Microbial transformations of aryltetralone and aryltetralin lignans by Cunninghamella echinulata and Beauveria bassiana

Article abstract of DOI:10.1021/np100607s

Microbiological transformation of the aryltetralone lignan (-)-8′-epi-aristoligone (1) with Cunninghamella echinulata ATCC 10028B afforded two known natural lignans, (-)-holostyligone (3) and (-)-arisantetralone (4). Incubation of the aryltetralin lignan

Full text of DOI:10.1021/np100607s

J. Nat. Prod. 2010, 73, 1933–1937
1933
Microbial Transformations of Aryltetralone and Aryltetralin Lignans by Cunninghamella
echinulata and BeauWeria bassiana
Gisele B. Messiano,†,§ E. M. Kithsiri Wijeratne,† Lucia M. X. Lopes,*,§ and A. A. Leslie Gunatilaka*,†
SW Center for Natural Products Research and Commercialization, Office of Arid Lands Studies, School of Natural Resources and the
EnVironment, College of Agriculture and Life Sciences, UniVersity of Arizona, 250 E. Valencia Road, Tucson, Arizona 85706-6800, United States, and
Instituto de Qu´ımica, UniVersidade Estadual Paulista, C.P. 355, 14801-970, Araraquara, SP, Brazil
ReceiVed August 29, 2010
Microbiological transformation of the aryltetralone lignan (-)-8′-epi-aristoligone (1) with Cunninghamella echinulata
ATCC 10028B afforded two known natural lignans, (-)-holostyligone (3) and (-)-arisantetralone (4). Incubation of
the aryltetralin lignan (-)-isogalbulin (2), obtained by chemical transformation of 1, with C. echinulata ATCC 10028B
afforded the known lignan aryltetralol (5) and seven new metabolites, (-)-8-hydroxyisogalbulin (6), (-)-7-
methoxyisogalbulin (7), (-)-4′-O-demethyl-8-hydroxyisogalbulin (8), (-)-7-methoxy-8-hydroxyisogalbulin (9), (-)-
4′-O-demethyl-7-methoxyisogalbulin (10), (-)-4′,5-O-didemethylcyclogalgravin (11), and (-)-4′-O-demethylcyclogal-
gravin (12). When 2 was subjected to biotransformation with BeauVeria bassiana ATCC 7159, (-)-8-hydroxyisogalbulin
(6) was the only isolable product. The structures of all new compounds were established by detailed analysis of their
spectroscopic data.
Lignans are a class of plant secondary metabolites with high
structural diversity and are biogenetically derived by the oxidative
dimerization of phenylpropenoids.1 The current interest in lignans
stems from their known medicinal potential as hepatoprotective,2
antioxidative,3 antiviral4 including anti-HIV,5 antibacterial,6 anti-
parasitic,7 antifungal,8 anti-inflammatory,9 and anticancer10 agents.
In addition, recent studies have shown that the presence of high
levels of lignans in humans reduces the risks of prostate, ovarian,
and breast cancers, osteoporosis, and cardiovascular disease.11
Lignans, especially those belonging to the classes of aryltetralins
and aryltetralones, have attracted attention due to the commercial
importance of podophyllotoxin as a raw material for the semisyn-
thesis of the anticancer drugs etoposide and teniposide.12 Significant
antiplasmodial activity of the aryltetralone lignan (-)-8′-epi-
aristoligone (1)13,14 and its potential as an antimalarial drug together
with its ready availability from Holostylis reniformis Duch.
(Aristolochiaceae)13 prompted us to investigate the application of
microbial biotransformation of 1 and the aryltetralin lignan (-)-
isogalbulin (2), obtained by chemical transformation of 1, to
generate their novel analogues for future biological testing. Although
there are a number of reports on metabolism of phenolic tetralin
and furofuranoid lignans including pinoresinol,15 deoxypodophyl-
lotoxin,16 and non-phenolic furofuranoid lignans (+)-eudesmin,15
(+)-magnolin,15 and (+)-yangabin15 by microbes, there are no
reports on microbial biotransformation of the non-phenolic arylte-
tralin and aryltetralone lignans. In continuing our studies on
microbial biotransformation of natural products,17 we have inves-
tigated the metabolism of 1 and 2 by Cunninghamella echinulata
ATCC 10028B and BeauVeria bassiana ATCC 7159, and here we
report the isolation and characterization of seven new and three
known analogues of these lignans.
lations during the biotransformation. Although C. echinulata is
known to effect de-O-methylation of alkaloids,18,19 and regiose-
lective de-O-methylation of lignans has been reported in biotrans-
formation of (+)-eudesmin and (+)-magnolin with Aspergillus
niger,15 this constitutes the first report of de-O-methylation of a
lignan by C. echinulata.
The known ability of C. echinulata to hydroxylate benzylic
positions of aromatic substrates20 prompted us to investigate the
metabolism of aryltetralin lignans by this fungus. To this end, the
aryltetralin lignan (-)-isogalbulin (2) was obtained in high overall
yield by the treatment of (-)-8′-epi-aristoligone (1) with NaBH4/
MeOH followed by hydrogenolysis (H2/10% Pd on C/EtOH). When
2 was incubated with C. echinulata for 20 days, the EtOAc extract
of the resulting culture broth afforded the known lignan aryltetralol
(5)14 and seven new metabolites, 6-12. Metabolite 6 was obtained
as a white, amorphous solid and was determined to have the
molecular formula C22H28O5 by a combination of HRMS and 13C
Results and Discussion
Incubation of the aryltetralone lignan (-)-8′-epi-aristoligone (1)
with C. echinulata afforded two polar products identified as (-)-
holostyligone (3)13 and (-)-arisantetralone (4),14 suggesting that
the substrate has undergone stepwise regioselective de-O-methy-
1
NMR data. The H NMR spectrum of 6 (Table 1) showed very
close resemblance to that of (-)-isogalbulin (2), the major difference
between them being due to the absence of one methine signal and
one secondary methyl signal in 6, and instead it showed the presence
of a quarternary methyl group attached to an oxygenated carbon
atom. The presence of this oxygenated aliphatic carbon in 6 was
also inferred from its 13C NMR spectrum (Table 2), which showed
a signal at δ 72.2. These data suggested that an oxygenation had
* To whom correspondence should be addressed. Tel: (520) 621-9932.
Fax: (520). E-mail: (A.A.L.G.) leslieg@ag.arizona.edu; (L.M.X.L.)
lopesxl@iq.unesp.br.
† University of Arizona.
§ Universidade Estadual Paulista.
10.1021/np100607s  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 10/20/2010

1934 Journal of Natural Products, 2010, Vol. 73, No. 11
Messiano et al.
Table 1. 1H NMR Data (δ) for Lignans 6-10 (CDCl3, 400 MHz, J in Hz)
H
6
7
8
9
10
3
6
6.17 (s)
6.53 (s)
6.25 (s)
6.75 (s)
6.18 (s)
6.52 (s)
6.28 (s)
6.66 (s)
6.25 (s)
6.74 (s)
7R
7ꢀ
8
2.81 (d, 15.2)
3.04 (d, 15.2)
2.81 (d, 15.2)
3.03 (d, 15.2)
3.99 (d, 3.2)
2.36 (m)
3.86 (s)
1.01 (s)
6.60 (d, 2.0)
6.78 (d, 8.0)
6.71 (dd, 8.0, 2.0)
3.32 (d, 10.4)
2.32 (m)
0.94 (d, 6.8)
3.78 (s)
3.60 (s)
3.89 (s)
3.86 (s)
3.98 (d, 3.6)
2.35 (m)
9
1.16 (s)
0.85 (d, 6.8)
6.58 (d, 2.0)
6.77 (d, 8.0)
6.67 (dd, 8.0, 2.0)
3.39 (d, 10.0)
2.23 (m)
0.84 (d, 6.8)
3.76 (s)
3.59 (s)
1.16 (s)
0.87 (d, 6.8)
6.54 (d, 2.0)
6.81 (d, 8.0)
6.64 (dd, 8.0, 2.0)
3.37 (d, 10.0)
2.23 (m)
0.84 (d, 6.8)
3.76 (s)
3.60 (s)
2′
5′
6′
7′
8′
9′
6.56 (d, 2.0)
6.78 (d, 8.0)
6.69 (dd, 8.0, 2.0)
3.41 (d, 10.8)
1.99 (m)
0.88 (d, 6.4)
3.78 (s)
3.56 (s)
6.52 (d, 2.0)
6.83 (d, 8.0)
6.65 (dd, 8.0, 2.0)
3.40 (d, 10.4)
1.97 (m)
0.88 (d, 6.8)
3.79 (s)
3.57 (s)
OCH3-3′
OCH3-4
OCH3-4′
OCH3-5
OCH3-7
OH
3.87 (s)
3.84 (s)
3.87 (s)
3.85 (s)
3.44 (s)
3.84 (s)
3.87 (s)
3.44 (s)
5.46 (s)
3.46 (s)
5.50 (s), 3.88 (s)
Table 2. 13C NMR Data (δ) for 6 and 8-10 (CDCl3, 100 MHz)
also showed a strong NOE correlation to CH3-9′ at δ 0.84. The
foregoing led to the identification of this metabolite as (-)-7-
methoxyisogalbulin [(7R,7′R,8S,8′S)-8,8′-dimethyl-3′,4,4′,5,7-pen-
tamethoxy-2,7′-cyclolignan] (7).
C
6
8
9
10
1
2
3
4
5
6
7
8
127.5 (s)
130.7 (s)
111.9 (d)
149.0 (s)
147.3 (s)
112.5 (d)
47.2 (t)
127.5 (s)
130.8 (s)
112.6 (d)
147.5 (s)
147.3 (s)
113.9 (d)
47.2 (t)
126.2 (s)
133.1 (s)
112.8 (d)
149.3 (s)
146.8 (s)
113.0 (d)
85.7 (d)
73.2 (s)
17.7 (q)
138.6 (s)
112.2 (d)
148.9 (s)
147.5 (s)
110.7 (d)
121.5 (d)
53.1 (d)
41.4 (d)
12.3 (q)
56.7 (q)
55.9 (q)
55.8 (q)
55.8 (q)
55.7 (q)
126.7 (s)
133.0 (s)
112.7 (d)
148.7 (s)
147.3 (s)
113.6 (d)
83.1 (s)
35.2 (d)
17.1 (q)
137.9 (s)
112.5 (d)
146.6 (s)
143.9 (s)
111.2 (d)
122.7 (d)
49.2 (d)
35.1 (d)
11.3 (q)
56.0 (q)
55.9 (q)
Metabolite 8, obtained as a white, amorphous solid, was
determined to have the molecular formula C21H26O5 by a combina-
tion of its HRFABMS and 13C NMR data. The 1H NMR data of 8
very closely resembled those of 6, the major difference between
them being due to the absence of one aromatic OCH3 group in 8.
Irradiation of the OCH3 signals at δH 3.57 (OCH3-4), 3.79 (OCH3-
3′), and 3.84 (OCH3-5) showed enhancement of the signals at δH
6.18 (H-3), 6.52 (H-2′), and 6.52 (H-6), respectively (Figure 1),
suggesting that these OCH3 groups and aromatic protons are in
close proximity to each other and the possible absence of an OCH3
group adjacent to H-5′. These data also suggested that de-O-
methylation had taken place at C-4′ of the C ring of the lignan.
Metabolite 8 was thus identified as (-)-4′-O-demethyl-8-hydroxy-
isogalbulin [(7′R,8S,8′S)-8,8′-dimethyl-4′-hydroxy-3′,4,5-trimethoxy-
2,7′-cyclolignan-8-ol].
72.2 (s)
72.2 (s)
9
20.4 (q)
138.2 (s)
111.1 (d)
147.5 (s)
147.5 (s)
110.8 (d)
121.6 (d)
53.1 (d)
45.9 (d)
12.6 (q)
55.9 (q)
55.8 (q)
55.8 (q)
55.8 (q)
20.4 (q)
137.6 (s)
111.1 (d)
146.7 (s)
144.1 (s)
111.0 (d)
122.6 (d)
53.2 (d)
45.9 (d)
12.6 (q)
55.9 (q)
55.8 (q)
1′
2′
3′
4′
5′
6′
7′
8′
9′
OCH3-3′
OCH3-4
OCH3-4′
OCH3-5
OCH3-7
The molecular formula of 9 was determined to be C23H30O6 by
1
1
a combination of HRFABMS and H and 13C NMR data. Its H
NMR spectrum was very similar to that of 7, the major difference
between them being due to the absence of one methine signal and
one secondary CH3 signal in 9 and instead the presence of one
CH3 group attached to an oxygenated quaternary carbon. These data
suggested that an additional OH group was present in 9. The
oxymethine proton at δ 3.86 of 9 showed HMBC correlations to
C-1, C-2, C-6, C-8, C-8′, and C-9 (Figure 2), placing it at C-7.
The OCH3 group at δ 3.46 showed HMBC correlations with the
oxymethine carbon at δ 85.7, suggesting its attachment to C-7.
Therefore, the additional OH group should be located at C-8 or
55.8 (q)
55.8 (q)
55.7 (q)
taken place at C-8 or C-8′ of the substrate to give an alcohol. The
coupling pattern observed for the two geminal protons at C-7 (δ
2.44 and 2.84) of 2 had changed from two double doublets to two
doublets (δ 2.81 and 3.04) during its biotransformation to 6,
suggesting that the oxygenation occurred at C-8. The methyl group
at C-8 of 6 showed NOE correlations with the C-8′ methyl group
and H-7′, confirming the ꢀ-orientation of these groups. Thus, this
metabolite was identified as (-)-8-hydroxyisogalbulin [(7′R,8S,8′S)-
8,8′-dimethyl-3′,4,4′,5-tetramethoxy-2,7′-cyclolignan-8-ol] (6). Al-
though the synthesis of 6 has been reported previously,21,22 none
of these reports have defined the relative configuration at C-8 and/
or C-8′.
The HRMS data for the metabolite 7 suggested the molecular
formula C23H30O5. Its 1H NMR spectroscopic data (Table 1)
indicated the presence of 1,2,4,5-tetrasubstituted and 1,3,4-trisub-
stituted benzene rings, four aromatic OCH3, one aliphatic OCH3,
four methine protons of which one is oxygenated [δ 3.99 (d, J )
3.2 Hz)], and two secondary CH3 groups. Comparison of these data
with those of aryltetralol (5) suggested that 7 had the same carbon
skeleton; the major difference was found to be the presence of an
OCH3 instead of an OH group on an aliphatic carbon. In order to
satisfy these data, this newly introduced OCH3 was placed at C-7.
The orientation of this OCH3 group was determined to be R on the
basis of NOE correlations observed between the oxymethine proton
(H-7) and the methine proton at δ 3.39 (H-7′); the latter proton
Figure 1. Selected NOE correlations of 8.

Aryltetralone and Aryltetralin Lignans
Journal of Natural Products, 2010, Vol. 73, No. 11 1935
Figure 4. Selected HMBC correlations of 11.
Figure 2. Selected HMBC (HfC) and NOE (HTH) correlations
of 9.
(d, J ) 3.2 Hz) and 2.32 (dq, J ) 7.2, 3.2 Hz)], a CH3 attached to
an olefinic carbon [(δ 1.76 (s)], a secondary CH3 group [1.05 (d, J
) 7.2 Hz)], and two OH groups [δ 5.43 (s) and 5.40 (s); D2O
exchangeable]. The 13C NMR spectrum of 11, when analyzed in
combination with the HSQC data, showed the presence of 14
aromatic/olefinic carbons, of which four were oxygenated (δ 146.2,
145.1, 144.2 and 143.8) and six were protonated (δ 121.1, 120.4,
113.9, 112.1, 111.7, and 110.1), two methine carbons, two OCH3
carbons, and two CH3 carbons. The aromatic/olefinic proton at δ
6.09 showed HMBC correlations with C-2 (δ 126.9), C-6 (δ 111.7),
and C-8′ (42.1), placing it at C-7. The D2O exchangeable singlet
at δ 5.40 (OH) showed HMBC correlations with C-4 and C-6,
locating it at C-5, and the other OH showed HMBC correlations
with C-3′ and C-5′, placing it at C-4′ (Figure 4). This metabolite
was thus identified as (-)-4′,5-O-didemethylcyclogalgravin
[(7′R,8′S)- 4′,5-dihydroxy-8,8′-dimethyl-3′,4-dimethoxy-2,7′-cyclo-
lignan-7-ene] (11).
The molecular formula of metabolite 12 was determined to be
1
C21H24O4 from its HRMS data. The H NMR spectroscopic data
(see Experimental Section) of this compound closely resembled
those of (-)-4′,5-O-didemethylcyclogalgravin (11) except for the
presence of an additional aromatic OCH3 group in 12. The presence
of a strong NOE of this OCH3 to H-6 confirmed that it was located
at C-5. Thus, this metabolite was identified as (-)-4′-O-demeth-
ylcyclogalgravin [(7′R,8′S)-8,8′-dimethyl-4′-hydroxy-3′,4,5-trimethoxy-
2,7′-cyclolignan-7-ene] (12).
Figure 3. Selected NOE correlations of 10.
C-8′. Since H-7′ appeared as a doublet (J ) 10.4 Hz), this OH
1
group should be located at C-8. The H NMR singlet at δ 3.86
(H-7) showed an NOE correlation to the CH3 signal at δ 1.01. Since
the CH3 groups are ꢀ-oriented, H-7 should have the same orienta-
tion. Thus, 9 was identified as (-)-7-methoxy-8-hydroxyisogalbulin
[(7S,7′R,8R,8′S)-8,8′-dimethyl-3′,4, 4′,5,7-pentamethoxy-2,7′-cy-
clolignan-8-ol].
Experimental Section
General Experimental Procedures. Optical rotations were measured
in CHCl3 with a JASCO Dip-370 digital polarimeter. 1D and 2D NMR
spectra were recorded in CDCl3 with a Bruker Avance III 400
Metabolite 10, obtained as a white, amorphous solid, was
determined to have the molecular formula C22H28O5 by a combina-
tion of HRFABMS and 13C NMR data. The H NMR data of 10
1
1
spectrometer at 400 MHz for H NMR and 100 MHz for 13C NMR
indicated the presence of five aromatic protons in two aromatic
rings, three OCH3 groups attached to olefinic/aromatic carbons, one
OCH3 group attached to an aliphatic carbon (δ 3.44), one oxyme-
thine proton [δ 3.98 (d, J ) 3.6 Hz)], three methine protons, and
two secondary CH3 groups (Table 1). Irradiation of the OCH3
signals at δH 3.87, 3.76, 3.60, and 3.44 showed enhancement of
those at δH 6.74 (H-6), 6.54 (H-2′), 6.25 (H-3), and 6.74 (H-6),
respectively (Figure 3), suggesting that demethylation occurred at
C-4′ and the OCH3 group attached to the aliphatic carbon is located
at C-7. The oxymethine proton at δ 3.98 showed NOE correlations
with CH3-8 protons, confirming its ꢀ-orientation. This compound
was thus identified as (-)-4′-O-demethyl-7-methoxyisogalbulin
[(7R,7′R,8S,8′S)-8,8′-dimethyl-4′-hydroxy-3′,4,5,7-tetramethoxy-
2,7′-cyclolignan] (10).
Metabolite 11, obtained as an off-white, amorphous solid, was
determined to have the molecular formula C20H22O4 by a combina-
tion of HRFABMS and 1H and 13C NMR data. Its 1H NMR
spectrum showed the presence of 1,2,4,5-tetrasubstituted and 1,3,4-
trisubstituted benzene rings, an olefinic/aromatic proton (δ 6.09),
two aromatic OCH3 (δ 3.76 and 3.75), two methine protons [δ 3.62
using residual CHCl3 resonances as internal reference. Low-resolution
and high-resolution MS were recorded on Shimadzu LCMS-QP8000R
and JEOL HX110A spectrometers, respectively. Analytical and pre-
parative thin-layer chromatography (TLC) were performed on precoated
0.25 mm thick plates of silica gel 60 F254; spraying with a solution of
7% phosphomolybdic acid in EtOH followed by heating was used to
visualize the spots on analytical TLC. Preparative HPLC was performed
on a Waters Delta Prep 4000 system equipped with a Waters 996
photodiode array detector and a Waters Prep LC controller utilizing
Empower Pro software and using a RP column (Phenomenex Luna 5
µm, C18, 100 Å, 250 × 10 mm) with a flow rate of 3.0 mL/min;
chromatograms were acquired at 254 and 270 nm. (-)-8′-epi-Aristo-
ligone (1) was isolated from Holostylis reniformis Duch. (Aristolochi-
aceae) as previously reported.13
Conversion of (-)-8′-epi-Aristoligone (1) to (-)-Isogalbulin (2).
To a stirred solution of 1 (200 mg, 0.5 mmol) in MeOH (7 mL) at 0
°C was added NaBH4 (100 mg, 2.6 mmol). After 1 h at 0 °C (TLC
control) excess NaBH4 was destroyed by adding ice. The resulting
solution was evaporated under reduced pressure to remove MeOH and
extracted with EtOAc (3 × 25 mL), and the organic extract was washed
with H2O, dried (Na2SO4), and evaporated under reduced pressure to
afford aryltetralol (5) (198 mg, 97%). Lignan 5 (195 mg, 0.5 mmol)

1936 Journal of Natural Products, 2010, Vol. 73, No. 11
Messiano et al.
was dissolved in ethanol (50 mL), and 10% Pd on C (ca.10 mg) was
added to the solution, which was stirred at room temperature under an
atmosphere of H2. After 2 h (TLC control), the reaction mixture was
filtered and the filtrate evaporated under reduced pressure to afford 2
(175 mg, 94%) as a white solid.
of MeOH, and combining fractions having similar TLC patterns
afforded three subfractions (F2A-F2C). Subfraction F2B was then
separated by RP-HPLC using 50% aqueous CH3CN to give 5 (1.0 mg)
and 6 (11.8 mg). Subfraction F2c was also separated by RP-HPLC, but
using 45% aqueous CH3CN to give an additional quantity of 6 (3.5
mg) and 7 (6.0 mg). Fraction F4 from the above Sephadex column was
directly subjected to RP-HPLC using 50% aqueous CH3CN to give 8
(3.6 mg), 9 (2.3 mg), and 11 (2.2 mg). RP-HPLC of F5 using 70%
aqueous CH3CN gave an additional quantity of 8 (8.4 mg), and a crude
fraction was separated by RP-HPLC using 60% aqueous CH3CN to
give 10 (1.3 mg). Fraction F8 from the Sephadex column on further
purification by RP-HPLC using 30% aqueous CH3OH as eluant afforded
12 (1.8 mg).
Aryltetralol (5): white, amorphous powder; [R]25 -38.2 (c 0.41,
D
CHCl3) [lit.14 -36.0 (c 0.83, CHCl3)]; 1H and 13C NMR, and MS data
were consistent with reported data.14
(-)-Isogalbulin (2): colorless needles (MeOH); mp 98-100 °C
(lit.23 101-102 °C); [R]D25 -35.8 (c 0.7, CHCl3) [lit.23 -36.7 (c 0.08,
23
1
CHCl3)]; H NMR and MS data were consistent with reported data.
Microbial Transformation of (-)-8′-epi-Aristoligone (1) by C.
echinulata ATCC 10028B. C. echinulata was grown in a two-stage
fermentation procedure in soybean meal/glucose medium [soybean meal
(5 g), glucose (20 g), K2HPO4 (5 g), NaCl (5 g), yeast extract (5 g),
distilled H2O (1 L)]. The pH of the medium was adjusted to 7.0 and
autoclaved at 121 °C for 15 min. Small-scale fermentations were
performed in 125 mL Erlenmeyer flasks, each holding 25 mL of the
culture medium on a rotary shaker operating at 220 rpm at 28 °C for
24 h. The substrate (1, 2.5 mg in 0.25 mL of DMF) was added to 24 h
old second-stage culture. Culture control consisted of fermentation broth
of C. echinulata without the substrate but with the same volume of
DMF, and the substrate control consisted of soybean meal/glucose
medium with the same amount of 1 in DMF. Both controls were
incubated under the same conditions. Samples (4 mL each) were taken
from all three flasks at various time intervals and individually extracted
with EtOAc (4 mL), and extracts were examined for the disappearance
of 1 by TLC. Large-scale biotransformation was performed under the
same conditions but in 4 × 250 mL flasks holding 50 mL of the medium
in each flask. A total of 21.1 mg of 1 was used, and after 20 days of
incubation, the fermentation broths were combined, mycelia were
removed by filtration and washed with H2O (3 × 250 mL), and the
washings were combined with the filtrate, neutralized with 1 M HCl,
and extracted with EtOAc (3 × 200 mL). Combined organic extracts
were dried over anhydrous Na2SO4, and the solvent was evaporated
under reduced pressure to give the EtOAc extract (20.1 mg) as a brown
syrup. This was separated by preparative TLC (silica gel) using hexanes/
EtOAc/CHCl3 (6:3:1) to give 1 (2.0 mg, Rf 0.53), 3 (0.5 mg, Rf 0.40),
and 4 (1.5 mg, Rf 0.22).
(-)-8-Hydroxyisogalbulin (6): white, amorphous powder; [R]25
D
1
-15.6 (c 0.11, CHCl3); H and 13C NMR data, see Tables 1 and 2,
respectively; HRAPCIMS m/z 371.1862 [M - H]+ (calcd for C22H27O5,
371.1864).
(-)-7-Methoxyisogalbulin (7): white, amorphous powder; [R]25
D
1
-51.5 (c 0.4, CHCl3); H NMR data, see Table 1; HRAPCIMS m/z
409.1987 [M + Na]+ (C23H30NaO5, 409.1985).
(-)-4′-O-Demethyl-8-hydroxyisogalbulin (8): white, amorphous
powder; [R]25 -15.0 (c 0.16, CHCl3); 1H and 13C NMR data, see
D
Tables 1 and 2, respectively; HRESIMS m/z 357.1707 [M - H]+ (calcd
for C21H25O5, 357.1707).
(-)-7-Methoxy-8-hydroxyisogalbulin (9): white, amorphous pow-
1
der; [R]25 -34.7 (c 0.18, CHCl3); H and 13C NMR data, see Tables
D
1 and 2, respectively; HRESIMS m/z 425.1936 [M + Na]+ (calcd for
C23H30NaO6, 425.1935).
(-)-4′-O-Demethyl-7-methoxyisogalbulin (10): white, amorphous
powder; [R]25D -16.1 (c 0.2, CHCl3); 1H and 13C NMR data, see Tables
1 and 2, respectively; HRAPCIMS m/z 371.1862 [M - H]+ (calcd for
C22H27O5, 371.1864).
(-)-4′,5-O-Didemethylcyclogalgravin (11): white, amorphous pow-
1
der; [R]25 -110.5 (c 0.18, CHCl3); H NMR (400 MHz, CDCl3) δ
D
6.74 (1H, d, J ) 8.0 Hz, H-5′), 6.64 (1H, s, H-6), 6.56 (1H, d, J ) 2
Hz, H-2′), 6.54 (1H, dd, J ) 8.0, 2.0 Hz, H-6′), 6.49 (1H, s, H-3), 6.09
(1H, s, H-7), 5.43 (1H, s, D2O exchangeable, OH-4′), 5.40 (1H, s, D2O
exchangeable, OH-5), 3.76 (3H, s, OMe), 3.75 (3H, s, OMe), 3.62 (1H,
d, J ) 3.2 Hz, H-7′), 2.32 (1H, dq, J ) 7.2, 3.2 Hz, H-8′), 1.76 (3H,
s, H3-9), 1.05 (1H, d, J ) 7.2 Hz, H3-9′); 13C NMR (100 MHz, CDCl3)
δ 146.2 (C, C-3′), 145.1 (C, C-5), 144.2 (C-4), 143.8 (C, C-4′), 138.8
(C, C-1′), 137.8 (C, C-8), 127.8 (C, C-1), 126.9 (C, C-2), 121.1 (CH,
C-7), 120.4 (CH, C-6′), 113.9 (CH, C-5′), 112.1 (CH, C-3), 111.7
(CH, C-6), 110.1 (CH, C-2′), 55.9 (CH3, OMe), 55.8 (CH3, OMe),
51.1 (CH, C-7′), 42.1 (C-8′), 22.2 (CH3, H3-9), 18.8 (CH3, H3-9′);
HRESIMS m/z 327.1597 [M + H]+ (calcd for C20H23O4, 327.1591).
(-)-Holostyligone (3): colorless, amorphous solid; [R]25D -38.2 (c
0.05, CHCl3) [lit.14 -27.0 (c 1.18, CHCl3)]; 1H and 13C NMR and MS
data were consistent with reported data.14
Arisantetralone (4): colorless, amorphous solid; [R]25 -35.4 (c
D
0.05, CHCl3) [lit.24 -35.0 (c 0.1, CHCl3)]; H and 13C NMR and MS
1
data were consistent with reported data.24
Microbial Transformation of (-)-Isogalbulin (2) by C. echinulata
ATCC 10028B. C. echinulata (ATCC 10028B) was grown in a two-
stage fermentation procedure in a soybean meal/glucose medium. Small-
scale microbial transformation of (-)-isogalbulin (2) by the organism
was carried out in 125 mL Erlenmeyer flasks containing 50 mL of
medium on a rotary shaker operating at 220 rpm and 28 °C. The
substrate (2, 5.0 mg in 0.25 mL of DMF) was added to 24 h old second-
stage culture. Culture control consisted of a fermentation broth of C.
echinulata without the substrate but with the same volume of DMF,
and the substrate control consisted of sterile soybean meal/glucose
medium with the same amount of 2 in DMF. Both controls were
incubated under the same conditions. Samples (4 mL each) were taken
from all three flasks at various time intervals and extracted separately
with EtOAc (4 mL), and extracts were examined for the formation of
metabolites by TLC. A preparative-scale microbial transformation was
performed under the same conditions in (5 × 1 L) Erlenmeyer flasks
holding 250 mL of medium. A total of 150 mg of 2 was used. After
20 days mycelia were separated by filtration and combined supernatant
was extracted with EtOAc (3 × 500 mL). The combined EtOAc layer
was washed with H2O, dried over anhydrous Na2SO4, and evaporated
under reduced pressure to give a crude extract (149.5 mg). This extract
was then subjected to gel permeation chromatography over Sephadex
LH-20 (4 g) made up in hexanes/CH2Cl2 (1:4) and eluted with hexanes/
CH2Cl2 (1:4, 180 mL), CH2Cl2/acetone (3:2, 90 mL), CH2Cl2/acetone
(1:4, 90 mL), CH2Cl2/MeOH (1:1, 90 mL), and finally MeOH (50 mL).
Fractions (6 mL each) were collected, and those having similar TLC
patterns were combined to give 14 major fractions (F1-F14). Further
separation of fraction F2 (41.4 mg) over a column of silica gel (1.5 g)
made up in CH2Cl2, elution with CH2Cl2 containing increasing amounts
(-)-4′-O-Demethylcyclogalgravin (12): white, amorphous powder;
1
[R]25 -108.6 (c 0.3, CHCl3); H NMR (400 MHz, CDCl3) δ 6.74
D
(1H, d, J ) 8.4 Hz, H-5′), 6.60 (1H, s, H-6), 6.56 (1H, d, J ) 2 Hz,
H-2′), 6.54 (1H, dd, J ) 8.4, 2.0 Hz, H-6′), 6.53 (1H, s, H-3), 6.12
(1H, d, J ) 1.2 Hz, H-7), 5.40 (1H, s, D2O exchangeable, OH-4′),
3.86 (3H, s, OMe), 3.76 (3H, s, OMe), 3.75 (3H, s, OMe), 3.64 (1H,
d, J ) 2.8 Hz, H-7′), 2.35 (1H, m, H-8′), 1.77 (3H, s, H3-9), 1.05 (1H,
d, J ) 7.2 Hz, H3-9′); 13C NMR (100 MHz, CDCl3) δ 147.6 (C, C-5),
147.5 (C, C-4), 146.2 (C, C-3′), 143.8 (C, C-4′), 138.8 (C, C-1′), 137.6
(C, C-8), 127.4 (C, C-2), 127.1 (C, C-1), 121.1 (CH, C-7), 120.4 (CH,
C-6′), 113.9 (CH, C-5′), 112.9 (CH, C-3), 110.1 (CH, C-2′), 108.9 (CH,
C-6), 55.9 (CH3, 2 × OMe), 55.8 (CH3, OMe), 50.9 (CH, C-7′), 42.2
(CH, C-8′), 22.2 (CH3, C-9), 18.7 (CH3, C-9′); HRESIMS m/z [M +
H]+ 341.1751 (calcd for C21H25O4, 341.1747).
Microbial Transformation of (-)-Isogalbulin (2) by B. bassiana
ATCC 7159. Screening-scale microbial transformation of 2 by B.
bassiana ATCC 7159 was carried out in a 125 mL Erlenmeyer flask
containing 25 mL of potato dextrose broth (PDB, Difco, Plymouth,
MN). The flask was placed on a rotary shaker operating at 220 rpm at
28 °C. Substrate (2, 2.5 mg in 0.25 mL of DMF) was added to 24 h
old second-stage culture broth. Culture control consisted of fermentation
broth of B. bassiana ATCC 7159 without the substrate but with the
same volume of DMF, and the substrate control consisted of sterile
PDB medium with the same amount of a solution of 2 in DMF. Both
controls were incubated under the same conditions. Formation of
microbial transformation metabolites was followed by TLC. Preparative-
scale fermentation was performed under the same conditions as small-
scale fermentations in 2 × 250 mL Erlenmeyer flasks, each holding

Aryltetralone and Aryltetralin Lignans
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50 mL of the culture broth. A total of 10 mg of 2 was used. After 20
days of incubation, the cultures were filtered; the combined filtrate was
neutralized with 1 M NaOH and extracted with EtOAc (3 × 200 mL).
The combined EtOAc layer was washed with H2O (200 mL), dried
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to yield (-)-8-hydroxyisogalbulin (6) (0.7 mg, Rf 0.6), identical (1H
NMR and LRMS) with previously obtained sample.
Acknowledgment. We thank Professor J. P. N. Rosazza (University
of Iowa) for providing the fungal strains used in this study, and
Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnolo´gico
(CNPq/MCT/MS/PRONEX, Brazil) for financial support and a graduate
research fellowship (to G.B.M.).
Supporting Information Available: This material is available free
of charge via the Internet at http://pubs.acs.org.
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NP100607S