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Use of a biosynthetic intermediate to explore the chemical diversity of
pseudo-natural fungal polyketides
Teigo Asai1,*, Kento Tsukada1, Satomi Ise1, Naoki Shirata1, Makoto Hashimoto2, Isao Fujii2
Katsuya Gomi3, Kosuke Nakagawara4, Eiichi N. Kodama4 and Yoshiteru Oshima1,*
1Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-yama, Aoba-ku, Sendai,
Japan. 2School of Pharmacy, Iwate Medical University, Nishitokuta, Yahaba, Iwate, Japan. 3Graduate School of Agricultural Science, Tohoku University, Sendai, Japan. 4Graduate School of
Medicine/Tohoku Medical Megabank Organization, Tohoku University, Sendai, Japan.
*e-mail: [email protected]
Table of Contents Supplementary Figure 1 S2 General Methods S3 Experimental Procedure and Characterization data Microbial Diversification The gene cluster containing pksCH-2 S3 Dimerization to 5 and 6 S4-S8 Supplementary Figure 2 S4 Supplementary Table 1 S6 Supplementary Figure 3 and 4 S7 Supplementary Figure 5 and Table 2 S8 Oligomerization to 7, 9 and 10 S8-S10 Supplementary Table 3 S10 Dimerization to 8 and chaetophenol E S11 Chemical Diversification Supplementary Table 4 S12 Conversion of 2 under acidic condition (11, 12, 19 and 38) S12-S15 Conversion of 2 under oxidative condition (39-44) S16-S18 Conversion of 2 under basic condition (13-18 and 34-37) S19-S25 Synthesis of indole-polyketide hybrid compounds (20-33) S25-S35 Supplementary Figure 6 S28 Evaluation of anti-adenovirus activity S36 NMR Spectra The 1H and 13C NMR spectra (2, 5-44) S37-S114
Use of a biosynthetic intermediate to explore the chemical diversity of pseudo-natural fungal polyketides
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Supplementary Figure 1. Structural diversity of natural and pseudo-natural polyketides based on fungal NR-PKS pathways. a, Several representative polyketides based on a NR-PKS in Chaetomium indicum are listed. The draft genome sequence analysis of C. indicum and feeding experiments with 13C-labeled sodium acetate suggested that 1 is a common intermediate of the chaetophenols and their related compounds (data not shown). b, A schematic illustration of the diversity-oriented semi-synthetic process in this study and whole structures produced in the process. Compounds 5–10 and 11-44 were generated through microbial diversification and chemical diversification, respectively. The new chemical entities were classified as polyketide oligomers (5-19), indole-polyketide hybrids (20–33) and azaphilone type compounds (34–44). Entities 19 and 38 were also categorized as isoquinoline alkaloid-like compounds.
O
O
O
HOHO
OOH
OH
O
H
5 : R = Me7 : R = H8 : R = CH2CHC(CH3)2
OO
O
O
O
OH
H
R1
R1
R2
R2
OHO
OH
O
O
OH
H
O
O
OHO
HO
O
R
R
O
OHO
OH
OH
O
OH
HO
O
H
OH
HO
O
O
O
OHH
N
O
O
HO
HO
O
O
O
OH
H
O
R1R2 R3
HO
OH
O
HO OH
H
O
HO
OH
NH
HO OH
H
O
O
O
O
O
O
N O
O
HO
NHBoc
OH
O
O
O
O
O
OHO OHHO
HO
6 9 11
12 13 : R1 = OH, R2 = Me, R3 = !-H 14 : R1 = Me, R2 = OH, R3 = "-H
15 : R1 = OH, R2 = Me 16 : R1 = Me, R2 = OH
34 35 36 37
O
O
O
OH
H
17 : 5S*,6S*,7S* 18 : 5R*,6R*,7R*
ON
O
HO
O
21
NH
O OH
O
23
32
NH
HO
OH
NH
N
HO
OH
26
NHO
HO OH
33
NH
OH
O
OH
22
NH
O
OH
OH
20 : R = H24 : R = Me
OH
HOR
O
O
O
OH
HO
O
HO
OH
OOH
O
O
O
O
O
OH
HO
H
H
OH
O
O
OHO
HO
O
chaetophenol C
chaetophenol E
chaetophenol D
chaetophenol A
O
O
HO
Ospiroindicumides
1
OH
HOR
O
Aspergillus oryzae
OH
HO
O
O
Enzyme ofheterologous
host
Key equivalent of o-QMs
Multipotent intermediate
N
N OH
OH
25
27 28 29
56
7
Polyketide oligomers
Indole-polyketide hybrid compounds
Azaphylone type compounds
DIversification
Heterologousbiosynthesis
Multipotent intermediates
O
ba
N
O
OMe
OMe
HON
OHC OMe
OMe30
N
HO
O
OMe
OMeI
H
OMe
OMe
O
OH
RN
HO
OH
10
SATKS
ATPT
ACPMT
R
NR-PKSHeterologousexpression
Isolated compounds Feeding
19(Isoquinoline alkaloid-like)
38(Isoquinoline alkaloid-like)
1: R = Me3 : R = HChaetophenol A : R = CH2CHC(CH3)2
2 : R = Me4 : R = HChaetophenol B : R = CH2CHC(CH3)2
NH
O
OMe
OMe
I31
39 40 41 42 43 44
O
OH
HO
chaetophenol B
O
O
O
OO
O
O
O
OO
O
O
O
O
OO
O
O
OO
O
O
O
O
O
O
O
OO
O
O
O
O
O
O
O
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General Methods
Analytical and preparative TLC were performed on silica gel 60 F254 (Merck) and RP-18 F254 (Merck).
Column chromatography was carried out on silica gel 60 (70–230 mesh, Merck). NMR spectra were
recorded on JEOL ECA-600. Chemical shifts for 1H and 13C NMR are given in parts per million (δ) relative
to tetramethylsilane (δH 0.00) and residual solvent signals (δC 77.0) for CDCl3, (δH 3.30) and (δC 49.0) for
CD3OD, (δH 7.19) and (δC 123.5) for py-d5, (δH 2.04) and (δC 29.8) for acetone-d6, (δH 1.93) and (δC 118.2)
for acetonitrile-d3, as internal standards. Mass spectra were measured on JEOL JMS-700 (EI-MS),
JMS-DX303 (FAB-MS), and Thermo Scientific Exactive Mass Spectrometer (ESI-MS). UV spectra were
recorded on a JASCO-V-550 spectrophotometer. IR spectra were recorded on a JASCO-FT/IR-4200
spectrometer. HPLC analysis was performed on HITACHI LaChrom Series HPLC equipped with L-7100
pump, L-7455 Diode Array Detector and D-7000 system manager.
Experimental Procedure and Characterization Data
Microbial Diversification
The gene cluster containing pksCH-2 in Chaetomium indicum
Gene Amino acid Protein homologue (origin) Identity/Similarity (%) orf1 (pksCH-2)
2655 pkeA/AN7903, polyketide synthase (Aspergillus nidulans FGSC A4)
45/62
orf2 494 Cytochrome P450 (Aspergillus flavus NRRL3357) 47/65 orf3 310 NmrA family transcriptional regulator
(Metarhizium acridum CQMa 102) 47/64
orf4 497 Aldehyde dehydrogenase (Aureobasidium melanogenum CBS 110374)
43/61
orf5 430 Tryptophan dimethylallyltransferase family protein (Metarhizium robertsii)
27/41
orf6 201 Hypothetical protein (Zymoseptoria tritici IPO323) 32/49 orf7 473 FAD dependent oxidoreductase family protein
(Metarhizium acridum CQMa 102) 36/54
orf8 409 O-Methyltransferase (Aspergillus fumigatus Af293)
29/47
orf9 575 RadR putative transcriptional regulator (Beauveria bassiana ARSEF 2860)
45/64
orf10 267 Citrinin biosynthesis oxydoreductase CtnB (Monascus purpureus)
52/66
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Cultivation of A. oryzae (OE::pksCH-2) and isolation of 2, 5 and 6
A. oryzae (OE::pksCH-2) was cultivated in CDS (Czapek-dox Broth 3.5 g, Polypepton 1.0 g, Soluble
starch 2.0 g dissolved in per 1 L of distilled water), YM (peptone 5.0 g, yeast 3.0 g, malt extract 3.0 g, and
glucose 10.0 g dissolved in per 1 L of distilled water), and potato dextrose PDB (Potato dextrose broth 24.0 g
dissolved in per 1 L of distilled water) liquid medium under shaking conditions (180 rpm) at 30 °C.
Maltose (1 %) was added in every condition to activate amyB promoter. Each culture media were
investigated by HPLC analysis on day 2 and 5 after inoculation. In order to identified 2, 5 and 6, A. oryzae
(OE::pksCH-2) was cultivated in CDS (1.5 L) and PDB (3.6 L) (7 days). Each culture medium was extracted
with ethyl acetate, and the extract was separated by silica gel column chromatography to afford 2 (48.7 mg)
from CDS, and 5 (11.8 mg) and 6 (4.8 mg) from PDB, respectively.
Supplementary Figure 2. HPLC profiles of a each culture media with A. oryzae (OE::pksCH-2): a, CDS; b,
YM; c, PDB. The data points were collected at incubation time of 2 and 5 days. The culture medium was
extracted with EtOAc and analyzed by reversed-phase HPLC (monitored at 215 nm) on a Mightysil RP-18
column (φ 250 mm x 4.6 mm, 1.0 mL/min) with methanol and water (0-5 min: 40:60, 5-20 min: from 40:60
to 80:20, 20-25 min: from 80:20 to 100:0, 25-35 min: 0:100).
pH 4.8 pH 4.3 pH 3.7
a b c
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Compound 1: HREIMS: m/z 222.0875 [M]+ (222.0892 calcd for C12H14O4). 1H NMR (600 MHz, CDCl3) δH
12.81 (s, 8-OH), 9.99 (s, H-10), 4.06 (s, H2-3), 2.25 (s, H3-1), 2.12 (s, H3-11), 2.12 (s, H3-12).
Compound 2: Colorless amorphous; UV (EtOH) λmax nm (log ε) 205 (4.24), 226 (4.36), 286 (4.03); IR
(KBr) νmax (cm–1) 3369, 2916, 2848, 1657, 1577, 1470, 1391, 1321, 1260, 1234, 1183, 1085, 1022;
HREIMS: m/z 206.0921 [M]+ (206.0943 calcd for C12H14O3). 1H NMR (600 MHz, acetone-d6) δH 7.11 (s,
8-OH), 6.98 (s, 6-OH), 5.75 (brs, H-3), 5.03 (s, H2-10), 2.12 (s, H3-11), 2.06 (s, H3-12), 1.87 (s, H3-1); 13C
NMR (150 MHz, acetone-d6) δC 154.9 (C-2), 153.6 (C-6), 148.8 (C-8), 129.4 (C-4), 110.3 (C-7), 109.6 (C-5),
107.2 (C-9), 99.5 (C-3), 64.7 (C-10), 19.8 (C-1), 10.7 (C-12), 9.3 (C-11).
Structure of 1 and key HMBC correlations of 2
Compound 5: Colorless amorphous; UV (EtOH) λmax nm (log ε) 205 (4.22), 283 (3.00); IR (KBr) νmax
(cm–1) 3420, 2964, 2928, 2869, 1717, 1596, 1458, 1270, 1174, 1118, 1067; HRFABMS: m/z 411.1789
[M–H]– (411.1807 calcd for C24H27O6). 1H and 13C NMR data, see Supplementary Table 1.
Compound 6: Colorless amorphous; UV (EtOH) λmax nm (log ε) 206 (4.50), 321 (4.10); IR (KBr) ν
max (cm-1) 3410, 2994, 2922, 1704, 1604, 1460, 1395, 1263, 1206, 1124, 1064; HRFABMS: m/z 413.1965
[M+H]+ (413.1964 calcd for C24H29O6). 1H and 13C NMR data, see Supplementary Table 1.
Key HMBC (red arrow), 1H-1H COSY (blue bold line) and NOE (purple arrow) correlations of 5 and 6
HO
OH
O
2
12
3
456
78 9
10
12
11
O
O
OH
HO
OH
O
1
23
4
56
78 9
10
12
11
1'
2'
3'4' 5'
6'
7'8'9'
10' 11'
12'
O
O
OH
HO
OH
OH
O
O
OHO
HO
O10
1
2 3
45
67
11
89
1'2'
3'
4'5'
12'
7'6'
11'
8'
9'
10'
O
O
O
OHHO
O
6
H
5
12H
HH
H
HH10
13
11'
11 7'
8' 9'
10'10
HO
OH
O
1
O
123
456
7
89 10
12
11
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Supplementary Table 1. 13C (150 MHz) and 1H NMR (600 MHz) data for 5 and 6.a ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ 5 b 6 c -------------------------------------------------------------------------------------------- ----------------------------------------------------------------------------------------- Position 13C 1H (multi, J in Hz) 13C 1H (multi, J in Hz) ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ 1 23.3 1.73 (3H, s) 29.3 2.12 (3H, s) 2 98.6 ------ 205.8 ------ 3 37.5 3.24 (1H, dd, 11.4, 5.5) 44.8 3.70 (1H, d, 17.4, Ha) 3.65 (1H, d, 17.4, Hb) 4 135.1 ------ 111.1 ------ 5 114.4 ------ 116.5 ------ 6 154.2 ------ 151.6 ------ 7 111.8 ------ 110.2 ------ 8 150.7 ------ 148.2 ------ 9 112.0 ------ 130.3 ------ 10 64.1 5.56 (1H, d, 15.4, Ha) 30.8 3.01 (1H, d, 17.3, Ha) 5.43 (1H, d, 15.4, Hb) 2.37 (1H, d, 17.3, Hb) 11 10.7 2.72 (3H, s) 8.4 2.14 (3H, s) 12 11.2 2.52 (3H, s) 12.1 2.10 (3H, s) 1ʹ′ 29.2 2.16 (3H, s) 20.8 2.01 (3H, s) 2ʹ′ 206.1 ------ 161.3 ------ 3ʹ′ 44.8 3.87 (1H, d, 17.3, Ha) 99.1 5.68 (1H, s) 3.80 (1H, d, 17.3, Hb) 4ʹ′ 130.0 ------ 120.2 ------ 5ʹ′ 117.7 ------ 147.8 ------ 6ʹ′ 153.5 ------ 196.6 ------ 7ʹ′ 112.0 ------ 46.3 2.82 (1H, q, 6.6) 8ʹ′ 149.5 ------ 100.4 ------ 9ʹ′ 112.8 ------ 38.4 ------ 10ʹ′ 28.9 3.09 (1H, dd, 15.4, 5.5, Ha) 66.8 4.32 (1H, dd, 11.4, 1.9, Ha) 2.83 (1H, dd, 15.4, 11.4, Hb) 4.08 (1H, d, 11.4, Hb) 11ʹ′ 10.1 2.64 (3H, s) 6.0 1.19 (3H, d, 6.6) 12ʹ′ 13.2 2.42 (3H, s) 10.1 1.83 (3H, s) OH 10.19 (1H, s) 6-OH 4.74 (1H, s) OH 10.00 (1H, s) OH 9.93 (1H, s) ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ a Assignment for all compounds were based on COSY, HMQC, HMBC experiments b in pyridine d5 c in CDCl3
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Supplementary Figure 3. Conversion of 1 and 2 in the culture medium of A. oryzae.
Compounds 1 and 2 were added to the A. oryzae cultivating medium (PDB) with or without boiling. After 24
h of incubation, the culture medium from each condition was extracted with EtOAc and the extract was
analyzed by reversed-phase HPLC (monitored at 215 nm) on a Mightysil RP-18 column (φ 250 mm x 4.6
mm, 1.0 mL/min) with methanol and water (0-5 min: 40:60, 5-20 min: from 40:60 to 80:20, 20-25 min: from
80:20 to 100:0, 25-35 min: 0:100): a, Both 1 and 2 were converted to dimers 5 and 6 in the PDB culture on
day 3 after inoculation of A. oryzae (wild type); b, 1 was not changed in the boiled culture medium. On the
other hands, 2 was dimerized to 5 and 6 in the same condition.
Supplementary Figure 4. Putative formation of polyketide dimers from isochromene precursors through
non-enzymatic dimerization in the culture media of Cordyceps indigotica and Chaetomium indicum.
O
HO
OH O
1
O
HO
OH
2
OHO
OH
O
O
OH
H
O
O
OHO
HO
O
5 6A. oryzaein PDB
0 5 10 15 20 25 (min)
0
1.0
2.0
AU (215 nm)
1
2
5
6
0 5 10 15 20 25 (min)
0
1.0
2.0
AU (215 nm)
2
5
6
1 + A. oryzae 2 + A. oryzae
a
b
OHO
OH
ORO
O
OH
ORH
O
O
OHO
HO
O
Indigotide C : R = 4-O-b-D-glucose
OHO
OH
ORO
O
OH
ORH
Indigotide D : R = 4-O-b-D-glucose
Chaetophenol E
O
OHRO
HO
Indigotide A : R = 4-O-b-D-glucose
O
HO
OH
Chaetophenol B
Cordyceps indigotica
Chaetomium indicum
O
HO
OH O
1
O
HO
OH
2
OHO
OH
O
O
OH
H
O
O
OHO
HO
O
5 6 Boiled A. oryzaeculture medium
XXO
0 5 10 15 20 25 (min)
0
1.0
AU (215 nm)1
2.0
0 5 10 15 20 25 (min)
0
1.0
AU (215 nm)
2.0
2
5
6
1 + A. oryzae (Δ) 2 + A. oryzae (Δ)
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Supplementary Figure 5. Confirmation of reversibility of dimerization to 5 and 6.
Dimmers 5 and 6 were incubated in the PDB culture medium (pH 3.5), respectively. After 24 h of incubation,
each culture medium was extracted with EtOAc and the analyzed reversed-phase HPLC (monitored at 215
nm) on a Mightysil RP-18 column (φ 250 mm x 4.6 mm, 1.0 mL/min) with methanol and water (0-5 min:
40:60, 5-20 min: from 40:60 to 80:20, 20-25 min: from 80:20 to 100:0, 25-35 min: 0:100): a, 5 was remained
without change in the culture medium.; b, 6 provided 2 and 5 in the culture medium, indicating that 6 was
converted to 2 through retro reaction, followed by dimerization of 2 yielded 5.
Supplementary Table 2. Conversion ration from 2 to 5 and 6, and residual ration of 2 (%).
The conversion ration from 2 to 5 and 6 under various pH conditions was investigated. The PDB culture
medium on day 3 after inoculation of A. oryzae (wild type) was filtrated. After pH value of the solution was
adjusted to 2.0, 3.0, 4.0, 5.0 and 6.0 using 1N HCl, 1 mg of 2 was fed into each medium (50 mL) and
incubated for 24 h under shaking condition (30 oC, 150 rpm). The reaction mixture was extracted with
EtOAc and analyzed by HPLC (monitored at 215 nm). The results summarized in Supplementary Table 2.
Conversion ration was calculated from peak area in HPLC chromatogram by compared to their authentic
samples.
Heterologous expression of tspks2 and cultivation of the transformant
Previously we constructed expression plasmid vector by introduction of tspks2 into fungal expression
plasmid pTAex3 possessing the α-amylase promoter (amyB) of A. oryzae. The constructed expression
plasmid pTA-tpks2 containing argB gene as selection marker was subsequently transformed into A. oryzae
M-2-3 by protoplast-PEG method for overexpression studies. Transformants selected on minimal medium. A.
oryzae (OE::tspks2) was cultivated in PDB medium (3.0 L) (30 oC, 180 rpm) for 6 days. The culture medium
(235.0 mg) was extracted by EtOAc and the extract was separated by silica gel column chromatography to
afford 4 (known, 12.2 mg), 7 (4.2 mg), 9 (3.2 mg) and 10 (2.2 mg) from PDB, respectively.
pH 2.0 3.0 4.0 5.0 6.0
5
6
2
55.4
0
0 0
0
53.9 18.5
28.1
13.7 36.2
7.3
0 0
0
17.8
5
0 5 10 15 20 25 (min)0
1.0
AU (215 nm)
2.0
0 5 10 15 20 25 (min)0
1.0
AU (215 nm)
2.0
2
5 6
a b
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Compound 7: Colorless amorphous; UV (EtOH) λmax nm (log ε) 212 (4.63), 283 (3.64); HRESIMS: m/z
383.1502 [M–H]– (383.1489 calcd for C22H23O6); 1H NMR (600 MHz, CDCl3 (10% CD3OD)) δH 6.25 (s,
H-5ʹ′), 6.16 (s, H-5), 4.93 (d, 14.7 Hz, Ha-10), 4.81 (d, 14.7 Hz, Hb-10), 3.60 (d, 15.1 Hz, Ha-3ʹ′), 3.46 (d,
15.1 Hz, Hb-3ʹ′), 3.02 (dd, 5.1, 4.4 Hz, H-3), 2.98 (dd, 16.1, 4.4 Hz, Ha-10ʹ′), 2.86 (dd, 16.1, 5.1 Hz, Hb-10ʹ′),
2.06 (s, H3-11ʹ′), 2.03 (s, H3-11), 2.02 (s, H3-1ʹ′), 1.60 (s, H3-1); 13C NMR (150 MHz, CDCl3 (10% CD3OD))
δC 210.4 (C-2ʹ′), 154.1 (C-6), 153.5 (C-6ʹ′), 152.7 (C-8ʹ′), 150.2 (C-8), 132.3 (C-4), 129.5 (C-4ʹ′), 112.0 (C-9),
111.2 (C-7ʹ′), 110.5 (C-9ʹ′), 109.9 (C-5ʹ′), 108.3 (C-7) 104.3 (C-5), 98.5 (C-2), 60.2 (C-10), 49.4 (C-3ʹ′), 36.8
(C-3), 28.6 (C-1ʹ′), 26.1 (C-10ʹ′), 25.3 (C-1), 8.0 (C-11ʹ′), 7.6 (C-11).
Structure of 7
Compound 9: Colorless amorphous; UV (EtOH) λmax nm (log ε) 224 (4.89), 285 (4.24); HRESIMS: m/z
575.2285 [M–H]– (575.2276 calcd for C33H35O9); 1H and 13C NMR data, see Supplementary Table 3.
Compound 10: Colorless amorphous; UV (EtOH) λmax nm (log ε) 210 (4.81), 331 (4.25); HRESIMS: m/z
575.2284 [M–H]– (575.2276 calcd for C33H35O9); 1H and 13C NMR data, see Supplementary Table 3.
Key HMBC (red arrow), 1H-1H COSY (blue bold line) and NOE (purple arrow) correlations of 9 and 10
OHO
OH
O
O
OH
1
23
4
5
67
89
1011
1'
2'
3'4'
5'
6'
7'8'9'
10' 11'
7
H
O
O
O
HOHO HO
O
OH
O
O
OHO
OH
OH
O
OH
HO
O
9 10
O
OHO
OH
OH
O
OH
HO
O
H
O
O
O
HOHO
O
H1
24
5
67 8 911
3
1'
2'
3' 4'5'
6'
7'8'9'
10'11'
1''
2''
3''4''
5''
6''
7''11''
8''9''
10'' 10
1
2 3
456
7
11
89
1'2'
3'4'
5'6'7'8'
9'
10'
11'
1''
2''3''
4'' 5''
6''7''
11''
8''9''
10''
13
H9'
10'
11'
11HHH
10
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Supplementary Table 3. 13C (150 MHz) and 1H NMR (600 MHz) data for 9 and 10.a ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ 9 b 10 b -------------------------------------------------------------------------------------------- ----------------------------------------------------------------------------------------- Position 13C 1H (multi, J in Hz) 13C 1H (multi, J in Hz) ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ 1 22.6 1.48 (3H, s) 29.4 2.17 (3H, s) 2 98.1 ------ 208.7 ------ 3 36.5 3.03 (1H, dd, 11.7, 2.7) 47.7 3.69 (1H, d, 16.9, Ha) 3.48 (1H, d, 16.9, Hb) 4 135.2 ------ 129.5 ------ 5 115.1 ------ 110.1 6.04 (1H, s) 6 152.2 ------ 153.1 ------ 7 110.4 ------ 109.9 ------ 8 149.5 ------ 150.0 ------ 9 111.0 ------ 111.1 ------ 10 62.8 4.98 (1H, d, 15.8, Ha) 26.2 3.03 (1H, d, 15.8, Ha) 4.87 (1H, d, 15.8, Hb) 2.25 (1H, d, 15.8, Hb) 11 8.2 2.10 (3H, s) 8.3 1.90 (3H, s) 1ʹ′ 28.9 2.03 (3H, s) 21.0 2.07 (3H, s) 2ʹ′ 208.6 ------ 165.3 ------ 3ʹ′ 47.5 3.39 (2H, s) 98.9 5.92 (1H, s) 4ʹ′ 129.2 ------ 145.1 ------ 5ʹ′ 109.8 6.22 (1H, s) 122.4 ------ 6ʹ′ 153.4 ------ 201.9 ------ 7ʹ′ 111.7 ------ 49.8 ------ 8ʹ′ 150.6 ------ 98.7 ------ 9ʹ′ 111.8 ------ 39.9 3.25 (1H, dd, 12.8, 4.7) 10ʹ′ 27.4 2.36 (1H, dd, 15.7, 2.7, Ha) 66.1 4.92 (1H, dd, 10.6, 4.7, Ha) 2.27 (1H, dd, 15.7, 11.7, Hb) 4.42 (1H, dd, 12.8, 10.6, Hb) 11ʹ′ 8.3 2.11 (3H, s) 21.6 1.22 (3H, d, 7.0) 1ʹ′ʹ′ 28.8 2.02 (3H, s) 29.1 2.04 (3H, s) 2ʹ′ʹ′ 208.9 ------ 208.2 ------ 3ʹ′ʹ′ 48.5 3.69 (1H, d, 16.1, Ha) 48.4 3.54 (1H, d, 16.5, Ha) 3.60 (1H, d, 16.1, Hb) 3.41 (1H, d, 16.5, Hb) 4ʹ′ʹ′ 130.9 ------ 131.0 ------ 5ʹ′ʹ′ 109.8 6.15 (1H, s) 109.8 6.07 (1H, s) 6ʹ′ʹ′ 153.8 ------ 154.0 ------ 7ʹ′ʹ′ 111.1 ------ 112.3 ------ 8ʹ′ʹ′ 154.2 ------ 154.2 ------ 9ʹ′ʹ′ 116.4 ------ 117.5 ------ 10ʹ′ʹ′ 22.9 3.91 (1H, d, 16.1, Ha) 22.4 3.39 (1H, d, 17.3, Ha) 3.82 (1H, d, 16.1, Hb) 3.35 (1H, d, 17.3, Hb) 11ʹ′ʹ′ 8.0 1.99 (3H, s) 8.7 2.05 (3H, s) ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ a Assignment for all compounds were based on COSY, HMQC, HMBC experiments. b in CDCl3 (10% CD3OD)
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Conversion of chaetophenol A to 8 and chaetophenol E
A. oryzae was cultivated in PDB medium (60 mL) for 3days, and then 0.6 mg of chaetophenol A was
added to the culture medium under shaking condition (30 oC, 180 rpm). After 24 h of incubation, the culture
medium was extracted with EtOAc and the extract was analyzed by reversed-phase HPLC (monitored at 215
nm) on a Mightysil RP-18 column (φ 250 mm x 4.6 mm, 1.0 mL/min) with acetonitrile and water (0-5 min:
20:80, 5-20 min: from 20:80 to 0:100, 20-30 min: 0:100). The conversion ratios of chaetophenol B (23.5%),
8 (10.7%) and chaetophenol E (14.8%) from chaetophenol A were calculated by peak area in the
chromatogram by comparing with the authentic samples. To characterize 8, the conversion experiment was
scaled up. A. oryzae was cultivated in PDB medium (150 mL x 4) for 3 days, and then 5 mg of isolated
chaetophenol A was added to the each culture medium and stirred at 30 oC (180 rpm) for 24 hours. The
culture medium was extracted with EtOAc and the extract was separated by silica gel column
chromatography to afford 8 (1.0 mg).
HPLC chromatogram of the conversion experiment of chaetophenol A in A. oryzae culture medium
and the structure of 8
Compound 8: Colorless amorphous; UV (EtOH) λmax nm (log ε) 212 (4.75), 284 (3.69); HRESIMS: m/z
519.2761 [M–H]‒ (519.2747 calcd for C32H39O6); 1H NMR (600 MHz, CDCl3) δH 5.27 (s, 6-OH), 5.08 (m,
H-13), 5.03 (m, H-13ʹ′), 5.02 (d, 15.8 Hz, Ha-10), 4.90 (d, 15.8 Hz, Hb-10), 4.61 (s, 8-OH), 3.67 (d, 17.6 Hz,
Ha-3ʹ′), 3.61 (d, 17.6 Hz, Hb-3ʹ′), 3.29 (m, H2-12), 3.24 (m, H2-12ʹ′), 2.99 (dd, 11.8, 5.5 Hz, H-3), 2.58 (dd,
15.8, 5.5 Hz, Ha-10ʹ′), 2.48 (dd, 15.8, 11.8 Hz, Hb-10ʹ′), 2.17 (s, H3-11ʹ′), 2.12 (s, H3-11), 2.09 (s, H3-1ʹ′), 1.84
(s, H3-15), 1.81 (s, H3-15ʹ′), 1.76 (s, H3-16), 1.70 (s, H3-16ʹ′), 1.56 (s, H3-1); 13C NMR (150 MHz, CDCl3) δC
206.3 (C-2ʹ′), 152.4 (C-6), 151.9 (C-6ʹ′), 148.8 (C-8ʹ′), 148.7 (C-8), 135.8 (C-14), 134.7 (C-4), 133.9 (C-14ʹ′),
128.4 (C-4ʹ′), 122.6 (C-13ʹ′), 122.1 (C-13), 118.9 (C-5ʹ′), 116.2 (C-5), 112.3 (C-9ʹ′), 111.2 (C-7ʹ′), 110.7 (C-9),
108.6 (C-7), 98.0 (C-2), 62.8 (C-10), 44.3 (C-3ʹ′), 36.8 (C-3), 29.3 (C-1ʹ′), 29.1 (C-10ʹ′), 26.2 (C-12ʹ′), 25.8
(C-15), 25.7 (C-15ʹ′), 24.4 (C-12), 22.7 (C-1), 18.1 (C-16), 18.0 (C-16ʹ′), 8.5 (C-11ʹ′), 7.9 (C-11).
0 5 10 15 20 25 (min)
0
1.0
2.0
AU (215 nm)
Chaetophenol E
Chaetophenol B
8
Chaetophenol AOHO
OH
O
O
OH
1
23
4
5
67
89
10
12
11
1'
2'
3'4' 5'
6'
7'8'9'
10' 11'
12'
13
1415
16
13'14'
15' 16'
8
H
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Chemical Diversification
Supplementary Table 4. Acid catalyzed dimerization of 2.
Conversion of 2 to 5 and 11
To a solution of 2 (30.0 mg, 0.15 mmol) in CH3CN (1.5 ml), BF3OEt2 (1.8 µl, 0.015 mmol) was added
dropwise at room temperature. After being stirred for 30 min at the same temperature, the reaction was
quenched with water and then extracted with EtOAc. The combined organic extracts were washed with
saturated aqueous solution of brine, dried over Na2SO2, and concentrated. The residue was purified by
silica-gel column chromatography to afford 5 quantitatively. A solution of 5 (10.0 mg, 0.024 mmol) in
CH3CN (2.0 mL) was treated with excessive BF3OEt (45.8 µl, 0.38 mmol) at room temperature for 30
min. The reaction was quenched by the addition of water and the mixture was extracted using EtOAc and
the residue was purified by silica-gel column chromatography and preparative TLC to afford 11 (6.6 mg).
Hypothetical reaction mechanism to 11 from 5
OHO
OH
O
O
OH
H
OH
HO
O
2 5
Lewis acid,r.t.
OHO
OH
O
O
OH
HOH
HO
O
O
O
OHH
OH
O
OH
O
OH
O
HOHHO
O
O
O
OH
H
OH
HO
O
OH
O
OH
115
2
BF3OEt2BF3OEt2
AlCl3Zn(OTf)2
Sc(OTf)3
Ti(OiPr)4
TsOHTFA
Amount (mol %)
10101010101010100
Solvent Time (min) Yield (%)(isolated)Acid
CH2Cl2
CH2Cl2CH2Cl2CH2Cl2CH2Cl2CH2Cl2CH2Cl2
CH3CN10
106010606010
10
quant.
quant.
80quant.
061
quant.
quant.
OHO
OH
O
O
OH
H
OH
HO
O
2 5
BF3OEt2 (10 mol%),CH3CN, r.t., quant.
OH
HO
O
O
O
OHH
11
BF3OEt2 (15 eq),CH3CN, r.t., 66%
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The 1H and 13C NMR spectra of 11 showed the two sets of signals in CDCl3 + CD3OD (10%) (ca. 3:1),
CD3CN (ca. 10:1) and CD3OD (ca. 20:1) due to the C-2 epimers. The structure determination of 11 was
established by using 1D and 2D NMR measured in CD3OD and CD3CN.
Compound 11: Colorless amorphous; UV (EtOH) λmax nm (log ε) 207 (4.59), 251 (4.12), 285 (3.75); IR
(KBr) νmax (cm-1) 3363, 3217, 3001, 2928, 1715, 1661, 1599, 1541, 1457, 1376, 1208, 1160, 1117, 1090,
1072, 1031; HRESIMS: m/z 411.1805 [M–H]– (411.1802 calcd for C24H27O6); 1H NMR (600 MHz, CD3OD)
δH 3.73 (d, 18.4 Hz, Ha-3ʹ′), 3.59 (d, 18.4 Hz, Hb-3ʹ′), 3.39 (brs, H-3), 2.86 (d, 17.9 Hz, Ha-10), 2.39 (d, 17.9
Hz, Hb-10), 2.37 (brd, 14.3 Hz, Ha-10ʹ′), 2.22 (s, H3-1ʹ′), 2.19 (s, H3-12), 2.11 (s, H3-11), 1.80 (brd, 14.3 Hz,
Hb-10ʹ′), 1.78 (s, H3-12ʹ′), 1.78 (s, H3-11ʹ′), 1.32 (s, H3-1); 13C NMR (150 MHz, CD3OD) δC 206.4 (C-2ʹ′),
189.7 (C-6ʹ′), 175.6 (C-8ʹ′), 152.7 (C-8), 151.9 (C-6), 151.3 (C-4ʹ′), 135.0 (C-5ʹ′), 133.5 (C-4), 117.7 (C-7ʹ′),
116.3 (C-5), 115.4 (C-9), 113.0 (C-7), 104.3 (C-2), 44.8 (C-3ʹ′), 41.1 (C-9ʹ′), 40.7 (C-3), 34.8 (C-10), 30.0
(C-1ʹ′), 29.1 (C-10ʹ′), 26.0 (C-1), 12.9 (C-12), 12.6 (C-12ʹ′), 9.4 (C-11), 8.0 (C-11ʹ′). 1H NMR (600 MHz,
CD3CN) δH 5.90 (OH), 5.82 (OH), 4.65 (1-OH), 3.63 (d, 18.6 Hz, Ha-3ʹ′), 3.52 (d, 18.4 Hz, Hb-3ʹ′), 3.36 (t,
3.0 Hz, H-3), 2.79 (d, 18.6 Hz, Ha-10), 2.32 (d, 18.6 Hz, Hb-10), 2.24 (dd, 12.6, 3.0 Hz, Ha-10ʹ′), 2.15 (s,
H3-1ʹ′), 2.13 (s, H3-12), 2.06 (s, H3-11), 1.76 (dd, 12.6, 3.0 Hz, Hb-10ʹ′), 1.73 (s, H3-11ʹ′), 1.72 (s, H3-12ʹ′),
1.27 (s, H3-1); 13C NMR (150 MHz, CD3CN) δC 204.9 (C-2ʹ′), 187.6 (C-6ʹ′), 172.8 (C-8ʹ′), 152.0 (C-8), 151.2
(C-6), 149.6 (C-4ʹ′), 134.6 (C-5ʹ′), 133.5 (C-4), 117.6 (C-7ʹ′), 115.1 (C-5), 114.7 (C-9), 110.5 (C-7), 103.4
(C-2), 44.4 (C-3ʹ′), 40.0 (C-9ʹ′), 39.9 (C-3), 34.7 (C-10), 30.1 (C-1ʹ′), 28.5 (C-10ʹ′), 26.0 (C-1), 12.9 (C-12),
12.5 (C-12ʹ′), 9.4 (C-11), 8.0 (C-11ʹ′).
Key HMBC (red arrow), 1H-1H COSY (blue bold line) and NOE (purple arrow) correlations of 11 in CD3OD
and CD3CN
HO
HO
O
O
O
OHH
11
OH
HO
O
O
O
OH1
23
45
6
7 8 9 1011
12
1' 2'
3'4'
5'
6'
7'8'9'
10'
1
12
11'
12'
HH
HHH H
10'
10
3'
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Conversion of 2 to 5, 11 and 12
Compound 2 (60.0 mg) was treated with AcOH (3 mL) at reflux for 15 min. The resulting mixture was
extracted with EtOAc, washed with saturated aqueous solution of NaHCO3 and brine, dried over Na2SO4,
and concentrated. The residue was purified with column chromatography and preparative TLC to afford
12, along with 5 (>90%) and 11 (1.8 mg). Compound 12 was further purified by preparative HPLC
(monitored at 215 nm) on a Mightysil RP-18 column (φ 250 mm x 4.6 mm, 1.0 mL/min) with CH3CN and
water (0-5 min: 20:80, 5-20 min: from 20:80 to 100:0, 20-30 min: 0:100) to afford 12 (1.2 mg) in pure form.
Addition of 2-bromoethylamine hydrobromide improved the yield of 12.
Compound 12: Colorless amorphous; UV (EtOH) λmax nm (log ε) 208 (4.57), 225 (4.21), 284 (3.54); IR
(KBr) νmax (cm-1) 3419, 3210, 2924, 2853, 1685, 1653, 1594, 1558, 1541, 1507, 1457, 1376, 1354, 1214,
1173, 1107; HRESIMS: m/z 411.1806 [M–H]– (411.1802 calcd for C24H27O6); 1H NMR (600 MHz, CDCl3
(10% CD3OD)) δH 4.80 (d, 15.4 Hz, Ha-10), 4.65 (d, 15.4 Hz, Hb-10), 3.98 (s, H-3ʹ′), 2.87 (d, 11.7 Hz,
Ha-10ʹ′), 2.57 (d, 11.7 Hz, Hb-10ʹ′), 2.57 (s, H-3), 2.18 (s, H3-11ʹ′), 2.13 (s, H3-11), 2.10 (s, H3-12), 1.97 (s,
H3-12ʹ′), 1.87 (s, H3-1ʹ′), 1.46 (s, H3-1); 13C NMR (150 MHz, CDCl3 (10% CD3OD)) δC 211.3 (C-2ʹ′), 151.2
(C-6), 151.0 (C-6ʹ′), 149.3 (C-8ʹ′), 147.8 (C-8), 133.8 (C-4), 130.4 (C-4ʹ′), 116.1 (C-9ʹ′), 114.3 (C-5ʹ′), 113.3
(C-5), 112.1 (C-9), 109.7 (C-7ʹ′), 108.7 (C-7), 74.3 (C-2), 63.2 (C-3ʹ′), 60.2 (C-10), 39.8 (C-3), 29.5 (C-1ʹ′),
27.3 (C-10ʹ′), 23.0 (C-1), 11.2 (C-12ʹ′), 9.8 (C-12), 8.7 (C-11ʹ′), 8.2 (C-11).
Key HMBC (red arrow), 1H-1H COSY (blue bold line) and NOE (purple arrow) correlations of 12
HO
OH
O
HO OH
O
12
HO
OH
O
HO OH
H O
1098
711
65
42
1
3 1'2'3'
4'5'6'7'
11'
12'
8'9'
10'
H
HH
3'3
110
OHO
OH
O
O
OH
H
OH
HO
O
O
O
OHH
5 ( >90%) 11 (3%)
HO
OH
O
HO OH
H
O
12 (2%)
OH
HO
OAcOH,reflux
2
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Conversion of 2 to 5, 19 and 38
The mixture of 2 (60.0 mg, 0.29 mmol) and NH4OAc (200 mg, 2.6 mmol) was treated with AcOH (3 mL) at
reflux for 15 min. The resulting mixture was extracted with EtOAc, washed with saturated aqueous
solution of NaHCO3 and brine, dried over Na2SO4, and concentrated. The residue was purified with
column chromatography and preparative TLC to afford 19 (4.8 mg), 38 (2.4 mg) and 5 (43.2 mg).
Compound 19: Colorless amorphous; UV (EtOH) λmax nm (log ε) 208 (4.60), 225 (4.26), 285 (3.51); IR
(KBr) νmax (cm-1) 3383, 1698, 1592, 1456, 1384, 1227, 1113, 1034; HRESIMS: m/z 412.2108 [M+H]+
(412.2118 calcd for C24H30O5N); 1H NMR (600 MHz, CD3OD) δH 4.28 (s, H-3ʹ′), 4.09 (d, 17.3 Hz, Ha-10),
4.00 (d, 17.3 Hz, Hb-10), 3.48 (t, 9.1 Hz, H-3), 3.38 (dd, 18.0, 9.1 Hz, Ha-10ʹ′), 2.34 (dd, 18.7, 9.1 Hz,
Hb-10ʹ′), 2.22 (s, H3-1ʹ′), 2.21(s, H3-12), 2.16 (s, H3-12ʹ′), 2.11 (s, H3-11ʹ′), 2.11 (s, H3-11), 1.31 (s, H3-1); 13C
NMR (150 MHz, CD3OD) δC 208.7 (C-2ʹ′), 154.2 (C-6), 153.6 (C-6ʹ′), 152.5 (C-8ʹ′), 151.1 (C-8), 135.7 (C-4),
127.6 (C-4ʹ′), 116.6 (C-5ʹ′), 115.7 (C-5), 115.1 (C-9), 113.9 (C-7ʹ′), 111.9 (C-7), 108.5 (C-9), 61.4 (C-3ʹ′), 55.5
(C-2), 39.9 (C-10), 34.7 (C-3), 31.5 (C-1ʹ′), 29.7 (C-10ʹ′), 21.0 (C-1), 12.1 (C-12ʹ′), 11.0 (C-12), 9.5 (C-11ʹ′),
9.2 (C-11).
Compound 38: Colorless amorphous; UV (EtOH) λmax nm (log ε) 206 (4.13), 246 (3.84); IR (KBr) ν
max (cm-1) 3363, 1741, 1698, 1600, 1557, 1457, 1375, 1293, 1083, 1041; HRESIMS: m/z 234.0769 [M–H]–
(234.0761 calcd for C12H12O4N); 1H NMR (600 MHz, CDCl3) δH 9.12 (s, H-10), 7.59 (s, H-3), 2.70 (s, H3-1),
1.83 (s, H3-12), 1.72 (s, H3-11); 13C NMR (150 MHz, CDCl3) δC 208.4 (C-6), 194.8 (C-8), 166.1 (C-2), 151.9
(C-4), 149.4 (C-10), 120.6 (C-9), 120.3 (C-3), 83.3 (C-7), 74.9 (C-5), 30.2 (C-12), 26.7 (C-11), 25.1 (C-1).
Key HMBC (red arrow), 1H-1H COSY (blue bold line) and NOE (purple arrow) correlations of 19 and 38
O
O
NHO
HO
38
O
O
NHO
HO1
2
3
45
12
6711
89
10
11
12
HO
OH
NH
HO OH
O
19
HO
OH
NH
HO OH
H
O10
987
11
65
42
1
3 1'2'3'
4'5'6'7'
11'
12'
8'9'
10'
1'1
3
HH
H
HH10'
OHO
OH
O
O
OH
H
5 (72%)
OH
HO
O
NH4OAcAcOH, reflux
2
N
O
O
HO
HO
38 (7%)
HO
OH
NH
HO OH
H
O
19 (8%)
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Conversion of 2 to 39-44
Compound 2 (100 mg, 0.49 mmol) was treated with silver oxide (Ag2O) (254 mg, 1.10 mmol) in CH2Cl2 (6.0
ml) at room temperature for 1 hour. The resulting solid was removed by filtration and the solution was
concentrated. The residue was purified with column chromatography, preparative TLC and preparative
HPLC to afford 39 (5.0 mg), 40 (7.1 mg), 41 (5.1 mg), 42 (2.6 mg), 43 (1.0 mg) and 44 2.1 mg). The
reaction was suppressed under argon atmosphere, indicating O2 involved in the generation of phenoxy
radical.
Compound 39: Colorless amorphous; UV (EtOH) λmax nm (log ε) 202 (3.82), 234 (3.66), 333 (4.19); IR
(KBr) νmax (cm–1) 2923, 2851, 1723, 1647, 1606, 1571, 1457, 1412, 1380, 1361, 1305, 1285, 1216, 1181,
1065; HRESIMS: m/z 431.1458 [M+Na]+ (431.1465 calcd for C24H24O6Na). 1H NMR (600 MHz, CDCl3) δH
5.79 (s, H-3, H-3ʹ′), 4.66 (d, 13.2 Hz, Ha-10, Ha-10ʹ′), 4.12 (d, 13.2 Hz, Hb-10, Hb-10ʹ′), 2.03 (s, H3-1, H3-1ʹ′),
1.87 (s, H3-12, H3-12ʹ′), 1.36 (s, H3-11, H3-11ʹ′); 13C NMR (150 MHz, CDCl3) δC 201.1 (C-8, C-8ʹ′), 192.6
(C-6, C-6ʹ′), 164.4 (C-2, C-2ʹ′), 143.1 (C-4, C-4ʹ′), 124.0 (C-5, C-5ʹ′), 99.6 (C-3, C-3ʹ′), 68.4 (C-7, C-7ʹ′), 65.9
(C-10, C-10ʹ′), 52.6 (C-9, C-9ʹ′), 21.1 (C-1, C-1ʹ′), 11.1 (C-12, C-12ʹ′), 9.9 (C-11, C-11ʹ′).
Compound 40: Colorless amorphous; UV (EtOH) λmax nm (log ε) 204 (4.41), 240 (4.10), 329 (4.48); IR
(KBr) νmax (cm–1) 2925, 1723, 1647, 1600, 1571, 1450, 1410, 1381, 1360, 1307, 1284, 1213, 1195, 1075;
HRESIMS: m/z 431.1454 [M+Na]+ (431.1465 calcd for C24H24O6Na). 1H NMR (600 MHz, CDCl3) δH 5.79
(s, H-3, H-3ʹ′), 4.66 (d, 13.2 Hz, Ha-10, Ha-10ʹ′), 4.12 (d, 13.2 Hz, Hb-10, Hb-10ʹ′), 2.03 (s, H3-1, H3-1ʹ′), 1.87
(s, H3-12, H3-12ʹ′), 1.36 (s, H3-11, H3-11ʹ′); 13C NMR (150 MHz, CDCl3) δC 201.1 (C-8, C-8ʹ′), 192.6 (C-6,
C-6ʹ′), 164.4 (C-2, C-2ʹ′), 143.1 (C-4, C-4ʹ′), 124.0 (C-5, C-5ʹ′), 99.6 (C-3, C-3ʹ′), 68.4 (C-7, C-7ʹ′), 65.9 (C-10,
C-10ʹ′), 52.6 (C-9, C-9ʹ′), 21.1 (C-1, C-1ʹ′), 11.1 (C-12, C-12ʹ′), 9.9 (C-11, C-11ʹ′).
39 (5%) 40 (7%) 41 (5%) 42 (3%)
43 (1%) 44 (2%)
O
O
O
OO
O
O
O
OO
O
O
O
O
OO
O
O
OO
O
O
O
O
O
O
O
OO
O
O
O
O
O
O
O
OH
HO
O
2
Ag2O,CH2Cl2,
r.t., 1 h
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17
Key HMBC (Red arrow) and NOE (Purple arrow) correlations of 39 and 40
Compound 41: Yellow amorphous; UV (EtOH) λmax nm (log ε) 203 (4.19), 236 (3.93), 339 (4.39); IR
(KBr) νmax (cm–1) 2923, 2852, 1725, 1649, 1612, 1577, 1542, 1447, 1404, 1385, 1358, 1301, 1185, 1071,
1048; HRESIMS: m/z 431.1454 [M+Na]+ (431.1465 calcd for C24H24O6Na). 1H NMR (600 MHz, CDCl3) δH
5.82 (s, H-3), 5.49 (s, H-3ʹ′), 4.96 (d, 12.6 Hz, Ha-10ʹ′), 4.85 (d, 12.6 Hz, Hb-10ʹ′), 4.71 (d, 13.2 Hz, Ha-10),
4.20 (d, 13.2 Hz, Hb-10), 2.05 (s, H3-1), 2.03 (s, H3-1ʹ′), 1.84 (s, H3-12), 1.44 (s, H3-12ʹ′), 1.23 (s, H3-12ʹ′),
1.21 (s, H3-12); 13C NMR (150 MHz, CDCl3) δC 202.2 (C-8), 202.0 (C-6ʹ′), 191.4 (C-6), 189.0 (C-8ʹ′), 167.4
(C-2ʹ′), 163.0 (C-2), 150.0 (C-4ʹ′), 142.4 (C-4), 124.9 (C-5), 115.8 (C-9ʹ′), 100.1 (C-3), 98.8 (C-3ʹ′), 68.4 (C-7),
68.3 (C-7ʹ′), 67.2 (C-10), 64.6 (C-10ʹ′), 57.9 (C-5ʹ′), 53.7 (C-9), 21.0 (C-1), 20.5 (C-1ʹ′), 12.2 (C-12ʹ′), 11.2
(C-12), 10.4 (C-11ʹ′), 10.4 (C-11).
Compound 42: Yellow amorphous; UV (EtOH) λmax nm (log ε) 202 (4.13), 220 (4.10), 273 (3.73), 344
(4.38); IR (KBr) νmax (cm–1) 2992, 2947, 2864, 1726, 1644, 1609, 1574, 1539, 1446 1407, 1382, 1360, 1303,
1183, 1079, 1052; HRESIMS: m/z 431.1455 [M+Na]+ (431.1465 calcd for C24H24O6Na). 1H NMR (600 MHz,
CDCl3) δH 5.78 (s, H-3), 5.31 (s, H-3ʹ′), 4.98 (d, 12.6 Hz, Ha-10ʹ′), 4.83 (d, 12.6 Hz, Hb-10ʹ′), 4.61 (d, 13.2 Hz,
Ha-10), 4.13 (d, 13.2 Hz, Hb-10), 2.02 (s, H3-1), 1.99 (s, H3-1ʹ′), 1.84 (s, H3-12), 1.38 (s, H3-11ʹ′), 1.32 (s,
H3-12), 1.30 (s, H3-12ʹ′); 13C NMR (150 MHz, CDCl3) δC 202.1 (C-8), 202.1 (C-6ʹ′), 192.2 (C-6), 190.0 (C-8ʹ′),
167.4 (C-2ʹ′), 163.9 (C-2), 151.7 (C-4ʹ′), 142.5 (C-4), 124.1 (C-5), 115.0 (C-9ʹ′), 99.7 (C-3ʹ′), 98.5 (C-3), 69.2
(C-7), 68.3 (C-7ʹ′), 66.0 (C-10), 64.5 (C-10ʹ′), 57.5 (C-5ʹ′), 52.6 (C-9), 21.1 (C-1), 20.5 (C-1ʹ′), 13.1 (C-12ʹ′),
11.4 (C-11), 11.1 (C-12), 9.9 (C-11ʹ′).
Key HMBC (Red arrow) and NOE (Purple arrow) correlations of 41 and 42
39 40
OOOO
O
O
O
O
O
O
O
O
12
3
45 6
78910 11
12
1'2'3'
4'5'6'
7' 8' 9' 10'11'
12'
O
O
O
OO
O
O
O
O
O
O
O
3
11'
H
H
H
10
12
3
45 6
78910 11
12
1' 2' 3'4' 5'
6'7'8'9'
10'11'
12'
H
H
H
3
10'
HOO
OO
O
OO
O
O
O
O
O
12
3
45 6
78910 11
12
7'8'9'
11'
3'
1' 2'
4'5'
12' 6'
10'
O
O
OO
O
O
OO
O
O
O
OH
H
H
10
3
12'
3'
12
3
45 6
78910 11
12
7'8' 9'
10'
11'
3'
1'2'
4'
5' 12'6' HH
H
H
3'
11
3
11'
10
41 42
© 2015 Macmillan Publishers Limited. All rights reserved
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18
Compound 43: Yellow amorphous; UV (EtOH) λmax nm (log ε) 205 (4.21), 268 (3.66), 366 (4.24); IR
(KBr) νmax (cm–1) 2981, 2921, 2850, 1734, 1636, 1542, 1525, 1445, 1382, 1297, 1242, 1096, 1048, 1006;
HRESIMS: m/z 431.1456 [M+Na]+ (431.1465 calcd for C24H24O6Na). 1H NMR (600 MHz, CDCl3) δH 5.29
(s, H-3, H-3ʹ′), 4.97 (d, 12.8 Hz, Ha-10, Ha-10ʹ′), 4.78 (d, 12.8 Hz, Hb-10, Hb-10ʹ′), 1.98 (s, H3-1, H3-1ʹ′), 1.34
(s, H3-11, H3-11ʹ′), 1.29 (s, H3-12, H3-12ʹ′); 13C NMR (150 MHz, CDCl3) δC 203.0 (C-6, C-6ʹ′), 189.8 (C-8,
C-8ʹ′), 167.1 (C-2, C-2ʹ′), 151.3 (C-4, C-4ʹ′), 115.0 (C-9, C-9ʹ′), 99.7 (C-3, C-3ʹ′), 69.0 (C-7, C-7ʹ′), 64.5 (C-10,
C-10ʹ′), 57.6 (C-5, C-5ʹ′), 20.5 (C-1, C-1ʹ′), 13.1 (C-12, C-12ʹ′), 11.4 (C-11, C-11ʹ′).
Compound 44: Yellow amorphous; UV (EtOH) λmax nm (log ε) 203 (4.16), 218 (4.08), 275 (3.60), 365
(4.10); IR (KBr) νmax (cm–1) 2990, 2923, 2853, 1726, 1648, 1544, 1460, 1384, 1296, 1244, 1200, 1097, 1052,
1005; HRESIMS: m/z 431.1454 [M+Na]+ (431.1465 calcd for C24H24O6Na). 1H NMR (600 MHz, CDCl3) δH
5.28 (s, H-3, H-3ʹ′), 4.96 (d, 12.8 Hz, Ha-10, Ha-10ʹ′), 4.78 (d, 12.8 Hz, Hb-10, Hb-10ʹ′), 1.99 (s, H3-1, H3-1ʹ′),
1.41 (s, H3-12, H3-12ʹ′), 1.21 (s, H3-11, H3-11ʹ′); 13C NMR (150 MHz, CDCl3) δC 202.9 (C-6, C-6ʹ′), 188.8
(C-8, C-8ʹ′), 166.7 (C-2, C-2ʹ′), 151.0 (C-4, C-4ʹ′), 115.3 (C-9, C-9ʹ′), 99.3 (C-3, C-3ʹ′), 68.2 (C-7, C-7ʹ′), 64.5
(C-10, C-10ʹ′), 58.5 (C-5, C-5ʹ′), 20.5 (C-1, C-1ʹ′), 13.8 (C-12, C-12ʹ′), 10.5 (C-11, C-11ʹ′).
Key HMBC (Red arrow) and NOE (Purple arrow) correlations of 43 and 44
O
O
OO
O
O
O
OO
O
O
O
1'2'3'
4'5'6'7'
8' 9'
10'
11' 12'
1112
1 2
345 6 7
89
10
1112
1 2
345 6 7
89
10
12' 11'6'
1'2'3'
4'5' 7'
8'9'
10'
O
O
O
OO
O
O
O
OO
O
O
HH
11'12'
10
43 44
HH
H
10
11'
3
12'
© 2015 Macmillan Publishers Limited. All rights reserved
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19
Conversion of 2 to 34 and 35
A solution of 2 (20.0 mg, 0.10 mmol) and cesium carbonate (81.5 mg, 0.25 mmol) in CH3CN (2.0 ml) was
stirred at room temperature for 30 min. The resulting mixture was extracted with EtOAc, washed with
saturated aqueous solution of brine, dried over Na2SO4, and concentrated. The residue was purified with
column chromatography to afford a mixture of 34 and 35 (5.8 mg). The structure elucidation was carried
out as a mixture because it was difficult to separate each other. The ratio was calculated by the signal
ration in the 1H NMR spectrum. This reaction was suppressed under argon atmosphere suggesting that
oxygen atoms came from air.
Mixture of 34 and 35: Colorless amorphous; HRESIMS: m/z 261.0720 [M+Na]+ (261.0733 calcd for
C12H14O5Na); 1H NMR (600 MHz, CDCl3) for 34, δH 5.82 (s, H-3), 4.21 (d, 11.4 Hz, Ha-10), 3.97 (d, 11.4
Hz, Hb-10), 2.22 (s, H3-11), 2.09 (s, H3-1), 1.81 (s, H3-12); 13C NMR (150 MHz, CDCl3) for 34. δC 204.7
(C-8), 199.2 (C-6), 164.9 (C-2), 159.4 (C-4), 125.2 (C-5), 96.4 (C-3), 87.7 (C-7), 74.1 (C-9), 70.0 (C-10),
27.1 (C-11), 21.2 (C-1), 7.9 (C-12); 1H NMR (600 MHz, CDCl3) for 35, δH 5.68 (s, H-3), 5.00 (d, 12.8 Hz,
Ha-10), 4.93 (d, 12.8 Hz, Hb-10), 2.02 (s, H3-1), 1.65 (s, H3-11), 1.60 (s, H3-12); 13C NMR (150 MHz,
CDCl3) for 35, δC 208.4 (C-6), 191.7 (C-8), 168.1 (C-2), 153.3 (C-4), 110.9 (C-9), 97.5 (C-3), 87.7 (C-7),
81.7 (C-5), 64.4 (C-10), 27.4 (C-12), 27.3 (C-11), 20.4 (C-1).
Key HMBC (red arrow) and NOE (purple arrow) correlations of 34 and 35.
O
O
O
O
O
OHO OH HO
HO
3534
O
O
O
O
O
OHO OH HO
HO1
2
3
456
711
12
8 9
10
12
3
45
12
6711
89
10
11
10
11
12
O
O
OHO OH
O
O
OHO
HO
OH
HO
O
Cs2CO3,CH3CN, r.t.
2 34 (20%) 35 (5%)
© 2015 Macmillan Publishers Limited. All rights reserved
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20
Conversion of 2 to 36 and 37
A solution of 2 (20.0 mg, 0.10 mmol), 45 (24.1 mg, 0.10 mmol) and cesium carbonate (81.5 mg, 0.20 mmol)
in CH3CN (3.0 ml) was stirred at room temperature for 1 hour. The resulting mixture was extracted with
EtOAc, washed with saturated aqueous solution of brine, dried over Na2SO4, and concentrated. The
residue was purified with column chromatography and preparative TLC to afford 36 (2.5 mg) and 37
(2.5 mg), as well as a mixture of 34 and 35 (5.6 mg).
Hypothetical reaction mechanism to 37
Compound 36: Colorless amorphous; UV (EtOH) λmax nm (log ε) 205 (4.41), 231 (4.08), 335 (4.19); IR
(KBr) νmax (cm-1) 3364, 2979, 2930, 1715, 1604, 1520, 1478, 1449, 1391, 1369, 1294, 1243, 1160, 1050;
HRESIMS: m/z 426.1919 [M–H]‒ (426.1911 calcd for C24H28O6N); 1H NMR (600 MHz, CDCl3) δH 7.57
(brd, 8.0 Hz, H-6ʹ′), 7.40 (s, NH), 7.09 (brt, 8.0 Hz, H-5ʹ′), 6.83 (brt, 8.0 Hz, H-4ʹ′), 6.80 (brd, 8.0 Hz, H-3ʹ′),
5.51 (brs, H-3), 4.56 (d, 12.5 Hz, Ha-10), 3.66 (d, 12.5 Hz, Hb-10), 3.31 (d, 13.0 Hz, Ha-1ʹ′), 3.06 (d, 14.0 Hz,
Hb-1ʹ′), 2.52 (s, 15-OH), 1.99 (s, H3-1), 1.72 (s, H3-12), 1.68 (s, H3-11), 1.55 (s, 3 x H3-t-Bu); 13C NMR (150
MHz, CDCl3) δC 207.4 (C-8), 200.0 (C-6), 162.9 (C-2), 153.9 (NCO, Boc), 141.4 (C-4), 136.1 (C-7ʹ′), 130.4
(C-3ʹ′), 128.2 (C-2ʹ′), 127.4 (C-6ʹ′), 125.3 (C-5), 123.8 (C-6ʹ′), 123.6 (C-4ʹ′), 97.6 (C-3), 80.0 (O-C-Me3, Boc),
70.3 (C-10), 66.9 (C-9), 59.7 (C-7), 36.8 (C-1ʹ′), 28.4 (3 x Me-Boc), 27.2 (C-11), 20.8 (C-1), 10.9 (C-12).
Compound 37: Colorless amorphous; UV (EtOH) λmax nm (log ε) 206 (4.46), 234 (4.13), 324 (4.17); IR
(KBr) νmax (cm-1) 3297, 2921, 1752, 1738, 1656, 1615, 1498, 1459, 1444, 1402, 1377, 1206, 1169,
1063 ;HRESIMS: m/z 352.1186 [M–H]– (352.1179 calcd for C20H18O5N); 1H NMR (600 MHz, CDCl3) δH
8.15 (brd, 7.7 Hz H-6ʹ′), 7.23 (brd, 7.7 Hz H-3ʹ′), 7.09 (brt, 7.7 Hz, H-5ʹ′), 7.06 (brt, 7.7 Hz, H-4ʹ′), 5.61 (brs,
H-3), 4.96 (d, 12.8 Hz, Ha-10), 4.27 (d, 12.8 Hz, Hb-10), 3.55 (d, 16.5 Hz, Ha-1ʹ′), 2.91 (d, 16.5 Hz, Hb-1ʹ′),
2.07 (s, H3-1), 1.78 (s, H3-12), 1.26 (s, H3-11); 13C NMR (150 MHz, CDCl3) δC 196.7 (C-6), 160.6 (C-2),
152.1 (C-8ʹ′), 137.6 (C-4), 131.5 (C-7ʹ′), 128.8 (C-3ʹ′), 127.1 (C-5ʹ′), 124.4 (C-4ʹ′), 123.6 (C-2ʹ′), 121.5 (C-5),
117.6 (C-6ʹ′), 97.1 (C-3), 86.7 (C-8), 76.0 (C-9), 68.7 (C-10), 47.2 (C-7), 31.0 (C-1ʹ′), 21.5 (C-11), 20.7 (C-1),
11.1 (C-12).
O
HO
OH
HN BocCl
O
HO
ONHBoc
O2
O
O
ONHBoc
O
O
N O
O
HO
37
OHO
O
N OHHO
OO
2
45
O
O
OHO
HOO
O
OHO OHOH
HO
O
Cs2CO3,CH3CN, r.t.
2 34 (16%) 35 (8%)
O
O
O
O
O
N O
O
HO
NHBoc
OH
36 (6%) 37 (7%)
HN Boc
Cl
45
© 2015 Macmillan Publishers Limited. All rights reserved
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21
Key HMBC (red arrow), 1H-1H COSY (blue bold line) and NOE (purple arrow) correlations of 36 and 37
Conversion of 5 to 13, 14, 15 and 16
Compound 5 (30 mg, 0.07 mmol) was treated with cesium carbonate (115 mg, 0.35 mmol) in CH3CN (3.0
ml) at 70 oC for 3 min, then cooled to room temperature and stirred for 30 min. The resulting mixture was
extracted with EtOAc, washed with saturated aqueous solution of brine, dried over Na2SO4, and
concentrated. The residue was purified with column chromatography and preparative TLC to afford 13
(1.5 mg), 14 (0.3 mg), 15 (3.6 mg) and 16 (1.0 mg).
Hypothetical mechanism of ring contraction in the pathway to 13 and 14
OHO
OH
O
O
OH
H
OO
O
O
O
OH
H
O
HO H
13
OHO
O
O
O
OH
H
O OH
OO
O
O
O
OH
H
OHHO
OO
OO
O
OH
H
CO
OH
O
O
O
OH
H
O
HOHO O
O
O
O
OH
H
O
HO H
14
5
O
O
O
O
O
N O
O
HO
NHBoc
OH
36 37
O
O
N O
O
HO
12
3
456
711
12
89
1'2'
3'4'
5'
6' 7'
10
12
3
456
7
12
89
11
101'
2'3'
4'
5'6'
7'8'
H
H
OHO
OH
O
O
OH
H
5
OO
O
O
O
OH
H
HO
HO
O
O
O
OH
H
O
HO H
13 ( 5%) 15 (11%)
OO
O
O
O
OH
H
HO
HO
16 (3%)
O
O
O
OH
H
O
HO H
14 (1%)
Cs2CO3,CH3CN,
70 oC to r.t.
© 2015 Macmillan Publishers Limited. All rights reserved
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22
Compound 13: Colorless amorphous; UV (EtOH) λmax nm (log ε) 206 (4.55), 247 (4.14), 290 (3.50); IR
(KBr) νmax (cm-1) 3445, 2925, 1706, 1651, 1593, 1457, 1166, 1107, 1062, 1030; HRESIMS: m/z
399.1803 [M–H]– (399.1802 calcd for C23H27O6); 1H NMR (600 MHz, CDCl3) δH 4.72 (s, OH), 4.29 (dd,
11.7, 8.0 Hz, Ha-9), 3.77 (t, 11.7 Hz, Hb-9), 3.75 (d, 17.6 Hz, Ha-3ʹ′), 3.69 (d, 17.6 Hz, Hb-3ʹ′), 3.18 (m, H-8),
3.14 (dd, 11.4, 6.2 Hz, H-3), 2.83 (dd, 15.4, 11.4 Hz, Ha-10ʹ′), 2.52 (dd, 15.4, 6.2 Hz, Hb-10ʹ′), 2.18 (s, H3-1ʹ′),
2.16 (s, H3-11ʹ′), 2.10 (s, H3-12ʹ′), 1.78 (s, H3-11), 1.54 (s, H3-1); 13C NMR (150 MHz, CDCl3) δC 209.3 (C-6),
205.8 (C-2ʹ′), 169.3 (C-4), 151.5 (C-6ʹ′), 147.8 (C-8ʹ′), 132.9 (C-5), 129.0 (C-4ʹ′), 115.9 (C-5ʹ′), 111.1 (C-9ʹ′),
110.2 (C-7ʹ′), 98.3 (C-2), 75.0 (C-7), 63.7 (C-9), 45.0 (C-8), 44.4 (C-3ʹ′), 40.1 (C-3), 29.5 (C-1ʹ′), 24.4 (C-10ʹ′),
23.8 (C-1), 21.4 (C-10), 12.1 (C-12ʹ′), 8.4 (C-11ʹ′), 7.6 (C-11).
Compound 14: Colorless amorphous; UV (EtOH) λmax nm (log ε) 206 (4.55), 247 (4.05), 290 (3.40); IR
(KBr) νmax (cm-1) 3428, 2925, 2854, 1707, 1633, 1593, 1456, 1385, 1359, 1302, 1223, 1160, 1108, 1039,
1009; HRESIMS: m/z 399.1819 [M–H]‒ (399.1802 calcd for C23H27O6); 1H NMR (600 MHz, CDCl3) δH
4.65 (s, OH), 4.11 (dd, 10.6, 6.2 Hz, Ha-9), 3.94 (t, 10.6 Hz, Hb-9), 3.89 (d, 17.2 Hz, Ha-3ʹ′), 3.66 (d, 17.2
Hz, Hb-3ʹ′), 3.35 (d, 16.9 Hz, Ha-10), 3.05 (brd, 5.2 Hz, H-3), 2.93 (dd, 10.6, 6.2 Hz, H-8), 2.92 (dd, 16.9,
5.2 Hz, Hb-10ʹ′), 2.18 (s, H3-1ʹ′), 2.13 (s, H3-11ʹ′), 2.07 (s, H3-12ʹ′), 1.50 (s, H3-11), 1.50 (s, H3-1), 1.12 (s,
H3-10).
Key HMBC (red arrow), 1H-1H COSY (blue bold line) and NOE (purple arrow) correlations of 13 and 14
Compound 15: Colorless amorphous; UV (EtOH) λmax nm (log ε) 206 (4.55), 233 (4.16), 284 (3.45); IR
(KBr) νmax (cm-1) 3444, 2992, 2925, 2854, 1736, 1682, 1594, 1459, 1378, 1343, 1221, 1182, 1131, 1107,
1078, 1030; HRESIMS: m/z 467.1658 [M+Na]+ (467.1676 calcd for C24H28O8Na); 1H NMR (600 MHz,
CDCl3 (10% CD3OD)) δH 4.78 (d, 17.9 Hz, Ha-10), 4.53 (d, 17.9 Hz, Hb-10), 3.69 (d, 16.9 Hz, Ha-3ʹ′), 3.64
(d, 16.9 Hz, Hb-3ʹ′), 3.07 (dd, 16.1, 5.9 Hz, Ha-10ʹ′), 2.42 (dd, 11.0, 5.9 Hz, H-3), 2.34 (dd, 16.1, 11.0 Hz,
Hb-10ʹ′), 2.16 (s, H3-11ʹ′), 2.12 (s, H3-12ʹ′), 2.10 (s, H3-1ʹ′), 1.67 (s, H3-12), 1.63 (s, H3-11), 1.60 (s, H3-1); 13C
NMR (150 MHz, CDCl3 (10% CD3OD)) δC 208.1 (C-6), 208.1 (C-2ʹ′), 194.9 (C-8), 158.4 (C-4), 151.8 (C-6ʹ′),
147.7 (C-8ʹ′), 128.7 (C-4ʹ′), 126.9 (C-9), 116.7 (C-5ʹ′), 111.4 (C-9ʹ′), 110.9 (C-7ʹ′), 96.9 (C-2), 80.9 (C-7), 75.8
(C-5), 62.1 (C-10), 44.7 (C-3ʹ′), 36.7 (C-3), 29.1 (C-1ʹ′), 28.8 (C-10ʹ′), 26.6 (C-12), 26.6 (C-11), 22.8 (C-1),
12.3 (C-12ʹ′), 8.4 (C-11ʹ′).
O
O
O
OH
H
O
HO
HO
O
O
OH
HO
HO H
14
O
O
O
OH
O
HO
13
1
9
8
234
5
67
11
10
1'2'
3'
4'
5'6'
7'8'9'
10' 11'
12'
H
H
H
H
97
10'
3
H H
© 2015 Macmillan Publishers Limited. All rights reserved
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23
Compound 16: Colorless amorphous; UV (EtOH) λmax nm (log ε) 206 (4.59), 232 (4.21), 286 (3.51); IR
(KBr) νmax (cm-1) 3433, 3010, 2925, 2854, 1736, 1685, 1594, 1461, 1383, 1347, 1219, 1186, 1132, 1108,
1054, 1032; HRESIMS: m/z 467.1659 [M+Na]+ (467.1676 calcd for C24H28O8Na); 1H NMR (600 MHz,
CDCl3) δH 4.68 (d, 18.0 Hz, Ha-10), 4.64 (d, 18.0 Hz, Hb-10), 3.73 (d, 16.5 Hz, Ha-3ʹ′), 3.66 (d, 16.5 Hz,
Hb-3ʹ′), 3.07 (dd, 9.9, 5.9 Hz, H-3), 3.01 (dd, 15.4, 5.9 Hz, Ha-10ʹ′), 2.70 (dd, 15.4, 9.9 Hz, Hb-10ʹ′), 2.15 (s,
H3-12ʹ′), 2.14 (s, H3-11ʹ′), 2.13 (s, H3-1ʹ′), 1.71 (s, H3-12), 1.70 (s, H3-11), 1.47 (s, H3-1); 13C NMR (150 MHz,
CDCl3) δC 207.7 (C-6), 206.2 (C-2ʹ′), 194.3 (C-8), 157.2 (C-4), 151.7 (C-6ʹ′), 148.0 (C-8ʹ′), 128.8 (C-4ʹ′),
127.5 (C-9), 115.8 (C-5ʹ′), 110.5 (C-9ʹ′), 110.1 (C-7ʹ′), 97.1 (C-2), 82.4 (C-7), 75.0 (C-5), 61.8 (C-10), 44.8
(C-3ʹ′), 35.7 (C-3), 29.3 (C-1ʹ′), 28.5 (C-10ʹ′), 27.3 (C-12), 27.2 (C-11), 22.6 (C-1), 12.2 (C-12ʹ′), 8.3 (C-11ʹ′).
Key HMBC (red arrow), 1H-1H COSY (blue bold line) and NOE (purple arrow) correlations of 15 and 16
Conversion of 5 to 17 and 18
Compound 5 (20 mg, 0.05 mmol) was treated with cesium carbonate (81.5 mg, 0.25 mmol) and 45 (36.0 mg,
31.3 mg, 0.13 mmol) in CH3CN (2.0 ml) at 70 oC for 3 min, then the reaction mixture was cooled to room
temperature and stirred for 1 hour. The resulting mixture was extracted with EtOAc, washed with saturated
aqueous solution of brine, dried over Na2SO4, and concentrated. The residue was purified with column
chromatography, preparative TLC and preparative HPLC to afford 17 (1.4 mg) and 18 (0.3 mg).
OO
O
O
O
OH
H
HO
HO
15
OO
O
O
O
OH
H
HO
HO
16
OO
O
O
O
OH
HO
HO1
1'
2'
3'4' 5'
6'
7'8'9'
10' 11'
12'
2345
6
78
910
12
11
15 and 16
1 1
3 310'
55
7 7
OHO
OH
O
O
OH
H
5
Cs2CO3,CH3CN,70 oC to r.t.
HN Boc
Cl
O
O
O
OH
H
ON
O
HO
O
17 (5%)
O
O
O
OH
H
ON
O
HO
O
18 (1%)45
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Hypothetical reaction pathway to 17 and 18
Compound 17: Colorless amorphous; UV (EtOH) λmax nm (log ε) 206 (4.62), 229 (4.28), 279 (3.37); IR
(KBr) νmax (cm-1) 3396, 2925, 2854, 1760, 1740, 1717, 1681, 1588, 1497, 1460, 1421, 1386, 1360, 1181,
1163, 1095; HRESIMS: m/z 558.2127 [M–H]– (558.2122 calcd for C32H32O8N); 1H NMR (600 MHz, CDCl3
(10% CD3OD)) δH 8.38 (brd, 8.0 Hz, H-6ʹ′ʹ′), 7.22 (brd, 8.0 Hz, H-3ʹ′ʹ′), 7.20 (brt, 8.0 Hz, H-5ʹ′ʹ′), 7.05 (brt, 8.0
Hz, H-4ʹ′ʹ′), 4.67 (d, 18.3 Hz, Ha-10), 4.39 (d, 18.3 Hz, Hb-10), 3.76 (s, H2-3ʹ′), 3.42 (d, 16.1 Hz, Ha-1ʹ′ʹ′), 3.11
(dd, 15.8, 5.9 Hz, Ha-10ʹ′), 3.04 (d, 16.1 Hz, Hb-1ʹ′ʹ′), 2.74 (dd, 11.0, 5.9 Hz, H-3), 2.34 (s, H3-1ʹ′), 2.29 (dd,
15.8, 11.0 Hz, Hb-10ʹ′), 2.14 (s, H3-11ʹ′), 2.06 (s, H3-12ʹ′), 1.80 (s, H3-12), 1.57 (s, H3-1), 1.34 (s, H3-11); 13C
NMR (150 MHz, CDCl3) δC 207.3 (C-2ʹ′), 195.7 (C-8), 151.8 (C-8ʹ′ʹ′), 151.3 (C-6ʹ′), 150.5 (C-4), 147.2 (C-8ʹ′),
132.7 (C-7ʹ′ʹ′), 129.0 (C-3ʹ′ʹ′), 128.8 (C-4ʹ′), 127.6 (C-9), 127.2 (C-5ʹ′ʹ′), 123.7 (C-4ʹ′ʹ′), 123.3 (C-2ʹ′ʹ′), 116.7
(C-5ʹ′), 116.5 (C-6ʹ′ʹ′), 110.9 (C-9ʹ′), 110.5 (C-7ʹ′), 96.3 (C-2), 88.4 (C-6), 82.4 (C-5), 62.0 (C-10), 47.2 (C-7),
44.0 (C-3ʹ′), 36.6 (C-3), 31.2 (C-1ʹ′ʹ′), 29.6 (C-1ʹ′), 29.4 (C-10ʹ′), 22.5 (C-1), 21.4 (C-11), 17.5 (C-12), 11.9
(C-12ʹ′), 8.3 (C-11ʹ′).
Compound 18: Colorless amorphous; UV (EtOH) λmax nm (log ε) 206 (4.63), 229 (4.28), 281 (3.42); IR
(KBr) νmax (cm-1) 3396, 2925, 2854, 1734, 1681, 1589, 1497, 1459, 1422, 1385, 1362, 1181, 1164, 1096;
HRESIMS: m/z 558.2134 [M–H]– (558.2122 calcd for C32H32O8N); 1H NMR (600 MHz, CDCl3 (10%
CD3OD)) δH 8.36 (brd, 8.0 Hz, H-6ʹ′ʹ′), 7.22 (brd, 8.0 Hz, H-3ʹ′ʹ′), 7.19 (brt, 8.0 Hz, H-5ʹ′ʹ′), 7.03 (brt, 8.0 Hz,
H-4ʹ′ʹ′), 4.55 (d, 18.3 Hz, Ha-10), 4.51 (d, 18.3 Hz, Hb-10), 3.73 (d, 16.1 Hz, Ha-3ʹ′), 3.66 (d, 16.1 Hz, Hb-3ʹ′),
3.45 (d, 16.1 Hz, Ha-1ʹ′ʹ′), 3.01 (d, 16.1 Hz, Hb-1ʹ′ʹ′), 2.95 (dd, 15.4, 5.5 Hz, Ha-10ʹ′), 2.77 (dd, 11.8, 5.5 Hz,
H-3), 2.47 (dd, 15.4, 11.8 Hz, Hb-10ʹ′), 2.16 (s, H3-1ʹ′), 2.15 (s, H3-11ʹ′), 2.14 (s, H3-12ʹ′), 1.84 (s, H3-12), 1.41
(s, H3-1), 1.39 (s, H3-11).
OHO
OH
O
O
OH
H
HN BocCl
OHO
O
O
O
OH
HNH
BocO
O
O
OH
H
OHN
O
HO
O
O2
OO
O
O
O
OH
HNH
BocHO
O
O
O
OH
H
ON
O
HO
O
O
17
O
O
O
OH
H
ON
O
HO
O
18
5
45
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Key HMBC (red arrow), 1H-1H COSY (blue bold line) and NOE (purple arrow) correlations of 17 and 18
Synthesis of 20, 21 and 22
a) To a solution of compound 2 (20.0 mg, 0.10 mmol) and indole (23.5 mg, 0.20 mmol) in acetonitrile (1
mL), catalytic amount of BF3OEt2 (1.2 µL, 0.01 mmol) was added, and the reaction mixture was stirred at
room temperature for 30 minutes. The resulting mixture was extracted with EtOAc, washed with water,
dried over Na2SO4, and concentrated. The residue was purified with column chromatography yielding 21
(17.2 mg). b) To a solution of compound 2 (10.0 mg, 0.05 mmol) and indole (11.7 mg, 0.10 mmol) in
CH2Cl2 (0.5 mL), catalytic amount of BF3OEt2 (0.6 µL, 0.005 mmol) was added, and the reaction mixture
was stirred at room temperature for 3 minutes, yielding 20 (3.3 mg) and 21 (2.7 mg). c) Compound 21 (10.0
mg, 0.02 mmol) was dissolved in acetonitrile (0.3 mL) under argon and excess amount of BF3OEt2 (12.5 µL,
0.10 mmol) was added and the solution was stirred at room temperature for 2 hours. The solution turned to
red soon after the addition of excess amount of BF3OEt2. The resulting mixture was extracted with
EtOAc, washed with water, dried over Na2SO4, and concentrated. The residue was purified with column
chromatography and preparative TLC yielding 22 (2.6 mg) and 23 (3.4 mg). Treatment of 2 and indole
with the excess amount of BF3OEt2 also afforded to 22 and 23 in the similar yield.
O
O
O
OH
H
ON
O
HO
O
17
O
O
O
OH
H
ON
O
HO
O
18
O
O
O
OH
ON
O
HO
O
17
1
2345
67
8 910
12
11
1'2'
3'4' 5'
6'
7'8'9'
10' 11'
12'
1''2''
3''4''
5''6''
7''
8''
H H
NH
O
OH
HO
N
HO
OH
22 (c : 34%)
a) BF3OEt2 (10 mol%), r.t., CH3CN, 30 minb) BF3OEt2 (10 mol%), r.t., CH2Cl2, 3 min
2
OHN
HO
OH
23 (c : 48%)
c) BF3OEt2 (10 eq), r.t., CH3CN, 2 h.
NH
O
OH
OH
20 (a : 0%, b : 21%)
NH
HO
OH
NH
21 (a : 42%, b : 13%)
+
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Compound 20: Yellow oil; UV (EtOH) λmax nm (log ε) 207 (4.47), 222 (4.38), 283 (3.73); IR (KBr) ν
max (cm-1) 3403, 3008, 2927, 1703, 1614, 1590, 1458, 1357, 1220, 1164, 1108; HREIMS: m/z 323.1543
[M]+ (323.1522 calcd for C20H21O3N); 1H NMR (600 MHz, CDCl3) δH 7.98 (s, NH), 7.62 (d, 8.0 Hz, H-4ʹ′),
7.35 (d, 8.0 Hz, H-7ʹ′), 7.22 (t, 8.0 Hz, H-6ʹ′), 7.14 (t, 8.0 Hz, H-5ʹ′), 6.65 (H-2ʹ′), 4.90 (s, OH), 4.71 (s, OH),
3.99 (s, H2-10), 3.82 (s, H2-3), 2.14 (s, H3-11), 2.10 (s, H3-12), 2.06 (s, H3-1); 13C NMR (150 MHz, CDCl3)
δC 206.6 (C-2), 151.2 (C-6), 151.0 (C-8), 136.7 (C-7ʹ′a), 130.6 (C-4), 127.0 (C-4ʹ′a), 122.5 (C-2ʹ′), 122.5
(C-6ʹ′), 119.6 (C-5ʹ′), 118.7 (C-4ʹ′), 117.4 (C-9), 115.0 (C-5), 113.4 (C-3ʹ′), 111.3 (C-7ʹ′), 109.6 (C-7), 45.2
(C-3), 29.5 (C-1), 23.1 (C-10), 12.3 (C-12), 8.7 (C-11).
Compound 21: Yellow oil; UV (EtOH) λmax nm (log ε) 208 (4.63), 224 (4.68), 284 (4.04); IR (KBr) ν
max (cm-1) 3403, 3052, 3005, 2969, 2927, 1614, 1587, 1460, 1335, 1297, 1231, 1190, 1100, 1012; HREIMS:
m/z 422.1988 [M]+ (422.1994 calcd for C28H26O2); 1H NMR (600 MHz, acetone-d6) δH 10.10 (s, NH), 9.30 (s,
NH), 7.68 (brd, 8.0 Hz, H-4ʹ′), 7.33 (brd, 8.0 Hz, H-7ʹ′ʹ′), 7.29 (s, H-2ʹ′ʹ′), 7.08 (m, H-4ʹ′ʹ′), 7.03 (brd, 8.0 Hz,
H-7ʹ′), 6.95 (brt, 8.0 Hz, H-6ʹ′ʹ′), 6.93 (brt, 8.0 Hz, H-5ʹ′), 6.89 (brt, 8.0 Hz, H-6ʹ′), 6.66 (brt, 8.0 Hz, H-5ʹ′ʹ′),
4.67 (d, 15.4 Hz, Ha-10), 4.12 (d, 15.4 Hz, Hb-10), 3.93 (d, 14.3 Hz, Ha-3), 3.21 (d, 14.3 Hz, Hb-3), 2.17 (s,
H3-11), 2.08 (s, H3-12), 1.74 (s, H3-1); 13C NMR (150 MHz, acetone-d6) δC 151.9 (C-8), 149.7 (C-6), 141.9
(C-2ʹ′), 138.2 (C-7ʹ′ʹ′a), 136.3 (C-7ʹ′a), 136.1 (C-4), 129.0 (C-4ʹ′a), 127.3 (C-4ʹ′ʹ′a), 124.8 (C-3ʹ′ʹ′), 123.4 (C-9),
122.1 (C-2ʹ′ʹ′), 121.8 (C-5ʹ′), 121.2 (C-6ʹ′), 121.0 (C-4ʹ′ʹ′), 119.2 (C-5ʹ′ʹ′), 118.8 (C-6ʹ′ʹ′), 118.7 (C-4ʹ′), 117.0 (C-5),
112.0 (C-7ʹ′ʹ′), 111.1 (C-7ʹ′), 110.6 (C-3ʹ′), 109.7 (C-7), 42.1 (C-3), 40.7 (C-2), 26.7 (C-1), 21.1 (C-10), 13.0
(C-12), 9.9 (C-11).
Key HMBC (red arrow) and 1H-1H COSY (blue bold line) correlations of 20 and 21
Compound 22: Colorless oil; UV (EtOH) λmax nm (log ε) 208 (4.54), 229 (4.55), 285 (3.89); IR (KBr)
νmax (cm-1) 3397, 3056, 3004, 2965, 2927, 2881, 1673, 1617, 1584, 1460, 1379, 1326, 1253, 1232, 1216,
1186; HRESIMS: m/z 322.1444 [M–H]‒ (322.1438 calcd for C20H20O3N), HREIMS: m/z 323.1507 [M]+
(323.1522 calcd for C20H21O3N); 1H NMR (600 MHz, CD3OD) δH 7.56 (brd, 8.0 Hz, H-4ʹ′), 7.17 (brd, 8.0 Hz
H-7ʹ′), 6.96 (brt, 8.0 Hz, H-6ʹ′), 6.94 (brt, 8.0 Hz, H-5ʹ′), 4.17 (d, 15.9 Hz, Ha-10), 4.05 (d, 15.9 Hz, Hb-10),
3.20 (m, Ha-3), 3.12 (m, H-2), 3.09 (m, Hb-3), 2.22 (s, H3-12), 2.10 (s, H3-11), 1.34 (d, 6.2 Hz, H3-1); 13C
NMR (150 MHz, CD3OD) δC 151.8 (C-6), 149.6 (C-8), 139.9 (C-2ʹ′), 137.1 (C-4), 136.5 (C-7ʹ′a), 129.5
(C-4ʹ′a), 125.0 (C-9), 121.2 (C-6ʹ′), 119.0 (C-5ʹ′), 118.4 (C-4ʹ′), 117.3 (C-5), 112.3 (C-7), 111.1 (C-7ʹ′), 110.4
(C-3ʹ′), 35.4 (C-3), 33.6 (C-2), 21.7 (C-1), 20.8 (C-10), 12.6 (C-12), 9.9 (C-11).
NH
O
OH
OH
20
NH
HO
OH
NH
21
1
2
34 5
6
78910 11
12
2'
3'4'
5'
6'
7'
4'a
7'a
2'
3'
4'5'
6'7'
4'a
7'a
11
7
6
5
12
4
9810
1
2 3
2''
3''4''a 4''
5''
6''7''7''a
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Compound 23: Red powder; UV (EtOH) λmax nm (log ε) 203 (4.23), 215 (4.23), 245 (4.13), 342 (4.50),
358 (4.52), 428 (3.82), 454 (3.84), 492 (3.78); IR (KBr) νmax (cm-1) 3169, 2956, 2928, 1730, 1576, 1541,
1466, 1354, 1272, 1216, 1119, 1071, 1035; HRESIMS: m/z 304.1332 [M+H]+ (304.1332 calcd for
C20H18O2N); 1H NMR (600 MHz, CD3OD) δH 9.89 (s, H-10), 8.46 (s, H-3), 8.16 (d, 8.0 Hz, H-4ʹ′), 7.68 (m,
H-7ʹ′), 7.67 (m, H-6ʹ′), 7.55 (brt, 8.0 Hz, H-5ʹ′), 2.84 (s, H3-1), 2.49 (s, H3-12), 2.32 (s, H3-11); 13C NMR (150
MHz, CD3OD) δC 165.3 (C-6), 157.7 (C-8), 150.4 (C-2ʹ′), 140.6 (C-3), 140.5 (C-7ʹ′a), 139.6 (C-10), 138.3
(C-3ʹ′), 132.2 (C-2), 130.7 (C-6ʹ′), 127.8 (C-4ʹ′a), 125.7 (C-5ʹ′), 125.5 (C-3ʹ′), 122.0 (C-9), 121.1 (C-4ʹ′), 118.6
(C-5), 118.0 (C-7), 114.4 (C-7ʹ′), 23.6 (C-1), 12.8 (C-12), 11.0 (C-11).
Key HMBC (red arrow) and 1H-1H COSY (blue bold line) correlations of 22 and 23
Hypothetical reaction scheme to 22 and 23
Confirmation of 1-hydroxy group in 22
Treatment of 22 (2.0 mg) with acetic anhydride (50 µL) in pyridine (100 µL) provided its diacetylated
derivative 22a (2.3 mg). The IR spectra of 1-hydroxyindole 22a showed broad peak around 3400 cm-1,
whereas its similar compound with indole NH group, 1,2,3,4-tetrahydrocarbazole, displayed a strong and
sharp peak at the same region, indicating the presence of a hydroxyl group in 22a.
N
HO
OH2'
3'4'
5'
6'7'
4'a7'a
11
7
6
5
12
4
9810
1
2 3
23
N
HO
OH
22
2'
3'
4'5'
6'7'
4'a7'a
11
7
6
5
12
4
9810
1
2 3
OH
OH
OH
ONH
NH
O
OH
OH NH OH
HO
OHNH
HO
OH
NH
21
NH
HO
OH
NH
22
N
HO
OH
20
2
OHNH
HO
OH
NH
H
NH
HO
OH
NH
HO
OH
H
O2
N
HO
OH
23
–H+H+
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Compound 22a: Yellow oil; IR spectra (KBr), see Supplementary figure 6; HREIMS: m/z 407.1734 [M]+
(407.1733 calcd for C24H25O5N); 1H NMR (600 MHz, CD3OD) δH 7.45 (brd, 7.8 Hz), 7.19 (brd, 7.8 Hz),
6.99 (m), 6.97 (m), 3.93 (s, 2H), 3.29–3.18 (3H), 2.42 (s, Me (Ac)), 2.32 (s, Me (Ac)), 2.18 (s, H3-12), 1.88
(s, H3-11), 1.39 (d, 6.2 Hz, H3-1).
Structure of 22a and key NOE correlations
Supplementary Figure 6. IR spectra of 22a (left) and 1,2,3,4-tetrahydrocarbazole (right).
N
AcO
OAc
OH22a
NH
1,2,3,4-Tetrahydrocarbazole
N
AcO
OAc
OH22a
2'
3'
4'5'
6'7'
4'a7'a
117
6
5
12
4
9810
1
2 3
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Synthesis of 24 and 25
To a solution of 2 (65 mg, 0.32 mmol) and 2-methylindole (84 mg, 0.64 mmol) in CH3CN (3 ml), BF3OEt2
(4.0 µl, 0.032 mmol) was added dropwise at room temperature and stirred for 30 min. The resulting mixture
was extracted with EtOAc, washed with water, dried over Na2SO4, and concentrated. The residue was
purified with column chromatography afforded 24 (91.4 mg). Compound 24 (100 mg) was treated with
TMS diazomethane 20% hexane solution (5 ml) and MeOH (0.5 ml) at room temperature for 2 days.
The residue was purified with column chromatography to afford 24a (32.4 mg). 24a (30.0 mg, 0.08
mmol) was treated with LHMDS 26% THF solution (154 µL, 0.20 mmol) in THF (1.5. mL) at -78 oC
and stirred for 30 min, and then the mixture was added I2 (25.4 mg, 0.10 mmol). After being slowly
warmed to room temperature, the reaction mixture was stirred for 2 hours. The mixture was added
saturated aqueous solution of Na2S2O3 and extracted with EtOAc. The combined organic extracts were
washed with brine, dried over Na2SO4, and concentrated. The residue was purified by silica-gel column
chromatography and preparative TLC to afford 25 (1.1 mg) and recovered 24a (6.4 mg, 21 %).
Compound 24: Yellow oil; UV (EtOH) λmax nm (log ε) 208 (4.53), 226 (4.51), 283 (3.87); HREIMS: m/z
337.1673 [M]+ (337.1678 calcd for C21H23O3N); IR (KBr) νmax (cm-1) 3394, 3007, 2921, 1703, 1616,
1589, 1459, 1429, 1358, 1339, 1302, 1224, 1107; 1H NMR (600 MHz, CDCl3) δH 7.82 (s, NH), 7.25 (d, 8.0
Hz, H-4ʹ′), 7.25 (d, 8.0 Hz, H-7ʹ′), 7.10 (t, 8.0 Hz, H-6ʹ′), 7.00 (t, 8.0 Hz, H-5ʹ′), 5.25 (s, 8-OH), 4.63 (6-OH),
3.97 (s, H2-10), 3.90 (s, H2-3), 2.24 (s, H3-8ʹ′), 2.12 (s, H3-12), 2.09 (s, H3-11); 13C NMR (150 MHz, CDCl3)
δC 206.7 (C-2), 151.5 (C-8), 151.1 (C-6), 135.3 (C-7ʹ′a), 132.3 (C-2ʹ′), 130.4 (C-4), 128.3 (C-4ʹ′a), 121.4
(C-6ʹ′), 119.6 (C-5ʹ′), 118.2 (C-4ʹ′), 117.4 (C-9), 114.9 (C-5), 110.3 (C-7ʹ′), 109.5 (C-7), 107.4 (C-3ʹ′), 45.4
(C-3), 29.1 (C-1), 22.9 (C-10), 12.3 (C-12), 11.7 (C-8ʹ′), 8.6 (C-11).
Key HMBC correlations of 24
Compound 24a: Colorless amorphous; HREIMS: m/z 365.2001 [M]+ (365.1991 calcd for C23H27O3N); 1H
NMR (600 MHz, CDCl3) δH 7.69 (s, NH), 7.32 (brd, 8.0 Hz), 7.20 (brd (8.0 Hz), 7.06 (brt, 8.0 Hz), 7.00 (brt,
8.0 Hz), 4.04 (s, 2H), 3.73 (s, OMe), 3.70 (s, OMe), 3.62 (s, 2H), 2.31 (s, Me), 2.04 (s, Me), 2.03 (s, Me),
1.49 (s, Me).
NH
O
OH
OH
1
2
34 5
6
78910 11
12
2'
3'4'
5'
6'7'
4'a
7'a8'
O
OH
HO
2
NH
O
OH
OH
24 2524aNH
BF3OEt2 (10 mol%),CH3CN, r.t., 86%
NH
O
OMe
OMe
TMSCH2N2,MeOH, r.t., 30%
LHMDS,THF, 0 oC,then I2, 4%
N
OMe
OMe
O
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Compound 25: Colorless amorphous; UV (EtOH) λmax nm (log ε) 208 (4.55), 221 (4.40), 273 (3.56); IR
(KBr) νmax (cm-1) 2929, 2854, 1713, 1573, 1466, 1458, 1423, 1404, 1377, 1358, 1339, 1271, 1248, 1191,
1160, 1113, 1002; HRESIMS: m/z 386.1725 [M+Na]+ (386.1727 calcd for C23H25O3NNa), 364.1907 [M+H]+
(364.1907 calcd for C23H26O3N); 1H NMR (600 MHz, CDCl3) δH 7.58 (brd, 7.8 Hz, H-7ʹ′), 7.36 (brt, 7.8 Hz,
H-6ʹ′), 7.19 (brt, 7.8 Hz, H-5ʹ′), 7.16 (brd, 7.8 Hz, H-4ʹ′), 4.49 (s, H-3), 3.75 (s, OMe), 3.73 (s, OMe), 3.34 (d,
15.6 Hz, Ha-10), 3.17 (d, 15.6 Hz, Hb-10), 2.26 (s, H3-11), 2.14 (s, H3-8ʹ′), 1.99 (s, H3-12), 1.81 (s, H3-1); 13C
NMR (150 MHz, CDCl3) δC 205.2 (C-2), 183.2 (C-2ʹ′), 157.5 (C-6), 153.9 (C-7ʹ′a), 153.5 (C-8), 144.2 (C-4ʹ′a),
138.6 (C-4), 129.2 (C-9), 128.6 (C-6ʹ′), 126.1 (C-5ʹ′), 123.9 (C-5), 123.9 (C-7), 120.8 (C-4ʹ′), 120.2 (C-7ʹ′),
66.5 (C-3ʹ′), 65.0 (C-3), 60.2 (OMe), 60.0 (OMe), 37.5 (C-10), 30.9 (C-1), 18.0 (C-8ʹ′), 13.6 (C-12), 9.6
(C-11).
Key HMBC (red arrow) and NOE (purple arrow) correlations of 25
Synthesis of 27
To a solution of 2 (10 mg, 0.05 mmol) and 3-methylindole (13.1 mg, 0.10 mmol) in CH3CN (0.5 ml),
BF3OEt2 (0.6 µl, 0.005 mmol) was added dropwise at room temperature. After being stirred for 30 min at the
same temperature, the reaction was quenched with water and then extracted with EtOAc. The combined
organic extracts were washed with saturated aqueous solution of brine, dried over Na2SO4, and
concentrated. The residue was purified by silica-gel column chromatography to afford 27 (10.2 mg).
N
OMe
OMe
O
N
OMe
OMe
O
2'
4'5'
6'7'
4'a7'a
3'
8'
1 23
45
12
6 7 11
89
10
N
OMe
OMeO
H
HH
33
3'
4' 4'
3'1010
8' 8'
1
1
1212
O
OH
HO
2
BF3OEt2 (10 mol%),CH3CN, r.t.
NH
OH
OH
O
N OH
OHNH
26 27 (66%)
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
31
Synthesis of 26, 28, 29 and 30
To a solution of 2 (100 mg, 0.49 mmol) and 3-methylindole (127 mg, 0.98 mmol) in CH2Cl2 (5 ml), BF3OEt2
(6.1 µl, 0.049 mmol) was added dropwise at room temperature and stirred 3 min. The reaction was quenched
with water and then extracted with EtOAc. The combined organic extracts were washed with saturated
aqueous solution of brine, dried over Na2SO4, and concentrated. The residue was purified by silica-gel
column chromatography to afford 26 (83.0 mg). Compound 27 was also observed as a minor component
in this reaction. Compound 26 (80.0 mg) was treated with TMS diazomethane 20% hexane solution (2.5
ml) and MeOH (0.25 ml) at room temperature for 2 days, affording its dimethyl derivative 26a (67.6
mg). Compound 26a (30.0 mg, 0.14 mmol) was treated with LHMDS 26% THF solution (158 µL, 0.35
mmol) in THF (2.5 mL) at -78 oC and stirred for 30 min, and then the mixture was added I2 (20.8 mg,
0.14 mmol). After being slowly warmed to room temperature, the reaction mixture was stirred for 2
hours. The mixture was added saturated aqueous solution of Na2S2O3 and extracted with EtOAc. The
combined organic extracts were washed with brine, dried over Na2SO4, and concentrated. The residue was
purified by silica-gel column chromatography and preparative TLC to afford 28 (3.2 mg), 29 (2.4 mg)
and 30 (1.1 mg) as well as recovered 26a (7.2 mg, 24%).
Hypothetical reaction scheme to 28–30 from 26a by the intramolecular oxidative coupling
NOMe
OMe
OHC
NOMe
OMe
O
N
HO
O
OMe
OMeIH
NH
OMe
OMe
O
N
OMe
OMe
O I
NOMe
OMe
O
NOMe
OMe
O
HO
N
H2O
I
O
30 29
2826a
I
O
OH
HO
2
BF3OEt2 (10 mol%),CH2Cl2, r.t, 3 min.
NH
OH
OH
ONH
26 (51%)
27 (trace)
NOMe
OMe
OHCN
OMe
OMe
O
HO
N
HO
O
OMe
OMeI
H
30 (3%)
TMSCH2N2,MeOH, r.t.,
LHMDS,THF, -78 oC,then I2, 5%
NH
OMe
OMe
O
26a (78%)
28 (10%) 29 (8%)
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32
Compound 26: Yellow oil; UV (EtOH) λmax nm (log ε) 208 (4.54), 227 (4.51), 285 (3.95); IR (KBr) ν
max (cm-1) 3401, 3007, 2919, 2860, 1704, 1589, 1459, 1315, 1240, 1217, 1164, 1110; HREIMS: m/z
337.1676 [M]+ (337.1678 calcd for C21H23NO3); 1H NMR (600 MHz, CDCl3) δH 8.49 (s, NH), 7.49 (brd, 7.8
Hz, H-4ʹ′), 7.22 (brd, 7.8 Hz, H-7ʹ′), 7.10 (brt, 7.8 Hz, H-6ʹ′), 7.06 (brt, 7.8 Hz, H-5ʹ′), 3.97 (s, H2-10), 3.95 (s,
H2-3), 2.40 (s, H3-8ʹ′), 2.27 (s, H3-1), 2.09 (s, H3-12), 2.09 (s, H3-11); 13C NMR (150 MHz, CDCl3) δC 207.7
(C-2), 151.5 (C-6), 151.3 (C-8), 135.8 (C-7ʹ′a), 131.7 (C-2ʹ′a), 130.1 (C-4), 128.8 (C-4ʹ′a), 121.6 (C-6ʹ′), 119.0
(C-5ʹ′), 118.2 (C-4ʹ′), 117.0 (C-9), 114.9 (C-5), 110.8 (C-7ʹ′), 110.1 (C-7), 106.9 (C-3ʹ′), 44.8 (C-3), 30.0 (C-1),
24.1 (C-10), 12.4 (C-12), 8.6 (C-11), 8.5 (C-8ʹ′).
Compound 26a: Colorless amorphous; HREIMS: m/z 365.1988 [M]+ (365.1991 calcd for C23H27O3N); 1H
NMR (600 MHz, CDCl3) δH 8.19 (s, NH), 7.46 (brd, 8.0 Hz), 7.19 (brd (8.0 Hz), 7.06 (brt, 8.0 Hz), 7.04 (brt,
8.0 Hz), 4.00 (s, 2H), 3.80 (s, 2H), 3.70 (s, OMe), 3.65 (s, OMe), 2.36 (s, Me), 2.29 (s, Me), 2.09 (s, Me),
2.08 (s, Me).
Compound 27: Yellow oil; UV (EtOH) λmax nm (log ε) 207 (4.54), 239 (4.48), 278 (4.29), 296 (4.13); IR
(KBr) νmax (cm-1) 3359, 2921, 1637, 1512, 1457, 1393, 1310, 1260, 1202, 1183, 1106, 1082, 1033;
HREIMS: m/z 319.1557 [M]+ (319.1572 calcd for C21H21O2N); 1H NMR (600 MHz, acetone-d6) δH 7.47 (brd,
8.0 Hz, H-4ʹ′), 7.41 (brd, 8.0 Hz, H-7ʹ′), 7.21 (OH), 7.04 (brt, 8.0 Hz, H-6ʹ′), 7.00 (brt, 8.0 Hz, H-5ʹ′), 6.55 (s,
H-3), 4.73 (d, 13.2 Hz, Ha-10), 3.19 (d, 13.2 Hz, Hb-10), 2.64 (s, H3-1), 2.29 (s, H3-8ʹ′), 2.16 (s, H3-11), 2.14
(s, H3-12); 13C NMR (150 MHz, acetone-d6) δC 152.2 (C-6), 149.8 (C-8), 142.6 (C-2ʹ′), 135.6 (C-2), 135.0
(C-7ʹ′a), 132.9 (C-4), 131.7 (C-4ʹ′a), 121.3 (C-6ʹ′), 120.1 (C-5ʹ′), 119.7 (C-9), 119.0 (C-4ʹ′), 118.7 (C-3), 114.2
(C-5), 112.5 (C-7ʹ′), 112.2 (C-7), 104.3 (C-3ʹ′), 23.0 (C-10), 22.4 (C-1), 12.4 (C-12), 9.9 (C-11), 8.6 (C-8ʹ′).
Key HMBC (red arrow) and NOE (purple arrow) correlation of 26 and 27
NH
OH
OH
O
N OH
OH
26 27
4'a7'a
7'
4'
6'
5'
2'3'
1 23
4
512
67
11
89
104'a7'a
7'
4'
6'
5'
2'3'
12
3 45
12
6 7
8910
8'8'
© 2015 Macmillan Publishers Limited. All rights reserved
NATURE CHEMISTRY | www.nature.com/naturechemistry 33
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
33
Compound 28: Colorless amorphous; UV (EtOH) λ:max nm (log ε) 208 (4.55), 226 (4.54), 281 (3.89); IR
(KBr) νmax (cm-1) 3412, 2361, 2926, 2855, 1723, 1574, 1457,1405, 1382, 1356, 1323, 1262, 1237, 1187,
1107, 1085, 1064, 1023; HRESIMS: m/z 378.1712 [M–H]‒ (378.1700 calcd for C23H24O4N); 1H NMR (600
MHz, CDCl3) δH 7.54 (brd, 7.8 Hz, H-4ʹ′), 7.32 (brd, 7.8 Hz, H-7ʹ′), 7.16 (brt, 7.8 Hz, H-5ʹ′), 7.13 (brt, 7.8 Hz,
H-6ʹ′), 5.47 (s, 3-OH), 4.32 (d, 22.2 Hz, Ha-10), 4.23 (d, 22.2 Hz, Hb-10), 3.85 (s, OMe), 3.70 (s, OMe), 2.36
(s, H3-8ʹ′), 2.32 (s, H3-11), 2.27 (s, H3-12), 1.54 (s, H3-1); 13C NMR (150 MHz, CDCl3) δC 201.5 (C-2), 157.5
(C-6), 154.9 (C-8), 133.9 (C-7ʹ′a), 130.5 (C-4ʹ′a), 129.2 (C-4), 128.4 (C-2ʹ′), 126.3 (C-5), 125.8 (C-7), 122.0
(C-9), 121.3 (C-6ʹ′), 120.5 (C-5ʹ′), 117.9 (C-4ʹ′), 112.9 (C-7ʹ′), 107.5 (C-3ʹ′), 87.2 (C-3), 60.0 (OMe), 60.0
(OMe), 22.6 (C-1), 21.1 (C-10), 12.4 (C-12), 10.0 (C-11), 8.5 (C-8ʹ′).
Compound 29: Yellow oil; UV (EtOH) λmax nm (log ε) 204 (4.46), 226 (4.42), 288 (4.12), 384 (3.49); IR
(KBr) νmax (cm-1) 2924, 2853, 1697, 1623, 1606, 1573, 1462, 1437, 1414, 1370, 1320, 1257, 1206, 1152,
1118, 1002; HRESIMS: m/z 384.1561 [M+Na]+ (384.1570 calcd for C23H23O3NNa); 1H NMR (600 MHz,
CDCl3) δH 9.88 (s, H-1), 7.56 (s, H-3), 7.49 (brd, 7.2 Hz, H-4ʹ′), 7.15 (brt, 7.2 Hz, H-6ʹ′), 7.12 (brt, 7.2 Hz,
H-5ʹ′), 7.06 (brd, 7.2 Hz, H-7ʹ′), 4.70 (d, 14.4 Hz, Ha-10), 3.79 (s, OMe), 3.66 (s, OMe), 3.30 (d, 14.4 Hz,
Hb-10), 2.38 (s, H3-8ʹ′), 2.32 (s, H3-12), 2.27 (s, H3-11), 1.54 (s, H3-1); 13C NMR (150 MHz, CDCl3) δC 187.8
(C-1), 156.3 (C-6), 154.2 (C-8), 139.8 (C-2ʹ′), 137.1 (C-3), 134.1 (C-7ʹ′a), 133.4 (C-2), 131.0 (C-9), 130.7
(C-4ʹ′a), 128.1 (C-4), 127.3 (C-7), 126.4 (C-5), 121.7 (C-6ʹ′), 120.2 (C-5ʹ′), 118.7 (C-4ʹ′), 111.9 (C-7ʹ′), 106.8
(C-3ʹ′), 61.4 (6-OMe), 60.2 (8-OMe), 22.9 (C-10), 12.5 (C-12), 10.2 (C-11), 8.8 (C-8ʹ′).
Compound 30: Colorless amorphous; UV (EtOH) λmax nm (log ε) 210 (4.76), 230 (4.44); IR (KBr) ν
max (cm-1) 3349, 2927, 2853, 1730, 1639, 1611, 1468, 1468, 1402, 1374, 1320, 1268, 1217, 1111, 1073,
1002; HRESIMS: m/z 528.0634 [M+Na]+ (528.0642 calcd for C23H24O4NINa); 1H NMR (600 MHz, CDCl3)
δH 7.43 (brd, 8.0 Hz, H-4ʹ′), 7.18 (brt, 8.0 Hz, H-6ʹ′), 6.89 (brt, 8.0 Hz, H-5ʹ′), 6.64 (brd, 8.0 Hz, H-7ʹ′), 4.62 (d,
1.4 Hz, H-2), 4.28 (s, 2ʹ′-OH), 4.17 (brs, H-3), 3.77 (s, 8-OMe), 3.70 (s, 6-OMe), 3.48 (d, 16.8 Hz, Ha-10),
3.10 (d, 16.8 Hz, Hb-10), 2.21 (s, H3-12), 2.21 (s, H3-11), 1.66 (s, H3-8ʹ′); 13C NMR (150 MHz, CDCl3) δC
160.6 (C-1), 157.5 (C-6), 153.4 (C-8), 147.4 (C-7ʹ′a), 138.0 (C-4), 131.4 (C-4ʹ′a), 129.9 (C-6ʹ′), 128.2 (C-9),
124.7 (C-4ʹ′), 123.9 (C-7), 123.3 (C-5), 120.2 (C-5ʹ′), 110.6 (C-7ʹ′), 93.3 (C-3ʹ′), 83.8 (C-2ʹ′), 61.7 (C-3), 60.0
(6-OMe), 59.9 (8-OMe), 43.0 (C-2), 35.2 (C-10), 22.3 (C-8ʹ′), 13.6 (C-12), 9.5 (C-11).
Key HMBC (red arrow), 1H-1H COSY (blue bold line) and NOE (purple arrow) correlation of 28–30, and
characteristic data of 30
28 29
N
O
OMe
OMe
HON
OMe
OMe
O
30
N
HO
O
OMe
OMeI
N
HO
O
OMe
OMeI H
4'a7'a
7'
4'
6'
5'
2'3'
8'
1 23
4
5 67
11
89
10 10
4'a7'a
7'
4'
6'
5'
2'3'
8'
12 3
4
512 12 11
6 7
89
H
4'a7'a
7'
4'
6'
5'
2'3'
8'10
9 8 711
6
5
432
12
3
12 12
10
8'
IR : !max 1730 cm-1
JH-2-H-3 = 1.4 Hz
NOMe
OMe
O2
12
HH
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34
Conversion of 30 to 31
When NMR measurement of 30 (1.0 mg) in CD3CN (150 µL) was carried out, conversion of 30 to 31 was
observed. The reaction was monitored in situ by 1H NMR spectroscopy and completed in 30 hours at room
temperature. This conversion could not be observed in CDCl3 in 48 h.
Compound 31: Yellow oil; UV (EtOH) λmax nm (log ε) 205 (4.56), 244 (4.13), 290 (3.91), 380 (3.85); IR
(KBr) νmax (cm-1) 3306, 2926, 2853, 1630, 1609, 1541, 1467, 1404, 1375, 1334, 1282, 1191, 1109, 1065,
1010; HREIMS: m/z 489.0772 [M]+ (489.0801 calcd for C23H24O3NI); 1H NMR (600 MHz, CD3CN) δH
10.19 (brs, NH), 7.40 (brd, 7.8 Hz, H-4ʹ′), 7.23 (brt, 7.8 Hz, H-6ʹ′), 7.10 (brd, 7.8 Hz, H-7ʹ′), 7.03 (brt, 7.8 Hz,
H-5ʹ′), 3.99 (d, 9.5 Hz, Ha-1), 3.78 (d, 9.5 Hz, Hb-1), 3.71 (s, 2 x OMe), 3.40 (d, 14.6 Hz, Ha-10), 2.61 (d,
14.6 Hz, Hb-10), 2.23 (s, H3-11), 2.16 (s, H3-12), 1.00 (s, H3-8ʹ′); 13C NMR (150 MHz, CD3CN) δC 192.8
(C-2), 169.1 (C-2ʹ′), 158.2 (C-6), 156.8 (C-8), 145.6 (C-7ʹ′a), 137.8 (C-4ʹ′a), 134.6 (C-4), 129.2 (C-5), 124.4
(C-5), 123.7 (C-4ʹ′), 123.2 (C-6ʹ′), 122.8 (C-7), 121.6 (C-9), 111.9 (C-7ʹ′), 102.6 (C-3), 61.2 (OMe), 60.3
(OMe), 48.7 (C-3ʹ′), 32.5 (C-10), 21.5 (C-8ʹ′), 15.3 (C-12), 9.8 (C-11), 7.5 (C-1).
Key HMBC (red arrow) and NOE (purple arrow) correlations of 31
N
HO
O
OMe
OMeI
H
30
CD3CN, r.t.NH
OI
OMe
OMe
31
NHO
I
OMe
OMe
4'a7'a
7'
4'
6'
5'
2'3'
1
23
45
6
789
1011
12
8'
4'
NH O I
H H
MeO
OMe8'
3'
10
1
12
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35
Synthesis of 32 and 33
To a solution of 2 (10.0 mg, 0.05 mmol) and 1,2,3,4-tetrahydrocarbazole (17.1 mg, 0.10 mmol) in CH3CN
(0.5 ml), BF3OEt2 (0.6 µl, 0.005 mmol) was added at room temperature. After being stirred for 30 min, the
reaction was quenched with water and then extracted with EtOAc. The combined organic extracts were
washed with saturated aqueous solution of brine, dried over Na2SO4, and concentrated. The residue was
purified by silica-gel column chromatography to afford 32 (2.6 mg) and 33 (13.0 mg).
Compound 32: Colorless amorphous; UV (EtOH) λmax nm (log ε) 208 (4.58), 289 (3.68); IR (KBr) ν
max (cm-1) 3361, 3008 2932, 2858, 1699, 1612, 1591, 1462, 1356, 1222, 1122, 1049, 1001; HRESIMS: m/z
378.2038 [M+H]+ (378.2064 calcd for C24H28O3N); 1H NMR (600 MHz, CDCl3) δH 7.00 (td, 7.3, 1.1 Hz,
H-6ʹ′), 6.92 (brd, 7.3 Hz, H-4ʹ′), 6.72 (td, 7.3, 1.1 Hz, H-5ʹ′), 6.68 (brd, 7.3 Hz, H-7ʹ′), 4.57 (s, OH), 4.39 (s,
NH), 3.73 (s, H2-3), 2.94 (d, 16.2 Hz, Ha-10), 2.82 (d, 16.2 Hz, Hb-10), 2.09 (s, H3-1), 2.07 (s, H3-12), 2.01
(m, Ha-11ʹ′), 2.00 (s, H3-11), 1.77 (m, Hb-11ʹ′), 1.76 (m, Ha-8ʹ′), 1.69 (m, Ha-10ʹ′), 1.63 (m, Hb-10ʹ′), 1.51 (m,
Hb-8ʹ′), 1.48 (m, Ha-9ʹ′), 1.41 (m, Hb-9ʹ′) 1.66 (s, H3-8ʹ′); 13C NMR (150 MHz, CDCl3) δC 207.0 (C-2), 150.9
(C-8), 150.7 (C-6), 147.3 (C-7ʹ′a), 136.6 (C-4ʹ′a), 129.3 (C-4), 127.3 (C-6ʹ′), 121.3 (C-4ʹ′), 119.6 (C-5ʹ′), 114.5
(C-5), 112.2 (C-9), 110.2 (C-7), 109.8 (C-7ʹ′), 98.1 (C-2ʹ′), 45.6 (C-3ʹ′), 44.8 (C-3), 37.7 (C-8ʹ′), 31.6 (C-11ʹ′),
29.1 (C-1), 28.1 (C-10), 22.8 (C-10ʹ′), 20.6 (C-9ʹ′), 12.1 (C-12), 8.5 (C-11).
Compound 33: Yellow oil; UV (EtOH) λmax nm (log ε) 205 (4.47), 231 (4.44), 284 (3.82); IR (KBr) ν
max (cm-1) 3394, 2930, 2854, 1637, 1706, 1616, 1445, 1358, 1326, 1234, 1162, 1110; HREIMS: m/z
377.1977 [M]+ (377.1991 calcd for C24H27O3N); 1H NMR (600 MHz, CDCl3) δH 7.57 (s, NH), 7.33 (d, 8.0
Hz, H-4ʹ′), 6.89 (dd, 8.0, 1.4 Hz, H-5ʹ′), 6.83 (d, 1.4 Hz, H-7ʹ′), 4.00 (s, H2-10), 3.76 (s, H2-3), 2.64 (m, H2-8ʹ′),
2.61 (m, H2-11ʹ′), 2.08 (s, H3-11), 2.07 (s, H3-12), 2.05 (s, H3-1), 1.83 (m, H2-10ʹ′ or 9ʹ′), 1.81 (m, H2-9ʹ′ or
10ʹ′); 13C NMR (150 MHz, CDCl3) δC 207.1 (C-2), 151.2 (C-8), 151.2 (C-6), 136.2 (C-7ʹ′a), 134.2 (C-2ʹ′),
131.7 (C-6ʹ′), 130.8 (C-4), 126.5 (C-4ʹ′a), 119.5 (C-5ʹ′), 118.6 (C-9), 117.9 (C-4ʹ′), 115.2 (C-5), 110.0 (C-7),
109.8 (C-3ʹ′), 109.6 (C-7ʹ′), 45.3 (C-3), 32.9 (C-10), 29.4 (C-1), 23.2 (C-11ʹ′), 23.1 (C-10ʹ′), 23.1 (C-9ʹ′), 20.9
(C-8ʹ′), 12.3 (C-12), 8.8 (C-11).
Key HMBC (red arrow), 1H-1H COSY (blue bold line) and NOE (purple arrow) correlation of 32 and 33
NH
O OH
O
NHO
OHHO
32 33
2'
4'5'
6'
7'
4'a7'a
3' 8'
9'10'
11'
1
23
45
12
6
7
11
8
9
10 2'
4'5'
6'
7'
4'a7'a
3'
8'9'
10'11'
O
OH
HO
2
BF3OEt2 (10 mol%),CH3CN, r.t.
NH
O OH
O
NHO
OHHO
32 (14%) 33 (71%)
+
NH
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36
Evaluation of anti-adenovirus activities.
A human T-cell leukemia cell line, MT-2, and a human lung caricinoma cell line, A549 were grown in
RPMI1640 and DMEM media, respectively, supplemented with 10% fatal calf serum (FCS), 2 mM
L-glutamine, 100 units/mL of penicillin, and 100 µg/mL of streptomycin. Cells were incubated at 37°C in a
humidified atmosphere with 5% CO2. Human Adenovirus type 19 was propagated in A549 cells and stored
at -80°C until use. Anti-adenovirus activity was evaluated with the MTT-colorimetric method as described
previously 4 with some modifications. Breifly, MT-2 cells (2 x 104 cells/well) were mixed with 103 TCID50
(50% tissue culture infectious dose) in the presence and absence of various concentrations of the compounds
in 96well plate and incubated for 5 days. At the end of the incubation period, the cytopathic effect (CPE) was
evaluated by the MTT assay. The 50% antiviral effective concentration (EC50) was defined as the
concentration that achieved 50% protection of virus-infected cells against virus-induced destruction.
Parallelly, we determined EC50 value of cidofovir as a control (EC50 = 24 µM).
2) Kodama, E., Shigeta, S., Suzuki, T. & De Clercq, E. Application of a gastric cancer cell line (MKN-28)
for anti-adenovirus screening using the MTT method. Antiviral Res 31, 159-164 (1996).
© 2015 Macmillan Publishers Limited. All rights reserved
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37
OH
HO
O
1 H N
MR
(ace
tone
-d6)
of 2
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
38
OH
HO
O
13C
NMR(
(ace
tone
-d6)
of 2
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
39
1 H N
MR
(pyr
idin
e-d 5
) of 5
OHO
OH
O
O
OH
H
© 2015 Macmillan Publishers Limited. All rights reserved
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
40
OHO
OH
O
O
OH
H
13C
NMR
(pyr
idin
e-d 5
) of 5
© 2015 Macmillan Publishers Limited. All rights reserved
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
41
O
O
OHO
HO O 1 H N
MR
(CDC
l 3) o
f 6
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
42
13C
NMR
(CDC
l 3) o
f 6
O
O
OHO
HO O
© 2015 Macmillan Publishers Limited. All rights reserved
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
43
1 H N
MR
(CDC
l 3 +
10%
CD 3
OD)
of 7
OHO
OH
O
O
OH
H
© 2015 Macmillan Publishers Limited. All rights reserved
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44
13C
NMR
(CDC
l 3 +
10%
CD 3
OD)
of 7
OHO
OH
O
O
OH
H
© 2015 Macmillan Publishers Limited. All rights reserved
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
45
1 H
NM
R (C
DCl 3
+ 10
% C
D 3O
D) o
f 8
OHO
OH
O
O
OH
H
© 2015 Macmillan Publishers Limited. All rights reserved
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
46
13C
NMR
(CDC
l 3 +
10%
CD 3
OD)
of 8
OHO
OH
O
O
OH
H
© 2015 Macmillan Publishers Limited. All rights reserved
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47
1 H N
MR
(CDC
l 3 +
10%
CD 3
OD)
of 9
O
OHO
OH
OH
O
OH
HO
O
H
© 2015 Macmillan Publishers Limited. All rights reserved
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
48
13C
NMR
(CDC
l 3 +
10%
CD 3
OD)
of 9
O
OHO
OH
OH
O
OH
HO
O
H
© 2015 Macmillan Publishers Limited. All rights reserved
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
49
1 H N
MR
(CDC
l 3 +
10%
CD 3
OD)
of 10
O
O
O
HOHO O
OH
OH
O
H
© 2015 Macmillan Publishers Limited. All rights reserved
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50
13C
NMR
(CDC
l 3 +
10%
CD 3
OD)
of 10
O
O
O
HOHO
OO
H
OH
O
H
© 2015 Macmillan Publishers Limited. All rights reserved
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
51
1H N
MR
(CD 3
OD)
of 11
OH
HO
O
O
O
OH
H
© 2015 Macmillan Publishers Limited. All rights reserved
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
52
13C
NMR
(CD 3
OD)
of 11
OH
HO
O
O
O
OH
H
© 2015 Macmillan Publishers Limited. All rights reserved
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
53
1 H N
MR
(CDC
l 3 +
10%
CD 3
OD)
of 12
HO
OH
O
HOO
H
H
O
© 2015 Macmillan Publishers Limited. All rights reserved
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
54
13C
NMR
(CDC
l 3 +
10%
CD 3
OD)
of 12
HO
OH
O
HOO
H
H
O
© 2015 Macmillan Publishers Limited. All rights reserved
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
55
1H N
MR
(CDC
l 3) o
f 13
O
O
O
OH
H
OHOH
© 2015 Macmillan Publishers Limited. All rights reserved
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
56
13
C NM
R (C
DCl 3)
of 13
O
O
O
OH
H
OHOH
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
57
1H N
MR
(CDC
l 3) o
f 14
O
O
O
OH
H
OHOH
© 2015 Macmillan Publishers Limited. All rights reserved
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
58
1 H N
MR
(CDC
l 3 +
10%
CD 3
OD)
of 15
OO
O
O
O
OH
H
HO
HO
© 2015 Macmillan Publishers Limited. All rights reserved
NATURE CHEMISTRY | www.nature.com/naturechemistry 59
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
59
13C
NMR
(CDC
l 3 +
10%
CD 3
OD)
of 15
OO
O
O
O
OH
H
HO
HO
© 2015 Macmillan Publishers Limited. All rights reserved
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
60
1 H N
MR
(CDC
l 3) o
f 16
OO
O
O
O
OH
H
HO
HO
© 2015 Macmillan Publishers Limited. All rights reserved
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
61
13C
NMR
(CDC
l 3) o
f 16
OO
O
O
O
OH
H
HO
HO
© 2015 Macmillan Publishers Limited. All rights reserved
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
62
1 H N
MR
(CDC
l 3) o
f 17
O
O
O
OH
H
ON
OHO
O
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
63
13C
NMR
(CDC
l 3) o
f 17
O
O
O
OH
H
ON
OHO
O
© 2015 Macmillan Publishers Limited. All rights reserved
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
64
1 H N
MR
(CDC
l 3) o
f 18
O
O
O
OH
H
ON
OHO
O
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
65
1H N
MR
(CD 3
OD)
of 19
HO
OH
NH
HOO
H
H
O
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
66
13C
NMR
(CD 3
OD)
of 19
HO
OH
NH
HOO
H
H
O
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
67
1 H N
MR
(CDC
l 3) o
f 20
N H
OH
O
OH
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
68
13C
NMR
(CDC
l 3) o
f 20
N H
OH
O
OH
© 2015 Macmillan Publishers Limited. All rights reserved
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
69
1 H
NM
R (a
ceto
ne-d
6) o
f 21
N H
HO
OH
N H
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
70
13C
NMR
(ace
tone
-d6)
of 21
N H
HO
OH
N H
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
71
1H N
MR
(CD 3
OD)
of 22
N
HO
OH
OH
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
72
13C
NMR
(CD 3
OD)
of 22
N
HO
OH
OH
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
73
1H N
MR
(CD 3
OD)
of 23
N
HO
OH
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
74
13C
NMR
(CD 3
OD)
of 23
N
HO
OH
© 2015 Macmillan Publishers Limited. All rights reserved
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
75
1 H N
MR
(CDC
l 3) o
f 24
N H
OH
O
OH
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NATURE CHEMISTRY | www.nature.com/naturechemistry 76
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
76
13C
NMR
(CDC
l 3) o
f 24
N H
OH
O
OH
© 2015 Macmillan Publishers Limited. All rights reserved
NATURE CHEMISTRY | www.nature.com/naturechemistry 77
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
77
1 H N
MR
(CDC
l 3) o
f 25
N
OM
e OM
e
O
© 2015 Macmillan Publishers Limited. All rights reserved
NATURE CHEMISTRY | www.nature.com/naturechemistry 78
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
78
13C
NMR
(CDC
l 3) o
f 25
N
OM
e OM
e
O
© 2015 Macmillan Publishers Limited. All rights reserved
NATURE CHEMISTRY | www.nature.com/naturechemistry 79
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
79
1 H N
MR
(CDC
l 3) o
f 26
N H
O
OH
OH
© 2015 Macmillan Publishers Limited. All rights reserved
NATURE CHEMISTRY | www.nature.com/naturechemistry 80
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
80
13C
NM
R (C
DCl 3)
of 26
N H
O
OH
OH
© 2015 Macmillan Publishers Limited. All rights reserved
NATURE CHEMISTRY | www.nature.com/naturechemistry 81
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
81
1 H N
MR
(ace
tone
-d6)
of 27
NO
H
OH
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NATURE CHEMISTRY | www.nature.com/naturechemistry 82
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
82
13C
NMR
(ace
tone
-d6)
of 27
NO
H
OH
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NATURE CHEMISTRY | www.nature.com/naturechemistry 83
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
83
1 H N
MR
(CDC
l 3) o
f 28
N
O
OM
e
OM
e
HO
© 2015 Macmillan Publishers Limited. All rights reserved
NATURE CHEMISTRY | www.nature.com/naturechemistry 84
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
84
13C
NMR
(CDC
l 3) o
f 28
N
O
OM
e
OM
e
HO
© 2015 Macmillan Publishers Limited. All rights reserved
NATURE CHEMISTRY | www.nature.com/naturechemistry 85
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
85
1 H N
MR
(CDC
l 3) o
f 29
NO
HCO
Me
OM
e
© 2015 Macmillan Publishers Limited. All rights reserved
NATURE CHEMISTRY | www.nature.com/naturechemistry 86
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
86
13C
NM
R (C
DC
l 3) o
f 29
NO
HC
OM
e
OM
e
© 2015 Macmillan Publishers Limited. All rights reserved
NATURE CHEMISTRY | www.nature.com/naturechemistry 87
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
87
1 H N
MR
(CDC
l 3) o
f 30
NHO
O
OM
e
OM
eI
H
© 2015 Macmillan Publishers Limited. All rights reserved
NATURE CHEMISTRY | www.nature.com/naturechemistry 88
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
88
13C
NMR
(CDC
l 3) o
f 30
NHO
O
OM
e
OM
eI
H
88
13C
NMR
(CDC
l 3) o
f 30
NHO
O
OM
e
OM
eI
H
© 2015 Macmillan Publishers Limited. All rights reserved
NATURE CHEMISTRY | www.nature.com/naturechemistry 89
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
89
N HO
OM
e
OM
e
I1 H
NM
R (C
D 3CN
) of 31
© 2015 Macmillan Publishers Limited. All rights reserved
NATURE CHEMISTRY | www.nature.com/naturechemistry 90
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
90
N HO
OM
e
OM
e
I13
C NM
R (C
D 3CN
) of 31
© 2015 Macmillan Publishers Limited. All rights reserved
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
91
N HO
OH
O
1 H N
MR
(CDC
l 3) o
f 32
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NATURE CHEMISTRY | www.nature.com/naturechemistry 92
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
92
N HO
OH
O
13C
NMR
(CDC
l 3) o
f 32
© 2015 Macmillan Publishers Limited. All rights reserved
NATURE CHEMISTRY | www.nature.com/naturechemistry 93
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
93
1 H N
MR
(CDC
l 3) o
f 33
N HO
HOO
H
© 2015 Macmillan Publishers Limited. All rights reserved
NATURE CHEMISTRY | www.nature.com/naturechemistry 94
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
94
13C
NM
R (C
DCl
3) o
f 33
N HO
HO
OH
© 2015 Macmillan Publishers Limited. All rights reserved
NATURE CHEMISTRY | www.nature.com/naturechemistry 95
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
95
1 H N
MR
(CDC
l 3) o
f 34
and
35
O
O
O
O
O
OH
OO
HH
O HO
© 2015 Macmillan Publishers Limited. All rights reserved
NATURE CHEMISTRY | www.nature.com/naturechemistry 96
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
96
13C
NMR
(CDC
l 3) o
f 34
and
35
O
O
O
O
O
OHO
OH
HO
HO
© 2015 Macmillan Publishers Limited. All rights reserved
NATURE CHEMISTRY | www.nature.com/naturechemistry 97
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
97
1 H N
MR
(CDC
l 3) o
f 36O
O
ONHBo
c
OH
© 2015 Macmillan Publishers Limited. All rights reserved
NATURE CHEMISTRY | www.nature.com/naturechemistry 98
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
98
13C
NMR
(CDC
l 3) o
f 36
O
O
ONHBo
c
OH
© 2015 Macmillan Publishers Limited. All rights reserved
NATURE CHEMISTRY | www.nature.com/naturechemistry 99
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
99
1 H N
MR
(CDC
l 3) o
f 37
O
O
NOO
HO
© 2015 Macmillan Publishers Limited. All rights reserved
NATURE CHEMISTRY | www.nature.com/naturechemistry 100
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
100
13C
NMR
(CDC
l 3) o
f 37
O
O
NOO
HO
© 2015 Macmillan Publishers Limited. All rights reserved
NATURE CHEMISTRY | www.nature.com/naturechemistry 101
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
101
1 H N
MR
(CDC
l 3) o
f 38
N
O
OHO
HO
© 2015 Macmillan Publishers Limited. All rights reserved
NATURE CHEMISTRY | www.nature.com/naturechemistry 102
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
102
13C
NMR
(CDC
l 3) o
f 38
N
O
OHO
HO
© 2015 Macmillan Publishers Limited. All rights reserved
NATURE CHEMISTRY | www.nature.com/naturechemistry 103
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
103
OOO
O O
O
1 H N
MR
(CDC
l 3) o
f 39
© 2015 Macmillan Publishers Limited. All rights reserved
NATURE CHEMISTRY | www.nature.com/naturechemistry 104
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
104
OOO
O O
O
13C
NMR
(CDC
l 3) o
f 39
© 2015 Macmillan Publishers Limited. All rights reserved
NATURE CHEMISTRY | www.nature.com/naturechemistry 105
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
105
1 H N
MR
(CDC
l 3) o
f 40
OOO
O
O
O
© 2015 Macmillan Publishers Limited. All rights reserved
NATURE CHEMISTRY | www.nature.com/naturechemistry 106
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
106
13C
NMR
(CDC
l 3) o
f 40
OOO
O
O
O
© 2015 Macmillan Publishers Limited. All rights reserved
NATURE CHEMISTRY | www.nature.com/naturechemistry 107
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
107
1 H N
MR
(CDC
l 3) o
f 41
OO
OOO
O
© 2015 Macmillan Publishers Limited. All rights reserved
NATURE CHEMISTRY | www.nature.com/naturechemistry 108
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
108
13C
NMR
(CDC
l 3) o
f 41
OO
OOO
O
© 2015 Macmillan Publishers Limited. All rights reserved
NATURE CHEMISTRY | www.nature.com/naturechemistry 109
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
109
1 H N
MR
(CDC
l 3) o
f 42
OOOO
O
O
© 2015 Macmillan Publishers Limited. All rights reserved
NATURE CHEMISTRY | www.nature.com/naturechemistry 110
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
110
13C
NMR
(CDC
l 3) o
f 42
OOOO
OO
© 2015 Macmillan Publishers Limited. All rights reserved
NATURE CHEMISTRY | www.nature.com/naturechemistry 111
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
111
1 H N
MR
(CDC
l 3) o
f 43
O
OOOO
O
© 2015 Macmillan Publishers Limited. All rights reserved
NATURE CHEMISTRY | www.nature.com/naturechemistry 112
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
112
13C
NMR
(CDC
l 3) o
f 43
O
OOOO
O
© 2015 Macmillan Publishers Limited. All rights reserved
NATURE CHEMISTRY | www.nature.com/naturechemistry 113
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
113
1 H N
MR
(CDC
l 3) o
f 44
O
OOO
O
O
© 2015 Macmillan Publishers Limited. All rights reserved
NATURE CHEMISTRY | www.nature.com/naturechemistry 114
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2308
114
13C
NMR
(CDC
l 3) o
f 44
O
OOO
O
O
© 2015 Macmillan Publishers Limited. All rights reserved