Synthesis of a trisaccharide related to the triterpenoid saponin kalopanaxsaponin I isolated from Nigella sativa

Article abstract of DOI:10.1080/07328303.2010.483040

Saponins are plant secondary metabolites important for growth and development, and they have immense potential as drug candidates for various diseases. Synthesis of the glycone part of the triterpenoid saponin kalopanaxsaponin I isolated from Nigella sati

Full text of DOI:10.1080/07328303.2010.483040

Journal of Carbohydrate Chemistry, 29:133–141, 2010
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Copyright Taylor & Francis Group, LLC
ISSN: 0732-8303 print / 1532-2327 online
DOI: 10.1080/07328303.2010.483040
Synthesis of a Trisaccharide
Related to the Triterpenoid
Saponin Kalopanaxsaponin I
Isolated from Nigella sativa
Santanu Mandal,1 Somnath Mukherjee,1
Balaram Mukhopadhyay,1 and Sumita Mukherjee2
1Indian Institute of Science Education and Research-Kolkata (IISER-K), Mohanpur
Campus, P.O. BCKV Campus Main Office, Mohanpur, Nadia 741 252, West Bengal,
India
2S. F. S. College, Seminary Hills, Nagpur University, Nagpur, Maharasthra
OH
O
OMP
HO
O
Me
O
HO
O
O
HO
HO
OH
OH
1
Graphical Abstract
Saponins are plant secondary metabolites important for growth and development, and
they have immense potential as drug candidates for various diseases. Synthesis of
the glycone part of the triterpenoid saponin kalopanaxsaponin I isolated from Nigella
sativa is reported as its p-methoxyphenyl glycoside. The synthesis of the target trisac-
charide has been accomplished by following concise protecting group manipulations
on commercially available L-arabinose, L-rhamnose, and D-xylose. Stereoselective gly-
cosylations are achieved through activation of thioglycoside by N-iodosuccinimide in
conjunction with H2SO4 silica.
Keywords Triterpenoid saponin; Glycosylation; H2SO4 silica
Received November 4, 2009; accepted March 31, 2010.
Address correspondence to Balaram Mukhopadhyay, Indian Institute of Science Edu-
cation and Research-Kolkata (IISER-K), Mohanpur Campus, P.O. BCKV Campus Main
Office, Mohanpur, Nadia 741 252, West Bengal, India. E-mail: sugarnet73@hotmail.com
133

S. Mandal et al.
134
INTRODUCTION
Triterpenoid saponins are synthesized by plants during their regular program
of growth and development. Many of them are exploited as a source of
drugs, for example, ginseng and liquorice, and also crop plants like legumes
and oats.[1] This class of molecules is important because of their antifungal
properties through preformed barriers in the healthy plants.[2] Despite the
commercial interest associated with this class of natural products, the genetic
machinery involved in the biosynthesis of these glycosylated secondary
metabolites are yet to be discovered completely. Glycosylation at the C-3
position of the triterpenoid backbone is a common feature shared by this class
of molecules. Since the C-3 position of the triterpenoid is important for saponin
bioactivity, the glycosylation pattern at this site is worthy of exploration.[3]
To obtain a better understanding of the glycosyltransferases involved and to
establish the order of events in saponin biosynthesis, synthetic saccharide
fragments would be very useful.[4]
In this communication, we report a concise route for the synthesis of the
glycone part of the triterpenoid saponin kalopanaxsaponin I isolated from
Nigella sativa[5] in the form of its p-methoxyphenyl glycoside (Fig. 1).
RESULT AND DISCUSSION
The glycone part of the triterpenoid saponin kalopanaxsaponin I isolated from
N. sativa consists of L-arabinose, L-rhamnose, and D-xylose units. The choice
of p-methoxyphenyl group as the reducing end glycoside was driven by the fact
that after the total synthesis, this glycoside can be hydrolyzed selectively and
coupled to suitable aglycon to generate biologically relevant glycoconjugate.
H
OH
O
O
OMP
HO
OH
OH
HO
Me
H
O
O
O
O
Me
O
H
HO
O
CH2OH
O
HO
HO
OH
O
OH
HO
O
O
HO
HO
OH
1
OH
kalopanaxsaponin I
Figure 1: Structure of kalopanaxsaponin I and the synthetic target.

Synthesis of Trisaccharide Related to Nigella Sativa
135
The axial OH group of the known p-tolyl 4-O-benzyl-1-thio-α-L-
rhamnopyranoside (2)[6] was selectively benzylated by phase-transfer tech-
nique using BnBr and 10% NaOH in the presence of Bu4NBr[7] to afford the
dibenzyl derivative 3 in 78% yield. Acetylation of the remaining OH group with
Ac2O and pyridine[8] furnished the rhamnosyl donor p-tolyl 3-O-acetyl-2,4-di-
O-benzyl-1-thio-α-L-rhamnopyranoside (4) in 92% yield.
Further, glycosylation of compound 4 with known p-methoxyphenyl
3,4-O-isopropylidene-β-L-arabinopyranoside (5)[9] was accomplished by using
N-iodosuccinimide (NIS) in the presence of H2SO4 immobilized on silica
(H2SO4-silica)[10] to furnish the disaccharide 6 in 86% yield. The required α-
linkage has been confirmed by the JC,H coupling constant 172 Hz, which is
normal for α-Rha linkage.[11] Removal of the acetyl group using NaOMe in
MeOH afforded the disaccharide acceptor 7 in 89% yield. Finally, the disaccha-
ride acceptor was coupled with the known donor p-tolyl 2,3,4-tri-O-acetyl-1-
thio-β-D-xylopyranoside (8) following the same glycosylation strategy as above
to afford the protected trisaccharide 9 in 81% yield. Next, hydrolysis of the
isopropylidene acetal with 80% AcOH at 80◦C[12] and catalytic hydrogenation
using Pd-C followed by Zemple´n de-O-acylation furnished the target trisaccha-
ride p-methoxyphenyl β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-
β-L-arabinopyranoside (1) in 73% yield over three steps (Sch. 1).
CONCLUSION
In conclusion, we have developed an efficient synthetic strategy for the trisac-
charide fragment related to the glycone part of the triterpenoid saponin
kalopanaxsaponin I isolated from N. sativa. The trisaccharide will be evalu-
ated for its biological activity in due course.
EXPERIMENTAL
General Methods
All reagents and solvents were dried prior to use according to standard
methods.[13] Commercial reagents were used without further purification un-
less otherwise stated. Analytical TLC was performed on silica gel 60-F254 with
detection by fluorescence and/or by charring following immersion in a 10%
ethanolic solution of sulfuric acid. An orcinol dip, prepared by the careful
addition of concentrated sulfuric acid (20 mL) to an ice-cold solution of 3,5-
dihydroxytoluene (360 mg) in EtOH (150 mL) and H2O (10 mL), was used
to detect deprotected compounds by charring. Flash chromatography was per-
formed with silica gel 60. Optical rotations were measured at the sodium D-
1
line at ambient temperature. H NMR and 13C NMR spectra were recorded

136

Synthesis of Trisaccharide Related to Nigella Sativa
137
on a spectrometer at 500 MHz and 125 MHz, respectively. Mass spectra were
recorded on a Micromass Q-TOF Micro TM spectrometer.
p-Tolyl 3-O-acetyl-2,4-di-O-benzyl-1-thio-α-L-
rhamnopyranoside (4)
Compound 2 (2.2 g, 6.2 mmol) was dissolved in CH2Cl2 (40 mL); 10% aq.
NaOH (7 mL) was added followed by Bu4NBr (2.9 g, 8.9 mmol) and BnBr (1.1
mL, 9.8 mmol), and the biphasic mixture was vigorously stirred at rt for 16
h. Then the mixture was washed with H2O (3 × 50 mL) and the organic layer
was collected, dried (Na2SO4), filtered, and evaporated. The crude product was
purified by flash chromatography using n-hexane-EtOAc (3:1) to afford pure
compound 3 (2.2 g, 78%) as a colorless gel. To a solution of compound 3 (2.0 g,
4.4 mmol) in dry pyridine (15 mL), Ac2O (10 mL) was added and the solution
was stirred at rt for 2 h. After evaporating the solvents in vacuo, the residue
was purified by flash chromatography using n-hexane-EtOAc (4:1) to afford
25
1
pure compound 4 (2.0g, 92%) as a colorless gel. [α]D +78 (c 1.1, CHCl3); H
NMR (500 MHz, CDCl3) δ: 7.36–7.10 (m, 14H, ArH), 5.39 (d, 1H, J1,2 = 1.5
Hz, H-1), 5.17 (dd, 1H, J2,3 = 3.5 Hz, J3,4 = 9.0 Hz, H-3), 4.73, 4.66, 4.64,
4.47 (4d, AB system, 4H, J = 12.5 Hz, 2 × CH2Ph), 4.23 (m, 1H, H-5), 4.08
(dd, 1H, J1,2 = 1.5 Hz, J2,3 = 3.5 Hz, H-2), 3.69 (t, 1H, J3,4, J4,5 = 9.0 Hz,
H-4), 2.32 (s, 3H, S-C6H4-CH3), 1.97 (s, 3H, COCH3), 1.34 (d, 3H, J = 6.5 Hz,
C-CH3). 13C NMR (125 MHz, CDCl3) δ: 170.2 (COCH3), 138.2, 137.7, 132.2(2),
130.5, 129.8(2), 128.4(3), 128.0(2), 127.9(2), 127.8(2), 127.7(2) (ArC), 85.6, 79.2,
75.0, 73.7, 72.4, 69.0, 21.1 (COCH3), 21.0 (S-C6H4-CH3), 18.0 (C-CH3). HRMS
calcd. for C29H32O5SNa (M+Na)+: 515.1868, found: 515.1866.
p-Methoxyphenyl 3-O-acetyl-2,4-di-O-benzyl-1-thio-α-L-
rhamnopyranosyl-(1→2)-3,4-O-isopropylidene-β-L-
arabinopyranoside (6)
A mixture of compound 4 (1.9 g, 3.9 mmol), compound 5 (1.0 g, 3.4 mmol),
´
˚
and MS 4A (1.5 g) in dry CH2CI2 (20 mL) was stirred under nitrogen atmo-
sphere for 1 h. NIS (1.1 g, 4.7 mmol) was added and the mixture was cooled
to 10◦C using ice-water bath. After stirring for 15 min, TMSOTf (20 µL, 1.2
mmol) was added and the mixture was allowed to stir at 10◦C until complete
conversion of acceptor 5 was evident by TLC (45 min). The mixture was im-
mediately filtered through a pad of Celite and the filtrate was washed succes-
sively with Na2S2O3 (2 × 30 mL), NaHCO3 (2 × 30 mL), and brine (30 mL).
The organic phase was collected, dried (Na2SO4), and evaporated to a syrup.
The crude product thus obtained was purified by flash chromatography using
n-hexane-EtOAc (3:1) to afford pure compound 6 (1.9 g, 86%) as white foam.
[α]D +134 (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3) δ: 7.36–7.24 (m, 12H,
25
ArH), 6.96, 6.77 (2d, 4H, O-C6H4-OCH3), 5.30 (d, 1H, J1 ,2 = 1.5 Hz, H-1ꢀ), 5.13
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S. Mandal et al.
138
(dd, 1H, J2 ,3 = 3.5 Hz, J3 ,4 = 9.5 Hz, H-3ꢀ), 4.91 (d, 1H, J1,2 = 7.5 Hz, H-1),
4.70, 4.67, 4.62, 4.57 (4d, AB system, 4H, J = 12.0 Hz, 2 × CH2Ph), 4.34 (m,
1H, H-4), 4.19 (t, 1H, J1,2, J2,3 = 7.5 Hz, H-2), 4.12–4.01 (m, 3H, H-3, H-5a, H-
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5ꢀ), 3.93 (dd, 1H, J1 ,2 = 1.5 Hz, J2 ,3 = 3.5 Hz, H-2ꢀ), 3.89 (dd, 1H, J4,5a = 5.0
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Hz, J5a,5b = 12.5 Hz, H-5b), 3.74 (s, 3H, O-C6H4-OCH3), 3.64 (t, 1H, J3 ,4 , J4 ,5
= 9.5 Hz, H-4ꢀ), 1.92 (s, 3H, COCH3), 1.54, 1.37 (2s, 6H, isopropylidene-CH3),
1.35 (d, 3H, J = 6.5 Hz, C-CH3). 13C NMR (125 MHz, CDCl3) δ: 170.3 (COCH3),
155.2, 151.1, 138.5, 138.0, 128.4(2), 128.3(2), 128.0, 127.8, 127.5(2), 127.4(2),
118.4(2), 114.6(2) (ArC), 110.6 (isopropylidene-C), 99.9 (C-1), 97.2 (C-1ꢀ), 79.0,
78.3, 76.4, 76.2, 74.5, 73.6, 73.0, 72.5, 67.9, 62.5 (C-5), 55.7 (O-C6H4-OCH3),
27.7, 25.9 (2 × isopropylidene-CH3), 21.1 (COCH3), 18.0 (C-CH3). HRMS calcd.
for C37H44O11Na (M+Na)+: 687.2781, found: 687.2778.
p-Methoxyphenyl 2,4-di-O-benzyl -α-L-rhamnopyranosyl-(1→2)-
3,4-O-isopropylidene-β-L-arabinopyranoside (7)
To a solution of compound 6 (1.8 g, 2.7 mmol) in dry MeOH (20 mL), 0.5N
NaOMe in MeOH (2 mL) was added and the solution was allowed to stir at
rt for 2 h. Then the solution was neutralized by DOWEX 50W H+ resin and
filtered. The filtrate was evaporated in vacuo and coevaporated with toluene to
remove residual MeOH. Flash chromatography using n-hexane-EtOAc (3:1) af-
25
forded pure compound 7 (1.3 g, 89%) as white foam. [α]D +96 (c 1.0, CHCl3);
1H NMR (500 MHz, CDCl3) δ: 7.38–7.24 (ArH), 6.95, 6.77 (2d, 4H, O-C6H4-
OCH3), 5.36 (s, 1H, H-1ꢀ), 4.87 (d, 1H, J1,2 = 7.0 Hz, H-1), 4.85, 4.77, 4.64,
4.60 (4d, AB system, 4H, J = 12.0 Hz, 2 × CH2Ph), 4.34 (m, 1H, H-4), 4.15
(t, 1H, J1,2, J2,3 = 7.0 Hz, H-2), 4.03–4.00 (m, 3H, H-3, H-3ꢀ, H-5a), 3.92 (m,
1H, H-5ꢀ), 3.89 (dd, 1H, J4,5a = 5.0 Hz, J5a,5b 12.5 Hz, H-5b), 3.78 (bd, 1H,
J2 ,3 = 3.5 Hz, H-2ꢀ), 3.73 (s, 3H, O-C6H4-OCH3), 3.34 (t, 1H, J3 ,4 , J4 ,5 = 9.0
Hz, H-4ꢀ), 2.39 (bs, 1H, OH), 1.54, 1.37 (2s, 6H, isopropylidene-CH3), 1.32 (d,
3H, J = 6.5 Hz, C-CH3). 13C NMR (125 MHz, CDCl3) δ: 155.2, 151.2, 138.7,
137.8, 128.6(2), 128.4(2), 128.1(2), 128.0, 127.8(2), 127.6, 118.3(2), 114.6(2)
(ArC), 110.7 (isopropylidene-C), 99.9 (C-1), 96.5 (C-1ꢀ), 82.2, 78.5, 78.2, 76.2,
74.7, 72.9, 72.4, 71.3, 67.5, 62.4 (C-5), 55.7 (O-C6H4-OCH3), 27.8, 25.9 (2 ×
isopropylidene-CH3), 18.1 (C-CH3). HRMS calcd. for C35H42O10Na (M+Na)+:
645.2676, found: 645.2673.
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p-Methoxyphenyl 2,3,4-tri-O-acetyl-β-D-xylopyranosyl-(1→3)-2,
4-di-O-benzyl -α-L-rhamnopyranosyl-(1→2)-3,4-O-
isopropylidene-β-L-arabinopyranoside (9)
A mixture of compound 7 (1.2 g, 1.9 mmol), compound 8 (950 mg, 2.5 mmol),
´
˚
and dried MS 4A (2 g) in dry CH2CI2 (20 mL) was stirred under nitrogen at-
mosphere for 1 h. NIS (672 mg, 3.0 mmol) was added and the mixture was

Synthesis of Trisaccharide Related to Nigella Sativa
139
cooled to 10◦C using ice-water bath. After stirring for 15 min, TMSOTf (10 µL,
0.8 mmol) was added and the mixture was allowed to stir at 10◦C until com-
plete conversion of acceptor 7 was evident by TLC (45 min). The mixture was
immediately filtered through a pad of Celite and the filtrate was washed suc-
cessively with Na2S2O3 (2 × 25 mL), NaHCO3 (2 × 25 mL), and brine (25 mL).
The organic phase was collected, dried (Na2SO4), and evaporated to a syrup.
The crude product thus obtained was purified by flash chromatography using
n-hexane-EtOAc (3:1) to afford pure compound 9 (1.4 g, 81%) as white foam.
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1
[α]D +107 (c 1.1, CHCl3); H NMR (500 MHz, CDCl3) δ: 7.43–7.26 (ArH),
6.95, 6.78 (2d, 4H, O-C6H4-OCH3), 5.22 (d, 1H, J1 ,2 = 2.0 Hz, H-1ꢀ), 5.15 (t,
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1H, J2 ,3 , J3 ,4 = 9.0 Hz, H-3ꢀꢀ), 5.03 (dd, 1H, J1 ,2 = 7.0 Hz, J2 ,3 = 9.0
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Hz, H-2ꢀꢀ), 4.92 (m, 1H, H-4ꢀꢀ), 4.90 (d, 1H, J1 ,2 = 7.0 Hz, H-1ꢀꢀ), 4.81 (d,
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1H, J1,2 = 7.5 Hz, H-1), 4.79, 4.77, 4.72, 4.55 (4d, 4H, J = 12.0 Hz, AB sys-
tem, 2 × CH2Ph), 4.33 (m, 1H, H-4), 4.15 (t, 1H, J1,2, J2,3 = 7.5 Hz, H-2),
4.03–4.00 (m, 3H, H-3, H-3ꢀ, H-5ꢀ, H-5a, H-5aꢀꢀ), 3.90 (dd, 1H, J4,5a = 5.0 Hz,
J5a,5b = 12.5 Hz, H-5b), 3.78 (dd, 1H, J1 ,2 = 2.0 Hz, J2 ,3 = 3.5 Hz, H-2ꢀ),
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3.76 (s, 3H, O-C6H4-OCH3), 3.58 (t, 1H, J3 ,4 , J4 ,5 = 9.0 Hz, H-4ꢀ), 3.24 (dd,
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1H, J4 ,5b = 9.5 Hz, J5a ,5b = 11.0 Hz, H-5bꢀꢀ), 2.04, 2.01, 1.83 (3s, 9H, 3 ×
COCH3), 1.52, 1.36 (2s, 6H, isopropylidene-CH3), 1.28 (d, 3H, J = 6.5 Hz, C-
CH3). 13C NMR (125 MHz, CDCl3) δ: 170.2, 169.9, 169.3 (3 × COCH3), 155.0,
151.1, 138.5, 138.3, 128.5(2), 128.3(2), 128.1(2), 127.7, 127.4(2), 127.3, 118.0(2),
114.5(2) (ArC), 110.6 (isopropylidene-C), 101.6 (C-1ꢀꢀ), 99.6 (C-1), 97.7 (C-1ꢀ),
80.3, 79.1, 78.0, 75.7, 74.6, 73.3, 72.3, 72.0, 71.5, 69.2, 68.1, 62.3 (C-5), 62.1
(C-5ꢀꢀ), 55.7 (O-C6H4-OCH3), 27.7, 25.8 (2 × isopropylidene-CH3), 20.7, 20.6,
20.5 (3 × COCH3), 17.8 (C-CH3). HRMS calcd. for C46H56O17Na (M+Na)+:
903.3415, found: 903.3416.
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p-Methoxyphenyl β-D-xylopyranosyl-(1→3)-α-L-
rhamnopyranosyl-(1→2)-β-L-arabinopyranoside (1)
A solution of compound 9 (1.0 g, 1.1 mmol) in 80% AcOH (10 mL) was
stirred at 80◦C for 2 h when the TLC showed complete conversion of the
starting material. Solvents were evaporated in vacuo and the syrupy mass
was dissolved in MeOH (100 mL). The dilute methanolic solution was then
passed through the flow hydrogenation assembly (H-cube, Thales Nano,
Hungary) that contained the Pd-C (10% Pd) cartridge. Complete hydrogena-
tion was achieved after three cycles at rt and 40 psi hydrogen pressure.
Solvents were evaporated and the residue was redissolved in dry MeOH
(10 mL); 0.5N NaOMe in MeOH (1 mL) was added and the solution was
stirred at rt for 12 h. The solution was neutralized with DOWEX 50W H+
resin, filtered, and evaporated in vacuo to afford pure trisaccharide 1 (440 mg,
25
1
73%) as amorphous white solid. [α]D +68 (c 0.8, H2O); H NMR (500 MHz,
CDCl3) δ: 6.92, 6.80 (2d, 4H, O-C6H4-OCH3), 4.95 (bs, 1H, H-1ꢀ), 4.89 (d, 1H,

S. Mandal et al.
140
J = 6.0 Hz, H-1), 4.40 (d, 1H, J1 ,2 = 7.6 Hz, H-1ꢀꢀ), 3.67 (s, 3H, C6H4OCH3),
1.04 (d, 3H, J = 6.0 Hz, C-CH3). 13C NMR (125 MHz, CDCl3) δ: 154.6, 150.3,
117.9(2), 115.1(2) (ArC), 104.5 (C-1ꢀꢀ), 101.0 (C-1), 99.8 (C-1ꢀ), 79.6, 77.2, 75.5,
73.2, 72.6, 71.0, 70.1, 69.2, 68.9, 68.4, 65.1, 65.0, 55.7 (O-C6H4-OCH3), 16.6
(C-CH3). HRMS calcd. for C23H34O14Na (M+Na)+: 557.1846, found: 557.1844.
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ACKNOWLEDGEMENT
Santanu Mandal and Somnath Mukherjee are thankful to the Council for
Scientific and Industrial Research for the research fellowships and Sumita
Mukherjee is thankful to the Indian Academy of Science for providing summer
fellowship. The work is funded by the Department of Science and Technology,
New Delhi, through the grant SR/S1/OC-67/2009.
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