Supplementary information
Experimental
General
All NMR spectra were measured with a JEOL ECA-600 spectrometer in CDCl3 (99.8
atom% enriched, ACROS) using TMS as an internal reference. HR ESIFT-MS spectra
were obtained on a ThermoFisher LTQ-Orbitrap XL mass spectrometer.
Optical rotation
and IR spectra were recorded on a JASCO P-1030 polarimeter and a Horiba FT-210
Fourier transform infrared spectrometer, respectively.
Isotope labeled pyruvate, sodium
[2,3-13C2]pyruvate (99 atom% enriched), was purchased from Shoko Co.
Fermentation
The amycolamicin production medium consisted of 0.5% glycerol, 0.5% dextrin,
0.2% soy bean meal, 0.05% yeast extract, 0.2% NaCl, 0.05% MgSO4·7H2O, 0.05%
(NH4)2SO4, 0.1% trace element solution and 0.1% CaCO3.
A slant culture of the
amycolamicin producing organism Amicolatopsis sp. MK575 was inoculated into a
500-mL baffled flask containing a 100 mL production medium.
The culture was grown
at 28 °C for 4 days with shaking at 180 rpm on a rotary shaker. Vegetative cultures
(100mL×10) of production medium with the same composition were inoculated with 2 mL
of the pre-culture.
Two days after inoculation under the same conditions, 1 mL of
filter-sterilized sodium [2,3-13C2]pyruvate solution (100 mg/mL) was added to each flask,
and fermentation continued for two days.
Isolation of amycolamicin
The resulting fermentation broth (1 L) was centrifuged to obtain supernatant.
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The
supernatant was extracted three times with ethyl acetate.
The combined organic layer
was washed with brine, dried over MgSO4, and concentrated in vacuo.
The resulting
crude extract was purified by centrifugal partition chromatography (CPC240, Senshu
Scientific Co.) with tBuOMe:MeCN:0.1M NaHCO3 using an ascending mode.
The
fractions containing amycolamicin were further purified by silica gel column
chromatography (Silica gel 60N, Kanto Chemical Co., 10% MeOH/AcOEt) to give pure
amycolamicin (1.24 mg).
Methanolysis of amycolamicin
The solution of amycolamicin (19.5 mg, 20.7 nmol) in methanol (1 mL) was treated
with 1 mL of 5 % hydrogen chloride methanol solution and stirred at room temperature for
3 h.
The reaction mixture was quenched with a saturated NaHCO3 solution, and the
resulting mixture was extracted three times with ethyl acetate.
The combined organic
layer was washed with brine, dried over MgSO4, and concentrated in vacuo.
The residue
was purified by preparative TLC (PLC Silica gel 60 F254, Merck, 5% MeOH/AcOEt) to
obtain the -anomeric derivative 3 (4.75 mg, 64%) and -anomeric derivative 4 (2.45 mg,
33%) as a colorless syrup respectively.
-isomer (3): [a]D23 = -113 (c 0.24, CHCl3); IR
(neat): 3398, 3215, 2973, 2927, 1624, 1535, 1271, 11630, 1074cm-1; 1H NMR (600 MHz,
CDCl3):  1.30 (3H, d, J = 7.3 Hz, 8-Me), 1.34 (3H, d, J = 6.2 Hz, 6-Me), 1.49 (1H, dd, J
= 9.3, 13.1 Hz, 2-CH), 1.92 (1H, dd, J = 2.1, 13.1 Hz, 2-CH), 2.28 (3H, s, Ar-Me), 2.50
(1H, brs, 4-OH), 3.17 (1H, brt, J = 7.9 Hz, 4-CH), 3.48 (3H, s, 1-OMe), 3.64 (1H, dq, J =
9.3, 6.2, Hz, 5-CH), 4.42 (1H, dq, J = 7.3, 6.2 Hz, 5-CH), 4.69 (1H, dd, J = 2.1, 9.3 Hz,
1-CH) , 5.22 (1H, brs, 3-OH), 6.63 (1H, d, J = 6.2 Hz, amide-NH), 9.5-9.6 (1H, brs,
pyrrole-NH); 13C NMR (150 Hz, CDCl3): 11.4 (q, Ar-Me), 16.1 (q, C-8), 18.0 (q, C-6),
36.3 (t, C-2), 52.6 (d, C-7), 55.6 (q, OMe), 70.4 (d, C-5), 74.2 (s, C-4), 76.2 (d, C-3), 99.7
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(d, C-1), 111.1 (s, Ar), 112.4 (s, Ar), 117.4 (s, Ar), 128.7 (s, Ar), 161.3 (s, C=O); HR
ESIFAB-MS (m/z) calcd for C15H22Cl2N2O5Na: m/z; 403.0798 (M+Na+). Found: m/z;
403.0788.-isomer (4): [a]D23 = +83 (c 0.13, CHCl3); IR (neat): 3389, 3215, 2974, 2931,
1630, 1535, 1271, 1130, 1053cm-1; 1H NMR (600 MHz, CDCl3):  1.27 (3H, d, J = 6.8
Hz), 1.33 (3H, d, J = 6.5 Hz), 1.82 (1H, dd, J = 3.8, 14.4 Hz), 1.99 (1H, dd, J = 1.1, 14.4
Hz), 2.27 (3H, s), 2.30 (1H, brs), 3.24 (1H, brd, J = 9.4 Hz), 3.38 (3H, s), 3.68 (1H, dq, J =
9.4, 6.5, Hz), 4.14 (1H, brs), 4.42 (1H, dq, J = 8.6, 6.8 Hz), 4.84 (1H, brd, J = 3.1 Hz),
6.84 (1H, d, J = 8.6 Hz), 9.74 (1H, brs); 13C NMR (150 Hz, CDCl3): 11.3 (q), 16.3 (q),
17.9 (q), 35.0 (t), 50.6 (d), 55.2 (q), 65.3 (d), 73.4 (s), 74.2 (d), 98.2 (d), 110.3 (s), 110.9
(s), 118.7 (s), 127.6 (s), 159.3 (s); HR ESIFAB-MS (m/z) calcd for C15H22Cl2N2O5Na: m/z;
403.0798 (M+Na+). Found: m/z; 403.0787.
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