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1. Experimental
Strain and culture media: A. ochraceus SIT34205 was obtained and screened by our laboratory. The composition of
the slant culture medium (g/L) was as follows: Corn steep liquor 10, tryptone 10, and agar 25. The composition of seed
culture medium (g/L) was as follows: Glucose 20, yeast extract 15, fish peptone 5, corn starch 5, corn steep liquor 0.5,
and KH2PO4 0.5. The composition of biotransformation medium (g/L) was as follows: glucose 25, yeast extract 15,
peptone 5, and KH2PO4 0.5.
Biotransformation of 2 from 1: Strain SIT34205 was cultured in slant medium at 28 8C for 5–7 days, and transferred
into sterile physiological saline to obtain a spore suspension at a concentration of 1 ꢀ 107 spores/mL. The suspension
was inoculated into a 100 mL seed culture medium in a 500 mL shake flask, cultured at 28 8C and 160 rpm/min for
20 h. The seed culture was inoculated into the biotransformation medium in an amount of 4% and cultured for 20 h,
followed by the addition of 10 g/L of 1. Incubation was continued for 50 h under the same conditions.
HPLC assay of 2 and 3: A RP C18 column (Dikma Diamonsil C18, 250 mm ꢀ 4.6 mm, 5 mm) was used as
chromatographic column. The wavelength for detection was 280 nm, mobile phase was methanol–water (7:3, v/v),
injection volume was 10 mL, column temperature was 25 8C, and flow rate was 1 mL/min.
Isolation and identification of 3: The biotransformation solution and the mycelia were extracted three times with
ethyl acetate. The organic phases were pooled, and concentrated under reduced pressure to obtain slurry. The slurry
was mixed with silica gel to homogeneity, dried completely, and then subjected to column chromatography. Gradient
elution was performed with dichloromethane–methanol (100:1; 50:1; 25:1) at 2 mL/min to obtain 3. The structure of 3
was identified by MS, 1H NMR, 13C NMR, DEPT135, and 1H–1H NOESY spectroscopy. Compound 3 was
30
crystallized from methanol; ½aꢁD ꢂ47 (c 0.11, CH3OH), 1H NMR (500 MHz, CDCl3): d 6.18 (d, 1H, J = 10 Hz), 6.09
(d, 1H, J = 10 Hz), 5.77 (s, 1H), 4.18 (dd, 1H, J = 10, 20 Hz), 4.18–4.13 (m, 1H), 3.49 (s, 1H), 2.70–2.52 (m, 4H),
2.43–2.30 (m, 3H), 2.18–2.00 (m, 1H), 1.98–1.90 (m, 3H), 1.59–1.56 (m, 1H), 1.48–1.42 (m, 3H), 1.22 (s, 3H), 0.88 (s,
3H). ESI-MS (m/z): 373.9 [M+H]+, 395.9 [M+Na]+.
2. Results and discussion
Compound 1 was biotransformed into 2 using A. ochraceus SIT34205 with 3 produced during the middle and late
stages of the biotransformation. The results of the TLC analysis of the sample after microbial transformation indicated
a small amounts of 1 with Rf = 0.73, 2 with Rf = 0.20, and 3 with Rf = 0.09 (data not shown). Therefore, the polarity of
3 was greater than that of 2 (Fig. 1).
The molecular weight of 2 was 356 [9], and that of 3 was m/z 373.9 in the ESI-MS result which appeared as a
[M+H]+ ion peak (Figure not shown), indicating that 3 had an additional hydroxyl group relative to 2. Thus, 3 was
speculated to be a dihydroxycanrenone. Compound 3 was structurally characterized using NMR (1H, 13C NMR,
DEPT135 and NOESY) according to the methods described previously [10,11].
The 1H NMR data of 3 revealed many similarities with those of 2 (Table not shown). The major differences were as
follows. For chemical shifts d 3.49, d 4.17, and d 4.28 in the low-field region, the strong peak at d 3.49 disappeared after
deuterium oxide exchange, suggesting the presence of a reactive hydrogen atom. Moreover, as demonstrated by
mAU
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Fig. 1. HPLC chromatogram of the biotransformation sample (left panel) and the purified compound 3 (right panel). In the left panel, the retention
time of 4.108, 4.379, and 6.779 min were correspond to compounds 3, 2 and 1, respectively.