Synthesis of ent-kaurane diterpene monoglycosides

Article abstract of DOI:10.3390/molecules16108402

Synthesis of two ent-kaurane diterpene glycosides, steviol 19-O-β-D-glucopyranosiduronic acid (steviol glucuronide, 5), and 13-hydroxy ent-kaur-16-en-19-oic acid-β-D-glucopyranosyl ester (7) has been achieved from a common starting material, steviol, usin

Full text of DOI:10.3390/molecules16108402

Molecules 2011, 16, 8402-8409; doi:10.3390/molecules16108402
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis of ent-Kaurane Diterpene Monoglycosides
Venkata Sai Prakash Chaturvedula *, Josef Klucik, Mani Upreti and Indra Prakash
Organic Chemistry Department, Global Research and Development, The Coca-Cola Company,
One Coca-Cola Plaza, Atlanta, GA 30313, USA
* Author to whom correspondence should be addressed; E-Mail: vchaturvedula@na.ko.com;
Tel.: +1-404-676-9257; Fax: +1-404-598-9257.
Received: 26 August 2011; in revised form: 28 September 2011 / Accepted: 29 September 2011 /
Published: 3 October 2011
Abstract: Synthesis of two ent-kaurane diterpene glycosides, steviol 19-O-β-D-
glucopyranosiduronic acid (steviol glucuronide, 5), and 13-hydroxy ent-kaur-16-en-19-oic
acid-β-D-glucopyranosyl ester (7) has been achieved from a common starting material,
steviol, using phase transfer catalyst. Also, synthesis of an additional 17-nor-ent-kaurane
glycoside, namely 13-methyl-16-oxo-17-nor-ent-kauran-19-oic acid-β-D-glucopyranosyl
ester (10) was performed using the starting material isosteviol and similar synthetic
methodology. Synthesis of all three steviol glycosides was performed using straightforward
chemistry and their structures were characterized on the basis of 1D and 2D NMR as well
as mass spectral (MS) data.
Keywords: ent-kaurane diterpene glycosides; nor ent-kaurane glycoside; steviol, isosteviol;
synthesis; structure characterization; spectral data
1. Introduction
The major constituents isolated from the leaves of Stevia rebaudiana Bertoni (family: Asteraceae)
are the potently sweet diterpenoid glycosides stevioside, and rebaudioside A. These compounds which
are known as Stevia sweeteners are glycosides of the diterpene steviol, ent-13-hydroxykaur-16-en-19-
oic acid [1]. Stevioside tastes about 150-250 times sweeter than sucrose, whereas rebaudioside A tastes
about 200-300 times sweeter than sucrose; both are non-caloric. In some parts of the world, including
Japan, South Korea, Israel, Mexico, Paraguay, Brazil, Argentina, and Switzerland, these steviol
glycosides are used to sweeten food products and beverages. As a part of our continuing research to

Molecules 2011, 16
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discover natural sweeteners, we have reported the isolation of several glycosides from the commercial
extract of S. rebaudiana [2-8]. Apart from isolating novel compounds from S. rebaudiana and utilizing
them as possible natural sweeteners or sweetness enhancers, we are also engaged in understanding the
physicochemical profiles of steviol glycosides in various systems of interest and their metabolites, as
well as their characterization [9]. Though many steviol glycosides have been reported in the literature,
focused synthetic studies were not carried out. In this article, we present the synthesis of three
ent-kaurane diterpene glycosides possessing steviol and isosteviol skeletons, and the characterization
of their structures based on extensive NMR and mass spectroscopic data.
2. Results and Discussion
2.1. Chemistry
The two compounds 5 and 7 were synthesized via the common intermediate 13-acetyloxy-ent-kaur-
16-en-19-oic acid (steviol acetate, 2), prepared by acetylation of steviol (1) with Ac2O and pyridine
(Scheme 1).
Scheme 1. Synthesis of steviol acetate (2).
Scheme 2. Synthesis of steviol glucuronide (5).

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8404
Compound 5 was prepared by esterification of 2 with bromo-2,3,4-tri-O-acetyl-α-D-glucopyran-
uronic acid methyl ester (methyl acetobromo-α-D-glucuronate) in the presence of the phase transfer
catalyst tetrabutylammonium bromide (TBAB) to yield 3, which upon deacetylation using Et3N,
followed by alkaline hydrolysis, furnished the final steviol glucuronide (5) (Scheme 2). Compound 7
was synthesized from 2 using the same esterification procedure as mentioned for 5 with 2,3,4,6-tetra-
O-acetyl-α-D-glucopyranosyl bromide (acetobromo-α-D-glucose) to furnish intermediate 6, which on
deacetylation using Et3N afforded the final compound 7 (Scheme 3).
Scheme 3. Synthesis of 13-hydroxy ent-kaur-16-en-19-oic acid-β-D-glucopyranosyl ester (7).
Compound 10 was prepared by esterification of isosteviol (8) with acetobromo-α-D-glucose in
the presence of phase transfer catalyst as described for 7, producing intermediate 9, which on
deacetylation with Et3N as reported above yielded compound 10 (Scheme 4).
Scheme 4. Synthesis of 13-methyl-16-oxo-17-nor-ent-kauran-19-oic acid-β-D-gluco-
pyranosyl ester (10).
CH3
CH3
O
O
CH3
CH3
acetobromo- -D-glucose
K2CO3/TBAB/CH2Cl2/H2O,
reflux, 48 hr
H
H
O
H3C
O
O
H3C
H3COCO
O
OH
H3COCO
H3COCO
OCOCH3
9
8
Et3N/MeOH/H2O/hexane,
rt, 48 hr
CH3
O
CH3
H
O
H3C
O
HO
HO
HO
O
OH
10

Molecules 2011, 16
8405
2.2. Spectroscopy
The structural characterization of 5, 7 and 10 was performed on the basis of one dimensional (1H,
13C), two-dimensional (1H-1H COSY, 1H-13C HMQC, 1H-13C HMBC) NMR and mass spectral data, as
well as in comparison with literature values [3,10]. The attachment of sugars at the C-19 position of
the steviol skeleton in 5 and 7, and the isosteviol skeleton in 10 was characterized by the key HMBC
correlation of the anomeric protons of the respective sugar moieties with the C-19 carbonyl group. The
1H- and 13C-NMR values for all the protons and carbons were assigned on the basis of COSY, HMQC
and HMBC correlations and are given in Tables 1 and 2. The anomeric protons in all three glycosides
5, 7 and 10 were observed as doublets at δ 5.44 (d, 7.8 Hz), 5.41 (d, 8.2 Hz) and 5.38 (d, 8.2 Hz)
respectively, suggesting their β-configuration similar to that of the steviol glycosides isolated from
S. rebaudiana [2-8]. This suggested that even though esterification was performed using their
corresponding α-derivatives; the configuration of the final products 5, 7, and 10 was observed to
correspond to their β-derivatives; an identical phenomenon was reported earlier for glycosylation of
triterpene acids under phase transfer catalytic conditions [11].
Table 1. 1H-NMR chemical shift values for compounds 5, 7 and 10 * recorded in CD3OD a.
Position
5
7
10
1
2
0.86 (m, 1H), 1.86 (m, 1H)
1.39 (m, 1H), 1.90 (m, 1H)
1.02 (m, 1H), 2.26 (d, 11.9, 1H)
1.08 (m, 1H)
0.86 (m, 1H), 1.88 (m, 1H)
1.43 (m, 1H),1.93 (m, 1H)
1.06 (m, 1H), 2.19 (d, 12.4, 1H)
1.12 (m, 1H)
0.96 (m, 1H), 1.68 (m, 1H)
1.36 (m, 1H), 1.92 (m, 1H)
1.05 (m, 1H), 2.16 (d, 13.2, 1H)
1.20 (m, 1H)
3
5
6
1.82 (m, 1H), 1.93 (m, 1H)
1.45 (m, 1H), 1.56 (m, 1H)
0.88 (m, 1H)
1.85 (m, 1H), 1.96 (m, 1H)
1.44 (m, 1H), 1.54 (m, 1H)
0.96 (m, 1H)
1.86 (m, 2H)
7
1.47 (m, 1H), 1.66 (m, 1H)
1.24 (m, 1H)
9
11
12
14
1.60 (m, 1H), 1.79 (m, 1H)
1.53 (m, 1H), 1.87 (m, 1H)
1.58 (m, 1H), 2.23 (d, 12.1, 1H)
1.61 (m, 1H), 1.77 (m, 1H)
1.46 (m, 1H), 1.74 (m, 1H)
1.28 (m, 1H), 2.11 (m, 1H)
1.25 (m, 1H), 1.70 (m, 1H)
1.42 (m, 1H), 1.56 (m, 1H)
1.44 (m, 1H), 1.58 (m, 1H)
1.80 (m, 1H), 2.65 (dd, 3.1,
17.2, 1H)
15
2.02 (m, 1H), 2.16 (d, J = 17.4, 1H) 2.06 (m, 1H), 2.17 (m, 1H)
17
18
20
1′
4.59 (s, 1H), 4.78 (br s, 1H)
1.23 (s, 3H)
4.77 (br s, 1H), 4.93 (s, 1H)
1.20 (s, 3H)
1.24 (s, 3H)
0.97 (s, 3H)
0.98 (s, 3H)
0.93 (s, 3H)
5.44 (d, 7.8, 1H)
5.41 (d, J = 8.2 Hz, 1H)
3.35 (dd, 7.1, 7.6, 1H)
3.44 (dd, 8.3, 9.1, 1H)
5.38 (d, 8.2, 1H)
3.32 (dd, 7.2, 7.8, 1H)
3.43 (dd, 8.1, 9.1, 1H)
2′
3.60 (dd, 8.2, 9.1, 1H)
3.42 (dd, 8.1, 8.9, 1H)
3′

Molecules 2011, 16
8406
Table 1. Cont.
4′
5′
3.49 (dd, 8.1, 9.2, 1H)
3.72 (d, 8.2, 1H)
3.34 (dd, 8.2, 9.4, 1H)
3.34 (dd, 8.1, 9.1, 1H)
3.37 (ddd, 8.2, 1.9, 7.2, 1H)
3.39 (ddd, 8.1, 2.1, 7.4, 1H)
3.66 (dd, 2.1, 12.1, 1H), 3.81 (dd, 3.68 (dd, 1.9, 12.1, 1H), 3.82
4.2, 12.1, 1H) (dd, 3.9, 12.1, 1H)
6′
a assignments made on the basis of COSY, HMQC and HMBC correlations; b Chemical shift values
are in δ (ppm); c Coupling constants are in Hz; * δH 0.80 (s, 3H): CH3-13 for compound 10.
Table 2. 13C-NMR chemical shift values for 5, 7 and 10 * recorded in CD3OD a.
Position
1
1
2
3
42.0
20.3
39.2
45.2
58.7
23.1
42.8
42.9
55.5
40.9
21.5
40.7
81.0
47.4
48.8
157.2
103.5
29.1
178.2
16.5
95.6
78.7
74.0
73.6
77.7
177.5
41.5
20.2
38.6
45.1
58.5
22.8
42.4
43.0
55.0
40.5
21.3
40.4
88.5
47.2
48.6
157.0
103.4
29.1
178.0
16.3
95.6
74.0
77.7
71.0
77.8
61.2
39.5
19.1
38.0
43.9
57.6
20.7
41.4
48.4
53.9
37.6
21.1
38.6
39.1
54.6
48.3
223.8
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
1′
27.6
176.8
19.1
95.5
73.8
77.3
70.8
77.6
61.1
2′
3′
4′
5′
6′
a
b
assignments made on the basis of HMQC and HMBC correlations; Chemical shift values are in
δ (ppm); * δC 12.8: CH3-13 for compound 10.
3. Experimental
3.1. General
Melting points were measured using a SRS Optimelt MPA 100 instrument and are uncorrected.
Optical rotations were recorded using a Rudolph Autopol V at 25 °C and IR spectral data was acquired

Molecules 2011, 16
8407
using a Perkin Elmer 400 Fourier Transform Infrared (FT-IR) spectrometer equipped with a universal
attenuated total reflectance (UATR) polarization accessory. NMR spectra were acquired on Bruker
Avance DRX 500 MHz using standard pulse sequences. Chemical shifts are given in δ (ppm), and
coupling constants are reported in Hz. HRMS and MS/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 mode and ThermoFisher Discovery OrbiTrap in the positive mode
of electrospray. Samples were diluted with water: acetonitrile (1:1) containing 0.1% formic acid and
introduced via infusion using the onboard syringe pump. HPLC was performed on an Agilent 1100
system using a Phenomenex Prodigy ODS (3) column (250 × 21.2 mm, 5 μm) or a Beckman Gold
system with a Zorbax amino (150 × 4.6 mm, 5 μm) column.
3.2. Isolation
Purification of compound 5 was carried out using the Beckman HPLC system using an isocratic
solvent method; UV Detection: 210 nm; Mobile Phase A: 75% CH3CN/25% H2O/0.05% AcOH;
Mobile Phase B: 100% CH3CN/0.05% AcOH; Flow Rate: 1.0 mL/min; Gradient ratio: Mobile Phase
A/B (60:40), by collecting the peak eluting at tR 1.33 min. Compound 7 was purified by using the
Agilent HPLC 1100 system. UV Detection: 210 nm; Mobile Phase A: H2O (0.02% AcOH, 0.08%
NH4OAc); Mobile Phase B: CH3CN; using gradient [25% B for 8.5 min, 25 to 29% B over 1.5 min, 29
to 30% B over 5.5 min, 30 to 34% B over 4.0 min, 34% B for 6 min, 34 to 52% B over 2.0 min, 52% B
for 3.0 min, 52 to 70% B over 1.0 min, 70% B for 5.5 min] at 5 mL/min flow rate. The peak eluting at
tR 28.3 min corresponds to 7. Compound 10 was also purified using the Agilent HPLC 1100 system.
UV Detection: 226 nm; Mobile Phase A: H2O (0.1% TFA); Mobile Phase B: CH3CN; gradient
increased from 95:5 (A:B) to 0:100 (A:B) over 30 min; Flow Rate: 5.0 mL/min. The peak eluting at
tR 18.2 min furnished 10.
Steviol acetate (2): Steviol acetate (2) was prepared from steviol (1) as reported in the literature and
1
characterized based on the H-NMR and mass spectral data and comparison with the spectral data
1
reported in the literature [12]. H-NMR (500 MHz, CDCl3): δ 4.92 (s, 1H, 17-H), 4.86 (s, 1H, 17-H),
2.03 (s, 3H, OCOCH3); HRMS (M+Na)+ m/z 383.2198 (calcd. for C22H32O4Na: 383.2208).
Steviol glucuronide (5): Compound 2 (0.633 g, 1.76 mmol) was dissolved in CH2Cl2 (25 mL) and
distilled water (3 mL). TBAB (10 mg), K2CO3 (0.62 g, 4.50 mmol), and methyl acetobromo-α-D-
glucuronate (0.80 g, 2.02 mmol) were added at room temperature. The reaction mixture was refluxed
for 48 h, cooled to room temperature and the layers were separated. The aqueous layer was extracted
with additional amounts of CH2Cl2 (2 × 20 mL) and the combined organic layer was washed with
water (2 × 30 mL) and brine (40 mL). Concentration of the CH2Cl2 layer under vacuum furnished a
residue (1.05 g), which showed a peak at m/z 677 in its mass spectrum corresponding to the (M+H)+
ion of 3. Compound 3 (1.00 g, 14.7 mmol) was dissolved in 10% solution of Et3N in MeOH-H2O-
hexane (10:2:1, 23 mL) and stirred at room temperature for 48 h. The MeOH and hexane were
evaporated under vacuum and the resulting product (0.73 g) was identified as 4 on the basis of its mass
spectrum, which showed a peak at m/z 509 corresponding to its (M+H)+ ion. Compound 4 (0.70 g) was
dissolved in H2O (20 mL) and KOH (0.16 g, 0.28 mmol) was added. The mixture was stirred at room

Molecules 2011, 16
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temperature for 48 h and the mixture was subjected to evaporation under reduced pressure. Purification
of the residue obtained after evaporation was performed using HPLC furnished 420 mg of 5. White
25
powder, mp 198.7 °C, [α]D −70.6 (c 1.0, MeOH); IR νmax: 3303, 2937, 1723, 1599, 1053, 970 cm−1;
13
1H-NMR (500 MHz, CD3OD, δ ppm) and C-NMR (125 MHz, CD3OD, δ ppm) spectroscopic data
see Tables 1 and 2; HRMS (M+NH4)+ m/z 512.2852 (calcd. for C26H42O9N: 512.2860); (M+Na)+ m/z
517.2405 (calcd. for C26H38O9Na: 517.2414).
13-Hydroxy-ent-kaur-16-en-19-oic acid-β-D-glucopyranosyl ester (7): To a solution of 2 (2.00 g,
5.56 mmol) in CH2Cl2 (35 mL) and distilled water (4 mL); TBAB (30 mg), K2CO3 (1.94 g, 14.06 mmol),
and acetobromo-α-D-glucose (2.88 g, 7 mmol) were added at room temperature. The reaction mixture
was refluxed for 48 h, cooled to room temperature and the layers were separated. The aqueous layer
was extracted with additional CH2Cl2 (2 × 50 mL) and the combined organic layer was washed with
water (2 × 60 mL) and brine (100 mL). Concentration of the CH2Cl2 layer under vacuum furnished a
residue (2.23 g) which was identified as 6 on the basis of its EIMS spectrum which showed a peak
at m/z 691 corresponding to its (M+H)+ ion. Compound 6 (2.00 g, 2.90 mmol) was dissolved in 10%
solution of Et3N in MeOH-H2O-hexane (10:2:1, 45 mL) and stirred at room temperature for 48 h. The
residue resulting after evaporation of the MeOH and hexane was purified by HPLC to furnish
25
850 mg of 7. White powder, [α]D −62.4 (c 1.0, EtOH); IR νmax: 3350, 2929, 1725, 1033, 890 cm−1;
13
1H-NMR (500 MHz, CD3OD, δ ppm) and C-NMR (125 MHz, CD3OD, δ ppm) spectroscopic data
see Tables 1 and 2; HRMS (M+Na)+ m/z 503.2608 (calcd. for C26H40O8Na: 503.2621).
13-Methyl-16-oxo-17-nor-ent-kauran-19-oic acid-β-D-glucopyranosyl ester (10): To a solution of 8
(0.70 g, 2.20 mmol) in CH2Cl2 (15 mL) and distilled water (4 mL) was added TBAB (15 mg), K2CO3
(1.00 g, 7.25 mmol), and acetobromo-α-D-glucose (1.25 g, 3.04 mmol). The reaction mixture was
refluxed for 48 h, cooled to room temperature and worked-up as described above to furnish a residue
(1.20 g) which showed a peak at m/z 649 in its EIMS spectrum corresponding to the (M+H)+ ion of 9.
Compound 9 (1.00 g, 1.54 mmol) was deacetylated as described above and upon usual work-up
afforded a residue which was purified by HPLC to yield 500 mg of 10. White powder, mp 172.5 °C,
[α]D25 −55.8 (c 1.0, EtOH); IR νmax: 3352, 2928, 1726, 1034, 891 cm−1; H-NMR (500 MHz, CD3OD,
1
δ ppm) and 13C-NMR (125 MHz, CD3OD, δ ppm) spectroscopic data see Tables 1 and 2; HRMS
(M+Na)+ m/z 503.2611 (calcd. for C26H40O8Na: 503.2621).
4. Conclusions
In conclusion, three ent-kaurane diterpene glycosides 5, 7 and 10 were synthesized from the natural
products steviol and isosteviol through simplified procedures. To the best of our knowledge, this is the
first report of the synthesis of C-19 glycosidic linkages on the steviol and istosteviol skeletons. The
structures of all the synthesized compounds were characterized on the basis of NMR (1D and 2D) and
mass spectral data, as well as in comparison with the data reported in the literature.

Molecules 2011, 16
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Acknowledgements
We wish to thank AMRI, Bothell Research Center, Bothell, WA for obtaining selected NMR
spectral data and Chris Mubarak, Analytical Sciences Department, The Coca-Cola Company, Atlanta,
GA for providing IR spectral data.
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