Supplementary Information for
Tolypoalbin, a new tetramic acid from Tolypocladium album TAMA 479
Takao Fukuda, Yuri Sudoh, Yuki Tsuchiya, Toru Okuda, Nobuyasu Matsuura, Atsuko
Motojima, Tsutomu Oikawa, Yasuhiro Igarashi*,
Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural
University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan. Hyphagenesis Inc.,
6-2-37 Tamagawa Gakuen, Machida, Tokyo 194-0041, Japan. Botanical Gardens, The
University of Tokyo, 3-7-1 Hakusan, Bunkyo, Tokyo 112-0001, Japan. Department of
Life Science, Faculty of Science, Okayama University of Science, 1-1-1 Tsushima-naka,
Kita-ku, Okayama 700-0005, Japan. Department of Nutritional Biochemistry, School of
Nutrition and Dietetics, Kanagawa University of Human Services, 1-10-1 Heisei-cho,
Yokosuka, Kanagawa 238-8522, Japan
Table of Contents
Experimental procedure
Figure S1. Marfey’s analysis of 1
Figure S2. Adipocyte differentiation-inducing activity in ST-13 cells
Figure S3. 1H NMR spectrum of 1 (500 MHz, CDCl3)
Figure S4. 1H−1H homonuclear decoupling experiment for 1 (500 MHz, CDCl3)
Figure S5. 1H NMR spectrum of 1 (500 MHz, DMSO-d6)
Figure S6. 13C NMR spectrum of 1 (125 MHz, CDCl3)
Figure S7. 13C NMR spectrum of 1 (125 MHz, DMSO-d6)
Figure S8. COSY spectrum of 1 (500 MHz, CDCl3)
Figure S9. HSQC spectrum of 1 (500 MHz, CDCl3)
Figure S10. HMBC spectrum of 1 (500 MHz, CDCl3)
Figure S11. HMBC spectrum of 1 (500 MHz, DMSO-d6)
Figure S12. 1H NMR spectrum of 2 (500 MHz, CDCl3)
Figure S13. 1H−1H homonuclear decoupling experiment for 2 (500 MHz, CDCl3)
Figure S14. 13C NMR spectrum of 2 (125 MHz, CDCl3)
Figure S15. COSY spectrum of 2 (500 MHz, CDCl3)
Figure S16. HSQC spectrum of 2 (500 MHz, CDCl3)
Figure S17. HMBC spectrum of 2 (500 MHz, CDCl3)
Experimental procedure
General experimental procedure
Optical rotations were measured using a JASCO P-1030 polarimeter (JASCO Co.,
Tokyo, Japan). UV spectra were recorded on a Hitachi U-3210 spectrophotometer
(Hitachi, Tokyo, Japan). IR spectra were measured on a Perkin-Elmer Spectrum 100
(Perkin-Elmer Inc., Fremont, CA, USA). NMR spectra were obtained on a Bruker
AVANCE 500 spectrometer or Bruker AVANCE 400 spectrometer (Bruker, Rheinstetten,
Germany). The 1H and 13C chemical shifts were referenced to the solvent signals (δH =
7.26 and δC = 77.0 in CDCl3; δH = 2.50 and δC = 39.5 in DMSO-d6). HR-ESITOFMS
and LC-MS were measured on a Bruker micrOTOF spectrometer. HPLC analyses were
carried out on an Agilent 1100 (Agilent Technologies, Santa Clara, CA, USA) using a
CAPCELL PAK C18 MGII S5 (Shiseido Co., Ltd., 4.6 × 250 mm).
Microorganism
The strain TAMA 479 was isolated from a soil sample collected at Machida, Tokyo.
The soil was treated by the washing filtration method, and then inoculated on LCA
medium (glucose 0.01%, KH2PO4 0.01%, MgSO4･7H2O 0.02%, KCl 0.02%, NaNO3
0.2%, yeast extract (Difco laboratories) 0.02%, agar 2%) supplemented with 0.05%
chloramphenicol, 0.0002% cyclosporin and 0.01% tetracycline. This strain was isolated
after incubation at room temperature and identified as Chaunopycnis alba W. Gams.
(Gams 1980) on the basis of morphological and cultural characteristics and 95-99%
similarity of internal transcribed spacer (ITS) sequence (545 nucreotides; GenBank
accession number AB987765) to Chaunopycnis alba (accession numbers JN628073,
AF389192, JQ272438, HQ608099, HQ608068, HQ608141, KF381088, AF389191, and
JN903552). This species was reclassified into the genus Tolypocladium in 2014 and was
renamed T. album. Therefore, this strain was identified as T. album.
Marfey’s Analysis
A solution of 1 (0.5 mg) in ethanolic 1 M HCl (200 μl) was heated at 110 °C for 15
mitutes and evaporated to dryness. The residue was hydrolyzed in 0.5 M KOH (200 μl)
at room temparature for 2 h, followed by neutralization with 2 M HCl and lyophilization.
The hydrolysate and 1-fluoro-2,4-dinitrophenyl-5-L-leucinamide (1% L-FDLA solution
in acetone, 50 μl) were dissolved in 0.1 M NaHCO3 (100 μl). Separately, L-Tyr was
dissolved in 0.1 M NaHCO3 (100 μl) and L- or D- FDLA solution. Each mixture was
heated at 55 °C for 30 min in a glass tube. After cooling, 0.2 M HCl (50 μl) was added
to each tube and then diluted with 50% MeOH/0.2% HCOOH (1:1, 100 μl). The FDLA
derivatives were analyzed by LC-MS using a Cadenza CD-C18 reverse-phase column (3
μm, 4.6 × 75 mm, 0.5 ml min-1) at 40 °C with a gradient of MeCN–10 mM NH4HCO3
(MeCN concentration: 25% for 0-10 min, 25-45% for 10-45 min). FDLA derivatives
were detected by UV absorption at 340 nm and by selective ion monitoring in the
negative ionization mode. The retention times for authentic L- and D-Tyr were 9.4 and
19.2 min, respectively. The FDLA-derivative from 1 was detected at 9.4 min.
Biological assays
Adipocyte differentiation assay was performed according to the procedure previously
described.1 Rosiglitazone, a thiazolidinedione-class antidiabetic agent, was used as a
positive control in the adipocyte differentiation assay. It enhanced differentiation in 80%
murine ST-13 preadipocyte cells at 0.02 μM. PPARγ agonist assay of 1 and troglitazone
was performed in a reporter gene assay (Promega, Madison, WI) according to the
manufacture’s instruction. Antimicrobial assy was performed with Escherichia coli
NIH-JC2, Micrococcus luteus ATCC9343 and Candida albicans IFO1594 according to
the procedures previously described.2
References and notes
1) kunimasa, K., Kuranuki, S., Matsuura, N., Iwasaki, N., Ikeda, M., Ito, A., Sashida,
Y., Mimaki, Y., Yano, M., Sato, M., Igarashi, Y., Oikawa, T. Identification of nobiletin, a
polymethoxyflavonoid, as an enhancer of adiponectin secretion. Bioorg. Med. Chem.
Lett. 19, 2062-2064 (2009)
2) Igarashi, Y., Ogura, H., Furihata, K., Oku, N., Indananda, C., Thamchaipenet, A.
Maklamicin, an antibacterial polyketide from an endophytic Micromonospora sp. J. Nat.
Prod. 74, 670-674 (2011)
Figure S1. Marfey’s analysis of 1
A) FDLA derivatized hydrolysate of 1
2 : 1
a)
EIC (m/z 474)
b)
L-Tyr
D -Tyr
EIC (m/z 474)
0
10
20
30
40
Time [min]
B) FDLA derivatives prepared from hydrolysate of alcohorized 1
a)
EIC (m/z 474)
b)
L-Tyr
D -Tyr
EIC (m/z 474)
0
10
20
30
40
Time [min]
Gradient system of MeCN-10 mM NH3HCO3 (MeCN concentration: A) 25% for 0-5 min, 25-45% for
5-45 min; B) 25% for 0-10 min, 25-45% for 10-45 min); a) Extracted ion chromatogram (EIC) m/z 474
of FDLA-derivatives of 1; b) EIC m/z 474 for FDLA standards corresponding to L- or D-Tyr.
C) Two-step degradation of tetramic acid
Figure S2. Adipocyte differentiation-inducing activity in ST-13 cells
16
Arbitrary units
12
8
4
0
ROS
(20 nM)
40
20
(ROS: rosiglitazone)
10
tolypoalbin (μM)
5
2.5
Figure S3. 1H NMR spectrum of 1 (500 MHz, CDCl3)
9
8
7
6
5
4
3
2
1
ppm
Figure S4. 1H−1H homonuclear decoupling experiment for 1 (500 MHz, CDCl3)
H-12
H-11
15.0 Hz
15.0 Hz
6.0 Hz
irradiation
ppm
H-12 H-11
CH3-13
ppm
H-12 H-11
7
6
5
4
3
2
1
ppm
Figure S5. 1H NMR spectrum of 1 (500 MHz, DMSO-d6)
9
8
7
6
5
4
3
2
1
ppm
Figure S6. 13C NMR spectrum of 1 (125 MHz, CDCl3)
200
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10
0 ppm
Figure S7. 13C NMR spectrum of 1 (125 MHz, DMSO-d6)
200
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
ppm
Figure S8. COSY spectrum of 1 (500 MHz, CDCl3)
ppm
1
2
3
4
5
6
7
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
ppm
Figure S9. HSQC spectrum of 1 (500 MHz, CDCl3)
ppm
20
40
60
80
100
120
140
160
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
ppm
Figure S10. HMBC spectrum of 1 (500 MHz, CDCl3)
ppm
20
40
60
80
100
120
140
160
180
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
200
ppm
Figure S11. HMBC spectrum of 1 (500 MHz, DMSO-d6)
ppm
20
40
60
80
100
120
140
160
180
10
9
8
7
6
5
4
3
2
1
200
ppm
Figure S12. 1H NMR spectrum of 2 (500 MHz, CDCl3)
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
ppm
Figure S13. 1H−1H homonuclear decoupling experiment for 2 (500 MHz, CDCl3)
H-12
H-11
15.3 Hz
15.3 Hz
6.1 Hz
irradiation
ppm
H-12 H-11
CH3-13
ppm
H-12 H-11
7
6
5
4
3
2
1
ppm
Figure S14. 13C NMR spectrum of 2 (125 MHz, CDCl3)
210
200
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20 ppm
Figure S15. COSY spectrum of 2 (500 MHz, CDCl3)
ppm
1
2
3
4
5
6
7
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
ppm
Figure S16. HSQC spectrum of 2 (500 MHz, CDCl3)
ppm
20
40
60
80
100
120
140
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
ppm
Figure S17. HMBC spectrum of 2 (500 MHz, CDCl3)
ppm
20
40
60
80
100
120
140
160
180
200
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
ppm