Synthesis of two trisaccharides related to the hepatoprotective phenylethanoids leonoside e and F isolated from Leonurus japonicus Houtt

Article abstract of DOI:10.1016/j.carres.2013.08.014

The chemical synthesis of two trisaccharides related to leonoside E and F is reported. The target oligosaccharides were prepared in the form of their p-methoxyphenyl glycosides using a common disaccharide acceptor. All reaction steps were high yielding (>80%) and the stereoselective glycosylations were achieved by activation of the thioglycoside donors using N-iodosuccinimide in the presence of La(OTf)₃.

Full text of DOI:10.1016/j.carres.2013.08.014

Accepted Manuscript
Synthesis of two trisaccharides related to the hepatoprotective phenylethanoids
leonoside E and F isolated from Leonurus japonicus Houtt
Darshita Budhadev, Balaram Mukhopadhyay
PII:
S0008-6215(13)00325-X
http://dx.doi.org/10.1016/j.carres.2013.08.014
CAR 6553
DOI:
Reference:
To appear in:
Carbohydrate Research
Received Date:
Revised Date:
Accepted Date:
14 May 2013
12 August 2013
14 August 2013
Please cite this article as: Budhadev, D., Mukhopadhyay, B., Synthesis of two trisaccharides related to the
hepatoprotective phenylethanoids leonoside E and F isolated from Leonurus japonicus Houtt, Carbohydrate
Research (2013), doi: http://dx.doi.org/10.1016/j.carres.2013.08.014
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Synthesis of two trisaccharides related to the hepatoprotective phenylethanoids
leonoside E and F isolated from Leonurus japonicus Houtt.
Darshita Budhadev, Balaram Mukhopadhyay*≠
Indian Institute of Science Education and Research-Kolkata, Mohanpur, Nadia 741252, India
Abstract: The chemical synthesis of two trisaccharides related to leonoside E and F is reported. The
target oligosaccharides were prepared in the form of their p-methoxyphenyl glycosides using a common
disaccharide acceptor. All reaction steps were high yielding (>80 %) and the stereoselective
glycosylations were achieved by activation of the thioglycoside donors using N-iodosuccinimide in the
presence of La(OTf)3.
1. Introduction
Plants of the genus Leonurus (labiatae) are widespread in western Europe and China. The extracts
of the plants are widely used as traditional medicine for increasing blood circulation, regulating menstrual
disorders and as diuretics.i Various types of metabolites obtained from these plants include irridoids,ii
flavonoids,iii labdane-type terpenoids,iv,v cyclic peptidesvi and phenylethanoid glycosides.vii Some of these
compounds have shown cytotoxic,viii anti-inflammatory2 and anti-platelet aggregationix properties.
Recently, Zhang et. al.x reported structures of two new phenylethanoid glycosides leonoside E and F and
showed that the compounds exibit potent hepatoprotective activity. Herein, we report the chemical
≠ Corresponding author’s E.mail: sugarnet73@hotmail.com; Tel: +91 9748 261742, Fax:

synthesis of the trisaccharides (1 and 2, Figure 1) related to leonoside E and F respectively in the form of
their p-methoxyphenyl glycosides.
Figure 1. Structures of the target oligosaccharides
2. Results and discussion
The disaccharide, p-methoxyphenyl α-L-rhamnopyranosyl-(1→3)-β-D-glucopyranoside is
common to both targets. Thus, we envisaged that preparation of a suitable protected disaccharide would
facilitate the synthesis of the trisaccharides. Therefore, known p-methoxyphenyl 4,6-O-benzylidene- β-D-
glucopyranoside (3) was benzylated with Bu2SnO in dry MeOH followed by BnBr in the presence of CsF
to afford corresponding 2-O-benzyl (4) and 3-O-benzyl (5) derivatives in 3:2 ratio (83% yield). The
required 2-O-benzyl derivative was isolated by flash chromatography. A portion of the compound was
acetylated (6) and analyzed by NMR to confirm the position of the free OH group. In a separate
experiment, known p-tolyl 3,4-di-O-benzyl-1-thio-α-L-rhamnopyranoside (7)xi was reacted with
chloroacetic anhydride in the presence of pyridinexii to afford the corresponding 2-chloroacetate
derivative 8 in 90% yield (Scheme 1).

Scheme 1: Synthesis of the monosaccharide acceptor and donor
Glycosylation of the acceptor 4 with the donor 8 using N-iodosuccinimide in the presence of
xiii
La(OTf)3 furnished the key disaccharide 9 in 89% yield. The chloroacetate group of compound 9 was
cleaved by using thiourea in the presence of 2,4,6-collidinexiv to afford the disaccharide acceptor 10 in
81% yield. It was then glycosylated with known, p-tolyl 2,3,4-tri-O-acetyl-1-thio-α-L-arabinopyranoside
(11)xv using NIS in the presence of La(OTf)3 to afford the protected trisaccharide 12 in 85% yield. The
benzylidene acetal of the trisaccharide 12 was hydrolysed by 80% AcOH at 80 °Cxvi to afford the
trisaccharide diol 13 in 88% yield. The benzyl groups of the trisaccharide 13 were removed by
hydrogenolysis using Pd-C under H2 atmosphere in a flow hydrogenation assembly (H-cube®,
ThalesNano, Hungary). Finally the acetate groups were removed by Zemplén de-O-acetylation using
NaOMe in MeOHxvii to afford the target trisaccharide 1 in 81% yield over two steps (Scheme 2).

Scheme 2: Synthesis of the trisaccharide 1
For the synthesis of target trisaccharide 2, the benzylidene acetal of disaccharide 9 was hydrolysed
using 80% AcOH at 80 ºC to afford disaccharide diol 14 in 92% yield. The glucosyl residue15 was
introduced into the 6-position using the same promoter system as in the previous couplings (16, 83 %).
Also the deprotection followed the elaborated route affording target trisaccharide 2 in 80% yield over two
steps (Scheme 3).

Scheme 3 . Synthesis of the trisaccharide 2
3. Conclusion
In conclusion, chemical synthesis of two trisaccharides related to the phenylethanoid glycosides
leonoside E and F isolated from Leonurus japonicus Houtt. has been accomplished. The target structures
were synthesised in the form of their p-methoxyphenyl glycosides leaving scope for selective removal of
the anomeric residue from the per-O-acetylated target trisaccharides followed by glycoconjugation with
suitable aglycons using e.g. trichloroacetimidate chemistry.
4. Experimental
4.1. General: All reagents and solvents were dried prior to use according to standard methods.xviii
Commercial reagents were used without further purification unless otherwise stated. Analytical TLC was
performed on Silica Gel 60-F254 (Merck or Whatman) with detection by fluorescence and/or by charring

following immersion in a 10 % ethanolic solution of sulfuric acid. An orcinol dip carefully prepared by
the 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 oligosaccharides by charring.
Flash chromatography was performed with Silica Gel 230-400 mesh (Merck, India). Optical rotations
1
were measured at the sodium D-line at ambient temperature, with a Perkin Elmer 141 polarimeter. H
NMR and 13C NMR spectra were recorded on Bruker Avance spectrometer at 500 and 125 MHz
respectively, using Me4Si or CH3OH as internal standards, as appropriate.
4.2. p-Methoxyphenyl 4,6-O-benzylidene-2-O-benzyl-β-D-glucopyranoside (4). A mixture of known p-
methoxyphenyl 4,6-O-benzylidene-β-D-glucopyranoside (3) (3.0 g, 8.0 mmol) and Bu2SnO (2.6 g, 10.4
mmol) in dry MeOH (30 mL) was refluxed for 3 h. The resulting clear solution was concentrated in
vacuo, co-evaporated with toluene and the residue was dried under vacuum for 30 min. The residue was
dissolved in dry DMF (20 mL) and BnBr (1.2 mL, 10.4 mmol) was added followed by CsF (1.3 g, 8.8
mmol) and the solution was stirred at 60 ºC for overnight. After evaporating the solvents in vacuo, the
residue was dissolved in CH2Cl2 (40 mL) and washed successively with H2O (50 mL) and brine (50 mL).
The organic layer was collected, dried over Na2SO4 and evaporated in vacuo. The crude product thus
obtained, was purified by flash chromatography using n-hexane-EtOAc (2:1) as eluent to afford
compound 4 (1.9 g) and compound 5 (1.2 g).
Compound 4: [α]D25 -19.3 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 7.54-7.27 (m, 10H, ArH), 7.01,
6.84 (2d, 4H, J 10.5 Hz, C6H4OCH3), 5.60 (s, 1H, CHPh), 4.99, 4.82 (2d, 2H, J 12 Hz, CH2Ph), 4.90 (d,
1H, J1,2 8.0 Hz, H-1), 4.37 (dd, 1H, J5,6a 4.5 Hz, J6a,6b 10.5 Hz, H-6a), 3.82 (m, 2H, H-6b, H-3), 3.79 (m,
1H, H-2), 3.78 (s, 3H, C6H4OCH3),3.73 (t, 1H,J3,4, J4,5 9.0 Hz,H-4), 3.54 (m, 1H, H-5), 2.17 (bs, 1H,
OH).13C NMR (CDCl3, 125 MHz) δ: 155.7, 137.2, 129.03,128.5(3), 128.3(2), 128.1(3), 127.9, 126.0(2),
6

118.8(2), 114.6(2) (ArC), 102.6 (C-1), 101.4 (CHPh), 81.2, 80.3, 74.7, 74.1, 68.7, 66.6, 55.6
(C6H4OCH3). HRMS calcd. for C27H28O7Na (M+Na)+: 487.1733, found: 487.1729.
4.3. p-Methoxyphenyl 3-O-acetyl-4,6-O-benzylidene-2-O-benzyl-β-D-glucopyranoside (6). To a
solution of compound 4 (100 mg, 0.2 mmol) in dry pyridine (2 mL), Ac2O (1 mL) was added and the
solution was stirred at rt for 2 h when TLC (n-hexane-EtOAc; 3:1) showed complete conversion of the
starting material to a faster moving spot. Solvents were evaporated in vacuo and co-evaporated with
toluene. The residue was dissolved in CH2Cl2 (5 mL) and washed successively with 1N HCl (10 mL),
NaHCO3 (10 mL) and brine (10 mL). The organic layer was collected, dried (Na2SO4), filtered and
evaporated to give compound 6 (105 mg, 96%). [α]D25 +112 (c 1.0).1H NMR (500 MHz, CDCl3) δ: 7.45-
6.85 (m, 14H, ArH), 5.48 (s, 1H, CHPh), 5.38 (t, 1H, J2,3, J3,4 9.5 Hz, H-3), 5.08 (d, 1H, J1,2 7.5 Hz, H-1),
4.95, 4.75 (2d, 2H, J 11.5 Hz, CH2Ph), 4.36 (dd, 1H, J5,6a 5.0 Hz, J6a,6b 10.5 Hz, H-6a), 3.80 (m, 1H, H-
6b), 3.78 (s, 3H, OC6H4OCH3), 3.70 (dd, 1H, J1,2 7.5 Hz, J2,3 9.5 Hz, H-2), 3.67 (t, 1H, J3,4, J4,5 9.5 Hz, H-
4), 3.59 (m, 1H, H-5), 1.99 (s, 3H, COCH3). 13C NMR (CDCl3, 125 MHz) δ: 169.8 (COCH3), 155.7,
150.9, 137.8, 136.9, 129.0, 128.4(2), 128.2(2), 128.1(2), 127.8, 126.1(2), 118.7(2), 114.7(2) (ArC), 103.3
(C-1), 101.4 (CHPh), 79.5, 78.5, 74.6, 72.5, 68.6, 66.2, 55.6 (OC6H4CH3), 20.9 (COCH3).HRMS calcd.
for C29H30O8Na (M+Na)+: 529.1838, found: 529.1833.
4.4. p-Tolyl 3,4-di-O-benzyl-2-O-chloroacetyl-1-thio-α-L-rhamnopyranoside (8): A solution of known
p-tolyl 3,4-di-O-benzyl-1-thio-α-L-rhamnopyranoside 711 (3.0 g, 6.7 mmol) in dry CH2Cl2 (20 mL) was
kept at -5 °C for 10 min. Pyridine (1 mL, 10.0 mmol) was added followed by chloroacetic anhydride (1.7
g, 10.0 mmol) and the reaction was stirred at -5 °C till the TLC (n-hexane-EtOAc; 5:1) showed complete
conversion of the starting material (1 h).The resulting solution was concentrated in vacuo, co-evaporated
with toluene and the residue was dried under vacuum. The crude product thus obtained was purified by
7

flash chromatography using n-hexane-EtOAc (6:1) as eluent to afford the compound 8 (3.2 g, 90%) as
1
colourless gel. [α]D25 +78 (c 1.1, CHCl3). H NMR (500 MHz, CDCl3) δ: 7.34-7.14 (m, 14 H, ArH), 5.7
(dd, 1H, J1,2 1.5 Hz, J2,33.0 Hz, H-2), 5.39 (d, 1H, J1,2 1.5 Hz, H-1), 4.95, 4.75, 4.67, 4.61 (4d, 4H, J 10.5
Hz, 2×CH2Ph), 4.28 (m, 1H, H-5), 4.17 (2d, 2H, J 15.5Hz, CH2Cl), 3.98 (dd, 1H, J2,3 3.0 Hz, J3,4 9.5 Hz,
13
H-3), 3.52 (t, 1H, J3,4, J4,5 9.5 Hz, H-4), 2.36 (s, 3H, SC6H4CH3), 1.38 (d, 3H, J5,6 6.5 Hz, C-CH3). C
NMR (CDCl3, 125 MHz) δ: 166.6 (COCH3), 138.2, 138.1, 137.4, 132.4(2), 129.9(2), 129.6, 128.4(2),
128.3(2), 128.2(2), 128.0(2), 127.9, 127.7 (ArC), 80.1 (C-1), 79.9, 78.1, 77.2, 72.5, 72.0, 69.0, 40.8
(CH2Cl), 21.1 (SC6H4CH3), 17.7 (C-CH3). HRMS calcd. for C29H31O5ClSNa (M+Na) +: 549.1478, found:
549.1481.
p-Methoxyphenyl
3,4-di-O-benzyl-2-O-chloroacetyl-α-L-rhamnopyranosyl-(1→3)-4,6-O-
benzylidene-2-O-benzyl-β-D-glucopyranoside (9): A mixture of compound 4 (1.0 g, 2.2 mmol),
compound 8 (1.2 g, 2.8 mmol) and MS 4 Å (1.5 g) in dry CH2Cl2 (15 mL) was stirred under
nitrogen for 1 h. NIS (820 mg, 3.6 mmol) was added and the mixture was cooled to 0 °C followed
by addition of La(OTf)3 (50 mg). The mixture was allowed to stir at the same temperature for 20
min when TLC (n-hexane-EtOAc; 2:1) revealed that entire acceptor 4 was consumed. The
reaction mixture was immediately filtered through a pad of celite. The filtrate was successively
washed with Na2S2O3 (2 × 50 mL), NaHCO3 (2 × 50 mL) and brine (50 mL). The organic layer
was collected, dried (Na2SO4) and evaporated in vacuo. The crude product thus obtained was
purified by flash chromatography using n-hexane-EtOAc (4:1) to give the pure disaccharide 9 (1.6
25
g, 89%) as a white foam. [α]D +86 (c 1.0, CHCl3).1H NMR (500 MHz, CDCl3) δ: 7.49-6.78 (m,
24H, ArH), 5.61 (s, 1H, CHPh), 5.51 (dd, 1H, J1′,2′ 1.5 Hz, J2′,3′3.0 Hz, H-2′), 5.37 (d, 1H, J1′,2′ 1.5
Hz, H-1′), 4.93 (d, 1H,J1,2 7.5 Hz, H-1), 4.99, 4.86, 4.75, 4.67, 4.60, 4.44 (6d, 6H, J 11.0 Hz,
OCH2Ph), 4.4 (dd, 1H, J3,4 5.0 Hz, J4,5 10.5 Hz, H-4), 4.25 (m, 1H, H-5′), 4.13, 4.07 (2d, 2H,J 15.5
8

Hz, OCOCH2Cl), 3.96 (t, 1H, J1,2 , J2,38.0 Hz, H-2), 3.89 (t, 1H, J2,3, J3,4 9.0 Hz, H-3), 3.87 (dd,
1H, J2',3' 3.0 Hz, J3',4' 9.5 Hz, H-3'), 3.80 (t, 1H, J3,4, J4,5 9.0 Hz, H-4), 3.77 (s, 3H, C6H4OCH3),
3.55 (m, 1H, H-5), 3.37 (t, 1H, J3′,4′,J4′,5′ 9.5 Hz, H-4′), 1.3 (d, 3H, J5′,6′ 6.0 Hz, C-CH3).13C NMR
(CDCl3, 125 MHz) δ: 166.6COCH2Cl), 155.5, 151.0, 138.7, 137.8, 137.7, 137.1, 129.1, 128.5(2),
128.4(2), 128.3(4), 128.2(2), 128.1(2), 127.9, 127.7, 127.5(2), 127.4, 125.9(2), 118(2), 114.6(2)
(ArC), 101.3 (CHPh), 100.9 (C-1), 97.9 (C-′) (1JC,H 169.0), 81.7, 81.6, 79.6, 77.8, 75.4, 75.0, 74.8,
71.9, 70.5, 68.7, 67.9, 66.1, 55.6 (C6H4OCH3) , 29.7 (CH2Cl), 17.8 (C-CH3). HRMS calcd. for
C49H51O12ClNa (M+Na)+: 889.2967, found: 889.2963.
p-Methoxyphenyl 3,4-di-O-benzyl-α-L-rhamnopyranosyl-(1→3)-4,6-O-benzylidene-2-O-benzyl-β-D-
glucopyranoside (10): Compound 9 (700 mg, 0.8 mmol) was dissolved in CH2Cl2-CH3OH (2:3, 40 mL).
Thiourea (310 mg, 4.0 mmol) and collidene (0.5 mL, 4.0 mmol) were consecutively added to the reaction
mixture and it was refluxed at 40 °C for 10 h. After evaporating the solvents in vacuo, the residue was
dissolved in CH2Cl2 (50 mL) and washed with H2O (2× 50 mL). The organic layer was collected, dried
(Na2SO4) and evaporated. The crude product thus obtained was purified by flash chromatography using n-
1
hexane-EtOAc (3:1) as eluent to afford the pure product (500 mg, 81%). [α]D25 +102 (c 1.1, CHCl3). H
NMR (500 MHz, CDCl3) δ: 7.51-6.77 (m, 24H, ArH), 5.60 (s,1H, CHPh), 5.39 (d, 1H, J1′,2′ 1.5 Hz, H-1′),
4.93 (d, 1H, J1,2 7.5 Hz, H-1), 4.96, 4.85 (2d, 2H, J 11.0 Hz, CH2Ph), 4.74,4.64 (2d, 2H, J11.5 Hz,
CH2Ph), 4.59 (s, 2H, CH2Ph), 4.39 (dd, 1H, J5,6a5.0 Hz, J6a,6b 10.5 Hz, H-6a), 4.13 (m, 1H, H-5′), 3.95 (t,
1H, J1,2, J2,37.5 Hz, H-2), 3.88 (bs, 1H, H-2′), 3.86-3.79 (m, 3H, H-3, H-4, H-6b), 3.77 (s, 3H,
C6H4OCH3), 3.74 (dd, 1H, J2′,3′ 3.5 Hz, J3′,4′ 9.0 Hz, H-3′), 3.55 (m, 1H, H-5), 3.45 (t, 1H, J3′,4′, J4′,5′ 9.0
Hz, H-4′), 1.61 (bs,1H, OH), 1.29 (d, 3H, J5′,6′ 6.0 Hz, C-CH3). 13C NMR (CDCl3, 125 MHz) δ: 155.3,
151.0, 138.7, 138.0, 137.9, 137.2, 129.0, 128.5 (2), 128.5 (2), 128.4 (2), 128.3 (2), 128.2 (2), 127.9(2),
127.7(2), 127.6 (2), 127.5, 126.0 (2), 117.9 (2), 114.6 (2) (ArC), 101.2 (CHPh), 100.9 (C-1), 99.9 (C-1′),
9

81.5, 81.4, 79.9, 79.8, 74.9, 74.8, 71.9(2), 68.7, 68.5, 67.6, 66.0, 55.6 (C6H4OCH3), 17.8 (C-CH3). HRMS
calcd. for C47H50O11Na (M+Na) +: 813.3251, found: 813.3254.
p-Methoxyphenyl
2,3,4-tri-O-acetyl-α-L-arabinopyranosyl-(1→2)-3,4-di-O-benzyl-α-L-
rhamnopyranosyl-(1→3)-4,6-O-benzylidene-2-O-benzyl-β-D-glucopyranoside (12): A mixture
of acceptor 10 (500 mg, 0.6 mmol), known compound p-tolyl 2,3,4-tri-O-acetyl-1-thio-α-L-
arabinopyranoside 915 (320 mg, 0.8 mmol) and MS 4 Å (700 mg) in dry CH2Cl2 (15 mL) was
stirred under nitrogen for one hour. To the reaction mixture, NIS (241.5 mg, 1.1 mmol) was added
and the mixture was cooled to 0 °C followed by addition of La(OTf)3 (25 mg). The mixture was
allowed to stir at the same temperature for 20 min when TLC (n-hexane-EtOAc; 3:1) showed
complete consumption of acceptor 10. The reaction mixture was immediately filtered through a
pad of celite. The filtrate was diluted with CH2Cl2 and was successively washed with Na2S2O3 (2
× 50 mL), NaHCO3 (2 × 50 mL) and brine (50 mL). The organic layers were together collected,
dried (Na2SO4) and evaporated in vacuo. The crude product thus obtained was purified by flash
chromatography using n-hexane-EtOAc (2.5:1) to give the pure trisaccharide 12 (550 mg, 83%) as
a white foam. [α]D25 +98 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 7.47-7.27 (m, 20H, ArH),
6.99, 6.78 (2d, 4H, J 9.0 Hz, C6H4OCH3), 5.57 (s, 1H, CHPh), 5.46 (s, 1H, H-1'), 5.24 (dd, 1H,
J1′′,2′′ 6.5 Hz, J2′′,3′′9.0 Hz, H-2′′), 5.15 (bs, 1H, H-4′′), 4.98 (dd, 1H, J 2′′,3′′9.0 Hz, J3′′,4′′ 3.0 Hz, H-
3′′), 4.92 (d, 1H, J1,2 8.5 Hz, H-1), 4.91, 4.82, 4.76, 4.61, 4.60, 4.54 (6d, 6H, J 11.5 Hz, 3×CH2Ph),
4.46 (d, 1H, J1′′,2′′ 6.5 Hz, H-1′′), 4.37 (dd, 1H, J5,6a5.0 Hz, J6a,6b11.0 Hz, H-6a), 4.12 (m, 1H, H-5′),
3.93 (m, 2H, H-2, H-2′), 3.80 (m, 4H, H-3, H-3′, H-4, H-6b), 3.66 (s, 3H, C6H4OCH3), 3.67 (dd,
1H, J4′′,5a′′ 3.0 Hz, J5a′′,5b′′ 13.0 Hz, H-5a′′), 3.53 (m, 1H, H-5), 3.42 (t, J3′,4′, J4′,5′9.5 Hz, H-4′), 3.27
(d, 1H, J5a′′,5b′′ 13.0 Hz, H-5b′′), 2.14, 2.03, 1.89 (3s, 9H, 3×COCH3), 1.27 (d, 3H, J5′,6′ 6.5 Hz, C-
CH3). 13C NMR (CDCl3, 125 MHz) δ: 170.3, 170.2, 169.4 (3×COCH3), 155.3, 151.0, 138.8,
138.4, 138.1, 137.1, 129.0, 128.4 (3), 128.3 (3), 128.2 (2), 127.9 (2), 127.7 (3), 127.6 (2), 127.4,
125.9 (3), 118.0 (2), 114.5 (2) (ArC), 102.5 (C-1′′), 101.2 (CHPh), 101.1 (C-1), 99.9 (C-1′), 81.5,
10

81.2, 80.4, 79.5, 74.9, 74.4, 72.5 (2), 69.9, 69.0, 68.7, 68.3, 67.4, 66.0, 62.5, 55.6 (C6H4OCH3),
+
29.7, 20.9, 20.7(2) (3×COCH3), 17.9 (C-CH3). HRMS calcd. for C58H64O18Na (M+Na) :
1071.3990, found: 1071.3993.
p-Methoxyphenyl
2,3,4-tri-O-acetyl-α-L-arabinopyranosyl-(1→2)-3,4-di-O-benzyl-α-L-
rhamnopyranosyl-(1→3)-2-O-benzyl-β-D-glucopyranoside (13): A suspension of the protected
trisaccharide 12 (550 mg, 0.5 mmol) in AcOH-H2O (9:1, 20 mL) was stirred at 90 °C for 2 h. The
solvents were then evaporated in vacuo, co-evaporated with toluene to ensure complete removal of
AcOH. The residue thus obtained was purified by flash chromatography using n-hexane-EtOAC (1:1) as
1
eluent to afford the pure product 13 (440 mg, 88%). [α]D25 +108 (c 0.9, CHCl3). H NMR (500 MHz,
CDCl3) δ: 7.36-7.27 (m, 15H, ArH), 6.97, 6.78 (2d, 4H, J 8.5 Hz, C6H4OCH3), 5.38 (s,1H, H-1′), 5.23
(dd, 1H, J1′′,2′′ 7.0 Hz, J2′′,3′′9.0 Hz, H-2′′), 5.13 (bs, 1H, H-4′′), 4.95 (dd, J2′′,3′′ 3.5 Hz, J3′′,4′′ 9.0 Hz, H-3′′),
4.84 (d, 1H, J1,2 7.5 Hz, H-1), 4.83, 4.82, 4.68, 4.63, 4.59, 4.54 (6d, 6H, J 11.0 Hz, 3×CH2Ph), 4.43 (d,
1H, J1′′,2′′ 7.0 Hz, H-1′′), 4.16 (m, 1H, H-5′), 3.91-3.79 (m, 5H, H-2, H-2', H-3′, H-6a, H-6b), 3.75 (s, 3H,
C6H4OCH3), 3.66 (t,1H, J3,4,J4,5 9.0 Hz, H-4), 3.54 (m, 2H, H-3, H-5a′′), 3.42 (m, 2H, H-4′,H-5), 3.24 (d,
J5a′′,5b′′ 12.5 Hz, H-5b′′), 2.74 (bs, 1H, OH), 2.32 (bs, 1H, OH), 2.12, 2.02, 1.88 (3s, 9H, 3×COCH3), 1.28
(d, 3H, J5′,6′ 6.0 Hz, C-CH3). 13C NMR (CDCl3, 125 MHz) δ: 170.3, 170.2, 169.5 (3×COCH3), 155.1,
151.0, 138.7, 138.3, 138.2, 128.6(2), 128.3(2), 128.2(2), 127.9, 127.7(2), 127.6(2), 127.5(2), 127.4(2),
117.5(2), 114.6(2) (ArC), 102.7 (C-1′′), 100.2 (C-1), 99.7 (C-1′), 85.5, 80.3, 79.5, 75.2, 74.9(2), 72.6(2),
70.4, 69.8, 68.9, 68.2, 67.3, 62.5, 62.2, 55.6 (C6H4OCH3), 20.9, 20.7(2) (3× COCH3), 17.8 (C-CH3).
HRMS calcd. for C51H60O18Na (M+Na)+: 983.3677, found: 983.3674.
p-Methoxyphenyl α-L-arabinopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→3)-β-D-glucopyranoside
(1): The trisaccharide 11 (440 mg, 0.5 mmol) was dissolved in MeOH (100 mL) and the solution was
11

passed through an H-cube flow hydrogenation assembly using a 10% Pd-C cartridge with a flow rate of 1
mL min-1. Complete removal of the three benzyl groups was achieved after three cycles as confirmed by
the mass spectra. MeOH was evaporated and the dried residue was re-dissolved in MeOH (10 mL).
NaOMe was added to the solution which was stirred at room temperature for 2 h. DOWEX 50W H+ resin
was added to neutralize excess NaOMe. The solution was filtered, evaporated and the residue was
purified by a short flash column using CH2Cl2-MeOH (5:1) to afford the desired trisaccharide 1 (210 mg,
25
1
81%). [α]D +68 (c 0.8, CH3OH). H NMR (500 MHz, CDCl3) δ: 6.96, 6.78 (2d, 4H, J 9.0 Hz,
C6H4OCH3), 5.50 (s, 1H, H-1′), 4.83 (d, 1H, J1,2 6.5 Hz, H-1), 4.33 (d, 1H, J1′′,2′′ 7 Hz, H-1′′), 3.95 (m, 2H,
H-2′, H-5′), 3.83 (m, 2H, H-6a, H-5a′′), 3.74 (s, 1H, H-4′′), 3.69 (s, 3H, C6H4OCH3), 3.66 (s, 1H, H-3′),
3.58 (m, 4H, H-3, H-4, H-2′′, H-3′′), 3.49 (m, 2H, H-2, H-5b′′), 3.35 (m, 3H, H-5, H-6b, H-4′), 1.21 (d, 3H,
J5′,6′ 6.5 Hz, C-CH3). 13C NMR (CDCl3, 125 MHz) δ: 156.5, 152.9, 118.7(2), 115.5(2) (ArC), 107.3 (C-
1′′), 101.3(2) (C-1, C-1′), 82.3, 79.5, 78.9, 77.9, 74.3(2), 72.8, 72.1, 71.4, 69.8(2), 67.3, 62.4, 56.1
(C6H4OCH3), 18.1 (C-CH3). HRMS calcd. for C24H36O15Na (M+Na) +: 587.1952, found: 587.1949.
p-Methoxyphenyl 3,4-di-O-benzyl-2-O-chloroacetyl-α-L-rhamnopyranosyl-(1→3)-2-O-benzyl-β-D-
glucopyranoside (14): Compound 9 (600 mg, 0.7 mmol) was dissolved in 90 % AcOH solution (5 mL)
and was stirred at 90 °C for 2 h. The solvents were then evaporated in vacuo, co-evaporated with toluene
to remove residual amount of AcOH. The crude product thus obtained was purified by flash
chromatography using n-hexane-EtOAc (2.5:1) as eluent to afford the disaccharide acceptor 14 (490 mg,
1
92%). [α]D25 +122 (c 1.1, CHCl3). H NMR (500 MHz, CDCl3) δ: 7.38-7.28 (m, 15H, ArH), 6.96, 6.78
(2d, 4H, J 9.5 Hz, C6H4OCH3), 5.48 (dd, 1H, J1′,2′ 3.0 Hz, J2′,3′ 3.5 Hz, H-2′), 5.28 (d, 1H, J1′,2′ 3.0 Hz, H-
1′), 4.87 (d, 1H, J1,2 8.0 Hz, H-1), 4.86 (d, 2H, J 11.0 Hz, CH2Ph), 4.81 (d, 1H, J 11.0 Hz, CH2Ph), 4.64,
4.61, 4.46 (3d, 3H, J 11.0 Hz, CH2Ph), 4.27 (m, 1H, H-5′), 4.09 (2d, 2H, J 15.0 Hz, OCOCH2Cl), 3.91
(dd, 1H, J2′,3′ 3.5 Hz, J3′,4′ 9.5 Hz, H-3′), 3.88-3.84 (m, 2H, H-2, H-6a), 3.78 (m, 1H, H-6b), 3.75 (s, 3H,
12

C6H4OCH3), 3.71 (t, 1H, J3,4, J4,5 9.0 Hz, H-4), 3.59 (t, 1H, J2,3, J3,4 9.0 Hz, H-3), 3.43-3.38 (m, 2H, H-5,
H-4′), 1.3 (d, 3H, J5′,6′ 6.5 Hz, C-CH3). 13C NMR (CDCl3, 125 MHz) δ: 166.7 (COCH2Cl), 155.3, 151.0,
138.6, 138.0, 137.7, 128.7(2), 128.3(2), 128.2(2), 128.1, 128.0(2), 127.8(2), 127.7, 127.6(2), 127.5,
117.6(2), 114.7(2) (ArC), 100.2 (C-1), 97.8 (C-1′), 85.5, 79.7, 77.7, 76.4, 75.4, 75.3, 74.9, 72.0, 70.8,
70.7, 68.0, 62.2 (C-6), 55.6 (C6H4OCH3), 40.8 (OCOCH2Cl), 17.8 (C-CH3). HRMS calcd. for
C42H47O12ClNa (M+Na) +: 801.2654, found: 801.2651.
p-Methoxyphenyl 3,4-di-O-benzyl-2-O-chloroacetyl-α-L-rhamnopyranosyl-(1→3)-2-O-benzyl-6-O-
(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)-β-D-glucopyranoside (16): To a mixture of disaccharide
acceptor 14 (490 mg, 0.6 mmol) and known compound p-tolyl 2,3,4,6-tetra-O-acetyl-1-thio-β-D-
glucopyranoside (15)15 (360 mg, 0.8 mmol) in dry CH2Cl2 (10 mL), MS 4 Å (500 mg) was added and the
mixture was stirred under nitrogen atmosphere for 1 h. NIS (225 mg, 1 mmol) was added to the mixture
and it was cooled to 0 °C. La(OTf)3 (25 mg) was added to the reaction mixture and it was stirred for 1 h
when TLC (toluene-EtOAc; 3:1) showed complete consumption of acceptor 14. The reaction mixture
was diluted and filtered through a pad of celite. The filtrate was then successively washed with aq
Na2S2O3 (2 × 50 mL), satd aq NaHCO3 (2 × 50 mL) and brine (50 mL). The organic layer was separated,
dried (Na2SO4) and evaporated in vacuo. The crude trisaccharide thus obtained was purified by flash
chromatography using toluene-EtOAc (3:1) as eluent to afford pure trisaccharide 16 (570 mg, 83%) as a
1
white compound. [α]D25 +96 (c 1.0, CHCl3). H NMR (500 MHz, CDCl3) δ : 7.36-7.26 (m, 15H, ArH),
6.97-6.82 (2d,4H, J 9.0 Hz, C6H4OCH3), 5.46 (dd, J1′,2′ 1.5 Hz, J2′,3′ 3.0 Hz, H-2′), 5.28 (d, 1H, J1′,2′ 1.5
Hz, H-1′), 5.16 (t, 1H, J2′′,3′′ , J3′′,4′′ 9.5 Hz, H-3′′), 5.07 (t, 1H, J3′′,4′′, J4′′,5′′ 9.5 Hz, H-4′′), 5.01 (dd, 1H,
J1′′,2′′ 8.0 Hz, J2′′,3′′ 9.5 Hz, H-2′′), 4.87, 4.86 (2d, 2H, J 11.0 Hz, CH2Ph), 4.81 (d, 1H, J1,2 8.0 Hz, H-1),
4.79 (d, 1H, J 11.0 Hz, CH2Ph), 4.64 (d, 1H, J 11.0 Hz, CH2Ph),4.63 (d, 1H,J1′′,2′′ 9.0 Hz, H-1′′), 4.61 (d,
1H, J 11.0 Hz, CH2Ph),4.46 (d, 1H, J 10.5 Hz, CH2Ph), 4.30 (m, 1H, H-5′), 4.23 (dd, 1H, J5′′,6a′′ 4.5
13

Hz,J6a′′,6b′′ 12.5 Hz, H-6a′′), 4.13 (m, 2H, H-6a, H-6b′′), 4.12, 4.07 (2d, 2H, J 15.5 Hz, OCOCH2Cl), 3.90
(dd, 1H, J2′,3′ 3.0 Hz, J3′,4′ 4.5 Hz, H-3′), 3.85 (m, 1H, H-2), 3.78 (s, 3H, C6H4OCH3),3.77 (m, 1H,H-6b),
3.59 (m, 4H, H-3, H-4, H-5, H-5′′), 3.39 (t, 1H, J3′,4′, J4′,5′ 9.5 Hz, H-4′),2.06, 2.02, 2.01, 1.93 (4s, 12H,
4×COCH3), 1.30 (d, 3H, J5′,6′ 6.5 Hz, C-CH3). 13C NMR (CDCl3, 125 MHz) δ: 170.7, 170.2, 169.3(2)
(4×COCH3), 166.7 (COCH2Cl), 155.3, 151.2, 138.6, 137.9, 137.7, 128.7(2), 128.3(2), 128.2(2), 128.1,
128.0(2), 127.9(2), 127.7, 127.5(2), 127.4, 117.5(2), 114.7(2) (ArC), 100.3 (C-1), 100.1 (C-1′′), 97.7 (C-
1′), 85.4, 79.7, 77.8, 76.0, 75.5, 75.0, 74.9, 72.7, 72.0, 71.8, 71.2, 71.1, 70.8, 68.4, 68.2, 67.9, 61.6, 55.6
(C6H4OCH3), 40.8 (COCH2Cl), 20.7, 20.6(2), 20.5 (4×COCH3), 17.8 (C-CH3). HRMS calcd. for
C56H65O21ClNa (M+Na) +: 1131.3605, found: 1131.3607.
p-Methoxyphenyl α-L-rhamnopyranosyl-(1→3)-6-O-(β-D-glucopyranosyl)-β-D-glucopyranoside (2):
A methanolic solution (100 mL) of the trisaccharide 14 (570 mg, 0.5 mmol) was prepared and was passed
through an H-cube flow hydrogenation assembly using a 10 % Pd-C cartridge with a flow rate of 1 mL
min-1. Complete debenzylation was achieved after three cycles, as confirmed by the mass spectrum.
Solvents were evaporated in vacuo and the residue was redissolved in MeOH (10 mL). NaOMe (1.0 mL,
0.5 M in MeOH) was added and the solution was stirred for 12 h. Then the solution was neutralized with,
DOWEX 50W H+ resin, filtered and the solvents were evaporated under reduced pressure and the residue
was purified by a short flash column using CH2Cl2-MeOH (5:1) to afford the target trisaccharide 2 (240
25
1
mg, 80%). [α]D +64 (c 0.8, CH3OH). H NMR (500 MHz, CD3OD) δ: 7.03, 6.84 (2d, 4H, J 9.0 Hz,
C6H4OCH3), 5.27 (s, 1H, H-1′), 4.90 (d, 1H,J1′′,2′′ 9.0 Hz,H-1′′), 4.37 (d, 1H, J1,2 8.0 Hz, H-1), 4.13 (dd,
1H, J5′′,6a′′1.0 Hz, J6a′′,6b′′11.0 Hz,H-6a′′), 4.04 (m, 1H, H-5′), 3.92 (bs, 1H, H-2′), 3.84 (dd, 1H, J5′′,6b′′2.0
Hz, J6a′′,6b′′ 11.0 Hz,H-6b′′),3.79 (dd, 1H, J5,6 6.0 Hz, J6a,6b 12.0 Hz, H-6a), 3.74 (s, 3H, C6H4OCH3), 3.63
(m, 4H, H-3, H-3′′, H-4′′, H-6a), 3.56 (m, 1H, H-6b), 3.39 (m, 2H, H-4′, H-5), 3.33, 3.26 (2d, J6a′′,6b′′ 9 Hz,
13
H-6a′′, H-6b′′), 3.20 (m, 2H, H-5′′, H-2), 1.27 (d, 3H, J5′,6′ 6 Hz, C-CH3). C NMR (CD3OD, 125 MHz)
14

δ: 156.5, 152.8, 118.6(2), 115.6(2) (ArC), 104.7 (C-1), 102.4 (C-1′′), 101.2 (C-1c′), 79.1, 78.9, 77.9(2),
77.2, 75.2, 73.9, 72.2(2), 71.6, 71.5, 69.9, 69.7, 62.7, 56.1 (C6H4OCH3), 18.1 (C-CH3). HRMS calcd. for
C25H38O16Na (M+Na)+: 617.2058, found: 617.2054.
Reference:
i.
Chinese Pharmacopoeia. 2005; Vol. 1, pp 203–204.
ii.
Tasdemir, D.; Scapozza, L.; Zerbe, O.; Linden, A.; Sticher, O.; Çalıs, I. J. Nat. Prod. 1999, 62,
811–816.
iii. Cong, Y.; Wang, J. H.; Li, X. J. Asian Nat. Prod. Res. 2005, 7, 273–277.
iv. Savona, G.; Piozzi, F.; Bruno, M.; Rodriguez, B. Phytochemistry 1982, 21, 2699–2701.
v.
Po, M. H.; En, S. W.; Stellak, M. L.; Yuen, M. C.; Chi, M. L.; Henry, N. C. Phytochemistry 1993,
33, 639–641.
vi. Morita, H.; Lizuka, T.; Gonda, A.; Itokawa, H.; Takeya, K. J. Nat. Prod. 2006, 69, 839–841.
vii. Calis, I.; Ersöz, T.; Tasdemir, D.; Ruedi, P. Phytochemistry 1992, 31, 357–359.
viii. Satoh, M.; Satoh, Y.; Isobe, K.; Fujimoto, Y. Chem. Pharm. Bull. 2003, 51, 341–342.
ix. Lee, C. M.; Jiang, L. M.; Shang, H. S.; Hon, P.-M.; He, Y.; Wong, H. N. C. Br. J. Pharmacol.
1991, 108, 1719–1724.
x.
Li, Y.; Chen, Z.; Feng, Z.; Yang, Y.; Jiang, J.; Zhang, P. Carbohydr. Res. 2012, 348, 42-46.
xi. Rajput, V. K.; Mukhopadhyay, B. J. Org. Chem. 2008, 73, 6924-6927.
xii. Bertolini, M.; Glaudemans, C. P. J. Carbohydr. Res. 1970, 15, 263-270.
xiii. Mukherjee, S.; Mukhopadhyay, B. Synlett 2010, 2853-2856.
xiv. Takeo, K.; Maki, K.; Wada, Y.; Kitamura, S. Carbohydr. Res. 1993, 245, 81-96.
xv. Mukhopadhyay, B.; Kartha, K. P. R.; Russell, D. A.; Field, R. A. J. Org. Chem. 2004, 69, 7758-
7760.
15

xvi. Gent, P. A.; Gigg, R. J. Chem. Soc. Perkin Trans. I 1974, 1446-1455.
xvii. Zemplén, G. Ber. Deutch. Chem. Ges. 1926, 59, 1254-1266.
xviii. Perrin, D. D.; Amarego, W. L.; Perrin, D. R. Purification of Laboratory Chemicals; Pergamon:
London, 1996.
16

Graphical Abstract:
Synthesis of two trisaccharides related to the hepatoprotective phenylethanoids leonoside E
and F isolated from Leonurus japonicus Houtt.
Darshita Budhadev and Balaram Mukhopadhyay*

Highlights
 Synthesis of two trisaccharides from a single disaccharide minimizing protecting
group manipulations
 Use of La(OTf)3 in conjunction with N-iodosuccinimide for the activation of
thioglycosides
 Use of p-methoxyphenyl glycoside that can be removed selectively from the per-O-
acetylated oligosaccharides