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(8 mL). The ether layer was dried over sodium sulfate and evaporated on a rotavpor at 30 °C under
reduced pressure to give crude steviol acetate. Recrystallization by acetone-water (5:1) gave 0.380 gm
of white needle like crystals of pure steviol acetate (56%). TLC (hexane: ethyl acetate, 1:1): Rf = 0.68;
1H-NMR (CDCl3) δ = 4.905 (s, 1H, H-17), 4.880 (s, 1H, H-17), 2.034 (s, 3H, OCOCH3), 1.261 (s, 3H,
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H-19) 1.01(s, 3H, H-20); C-NMR: δ = 152.679 (C=CH2, C-16), 103.640 (C=CH2, C-17); ESI-MS
m/z: 383.4 [M+Na]+, Calcd. 383.48 for C22H32O4[M+Na] +; HPLC (tR, 34.8 min).
Steviol Acetate Ketone (3) [3]. Steviol acetate (2, 320 mg, 0.88 mmol) was dissolved in a mixture of
THF (3.2 ml) and water (2.8 mL). To this was added freshly made 4% aqueous solution of OsO4
(0.4 mL). After 10 min the reaction mixture had become deep brown and to this NaIO4 (672 mg) was
added slowly over a period of 2–3 min and the mixture was left for stirring at room temperature for 18 h.
It had become a thick white suspension by now and was extracted with ethyl acetate (3 × 30 mL). The
organic layer was dried over sodium sulfate and evaporated on a rotavapor at 30 °C under reduced
pressure to get crude steviol acetate ketone, which was flash chromatographed on silica gel 60 (70–230 mesh)
with a stepwise gradient from 10% ethyl acetate in hexane to 40% ethyl acetate in hexane. Pure steviol
acetate ketone came out in fractions collected at 35% ethyl acetate in hexane, yield = 230 mg (71%).
TLC (hexane: ethyl acetate, 1:1): Rf = 0.41; 1H-NMR (CDCl3) δ = 2.069 (s, 3H, OCOCH3), 1.273 (s,
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3H, H-19) 1.038(s, 3H, H-20); C-NMR: δ = 214.106 (C=O, C-16), peaks at 152.679 and 103.640
present in 2 disappear here; ESI-MS m/z: 385.4 [M+Na]+, Calcd. 385.45 for C21H30O5 [M+Na]+, HPLC
(tR, 25 min).
Methylenation of Steviol Acetate Ketone [4]. Steviol acetate ketone (3, 100 mg, 0.27 mmol) was taken
up in anhydrous dichloromethane (4 mL) and to this solution was slowly added Zn/CH2Br2/TiCl4
reagent (5 mL, 7.1 equivalents, cold reagent). The reaction mixture was stirred for five days at room
temperature and then quenched by pouring into a biphasic 2:1 mixture of sodium bicarbonate-water
(10 mL) and ether (10 mL). The aqueous layer was extracted two more times with ether (10 mL ea.).
The combined ether layer was dried over sodium sulfate and evaporated on a rotavapor at 30 °C under
reduced pressure to afford a crude product that was chromatographed on silica gel 60 (70–230 mesh)
with a stepwise gradient from 5% ethyl acetate in hexane to 20% ethyl acetate in hexane. First an
impurity eluted at 15% ethyl acetate/hexane and after this at 20% ethyl acetate /hexane came a product
similar to steviol acetate (2) on TLC, yield = 60 mg (60%). TLC (hexane-ethyl acetate, 1:1): Rf = 0.68;
1H-NMR (CDCl3) δ = 4.916 (s, 1H, H-17), 4.876 (s, 1H, H-17), 2.060 (s, 3H, OCOCH3), 1.292 (s, 3H,
H-19), 1.001(s, 3H, H-20); 13C-NMR: δ = 152.604 (C=CH2, C-16), 103.867 (C=CH2, C-17);. ESI-MS
m/z: 383.4 [M+Na]+, Calcd. 383.48 for C22H32O4[M+Na]+, HPLC (tR, 34.8 min).
Steviol nor-ketone (4). Steviol acetate nor-ketone (40 mg, 0.11 mmol) was refluxed in 2 M KOH
(20 mL) for one hour and then cooled. The cooled reaction mixture was then acidified with 30% HCl
to pH = 2.5 whereupon the solution becomes milky and a white precipitate appears. It was extracted
with ethyl acetate (3 × 15 mL), dried over sodium sulfate and then evaporated on a rotavapor at 30 °C
1
under reduced pressure, yield = 30 mg, 85%. TLC (hexane-ethyl acetate, 1:1): Rf = 0.28; H-NMR
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(CDCl3) δ = 1.205 (s, 3H, H-19), 0.940 (s, 3H, H-20); C-NMR: δ = 214.201 (C=O, C-16); ESI-MS