Total synthesis and structural revision of rebaudioside S, a steviol glycoside

Article abstract of DOI:10.1021/acs.joc.0c00442

The total synthesis of rebaudioside S, a minor steviol glycoside from the leaves of Stevia rebaudiana, was investigated via a modular strategy, culminating not only in the first and highly efficient synthesis of Reb-S and analogues thereof but also in the revision of the originally proposed structure. The modular strategy dictated the application of C2-branched disaccharide Yu donors to forge C-13 steviol glycosidic linkages, posing considerable challenges in stereoselectivity control. Through systematic investigations, the effect of the internal glycosidic linkage configuration on the glycosylation stereoselectivity of 1,2-linked disaccharide donors was disclosed, and the intensified solvent effect by the 4,6-O-benzylidene protecting group was also observed with glucosyl donors. Through the orchestrated application of these favorable effects, the stereoselectivity problems were exquisitely tackled.

Full text of DOI:10.1021/acs.joc.0c00442

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Article
Total Synthesis and Structural Revision
of Rebaudioside S, A Steviol Glycoside
Guo-En Wen, Hui Liu, Qi-Shuang Yin, Jin-Xi Liao, Yuan-Hong Tu, Qing-Ju Zhang, and Jian-Song Sun
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.0c00442 • Publication Date (Web): 12 Apr 2020
Downloaded from pubs.acs.org on April 16, 2020
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Total Synthesis and Structural Revision of Rebaudioside S, A Steviol
Glycoside
Guo-En Wen, Hui Liu, Qi-Shuang Yin, Jin-Xi Liao, Yuan-Hong Tu, Qing-Ju Zhang,*
and Jian-Song Sun*
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National Research Centre for Carbohydrate Synthesis, Jiangxi Normal University, 99
Ziyang Avenue, Nanchang 330022, China.
zhangq@jxnu.edu.cn; jssun@jxnu.edu.cn
Table of content
HO
OH
HO
OH
OH
O
OH
OH
O
O
O
HO
HO
OH
OH
HO
HO
O
O
O
1
OH
3
O
1
3
OH
H
OH
O
H
HO
HO
O
1
HO
HO
O
H
O
1
9
H
O
9
O
O
O
O
HO
O
HO
HO
OH Rebaudioside S
originally proposed)
Rebaudioside S
HO
(
OH
(revised)
Abstract
The total synthesis of rebaudioside S, a minor steviol glycoside from the leaves of
Stevia rebaudiana was investigated via a modular strategy, culminating not only in
the first and highly efficient synthesis of Reb-S and analogues thereof but also in the
revision of the originally proposed structure. The modular strategy dictated the
application of C2-branched disaccharide Yu donors to forge C-13 steviol glycosidic
linkages, posing considerable challenging in stereoselectivity control. Through
systematic investigations the effect of the internal glycosidic linkage configuration on
the glycosylation stereoselectivity of 1,2-linked disaccharide donors was disclosed
and the intensified solvent effect by the 4,6-O-benzylidene protecting group was also
observed with glucosyl donors. Orchestrated application these favorable effects, the
stereoselectivity problems were tackled exquisitely.
Introduction
The acceptance of the statement that steviol glycosides (rebaudiosides, Figure 1)
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are ‘Generally Recognized As Safe’ (GRAS) by the FDA in 2010 boosts the
1
development of steviol glycoside industry rapidly in the past decade. The natural
2
origin, high sweetness, as well as reliably safety renders steviol glycosides as the
3
most popular sweeteners all over the world. Furthermore, the beneficial effects of
0
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steviol glycosides on human health were also disclosed recently, thus the
pharmaceutical use of rebaudiosides with high added-value is also looming. Along
with these factors beneficial to the development of stevioside industry, impediments
retarding the advance also exist, among which the unpleasant after-taste bitter of
available rebaudioside sweeteners as well as the difficulties associated with precise
quality-control of feedstock for stevioside sweeteners are prominent. The tackling of
these bottle-necks and the searching for ideal lead compounds for stevioside-drug
development are highly dependent on the easy access of steviosides and analogues
thereof with definite chemical structures, which heavily relies on conventional
phytochemical isolation from the crude extracts of S. rebaudiana at present.5
However, the microheterogeneities of naturally occurring steviosides make the
separation of individual steviosides from natural sources a formidable task, especially
6
for members with extremely low natural content. With easily accessible steviosides
as starting materials, the enzymatic synthesis has also been tried, but the tedious
purification process could not be avoided because of the complex product mixtures.7
Chemical synthesis provides a reliable means for steviosides supply; nevertheless, this
approach has been being seriously under-developed due to the lack of efficient
strategies for steviosides synthesis.8
Our recent studies on chemical synthesis of steviosides have led to the
establishment of efficient strategies for syntheses of different type of steviol
glycosides,9 which encourages us to investigate the chemical synthesis of
rebaudioside S (Figure 1), a minor steviol glycoside isolated from the leaves of S.
1
0
rebaudiana in 2016. The studies finally culminate in not only the first total synthesis
of Reb-S via a modular strategy but also the revision of the originally proposed
chemical structure.
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HO
OHOH
OH
OH
OH
HO
O
O
HO
HO
OH
O
O
OH
O
O
HO
HO
OH
OH
O
O
O
13
OH
O
HO
13
OH
OH
H
O
HO
HO
O
O
19
1
1
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HO
H
O
R2O
O
19
OR1
R1 = R2 = H, Rebaudioside A
O
O
O
HO
Rebaudioside S
OH (originally proposed)
HO
R1 = -D-glucopyranosyl, R2 = H, Rebaudioside D
R1 = R2 = -D-glucopyranosyl, Rebaudioside M
Figure 1. The chemical structures of rebaudiosides.
Results and discussion
The structural characters of the proposed Reb-S, especially the 1,2-cis-glucosidic
linkage in C-13 sugar chain, promoted us to adopt a modular synthetic strategy, and
the target molecule could be assembled by building blocks disaccharide Yu donor 2
9
(
or 2’), steviol aglycon 3, and disaccharide bromide 4 (Figure 2). In the forward
1
1
sense, the C-13 sugar chain could be installed to the acid-sensitive steviol core with
1
2
glycosyl o-alkynylbenzoate 2 or 2’ via Yu glycosylation, wherein the challenging
1,2-cis-glycosidic linkage would be selectively forged through the long-distance
1
3
participation (LDP) effect of C-6 located acyl group of the distal glucosyl donor
while the pivotal C-13 glycosidic linkage was conceived to be constructed
β-selectively either by the nitrile solvent effect14 or by the C2-branched donor.15
9
Analogous to the syntheses of Reb-A, D, M, and S congeners, the introduction of
C-19 sugar residues was envisioned to be achieved via phase-transfer catalysis (PTC)
protocol with disaccharide bromide 4 as donor, which could not only ensure the
1
,2-trans-selectivity of the C-19 ester-type glycosidic linkage but also guarantee the
modularity of the synthesis.
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BzO
BzO
RO
O
O
HO
OH
O
O
BnO
OH
BnO
BnO
BzO
O
O
OH
O
O
HO
HO
OH
2 R = H (or 2' R = Bz)
O
1
+
OH
13
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0
OH
H
H
O
HO
HO
O
1
TBDPSO
H
H
9
19
O
3
O
O
O
+
HO
OAc
O
Rebaudioside S (1)
OH (originally proposed)
HO
AcO
AcO
O
Br
O
BzO
BzO
OBz
4
Figure 2. Retrosynthetic analysis of the proposed structure of rebaudioside S.
The synthetic investigation commenced with the syntheses of donors 2, 2’, and 4
(
Scheme 1). Under the combined effect of NIS/TMSOTf, the coupling between 516
and 617 proceeded smoothly affording disaccharide 7 as a mixture of α/β
stereoisomers with the desired α-isomer predominating remarkably due to the
favorable LDP effect of the C-6 benzoyl (Bz) group in 5 (90%, α/β = 7 : 1, J = 3.6 Hz
for the α-isomer). Unifying the protecting groups (PGs) of 5 to benzoyl group was
conducted via a sequence of benzyl and benzylidene groups removal under
hydrogenolysis conditions and benzoylation under conventional conditions (BzCl,
o
pyridine, 0 C to rt). Surprisingly, the desired fully benzoylated 8’ was isolated only
as a minor product with the partially blocked disaccharide 8 bearing a free C3-OH as
the major product. The reluctance of C3-OH of 8 to further benzoylation reveals that
the reactivity of C3-OH is rather low, presumably due to the bulky and
electron-withdrawing property of the α-linked vicinal perbenzoylated glucosyl
1
8
substituent. Considering that the inert C3-OH may not interfere with the following
reactions, both 8 and 8’ were advanced to o-alkynylbenzoate donors 2 and 2’
separately (62% and 42% yields, respectively) without the overacylation byproduct
being observed for donor 2.
The synthesis of 4 entailed the glycosylation of 1019 with rhamnosyl
2
0
21
trichloroacetimidate donor 9 under Schmidt glycosylation conditions to provide
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disaccharide 11 (90%), which was then successively subjected to deallylation,
acetylation, and bromination conditions to deliver 4 fluently (40%, for 3 steps).
Ph
O
OBz
O
O
BnO
O
Ph
O
O
BnO
OMP
1) H , Pd/C, EA/MeOH, rt
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
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0
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9
0
BnO
BnO
O
NIS, TMSOTf
O
2
STol
+
OMP
BnO
2) BzCl, pyridine, 0 o
C
BnO
BnO
BzO
OBn
OH
4A MS, CH2Cl2
-30 oC, 90%
5
6
O
to rt
7
(/ = 7 : 1)
BzO
BzO
BzO
BzO
RO
O
O
O
OMP
OABz
) CAN, CH3CN, H2O, 0 o
RO
1
C
O
BzO
BzO
BzO
BzO
BzO
2) ABzOH, EDCI, DMAP
BzO
BzO
BzO
i
O
Pr NEt, CH Cl
2
2
2
O
8
8
R = H, 71% (2 steps)
' R = Bz, 18% (2 steps)
2 R = H (62%, 2 steps)
2' R = Bz (42%, 2 steps)
OTCAI
O
OAc
O
OAc
O
BzO
BzO
AcO
AcO
AcO
OAll 1) PdCl2, CH2Cl2/MeOH
2
AcO
OBz TMSOTf, 4A MS
2) Ac O, pyridine
9
O
O
+ OAc
Br
CH2Cl2, 0 oC to rt
BzO
BzO
3) HBr/HOAc, CH2Cl2
0 oC to rt
O
O
BzO
BzO
O
90%
AcO
AcO
OAll
OBz
1
40% (3 steps)
OBz
4
10 OH
1
Scheme 1. Synthesis of donors 2, 2’, and 4.
With all donors in hand, now the stage was set for the assembly of the proposed
structure of Reb-S (Scheme 2). The major 2 was chosen as donor to condense with 3
under the effect of Au(I) complex. In order to take advantage of the solvent effect to
enhance the β-stereoselectivity of the Yu glycosylation reaction, acetonitrile was
selected as solvent. Although the reaction proceeded sluggishly under the effect of
catalytic amount of Ph PAuNTf (0.2 eq), stoichiometric amount of Au(I) complex
3
2
promoted the reaction to reach completion with reasonable rate as well as excellent
2
2
yield and stereoselectivity (91%, only β-isomer was isolated), and no detrimental
effect of the free C3-OH was detected. The subsequent cleavage of silyl ester was
effected under buffered conditions, delivering the carboxylic acid acceptor 13
efficiently (90%), which is ready for C-19 sugar chain incorporation under PTC
conditions. Under the previously optimized conditions (K CO , TBAB, CHCl /H O,
2
3
3
2
o
23
4
0 C), all 13 was consumed leading to three new compounds, among which the
desired 14 was isolated in 41% yield, accompanied by C4-O-benzoyl group migrated
and cleaved byproducts (14a and 14b) elicited by the free OH in C-13 sugar residue.
Global saponification of all ester-type PGs of 14 gave the proposed Reb-S (1), yet
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with compromised overall efficiency because of the undesired intervention of the free
OH of 13 (36%, 2 steps). Nevertheless, the low overall yield was remedied by putting
the PTC glycosylation products mixture to the final deprotection step all together,
providing the proposed Reb-S (1) with 71% yield (2 steps). The comparison of the
spectroscopic data of the synthetic sample with those reported in literature was
subsequently made, and evident discrepancy was observed especially for the 13C
NMR signals corresponding to the four anomeric sugar carbons, which were reported
to have the chemical shifts of 94.1, 98.5, 102.0, and 107.0 ppm while being proved to
be 93.9, 99.2, 101.8, and 99.9 ppm for the corresponding carbons of the synthetic
sample.
0
1
2
3
4
5
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7
8
9
0
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BzO
BzO
HO
O
BzO
BzO
HO
OH
13
BzO
BzO
BzO
O
OBzO
O
OABz
O
TBAF, HOAc
THF, rt, 90%
Ph3PAuNTf2, 4A MS
O
BzO
+
H
CH3CN, -40 oC to rt
91%
BzO
BzO
BzO
TBDPSO
H
H
O
H
19
2
3
TBDPSO
O
1
2
O
1
BzO
R O
2
BzO
BzO
R O
O
HO
BzO
BzO
BzO
O
OBz
O
O
BzO
BzO
BzO
OBzO
O
4, K CO , TBAB
2
3
O
NaOMe
Proposed Reb S (1)
O
CHCl3/H2O, 40 o
MeOH, rt
89%
C
OAc
H
O
AcO
AcO
H
O
H
HO
O
H
O
14 R1 = Bz, R2 = H (41%)
13
O
NaOMe, MeOH, rt
71% (for 2 steps)
O
14a R1 = H, R2 = Bz (24%)
BzO
BzO
14b R1 = R2 = H (17%)
OBz
Scheme 2. Synthesis of the proposed structure of Reb S (1).
The evident downfield shift of the anomeric carbons of the C-13 branch glucosyl
moiety (107.0 ppm) in authentic Reb-S promote us to conjecture that the
1,2-cis-glucosidic linkage was incorrectly assigned. In addition, careful scrutiny of the
original spectra provided in the supporting information of the isolation literature also
laid suspicion on the linkage position between the two glucosyl moieties because of
1
0
the severe coalescence of proton signals between 4.50-3.50 ppm. In combination
with the prevailing C-13 glycoform of 2,3-dibranched triglucoside such as in Reb-A,
D, and M, the authentic structure of Reb-S was tentatively proposed to 24 with a
1
,3-β-linked C-13 disaccharide moiety (Scheme 3).
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Similar to the synthesis of 1, a modular strategy was also applied to assemble 24
(
Scheme 3). Thus, the synthesis commenced with the preparation of 1,3-linked
2
4
disaccharide Yu donor 20. Benzoylation of 15 was followed by palladium-mediated
deallylation to afford 17 via 16 (60%, for 2 steps). Acceptor 17 was then glycosylated
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
5
with Schmidt donor 18 to furnish disaccharide 19, which was then subjected to
anomeric MP group removal and o-alkynylbenzoate installation conditions
sequentially to provide disaccharide alkynylbenzoate donor 20 (60%, for 2 steps).
With the assistance of the anchimeric effect of C2-OBz of the reducing-end sugar in
2
0, the condensation between 3 and 20 proceeded stereoselectively in
dichloromethane to afford 21 exclusively under the promotion of Au(I) complex (84%
yield, for the anomeric proton of sugar residue directly appended to C13-OH of
steviol: 5.05 ppm, J = 7.8 Hz). Desilylation of 21 provided 22, which was then
exposed to the PTC glycosylation conditions in the presence of disaccharide bromide
4. The obtained fully protected tetrasaccharide glycoside 23 (86%) was then put to
1
3
deprotection process to give 24 (60%, 2 steps). Although the comparison of the C
NMR data of 24 with the reported ones revealed a narrowed chemical shift
discrepancy for the anomeric carbons (93.9, 99.3, 101.7, and 106.1 ppm,
respectively), especially for the anomeric carbon of C-13 branched glucosyl residue
(
106.1 ppm vs 107.0 ppm), the desired good accordance was still not observed.
To further corroborate the structure of 24, an alternative route to produce it from
9
known compound 25 was also conducted (Scheme 3). Similar procedures as those
applied for the synthesis of 24 from 21 were adopted to convert 25 to 24 via a
sequence of desilylation, PTC glycosylation, and deprotection (48%, for 4 steps). The
1H NMR of thus obtained 24 was proved to essentially identical to that of the
compound derived from 21 via a modular strategy, further testifying the correctness
of the structure of 24.
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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
OBz
O
NH
CCl3
BzO
BzO
O
PdCl2
CH2Cl2
MeOH
Ph
O
OBz
18
BzO
BzO
BzO
Ph
O
O
Ph
O
O
O
O
O
O
O
OMP
O
OMP
AllO
OMP
rt, 65%
HO
TMSOTf, CH2Cl2
0 oC to rt, 84%
OBz
OR
1
OBz
OBz
BzCl, pyridine
0 oC to rt, 92%
5 R = H
17
19
16 R = Bz
BzO
BzO
BzO
Ph
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
O
O
BzO
, Ph3PAuNTf2, 4A MS
O
BzO
BzO
BzO
Ph
O
O
1) CAN, CH3CN, H2O, 0 o
C
3
O
O
BzO
H
O
OABz
O
13
i
CH2Cl2, -40 oC to 0 o
C
2
) ABzOH, EDCI, Pr2NEt
OBz
OBz
2
CH2Cl2, rt, 60% (2 steps)
84%
0 (/ = 1 : 2)
RO
H
19
O
TBAF, HOAc
THF, rt, 94%
21 R = TBDPS
22 R = H
Ph
O
HO
HO
HO
OH
BzO
BzO
BzO
OH
O
O
HO
O
O
O
O
O
OBz
BzO
HO
O
O
1) p-TsOH, DCM/MeOH, rt
4
2 3
, K CO , TBAF
CHCl3/H2O, 40 o
86%
C
2) NaOMe, MeOH, rt, 60%
(2 steps)
OAc
O
OH
H
H
H
O
O
AcO
AcO
HO
HO
O
H
O
O
O
O
O
O
24
BzO
BzO
23
HO
HO
OBz
OH
1)
p-TsOH
2) NaOMe, MeOH
rt, 71%, (2 steps)
DCM/MeOH, rt
Ph
BzO
BzO
BzO
O
BzO
O
BzO
BzO
O
O
Ph
O
O
O
O
OBz
BzO
O
4, K CO , TBAF
LevO
O
2
3
O
CHCl3/H2O, 40 oC, 82%
LevO
O
13
AcO
AcO
AcO
H
O
H
O
H
O
RO
H
O
19
O
TBAF, HOAc
THF, rt, 83%
25 R = TBDPS
26 R = H
27
O
BzO
BzO
OBz
Scheme 3. Synthesis of the proposed Reb-S (24) via two different routes.
The diminished spectroscopic divergence between 24 and the authentic Reb-S
verifies our previous assumption that 1,2-trans glucosidic linkage is contained in C-13
sugar chain. Along this direction, we then turned to 33 with a 1,2-connected
interglucosyl linkage (Scheme 4). To secure 33, the required disaccharide
alkynylbenzoate 29 was prepared from Schmidt donor 18 and acceptor 15 via
switching the anomeric substituent of 28 from MP to ABz (29, 79% for 3 steps). The
coupling between 29 and 3 proceeded stereoselectively in acetonitrile to deliver 30
efficiently (82%), albeit with stoichiometric amount of Au(I) complex as promoter.
The releasing of the C-19 carboxylic group (31, 81%) was followed by PTC-mediated
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glycosylation to install the C-19 disaccharide moiety to furnish 32 (88%).
Deprotection of 32 was eventually realized by successive deallylation,
debenzylidenation, and saponification to give 33 (77%, 3 steps). Fortunately, the
spectroscopic data of 33 were shown to be in good accordance with those of the
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
6
authentic sample, demonstrating that the true structure of Reb-S should be 33.
OBz
O
NH
CCl3
BzO
BzO
O
Ph
O
O
O
Ph
O
OBz
1
OMP
O
AllO
1) CAN, CH CN, H O, 0 oC
O
3
2
8
O
O
AllO
OMP
BzO
BzO
i
TMSOTf, CH2Cl2, -30 o
2) ABzOH, EDCI, Pr NEt
OH
5
C
2
OBz
1
BzO
CH2Cl2, rt, 83% (2 steps)
95%
28
Ph
O
O
AllO
BzO
O
O
Ph
O
O
BzO
BzO
O
O
O
OABz
AllO
3, Ph PAuNTf , 4A MS
3
2
4, TBAB, K2CO3
OBz
O
O
BzO
BzO
CH3CN, -40 oC to 0 o
CHCl3/H2O, 40 o
C
C
OBz
82%
H
88%
BzO
2
9
RO
H
Ph
O
O
TBAF/HOAc
THF, rt, 81%
30 R = TBDPS
31 R = H
O
OH
HO
AllO
BzO
BzO
BzO
O
HO HO
HO
HO
O
O
O
O
O
OBz
H
O
H
O
OH
OAc
H
OH
O
AcO
AcO
2 3 2 2
1) PdCl , CH OH/CH Cl , rt
O
O
HO
HO
2) p-TsOH, CH3OH/CH2Cl2
O
H
O
O
3
) NaOMe, MeOH, rt
77% (3 steps)
O
O
O
BzO
BzO
32
( )
Revised Reb S 33
O
HO
OBz
HO
OH
Scheme 4. Synthesis of the revised Reb-S (33).
To secure satisfactory efficiency in the synthesis of Reb-S and its analogues via the
modular strategy, the installation of C-13 disaccharide moiety with C2-branched
disaccharide alnylbenzoate donors were optimized systematically in order to achieve
high stereoselectivity (Table 1). In fact, dichloromethane was first chosen as media
for the coupling between 2 and 3 under the promotion of Au(I) catalyst. However,
although excellent yield was recorded (93%), almost no stereoselectivity was secured
(
12, α/β = 1.5 : 1, entry 1). Similarly, the glycosylation of 3 with 2’, the fully
benzoylated variant of 2, in dichloromethane afforded 12’ also nonstereoselectively
95%, α/β = 1.2 : 1, entry 2). Surprisingly, when the same reaction was conducted in
(
acetonitrile, the only detected products was N-acetyl-glucosylamine 35, derived from
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the hydrolysis of glycosyl acetonitrilium intermediate responsible for the β-eliciting
effect of the nitrile solvent (80%, α/β = 6.5 : 1, entry 3). Donor 29 with a β internal
glycosidic linkage provided 30α exclusively when dichloromethane was selected as
solvent, while affording 30β selectively with acetonitrile as reaction media (88% and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
2% yields, respectively, entries 4 and 5). The α/β ratio of 35 (6.5 : 1) reflects the
capability of the nitrile effect in stereoselective control of the forged disaccharide
glycosidic linkages, at most biasing the β isomers with a β to α ratio of 6.5 : 1. Thus,
the sole production of 12β as well as 30β with donors 2 and 29 in acetonitrile cannot
be attributed to the solvent effect exclusively. Presumably, the excellent
β-stereoselectivities are results of the cooperative influences of solvent effect and
favorable C2-OH appended bulkiness resulted from the 1,2-linked perbenzoylated
glucosyl residues, as it is well known that donors with steric demanding PGs at
2
7
C2-OH tend to give 1,2-trans glycosylation products preferentially. However, the
strikingly different stereoselectivity of 2 (α/β = 1.5 : 1) and 29 (α only) in CH Cl
2
2
implies that the anomeric configuration of the C2-O-linked glucosyl moiety may
affect the stereoselectivity of the C2-branched donors, and the α-linked
perbenzoylated glucosyl residue favors the formation of β glycosylation product more
robustly than its β-linked counterpart. To exclude the possible stereo-steering effect of
the 4,6-O-benzylidene PG in 29, donor 34 with two benzoyl groups replacing the
original 4,6-O-benzylidene PG was prepared26 and was subjected to react with
acceptor 3. The condensation between 29 and 3 in dichloromethane afforded 36α
predominately but not exclusively (90%, α/β = 15 : 1, entry 6); while only a 1 : 6 ratio
of α to β isomers was recorded when the same reaction was conducted in acetonitrile
(83%, entry 7).
The results listed in Table 1 firmly corroborate the assumption that the anomeric
configuration of C2-O-linked glucosyl residue has a profound effect on the
stereoselectivity of the C2-branched disaccharide donor, and the α-oriented internal
glycosidic linkage favors the formation of 1,2-trans-glycoside products while the
same effect of the β-counterpart can be negligible (entries 1 vs 6). The desired
β-inducing influence of the internal C2-O-α-glycosidic linkage could be ascribed to
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the close proximity of the two glucosyl residues imposed by the α anomeric
configuration. The stereoselectivity obtained in entry 7 (α/β = 1 : 6) coincides quite
1
4
well with that of entry 3 (α/β = 6.5 : 1), further unveiling the β-stereoselective
contribution made by the favorable solvent effect. The excellent α- and β-selectivity
for 29 in dichloromethane and acetonitrile, respectively, can be attributed to the
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
8
α-biased effect of 4,6-O-benzylidene group, which also guaranteed the generation of
0β in acetonitrile by promoting the formation of α-glucosyl acetonitrium species
entries 4 and 5).
3
(
Table 1. Optimization of conditions for the glycosylation of 3 with C2-branched
disaccharide o-alkynylbenzoates as donors.
BzO
BzO
Product
BzO
BzO
BzO
Donors
BzO
BzO
RO
RO
O
O
O
BzO
OABz
BzO
OBzO
BzO
O
O
BzO
O
OAll
BzO
Acceptor
OH
O
O
OR1
OR2
BzO
BzO
O
O
BzO
H
2
R = H
2' R = Bz
Conditions TBDPSO
H
+
R1O
R2O
AllO
O
12 R = H
12' R = Bz
O
OTBDPS 36 R1 = R2 = Bz
1 2
30 R , R = PhCH
O
O
OABz
BzO
BzO
BzO
O
O
OTBDPS
3
O
H
BzO
BzO
N
BzO
2
OBz
9 R1, R2 = PhCH
34 R1 = R2 = Bz
O
BzO
O
BzO
BzO
BzO
O
35
Entry
Donors
Acceptor
Conditions
Resultsa
12 93% (/ = 1.5 : 1)b
Ph3PAuNTf2 (0.4 eq), CH2Cl2, -40 to 0 o
C
1
2
3
4
5
6
7
2
3
3
3
3
3
3
3
o
o
c
2'
P
h
3
P
A
u
N
T
f
2
(
0
.
6
e
q
)
,
C
H
2
C
l
2
,
-
4
0
C
t
o
0
C
12' 95% (/ = 1.2 : 1)
b
2'
Ph3PAuNTf2 (1.0 eq), CH3CN, 0 oC to rt
35 80% (/ = 6.5 : 1)
30 88% (only  isomer)
30 82% (only )
o
o
29
29
34
34
P
h
3
P
A
u
N
T
f
2
(
0
.
4
e
q
)
,
C
H
2
C
l
2
,
-
4
0
C
t
o
0
C
Ph3PAuNTf2 (1.0 eq), CH3CN, -40 oC to rt
Ph3PAuNTf2 (0.5 eq), CH2Cl2, -40 to 0 o
b
C
36 90% (/ = 15 : 1)
36 83% (/ = 1 : 6)
Ph3PAuNTf2 (1.0 eq), CH3CN, -40 oC to rt
b
a
b
1
c
Isolated yield, α/β ratio were determined by H NMR, α/β ratio was determined by
separation of both isomers.
In summary, a modular and highly efficient route to synthesize Reb-S and
analogues thereof were established, through which the first total synthesis as well as
structural revision of Reb-S was achieved. With C2-branched disaccharide
o-alkynylbenzoates as donors, the effect of internal glycosidic linkage configuration
on the glycosylation stereoselectivity of the 1,2-linked disaccharide donors were
disclosed, and the potential of nitrile solvent effect, which was also proved intensified
by the 4,6-O-benzylidene PG of the glucosyl donor, in β-stereoselective control of
glycosylations was also evaluated. The established modular synthetic strategy is
applicable to the preparation of Reb-S analogues, thus holding the promise to speed
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up the process of searching for ideal sweeteners and promising lead compounds.
Experimental Section
All reagents were of commercial grade and used as received. All moisture sensitive reactions were
performed under an argon atmosphere. DCM and acetonitrile used in the glycosylation was distilled
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
over CaH and stored on activated 4Å molecular sieves before being used. Analytical thin-layer
chromatography (TLC) was conducted with silica gel 60 F254 precoated glass plates (0.25 mm) and
visualized by exposure to UV light (254 nm) or by staining with sulfuric acid. Column chromatography
was performed using silica gel (Qingdao Marine Chemical Inc., China), and Sephadex LH-20 (GE
Healthcare Bio-Sciences AB, Sweden). NMR spectra were recorded on a Bruker AM-400 spectrometer
1
(400 MHz). Chemical shifts (δ) are given in ppm relative to tetramethylsilane as internal standard ( H
3
NMR in CDCl ) or the residual signals of the deuterated solvents. Coupling constants (J) are given in
Hz. All 13C spectra are proton decoupled. Optical rotations were measured at 20 °C with a Rudolph
Autopol IV automatic polarimeter using a quartz cell with 2 mL capacity and a 1 dm path length.
Concentrations (c) are given in g/100 mL. High resolution mass spectra (HRMS) were recorded on a
Bruker micrOTOF II spectrometer using electrospray ionization (ESI).
p-Methoxyphenyl
2-O-(2,3,4-tri-O-benzyl-6-O-benzoyl-α-D-glucopyranosyl)-3-O-benzyl-4,6-
O-benzylidene-β-D-glucopyranoside (7)
A solution of 5 (1066 mg, 1.6 mmol) and 6 (500 mg, 1.11 mmol) in dry dichloromethane (20 mL) was
stirred at room temperature in the presence of powdered 4A molecular sieves for 30 min under N
2
o
atmosphere. After being chilled to -30 C, NIS (453 mg, 2.01 mmol) and TMSOTf (56 µL, 0.31 mmol)
were added to the suspension successively under N
2
atmosphere. The resultant mixture was stirred at
the same temperature for another 1 h, then Et N was added to quench the reaction. Filtration through a
3
pad of Celite and filtrate was concentrated under reduced pressure to give the crude product, which was
further purified by silica gel column chromatography (petroleum ether/ethyl acetate = 5 : 1) to provide
7 (990 mg, 1.0 mmol, 90%) as a mixture of α/β isomers, and the α/β isomers can be completely
separated by silica gel column chromatography with toluene/ethyl acetate as eluent (toluene/ethyl
2
5
1
o
acetate = 40 : 1). For 7α: [α]
(dd, J = 8.4, 1.2 Hz, 2H), 7.68 – 7.64 (m, 1H), 7.53 (t, J = 8.0 Hz, 2H), 7.48 – 7.38 (m, 5H), 7.34 – 7.26
m, 12H), 7.18 – 7.05 (m, 8H), 6.87 – 6.83 (m, 4H), 5.70 (d, J = 3.6 Hz, 2H), 5.47 (d, J = 7.6 Hz, 1H),
D
3 6
= +30.5 (c 1.0, CHCl ); H NMR (400 MHz, DMSO-d , 50 C) δ 7.98
(
4
4
.89 – 4.82 (m, 3H), 4.77 (d, J = 11.2 Hz, 1H), 4.72 (d, J = 10.8 Hz, 1H), 4.59 (d, J = 11.2 Hz, 1H),
.55 – 4.51 (m, 1H), 4.44 (d, J = 11.6 Hz, 1H), 4.38 (d, J = 11.6 Hz, 1H), 4.32 – 4.25 (m, 2H), 4.10 (dd,
J = 12.0, 4.8 Hz, 1H), 3.95 – 3.88 (m, 2H), 3.86 – 3.80 (m, 2H), 3.78 – 3.71 (m, 2H), 3.69 (s, 3H), 3.63
1
3
1
o
–
6
3.56 (m, 2H); C{ H} NMR (100 MHz, DMSO-d , 50 C) δ 165.2, 154.6, 149.9, 138.5, 138.1, 137.8,
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7
37.4, 137.3, 133.0, 129.6, 129.1, 128.6, 128.5, 128.4, 128.0, 127.9 (2 C), 127.8, 127.7, 127.4 (2 C),
27.3, 127.2, 127.1, 127.0, 125.7, 116.6, 114.6, 100.1, 100.0, 94.3, 81.4, 81.0, 79.4, 77.8, 77.4, 74.6,
61
4.4, 74.1, 73.9, 71.3, 68.2, 67.7, 64.8, 62.8, 55.3; HRMS (ESI) calcd for C61H O
13 [M+H] +
2
5
1
001.4107, found 1001.4108. For 7β: [α]
D
3 6
= +17.8 (c 0.5, CHCl ); H NMR (400 MHz, DMSO-d ) δ
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
.98 (d, J = 7.2 Hz, 2 H), 7.67 (t, J = 7.6 Hz, 1 H), 7.52 (t, J = 7.6 Hz, 2 H), 7.40-7.37 (m, 5 H),
.30-7.22 (m, 20 H), 6.89 (d, J = 8.8 Hz, 2 H), 6.64 (d, J = 9.2 Hz, 2 H), 5.71 (s, 1 H), 5.27 (d, J = 7.2
Hz, 1 H), 5.00 (d, J = 4.8 Hz, 1 H), 4.99 (d, J = 7.2 Hz, 1 H), 4.87 (d, J = 11.2 Hz, 1 H), 4.77 (dd, J =
1.2, 3.2 Hz, 2 H) , 4.74-4.64 (m, 3 H), 4.57 (d, J = 11.2 Hz, 1 H), 4.31-4.23 (m, 3 H), 3.97 (t, J = 8.4
Hz, 1 H), 3.88 (t, J = 7.6 Hz, 1 H), 3.84-3.73 (m, 3 H), 3.72-3.66 (m, 2 H), 3.63 (s, 3 H), 3.61 (d, J =
1
.4 Hz, 1 H), 3.38 (d, J = 7.6 Hz, 1 H); 13C{ H} NMR (100 MHz, DMSO-d
1
8
1
6
) δ 165.4, 154.4, 150.8,
38.5, 138.4, 138.3, 138.0, 137.5, 133.4, 129.5, 129.3, 128.8, 128.2 (2 C), 128.1, 128.0 (2 C), 127.8 (2
C), 127.7 (2 C), 127.5, 127.4, 127.3, 125.9, 117.2, 114.3, 102.2, 100.1, 99.4, 83.9, 82.1, 81.1, 80.4,
60 13
78.9, 77.1, 74.6, 74.0, 73.9, 73.8, 71.9, 67.8, 64.8, 62.9, 55.3; HRMS (ESI) calcd for C61H O K
+
[M+K] 1039.3665, found 1039.3664.
p-Methoxyphenyl
2-O-(2,3,4,6-tetra-O-benzoyl-α-D-glucopyranosyl)-4,6-di-O-benzoyl-β-D-glucopyranoside (8) and
p-methoxyphenyl
2
-O-(2,3,4,6-tetra-O-benzoyl-α-D-glucopyranosyl)-3,4,6-tri-O-benzoyl-β-D-glucopyranoside (8’)
To a solution of 7 (1.15 g, 1.15 mmol) in a mixed solvent of ethyl acetate and MeOH (60 mL, v/v = 1 :
) was added 10% Pd/C (124 mg). The flask containing the resultant black suspension was evacuated
under reduced pressure and refilled with H . After this process was repeated for 3 times, the mixture
1
2
was stirred at room temperature for another 17 h. Filtration through a pad of Celite and silica gel was
followed by filtrate concentration under reduced pressure to afford the crude product, which was
3 2 2
briefly purified by flash chromatography (CH OH/CH Cl = 1 : 5). The crude product was used for the
next step without further characterization. To a solution of above obtained intermediate (600 mg, 1.08
o
mmol) in dry pyridine was added BzCl (1.2 mL, 10.3 mmol) dropwise at 0 C under N
2
atmosphere.
After the addition was completed, the reaction mixture was warmed up to room temperature gradually
and the stirring was continued for another 10 h. Ethyl acetate was added to dilute the reaction mixture
and the resulting solution was washed with water, 1N HCl, saturated aqueous NaHCO
3
, and brine
successively. After being dried over anhydrous Na SO and filtrating to remove the desiccant, the
2
4
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filtrate was concentrated in vacuo to provide the crude product, which was further purified by silica gel
chromatography (petroleum ether/ethyl acetate = 2 : 1) to afford 8 (886 mg, 0.82 mmol, 71%) as a
2
5
1
white foam and 8’ (250 mg, 0.21 mmol, 18%) as a white foam. For 8: [α]
NMR (400 MHz, CDCl ) δ 8.08 (t, J = 8.3 Hz, 4H), 7.92 (dd, J = 13.9 Hz, 8.2 Hz, 4H), 7.85 (d, J = 8.2
Hz, 2H), 7.72 (d, J = 8.2 Hz, 2H), 7.61 – 7.24 (m, 16H), 7.14 (t, J = 7.7 Hz, 2H), 6.66 (d, J = 9.1 Hz,
D
3
= +40.3 (c 1.0, CHCl ); H
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2H), 6.33 (d, J = 9.1 Hz, 2H), 6.21 (t, J = 10.0 Hz, 1H), 5.91 (d, J = 3.8 Hz, 1H), 5.72 (t, J = 10.0 Hz,
1H), 5.46 (dd, J = 10.4, 3.8 Hz, 1H), 5.25 (t, J = 9.6 Hz, 1H), 5.11 – 5.06 (m, 1H), 4.98 (d, J = 7.8 Hz,
1H), 4.62 (t, J = 12.2 Hz, 2H), 4.47 (dd, J = 12.3, 4.8 Hz, 1H), 4.37 (dd, J = 12.0, 7.1 Hz, 1H), 4.21 (t,
J = 9.2 Hz, 1H), 4.10 – 4.03 (m, 2H), 3.59 (s, 3H). 13C{ H} NMR (100 MHz, CDCl
1
) δ 166.6, 166.5,
3
1
1
1
C
66.1, 166.0, 165.5, 165.4, 155.0, 150.2, 133.9, 133.4, 133.3, 133.2 (2 C), 133.0, 130.1, 129.9 (2 C),
29.8 (2 C), 129.7, 129.6, 129.2, 129.0, 128.9, 128.8, 128.7, 128.5, 128.4 (2 C), 128.1, 117.5, 114.2,
01.0, 96.3, 78.9, 74.2, 72.8, 71.9, 71.4, 70.4, 69.6, 68.2, 63.5, 63.1, 55.5. HRMS (ESI) calcd for
+
25
1
61
H
53
O
18 [M+H] 1073.3226, found 1073.3220. For 8’: [α]
D
3
= +83.4 (c 2.2, CHCl ); H NMR (400
MHz, CDCl
3
) δ 8.09 (d, J = 7.8 Hz, 2H), 8.05 (m, J = 7.8 Hz, 2H), 7.99 – 7.93 (m, 4H), 7.86 (dd, J =
8.4, 1.6 Hz, 2H), 7.72 – 7.69 (m, 4H), 7.59 – 7.47 (m, 6H), 7.40 – 7.32 (m, 9H), 7.25 – 7.16 (m, 6H),
6.72 (d, J = 9.2 Hz, 2H), 6.40 (d, J = 9.2 Hz, 2H), 6.12 – 6.03 (m, 3H), 6.68 – 5.59 (m, 2H), 5.42 (dd, J
= 10.4, 3.8 Hz, 1H), 5.16 (d, J = 7.8 Hz, 1H), 4.60 (dd, J = 12.0, 2.8 Hz, 1H), 4.47 (dd, J = 12.4, 7.2
Hz, 1H), 4.41 (dd, J = 9.6, 7.6 Hz, 1H), 4.25 – 4.11 (m, 3H), 3.71 (dd, J = 12.4, 4.0 Hz, 1H), 3.62 (s,
1
3
1
3
1
1
7
1
2
3
H); C{ H} NMR (100 MHz, CDCl ) δ 166.0 ( 2 C), 165.7, 165.6, 165.5, 165.4, 164.9, 155.2, 149.9,
33.7, 133.6, 133.4, 133.2, 133.2, 133.1, 133.0, 129.9, 129.8 (4 C), 129.7 (2 C), 129.6, 129.2, 128.9,
28.8, 128.7 (2 C), 128.6 (2 C), 128.4, 128.3 (2 C), 128.1, 117.6, 114.2, 101.4, 95.8, 77.4, 75.7, 73.2,
57
H O
19 [M+H]+
2.2, 70.9, 70.1, 70.0, 68.8, 68.2, 63.2, 61.9, 55.4; HRMS (ESI) calcd for C68
177.3488, found 1177.3486.
-O-(2,3,4,6-Tetra-O-benzoyl-α-D-glucopyronosyl)-4,6-di-O-benzoyl-D-glucopyranosyl
ortho-cyclopropylethynylbenzoate (2)
3 2
To a solution of 8 (886 mg, 0.826 mmol) in a mixed solvent of CH CN (23.7 mL) and H O (5.5 ml)
o
was added CAN (1.36 g, 2.48 mmol) at 0 C under N
2
atmosphere. The resulting mixture was stirred at
same temperature for 10 min, then ethyl acetate was added to dilute the reaction. The resultant mixture
was washed with water, saturated aqueous Na and NaHCO , and brine successively, and dried
2
S
2
O
3
3
over anhydrous Na
2
SO
4
. Filtration was followed by concentration to provide a residue, which was
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further subjected to silica gel column chromatography to give the hemiacetal intermediate, which was
used for the next step without further characterization. The above obtained hemiacetal intermediate
2 2
(740 mg, 0.765 mmol) was dissolved in dry CH Cl (5 mL), to which ABzOH (220 mg, 1.18 mmol),
i
DMAP (188 mg, 1.54 mmol), Pr
2
NEt (0.19 mL, 1.15 mmol), and EDCI (439.6 mg, 2.29 mmol) were
atmosphere. The resulting mixture was stirred at the
same temperature for 8 h, then ethyl acetate was added to dilute the reaction mixture. The resultant
solution was washed with water, 1N HCl, saturated aqueous NaHCO , and brine successively, and
dried over anhydrous Na SO . Filtration and concentration gave a residue, which was further subjected
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
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4
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4
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5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
added successively at room temperature under N
2
3
2
4
to silica gel column chromatography (petroleum ether/ethyl acetate = 3 : 1) to yield 2 (580 mg, 0.51
mmol, 62% for 2 steps) as a mixture of α/β isomers (α/β = 1 : 3). An aliquot of pure 2β was obtained
25
1
for detailed characterization: [α]
D
3 3
= +65.2 (c 1.0, CHCl ); H NMR (400 MHz, CDCl ) δ 8.10 – 8.05
(m, 4H), 7.96 (d, J = 8.2 Hz, 2H), 7.91 (d, J = 7.6 Hz, 2H), 7.80 (d, J = 7.6 Hz, 2H), 7.64 – 7.12 (m,
24H), 6.17 (t, J = 10.0 Hz, 1H), 5.94 (d, J = 8.0 Hz, 1H), 5.86 (d, J = 3.8 Hz, 1H), 5.68 (t, J = 10.0 Hz,
1H), 5.38 – 5.30 (m, 2H), 5.11 – 5.06 (m, 1H), 4.63 (dd, J = 12.4, 2.8 Hz, 1H), 4.56 (dd, J = 12.0, 2.4
Hz, 1H), 4.48 – 4.39 (m, 2H), 4.25 (t, J = 9.1 Hz, 1H), 4.20 – 4.06 (m, 2H), 1.48-1.40 (m, 1H),
1
3
1
0.86-0.74 (m, 4H); C{ H} NMR (100 MHz, CDCl
3
) δ 166.6 (2 C), 166.2, 165.9, 165.4, 165.2, 163.3,
134.6, 133.9, 133.4, 133.3, 133.2 (2 C), 133.1, 132.5, 131.4, 130.1, 130.0, 129.9, 129.8, 129.7 (3 C),
1
7
29.2, 129.0, 128.9, 128.8, 128.7, 128.6, 128.4 (2 C), 128.3 (2 C), 126.9, 125.7, 100.2, 96.6, 94.0, 78.4,
54 18
5.2, 74.4, 72.6, 72.5, 71.3, 70.4, 69.6, 68.2, 63.1, 63.0, 8.9, 0.8; HRMS (ESI) calcd for C66H O K
+
[
M+K] 1173.2942, found 1173.2952.
2
-O-(2,3,4,6-Tetra-O-benzoyl-α-D-glucopyronosyl)-3,4,6-tri-O-benzoyl-D-glucopyranosyl
ortho-cyclopropylethynylbenzoate (2’)
Similar procedure as that used for the synthesis of 2 was adopted to convert 8’ (340 mg, 0.289 mmol)
to 2’ (150 mg, 0.121 mmol, 42% for 2 steps) as a mixture of α/β epimers (α/β = 1 : 2). An aliquot of
2
5
1
pure 2’ β was obtained for detailed characterization: [α]
MHz, CDCl ) δ 8.09 – 8.07 (m, 2H), 8.03 – 7.98 (m, 4H), 7.93 – 7.90 (m, 2H), 7.80 – 7.78 (m, 2H),
.72 – 7.67 (m, 5H), 7.54 – 7.12 (m, 24H), 6.11 – 5.98 (m, 4H), 5.80 (t, J = 9.6 Hz, 1H), 5.55 (t, J =
D
3
= +113.7 (c 2.3, CHCl ); H NMR (400
3
7
1
–
1
0.0 Hz, 1H), 5.23 (dd, J = 10.4, 4.0 Hz, 1H), 4.55 (dd, J = 12.4, 3.2 Hz, 1H), 4.50 – 4.43 (m, 2H), 4.34
4.30 (m, 1H), 4.23 – 4.19 (m, 1H), 4.10 (dd, J = 12.4, 2.8 Hz, 1H), 3.70 (dd, J = 12.4, 4.4 Hz, 1H),
.49 – 1.42 (m, 1H), 0.87 – 0.75 (m, 4H); 13C{ H} NMR (100 MHz, CDCl
1
) δ 166.1, 166.0, 165.8,
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65.5, 165.3 (2 C), 164.9, 163.2, 134.6, 133.7, 133.6, 133.4 (2 C), 133.3, 133.1 (2 C), 132.6, 131.4,
29.9 (3 C), 129.8 (2 C), 129.7 (2 C), 129.2, 129.0, 128.9, 128.8 (2 C), 128.7, 128.6 (2 C), 128.5, 128.4
(2 C), 128.3 (2 C), 127.0, 125.6, 100.4, 95.8, 94.4, 75.1, 74.8, 73.6, 72.8, 71.1, 70.1, 69.4, 68.8, 68.2,
+
6
2.7, 62.0, 29.8, 8.9, 0.8; HRMS (ESI) calcd for C73
H
58
O
19Na [M+Na] 1261.3464, found 1261.3468.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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9
0
Allyl 2-O-(2,3,4-tri-O-benzoyl-α-L-rhamnopyranosyl)-3,4,6-tri-O-acetyl-β-D-glucopyranoside (11)
A solution of 9 (2.68 g, 4.32 mmol) and 10 (1.0 g, 2.89 mmol) in dry CH Cl (10 mL) was stirred in the
atmosphere. After being chilled
2
2
presence of powdered 4A MS for 30 min at room temperature under N
2
o
to 0 C, TMSOTf (160 µL, 0.884 mmol) was added. The reaction mixture was then allowed to warm up
3
to room temperature gradually, and the stirring was continued for 1 h before Et N was added to quench
the reaction. Filtration and the filtrate was concentrated under reduced pressure to provide a residue,
which was further purified by silica gel column chromatography (petroleum ether/ethyl acetate = 3 : 1)
25
1
to yield 11 (2.1 g, 2.61 mmol, 90%) as a white foam: [α]
D
3
= +192.8 (c 1.0, CHCl ); H NMR (400
MHz, CDCl ) δ 8.19 (d, J = 8.0 Hz, 2H), 7.95 (d, J = 7.8 Hz, 2H), 7.83 (d, J = 7.8 Hz, 2H), 7.61 – 7.58
3
(m, 1H), 7.52 – 7.45 (m, 3H), 7.40 – 7.35 (m, 3H), 7.24 (t, J = 7.8 Hz, 1H), 6.05 – 5.95 (m, 1H), 5.79
(dd, J = 10.2, 3.4 Hz, 1H), 5.68 (t, J = 10.0 Hz, 1H), 5.43 – 5.34 (m, 3H), 5.25 (dd, J = 7.1, 1.7 Hz,
2H), 5.03 (t, J = 9.7 Hz, 1H), 4.64 (d, J = 7.8 Hz, 1H), 4.55 (dd, J = 9.9, 6.1 Hz, 1H), 4.49 – 4.44 (m,
1H), 4.31 (dd, J = 12.3, 4.7 Hz, 1H), 4.23 – 4.13 (m, 2H), 3.86 (dd, J = 9.5, 7.8 Hz, 1H), 3.76 (m, 1H),
.17 (s, 3H), 2.09 (s, 3H), 2.03 (s, 3H), 1.29 (d, J = 6.0 Hz, 3H); 13C{ H} NMR (100 MHz, CDCl
1
) δ
2
1
1
6
2
3
70.7, 170.5, 169.7, 165.8, 165.5 (2 C), 133.6, 133.3, 133.2, 129.9, 129.7 (2 C), 129.4, 129.3, 129.2,
28.6, 128.4, 128.3, 118.8, 100.6, 97.8, 77.4, 76.3, 74.8, 71.9, 71.8, 71.3, 71.1, 70.8, 69.5, 68.8, 67.0,
+
2.1, 20.8 (2 C), 20.7, 17.5; HRMS (ESI) calcd for C42
H
44
O
16K [M+K] 843.2261, found 843.2261.
-O-(2,3,4-Tri-O-benzoyl-α-L-rhamnopyranosyl)-3,4,6-tri-O-acetyl-α-D-glucopyranosyl bromide
4)
To a solution of 11 (1.0 g, 1.24 mmol) in a mixed solvent of CH
was added PdCl (65.06 mg, 0.37 mmol) at room temperature. The black suspension was stirred at the
(
2
2
Cl and methanol (40 mL, v/v = 1 : 1)
2
same temperature for 1 h before filtration through a pad of Celite and silica gel was adopted to remove
the catalyst. The filtrate was condensed in vacuo and the obtained residue was co-evaporated with
toluene for three times before being used for next step. The above obtained residue was dissolved in
o
dry pyridine (4 mL), to which Ac
2
O (0.1 mL, 1.06 mmol) was added dropwise at 0 C under N
2
atmosphere. After the addition was completed, the reaction mixture was gradually warmed up to room
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temperature and the stirring was continued for 10 h, at which time TLC showed that all starting
materials were consumed completely. Ethyl acetate was added to dilute the reaction mixture and the
resulting solution was washed successively with water, 1N HCl, saturated aqueous NaHCO
and was then dried over anhydrous Na SO . Filtration was followed by concentration to provide a
residue, which was further co-evaporated with toluene for 3 times. The thus obtained residue was put to
3
, and brine,
2
4
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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8
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8
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3
4
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8
9
0
1
2
3
4
5
6
7
8
9
0
2 2
next step without further purification. To a solution of the above obtained residue in dry CH Cl (5 mL)
o
was added HBr/HOAc (2 mL) at 0 C. The resulting solution was gradually warmed up to room
temperature and the stirring was continued at the same temperature for another 3 h. Ethyl acetate was
added to dilute the reaction mixture and the resulting solution was thoroughly washed with water,
saturated aqueous NaHCO
3 2 4
, and brine, and was then dried over anhydrous Na SO . Filtration,
concentration in vacuo, and purification by silica gel chromatography (petroleum ether/ethyl acetate =
25
3
: 1) delivered 4 (408 mg, 0.493 mmol, 40% for 3 steps) as a white foam: [α]
D
= +156.3 (c 1.0,
1
CHCl
3
); H NMR (400 MHz, CDCl
3
) δ 8.09 (dd, J = 8.0, 1.2 Hz, 2H), 8.00 (dd, J = 8.0, 1.2 Hz, 2H),
7.83 (dd, J = 8.0, 1.2 Hz, 2H), 7.64 – 7.60 (m, 1H), 7.53 – 7.48 (m, 3H), 7.44 – 7.38 (m, 3H), 7.28 –
7.24 (m, 2H), 6.51 (d, J = 4.0 Hz, 1H), 5.76 – 5.67 (m, 3H), 5.61 (t, J = 9.6 Hz, 1H), 5.51 (dd, J = 3.1,
1.7 Hz, 1H), 5.19 – 5.14 (m, 2H), 4.38 – 4.35 (m, 2H), 4.16 – 4.11 (m, 1H), 3.88 (dd, J = 9.8, 3.9 Hz,
1H), 2.21 (s, 3H), 2.10 (s, 3H), 2.08 (s, 3H), 1.35 (d, J = 6.0 Hz, 3H); 13C{ H} NMR (100 MHz,
1
CDCl
3
) δ 170.6, 170.3, 169.8, 166.0, 165.7, 165.3, 133.8, 133.6, 133.3, 130.0 (2 C), 129.8, 129.3,
1
28.8, 128.6, 128.4, 100.2, 88.9, 78.8, 72.5, 71.7, 71.4, 70.9, 69.6, 68.6, 67.3, 61.2, 20.8 (2 C), 17.7;
+
HRMS (ESI) calcd for C39
H39BrO15K [M+K] 865.1104, found 865.1096.
1
3-O-[2-O-(2,3,4,6-Tetra-O-benzoyl-α-D-glucopyranosyl)-4,6-di-O-benzoyl-β-D-glucopyranosyl]-st
eviol tert-butyldiphenylsilyl ester (12β)
A solution of 2 (68.1 mg, 0.06 mmol) and 3 (28 mg, 0.05 mmol) in dry CH
3
CN (3 mL) was stirred in
the presence of powdered 4A MS at room temperature for 30 min under N
2
atmosphere. After being
o
chilled to -40 C, Ph
3
PAuNTf
2
(44.3 mg, 0.06 mmol) was added. The resultant reaction mixture was
then gradually warmed up to room temperature and the stirring was continued for another 4 h at the
same temperature. After the 4A MS was removed by filtration, the filtrate was condensed in vacuo. The
obtained residue was further subjected to purification by silica gel column chromatography (petroleum
25
ether/ethyl acetate = 4 : 1) to provide 12 (69 mg, 0.458 mmol, 91%) as a white foam: [α]
D
= +28.2 (c
1
1
.0, CHCl
3
); H NMR (400 MHz, CDCl
3
) δ 8.08 – 8.03 (m, 4H), 7.93 – 7.87 (m, 6H), 7.76 (d, 7.6 Hz,
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H), 7.67 (t, 6.8 Hz, 4H), 7.57 (t, J = 7.4 Hz, 1H), 7.49 – 7.19 (m, 22H), 6.20 (t, J = 9.9 Hz, 1H), 5.81
(d, J = 3.7 Hz, 1H), 5.65 – 5.56 (m, 2H), 5.25 – 5.20 (m, 1H), 5.13 (t, J = 9.5 Hz, 1H), 4.88 (s, 1H),
4
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3
1
.69 – 4.67 (m, 2H), 4.55 (dd, J = 12.2, 2.6 Hz, 1H), 4.47 (dd, J = 12.3, 5.2 Hz, 1H), 4.41 – 4.36 (m,
H), 4.18 (t, J = 9.2 Hz, 1H), 3.95 – 3.89 (m, 2H), 3.29 (s, 1H), 2.20 (d, J = 13.3 Hz, 1H), 1.99 (d, J =
6.2 Hz, 1H), 1.84 – 1.73 (m, 4H), 1.62 (p, J = 8.4, 6.6 Hz, 3H), 1.46 – 1.34 (m, 2H), 1.25 (m, 3H),
0
1
2
3
4
5
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7
8
9
0
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2
3
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7
8
9
0
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2
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
.18 (s, 3H), 1.11 (s, 9H), 1.06 – 0.75 (m, 5H), 0.64 (d, J = 8.0 Hz, 1H), 0.57 – 0.49 (m, 1H), 0.30 (s,
H); 13C{ H} NMR (100 MHz, CDCl
1
) δ 176.6, 166.6, 166.2, 166.1, 165.6, 165.4, 152.4, 135.8 (2 C),
3
33.2, 132.3, 130.1 (2 C), 130.0 (2 C), 129.9, 129.8 (2 C), 129.7, 129.2 (3 C), 129.1, 128.7, 128.6 (2
C), 128.4 (2 C), 128.3, 127.7 (2 C), 104.8, 98.8, 95.4, 86.4, 76.7, 74.7, 73.4, 71.4, 71.0, 70.7, 70.0,
6
1
1
7.9, 64.0, 63.4, 56.9, 53.5, 47.2, 45.3, 42.6, 41.2, 40.2, 39.2, 38.7, 37.6, 29.8, 29.3, 27.2, 22.0, 19.9,
]+ 1522.6340, found 1522.6386.
9.4, 19.3, 16.2; HRMS (ESI) calcd for C90H96NO19Si [M+NH
4
3-O-[2-O-(2,3,4,6-Tetra-O-benzoyl-α-D-glucopyranosyl)-4,6-di-O-benzoyl-β-D-glucopyranosyl]-st
eviol (13)
To a solution of 12 (132 mg, 0.088 mmol) in dry THF (5 mL) were added HOAc (10.03 µL, 0.175
mmol) and TBAF (1 N solution in THF, 98 µL, 0.098 mmol) at room temperature. After the mixture
was stirred at the same temperature for 30 min, ethyl acetate was added to dilute the reaction mixture.
The resulting solution was successively washed with water and brine and dried over anhydrous
Na
2 4
SO . Filtration was followed by concentration under reduced pressure to deliver a residue, which
was further purified by silica gel column chromatography (petroleum ether/ethyl acetate = 2 : 1) to
2
5
1
provide 13 (100 mg, 0.079 mmol, 90%) as a white solid: [α]
MHz, CDCl ) δ 8.11 (d, J = 7.6 Hz, 2H), 8.04 (d, J = 8.0 Hz, 2H), 7.95 (dd, J = 11.2, 7.6 Hz, 4H), 7.77
t, J = 7.1 Hz, 4H), 7.55 – 7.43 (m, 7H), 7.40 – 7.26 (m, 8H), 7.18 – 7.12 (m, 4H), 6.26 (t, J = 9.9 Hz,
D
3
= +66.7 (c 0.5, CHCl ); H NMR (400
3
(
1
H), 5.89 (d, J = 3.7 Hz, 1H), 5.68 – 5.63 (m, 2H), 5.30 – 5.25 (m, 1H), 5.09 (t, J = 9.6 Hz, 1H), 4.94
(s, 1H), 4.76 (d, J = 8.2 Hz, 2H), 4.56 (dd, J = 12.4, 2.5 Hz, 1H), 4.50 – 4.45 (m, 2H), 4.32 (dd, J =
1
1
0
2.1, 7.2 Hz, 1H), 4.13 (t, J = 9.2 Hz, 1H), 3.99 – 3.93 (m, 2H), 2.14 (d, J = 13.2 Hz, 1H), 2.02 (d, J =
6.8 Hz, 1H), 1.89 – 1.81 (m, 2H), 1.71 (s, 1H), 1.52 – 1.35 (m, 5H), 1.27 (s, 2H), 1.19 (s, 4H), 0.97 –
.86 (m, 5H), 0.68 (d, J = 8.0 Hz, 1H), 0.60 – 0.54 (m, 1H), 0.45 (s, 3H); 13C{ H} NMR (100 MHz,
1
CDCl
3
) δ 183.2, 166.6, 166.5, 166.4, 166.2, 165.5, 165.4, 152.3, 133.6, 133.5, 133.3, 133.2, 133.1 (2
C), 130.1, 129.9 (2 C), 129.8, 129.7 (2 C), 129.3, 129.1, 129.0, 128.9, 128.7, 128.5 (2 C), 128.3, 104.9,
9
8.6, 95.2, 86.3, 76.7, 74.2, 73.2, 71.4, 71.0, 70.9, 69.9, 67.9, 64.2, 63.4, 56.6, 53.5, 47.2, 43.7, 42.5,
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74
2.4, 41.0, 40.2, 39.2, 37.8, 29.8, 29.1, 21.6, 19.9, 18.9, 15.4; HRMS (ESI) calcd for C74H O19Na
+
[M+Na] 1289.4716, found 1289.4744.
1
3-O-[2-O-(2,3,4,6-Tetra-O-benzoyl-α-D-glucopyranosyl)-4,6-di-O-benzoyl-β-D-glucopyranosyl]-st
eviol 2-O-(2,3,4-tri-O-benzoyl-α-L-rhamnosyl)-3,4,6-tri-O-acetyl-β-D-glucopyranosyl ester (14),
14a) and (14b)
To a solution of 13 (80 mg, 0.063 mmol) in a mixed solvent of CHCl
was added K CO (26 mg, 0.188 mmol) and TBAB (40.6 mg, 0.126 mmol) at room temperature. After
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8
9
0
(
3
2
and H O (3 mL, v/v = 1.5 : 1.5)
2
3
the mixture was stirred at the same temperature for 20 min, disaccharide bromide donor 4 (104 mg,
o
0
.126 mmol) was added. After being stirred at 40 C in oil bath for another 16 h, the reaction mixture
was cooled to room temperature. Ethyl acetate was added to dilute the reaction mixture, and the
resulting solution was washed successively with water, 1N HCl, saturated aqueous NaHCO , and brine,
and dried over anhydrous Na SO . Filtration, filtrate concentration in vacuo, and purification by silica
3
2
4
gel column chromatography (petroleum ether/ethyl acetate = 2 : 1) provided 14 (52 mg, 0.0258 mmol,
41%) as a white solid, 14a (30 mg, 0.0149 mmol, 24%) as a white solid, and 14b (20 mg, 0.0105
2
5
1
mmol, 17%) as a white solid. For 14: [α]
D
3 3
= +61.8 (c 1.0, CHCl ); H NMR (400 MHz, CDCl ) δ 8.11
– 8.07 (m, 6H), 8.01 – 7.88 (m, 9H), 7.83 – 7.78 (m, 4H), 7.63 – 7.16 (m, 26H), 6.20 (t, J = 10.0 Hz,
1H), 5.84 (d, J = 3.7 Hz, 1H), 5.80 (d, J = 7.8 Hz, 1H), 5.69 – 5.59 (m, 5H), 5.52 (s, 1H), 5.44 (t, J =
9
.0 Hz, 1H), 5.32 (s, 1H), 5.26 – 5.19 (m, 2H), 5.14 (t, J = 9.5 Hz, 1H), 4.91 (s, 1H), 4.79 (d, J = 7.8
Hz, 1H), 4.73 (s, 1H), 4.57 (dd, J = 12.4, 2.8 Hz, 1H), 4.52 – 4.48 (m, 2H), 4.42 – 4.29 (m, 4H), 4.19 (t,
J = 9.1 Hz, 1H), 4.08 (t, J = 8.2 Hz, 2H), 3.94 (t, J = 8.0 Hz, 1H), 2.20 (s, 3H), 2.17 (s, 3H), 2.17 – 2.12
(m, 1H), 2.09 (s, 3H), 2.03 (d, J = 17.5 Hz, 1H), 1.95 (d, J = 11.0 Hz, 1H), 1.86 – 1.65 (m, 5H), 1.54 –
1
.46 (m, 13H), 0.94 – 0.84 (m, 3H), 0.78 – 0.73 (m, 1H), 0.67 (d, J = 8.0 Hz, 1H), 0.52 (s, 3H);
1
3
1
C{ H} NMR (100 MHz, CDCl
3
) δ 174.5, 170.5 (2 C), 169.9, 166.6 (2 C), 166.4, 166.0, 165.8, 165.7,
1
65.6, 165.4, 152.2, 133.8 (2 C), 133.6, 133.4, 133.3, 133.2 (2 C), 130.2, 130.1, 130.0, 129.9, 129.8 (3
C), 129.7, 129.3 (2 C), 129.2 (2 C), 129.0, 128.8, 128.7, 128.6, 128.5, 128.4, 128.3, 105.0, 98.5, 97.9,
9
5
1
5.4, 92.4, 86.4, 76.2, 74.5, 73.4, 72.2, 71.5, 71,0, 70.6, 69.9, 69.6, 68.5, 67.9, 67.8, 64.3, 63.4, 61.8,
7.8, 53.5, 47.3, 44.0, 42.6, 41.1, 40.0, 39.3, 37.6, 37.4, 29.8, 29.3, 21.5, 21.1, 21.0, 20.9, 19.8, 19.2,
7.9, 16.2; HRMS (ESI) calcd for C113
H
116NO34 [M+NH
4
]+ 2030.7373, found 2030.7418. For 14a:
25
1
[α]
D
= +78.1 (c 1.0, CHCl ); H NMR (400 MHz, CDCl ) δ 8.15 (d, J = 7.6 Hz, 2H), 8.11 – 8.08 (m,
3
3
6
H), 7.97 (t, J = 8.4 Hz, 4H), 7.83 (d, J = 8.0 Hz, 2H), 7.73 (d, J = 8.0 Hz, 2H), 7.4 – 7.58 (m, 2H),
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.55 – 7.18 (m, 27H), 5.95 (d, J = 3.6 Hz, 1H), 5.90 (t, J = 9.9 Hz, 1H), 5.79 – 5.68 (m, 4H), 5.58 –
.42 (m, 4H), 5.32 (s, 1H), 5.25 (t, J = 9.7 Hz, 1H), 4.92 (s, 1H), 4.86 (d, J = 7.7 Hz, 1H), 4.77 (s, 1H),
.63 (d, J = 4.1 Hz, 2H), 4.49 – 4.25 (m, 5H), 4.17 – 4.07 (m, 3H), 3.93 – 3.87 (m, 2H), 3.68 (t, J = 9.5
Hz, 1H), 2.26 (s, 3H), 2.21 (s, 3H), 2.14 (d, J = 14.6 Hz, 1H), 2.10 (s, 3H), 2.05 (d, J = 17.2 Hz, 1H),
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1
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1
9
6
2
C
.99 (d, J = 11.2 Hz, 1H), 1.84 (d, J = 17.2 Hz, 1H), 1.77 – 0.57 (m, 31H), 0.55 (s, 3H), 0.51 (d, J =
1.6 Hz, 1H); 13C{ H} NMR (100 MHz, CDCl
1
) δ 174.5, 171.1, 170.5, 169.9, 167.1, 166.6, 166.2,
3
65.9, 165.7, 165.6, 165.5, 165.4, 165.1, 152.2, 133.8, 133.6, 133.5, 133.4, 133.3, 133.0, 130.2, 130.1,
30.0, 129.9, 129.8 (3 C), 129.7 (2 C), 129.4, 129.2 (3 C), 128.8, 128.6 (3 C), 128.4, 128.3, 104.9,
9.0, 97.9, 94.8, 92.8, 86.7, 77.4, 76.5, 76.4, 74.5, 73.9, 73.1, 72.0, 71.5, 71.0 (2 C), 70.5, 70.3, 69.6,
9.1, 68.6, 67.9, 67.8, 64.7, 62.9, 61.9, 57.8, 53.3, 47.2, 43.9, 42.6, 41.0, 39.9, 39.2, 37.2, 37.1, 30.0,
9.8, 29.6, 22.8, 21.5, 21.1, 21.0, 20.8, 19.7, 19.2, 18.0, 16.0, 14.2; HRMS (ESI) calcd for
+
25
1
113
H
112
O
34K [M+K] 2051.6666, found 2051.6623. For 14b: [α]
D
3
= +58.0 (c 1.0, CHCl ); H NMR
(400 MHz, CDCl
3
) δ 8.09 – 8.04 (m, 6H), 7.96 – 7.92 (m, 6H), 7.83 – 7.80 (m, 4H), 7.64 – 7.23 (m,
24H), 6.22 (t, J = 10.0 Hz, 1H), 5.79 (t, J = 5.6 Hz, 2H), 5.68 (d, J = 6.8 Hz, 2H), 5.64 – 5.55 (m, 2H),
5.51 (s, 1H), 5.42 (t, J = 8.8 Hz, 1H), 5.31 (s, J = 2.0 Hz, 1H), 5.21 – 5.16 (m, 2H), 4.85 (s, 1H), 4.70
(d, J = 7.2 Hz, 2H), 4.65 – 4.61 (m, 2H), 4.50 (dd, J = 12.4, 2.0 Hz, 1H), 4.42 (dd, J = 12.0, 5.6 Hz,
1H), 4.32 – 4.26 (m, 3H), 4.12 – 4.05 (m, 2H), 3.92 – 3.90 (m, 1H), 3.82 – 3.70 (m, 2H), 3.65 – 3.61
(
m, 1H), 3.27 (t, J = 9.2 Hz, 1H), 2.19 (s, 3H), 2.17 – 2.11 (m, 4H), 2.08 (s, 3H), 2.03 (brs, 1H), 1.97
d, J = 11.2 Hz, 1H), 1.87 – 0.75 (m, 31H), 0.69 (d, J = 8.0 Hz, 1H), 0.58 (s, 3H); 13C{ H} NMR (100
1
(
MHz, CDCl
3
) δ 174.7, 170.6, 170.5, 169.9, 167.5, 166.5, 166.1, 165.9, 165.7 (2 C), 165.5, 165.4,
1
1
7
6
2
1
52.3, 133.8, 133.6 133.4, 133.3, 133.2, 133.1, 130.2, 130.1, 130.0, 129.9 (2 C), 129.8, 129.7, 129.3,
29.2 (3 C), 129.1, 128.8, 128.6 (2 C), 128.5 (2 C), 128.4 (3 C), 104.9, 98.5, 97.9, 95.5, 92.5, 86.2,
7.4, 76.7, 76.3, 75.6, 74.4, 73.4, 72.1, 71.5, 71.1, 70.9, 70.6, 70.0, 69.6, 68.6, 68.0, 67.8, 64.6, 63.4,
2.0, 57.8, 53.5, 50.7, 47.3, 44.0, 43.0, 42.6, 41.1, 40.1, 39.3, 37.3, 37.2, 30.0, 29.8, 29.4, 22.8, 22.7,
108
H O
33K [M+K]+
1.5, 21.1, 21.0, 20.8, 19.8, 19.2, 18.0, 16.2, 14.2; HRMS (ESI) calcd for C106
947.6404, found 1947.6358.
Proposed Reb-S (1)
To a solution of 14 (52 mg, 0.0258 mmol) in absolute MeOH (4 mL) was added freshly prepared
NaOMe (in absolute MeOH, 1 mL) at room temperature. The resulting solution was stirred at the same
+
temperature for another 1 h before resins (H form) was added to adjust the pH value of the reaction
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mixture to around 7.0. Filtration, washing the resins thoroughly with methanol, and filtrate
concentration in vacuo provided a residue, which was further purified by RP-18 silica gel column
chromatography (H
2
O/MeOH = 1 : 2) to furnish the proposed Reb-S (1) (22 mg, 0.023 mmol, 89%) as
2
5
1
a white solid: [α]
D
= − 13.6 (c 1.0, CH
3
OH); H NMR (400 MHz, pyridine-d
5
) δ 6.45 (s, 1H), 6.28 (d,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
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0
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0
1
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9
0
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7
8
9
0
J = 7.7 Hz, 1H), 6.16 (d, J = 3.8 Hz, 1H), 5.69 (s, 1H), 5.23 – 5.19 (m, 1H), 5.13 (d, J = 7.5 Hz, 1H),
.05 (s, 1H), 4.75 (d, J = 3.2 Hz, 1H), 4.66 (t, J = 9.1 Hz, 1H), 4.55 – 4.14 (m, 17H), 3.88 (s, 1H), 3.67
s, 1H), 2.68 (d, J = 13.2 Hz, 1H), 2.57 – 2.51(m, 2H), 2.30 – 2.23 (m, 1H), 2.15 – 2.04 (m, 4H), 1.93 –
5
(
1
0
1
7
2
.85 (m, 2H), 1.77 – 1.62 (m, 6H), 1.49 (s, 3H), 1.44 – 1.24 (m, 2H), 1.16 (s, 3H), 1.01 – 0.97 (m, 3H),
.89 (d, J = 5.2 Hz, 1H), 0.74 – 0.68 (m, 1H); 13C{ H} NMR (100 MHz, pyridine-d
1
) δ 176.0, 155.0,
5
04.8, 101.8, 99.9, 99.2, 93.9, 86.4, 79.8, 79.7, 79.0, 77.7, 77.4, 76.7, 75.8, 74.5, 74.1, 73.8, 72.7, 72.5,
2.1, 71.3, 70.1, 62.8, 62.7, 62.4, 58.2, 54.1, 48.0, 46.5, 44.6, 44.4, 42.7, 41.8, 40.8, 39.8, 37.8, 37.1,
]+ 968.4697,
9.5, 22.2, 20.7, 20.0, 19.1, 16.9, 11.6, 7.6; HRMS (ESI) calcd for C44H74NO22 [M+NH
4
found 968.4708.
p-Methoxyphenyl 2-O-benzoyl-3-O-allyl-4,6-O-benzylidene-β-D-glucopyranoside (16)
To a solution of 15 (1.3 g, 3.14 mmol) in dry pyridine was added BzCl (0.6 mL, 5.17 mmol) dropwise
o
at 0 C under N
2
atmosphere. After the addition was completed, the reaction mixture was warmed up to
room temperature gradually and the stirring was continued for another 4 h. Ethyl acetate was added to
dilute the reaction mixture and the resulting solution was washed with water, 1N HCl, saturated
3 2 4
aqueous NaHCO , and brine successively. After being dried over anhydrous Na SO and filtrating to
remove the desiccant, the filtrate was concentrated in vacuo to provide the crude product, which was
further purified by silica gel chromatography (petroleum ether/ethyl acetate = 5 : 1) to afford 16 (1.5 g,
2
5
1
2
.89 mmol, 92%) as a white solid: [α]
dd, J = 8.4, 1.2 Hz, 2H), 7.68 – 7.64 (m, 1H), 7.55 – 7.53 (m, 4H), 7.42 – 7.37 (m, 3H), 6.94 (d, J =
.2 Hz, 2H), 6.79 (d, J = 8.8 Hz, 2H), 5.76 – 5.72 (m, 2H), 5.45 – 5.38 (m, 2H), 5.15 (dd, J = 17.2, 2.0
Hz, 1H), 4.96 (dd, J = 10.4, 1.6 Hz, 1H), 4.40 – 4.32 (m, 2H), 4.18 – 4.12 (m, 1H), 4.06 (t, J = 9.2 Hz,
D
3 6
= −0.9 (c 1.0, CHCl ); H NMR (400 MHz, acetone-d ) δ 8.11
(
9
1
3
1
1
1
6
H), 3.94 – 3.88 (m, 2H), 3.85 – 3.79 (m, 1H), 3.71 (s, 3H); C NMR (100 MHz, acetone-d ) δ 165.7,
56.6, 152.2, 139.0, 136.2, 134.3, 131.1, 130.5, 129.7, 129.6, 129.0, 127.2, 119.2, 116.5, 115.4, 102.0,
+
01.7, 82.1, 79.6, 74.4, 73.8, 69.2, 67.3, 55.9; HRMS (ESI) calcd for C30
H
30
O
8
K [M+K] 557.1572,
found 557.1582.
p-Methoxyphenyl 2-O-benzoyl-4,6-O-benzylidene-β-D-glucopyranoside (17)
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To a solution of 16 (1.5 g, 2.89 mmol) in a mixed solvent of CH
2 2
Cl and MeOH (40 mL, v/v = 1 : 1)
was added PdCl (154 mg, 0.87 mmol) at room temperature. The black suspension was stirred at the
2
same temperature for 1 h before filtration through a pad of Celite and silica gel was adopted to remove
the catalyst. The filtrate was condensed in vacuo to yield a residue, which was further purified by silica
gel chromatography (petroleum ether/ethyl acetate = 3 : 1) to afford 17 (900 mg, 1.88 mmol, 65%) as a
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1
white solid: [α]
H), 7.64 (t, J = 7.6 Hz, 1H), 7.54 – 7.50 (m, 4H), 7.42 – 7.35 (m, 3H), 6.94 (d, J = 9.2 Hz, 2H), 6.80
d, J = 9.2Hz, 2H), 5.71 (s, 1H), 5.41 – 5.35 (m, 2H), 5.05 (d, J = 4.8 Hz, 1H), 4.36 (dd, J = 10.0, 3.6
D
3 6
= − 12.3 (c 0.5, CHCl ); H NMR (400 MHz, acetone-d ) δ 8.09 (dd, J = 8.0, 1.6 Hz,
2
(
13
1
Hz, 1H), 4.22 – 4.16 (m, 1H), 3.92 – 3.85 (m, 1H), 3.82 – 3.75 (m, 2H), 3.71 (s, 3H). C{ H} NMR
100 MHz, acetone-d ) δ 165.9, 156.6, 152.3, 139.1, 134.1, 131.3, 130.5, 129.7, 129.5, 128.9, 127.4,
19.2, 115.4, 102.4, 101.8, 81.9, 75.9, 72.6, 69.2, 67.5, 55.9; HRMS (ESI) calcd for
[M+NH
]+ 496.1966, found 496.1962.
p-Methoxyphenyl
(
6
1
C
27
H
30NO
8
4
2-O-benzoyl-3-O-(2,3,4,6-tetra-O-benzoyl-β-D-glucopyranosyl)-4,6-O-benzylidene-β-D-glucopyran
oside (19)
Similar procedure as that used for the synthesis of 11 was adopted to conduct the coupling between 17
(380 mg, 0.794 mmol) and 18 (882.5 mg, 1.191 mmol) to provide 19 (707 mg, 0.669 mmol, 84%) as a
2
5
1
white foam: [α]
D
3 6
= +26.0 (c 1.0, CHCl ); H NMR (400 MHz, acetone-d ) δ 8.00 (dd, J =8.4, 1.2 Hz,
2
7
H), 7.84 (dd, J =7.2, 1.2 Hz, 4H), 7.72 (dd, J =8.0, 1.2 Hz, 2H), 7.63 – 7.37 (m, 15H), 7.32 – 7.23 (m,
H), 6.84 (d, J =9.2 Hz, 2H), 6.74 (d, J =9.2 Hz, 2H), 5.87 (t, J =9.6 Hz, 1H), 5.80 (s, 1H), 5.70 (t, J
=
9.6 Hz, 1H), 5.53 – 5.46 (m, 3H), 5.33 (d, J = 7.6 Hz, 1H), 4.59 (t, J =9.2 Hz, 1H), 4.54 (dd, J =12.0,
3
.2 Hz, 1H), 4.44 (dd, J =12.0, 4.4 Hz, 1H), 4.38 (dd, J =10.0, 4.8 Hz, 1H), 4.34 – 4.30 (m, 1H), 4.06
1
3
1
(
t, J =9.2 Hz, 1H), 3.94 (t, J =9.6 Hz, 1H), 3.89 – 3.79 (m, 1H), 3.68 (s, 3H); C{ H} NMR (100 MHz,
acetone-d ) δ 166.4, 166.1, 165.8 165.5, 165.3, 156.5, 152.1, 138.8, 134.4, 134.2, 134.0 (2 C), 133.8,
30.8, 130.6, 130.5 (2 C), 130.4, 130.3, 130.2 (2 C), 130.1, 130.0, 129.6, 129.4, 129.3 (3 C), 129.1,
6
1
1
6
2
28.8, 127.2, 119.1, 115.3, 108.4, 102.0, 101.7, 101.4, 80.0, 79.6, 74.3, 74.2, 73.1, 72.5, 70.8, 69.1,
+
7.6, 67.4, 64.0, 55.8, 24.6; HRMS (ESI) calcd for C61
H
52
O
17K [M+K] 1095.2836, found 1095.2859.
-O-benzoyl-3-O-(2,3,4,6-tetra-O-benzoyl-β-D-glucopyranosyl)-4,6-O-benzylidene-D-glucopyranos
yl o-cyclopropylethynylbenzoate (20)
Similar procedure as that used for the synthesis of 2 was adopted to convert 19 (707 mg, 0.669 mmol)
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to 20 (450 mg, 0.402 mmol, 60% for 2 steps) as a mixture of α/β epimers (α/β = 1 : 2). An aliquot of
2
5
1
pure 20β was obtained for detailed characterization as a white foam: [α]
NMR (400 MHz, acetone-d ) δ 8.02 (dd, J = 8.0, 1.2 Hz, 2H), 7.86 (dd, J = 8.0, 1.2 Hz, 2H), 7.81 (dd,
J = 8.4, 1.2 Hz, 2H), 7.75 (d, J = 8.0 Hz, 1H), 7.72 (dd, J = 8.4, 1.2 Hz, 2H), 7.64 – 7.54 (m, 7H), 7.50
7.35 (m, 10H), 7.32 – 7.21 (m, 8H), 6.21 (d, J = 7.6 Hz, 1H), 5.92 – 5.87 (m, 1H), 5.83 (s, 1H), 5.72
D
3
= +21.6 (c 1.0, CHCl ); H
6
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
–
(t, J = 9.6 Hz, 1H), 5.58 – 5.50 (m, 3H), 4.74 (t, J = 8.8 Hz, 1H), 4.58 (dd, J = 12.0, 3.2 Hz, 1H), 4.47
(dd, J = 12.0, 5.2 Hz, 1H), 4.42 – 4.36 (m, 2H), 4.12 (t, J = 9.2 Hz, 1H), 3.99 – 3.90 (m, 2H), 1.51 –
13
1
1
1
1
1
1
6
.44 (m, 1H), 0.91 – 0.85 (m, 2H), 0.79 – 0.72 (m, 2H); C{ H} NMR (100 MHz, acetone-d ) δ 166.5,
66.1, 165.8, 165.6, 165.5, 163.8, 138.7, 135.0, 134.4, 134.2, 134.0, 133.8, 133.5, 131.3, 130.8, 130.6,
30.5, 130.4, 130.3, 130.2, 130.1 (2 C), 130.0, 129.6, 129.4 (3 C), 129.3, 129.1, 128.8, 128.2, 127.2,
26.2, 102.0, 101.4, 101.0, 93.7, 79.7, 79.4, 74.8, 74.4, 73.6, 73.1, 72.6, 70.8, 69.0, 68.1, 64.0, 9.3, 9.2,
+
54
.2; HRMS (ESI) calcd for C66H O17K [M+K] 1157.2993, found 1157.3006.
13-O-[2-O-Benzoyl-3-O-(2,3,4,6-tetra-O-benzoyl-β-D-glucopyranosyl)-4,6-O-benzylidene-β-D-gluc
opyranosyl]-steviol tert-butyldiphenylsilyl ester (21)
A solution of 20 (36.3 mg, 0.0324 mmol) and 3 (15 mg, 0.027 mmol) in dry CH
2
2
Cl (1.5 mL) was
stirred in the presence of powered 4A MS at room temperature for 30 min under N
2
atmosphere. After
o
being chilled to -40 C, Ph
3
PAuNTf
2
(9.6 mg, 0.013 mmol) was added. The resultant reaction mixture
was then gradually warmed up to room temperature and the stirring was continued for another 5 h at
the same temperature. After the MS was removed by filtration, the filtrate was condensed in vacuo. The
obtained residue was further subjected to purification by silica gel column chromatography (petroleum
2
5
ether/ethyl acetate = 4 : 1) to provide 21 (34 mg, 0.0228 mmol, 84%) as a white solid: [α]
D
= +17.2 (c
1
1
7
2
1
9
1
.0, CHCl
3
); H NMR (400 MHz, acetone-d
6
) δ 7.98 (dd, J = 8.0, 1.2 Hz, 2H), 7.84 – 7.69 (m, 10H),
.63 – 7.22 (m, 28H), 5.84 (t, J = 9.6 Hz, 1H), 5.77 (s, 1H), 5.68 (t, J = 9.6 Hz, 1H), 5.49 – 5.42 (m,
H), 5.25 (dd, J = 9.2, 7.6 Hz, 1H), 5.04 (d, J = 8.0 Hz, 1H), 4.74 (s, 1H), 4.61 (s, 1H), 4.53 (dd, J =
2.0, 3.6 Hz, 1H), 4.50 – 4.42 (m, 2H), 4.32 – 4.28 (m, 1H), 4.17 (dd, J = 10.4, 4.8 Hz, 1H), 3.91 (t, J =
.2 Hz, 1H), 3.85 (t, J = 10.0 Hz, 1H), 3.64 – 3.58 (m, 1H), 2.22 (d, J = 13.2 Hz, 1H), 2.01 (s, 1H),
.97 – 1.90 (m, 2H), 1.86 – 1.59 (m, 6H), 1.47 – 1.28 (m, 5H), 1.27 (s, 3H), 1.15 (s, 9H), 1.08 – 1.04
m, 2H), 0.89 (dd, J = 13.6, 6.8 Hz, 2H), 0.78 (dd, J = 13.2, 3.6 Hz, 1H), 0.72 (s, 3H); 13C{ H} NMR
1
(
(100 MHz, acetone-d
6
) δ 177.0, 166.5, 166.2, 165.8, 165.6, 165.3, 153.5, 139.0, 136.6, 136.5, 134.4,
1
34.3, 134.0 (2 C), 133.8, 133.2, 133.1, 131.1 (2 C), 131.0, 130.9, 130.6, 130.4 (2 C), 130.3, 130.2 ( 2
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C), 130.1, 129.6, 129.5, 129.4 (2 C), 129.1, 128.9, 128.6, 127.3, 105.0, 102.0, 101.4, 97.5, 86.7, 80.4,
7
3
1
1
9.9, 74.8, 74.4, 73.2, 72.6, 71.0, 69.5, 67.4, 64.1, 57.6, 54.5, 48.0, 46.0, 45.1, 43.2, 42.1, 41.3, 40.2,
92
H O
18SiNa [M+Na]+
9.4, 37.4, 27.6, 22.9, 20.9, 20.2, 19.9, 17.0; HRMS (ESI) calcd for C90
511.5945, found 1511.5981.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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2
3
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7
8
9
0
1
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9
0
3-O-[2-O-Benzoyl-3-O-(2,3,4,6-tetra-O-benzoyl-β-D-glucopyranosyl)-4,6-O-benzylidene-β-D-gluc
opyranosyl]-steviol (22)
Similar procedure as that used for the synthesis of 13 was adopted to convert 21 (190 mg, 0.128 mmol)
25
1
to 22 (150 mg, 0.12 mmol, 94%) as a white foam: [α]
acetone-d ) δ 7.99 (dd, J = 8.4, 1.2 Hz, 2H), 7.85 – 7.82 (m, 4H), 7.70 (dd, J = 8.4, 1.2 Hz, 2H), 7.65 –
.47 (m, 8H), 7.45 – 7.36 (m, 7H), 7.31 – 7.22 (m, 7H), 5.84 (t, J = 9.6 Hz, 1H), 5.76 (s, 1H), 5.68 (t, J
D
3
= +16.1 (c 3.5, CHCl ); H NMR (400 MHz,
6
7
=
9.6 Hz, 1H), 5.50 – 5.41 (m, 2H), 5.26 (dd, J = 9.2, 7.6 Hz, 1H), 5.05 (d, J = 8.0 Hz, 1H), 4.90 (s,
1
H), 4.71 (s, 1H), 4.53 (dd, J = 13.4, 3.6 Hz, 1H), 4.48 (t, J = 9.6 Hz, 1H), 4.44 (dd, J = 12.0, 4.4 Hz,
1H), 4.32 – 4.28 (m, 1H), 4.24 (dd, J = 10.4, 4.8 Hz, 1H), 3.90 (q, J = 9.2 Hz, 2H), 3.65 – 3.59 (m, 1H),
2.09 – 1.29 (m, 15H), 1.16 – 1.12 (m, 4H), 1.01 – 0.92 (m, 2H), 0.88 (d, J = 8.0 Hz, 1H), 0.79 – 0.71
1
3
1
6
(m, 1H), 0.67 (s, 3H); C{ H} NMR (100 MHz, acetone-d ) δ 179.0, 166.4, 166.1, 165.7, 165.4, 165.1,
152.9, 138.9, 134.3, 134.2, 134.0, 133.9, 133.7, 130.8 (2 C), 130.5, 130.4, 130.3 (2 C), 130.2, 130.1 (2
C), 129.9, 129.5, 129.4, 129.3, 129.2, 129.0, 128.8, 127.2, 105.3, 101.9, 101.3, 97.3, 87.3, 80.3, 79.7,
7
3
1
4.7, 74.3, 73.1, 72.4, 70.8, 69.3, 67.3, 64.0, 57.4, 54.5, 48.3, 44.6, 43.9, 42.7, 42.1, 41.3, 40.0, 38.8,
+
8.4, 22.5, 20.7, 19.9, 15.7; HRMS (ESI) calcd for C74
H
74
O
18K [M+K] 1289.4507, found 1289.4531.
3-O-[2-O-Benzoyl-3-O-(2,3,4,6-tetra-O-benzoyl-β-D-glucopyranosyl)-4,6-O-benzylidene-β-D-gluc
opyranosyl]-steviol
-O-(2,3,4-tri-O-benzoyl-α-L-rhamnopyranosyl)-3,4,6-tri-O-acetyl-β-D-glucopyranosyl ester (23)
Similar procedure as that used for the synthesis of 14 was applied to mediate the coupling between 22
2
(80 mg, 0.064 mmol) and 4 (107.6 mg, 0.13 mmol) to provide 23 (110 mg, 0.055 mmol, 86%) as a
2
5
1
white solid: [α]
D
3 6
= +59.4 (c 1.0, CHCl ); H NMR (400 MHz, acetone-d ) δ 8.09 (dd, J = 8.4, 1.2 Hz,
2
6
5
1
3
H), 7.98 (td, J = 8.0, 1.2 Hz, 4H), 7.86 – 7.80 (m, 6H), 7.73 – 7.68 (m, 4H), 7.62 – 7.23 (m, 29H),
.14 (d, J = 8.0 Hz, 1H), 5.84 (t, J = 9.2 Hz, 1H), 5.76 (s, 1H), 5.71 – 5.59 (m, 5H), 5.49 – 5.42 (m, 3H),
.27 (dd, J = 9.2, 7.6 Hz, 1H), 5.12 (t, J = 9.2 Hz, 1H), 5.04 (d, J = 8.0 Hz, 1H), 4.92 (s, 1H), 4.72 (s,
H), 4.53 (dd, J = 12.4, 3.6 Hz, 1H), 4.48 – 4.40 (m, 3H), 4.32 – 4.23 (m, 4H), 4.19 – 4.13 (m, 2H),
.94 – 3.86 (m, 2H), 3.62 (td, J = 9.6, 4.8 Hz, 1H), 2.25 (d, J = 13.2 Hz, 1H), 2.14 (s, 3H), 2.06 – 1.00
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(
m, 26H), 0.89 (d, J = 8.4 Hz, 1H), 0.77 – 0.71 (m, 1H), 0.68 (s, 3H); 13C{ H} NMR (100 MHz,
DMSO) δ 173.8, 169.9, 169.7, 169.5, 165.4, 165.0 (2 C), 164.8, 164.6 (2 C), 164.5, 164.2, 151.7, 137.5,
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1
7
5
34.1, 133.9, 133.8, 133.6, 133.3, 133.1, 129.3, 129.2 (3 C), 129.1, 129.0, 128.9 (2 C), 128.8, 128.7,
28.6 (2 C), 128.5 (2 C), 128.4, 128.3, 128.2, 127.8, 126.0, 104.5, 100.1, 99.8, 96.9, 95.8, 91.1, 86.0,
8.5, 78.4, 75.2, 73.6, 73.5, 73.2, 72.0, 71.1, 71.0, 70.5, 70.0, 69.4, 68.5, 67.9, 66.7, 65.8, 62.8, 61.9,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
6
7
8
9
0
1
2
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6.8, 52.8, 47.1, 43.3, 41.5, 40.8, 38.7, 37.3, 36.7, 29.0, 28.5, 20.9, 20.6, 20.5, 19.6, 18.8, 17.5, 15.5;
+
HRMS (ESI) calcd for C113
H
112
O
33Na [M+Na] 2019.6978, found 2019.7010.
Reb-S analogue (24)---derived from 23
2 2
To a solution of 23 (56 mg, 0.028 mmol) in a mixed solvent of CH Cl and MeOH (2 mL, v/v = 1 : 1)
was added p-TsOH (26.7 mg, 0.155 mmol) at room temperature. The resultant solution was stirred at
the same temperature overnight before Et N was added to quench the reaction. The solvent was
3
removed under reduced pressure, and the obtained residue was briefly purified by flash silica gel
column chromatography (petroleum ether/ethyl acetate = 1 : 1) to provide diol intermediate, which was
not characterized in detail and was applied directly to the next step.
The above obtained diol intermediate was then dissolved in absolute methanol (4 mL), to which the
freshly prepared NaOMe (in absolute methanol, 1 mL) was added at room temperature. The reaction
mixture was stirred at the same temperature for 1 h, which was followed by addition of resins (H+
form) to adjust the pH value of the reaction mixture to around 7. Filtration was followed by a through
washing of the resins with methanol. The filtrate was then condensed in vacuo, and the resulting
residue was purified by RP-C18 silica gel column chromatography (H
2
O/MeOH = 1 : 2) to yield 24 (16
2
5
1
mg, 0.0168 mmol, 60% for 2 steps) as a white solid: [α]
D
= − 36 (c 0.5, CH
3
OH); H NMR (400
MHz, pyridine-d
5
) δ 6.47 (s, 1H), 6.31 (d, J = 7.9 Hz, 1H), 5.66 (s, 1H), 5.35 (d, J = 7.8 Hz, 1H), 5.07
(
s, 1H), 5.04 (s, 1H), 4.76 (s, 1H), 4.63 – 4.49 (m, 3H), 4.45 (t, J = 7.9 Hz, 1H), 4.40 – 4.02 (m, 15H),
3
.94 (d, J = 8.7 Hz, 1H), 3.75 (s, 1H), 2.69 (d, J = 13.2 Hz, 1H), 2.48 (d, J = 11.0 Hz, 1H), 2.24 – 2.01
(
m, 6H), 1.90 (m, 2H), 1.78 (d, J = 6.1 Hz, 3H), 1.65 – 1.56 (m, 3H), 1.51 (s, 3H), 1.48 – 1.23 (m, 3H),
1
1
.12 (brs, 4H), 1.01 (d, J = 11.8 Hz, 1H), 0.91 (brs, 1H), 0.80 – 0.66 (m, 1H); 13C{ H} NMR (100
MHz, pyridine-d
5
) δ 175.9, 153.7, 106.1, 105.2, 101.7, 99.3, 93.9, 88.9, 87.0, 79.7, 78.9, 78.8, 78.4,
7
4
7.8, 76.6, 75.8, 74.3, 74.1, 72.7, 72.5, 71.78, 71.3, 70.1, 70.0, 62.7, 62.5, 62.3, 58.2, 54.1, 48.4, 44.7,
4.4, 42.2, 41.8, 40.8, 39.9, 38.7, 37.9, 29.4, 22.2, 20.7, 20.0, 19.1, 16.8; HRMS (ESI) calcd for
]+ 968.4697, found 968.4696.
44 4
C H74NO22 [M+NH
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3-O-[2-O-Levulinyl-3-O-(2,3,4,6-tetra-O-benzoyl-β-D-glucopyranosyl)-4,6-O-benzylidene-β-D-glu
copyranosyl]-steviol (26)
Similar procedure as that used for the synthesis of 13 was applied to transform 25 (200 mg, 0.135
2
5
1
mmol) to 26 (140 mg, 0.112 mmol, 83%) as a white solid: [α]
MHz, CDCl ) δ 8.00 (d, J = 8.0 Hz, 2H), 7.95 (d, J = 7.7 Hz, 2H), 7.86 (d, J = 8.0 Hz, 2H), 7.79 (d, J =
.4 Hz, 2H), 7.69 – 7.21 (m, 17H), 6.06 (t, J = 9.2 Hz, 1H), 5.75 – 5.70 (m, 2H), 5.48 – 5.43 (m, 2H),
D
3
= +17.6 (c 1.0, CHCl ); H NMR (400
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
8
4
1
2
1
.99 (t, J = 8.3 Hz, 1H), 4.89 (d, J = 8.0 Hz, 2H), 4.76 (s, 1H), 4.56 – 4.44 (m, 2H), 4.36 – 4.32 (m,
H), 4.28 (t, J = 9.1 Hz, 1H), 4.19 (dd, J = 10.3, 4.8 Hz, 1H), 3.80 (m, 2H), 3.58– 3.52 (m, 1H), 2.68 –
.40 (m, 4H), 2.14 (brs, 4H), 2.08 (d, J = 2.8 Hz, 1H), 2.05 – 2.02 (m, 1H), 2.01 – 1.74 (m, 7H), 1.67 –
13
1
.25 (m, 6H), 1.19 (s, 3H), 1.12 – 0.96 (m, 2H), 0.97 (s, 3H), 0.93 – 0.80 (m, 2H); C{ H} NMR (100
) δ 206.5, 179.1, 171.4, 166.3, 166.1, 165.7, 153.3, 138.8, 134.3, 134.1 (2 C), 133.9,
30.7, 130.5 (2 C), 130.4, 130.3, 130.1, 130.0 (2 C), 129.4, 129.3, 129.2, 128.7, 127.1, 105.1, 101.8,
100.7, 97.1, 86.6, 80.2, 79.0, 74.4, 74.3, 73.5, 72.4, 70.9, 69.2, 66.9, 63.9, 57.3, 54.5, 48.1, 44.4, 44.0,
43.0, 42.0, 41.4, 40.1, 38.7, 38.2, 37.8, 28.5, 22.6, 20.9, 19.9, 16.1; HRMS (ESI) calcd for C72
[M+NH
]+ 1262.5319, found 1262.5317.
MHz, CDCl
3
1
H
80NO19
4
13-O-[2-O-Levulinyl-3-O-(2,3,4,6-tetra-O-benzoyl-β-D-glucopyranosyl)-4,6-O-benzylidene-β-D-glu
copyranosyl]-steviol
2
-O-(2,3,4-tri-O-benzoyl-α-L-rhamnosyl)-3,4,6-tri-O-acetyl-β-D-glucopyranosyl ester (27)
Similar procedure as that used for the synthesis of 14 was applied to mediate the coupling between 26
(76 mg, 0.061 mmol) and 4 (101 mg, 0.122 mmol) to provide 27 (100 mg, 0.05 mmol, 82%) as a white
2
5
1
solid: [α]
D
3 3
= +70.3 (c 1.0, CHCl ); H NMR (400 MHz, CDCl ) δ 8.09 (d, J = 7.7 Hz, 2H), 8.01 –
7
5
1
7
3
2
1
1
8
.94 (m, 6H), 7.87 (d, J = 7.8 Hz, 2H), 7.81 – 7.78 (m, 4H), 7.71 – 7.24 (m, 26H), 6.10 – 6.05 (m, 2H),
.75 – 5.68 (m, 4H), 5.64 (s, 1H), 5.58 (t, J = 9.1 Hz, 1H), 5.51 – 5.44 (m, 3H), 5.13 (t, J = 9.5 Hz,
H), 5.00 (t, J = 8.3 Hz, 1H), 4.92 – 4.89 (m, 2H), 4.76 (s, 1H), 4.57 – 4.42 (m, 3H), 4.36 – 4.17 (m,
H), 3.81 (t, J = 9.0 Hz, 2H), 3.60 – 3.54 (m, 1H), 2.68 – 2.44 (m, 4H), 2.32 – 2.28 (m, 1H), 2.16 (s,
H), 2.15 (s, 3H), 2.11 (d, J = 6.4 Hz, 1H), 2.04 – 0.76 (m, 30H); 13C{ H} NMR (100 MHz, CDCl
1
) δ
3
06.6, 175.3, 171.6, 170.7, 170.6, 170.3, 166.5, 166.2, 166.1, 165.9, 165.8, 153.5, 138.9, 134.7, 134.5,
34.4, 134.3, 134.2 (2 C), 134.0, 130.8, 130.6 (2 C), 130.5 (2 C), 130.4 (2 C), 130.3, 130.2 (4 C),
30.1, 129.8, 129.6, 129.5, 129.4 (3 C), 129.3, 128.8, 127.3, 105.1, 101.9, 100.9, 98.5, 97.4, 93.0, 86.8,
0.3, 79.2, 77.0, 75.0, 74.5, 74.4, 73.6, 72.8, 72.6, 72.4, 71.5, 71.0, 70.8, 69.6, 69.4, 68.3, 67.0, 64.0,
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2.9, 58.5, 54.5, 48.3, 44.7, 44.2, 43.2, 42.1, 41.2, 40.3, 38.4, 38.3, 38.1, 29.6 28.7, 22.3, 21.1, 21.0,
+
0.8, 20.1, 18.4, 17.2; HRMS (ESI) calcd for C111
H
115
O
34 [M+H] 1991.7264, found 1991.7267.
Reb-S analogue (24)---derived from 27
Similar procedures as those applied for the synthesis of 24 from 23 were applied to convert 27 (53 mg,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
3
3
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3
4
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4
4
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4
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5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
0
.0266 mmol) to 24 (18 mg, 0.0189 mmol, 71% for 2 steps), whose H NMR data were shown to be
identical to those of 24 derived from 23.
p-Methoxyphenyl
2
-O-(2,3,4,6-tetra-O-benzoyl-β-D-glucopyranosyl)-3-O-allyl-4,6-O-benzylidene-β-D-glucopyranosi
de (28)
o
Except for the reaction temperature (-30 C), similar procedure as that used for the synthesis of 11 was
applied to mediate the coupling between 15 (50 mg, 0.121 mmol) and 18 (134.8 mg, 0.182 mmol) to
25
provide disaccharide 28 (114 mg, 0.115 mmol, 95%) as a white foam: [α]
D
3
= −2.4 (c 2.0, CHCl );
1H NMR (400 MHz, acetone-d
6
) δ 8.12 (dd, J = 8.4, 1.6 Hz, 2H), 8.01 (dd, J = 8.0, 1.2 Hz, 2H), 7.93
(dd, J = 8.0, 1.2 Hz, 2H), 7.83 (dd, J = 8.4, 1.2 Hz, 2H), 7.64 (t, J = 7.6 Hz, 1H), 7.58 – 7.31 (m, 16H),
7.11 (d, J = 8.8 Hz, 2H), 6.72 (d, J = 9.0 Hz, 2H), 6.09 (t, J = 9.6 Hz, 1H), 5.93 – 5.80 (m, 2H), 5.66 –
5.59 (m, 3H), 5.22 (d, J = 7.6 Hz, 1H), 5.13 (dd, J = 17.2, 2.0 Hz, 1H), 4.99 (dd, J = 10.4, 1.6 Hz, 1H),
4.53– 4.42 (m, 3H), 4.33 (dd, J = 10.0, 4.0 Hz, 1H), 4.20 – 4.15 (m, 1H), 4.07 – 3.99 (m, 2H), 3.78 (t, J
9.2 Hz, 1H), 3.71 (dd, J = 5.6, 4.0 Hz, 3H), 3.68 (s, 3H); 13C{ H} NMR (100 MHz, acetone-d
1
) δ
=
6
1
1
8
66.3, 166.1, 165.7, 155.9, 152.3, 138.8, 136.4, 134.2, 134.1, 133.9, 130.8, 130.4 (2 C), 130.3 (2 C),
30.1, 130.0, 129.9, 129.5, 129.3 (2 C), 129.2, 128.8, 126.9, 118.3, 116.3, 115.2, 101.8, 101.6, 101.2,
53 16
1.9, 81.4, 81.05, 74.4, 74.0, 73.4, 72.4, 70.2, 69.1, 66.2, 63.2, 55.8; HRMS (ESI) calcd for C57H O
+
[M+H] 993.3328, found 993.3365.
2
-O-(2,3,4,6-Tetra-O-benzoyl-β-D-glucopyranosyl)-3-O-allyl-4,6-O-benzylidene-D-glucopyranosyl
o-cyclopropylethynylbenzoate (29)
Similar procedure as that applied for the synthesis of 2 was applied to convert 28 (400 mg, 0.403
mmol) to 29 (352 mg, 0.334 mmol, 83% for two steps) as a mixture of α/β isomers (α/β = 1 : 1.3). An
2
5
1
aliquot of pure 29α was obtained for detailed characterization: [α]
400 MHz, acetone-d ) δ 8.07 (dd, J = 8.4, 1.6 Hz, 2H), 8.01 – 7.91 (m, 5H), 7.81 (dd, J = 8.0, 1.2 Hz,
H), 7.65 – 7.53 (m, 3H), 7.49 – 7.27 (m, 18H), 6.82 (d, J = 3.6 Hz, 1H), 6.07 (t, J = 9.2 Hz, 1H), 5.77
D
3
= −32.8 (c 1.0, CHCl ); H NMR
(
6
1
(t, J = 9.6 Hz, 1H), 5.66 (s, 1H), 5.64 – 5.52 (m, 3H), 5.00 (dd, J = 17.6, 2.0 Hz, 1H), 4.82 (dd, J = 10.4,
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.0 Hz, 1H), 4.69 (t, J = 4.0 Hz, 2H), 4.59 (dt, J = 10.0, 3.6 Hz, 1H), 4.27 (dd, J = 10.0, 4.8 Hz, 1H),
.19– 3.96 (m, 5H), 3.78 (t, J = 10.0 Hz, 1H), 3.74 (t, J = 9.6 Hz, 1H), 1.68 – 1.61 (m, 1H), 0.79 – 0.72
13
1
(
m, 4H); C{ H} NMR (100 MHz, acetone-d
34.4, 134.3, 133.9, 132.8, 132.2, 131.9, 131.0, 130.5 (2 C), 130.4 (2 C), 130.2, 130.1, 129.9, 129.6,
29.4 (2 C), 129.3, 128.9, 128.2, 126.9, 125.3, 116.1, 102.8, 101.8, 100.3, 93.2, 82.1, 80.4, 77.8, 75.7,
6
) δ 166.5, 166.2, 165.8, 165.6, 165.2, 138.8, 136.3, 135.2,
1
1
7
1
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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6
7
8
9
0
54
H O
16K [M+K]+
4.2, 74.1, 73.0, 72.7, 70.3, 69.2, 65.7, 63.5, 9.5, 9.4, 1.4; HRMS (ESI) calcd for C62
093.3044, found 1093.3055.
3-O-[2-O-(2,3,4,6-Tetra-O-benzoyl-β-D-glucopyranosyl)-3-O-allyl-4,6-O-benzylidene-β-D-glucop
yranosyl]-steviol tert-butyldiphenylsilyl ester (30β)
Similar procedure as that used for the synthesis of 12β was applied to mediate the coupling between 29
(22.7 mg, 0.0215 mmol) and 3 (10 mg, 0.0179 mmol) to provide 30β (21 mg, 0.0147 mmol, 82%) as a
white solid. The data was in good accordance with reported data.9a
13-O-[2-O-(2,3,4,6-Tetra-O-benzoyl-β-D-glucopyranosyl)-3-O-allyl-4,6-O-benzylidene-β-D-glucop
yranosyl]-steviol (31)
Similar procedure as that used for the synthesis of 13 was applied to convert 30 (208 mg, 0.146 mmol)
2
5
1
to 31 (140 mg, 0.118 mmol, 81%) as a white solid: [α]
acetone-d ) δ 8.06(dd, J = 8.2, 1.6 Hz, 2H), 7.98 (dd, J = 8.0, 1.2 Hz, 2H), 7.93 (dd, J = 8.4, 1.2 Hz,
H), 7.88 (dd, J = 8.4, 1.2 Hz, 2H), 7.65 – 7.31 (m, 17H), 6.06 – 5.96 (m, 2H), 5.75 (t, J = 9.6 Hz, 1H),
D
3
= − 33.1 (c 0.5, CHCl ); H NMR (400 MHz,
6
2
5
1
.60 – 5.54 (m, 3H), 5.22 – 5.16 (m, 2H), 5.06 – 5.02 (m, 1H), 4.82 (d, J = 7.6 Hz, 1H), 4.69 (dd, J =
2.0, 2.8 Hz, 2H), 4.58 (dd, J = 12.0, 4.4 Hz, 1H), 4.52 – 4.48 (m, 1H), 4.28 – 4.23 (m, 1H), 4.16 (dd, J
=
10.4, 4.8 Hz, 1H), 4.04 – 3.98 (m, 1H), 3.85 – 3.76 (m, 1H), 3.72 (t, J = 10.2 Hz, 1H), 3.62 – 3.53 (m,
2
7
H), 3.46 – 3.40 (m, 1H), 2.28 (dd, J = 11.6, 2.8 Hz, 1H), 2.17 (d, J = 13.2 Hz, 1H), 2.03 – 1.68 (m,
H), 1.64 – 1.37 (m, 6H), 1.29 (s, 1H), 1.22 (s, 3H), 1.11 (dd, J = 12.0, 2.8 Hz, 1H), 1.04 (s, 3H), 0.98
d, J = 8.0 Hz, 1H), 0.91 – 0.82 (m, 2H); 13C{ H} NMR (100 MHz, Acetone) δ 178.9, 166.4, 166.1,
1
(
1
1
8
4
1
1
66.0, 165.5, 163.8, 153.6, 138.9, 136.6, 134.4, 134.2 ( 2 C), 133.8, 130.8, 130.4 ( 3 C), 130.3 ( 2 C),
30.0, 129.9, 129.4, 129.3 ( 2 C), 129.2, 128.7, 126.9, 116.1, 108.2, 105.0, 101.6, 101.5, 97.4, 86.7,
2.4, 79.8, 74.3, 73.9, 73.5, 72.4, 70.9, 69.4, 67.5, 66.1, 64.0, 57.4, 54.7, 48.2, 44.6, 44.0, 42.9, 42.2,
]+
1.4, 40.2, 38.8, 38.1, 24.5, 22.7, 21.0, 19.9, 16.3; HRMS (ESI) calcd for C70
204.5254, found 1204.5260.
H78NO17 [M+NH
4
3-O-[2-O-(2,3,4,6-Tetra-O-benzoyl-β-D-glucopyranosyl)-3-O-allyl-4,6-O-benzylidene-β-D-glucop
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yranosyl]-steviol 2-O-(2,3,4-tri-O-benzoyl-α-L-rhamnosyl)-3,4,6-tri-O-acetyl-β-D-glucopyranosyl
ester (32)
Similar procedure as that used for the synthesis of 14 was adopted to mediate the condensation between
3
1 (80 mg, 0.067 mmol) and 4 (110.9 mg, 0.134 mmol) to provide 32 (115 mg, 0.059 mmol, 88%) as a
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
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0
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9
0
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3
4
5
6
7
8
9
0
2
5
1
white solid: [α]
D
3 6
= +31.0 (c 1.0, CHCl ); H NMR (400 MHz, acetone-d ) δ 8.11 – 8.05 (m, 4H), 8.02
(dd, J = 8.4, 1.6 Hz, 2H), 7.99 – 7.91 (m, 4H), 7.86 (d, J = 7.6 Hz, 2H), 7.80 (dd, J = 8.0, 1.2 Hz, 2H),
7
.72 – 7.29 (m, 26H), 6.15 (d, J = 7.8 Hz, 1H), 6.08– 5.95 (m, 2H), 5.79 (t, J = 9.6 Hz, 1H), 5.72 – 5.70
(
m, 2H), 5.65 – 5.53 (m, 5H), 5.50 (brs, 1H), 5.22 – 5.21 (m, 2H), 5.12 (t, J = 9.6 Hz, 1H), 5.06 – 5.02
m, 1H), 4.81 (d, J = 7.4 Hz, 1H), 4.70 – 4.66 (m, 2H), 4.60 (dd, J = 12.1, 4.4 Hz, 1H), 4.53 – 4.45 (m,
(
2
–
H), 4.29 – 4.16 (m, 6H), 4.04 – 3.98 (m, 1H), 3.80 (t, J = 7.9 Hz, 1H), 3.72 (t, J = 10.2 Hz, 1H), 3.64
3.52 (m, 2H), 3.48 – 3.42 (m, 1H), 2.35 – 2.27 (m, 2H), 2.14 (s, 3H), 2.03 – 0.80 (m, 33H); 13C{ H}
1
6
NMR (100 MHz, acetone-d ) δ 175.3, 170.7, 170.6, 170.2, 166.6, 166.2 (2 C), 166.1, 166.0, 165.8,
165.6, 153.8, 139.0, 136.8, 134.7, 134.4 (2 C), 134.3 (3 C), 133.9, 131.0, 130.6, 130.5 (2 C), 130.4 (3
C), 130.3, 130.2 (3 C), 130.0, 129.8, 129.5, 129.4 (3 C), 128.9, 127.0, 116.2, 105.1, 101.8, 98.6, 97.7,
92.9, 86.8, 82.6 (2 C), 80.0, 76.7, 75.2, 74.5, 74.0, 73.5, 72.8, 72.5, 72.4, 71.5, 71.0, 70.8, 69.5, 68.2,
66.2, 64.1, 62.6, 58.7, 54.7, 48.4, 44.8, 44.3, 43.2, 42.3, 41.3, 40.3, 38.3, 38.2, 22.4, 21.1, 21.0, 20.8,
+
20.2, 18.3, 17.3; HRMS (ESI) calcd for C109
H
112
O
32Na [M+Na] 1955.7029, found 1955.7048.
Revised Reb-S (33)
To a solution of 32 (65 mg, 0.0336 mmol) in a mixed solvent of CH
2 2
Cl and MeOH (4 mL, v/v = 2 : 2)
was added PdCl (1.8 mg, 0.010 mmol) at room temperature. The resultant black suspension was
2
stirred at the same temperature for 35 min before filtrating through a pad of Celite and silica gel. The
filtrate was concentrated under reduced pressure and the resulting residue was then subjected to flash
silica gel column chromatography (petroleum ether/ethyl acetate = 2 : 1) to give the alcohol
intermediate. The above obtained alcohol intermediate was dissolved in a mixed solvent of
CH
2
Cl
2
/MeOH (2 mL, v/v = 1 : 1), to which p-TsOH (25 mg, 0.145 mmol) was added at room
N was
temperature. The resultant mixture was stirred at the same temperature for overnight before Et
3
added to quench the reaction. The solvent was removed under reduced pressure, and the obtained
residue was briefly purified by flash silica gel column chromatography (petroleum ether/ethyl acetate =
1
: 1) to provide triol intermediate, which was not characterized in detail and was applied directly to the
next step. The above obtained triol intermediate was then dissolved in absolute methanol (4 mL), to
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