Mapping mechanisms in glycosylation reactions with donor reactivity: Avoiding generation of side products

Article abstract of DOI:10.1021/acs.joc.0c01313

The glycosylation reaction, which is key for the studies on glycoscience, is challenging due to its complexity and intrinsic side reactions. Thioglycoside is one of the most widely used glycosyl donors in the synthesis of complex oligosaccharides. However, one of the challenges is its side reactions, which lower its yield and limits its efficiency, thereby requiring considerable effort in the optimization process. Herein, we reported a multifaceted experimental approach that reveals the behaviors of side reactions, such as the intermolecular thioaglycon transformation and N-glycosyl succinimides, via the glycosyl intermediate. Our mechanistic proposal was supported by low temperature NMR studies that can further be mapped by utilizing relative reactivity values. Accordingly, we also presented our findings to suppress the generation of side products in solving this particular problem for achieving high-yield glycosylation reactions.

Full text of DOI:10.1021/acs.joc.0c01313

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Article
Mapping Mechanisms in Glycosylation Reactions with
Donor Reactivity: To Avoid Generation of Side Products
Chun-Wei Chang, Mei-Huei Lin, Chia-Hui Wu, Tsun-Yi Chiang, and Cheng-Chung Wang
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.0c01313 • Publication Date (Web): 14 Aug 2020
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Mapping Mechanisms in Glycosylation Reactions with Donor Reactivity: To
Avoid Generation of Side Products
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Chun-Wei Chang,a,b,c Mei-Huei Lin,a Chia-Hui Wu,a Tsun-Yi Chiang,a Cheng-Chung
Wang*a,b
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a Institute of Chemistry, Academia Sinica Taipei 115 (Taiwan)
b Chemical Biology and Molecular Biophysics Program, Taiwan International
Graduate Program (TIGP), Academia Sinica, Taipei 115 (Taiwan)
c Department of Chemistry, National Taiwan University Taipei 106 (Taiwan)
wangcc@chem.sinica.edu.tw
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11 Table of contents
O
NXS (X= I, Br, Cl)/
TfOH
HO
STol
O
O
PGO
PGO
STol
OTf
(Reactivity was quantified by RRV)
byproducts
X
Directing
S
H
N
+
O
O
O
PGO
O
O
STol
X= I, Br, Cl
Intermolecular side reaction
O
O
or
O
N
PGO
PGO
STol
O
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ABSTRACT
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The glycosylation reaction, which is key for the studies on glycoscience, is
challenging due to its complexity and intrinsic side reactions. Thioglycoside is one
of the most widely used glycosyl donors in the synthesis of complex
oligosaccharides. However, one of the challenges is its side reactions, which lowers
its yield and limits its efficiency, thereby requiring considerable effort in the
optimization process. Herein, we reported a multifaceted experimental approach
that reveals the behaviors of side reactions, such as the intermolecular thioaglycon
transformation and N-glycosyl succinimides, via the glycosyl intermediate. Our
mechanistic proposal was supported by low temperature NMR studies that can
further be mapped by utilizing relative reactivity values (RRVs). Accordingly, we
also presented our findings to suppress the generation of side products in solving
this particular problem for achieving high-yield glycosylation reactions.
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INTRODUCTION
In the past decades, chemical approaches have provided a significant advantage
for accessing high-quality carbohydrate-based molecules in large quantities.1-4 The
creation and development of newly synthetic methodologies have given a direct
approach to prepare and manipulate a vast variety of glycoconjugate molecules for
glycoscience.5-8 Glycans have been built sequentially by linking numerous saccharide
building blocks to construct O-glycosidic linkages through chemical or enzymatical
glycosylation reactions.9-13 Chemical approaches show higher flexibility on the variety
of sugar types and functional group modifications; however, the main challenges of
chemical glycosylation between the glycosyl donor and glycosyl acceptor are to achieve
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high yield and stereoselectivity, which are known to be affected by numerous factors.2-
4,14-20
Among the glycosyl donors reported, thioglycosides 1 are one of the most
commonly used donors as they exhibit a high tolerance toward protecting group
manipulations (Scheme 1).21-24 Meanwhile, thioglycoside donors can be
chemoselectively activated by various electrophilic promoters to give the desired
product in good yield, such as N-halosuccinimide (NXS)/trifluoromethanesulfonic acid
(TfOH), 1-benzenesulfinyl piperidine (BSP)/trifluoro-methanesulfonic anhydride
(Tf2O), and p-toluenesulfenyl chloride (TolSCl)/silver triflate (AgOTf).23-25 The
reactivity of thioglycosides 1 are highly associated with their protecting group patterns
and conformation. Wong developed a relative reactivity values (RRVs) system to
optimize the combination of building blocks for one-pot oligosaccharide synthesis.26-30
Subsequently, Yeh, Huang and Yoshida developed a preactivation strategy that allows
an iterative activation after the addition of 3 to give the product 4.31-40
Despite the significant success of thioglycosides 1 documented in the literature,41-
44
several side reactions are frequently formed depending on various factors, such as
the type of promotor, solvent, temperature, and particular donor/acceptor pair.16,45-47
There are two major problems. First, intermolecular aglycon transfer 7 may be formed
again from triflate intermediate 2 to significantly decrease the yield of the desired O-
glycoside product and quench the reaction.16,33,48-50 Second, an undesired side reaction
of glycosyl triflate 2 to the corresponding stable N-glycosyl succinimide 8 has been
noted upon the promotion using NXS/TfOH and requires a tedious separation
process.16,45-47
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Scheme 1. The suppression of side products for achieving high yield
glycosylation reactions using RRV as an indicator.
O
HO
STol
NXS (X= I, Br, Cl)/
TfOH
O
3
O
PGO
PGO
STol
OTf
2
1
10
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(Reactivity was quantified by RRV)
byproducts
X
H
N
S
O
5-I
5-Br
5-Cl
, X= I
,X = Br
,X = Cl
+
O
O
PGO
O
O
STol
4
6
5
Intermolecular side reaction
O
O
O
or
N
PGO
PGO
STol
O
7
8
Regenerated donor
)
(lower RRV
3
4
5
6
Unfortunately, a suitable explanation that answers how these side reactions will
happen is unclear, which can be attributed to the unclearness of the mechanism of the
reaction itself. The central obstacle is that glycosyl triflate 2, which serves as the main
intermediate in TfOH-mediated thioglycoside reactions, is highly unstable to
analyze.46,51,52 By-products 5 and 6, which are generated through the stoichiometric
amount of NXS 5 required for anomeric leaving group activation,25,53-55 induce the
formation of side products 7-8 and lower the yield of desired 4. Therefore, the lack of
precise mechanistic study leads to indeterminacy, as the reaction yields are drastically
fluctuant, even under slight changes of the protecting group, promotor or temperature.
The unique glycosylation reactions must always be optimized individually, and the
outcome is usually unpredictable.
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Capitalizing on this viewpoint, our laboratory discovered that the RRVs of
thioglycosides 1 provide a new angle to correlate the stereoselectivities and
intermediate changes.42 Herein, we further reported systematically mechanistic studies
that connect the glycosyl triflate intermediate 2 to side products 7-8. The detailed
behavior of the side products can be successfully elucidated, as indicated by the RRVs
of thioglycosides 1. The generation of thioaglycon-transferred thioglycoside 7 is
characterized through the intermolecular process between glycosyl triflate 2 and
tolylsulfenyl halide (TolSX, 5) in each NXS/TfOH system. The RRV platform can be
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utilized for data organizations by a statistical approach. Additionally, it helps with the
establishment of a general protocol to eliminate the formation of side products on
numerous building blocks of thioglycosides to achieve a higher yielding glycosylation
reaction of the desired product 4.
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RESULTS AND DISCUSSION
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Since the presence of glucosamine derivative is essential to the activity of many
natural products,50,56 our work began by controlling preactivation-based glycosylation
on thioglycoside donor 9-α (α/β = 1/0) on the promotion of NIS/TfOH system.40 We
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pre-mixed donor 9-α with NIS (1.0 equiv.) and TfOH (1.0 equiv.) in DCM at -70 C
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for 10 min (Table 1). The acceptor (MeOH, 10) was then introduced in the next step
and further stirred for 4-5 hours (Entry 1). However, the glycosylation reaction only
furnished methyl glycoside 13 with a 31% yield and an α/β ratio of 1/9. Moreover, 44%
of donor 9-β (α/β = 0/1) was recovered with exclusive β-form even after a complete
consumption of the donor 9-α during preactivation. Similar situations also occurred in
other activation systems, such as NBS/TfOH, NCS/TfOH, and TolSCl/AgOTf
promotor systems, as the related glycosylation still recovered large amount of β-donor
9-β in 30-40% yield (Entries 2-4). Even after excess amounts (2.0 equiv.) of
TolSCl/AgOTf promotor was introduced, the regeneration of 9-β increased to 42%
(Entry 5). We also noticed that adding other acceptors still gave similar results, such as
6-OH glucoside 11 and 4-OH glucoside 12. Apart from the corresponding product 14-
15, a considerable amount of 9-β was observed in 43-45% yield.
We initially assumed that generation of 9-β was derived from thioglycoside
epimerization or during aglycon transfer of thioglycosides.16,33,48-50 However, we found
that in a preactivation manner after complete consumption of the donor 9-α the
regeneration of 9-β could still be observed after the introduction of the acceptor. The
regeneration of 9-β was highly associated with the presence of acceptor, because before
the addition of acceptor only the corresponding glycosyl triflate could be observed in
NMR. Therefore, it showed that generation of 9-β underwent a different pathway rather
than a simple thioglycoside epimerization or aglycon transfer of thioglycosides. This
result raised our interest in understanding how the thioglycoside donor is regenerated
and how this side product can be eliminated so as to achieve a high-yield glycosylation
reaction.
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Table 1. Preactivation-based glycosylation on donor 9-α.
1) Promotor
2) Acceptor
Ph
O
Ph
O
Ph
O
O
O
O
O
O
O
+
STol
BnO
BnO
BnO
N3
STol
N3
N3
DCM
-70 oC, 4-5 h
OR
Regenerated donor
9-
Product
9-
 (/ = 1/0)
 (/ = 0/1)
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Entry
1
Promotor (equiv.)
NIS (1),
Acceptor
MeOH 10
Product, yielda(α/β)b 9-β, yielda
13, 31% (1/9.0)
13, 33% (1/11)
13, 34% (1/8.0)
13, 44% (1/8.1)
13, 33% (1/8.0)
14, 30% (1/6.7)
15, 30% (1/3.4)
44%
40%
36%
30%
42%
45%
43%
TfOH (1)
NBS (1),
TfOH (1)
NCS (1),
2
3
4
5
6
7
MeOH 10
MeOH 10
MeOH 10
MeOH 10
TfOH (1)
TolSCl (1),
AgOTf (1)
TolSCl (2),
AgOTf (2)
TolSCl (2),
AgOTf (2)
TolSCl (2),
AgOTf (2)
6-OH Glc 11
4-OH Glc 12
3
aIsolated yield. bDetermined by HPLC
Ph
O
O
O
Ph
O
O
OH
OBn
O
OBn
BnO
O
N3
Ph
O
O
BnO
O
O
O
O
N3
O
BnO
BnO
HO
BnO
O
BnO
BnO
BnO
BnO
N3
BnO
BnO
BnO
OMe
OMe
OMe
OMe
BnO
11
12
13
14
OMe
15
4
5
In light of donor regeneration, we further investigated the glycosylation reactions
on more donors including 16-α (α/β = 1/0), 17-α (α/β = 1/0), 18-β (α/β = 0/1), 19-β (α/β
= 0/1), 9-α (α/β = 1/0) and 9-β (α/β = 0/1) (Table 2). The main purpose of this study
was to clarify the relationship between donor reactivity and the amount of regenerated
donors and other side products. Therefore, we applied RRV of donor as the general
parameter to outline the amount of regenerated donor produced on different donor
building blocks. To simplify the analysis in this investigation, each donor was promoted
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9
10
11
12
13
14
15
16
17
18
19
o
under NIS/TfOH condition in DCM at -70 C, and methyl alcohol (MeOH, 10) was
introduced as the modeling acceptor.
As our expectation, apart from the desired O-methyl glycosides 13, 20-23,
substantial amounts of donors 9-β, 17-α, 18-α, 19-α were regenerated back in 1 hour.
The amount of regenerated donor was gradually eliminated as the reactivity of the donor
kept on increasing (increasing RRVs of donors). It indicated that armed donor under
the reaction conditions transfers faster into corresponding product than disarmed donor.
Therefore, the side effect of donor regeneration was remarkably reduced. For example,
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2-deoxy donors, 16-α and 17-α (Entries 2-3), which had the highest RRV of 1 × 106
and 5 × 105 respectively gave a minimal amount of regenerated donor 17-β (0-8%) and
the isolated yield of its corresponding product 20-21 was up to 83-84%. The activation
of thioglucosides, 18-β and 19-β with a moderated RRV of 17000 and 2656 respectively
turned out the desired O-methyl glucoside 22-23 in a moderate yield (55-64%) due to
the generation of higher amount of regenerated donor 18-α and 19-α (25-36%) (Entries
3-4). With regard to 2-azido-2-deoxy glycosides, 9-α and 9-β (Entries 5-6), abundance
of β-donor, 9-β was recovered with a 42-44% yield and the lowest glycosylation yield
of 13 was given with a 31-34% yield.
3
4
10
11
12
13
14
15
16
17
18
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9
10
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The anomeric configuration of the regenerated donor is associated with the RRV
of both anomers. On 4,6-O-benzylidene donors (Entries 5-6), even when -donor 9-β
(α/β = 0/1) or α-donor 9-α (α/β = 1/0) could be activated individually in the beginning,
a consistent result of β-counterpart 9-β (α/β = 0/1) was regenerated. The RRV clearly
showed that 9-β (RRV = 5.4) was more stable than α-counterpart 9-α (RRV = 314),
suggesting that a regenerated donor towards -anomer 9- was a relatively stable
configuration.57 The influence of benzylidene acetals on anomeric equilibria has been
reported in the literature, but only of O-glycosides.58 For the stability of thioglycosides,
according to the RRV platform, the β-counterpart 9-β is indeed more stable than its α-
isomer 9-α.
To further clarify the anomeric reactivity, Bols et al. have reported that β-anomer
of 9-β is more stable than the 9-α in such conformationally fixed systems.57 Upon
activation of thioglycoside, the sulfonium ion was generated in situ via an
intermolecular iodination reaction from iodonium ion (NIS). Since α-sulfonium ion
performed an axial/pseudoaxial configuration at C1, the lone pairs of electrons present
on O5 can easily participate to the C1 for the activation. The oxocarbenium ion was
then formed together with TolSI via E1 elimination reaction for forming the
oxocarbenium ion.57 However, the activation of β-sulfonium ion was very limited. The
β-sulfonium ion in turn showed an equatorial/pseudoequatorial configuration, which is
not favorable for the E1 elimination.57
The story was completely different with regard to per-O-benzylated donor
(conformationally flexible system) such as galactoside 18-β (Entry 3). The anomeric
configuration of the regenerated donor would turn into the thermodynamically favored
-form [18-α (α/β = 1/0), RRV = 3646) rather than the β-anomer [18-β (α/β = 0/1),
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RRV = 17000). Similarly, α-thioglucoside [19-α (α/β = 1/0), RRV = 727] was more
stable than its β-counterpart [19-β (α/β = 0/1), RRV = 2656] (Entry 4). This is in
agreement with the recent work conducted by Zhu, Demchenko, Boons, and Jensen.59-
62 Their competitive study showed that α-thioglycoside demonstrates a noticeably low
reactivity compared to β-thioglycoside on full benzylated donors. The electron density
of O5 increased due to the electron donating effect of benzyl (Bn) functionality and it
therefore enhances the nucleophilicity of thiotolyl group (-STol) of equatorial anomer
(β-glycoside) to interact with the electrophilic promotor under NIS/TfOH condition.
3
4
10
11
12
13
14
15
16
17
18
19
20
21
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6
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9
10
Table 2. Organization of thioglycoside regeneration using RRV
1) NIS (1.0 equiv.);
TfOH (0.4 equiv.)
O
10
2) MeOH
(1.0 equiv.)
O
O
PGO
PGO
PGO
+
OMe
STol
STol
DCM, - 70 oC, 1h
Donor
Regenerated donor
(lower RRV)
Product
11
Entry
1
Product,
Regenerated donor,
(RRV), yielda
Donor, (RRV)
yielda
OBn
OBn
O
O
BnO
BnO
BnO
BnO
b
-
OMe
STol
/ = 1/0
/ = 1/1.5
16-α, (1×106)
20, 83%
BnO
OBn
BnO
OBn
OBn
BnO
O
O
O
STol
BnO
BnO
BnO
2
3
4
OMe
/ = 1/1.5
STol
/ = 1/0
/ = 0/1
17-β, (3×105), 8%
OBn
17-α, (5×105)
21, 84%
OBn
BnO
OBn
BnO
BnO
O
O
O
STol
BnO
BnO
BnO
OMe
BnO
BnO
STol
/ = 1/0
BnO
/ = 0/1
/ = 1/2.0
18-β, (17000)
OBn
22, 64%
18-α, (3646), 25%
OBn
OBn
O
O
O
BnO
BnO
BnO
BnO
BnO
STol
BnO
BnO
BnO
STol
/ = 1/0
OMe
BnO
/ = 0/1
/ = 1/3.0
19-β, (2656)
23, 55%
19-α, (727), 36%
Ph
Ph
O
Ph
O
O
BnO
O
Ph
O
O
O
O
O
STol
BnO
BnO
N3
N3
N3
OMe
OMe
5
6
STol
/ = 1/0
/ = 0/1
/ = 1/5.7
9-α, (314)
9-β, (5.4), 42%
13, 34%
O
Ph
O
Ph
O
O
O
O
O
O
O
STol
STol
BnO
N3
BnO
N3
/ = 0/1
BnO
N3
/ = 0/1
/ = 1/5.7
9-β, (5.4)
9-β, (5.4), 44%
13, 31%
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9
1
2
Considering the reaction mechanism, we proposed that the formation of
regenerated donor 7 substantially undergoes an intermolecular process (Scheme 2). The
NIS initiated thioglycoside donor 1 under TfOH catalyzation and then furnished the β-
sulfonium ion 25-β. The α-triflate intermediate 2 was then generated in situ as the most
plausible intermediate with the departure of the TolSI 5-I by-product.46,51,52 Both
glycosyl triflate 2 and oxocarbenium ion 26 determined the glycosidic bond formation
with acceptor [ROH, (27)] to produce glycoside 30 through the nucleophilic
substitution.18,63-67
3
4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
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44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5
6
7
8
9
In our present research, we also observed the regenerated donor 7 in the reaction
mixture. Since glycosyl triflate 2 and the corresponding TolSI 5-I byproduct
accumulated using the NIS/TfOH promoter system under pre-mix conditions, we
proposed that 7 was derived from glycosyl sulfonium ion 25. After introducing protic
reagent (either water or acceptor 27), the protic reagent terminated 25 and expelled
either hypoiodate [RO-I, (28-I)] or hypoiodous acid [HO-I (29-I)] to produce the
thioaglycon-transferred side-product 7 (see Scheme 3 and Scheme 4). It was likely that
the protic reagent (water or the acceptor 27) reacted with TolSI 5-I to give either
hypoiodate [RO-I, (28-I)] or hypoiodous acid [HO-I (29-I)] and produced p-thiocresol
[TolSH, (5-H)], which was the nucleophile; nevertheless, we found that 5-H could not
react with glycosyl triflate 2. Therefore, the participation of 5-H as the source of side
product 7 was excluded (see Scheme 5).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
Since the anomeric configuration of 7 was independent from that of 1, an
intermolecular reaction taking place between oxocarbenium ion 26 and TolSI 5-I was
presumably suggested. Interestingly, the regenerated 7 gave exclusive anomeric
selectivities, and a more stable configuration of 7 (lower RRV) was eventually observed
(as summarized in Table 1 and Table 2). However, despite the notable differences in
the RRVs between the α- and β-thioglycoside, the complete selectivity far exceeded the
ratio that would be expected from the thermodynamic control. Since the RRVs of the
regenerated 7-α and 7-β could defer for an order, a plausible explanation was that, after
the reaction between oxocarbenium ion 26 and TolSI 5-I, upon preactivation condition,
the reformed 25 that gave 7 anomer of higher RRV may equilibrate back to 26 faster;
in contrast, the reformed 25 that resulted in 7 anomer of lower RRV equilibrated more
slowly, of which the sulfonium salt was further quenched by protic reagent (water or
the acceptor 27) to give the regenerated 7 of lower RRV (as shown in Figure 1 and
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Table 3). Therefore, the regenerated 7 performed an exclusive anomeric configuration
in the form of lower RRV. The formation of the side product N-glycosyl succinimide 8
was also confirmed as a result of the side reaction of NIS and 2 (see Table 4 and Table
5).
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11
12
13
14
15
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19
20
21
22
23
24
25
26
27
28
29
30
31
32
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35
36
37
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 2. Proposed mechanism for the generation of side products on the
promotion of NIS/TfOH.
#Reactivity was quantified by RRV
O
1#
I
PGO
S
Tol
N
O
O
TfOH
OTf
O
I
NIS
N
O
H
H
O
PGO
N
O
O
24
STol
7
6
Regenerated donor
28-I
29-I
) or HOI (
ROI (
) + TfOH
lower RRV
27
ROH ( ) or H2O
O
PGO
Tol
S
Higher RRV
TolSI
5-I
25
I
TolSI
5-I
TolSI
5-I
TolSI
5-I
Tol
O
O
O
PGO
PGO
PGO
S
I
26
26
OTf
OTf
25-

TolSI
5-I
O
PGO
2
OTf
TolSH
5-H
ROH
27
O
NIS
O
N
PGO
O
O
PGO
8
7
OR
30
8
9
To further substantiate our results associated with the intermolecular reaction
between glycosylation triflate and TolSI by-product, we further conducted cross-over
experiments on the promotion of the tolylsulfenyl iodide (TolSI)/TfOH condition
(Scheme 3).68 The experimental evidence was supported by low-temperature nuclear
magnetic resonance (NMR) experiments at -70 oC. When 0.5 equivalent of TolSI/TfOH
was introduced in the reaction as the combined-promotor, our NMR spectrum showed
that 50% of donor 31-β still remained and a mixture of glycosyl triflate 32 and
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thioaglycon-transferred thioglycoside 31-α was detected. The NMR yield of triflate 32
was determined to be 22%, while 31-α was 28%. The anomeric proton of glycosyl
1
13
3
triflate 32 was detected at 6.18 ppm in H NMR and the corresponding C signal
appeared at 104.8 ppm in 13C NMR as demonstrated in previous literature.42
Subsequently, a clear conversion from 32 to 31-α was discovered when the temperature
4
10
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23
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25
26
27
28
29
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31
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36
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52
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54
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5
o
6
was warmed to 0 C. This information suggested that TolSI would trap the triflate
7
intermediate 32 to yield thioaglycon-transferred thioglycoside 31-α under an
intermolecular process (see spectra in supporting information). This result was similar
to the works by Kartha and Field, in which intermolecular thioaglycon-transformation
was observed as well on the activation of methyl thioglycosides via the participation of
methylsulfenyl iodide.68
8
9
10
11
12
13
14
15
16
17
18
19
20
21
Moreover, using an alternative promotor of phenylsulfenyl iodide (PhSI)/TfOH
turned out a mixture of thiotolyl-transferred thioglycoside 31-α and thiophenyl-
transferred thioglycoside 33-α at a ratio of 1.9/1. The generation of 33-α again indicated
that reaction underwent a rapid transformation between glycosyl triflate intermediate
and PhSI. We also observed the corresponding by-products tolyl disulfide (TolSSTol,
34) and phenyl tolyl disulfide (TolSSPh, 35), which indicated a spontaneous
dimerization and disulfide exchange from PhSI and TolSI (see spectra in supporting
information).69
Scheme 3. Cross-over experiments in support of intermolecular transformation.
TolSI (0.5 equiv.)
OAc
O
OAc
O
OAc
O
TfOH (0.5 equiv.)
AcO
MeO
AcO
MeO
AcO
MeO
STol
d-DCM, -70 o
C
MeO
MeO
MeO
OTf
, 22%
observed by NMR
STol
, 28%
31-

32
31-
PhSI (0.5 equiv.)
TfOH (0.5 equiv.)
OAc
OAc
O
O
TolSSTol
TolSSPh
AcO
MeO
AcO
MeO
+
+
+
34
35
DCM, -70 oC, 42%
31- 33-
MeO
MeO
SPh
STol
(
/  = 1.9/1)
33-

31-

22
23
24
25
26
27
A detailed investigation was further studied between glycosyl triflate 36D and
TolSCl 5-Cl to precisely study the thioaglycon-transformation (Figure 1). The
deuterium-labeled functionalizations were designed to remove peaks overlapping from
the benzylic protons (4.4-5.0 ppm) in the 1H NMR spectra to simplify the analysis. We
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initially prepared pure α-glycosyl triflate 36D (α/β = 1/0) on the promotion of BSP/Tf2O
at -70 oC as reported in the literatures (Figure 1A).18,66,70,71 The observed chemical shift
(δ) of the anomeric proton (H-1) signal was 6.05 ppm and the coupling constant value
of 3.5 Hz suggested it to be an α-anomer. In 19F NMR spectra, the characteristic peak
of covalent anomeric triflates was -75.7 ppm and these results are in agreement with
the NMR analysis observed in previous literature.72
Accompanied by the additional reagents of TolSCl 5-Cl (1.0 equiv.) and TfOH
(0.4 equiv.), as well as the corresponding acceptor (MeOH, 10), a substantial amount
of thioaglycon-transferred thioglycoside 9D- (α/β = 0/1) was regenerated with a
decreasing amount of glycosyl triflate 36D (Figure 1B). The NMR yield of thioaglycon-
transferred thioglycoside 9D- was 30% in 5 mins, while the corresponding yield for
methyl glycoside product 13D (α/β = 1/3) was determined to be 69%. To specifically
look for the characteristic peak on the sugar ring, a selective 1D-TOCSY spectrum of
the peak at 4.46 ppm was observed (Figure 1C). The anomeric proton atom of 9D-
was observed at 4.46 ppm with a 3JH–H value of 10.2 Hz, which was determined to be
the β-anomer. The anomeric configuration of the transferred donor was consistent with
observations of the NIS/TfOH activation system (Table 1). In addition, the identity of
the thioaglycon-transferred thioglycoside 9D- could also be isolated and further
confirmed by high-resolution mass spectrometry (HRMS). We noticed that the quantity
of 9D- highly depended on the amount of TolSCl 5-Cl. However, without adding the
acceptor, glycosyl triflate 36D and TolSCl 5-Cl could not interact, thereby suggesting
that acceptor provided an essential driving force for this intermolecular transformation
between glycosyl triflate 36D and TolSCl 5-Cl.
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
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52
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11
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13
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17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
After introducing the acceptor (MeOH 10), the glycosyl triflate 36D was
immediately converted into 9D-, which was accompanied with the decrease of TolSCl
5-Cl at -70 oC in 5 mins. On the contrary, without adding the acceptor, the combination
of glycosyl triflate, TfOH (0.2-1.0 equiv.), and excess TolSCl 5-Cl (1.0-3.0 equiv.)
could not result in 9D-, even after stirring for an additional 10-12 h and warming up
o
to 0 C. Therefore, it showed that an equimolar amount of acceptor was essential to
promote this aglycon transformation under acidic conditions, as it quenched the
glycosyl sulfonium ion to result in regenerated 9D-. Without adding the acceptor, the
equilibration loop among glycosyl triflate 36D, oxocarbenium ion 36D-TS, and
sulfonium ion 36D-SI would continue (Scheme 4); therefore, only 36D could be
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observed in the NMR spectroscopy shown in Figure 1A, and the immediate formation
of 9D- after the addition of MeOH implied that the regeneration of 9D- went through
the sulfonium ion intermediate 36D-SI.
10
11
12
13
14
15
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17
18
19
20
21
22
23
24
25
26
27
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36
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. Overall mechanistic studies of the intermolecular thioglycon
transformation between triflate 36D and TolSCl: (A) 1H-NMR of glycosyl triflate
1
36D on the promotion of BSP/Tf2O system; (B) H-NMR of thioaglycon-
transferred thioglycoside 9D- in the presence of TolSCl, TfOH, MeOH; (c) 1D-
TOCSY spectrum of thioaglycon-transferred thioglycoside 9D-.
5-Cl
1) TolSCl (
) (1.0 equiv,)
D
D
D
2) TfOH (0.4 equiv.)
O
O
O
10
3) MeOH ( ) (1.0 equiv.)
d5-Ph
d5-Ph
d7-BnO
d5-Ph
d7-BnO
O
O
O
O
O
O
STol
+
d7-BnO
N3
OMe
N3
N3
CD2Cl2
-70 oC, 5 min
OTf
36D,  
9D-  
 ( / = 0/1), 30%
13D  
, ( / = 1/4), 70%
( / = 1/0)
1
36D
A)
( H NMR)
36D
H-1,
1
13D
H-1,
B) 13D + 9D-
 ( H NMR)
13D
H-1,
9D-
H-1,

36D
H-1,
C) 9D- ,

(1D-TOCSY)
10
11
12
Scheme 4. The plausible mechanism of the aglycon transfer reaction on 9D-.
D
D
D
O
O
O
d5-Ph
d7-BnO
O
d5-Ph
d7-BnO
d5-Ph
d7-BnO
O
O
O
O
O
Tol
N3
OTf
S
N3
N3
OTf
Cl
36D,
(Observed by NMR)
(/ = 1/0)
36D-TS
36D-SI
D
TfOH
O
d5-Ph
d7-BnO
O
O
STol
N3
9D- ,
 (/ = 0/1)
28-C
ROCl (
l)
27)
ROH (
or
or
29-Cl
HOCl (
)
H2O
13
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To further confirm the role of protic reagents (acceptor or water), we extended our
scope of the study to discuss the effect of solvent moisture (Table 3, Figure S1). Herein,
the solvent moisture of DCM was dried to 5 ppm and different amount of H2O was
introduced in the reaction to precisely understand the effects of water. Indeed, we
discovered that water did have an influence on the formation of thioaglycon-transferred
thioglycoside 9D-, while the corresponding hydrolyzed side product could not be
o
detected via low temperature NMR at -70 C (Entries 1-5). As the quantity of 9D-
accumulated, the yield of corresponding product 13D gradually decreased. For acceptor
dosage effect, we originally thought that generation of 9D- could be simply suppressed
by using excess of acceptor, as high amount of acceptor may trap activated donor in
order to improve the yield. Nonetheless, we noticed that the amount of 9D- rather
increased with increasing acceptor dosage (Entries 6-11). Based on Table 3, all the
above-mentioned reagents [TolSCl 5-Cl, TfOH, protic reagent (MeOH 10, H2O)] were
indispensable for the formation of 9D-. Without adding additional TfOH, the yield of
9D- was reduced to 13% (Entry 12).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
With these experiments, we proposed that glycosyl sulfonium ion 36D-SI could
be the source of the regenerated donor 9D- (Scheme 4), which was formed in situ by
mixing glycosyl triflate 36D and TolSCl 5-Cl. Next, protic reagents (H2O or acceptor
27) quenched the glycosyl sulfonium ion 36D-SI by attacking the chloronium ion (Cl+)
and expelled the hypoiodate [RO-Cl (28-Cl)] or hypochlorous acid [HO-Cl (29-Cl)],
resulting in a large quantity of thioaglycon-transferred side-product 9D-β.
Table 3. The mechanism study to investigate the proton effect (H2O, MeOH) in the
formation of regenerated thioglycoside.
5-Cl
1) TolSCl (
) (1.0 equiv.)
2) TfOH (0.4 equiv,)
3) H2O (x equiv.)
D
D
D
O
O
O
10
4) MeOH ( ) (y equiv.)
d5-Ph
d5-Ph
d7-BnO
d5-Ph
d7-BnO
O
O
O
O
O
O
STol
+
d7-BnO
N3
OMe
N3
N3
CD2Cl2
-70 oC, 5 min
OTf
36D,  
9D-  
 (
13D  
, (
(
/
= 1/0)
/
= 0/1)
/
= 1/4)
25
Entry
H2O
MeOH (10)
(y equiv.)
1.0
9D-β,
yielda
30%
35%
42%
46%
47%
13D,
yielda
70%
51%
45%
44%
45%
(x equiv.)
b
1
2
3
4
5
-
1.0
1.5
2.0
2.5
1.0
1.0
1.0
1.0
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b
b
b
b
b
b
6
7
8
-
-
-
-
-
-
0.5
0.7
1.0
2.0
3.0
3.0
1.0
10%
34%
45%
70%
41%
47%
47%
80%
16%
30%
44%
44%
44%
13%
9
10
11
12c
10
11
12
13
14
15
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22
23
24
25
26
27
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32
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60
2.5
1
2
aThe yield was determined by NMR. bThe solvent moisture was 5 ppm. cWithout adding additional TfOH.
3
4
TolSH is obviously a possible nucleophile that leads to the occurrence of
thioaglycon-transferred thioglycoside 9D-. However, in our modeling reaction
(Scheme 5A), the addition of TolSH (1.0 equiv.) to glycosyl triflate 36D did not
transform 36D into 9D-. Moreover, the combination of TolSCl 5-Cl (1.0 equiv.) and
MeOH 10 (1.0 equiv.) did not result in the corresponding TolSH at -70 oC, regardless
if the TfOH (0.4 equiv.) was introduced or not (Scheme 5B, Scheme 5C). These results
suggested that TolSH was not involved in aglycon transformation.
5
6
7
8
9
10
11
Scheme 5. The mechanism study to investigate aglycon transformation.
A)
TolSH
(1.0 equiv.)
D
D
O
O
d5-Ph
d7-BnO
d5-Ph
d7-BnO
O
O
O
O
STol
N3
OTf
N3
9D-

36D,  
/ = 1/0)
(
B)
C)
5-Cl
10
)
TfOH
TolSCl (
)
MeOH (
(1.0 equiv.)
+
+
+
n.r
(0.4 equiv.)
(1.0 equiv.)
-70 o
C
5-Cl
TolSCl (
(1.0 equiv.)
10
)
)
MeOH (
(1.0 equiv.)
n.r
-70 o
C
12
13
14
15
16
17
18
19
Having observed the thioaglycon-transformation, we attempted to determine
whether the intermediate conversion could influence the stereoselectivity (Figure 2,
Figure S2). The reaction was continuously monitored in the same reaction batch using
o
o
variable-temperature (VT) NMR experiments from -70 C to 0 C in 85 mins. We
noticed that the glycosyl triflate 36D (black square) immediately converted into a
mixture of methyl glycoside 13D- (blue triangle) and 13D-β (green triangle) from -70
20 oC to -50 oC. However, the thioaglycon-transferred thioglycoside 9D-β (red circle) was
21
gradually consumed with the major increase of β-methyl glycoside product 13D-β from
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8
9
-40 C to 0 C after the combination of NIS (0.5 equiv.) and TfOH (0.5 equiv.) was
added. This information indicated that both glycosyl triflate 36D and thioaglycon-
transferred thioglycoside 9D-β were involved in controlling the rate of the
glycosylation reactions. Once the thioaglycon-transferred thioglycoside 9D-β was
regenerated, 9D-β would be further activated into α-triflate 36D, which contributed
selective β-glycosylation in the presence of a strong nucleophile such as MeOH.63-
65,71,73
Figure 2. Transformation profile of glycosyl triflate and thioaglycon-transferred
thioglycoside from -70 oC to 0 oC, in which all of the data points were continuously
obtained in the same reaction batch.
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11
19
20
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36
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D
D
NIS (0.5 equiv.)
TfOH (0.5 equiv.)
O
O
d5-Ph
d7-BnO
d5-Ph
d7-BnO
O
O
O
O
STol
-70 oC ~ 0 o
0-85 min
C
N3
N3
OTf
9D-

36D
MeOH
10
D
D
O
O
d5-Ph
d5-Ph
d7-BnO
O
O
O
O
OMe
d7-BnO
N3
OMe
N3
13D-
13D-


70
36D
9D-
13D-
-OTf,
-STol,
-OMe,

60
50
40


13D-
-OMe,
(%)
30
20
10
Glycoside
0
-60
-50
-40
-30
-20
-10
o
C,
-70
0
o
C,
o
C,
o
C,
o
C,
o
C,
o
C,
o
C,
85
12
23
34
47
60
75
0
min
min
min
min
min
min
min
min
Temperature (oC), time (min)
12
13
14
15
16
Next, this study clarified the side reaction of N-glycosyl succinimide, as it is
usually observed in NXS/TfOH-activated thioglycoside systems (Table 4).45 Followed
by the preactivation manner, we investigated the glycosylation reaction using 18-β as
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the donor in the absence of an acceptor at -40 C on the promotion of NIS/TfOH,
NBS/TfOH, and NCS/TfOH systems individually. The galactosyl donor 18-β was
preactivated completely in 15-30 mins to afford galactosyl succinimide 37 with a yield
of 24-52%, digalactoside 38 with a yield of 11-23%, and cyclic galactoside 39 with a
yield of 18-21%. During preactivation in the absence of acceptor, the galactosyl triflate
intermediate 40, which was generated in in situ, gradually decomposed without
molecular sieve due to the detrimental effect of water from the organic solvent, and side
products such as digalactoside 38 and cyclic galactoside 39 were further produced. The
trehalose-type digalactoside 38 originated from the 1,1-glycosylayion due to the
reaction occurring between hydrolyzed glycoside and remaining galactosyl donor 18-
β. The identity of 37 was confirmed by NMR. The observed chemical shift (δ) of the
3
4
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37
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5
6
7
8
9
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14
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17
18
19
20
21
22
13
anomeric proton signal was 6.06 ppm, and corresponding anomeric C signal was
observed at 75.8 ppm as reported.74 The coupling constant of anomeric proton (H-1)
was determined to be 7.6 Hz, which referred to the β-anomer (see spectra in supporting
information). We noticed that a substantial amount of N-galactosyl succinimide 37
(52%) was observed, especially in the NCS/TfOH system (Entry 1). However, the
amount of 37 was gradually decreased to 24%-32% in the case of NBS/TfOH and
NIS/TfOH, respectively (Entries 2-3). This result addressed that using a NIS/TfOH
promotor system may slightly reduce the generation of N-galactosyl succinimide 37
compared to NBS/TfOH and NCS/TfOH systems.
Table 4. Study of N-galactosyl succinimide on the promotion of NXS/TfOH.
X
N
Intermediate:
NXS (1.2 equiv.)
TfOH (0.5 equiv.)
OBn
O
BnO
BnO
BnO
OBn
O
O
STol
O
DCM
-40 °C, 15-30 min
BnO
BnO
BnO
X = I, NIS
X = Br, NBS
X = Cl, NCS
18-

OTf
40
OBn
O
BnO
BnO
OBn
O
BnO
BnO
O
OBn
O
BnO
BnO
OBn
BnO
O
+
+
N
O
O
O
OBn
OBn
BnO
39
37
38
(-only)
(/ = 1/1)
BnO
(H-1, C-1) = (6.06, 75.8)
23
Entry
NXS
NCS
NBS
NIS
37, yielda
52%
38, yielda
-
39, yielda
1
2
3
-
32%
24%
11%
23%
21%
18%
24
25
aIsolated yield.
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Since N-galactosyl succinimide 37 was obtained during preactivation, we
proposed that glycosyl triflate 40, which was in situ generated in the reaction, was the
key intermediate (Table 5). To this point, we initially prepared exclusive galactosyl
triflate 40 through the known procedure on the BSP/Tf2O condition.18,66,75
Subsequently, a 1.0 equivalent of NHS 6 (Entry 1) and 1.0 equivalent of NIS (Entry 2)
were then introduced into the reaction individually. Interestingly, our result showed that
galactosyl succinimide 37 was only observed in the presence of NIS (Entry 2), instead
of NHS 6. This result indicated that NHS 6 was not sufficiently reactive to displace the
triflate of α-glycosyl triflate 40. Consistent results of the intermediate change were also
observed in other halide-containing promotor systems such as NBS (32%, Entry 3) and
NCS (22%, Entry 4). Since succinimide 37 formation occurred in acidic condition, it
was also reasonable that its transformation stopped when 1.0 equiv. of acid scavenger
such as triethylamine (TEA) was added (Entries 5-7). Moreover, the condensation
between glycosyl triflate 40 and NHS 6 was unsuccessful, even when iodine (I2) or H2O
(Entries 8-9) were involved, confirming that N-halosuccinimide [NXS (X = I, Br, Cl)]
was essential for the transformation from glycosyl triflate 40 into N-galactosyl
succinimide 33.
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We proposed that the iodonium ions (I+) from NIS could increase the reactivity of
glycosyl triflate 40 with the release of iodine monotriflate (I-OTf, 41-I) under acidic
conditions.76 Therefore, N-galactosyl succinimide 33 was afforded in 21%, and it was
found that employing more NIS increased the amount of succinimide 37. This agreed
with the work of Madsen et al., in which introducing NIS significantly promoted
glycosyl bromide by expelling iodine monobromide (I-Br) to result in a higher yield
and faster conversion.76 On the other hand, Cienfuegos et al. applied DFT calculations
to investigate the mechanism of NIS-mediated nucleophilic additions to glycal, in
which the reaction might not begin with the N-I bond cleavage due to a highly
unfavorable charge separation. In contrast, a concerted I-OTf cleavage and succinimide
addition without charge separation might be more favorable.77 This could be the reason
why NHS 6 could not directly displace triflate 40 to produce side product 37, even in
the presence of I2 (Entry 8, Table 5), and the formation of β exclusive 37 could also be
a result of a concerted reaction without charge separation between α-triflate 40 and
NXS.
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Table 5. Model studies to substantiate the formation of N-galactosyl succinimide.
R
N
O
N
O
O
BnO
BnO
BnO
BnO
OBn
O
X
OTf
OBn
O
Condition
CD2Cl2
6
41-I
NHS ( ), R = H
NIS, R = I
, R = I
BnO
41-Br
41-Cl
, R = Br
, R = Cl
BnO
OTf
O
NBS, R = Br
NCS, R = Cl
40
37
(-only)
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Entry
Condition (equiv.)
NHS (1.0)
Temperature (oC)
-70 to rt
-70
Time
37, yield
1
2
3
4
5
6
7
8
9
6 h
5 min
5 min
5 min
6 h
6 h
6 h
6 h
6 h
n.r.b
21%a
32%a
22%a
n.r.b
NIS (1.0)
NBS (1.0)
NCS (1.0)
-70
-70
NIS (1.0), TEA (1.0)
NBS (1.0), TEA (1.0)
NCS (1.0), TEA (1.0)
NHS (1.0), I2 (1.0)
NHS (1.0), H2O (1.0)
-70 to rt
-70 to rt
-70 to rt
-70 to rt
-70 to rt
n.r.b
n.r.b
n.r.b
n.r.b
aNMR yield. bNo reaction.
3
4
A suggested protocol was noted in this work to suppress the formation of side
products 7-8 for achieving higher-yielding reactions of corresponding product 30
(Figure 3). We considered three major factors in chemical glycosylation, including
donor reactivity (RRV), promotor system, and temperature. Frist, it was necessary to
know the reactivity (RRV) of the glycosyl donor 1 as this would determine the side
products, such as thioaglycon-transferred thioglycoside 7 and N-glycosyl succinimides
8. The corresponding RRV of the thioglycoside 1 could be determined through the
competition experiment or Auto-CHO software, as established by Wong and
coworker.26-30
5
6
7
8
9
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23
When the RRV of thioglycoside 1 is lower than 18.6, a high proportion of
thioaglycon-transferred thioglycoside 7 may regenerate as the main side product, which
significantly reduces the glycosylation yield and further limits one-pot glycosylation,
especially in an NXS/TfOH or TolSCl/AgOTf system. Since the formation of 7 was
driven by an intermolecular process from the TolSX by-product, using an alternative
promotor system was a direct approach to solve this problem such as BSP/Tf2O.
Moreover, it was shown that 7 could also be fully activated by manipulating a higher
temperature from -40 oC to 0 oC.
The highly active thioglycoside 1 (RRV more than 17000) turned out N-glycosyl
succinimides 8 as the main side product. Our finding revealed that the NIS/TfOH
promotor system could slightly regress the formation of N-glycosyl succinimides 8
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compared to an NBS/TfOH system or NCS/TfOH system. It is to be noted that usage
of other promotor systems such as BSP/Tf2O and TolSCl/AgOTf, can avoid 8.
Figure 3. Suggested protocol for suppressing the formation of side products in
chemical glycosylation.
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promotor
DCM
27
ROH (
)
30
1
1
RRV of > 17000?
Yes
No
O
O
PGO
O
N
PGO
STol
7
8
(major)
O
(major)
NXS/TfOH or
TolSCl/AgOTf system?
NXS/TfOH system?
No
Yes
No
Yes
O
O
O
PGO
O
O
PGO
PGO
N
PGO
OR
STol
30
(Exclusive)
OR
7
8
O
30
(Exclusive)
(major)
(major)
NIS/TfOH system?
No Yes
Temperature > -40 oC?
No
Yes
O
O
PGO
O
O
PGO
OR
O
30
(Exclusive)
PGO
N
PGO
OR
30
(Exclusive)
STol
7
8
O
(major)
(major)
6
7
8
9
CONCLUSIONS
The use of thioglycosides as glycosyl donors in the chemical synthesis of
10 glycans and glycoconjugates has gained great popularity over the past years.
11 Their distinct advantages are their high stability during building block
12 manipulation and their ease of transformation into reactive intermediates using
13 various promotors. Such features have facilitated the development of advanced
14 methodologies such as one-pot protocols47 and automated solid-phase
15 synthesis.16 However, side products are often created, which quench the reaction
16 and increase the uncertainty in chemical glycosylation due to the requirement of
17 stoichiometric or excess amounts of promoters. The unclear mechanism has
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rendered it difficult to optimize the combination for achieving high-yield
reactions. Herein, we established a series of mechanistic studies to rule out the
generation of side products. Our low temperature NMR experiments revealed
that the formation of thioaglycon-transferred thioglycoside and N-glycosyl
succinimides follows an intermolecular process from glycosyl triflate. Therefore,
the glycosylation reaction faces an unavoidable competition between side
reactions and glycoside bond formation with the acceptors. Although both steric
and electronic effects highly influence the glycosylation result, a thorough
understanding to the mechanisms and the new factors discovered based on the
10
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10 mechanism studies could provide new ideas and solutions to increase the
11 glycosylation yield. Eventually, a general guideline of how to suppress the side
12 reactions was provided using the RRVs of the thioglycosides as an indicator
13 under certain reaction conditions. Further studies of acceptor effects, solvent
14 effect and other promotor systems are currently underway.
15
16 EXPERIMENTAL SECTION
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General Methods
All reactions were conducted in flame-dried glassware, under nitrogen atmosphere.
All solvents were purified and dried from a safe purification system containing
activated Al2O3. All reagents obtained from commercial sources were used without
purification, unless otherwise mentioned. Flash column chromatography was carried
out by Silica Gel Geduran Si 60 (0.040-0.063 mm, E. Merck). TLC was performed on
pre-coated glass plates of Silica Gel 60 F254 (0.25 mm, E. Merck); detection was
executed by spraying with a solution of Ce(SO4)2, (NH4)2MoO4, and H2SO4 in water
and subsequently heating on a hot plate. UV light for TLC analysis was UVGL-25
compact UV lamp (4 watt/ 254 nm), UVP. High performance liquid chromatography
(HPLC) reactions were carried out by Agilent Technologies 1200 Serise. 1H, 13C NMR,
DEPT and 2D-HSQC spectra were recorded by Bruker AV400, DRX500, AVIII 500,
N600 MHz. Chemical shifts are in ppm from Me4Si, generated from the CDCl3 lock
signal at δ 7.26 and CD2Cl2 lock signal at δ 5.32. Multiplicities are reports by using the
following abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet,
br = broad, ABq = AB quartets, dd = doublet of doublets, dt = doublet of triplets, td =
triplet of doublets; J = coupling constant values in Hertz. Mass spectra were analyzed
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by a Waters Premier XE instrument with ESI source. Structural assignments were made
with additional information from selective 1D-TOCSY and 2D-HSQC experiments
General Procedure for RRV Experiment of Thioglycosides
Two of thioglycoside donors (0.01 mmol) were mixed with molecular sieves in
MeOH (0.05 mmol) and CH2Cl2 (1.0 ml). One of the thioglycoside performed known
RRV, which was controlled as the reference donor, and the other one with unknown
RRV. The reaction mixture was stirred at room temperature for 10 mins. The prepared
mixture (30 μl) was through a filter, and 10-μl filtrate was then injected into an HPLC
to determine the time absorbance (Ax)0 and (Aref)0 at 254 nm. Next, a solution of 0.5
M NIS in MeCN (20 ml, 0.01 mmol) was injected into the remained prepared mixture
and then treated 0.1 M TfOH (10 ml, 0.001 mmol) for the thioglycoside activation.
After that, the mixture was stirred at room temperature for 2 h. Next, the CH2Cl2 (2.0
ml) was added to diluted the reaction and then extract with saturated Na2S2O3 (aq)
containing 10 wt % NaHCO3. After all of the organic layer was collected, the solution
was dried over MgSO4, and concentrated with rotary evaporator. Eventually, the
residue dissolved in CH2Cl2 (1.0 ml) was measured absorbance at 254 nm to determine
the ratio of the remaining unreacted donors (Ax)t and (Aref)t by HPLC with the same
procedure as previously mentioned for (Ax)0 and (Aref)0. The ratio of RRVs, kx/kref,
10
11
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kx
ln (Ax)t - ln (Ax)0
.
ln (Aref)t - ln (Aref)0
26,28-30
19
was referred to the following equation:
=
kref
20
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22
23
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25
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29
30
31
32
33
Preactivation-based glycosylation in Table 1
Donor 9-α42,78 (100 mg, 0.204 mmol, α/β = 0/1, 1.0 equiv.) and molecular sieves
(3Å, 100 mg) were dissolved in DCM (4 mL) to remove the solvent moisture at -70 oC
under N2 atmosphere for 10 mins. 1.0-2.0 equivalent of combined-promotor
(NIS/TfOH, NBS/TfOH, NCS/TfOH, TolSCl/AgOTf) was then introduced
individually in the reaction for the preactivation. After five minutes when TLC
indicated that donor was completely activated, 1.0 equivalent of acceptor 10 (8 μL,
0.204 mmol, 1.0 equiv.), acceptor 11 (94 mg, 0.204 mmol, 1.0 equiv.), acceptor 12 (94
mg, 0.204 mmol, 1.0 equiv.) was added into the reaction mixture respectively at -70 oC
and stirred for 4-5 hour. After completion of the reaction the solution was quenched by
Et3N (1 mL). The solution was filtered through celite and washed with DCM. The
filtrate was evaporated in vacuo to furnish the crude oil, which was purified by flash
column chromatography to give the corresponding product 1342 (25-36 mg, 31-44%,
α/β = 1/8 ~ 1/11), 1471,79 (51 mg, 30%, α/β = 1/6.7), 1571,79 (51 mg, 30%, α/β = 1/3.4)
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and thio-aglycon transferred thioglycoside 9-β42,78 (30-45 mg, 30-45%, α/β = 0/1).
5
6
7
8
Ph
O
O
O
BnO
STol
N3
9
9-
 for -anomer, RRV = 5.4
 for a-anomer, RRV = 314
9
9-
2
3
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p-Tolyl 2-azido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-D-thioglucopyranoside (9).42
The preparation of 9 was followed by the general procedure as literature.42
Compound p-tolyl 2-azido-2-deoxy-D-thioglucopyranoside42,80 (100 mg, 0.321 mmol)
was mixed with HMDS (140 μL, 0.642 mmol) in CH2Cl2 (1.0 mL) under N2, and
TMSOTf (6 μL, 0.032 mmol) was added at room temperature and further stirred for 3
h. Next, CH2Cl2 (1 mL), benzaldehyde (65 μL, 0.642 mmol) and TMSOTf (6 μL, 0.032
mmol) were added in the reaction, and the mixture was kept stirring at the same
temperature for 4 h. Next, TBAF (385 mL, 0.385 mmol) was added, and the mixture
was allowed to warm to room temperature and kept stirring for 1 h. Next, DMF (1 mL),
BnBr (45 μL, 0.385 mmol), NaH (25.7 mg, 0.642 mmol) were added with stirring. The
mixture was stirred at room temperature for 15 min. Eventually, the reaction solution
was quenched with H2O (10 mL). The aqueous layer was extracted with EtOAc (3 × 5
mL), dried with anhydrous MgSO4, filtered, and concentrated in vacuo.80,81 The mixture
was purified by flash column chromatography (n-Hexane/EtOAc 15:1) on silica gel to
furnish the - and - anomer of 8 individually (152 mg, 96%, / = 1/1.3). The
preparation of 9-β (β-anomer) was followed by the general procedure as Preactivation-
based glycosylation in Table 1, and compound 9-β (β-anomer) was obtained with the
yield of 36% (36 mg). The RRV of β-anomer referring to previous report is 5.4.42 The
RRV of α-anomer followed General Procedure for RRV Experiment of
Thioglycosides is 314. []25D -3.3 (c 0.8, CHCl3); IR ν 2106, 1492, 1453, 1274, 1089,
991, 809, 748, 697 cm-1; 1H NMR (400 MHz, CDCl3) δ 7.52-7.13 (m, 28H, Ar-H), 5.60
(s, 1H, CHPh), 5.56 (s, 1H, CHPh), 5.50 (d, J = 2.3 Hz, 1H, H-1), 4.99-4.76 (m, 4H,
CH2Ph), 4.44 (td, J = 10.6, 4.8 Hz, 1H, H-5), 4.43 (d, J = 10.0 Hz, 1H, H-1), 4.38
(dd, J = 9.9, 4.6 Hz, 1H, H-6ax), 4.23 (dd, J= 9.9, 4.6 Hz, 1H, H-6eq), 4.00-3.93 (m,
2H, H-2, H-3), 3.81-3.73 (m, 3H, H-4, H-6ax, H-6eq), 3.67-3.58 (m, 2H, H-3,
H-4), 3.44 (td, J = 9.8, 5.0 Hz, 1H, H-5), 3.32 (dd, J = 10.0, 8.4 Hz, 1H, H-2), 2.36
(s, 3H, ArCH3), 2.34 (s, 3H, ArCH3); 13C{1H} NMR (100 MHz, CDCl3)  139.1 (C),
138.3 (C), 137.7 (C), 137.6 (C), 137.1 (C), 134.5 (CH), 133.1 (CH), 130.0 (CH), 129.9
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(CH), 129.1 (CH), 128.4 (CH), 128.32 (CH), 128.29 (CH), 128.2 (CH), 128.0 (CH),
127.9 (CH), 126.0 (CH), 125.95 (CH), 101.5 (CH), 101.3 (CH), 88.2 (CH), 86.6 (CH),
82.8 (CH), 81.3 (CH), 81.0 (CH), 77.8 (CH), 75.21 (CH2), 75.17 (CH2), 70.46 (CH),
1
68.6 (CH2), 68.5 (CH2), 64.5 (CH), 63.7 (CH), 63.6 (CH), 21.2 (CH), 21.1 (CH); H
NMR (500 MHz, CD2Cl2) δ 7.51-7.15 (m, 28H, Ar-H), 5.61 (s, 1H, CHPh), 5.58 (s,
1H, CHPh), 4.97-4.76 (m, 4H, CH2Ph), 4.46 (d, J = 10.2 Hz, 1H, H-1), 4.42 (td, J =
10.5, 5.0 Hz, 1H, H-5), 4.35 (dd, J = 10.4, 4.9 Hz, 1H, H-6ax), 4.20 (dd, J = 9.9, 5.0
Hz, 1H, H-6eq), 3.99-3.92 (m, 3H, H-2, H-3, H-6eq), 3.79-3.76 (m, 3H, H-4,
H-6ax, H-6eq), 3.66-3.63 (m, 2H, H-3, H-4), 3.44 (m, 1H, H-5), 3.32 (dd, J =
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15
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10.2, 8.7 Hz, 1H, H-2), 2.35 (s, 3H, ArCH3), 2.34 (s, 3H, ArCH3); C NMR (125
MHz, CDCl3)  139.5 (C), 138.9 (C), 138.5 (C), 138.4 (C), 137.9 (C), 137.8 (C), 134.4
(CH), 133.6 (CH), 130.33 (CH), 130.25 (CH), 129.4 (CH), 128.7 (CH), 128.7 (CH),
128.6 (CH), 128.2 (CH), 126.5 (CH), 126.4 (CH), 101.9 (CH), 101.6 (CH), 88.7 (CH),
87.0 (CH), 83.1 (CH), 81.7 (CH), 81.4 (CH), 78.2 (CH), 75.3 (CH2), 70.9 (CH), 69.0
(CH2), 68.9 (CH2), 65.2 (CH), 64.14 (CH), 64.09 (CH), 21.3 (CH3), 21.2 (CH3); HRMS
(ESI) calcd for C27H27N3O4NaS [M+Na]+ 512.1620, found 512.1617.
Ph
O
O
O
BnO
BnO
OMe
S2
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To a solution of methyl 4,6-O-benzylidene-α-D-glucopyranoside S1 (1.5 g, 5.31
mmol) in DMF (20 mL) was added benzyl bromide (2.7 mL, 0.025 mol). The reaction
was cooled in an ice bath, and sodium hydride (1.28 g, 0.032 mol) was added. The
reaction stirred at room temperature overnight and quenched by water (5 mL) and
extracted with EtOAc (20 mL 2). The organic layers were combined, dried over
anhydrous MgSO4, filtered, and evaporated under reduced pressure. The crude product
was purified by flash column chromatography on silica gel using EtOAc/ hexane 1/3 as
the eluent to obtain S2 as a colorless oil (2.1 g, 86%). [α]28D 4.6 (c 0.3, CHCl3); IR ν
2912, 1088, 1071, 1048, 1028, 736, 697 cm-1; 1H NMR (400 MHz, CDCl3) δ 7.48-7.46
(m, 2H, Ph-H), 7.38-7.27 (m, 13H, Ph-H), 5.54 (s, 1H, CHPh), 4.90 (d, J = 11.4 Hz,
1H, CH2Ph), 4.84 (d, J = 12.1 Hz, 1H, CH2Ph), 4.82 (d, J = 11.4 Hz, 1H, CH2Ph), 4.70
(d, J = 12.1 Hz, 1H, CH2Ph), 4.59 (d, J = 3.6 Hz, 1H, H-1), 4.24 (dd, J = 10.1, 4.8 Hz,
1H, H-6eq), 4.02 (t, J = 9.5 Hz, 1H, H-3), 3.81 (ddd, J = 10.1, 9.5, 4.8 Hz, 1H, H-5),
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3.70 (t, J = 10.1, 4.8 Hz, 1H, H-6ax), 3.58 (t, J = 9.5 Hz, 1H, H-4), 3.54 (dd, J = 9.5, 3.6
Hz, 1H, H-2), 3.39 (s, 3H, CH3); 13C{1H} NMR (100 MHz, CDCl3) δ 138.8 (C), 138.2
(C), 137.5 (C), 128.9 (CH), 128.8 (CH), 128.7 (CH), 128.6 (CH), 128.2 (CH), 128.0
(CH), 127.7 (CH), 127.4 (CH), 126.0 (CH), 101.3 (CH), 99.3 (CH), 82.2 (CH), 79.3
(CH), 78.6 (CH), 75.3 (CH2), 73.8 (CH2), 69.1 (CH2), 62.4 (CH), 55.3 (CH), 29.7(CH3);
HRMS (ESI) calcd for C28H30O6Na [M + Na]+ 485.1940, found 485.1937.
OH
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O
BnO
BnO
BnO
OMe
11
7
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Methyl 2,3,4-tri-O-benzyl-α-D-glucopyranoside (11).83
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To a solution of compound S2 (0.3 g, 0.65 mmol) in BH3/THF (0.47 mL, 4.86
mmol). The reaction was stirred for 10 min, and then TMSOTf (0.024 mL, 0.13 mmol)
o
was added. The reaction stirred at 0 C gradually to room temperature for 2 h, and
quenched by MeOH (5 mL) and evaporated under reduced pressure. The crude product
was purified by flash column chromatography on silica gel using EtOAc/ hexane 1/2 as
the eluent to obtain 11 as a colorless oil (0.316 g, 99%). [α]28D -204.2 (c 1.3, CHCl3);
1
IR ν 2959, 1069, 1051, 737, 697 cm-1; H NMR (500 MHz, CDCl3) δ 7.33-7.26 (m,
15H, Ph-H), 4.98 (d, J = 10.9 Hz, 1H, CH2Ph), 4.85 (d, J = 10.9 Hz, 1H, CH2Ph), 4.82
(d, J = 12.2 Hz, 1H, CH2Ph), 4.68 (d, J = 10.9 Hz, 1H, CH2Ph), 4.66 (d, J = 11.0 Hz,
1H, CH2Ph), 4.59 (d, J = 3.6 Hz, 1H, H-1), 4.03 (dd, J = 9.7, 9.3 Hz, 1H, H-3), 3.79
(dd, J = 11.7, 6.5 Hz, 1H, H-6a), 3.71 (dd, J = 11.7, 3.8 Hz, 1H, H-6b), 3.69 (t, J = 4.2
Hz, 1 H, H-5), 3.54 (dd, J = 9.3, 5.2 Hz, 1H, H-4), 3.52 (dd, J = 9.7, 3.6 Hz, 1H, H-2),
13
3.35 (s, 3 H, CH3); C{1H} NMR (100 MHz, CDCl3) δ 138.8 (C), 138.2 (C), 138.0
(C), 128.6 (CH), 128.5 (CH), 128.3 (CH), 128.1 (CH), 128.0 (CH), 127.9 (CH), 127.7
(CH), 127.4 (CH), 126.0 (CH), 98.2 (CH), 82.0 (CH), 80.0 (CH), 77.5 (CH), 75.7 (CH2),
75.0 (CH2), 73.4 (CH2), 70.7 (CH), 61.9 (CH), 55.2 (CH3); HRMS (ESI) calcd for
C28H32O6Na [M + Na]+ 487.2097, found 487.2049.
OBn
O
HO
BnO
BnO
OMe
12
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Methyl 2,3,6-tri-O-benzyl-α-D-glucopyranoside (12).83
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To a solution of compound S2 (0.4 g, 0.865 mmol) in DCM was added Et3SiH
(1.24 mL, 7.78 mmol), TFA (0.5 mL, 6.48 mmol) and TFFA (0.025 mL, 0.173 mmol).
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The reaction stirred at -40 oC for 6 h and quenched by Et3N (5 mL). The mixture was
extracted with EtOAc (20 mL) and NaHCO3 (20 mL2). The organic layers were
combined, dried over anhydrous MgSO4, filtered, and evaporated under reduced
pressure. The crude product was purified by flash column chromatography on silica gel
using EtOAc/ hexane 1/3 as the eluent to obtain 12 as a colorless oil (0.365 g, 91 %).
[α]28D -290.6 (c 0.5, CHCl3); IR ν 2922, 1055, 1028, 736, 697 cm-1; 1H NMR (400 MHz,
CDCl3) δ 7.35-7.28 (m, 15H, Ar-H), 4.98 (d, J =11.4 Hz, 1H, CH2Ph), 4.78 (d, J = 12.1
Hz, 1H, CH2Ph), 4.75 (d, J = 11.4 Hz, 1H, CH2Ph), 4.67 (d, J = 12.1 Hz, 1H, CH2Ph),
4.64 (d, J = 3.6 Hz, 1H, H-1), 4.60 (d, J = 12.2 Hz, 1H, CH2Ph), 4.55 (d, J = 12.2 Hz,
1H, CH2Ph), 3.79 (t, J = 9.1 Hz, 1H, H-3), 3.74-3.67 (m, 3H, H-5, H-6a, H-6b), 3.61
(td, J = 9.1, 2.1 Hz, 1H, H-4), 3.54 (dd, J = 9.1, 3.6 Hz, 1H, H-2), 3.39 (s, 3H, CH3),
2.30 (d, J = 2.1 Hz, 1H, OH); 13C{1H} NMR (100 MHz, CDCl3) δ 138.8 (C), 138.2 (C),
138.0 (C), 128.6 (CH), 128.5 (CH), 128.3 (CH), 128.1 (CH), 128.0 (CH), 127.9 (CH),
127.7 (CH), 127.4 (CH), 126.0 (CH), 98.2 (CH), 81.4 (CH), 79.6 (CH), 75.4 (CH2),
73.6 (CH2), 73.2 (CH2), 70.7 (CH), 69.9 (CH), 69.5 (CH), 55.2 (CH3); HRMS (ESI)
calcd for C28H32O6Na [M + Na]+ 487.2097, found 487.2100.
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Ph
O
O
O
BnO
N3
OMe
13
17
18
Methyl 2-azido-3-O-benzyl-2-deoxy-4,6-O-benzylidine-D-glucopyranoside (13).42
Synthesis procedure was shown as Preactivation-based glycosylation in Table
1 to afford compound 13 (25 mg, 31%, α/β = 1/9.0) with NIS/TfOH; compound 13 (27
mg, 33%, α/β = 1/11) with NBS/TfOH; compound 13 (28 mg, 34%, α/β = 1/8.0) with
NCS/TfOH; compound 13 (36 mg, 44%, α/β = 1/8.1) with TolSCl (1.0 equiv.)/AgOTf
(1.0 equiv.); compound 13 (27 mg, 33%, α/β = 1/8.0) with TolSCl (2.0 equiv.)/AgOTf
(2.0 equiv.). []25D -10.1 (c 1.4, CHCl3); IR ν 2109, 1093, 995, 749, 697 cm-1; 1H NMR
(400 MHz, CDCl3) δ 4.50-4.26 (m, 20H, Ph-H), 5.59 (s, 1H, CHPh), 5.58 (s, 1H, CHPh),
4.99-4.91 (m, 2H, CH2Ph), 4.82-4.78 (m, 3H, H-1, CH2Ph), 4.36 (dd, J = 10.5, 5.0
Hz, 1H, H-6a), 4.30 (dd, J = 10.0, 4.7 Hz, 1H, H-6a), 4.07 (t, J = 9.0 Hz, 1H, H-3),
3.87 (dt, J = 10.0, 4.8 Hz, 1H, H-5), 3.83-3.74 (m, 2H, H-6b, H-6b), 3.73-3.69 (m,
2H, H-4, H-4), 3.58 (t, J = 8.4 Hz, 1H, H-3), 3.58 (s, 3H, OCH3), 3.46-3.37 (m, H,
H-2, H-3, H-5, OCH3); 13C{1H} NMR (100 MHz, CDCl3)  137.8 (C), 137.2 (C),
137.1 (C), 129.2 (C), 129.1 (CH), 128.40 (CH), 128.37 (CH), 128.3 (CH), 128.22 (CH),
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128.20 (CH), 128.1 (CH), 127.9 (CH), 126.0 (CH), 103.4 (CH), 101.5 (CH), 101.3 (CH),
99.4 (CH), 82.7 (CH), 81.6 (CH), 79.0 (CH), 76.3 (CH), 75.03 (CH2), 75.93 (CH2), 68.9
(CH2), 68.6 (CH2), 66.14 (CH), 66.11 (CH), 63.2 (CH), 62.6 (CH), 57.5 (CH3), 55.4
(CH3); HRMS (ESI) calcd for C21H23N3O5Na [M + Na]+ 420.1535, found 420.1527.
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Ph
O
O
O
O
BnO
N3
BnO
BnO
O
BnO
OMe
14-

5
6
Methyl 2,3,4-tri-O-benzyl-6-O-(2-azido-3-O-benzyl-4,6-O-benzyli-dene-2-deoxy-β-D-
glucopyranosyl)-α-D-glucopyranoside (14-β).71,79
7
8
Synthesis procedure was shown as Preactivation-based glycosylation in Table
1 to afford compound 14 (51 mg, 30%, α/β = 1/6.7) with TolSCl (2.0 equiv.)/AgOTf
(2.0 equiv.). [α]25D -29.5 (c 1.2, CHCl3); IR ν 2109, 1453, 1088, 735, 695 cm-1; 1H NMR
(500 MHz, CDCl3) δ 7.49-7.29 (m, 25H, Ph-H), 5.55 (s, 1H, CHPh), 5.01-4.91 (m, 3H,
CH2Ph), 4.85-4.78 (m, 3H, CH2Ph), 4.67 (dd, J = 11.0, 9.3 Hz, 2H, CH2Ph), 4.63 (d, J
= 3.6 Hz, 1H, H-1), 4.31 (dd, J = 9.9, 5.0 Hz, 1H, H-6a’), 4.25 (d, J = 8.0 Hz, 1H, H-
1’), 4.08 (dd, J = 10.5, 1.6 Hz, 1H, H-6a), 4.01 (t, J = 9.4 Hz, 1H, H-3), 3.80-3.68 (m,
4H, H-4’, H-5, H-6b, H-6’b), 3.60 (t, 1H, J = 9.4 Hz, H-4), 3.59-3.48 (m, 3H, H-2, H-
2’, H-3’), 3.39 (s, 3H, OCH3), 3.34 (dt, J = 9.9, 5.1 Hz, 1H, H-5’); 13C{1H} NMR (125
MHz, CDCl3)  138.7 (C), 138.4 (C), 138.1 (C), 137.7 (C), 137.1 (C), 129.0 (CH),
128.43 (CH), 128.36 (CH), 128.4 (CH), 128.2 (CH), 128.1 (CH), 128.0 (CH), 127.90
(CH), 127.86 (CH), 127.98 (CH), 127.72 (CH), 127.69 (CH), 127.6 (CH), 126.0 (CH),
102.4 (CH), 101.3 (CH), 98.2 (CH), 82.1 (CH), 81.4 (CH), 79.7 (CH), 79.2 (CH), 77.6
(CH), 75.7 (CH2); 74.9 (CH2), 74.8 (CH2), 69.6 (CH), 68.7 (CH2), 68.5 (CH2), 66.2
(CH), 66.1 (CH), 55.2 (CH3); HRMS (ESI) calcd for C48H51N3O10Na [M + Na]+
852.3472, found 852.3452.
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Ph
O
O
OBn
O
O
BnO
N3
O
BnO
BnO
OMe
15
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2,3,4-tri-O-benzyl-4-O-(2-azido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-D-
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29
glucopyranosyl)-α-D-glucopyranoside (15).71,79
Synthesis procedure was shown as Preactivation-based glycosylation in Table
1 to afford compound 15 (51 mg, 30%, α/β = 1/3.4) with TolSCl (2.0 equiv.)/AgOTf
(2.0 equiv.). β-anomer (15β): [α]25D -0.9 (c 0.9, CHCl3); IR ν 2922, 2109, 1453, 1092,
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771, 697 cm-1; 1H NMR (500 MHz, CDCl3) δ 7.48-7.26 (m, 25H, Ph-H), 5.47 (s, 1H,
CHPh), 4.88 (t, J = 11.2 Hz, 2H, CH2Ph), 4.82-4.70 (m, 4H, CH2Ph), 4.64-4.61 (m, 2H,
H-1, CH2Ph), 4.42 (d, J = 12.0 Hz, 1H, CH2Ph), 4.21 (d, J = 8.0 Hz, 1H, H-1’), 4.11
(dd, J = 8.8, 5.3 Hz, 1H, H-6’a), 3.98 (dd, J = 10.1, 3.0 Hz, 1H, H-6a), 3.94 (t, J = 10.1
Hz, 1H, H-4), 3.85 (t, J = 10.1 Hz, 1H, H-3), 3.76 (bd, J = 10.1 Hz, 1H, H-5), 3.70 (dd,
J = 10.1, 3.0 Hz, 1H, H-6b), 3.65 (t, J = 8.8 Hz, 1H, H-4’), 3.52 (dd, J = 10.1, 4.0 Hz,
1H, H-2), 3.44-3.40 (m, 1H, H-6’b), 3.40 (s, 3H, OCH3), 3.37-3.29 (m, 2H, H-2’, H-
3’), 3.01 (dt, J = 8.8, 5.0 Hz, 1H, H-5’); 13C{1H} NMR (125 MHz, CDCl3)  139.3 (C),
138.4 (C), 137.9 (C), 137.8 (C), 137.3 (C), 129.0 (CH), 128.5 (CH), 128.4 (CH), 128.3
(CH), 128.2 (CH), 128.1 (CH), 128.04 (CH), 127.99 (CH), 127.9 (CH), 127.8 (CH),
127.5 (CH), 127.3 (CH), 126.0 (CH), 101.2 (CH), 101.2 (CH), 98.3 (CH), 81.7 (CH),
80.1 (CH), 79.2 (CH), 79.1 (CH), 77.0 (CH), 75.4 (CH2); 74.7 (CH2), 73.53 (CH2),
73.50 (CH), 69.7 (CH), 68.5 (CH2), 68.0 (CH2), 66.6 (CH), 65.8 (CH), 55.3 (CH3);
HRMS (ESI) calcd for C48H51N3O10Na [M + Na]+ 852.3472, found 852.3490. α-anomer
(15α): [α]25D 32.0 (c 0.5, CHCl3); IR ν 2923, 2107, 1453, 1094, 1050, 772, 697 cm-1;
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15
16 1H NMR (400 MHz, CDCl3) δ 7.47-7.23 (m, 25H, Ph-H), 5.71 (d, J = 4.0 Hz, 1H, H-
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1), 5.54 (s, 1H, CHPh), 5.10 (d, J = 10.6 Hz, 1H, CH2Ph), 4.95 (d, J = 10.9 Hz, 1H,
CH2Ph), 4.85 (d, J = 10.9 Hz, 1H, CH2Ph), 4.77 (d, J = 10.3 Hz, 1H, CH2Ph), 4.75 (d,
J = 11.5 Hz, 1H, CH2Ph), 4.65-4.55 (m, 4H, H-1’, CH2Ph), 4.11-4.05 (m, 2H, H-5, H-
6a), 3.99 (t, J = 9.6 Hz, 1H, H-3), 3.96 (dd, J = 9.4, 7.1 Hz, 1H, H-4’), 3.85-3.78 (m,
3H, H-4, H-5’, H-6’a), 3.70-3.63 (m, 2H, H-3’, H-6’b), 3.60-3.56 (m, 2H, H-2’, H-6b),
13
3.39 (s, 3H, OCH3), 3.29 (dd, J = 10.1, 4.0 Hz, 1H, H-2); C{1H} NMR (100 MHz,
CDCl3) δ 138.7 (C), 138.09 (C), 137.95 (C), 137.4 (C), 129.0 (CH), 128.5 (CH), 128.4
(CH), 128.3 (CH), 128.2 (CH), 128.1 (CH), 128.0 (CH), 127.9 (CH), 127.6 (CH), 127.5
(CH), 127.4 (CH), 126.0 (CH), 101.3 (CH), 98.1 (CH), 97.8 (CH), 76.2 (CH), 75.0
(CH2), 73.5 (CH2), 73.2 (CH2), 69.4 (CH), 69.1 (CH2), 68.7 (CH2), 66.2 (CH), 63.4
(CH), 62.9 (CH), 55.3 (CH3); HRMS (ESI) calcd for C48H51N3O10Na [M + Na]+
852.3472, found 852.3463
Preactivation-based glycosylation in Table 2
To a suspension of the donor 9-α,78 9-β,78 16-α,41,42 17-α,42 18-β,42 19-β42 (100
mg, 1.0 equiv.), molecular sieves (3Å, 100 mg) and NIS (1.0 equiv.) in CH2Cl2 (4 mL)
was stirred at -70 oC under N2 atmosphere for 1 h. TfOH (0.4 equiv.) was then added
o
into the reaction mixture at -70 C and stirred for 10 min at same temperature. After
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five minutes when TLC indicated that donor was completely activated, the methanol
10 (1.0 equiv.) was injected into the reaction mixture and further stirred for 1 h judged
by TLC (Hexane/EtOAc 6:1). The solution was filtered through celite and washed with
EtOAc. The filtrate was evaporated in vacuo to furnish the crude product, which was
purified by flash chromatography to give the product 1342 (25-28 mg, 31-34%), 2041
(69 mg, 83%), 2142 (70 mg, 84%), 2242 (53 mg, 64%), 2342 (46 mg, 55%) and thio-
aglycon transferred thioglycoside donor 9-β78 (42-44 mg, 42-44%), 17-β42 (8 mg, 8%),
18-α84 (25 mg, 25%), 19-α85,86 (36 mg, 36%).
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OBn
O
BnO
BnO
STol
17
17-
17-
 for -anomer, RRV = 300000
 for a-anomer, RRV = 500000
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p-Tolyl-3,4,6-tri-O-benzyl-2-deoxy-D-thiogalactopyranoside (17).
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The preparation of 17 was followed by the general procedure as literature.42
Compound p-Tolyl 3,4,6-tri-O-acetyl 2-deoxy-D-thiogalactopyranoside87 (8.58 g,
0.0216 mol) and were dissolved in MeOH (100 mL), and the reaction mixture was
stirred at room temperature for 1 h. After reaction was neutralized using acidic
Amberlite resin IR-120, the mixture was filtered, and the solution was evaporated to
give colorless oil. The colorless oil and BnBr (11 mL, 0.104 mol) were dissolved in
DMF (60 mL), and NaH (5.2 g, 0.13 mol) was then added slowly at 0 oC. The reaction
was stirred at room temperature overnight, and quenched by water (10 mL). The
mixture was extracted with EtOAc (50 mL  2). The organic layers were combined,
dried over anhydrous MgSO4, filtered, and evaporated under reduced pressure. The
crude product was purified by flash column chromatography on silica gel using
EtOAc/n-Hexane 1/6 as the eluent to obtain 17 as a colorless oil (9.85 g, 85%, α/β =
1.8/1). The preparation of 17-β (β-anomer) was followed by the general procedure as
Preactivation-based glycosylation in Table 2, and compound 17-β (β-anomer) was
obtained with the yield of 36% (36 mg). The RRV of 17-β (β-anomer) obtained by
General Procedure for RRV Experiment of Thioglycosides is 300000.42 The RRV
of 17-α (α-anomer) obtained by General Procedure for RRV Experiment of
Thioglycosides is 500000. -anomer (17-α):88 [α]28D +128.7 (c 0.4, CHCl3); IR ν 2865,
1093, 1062, 734, 696 cm-1; 1H NMR (400 MHz, CDCl3) δ 7.37-7.24 (m, 17H, Ar-H),
7.00 (d, J = 8.4 Hz, 2H, Ar-H), 5.65 (d, J = 5.3 Hz, 1H, H-1), 4.93 (d, J = 11.8 Hz, 1H,
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CH2Ph), 4.65-4.55 (m, 4H, CH2Ph), 4.46-4.38 (m, 3H, H-5, CH2Ph), 3.95 (d, J = 2.6
Hz, 1H, H-4), 3.91 (ddd, J = 13.0, 5.3, 2.6 Hz, 1H, H-3), 3.65-3.56 (m, 2H, H-6a, H-
6b), 2.60 (td, J = 13.0, 5.3 Hz, 1H, H-2ax), 2.28 (s, 3H, CH3), 2.15 (dd, J = 13.0, 5.3
Hz, 1H, H-2eq); 13C{1H} NMR (100 MHz, CDCl3) δ 136.8 (C), 138.2 (C), 138.1 (C),
131.8 (CH), 129.6 (CH), 129.4 (CH), 128.4 (CH), 128.1 (CH), 127.8 (CH), 127.6 (CH),
127.4 (CH), 84.7 (CH), 75.3 (CH), 74.3 (CH2), 73.3 (CH), 72.1 (CH), 70.7 (CH), 70.5
(CH2), 69.4 (CH2), 31.8 (CH2), 29.7 (CH2), 21.0 (CH3). β-anomer(17-β): [α]29 -20.7
D
(c 0.2, CHCl3); IR ν 2918, 1100, 1064, 734, 696 cm-1; 1H NMR (400 MHz, CDCl3) δ
7.40 (d, J = 6.4 Hz, 2H, Ar-H), 7.35-7.24 (m, 15H, Ar-H), 7.02 (d, J = 8.0 Hz, 2H,
Ph-H), 4.92 (d, J = 11.7 Hz, 2H, CH2Ph), 4.67 (dd, J = 11.8, 2.2 Hz, 1H, H-1), 4.63-
4.54 (m, 3H, CH2Ph), 4.45 (d, J = 11.6 Hz, 1H, CH2Ph), 4.39 (d, J = 11.6 Hz, 1H,
CH2Ph), 3.84 (t, J = 4.6 Hz, 1H, H-4), 3.67-3.62 ( m, 2H, H-6a, H-6b), 3.57 (ddd, J =
11.8, 4.6, 2.2 Hz, 1H, H-3), 3.51 (t, J= 4.6 Hz, 1H, H-5), 2.16 (q, J = 11.8 Hz, 1H, H-
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2ax), 2.28 (s, 3H, CH3), 2.13 (dd, J = 11.8, 2.2 Hz, 1H, H-2eq); C{1H} NMR (100
MHz, CDCl3) δ 138.3 (C), 138.1 (C), 137.6 (C), 131.9 (CH), 130.8 (CH), 130.2 (CH),
129.4 (CH), 128.4 (CH), 128.1 (CH), 127.8 (CH), 127.6 (CH), 127.4 (CH), 127.3 (CH),
83.2 (CH), 77.0 (CH), 76.7 (CH), 74.1 (CH2), 73.5 (CH2), 71.7 (CH), 70.2 (CH2), 69.5
(CH2), 32.4 (CH2), 21.1 (CH3); HRMS (ESI) calcd for C34H36O5NaS [M + Na]+
563.2232, found 563.2235.
OBn
O
BnO
BnO
STol
BnO
18
18-
 for -anomer, RRV = 17000
18-
 for a-anomer, RRV = 3646
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p-Tolyl 2,3,4,6-tetra-O-benzyl-1-thio-D-galactopyranoside (18).
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The preparation of 18-β was followed by the general procedure as literature.42
1,2,3,4,6-Penta-O-acetyl-thio-β-D-glucopyranose (5.0 g, 11.00 mmol) and NaOMe
(59.4 mg, 1.10 mmol) were dissolved in MeOH (50 mL), and the reaction was
processed at room temperature for 1 h. After that, amberlite 120 (H+) was used to
neutralize the reaction, and reaction was evaporated under high vacuum system to give
white-solid compound. The white-solid compound was next mixed with BnBr (6.4 ml,
52.80 mmol) and stirred for 1 h at 0 °C in DMF (32 ml), and NaH (3.5 g, 88.00 mmol)
was added into the mixture slowly at 0 °C. The reaction underwent at room temperature
for 12 h. To workup the reaction, water was added into reaction in ice-bath, and aqueous
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