electronic supporting information (esi) · s5 iii. synthesis of citreorosein (19)2: scheme s1:...
TRANSCRIPT
S1
Electronic Supporting Information (ESI)
Chemoenzymatic, Biomimetic Total Synthesis of (‒)-Rugulosin B, C and
Rugulin Analogues and its Biosynthetic Implications
Amit Mondal, Shailesh Kumar Singh, Tanaya Manna, Syed Masood Husain*
I. General Remarks S2
II. Cloning, Expression and Purification of Enzymes S3–S4
III. Synthesis of citreorosein (19) S5–S6
IV. Chemoenzymatic reduction of anthraquinones S6–S9
V. Synthesis of (–)-flavoskyrin C (24) and (–)-flavoskyrin B/B’ (26/27) S10–S13
VI. “Cascade” conversion of (–)-flavoskyrin C (24) to
(–)-rugulosin C (ent-2) S13–S16
VII. “Cascade” conversion of (–)-flavoskyrin B/B’ (26/27) to
(–)-rugulosin B (ent-8) S17–S21
VIII. Mechanistic insight into the “Cascade” reaction of
(–)-flavoskyrin B/B’ (26/27) S22–S24
IX. Synthesis of rugulin analogues S24–S27
X. NMR Spectra S28–S60
XI. Circular Dichroism (CD) Spectra S61–S63
XII. HPLC Chromatograms S64
XIII. References S65
Electronic Supplementary Material (ESI) for ChemComm.This journal is © The Royal Society of Chemistry 2020
S2
I. General Remarks
All commercial reagents were obtained from Sigma-Aldrich Chemical Co., Sisco Research
Laboratories and Avra synthesis Pvt. Ltd, India. Reactions were monitored by thin-layer
chromatography (TLC, 0.25 mm E. Merck silica gel plates, 60F254) and the plates were
visualized by using UV light. Column chromatography was performed on silica gel 60–
120/230–400 mesh obtained from S. D. Fine Chemical Co., India. 0.2(N) Oxalic acid
impregnated silica gel was prepared by adding silica gel (230–400 mesh size) to a solution of
oxalic acid in H2O, filtered the resulting suspension to dryness under reduced pressure and then
activating at 120 °C overnight, and then finally cooling under argon. Evaporation of solvent
was achieved using a Büchi water bath B-481 rotary evaporator under reduced pressure (0 –
1000 mbar) with a bath temperature of 40 °C. Yields represent to chromatographically pure
materials; conversions were calculated from the 1H NMR spectra of the crude products. 1H
NMR spectra were recorded on Bruker 400 MHz and 800 MHz Ultra Shield instrument using
deuterated solvents. Proton coupling constants (J) are reported as absolute values in Hz. 13C
NMR spectra were recorded on Bruker 400 MHz and 800 MHz Ultra Shield instrument
operating at 100 MHz and 200 MHz respectively. Chemical shifts (δ) of the 1H and 13C NMR
spectra are reported in ppm with a solvent resonance as an internal standard. For 1H NMR:
chloroform-d1 7.26, acetone-d6 2.05, DMSO-d6 2.50, Pyridine-d5 (7.22) THF-d8 (δ 1.72, 3.58);
for 13C NMR: chloroform-d1 77.16, acetone-d6 29.84, THF-d8 (δ 25.3, 67.21). The following
abbreviations were used to explain the multiplicities: s = singlet, d = doublet, dd = doublet of
a doublet, ddd = doublet of a doublet of doublet, t = triplet, dt = doublet of a triplet, q = quartet,
quint = quintet, m = multiplet, br = broad, ar = aromatic. IR spectra were recorded on a
PerkinElmer Spectrum Two FT-IR Spectrometer. Electrospray ionization (ESI) mass
spectrometry (MS) experiments were performed on an Agilent 6530 Accurate-Mass Q-TOF
LC/MS system (Agilent Technologies). Optical rotations were measured on a DigiPol 781
M6U Automatic Polarimeter. CD spectroscopy was carried out on a Jasco J-1500
spectrophotometer (Jasco International Co.) equipped with Spectra ManagerTM software. UV
spectroscopy and activity measurements were performed on Cary 300 UV/Vis
spectrophotometer (Agilent Technologies). For determination of the enantiomeric excess (ee)
the chiral phases Chiralcel OD-H (Daicel Inc., 250 × 4.6 mm, 5 µm) and Chiralpak IC (Daicel
Inc., 250 × 4.6 mm, 5 µm) were used on Agilent Technologies 1260 Infinity HPLC system
equipped with OpenLAB CDS v2.3 software.
S3
II. Cloning, Expression and Purification of Enzymes1
Gene synthesis and expression vector
The synthesis of codon-optimized gene encoding ARti (NCBI GenBank accession:
CRG86682.1) was ordered from Biomatik Company (Biomatik, Ontario, Canada). The gene
was cloned into pET-19b vector using 5’-NdeI and 3’-XhoI yielding (N)his10-tagged ARti.
Plasmid encoding Glucose dehydrogenase (GDH) was obtained from Prof. Werner Hummel
(University of Bielefeld, Germany).
Transformation of the plasmid to E. coli cells
Transformation of plasmid DNA to competent E. coli BL21 (DE3) cells was performed by
applying a heat shock at 42 °C for 50 s. The transformed cells were grown overnight on SOB-
agar medium containing 100 µg/mL ampicillin.
Media and growth conditions
One clone was picked and dispersed in 5 mL of LB-media (Lennox), followed by incubation
overnight (37 °C, 220 rpm). Ampicillin (100 µg/mL) was added as required.
Cultivation and expression
ARti: The overnight cultures were diluted to 500 mL of medium each (ampicillin 100 µg/mL)
and incubated at 37 °C, 160 rpm. IPTG (0.2 mM) was added to the mid-log phase (OD600 =
0.6) was reached, and cultures were incubated for 20 h at 18 °C, 160 rpm.
GDH: The overnight cultures were diluted to 500 mL of medium each (ampicillin 100 µg/mL)
and incubated at 37 °C, 160 rpm. IPTG (0.2 mM) was added to the mid-log phase (OD600 =
0.6) was reached, and cultures were incubated for 4 h at 37 °C, 160 rpm.
Workup and storage
ARti: The harvested E. coli cells were resuspended in lysis buffer (20 mM Tris-HCl, pH 8.0;
2.5 mL per harvested cells of 500 mL medium).
GDH: The harvested E. coli cells were resuspended in lysis buffer (50 mM HEPES, pH = 8.0;
2.5 mL per harvested cells of 500 mL medium).
The cells were disrupted by sonication (8 x 10 s, Vibra-Cell Processors, model number
VCX500, Sonics), followed by centrifugation (30 min, 12000×g, 4 °C). Glycerol (20% v/v)
was added, and the crude enzyme preparation was frozen at –20 °C.
S4
Enzyme purification
ARti was purified by Ni-NTA affinity chromatography. Non-specifically bound proteins were
washed off with 5 mM and 20 mM imidazole in Tris buffer (25 mM Tris-HCl, pH 8.0). Elution
was performed with 25 mM Tris buffer (pH = 8.0) containing 50 and 250 mM imidazole. The
fractions containing purified proteins are collected and desalted by gel filtration (Econo-Pac
10DG desalting gel column, Bio-Rad). The concentration of the protein was performed by
ultrafiltration (Vivaspin 20R centrifugal filter units, 10 kDa nominal molecular weight limit,
Sartorius). The concentration of the protein was determined by measuring the UV absorption
at 280 nm (NanoVue, GE Healthcare; extinction coefficient 16055 M–1·cm–1, molecular weight
30.95 kDa).
S5
III. Synthesis of citreorosein (19)2:
Scheme S1: Synthesis of citreorosein (19)
1,3,8-trihydroxy-6-(hydroxymethyl) anthracene-9,10-dione (19)
C15H10O6: 286.23 g/mol
The synthesis of citreorosein (19) was achieved by the four-step process by using commercially
available emodin (15). At first acetylation of emodin (15) at phenolic hydroxy group and then
chromium oxide (CrO3) oxidation converted it to triacetyl emodic acid (TAEA). Finally,
reduction of the carboxylic acid with boron dimethyl sulphate (BMS) and deprotection of acetyl
group with basic hydrolysis provides citreorosein 19 with overall yield 33% in four steps.
TLC (CHCl3/MeOH, 9:1 v/v): Rf = 0.46.
Column chromatography: silica gel (230-400 mess size), CH2Cl2: MeOH :: 19:1.
Yield: 33%
1H NMR (400 MHz, DMSO-d6): δ (ppm) 4.57 (s, 2H, CH2), 6.51 (d, J = 2.4 Hz, 1H, CHar),
7.04 (d, J = 2.4 Hz, 1H, CHar), 7.17 (s, 1H, CHar), 7.55 (s, 1H, CHar), 12.00 (s, 2H, phenolic
OH).
S6
13C NMR (100 MHz, DMSO-d6): δ (ppm) 62.4, 108.3, 109.2, 109.3, 114.4, 117.4, 121.1,
133.2, 135.4, 153.2, 161.9, 164.9, 166.1, 181.6, 190.0.
FT-IR (neat) νmax: 3412, 2923, 2853, 1676, 1628, 1476, 1436, 1383, 1314, 1258, 1173, 1050,
1026, 989, 875, 758, 703, 566 cm‒1
IV. Chemoenzymatic reduction of anthraquinones
Scheme S2: Stereo- and regioselective reduction of emodin (15) and citreorosein (19).
General procedure: 50 mM Potassium phosphate buffer (mixed with 1 mM EDTA, 1 mM
DTT, pH 7; 100 mL) was degassed under reduced pressure for 20 min using argon medium.
Under argon counterflow, D-glucose (333.7 mg, 1.85 mmol, 5.0 equiv.), NADP+ (29 mg, 37.0
µmol, 0.1 equiv.), Na2S2O4 (1289 mg, 7.4 mmol, 20.0 equiv.), and substrate (15 & 19); (100.0
mg, 370 µmol) in DMSO (10 mL), GDH (150 U), and ARti (5 mL, 2 mg/mL) were added to
the buffer and the mixture was stirred under argon atmosphere for 14 h. The solution was
extracted with ethyl acetate (EtOAc, 3 x 50 mL), dried over Na2SO4, and the solvent was
removed under reduced pressure. The crude reaction mixture was subjected for the column
chromatography to afford the pure product as brown solid.
(R)-3,8,9,10-Tetrahydroxy-6-methyl-3,4-dihydroanthracen-1(2H)-one (17)
C15H14O5: 274.27 g·mol-1
TLC: (EtOAc/hexane, 1:1 v/v): Rf = 0.29.
Column chromatography: silica gel (60-120 mess size), EtOAc:hexane :: 4:1.
Yield: 74%
S7
1H NMR (400 MHz, acetone-d6): δ (ppm) 2.44 (s, 3H, CH3), 2.80 (ddd, J = 17.1 Hz, J = 7.1
Hz, J = 1.1 Hz, 1H), 3.0 (dd, J = 17.1 Hz, J = 2.9 Hz, 1H), 3.07 (dd, J = 16.4 Hz, J = 6.8 Hz,
1H), 3.26 (dd, J = 16.4 Hz, J = 3.6 Hz, 1H), 4.37 (bs, 1H, OH), 4.42–4.48 (m, 1H), 6.69 (s, 1H,
CHar), 7.47 (s, 1H, CHar), 7.64 (s, 1H, phenolic OH), 9.78 (s, 1H, phenolic OH), 15.94 (s, 1H,
phenolic OH).
13C NMR (100 MHz, acetone-d6): δ (ppm) 21.5, 31.7, 45.8, 65.0, 109.1, 110.7, 112.5, 112.6,
116.8, 133.1, 140.7, 142.8, 158.0, 159.3, 204.0.
FT-IR (neat) νmax: 3331, 2920, 2855, 1703, 1635, 1597, 1560, 1511, 1396, 1370, 1322, 1257,
1183, 1044, 827, 740, 692 cm‒1
[α]D27 = + 45.0 (c = 0.05 g/100 mL in acetonitrile).
CD (c 50 µM, 1,4-dioxane), λ [nm] (mdeg): 220 (0.13), 224 (–1.14), 231 (–1.90), 240 (–0.73),
245 (–0.41), 260 (–1.31), 267 (–1.76), 280 (–0.44), 288 (–0.16), 295 (–0.21), 323 (0.27), 350
(0.00), 418 (0.19).
Exact Mass [M+H]+: 275.0914 (calculated), 275.0909 (found).
HPLC [Flow rate: 1 mL/min; Typical injection volume: 5 µL; Isocratic: 95% n-Hexane, 5%
Isopropanol; DAD: 280 nm (bandwidth = 4nm); Column: Chiralcel OD-H, 5 µm, 4.6 mm (ɸ)
x 250 mm (L) mm, Temperature: 20 °C.]: Retention time (Rt), (17) = 62.45 min.; >99% ee
(determined by comparison to rac-17, Rt [(S)-17] = 52.27 min, Rt [(R)-17] = 63.23 min).
C15H10O4: 254.24 g.mol-1
TLC: (EtOAc/hexane, 1:1 v/v): Rf = 0.8.
Column chromatography: silica gel (60-120 mess size), EtOAc:hexane :: 1:9.
Yield: 12%
1H NMR (400 MHz, CDCl3): δ (ppm) 2.47 (CH3), 7.10 (brs, 1H), 7.29 (dd, J = 5.0 Hz,
J = 2.4 Hz, 1H, CHar), 7.65–7.69 (m, 2H, CHar), 7.82 (dd, J = 7.5 Hz, J = 1.1 Hz, 1H, CHar),
11.92 (s, 1H, phenolic OH), 12.12 (s, 1H, phenolic OH).
S8
13C NMR (100 MHz, CDCl3): δ (ppm) 22.4, 113.9, 116.0, 120.1, 121.5, 124.5, 124.7, 133.4,
133.8, 137.1, 149.5, 162.6, 162.9, 182.1, 192.7.
FT-IR (neat) νmax: 2916, 2849, 1707, 1675, 1626, 1476, 1452, 1365, 1271, 1210, 1160, 839,
751 cm‒1
Exact Mass [M+H]+: 255.0652 (calculated), 255.0654 (found).
(R)-3,8,9,10-tetrahydroxy-6-(hydroxymethyl)-3,4-dihydroanthracen-1(2H)-one (21)
C15H14O6: 290.27 g.mol-1
TLC (CHCl3/MeOH, 9:1 v/v): Rf = 0.2.
Column chromatography: silica gel (230-400 mess size), CH2Cl2 : MeOH :: 4:1.
Yield: 79%
1H NMR (400 MHz, acetone-d6): δ (ppm) 2.82 (dd, J = 17.5 Hz, J = 7.5 Hz, 1H), 3.01 (dd, J
= 17.1 Hz, J = 3.3 Hz, 1H), 3.09 (dd, J = 16.3 Hz, J = 6.7 Hz, 1H), 3.28 (dd, J = 16.4 Hz, J =
3.7 Hz, 1H), 4.39 (brs, 1H, OH), 4.42–4.49 (m, 1H), 4.75 (d, J = 5.6 Hz, 2H), 6.84 (s, 1H,
CHar), 7.67 (s, 1H, CHar), 7.69 (s, 1H, phenolic OH), 9.81 (s, 1H, phenolic OH), 15.90 (s, 1H,
phenolic OH).
13C NMR (100 MHz, acetone-d6): δ (ppm) 32.6, 46.8, 64.7, 66.0, 110.0, 110.3, 110.6, 112.4,
117.8, 133.9, 142.1, 148.1, 159.0, 160.0, 205.1.
FT-IR (neat) νmax: 3346, 2961, 2884, 1769, 1636, 1459, 1441, 1370, 1188, 1067, 1036, 961,
926, 850, 559 cm‒1
[α]D27 = + 22.4 (c = 0.025 g/100 mL, acetonitrile).
S9
CD (c 50 µM, 1,4-dioxane) λ [nm] (mdeg): 215 (–1.39), 228 (–1.15), 240 (–0.45), 250 (–
1.10), 259 (–1.44), 270 (–1.34), 300 (–0.09), 310 (0.41), 320 (0.60), 348 (0.22), 360 (0.35),
380 (0.78), 419 (1.53), 450 (1.09), 497 (0.09).
Exact Mass [M+H]+: 290.0790 (calculated), 290.0795 (found).
HPLC [Flow rate: 0.5 mL/min; Typical injection volume: 5 µL; Isocratic: 85% n-Hexane, 15%
Isopropanol; DAD: 280 nm (bandwidth = 4 nm); Column: Chiralpak IC, 5 µm, 4.6 mm (ɸ) x
250 mm (L) mm, Temperature: 20 °C.]: Retention time (Rt), (21) = 47.94 min.; >99% ee
(determined by comparison to rac-21, Rt [(S)-21] = 41.29 min, Rt [(R)-21] = 47.94 min).
C15H10O5: 270.05 g.mol-1
TLC (CHCl3/MeOH, 9:1 v/v): Rf = 0.59.
Column chromatography: silica gel (230-400 mess size), MeOH : CH2Cl2 :: 1:9.
Yield: 10%
1H NMR (800 MHz, DMSO-d6): δ (ppm) 4.63 (d, J = 5.6 Hz, 2H, CH2), 5.62 (t, J = 5.6 Hz,
1H, OH), 7.29 (s, 1H, CHar), 7.39 (d, J = 8.2 Hz, 1H, CHar), 7.69 (s, 1H, CHar), 7.72 (d, J = 7.6
Hz, 1H, CHar), 7.81 (t, J = 7.9 Hz, 1H, CHar), 11.96 (s, 2H, phenolic OH).
13C NMR (100 MHz, DMSO-d6): δ (ppm) 62.1, 114.5, 116.0, 117.1, 119.4, 120.7, 124.5,
133.2, 133.4, 137.4, 153.7, 161.4, 131.6, 181.5, 191.7.
FT-IR (neat) νmax: 3322, 2924, 2853, 1713, 1674, 1625, 1458, 1376, 1274, 1204, 1157, 1087,
753 698 cm‒1
Exact Mass [M+H]+: 271.0601 (calculated), 271.0603 (found).
S10
V. Synthesis of (–)-flavoskyrin C (24)
To a solution of 21 (42.0 mg, 144.0 μmol, 1.0 equiv.) in AcOH (0.600 mL) cooled at 0 ºC, a
suspension of Pb(OAc)4 (65.0 mg, 144.0 μmol, 1.0 equiv.) in AcOH (1.0 mL) was added. The
reaction mixture was stirred at room temperature and the reaction mixture become dark brown
colour. After 20 min, ice-cold water was added to the dark brown solution. Then, the reaction
mixture was extracted with EtOAc (3 x 40 mL), and the organic layer was removed under
reduced pressure. The crude compound was dissolved in CHCl3/MeOH (9:1, 4.0 mL) and
spread onto a preparative TLC plate. After 2 h, the silica was scratched off the plate and washed
with CHCl3/MeOH (8:2) to obtain a filtrate. After that removal of the solvent under reduced
pressure, column chromatography using silica gel impregnated with 0.2 (N) oxalic acid and
benzene/acetone (7:3) as eluent afforded pure (–)-flavoskyrin C (24) (20.0 mg, 48 %) as a
yellow solid. During this transformation aloe-emodin (22) (9 mg, 23%) isolated as a side
product.
C30H24O12: 576.12 g.mol-1
TLC: (CHCl3/MeOH, 9:1 v/v): Rf = 0.46.
Column chromatography: silica gel (230-400 mess size) impregnated with oxalic acid;
benzene:acetone :: 7:3.
Yield: 48%
1H NMR (800 MHz, THF-d8): δ (ppm) 2.76 (dd, J = 17.6 Hz, J = 10.6 Hz, 1H, H'-5), 2.87
(dd, J = 17.2 Hz, J = 11.5 Hz, 1H, H-2), 2.97 (dd, J = 17.5 Hz, J = 5.2 Hz, 1H, H-5), 3.07–3.11
(m, 2H, H'-2 and H-3a), 3.20 (d, J = 10.8 Hz, 1H, H-3b), 4.05 (d, J = 15.0 Hz, 1H), 4.06–4.11
(m, 1H), 4.22 (d, J = 13.5 Hz, 1H), 4.26 (d, J = 15.0 Hz, 1H), 4.39–4.35 (m, 1H), 4.41 (d, J =
S11
13.5 Hz, 1H), 6.21 (s, 1H, H-16, CHar), 6.41 (s, 1H, H-9, CHar), 6.82 (s, 1H, H-11, CHar), 7.22
(s, 1H, H-14, CHar), 9.62 (s, 1H, phenolic OH), 11.34 (s, 1H, phenolic OH), 14.38 (s, 1H,
phenolic OH), 16.01 (s, 1H, phenolic OH).
Assignment of proton is done by the help of 2D-COSY and 2D-HSQC.
13C NMR (100 MHz, THF-d8): δ (ppm) 39.8, 41.4, 43.4, 47.8, 63.2, 64.3, 66.5, 68.9, 83.6,
106.0, 108.3, 109.5, 110.6, 112.2, 115.4, 116.5, 120.0, 122.0, 133.1, 136.2, 140.4, 148.7, 154.1,
159.0, 161.9, 162.4, 181.1, 193.5, 195.9, 202.8.
FT-IR (neat) νmax: 3411, 2923, 2854, 1698, 1665, 1620, 1574, 1482, 1365, 1293, 1251, 1056,
855, 760, 721 cm‒1
[α]D27 = – 55.80 (c = 0.025 g/100 mL in 1,4-dioxane).
CD (c 50 µM, 1,4-dioxane), λ [nm] (mdeg): 216 (0.42), 225 (44.69), 239 (7.57), 255 (44.45),
263 (−8.44), 274 (−82.39), 290 (−20.53), 308 (−6.38), 322 (−1.20), 331 (0.52), 344 (−1.71),
360 (3.18), 373 (5.75), 392 (1.64), 407 (−1.11), 421 (−1.48).
Exact Mass [M+H]+: 576.1268 (calculated), 576.1254 (found).
Synthesis of (–)-flavoskyrin B/B’ (26/27)
To a mixture of 21 (20 mg, 68.96 μmol, 1 equiv.) and 17 (38 mg, 138.6 μmol, 2 equiv.) in
AcOH (1 mL) cooled at 0 ºC, a suspension of Pb(OAc)4 (93 mg, 207.56 μmol, 3 equiv.) in
AcOH (1.6 mL) was added. The reaction mixture was stirred at room temperature and changes
its colour to dark brown colour. After 20 min, ice-cold water was added to the dark brown
solution. Then, the reaction mixture was extracted with EtOAc (3 x 30 mL), and the organic
layer was removed under reduced pressure. The crude compound was dissolved in
CHCl3/MeOH (8:2, 4 mL) and spread onto a preparative TLC plate. After 2 h, the silica was
scratched off the plate and washed with CHCl3/MeOH (8:2) to obtain a filtrate. TLC of the
filtrate shows four individual spots along with (−)-flavoskyrin A. After that removal of the
solvent under reduced pressure, column chromatography afforded flavoskyrin analogue of
rugulosin B (26/27) as yellow solid as two diastereomeric mixture as a major product.
S12
C30H24O11: 560.51 g.mol-1
TLC: (CHCl3/MeOH, 9:1 v/v): Rf = 0.28.
Column chromatography: silica gel (230-400 mess size) impregnated with oxalic acid;
benzene:acetone :: 4:1.
Yield: 55 %
1H NMR (400 MHz, THF-d8): δ (ppm) 1.95 (s, 3H, CH3), 2.08 (s, 3H, CH3), 2.70 (dd, J =10.5
Hz, 3 Hz, 1H), 2.74 (dd, J =10.5 Hz, 3 Hz, 1H), 2.78−2.86 (m, 5H), 2.90−2.96 (m, 5H),
3.03−3.09 (m, 5 H), 3.13 (dd, J = 8 Hz, 2Hz, 1H), 3.16 (dd, J = 8 Hz, 2Hz, 1H), 4.06−4.09 (m,
2H), 4.09 (d, J = 15.4 Hz, 2H), 4.23 (d, J = 15.4 Hz, 2H), 4.34 (ddd, J = 20.3 Hz, 13.7 Hz, 8.1
Hz, 3H), 6.12 (d, J = 1.1 Hz, 1H, CHar), 6.26 (d, J = 1.3 Hz, 1H, CHar), 6.33 (s, 1H, CHar), 6.46
(s, 1H, CHar), 6.52 (d, J = 1.2 Hz, 1H, CHar), 6.78 (d, J = 1.0 Hz, 1H, CHar), 6.93 (s, 1H, CHar),
7.18 (d, J = 1.2 Hz, 1H, CHar), 9.55 (s, 1H, phenolic OH), 9.60 (s, 1H, phenolic OH), 11.26 (s,
1H, phenolic OH), 11.33 (s, 1H, phenolic OH), 14.31 (s, 1H, phenolic OH), 15.99 (s, 1H,
phenolic OH).
13C NMR (100 MHz, THF-d8): δ (ppm) 21.5, 22.0, 39.9, 41.6, 43.6, 43.6, 47.9, 48.0, 63.3,
64.3, 66.7, 69.0, 83.6, 83.7, 106.2, 106.3, 108.2, 108.4, 109.4, 110.6, 111.4, 112.2, 112.3,
113.7, 114.8, 115.6, 116.6, 119.9, 120.0, 120.3, 121.9, 123.5, 133.3, 133.4, 136.3, 137.8, 140.2,
144.4, 149.2, 149.4, 154.3, 158.9, 159.2, 161.8, 162.1, 162.7, 162.8, 181.0, 181.1, 193.6, 193.7,
196.1.
FT-IR (neat) νmax: 3405, 2926, 2855, 1707, 1614, 1583, 1389, 1333, 1246, 1180, 1044, 998,
843, 720 cm‒1
S13
[α]D27= – 232.60 (c = 0.1 g/100 mL in 1,4-dioxane).
CD (c 50 µM, 1,4-dioxane), λ [nm] (mdeg): 215 (0.14), 224 (24.53), 231(14.84), 239 (3.53),
248 (14.43), 255 (24.53), 265 (−8.38), 275 (−43.72), 288 (−11.87), 308 (−3.09), 321 (−0.38),
331 (0.31), 343 (−0.83), 356 (0.89), 372 (3.15), 388 (1.11), 405 (−0.90), 428 (−0.90).
Exact Mass [M+H]+: 561.1391 (calculated), 561.1362 (found).
VI. “Cascade” conversion of (−)-flavoskyrin C (24) to (−)-rugulosin C (ent-2)
A solution of 24 (22.0 mg, 38.0 µmol) dissolved in pyridine (2.5 mL) could stand at room temp
under oxygen for 16 h. After 16 h TLC shows complete consumption of starting material and
two new spots was found. Then, 10 % HCl (15 mL) was added into the reaction mixture to
quenched pyridine and extracted with EtOAc (3 x 25 mL). The organic layer was washed with
brine and dried under reduced pressure. The residue, obtained by evaporation of the solvent,
was separated by column chromatography to obtain (–)-rugulosin C (ent-2) as a pure product.
C30H22O12: 574.11 g.mol-1
TLC: (CHCl3/MeOH, 7:3 v/v): Rf = 0.11.
Column chromatography: silica gel (230-400 mess size) impregnated with oxalic acid;
benzene:acetone :: 7:3.
Yield: 50%
1H NMR (400 MHz, DMSO-d6): δ (ppm) 2.77 (d, J = 5.9 Hz, 2H), 3.37 (s, 2H), 4.39 (s, 2H),
4.62 (s, 4H), 5.49 (d, J = 3.5 Hz, 2H), 5.58 (s, 2H), 7.29 (d, J = 1.4 Hz, 2H, CHar), 7.59 (d, J =
1.4 Hz, 2H, CHar), 11.43 (s, 2H, phenolic OH).
S14
13C NMR (100 MHz, DMSO-d6): δ (ppm) 47.8, 55.6, 58.5, 62.1, 68.6, 106.2, 114.9, 117.2,
120.8, 132.1, 152.2, 160.2, 179.9, 186.8, 194.1.
FT-IR (neat) νmax: 3364, 2920, 2855, 2689, 1769, 1612, 1571, 1481, 1368, 1287, 1257, 1239,
1177, 1131, 1044, 754 cm‒1
[α]D27 = – 298.6 (c = 0.1 g/100 mL in tetrahydrofuran).
CD (c 50 µM, 1,4-dioxane), λ [nm] (mdeg): 221 (17.92), 232 (−0.35), 245 (−21.27), 260
(−10.30), 207 (9.49), 280 (25.34), 290 (5.67), 300 (−1.10), 325 (3.73), 347 (7.60), 402 (−11.79).
Exact Mass [M+H]+: 574.1111 (calculated), 574.1111 (found).
Comparison between synthesised (−)-rugulosin C (ent-2) with isolated (+)-rugulosin C
(2)3.
13C
position
Synthesised (−)-
rugulosin C
(400 MHz,
DMSO-d6) δH
(ppm)
Isolated
(+)-rugulosin C3
(400 MHz, DMSO-
d6) δH (ppm)
Synthesised
(−)-rugulosin C
(400 MHz,
DMSO-d6) δc
(ppm)
Isolated
(+)-rugulosin C3
(400 MHz,
DMSO-d6) δc
(ppm)
1 186.8 186.7
1-OH 14.7 brs
2 2.77 (d, J = 5.9 Hz,
2H)
2.76 (d, J = 6.0 Hz) 58.5 58.5
3 4.39 (s, 2H) 4.38 (dd, J = 6.0, 2.0
Hz)
68.6 68.5
3-OH 5.58 (s, 2H)
4 3.37 (brs, 2H) 3.37 brs 47.8 47.8
5 56.6 56.5
6 7.57 (d, J = 1.4 Hz) 7.57 (d, J = 2.0 Hz) 117.2 117.2
7 152.2 152.2
8 7.29 (d, J = 1.4 Hz) 7.27 (d, J = 2.0 Hz) 120.8 120.7
9 160.2 160.1
9-OH 11.4 s 11.4 s
10 114.9 114.9
S15
11 179.9 181.3
12 106.2 106.8
13 194.1 194.0
14 132.1 132.1
15 4.62 s 4.60 s 62.1 62.0
During the purification of (−)-rugulosin C (ent-2) the above title compound (−)-
dianhydrorugulosin C (31) isolated due to dehydration and aromatization from 24 as an orange
solid after column chromatography.
C30H18O10: 538.09 g.mol-1
TLC: (CHCl3/MeOH, 17:3 v/v): Rf = 0.52.
Column chromatography: silica gel (230-400 mess size) impregnated with oxalic acid;
benzene:acetone :: 9:1.
Yield: 23%
1H NMR (400 MHz, DMSO-d6): δ (ppm) 4.52 (s, 4H), 5.50 (s, 2H), 7.26 (d, J = 1.4 Hz, 2H,
CHar), 7.36−7.45 (m, 6H, CHar), 11.85 (s, 2H, phenolic OH), 12.53 (s, 2H, phenolic OH).
1H NMR (400 MHz, acetone-d6): δ (ppm) 4.62 (t, J = Hz, OH, 2H), 4.71 (d, J = 5.7 Hz, 4H),
7.33 (d, J = 1.4 Hz, 2H), 7.40 (d, J = 8.7 Hz, 2H, CHar), 7.49 (d, J = 1.4 Hz, 2H, CHar), 7.51
(d, J = 8.7 Hz, 2H, CHar), 12.00 (s, 2H, phenolic OH), 12.60 (s, 2H, phenolic OH).
13C NMR (100 MHz, DMSO-d6): δ (ppm) 62.0, 114.1, 115.9, 117., 120.3, 124.1, 129.5, 133.6,
136.4, 139.5, 153.8, 161.3, 161.4, 182.0, 191.8.
FT-IR (neat) νmax: 3405, 2918, 2850, 1719, 1671, 1627, 1453, 1405, 1287, 1239, 1183, 1111,
906, 782, 742 cm‒1
S16
[α]D27 = – 88.0 (c = 0.05 g/100 mL in 1,4-dioxane).
Exact Mass [M+H]+: 538.0900 (calculated), 538.0904 (found).
Isolation and characterization of intermediate I (29)
A solution of 24 (15 mg, 26 µmol) dissolved in pyridine (2 mL) and stand at room temperature
under argon for 10 h. After 10 h TLC shows no starting compound present and newly generated
spot was found in TLC. Then, 10 % HCl (10 mL) was added into the reaction mixture to
quenched pyridine and extracted crude mixture with EtOAc (3 x 20 mL). The organic layer
was washed with brine and dried under reduced pressure. The residue, obtained by evaporation
of the solvent, was separated by column chromatography to afford the pure product.
C30H24O12: 576.12 g.mol-1
TLC: (CHCl3/MeOH, 17:3 v/v): Rf = 0.25.
Column chromatography: silica gel (230-400 mess size) impregnated with oxalic acid;
benzene:acetone :: 7:3.
Yield: 53%
1H NMR (400 MHz, acetone-d6): δ (ppm) 1.23 (dd, J = 18 Hz, 3.8 Hz, 1H), 2.0 (dt, J = 18
Hz, 2.2 Hz, 1H), 3.06 (d, J = 4.4 Hz, 1H), 4.19 (dt, J = 5.9 Hz, 2 Hz, 1H), 4.41 (t, J = 5.2 Hz,
1H), 4.45 (m, 1H), 4.58 (t, J = 4.7 Hz, 1H), 4.78 (s, 2H), 4.79 (s, 2H), 6.92 (s, 1H, CHar), 7.33
(s, 1H, CHar), 7.61 (s, 1H, CHar), 7.73 (s, 1H, CHar), 9.60 (s, 1H, phenolic OH), 11.95 (s, 1H,
phenolic OH), 14.30 (s, 1H, phenolic OH), 14.80 (s, 1H, phenolic OH).
13C NMR (100 MHz, acetone-d6): δ (ppm) 32.0, 36.5, 42.3, 44.4, 56.2, 63.4, 63.7, 64.5, 64.9,
73.8, 105.4, 110.0, 110.9, 111.0, 116.0, 117.9, 119.5, 121.3, 133.7, 134.1, 143.3, 148.8, 154.0,
159.0, 159.1, 163.0, 178.9, 191.7, 195.1, 204.0.
Exact Mass [M+H]+: 577.1341 (calculated), 577.1335 (found).
S17
VII. “Cascade” conversion of (−)-flavoskyrin B/B’ (26/27) to (−)-rugulosin
B (ent-8)
A solution of two diastereomeric mixture of (26/27) (20 mg, 35.68 µmol) dissolved in pyridine
(2.5 mL) and kept at room temperature under oxygen for 24 h. After 24 h TLC shows no starting
compound present and two new spots was present. Then, 10 % HCl (8 mL) was added into the
reaction mixture to quenched pyridine and extracted crude mixture with EtOAc (3 x 20 mL).
The organic layer was washed with brine and dried under reduced pressure. The residue,
obtained by evaporation of the solvent, was separated by column chromatography. A yellow
pigment obtained from the second band was assigned to (‒)-rugulosin B (ent-8).
C30H22O11: 558.49 g.mol-1
TLC: (CHCl3/MeOH, 17:3 v/v): Rf = 0.15.
Column chromatography: silica gel (230-400 mess size) impregnated with oxalic acid;
benzene:acetone :: 4:1.
Yield: 60 %
1H NMR (400 MHz, DMSO−d6): δ (ppm) 2.43 (s, 3H), 2.78 (m, 2H), 3.37 (s, 2H) 4.39 (s,
1H), 4.62 (s, 2H), 5.47 (s, 1H), 5.56 (s, 1H), 7.21 (d, J = 1.1 Hz, 1H, CHar), 7.28 (d, J = 1.4 Hz,
1H, CHar), 7.46 (d, J = 1 Hz, 1H, CHar), 7.59 (d, J = 1.4 Hz, 1H, CHar), 11.40 (s,1H, phenolic
OH), 14.73 (s, 1H, phenolic OH).
1H NMR (400 MHz, Pyridine−d5): δ (ppm) 2.20 (s, 3H, CH3), 3.58 (t, J = 6.8 Hz, 2H), 4.07
(s, 2H), 4.96 (S, 2H, CH2), 5.20−5.24 (m, 2H), 7.05 (s, 1H, CHar), 7.52 (s, 1H, CHar), 7.99 (s,
1H, CHar).
S18
13C NMR (100 MHz, DMSO−d6): δ (ppm) 21.5, 47.8, 55.6, 55.6, 58.3, 58.5, 62.0, 68.5, 106.1,
106.2, 114.1, 114.9, 117.2, 120.5, 120.8, 124.1, 132.0, 132.1, 147.6, 152.2, 160.1, 160.1, 179.9,
180.6, 186.0, 186.8, 194.0, 194.0.
FT-IR (neat) νmax: 3387, 3239, 2920, 2855, 1731, 1689, 1616, 1571, 1482, 1364, 1293, 1245,
1129, 1068, 873 cm‒1
[α]D27 = – 421.4 (c = 0.1 g/100 mL in tetrahydrofuran).
CD (c 50 µM, 1,4-dioxane), λ [nm] (mdeg): 221 (38.54), 230 (1.78), 244 (−43.53), 260
(−19.45), 270 (17.39), 281 (55.18), 290 (15.13), 300 (−2.81), 325 (8.04), 347 (14.26), 375
(−6.81), 402 (−25.35), 442 (−0.63), 456 (−0.19).
HRMS [M+H]+: 558.1162 (calculated), 558.1165 (found).
Comparison between synthesised (−)-rugulosin B (ent-8) with isolated (+)-rugulosin B (8)3
13C
position
Synthesised (−)-
rugulosin B
(400 MHz, DMSO-
d6) δH (ppm)
Isolated
(+)-rugulosin B3
(400 MHz,
DMSO-d6) δH
(ppm)
Synthesised (−)-
rugulosin B
(400 MHz, DMSO-
d6) δc (ppm)
Isolated (+)-
rugulosin B3
(400 MHz,
DMSO-d6) δc
(ppm)
1 186.0 185.7
1-OH 14.7 brs 14.7 brs
2 2.78 m 2.77 m 58.3 58.3 or 58.4
3 4.39 m 4.38 m 68.5 68.5
3-OH 5.47 brs 5.39 brs
4 3.37 brs 3.36 brs 47.8 47.8
5 55.6 55.5 or 55.6
6 7.46 (d, J = 1.2 Hz) 7.44 (d, J = 1.2 Hz) 120.5 120.5
7 147.6 147.6
8 7.21 (d, J = 1.2 Hz) 7.18 (d, J = 1.2 Hz) 124.1 124.0
9 160.1 160.1
9-OH 11.4 s 11.4 s
10 114.1 114.1
11 179.9 180.0
12 106.1 106.1 or 106.2
S19
13 194.0 193.9
14 132.0 132.0
15 2.43 (s, 3H) 2.41 (s, 3H) 21.5 21.5
1’ 186.8 186.7
1’-OH 14.7 brs 14.7 brs
2’ 2.78 m 2.77 m 58.3 58.3 or 58.4
3’ 4.39 m 4.38 m 68.5 68.5
3’-OH 5.47 brs 5.39 brs
4’ 3.36 brs 3.36 m 47.8 47.8
5’ 55.6 55.5 or 55.6
6’ 7.59 (d, J = 1.2 Hz) 7.58 (d, J = 1.2 Hz) 117.2 117.2
7’ 152.2 152.2
8’ 7.28 (d, J = 1.2 Hz) 7.27 (d, J = 1.2 Hz) 120.8 120.8
9’ 160.1 160.1
9’-OH 11.4 s 11.4 s
10’ 114.9 114.9
11’ 180.6 180.7
12’ 106.2 106.1 or 106.2
13’ 194.0 194.0
14’ 132.1 132.1
15’ 4.62 s 4.60 s 62.0 62.0
During the conversion of (−)-rugulosin B (ent-8) the above title compound (−)-
dianhydrorugulosin B (38) was formed due to dehydration and aromatisation from 26/27 and
isolated as orange solid after column chromatography.
C30H18O9: 522.46 g.mol-1
S20
TLC: (CHCl3/MeOH, 17:3 v/v): Rf = 0.7.
Column chromatography: silica gel (230-400 mess size) impregnated with oxalic acid;
benzene:acetone :: 9:1.
Yield: 18 %
1H NMR (400 MHz, acetone-d6): δ (ppm) 2.40 (s, 4H), 4.70 (s, 2H), 7.16 (d, J = 0.8 Hz, 1H,
CHar), 7.32 (d, J = 5.6 Hz, 2H, CHar), 7.39−7.41 (dd, J = 8.6 Hz, 3.3 Hz, 2H, CHar), 7.49−7.51
(dd, J = 8.0 Hz, J = 2.0 Hz, 3H, CHar), 11.95 (s, 1H, phenolic OH), 12.00 (s, 1H, phenolic OH),
12.60 (s, 1H, phenolic OH).
1H NMR (400 MHz, DMSO-d6): δ (ppm) 2.33 (s, 3H), 4.53 (s, 1H), 5.51 (s, 2H), 7.19 (s, 1H,
CHar), 7.27 (s, 2H, CHar), 7.38−7.42 (m, 5H, CHar), 11.82 (s, 1H, phenolic OH), 11.86 (s, 1H,
phenolic OH), 12.53 (s, 2H, phenolic OH).
13C NMR (100 MHz, DMSO-d6): δ (ppm) 21.6, 62.0, 113.4, 114.1, 115.9, 115.9, 117.1, 120.3,
120.6, 123.7, 124.1, 129.5, 129.5, 133.4, 133.6, 136.4, 136.4, 139.5, 149.3, 153.8, 161.3, 161.3,
161.4, 181.9, 191.8.
FT-IR (neat) νmax: 3429, 2926, 2849, 1712, 1671, 1627, 1487, 1452, 1405, 1382, 1352, 1289,
1243, 1185, 1111, 761 cm‒1
[α]D27 = – 4.40 (c = 0.05 g/100 mL in 1,4-dioxane).
Exact Mass [M+H]+: 538.0951 (calculated), 538.0938 (found).
Isolation and characterization of intermediate I/I’ (34/35)
C30H24O11: 560.51 g.mol-1
A solution of two diastereomeric mixture of (26/27) (15 mg, 26 µmol) dissolved in pyridine (2
mL) could stand at room temperature under argon for 10 h. After 10 h TLC shows newly
generated is present and starting material consumed totally. Then, 10 % HCl (10 mL) was
S21
added into the reaction mixture to quenched pyridine and extracted crude mixture with EtOAc
(3 x 20 mL). The organic layer was washed with brine and dried under reduced pressure. The
residue, obtained by evaporation of the solvent, was separated by column chromatography to
afford the pure product.
TLC: (CHCl3/MeOH, 9:1 v/v): Rf = 0.2.
Column chromatography: silica gel (230-400 mess size) impregnated with oxalic acid;
benzene:acetone :: 4:1.
Yield: 40 %
1H NMR (400 MHz, acetone-d6): δ (ppm) 1.21 (dd, J = 19 Hz, J = 4.3 Hz, 1H), 1.23 (dd. J =
19 Hz, 4.3 Hz, 1H), 1.99 (dt, J = 19 Hz, 2Hz, 2H), 2.47 (s, 3H), 2.48 (s, 3H), 3.05 (t, J = 3.9
Hz, 2H), 4.19 (dt, J = 5.8, 1.9 Hz, 2H), 4.37–4.42 (m, 2H), 4.42–4.45 (m, 2H), 4.57 (t, J = 4.8
Hz, 2H), 4.78 (s, 2H), 4.79 (s, 2H), 6.78 (d, J = 1.0 Hz, 1H, CHar), 6.92 (d, J = 1.3 Hz, 1H,
CHar), 7.16 (s, 1H, CHar), 7.33 (s, 1H, CHar), 7.42 (d, J = 1.0 Hz, 1H, CHar), 7.53 (s, 1H, CHar),
7.61 (s, 1H, CHar), 7.73 (s, 1H, CHar), 9.56 (s, 1H, phenolic OH), 9.60 (s, 1H, phenolic OH),
11.90 (s, 1H, phenolic OH), 11.95 (s, 1H, phenolic OH), 14.27 (s, 1H, phenolic OH), 14.30 (s,
1H, phenolic OH), 14.80 (s, 1H, phenolic OH), 14.82 (s, 1H, phenolic OH).
1H NMR (400 MHz, pyridine-d5): δ (ppm) 1.86 (dd, J = 18.6 Hz, 4 Hz, 2H), 2.22 (s, 3H,
CH3), 2.36 (s, 3H, CH3), 2.47 (dt, J = 19 Hz, 2.4 Hz, 2H), 3.70 (dt, J = 5 Hz, 2H), 4.92 (d, J =
5 Hz, 2H), 4.99 (s, 2H, CH2), 5.06–5.11 (m, 4H), 5.13 (s, 2H, CH2), 5.21 (t, J = 4.6 Hz,1H),
5.24 (t, J = 4.6 Hz,1H), 6.95 (s, 1H, CHar), 7.06 (s, 1H, CHar), 7.34 (s, 1H, CHar), 7.49 (s, 1H,
CHar), 7.97 (s, 2H, CHar), 8.51 (s, 1H, CHar).
13C NMR (100 MHz, acetone-d6): δ (ppm) 22.0, 22.4, 36.5, 43.3, 44.4, 56.2, 63.4, 63.7, 64.5,
64.9, 73.8, 105.3, 109.8, 110.1, 110.9, 111.0, 113.9, 114.2, 115.2, 117.9, 119.4, 119.5, 121.2,
124.5, 133.6, 134.0, 134.1, 142.9, 143.3, 144.4, 148.8, 149.6, 154.0, 159.0, 159.1, 159.3, 162.9,
178.7, 178.9, 191.7, 195.1, 203.8.
Exact Mass [M+H]+: 561.1391 (calculated), 561.1391 (found).
S22
VIII. Mechanistic insight into the “Cascade” reaction of (–)-flavoskyrin
B/B’
Scheme S3: Cascade conversion of (−)-flavoskyrin B/B’ (26/27) into a single enantiomer (−)-rugulosin
B (ent-8)
The conversion of (−)-flavoskyrin B/B’ (26/27) into a single enantiomer (−)-rugulosin B (ent-
8) is investigated in detail by following a cascade using 1H NMR spectra in pyridine-d5 as
solvent (Scheme S3). At first, we isolated and characterized the inseparable mixture of
diastereomeric intermediates I/I’ (34/35) which might have been formed by the Michael
reaction of putative biosynthetic intermediates 32/33 in the presence of pyridine under argon
atmosphere (Scheme 4B). Next, we incubated the purified mixture of intermediate I/I’ (34/35)
(6 mg, 10.7 mol) and 0.5 mL of pyridine-d5 added in the NMR tube flushed with argon and
recorded the 1H NMR spectra at regular intervals. At the starting point, the reaction is very
slow as there was no conversion to the products was observed (Figure S1A). After two hours
it only shows12% to (−)-rugulosin B (ent-8) (Figure S1B). To complete the cascade and start
the second Michael addition oxidation of the hydroquinone moiety in intermediate I/I’ (34/35)
was necessary. Therefore, molecular oxygen was bubbled into the NMR tube after 3 h. 1H
NMR spectra recorded after 4 h shows the 56% conversion of 34/35 (proton shown in red) to
(−)-rugulosin B (protons shown in blue and green) (Figure S1C). After 10 h, the 1H NMR
spectra shows complete conversion of intermediate I/I’ into a single product, (−)-rugulosin B
(ent-8) (Figure S1D). As shown no oxidized intermediate II/II’ (36/37) was observed, this may
be due to fast conversion to the final product ent-8. It is to be noted that once the third C-C
bond is formed both intermediate II/II’ (36/37) will lead to form the same enantiomer, which
is (−)-rugulosin B (ent-8).
S23
Figure S1: 1H NMR spectra showing in situ conversion of intermediate I/I’ (34/35) into (−)-rugulosin
B (ent-8) in pyridine-d5. A) Diastereomeric mixture of Intermediate I/I’ (34/35). B) 1H NMR shows
12% conversion to product after 2 h. C) 1H NMR shows 56% conversion after bubbling oxygen at 4 h.
D) 1H NMR shows quantitative conversion to product after 10 h.
S24
Comparison of optical rotation and CD data of (‒)-rugulosin B, C and that of (+)-rugulosin B, C
Compound Optical Rotation CD (Dioxane)
(‒)-rugulosin B
(ent-8)
– 421.4 ⁰ (c = 0.1, tetrahydrofuran). λ [nm] (mdeg) 221 (38.54), 244
(-43.53), 260 (-19.45), 270
(17.39), 281 (55.18), 290
(15.13), 347 (14.26), 402 (-
25.35).
(+)-rugulosin B3 +436.6 ⁰ (c 0.1, dioxane) λex termum (∆ε) 243 (24.4), 280
(-34.1), 349 (-10.6), 404 (12.4)
(‒)-rugulosin C
(ent-2)
– 298.6 ⁰ (c = 0.1, tetrahydrofuran) λ [nm] (mdeg) 221 (17.92), 245
(-21.27), 260 (-10.30), 280
(25.34), 347 (7.60), 402 (-11.79).
(+)-rugulosin C3 +289.9.6 ⁰ (c 0.1, dioxane) λex termum (∆ε) 243 (25.1), 279
(-28.6), 347 (-7.73), 403 (14.6)
IX. Synthesis of rugulin analogue
Scheme S4: Synthesis of rugulin cages from rugulosin like molecules
In a 10 mL two necked round bottom flask substrate (8 mg, 14 μmol, 1 equiv.) was taken and
4 mL mixture of acetonitrile: water (1:1) added into the reaction mixture. After that cerium
ammonium nitrate (CAN) (15 mg, 28 μmol, 2 equiv.) was added to the reaction mixture and
stirred at room temperature for two hours. After 2 h, TLC was check-in 10% MeOH in CHCl3
and it shows only one UV active spot and starting material was totally consumed. Then, the
whole reaction mixture was extracted crude NMR was recorded in CDCl3 it shows an only
product was present. Finally, it was purified using column chromatography to obtain pure four
bonded rugulin analogue product as white solid. Substrates (–)-rugulosin (5) and (–)-2,2’-epi-
cytoskyrin A (ECA, 6) were synthesized as reported elsewhere.2
S25
(−)-rugulin analogue of (−)-rugulosin (39)
C30H20O10: 540.48 g.mol-1
TLC (CHCl3/MeOH, 9:1 v/v): Rf = 0.32.
Column chromatography: silica gel (230-400 mess size) impregnated with oxalic acid;
benzene:acetone :: 9:1.
Yield: 84 %
1H NMR (400 MHz, CDCl3): δ (ppm) 2.44 (s, 6H), 3.10 (d, J = 7.4 Hz, 2H), 3.90 (d, J = 1.2
Hz, 2H), 5.13 (d, J = 6.4 Hz, 2H), 7.14 (s, 2H, CHar), 7.38 (s, 2H, CHar), 12.00 (s, 2H, phenolic
OH).4
1H NMR (400 MHz, 96 %CDCl3 + 4% DMSO-d6): δ (ppm) 2.44 (s, 6H), 3.03 (d, J = 6.6 Hz,
2H), 3.82 (s, 2H), 5.01 (d, J = 6.6 Hz, 2H), 5.59 (brs, 2H), 7.11 (s, 2H, CHar), 7.38 (s, 2H,
CHar), 12.10 (s, 2H, phenolic OH).
13C NMR (100 MHz, 96 %CDCl3 + 4% DMSO-d6): δ (ppm) 22.1, 49.5, 59.8, 64.2, 74.0,
75.6, 116.6, 119.9, 124.3, 133.3, 149.1, 162.3, 190.1, 191.1, 195.8.
FT-IR (neat) νmax: 3407, 2920, 2855, 1766, 1689, 1630, 1565, 1489, 1449, 1366, 1255, 1145,
1049, 868, 751 cm‒1
[α]D27 = – 292.8 (c = 0.05 g/100 mL in CHCl3).
Exact Mass [M+H]+: 541.1129 (calculated), 541.1126 (found).
(−)-rugulin analogue of (−)-2,2’-epi-cytoskyrin A (40)
S26
C30H20O12: 572.48 g.mol-1
TLC (CHCl3/MeOH, 19:1 v/v): Rf = 0.28.
Column chromatography: silica gel (230-400 mess size) impregnated with oxalic acid;
benzene:acetone :: 4:1.
Yield: 57 %
1H NMR (400 MHz, DMSO-d6): δ (ppm) 3.14 (d, J = 8 Hz, 2H), 3.60 (d, J = 3.2 Hz, 2H),
3.92 (s, 6H), 4.90 (dd, J = 8 Hz, 2.2 Hz, 2H), 6.07 (brs, 2H), 6.93 (d, J = 2.5 Hz, 2H, CHar),
6.98 (d, J = 2.5 Hz, 2H, CHar), 12.24 (brs, 2H, phenolic OH).
13C NMR (100 MHz, DMSO-d6): δ (ppm) 50.0, 56.4, 59.1, 63.7, 73.6, 75.2, 106.2, 107.0,
113.2, 135.3, 163.7, 165.9, 188.7, 190.6, 196.1.
FT-IR (neat) νmax: 3411, 2914, 2852, 1765, 1689, 1612, 1565, 1441, 1387, 1251, 1204, 1142,
1038, 873, 580 cm‒1
[α]D27 = – 46.8 (c = 0.1 g/100 mL in CHCl3).
Exact Mass [M+H]+: 573.1028 (calculated), 573.1019 (found).
(−)-rugulin analogue of (−)-rugulosin B (41)
S27
C30H18O11: 554.46 g.mol-1
TLC (CHCl3/MeOH, 9:1 v/v): Rf = 0.24.
Column chromatography: silica gel (230-400 mess size) impregnated with oxalic acid;
benzene:acetone :: 9:1.
Yield: 52 %
1H NMR (400 MHz, DMSO-d6): δ (ppm) 2.37 (s, 3H, CH3), 3.02 (d, J = 8 Hz, 1H), 3.09 (d, J
= 8 Hz, 1H), 3.26 (t, J = 3 Hz, 1H), 3.33 (t, J = 3 Hz, 1H), 4.90 (t, J = 8.2 Hz, 2H), 5.96 (bs,
2H), 7.16 (s, 1H, CHar), 7.22 (s, 1H, CHar), 7.69 (s, 1H, CHar), 7.79 (s, 1H, CHar), 10.04 (s, 1H,
CHO), 10.84 (bs, 1H, OHph), 11.18 (bs, 1H, OHph).
13C NMR (100 MHz, DMSO-d6): δ (ppm) 21.4, 54.6, 55.5, 58.0, 58.0, 64.7, 65.9, 73.6, 74.2,
76.0,76.0, 118.3,118.7, 120.4, 120.9,123.2, 128.7, 134.8, 136.7, 139.7, 146.6, 156.7, 157.5,
189.3, 190.4, 192.5, 192.6, 192.7, 197.7, 198.2.
FT-IR (neat) νmax: 3373, 2920, 2850, 1766, 1694, 1642, 1613, 1571, 1352, 1300, 1245, 1147,
1024, 997, 825, 760 cm‒1
[α]D27 = – 52.7 (c = 0.033 g/100 mL in CHCl3).
Exact Mass [M+H]+: 555.0922 (calculated), 555.0923 (found).
S28
X. NMR Spectra
1H NMR (400 MHz, DMSO-d6)
13C NMR (100 MHz, DMSO-d6)
19
19
S29
1H NMR (400 MHz, Acetone-d6)
13C NMR (100 MHz, Acetone-d6)
17
17
S30
1H NMR (400 MHz, CDCl3)
13C NMR (100 MHz, CDCl3)
18
18
S31
1H NMR (400 MHz, Acetone-d6)
13C NMR (100 MHz, Acetone-d6)
21
21
S32
1H NMR (400 MHz, DMSO-d6)
13C NMR (100 MHz, DMSO-d6)
22
22
S33
1H NMR (800 MHz, THF-d8)
13C NMR (200 MHz, THF-d8)
(–)-flavoskyrin C (24)
(–)-flavoskyrin C (24)
S34
DEPT 135 NMR (200 MHz, THF-d8)
2DCOSY NMR (400 MHz, THF-d8)
(–)-flavoskyrin C (24)
(–)-flavoskyrin C (24)
S35
2DHSQC NMR (200 MHz, THF-d8)
(–)-flavoskyrin C (24)
S36
1H NMR (400 MHz, THF-d8)
13C NMR (100 MHz, THF-d8)
(–)-flavoskyrin B (26) (–)-flavoskyrin B’ (27)
(–)-flavoskyrin B’ (27) (–)-flavoskyrin B (26)
S37
DEPT 135 NMR (100 MHz, THF-d8)
2DCOSY NMR (400 MHz, THF-d8)
(–)-flavoskyrin B’ (27) (–)-flavoskyrin B (26)
(–)-flavoskyrin B (26) (–)-flavoskyrin B’ (27)
S38
2DHSQC NMR (100 MHz, THF-d8)
(–)-flavoskyrin B (26) (–)-flavoskyrin B’ (27)
S39
1H NMR (400 MHz, DMSO-d6)
13C NMR (100 MHz, DMSO-d6)
(–)-rugulosin C (ent-2)
(–)-rugulosin C (ent-2)
S40
DEPT 135 NMR (100 MHz, DMSO-d6)
2DCOSY NMR (100 MHz, DMSO-d6)
(–)-rugulosin C (ent-2)
(–)-rugulosin C (ent-2)
S41
2DHSQC NMR (100 MHz, DMSO-d6)
(–)-rugulosin C (ent-2)
S42
1H NMR (400 MHz, DMSO-d6)
13C NMR (100 MHz, DMSO-d6)
(–)-dianhydro rugulosin C (31)
(29)
(–)-dianhydro rugulosin C (31)
(29)
S43
1H NMR (400 MHz, Acetone-d6)
1H NMR (100 MHz, Acetone-d6)
intermediate I (29)
intermediate I (29)
S44
DEPT 135 NMR (100 MHz, Acetone-d6)
2DCOSY NMR (400 MHz, Acetone-d6)
intermediate I (29)
intermediate I (29)
S45
2DHSQC NMR (100 MHz, Acetone-d6)
intermediate I (29)
S46
1H NMR (400 MHz, DMSO-d6)
13C NMR (100 MHz, DMSO-d6)
(–)-rugulosin B (ent-8)
(–)-rugulosin B (ent-8)
S47
DEPT 135 NMR (100 MHz, DMSO-d6)
2DCOSY NMR (400 MHz, DMSO-d6)
(–)-rugulosin B (ent-8)
(–)-rugulosin B (ent-8)
S48
2DHSQC NMR (100 MHz, DMSO-d6)
1H NMR (400 MHz, Pyridine-d5)
(–)-rugulosin B (ent-8)
(–)-rugulosin B (ent-8)
S49
1H NMR (400 MHz, DMSO-d6)
13C NMR (100 MHz, DMSO-d6)
(–)-dianhydro rugulosin B (38)
(29)
(–)-dianhydro rugulosin B (38)
(29)
S50
1H NMR (400 MHz, Acetone-d6)
13C NMR (100 MHz, Acetone-d6)
int I (34) int I’ (35)
int I (34) int I’ (35)
S51
DEPT 135 NMR (100 MHz, Acetone-d6)
2DCOSY NMR (400 MHz, Acetone-d6)
int I (34) int I’ (35)
int I (34) int I’ (35)
S52
2DHSQC NMR (100 MHz, Acetone-d6)
1H NMR (400 MHz, pyridine-d5)
int I (34) int I’ (35)
int I (34) int I’ (35)
S53
1H NMR (400 MHz, CDCl3)
1H NMR (400 MHz, 96% CDCl3 + 4% DMSO-d6)
(–)-rugulin analogue A (39)
(–)-rugulin analogue A (39)
S54
13C NMR (100 MHz, 96% CDCl3 + 4% DMSO-d6)
DEPT 135 NMR (400 MHz, 96% CDCl3 + 4% DMSO-d6)
(–)-rugulin analogue A (39)
(–)-rugulin analogue A (39)
S55
2DCOSY NMR (400 MHz, 96% CDCl3 + 4% DMSO-d6)
(–)-rugulin analogue A (39)
S56
1H NMR (400 MHz, DMSO-d6)
13C NMR (100 MHz, DMSO-d6)
(–)-rugulin analogue ECA (41)
(–)-rugulin analogue ECA (41)
S57
DEPT 135 NMR (100 MHz, DMSO-d6)
2DCOSY NMR (400 MHz, DMSO-d6)
(–)-rugulin analogue ECA (41)
(–)-rugulin analogue ECA (41)
S58
2DHSQC NMR (100 MHz, DMSO-d6)
(–)-rugulin analogue ECA (41)
S59
1H NMR (400 MHz, DMSO-d6)
13C NMR (200 MHz, DMSO-d6)
(–)-rugulin analogue B (40)
(–)-rugulin analogue B (40)
S60
2DCOSY NMR (400 MHz, DMSO-d6)
2DHSQC NMR (100 MHz, DMSO-d6)
(–)-rugulin analogue B (40)
(–)-rugulin analogue B (40)
S61
XI. Circular Dichroism (CD) Spectra
200 250 300 350 400 450 500-5.7
-3.8
-1.9
0.0
1.9
3.8
5.7
(17)
CD
(m
deg
)
Wavelength (nm)
250 300 350 400 450 500
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
CD
(m
de
g)
Wavelength (nm)
(21)
S62
250 300 350 400 450
-100
-80
-60
-40
-20
0
20
40
60C
D (
mdeg)
Wavelength (nm)
(24)
250 300 350 400 450
-45
-30
-15
0
15
30
45
CD
(m
de
g)
Wavelength (nm)
(26 + 27)
S63
250 300 350 400 450 500
-30
-15
0
15
30C
D (
mdeg)
Wavelength (nm)
(ent-2)
250 300 350 400 450 500
-60
-40
-20
0
20
40
60
CD
(m
de
g)
Wavelength (nm)
(ent-8)
S64
XII. HPLC Chromatogram
rac-17
rac-21
17
21
S65
XIII. References
1 S. K. Singh, A. Mondal, N. Saha and S. M. Husain, Green Chem., 2019, 18, 6594.
2 A. Mondal, N. Saha, A. Rajput, S. K. Singh, B. Roy and S. M. Husain, Org. Biomol. Chem.,
2019, 17, 8711.
3 H. Yamazaki, N. Koyama, S. Oura and H. Tomoda, Org. Lett., 2010, 12, 1572.
4 K. C. Nicolaou, H. L. Yee, J. L. Piper and C. D. Papageorgiou, J. Am. Chem. Soc., 2007, 129,
4001.