Synthetic study on the relationship between structure and sweet taste properties of steviol glycosides

Article abstract of DOI:10.3390/molecules17044186

The structure activity relationship between the C₁₆-C ₁₇ methylene double bond on the aglycone of steviol glycosides and the corresponding impact on their sweet taste has been reported here for the first time. It has been observed that converting stevioside and rebaudioside A to their corresponding ketones by switching the doubly bonded methylene on C-17 for a ketone group actually removes the sweet taste properties of these molecules completely. Regenerating the original molecules tends to restore the sweet taste of both the steviol glycosides. Thus this C₁₆-C ₁₇ methylene double bond in rebaudioside A and stevioside can be regarded as a pharmacophore essential for the sweetness property of these molecules.

Full text of DOI:10.3390/molecules17044186

Molecules 2012, 17, 4186-4196; doi:10.3390/molecules17044186
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthetic Study on the Relationship Between Structure and
Sweet Taste Properties of Steviol Glycosides
Mani Upreti *, Grant Dubois and Indra Prakash
Organic Chemistry Department, Global Research and Development, the Coca-Cola Company,
One Coca-Cola Plaza, Atlanta, GA 30313, USA; E-Mails: gdubois@coca-cola.com (G.D.);
iprakash@coca-cola.com (I.P.)
* Author to whom correspondence should be addressed; E-Mail: mupreti@coca-cola.com;
Tel.: +1-404-676-2923; Fax: +1-404-598-2923.
Received: 2 March 2012; in revised form: 24 March 2012 / Accepted: 30 March 2012 /
Published: 5 April 2012
Abstract: The structure activity relationship between the C16-C17 methylene double bond
on the aglycone of steviol glycosides and the corresponding impact on their sweet taste has
been reported here for the first time. It has been observed that converting stevioside and
rebaudioside A to their corresponding ketones by switching the doubly bonded methylene
on C-17 for a ketone group actually removes the sweet taste properties of these molecules
completely. Regenerating the original molecules tends to restore the sweet taste of both
the steviol glycosides. Thus this C16-C17 methylene double bond in rebaudioside A and
stevioside can be regarded as a pharmacophore essential for the sweetness property of
these molecules.
Keywords: reb A; stevioside; steviol; reb A ketone; stevioside ketone and steviol nor
ketone, methylenation
1. Introduction
Rebaudioside A (reb A) and stevioside (Figure 1) are sweet ent-kaurene-type diterpenoid glycosides
found in high concentration levels in the leaves of Stevia rebaudiana. Steviol (Figure 1) is the
aglycone diterpene, while reb A and stevioside are glycosides based on this aglycone’s carbon
skeleton. Both stevioside and reb A have a D-glucose group affixed at C19 and in addition stevioside
has a di-glucosyl, while reb A has a tri-glucosyl sugar moiety affixed at C13. Steviol is not sweet at all

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and it is known that with increasing number of attached glucose moieties the sweetness potency of
steviol glycosides tends to increase.
Figure 1. Ketone derivatives of steviol and steviol glycosides.
However it is not clear which part of these steviol glycoside molecules is essential for the sweetness
properties. The concentration/response (C/R) function of stevioside and reb A has been reported by
Dubois et al. in 1991 [1]. Based on the C/R functions, R = 10.0C/(200 + C) for reb A and
R = 9.9C/(410 + C) for stevioside, the potency (against 5% sugar solution) has been reported to be 120
for stevioside and 250 for reb A [1,2]. The in-house data in Table 1 for reb A in water at 4–6 °C also
complies with this reported C/R function:
Table 1. Sweetness potency data of reb A.
R (% Sugar Equivalence) C (Concentration)
Potency (w/w sugar vs. reb A)
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
26.51
58.23
96.85
145
377
343
310
276
242
209
175
141
206.25
287.42
399.81
566
Due to our interest in the chemistry of these steviol glycosides, we have been investigating various
ways of modifying these natural molecules in order to find relations between various parts of their
structures and the sweet taste properties. We have found that converting stevioside and reb A into their
corresponding ketones by switching the doubly bonded methylene on the C17 with a ketone actually
removes the sweet taste properties of these molecules completely. Interestingly, upon regenerating the
original molecules back, the sweet taste of both the steviol glycosides were found to be restored. This
finding indicates that the C16-C17 parts of reb A and stevioside are crucial for the sweetness properties
of these molecules. We describe here in detail the various chemical manipulations we carried have out
to arrive at these findings.

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2. Results and Discussion
Preparation of steviol ketone has previously been reported in the literature [3]. This Phytochemistry
article has described use of radiolabeled steviol for the synthesis of radiolabeled gibberellins using the
fungus Gibberella Fujikuroi. In this article the labeled steviol was synthesized from steviol acetate
nor-ketone by the Wittig reaction using [14C](methyl)triphenyl phosphonium iodide. However there
are no other similar literature reports available for similar synthetic transformations on reb A or
stevioside molecules.
We first repeated the literature reported Wittig methylenation reaction procedure on steviol acetate
ketone to see if it worked in our hands, but we were not very successful with this procedure. Further
Wittig reaction was also tried on reb A peracetate nor-ketone and stevioside peracetate nor-ketone. The
reaction conditions seemed to deacetylate the starting molecules and the resulting reaction mixtures
seemed to be mixtures of several products. Apparently this reaction did not seem to work for any of
these molecules.
We therefore designed a four step novel approach (Scheme 1 and 2) taking advantage of the
presence of the similar diterpene aglycone in all three of these compounds: Steviol, stevioside and
reb A. Briefly the starting molecule was first acetylated and then was oxidized at the aglycone part to
the 17-nor-16-ketone. This intermediate ketone was then converted back to C17 methylene form using
TiCl4-Zn-CH2Br2 reagent [4] in dry THF as solvent, (Lombardo’s procedure) which is a mild
non-basic method for the methylenation of ketones.
Scheme 1. From stevioside to steviol acetate nor-ketone and steviol re-generation.

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Scheme 2. The synthetic transformations of stevioside and rebaudioside A molecules.
4189
The methylenation reaction required anhydrous conditions, therefore reb A and stevioside
diterpenoid glycosides and steviol were first converted to their non-polar per-acetate forms. For this
purpose hydroxy protection methods such as acetylation and silylation were tried. Silylation did not
seem to work well as in case of reb A it resulted in an oily substance which was hard to dry and did not
give the right molecular weight expected for the desired per-silylated reb A. Instead it was a mixture of
partially silylated reb A derivatives. Acetylation under traditional pyridine-acetic anhydride conditions
seemed to work well for both reb A and stevioside and was a quantitative reaction. Steviol acetate
however was prepared in presence of dimethyl amino pyridine and triethylamine in order to acetylate
the tertiary hydroxy group [5]. Hydroxyl functionalities thus protected with base labile acetyl groups
survived the ketone formation conditions and further methylenation step and de-protection were
carried out by basic hydrolysis under sodium methoxide-methanol conditions.

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To prepare the intermediate ketones, the most obvious method seemed to be ozonolysis. However
this seemingly common reaction could not be optimized for these diterpenoid molecules as it did not
give consistent results under the present laboratory conditions. At the same time the oxidation reaction
using osmium tetroxide and sodium periodate was attempted and it worked for all the three molecules.
This method always resulted in per-acetylated ketone, which is the desired reaction product, along with
unreacted starting material. It was difficult to resolve reb A and stevioside peracetate nor-ketones on
silica-gel column/TLC by any variation in the solvent systems, hence extensive purification was
followed here to get pure intermediate ketones out of these oxidation reactions. Scaling-up of the
ketone formation reaction or increase in equimolar amounts of osmium tetroxide and sodium periodate
did not result in any enhanced ketone yields as well.
The intermediates, stevioside and reb A per-acetate and its ketones prepared here are novel
compounds being reported for the first time and similarly TiCl4-Zn-CH2Br2 reagent’s methylenation
reaction with steviol acetate nor-ketone, stevioside and reb A per-acetate ketones are also the first such
reports in the literature. During analytical characterization of these novel ketone derivatives, we found
that in the carbon NMR spectra, the carbonyl carbon of steviol and reb A ketones tends to appear
downfield around δ 214–215 while in stevioside ketone, it appears a little upfield around δ 192. This
difference in signal values tells that the carbonyl carbon at C-16 is deshielded to a different degree in
reb A and steviol as compared to stevioside. The stevioside and reb A ketone derivatives in purified
form and their regenerated stevioside and reb A were taste tested by authors (Dubois and Prakash) who
are also in-house sensory experts following an in-house developed “sip and spit protocol” at 100 ppm
concentrations. It was concluded that ketone formation thus led to absence of sweet taste for
corresponding stevioside and reb A derivatives.
3. Experimental Section
3.1. General
The starting materials used for steviol and stevioside work were not readily available. Preparation of
the starting material steviol was carried out in-house by slightly modifying a chemical method reported
in a Japanese patent [6] involving sodium periodate treatment followed by alkaline hydrolysis and by
an enzymatic method by hydrolysis of stevioside with crude pectinase from Aspergillus Niger. Both
methods gave satisfactory results upon comparing with a commercial sample of steviol from
Sigma-Aldrich. The starting material for stevioside work was a crude stevioside mixture containing
only 55% stevioside. Pure stevioside peracetate was purified from this mixture after the acetylation
reaction. The +99% pure reb A were obtained from in-house crystallization work. NMR spectra were
acquired on Varian Unity Plus 600 MHz instruments using standard pulse sequences. The chemical
shifts (δ) are expressed in ppm in relation to tetramethylsilane as a standard and the coupling constants
(J) are given in Hz. MS data were generated with a Waters Premier Quadrupole Time-of-Flight
(Q-Tof) mass spectrometer equipped with an electrospray ionization source operated in the positive-
ion and negative-ion mode. Samples were diluted with water: acetonitrile (1:1) containing 0.1% formic
acid and introduced via infusion using the onboard syringe pump. HPLC analysis was carried out for
steviol and its derivatives via Phenomenex Hydro-RP, 150 × 4.6 mm, eluent A: 10% CH3CN-50 mM

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H3PO4, eluent B: 100% CH3CN, t0 A:B = 100:0, t45 A:B = 0:100, flow 1.0 mL/min, UV 210 nm, and
for stevioside, reb A and their derivatives via Zorbax amino column, 4.6 × 150 mm using an isocratic
gradient of A = 75% acetonitrile-25% water-0.05% acetic acid and B = 100% acetonitrile-0.05% acetic
acid (A:B = 60:40), 1.0 mL per minute using UV detection at 210 nm.
3.2. 4% Aqueous Solution of OsO4
A 250 mg capsule of OsO4 was broken into a wide mouth glass bottle containing 6.25 mL of water.
The bottle was immediately capped and then swirled for 15–20 min in order to dissolve all the OsO4
crystals in water.
3.3. Zn/CH2Br2/TiCl4 Reagent
TiCl4 (2.3 mL) was added dropwise over 10 min to a stirred suspension of zinc dust (5.75 g) in
CH2Br2 (2 mL) and THF (50 mL, freshly distilled over sodium and benzophenone) at −40 °C. The
mixture was allowed to warm to 5 °C and was then stirred at this temperature for 3 days to give a thick
grey slurry of an active species. It was stored at −20 °C.
Steviol (1) - (i) Chemical Method [6]: Stevioside (1.0 gm, 1.24 mmol) was suspended in water
(75 mL) and treated with sodium periodate (1.5 g, 7.0 mmol) for 21 h at room temperature. The white
frothy solution was then freeze-dried and was redissolved in 10% KOH solution (50 mL). The clear
solution thus obtained was heated at 85 °C in an oil bath fitted with a reflux condenser. It was cooled
and neutralized with 2% aqueous HCl to pH = 5.0. Heavy precipitation appeared at this time. The
reaction mixture was then extracted with ethyl acetate three times (30 mL ea.). The combined ethyl
acetate solution was dried over sodium sulfate and evaporated under reduced pressure to get 270 mg of
crude steviol, which was absorbed on silica and purified by column chromatography with 35–50%
ethyl acetate in hexane as eluent. 150 mg of pure steviol was obtained (1, yield = 35%). TLC (hexane:
ethyl acetate, 1:1): Rf = 0.29; ESI-MS m/z: 317.2 [M−H]−, Calcd. 317.44 for C20H30O3[M−H]−; HPLC
(tR, 26.85 min).
Steviol (1) - (ii) Enzymatic Method [3]: Stevioside (0.5 g, 0.62 mmol) was hydrolyzed at 45 °C with
crude pectinase from Aspergillus Niger (1.0 mL, Sigma-Aldrich, P2736) in 0.1 M phosphate buffer
(50 mL), pH = 4.5 for 24 h. The crude steviol precipitated out during the reaction. It was filtered and
then dissolved in acetonitrile. The acetonitrile layer was left overnight at room temperature when some
more material precipitation occurred. It was again filtered and the acetonitrile filtrate was evaporated
under reduced pressure. Crude product thus obtained was crystallized from methanol to give white
needle like crystals of pure steviol (1, yield = 25%).
Steviol Acetate (2). Steviol (1, 0.954 g, 3 mmol), 4-N,N-dimethylaminopyridine (96 mg) and acetic
anhydride (1.8 mL) were dissolved in anhydrous triethylamine (5 mL) and the reaction mixture was
stirred at room temperature for 48 h [5]. To this methanol (2 mL) was added in order to remove the
excess of acetic anhydride followed by evaporation on a rotavapor at 30 °C under reduced pressure.
The residue was dissolved in ether (20 mL) and washed with water (3 × 8 mL), 2% HCl (3 × 8 mL)
followed by washing with dilute sodium bicarbonate (5%, 3 × 8 mL) and one final wash with brine

Molecules 2012, 17
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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,
13
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,
13
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
13
(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

Molecules 2012, 17
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m/z: 319.4 [M−H]−, Calcd. 319.42 for C19H28O4[M−H]−, HPLC (tR, 27 min). A similar procedure was
followed for de-acetylation of 2 to 1.
Stevioside peracetate (5). In a 500 mL round bottom flask, stevioside (55% stevioside by HPLC
analysis, 15 g, nearly 10.25 mmol) was dried overnight in a vacuum oven at 75 °C. To this was added
dry pyridine (200 mL) followed by cooling to 0 °C and then addition of acetic anhydride (350 mL). It
was allowed to warm up to room temperature and then stirred at room temperature for 48 h. The
reaction was then poured on a beaker containing ice and shaken vigorously. It was then extracted with
diethyl ether (3 × 50 mL) followed by washing the organic layer with cold water (3 × 30 mL), 5%
hydrochloric acid (3 × 30 mL), cold saturated sodium bicarbonate till no further evolution of CO2 was
seen (3 × 40 mL) and then finally by brine (30 mL). The organic layer was dried over sodium sulfate
and then evaporated to get the crude per-acetylated stevioside which was purified by silica gel flash
chromatography (230–400 mesh) using a stepwise gradient of dichloromethane-methanol from
pure dichloromethane up to 10% methanol in dichloromethane, yield = 10.00 g (77%). TLC
(dichloromethane-methanol, 10:0.5): Rf = 0.52; 1H-NMR (DMSO) δ = 6.016 (d, 1H, H-21, J = 8.4 Hz);
13C NMR δ = 153.016 (C=CH2, C-16), 103.792 (C=CH2, C-17); ESI-MS m/z: 1289.8 [M+Na]+, Calcd.
1290.27 for C60H82O29 [M+Na]+.
Stevioside per acetate Ketone (7). Stevioside per-acetate (5, 866 mg, 1.1 mmol) was taken up in a
mixture of THF (4.0 ml) and water (3.5 mL). To this was added freshly made 4% aqueous solution of
OsO4 (0.5 mL). After 10 min the reaction mixture had become deep brown and to this NaIO4
(840 mg) was added slowly over a period of 2–3 min and the mixture was left for stirring at room
temperature for 17 h. It had become a thick white suspension by now and was extracted with ethyl
acetate (2 × 40 mL). The organic layer was dried over sodium sulfate and evaporated on arotavapor
at 30 °C under reduced pressure to give the crude stevioside per-acetate ketone, which was
chromatographed on silica gel 60 (70–230 mesh) with a stepwise gradient from pure dichloromethane
to 2.5% methanol in dichloromethane. Pure ketone started coming out in fractions collected at 2%
dichloromethane in methanol, yield = 378 mg (27%). TLC (DCM–MeOH, 10:0.5): Rf = 0.42;
1H-NMR (DMSO) δ = 6.024 (d, 1H, H-21, J = 8.4 Hz); 13C-NMR δ = (C=O, C-16), peaks at
153.016 & 103.792 present in 5 disappear here; ESI-MS m/z: 1292.0 [M+Na]+, Calcd. 1292.24 for
C59H80O30 [M+Na] +.
Methylenation of stevioside acetate ketone and re-generation of stevioside. Stevioside peracetate
ketone (500 mg, 0.393 mmol) was taken up in anhydrous dichloromethane (10.5 mL) and to this
solution was slowly added, under nitrogen, Zn/CH2Br2/TiCl4 reagent (10 mL, 8.3 equivalents, cold
reagent). The reaction mixture was stirred for 18 h. at room temperature when the TLC in
DCM–MeOH (20:1) showed disappearance of ketone. The reaction was further monitored by HPLC
on an amino column. On the basis of HPLC and LC-MS of t = 0 and t = 18 h. samples, the reaction
appeared complete. It was then quenched by pouring into a biphasic mixture of saturated sodium
bicarbonate (20 mL) and ethyl acetate (10 mL). The aqueous layer was extracted two more times with
ethyl acetate (10 mL ea.). The combined ethyl acetate layer was dried over sodium sulfate and
evaporated on a rotavapor at 30 °C under reduced pressure to afford a crude product that was taken up

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in anhydrous methanol (10 mL). To this was added anhydrous sodium methoxide (200 mg). It was
stirred at room temperature for 2 h and then neutralized by 0.5% HCl to pH ~ 6.5–7.0. The methanol
was removed on a rotavapor under reduced pressure. A 1.0 mL size short column of reverse phase
silica, C18, was packed in 50% acetonitrile-water and then washed with 50–100 mL double distilled
water. The de-acetylated product was dissolved in minimum amount of water and loaded on this C18
column followed by washing with water (100 mL). Elution with 90% acetonitrile-water (50 mL)
resulted in pure stevioside, as shown by HPLC (tR, 3.51, same as standard stevioside). The acetonitrile
solution was evaporated and dried to get pure stevioside, yield = 208 mg (68%).
Stevioside ketone (9). Stevioside peracetate ketone (500 mg, 0.393 mmol) was taken up in anhydrous
methanol (10 mL). To this was added anhydrous sodium methoxide (200 mg). It was stirred at room
temperature for 2 h. and then neutralized by 0.5% HCl to pH ~ 6.5–7.0. The methanol was removed on
a rotavapor under reduced pressure followed by desalting on a pre-packed C-18 cartridge (500 mg)
from Varian. The de-acetylated product was further purified via preparative HPLC on a C-18 column
to get pure stevioside ketone, yield = 158 mg (55%). 1H-NMR (DMSO) δ = 1.212 (s, 3H, H-19), 0.983
13
(s, 3H, H-20); C-NMR: δ = 191.230 (C=O, C-16); ESI-MS m/z: 809.1 [M+H]+, Calcd. 807.85 for
C37H58O19 [M+H] +.
Per-acetylated reb A (6). In a 500 mL round bottom flask, rebaudioside A (+99% by crystallization,

5 g, 5.17 mmol) was dried overnight in a vacuum oven at 75 C. To this was added dry pyridine
(150 mL) followed by cooling it to 0 C and then addition of acetic anhydride (250 mL). It was allowed
to warm up to room temperature and then stirred for 48 h. The reaction was then poured on a beaker
containing ice and shaken vigorously. It was then extracted with diethyl ether (3 × 50 mL) followed by
washing the organic layer with cold water (3 × 30 mL), 5% hydrochloric acid (3 × 30 mL), cold saturated
sodium bicarbonate till no further evolution of CO2 was seen (3 × 40 mL) and then finally by brine
(30 mL). The organic layer was dried over sodium sulfate and then evaporated to get the per-acetylated
rebaudioside A as a single spot on TLC, yield = 6.5 g (80%). TLC (DCM–MeOH, 10:0.5): Rf = 0.36;
1H-NMR (DMSO) δ = 5.982 (d, 1H, H-21, J = 7.8 Hz); 13C-NMR δ = 152.890 (C=CH2, C-16),
104.292 (C=CH2, C-17); ESI-MS m/z: 1578.5 [M+Na]+, Calcd. 1578.52 for C72H98O37[M+Na]+.
Rebaudioside A Per-Acetate Ketone (8). Rebaudioside A peracetate (800 mg, 0.51 mmol) was taken up
in a mixture of THF (4.0 mL) and water (3.5 mL). To this was added freshly made 4% aqueous
solution of OsO4 (0.5 mL). After 15 minthe reaction mixture had become deep brown and to this
NaIO4 (840 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 (2 × 40 mL). Organic layer was dried over sodium sulfate and evaporated on a rotavapor
at 30 °C under reduced pressure to get crude rebaudioside A per-acetate ketone that was
chromatographed on silica gel 60 (70–230 mesh) with a slow stepwise gradient from pure
dichloromethane to 2.5% methanol in dichloromethane, yield = 220 mg (30%). TLC (DCM–MeOH,
1
13
10:0.5): Rf = 0.28; H-NMR (DMSO) δ = 5.982 (d, 1H, H-21, J = 7.8 Hz), C-NMR δ = 215.749
(C=O, C-16), peaks at 152.890 and 104.292 present in 6 disappear here; ESI-MS m/z: 1580.0
[M+Na]+, Calcd. 1580.5 for C71H96O38[M+Na]+.

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Methylenation of Rebaudioside A Acetate Ketone and re-generation of reb A. Rebaudioside A per-
acetate ketone (8, 233 mg, 0.149 mmol) was taken up in anhydrous dichloromethane (4 Ml). To this
solution was slowly added under nitrogen, Zn/CH2Br2/TiCl4 reagent (4 mL, 8.7 equivalents, cold
reagent). The reaction mixture was stirred at room temperature and was monitored by HPLC on an
amino column (it was hard to interpret the TLC data due to the appearance of several spots below the
actual ketone spot, which were probably the de-acetylation products). A series of aliquots of the
reaction mixture were taken out at t = 18, t = 41 and t = 72 h (each ~400 L, 7 mol) to monitor the
reaction. They were first quenched with sat. sodium bicarbonate and ethyl acetate (1:1) followed by
isolation, drying and evaporation of ethyl acetate layer and then de-acetylation. On the basis of HPLC
and LC-MS of t = 0 and t = 18, 41, 72 h. samples the reaction seemed complete after 72 h. The
reaction was then quenched by pouring into a biphasic mixture of saturated sodium bicarbonate
(10 mL) and ethyl acetate (5 mL). The aqueous layer was extracted two more times with ethyl acetate
(5 mL ea.). The combined ethyl acetate layer was dried over sodium sulfate and evaporated on a

rotavapor at 30 C under reduced pressure to get a crude product which was only 37 mg. The crude
was taken up in anhydrous methanol (4 mL). To this was added anhydrous sodium methoxide (19 mg).
It was stirred at room temperature for 2 h. and then neutralized by 0.5% HCl to pH ~ 6.5–7.0. The
methanol was removed on a rotavapor under reduced pressure. A 500 mg pre-packed C-18 cartridge
from Varian was first treated with 50% acetonitrile-water and then washed with 15–20 mL double
distilled water. The de-acetylated product was dissolved in minimum amount of water and loaded on
C-18 column followed by washing with water (15 mL). Elution with 90% acetonitrile-water (15 mL)
resulted in pure reb A as shown by HPLC. The acetonitrile solution was evaporated and dried to get
pure reb A, yield = 17 mg (14.5% yield).
Reb A ketone (10). Rebaudioside A peracetate ketone (8, 233 mg, 0.149 mmol) was taken up in
anhydrous methanol (4 mL). To this was added anhydrous sodium methoxide (19 mg). It was stirred at
room temperature for 2 h. and then neutralized by 0.5% HCl to pH ~ 6.5–7.0. The methanol was
removed on a rotavapor under reduced pressure followed by desalting on a pre-packed C-18 cartridge
(500 mg) from Varian. The de-acetylated product was further purified via preparative HPLC on a C-18
1
column to get pure reb A ketone, yield = 31 mg (22%). H-NMR (DMSO) δ = 1.204 (s, 3H, H-19),
0.979 (s, 3H, H-20); 13C-NMR: δ = 215.457 (C=O, C-16); ESI-MS m/z: 992.1 [M+Na]+, Calcd. 991.98
for C43H68O24[M+Na]+.
4. Conclusions
We have investigated the relationship between the presence or absence of the methylene double
bond on the aglycone of steviol glycosides and the corresponding impact on taste. We have found that
converting stevioside and reb A to their corresponding ketones by switching the doubly bonded
methylene on the C17 with a ketone actually removes the sweet taste properties of these molecules
completely. Upon regenerating the original molecules, the sweet taste of both the steviol glycosides
were found to be restored. These findings indicate that the C16-C17 part of reb A and stevioside is an
essential pharmacophore for the sweetness properties of these molecules. This is also the first literature

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report of stevioside and reb A peracetate and its novel ketones derivatives. The methylenation reaction
of these ketone derivatives using TiCl4-Zn-CH2Br2 reagent is also the first such report in the literature.
References and Notes
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5
McMurry, J.E.; Musser, J.H.; Ahmad, M.S.; Blaszcak, L.C. Total synthesis of Eremophilone.
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Sample Availability: Samples of the ketone derivatives are available from the authors.
© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).