Influence of intramolecular hydrogen bonds on regioselectivity of glycosylation. Synthesis of lupane-type saponins bearing the OSW-1 saponin disaccharide unit and its isomers

Article abstract of DOI:10.1016/j.carres.2016.01.010

A series of lupane-type saponins bearing OSW-1 disaccharide unit as well as its regio- and stereoisomers were prepared and used for the structure-activity relationships (SAR) study. Unexpected preference for 1→4-linked regioisomers and an unusual inversio

Full text of DOI:10.1016/j.carres.2016.01.010

Carbohydrate Research 423 (2016) 49–69
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Carbohydrate Research
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Influence of intramolecular hydrogen bonds on regioselectivity of
glycosylation. Synthesis of lupane-type saponins bearing the OSW-1
saponin disaccharide unit and its isomers
Kinga Kuczynska a, Piotr Cmoch a, Lucie Rárová b, Jana Okleštková c, Anna Korda a,
̓
Zbigniew Pakulski a,*, Miroslav Strnad c,**
a Institute of Organic Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland
b Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University, Šlechtitelu˚
27, 783 71 Olomouc, Czech Republic
c Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany ASCR &
Palacký University, Šlechtitelu˚ 27, 783 71 Olomouc, Czech Republic
A R T I C L E
I N F O
A B S T R A C T
Article history:
Received 17 December 2015
Received in revised form 14 January 2016
Accepted 21 January 2016
Available online 26 January 2016
A series of lupane-type saponins bearing OSW-1 disaccharide unit as well as its regio- and stereoiso-
mers were prepared and used for the structure–activity relationships (SAR) study. Unexpected preference
for 1→4-linked regioisomers and an unusual inversion of the conformation of the sugar rings were noted.
Cytotoxic activity of new lupane compounds was evaluated in vitro and revealed that some saponins ex-
hibited an interesting bioactivity profile against human cancer cell lines. Influence of the protecting groups
on the cytotoxicity was investigated. These results open the way to the synthesis of various lupane-
type triterpene and saponin derivatives as potential anticancer compounds.
Keywords:
OSW-1 disaccharide
Glycosylation
Lupane saponins
SAR
© 2016 Elsevier Ltd. All rights reserved.
Hydrogen bond network
1. Introduction
O
Saponins are a large family of steroid or triterpenoid glyco-
sides, widely distributed in plants and in some marine organisms,1
in which hydrophilic mono- or oligosaccharides are attached to a
hydrophobic sapogenin backbone. They possess interesting biolog-
ical properties including antitumor, antiviral, antifungal, and anti-
inflammatory activity, which are extensively studied and reviewed
in the last years.2 Natural saponins based on betulin scaffold occur
less frequently3 than those having other triterpene-type agly-
cones, although growing interest in their synthesis is noticeable.4
Saponin OSW-1 (1) was isolated by Sashida et al in 1992 from
the bulbs of Ornithogalum saudersiae together with a number of other
cholestane glycosides (Fig. 1).5 It was found that OSW-1 exhibited
extremely potent cytotoxicity against a wide range of malignant
OH
O
O
AcO
O
O
OH
HO
HO
HO
MBzO
1
Fig. 1. OSW-1 saponin.
tumor cells in nanomolar concentration (IC50 = 0.1–0.7 nM), about
10–100 times more potent than that of the clinically applied an-
ticancer agents.6 Structure, biological activity, chemical synthesis,
and modifications of saponin OSW-1 and its congeners have been
recently exhaustively reviewed.7
*
Corresponding author. Institute of Organic Chemistry, Polish Academy of Sciences,
The disaccharide part is crucial to the antitumor activities of the
molecule. SAR studies revealed that removal of the acetyl (Ac) and/
or the 4-methoxybenzoyl (MBz) groups located at the disaccharide
moiety diminished the cytotoxicity by three orders of magnitude.6,8
On the other hand, steroidal part of OSW-1 can tolerate some modi-
fications without significant loss of activity.9 Therefore, we speculated
Kasprzaka 44/52, 01-224 Warsaw, Poland. Tel.: +48 22 343 2005; fax: +48 22 632 6681.
E-mail address: zbigniew.pakulski@icho.edu.pl (Z. Pakulski).
** Corresponding author. Laboratory of Growth Regulators & Department of Chemical
Biology and Genetics, Centre of the Region Haná for Biotechnological and Agricultural
Research, Institute of Experimental Botany ASCR & Palacký University, Šlechtitelu˚ 27,
783 71 Olomouc, Czech Republic. Tel.: +420 585 634 850; fax: +420 585 634 870.
E-mail address: miroslav.strnad@upol.cz (M. Strnad).
http://dx.doi.org/10.1016/j.carres.2016.01.010
0008-6215/© 2016 Elsevier Ltd. All rights reserved.

50
K. Kuczynska et al./Carbohydrate Research 423 (2016) 49–69
Path A:
H
BzO
BzO
HO
1. TMSOTf
O
R2
O
+
Lup = lupene
H
OLup
HO
28
2. protective groups
BzO
AcO
OTCA
H
manipulation
3
R1O
H
Path B:
O
O
MBzO
HO
HO
HO
HO
O
1. BF3.Et2O
R3O
R3O
O
O
O
+
+
O
OLup
OLup
HO
HO
HO
O
MBzO
OLup
OTCA
HO
2. protective groups
manipulation
AcO
AcO
MBzO
1
AcO
3 - linked
1
4 - linked
Scheme 1. General methodology for the synthesis of lupane-type saponins bearing regio- and stereoisomers of OSW-1 disaccharide moiety.
that betulin analogues of OSW-1 might also show interesting
bioactivity.4a,f,g,10,11 As a part of our study directed to the search of
new anticancer drugs based on lupanes isolated from white birch
bark, we decided to prepare lupane saponins bearing OSW-1 di-
saccharide fragment.
In this paper we report on the synthesis of lupane saponins
bearing regio- and stereoisomers of OSW-1 disaccharide unit. The
synthetic strategy is directed toward glycosylation of lupeol and
betulin derivatives with l- and d-arabinopyranose donors. Subse-
quent protective groups manipulation and glycosylation with d- or
l-xylose derivatives followed by saponification afforded free sa-
ponins which cytotoxicity was tested against series of normal and
cancer cell lines. Strong influence of intramolecular hydrogen bond
on the regioselectivity of glycosylation was observed.
built onto the lupane framework stepwise. For the synthesis of lupenyl
arabinopyranoside diols (Path A) perbenzoylated Schmidt donors, for
which acyl migration was not observed, were used. Further cou-
pling with xylopyranoside donors (Path B) gave the required
disaccharides in which the 1→3 and 1→4 connections within sugars
are present. Usually slight preference for the 1→4 connected disac-
charide was observed. No optimization was performed because both
regioisomers were indispensable for further biological tests.
Betulin derivatives 2–5, lupeol (6), and glycosyl donors 7–11 were
used as starting materials (Fig. 2). Regardless of the method used for
their preparation, donors 7 (l-arabino) and 8 (d-arabino) were con-
taminated by variable amounts of inseparable corresponding furanosyl
trichloroacetimidates. Therefore, minute amounts of furanosides were
isolated as side products after some glycosylation reaction.
2. Results and discussion
2.1. Synthesis of lupenyl L-arabinopyranosides
Coupling of the aglycon with the OSW-1 disaccharide donor is con-
sidered as one of the key steps in all known approaches to OSW-1
saponin and its congeners.7 In the first attempt to lupane-type ana-
logues of OSW-1 saponin we tried to couple appropriately protected
OSW-1 disaccharide with lupanes. However, glycosylation of lupanes
with OSW-1 disaccharide unit donor12,13 failed due to the presence
of acetyl group at the 2-O position of the sugar. It is well known that
during glycosylation of lupane triterpenes with acetylated glycosyl
donors, acetyl migration occurs as a main process instead of the for-
mation of a glycoside. Recently we developed a modified methodology
allowing us to use acetylated donors for glycosylation of betulin;
however, it was ineffective in this case.14 Therefore, we proposed an
approach shown in Scheme 1 in which the disaccharide moiety was
In the first step we focused on the synthesis of betulin deriva-
tives. Reaction of 3-O-acetylbetulin (2)15 with trichloroacetimidate
7
16 afforded glycoside 12 (92%). Small amounts of furanoside 13 (7%)
was also isolated. Deesterification of 12 with K2CO3 in methanol gave
a mixture of products; due to known resistance of betulin acetate
to basic hydrolysis, 3-O-acetyl group was only partially removed.4f,g
The above crude mixture was treated with dimethoxypropane; sub-
sequent reacetylation under the standard conditions (Ac2O, pyridine)
afforded diacetate 14 in 88% total yield after three steps. Final hy-
drolysis of the 3,4-O-acetonide yielded the desired acceptor 15 (92%,
Scheme 2).
Recently, we found that allylation of the 3-OH group of se-
lected betulin derivatives significantly increases cytotoxicity.17 Thus,
Fig. 2. Structures of lupane triterpenes and glycosyl donors.

K. Kuczynska et al./Carbohydrate Research 423 (2016) 49–69
51
Scheme 2. Reagents and conditions: (i) 7, CH2Cl2, molecular sieves, TMSOTf; (ii) K2CO3, methanol; (iii) DMF, dimethoxypropane, p-TsOH; (iv) Ac2O, Py; (v) p-TsOH, meth-
anol, ethyl acetate.
we also prepared 3-O-allyl betulin derivative 20 for comparison of
its cytotoxic activity with the corresponding lupane 3-O-acetyl and
3-OH derivatives. Starting from 3-O-allyl betulin (3)4a and using the
same methodology as described for 15, glycoside 20 was obtained
in 55% total yield (Scheme 2).
Attempts to reacetylation of 24 failed; starting material was recov-
ered and partial decomposition was also observed. It is likely that
such phenomenon was caused by rarely observed influence of a sub-
stituent distant to the reacting center of lupane-type triterpenes.4a,c
Its origin is unclear, but it must be considered as an explanation
for some unsuccessful trials. Treatment of 23 with p-TsOH gave re-
quired diacetate 25 in good yield (71%). However, due to low yield
of 23 and unpredictability of the above reactions, further tests have
been suspended.
A new approach to the synthesis was initiated from silylated
betulin 5.20 According to the procedure described for 15, com-
pound 5 was easily converted into 30 by glycosylation with 7
followed by standard deprotection/protection steps in 79% total yield
(Scheme 3).
The first attempt to betulin derivatives bearing sugar moiety at
the lupane O-3 position started from 28-O-acetyl-betulin (4).18 Its
glycosylation with 7 gave l-arabinopyranoside 21 and the corre-
sponding l-furanoside 22 in 87% and 6% yield, respectively.
Debenzoylation of 21 with potassium carbonate followed by
acetonide formation and reacetylation afforded, however, a complex
mixture of products. It is likely that unidentified side-products were
formed by the rearrangement of the lupane core under acidic
conditions.19 Essential for further synthesis derivative 23 was iso-
lated in moderate yield (20–53%). Significant amount of monoacetate
24 (10–41%) was also obtained. This reaction was capricious and
difficult to control, and provided the desired product in low yield
together with large amounts of unidentified contaminants. Com-
pound 24 was also obtained independently by desilylation of 29.
Saponin 31 was prepared in 86% yield by glycosylation of lupeol
(6)4f with l-arabinopyranosyl trichloroacetimidate 7. Small amount
of furanosyl derivative 32 (6%) was also isolated. Deesterification
of 31 with potassium carbonate in methanol led to free saponin 33
in 96% yield. Reaction with dimethoxypropane in the presence of
Scheme 3. Reagents and conditions: (i) 7, CH2Cl2, molecular sieves, TMSOTf; (ii) K2CO3, methanol; (iii) DMF, dimethoxypropane, p-TsOH; (iv) Ac2O, Py; (v) p-TsOH, meth-
anol, ethyl acetate; (vi) Bu4NF, AcOH, THF.

52
K. Kuczynska et al./Carbohydrate Research 423 (2016) 49–69
Scheme 4. Reagents and conditions: (i) 7, CH2Cl2, molecular sieves, TMSOTf; (ii) K2CO3, methanol; (iii) DMF, dimethoxypropane, p-TsOH; (iv) Ac2O, Py; (v) p-TsOH, meth-
anol, ethyl acetate.
p-TsOH (34, 95%) followed by acetylation (35, 95%), and acetal hy-
drolysis gave required acetate 36 (72% total yield after five steps;
Scheme 4).
Similarly, reaction of allylated arabinoside 20 with donor 10
gave the mixture of 47 and 48 in 71% yield. Desilylation and
chromatographic separation afforded two fractions. The first one
(50, 36%) comprised a mixture of 1→4 linked disaccharides acety-
lated at the O-2 or O-3 positions; the latter was formed by a
migration of an acetyl group, in which the process was already
observed in our earlier studies.12 The second fraction was identi-
fied as 1→3 connected disaccharide 49 (30%). It must be noted
that in most cases studied here, acetyl migration was observed
and at least traces of 3-O-acetates were detected in the NMR
spectra of 1→4 connected disaccharides. Deallylation of 49 af-
forded free disaccharide 51, although in moderate yield only
(40%; Scheme 6).
Glycosylation of 30 with 10 afforded disaccharides 52 and 53 in
78% total yield; further deprotection as described above gave sa-
ponins 54 (25%, slightly contaminated by unknown compound) and
55 (62%). Deprotection of 54 yielded pure disaccharide 56 in 57%.
Similar desilylation of 55 afforded 1→4 linked disaccharides as in-
separable mixture (80%) comprising unexpected 3-OAc derivative
57, formed by acetyl migration, as main component and 58 present
in smaller amounts (Scheme 7).
2.2. Synthesis of saponins bearing the OSW-1 disaccharide unit
Our first synthesis of the desired disaccharides in this series began
with a TMSOTf catalyzed coupling of 15 with glycosyl donor 912,21
protected with butane-2,3-diacetal (BDA-acetal) which afforded a
mixture of 37 (16%) and 38 (53%) easily separated by chromatog-
raphy. Structure of these products was assigned by the NMR spectra
of their di-acetates 39 and 40 (Scheme 5). The 1H–13C HMBC spec-
tral data showed that glycosylation preferentially took place at the
4-OH to give 1→4-linked disaccharide 38 as the main product,
whereas 1→3-linked disaccharide 37 was observed as minor com-
ponent. Origin of this unusual regioselectivity will be discussed later
in the text. Surprisingly, all attempts to deprotect the BDA-acetal
caused decomposition of the starting materials. This issue has forced
us to use donor 10.13
Coupling of arabinoside 15 with 10 afforded a mixture of 41 and
42 (total yield 65%), which was only partially separable. Desilylation
of this mixture with CSA allowed us to isolate pure disaccharides
43 (39%) and 44 (51%). Type of connection was confirmed inde-
pendently by acetylation and desilylation of analytical samples of
41 and 42; corresponding diacetates (45, 58% and 46, 90% after two
steps, respectively) were obtained and used for NMR analysis
(Scheme 6).
Finally, coupling of 36 with d-xylopyranosyl trichloroacetimidate
10, carried out in the presence of BF3 × OEt2 at −40 °C, afforded a
mixture of products (59/60) in 76% total yield. Desilylation of crude
59/60 mixture by acidic hydrolysis gave desired disaccharides 61
(17%) and 62 (67%). The position of the connection was addition-
ally confirmed by acetylation of 59/60 mixture (to give 63/64; 74%
Scheme 5. Reagents and conditions: (i) 9, CH2Cl2, molecular sieves, TMSOTf; (ii) Ac2O, Py.

K. Kuczynska et al./Carbohydrate Research 423 (2016) 49–69
53
Scheme 6. Reagents and conditions: (i) 10, CH2Cl2, molecular sieves, BF3.OEt2; (ii) CSA, MeOH, CH2Cl2; (iii) Ac2O, Py; (iv) (a) [Ir]; (b) p-TsOH.
yield) followed by hydrolysis to afford 66 as the main product (55%).
Compounds 67 (8%), in which p-methoxybenzoyl group migrated
to position 4-OH of xylopyranoside ring, and 65 (7%) were iso-
lated as minor components (Scheme 8). It is likely that significant
decomposition of 63 in acidic media took place causing lowering
of the yield of 1→3-linked disaccharide 65. The above results show
that compounds in which disaccharide is connected to the lupane
O-3 position are unstable and easily rearrange or decompose during
deprotection even under very mild conditions.
2.3. Saponins bearing stereoisomers of OSW-1 disaccharide unit
The same methodology was used for preparation of saponins
bearing stereoisomers of OSW-1 disaccharide unit. The details of
Scheme 7. Reagents and conditions: (i) 10, CH2Cl2, molecular sieves, BF3.OEt2; (ii) CSA, MeOH, CH2Cl2; (iii) Bu4NF, AcOH, THF.
Scheme 8. Reagents and conditions: (i) 10, CH2Cl2, molecular sieves, BF3.OEt2; (ii) CSA, CH2Cl2, methanol; (iii) Ac2O, Py.

54
K. Kuczynska et al./Carbohydrate Research 423 (2016) 49–69
Scheme 9. Synthesis of 28-O-(2-O-acetyl-α-d-arabinopyranosyl) betulins.
these preparations including analytical data are presented in the
Supplementary material. Thus, acceptor 68 was prepared from betulin
2 and d-arabinopyranosyl trichloroacetimidate 816 in 42% total yield.
Arabinoside 69 was prepared in 27% total yield after 5 steps, from
acceptor 3 (Scheme 9).
Corresponding disaccharides consisted of l-xylose and
d-arabinose (70–73) were obtained by glycosylation of 68 or 69 with
l-xylose glycosyl donor 1112 (Scheme 10). Glycosylation of 68 or 69
with 10 afforded the corresponding disaccharides bearing d-xylose
and d-arabinose with slight preference for the 1→3 linked prod-
ucts (74 and 76); 1→4 linked disaccharides 75 and 77 were isolated
as minor products (Scheme 11). Those having l-xylose and
l-arabinose in sugar part (78 and 79) were prepared by glycosylation
of 20 with donor 11 (Scheme 12).
Derivatives 80–84 bearing d-arabinopyranoside moiety were pre-
pared by glycosylation of lupeol (6) with donor 8 and subsequent
manipulation of protecting groups; they were used for NMR studies
(Scheme 13).
2.4. Configurational assignments
The structures of all new compounds were confirmed by ex-
tended 1D and 2D NMR experiments, as well as elemental analysis
and HRMS. Structures of 26–28 were confirmed by comparison with
Scheme 10. Synthesis of OSW-1 analogues bearing l-xylose and d-arabinose in disaccharide part.
Scheme 11. Synthesis of OSW-1 analogues bearing d-xylose and d-arabinose in disaccharide part.

K. Kuczynska et al./Carbohydrate Research 423 (2016) 49–69
55
Scheme 12. Synthesis of OSW-1 analogues bearing l-xylose and l-arabinose in disaccharide part.
the literature data. As expected, in all cases studied in this report
1,2-trans-glycosides were formed exclusively due to the presence
of benzoyl and p-methoxybenzoyl protecting groups in the donor
molecules, which directed the anomeric selectivity of the
glycosidation reaction.22 These observations were strongly sup-
that clearly showed that the regioselectivity of the glycosylation of
arabinopyranoside 3,4-diols depends on the configuration at the
anomeric center of acceptor. Acceptors bearing the axially ori-
ented substituent at the anomeric position provided the expected
1→3 linked regioisomers, whereas in case of equatorially oriented
aglycon part, the 1→4 linked regioisomers were obtained as
slightly preferred products.12 Reversed selectivity of some
l-arabinopyranoside derivatives was reported in literature24 and was
explained by an influence of a bulky substituent at the O-2 position25
or inversion of the pyranoside ring, which adopts unusual 1C4
conformation.20,26 Inversion process was confirmed by the NMR data
in which low values of J1,2 coupling constant were observed. As a
result of conformational mobility, the 4-OH group occupies more
reactive equatorial position. Ring inversion and its consequences were
discussed in the literature; however, the origin of this phenome-
non was not defined yet.20,26a Such ring-mobility was observed only
for the corresponding 3,4-diols; the pyranoside ring returns to the
1
ported by the JC1-H1 coupling constants.23 The classification of the
glycosylation position within disaccharide moiety was readily con-
firmed by HMBC analyses that showed the correlation between the
1H signals of H-3 (or H-4) and 13C signals of C-1′. Additionally, the
13C NMR spectra of disaccharides indicated characteristic deshielding
effects of the C-3 (or C-4) signals. In some cases, free hydroxyl groups
were acetylated and the 1H NMR spectra of acetates 45, 46, 65 and
66 showed characteristic “acylation shift”; the H-3 or H-4 reso-
nances (depending on the glycosylation position) of the arabinose
residue were deshielded with comparison to the free hydroxyl de-
rivatives. These observations clearly proved the position of the
glycosidic linkages.
4
normal C1 chair conformation after protection or glycosylation.
2.5. Influence of the hydrogen bond on the regioselectivity
of glycosylation
We suppose that in case of α-l-arabinoside A (4C1 conforma-
tion) ring inversion to conformer A′ (1C4 conformation) is strongly
supported by the formation of the hydrogen bond between the 3-OH
and the anomeric oxygen atom and is additionally strengthened by
the anomeric effect. Corresponding coupling constants measured
in CDCl3 solution for diols 15, 30 and 36—J1,2 = 2.0–3.9 Hz and
1JC1,H1 = 165.5–166.9 Hz—prove the equatorial position of the anomeric
hydrogen atom and the preference for conformer A′. Similar effect
was observed for α-d-arabinopyranoside B (1C4), which was
It is commonly believed that hydroxyl groups in equatorial po-
sitions are more reactive than axially oriented ones in six-membered
rings. However, in almost all cases presented above, glycosylation
took place preferentially at the axially oriented 4-OH group of
arabinopyranoside ring, leading to unexpected 1→4 connected di-
saccharides. These results are in line with our previous observations
Scheme 13. Reagents and conditions: (i) 8, CH2Cl2, molecular sieves, TMSOTf; (ii) K2CO3, methanol; (iii) DMF, dimethoxypropane, p-TsOH; (iv) Ac2O, Py; (v) p-TsOH, meth-
anol, ethyl acetate.

56
K. Kuczynska et al./Carbohydrate Research 423 (2016) 49–69
Table 1
1
Selected J1,2 and JC1,H1 coupling constants values for anomeric atoms
Compound
Solvent
Atom
1H
J1,2
Atom
13C
1JC1-H1
[ppm]
[Hz]
[ppm]
[Hz]
Arabinosides:
12
15
15
26
30
36
36
68
80
84
84
CDCl3
CDCl3
CD3OD
CDCl3
CDCl3
CDCl3
CD3OD:CDCl3
CDCl3
CDCl3
CDCl3
H-1
H-1
H-1
H-1
H-1
H-1
H-1
H-1
H-1
H-1
H-1
4.70
4.55
4.32
4.77
4.59
4.63
4.37
4.55
4.84
4.69
4.40
6.2
3.7
7.5
6.4
2.0
3.9
7.5
3.5
5.7
2.1
7.2
C-1
C-1
C-1
C-1
C-1
C-1
C-1
C-1
C-1
C-1
C-1
101.6
99.0
159.9
166.9
160.5
159.0
165.5
166.6
160.7
166.9
162.3
166.9
157.8
102.1
103.0
100.7
100.7
103.4
99.0
97.6
94.9
98.4
a
a
CD3OD:CDCl3
(1→3) Connected disaccharides:
43
45
45
61
65
65
CDCl3
H-1
H-1′
H-1
H-1′
H-1
H-1′
H-1
H-1′
H-1
H-1′
H-1
H-1′
4.23
4.68
4.32
4.94
4.26
4.65
4.26
4.66
4.38
4.95
4.34
4.65
6.1
6.7
6.4
4.1
7.8
~6.3
7.4
6.7
7.1
C-1
C-1′
C-1
C-1′
C-1
C-1′
C-1
C-1′
C-1
C-1′
C-1
C-1′
101.1
101.8
101.6
99.6
102.3
102.5
103.3
101.8
103.3
99.6
159.7
162.0
162.2
169.4
157.6
161.0
156.3
161.4
159.0
168.0
159.4
161.4
CDCl3
b
CD3OD:CDCl3
CDCl3
CDCl3
3.4
7.9
7.5
b
CD3OD:CDCl3
103.5
102.4
(1→4) Connected disaccharides:
38
40
40
CDCl3
H-1
H-1′
H-1
H-1′
H-1
H-1′
4.34
4.72
4.45
4.59
4.37
4.61
5.2
6.8
2.4
7.3
5.5
7.4
C-1
C-1′
C-1
C-1′
C-1
C-1′
100.6
103.2
98.4
101.9
100.3
102.9
162.0
163.6
166.2
162.1
160.8
162.4
CDCl3
b
CD3OD:CDCl3
a
85:15 v/v solution.
3:1 v/v solution.
b
flipping into conformer B’ (4C1). Ring inversion was confirmed by
coupling constants measured in CDCl3 solution for compounds 68
and 84—J1,2 = 2.1–3.5 Hz and 1JC1,H1 = 166.9 Hz. Ring-flipping was ob-
served only for the corresponding 3,4-diols; for protected derivatives
12, 26 and 80, the measured coupling constant values (J1,2 = 5.7–
6.4 Hz and 1JC1,H1 = 159.0–162.3 Hz) remain in the expected range for
A (or B) conformer. Addition of methanol, which breaks intramo-
lecular hydrogen bonds, to the solution of 15, 36 and 84 significantly
changed the coupling constant values giving J1,2 = 7.2–7.5 Hz and
1JC1,H1 = 157.8–160.7 Hz. Such values are characteristic for the axially
oriented anomeric hydrogen atom and correspond to conformers
A and B. These data clearly confirm the participation of the hydro-
gen bond in stabilization of preferred conformers A′ and B′ in aprotic
solvents and return to conformers A (or B) by breaking the
hydrogen bond in the presence of methanol (Table 1, Scheme 14).
Recently, we observed the same phenomenon in the case of allyl
arabinopyranosides.12
It is known that intramolecular hydrogen bonds increase reac-
tivity of hydroxyl group in glycosylation reaction.27 The above results
show that relative position of the hydroxyl group has a much greater
impact on reactivity than intramolecular hydrogen bond in which
the hydroxyl is involved. As observed, hydroxyl group in equatori-
al position is still more reactive than hydroxyl group involved in the
hydrogen bond as hydrogen donor. Question about the possible role
of ring inversion in regioselective glycosylation of the sugar diols
remains unanswered. Earlier observations of reversed regioselectivity
during glycosylation of some galactopyranoside diols28 may suggest,
however, that the hydrogen bond supported ring inversion plays an
important role. This is only the plausible assumption and pro-
posed mechanism should be carefully investigated before its final
approval.
An another type of interactions was observed for some 1→3
linked disaccharides. For compounds 43 and 61 having free 4-OH
hydroxyl group at the l-arabinopyranoside part, both sugars
Scheme 14. Proposed formation of the hydrogen bond under aprotic conditions.

K. Kuczynska et al./Carbohydrate Research 423 (2016) 49–69
57
Scheme 15. Observed ring inversion in disaccharide derivatives (Lup = lupene).
occurred in the expected 4C1 conformation, which was confirmed
by the corresponding coupling constants: J1,2 = 6.1–7.4 Hz and
1JC1,H1 = 156.3–162.0 Hz. Acetylation of the 4-OH hydroxyl group in
the l-arabinopyranoside ring caused substantial changes in xyloside
ring. Observed coupling constants for d-xylopyranoside part of 45
and 65 reached J1,2 = 3.4–4.1 Hz and 1JC1,H1 = 168.0–169.4 Hz, which
correspond to the theorethically unfavorable 1C4 conformation. Values
of the considered coupling constants for l-arabinopyranoside part
derivatives connected to 1→3-linked disaccharides and having
triterpene 3-OH group free or protected as acetate were highly active
(IC50 up to 6.0 μM). Compounds protected as allyl ether were only
moderately active (IC50 approx. 26.0–45.3 μM), except 78, which was
slightly selective against CEM and HeLa cell lines (IC50 13.6–
16.5 μM). No significant differences between 1→3- and 1→4-
linked disaccharides were observed. The complete results obtained
from Calcein AM assays are presented in the Supplementary material.
A striking observation from these data was that on the human
BJ fibroblasts, in most cases, new derivatives have similar or higher
cytotoxicity in comparison with cancer cell lines. These results
suggest that cancer and normal cells respond similarly to certain
structure of the lupane saponins. Lupane saponins bearing OSW-1
disaccharide moiety are also less cytotoxic than the natural OSW-1
saponin. As we mentioned in Section 1, disaccharide moiety is nec-
essary for activity of OSW-1 saponin, whereas steroidal part can
tolerate some modifications without loss of activity. Our results
clearly show, however, that it is also necessary for efficient and strong
bonding to the receptors. It is obvious that lupane (pentacyclic
triterpene) core is structurally too far from original steroid and does
not fit to the receptor harbor.
1
remained in the same range: J1,2 = 6.4–7.1 Hz and JC1,H1 = 159.0–
162.2 Hz. Addition of methanol to the solution of 45 and 65
promoted recovery of the xylopyranoside ring to the expected 4C1
conformation of the xyloside ring. Observed coupling constant values
1
J1,2 = 6.3–7.5 Hz and JC1,H1 = 161.0–161.4 Hz are in accordance with
the values expected for sugar ring in the 4C1 conformation. It is likely
that the above process is also supported by a formation of any kind
of hydrogen bonds (Table 1, Scheme 15).
An interesting ring flipping of the l-arabinose fragment was ob-
served for 1→4 linked BDA protected derivatives 38 and 40. Whereas
both sugar parts in 38 (bearing free OH group at the C-3 position
of arabinoside) were in 4C1 conformations, acetylation of the 3-OH
position caused flipping of the arabinose ring to 1C4 conformation
in 40. Also in this case, inversion process was reversible and the ad-
dition of methanol restored the 4C1 conformation. The origin of the
above phenomenon is unknown, but steric factors may play sig-
nificant role (Table 1, Scheme 15).
3. Conclusion
In conclusion, a series of lupane-type saponins bearing regio-
and stereoisomers of OSW-1 disaccharide were synthesized and
evaluated for their cytotoxic activities toward normal and cancer
cell lines. Selected saponins showed interesting cytotoxic activity
profiles against human cancer cell lines. Required derivatives were
obtained by a glycosylation of suitably protected arabinopyranosides
having the 3,4-diol function. Both regioisomers, usually easily
2.6. Results of in vitro anticancer activity assays
Anticancer activities of the studied lupane saponins with a modi-
fied sugar part were tested in vitro against normal human BJ
fibroblasts and cancer cell lines of various histopathological origins,
including T-lymphoblastic leukemia CEM, breast adenocarcinoma
MCF7, cervical carcinoma HeLa and malignant melanoma G361 lines.
Detailed procedure for the cytotoxicity assay was described
previously.11
All tested arabinosides were active except fully deprotected fu-
ranoside 85 (obtained by treatment of furanoside 13 with KOH in
ethanol; Scheme 16). Derivatives of lupeol were moderately cyto-
toxic, although acetal 34 was highly selective against HeLa and G-361
cell lines (IC50 7.9 and 16.0 μM, respectively). Betulin derivatives were
usually highly active. The most cytotoxic was acetal 24 (IC50 1.5–
5.5 μM). No significant differences between saponins bearing α-l-
Arap (15) and α-d-Arap (68) were observed. Disaccharides with
lupeol as aglycon were inactive. This observation was in accor-
dance with our previously reported suggestions that oxygen atom
in the triterpene C-28 position is necessary for cytotoxicity. Betulin
Scheme 16. Reagents and conditions: (i) KOH, ethanol.

58
K. Kuczynska et al./Carbohydrate Research 423 (2016) 49–69
separated, were prepared, thus opening an easy access to all pos-
sible derivatives in which sugar components were 1→3-linked or
1→4-linked. To our best knowledge, migration of acetyl and
p-methoxybenzoyl groups within OSW-1 disaccharide part was ob-
served for the first time. Unexpected preference for 1→4-linked
regioisomers was noted. Plausible mechanism, based on the NMR
studies explaining such regioselectivity, was presented. Unusual ring
mobility and inversion of the conformation of the sugar rings were
detected for some arabinopyranosides and xylopyranosides. We
suppose that observed ring inversion may affect the relative reac-
tivity of the hydroxyl groups and, as a result, influence regioselectivity
of the glycosylation reaction.
0.64 (m, 1 H, lupane H-5). 13C NMR (600 MHz, CDCl3) δ: 171.0, 165.7,
165.6, 165.1, 150.4 (lupane C-20), 128.3–133.3 (Ar), 109.6 (lupane
C-29), 101.6 (C-1), 80.9 (lupane C-3), 70.5 (C-3), 70.0 (C-2), 68.6
(lupane C-28), 68.4 (C-4), 62.5 (C-5), 55.3, 50.2, 48.7, 47.9, 47.0 (C),
42.5 (C), 40.7 (C), 38.3 (CH2), 37.8 (C), 37.6, 37.0 (C), 34.8 (CH2), 33.8
(CH2), 29.7 (CH2), 29.2 (CH2), 27.9, 27.0 (CH2), 25.0 (CH2), 23.7 (CH2),
21.3, 20.8 (CH2), 19.0, 18.1 (CH2), 16.5, 16.1, 15.9, 14.7. Anal. Calcd
for C58H72O10 (929.21): C, 74.97; H, 7.81. Found: C, 74.73; H, 7.83.
Data for 13. White foam. [α]D20 23.1 (c 0.3, chloroform). 1H NMR
(600 MHz, CDCl3) δ: 7.29–8.08 (m, 15 H, Ar), 5.56 (d, 1 H, J 4.6 Hz,
H-3), 5.54 (s, 1 H, H-2), 5.27 (s, 1 H, H-1), 4.84 (dd, 1 H, J5,4 3.6, J5,5′
11.9 Hz, H-5), 4.66–4.70 (m, 2 H, H-5, lupane 29), 4.58 (br s, 1 H,
lupane 29), 4.53–4.55 (m, 1 H, H-4), 4.47 (dd, 1 H, J 5.4, 11.0 Hz, lupane
H-3), 3.60 (d, 1 H, J 9.2 Hz, lupane 28), 3.56 (d, 1 H, J 9.2 Hz, lupane
28), 2.43–2.48 (m, 1 H, lupane H-19), 2.06–2.08 (m, 1 H), 2.04 (s, 3
H, CH3), 1.95–2.01 (m, 1 H), 1.68 (s, 3 H, lupane C-30), 1.01 (s, 3 H,
CH3), 0.97 (s, 3 H, CH3), 0.84 (s, 3 H, CH3), 0.84 (s, 3 H, CH3), 0.83 (s,
3 H, CH3), 0.77–0.79 (m, 1 H, lupane H-5), 0.86–1.75 (m, 21 H, lupane
protons). 13C NMR (150 MHz, CDCl3) δ: 171.0, 166.2, 165.7, 165.4,
150.4 (lupane C-20), 128.3–133.5 (Ar), 109.6 (lupane C-29), 105.9
(C-1), 81.6 (C-2), 81.4 (C-4), 80.9 (lupane C-3), 77.9 (C-3), 65.3 (lupane
28), 63.9 (C-5), 55.3 (lupane C-5), 50.3, 48.7, 47.8, 47.0 (C), 42.7 (C),
40.9 (C), 38.4 (CH2), 37.8 (C), 37.5, 37.0 (C), 34.8 (CH2), 34.1 (CH2),
29.8 (CH2), 29.7 (CH2), 27.9, 27.2 (CH2), 25.2 (CH2), 23.7 (CH2), 21.3,
20.8 (CH2), 19.2, 18.1 (CH2), 16.5, 16.1, 16.0, 14.7. Anal. Calcd for
C58H72O10 × ½H2O (938.22): C, 74.25; H, 7.84. Found: C, 74.26; H, 7.94.
4. Experimental section
4.1. General
Silica gel HF254 and Silica gel 230–400 mesh (E. Merck) were used
for TLC and column chromatography, respectively. The following mix-
tures of solvents were used as eluents: eluent A: hexane–ethyl
acetate, 40:1 → 9:1; eluent B: hexane–ethyl acetate, 40:1 → 5:1;
eluent C: hexane–ethyl acetate, 5:1 then hexane–ethyl acetate–
methanol, 5:3:1; eluent D: hexane–ethyl acetate, 40:1 → 7:3; eluent
E: hexane–ethyl acetate, 20:1 → 5:1 then hexane–ethyl acetate–
methanol, 5:3:0.5; eluent F: hexane–ethyl acetate, 5:1 → 7:3; eluent
G: hexane–ethyl acetate, 10:1 → 1:1; eluent H: hexane–ethyl
acetate–methanol, 5:3:1. 1H and 13C NMR spectra were recorded at
298 K with a Varian NMR-vnmrs600 or vnmrs500 spectrometer using
standard experimental conditions and Varian software (ChemPack
4.1). Configurational assignments were based on the NMR mea-
surements, generated using two-dimensional techniques like COSY
and 1H–13C gradient selected HSQC (g-HSQC), as well as 1H–13C gra-
dient selected HMBC (g-HMBC) in several cases. Internal TMS was
used as the 1H and 13C NMR chemical shift standard. J values are
given in hertz (Hz). High-resolution mass spectra (HRMS ESI) were
acquired with MARINER and MaldiSYNAPT G2-S HDMS (Waters)
mass spectrometers. Optical rotations were measured with a JASCO
P-2000 automatic polarimeter.
4.2.2. 3β-O-Allyl-28-O-(2,3,4-tri-O-benzoyl-α-l-
arabinopyranosyl) betulin (16)
20
Eluent A. White foam. Yield 82%. [α]D 96.2 (c 0.4, chloro-
form). νmax (film): 2942, 2869, 1730, 1452, 1281, 1262, 1093, 1069,
1027, 710 cm−1 1H NMR (600 MHz, CDCl3) δ: 7.31–8.07 (m, 15 H,
.
Ar), 5.89–5.96 (m, 1 H, allyl =CH), 5.73 (dd, 1 H, J2,1 6.3, J2,3 8.6 Hz,
H-2), 5.68–5.69 (m, 1 H, H-4), 5.62 (dd, 1 H, J3,2 8.6, J3,4 3.6 Hz, H-3),
5.24–5.28 (m, 1 H, allyl =CH2), 5.11–5.13 (m, 1 H, allyl =CH2), 4.69
(d, 1 H, J1,2 6.3 Hz, H-1), 4.65–4.66 (m, 1 H, lupane H-29), 4.56 (br
s, 1 H, lupane H-29), 4.32 (dd, 1 H, J5,4 4.1, J5,5′ 12.9 Hz, H-5), 4.10–
4.13 (m, 1 H, allyl OCH2), 3.86–3.92 (m, 2 H, H-5, allyl OCH2), 3.67
(d, 1 H, J 9.0 Hz, lupane H-28), 3.58 (d, 1 H, J 9.0 Hz, lupane H-28),
2.78 (dd, 1 H, J 4.3, 11.7 Hz, lupane H-3), 2.33–2.38 (m, 1 H, lupane
H-19), 1.95–2.01 (m, 2 H), 1.81–1.83 (m, 1 H), 1.68–1.72 (m, 1 H),
1.65 (s, 3 H, CH3), 0.95 (s, 3 H, CH3), 0.90 (s, 3 H, CH3), 0.81 (s, 3 H,
CH3), 0.79 (s, 3 H, CH3), 0.78–1.67 (m, 22 H, lupane protons), 0.62–
0.64 (m, 1 H, lupane H-5). 13C NMR (150 MHz, CDCl3) δ: 165.7, 165.6,
165.1, 150.4 (lupane C-20), 135.9, 128.3–133.4 (Ar), 115.9 (allyl CH2),
109.6 (lupane C-29), 101.7 (C-1), 86.3 (lupane C-3), 70.6 (CH2), 70.6,
70.0, 68.7 (CH2), 68.5, 62.6 (C-5), 55.8, 50.3, 48.7, 47.9, 47.0 (C), 42.5
(C), 40.8 (C), 38.8 (C), 38.6 (CH2), 37.6, 37.1 (C), 34.8 (CH2), 33.9 (CH2),
29.8 (CH2), 29.2 (CH2), 28.1, 27.0 (CH2), 25.1 (CH2), 23.1 (CH2), 20.8
(CH2), 19.0, 18.1 (CH2), 16.3, 16.1, 15.9, 14.7. Anal. Calcd for C59H74O9
(927.24): C, 76.43; H, 8.04. Found: C, 76.33; H, 8.23.
4.2. General method for glycosylation—synthesis of
arabinopyranosides
A solution of glycosyl donor (2.10 mmol) and the correspond-
ing triterpene (2.00 mmol) in CH2Cl2 (25 mL) was stirred for 20 min
at rt over molecular sieves (4 Å, 500 mg, finely ground), then cooled
to −40 °C and TMSOTf (100 μL, 0.55 mmol) was added. After 30 min
the reaction was quenched with Et3N (1 mL), and the solvents were
evaporated under diminished pressure. Column chromatography of
the residue gave the protected arabinopyranoside.
4.2.1. 3β-O-Acetyl-28-O-(2,3,4-tri-O-benzoyl-α-l-
arabinopyranosyl) betulin (12) and 3β-O-acetyl-28-
O-(2,3,5-tri-O-benzoyl-α-l-arabinofuranosyl) betulin (13)
Eluent B. Arabinofuranoside 13 (7%) was eluted as the first
product; the second fraction contained arabinopyranoside 12 (92%).
Data for 12. White foam. [α]D20 103.4 (c 0.8, chloroform). 1H NMR
(600 MHz, CDCl3) δ: 7.31–8.07 (m, 15 H, Ar), 5.73 (dd, 1 H, J2,1 6.2,
J2,3 8.6 Hz, H-2), 5.68–5.69 (m, 1 H, H-4), 5.63 (dd, 1 H, J3,2 8.6, J3,4
3.5 Hz, H-3), 4.70 (d, 1 H, J1,2 6.2 Hz, H-1), 4.66–4.67 (m, 2 H, lupane
H-29), 4.56 (br s, 1 H, lupane H-29), 4.46 (dd, 1 H, J 5.3, 10.8 Hz,
lupane H-3), 4.32 (dd, 1 H, J5,4 4.2, J5,5′ 12.8 Hz, H-5), 3.91 (dd, 1 H,
J5,4 2.1, J5,5′ 12.8 Hz, H-5), 3.67 (d, 1 H, J 9.0 Hz, lupane H-28), 3.58
(m, 1 H, J 9.0 Hz, lupane H-28), 2.33–2.38 (m, 1 H, lupane H-19),
2.04 (s, 3 H, CH3), 1.95–2.01 (m, 1 H), 1.81–1.83 (m, 1 H), 1.65 (s, 3
H, lupane C-30), 0.91 (s, 3 H, CH3), 0.86 (s, 3 H, CH3), 0.84 (s, 6 H,
2 × CH3), 0.83 (s, 3 H, CH3), 0.74–1.62 (m, 21 H, lupane protons), 0.62–
4.2.3. 28-O-Acetyl-3β-O-(2,3,4-tri-O-benzoyl-α-l-
arabinopyranosyl) betulin (21) and 28-O-acetyl-3-β-O-(2,3,5-tri-
O-benzoyl-α-l-arabinofuranosyl) betulin (22)
Eluent B. Arabinofuranoside 22 (6%) was eluted as the first
product; the second fraction contained arabinopyranoside 21 (87%).
20
Data for 21. Yield 87%. [α]D 99.1 (c 0.5 chloroform). 1H NMR
(600 MHz, CDCl3) δ: 7.30–8.07 (m, 15 H, Ar), 5.75 (dd, 1H, J2,1 6.3,
J2,3 8.7 Hz, H-2), 5.66–5.68 (m, 1 H, H-4), 5.59 (dd, 1 H, J3,2 8.7, J3,4
3.6 Hz, H-3), 4.78 (d, 1 H, J1,2 6.3 Hz, H-1), 4.68–4.69 (m, 1 H, lupane
H-29), 4.59 (br s, 1 H, lupane H-29), 4.33 (dd, 1 H, J5,4 4.0, J5,5′ 12.9 Hz,
H-5), 4.24 (d, 1 H, J 11.0 Hz, lupane H-28), 3.84–3.88 (m, 2 H, H-5,
lupane H-28), 3.13 (dd, 1 H, J 4.7, 11.7 Hz, lupane H-3), 2.41–2.46
(m, 1 H, lupane H-19), 2.06 (s, 3 H, CH3), 1.92–1.99 (m, 1 H), 1.80–
1.86 (m, 2 H), 1.74–1.78 (m, 2 H), 1.68 (s, 3 H, lupane C-30), 1.00 (s,

K. Kuczynska et al./Carbohydrate Research 423 (2016) 49–69
59
3 H, CH3), 0.95 (s, 3 H, CH3), 0.81 (s, 3 H, CH3), 0.77 (s, 3 H, CH3),
0.64 (s, 3 H, CH3), 0.74–1.67 (m, 18 H, lupane protons), 0.63 (br s,
1 H, lupane H-5). 13C NMR (150 MHz, CDCl3) δ: 171.6, 165.8, 165.6,
165.2, 150.1 (lupane C-20), 128.3–133.3 (Ar), 109.8 (lupane C-29),
103.0 (C-1), 90.1 (lupane C-3), 70.7, 70.2, 68.6, 62.8 (lupane C-28),
62.4 (C-5), 55.5 (lupane C-5), 50.3, 48.8, 47.7, 46.3 (C), 42.6 (C), 40.8
(C), 39.0 (C), 38.7 (CH2), 37.5, 36.8 (C), 34.5 (CH2), 34.1 (CH2), 29.7
(CH2), 29.6 (CH2), 27.7, 27.0 (CH2), 26.1 (CH2), 25.2 (CH2), 21.0, 20.8
(CH2), 19.1, 18.1 (CH2), 16.0, 16.0, 14.7. Anal. Calcd for C58H72O10
(929.21): C, 74.97; H, 7.81. Found: C, 74.96; H, 7.82.
CH3), 0.87 (s, 6 H, 2 × CH3), 0.80 (s, 3 H, CH3), 0.73–0.75 (m, 1 H, lupane
H-5), 0.89–1.78 (m, 21 H, lupane protons). 13C NMR (125 MHz, CDCl3)
δ: 166.2, 165.7, 165.5, 151.0 (lupane C-20), 128.3–133.4 (Ar), 109.3
(lupane C-29), 107.5 (C-1), 87.8 (lupane C-3), 82.2, 80.7, 77.7, 64.0
(C-5), 55.5 (lupane C-5), 50.4, 48.3, 48.0, 43.0 (C), 42.8 (C), 40.9 (C),
40.0 (CH2), 39.0 (C), 38.6 (CH2), 38.0, 36.9 (C), 35.6 (CH2), 34.3 (CH2),
29.8 (CH2), 28.1, 27.4 (CH2), 25.6 (CH2), 25.1 (CH2), 20.9 (CH2), 19.3,
18.3 (CH2), 18.0, 16.2, 16.2, 16.0, 14.5. Anal. Calcd for C56H70O8 × H2O
(889.18): C, 75.64; H, 8.16. Found: C, 75.28; H, 7.97. HR-MS (ESI) calc.
for C56H70NaO8 [M + Na]+: 893.4968. Found: 893.4960.
Data for 22. Yield 6%; mainly α-anomer. 1H NMR (600 MHz, CDCl3)
δ: 7.30–8.08 (m, 15 H, Ar), 5.57 (d, 1 H, J 4.8 Hz, H-3), 5.50 (br s, 1 H,
H-2), 5.34 (s, 1 H, H-1), 4.79 (dd, 1 H, J5,4 3.6, J5,5′ 11.8 Hz, H-5),
4.68–4.69 (m, 1 H, lupane 29), 4.67 (dd, 1 H, J5,4 5.1, J5,5′ 11.8 Hz, H-5),
4.60–4.63 (m, 1 H, H-4), 4.59 (br s, 1 H, lupane 29), 4.25 (d, 1 H, J
10.9 Hz, lupane 28), 3.86 (d, 1 H, J 10.9 Hz, lupane 28), 3.19 (dd, 1
H, J 4.6, 11.7 Hz, lupane H-3), 2.42–2.47 (m, 1 H, lupane H-19), 2.07
(s, 3 H, CH3), 1.92–2.00 (m, 1 H), 1.83–1.88 (m, 2 H), 1.75–1.79 (m, 1
H), 1.68 (s, 3 H, lupane C-30), 1.03 (s, 3 H, CH3), 1.02 (s, 3 H, CH3), 0.97
(s, 3 H, CH3), 0.86 (s, 3 H, CH3), 0.85 (s, 3 H, CH3), 0.77–1.73 (m, 19
H, lupane protons), 0.71–0.73 (m, 1 H, lupane H-5). 13C NMR (150 MHz,
CDCl3) δ: 171.6, 166.2, 165.7, 165.5, 150.1 (lupane C-20), 128.3–
133.4 (Ar), 109.8 (lupane C-29), 107.5 (C-1), 87.8, 82.2, 80.7, 77.7, 63.9
(CH2), 62.9 (CH2), 55.5 (lupane C-5), 50.3, 48.8, 47.7, 46.3 (C), 42.7 (C),
40.9 (C), 39.0 (C), 38.6 (CH2), 37.6, 36.9 (C), 34.5 (CH2), 34.2 (CH2), 29.7
(CH2), 29.6 (CH2), 28.0, 27.0 (CH2), 25.6 (CH2), 25.2 (CH2), 21.0, 20.8
(CH2), 19.1, 18.2 (CH2), 16.2, 16.1, 16.0, 14.7. Anal. Calcd for C58H72O10
(929.21): C, 74.97; H, 7.81. Found: C, 74.95; H, 7.81.
4.2.6. 3β-O-(2,3,4-Tri-O-benzoyl-α-d-arabinopyranosyl)
lupeol (80)
20
1
Eluent A. White foam. Yield 93%. [α]D −60.6 (c 0.3, CHCl3). H
NMR (600 MHz, CDCl3) δ: 7.32–8.06 (m, 15 H, Ar), 5.67–5.69 (m, 1
H, H-4), 5.65 (dd 1 H, J2,1 5.7, J2,3 8.2 Hz, H-2), 5.59 (dd, 1 H, J3,2 8.2,
J3,4 3.6 Hz, H-3), 4.84 (d, 1 H, J1,2 5.7 Hz, H-1), 4.68–4.69 (m, 1 H, lupane
H-29), 4.56 (br s, 1 H, lupane H-29), 4.34 (dd, 1 H, J5,4 4.6, J5,5′ 12.7 Hz,
H-5), 3.86 (dd, 1 H, J5,4 2.4, J5,5′ 12.7 Hz, H-5), 3.28 (dd, 1 H, J 4.4,
11.9 Hz, lupane H-3), 2.34–2.39 (m, 1H, lupane H-19), 1.88–1.95 (m,
1 H), 1.68 (s, 3 H, CH3), 1.04 (s, 3 H, CH3), 1.00 (s, 3 H, CH3), 0.92 (s,
3 H, CH3), 0.77 (s, 3 H, CH3), 0.72 (s, 3 H, CH3), 0.67 (s, 3 H, CH3),
0.67–1.67 (m, 23 H, lupane protons). 13C NMR (150 MHz, CDCl3) δ:
165.8, 165.6, 165.2, 151.0 (lupane C-20), 128.3–133.3 (Ar), 109.3
(lupane C-29), 97.6 (C-1), 84.7 (lupane C-3), 70.6, 70.4, 68.4, 62.0
(C-5), 55.8, 50.4, 48.3, 48.0, 43.0 (C), 42.8 (C), 40.9 (C), 40.0 (CH2),
38.4 (CH2), 38.3, 38.0, 37.0 (C), 35.6 (CH2), 34.3 (CH2), 29.8 (CH2),
28.1, 27.4 (CH2), 26.1 (CH2), 22.6 (CH2), 20.9 (CH2), 19.3, 18.3 (CH2),
18.0, 16.0, 15.9, 14.5. Anal. Calcd for C56H70O8 × 1½ H2O (898.18):
C, 74.88; H, 8.19. Found: C, 74.68; H, 8.29.
4.2.4. 28-O-tert-Butyldiphenylsilyl-3β-O-(2,3,4-tri-O-
benzoyl-α-l-arabinopyranosyl) betulin (26)
20
20
Eluent B. Yield 89%. [α]D 77.4 (c 0.5 chloroform); lit.:29 [α]D
4.3. General procedure for debenzoylation of lupenyl arabinosides
71.0 (c 1.0, chloroform). NMR data of compound 26 match the
literature.29 Anal. Calcd for C72H88O9Si (1125.58): C, 76.83; H, 7.88.
Found: C, 76.66; H, 7.84.
A suspension of protected saponin (1.50 mmol) and K2CO3 (50 mg)
in MeOH (40 mL) was stirred for 1 h, then neutralized with Amberlyst
15 resin (H+ form), filtered through a PTFE syringe filter, and con-
centrated. The residue was purified by column chromatography to
afford the title compound.
4.2.5. 3β-O-(2,3,4-Tri-O-benzoyl-α-l-arabinopyranosyl) lupeol
(31) and 3β-O-(2,3,5-tri-O-benzoyl-α-l-arabinofuranosyl)
lupeol (32)
Eluent A. Arabinofuranoside 32 (6%) was eluted as the first
product; the second fraction contained arabinopyranoside 31 (86%).
Data for 31. White foam. [α]D20 109.7 (c 0.5, chloroform). 1H NMR
(600 MHz, CDCl3) δ: 7.26–8.07 (m, 15 H, Ar), 5.76 (dd, 1 H, J2,1 6.4,
J2,3 8.9 Hz, H-2), 5.66–5.68 (m, 1 H, H-4), 5.59 (dd, 1 H, J3,2 8.9, J3,4
3.6 Hz, H-3), 4.78 (d, 1 H, J1,2 6.4 Hz, H-1), 4.68–4.69 (m, 1 H, lupane
H-29), 4.56–4.57 (m, 1 H, lupane H-29), 4.33 (dd, 1 H, J5,4 4.0, J5,5′
13.0 Hz, H-5), 3.87 (dd, 1 H, J5,4 2.0, J5,5′ 13.0 Hz, H-5), 3.13 (dd, 1 H,
J 4.8, 11.6 Hz, lupane H-3), 2.34–2.40 (m, 1 H, lupane H-19), 1.88–
1.95 (m, 1 H), 1.83–1.86 (m, 1 H), 1.74–1.81 (m, 1 H), 1.68 (s, 3 H,
lupane C-30), 1.00 (s, 3 H, CH3), 0.92 (s, 3 H, CH3), 0.81 (s, 3 H, CH3),
0.78 (s, 3 H, CH3), 0.77 (s, 3 H, CH3), 0.64 (s, 4 H, lupane H-5, CH3),
0.79–1.67 (m, 21 H, lupane protons). 13C NMR (150 MHz, CDCl3) δ:
165.8, 165.6, 165.2, 151.0 (lupane C-20), 133.3–128.3 (Ar), 109.3
(lupane C-29), 103.0 (C-1), 90.1 (lupane C-3), 70.7 (C-3), 70.3 (C-
2), 68.7 (C-4), 62.6 (C-5), 55.6 (lupane C-5), 50.4, 48.3, 48.0, 43.0
(C), 42.8 (C), 40.8 (C), 40.0 (CH2), 39.0 (C), 38.7 (CH2), 38.0, 36.9 (C),
35.6 (CH2), 34.2 (CH2), 29.8 (CH2), 27.7, 27.4 (CH2), 26.1 (CH2), 25.1
(CH2), 20.9 (CH2), 19.3, 18.1 (CH2), 18.0, 16.1, 15.9, 14.5. Anal. Calcd
for C56H70O8 (871.18): C, 77.21; H, 8.10. Found: C, 77.11; H, 8.28.
Data for 32. White foam. [α]D20 23.4 (c 0.3, chloroform). 1H NMR
(500 MHz, CDCl3) δ: 7.31–8.09 (m, 15 H, Ar), 5.58 (d, 1 H, J1,2 4.8 Hz,
H-1), 5.51–5.52 (m, 1 H, lupane H-29), 5.36 (br s, 1 H, lupane H-29),
4.81 (dd, 1 H, J5,4 3.6, J5,5′ 11.7 Hz, H-5), 4.67–4.70 (m, 2 H, H-5), 4.62–
4.64 (m, 1 H), 4.58–4.59 (m, 1 H), 3.21 (dd, 1 H, J 4.5, 11.5 Hz, lupane
H-3), 2.36–2.42 (m, 1 H, lupane H-19), 1.86–1.97 (m, 2 H), 1.70 (s,
3 H, lupane C-30), 1.05 (s, 3 H, CH3), 1.03 (s, 3 H, CH3), 0.96 (s, 3 H,
4.3.1. 3β-O-Allyl-28-O-α-l-arabinopyranosyl betulin (17)
20
Eluent E. Yield 98%, amorphous powder. [α]D 7.7 (c 0.4, chlo-
roform). νmax (film): 3414, 2942, 2869, 1454, 1137, 1087, 1070, 999,
757 cm−1. 1H NMR (500 MHz, CDCl3) δ: 5.88–5.97 (m, 1 H, allyl =CH),
5.24–5.28 (m, 1 H, allyl =CH2), 5.11–5.13 (m, 1 H, allyl =CH2), 4.67
(br s, 1 H, lupane H-29), 4.58 (s, 1 H, lupane H-29), 4.27 (d, 1 H, J1,2
5.5 Hz, H-1), 4.10–4.14 (m, 1 H, allyl OCH2), 3.93–3.95 (m, 1 H, H-4),
3.86–3.92 (m, 2 H), 3.72–3.77 (m, 2 H), 3.64 (d, 1 H, J 9.4 Hz, lupane
H-28), 3.59 (dd, 1 H, J5,4 2.2, J5,5′ 12.4 Hz, H-5), 3.53 (d, 1 H, J 9.4 Hz,
lupane H-28), 2.80 (dd, 1 H, J 4.1, 11.7 Hz, lupane H-3), 2.36–2.44
(m, 1 H, lupane H-19), 1.86–2.00 (m, 3 H), 1.68 (s, 3 H, CH3), 1.02
(s, 3 H, CH3), 0.97 (s, 3 H, CH3), 0.96 (s, 3 H, CH3), 0.83 (s, 3 H, CH3),
0.78 (s, 3 H, CH3), 0.79–1.73 (m, 23 H, lupane protons), 0.67–0.69
(m, 1 H, lupane H-5). 13C NMR (125 MHz, CDCl3) δ: 150.3 (lupane
C-20), 135.9, 115.9 (CH2), 109.7 (lupane C-29), 102.9 (C-1), 86.2
(lupane C-3), 72.3, 71.4, 70.6 (CH2), 68.0 (CH2), 67.0, 64.3 (C-5), 55.8
(lupane C-5), 50.3, 48.8, 47.7, 47.0 (C), 42.7 (C), 40.9 (C), 38.8 (C),
38.6 (CH2), 37.6, 37.1 (C), 34.8 (CH2), 34.2 (CH2), 29.7 (CH2), 29.7 (CH2),
28.1, 27.1 (CH2), 25.2 (CH2), 23.1 (CH2), 20.8 (CH2), 19.1, 18.2 (CH2),
16.3, 16.1, 16.1, 14.7. Anal. Calcd for C38H62O6 × ½ H2O (623.92): C,
73.15; H, 10.18. Found: C, 72.94; H, 10.22.
4.3.2. 28-O-tert-Butyldiphenylsilyl-3β-O-α-l-arabinopyranosyl
betulin (27)
Eluent C. Yield 98%, amorphous powder. [α]D20 −12.9 (c 0.3, chlo-
20
1
roform); lit.:20 [α]D −10.2 (c 0.5, chloroform). H NMR (600 MHz,
CDCl3) δ: 7.34–7.70 (m, 10 H, Ar), 4.59–4.60 (m, 1 H, lupane H-29),

60
K. Kuczynska et al./Carbohydrate Research 423 (2016) 49–69
4.53 (br s, 1 H, lupane H-29), 4.39 (d, 1 H, J1,2 5.6 Hz, H-1), 3.88–
3.91 (m, 2 H), 3.75–3.78 (m, 1 H), 3.71–3.72 (m, 1 H), 3.68 (d, 1 H,
J 9.9 Hz, lupane H-28), 3.56 (dd, 1 H, J5,4 1.9, J5,5′ 11.9 Hz, H-5), 3.32
(d, 1 H, J 9.9 Hz, lupane H-28), 3.11 (dd, 1 H, J 4.5, 11.7 Hz, lupane
H-3), 2.24–2.28 (m, 1 H, lupane H-19), 2.11–2.14 (m, 2 H), 1.80–
1.85 (m, 2 H), 1.65 (s, 3 H, CH3), 1.06 (s, 9 H, tBu), 0.96 (s, 3 H, CH3),
0.92 (s, 3 H, CH3), 0.79 (s, 3 H, CH3), 0.77 (s, 3 H, CH3), 0.70 (s, 3 H,
CH3), 0.82–1.70 (m, 22 H, lupane protons), 0.66–0.67 (m, 1 H, lupane
H-5). 13C NMR (150 MHz, CDCl3) δ: 150.8 (lupane C-20), 127.6–
135.7 (Ar), 109.4 (lupane C-29), 104.2 (C-1), 89.9 (lupane C-3), 72.2,
71.6, 66.8, 63.8 (CH2), 61.0 (CH2), 55.5, 50.3, 48.4, 48.4 (C), 47.8, 42.6
(C), 40.8 (C), 39.1 (C), 38.6 (CH2), 37.2, 36.8 (C), 34.5 (CH2), 34.1 (CH2),
29.8 (CH2), 29.5 (CH2), 28.1, 27.0 (CH2), 26.9 (tBu), 25.9 (CH2), 25.1
(CH2), 20.7 (CH2), 19.4, 19.1 (C), 18.2 (CH2), 16.4, 16.0, 15.7, 14.6, 14.2
(C). Anal. Calcd for C51H76O6Si × ½H2O (822.26): C, 74.50; H, 9.44.
Found: C, 74.35; H, 9.44.
48.8, 47.8, 47.1 (C), 42.7 (C), 40.9 (C), 38.8 (C), 38.6 (CH2), 37.6, 37.1,
34.7 (CH2), 34.2 (CH2), 29.7 (CH2), 28.1, 28.0, 27.1 (CH2), 26.0, 25.2
(CH2), 23.1 (CH2), 20.8 (CH2), 19.1, 18.2 (CH2), 16.3, 16.1, 16.1, 14.8.
Anal. Calcd for C41H66O6 × ½ H2O (663.99): C, 74.17; H, 10.17. Found:
C, 74.08; H, 10.11.
4.4.2. 28-O-tert-Butyldiphenylsilyl-3β-O-(3,4-O-isopropylidene-
α-l-arabinopyranosyl) betulin (28)
20
Eluent D. Yield 96%, amorphous powder. [α]D 3.1 (c 0.3, chlo-
roform); lit.:20 [α]D20 1.7 (c 1.0, chloroform). NMR data of compound
28 match the literature.20 Anal. Calcd for C54H80O6Si (853.32): C, 76.01;
H, 9.45. Found: C, 75.77; H, 9.45.
4.4.3. 3β-O-(3,4-O-Isopropylidene-α-l-arabinopyranosyl) lupeol
(34)
Eluent D. Yield 95%, amorphous powder. [α]D20 31.0 (c 0.5, chlo-
roform). 1H NMR (500 MHz, CDCl3) δ: 4.68–4.69 (m, 1 H, lupane
H-29), 4.56–4.57 (m, 1 H, lupane H-29), 4.17–4.22 (m, 3 H), 4.06
(dd, 1 H, J 5.7, 7.6 Hz), 3.74–3.77 (m, 1 H), 3.63 (t, 1 H, J 7.7 Hz), 3.10
(dd, 1 H, J 4.7, 11.5 Hz), 2.35–2.40 (m, 1 H, lupane H-19), 1.88–1.96
(m, 1 H), 1.77–1.83 (m, 1 H), 1.68 (s, 3 H, CH3), 1.54 (s, 3 H, CH3),
1.36 (s, 3 H, CH3), 1.02 (s, 3 H, CH3), 0.97 (s, 3 H, CH3), 0.93 (s, 3 H,
CH3), 0.84 (s, 3 H, CH3), 0.81 (s, 3 H, CH3), 0.79 (s, 3 H, CH3), 0.85–
4.3.3. 3β-O-(α-l-Arabinopyranosyl) lupeol (33)
20
Eluent C. Yield 96%, amorphous powder. [α]D 9.3 (c 0.5, chlo-
roform). 1H NMR (600 MHz, CDCl3) δ: 4.67–4.68 (m, 1 H, lupane
H-29), 4.55–4.56 (m, 1 H, lupane H-29), 4.37 (d, 1 H, J1,2 5.9 Hz, H-1),
3.87–3.92 (m, 2 H, H-4, lupane H-3), 3.75 (dd, 1 H, J2,1 5.9, J2,3 7.5 Hz,
H-2), 3.69 (dd, 1 H, J3,2 7.5, J3,4 3.0 Hz, H-3), 3.53 (dd, 1 H, J5,4 1.8, J5,5′
11.7 Hz, H-5), 3.11 (dd, 1 H, J5,4 4.5, J5,5′ 11.7 Hz, H-5), 2.33–2.39 (m,
1 H, lupane H-19), 1.87–1.94 (m, 1 H), 1.79–1.84 (m, 1 H), 1.67 (s, 3
H, CH3), 1.01 (s, 3 H, CH3), 0.95 (s, 3 H, CH3), 0.92 (s, 3 H, CH3), 0.82
(s, 3 H, CH3), 0.78 (s, 3 H, CH3), 0.77 (s, 3 H, CH3), 0.76–1.71 (m, 24
H, lupane protons), 0.67–0.69 (m, 1 H, lupane H-5). 13C NMR
(150 MHz, CDCl3) δ: 150.9 (lupane C-20), 109.3 (lupane C-29), 104.3
(C-1), 89.9 (lupane C-3), 72.3, 71.6, 66.9, 64.0 (C-5), 55.5, 50.4, 48.3,
47.9, 43.0 (C), 42.8 (C), 40.8 (C), 40.0 (CH2), 39.1 (C), 38.7 (CH2), 38.0,
36.9 (C), 35.6 (CH2), 34.2 (CH2), 29.8 (CH2), 28.1, 27.4 (CH2), 25.9 (CH2),
25.1 (CH2), 20.9 (CH2), 19.3, 18.2 (CH2), 18.0, 16.4, 16.1, 16.0, 14.5.
Anal. Calcd for C35H58O5 (558.85): C, 75.22; H, 10.46. Found: C, 75.12;
H, 10.35.
1.67 (m, 22 H, lupane protons), 0.68–0.70 (m, 1 H, lupane H-5). 13
C
NMR (125 MHz, CDCl3) δ: 150.9 (lupane C-20), 110.0, 109.3 (lupane
C-29), 104.3 (C-1), 89.0 (lupane C-3), 78.1, 74.3, 73.2, 63.1 (C-5), 55.6,
50.4, 48.3, 48.0, 43.0 (C), 42.8 (C), 40.8 (C), 40.0 (CH2), 39.1 (C), 38.7
(CH2), 38.0, 36.9 (C), 35.6 (CH2), 34.3 (CH2), 29.8 (CH2), 28.2, 28.0,
27.4 (CH2), 26.1 (CH2), 26.1, 25.1 (CH2), 20.9 (CH2), 19.3, 18.2 (CH2),
18.0, 16.5, 16.1, 16.0, 14.5. Anal. Calcd for C38H62O5 (598.91): C, 76.21;
H, 10.43. Found: C, 76.54; H, 10.68.
4.4.4. 3β-O-(3,4-O-Isopropylidene-α-d-arabinopyranosyl)
lupeol (82)
Eluent D. Yield 94%, amorphous powder. [α]D20 24.3 (c 0.2, chlo-
roform). 1H NMR (600 MHz, CDCl3) δ: 4.68–4.69 (m, 1 H, lupane
H-29), 4.56–4.57 (m, 1 H, lupane H-29), 4.21–4.23 (m, 2 H, H-1, H-4),
4.17 (dd, 1 H, J5,4 2.7, J5,5′ 13.3 Hz, H-5), 4.07 (dd, 1 H, J3,4 6.0, J3,2 7.7 Hz,
H-3), 3.75 (dd, 1 H, J5,4 3.4, J5,5′ 13.3 Hz, H-5), 3.57 (t, 1 H, J 7.7 Hz,
H-2), 3.23 (dd, 1 H, J 4.3, 11.9 Hz, lupane H-3), 2.35–2.40 (m, 1 H,
lupane H-19), 1.88–1.95 (m, 2 H), 1.68 (s, 3 H, CH3), 1.55 (s, 3 H, CH3),
1.37 (s, 3 H, CH3), 1.03 (s, 3 H, CH3), 0.99 (s, 3 H, CH3), 0.92 (s, 3 H,
CH3), 0.84 (s, 3 H, CH3), 0.79 (s, 3 H, CH3), 0.77 (s, 3 H, CH3), 0.81–
4.3.4. 3β-O-(α-d-Arabinopyranosyl) lupeol (81)
Eluent C. Yield 86%, amorphous powder. [α]D20 47.5 (c 0.3, chlo-
20
roform); lit.:16 [α]D 26.8 (c 1.25, chloroform). NMR spectral data
of 81 were in agreement with those published in the literature.16
4.4. Synthesis of isopropylidene derivatives—general method
To a solution of deprotected arabinopyranoside (1.00 mmol) in
DMF (15 mL), 2,2-dimethoxypropane (5 mL) and p-TsOH (80 mg)
were added. A solution was stirred for 3 h, then neutralized with
triethylamine (1 mL) and concentrated. The residue was purified by
column chromatography to afford the title compound.
1.70 (m, 22 H, lupane protons), 0.69–0.70 (m, 1 H, lupane H-5). 13
C
NMR (150 MHz, CDCl3) δ: 151.0 (lupane C-20), 110.1 (C), 109.3
(lupane C-29), 99.1 (C-1), 83.9 (lupane C-3), 78.1, 73.7, 73.2, 63.2 (C-
5), 55.9, 50.4, 48.3, 47.9, 43.0 (C), 42.8 (C), 40.9 (C), 40.0 (CH2), 38.4
(CH2), 38.2 (C), 38.0, 37.0 (C), 35.6 (CH2), 34.3 (CH2), 29.8 (CH2), 28.0,
27.4 (CH2), 26.0, 25.1 (CH2), 23.1 (CH2), 20.9 (CH2), 19.3, 18.3 (CH2),
18.0, 16.3, 16.1, 16.0, 14.4. HRMS (ESI) calc. for C38H62NaO5 [M + Na]+:
621.4495. Found: 621.4501. Anal. Calcd for C38H62O5 × ½ H2O (607.92):
C, 75.08; H, 10.45. Found: C, 75.45; H, 10.03.
4.4.1. 3β-O-Allyl-28-O-(3,4-O-isopropylidene-α-l-
arabinopyranosyl) betulin (18)
Eluent A. Yield 95%, amorphous powder. [α]D20 23.7 (c 0.4, chlo-
roform). νmax (film): 3466, 2942, 2870, 1455, 1374, 1218, 1123, 1086,
1070, 1042, 757 cm−1
.
1H NMR (500 MHz, CDCl3):δ: 5.89–5.97 (m,
4.5. Acetylation—general method
1 H, allyl =CH), 5.24–5.28 (m, 1 H, allyl =CH2), 5.11–5.13 (m, 1 H,
allyl =CH2), 4.66–4.67 (m, 1 H, lupane H-29), 4.57 (br s, 1 H, lupane
H-29), 4.18–4.24 (m, 2 H), 4.07–4.14 (m, 3 H), 3.86–3.90 (m, 1 H,
allyl OCH2), 3.79 (dd, 1 H, J5,4 3.2, J5,5′ 13.2 Hz, H-5), 3.61–3.64 (m, 2
H), 3.51 (d, 1 H, J 9.2 Hz, lupane H-28), 2.80 (dd, 1 H, J 4.1, 11.7 Hz,
lupane H-3), 2.37–2.43 (m, 1 H, lupane H-19), 1.92–2.00 (m, 3 H),
1.67 (s, 3 H, CH3), 1.55 (s, 3 H, CH3), 1.37 (s, 3 H, CH3), 1.01 (s, 3 H,
CH3), 0.97 (s, 3 H, CH3), 0.96 (s, 3 H, CH3), 0.83 (s, 3 H, CH3), 0.78 (s,
3 H, CH3), 0.79–1.73 (m, 21 H, lupane protons), 0.67–0.68 (m, 1 H,
lupane C-5). 13C NMR (125 MHz, CDCl3) δ: 150.5 (lupane C-20), 135.9,
115.9 (CH2), 110.1 (C), 109.6 (lupane C-29), 102.7 (C-1), 86.3 (lupane
C-3), 78.0, 73.8, 73.1, 70.6 (CH2), 67.6 (CH2), 63.1 (CH2), 55.8, 50.4,
A solution of saponin (0.80 mmol) in pyridine (5 mL) and acetic
anhydride (2 mL) was stirred for 3 h. Solvents were coevaporated
with toluene twice and the residue was purified by column chro-
matography to afford the title compound.
4.5.1. 3β-O-Acetyl-28-O-(2-O-acetyl-3,4-O-isopropylidene-α-
l-arabinopyranosyl) betulin (14)
Arabinoside 12 was debenzoylated using the procedure 4.3. Crude
product (a mixture of 3β-O-acetyl-28-O-α-l-arabinopyranosyl betulin
and 3β-28-O-α-l-arabinopyranosyl betulin) was protected as 3,4-
O-isopropylidene derivative using the procedure 4.4 and then

K. Kuczynska et al./Carbohydrate Research 423 (2016) 49–69
61
acetylated as described above. Column chromatography (eluent B)
gave the title compound as amorphous powder. Yield 88% after 3
lupane H-19), 2.09 (s, 3 H, CH3), 2.07 (s, 3 H, CH3), 1.92–2.00 (m, 1
H), 1.83–1.85 (m, 1 H), 1.75–1.78 (m, 2 H), 1.68 (s, 3 H, lupane C-30),
1.56 (s, 3 H, CH3), 1.35 (s, 3 H, CH3), 1.02 (s, 3 H, CH3), 0.96 (s, 3 H,
CH3), 0.89 (s, 3 H, CH3), 0.81 (s, 3 H, CH3), 0.73 (s, 3 H, CH3), 0.84–
20
1
steps. [α]D 18.8 (c 0.5, chloroform). H NMR (600 MHz, CDCl3) δ:
5.03 (t, 1 H, J2,1 = J2,3 = 6.6 Hz, H-2), 4.65–4.66 (m, 1 H, lupane H-29),
4.56 (br s, 1 H, lupane H-29), 4.47 (dd, 1 H, J 5.2, 11.1 Hz, lupane
H-3), 4.32 (d, 1 H, J1,2 6.6 Hz, H-1), 4.25–4.27 (m, 1 H, H-4), 4.16 (dd,
1 H, J3,2 6.6, J3,4 5.8 Hz, H-3), 4.11 (dd, 1 H, J5,4 4.1, J5,5′ 13.0 Hz, H-5),
3.77 (dd, 1 H, J5,4 4.0, J5,5′ 13.0 Hz, H-5), 3.57 (d, 1 H, J 9.1 Hz, lupane
H-28), 3.42 (m, 1 H, J 9.1 Hz, lupane H-28), 2.36–2.40 (m, 1 H, lupane
H-19), 2.08 (s, 3 H, CH3), 2.04 (s, 3 H, CH3), 1.87–1.98 (m, 3 H), 1.67
(s, 3 H, CH3), 1.56 (s, 3 H, CH3), 1.36 (s, 3 H, CH3), 1.02 (s, 3 H, CH3),
0.96 (s, 3 H, CH3), 0.85 (s, 6 H, 2 × CH3), 0.84 (s, 3 H, CH3), 0.86–
1.70 (m, 19 H, lupane protons), 0.64–0.66 (m, 1 H, lupane H-5). 13
C
NMR (150 MHz, CDCl3) δ: 171.5, 169.4, 150.1 (lupane C-20), 110.5
(C), 109.8 (lupane C-29), 102.3 (C-1), 89.3 (lupane C-3), 76.4, 73.3,
72.9, 62.7 (CH2), 62.3 (CH2), 55.6, 50.4, 48.8, 47.6, 46.3 (C), 42.6 (C),
40.9 (C), 39.0 (C), 38.7 (CH2), 37.5, 36.9 (C), 34.5 (CH2), 34.2 (CH2),
29.7 (CH2), 29.6 (CH2), 27.7, 27.6, 27.0 (CH2), 26.2, 26.0 (CH2), 25.2
(CH2), 21.1, 21.0, 20.8 (CH2), 19.1, 18.1 (CH2), 16.1, 16.0, 16.0, 14.7.
Anal. Calcd for C42H66O8 (698.99): C, 72.17; H, 9.52. Found: C, 72.19;
H, 9.68.
1.64 (m, 20 H, lupane protons), 0.78–0.80 (m, 1 H, lupane H-5). 13
C
20
NMR (150 MHz, CDCl3) δ: 170.9, 169.4, 150.5 (lupane C-20), 110.3,
109.6 (lupane C-29), 100.6 (C-1), 80.9 (lupane C-3), 75.9, 72.5, 72.3,
67.7 (CH2), 62.0 (CH2), 55.3, 50.2, 48.6, 47.9, 47.0 (C), 42.7 (C), 40.9
(C), 38.3 (CH2), 37.8 (C), 37.6, 37.0 (C), 34.7 (CH2), 34.1 (CH2), 29.7
(CH2), 29.5 (CH2), 27.9, 27.7, 27.1 (CH2), 26.1, 25.1 (CH2), 23.7 (CH2),
21.3, 20.9, 20.8 (CH2), 19.0, 18.1 (CH2), 16.5, 16.1, 16.0, 14.8. Anal.
Calcd for C42H66O8 (698.99): C, 72.17; H, 9.52. Found: C, 72.29; H,
9.52.
Data for 24. Yield 92%. [α]D 32.7 (c 0.3, chloroform). 1H NMR
(600 MHz, CDCl3) δ: 5.05 (t, 1 H, J2,1 = J2,3 = 7.4 Hz, H-2), 4.68–4.69
(m, 1 H, lupane H-29), 4.58 (br s, 1 H, lupane H-29), 4.36 (d, 1 H,
J1,2 7.4 Hz, H-1), 4.23–4.25 (m, 1 H, H-4), 4.16 (dd, 1 H, J5,4 3.3, J5,5′
13.2 Hz, H-5), 4.13 (dd, 1 H, J3,2 7.4, J3,4 5.8 Hz, H-3), 3.79 (d, 1 H, J
10.8 Hz, lupane H-28), 3.75 (dd, 1 H, J5,4 3.7, J5,5′ 13.2 Hz, H-5), 3.33
(d, 1 H, J 10.8 Hz, lupane H-28), 3.00 (dd, 1 H, J 4.7, 11.6 Hz, lupane
H-3), 2.35–2.41 (m, 1 H, lupane H-19), 2.09 (s, 3 H, CH3), 1.84–1.93
(m, 3 H), 1.71–1.78 (m, 1 H), 1.68 (s, 3 H, lupane C-30), 1.56 (s, 3 H,
CH3), 1.35 (s, 3 H, CH3), 1.01 (s, 3 H, CH3), 0.96 (s, 3 H, CH3), 0.89 (s,
3 H, CH3), 0.81 (s, 3 H, CH3), 0.73 (s, 3 H, CH3), 0.84–1.65 (m, 20 H,
lupane protons), 0.65–0.67 (m, 1 H, lupane H-5). 13C NMR (150 MHz,
CDCl3) δ: 169.5, 150.4 (lupane C-20), 110.5 (C), 109.7 (lupane C-29),
102.3 (C-1), 89.3 (lupane C-3), 76.4 (C-3), 73.3 (C-2), 73.0 (C-4), 62.4
(C-5), 60.5 (lupane C-28), 55.6, 50.4, 48.8, 47.8, 42.7 (C), 40.9 (C),
39.0 (C), 38.7 (CH2), 37.3, 36.9 (C), 34.2 (CH2), 34.0 (CH2), 29.7 (CH2),
29.2 (CH2), 27.7, 27.6, 27.0 (CH2), 26.2, 26.0 (CH2), 25.2 (CH2), 21.1,
20.8 (CH2), 19.1, 18.2 (CH2), 16.1, 16.1, 16.0, 14.7. Anal. Calcd for
C40H64O7 (656.95): C, 73.13; H, 9.82. Found: C, 73.29; H, 9.67.
4.5.2. 3β-O-Allyl-28-O-(2-O-acetyl-3,4-O-isopropylidene-
α-l-arabinopyranosyl) betulin (19)
Eluent A. Yield 92%, amorphous powder. [α]D20 19.2 (c 0.3, chlo-
roform). νmax (film): 2942, 2870, 1755, 1372, 1221, 1124, 1090, 1055,
758 cm−1. 1H NMR (500 MHz, CDCl3) δ: 5.88–5.98 (m, 1 H, allyl =CH),
5.24–5.28 (m, 1 H, allyl =CH2), 5.11–5.13 (m, 1 H, allyl =CH2), 5.03
(t, 1 H, J2,1 = J2,3 = 6.6 Hz, H-2), 4.66 (br s, 1 H, lupane H-29), 4.56 (br
s, 1 H, lupane H-29), 4.31 (d, 1 H, J1,2 6.6 Hz, H-1), 4.24–4.27 (m, 1
H), 4.15–4.17 (m, 1 H), 4.10–4.14 (m, 2 H), 3.86–3.90 (m, 1 H, OCH2),
3.77 (dd, 1 H, J5,4 3.9, J5,5′ 13.0 Hz, H-5), 3.57 (d, 1 H, J 9.2 Hz, lupane
H-28), 3.42 (d, 1 H, J 9.2 Hz, lupane H-28), 2.80 (dd, 1 H, J 4.1, 11.7 Hz,
lupane H-3), 2.35–4.40 (m, 1 H, lupane H-19), 2.07 (s, 3 H, CH3), 1.86–
1.98 (m, 3 H), 1.66 (s, 3 H, CH3), 1.56 (s, 3 H, CH3), 1.36 (s, 3 H, CH3),
1.02 (s, 3 H, CH3), 0.96 (s, 3 H, CH3), 0.96 (s, 3 H, CH3), 0.83 (s, 3 H,
CH3), 0.78 (s, 3 H, CH3), 0.79–1.73 (m, 20 H, lupane protons), 0.67–
0.69 (m, 1 H, lupane C-5). 13C NMR (125 MHz, CDCl3) δ: 169.4, 150.6
(lupane C-20), 135.9, 115.9 (CH2), 110.3 (C), 109.5 (lupane C-29),
100.6 (C-1), 86.3 (lupane C-3), 75.9, 72.5, 72.4, 70.6 (CH2), 67.8 (CH2),
62.1 (CH2), 55.8 (lupane C-5), 50.4, 48.7, 47.9, 47.0 (C), 42.7 (C), 40.9
(C), 38.8 (C), 38.6 (CH2), 37.6, 37.1 (C), 34.7 (CH2), 34.2 (CH2), 29.7
(CH2), 29.5 (CH2), 28.1, 27.8, 27.1 (CH2), 26.2, 25.2 (CH2), 23.1 (CH2),
20.9, 20.8 (CH2), 19.1, 18.2 (CH2), 16.3, 16.1, 16.1, 14.8. Anal. Calcd
for C43H68O7 (697.02): C, 74.10; H, 9.83.Found: C, 74.17; H, 9.71.
4.5.4. 28-O-tert-Butyldiphenylsilyl-3β-O-(2-O-acetyl-3,4-
O-isopropylidene-α-l-arabinopyranosyl) betulin (29)
20
Eluent B. Yield 98%, amorphous powder. [α]D 7.8 (c 0.3, chlo-
roform). 1H NMR (600 MHz, CDCl3) δ: 7.36–7.70 (m, 10 H, Ar), 5.05
(t, 1 H, J2,1 = J2,3 = 7.4 Hz, H-2), 4.59–4.60 (m, 1 H, lupane H-29), 4.52–
4.53 (m, 1 H, lupane H-29), 4.35 (d, 1 H, J1,2 7.4 Hz, H-1), 4.23–4.25
(m, 1 H, H-4), 4.15 (dd, 1 H, J5,4 2.8, J5,5′ 13.2 Hz, H-5), 4.12 (dd, 1 H,
J3,2 7.4, J3,4 1.7 Hz, H-3), 3.75 (dd, 1 H, J5,4 3.7, J5,5′ 13.2 Hz, H-5), 3.68
(d, 1 H, J 9.9 Hz, lupane H-28), 3.32 (d, 1 H, J 9.9 Hz, lupane H-28),
2.98 (dd, 1 H, J 4.7, 11.6 Hz, lupane H-3), 2.23–2.28 (m, 1 H), 2.11–
2.14 (m, 2 H), 2.09 (s, 3 H, CH3), 1.80–1.87 (m, 1 H), 1.73–1.76 (m, 1
H), 1.67–1.69 (m, 1 H), 1.64 (s, 3 H, lupane C-30), 1.56 (s, 3 H, CH3),
1.35 (s, 3 H, CH3), 1.06 (s, 9 H, tBu), 0.91 (s, 3 H, CH3), 0.88 (s, 3 H,
CH3), 0.75 (s, 3 H, CH3), 0.72 (s, 3 H, CH3), 0.69 (s, 3 H, CH3), 0.73–
4.5.3. 28-O-Acetyl-3β-O-(2-O-acetyl-3,4-O-isopropylidene-
α-l-arabinopyranosyl) betulin (23) and 3β-O-(2-O-acetyl-3,4-O-
isopropylidene-α-l-arabinopyranosyl) betulin (24)
Arabinoside 21 was transformed into the title compounds using
procedure described for 14 (Method A). Column chromatography
(eluent B) gave 23 (20–53%) and 24 (10–41%) as amorphous powders
in variable proportion depending on batch.
1.64 (m, 18 H, lupane protons), 0.61–0.63 (m, 1 H, lupane H-5). 13
C
NMR (150 MHz, CDCl3) δ: 169.4, 150.8 (lupane C-20), 127.6–135.7
(Ar), 110.5 (C), 109.4 (lupane C-29), 102.3 (C-1), 89.3 (lupane C-3),
76.4, 73.3, 73.0, 62.4 (CH2), 61.1 (CH2), 55.5, 50.3, 48.5, 48.4 (C), 47.8,
42.6 (C), 40.8 (C), 39.0 (C), 38.6 (CH2), 37.2, 36.8 (C), 34.5 (CH2), 34.1
(CH2), 29.8 (CH2), 29.5 (CH2), 27.7, 27.6, 27.0 (CH2), 26.9 (tBu), 26.2,
26.0 (CH2), 25.2 (CH2), 21.1, 20.7 (CH2), 19.1, 18.2 (CH2), 16.1, 16.0,
15.7, 14.7. Anal. Calcd for C56H82O7Si (895.36): C, 75.12; H, 9.23. Found:
C, 75.09; H, 9.38.
Synthesis of 24—Method B. To a solution of 29 (100 mg,
0.11 mmol) in THF (3 mL), tetrabutylammonium fluoride (1 M in THF,
1.00 mL, 1.00 mmol) was added followed by acetic acid (57 μL,
1.00 mmol) and the mixture was stirred in a sealed tube at 60 °C
for 24 h. The mixture was then concentrated and the residue was
purified by column chromatography (eluent F) to give the title com-
pound (67 mg, 92%) as a foam.
4.5.5. 3β-O-(2-O-Acetyl-3,4-O-isopropylidene-α-
l-arabinopyranosyl) lupeol (35)
Eluent B. Yield 95%. M.p. 177–178 °C. [α]D20 38.5 (c 0.6, chloro-
form). 1H NMR (600 MHz, CDCl3) δ: 5.03 (t, 1 H, J2,1 = J2,3 = 7.4 Hz, H-2),
4.67–4.68 (m, 1 H, lupane H-29), 4.55–4.56 (m, 1 H, lupane H-29),
4.35 (d, 1 H, J1,2 7.4 Hz, H-1), 4.22–4.24 (m, 1 H, H-4), 4.10–4.16 (m,
2 H, H-3, H-5), 3.74 (dd, 1 H, J5,4 3.7, J5,5′ 13.2 Hz, H-5), 2.98 (dd, 1
H, J 4.7, 11.7 Hz, lupane H-3), 2.34–2.38 (m, 1 H, lupane H-19), 2.08
(s, 3 H, CH3), 1.87–1.94 (m, 1 H), 1.73–1.77 (m, 1 H), 1.67 (s, 3 H, CH3),
20
Data for 23. [α]D 24.8 (c 0.2, chloroform). 1H NMR (600 MHz,
CDCl3) δ: 5.05 (t, 1 H, J2,1 = J2,3 = 7.3 Hz, H-2), 4.68–4.69 (m, 1 H, lupane
H-29), 4.59 (br s, 1 H, lupane H-29), 4.36 (d, 1 H, J1,2 7.3 Hz, H-1),
4.23–4.25 (m, 2 H, H-4, lupane H-28), 4.11–4.17 (m, 2 H, H-3, H-5),
3.86 (d, 1 H, J 11.0 Hz, lupane H-28), 3.75 (dd, 1 H, J5,4 3.7, J5,5′ 13.2 Hz,
H-5), 2.99 (dd, 1 H, J 4.7, 11.5 Hz, lupane H-3), 2.42–2.46 (m, 1 H,

62
K. Kuczynska et al./Carbohydrate Research 423 (2016) 49–69
1.54 (s, 3 H, CH3), 1.34 (s, 3 H, CH3), 1.00 (s, 3 H, CH3), 0.91 (s, 3 H,
CH3), 0.87 (s, 3 H, CH3), 0.80 (s, 3 H, CH3), 0.77 (s, 3 H, CH3), 0.71 (s,
3 H, CH3), 0.83–1.71 (m, 21 H, lupane protons), 0.64–0.65 (m, 1 H,
lupane H-5). 13C NMR (150 MHz, CDCl3) δ: 169.4, 150.9 (lupane C-20),
110.4, 109.3 (lupane C-29), 102.3 (C-1), 89.3 (lupane C-3), 76.4, 73.3,
73.0, 62.4 (C-5), 55.6, 50.4, 48.3, 47.9, 43.0 (C), 42.8 (C), 40.8 (C),
40.0 (CH2), 39.0 (C), 38.7 (CH2), 38.0, 36.9 (C), 35.6 (CH2), 34.3 (CH2),
29.8 (CH2), 27.7, 27.4 (CH2), 26.2 (CH2), 26.0, 25.1 (CH2), 21.1, 20.9
(CH2), 19.3, 18.2 (CH2), 18.0, 16.1, 16.1, 15.9, 14.5. Anal. Calcd for
C40H64O6 (640.95): C, 74.96; H, 10.07. Found: C, 74.84; H, 10.27.
1068, 1014, 758 cm−1 1H NMR (500 MHz, CDCl3) δ: 5.89–5.97 (m,
.
1 H, allyl =CH), 5.24–5.28 (m, 1 H, allyl =CH2), 5.11–5.13 (m, 1 H,
allyl =CH2), 4.98 (dd, 1 H, J2,1 3.7, J2,3 5.6 Hz, H-2), 4.68–4.69 (m, 1
H, lupane H-29), 4.58 (br s, 1 H, lupane H-29), 4.55 (d, 1 H, J1,2 3.7 Hz,
H-1), 4.10–4.14 (m, 1 H, allyl O = CH2), 3.84–3.91 (m, 3 H), 3.72 (dd,
1 H, J5,4 7.7, J5,5′ 11.9 Hz, H-5), 3.65 (dd, 1 H, J5,4 4.3, J5,5′ 11.9 Hz, H-5),
3.59 (d, 1 H, J 9.4 Hz, lupane H-28), 3.49 (d, 1 H, J 9.4 Hz, lupane
H-28), 2.80 (dd, 1 H, J 4.1, 11.7 Hz, lupane H-3), 2.35–2.42 (m, 1 H,
lupane H-19), 2.11 (s, 3 H, CH3), 1.89–1.98 (m, 2 H), 1.75–1.79 (m,
1 H), 1.68 (s, 3 H, CH3), 1.01 (s, 3 H, CH3), 0.97 (s, 3 H, CH3), 0.96 (s,
3 H, CH3), 0.83 (s, 3 H, CH3), 0.78 (s, 3 H, CH3), 0.79–1.73 (m, 20 H,
lupane protons), 0.67–0.69 (m, 1 H, lupane C-5). 13C NMR (125 MHz,
CDCl3) δ: 170.2, 150.1 (lupane C-20), 135.9, 115.9 (CH2), 109.8 (lupane
C-29), 99.0 (C-1), 86.3 (lupane C-3), 71.0, 70.6 (CH2), 69.6, 67.8 (CH2),
65.3, 61.2 (CH2), 55.8 (lupane C-5), 50.3, 48.7, 47.7, 46.9 (C), 42.7 (C),
40.9 (C), 38.8 (C), 38.6 (CH2), 37.6, 37.1 (C), 34.9 (CH2), 34.2 (CH2),
29.7 (CH2), 29.5 (CH2), 28.1, 27.0 (CH2), 25.2 (CH2), 23.1 (CH2), 20.9,
20.8 (CH2), 19.1, 18.2 (CH2), 16.2, 16.1, 16.1, 14.8. Anal. Calcd for
C40H64O7 × ½ H2O (665.96): C, 72.14; H, 9.84. Found: C, 72.37; H, 9.79.
4.5.6. 3β-O-(2-O-Acetyl-3,4-O-isopropylidene-α-d-
arabinopyranosyl) lupeol (83)
Eluent B. Yield 98%, amorphous powder. [α]D20 23.1 (c 0.3, chlo-
roform). 1H NMR (600 MHz, CDCl3) δ: 4.99 (t, 1 H, J 7.0 Hz, H-2), 4.68–
4.69 (m, 1 H, lupane H-29), 4.56–4.57 (m, 1 H, lupane H-29), 4.44
(d, 1 H, J1,2 6.8 Hz, H-1), 4.25–4.27 (m, 1 H, H-4), 4.11–4.16 (m, 2 H,
H-3, H-5), 3.74 (dd, 1 H, J5,4 4.1, J5,5′ 13.0 Hz, H-5), 3.16 (dd, 1 H, J
4.4, 11.8 Hz, lupane H-3), 2.35–2.40 (m, 1 H, lupane H-19), 2.07 (s,
3 H, CH3), 1.88–1.95 (m, 1 H), 1.68 (s, 3 H, CH3), 1.57 (s, 3 H, CH3),
1.35 (s, 3 H, CH3), 1.02 (s, 3 H, CH3), 0.97 (s, 3 H, CH3), 0.92 (s, 3 H,
CH3), 0.82 (s, 3 H, CH3), 0.78 (s, 3 H, CH3), 0.72 (s, 3 H, CH3), 0.73–
4.6.3. 28-O-Acetyl-3β-O-(2-O-acetyl-α-l-arabinopyranosyl)
betulin (25)
20
1.67 (m, 22 H, lupane protons), 0.66–0.68 (m, 1 H, lupane H-5). 13
C
Eluent E. Yield 71%, amorphous powder. [α]D 2.1 (c 0.5, chlo-
NMR (150 MHz, CDCl3) δ: 169.4, 151.0 (lupane C-20), 110.4 (C), 109.2
(lupane C-29), 96.8 (C-1), 83.7 (lupane C-3), 76.1, 72.9, 72.6, 62.2
(C-5), 55.8, 50.4, 48.3, 48.0, 43.0 (C), 42.8 (C), 40.9 (C), 40.0 (CH2),
38.4 (CH2), 38.2 (C), 38.0, 37.0 (C), 35.5 (CH2), 34.3 (CH2), 29.8 (CH2),
28.0, 27.7, 27.4 (CH2), 26.1, 25.1 (CH2), 22.6 (CH2), 21.0, 20.9 (CH2),
19.3, 18.3 (CH2), 18.0, 16.1, 16.1, 16.0, 14.5. Anal. Calcd for C40H64O6
(640.95): C, 74.96; H, 10.07. Found: C, 74.81; H, 10.17.
roform). 1H NMR (500 MHz, CDCl3) δ: 4.97 (dd, 1 H, J2,1 3.8, J2,3 5.8 Hz,
H-2), 4.68–4.69 (m, 1 H, lupane H-29), 4.64 (d, 1 H, J1,2 3.8 Hz, H-1),
4.59 (br s, 1 H, lupane H-29), 4.24 (d, 1 H, J 11.0 Hz, lupane H-28),
3.88–3.91 (m, 1 H, H-4), 3.80–3.86 (m, 3 H, H-3, H-5, lupane H-28),
3.60 (dd, 1 H, J5,4 3.9, J5,5′ 11.9 Hz, H-5), 3.10 (dd, 1 H, J 4.6, 11.6 Hz,
lupane H-3), 2.41–2.47 (m, 1 H, lupane H-19), 2.11 (s, 3 H, CH3), 2.07
(s, 3 H, CH3), 1.92–2.01 (m, 1 H), 1.75–1.86 (m, 2 H), 1.68 (s, 3 H,
lupane C-30), 1.03 (s, 3 H, CH3), 0.96 (s, 3 H, CH3), 0.95 (s, 3 H, CH3),
0.83 (s, 3 H, CH3), 0.77 (s, 3 H, CH3), 0.86–1.70 (m, 22 H, lupane
protons), 0.67–0.69 (m, 1 H, lupane H-5). 13C NMR (125 MHz, CDCl3)
δ: 171.6, 170.3, 150.1 (lupane C-20), 109.8 (lupane C-29), 100.7 (C-
1), 90.4 (lupane C-3), 71.4, 69.9, 65.6, 62.8 (CH2), 61.4 (CH2), 55.5,
50.3, 48.8, 47.7, 46.3 (C), 42.7 (C), 40.9 (C), 39.0 (C), 38.6 (CH2), 37.5,
36.8 (C), 34.5 (CH2), 34.1 (CH2), 29.7 (CH2), 29.6 (CH2), 27.9, 27.0 (CH2),
25.8 (CH2), 25.2 (CH2), 21.0, 20.9, 20.8 (CH2), 19.1, 18.2 (CH2), 16.2,
16.0, 16.0, 14.7. Anal. Calcd for C39H62O8 (658.92): C, 71.09; H, 9.48.
Found: C, 70.88; H, 9.61.
4.6. Hydrolysis of isopropylidene protection—general method
To a solution of isopropylidene derivative (0.75 mmol) in ethyl
acetate (10 mL) and methanol (10 mL), a solution of p-TsOH (80 mg)
in ethyl acetate (1 mL) and methanol (1 mL) was added. A solu-
tion was stirred for 3–4 h until no more starting material was
detected on TLC, then neutralized with triethylamine (1 mL) and con-
centrated. The residue was purified by column chromatography to
afford the title compound.
4.6.1. 3β-O-Acetyl-28-O-(2-O-acetyl-α-l-arabinopyranosyl)
betulin (15)
4.6.4. 28-O-tert-Butyldiphenylsilyl-3β-O-(2-O-acetyl-α-
l-arabinopyranosyl) betulin (30)
20
Eluent E. Yield 92%, amorphous powder. [α]D 5.2 (c 0.4, chlo-
Eluent E. Yield 96%, amorphous powder. [α]D20 −11.8 (c 0.4, chlo-
roform). 1H NMR (600 MHz, CDCl3) δ: 7.36–7.70 (m, 10 H, Ar), 4.97
(dd, 1 H, J2,1 3.9, J2,3 5.8 Hz, H-2), 4.63 (d, 1 H, J1,2 3.9 Hz, H-1), 4.59–
4.60 (m, 1 H, lupane H-29), 4.52 (br d, 1 H, lupane H-29), 3.87–
3.91 (m, 1 H, H-4), 3.79–3.82 (m, 2 H), 3.68 (d, 1 H, J 9.9 Hz, lupane
H-28), 3.59 (dd, 1 H, J5,4 3.9, J5,5′ 11.9 Hz, H-5), 3.29–3.33 (m, 2 H),
3.08 (dd, 1 H, J 4.6, 11.7 Hz, lupane H-3), 2.24–2.28 (m, 1 H), 2.11–
2.14 (m, 1 H), 2.11 (s, 3 H, CH3), 1.77–1.87 (m, 2 H), 1.65 (s, 3 H, lupane
C-30), 1.06 (s, 9 H, tBu), 0.95 (s, 3 H, CH3), 0.91 (s, 3 H, CH3), 0.77
(s, 3 H, CH3), 0.76 (s, 3 H, CH3), 0.70 (s, 3 H, CH3), 0.79–1.70 (m, 21
H, lupane protons), 0.64–0.66 (m, 1 H, lupane H-5). 13C NMR
(150 MHz, CDCl3) δ: 170.3, 150.8 (lupane C-20), 127.6–135.7 (Ar),
109.4 (lupane C-29), 100.7 (C-1), 90.4 (lupane C-3), 71.4, 69.9, 65.6,
61.5 (CH2), 61.0 (CH2), 55.4, 50.3, 48.5, 48.4 (C), 47.8, 42.6 (C), 40.8
(C), 39.0 (C), 38.6 (CH2), 37.2, 36.8 (C), 34.5 (CH2), 34.1 (CH2), 29.8
(CH2), 29.5 (CH2), 27.9, 27.0 (CH2), 26.9 (tBu), 25.8 (CH2), 25.1 (CH2),
21.1, 20.8 (CH2), 19.4 (C), 19.1, 18.2 (CH2), 16.2, 16.0, 15.7, 14.7, 14.2
(C). Anal. Calcd for C53H78O7Si × H2O (873.31): C, 72.89; H, 9.23. Found:
C, 73.16; H, 9.43.
roform). 1H NMR (600 MHz, CDCl3) δ: 4.99 (t, 1 H, J2,1 3.6, J2,3 5.5 Hz,
H-2), 4.67–4.68 (m, 1 H, lupane H-29), 4.59 (br s, 1 H, lupane H-29),
4.57 (d, 1 H, J1,2 3.6 Hz, H-1), 4.47 (dd, 1 H, J 5.1, 11.1 Hz, lupane H-3),
3.90–3.93 (m, 1 H, H-4), 3.84 (dd, 1 H, J3,2 5.5, J3,4 3.6 Hz, H-3), 3.71
(dd, 1 H, J5,4 7.9, J5,5′ 11.9 Hz, H-5), 3.65 (dd, 1 H, J5,4 4.4, J5,5′ 11.9 Hz,
H-5), 3.59 (d, 1 H, J 9.4 Hz, lupane H-28), 3.49 (m, 1 H, J 9.4 Hz, lupane
H-28), 2.37–2.42 (m, 1 H, lupane H-19), 2.12 (s, 3 H, CH3), 2.04 (s,
3 H, CH3), 1.89–1.98 (m, 2 H), 1.75–1.79 (m, 1 H), 1.68 (s, 3 H, CH3),
1.01 (s, 3 H, CH3), 0.97 (s, 3 H, CH3), 0.85 (s, 6 H, 2 × CH3), 0.84 (s, 3
H, CH3), 0.95–1.67 (m, 22 H, lupane protons), 0.78–0.80 (m, 1 H,
lupane H-5). 13C NMR (150 MHz, CDCl3) δ: 171.0, 170.1, 150.1 (lupane
C-20), 109.9 (lupane C-29), 99.0 (C-1), 80.9 (lupane C-3), 70.9, 69.5,
67.8 (CH2), 65.2, 61.0 (CH2), 55.3, 50.2, 48.7, 47.7, 46.9 (C), 42.7 (C),
40.9 (C), 38.3 (CH2), 37.8 (C), 37.6, 37.0 (C), 34.9 (CH2), 34.1 (CH2),
29.7 (CH2), 29.5 (CH2), 27.9, 27.0 (CH2), 25.1 (CH2), 23.6 (CH2), 21.3,
20.9, 20.8 (CH2), 19.1, 18.1 (CH2), 16.5, 16.1, 16.0, 14.8. Anal. Calcd
for C39H62O8 (658.93): C, 71.09; H, 9.48. Found: C, 70.97; H, 9.29.
4.6.2. 3β-O-Allyl-28-O-(2-O-acetyl-α-l-arabinopyranosyl)
betulin (20)
Eluent F. Yield 79%, amorphous powder. [α]D 9.5 (c 0.3, chlo-
roform). νmax (film): 3410, 2943, 2869, 1739, 1455, 1371, 1238, 1137,
4.6.5. 3β-O-(2-O-Acetyl-α-L-arabinopyranosyl) lupeol (36)
20
20
Eluent E. Yield 97%, amorphous powder. [α]D 9.1 (c 0.4, chlo-
roform). 1H NMR (600 MHz, CDCl3) δ: 4.96 (dd, 1 H, J2,1 3.8, J2,3 5.7 Hz,

K. Kuczynska et al./Carbohydrate Research 423 (2016) 49–69
63
H-2), 4.67–4.68 (m, 1 H, lupane H-29), 4.63 (d, 1 H, J1,2 3.8 Hz, H-1),
4.55–4.56 (m, 1 H, lupane H-29), 3.87–3.90 (m, 1 H, H-4), 3.78–
3.82 (m, 2 H, H-3, H-5), 3.59 (dd, 1 H, J5,4 4.0, J5,5′ 11.9 Hz, H-5), 3.09
(dd, 1 H, J 4.6, 11.5 Hz, lupane H-3), 2.34–2.38 (m, 1 H, lupane H-19),
2.10 (s, 3 H, CH3), 1.87–1.94 (m, 1 H), 1.78–1.81 (m, 1 H), 1.67 (s, 3
H, CH3), 1.01 (s, 3 H, CH3), 0.94 (s, 3 H, CH3), 0.92 (s, 3 H, CH3), 0.82
(s, 3 H, CH3), 0.77 (s, 3 H, CH3), 0.75 (s, 3 H, CH3), 0.78–1.71 (m, 23
H, lupane protons), 0.66–0.68 (m, 1 H, lupane H-5). 13C NMR
(150 MHz, CDCl3) δ: 170.3, 150.9 (lupane C-20), 109.3 (lupane C-29),
100.7 (C-1), 90.4 (lupane C-3), 71.3, 69.9, 65.6, 61.4 (C-5), 55.5, 50.4,
48.3, 47.9, 43.0 (C), 42.8 (C), 40.8 (C), 40.0 (CH2), 39.0 (C), 38.6 (CH2),
38.0, 36.8 (C), 35.5 (CH2), 34.2 (CH2), 29.8 (CH2), 27.9, 27.4 (CH2), 25.8
(CH2), 25.1 (CH2), 20.9 (CH2), 19.3, 18.2 (CH2), 18.0, 16.2, 16.0, 15.9,
14.5. Anal. Calcd for C37H60O6 (600.89): C, 73.96; H, 10.07. Found:
C, 73.76; H, 10.09.
37.5, 37.1, 34.3, 34.2, 30.1, 29.7, 28.1, 27.1, 25.2, 23.1, 20.9, 20.8, 19.1,
18.2, 16.3, 16.1, 16.0, 14.8. Anal. Calcd for C40H64O7 × 2 H2O (692.92):
C, 69.33; H, 9.89. Found: C, 69.50; H, 9.31.
4.6.8. 3β-O-(2-O-Acetyl-α-d-arabinopyranosyl) lupeol (84)
Eluent E. Yield 97%, amorphous powder. [α]D20 48.6 (c 0.3, chlo-
roform). 1H NMR (600 MHz, CDCl3) δ: 4.90 (dd, 1 H, J2,1 3.4, J2,3 5.4 Hz,
H-2), 4.74 (d, 1 H, J1,2 3.4 Hz, H-1), 4.68–4.69 (m, 1 H, lupane H-29),
4.56–4.57 (m, 1 H, lupane H-29), 3.89–3.91 (m, 1 H, H-4), 3.85 (br
s, 1 H, H-3), 3.81 (dd, 1 H, J5,4 8.3, J5,5′ 11.8 Hz, H-5), 3.62 (dd, 1 H,
J5,4 4.3, J5,5′ 11.8 Hz, H-5), 3.26 (dd, 1 H, J 4.1, 11.8 Hz, lupane H-3),
2.35–2.40 (m, 1 H, lupane H-19), 2.10 (s, 3 H, CH3), 1.88–1.95 (m, 1
H), 1.68 (s, 3 H, CH3), 1.03 (s, 3 H, CH3), 1.01 (s, 3 H, CH3), 0.93 (s, 3
H, CH3), 0.84 (s, 3 H, CH3), 0.79 (s, 3 H, CH3), 0.76 (s, 3 H, CH3), 0.77–
1.71 (m, 24 H, lupane protons), 0.70–0.71 (m, 1 H, lupane H-5). 13
C
NMR (150 MHz, CDCl3) δ: 170.2, 150.9 (lupane C-20), 109.3 (lupane
C-29), 94.9 (C-1), 84.9 (lupane C-3), 71.3, 69.5, 65.2, 61.2 (C-5), 55.6,
50.4, 48.3, 47.9, 43.0 (C), 42.8 (C), 40.8 (C), 40.0 (CH2), 38.2 (CH2),
38.2 (C), 38.0, 37.0 (C), 35.5 (CH2), 34.2 (CH2), 29.8 (CH2), 28.5, 27.4
(CH2), 25.1 (CH2), 22.4 (CH2), 20.9 (CH2), 20.9, 19.3, 18.2 (CH2), 18.0,
16.4, 16.0, 16.0, 14.5. Anal. Calcd for C37H60O6 (600.89): C, 73.96; H,
10.07. Found: C, 73.99; H, 10.07.
4.6.6. 3β-O-Acetyl-28-O-(2-O-acetyl-α-d-arabinopyranosyl)
betulin (68)
Hydrolysis of 89 (see Supplementary material) afforded the title
20
compound. Eluent E. Yield 94%, amorphous powder. [α]D 25.6 (c
0.2, chloroform). 1H NMR (500 MHz, CDCl3) δ: 4.99 (dd, 1 H, J2,1 3.5,
J2,3 5.5 Hz, H-2), 4.67–4.68 (m, 1 H, lupane H-29), 4.59 (br s, 1 H,
lupane H-29), 4.56 (d, 1 H, J1,2 3.5 Hz, H-1), 4.47 (dd, 1 H, J 5.4, 10.8 Hz,
lupane H-3), 4.03 (d, 1 H, J 9.5 Hz, lupane H-28), 3.91–3.93 (m, 1
H, H-4), 3.84 (dd, 1 H, J3,2 5.3, J3,4 3.5 Hz, H-3), 3.74 (dd, 1 H, J5,4 8.0,
J5,5′ 11.8 Hz, H-5), 3.65 (dd, 1 H, J5,4 4.3, J5,5′ 11.8 Hz, H-5), 3.07 (m, 1
H, J 9.5 Hz, lupane H-28), 2.32–2.39 (m, 1 H, lupane H-19), 2.11 (s,
3 H, CH3), 2.04 (s, 3 H, CH3), 1.80–1.95 (m, 3 H), 1.68 (s, 3 H, CH3),
1.03 (s, 3 H, CH3), 0.97 (s, 3 H, CH3), 0.85 (s, 6 H, 2 × CH3), 0.84 (s, 3
H, CH3), 0.86–1.72 (m, 22 H, lupane protons), 0.76–0.80 (m, 1 H,
lupane H-5). 13C NMR (125 MHz, CDCl3) δ: 171.0, 170.2, 150.1 (lupane
C-20), 109.9 (lupane C-29), 99.0 (C-1), 80.9 (lupane C-3), 71.0, 69.6,
67.8 (CH2), 65.3, 61.0 (CH2), 55.4, 50.3, 48.8, 47.9, 46.9 (C), 42.7 (C),
40.9 (C), 38.4 (CH2), 37.8 (C), 37.6, 37.1 (C), 34.3 (CH2), 34.1 (CH2),
30.0 (CH2), 29.6 (CH2), 27.9, 27.1 (CH2), 25.1 (CH2), 23.7 (CH2), 21.3,
20.9, 20.8 (CH2), 19.0, 18.1 (CH2), 16.5, 16.1, 16.0, 14.7. Anal. Calcd
for C39H62O8 (658.93): C, 71.09; H, 9.48. Found: C, 71.15; H, 9.51.
4.7. Synthesis of disaccharides—general methods
Method A. A solution of 2-O-acetyl-arabinoside (1.00 equiv) and
the corresponding glycosyl donor (1.20 equiv) in CH2Cl2 (15 mL) was
stirred for 20 min at rt over molecular sieves (4 Å, 500 mg, finely
ground), then cooled to −40 °C and BF3 × OEt2 (0.50 equiv) in CH2Cl2
(4 mL) was added over 10 min via syringe pump. The mixture was
stirred for 30 min, reaction was quenched with Et3N (1 mL), and the
solvents were evaporated under diminished pressure. Column chro-
matography of the residue gave the protected disaccharides.
Method B. A solution of 2-O-acetyl-arabinoside (1.00 equiv) and
the corresponding glycosyl donor (1.10 equiv) in CH2Cl2 (10 mL) was
stirred for 20 min at rt over molecular sieves (4 Å, 300 mg, finely
ground), then cooled to −40 °C and TMSOTf (0.50 equiv) was added.
The mixture was stirred for 30 min, reaction was quenched with
Et3N (1 mL), and the solvents were evaporated under diminished
pressure. Column chromatography of the residue gave the pro-
tected disaccharides.
4.6.7. 3β-O-Allyl-28-O-(2-O-acetyl-α-d-arabinopyranosyl)
betulin (69)
Glycosylation of 3 with 8 was performed according to the pro-
cedure 4.2. to yield crude 3β-O-allyl-28-O-(2,3,4-tri-O-benzoyl-α-
d-arabinopyranosyl) betulin. Debenzoylation using the procedure
4.3. afforded 3β-O-allyl-28-O-(α-d-arabinopyranosyl) betulin which,
without further purification, was transformed into 3β-O-allyl-28-
O-(3,4-O-isopropylidene-α-d-arabinopyranosyl) betulin as described
in procedure 4.4. It was further acetylated (general method 4.5.) to
afford crude 3β-O-allyl-28-O-(2-O-acetyl-3,4-O-isopropylidene-α-
d-arabinopyranosyl) betulin. Hydrolysis of isopropylidene group gave
the title compound as amorphous powder in 27% total yield (after
five steps). Eluent B. [α]D20 29.4 (c 0.3, chloroform). νmax (film): 3360,
2943, 2869, 1738, 1725, 1374, 1258, 1106, 1085, 1066, 1017,
756 cm−1. 1H NMR (500 MHz, CDCl3): δ: 5.88–5.96 (m, 1 H, allyl =CH),
5.23–5.27 (m, 1 H, allyl =CH2), 5.10–5.12 (m, 1 H, allyl =CH2), 4.97
(dd, 1 H, J2,1 3.6, J2,3 5.6 Hz, H-2), 4.66 (br s, 1 H, lupane H-29), 4.58
(br s, 1 H, lupane H-29), 4.54 (d, 1 H, J1,2 3.6 Hz, H-1), 4.09–4.13 (m,
1 H, allyl OCH2), 4.02 (d, 1 H, J 9.3 Hz, lupane H-28), 3.82–3.91 (m,
3 H, H-3, H-4, allyl OCH2), 3.74 (dd, 1 H, J5,4 7.9, J5,5′ 11.8 Hz, H-5),
3.64 (dd, 1 H, J5,4 4.2, J5,5′ 11.8 Hz, H-5), 3.06 (d, 1 H, J 9.3 Hz, lupane
H-28), 2.79 (dd, 1 H, J 4.1, 11.7 Hz, lupane H-3), 2.33–2.39 (m, 1 H,
lupane H-19), 2.11 (s, 3 H, CH3), 1.67 (s, 3 H, CH3), 1.02 (s, 3 H, CH3),
0.96 (s, 3 H, CH3), 0.95 (s, 3 H, CH3), 0.82 (s, 3 H, CH3), 0.78 (s, 3 H,
CH3), 0.84–1.72 (m, 25 H, lupane protons), 0.66–0.68 (m, 1 H, lupane
C-5). 13C NMR (125 MHz, CDCl3): δ: 170.2, 150.1 (lupane C-20), 135.9,
115.9, 109.8 (lupane C-29), 99.0 (C-1), 86.3 (lupane C-3), 71.0, 70.6,
69.7, 67.8, 65.3, 61.1, 55.8, 50.4, 48.8, 47.9, 46.9, 42.7, 40.9, 38.8, 38.6,
4.7.1. 3β-O-Acetyl-28-O-[3,4-O-(2′,3′-dimethoxybutane-2′,3′-
diyl)-2-O-(4-methoxybenzoyl)-β-d-xylopyranosyl-(1→3)-2-O-
acetyl-β-l-arabinopyranosyl] betulin (37) and 3β-O-acetyl-28-O-
[3,4-O-(2′,3′-dimethoxybutane-2′,3′-diyl)-2-O-(4-
methoxybenzoyl)-β-d-xylopyranosyl-(1→4)-2-O-acetyl-β-l-
arabinopyranosyl] betulin (38)
Method B. Eluent G. (1→4)-Linked disaccharide 38 was eluted
as the first product; the second fraction contained (1→3)-linked di-
saccharide 37.
Data for 37. Amorphous powder. Yield 16% (50 mg). [α]D20 79.0
(c 0.2, chloroform). 1H NMR (600 MHz, CDCl3) δ: 7.98–7.99 (m, 2 H,
Ar), 6.91–6.93 (m, 2 H, Ar), 5.15 (dd, 1 H, J2,1 7.6, J2,3 9.8 Hz, H-2′),
5.00 (dd, 1 H, J2,1 6.1, J2,3 7.8 Hz, H-2), 4.65 (d, 1 H, J1,2 7.6 Hz, H-1′),
4.62–4.63 (m, 1 H, lupane H-29), 4.54 (br s, 1 H, lupane H-29), 4.46
(dd, 1 H, J 5.2, 11.0 Hz, lupane H-3), 4.24 (d, 1 H, J1,2 6.1 Hz, H-1),
3.98–4.00 (m, 1 H, H-4), 3.94–3.97 (m, 2 H, H-5, H-5′), 3.84–3.91
(m, 5 H, H-3′, H-4′, OCH3), 3.45–3.50 (m, 3 H, H-5, H-5′, lupane H-28),
3.31 (d, 1 H, J 9.5 Hz, lupane H-28), 3.26 (s, 3 H, OCH3), 3.17 (s, 3
H, OCH3), 2.30–2.35 (m, 1 H, lupane H-19), 2.04 (s, 3 H, CH3), 1.82–
1.93 (m, 3 H), 1.65 (s, 3 H, CH3), 1.64 (s, 3 H, CH3), 1.27 (s, 3 H, CH3),
1.22 (s, 3 H, CH3), 0.96 (s, 3 H, CH3), 0.91 (s, 3 H, CH3), 0.84 (s, 3 H,
CH3), 0.83 (m, 6 H, 2 × CH3), 0.88–1.68 (m, 17 H, lupane protons),
0.76–0.78 (m, 1 H, lupane H-5). 13C NMR (150 MHz, CDCl3) δ: 170.9,
168.9, 164.2, 163.4 (Ar-OMe), 150.6 (lupane C-20), 131.8 (Ar), 122.3

64
K. Kuczynska et al./Carbohydrate Research 423 (2016) 49–69
1
(Ar), 113.6 (Ar), 109.5 (lupane C-29), 103.4 (C-1′, JC1,H1 161.8 Hz),
101.0 (C-1, 1JC1,H1 160.0 Hz), 99.8 (C), 99.6 (C), 80.9 (lupane C-3), 80.2
(C-3), 70.9 (C-2′), 70.7 (C-3′), 70.6 (C-2), 67.3 (lupane C-28), 67.0 (C-
4), 65.6 (C-4′), 64.2 (C-5′), 63.5 (C-5), 55.4 (OCH3), 55.3 (lupane C-5),
50.3, 48.6, 48.0, 47.9, 47.7, 46.8, 42.6, 40.8, 38.3, 37.8, 37.6, 37.0, 34.6,
34.1, 29.7, 29.4, 27.9, 27.0, 25.1, 23.7, 21.3, 20.8, 20.4, 19.0, 18.2, 17.6,
17.5, 16.5, 16.1, 16.0, 14.7. Anal. Calcd for C58H86O16 × 3/2 H2O (1066.35):
C, 65.33; H, 8.41. Found: C, 65.42; H, 8.18.
4.8. 3β-O-Acetyl-28-O-[3,4-O-(2′,3′-dimethoxybutane-2′,3′-diyl)-
2-O-(4-methoxybenzoyl)-β-d-xylopyranosyl-(1→3)-2,4-di-O-
acetyl-β-l-arabinopyranosyl] betulin (39)
Disaccharide 37 (0.05 mmol) was acetylated according to general
method 4.5. Preparative TLC of the residue (eluent F) gave the title
20
saponin (32 mg, 65%). [α]D 82.1 (c 0.20, chloroform). 1H NMR
(600 MHz, CDCl3) δ: 7.95–7.97 (m, 2 H, Ar), 6.91–6.92 (m, 2 H, Ar),
5.22 (br s, 1 H, H-4), 5.01–5.11 (m, 2 H, H-2, H-2′), 4.62–4.63 (m, 2
H, H-1′, lupane H-29), 4.46 (dd, 1 H, J 5.2, 11.1 Hz, lupane H-3), 4.19
(d, 1 H, J1,2 7.5 Hz, H-1), 4.01 (m, 1 H, J5,4 2.5, J5,5′ 13.2 Hz, H-5), 3.84–
3.92 (m, 3 H, H-3′, H-4′, H-5′), 3.85 (s, 3 H, OCH3), 3.79 (dd, 1 H, J3,2
9.6, J3,4 3.7 Hz, H-3), 3.51–3.55 (m, 2 H, H-5, lupane H-28), 3.39–
3.42 (m, 2 H, H-5′, lupane H-28), 3.25 (s, 3 H, OCH3), 3.18 (s, 3 H,
OCH3), 2.29–2.33 (m, 1 H, lupane H-19), 2.15 (s, 3 H, CH3), 2.04 (s,
3 H, CH3), 1.89–1.93 (m, 1 H), 1.82–1.85 (m, 1 H), 1.71–1.75 (m, 1
H), 1.64 (s, 3 H, CH3), 1.63 (s, 3 H, CH3), 1.27 (s, 3 H, CH3), 1.21 (s, 3
H, CH3), 0.98 (s, 3 H, CH3), 0.92 (s, 3 H, CH3), 0.84 (s, 3 H, CH3), 0.84
(m, 6 H, 2 × CH3), 0.90–1.65 (m, 21 H, lupane protons), 0.77–0.79
(m, 1 H, lupane H-5). 13C NMR (150 MHz, CDCl3) δ: 170.9, 170.8,
168.6, 164.2, 163.3, 150.4 (lupane C-20), 131.7 (Ar), 126.0 (Ar), 122.4
Data for 38. Amorphous powder. Yield 53% (165 mg). [α]D20 72.3
(c 0.3, chloroform). 1H NMR (600 MHz, CDCl3) δ: 7.98–8.00 (m, 2 H,
Ar), 6.90–6.92 (m, 2 H, Ar), 5.18 (dd, 1 H, J2,1 6.8, J2,3 9.5 Hz, H-2′),
4.91 (dd, 1 H, J2,1 5.2, J2,3 7.4 Hz, H-2), 4.72 (d, 1 H, J1,2 6.8 Hz, H-1′),
4.65 (br s, 1 H, lupane H-29), 4.56 (br s, 1 H, lupane H-29), 4.46 (dd,
1 H, J 5.1, 11.1 Hz, lupane H-3), 4.34 (d, 1 H, J1,2 5.2 Hz, H-1), 3.91–
4.01 (m, 4 H, H-3′, H-4′, H-5, H-5′), 3.86–3.90 (m, 1 H, H-4), 3.85
(s, 3 H, OCH3), 3.66–3.70 (m, 1 H, H-3), 3.50–3.56 (m, 3 H, H-5, H-5′,
lupane H-28), 3.41 (d, 1 H, J 9.3 Hz, lupane H-28), 3.26 (s, 3 H, OCH3),
3.23 (s, 3 H, OCH3), 2.33–2.37 (m, 1 H, lupane H-19), 2.04 (s, 3 H,
CH3), 2.04 (s, 3 H, CH3), 1.82–1.93 (m, 3 H), 1.66 (s, 3 H, CH3), 1.27
(s, 3 H, CH3), 1.25 (s, 3 H, CH3), 1.00 (s, 3 H, CH3), 0.95 (s, 3 H, CH3),
0.84 (s, 3 H, CH3), 0.84 (s, 3 H, CH3), 0.83 (s, 3 H, CH3), 0.89–1.64
(m, 21 H, lupane protons), 0.77–0.79 (m, 1 H, lupane H-5). 13C NMR
(150 MHz, CDCl3) δ: 171.0, 169.7, 165.2, 163.6 (Ar-OMe), 150.5
(lupane C-20), 132.8 (Ar), 122.0 (Ar), 113.7 (Ar), 109.6 (lupane C-29),
103.2 (C-1′), 100.6 (C-1), 99.8 (C), 99.5 (C), 80.9 (lupane C-3), 75.6
(C-4), 72.0 (C-2), 71.7 (C-2′), 70.2 (C-3′), 69.6 (C-3), 67.8 (lupane C-28),
65.3 (C-4′), 64.1 (C-5′), 62.1 (C-5), 55.4 (OCH3), 55.3 (lupane C-5),
50.3, 48.7, 48.0, 47.8, 47.7, 47.0, 42.7, 40.9, 38.3, 37.8, 37.6, 37.1, 34.7,
1
(Ar), 113.5 (Ar), 109.5 (lupane C-29), 103.4 (C-1′, JC1,H1 161.8 Hz),
102.5 (C-1, 1JC1,H1 160.0 Hz), 99.7 (C), 99.5 (C), 80.8 (lupane C-3), 76.9
(C-3), 71.4 (C-2′), 70.6 (C-2), 70.5 (C-3′ or C-4′), 70.2 (C-4), 68.1
(lupane C-28), 65.3 (C-3′ or C-4′), 63.9 (C-5′), 63.7 (C-5), 55.4 (OCH3),
55.3 (lupane C-5), 50.2, 48.6, 47.9, 47.8, 47.6, 46.9, 42.6, 40.8, 38.3,
37.8, 37.6, 37.0, 34.5, 34.1, 29.6, 29.3, 27.9, 27.0, 25.0, 23.7, 21.3, 21.1,
20.8, 20.3, 19.0, 18.1, 17.6, 17.5, 16.5, 16.1, 16.0, 14.8. Anal. Calcd for
C60H88O17 (1081.36): C, 66.64; H, 8.20. Found: C, 66.42; H, 8.04.
34.1, 29.8, 29.5, 27.9, 27.1, 25.1, 23.7, 21.3, 21.0, 20.8, 19.1, 18.2, 17.6,
17.5, 16.5, 16.2, 16.0, 14.8. Anal. Calcd for C58H86O16
(1066.35): C, 65.33; H, 8.41. Found: C, 65.07; H, 8.23.
3
×
/
H2O
2
4.9. 3β-O-Acetyl-28-O-[3,4-O-(2′,3′-dimethoxybutane-2′,3′-diyl)-
2-O-(4-methoxybenzoyl)-β-d-xylopyranosyl-(1→4)-2,3-di-O-
acetyl-β-l-arabinopyranosyl] betulin (40)
4.7.2. 3β-O-Acetyl-28-O-[2-O-(4-methoxybenzoyl)-3,4-di-
O-triethylsilyl-β-d-xylopyranosyl-(1→3)-2-O-acetyl-α-
Disaccharide 38 (0.05 mmol) was acetylated according to general
method 4.5. Preparative TLC of the residue (eluent F) gave the title
l-arabinopyranosyl] betulin (41) and 3β-O-Acetyl-28-O-[2-O-(4-
methoxybenzoyl)-3,4-di-O-triethylsilyl-β-d-xylopyranosyl-
(1→4)-2-O-acetyl-α-l-arabinopyranosyl] betulin (42)
Method A. Eluent D. Isolated as a mixture of regioisomers
(450 mg, 65%) as amorphous powder.
20
saponin (40 mg, 75%). [α]D 37.4 (c 0.2, chloroform). 1H NMR
(600 MHz, CDCl3) δ: 7.97–7.98 (m, 2 H, Ar), 6.90–6.92 (m, 2 H, Ar),
5.14 (dd, 1 H, J2,1 7.3, J2,3 9.8 Hz, H-2′), 4.93–4.97 (m, 2 H, H-2, H-3),
4.66–4.67 (m, 1 H, lupane H-29), 4.59 (d, 1 H, J1,2 7.3 Hz, H-1′), 4.57
(br s, 1 H, lupane H-29), 4.44–4.47 (m, 2 H, J1,2 2.4 Hz, H-1, lupane
H-3), 4.04–4.08 (m, 1 H, H-4), 3.85–4.00 (m, 4 H, H-3′, H-4′, H-5,
H-5′), 3.85 (s, 3 H, OCH3), 3.58 (dd, 1 H, J5,4 3.7, J5,5′ 11.2 Hz, H-5),
3.45–3.48 (m, 2 H, H-5′, lupane H-28), 3.36 (d, 1 H, J 8.9 Hz, lupane
H-28), 3.26 (s, 3 H, OCH3), 3.18 (s, 3 H, OCH3), 2.36–2.40 (m, 1 H,
lupane H-19), 2.06 (s, 3 H, CH3), 2.03 (s, 3 H, CH3), 1.91–1.95 (m, 2
H), 1.83–1.86 (m, 1 H), 1.67 (s, 3 H, CH3), 1.28 (s, 3 H, CH3), 1.22 (s,
3 H, CH3), 0.98 (s, 3 H, CH3), 0.94 (s, 3 H, CH3), 0.84 (s, 3 H, CH3),
0.83 (s, 3 H, CH3), 0.83 (s, 3 H, CH3), 0.92–1.69 (m, 23 H, lupane
protons), 0.77–0.78 (m, 1 H, lupane H-5). 13C NMR (150 MHz, CDCl3)
δ: 171.0, 170.2, 169.0, 164.5, 163.4, 150.5 (lupane C-20), 131.7 (Ar),
122.3 (Ar), 113.6 (Ar), 109.6 (lupane C-29), 101.9 (C-1′), 99.7 (C),
99.5 (C), 98.5 (C-1), 80.9 (lupane C-3), 70.9 (C-2′), 70.8 (C-4), 70.6
(C-3′), 69.1 and 68.0 (C-2, C-3), 66.7 (lupane C-28), 65.7 (C-4′), 64.1
(C-5′), 60.0 (C-5), 55.4 (OCH3), 55.3 (lupane C-5), 50.2, 48.7, 47.9,
47.8, 47.6, 46.9, 42.6, 40.8, 38.3, 37.8, 37.5, 37.0, 34.6, 34.1, 29.8, 29.4,
27.9, 27.0, 25.1, 23.7, 21.3, 21.3, 20.9, 20.8, 19.1, 18.1, 17.6, 17.5, 16.5,
16.1, 16.0, 14.7. Anal. Calcd for C60H88O17 (1081.36): C, 66.64; H, 8.20.
Found: C, 66.54; H, 8.16.
4.7.3. 3β-O-Allyl-28-O-[2-O-(4-methoxybenzoyl)-3,4-di-
O-triethylsilyl-β-d-xylopyranosyl-(1→3)-2-O-acetyl-α-
l-arabinopyranosyl] betulin (47) and 3β-O-allyl-28-O-[2-O-(4-
methoxybenzoyl)-3,4-di-O-triethylsilyl-β-d-xylopyranosyl-
(1→4)-2-O-acetyl-α-l-arabinopyranosyl] betulin (48)
Method A. Eluent A. Isolated as a mixture of regioisomers
(265 mg, 71%) as amorphous powder.
4.7.4. 28-O-tert-Butyldiphenylsilyl-3β-O-[2-O-(4-
methoxybenzoyl)-3,4-di-O-triethylsilyl-β-d-xylopyranosyl-
(1→3)-2-O-acetyl-α-l-arabinopyranosyl] betulin (52) and
28-O-tert-butyldiphenylsilyl-3β-O-[2-O-(4-methoxybenzoyl)-3,
4-di-O-triethylsilyl-β-d-xylopyranosyl-(1→4)-2-O-acetyl-α-l-
arabinopyranosyl] betulin (53)
Method A. Eluent D. Isolated as a mixture of regioisomers
(633 mg, 78%) as amorphous powder.
4.7.5. 3β-O-[2-O-(4-methoxybenzoyl)-3,4-di-O-triethylsilyl-β-d-
xylopyranosyl-(1→3)-2-O-acetyl-α-l-arabinopyranosyl] lupeol
(59) and 3β-O-[2-O-(4-methoxybenzoyl)-3,4-di-O-triethylsilyl-β-
d-xylopyranosyl-(1→4)-2-O-acetyl-α-l-arabinopyranosyl]
lupeol (60)
4.10. Hydrolysis of triethylsilyl group—general methods
Method A. To a solution of silylated disaccharide (0.10 mmol) in
methanol (4 mL) and dichloromethane (2 mL) camphorsulfonic acid
(CSA, 10 mg) was added and the mixture was stirred at rt for 30–
60 min. Reaction was quenched with Et3N (0.5 mL), and the solvents
Method A. Eluent D. Isolated as a mixture of regioisomers in ap-
proximate ratio 3.9:1.0 (496 mg, 76%) as amorphous powder.

K. Kuczynska et al./Carbohydrate Research 423 (2016) 49–69
65
were evaporated under diminished pressure. Preparative TLC of the
residue (hexane–ethyl acetate–methanol, 5:3:0.5) gave the title
saponin.
(CH2), 29.7 (CH2), 29.4 (CH2), 27.9, 27.0 (CH2), 25.1 (CH2), 23.6 (CH2),
21.3, 20.9, 20.8 (CH2), 19.0, 18.1 (CH2), 16.5, 16.1, 16.0, 14.7. Anal.
Calcd for C52H76O14 × 1½ H2O (952.20): C, 65.59; H, 8.36. Found: C,
65.42; H, 8.54. HR-MS (ESI) calc. for C52H76NaO14 [M + Na]+: 947.5133.
Found: 947.5146.
Method B. Product of glycosylation (disaccharide, 0.05 mmol) was
acetylated under standard conditions (pyridine, Ac2O), then
coevaporated with toluene (3–4 times), filtered through short silica
column (hexane–ethyl acetate, 5:1 containing 0.5% of Et3N) and
evaporated to dryness. The residue was dissolved in methanol
(4 mL)–dichloromethane (2 mL) mixture and camphorsulfonic acid
(CSA, 10 mg) was added. The stirring was continued for 30–
60 min. Reaction was quenched with Et3N (0.5 mL), and the solvents
were evaporated under diminished pressure. Preparative TLC of the
residue (hexane–ethyl acetate–methanol, 5:3:0.5) gave the title
saponin.
4.10.2. 3β-O-Acetyl-28-O-[2-O-(4-methoxybenzoyl)-β-d-
xylopyranosyl-(1→3)-2,4-di-O-acetyl-α-l-arabinopyranosyl]
betulin (45) and 3β-O-acetyl-28-O-[2-O-(4-methoxybenzoyl)-β-
d-xylopyranosyl-(1→4)-2,3-di-O-acetyl-α-l-arabinopyranosyl]
betulin (46)
An analytical sample of 41/42 mixture was separated by pre-
parative TLC (hexane–ethyl acetate, 5:1 containing 0.5% Et3N) to
afford pure disaccharides 41 and 42. They were transformed into
acetates 45 and 46 according to Method B.
20
4.10.1. 3β-O-Acetyl-28-O-[2-O-(4-methoxybenzoyl)-β-d-
xylopyranosyl-(1→3)-2-O-acetyl-α-l-arabinopyranosyl] betulin
(43) and 3β-O-acetyl-28-O-[2-O-(4-methoxybenzoyl)-β-d-
xylopyranosyl-(1→4)-2-O-acetyl-α-l-arabinopyranosyl]
betulin (44)
Data for 45. Yield 58%. [α]D −0.4 (c 0.3, chloroform). 1H NMR
(600 MHz, CDCl3) δ: 7.94–7.96 (m, 2 H, Ar), 6.92–6.93 (m, 2 H, Ar),
5.21–5.23 (m, 1 H, H-4), 5.18 (dd, 1 H, J2,1 6.4, J2,3 8.5 Hz, H-2), 4.94
(d, 1 H, J1,2 4.1 Hz, H-1′), 4.89 (dd, 1 H, J2,1 4.1, J2,3 5.3 Hz, H-2′), 4.64–
4.65 (m, 1 H, lupane H-29), 4.56 (br s, 1 H, lupane H-29), 4.46 (dd,
1 H, J 5.1, 11.1 Hz, lupane H-3), 4.32 (d, 1 H, J1,2 6.4 Hz, H-1), 4.16–
4.18 (m, 1 H, H-5′), 4.03 (dd, 1 H, J5,4 4.0, J5,5′ 12.8 Hz, H-5), 3.95
(dd, 1 H, J3,2 8.5, J3,4 3.3 Hz, H-3), 3.87 (s, 3 H, OCH3), 3.81–3.83 (m,
1 H, H-3′), 3.74–3.76 (m, 1 H, H-4′), 3.56–3.59 (m, 2 H, H-5, lupane
H-28), 3.52 (dd, 1 H, J5,4 5.2, J5,5′ 12.2 Hz, H-5′), 3.44 (d, 1 H, J 9.3 Hz,
lupane H-28), 2.32–2.36 (m, 1 H, lupane H-19), 2.14 (s, 3 H, CH3),
2.04 (s, 3 H, CH3), 2.01 (s, 3 H, CH3), 1.78–1.98 (m, 3 H), 1.66 (s, 3
H, CH3), 1.00 (s, 3 H, CH3), 0.94 (s, 3 H, CH3), 0.84 (s, 3 H, CH3), 0.84
(s, 3 H, CH3), 0.84 (s, 3 H, CH3), 0.95–1.64 (m, 22 H, lupane protons),
0.78–0.79 (m, 1 H, lupane H-5). 13C NMR (150 MHz, CDCl3) δ: 171.0,
170.8, 169.4, 163.9 (Ar-OMe), 150.4 (lupane C-20), 131.9 (Ar), 121.3
(Ar), 113.8 (Ar), 109.6 (lupane C-29), 101.6 (C-1), 99.6 (C-1′), 80.8
(lupane C-3), 75.3 (C-3), 74.4 (C-2′), 71.0 (C-3′), 70.3 (C-2), 69.3
(C-4), 69.0 (C-4′), 68.2 (lupane C-28), 62.3 (C-5, C-5′), 55.5 (OCH3),
55.3 (lupane C-5), 50.2, 48.6, 47.9, 46.9, 42.6, 40.8, 38.3,
37.8, 37.6, 37.0, 34.6, 34.1, 29.7, 29.3, 27.9, 27.0, 25.0, 23.621.3, 21.1,
20.8, 20.7, 19.0, 18.1, 16.5, 16.1, 16.0, 14.8. Anal. Calcd for
C54H78O15 × 1½ H2O (994.24): C, 65.24; H, 8.21. Found: C, 65.30; H,
8.18. HR-MS (ESI) calc. for C54H78NaO15 [M + Na]+: 989.5238. Found:
989.5236.
Method A. Starting from mixture of 41/42 saponins 44 (51%) and
43 (39%) were obtained.
20
Data for 43. [α]D 0.6 (c 0.2, chloroform). 1H NMR (600 MHz,
CDCl3) δ: 7.93–7.95 (m, 2 H, Ar), 6.86–6.88 (m, 2 H, Ar), 5.06 (dd, 1
H, J2,1 6.6, J2,3 8.4 Hz, H-2), 4.98 (dd, 1 H, J2,1 6.8, J2,3 8.3 Hz, H-2′), 4.67
(d, 1 H, J1,2 6.8 Hz, H-1′), 4.63 (br s, 1 H, lupane H-29), 4.54 (br s, 1
H, lupane H-29), 4.46 (dd, 1 H, J 5.2, 10.9 Hz, lupane H-3), 4.22 (d,
1 H, J1,2 6.6 Hz, H-1), 4.09 (dd, 1H, J5,4 4.6, J5,5′ 11.6 Hz, H-5′), 4.05 (br
s, 1 H, H-4), 3.99 (dd, 1 H, J5,4 3.4, J5,5′ 12.4 Hz, H-5), 3.81–3.81 (s, 4
H, H-4′, OCH3), 3.71–3.74 (m, 2 H, H-3, H-3′), 3.54 (d, 1 H, J 9.2 Hz,
lupane H-28), 3.48 (d, 1 H, J5,5′ 12.4 Hz, H-5), 3.34–3.37 (m, 2 H, H-5′,
lupane H-28), 2.29–2.36 (m, 1 H, lupane H-19), 2.04 (s, 3 H, CH3),
1.87–1.94 (m, 1 H), 1.80–1.84 (m, 1 H), 1.64 (s, 3 H, CH3), 1.62 (s, 3
H, CH3), 0.97 (s, 3 H, CH3), 0.91 (s, 3 H, CH3), 0.84 (s, 3 H, CH3), 0.83
(s, 6 H, 2 × CH3), 0.88–1.66 (m, 24 H, lupane protons), 0.76–0.78 (m,
1 H, lupane H-5). 13C NMR (150 MHz, CDCl3) δ: 171.0, 169.5, 165.9,
163.7 (Ar-OMe), 150.5 (lupane C-20), 132.1 (Ar), 121.6 (Ar), 113.6
(Ar), 109.5 (lupane C-29), 102.0 (C-1′), 101.4 (C-1), 80.9 (lupane C-3),
80.1 (C-3), 74.5 (C-3′), 73.7 (C-2′), 70.5 (C-2), 69.6 (C-4′), 67.7 (lupane
C-28), 67.3 (C-4), 64.9 (C-5′), 64.3 (C-5), 55.4 (OCH3), 55.3 (lupane
C-5), 50.2, 48.6, 47.8, 46.9, 42.6, 40.8, 38.3, 37.7, 37.5, 37.0, 34.6, 34.1,
29.6, 29.3, 27.9, 26.9, 25.0, 23.6, 21.3, 20.8, 20.3, 19.0, 18.1, 16.5, 16.1,
16.0, 14.8. Anal. Calcd for C52H76O14 (925.18): C, 67.51; H, 8.28. Found:
C, 67.33; H, 8.43. HR-MS (ESI) calc. for C52H76NaO14 [M + Na]+:
947.5133. Found: 947.5121.
Data for 46. Yield 90%. [α]D20 −24.0 (c 0.3, chloroform). 1H NMR
(600 MHz, CDCl3) δ: 7.97–7.99 (m, 2 H, Ar), 6.91–6.93 (m, 2 H, Ar),
5.00–5.04 (m, 2 H, H-2, H-3), 4.93 (dd, 1 H, J2,1 6.3, J2,3 7.9 Hz, H-2′),
4.72 (d, 1 H, J1,2 6.3 Hz, H-1′), 4.65–4.66 (m, 1 H, lupane H-29), 4.57
(br s, 1 H, lupane H-29), 4.46 (dd, 1 H, J 5.4, 10.9 Hz, lupane H-3),
4.43 (d, 1 H, J1,2 3.9 Hz, H-1), 4.09–4.12 (m, 2 H, H-4, H-5′), 4.02 (dd,
1 H, J5,4 6.9, J5,5′ 11.7 Hz, H-5), 3.86 (s, 3 H, OCH3), 3.78–3.82 (m, 1
H, H-4′), 3.73 (t, 1 H, J3,2 = J3,4 = 7.9 Hz, H-3′), 3.61 (dd, 1 H, J5,4 3.2,
J5,5′ 11.7 Hz, H-5), 3.52 (d, 1 H, J 9.2 Hz, lupane H-28), 3.38–3.41 (m,
2 H, H-5′, lupane H-28), 2.34–2.39 (m, 1 H, lupane H-19), 2.06 (s,
3 H, CH3), 2.04 (s, 3 H, CH3), 1.83–1.98 (m, 3 H), 1.78 (s, 3 H, CH3),
1.67 (s, 3 H, CH3), 1.00 (s, 3 H, CH3), 0.94 (s, 3 H, CH3), 0.84 (s, 6 H,
2 × CH3), 0.83 (s, 3 H, CH3), 0.97–1.63 (m, 22 H, lupane protons), 0.77–
0.79 (m, 1 H, lupane H-5). 13C NMR (150 MHz, CDCl3) δ: 171.0, 170.3,
169.1, 166.2, 163.8 (Ar-OMe), 150.4 (lupane C-20), 132.0 (Ar), 121.6
Data for 44. [α]D20 −13.5 (c 0.3, chloroform). 1H NMR (600 MHz,
CDCl3) δ: 7.96–7.98 (m, 2 H, Ar), 6.89–6.91 (m, 2 H, Ar), 5.00 (dd, 1
H, J2,1 6.2, J2,3 7.8 Hz, H-2′), 4.89 (dd, 1 H, J2,1 5.2, J2,3 7.3 Hz, H-2),
4.82 (d, 1 H, J1,2 6.2 Hz, H-1′), 4.64–4.65 (m, 1 H, lupane H-29),
4.56 (br s, 1 H, lupane H-29), 4.46 (dd, 1 H, J 5.3, 11.0 Hz, lupane
H-3), 4.36 (d, 1 H, J1,2 5.2 Hz, H-1), 4.13 (dd, 1H, J5,4 4.5, J5,5′ 11.7 Hz,
H-5′), 4.02 (dd, 1 H, J5,4 5.3, J5,5′ 12.2 Hz, H-5), 3.93–3.95 (m, 1 H,
H-4), 3.85 (s, 3 H, OCH3), 3.78–3.83 (m, 1 H, H-4′), 3.74–3.77 (m, 2
H, H-3, H-3′), 3.55–3.57 (m, 2 H, H-5, lupane H-28), 3.39–3.43 (m,
2 H, H-5′, lupane H-28), 2.31–2.37 (m, 1 H, lupane H-19), 2.04 (s,
3 H, CH3), 2.04 (s, 3 H, CH3), 1.79–1.93 (m, 2 H), 1.66 (s, 3 H, CH3),
1.00 (s, 3 H, CH3), 0.95 (s, 3 H, CH3), 0.84 (s, 6 H, 2 × CH3), 0.83 (s, 3
H, CH3), 0.97–1.63 (m, 25 H, lupane protons), 0.77–0.79 (m, 1 H,
lupane H-5). 13C NMR (150 MHz, CDCl3) δ: 170.1, 170.0, 166.4, 163.8
(Ar-OMe), 150.3 (lupane C-20), 132.0 (Ar), 121.4 (Ar), 113.8 (Ar),
1
(Ar), 113.8 (Ar), 109.6 (lupane C-29), 100.5 (C-1′, JC1,H1 164.0 Hz),
1
99.5 (C-1, JC1,H1 164.2 Hz), 80.9 (lupane C-3), 74.2 (C-3′), 73.5 (C-
2′), 71.6 (C-4), 69.9 (C-4′), 69.2 (C-2, C-3), 67.3 (lupane C-28), 64.3
(C-5′), 61.3 (C-5), 55.4 (OCH3), 55.3 (C-5), 50.2, 48.6, 47.8, 46.9 (C),
42.6 (C), 40.8 (C), 38.3 (CH2), 37.8 (C), 37.5, 37.0 (C), 34.6 (CH2), 34.1
(CH2), 29.7 (CH2), 29.4 (CH2), 27.9, 27.0 (CH2), 25.1 (CH2), 23.7 (CH2),
21.3, 20.8, 20.8 (CH2), 20.4, 19.1, 18.1 (CH2), 16.5, 16.1, 16.0, 14.7. Anal.
Calcd for C54H78O15 × H2O (985.23): C, 65.83; H, 8.18. Found: C, 65.94;
H, 8.07. HR-MS (ESI) calc. for C54H78NaO15 [M + Na]+: 989.5238. Found:
989.5255.
1
1
109.7 (lupane C-29), 101.3 (C-1′, JC1,H1 162.5 Hz), 100.5 (C-1, JC1,H1
163.0 Hz), 80.9 (lupane C-3), 75.3 (C-4), 74.1 (C-3), 73.8 (C-2′), 72.0
(C-2), 69.7 (C-4′), 69.6 (C-3′), 68.0 (lupane C-28), 64.4 (C-5′), 62.0
(C-5), 55.4 (OCH3), 55.3 (lupane C-5), 50.2, 48.6, 47.8, 46.9 (C), 42.6
(C), 40.8 (C), 38.3 (CH2), 37.8 (C), 37.5, 37.0 (C), 34.7 (CH2), 34.1

66
K. Kuczynska et al./Carbohydrate Research 423 (2016) 49–69
4.10.3. 3β-O-Allyl-28-O-[2-O-(4-methoxybenzoyl)-β-d-
xylopyranosyl-(1→3)-2-O-acetyl-α-l-arabinopyranosyl]
betulin (49)
Method A. Starting from mixture of 47/48, saponins 50 (in approx.
ratio 1:1, 36%) and 49 (30%) were obtained. It is likely that 50 com-
prised a mixture of (1→4)-linked disaccharides acetylated at O-2
or O-3 positions of the arabinopyranoside ring.
2 H, Ar), 4.99 (dd, 1 H, J2,1 5.9, J2,3 7.5 Hz, H-2′), 4.90 (dd, 1 H, J2,1 5.2,
J2,3 7.4 Hz, H-2), 4.84 (d, 1 H, J1,2 5.9 Hz, H-1′), 4.59–4.60 (m, 1 H,
lupane H-29), 4.52 (br s, 1 H, lupane H-29), 4.46 (d, 1 H, J1,2 5.2 Hz,
H-1), 4.14 (dd, 1H, J5,4 4.3, J5,5′ 11.8 Hz, H-5′), 4.04 (dd, 1 H, J5,4 5.6,
J5,5′ 12.3 Hz, H-5), 3.92–3.94 (m, 1 H, H-4), 3.85 (s, 3 H, OCH3), 3.79–
3.83 (m, 1 H, H-4′), 3.74–3.77 (m, 2 H, H-3, H-3′), 3.68 (d, 1 H, J
10.0 Hz, lupane H-28), 3.53 (dd, 1 H, J5,4 2.7, J5,5′ 12.3 Hz, H-5), 3.42
(dd, 1H, J5,4 7.9, J5,5′ 11.8 Hz, H-5′), 3.32 (d, 1 H, J 10.0 Hz, lupane H-28),
3.01 (dd, 1 H, J 4.6, 11.7 Hz, lupane H-3), 2.23–2.28 (m, 1 H, lupane
H-19), 2.11–2.14 (m, 2 H), 2.05 (s, 3 H, CH3), 1.72–1.87 (m, 2 H), 1.64
(s, 3 H, CH3), 1.06 (m, 9 H, tBu), 0.90 (s, 3 H, CH3), 0.90 (s, 3 H, CH3),
0.73 (s, 3 H, CH3), 0.70 (s, 3 H, CH3), 0.68 (s, 3 H, CH3), 0.75–1.67
(m, 22 H, lupane protons), 0.60–0.62 (m, 1 H, lupane H-5). 13C NMR
(150 MHz, CDCl3) δ: 170.1, 166.4, 163.9 (Ar-OMe), 150.8 (lupane
Data for 49. [α]D20 0.1 (c 0.3, chloroform). νmax (film): 3445, 2941,
2869, 1721, 1605, 1257, 1171, 1131, 1087, 1071, 1034, 757 cm−1. 1H
NMR (600 MHz, CDCl3): δ:7.96–7.97 (m, 2 H, Ar), 6.89–6.91 (m, 2
H, Ar), 5.89–5.96 (m, 1 H, allyl =CH), 5.24–5.27 (m, 1 H, allyl =CH2),
5.11–5.13 (m, 1 H, allyl =CH2), 5.06 (dd, 1 H, J2,1 6.0, J2,3 7.8 Hz, H-2),
4.97 (dd, 1 H, J2,1 6.6, J2,3 8.2 Hz, H-2′), 4.71 (d, 1 H, J1,2 6.6 Hz, H-1′),
4.63 (br s, 1 H, lupane H-29), 4.54 (br s, 1 H, lupane H-29), 4.26 (d,
1 H, J1,2 6.0 Hz, H-1), 4.10–4.14 (m, 2 H), 4.00 (br s, 1 H), 3.96 (dd, 1
H, J 4.4, 12.2 Hz), 3.86–3.89 (m, 1 H, allyl CH2O), 3.82–3.85 (m, 4
H), 3.78 (dd, 1 H, J 3.2, 8.0 Hz), 3.73 (t, 1 H, J 8.2 Hz), 3.49–3.53 (m,
2 H), 3.38 (dd, 1 H, J 9.1, 11.6 Hz), 3.33 (d, 1 H, J 9.2 Hz), 2.79 (dd, 1
H, J 4.2, 11.7 Hz, lupane H-3), 2.30–2.35 (m, 1 H, lupane H-19), 1.87–
1.94 (m, 1 H), 1.81–1.84 (m, 1 H), 1.71 (m, 3 H, CH3), 1.64 (s, 3 H,
CH3), 0.96 (s, 3 H, CH3), 0.95 (s, 3 H, CH3), 0.90 (m, 3 H, CH3), 0.81
(m, 3 H, CH3), 0.78 (s, 3 H, CH3), 0.82–1.67 (m, 24 H, lupane protons),
0.65–0.67 (m, 1 H, lupane H-5). 13C NMR (150 MHz, CDCl3):169.5,
166.1, 163.9, 150.6 (lupane C-20), 135.9, 132.2, 121.5, 115.9 (CH2),
113.7, 109.5 (lupane C-29), 101.8, 101.0 (C-1), 86.3 (lupane C-3), 80.0,
74.6, 73.9, 70.6 (CH2), 70.5, 69.8, 67.5 (CH2), 66.9, 64.7 (CH2), 63.7
(CH2), 55.8, 55.5, 50.4, 48.6, 47.9, 46.9 (C), 42.6 (C), 40.9 (C), 38.8
(C), 38.6 (CH2), 37.6, 37.1 (C), 34.6 (CH2), 34.2 (CH2), 29.7 (CH2), 29.3
(CH2), 28.1, 27.0 (CH2), 25.2 (CH2), 23.1 (CH2), 20.8 (CH2), 20.5, 19.1,
18.2 (CH2), 16.3, 16.1, 16.0, 14.8. Anal. Calcd for C53H78O13 (923.21):
C, 68.25; H, 8.50. Found: C, 68.13; H, 8.44.
1
C-20), 113.8–135.7 (Ar), 109.4 (lupane C-29), 101.8 (C-1, JC1,H1
1
165.0 Hz), 101.2 (C-1′, JC1,H1 165.6 Hz), 90.2 (lupane C-3), 75.4 (C-
4), 73.9 (C-3′), 73.7 (C-2′), 72.2 (C-2), 69.8 (C-3), 69.7 (C-4′), 64.1
(C-5′), 62.0 (C-5), 61.1 (lupane C-28), 55.5 (lupane C-5), 55.4 (OCH3),
50.3, 48.5, 48.4, 47.8, 42.6, 40.8, 39.0, 38.6, 37.2, 36.8, 34.5, 34.1, 29.9,
29.5, 27.8, 27.0, 26.9 (tBu), 25.8, 25.2, 21.0, 20.7, 19.4, 19.1, 18.2, 16.2,
16.0, 15.7, 14.7. Anal. Calcd for C66H92O13Si × 1½ H2O (1148.57): C,
69.02; H, 8.34. Found: C, 69.05; H, 8.35. HR-MS (ESI) calc. for
C66H96NO13Si [M + NH4]+: 1138.6651. Found: 1138.6658.
4.10.5. 3β-O-[2-O-(4-Methoxybenzoyl)-β-d-xylopyranosyl-
(1→3)-2-O-acetyl-α-l-arabinopyranosyl] lupeol (61) and 3β-O-
[2-O-(4-methoxybenzoyl)-β-d-xylopyranosyl-(1→4)-2-O-acetyl-
α-l-arabinopyranosyl] lupeol (62)
Method A. Starting from mixture of 59/60, saponins 62 (237 mg,
67%) and 61 (60 mg, 17%) were obtained.
20
Data for 61: [α]D 11.6 (c 0.1, chloroform). 1H NMR (600 MHz,
CDCl3) δ: 7.94–7.96 (m, 2 H, Ar), 6.86–6.88 (m, 2 H, Ar), 5.08 (dd, 1
H, J2,1 7.4, J2,3 9.2 Hz, H-2), 4.99 (dd, 1 H, J2,1 6.7, J2,3 8.1 Hz, H-2′), 4.68–
4.69 (m, 1 H, lupane H-29), 4.66 (d, 1 H, J1,2 6.7 Hz, H-1′), 4.56 (br
s, 1 H, lupane H-29), 4.26 (d, 1 H, J1,2 7.4 Hz, H-1), 4.05–4.08 (m, 2
H, H-4, H-5′), 3.99 (dd, 1 H, J5,4 1.9, J5,5′ 12.0 Hz, H-5), 3.80–3.83 (m,
4 H, H-4′, OCH3), 3.76 (t, 1 H, J3,2 = J3,4 = 8.1 Hz, H-3′), 3.68 (dd, 1 H,
J3,2 9.2, J3,4 3.1 Hz, H-3), 3.44 (d, 1 H, J5,5′ 12.0 Hz, H-5), 3.35 (dd, 1
H, J5,4 9.2, J 11.5 Hz, H-5′), 2.95 (dd, 1 H, J 4.5, 11.4 Hz, lupane H-3),
2.34–2.39 (m, 1 H, lupane H-19), 1.73–1.95 (m, 2 H), 1.68 (s, 3 H,
CH3), 1.57 (s, 3 H, CH3), 1.01 (s, 3 H, CH3), 0.99 (s, 3 H, CH3), 0.91 (s,
3 H, CH3), 0.78 (s, 3 H, CH3), 0.78 (s, 3 H, CH3), 0.77 (s, 3 H, CH3),
0.80–1.66 (m, 24 H, lupane protons), 0.60–0.61 (m, 1 H, lupane H-5).
13C NMR (150 MHz, CDCl3) δ: 169.5, 165.8, 163.7 (Ar-OMe), 150.9
(lupane C-20), 132.2 (Ar), 121.8 (Ar), 113.6 (Ar), 109.3 (lupane C-29),
103.3 (C-1), 101.8 (C-1′), 89.5 (lupane C-3), 80.4 (C-3), 74.1 (C-3′),
73.5 (C-2′), 70.7 (C-2), 69.4 (C-4′), 67.8 (C-4), 64.9 (C-5), 64.9 (C-
5′), 55.6, 55.4, 50.4, 48.3, 47.9, 43.0, 42.7, 40.8, 40.0, 38.9, 38.6, 38.0,
36.8, 35.6, 34.2, 29.8, 27.6, 27.4, 26.0, 25.1, 20.9, 20.4, 19.7, 19.3, 18.1,
18.0, 16.1, 16.1, 15.9, 14.5. Anal. Calcd for C50H74O12 × 1½ H2O (894.17):
C, 67.16; H, 8.68. Found: C, 67.01; H, 8.35.
4.10.4. 28-O-tert-Butyldiphenylsilyl-3β-O-[2-O-
(4-methoxybenzoyl)-β-d-xylopyranosyl-(1→3)-2-O-acetyl-α-l-
arabinopyranosyl] betulin (54) and 28-O-tert-butyldiphenylsilyl-
3β-O-[2-O-(4-methoxybenzoyl)-β-d-xylopyranosyl-(1→4)-2-O-
acetyl-α-l-arabinopyranosyl] betulin (55)
Method A. Starting from mixture of 52/53, saponins 55 (62%) and
54 (25%, slightly contaminated by unknown component) were
obtained.
20
Data for 54. [α]D −0.5 (c 0.2, chloroform). 1H NMR (600 MHz,
CDCl3) δ: 7.92–7.94 (m, 2 H, Ar), 7.36–7.69 (m, 10 H), 6.86–6.87 (m,
2 H, Ar), 5.08 (dd, 1 H, J2,1 7.2, J2,3 8.9 Hz, H-2), 4.98 (dd, 1 H, J2,1 6.4,
J2,3 7.5 Hz, H-2′), 4.66 (d, 1 H, J1,2 6.4 Hz, H-1′), 4.59 (br s, 1 H, lupane
H-29), 4.52 (br s, 1 H, lupane H-29), 4.26 (d, 1 H, J1,2 7.2 Hz, H-1),
4.08 (dd, 1H, J5,4 4.1, J5,5′ 11.4 Hz, H-5’), 4.03 (br s, 1 H, H-4), 3.97 (d,
1 H, J 12.1 Hz, lupane H-28), 3.81–3.83 (m, 4 H, H-4′, OCH3), 3.71–
3.74 (m, 1 H, H-3′), 3.65–3.68 (m, 2 H, H-3, H-5), 3.43 (d, 1 H, J
12.1 Hz, lupane H-28), 3.30–3.36 (m, 2 H, H-5, H-5′), 2.93 (dd, 1 H,
J 4.3, 11.7 Hz, lupane H-3), 2.22–2.27 (m, 1 H, lupane H-19), 2.10–
2.13 (m, 2 H), 1.70–1.86 (m, 2 H), 1.64 (s, 3 H, CH3), 1.59 (s, 3 H, CH3),
1.05 (m, 9 H, tBu), 0.89 (s, 3 H, CH3), 0.77 (s, 3 H, CH3), 0.69 (s, 3 H,
CH3), 0.67 (s, 3 H, CH3), 0.60 (s, 3 H, CH3), 0.73–1.53 (m, 22 H, lupane
protons), 0.56–0.57 (m, 1 H, lupane H-5). 13C NMR (150 MHz, CDCl3)
δ: 169.6, 165.8, 163.7 (Ar-OMe), 150.8 (lupane C-20), 113.7–135.7
20
Data for 62: [α]D −5.2 (c 0.2, chloroform). 1H NMR (600 MHz,
CDCl3) δ: 7.96–7.98 (m, 2 H, Ar), 6.88–6.90 (m, 2 H, Ar), 5.00 (dd, 1
H, J2,1 6.1, J2,3 7.5 Hz, H-2′), 4.89 (dd, 1 H, J2,1 5.4, J2,3 7.6 Hz, H-2), 4.81
(d, 1 H, J1,2 6.1 Hz, H-1′), 4.68–4.69 (m, 1 H, lupane H-29), 4.57 (br
s, 1 H, lupane H-29), 4.44 (d, 1 H, J1,2 5.4 Hz, H-1), 4.12 (dd, 1 H, J5,4
4.4, J 11.7 Hz, H-5′), 4.06 (dd, 1 H, J5,4 5.4, J5,5′ 12.4 Hz, H-5), 3.80–
3.84 (m, 4 H, H-4′, OCH3), 3.73–3.77 (m, 2 H, H-3, H-3′), 3.52 (dd,
1 H, J5,4 2.6, J5,5′ 12.4 Hz, H-5), 3.40 (dd, 1 H, J5,4 8.1, J5,5′ 11.7 Hz, H-5′),
3.02 (dd, 1 H, J 4.6, 11.7 Hz, lupane H-3), 2.35–2.40 (m, 1 H, lupane
H-19), 2.04 (s, 3 H, CH3), 1.88–1.95 (m, 1 H), 1.73–1.77 (m, 1 H), 1.68
(s, 3 H, CH3), 1.01 (s, 3 H, CH3), 0.92 (s, 3 H, CH3), 0.89 (s, 3 H, CH3),
0.80 (s, 3 H, CH3), 0.78 (s, 3 H, CH3), 0.70 (s, 3 H, CH3), 0.83–1.67
(m, 25H, lupane protons), 0.63–0.65 (m, 1 H, lupane H-5). 13C NMR
(150 MHz, CDCl3) δ: 170.1, 166.3, 163.8 (Ar-OMe), 150.9 (lupane
C-20), 132.2 (Ar), 121.5 (Ar), 113.8 (Ar), 109.3 (lupane C-29), 101.9
1
(Ar), 109.4 (lupane C-29), 103.2 (C-1, JC1,H1 161.1 Hz), 101.8 (C-1′,
1JC1,H1 162.0 Hz), 89.5 (lupane C-3), 80.5 (C-3), 74.2 (C-3′), 73.6 (C-
2′), 70.7 (C-2), 69.5 (C-4′), 67.9 (C-4), 64.8 (C-5′, lupane C-28), 61.1
(C-5), 55.5 (lupane C-5), 55.4 (OCH3), 50.3, 48.5, 48.4, 47.8, 42.6, 40.8,
38.9, 38.6, 37.2, 36.8, 34.5, 34.2, 29.9, 29.5, 27.6, 27.0, 26.9 (tBu), 25.9,
25.2, 20.7, 20.4, 19.4, 19.1, 18.1, 16.1, 16.0, 15.7, 14.7. Anal. Calcd for
C66H92O13Si × 2 H2O (1157.58): C, 68.48; H, 8.36. Found: C, 68.41; H,
8.18. HR-MS (ESI) calc. for C66H92NaO13Si [M + Na]+: 1143.6205. Found:
1143.6204.
Data for 55. [α]D20 −26.7 (c 0.2, chloroform). 1H NMR (600 MHz,
CDCl3) δ: 7.97–7.98 (m, 2 H, Ar), 7.36–7.70 (m, 10 H), 6.90–6.91 (m,

K. Kuczynska et al./Carbohydrate Research 423 (2016) 49–69
67
(C-1, 1JC1,H1 157.1 Hz), 101.4 (C-1′, 1JC1,H1 163.1 Hz), 90.2 (lupane C-3),
75.7 (C-4), 73.9 (C-3′), 73.7 (C-2′), 72.3 (C-2), 69.9 (C-3), 69.6 (C-
4′), 64.3 (C-5′), 62.2 (C-5), 55.5, 55.4, 50.4, 48.3, 47.9, 43.0, 42.8, 40.8,
40.0, 39.0, 38.6, 38.0, 36.8, 35.6, 34.2, 29.8, 27.7, 27.4, 25.8, 25.1, 21.0,
20.9, 19.3, 18.2, 18.0, 16.2, 16.0, 15.9, 14.5. Anal. Calcd for
C50H74O12 × H2O (885.16): C, 67.85; H, 8.65. Found: C, 67.82; H, 8.73.
4 H, H-3′, OCH3), 3.55–3.59 (m, 2 H, H-2′, H-5), 3.38 (dd, 1 H, J5,4
9.0, J5,5′ 11.7 Hz, H-5′), 3.04 (dd, 1 H, J 4.7, 11.6 Hz, lupane H-3), 2.35–
2.40 (m, 1 H, lupane H-19), 2.10 (s, 3 H, CH3), 2.08 (s, 3 H, CH3), 1.88–
1.95 (m, 1 H), 1.79–1.82 (m, 1 H), 1.68 (s, 3 H, CH3), 1.02 (s, 3 H, CH3),
0.93 (s, 3 H, CH3), 0.91 (s, 3 H, CH3), 0.83 (s, 3 H, CH3), 0.79 (s, 3 H,
CH3), 0.75 (s, 3 H, CH3), 0.74–1.73 (m, 23 H, lupane protons), 0.66–
0.68 (m, 1 H, lupane H-5). 13C NMR (150 MHz, CDCl3) δ: 170.9, 169.4,
165.7, 163.7, 150.9 (lupane C-20), 131.8 (Ar), 121.7 (Ar), 113.7 (Ar),
109.3 (lupane C-29), 104.5 (C-1′), 101.6 (C-1), 89.8 (lupane C-3), 74.2
(C-4), 73.1 (C-2, C-3′), 71.4 (C-4′), 70.8 (C-3), 69.9 (C-2), 62.8 (C-5),
62.6 (C-5′), 55.6, 55.4, 50.4, 48.3, 47.9, 43.0, 42.8, 40.8, 40.0, 39.0,
38.6, 38.0, 36.9, 35.6, 34.3, 29.8, 27.8, 27.4, 25.8, 25.1, 20.9, 20.9, 19.3,
18.2, 18.0, 16.2, 16.1, 15.9, 14.5. HR-MS (ESI) calc. for C52H76NaO13
[M + Na]+: 931.5184. Found: 931.5175.
4.10.6. 3β-O-[2-O-(4-Methoxybenzoyl)-β-d-xylopyranosyl-(1→3)
-2,4-di-O-acetyl-α-l-arabinopyranosyl] lupeol (65), 3β-O-[2-O-(4-
methoxybenzoyl)-β-d-xylopyranosyl-(1→4)-2,3-di-O-acetyl-α-l-
arabinopyranosyl] lupeol (66) and 3β-O-[4-O-(4-
methoxybenzoyl)-β-d-xylopyranosyl-(1→4)-2,3-di-O-acetyl-α-l-
arabinopyranosyl] lupeol (67)
Method B. Acetylation of 59/60 mixture gave inseparable 63/
64 mixture. Its desilylation afforded saponins 67 (8 mg, 8%), 66
(53 mg, 55%) and 65 (7 mg, 7%).
4.11. Deallylation—general method
20
Data for 65. [α]D 16.1 (c 0.2, chloroform). 1H NMR (600 MHz,
A
solution of hydrogen activated iridium complex
CDCl3) δ: 7.93–7.95 (m, 2 H, Ar), 6.92–6.94 (m, 2 H, Ar), 5.21–5.24
(m, 2 H, H-2, H-4), 4.95 (d, 1 H, J1,2 3.4 Hz, H-1′), 4.88 (dd, 1 H, J2,1
3.4, J2,3 4.5 Hz, H-2′), 4.68–4.69 (m, 1 H, lupane H-29), 4.56 (br s, 1
H, lupane H-29), 4.38 (d, 1 H, J1,2 7.1 Hz, H-1), 4.20 (dd, 1 H, J5,4 3.0,
J 12.3 Hz, H-5′), 4.02 (dd, 1 H, J5,4 3.1, J5,5′ 13.1 Hz, H-5), 3.90 (dd, 1
H, J3,2 9.3, J3,4 3.6 Hz, H-3), 3.87 (s, 3 H, OCH3), 3.83 (br s, 1 H, H-3′),
3.75 (br s, 1 H, H-4′), 3.52–3.55 (m, 2 H, H-5, H-5′), 3.02 (dd, 1 H, J
5.1, 11.3 Hz, lupane H-3), 2.35–2.39 (m, 1 H, lupane H-19), 2.13 (s,
3 H, CH3), 2.03 (s, 3 H, CH3), 1.88–1.94 (m, 1 H), 1.69–1.76 (m, 1 H),
1.68 (s, 3 H, CH3), 1.01 (s, 3 H, CH3), 0.92 (s, 3 H, CH3), 0.88 (s, 3 H,
CH3), 0.81 (s, 3 H, CH3), 0.78 (s, 3 H, CH3), 0.72 (s, 3 H, CH3), 0.73–
[Ir(COD)(MePPh2)2]PF6 (5 mg)10 was transferred into solution of the
corresponding allyl ether (0.10 mmol) in THF (3 mL) and stirred at
rt for 4 h. Then, methanol (2 mL) and p-TsOH (30 mg) were added;
the mixture was stirred at rt for an additional 6 h, and concen-
trated under reduced pressure. The residue was purified by
preparative TLC (eluent H) to afford the title compound.
4.11.1. 3β-28-O-[2-O-(4-Methoxybenzoyl)-β-d-xylopyranosyl-
(1→3)-2-O-acetyl-α-l-arabinopyranosyl] betulin (51)
20
Starting from 49 the title compound (40%) was obtained. [α]D
16.6 (c 0.3, chloroform). νmax (film): 3418, 2941, 2869, 1734, 1719,
1.65 (m, 23 H, lupane protons), 0.64–0.66 (m, 1 H, lupane H-5). 13
C
1605, 1371, 1256, 1171, 1100, 1073, 1046, 757 cm−1 1H NMR
.
NMR (150 MHz, CDCl3) δ: 170.9, 169.4, 165.1, 163.9 (Ar-OMe), 150.9
(lupane C-20), 131.9 (Ar), 121.3 (Ar), 113.8 (Ar), 109.3 (lupane C-29),
103.3 (C-1), 99.6 (C-1′), 90.1 (lupane C-3), 76.1 (C-3), 71.1 (C-2′), 70.8
(C-2), 70.5 (C-3′), 69.7 (C-4), 68.9 (C-4′), 63.0 (C-5), 62.1 (C-5′), 55.6,
55.5, 50.4, 48.3, 47.9, 43.0, 42.8, 40.8, 40.0, 39.0, 38.7, 38.0, 36.9, 35.6,
34.3, 29.8, 27.7, 27.4, 26.0, 25.1, 21.1, 20.9, 20.9, 19.3, 18.2, 18.0, 16.1,
16.0, 15.9, 14.7. HR-MS (ESI) calc. for C52H76NaO13 [M + Na]+: 931.5184.
Found: 931.5167.
(500 MHz, CDCl3/CD3OD): δ: 8.01–8.03 (m, 2 H, Ar), 6.97–6.98 (m,
2 H, Ar), 5.05–5.08 (m, 1 H), 4.94 (t, 1 H, J 8.1 Hz), 4.54 (br s, 1 H),
4.20 (d, 1 H, J 7.6 Hz), 4.08 (br s, 1 H), 4.00 (dd, 1 H, J 4.2, 11.5 Hz),
3.97 (br s, 1 H), 3.89 (s, 3 H, OCH3), 3.75 (dd, 1 H, J 3.0, 9.5 Hz), 3.63–
3.71 (m, 2 H), 3.59 (d, 1 H, J 9.2 Hz), 3.55 (d, 1 H, J 12.7 Hz), 3.31
(d, 1 H, J 11.5 Hz), 3.15 (dd, 1 H, J 5.8, 10.3 Hz, lupane C-3), 2.31–
2.33 (m, 1 H, lupane C-19), 1.68–1.92 (m, 4 H), 1.65 (s, 3 H, CH3),
1.53 (s, 3 H, CH3), 1.01 (s, 3 H, CH3), 0.96 (s, 3 H, CH3), 0.95 (s, 3 H,
CH3), 0.84 (s, 3 H, CH3), 0.76 (s, 3 H, CH3), 0.85–1.63 (m, 26 H, lupane
protons), 0.68–0.70 (m, 1 H, lupane C-5). HRMS (ESI): m/z calcd for
C50H74NaO13 [M + Na]+: 905.5027. Found: 905.5021.
Data for 66. [α]D20 −13.9 (c 0.2, chloroform). 1H NMR (600 MHz,
CDCl3) δ: 7.97–7.99 (m, 2 H, Ar), 6.91–6.93 (m, 2 H, Ar), 5.08 (dd, 1
H, J2,1 5.3, J2,3 7.6 Hz, H-2), 4.96 (dd, 1 H, J3,2 7.6, J3,4 3.0 Hz, H-3), 4.93
(dd, 1 H, J2,1 5.9, J2,3 7.4 Hz, H-2′), 4.73 (d, 1 H, J1,2 5.9 Hz, H-1′), 4.68–
4.69 (m, 1 H, lupane H-29), 4.57 (br s, 1 H, lupane H-29), 4.46 (d, 1
H, J1,2 5.3 Hz, H-1), 4.07–4.12 (m, 3 H, H-4, H-5, H-5′), 3.86 (s, 3 H,
OCH3), 3.76–3.80 (br s, 1 H, H-4′), 3.73–3.75 (br s, 1 H, H-3′), 3.55
(dd, 1 H, J5,4 4.6, J5,5′ 14.0 Hz, H-5), 3.39 (dd, 1 H, J5,4 7.9, J 11.9 Hz,
H-5′), 2.99 (dd, 1 H, J 4.6, 11.4 Hz, lupane H-3), 2.35–2.40 (m, 1 H,
lupane H-19), 2.04 (s, 3 H, CH3), 1.88–1.93 (m, 1 H), 1.83 (s, 3 H, CH3),
1.73–1.75 (m, 1 H), 1.68 (s, 3 H, CH3), 1.01 (s, 3 H, CH3), 0.92 (s, 3 H,
CH3), 0.87 (s, 3 H, CH3), 0.80 (s, 3 H, CH3), 0.78 (s, 3 H, CH3), 0.69 (s,
3 H, CH3), 0.82–1.67 (m, 23 H, lupane protons), 0.63–0.65 (m, 1 H,
lupane H-5). 13C NMR (150 MHz, CDCl3) δ: 170.4, 169.1, 166.2 (C = O),
163.8 (Ar-OMe), 150.9 (lupane C-20), 132.1 (Ar), 121.6 (Ar), 113.8
4.12. 3β-O-[2-O-(4-methoxybenzoyl)-β-d-xylopyranosyl-
(1→3)-2-O-acetyl-α-l-arabinopyranosyl] betulin (56)
To a solution of 54 (78 mg, 0.069 mmol) in THF (2 mL),
tetrabutylammonium fluoride (1 M in THF, 0.59 mL, 0.59 mmol) was
added followed by acetic acid (34 μL, 0.59 mmol) and the mixture
was stirred in a screw tap tube at 60 °C for 24 h. The mixture was
then concentrated and the residue was purified by preparative TLC
(hexane–ethyl acetate–methanol, 5:3:1) to afford the title com-
pound (35 mg, 57%) as a foam. [α]D20 2.5 (c 0.2, chloroform). 1H NMR
(600 MHz, CDCl3) δ: 7.93–7.95 (m, 2 H, Ar), 6.86–6.87 (m, 2 H, Ar),
5.08 (m, 1 H, H-2), 4.98 (m, 1 H, H-2′), 4.68 (br s, 1 H, lupane H-29),
4.65 (d, 1 H, J1,2 6.5 Hz, H-1′), 4.58 (br s, 1 H, lupane H-29), 4.26 (d,
1 H, J1,2 7.3 Hz, H-1), 4.04–4.08 (m, 2 H, H-4, H-5′), 3.98 (br d, 1 H,
J5,5′ 12.0 Hz, H-5), 3.78–3.84 (m, 5 H, H-4′, lupane H-28, OCH3), 3.72–
3.74 (m, 1 H, H-3′), 3.67 (br d, 1 H, J 9.4 Hz, H-3), 3.44 (br d, 1 H,
J5,5′ 12.0 Hz, H-5), 3.31–3.35 (m, 2 H, H-5′, lupane H-28), 2.95 (dd,
1 H, J 4.1, 11.3 Hz, lupane H-3), 2.35–2.39 (m, 1 H, lupane H-19), 1.83–
1.97 (m, 3 H), 1.67 (s, 3 H, CH3), 1.56 (s, 3 H, CH3), 0.98 (s, 3 H, CH3),
0.94 (s, 3 H, CH3), 0.78 (s, 3 H, CH3), 0.76 (s, 3 H, CH3), 0.61 (m, 4 H,
lupane H-5, CH3), 0.87–1.74 (m, 24 H, lupane protons). 13C NMR
(150 MHz, CDCl3) δ: 169.6, 165.8, 163.7 (Ar-OMe), 150.4 (lupane
C-20), 132.1 (Ar), 121.7 (Ar), 113.7 (Ar), 109.7 (lupane C-29), 103.3
(C-1, 1JC1,H1 159.5 Hz), 101.9 (C-1′, 1JC1,H1 163.0 Hz), 89.5 (lupane C-3),
1
(Ar), 109.3 (lupane C-29), 101.8 (C-1, JC1,H1 162.6 Hz), 100.7 (C-1′,
1JC1,H1 164.5 Hz), 89.7 (lupane C-3), 73.8 (C-3′), 73.4 (C-2′), 72.5 (C-
4), 70.3 (C-3), 69.9 (C-4′), 69.7 (C-2), 64.0 (C-5′), 62.6 (C-5), 55.5,
55.4, 50.4, 48.3, 47.9, 43.0, 42.8, 40.8, 40.0, 39.0, 38.6, 38.0, 36.8, 35.6,
34.2, 29.8, 27.7, 27.4, 25.8, 25.1, 20.9, 20.9, 20.5, 19.3, 18.2, 18.0, 16.1,
16.0, 15.9, 14.5. Anal. Calcd for C52H76O13 (909.18): C, 68.70; H, 8.43.
Found: C, 68.59; H, 8.38.
Data for 67. 1H NMR (600 MHz, CDCl3) δ: 7.97–7.99 (m, 2 H, Ar),
6.91–6.92 (m, 2 H, Ar), 5.14 (dd, 1 H, J2,1 5.5, J2,3 7.8 Hz, H-2), 5.01–
5.05 (m, 2 H, H-3, H-4′), 4.68–4.69 (m, 1 H, lupane H-29), 4.56–
4.57 (m, 1 H, lupane H-29), 4.49 (d, 1 H, J1,2 5.5 Hz, H-1), 4.38 (d, 1
H, J1,2 6.9 Hz, H-1′), 4.18 (dd, 1 H, J5,4 5.0, J 11.7 Hz, H-5′), 4.11 (dd,
1 H, J5,4 5.3, J5,5′ 11.8 Hz, H-5), 4.08–4.09 (m, 1 H, H-4), 3.83–3.87 (m,

68
K. Kuczynska et al./Carbohydrate Research 423 (2016) 49–69
80.5 (C-3), 74.2 (C-3′), 73.6 (C-2′), 70.7 (C-2), 69.5 (C-4′), 67.9 (C-
4), 64.9 (C-5, C-5′), 60.5 (lupane C-28), 55.6 (lupane C-5), 55.5 (OCH3),
50.4, 48.8, 47.8, 42.6, 40.9, 38.9, 38.7, 37.3, 36.8, 34.2, 34.0, 29.8, 29.2,
27.6, 27.0, 26.0, 25.2, 20.8, 20.4, 19.1, 18.1, 16.1, 16.1, 16.0, 15.0.HR-
MS (ESI) calc. for C50H74NaO13 [M + Na]+: 905.5027. Found: 905.5015.
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(1→4)-3-O-acetyl-α-l-arabinopyranosyl] betulin (57)
Starting from 55 (245 mg, 0.218 mmol) and using procedure de-
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Product contained minute amounts of 2-OAc isomer 58. Data for 57.
1H NMR (600 MHz, CDCl3) δ: selected signals—4.99–5.01 (m, 1 H,
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4.58–4.59 (m, 2 H, H-1′, lupane H-29), 4.26 (d, 1 H, J1,2 6.7 Hz, H-1),
4.05–4.10 (m, 1 H, H-5), 4.03 (dd, 1 H, J5,4 4.8, J5,5′ 11.9 Hz, H-5′), 4.00
(br s, 1 H, H-4), 3.73–3.79 (m, 3 H, H-2, H-3′, H-4′), 3.50 (d, 1 H,
J5,5′ 11.5 Hz, H-5), 3.19–3.22 (m, 2 H, lupane H-28), 3.07 (dd, 1 H, J
4.5, 11.7 Hz, lupane H-3), 1.72 (s, 3 H, CH3), 1.68 (s, 3 H, CH3). 13C
NMR (150 MHz, CDCl3) δ: selected signals—109.6 (lupane C-29), 105.1
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74.6 (C-3′), 74.5 (C-4), 73.7 (C-2′), 73.2 (C-3), 70.1 (C-4′), 69.5 (C-
2), 64.9 (C-5′), 64.8 (C-5), 60.4 (lupane C-28). HR-MS (ESI) calc. for
C50H74NaO13 [M + Na]+: 905.5027. Found: 905.5005.
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20
(dec). [α]D −37.0 (c 0.3, chloroform). 1H NMR (600 MHz, CDCl3/
CD3OD) δ: 4.96 (s, 1 H, H-1), 4.68 (br s, 1 H, lupane H-29), 4.58 (br
s, 1 H, lupane H-29), 4.10–4.11 (m, 1 H), 4.00 (s, 1 H), 3.97 (br s, 1
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(CH2), 25.1 (CH2), 20.8 (CH2), 19.0, 18.2 (CH2), 16.0, 15.9, 15.3, 14.7.
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Appendix: Supplementary material
Supplementary data to this article can be found online at
doi:10.1016/j.carres.2016.01.010.

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