1

Production of a New Tetracyclic Triterpene Sulfate Metabolite Sambacide by

Solid-state Cultivated Fusarium sambucinum B10.2 using Potato as Substrate

Jian-Wei Dong1, Le Cai

1, Xue-Jiao Li, Rong-Ting Duan, Yan

Shu, Feng-Yun Chen,

Jia-Peng Wang, Hao Zhou, Zhong-Tao Ding*

School of Chemical Science and Technology, Yunnan University, Kunming 650091,

China.

Supplementary Data

* Corresponding Authors. Tel./Fax. +86 871 65033910;

E-mail address: ztding@ynu.edu.cn (Z. T. Ding)

1 The first two authors contributed equally to this paper.

2

Supplementary Experimental Procedures

General

UV-Vis spectra were record using a Shimadzu UV-2550 PC spectrometer

(SHIMADZU Co., Ltd., Tokyo, Japan). Electronic circular dichroism (ECD) spectra

were recorded using a Chirascan circular dichroism spectrometer (Applied Photophysics

Ltd., UK). The nuclear magnetic resonance (NMR) spectra were recorded on a Bruker

Avance 400 MHz spectrometer (Bruker, Karlsruhe, Germany) using tetramethylsilane

(TMS) as the internal reference. High-resolution electrospray ionization mass

spectrometry (HRESIMS) and electrospray ionization mass spectrometry (ESIMS)

analyses were conducted using an Agilent G3250AA (Agilent, Santa Clara, CA, USA).

Silica gel (200–300 mesh, Qingdao Marine Chemical Group Co., Qingdao, China) and

Sephadex LH-20 (GE Healthcare Bio-Science AB, Uppsala, Sweden) were used for

column chromatography (CC).

Acetonitrile (HPLC grade) were purchased from Fisher Scientific Co., Ltd. (Fair

Lawn, NJ, USA). The water (resistivity ≥ 18.25 MΩ/cm) used was purified with a

water-purifying system (Chengdu, China). All other chemicals used were of analytical

grade.

Determination of sambacide (1) using HPLC

The content of sambacide was determined using a Waters HPLC system equipped

with a Waters 600 quaternary pump, a Waters 2487 dual-λ absorbance detector, an

Agilent Zorbax SB-C18 (250 × 4.6 mm i.d., 5 μm) column (SN: USCL054040), and an

Empower data-processing station. All samples and standards were filtered through a

0.45-μm filter before being injected into the aforementioned Waters HPLC system. A

gradient elution system consisting of solvents A (water) and B (acetonitrile) was used

for the analysis, and the gradient program was as follows: 0–10 min, 30–60% B; 10–20

3

min, 60% B; 20–25, 60–30% B. The compound was confirmed by adding standard

method and the retention time of 9.38 min from chromatograms by ultraviolet

absorption at a wavelength of 247 nm. The flow rate was 1.5 mL/min, the column

temperature was set to 25 °C, and the injection volume was 10 μL. Each sample was

repeated in triplicate. The calibration curve and method validation are described in

Figure S2 and Table S1-S3. The content of 1 was calculated according to the

calibration curve and expressed as g/Kg dry weight (dw).

Figure S1. HPLC chromatograms of compound 1 (A) and the F. sambucinum-SSF

extract (B).

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Figure S2 The standard curve for quantifying sambacide (1).

Table S1 LOD, LOQ, and linearity range for quantitation of sambacide (1)

LOD LOQ Linearity range

0.0015 μg 0.02 μg 0.02-20 μg

Table S2 Precision for quantifying sambacide (1)

A1 A2 A3 A4 A5 A SD%

1093249 1094797 1091376 1088089 1077706 1089043 0.63

Table S3 Recoveries of sambacide (1)

Sample (μg) Added (μg) Determined (μg) Recovery % SD%

3.29

2 5.36 103.6 2.52

4 7.19 97.5 4.13

6 9.47 102.9 1.62

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UV-Vis and NMR analysis

UV-Vis spectra of samples (0.10 and 0.05 mg/mL in methanol for F. sambucinum

SSF extract and sambacide, respectively) were recorded using a SHIMADZU-2550 PC

spectrophotometer (SHIMADZU, Tokyo, Japan) with a range of 200-800 nm.

NMR spectra employed to estimate the chemical constituents of extracts were

obtained using a Bruker Avance 400 MHz spectrometer (Bruker, Karlsruhe, Germany)

with the 5 mm NMR tube (TMS as an internal standard). All samples were dissolved in

DMSO-d6–D2O (1:1, v/v). 1H NMR spectra were acquired using the Bruker zg30 pulse

program with following settings: relaxation delay (D1) = 1 s, size of fid (TD) = 65,536,

number of scans (NS) = 32, spectral width (SW) = 16.4990 ppm, acquisition time (AQ)

= 4.96 s, requested probe temperature (TE) = 298.0 K. 13

C NMR, DEPT135, and

DEPT90 spectra were acquired using the Bruker zgpg30, dept135, and dept90 pulse

program, respectively, with following settings: D1 = 2 s, TD = 65,536, NS = 2048 (1024

and 512 for DEPT135 and DEPT90 measurements, respectively), SW = 241.9859 ppm,

AQ = 1.35 s, TE = 298.0 K. Data acquisition and processing were done with Bruker

Topspin 2.1.

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Figure S3 A) 1H NMR spectrum for crude extract of F. sambucinum-SSF; B)

13C and

DEPT NMR spectra for crude extract of F. sambucinum-SSF; C) UV-Vis spectra

(200-400 nm)of crude extract of F. sambucinum-SSF (0.10 mg/mL) and sambacide (1)

(0.05 mg/mL).

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Calculation

The theoretical calculation of compound 1 were performed using Gaussian

Program by Yunnan Electronic Computing Center. Two geometries of Compound 1

were previously optimized by Density Functional Theory (DFT) method at the

B3LYP/6-31G(d,p) level (Ding et al., 2009) and excitation energies and rotational

strengths were calculated using time-dependent Density Functional Theory (TDDFT) at

the B3LYP/6-31 G(d,p) level in methanol with PCM model (Liao et al., 2015). The

ECD spectrum is simulated from electronic excitation energies and velocity rotational

strengths.

Spectroscopic data

Sambacide (1): white amorphous powder; m.p.191-194 °C; ESIMS m/z 577 [M-H]-;

HRESIMS m/z 577.3222 [M-H]- (Calcd. for C32H49O7S 577.3204) ;UV λ

CH3OH

max nm (ε) 241

(8890, sh), 247 (9664), 258 (6659, sh); FT-IR: νKBr

max (cm-1

) 3426 (O–H), 2961~2877

(−C–H), 1717 (C=O), 1639 (C=CH–CH=C),1464, 1375, 1245(R–OSO2OH), 1047

(C–O–C) ; 1H and

13C NMR spectroscopic data, see Table 1.

Reference

Ding, S., Jia, L., Durandin, A., Crean, C., Kolbanovskiy, A., Shafirovich, V., Broyde, S.,

Geacintov, N.E. 2009. Absolute Configurations of Spiroiminodihydantoin and

Allantoin Stereoisomers: Comparison of Computed and Measured Electronic

Circular Dichroism Spectra. Chem. Res. Toxicol., 22, 1189-1193.

Liao, Y., Liu, X., Yang, J., Lao, Y.Z., Yang, X.W., Li, X.N., Zhang, J.J., Ding, Z., J., Xu,

H.X., Xu, G. 2015. Hypersubones A and B, New Polycyclic Acylphloroglucinols

with Intriguing Adamantane Type Cores from Hypericum subsessile. Org. Lett.,

17, 1172-1175.

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Figure S4 A) Key 1H-

1H COSY ( ), HMBC ( ) and NOESY ( ) correlations

of 1b; B) The Newman projections of the C-17–C-15 bond illustrating the assigned

C-20 relative configuration ( NOE correlations).

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Table S4 The MIC values (μg/mL) of 1.

Sample S. aureus B. subtilis E. coli C. albicans

sambacide (1) 16 32 16 64

Kanamycina a

8 1 8

Nystatin a

2

a Positive controls.

Figures for optimizing SSF conditions.

potato rice rice bran wheat bran cornmeal

0

5

10

15

20

Yie

ld (

g/K

g d

w)

Substrate

Figure S5. Yields of sambacide (1) from the SSFs using different substrates;

10

AT 20 28 370

5

10

15

20

Con

ten

t (g

/Kg d

w)

Temperature (oC)

Figure S6. Yields of sambacide (1) from the SSFs at different temperature (AT, ambient

temperature).

0 10 20 30 400

5

10

15

20

25

Yie

ld (

g/K

g d

w)

Time (days)

Figure S7. Yields of sambacide (1) from the SSFs undergone different fermentation

time.

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Supplementary spectra for sambacide (1)

Figure S8 HRESIMS spectrum for sambacide (1)

Sample : TMB101 Frequency Range : 399.246 - 3996.32 Measured on : 02/02/2016

Technique : KBr压片

Customer : 160202IR5

Resolution : 4

Zerofilling : 2

Instrument : Tensor27

Acquisition : Double Sided,Forward-Backward

Sample Scans : 16

2961.0

62930.6

52876.8

7

1731.5

51717.7

0

1638.8

0

1464.3

2

1375.1

7

1244.6

71209.6

8

1124.9

9

1061.4

01047.7

11030.9

9

967.8

1933.1

5

643.6

4

588.3

6569.7

4

500100015002000250030003500

Wavenumber cm-1

30

40

50

60

70

80

90

Tra

nsm

itta

nce [

%]

Figure S9 FT-IR spectrum for sambacide (1)

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Figure S10 1H NMR spectrum (400 MHz) for sambacide (1) in pyridine-d5.

Figure S11 13

C NMR and DEPT spectra (100 MHz) for sambacide (1) in pyridine-d5.

13

Figure S12 1H-

1H COSY spectrum (400 MHz) for sambacide (1) in pyridine-d5.

Figure S13 HSQC spectrum (400 MHz) for sambacide (1) in pyridine-d5.

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Figure S14 HMBC spectrum (400 MHz) for sambacide (1) in pyridine-d5.

Figure S15 NOESY spectrum (400 MHz) for sambacide (1) in pyridine-d5.