electronic supporting information (esi) · s5 iii. synthesis of citreorosein (19)2: scheme 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

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.

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.

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).

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

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%

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).

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

(R)-3,8,9,10-tetrahydroxy-6-(hydroxymethyl)-3,4-dihydroanthracen-1(2H)-one (21)

C15H14O6: 290.27 g.mol-1

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).

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).

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

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

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 =

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).

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.

C30H24O11: 560.51 g.mol-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

[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).

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

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).

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).

Comparison between synthesised (−)-rugulosin C (ent-2) with isolated (+)-rugulosin C

position

Synthesised (−)-

rugulosin C

(400 MHz,

DMSO-d6) δH

Isolated

(+)-rugulosin C3

(400 MHz, DMSO-

d6) δH (ppm)

Synthesised

(−)-rugulosin C

(400 MHz,

DMSO-d6) δc

Isolated

(+)-rugulosin C3

(400 MHz,

DMSO-d6) δc

1 186.8 186.7

1-OH 14.7 brs

2 2.77 (d, J = 5.9 Hz,

2.76 (d, J = 6.0 Hz) 58.5 58.5

3 4.39 (s, 2H) 4.38 (dd, J = 6.0, 2.0

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

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

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

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

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.

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

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).

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

position

Synthesised (−)-

rugulosin B

(400 MHz, DMSO-

d6) δH (ppm)

Isolated

(+)-rugulosin B3

(400 MHz,

DMSO-d6) δH

Synthesised (−)-

rugulosin B

(400 MHz, DMSO-

d6) δc (ppm)

Isolated (+)-

rugulosin B3

(400 MHz,

DMSO-d6) δc

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

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

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).

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

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

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.

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.

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).

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.

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

(−)-rugulin analogue of (−)-rugulosin (39)

C30H20O10: 540.48 g.mol-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

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).

(−)-rugulin analogue of (−)-2,2’-epi-cytoskyrin A (40)

C30H20O12: 572.48 g.mol-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).

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).

(−)-rugulin analogue of (−)-rugulosin B (41)

C30H18O11: 554.46 g.mol-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).

X. NMR Spectra

1H NMR (400 MHz, DMSO-d6)

13C NMR (100 MHz, DMSO-d6)

1H NMR (400 MHz, Acetone-d6)

13C NMR (100 MHz, Acetone-d6)

1H NMR (400 MHz, CDCl3)

13C NMR (100 MHz, CDCl3)

1H NMR (800 MHz, THF-d8)

13C NMR (200 MHz, THF-d8)

(–)-flavoskyrin C (24)

DEPT 135 NMR (200 MHz, THF-d8)

2DCOSY NMR (400 MHz, THF-d8)

2DHSQC NMR (200 MHz, THF-d8)

1H NMR (400 MHz, THF-d8)

13C NMR (100 MHz, THF-d8)

(–)-flavoskyrin B (26) (–)-flavoskyrin B’ (27)

(–)-flavoskyrin B’ (27) (–)-flavoskyrin B (26)

DEPT 135 NMR (100 MHz, THF-d8)

2DCOSY NMR (400 MHz, THF-d8)

(–)-flavoskyrin B’ (27) (–)-flavoskyrin B (26)

2DHSQC NMR (100 MHz, THF-d8)

(–)-rugulosin C (ent-2)

DEPT 135 NMR (100 MHz, DMSO-d6)

2DCOSY NMR (100 MHz, DMSO-d6)

2DHSQC NMR (100 MHz, DMSO-d6)

(–)-dianhydro rugulosin C (31)

intermediate I (29)

DEPT 135 NMR (100 MHz, Acetone-d6)

2DCOSY NMR (400 MHz, Acetone-d6)

intermediate I (29)

2DHSQC NMR (100 MHz, Acetone-d6)

intermediate I (29)

(–)-rugulosin B (ent-8)

1H NMR (400 MHz, Pyridine-d5)

(–)-dianhydro rugulosin B (38)

int I (34) int I’ (35)

DEPT 135 NMR (100 MHz, Acetone-d6)

2DCOSY NMR (400 MHz, Acetone-d6)

int I (34) int I’ (35)

2DHSQC NMR (100 MHz, Acetone-d6)

1H NMR (400 MHz, pyridine-d5)

int I (34) int I’ (35)

1H NMR (400 MHz, CDCl3)

1H NMR (400 MHz, 96% CDCl3 + 4% DMSO-d6)

(–)-rugulin analogue A (39)

13C NMR (100 MHz, 96% CDCl3 + 4% DMSO-d6)

DEPT 135 NMR (400 MHz, 96% CDCl3 + 4% DMSO-d6)

2DCOSY NMR (400 MHz, 96% CDCl3 + 4% DMSO-d6)

(–)-rugulin analogue ECA (41)

(–)-rugulin analogue B (40)

XI. Circular Dichroism (CD) Spectra

200 250 300 350 400 450 500-5.7

Wavelength (nm)

250 300 350 400 450 500

Wavelength (nm)

250 300 350 400 450

Wavelength (nm)

250 300 350 400 450

Wavelength (nm)

(26 + 27)

250 300 350 400 450 500

Wavelength (nm)

(ent-2)

250 300 350 400 450 500

Wavelength (nm)

(ent-8)

XII. HPLC Chromatogram

rac-17

rac-21

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.

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