signal at δC 39.2 (C-2)
(Figure 1), suggesting that the newly introduced hydroxyl
group is at C-6, allowing this biotransformation product
to be identified as 6-hydroxycurvularin-4′-O-methyl-6-O-
â-D-glucopyranoside (4).
In an attempt to determine the nature of the enzymes
of B. bassiana ATCC 7159 involved in the biotransforma-
tion of 1 to above glucosidation, methylglucosidation, and
hydroxylation products, 2-4, we investigated the fermen-
tation broth of this fungus in the absence of any added
substrate. TLC investigation of the EtOAc extract of the
fermentation broth indicated the presence of a major
metabolite, and this was isolated by preparative TLC and
identified as 6-methyl-2,4-dihydroxyphenyl 4′-O-methyl-1-
O-â-D-glucopyranoside (5). Although this is the first report
of 5 as a metabolite of B. bassiana, it has previously been
isolated from the entomogenous deuteromycete, Beauveria
amorpha,14 and the presence of novel 4′-O-methylglucose
derivatives of aromatic compounds in dried silkworm larva
stiffened due to B. bassiana infection has recently been
reported.15 Occurrence of 5 in the fermentation broth of B.
bassiana ATCC 7159 suggests that this strain is capable
of hydroxylation and 4′-O-methylglucosidation even in the
absence of the substrate, indicating that hydroxylase,
glucosyltransferase, and methylase in these biotransfor-
mation processes are not inducible enzymes, but constitu-
tive enzymes. Compounds 2-4 were tested for their
cytotoxicity against several cancer cell lines [NCI-H460
(nonsmall cell lung), MCF-7 (breast), and SF-268 (CNS
glioma)] using the MTT assay7 and were found to be
inactive at concentrations up to 10 µg/mL.
Curvularin-7-O-â-D-glucopyranoside (2): Colorless crys-
talline powder; mp 155-156 °C; [R]25 -45.7° (c 0.2, MeOH);
D
UV (MeOH) λmax (log ꢀ) 270 (3.74), 218 (4.03) nm; IR (KBr)
νmax 3379, 2928, 1705, 1609, 1458, 1312, 1265, 1173, 1076,
1034, and 806 cm-1 1H NMR (d6-acetone, 500 MHz) δ 8.96
;
(1H, s, OH-5), 6.68 (1H, d, J ) 2.1 Hz, H-6), 6.51 (1H, d, J )
2.1 Hz, H-4), 4.97 (1H, m, H-15), 4.94 (1H, d, J ) 7.6 Hz, H-1′),
4.60 (1H, d, J ) 4.7 Hz, OH-2′), 4.42 (1H, d, J ) 4.0 Hz, OH-
3′), 4.33 (1H, d, J ) 4.4 Hz, OH-4′), 3.89 (1H, m, H-6′), 3.72
(2H, m, H-2), 3.69 (1H, m, H-6′), 3.51 (1H, m, H-3′), 3.49 (1H,
m, H-5′), 3.42 (1H, m, H-2′), 3.39 (1H, m, H-4′), 3.15 (1H, m,
H-10), 2.86 (1H, m, H-10), 1.79 (1H, m, H-11), 1.65 (1H, m,
H-14), 1.44 (1H, m, H-13), 1.43 (1H, m, H-12), 1.41 (1H, m,
H-14), 1.35 (1H, m, H-11), 1.27 (1H, m, H-13), 1.26 (1H, m,
H-12), 1.13 (1H, d, J ) 6.3 Hz, CH3-15); 13C NMR (d6-acetone,
125 MHz) δ 207.0 (C, C-9), 171.0 (C, C-1), 159.8 (C, C-5), 157.4
(C, C-7), 135.5 (C, C-3), 124.7 (C, C-8), 113.9 (CH, C-4), 102.7
(CH, C-6), 102.3 (CH, C-1′), 78.2 (CH, C-5′), 77.8 (CH, C-3′),
74.6 (CH, C-2′), 72.8 (CH, C-15), 71.3 (CH, C-4′), 62.6 (CH2,
C-6′), 44.0 (CH2, C-10), 39.1 (CH2, C-2), 33.4 (CH2, C-14), 27.8
(CH2, C-12), 24.7 (CH2, C-13), 23.4 (CH2, C-11), 20.7 (CH3, CH3-
15); HRFABMS m/z 455.1905 [M + 1]+ (calcd for C22H31O10
455.1917).
Experimental Section
General Experimental Procedures. Melting points were
determined on a Gallenkamp micromelting point apparatus
and were uncorrected. Optical rotations were measured in
MeOH with a Jasco DIP-370 digital polarimeter. IR spectra
were recorded on a Shimadzu FTIR-8300 spectrometer in KBr
disks and UV spectra in MeOH on a Shimadzu UV-1601
spectrometer. 1D and 2D NMR spectra were recorded in d6-
1
acetone on a Bruker DRX-500 instrument at 500 MHz for H
Curvularin-4′-O-methyl-7-O-â-D-glucopyranoside (3):
Colorless crystalline powder; mp 123-124 °C; [R]25D -40.1° (c
0.2, MeOH); UV (MeOH) λmax (log ꢀ) 269 (3.63), 219 (3.94) nm;
IR (KBr) νmax 3422, 2925, 1728, 1659, 1616, 1312, 1273, 1180,
NMR and 125 MHz for 13C NMR. The chemical shift values
(δ) are given in parts per million (ppm) and the coupling
constants are given in hertz. Abbreviations for NMR signals
are as follows: s ) singlet, d ) doublet, t ) triplet, m )
multiplet. High-resolution FABMS was obtained with a JEOL
HX110A mass spectrometer.
1
1087, 1045, and 806 cm-1; H NMR (d6-acetone, 500 MHz) δ
8.93 (1H, s, OH-5), 6.66 (1H, d, J ) 2.2 Hz, H-6), 6.51 (1H, d,
J ) 2.2 Hz, H-4), 4.97 (1H, m, H-15), 4.92 (1H, d, J ) 7.8 Hz,
H-1′), 4.64 (1H, d, J ) 4.9 Hz, OH-2′), 4.49 (1H, d, J ) 4.5 Hz,
OH-3′), 3.80 (1H, m, H-6′), 3.72 (2H, m, H-2), 3.67 (1H, m,
H-6′), 3.61 (1H, m, H-3′), 3.55 (3H, s, OCH3-4′), 3.46 (1H, m,
H-5′), 3.45 (1H, m, H-2′), 3.18 (1H, t, J ) 9.3 Hz, H-4′), 3.12
(1H, m, H-10), 2.86 (1H, m, H-10), 1.79 (1H, m, H-11), 1.63
(1H, m, H-14), 1.45 (1H, m, H-13), 1.43 (1H, m, H-12), 1.37
(1H, m, H-14), 1.26 (1H, m, H-11), 1.25 (1H, m, H-12), 1.24
(1H, m, H-13), 1.13 (3H, d, J ) 6.3 Hz, CH3-15); 13C NMR (d6-
acetone, 125 MHz) δ 207.0 (C, C-9), 171.0 (C, C-1), 159.8 (C,
C-5), 157.3 (C, C-7), 135.5 (C, C-3), 124.7 (C, C-8), 113.9 (CH,
C-4), 102.6 (CH, C-6), 102.0 (CH, C-1′), 80.1 (CH, C-4′), 78.2
(CH, C-3′), 77.0 (CH, C-5′), 74.8 (CH, C-2′), 72.8 (CH, C-15),
62.1 (CH2, C-6′), 60.5 (CH3, OCH3-4′), 44.0 (CH2, C-10), 39.1
(CH2, C-2), 33.4 (CH2, C-14), 27.8 (CH2, C-12), 24.7 (CH2, C-13),
Curvularin (1). Dehydrocurvularin (6) (49.6 mg), obtained
from an unidentified Penicillium sp.,7a was dissolved in EtOAc
(2.0 mL) and subjected to catalytic hydrogenation with 10%
Pd-C for 3 h at 25 °C. Filtration followed by evaporation of
1
EtOAc afforded 1 (49.8 mg) with H and 13C NMR and mass
spectral data identical to those reported in the literature.2a
Culture and Biotransformation Procedures. Screening
scale biotransformation of 1 by B. bassiana ATCC 7159 was
carried out in a 125-mL flask containing 50 mL of potato
dextrose broth (PDB, Difco, Plymouth, MN). The flask was
placed in a rotary shaker at 150 rpm at 25 °C. After 3 days,
the fermentation broth turned red, and to this was added 1
(0.1 mL of a solution of 10 mg/mL in acetone). After substrate
administration, the flask was maintained under the same

Notes
Journal of Natural Products, 2005, Vol. 68, No. 8 1273
References and Notes
23.2 (CH2, C-11), 20.7 (CH3, CH3-15); HRFABMS m/z 469.2075
[M + 1]+ (calcd for C23H33O10 469.2074).
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6-Hydroxycurvularin-4′-O-methyl-6-O-â-D-glucopyra-
noside (4): Colorless crystalline powder; mp 127-128 °C;
[R]25D -17.8° (c 0.2, MeOH); UV (MeOH) λmax (log ꢀ) 270 (3.74),
226 (4.06) nm; IR (KBr) νmax 3422, 2936, 1713, 1663, 1589,
1454, 1358, 1258, 1200, 1076, and 806 cm-1 1H NMR (d6-
;
acetone, 500 MHz) δ 6.44 (1H, s, H-4), 4.93 (1H, m, H-15), 4.64
(1H, d, J ) 8.0 Hz, H-1′), 3.88 (1H, m, H-6′), 3.72 (2H, m, H-2),
3.71 (1H, m, H-6′), 3.66 (1H, t, J ) 9.0 Hz, H-3′), 3.55 (3H, s,
OCH3-4′), 3.54 (1H, m, H-2′), 3.51 (1H, m, H-5′), 3.28 (1H, t, J
) 9.0 Hz, H-4′), 3.05 (1H, m, H-10), 2.81 (1H, m, H-10), 1.77
(1H, m, H-11), 1.60 (1H, m, H-14), 1.51 (1H, m, H-11),1.45 (1H,
m, H-13), 1.44 (1H, m, H-14), 1.43 (1H, m, H-12), 1.27 (1H, m,
H-12), 1.25 (1H, m, H-13), 1.12 (3H, d, J ) 6.3 Hz, CH3-15);
13C NMR (d6-acetone, 125 MHz) δ 205.6 (C, C-9), 170.9 (C, C-1),
151.8 (C, C-5), 149.6 (C, C-7), 133.1 (C, C-6), 132.3 (C, C-3),
121.7 (C, C-8), 112.5 (CH, C-4), 107.6 (CH, C-1′), 79.7 (CH,
C-4′), 77.7 (CH, C-5′), 77.1 (CH, C-3′), 74.8 (CH, C-2′), 72.6
(CH, C-15), 61.5 (CH2, C-6′), 60.7 (CH3, OCH3-4′), 43.9 (CH2,
C-10), 39.2 (CH2, C-2), 33.0 (CH2, C-14), 27.5 (CH2, C-12), 24.7
(CH2, C-13), 23.2 (CH2, C-11), 20.6 (CH3, CH3-15); HRFABMS
m/z 485.2047 [M + 1]+ (calcd for C23H33O11 485.2023).
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6-Methyl-2,4-dihydroxyphenyl-4′-O-methyl-1-O-â-D-glu-
copyranoside (5): Colorless amorphous solid; 1H and 13C
NMR spectral data were consistent with those reported in the
literature;5 APCIMS (+) ve mode m/z 317 [M + 1]+.
Acknowledgment. This work was supported by the
National Cancer Institute Grant No. 1RO1CA90265-01A1, and
this support is gratefully acknowledged. We also thank Profes-
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fungal strains used in this study.
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NP0580309