Effects of frozen conditions on stereoselectivity and velocity of O-glycosylation reactions

Article abstract of DOI:10.1016/j.bmc.2010.04.013

Rate acceleration of O-glycosylation had been observed in p-xylene under frozen conditions, when thioglycosides were activated by methyl trifluoromethane sulfonate. Curiously, significant perturbation of stereoselectivity was observed. Effects of various

Full text of DOI:10.1016/j.bmc.2010.04.013

Bioorganic & Medicinal Chemistry 18 (2010) 3687–3695
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Effects of frozen conditions on stereoselectivity and velocity of O-glycosylation
reactions
a
a
a,b,
Akihiro Ishiwata a, , Ayaka Sakurai , Katharina Dürr , Yukishige Ito
*
*
a RIKENꢀAdvanced Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
b ERATO, JST, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Rate acceleration of O-glycosylation had been observed in p-xylene under frozen conditions, when
thioglycosides were activated by methyl trifluoromethane sulfonate. Curiously, significant perturbation
of stereoselectivity was observed. Effects of various factors, such as solvent, concentration, anomeric con-
figuration and protective groups of the donor, were systematically examined to clarify the mechanistic
implications of stereoselectivity on glycosylation under frozen system. Our study revealed that the
stereoselectivity was affected by concentration both in liquid as well as in frozen conditions, indicating
that rate acceleration effect in frozen solvent was caused by highly concentrated environments.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 20 January 2010
Revised 2 April 2010
Accepted 6 April 2010
Available online 9 April 2010
Keywords:
Rate acceleration
Frozen conditions
Glycosylation
Stereoselectivity
Concentration effect
1. Introduction
point. A similar effect was also observed in the synthesis of larger
oligosaccharides, extending its practical utility to facile prepara-
It is widely perceived that chemical reactions proceed most
smoothly in homogeneous solutions, in which molecules diffuse
without impediment. Therefore, it is almost a golden rule to keep
the reaction temperature above melting point of the solvent. Most
researchers probably believe that reactions in frozen solvents barely
proceed, because diffusion of molecules must be severely restricted.
However, several reports have shown that certain reactions, both
chemical1 and enzymatic,2 proceed under frozen conditions. Con-
trary to the general belief, these reactions were shown to proceed
with higher velocity compared to solution conditions. These obser-
vations may well be explained by the concentration effect.3 Below
freezing point, but above the eutectic temperature, frozen mixture
contains liquid particles that are dispersed in a seemingly solidified
mixture.4 Freezing point-composition relationship predicts that sol-
utes (reactants and reagents) are highly concentrated in liquid par-
ticles, whichmay be seen micropockets. If the effect of concentration
overrides the rate reduction caused by reduced temperature associ-
ated with freezing, reactions would be accelerated.5
tion of oligomannoside probes,6 as well as block condensation of
oligosaccharide fragments to provide a high-mannose type glycan
analogue.6,8 In addition, this technique was applied to the intro-
duction of a fucose residue to highly hindered site of octasaccha-
ride acceptor, completing the synthesis of complex-type glycans
derived from parasitic helminthes.9 As an additional example,
arabinofuranosylation under frozen conditions was applied to the
synthesis of mycobacterial arabinan fragment.10
In order to accelerate reactions, various types of conditions have
proven useful, the most prominent being microwave irradia-
tion,11,12 ultrasound sonication,13 high pressure compression,14,15
and microfluidic conditions.16,17 Compared to these techniques,
which require special devices, freezing conditions are readily
accessible to any researchers.
Incidentally, we came across to the phenomenon that stereose-
lectivity of glycosylation was significantly perturbed under frozen
conditions. We anticipated that correlation of the rate enhance-
ment with selectivity would provide a clue to understand the
origin of the effects of frozen conditions.
Our previous study provided the first example of the rate accel-
eration of O-glycosylation in frozen medium.6 Namely, mannopyr-
anosylation in the presence of methyl trifluoromethanesulfonate
(MeOTf)7 and 2,6-di-tert-butyl-4-methylpyridine (DTBMP),
enormous rate acceleration was observed, when the glycosylation
was conducted in p-xylene (mp = 12–13 °C) below its freezing
2. Results and discussion
2.1. Effect of additive solvents in xylene under frozen conditions
As an exemplary reaction of our study, we revisited mannopyr-
* Corresponding authors. Tel.: +81 48 467 9430; fax: +81 48 462 4680 (A.I.).
E-mail addresses: aishiwa@riken.jp (A. Ishiwata), yukito@riken.jp (Y. Ito).
anosylation of 118 through activation of 219 with MeOTf and
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.013

3688
A. Ishiwata et al. / Bioorg. Med. Chem. 18 (2010) 3687–3695
enhance the yield by lowering the temperature (entry 9) or using
OBn
O
BnO
BnO
BnO
aq NaCl in order to attain better segregation of water (entries 8
and 10) did not give any improvement. The result suggested that,
even under frozen condition, H2O was not completely separated
from organic solvent. Curiously, however, a significant reduction
of stereoselectivity was seen when aq. NaCl was included.
We subsequently examined the effects of organic co-solvents
such as toluene, Et2O, CH3CN, CHCl3, and Et2O–CHCl3 (1:1),20 antic-
ipating that these co-solvents are concentrated in the liquid phase
of frozen mixture and affect the selectivity (entries 11–15). How-
ever their effects on stereoselectivity were only marginal. Instead,
the rate accelerating effect was deteriorated in these cases; even
inclusion of 1% toluene was detrimental (entry 11), indicating that
rigidly ordered frozen state of the mixture is crucial in order to
optimally accelerate reactions.
OBn
O
SMe
BnO
BnO
BnO
Donor
2
OH
O
BnO
BnO
BnO
MeOTf (2.5 eq.)
DTBMP (1.5 eq.)
MS4A
O
BnO
BnO
BnO
O
BnO
6 h
Acceptor
BnO
1
3
Scheme 1.
2.2. Glycosylation in various solvents under frozen conditions
DTBMP (Scheme 1), because this reaction only proceeds slowly at
room temperature in p-xylene or toluene. Selectivity was evalu-
ated through 1H NMR analysis of disaccharide fractions, which
were separated from crude mixtures by size exclusion column
chromatography. In some cases, deuterated benzyl protected sub-
strates were used to evaluate selectivity with higher precision
(Table 1). In accordance with our previous report, enormous rate
acceleration was observed under frozen conditions at 4 °C and
ꢁ20 °C (entries 1–6).6 This reaction was thus further studied to
explore factors that govern the outcome of glycosylation under
frozen conditions. Glycosylations are generally conducted under
anhydrous conditions. From pragmatic point of view, it would be
of some merit, if such reactions are tolerant to the presence of
water. Our initial anticipation was that water molecules are segre-
gated from organic solvent when the latter was frozen. We thus
attempted the glycosylation in p-xylene in the presence of deliber-
ately added H2O (1%) under frozen conditions (entry 7). It was ob-
served that the disaccharide 3 was formed in a substantial yield
(40%) indeed, indicating that the reactivity of the acceptor 1, to
some extent, competed successfully with that of H2O under such
circumstances, even though the amount of the water is more than
ten times higher (0.56 mol/L) than 1. However, further attempts to
In order to clarify the generality of the frozen effect which was
clearly observed in p-xylene,21,22 we examined the glycosylation in
other solvents which can be frozen at 4 °C and we compared them
with non-freezing solvents possessing similar chemical property
(Table 2). In the case of p-xylene, rate enhancement was observed
compared to the reactions in toluene (Table 1) and m-xylene (entry
1). As an ethereal and a nitrile solvents, dioxane (mp = 10–12 °C)
and t-BuCN (mp = 15–16 °C) were compared with tetrahydropyra-
ne (mp = ꢁ45 °C) and MeCN (mp = ꢁ48 °C), respectively (entries
2–5). In these cases, rate acceleration was also seen, although the
extents were smaller. Since proportion of the b-isomer was
increased in frozen p-xylene (Table 1), we initially hoped that
b-directing effect of nitrile solvent would be augmented. However
the selectivity in frozen t-BuCN was nearly identical with that in
MeCN. Interestingly, Mong et al reported that glycosylation with
2-O-benzylated galactopyranosyl or glucopyranosyl donor under
diluted conditions in nitrile solvent gave higher 1,2-trans (b) selec-
tivity under diluted conditions. These authors rationalized this
intriguing results by enhanced formation of nitrilium ion interme-
diates with low concentration of substrates.23
Table 1
Glycosylation of 1 with 2 under frozen conditions:a effect of additive solvents and concentration
Entry
Solvent
Concnb (mol/L)
Temperature
Additive solvents (1%)
Yield (%)
Ratio (
a
:b)
Recovery
D (H)
A
1
2
p-Xylene
p-Xylened,e
Toluene
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.15
0.3
rt
rt
4 °C
None
None
None
None
None
None
H2O
Satd NaCl aq
H2O
Satd NaCl aq
Toluene
CH3CN
Et2O
CHCl3
Et2O–CHCl3(1:1)
None
33
83
25
90
<1
66
40
24
25
17
66
71
50
54
56
78
84
90
51:1
55:1
55:1
4.3:1
-
49
7
65
8
85
-
72
23
87
46
26 (32)
43 (42)
41 (41)
35 (49)
57
49
68
73
70
3
4c
p-Xylened
Toluenef,6
p-Xylenef,6
p-Xylene
p-Xylene
p-Xylene
p-Xylene
p-Xylene
p-Xylene
p-Xylene
p-Xylene
p-Xylene
Toluened
Toluened
Toluened
4 °C
ꢁ20 °C
ꢁ20 °C
4 °C
4 °C
ꢁ20 °C
ꢁ20 °C
4 °C
4 °C
4 °C
4 °C
4 °C
4 °C
4 °C
4 °C
5
89
28
59
76
76
79
33
22
48
43
39
10
6
6c
4.5:1
3.4:1
1.5:1
4.2:1
3.9:1
7.1:1
5.9:1
5.8:1
6.6:1
6.1:1
13:1
7.9:1
4.5:1
7c
8c
9c
10c
11c
12c
13c
14c
15c
16
17
18
39
30
14
None
None
1.5
10
a
b
c
The yield and stereoselectivity were determined after gel filtration.
Concentration of acceptor before freezing.
Frozen conditions.
d
The yield was determined by MALDI-TOF MASS quantitative analysis of crude sample. The stereoselectivity was determined by 1H NMR of crude sample. Methyl 2,3,4,
6-tetra-O-[D7]benzyl-1-thio-
a
-
D
-mannopyranoside [D28]2 was used instead of 2.
e
For 14 days.
f
The yield and stereoselectivity were determined based on isolated yield.

A. Ishiwata et al. / Bioorg. Med. Chem. 18 (2010) 3687–3695
3689
Table 2
frozen p-xylene (0.03 mol/L, entry 4), in terms of both the product
yield (90%) and selectivity (4.5:1 vs 4.3:1). These results support
our hypothesis that the decrease of stereoselectivity in frozen sys-
tems was caused by increased concentration, and provided the
estimate of substrate concentrations under frozen conditions,
which seems to reached as high as ꢂ1.5 mol/L.
Glycosylation of 1 with 2 at 4 °C: effect of solventsa,b
Entry
Solvent
Yield (%)
Ratio (
a
:b)
Recovery
A
D
1
m-Xylene
Dioxane
THP
t-BuCN
MeCN
8
87
61
84
54
100:1
3.7:1
7.1:1
2.5:1
2.1:1
78
11
33
19
39
97
34
75
28
61
2c
3
4c
5
2.4. Effect of anomeric stereochemistry of donor on reactivity of
donors and stereoselectivity of their glycosylation
a
The reactions were carried out in 0.03 mol/L solution of acceptor before
freezing.
Our study next addressed effect of the donor anomericity. That
the glycosylation in frozen media proceeded with reduced a-selec-
b
Compound [D28]2 was used. The yield was determined by MALDI-TOF MASS
quantitative analysis of crude sample. The stereoselectivity was determined by 1H
NMR of crude sample.
tivity may be explained by two different ways. As the first possibil-
ity, higher concentrations of substrates would perturb the nature
(e.g., polarity) of the solvent, and affect the stereoselectivity. Alter-
natively, direct SN2 type displacement of activated thioglycoside,
which gives b-glycoside, might become functional as an alternative
pathway that compete to oxocarbenium ion pathway, when con-
centrations of 1 and 2 are high. To gain a clue to distinguish these
possibilities, we conducted reactions with b-thioglycoside 2b as the
donor, which was prepared from 10b24 (Scheme 3). As shown in
Table 3 (entries 1–3), the reactivity of the b-isomer 2b was much
c
Solvent was freezing.
2.3. Effect of concentration on stereoselectivity of glycosylation
Rate-enhancing effect of the frozen system may be explained by
its ability to generate highly concentrated micropockets. However,
it is rather difficult to experimentally assess their substrate con-
centrations. On the other hand, our study has shown that rate
acceleration in frozen systems was associated with alternation of
stereoselectivity, leading us to explore the correlation between
the selectivity and concentration. We expected that, by extrapolat-
ing the concentration-selectivity relationship of non-frozen reac-
tions, reasonable estimate of the degree of concentration in
frozen mixtures.
To this end, the effect of the concentration of the substrate in
toluene (non-frozen) at 4 °C was evaluated (Table 1, entries 3
and 16–18). In toluene, the reaction was accelerated in a concen-
tration dependent manner, while the a-selectivity of the glycoside
3 was reduced at higher concentrations. The result in concentrated
faster than that of the
pletion within 1 h both at rt and at 4 °C. In frozen p-xylene (entry
2), the -selectivity was approximately two times higher than the
same reaction using the -thioglycoside 2 (Table 1, entry 4), while
differences in the selectivity under non-frozen conditions are less
marked and, instead, slightly shifted to less -selective direction
a-isomer (2), reactions proceeding to com-
a
a
a
(entries 1 and 3). These results seem to suggest the occurrence of
SN2 type displacement of the activated intermediate in association
with more dominant oxocarbenium ion like intermediate under
highly concentrated conditions. However, it is certainly possible
that the presence of substrates and reagents in high concentrations
gave an impact and altered selectivity, although our current exper-
imental setup is not able to address this possibility.
toluene solution (1.5 mol/L, entry 18) was very similar to that in
Table 3
Glycosylation of various donors under frozen conditionsa,b
Entry
Donor
Solvent
Temp
Time
Yield (%)
Ratio (
a
:b)
Recovery
A
D (H)
1
2b
2b
2b
4
4
4
4
4
4
p-Xylene
p-Xylene
Toluene
p-Xylene
p-Xylene
Toluene
p-Xylene
p-Xylene
Toluene
p-Xylene
p-Xylene
Toluene
p-Xylene
p-Xylene
Toluene
p-Xylene
p-Xylene
Toluene
p-Xylene
p-Xylene
Toluene
p-Xylene
p-Xylene
Toluene
rt
1 h
1 h
1 h
6 h
6 h
6 h
14 d
14 d
14 d
14 d
14 d
14 d
6 h
6 h
6 h
5 d
5 d
5 d
6 h
95
100
73
1
5
0
19
51
14
25
60
18
5
19
0
26
92
17
2
37:1
8.3:1
26:1
1.8:1
1:1.3
—
2.4:1
1.2:1
1.4:1
2.1:1
1:1.3
1.1:1
3.1:1
1:1.5
—
3.8:1
1.5:1
2.1:1
a
41:1
—
a
46:1
a
1
—
2
—
37
130
113
126
113
49
97
98
55
100
120
93
128
90
2c
4 °C
4 °C
rt
4 °C
4 °C
rt
4 °C
4 °C
rt
4 °C
4 °C
rt
4 °C
4 °C
rt
4 °C
4 °C
rt
4 °C
4 °C
rt
4 °C
4 °C
3
20
87
80
85
80
32
82
73
25
83
87
80
89
64
5
74
90
57
93
73
10
90
4
5c
6
7
8c
9
10
11c
12
13
14c
15
16
17c
18
19
20c
21
22
23c
24
4d
4d
4d
5e
5e
5e
5e
5e
5e
6
—
112
110
77
123
95
6
6
6
6
6 h
6 h
2 d
2 d
2 d
32
Trace
16
82
5
33
110
6
a
b
c
The reactions were carried out in 0.03 mol/L solution of acceptor before freezing.
The yield and stereoselectivity were determined after gel filtration.
Solvent was freezing.
d
e
2.5 equiv of MeOTf and 3.0 equiv of DTBMP were used.
The yield was determined by MALDI-TOF MASS quantitative analysis of crude sample. The stereoselectivity was determined by 1H NMR of crude sample.

3690
A. Ishiwata et al. / Bioorg. Med. Chem. 18 (2010) 3687–3695
OR1
R2O
Acceptor
O
R2O
MeOTf (2.5 eq.)
DTBMP (1.5 eq.)
1
BnO
R1 R2
+
3
7
8
9
Bn Bn
MS4A
O
BnO
Donors
Bn PhCH<
Bn H10C6<
Bz Bn
O
BnO
2,4~6
BnO
BnO
OBn
OBn
O
OBz
BnO
BnO
BnO
BnO
O
Ph
O
O
BnO
O
BnO
BnO
SMe
SMe
SMe
2α
4
5
6
OBn
O
OBn
O
BnO
BnO
BnO
O
O
SMe
BnO
SMe
2β
Scheme 2.
2.5. Effect of protective groups
ene group,26,28 was revealed to be more reactive than 4 (entries
13–18). For instance, disaccharide was obtained in 92% ( :b =
a
In addition to the perbenzylated thioglycoside 2 (see Scheme 2),
mannosyl donors with 4,6-O-cyclic (4, 5) or 2-O-acyl (6) protection
were studied, which in turn were prepared from 12,25 13,26 and
11,24 respectively (Scheme 3). Results of reactions with 1 are tabu-
lated in Table 3 (entries 4–24). In the case of 4,6-O-benzylidene
protected donor 4, which has been widely used for direct b-man-
nosylation,27 the reaction was extremely slow under our standard
conditions (entries 4–12). However, rate-enhancing effect of the
frozen system is still evident. After 14 days under frozen condi-
tions, 51% of the product was obtained with low selectivity
1.5:1) under frozen conditions after 5 days (entry 17). Since we
perceived that 4,6-O-cyclohexylidene group would be more deacti-
vating than 4,6-O-benzylidene acetal, because the former would
likely to confer higher degree of rigidity to the pyranose ring
system, these results were somehow unexpected. In any event,
we observed that the proportion of the b-isomer was uniformly
increased when 4,6-O-cyclic protected donors 4 and 5 were used
and that tendency was more prominent under frozen conditions.
The reactivity of 2-O-Bz protected donor 6 was revealed to be in
between 2 and 4/5, (entries 19–24), although much higher activity
was initially expected due to the ‘super arming’ effect proposed by
Demchenko.29 Quite dramatic rate acceleration was observed in its
(a:b = 1.2:1) (entry 8), although use of excess amount of DTBMP
resulted in improvement of the yield slightly (60%) and the selec-
tivity was slightly in favor of the b-glycoside (entry 11). On the
other hand, the compound 5, which carried a 4,6-O-cyclohexylid-
reactions with 1 under frozen conditions. In these cases, the a-gly-
coside was obtained stereoselectively as expected, although forma-
tion of a small amount of the b-isomer was revealed, implicating
the high precision of our setup in evaluating the selectivity (entries
20 and 23).
OAc
O
OBn
O
a,b
BnO
BnO
BnO
BnO
SMe
SMe
3. Conclusion
BnO
BnO
10β
2β
The aim of our study was to reveal the origin of rate-enhancing
effect of frozen conditions on O-glycosylation. It first revealed that
inclusion of small proportion of co-solvent was detrimental,
indicating that rigidly frozen conditions are necessary to cause
optimum rate enhancement. Secondly, the stereoselectivities
under frozen and non-frozen conditions were compared. Concentra-
tion-selectivity relationship suggested that highly concentrated
particles are generated in frozen solvents. Since selectivity and yield
in frozen p-xylene (0.03 mol/L) were nearly identical with those in
highly concentrated solution (1.5 mol/L) in toluene, frozen condi-
tions seem to give rise to ꢂ50 times increase of concentration.
Rate enhancements were also observed in other solvents such
as dioxane and t-BuCN, although the extent of acceleration was
less marked. In addition, reactions with donors having 4,6-O-cyclic
as well as 2-O-acyl protection were accelerated also, indicating
that rate enhancement effect of frozen p-xylene has some general-
ity. However, our current study is not able to pin-down the domi-
nant factor that attenuates stereoselectivity in a concentrated-
dependent manner. Further study is underway to clarify this
intriguing issue.
OH
O
OBn
O
RO
RO
c
RO
HO
RO
BnO
SMe
SMe
R
R
PhCH<
H10C6<
11
12
PhCH<
10C6<
4
5
H
OAc
O
OBz
BnO
BnO
a,d
O
BnO
BnO
BnO
BnO
SMe
SMe
10α
6
Scheme 3. Reagents and conditions: (a) NaOMe, MeOH, (b) NaH, BnBr, DMF, 0 °C to
rt, 3 h, 71% in two steps from 10b; (c) NaH, BnBr, DMF, 0 °C to rt, 3 h, 88%, (4) 78%
(5); (d) BzCl, pyridine, DMAP, 0 °C to rt, 1.5 h, 92% in two steps from 10a.

A. Ishiwata et al. / Bioorg. Med. Chem. 18 (2010) 3687–3695
3691
NaH (0.563 g, 8.31 mmol) at 0 °C under Ar atmosphere and the
mixture was stirred for 30 min at the same temperature. To the
reaction mixture was added benzyl bromide (1.47 mL, 12.1 mmol),
and the mixture was stirred for 3 h at room temperature under Ar
atmosphere. After addition of triethylamine to the mixture fol-
lowed by quenching by brine, the product was extracted with
CHCl3. Combined solutions were washed with brine and dried over
Na2SO4. After concentration, the residue was purified by flash col-
umn chromatography using gradient solvent system (toluene/ethyl
acetate = 100/1 to 50/1 to 20/1 to 10/1 to 5/1) to give the title com-
pound (2.11 g, 88%).
4. Experimental section
4.1. General procedures
All reactions sensitive to air or moisture were carried out under
nitrogen or argon atmosphere with anhydrous solvent. All reagents
were purchased from commercial suppliers and used without fur-
ther purification unless otherwise noted. Thin-layer chromatogra-
phy was performed using silica gel 60 F254 precoated plates
(0.25 mm thickness) with a fluorescent indicator. Visualization
on TLC was achieved by UV light (254 nm) and a typical TLC indi-
cation solution. Column chromatography was performed on silica
gel 60 N, 100–210 mesh (Kanto Kagaku Co., Ltd.). 1H NMR spectra
were recorded at 400 MHz on a JEOL ECX 400 spectometer and
chemical shifts were referred to internal tetramethylsilane
(0 ppm) or CDCl3 (7.26 ppm). 13C NMR spectra were recorded at
100 MHz on the same instruments and chemical shifts were re-
ferred to internal CDCl3 (77.0 ppm). Melting points were deter-
mined with Büchi 510 melting point apparatus. Optical rotations
were measured with a JASCO DIP 370 polarimeter. MALDI-TOF
mass spectra were recorded on a SHIMADZU Kompact MALDI AX-
IMA-CFR spectrometer with 2,5-dihydroxybenzoic acid as the
matrix. ESI-TOF mass spectra were recorded on a JEOL AccuTOF
JMS-T700LCK with CF3CO2Na as the internal standard.
½
a 2D8 81.4 (c 1.0, CHCl3); 1H NMR (CDCl3, 400 MHz): d 2.08 (s,
ꢃ
SMe, 3H), 3.87 (dd, J = 3.2, 0.9 Hz, 2-H, 1H), 3.88 (dd, J = 10.1,
1.4 Hz, 6a-H, 1H), 3.91 (dd, J = 9.6, 3.2, 3-H, 1H), 4.15 (ddd,
J = 9.6, 4.6, 1.4 Hz, 5-H, 1H), 4.22 (dd, J = 10.1, 4.6 Hz, 6b-H, 1H),
4.27 (t, J = 9.6 Hz, 4-H, 1H), 4.60 (d, J = 12.4, –CH2Ph, 1H), 4.73–
4.82 (m, –CH2Ph, 3H), 5.19 (d, J = 0.9 Hz, 1-H, 1H), 5.63 (s, >CHPh,
1H), 7.24–7.39 (m, Ar, 13H), 7.48–7.51 (m, Ar, 2H); 13C NMR
(CDCl3, 100 MHz): d 13.8, 64.6, 68.7, 72.99, 73.04, 76.4, 77.8,
79.2, 84.9, 101.5, 126.1, 127.55, 127.61, 127.8, 128.1, 128.2,
128.3, 128.4, 128.8, 137.6, 137.8, 138.4; MALDI-TOF MS: [M+Na]+
calcd for C28H30O5S1Na1, 501.17, found 501.20; HRMS ESI-TOF:
[M+Na]+ calcd for C28H30O5S1Na1, 501.1712, found 501.1706.
4.4. Methyl 4,6-O-cyclohexylidenel-2,3-di-O-benzyl-1-thio-a-D-
mannopyranoside (5)
4.2. Methyl 2,3,4,6-tetra-O-benzyl-1-thio-b-
(2b)
D-mannopyranoside
The title compound 5 was synthesized from methyl 4,6-O-
cyclohexylidene-1-thio- -mannopyranoside (12) according to
the procedure for the synthesis of 4 from 11 (78%).
114.0 (c 1.0, CHCl3); 1H NMR (CDCl3, 400 MHz): d 1.98 (s,
To a solution of methyl 2-O-acetyl-1-thio-3,4,6-tri-O-benzyl-
D-mannopyranoside (10b) (128.2 mg, 0.245 mmol) in dry metha-
a-D
b-
½ ꢃ
a 2D8
nol (2 mL) was added NaOMe to justify to alkaline, indicated by
phenolphthalein, at room temperature, and the mixture was stir-
red for 1 h at the same temperature. Amberlist 15H+ was added
to the mixture to quench excess NaOMe. The resin was filtered
off and concentrated. Crude mixture was then dissolved in dry
DMF (2 mL) was added NaH (13.7 mg, 0.343 mmol) at 0 °C under
Ar atmosphere and the mixture was stirred for 30 min at the same
temperature. To the reaction mixture was added benzyl bromide
SMe, 3H), 3.69 (dd, J = 9.6, 3.2 Hz, 3-H, 1H), 3.72 (dd, J = 10.1,
4.6 Hz, 6a-H, 1H), 3.77 (dd, J = 3.2, 1.4, 2-H, 1H), 3.85 (dd,
J = 10.6, 10.1 Hz, 6b-H, 1H), 3.91 (ddd, J = 10.6, 9.6, 4.6 Hz, 5-H,
1H), 4.21 (t, J = 9.6 Hz, 4-H, 1H), 4.55 (d, J = 12.4, –CH2Ph, 1H),
4.61–4.70 (m, –CH2Ph, 2H), 4.79 (d, J = 12.4 Hz, –CH2Ph, 1H), 5.06
(s, 1-H, 1H), 7.12–7.31 (m, Ar, 10H); 13C NMR (CDCl3, 100 MHz):
d 13.7, 22.6, 23.0, 25.6, 28.0, 38.2, 61.7, 65.6, 71.4, 73.0, 73.1,
76.8, 78.0, 85.0, 100.0, 127.4, 127.7, 128.0, 128.2, 128.3, 138.0,
138.9; MALDI-TOF MS: [M+Na]+ calcd for C27H34O5S1Na1, 493.20,
found 492.50; ESI-TOF MS: [M+Na]+ calcd for C27H34O5S1Na1,
493.20, found 493.16; HRMS ESI-TOF: [M+Na]+ calcd for
C27H34O5S1Na1, 493.2025, found 493.2039.
(35.7 lL, 0.294 mmol), and the mixture was stirred for 3 h at room
temperature under Ar atmosphere. After addition of triethylamine
to the mixture followed by quenching by brine, the product was
extracted with CHCl3. Combined solutions were washed with brine
and dried over Na2SO4. After concentration, the residue was puri-
fied by flash column chromatography using gradient solvent sys-
tem (toluene/ethyl acetate = 50/1 to 20/1 to 10/1 to 5/1) to give
the title compound (99.8 mg, 71%).
4.5. Methyl 2-O-benzoyl-3,4,6-O-benzyl-1-thio-a-D-mannopyra-
noside (6)
½
a 2D8
ꢃ
ꢁ13.3 (c 1.0, CHCl3); 1H NMR (CDCl3, 400 MHz): d 2.17
(s, SMe, 3H), 3.42 (ddd, J = 11.4, 6.0, 1.4 Hz, 5-H, 1H), 3.53 (dd,
J = 9.6, 2.8 Hz, 3-H, 1H), 3.65 (dd, J = 11.0, 6.0 Hz, 6a-H, 1H), 3.73
(dd, J = 11.4, 1.4 Hz, 6b-H, 1H), 3.84 (dd, J = 10.1, 9.6 Hz, 4-H, 1H),
3.91 (d, J = 2.8 Hz, 2-H, 1H), 4.38 (s, 1-H, 1H), 4.48–4.65 (m,
–CH2Ph, 5H), 4.76 (d, J = 11.9 Hz, –CH2Ph, 1H), 4.80 (d, J = 11.0 Hz,
–CH2Ph, 1H), 4.89 (d, J = 11.9 Hz, –CH2Ph, 1H), 7.11–7.13 (m, Ar,
2H), 7.18–7.28 (m, Ar, 16H), 7.37 (d, J = 6.9 Hz, Ar, 2H); 13C NMR
(CDCl3, 100 MHz): d 15.4, 70.5, 73.1, 74.2, 75.5, 75.6, 75.9, 81.0,
85.0, 86.4, 128.1, 128.3, 128.4, 128.5, 128.8, 128.96, 129.03,
129.1, 138.77, 138.84; MALDI-TOF MS: [M+Na]+ calcd for
C35H38O5SNa, 593.23, found 593.2; ESI-TOF MS: [M+Na]+ calcd
for C35H38O5S1Na1, 593.23, found 593.21; HRMS ESI-TOF:
[M+Na]+ calcd for C35H38O5S1Na1, 593.2338, found 593.2339.
To a solution of methyl 2-O-acetyl-3,4,6-O-benzyl-1-thio-a-D-
mannopyranoside (10b) (300 mg, 0.57 mmol) in dry methanol
(3 mL) was added NaOMe to justify to alkaline, indicated by phe-
nolphthalein, at room temperature, and the mixture was stirred
for 1 h at the same temperature. Amberlist 15H+ was added to
the mixture to quench excess NaOMe. Resin was filtered off and
concentrated. Crude mixture was then dissolved in dry pyridine
(3 mL), benzoyl chloride (66.2 mL, 0.57 mmol) and DMAP
(7.0 mg, 0.057 mmol) were added to the mixture at 0 °C under Ar
atmosphere. After being stirred at room temperature for 1.5 h,
the reaction was quenched with methanol and co-evaporated with
toluene. The residue was diluted with dichloromethane, washed
with 1 N HCl, water, sat. NaHCO3, water, and brine. The organic
layer was dried with MgSO4 and concentrated in vacuo. The resi-
due was purified by flash column chromatography using gradient
solvent system (toluene/ethyl acetate = 50/1 to 20/1 to 10/1 to
5/1) to give the title compound (325.1 mg, 94%).
4.3. Methyl 2,3-di-O-benzyl-4,6-O-benzylidene-1-thio-a-D-man-
nopyranoside (4)
a 2D8
31.9 (c 1.0, CHCl3); 1H NMR (CDCl3, 400 MHz): d 2.19 (s,
SMe, 3H), 3.81 (dd, J = 11.0, 1.4 Hz, 6a-H, 1H), 3.96 (dd, J = 11.0,
To a solution of methyl 4,6-O-benzylidene-1-thio-
a-D-manno-
½ ꢃ
pyranoside (11) (1.50 g, 5.03 mmol) in dry DMF (10 mL) was added

3692
A. Ishiwata et al. / Bioorg. Med. Chem. 18 (2010) 3687–3695
3.7 Hz, 6b-H, 1H), 4.08 (dd, J = 9.2, 3.2 Hz, 3-H, 1H), 4.17 (dd,
J = 10.1, 9.2 Hz, 4-H, 1H), 4.23 (ddd, J = 10.1, 3.7, 1.4 Hz, 5-H, 1H),
4.55–4.62 (m, –CH2Ph, 3H), 4.77 (d, J = 12.4 Hz, –CH2Ph, 1H), 4.81
(d, J = 11.9 Hz, –CH2Ph, 1H), 4.92 (d, J = 11.0 Hz, –CH2Ph, 1H), 5.36
(d, J = 1.4 Hz, 1-H, 1H), 5.75 (dd, J = 3.2, 1.4 Hz, 2-H, 1H), 7.20–
7.42 (m, Ar, 17H), 7.56–7.60 (m, Ar, 1H), 8.11–8.13 (m, Ar, 2H);
13C NMR (CDCl3, 100 MHz): d 13.9, 69.0, 70.5, 71.6, 71.9, 73.2,
74.4, 75.2, 78.6, 83.9, 127.4, 127.6, 127.7, 127.9, 128.1, 128.27,
128.34, 129.9, 133.1, 137.6, 138.27, 138.31, 165.6; MALDI-TOF
MS: [M+Na]+ calcd for C35H36O6SNa1, 607.21, found 607.20; ESI-
TOF MS: [M+Na]+ calcd for C35H36O6SNa1, 607.21, found 607.16;
HRMS ESI-TOF: [M+Na]+ calcd for C35H36O6SNa1, 607.2130, found
607.2149.
78.0, 81.5, 96.5 (C1Man1 JC–H = 169.8 Hz), 99.3 (C1Man2
, , JC–
H = 157.4 Hz), 127.2, 127.5, 127.78, 127.84, 128.0, 128.1, 128.19,
128.21, 128.3, 128.4, 137.1, 137.87, 137.94, 138.2, 138.4, 138.6,
138.9; MALDI-TOF MS: [M+Na]+ calcd for C68H42D28O11Na1,
1113.66, found 1113.61; ESI-TOF MS: [M+Na]+ calcd for
C68H42D28O11Na1, 1113.66, found 1113.65; HRMS ESI-TOF:
[M+Na]+ calcd for C68H42D28O11Na1, 1113.6573, found 1113.6596.
4.8. Benzyl 2,3,4,6-tetra-O-benzyl-
D-mannopyranosyl-(1?2)-3,
4,6-tri-O-benzyl- -mannopyranoside (3)
a
-D
Compound 3
a
: ½a 2D7
ꢃ
24.5° (c 1.0, CHCl3); 1H NMR (400 MHz,
CDCl3): d 3.52–3.88 (m, 3-HMan1, 4-HMan1, 5-HMan1, 6a-HMan1, 6b-
H
Man1, 2-HMan2, 3-HMan2, 4-HMan2, 5-HMan2, 6a-HMan2, 6b-HMan2
,
4.6. General procedure for frozen conditions
11H), 4.02 (dd, J = 2.4, 2.3 Hz, 2-HMan1, 1H), 4.27 (d, J = 11.9 Hz, –
CH2Ph, 1H), 4.38–4.61 (m, –CH2Ph, 13H), 4.74–4.78 (m, –CH2Ph,
After azeotropic removal of water with toluene, to the mixture
2H), 4.89 (d, J = 1.8 Hz, 1-HMan1, 1H), 5.10 (d, J = 1.8 Hz, 1-HMan2
,
of an acceptor 1 (8.1 mg, 15.0
20.3 mol, 1.35 equiv) in dry solvent (500
(ca. 0.3 g/0.1 mmol of acceptor) and DTBMP (5.7 mg, 22.5
1.5 equiv) and mixture was stirred under Ar for at room tempera-
ture for 30 min. MeOTf (4.2 L, 37.5 mol, 2.5 equiv) was added to
l
mol) and a donor [D28]2 (12.2 mg,
L) were added MS 4A
mol,
1H), 7.26–7.96 (m, Ar, 40H); 13C NMR (100 MHz, CDCl3): d 69.0,
l
l
69.2, 72.0, 72.1, 72.4, 73.3, 74.5, 74.9, 75.0, 79.6, 79.9, 98.2
l
(C1Man1 JC–H = 172.6 Hz), 99.5 (C1Man2
, , JC–H = 176.4 Hz), 127.4,
127.6, 127.7, 127.8, 128.2, 137.2, 138.2, 138.2, 138.4, 138.5; MALDI
TOF MS: calcd for C68H70NaO11 [M+Na]+, 1085.48; found 1085.33;
ESI-TOF MS: [M+Na]+ calcd for C68H70O11Na1, 1085.48, found
1085.43; HRMS ESI-TOF: [M+Na]+ calcd for C68H70O11Na1,
1085.4816, found 1085.4837.
l
l
mixture, and the mixture was rapidly mixed and frozen by liquid
nitrogen. The mixture was stored in refrigerator at 4 °C for 6 h
and was defrosted at temperature. The reaction quenched with tri-
ethylamine followed by filtration through Celite pad and washing
of pad with ethyl acetate. The combined solutions were washed
with brine, dried over MgSO4, and evaporated in vacuo. The residue
was purified by gel filtration (Bio beads SX-3, toluene/ethyl ace-
tate = 1:1) to give the mixture of the isomers (14.7 mg, 90%,
Compound 3b: ½a 2D7
ꢃ
ꢁ57.8° (c 1.0, CHCl3); 1H NMR (400 MHz,
CDCl3): d 3.33–3.43 (m, 5-HMan1, 3-HMan2, 2H), 3.54–3.73 (m, 6a-
H
Man1, 6b-HMan1, 5-H Man2, 6a-H Man2, 6b-HMan2, 5H), 3.77–3.84
(m, 4-HMan1, 4-HMan2, 2H), 3.91–3.94 (m, 3-HMan1, 2-HMan2, 2H),
4.28 (d, J = 11.5 Hz, –CH2Ph, 1H), 4.57–4.28 (m, 2-HMan1, 1-HMan2
,
a
:b = 4.3:1).
–CH2Ph, 10H), 4.64–4.70 (m, –CH2Ph, 3H), 4.76 (d, J = 11.9 Hz, –
CH2Ph, 1H), 4.83 (d, J = 11.0 Hz, –CH2Ph, 1H), 4.92 (d, J = 11.4 Hz,
–CH2Ph, 1H), 4.95 (d, J = 1.8 Hz, 1-HMan1, 1H), 5.03 (d, J = 11.9 Hz,
–CH2Ph, 1H), 6.95–7.44 (m, Ar, 40H); 13C NMR (100 MHz, CDCl3):
d 68.9, 69.2, 70.1, 70.8, 71.4, 71.5, 73.3, 73.4, 74.0, 74.2, 74.8,
74.9, 75.1, 75.8, 78.0, 81.6, 96.4 (C1Man1, JC–H = 172.6 Hz), 99.31
(C1Man2, JC–H = 158.3 Hz), 127.5, 127.5, 127.7, 127.7, 127.9, 128.0,
128.2, 128.4, 138.1, 138.4, 138.8; MALDI TOF MS: calcd for
C68H70NaO11 [M+Na]+, 1085.48; found 1085.32; ESI-TOF MS:
[M+Na]+ calcd for C68H70O11Na1, 1085.48, found 1085.44; HRMS
ESI-TOF: [M+Na]+ calcd for C68H70O11Na1, 1085.4816, found
1085.4844.
The residue was purified by preparative TLC (toluene/ethyl ace-
tate = 5/1) to afford 14.7 mg (90%) of compound. Detailed reaction
conditions, yield and stereoselectivity of the products for each
reaction were listed in Tables.
4.7. Benzyl 2,3,4,6-tetra-O-[D7]benzyl-
3,4,6-tri-O-benzyl- -mannopyranoside ([D28]3)
D-mannopyranosyl-(1?2)-
a
-D
Compound [D28]3
a
:
½
a 2D6
ꢃ
19.8° (c 1.0, CHCl3); 1H NMR
(400 MHz, CDCl3): d 3.51–3.88 (m, 3-HMan1, 4-HMan1, 5-HMan1, 6a-
Man1, 6b-HMan1, 2-HMan2, 3-HMan2, 4-HMan2, 5-HMan2, 6a-HMan2
H
,
6b-HMan2, 11H), 4.02 (dd, J = 2.4, 2.3 Hz, 2-HMan1, 1H), 4.27 (d,
J = 12.4 Hz, –CH2Ph, 1H), 4.47 (d, J = 11.9 Hz, –CH2Ph, 2H), 4.52–
4.62 (m, –CH2Ph, 4H), 4.76 (d, J = 11.0 Hz, –CH2Ph, 1H), 4.89 (d,
J = 1.4 Hz, 1-HMan1, 1H), 5.10 (d, J = 1.4 Hz, 1-HMan2, 1H), 7.08–
7.26 (m, Ar, 20H); 13C NMR (100 MHz, CDCl3): d 69.0, 69.1, 69.2,
72.1, 72.5, 73.3, 74.5, 74.7, 74.9, 75.1, 79.6, 79.9, 98.2, 99.5,
127.4, 127.5 127.7, 127.9, 128.0, 128.25, 128.32, 128.36, 128.40,
137.3, 138.0, 138.2, 138.3, 138.5; MALDI-TOF MS: [M+Na]+ calcd
for C68H42D28O11Na1, 1113.66, found 1113.68; ESI-TOF MS:
[M+Na]+ calcd for C68H42D28O11Na1, 1113.66, found 1113.66;
HRMS ESI-TOF: [M+Na]+ calcd for C68H42D28O11Na1, 1113.6573,
found 1113.6536.
4.9. Benzyl 4,6-O-benzylidene-2,3-di-O-benzyl-
D-mannopyrano-
syl-(1?2)-3,4,6-tri-O-benzyl- -mannopyranoside (7)
a
-D
Compound 7
a
: ½a 2D4
ꢃ
27.2° (c 1.0, CHCl3); 1H NMR (CDCl3,
400 MHz): d 3.60–3.81 (m, 2-HMan1, 5-HMan1, 6-HMan1, 4-HMan2
,
,
5-HMan2, 6a-HMan2, 6b-HMan2, 7H), 3.86–3.91 (m, 3-HMan2, 3-HMan1
2H), 3.98 (t, J = 2.3 Hz, 2-HMan2, 1H), 4.02 (dd, J = 9.2, 3.2 Hz, 6a-
H
Man1, 1H), 4.13 (t, J = 9.6 Hz, 4-HMan1, 1H), 4.39–4.76 (m, –CH2Ph,
12H), 4.85 (d, J = 1.8 Hz, 1-HMan2, 1H), 5.08 (d, J = 1.4 Hz, H-1Man1
,
1H), 5.53 (s, >CHPh, 1H), 7.14–7.45 (m, Ar, 35H); 13C NMR (CDCl3,
100 MHz): d 64.6, 68.7, 69.1, 69.2, 72.2, 72.7, 73.0, 73.3, 73.4,
Compound [D28]3b:
½
a 2D7
ꢃ
ꢁ30.8° (c 1.0, CHCl3); 1H NMR
74.4, 74.9, 75.2, 76.2, 76.4, 77.2, 79.2, 80.1, 98.2 (C1Man2
,
(400 MHz, CDCl3): d 3.32–3.37 (m, 5-HMan1, 1H), 3.40 (dd, J = 9.6,
JC–H = 171.7 Hz), 100.5 (C1Man1
, JC–H = 179.3 Hz), 101.5 (JC–H =
3.2 Hz, 3-HMan1, 1H), 3.55–3.67 (m, 6a-HMan1, 6b-HMan1, 6a-HMan2
,
166.9 Hz), 126.1, 127.49, 127.52, 127.88, 127.93, 128.0, 128.1,
128.17, 128.21, 128.3, 128.4, 128.5, 137.1, 137.7, 138.09, 138.14,
138.3, 138.5, 138.8; MALDI-TOF MS: [M+Na]+ calcd for
C61H62O11Na, 993.42, found 993.57; ESI-TOF MS: [M+Na]+ calcd
for C61H62O11Na1, 993.42, found 993.39; HRMS ESI-TOF: [M+Na]+
calcd for C61H62O11Na1, 993.4190, found 993.4148.
6b-HMan2, 4H), 3.70–3.74 (m, 5-HMan2, 1H), 3.79 (dd, J = 10.1,
9.6 Hz, 4-HMan1, 1H), 3.82 (dd, J = 10.1, 9.2 Hz, 4-HMan2, 1H),
3.92–3.95 (m, 3-HMan2, 2-HMan1, 2H), 4.29 (d, J = 11.0 Hz, –CH2Ph,
1H), 4.36–4.43 (m, 2-HMan2 1-HMan1
, , –CH2Ph, 5H), 4.53 (d,
J = 12.4 Hz, –CH2Ph, 1H), 4.66 (d, J = 11.9 Hz, –CH2Ph, 1H), 4.68 (d,
J = 11.0 Hz, –CH2Ph, 1H), 4.92 (d, J = 11.0 Hz, –CH2Ph, 1H), 4.95 (d,
J = 1.8 Hz, 1-HMan2, 1H), 6.95–6.98 (m, Ar, 2H), 7.11–7.26 (m, Ar,
16H), 7.36–7.38 (m, Ar, 2H); 13C NMR (100 MHz, CDCl3): d 68.9,
69.3, 69.9, 70.1, 71.5, 71.6, 73.3, 73.7, 74.1, 74.8, 74.0, 75.9, 77.2,
Compound 7b: ½a 2D3
ꢃ
ꢁ44.4° (c 1.0, CHCl3); 1H NMR (400 MHz,
CDCl3):
d , 1H), 3.57 (dd,
3.46 (dd, J = 10.1, 3.2 Hz, 3-HMan1
J = 11.0, 1.8 Hz, 6a-HMan2, 1H), 3.63 (dd, J = 11.0, 4.6 Hz, 6b-HMan2
1H), 3.71–3.76 (m, 6a-HMan1, 5-HMan2, 2H), 3.84 (dd, J = 9.6,
,

A. Ishiwata et al. / Bioorg. Med. Chem. 18 (2010) 3687–3695
3693
9.2 Hz, 4-HMan2, 1H), 3.90–3.93 (m, 2-HMan2, 3-HMan1, 2H), 4.12 (dd,
6b-HMan1, 3H), 3.80 (dd, J = 9.6, 9.2 Hz, 4-HMan2, 1H), 3.86–3.98
(m, 2-HMan2, 4-HMan1, 3-HMan2, 5-HMan1, 4H), 4.02 (dd, J = 99.2,
2.8 Hz, 3-HMan1, 1H), 4.31 (d, J = 11.9, –CH2Ph, 1H), 4.36–4.44 (m,
–CH2Ph, 3H), 4.48 (d, J = 11.9 Hz, –CH2Ph, 2H), 4.57–4.64 (m, 5H),
4.68 (d, J = 11.0 Hz, –CH2Ph, 1H), 4.68 (d, J = 11.0 Hz, –CH2Ph, 2H),
J = 10.1, 9.2 Hz, 6b-HMan1, 1H), 4.14 (dd, J = 10.5, 5.0 Hz, 6b-HMan1
1H), 4.21–4.24 (m, 2-HMan2, –CH2Ph, 2H), 4.37–4.78 (m, 1-HMan1
,
,
–CH2Ph, 11H), 4.93 (d, J = 1.8 Hz, 1-HMan2
, 1H), 5.00 (d,
J = 11.9 Hz, –CH2Ph, 1H), 5.51 (s, >CHPh, 1H), 6.87–7.01 (m, Ar,
2H), 7.10–7.36 (m, Ar, 27H), 7.36–7.45 (m, Ar, 6H); 13C NMR
(100 MHz, CDCl3): d 67.6, 68.5, 68.9, 69.3, 70.7, 71.6, 71.8, 72.9,
4.75–4.80 (d, J = 11.0 Hz, –CH2Ph, 2H), 4.91 (d, J = 1.8 Hz, 1-HMan2
,
1H), 5.11 (d, J = 1.8 Hz, 1-HMan1, 1H), 5.70 (dd, J = 2.7, 2.3 Hz, 2-
H
Man1, 1H), 7.10–7.30 (m, Ar, 37H), 7.48 (dd, J = 7.8, 1.4 Hz, Ar,
73.3, 73.5, 74.3, 74.95, 75.04, 76.1, 77.2, 78.1, 78.3, 96.6 (C1Man2
,
JC–H = 170.7 Hz), 100.3 (C1Man1
,
JC–H = 154.5 Hz), 101.3 (JC–H
=
1H), 7.89–8.00 (dd, J = 9.6, 1.4 Hz, Ar, 2H); 13C NMR (CDCl3,
100 MHz): d 69.0, 69.2, 71.7, 72.96, 72.04, 72.2, 73.3, 73.4, 74.3,
74.7, 75.2, 77.2, 78.1, 80.0, 98.0 (C1Man2, JC–H = 172.6 Hz), 99.6
(C1Man1, JC–H = 178.4 Hz), 127.4, 127.5, 127.6, 127.7, 127.9, 128.1,
128.2, 128.30, 128.32, 128.4, 129.9, 133.0, 137.2, 138.3, 138.4,
138.45, 138.50, 165.4; MALDI-TOF MS: [M+Na]+ calcd for
C68H68O12Na1, 1099.46, found 1099.48; ESI-TOF MS: [M+Na]+ calcd
for C68H68O12Na1, 1099.46, found 1099.47; HRMS ESI-TOF:
[M+Na]+ calcd for C68H68O12Na1, 1099.4609, found 1099.4575.
164.0 Hz), 126.0, 127.5, 127.8, 127.9, 128.1, 128.15, 128.24,
128.27, 128.30, 128.4, 128.7, 137.1, 137.6, 138.1, 138.3, 138.7;
MALDI-TOF MS: [M+Na]+ calcd for C61H62O11Na, 993.42, found
993.42; ESI-TOF MS: [M+Na]+ calcd for C61H62O11Na1, 993.42,
found 993.36; HRMS ESI-TOF: [M+Na]+ calcd for C61H62O11Na1,
993.4190, found 993.4151.
4.10. Benzyl 4,6-O-cyclohexylidene-2,3-di-O-benzyl-
D-mannopy-
Compound 9b: ½a 2D6
ꢃ
ꢁ55.7 (c 1.0, CHCl3); 1H NMR (400 MHz,
ranosyl-(1?2)-3,4,6-tri-O-benzyl- -mannopyranoside (8)
a-D
CDCl3): d 3.40–3.54 (m, 5-HMan1, 4-HMan2, 6a-HMan2, 6b-HMan2
,
4H), 3.67–3.73 (m, 3-HMan1, 5-HMan2, 6a-HMan1, 6b-HMan1, 4H),
3.80–3.85 (m, –CH2Ph, 3-HMan2, 2H), 3.91 (dd, J = 10.1, 9.6 Hz,
4-HMan1, 1H), 4.21 (d, J = 11.5 Hz, –CH2Ph, 1H), 4.26 (d, J = 11.5,
–CH2Ph, 1H), 4.33 (t, J = 2.3 Hz, 2-HMan2, 1H), 4.39–4.43 (m,
–CH2Ph, 3H), 4.47–4.53 (m, –CH2Ph, 4H), 4.66 (d, J = 9.6 Hz,
–CH2Ph, 1H), 4.68 (s, 1-HMan1, 1H), 4.75 (d, J = 11.4 Hz, –CH2Ph,
1H), 4.82 (d, J = 11.9 Hz, –CH2Ph, 1H), 4.83 (d, J = 11.0 Hz, –CH2Ph,
Compound 8
a
: ½a 2D5
ꢃ
48.2° (c 1.0, CHCl3); 1H NMR (400 MHz,
CDCl3): d 1.30–2,25 (m, 10H), 3.68–3.87 (m, 2-HMan1, 3-HMan1
,
5-HMan1, 6a-HMan1, 6b-HMan1, 4-HMan2, 5-HMan2, 6a-HMan2, 6b-
Man2, 9H), 3.94 (dd, J = 9.2, 3.2 Hz, 3-HMan2, 1H), 4.04 (dd, J = 2.8,
H
1.8 Hz, 2-HMan2, 1H), 4.22 (t, J = 9.6 Hz, 4-HMan1, 1H), 4.46–4.65
(m, –CH2Ph, 8H), 4.71 (d, J = 12.8 Hz, –CH2Ph, 2H), 4.80 (d,
J = 11.0 Hz, –CH2Ph, 1H), 4.82 (d, J = 11.9 Hz, –CH2Ph, 1H), 4.92 (d,
J = 1.8 Hz, 1-HMan2, 1H), 5.11 (d, J = 1.4 Hz, 1-HMan1, 1H), 7.19–
7.38 (m, Ar, 30H); 13C NMR (100 MHz, CDCl3): d 22.6, 23.0, 25.6,
28.0, 38.2, 61.7, 65.5, 69.1, 71.3, 72.1, 72.6, 72.9, 73.3, 73.4, 74.0,
74.8, 75.2, 76.5, 77.2, 80.0, 98.4, 99.8 (C1Man2, JC–H = 171.7 Hz),
100.6 (C1Man1, JC–H = 179.3 Hz), 127.2, 127.4, 127.5, 127.7, 127.87,
127.92, 127.95, 128.10, 128.3, 128.36, 128.42, 128.5, 137.1, 138.1,
138.25, 138.31, 138.5, 139.3; MALDI-TOF MS: [M+Na]+ calcd for
C60H66O11Na1, 985.45. found 985.29; ESI-TOF MS: [M+Na]+ calcd
for C60H66O11Na1, 985.45, found 985.40; HRMS ESI-TOF: [M+Na]+
calcd for C60H66O11Na1, 985.4503, found 985.4527.
1H), 4.93 (d, J = 1.8 Hz, 1-HMan2, 1H), 5.81 (d, J = 3.2 Hz, 2-HMan1
,
1H), 6.81–6.83 (m, Ar, 2H), 7.02–7.34 (m, Ar, 36H), 8.04 (d,
J = 8.2, Ar, 2H); 13C NMR (100 MHz, CDCl3): d 68.7, 69.2, 69.3,
69.6, 70.6, 71.0, 71.4, 71.8, 73.1, 73.4, 74.3, 74.5, 75.3, 75.4, 77.2,
78.3, 80.0, 96.4 (x2, C1Man1, C1Man2, JC-H = 166.0 Hz), 127.1, 127.4,
127.5, 127.7, 127.8, 128.0, 128.1, 128.19, 128.23, 128.3, 128.4,
130.3, 130.4, 132.6, 137.2, 137.6, 138.2, 138.3, 138.4, 138.9; MAL-
DI-TOF MS: [M+Na]+ calcd for C68H68O12Na1, 1099.46, found
1099.46; ESI-TOF MS: [M+Na]+ calcd for C68H68O12Na1, 1099.46,
found 1099.44; HRMS ESI-TOF: [M+Na]+ calcd for C68H68O12Na1,
1099.4609, found 1099.4619.
Compound 8b: ½a 2D6
ꢃ
ꢁ26.0° (c 1.0, CHCl3); 1H NMR (400 MHz,
CDCl3): d 1.28–2.26 (m, 10H), 3.10 (ddd, 5-HMan1, J = 9.6, 6.4,
3.2 Hz, 1H), 3.38 (dd, J = 9.6, 3.7 Hz, 3-HMan1, 1H), 3.63 (dd,
4.12. General procedure for high through-put screening for
O-glycosylation
J = 10.6, 1.8 Hz, 6a-HMan2, 1H), 3.68 (dd, J = 11.0, 4.6 Hz, 6b-HMan2
,
1H), 3.75–3.82 (m, 5-HMan2, 6a-HMan1, 6b-HMan1, 3H), 3.88 (dd,
J = 10.1, 8.7 Hz, 4-HMan2, 1H), 3.93 (d, J = 3.2 Hz, 2-HMan1, 1H),
3.97 (dd, J = 8.7, 3.2 Hz, 3-HMan2, 1H), 4.19 (dd, J = 10.1, 9.6 Hz,
4-HMan1, 1H), 4.26 (s, 2-HMan2, 1H), 4.58 (d, J = 12.4 Hz, –CH2Ph,
1H), 4.63 (d, J = 12.8 Hz, –CH2Ph, 1H), 4.70–4.76 (m, –CH2Ph, 3H),
4.81–4.87 (m, –CH2Ph, 2H), 4.98 (d, J = 1.8 Hz, 1-HMan1, 1H), 5.03
(d, J = 11.9 Hz, –CH2Ph, 1H), 7.05–7.09 (m, Ar, 2H), 7.15–7.37 (m,
Ar, 24H), 7.43–7.45 (m, Ar, 2H), 7.51–7.53 (m, Ar, 2H); 13C NMR
(100 MHz, CDCl3): d 22.6, 23.0, 25.6, 28.0, 38.1, 61.5, 68.8, 68.9,
69.2, 70.5, 70.7, 71.6, 71.8, 72.9, 73.3, 74.3, 74.9, 77.7, 78.1, 96.7,
Each 12.0 mL of solution of acceptor 1 (48.6 mg, 90.0
donor [D7]2 (72.8 mg, 122 mol) and DTBMP (34.5 mg, 140
in 2.0 mL of CH2Cl2 were pipetted into multiplicate tubes, and the
l
mol),
l
lmol)
mixtures were evaporated by flashing with N2 gas. In each tube,
15.0
DTBMP were prepared for the reaction. After MS4A (45 mg) and
each solvent (500 L) were added to the mixture, methyl trifluoro-
methanesulfonate (4.2 L, 38.0 mol) was added to each tube. the
lmol of acceptor, 20.3 lmol of donor and 22.5 lmol of
l
l
l
mixture was rapidly mixed and frozen by liquid nitrogen. The mix-
ture was stored in refrigerator at 4 °C for 6 h and was defrosted at
temperature. The reaction quenched with triethylamine followed
by filtration through Celite pad and washing of pad with ethyl ace-
tate. The combined solutions were washed with brine, dried over
MgSO4, and evaporated in vacuo. The residue was purified by gel
filtration (Bio beads SX-3, toluene/ethyl acetate = 1:1) to give the
99.9 (C1Man2
, , JC–H = 157.4 Hz),
JC–H = 169.8 Hz), 100.4 (C1Man1
127.2, 127.3, 127.5, 127.8, 127.87, 127.92, 128.0, 128.06, 128.12,
128.2, 128.4, 128.6, 137.1, 138.1, 138.4, 138.78, 138.83, 138.9;
MALDI-TOF MS: [M+Na]+ calcd for C60H66O11Na1, 985.45. found
985.55; ESI-TOF MS: [M+Na]+ calcd for C60H66O11Na1, 985.45,
found 985.41; HRMS ESI-TOF: [M+Na]+ calcd for C60H66O11Na1,
985.4503, found 985.4537.
mixture of the isomers (14.7 mg, 90%,
a:b = 4.3:1). Results of
glycosylations are listed in Table 1 (entries 15–17) and Table 2
which were obtained by following two analytical methods.
4.11. Benzyl 2-O-benzoyl-3,4,6-tri-O-benzyl-
D-mannopyranosyl-
4.12.1. Determination of the stereoselectivities by 1H-NMR
analysis
(1?2)-3,4,6-tri-O-benzyl- -mannopyranoside (9)
a-D
Compound 9
a
: ½a 2D5
ꢃ
11.0 (c 1.0, CHCl3); 1H NMR (CDCl3,
The anomeric ratios of products were estimated from the rela-
400 MHz): d 3.55 (dd, J = 11.0, 1.8 Hz, 6a-HMan1, 1H), 3.63 (dd,
tive intensities of H-1 signals (in C6D6) of
lyzing the crude mixture.
a- and b-isomers by ana-
J = 10.5, 1.4 Hz, 6a-HMan2, 1H), 3.68–3.75 (m, 6b-HMan1, 5-HMan1
,

3694
A. Ishiwata et al. / Bioorg. Med. Chem. 18 (2010) 3687–3695
4.12.2. Determination of the yield by the quantitative MALDI-
TOF MASS analysis
O
11Na1, 999.54, found 999.53; HRMS ESI-TOF: [M+Na]+ calcd for
C60H52D14O11Na1, 999.5382, found 999.5343.
The crude mixtures were diluted with 15 mL of CH3CN. A 50
measure of 1.0 mM standard solution of each of the three non-la-
beled compounds for the reaction was pre-mixed with 50 L of
lL
Acknowledgments
l
the crude solutions for MS analysis. The resulting solutions were
measured by MALDI-TOF MASS using the RASTER function. The
molar ratio of labeled to non-labeled compound was obtained
from the ratio of each value (mV) at the apex of the ion peak of
[M+Na]+. For Table 1 (entries 15–17) and Table 2, [D7]1, 2 and 3
were used as standard for 1, [D28]2 and [D28]3. For Table 3 (en-
tries 16–21), [D7]1, [D14]5 and [D14]8 were used as standard for
This work was partly supported by ‘Clean Chemistry’ program
and Incentive Research Grant (2009) in RIKEN. We thank Ms. A.
Takahashi for technical assistance.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.04.013.
1,
5 and 8. [D7]Bn protected compounds [D28]2, [D7]1 and
[D14]5 were synthesized according to the synthesis of correspond-
ing Bn protected compounds 2, 1 and 5, respectively; [D28]2:
HRMS ESI-TOF: [M+Na]+ calcd for C35H10D28O5S1Na1, 621.4095,
found 621.4068; [D7]1: HRMS ESI-TOF: [M+Na]+ calcd for
C34H29D7O6Na1, 570.2849, found 570.2831; [D14]5: HRMS ESI-
TOF: [M+Na]+ calcd for C27H20D14O5S1Na1, 507.2903, found
507.2926.
References and notes
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4.12.3. Benzyl 4,6-O-cyclohexylidene-2,3-di-O-[D7]benzyl-
D-man-
nopyranosyl-(1?2)-3,4,6-tri-O-benzyl- -mannopyranoside
a
-D
([D14]8) for Table 3 (entries 13–18) as the standard
Compound [D14]8 was synthesized from methyl 4,6-O-cyclo-
hexylidene-2,3-di-O-[D7]benzyl-1-thio-D-mannopyranoside [D7]5
4. Pincock, R. E. Acc. Chem. Res. 1969, 2, 97.
according to the general procedure for glycosylation under frozen
conditions. The results of the reaction of 5 were obtained by two
analytical methods in Sections 4.12.1 and 4.12.2.
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Compound [D14]8
a
:
½
a 2D6 57.2° (c 1.0, CHCl3); 1H NMR
ꢃ
(400 MHz, CDCl3): d 1.31–2,20 (m, 10H), 3.58–3.80 (m, 2-HMan1
,
,
3-HMan1, 5-HMan1, 6a-HMan1, 6b-HMan1, 4-HMan2, 5-HMan2, 6a-HMan2
6b-HMan2, 9H), 3.86 (dd, J = 9.2, 2.7 Hz, 3-HMan2, 1H), 3.96 (t,
J = 2.3 Hz, 2-HMan2, 1H), 4.15 (dd, J = 10.1, 9.6 Hz, 4-HMan1, 1H),
4.38–4.58 (m, –CH2Ph, 5H), 4.63 (d, J = 12.8 Hz, –CH2Ph, 2H),
4.72 (d, J = 11.0 Hz, –CH2Ph, 1H), 4.84 (d, J = 1.8 Hz, 1-HMan2, 1H),
5.03 (d, J = 1.4 Hz, 1-HMan1, 1H), 7.10–7.31 (m, Ar, 20H); 13C
NMR (100 MHz, CDCl3): d 22.6, 23.0, 25.7 28.0, 38.2, 61.7, 65.5,
69.1, 71.3, 72.1, 72.6, 73.4, 74.0, 74.8, 75.2, 76.5, 80.1, 98.4, 99.8
(C1Man2, JC–H = 173.6 Hz), 100.7 (C1Man1, JC–H = 174.5 Hz), 127.4,
127.5, 127.7, 127.9, 128.0, 128.3, 128.37, 128.43, 128.5, 137.1,
138.0, 138.1, 138.3, 138.5; MALDI-TOF MS: [M+Na]+ calcd for
C60H52D14O11Na1, 999.54, found 999.66; ESI-TOF MS: [M+Na]+
calcd for C60H52D14O11Na1, 999.54, found 999.53; HRMS ESI-
TOF: [M+Na]+ calcd for C60H52D14O11Na1, 999.5382, found
999.5343.
Compound [D14]8b:
½
a 2D6
ꢃ
ꢁ30.6° (c 1.0, CHCl3); 1H NMR
(400 MHz, CDCl3): d 1.22–2.16 (m, 10H), 3.02 (td, J = 9.6, 6.0 Hz,
5-HMan1, 1H), 3.29 (dd, J = 9.2, 2.8 Hz, 3-HMan1, 1H), 3.55 (dd,
J = 8.7, 1.8 Hz, 6a-HMan2, 1H), 3.55 (dd, J = 8.1, 4.6 Hz, 6b-HMan2
,
1H), 3.67–3.74 (m, 5-HMan2, 6a-HMan1, 6b-HMan1, 3H), 3.80 (dd,
J = 10.1, 8.7 Hz, 4-HMan2, 1H), 3.84 (d, J = 3.2 Hz, 2-HMan1, 1H),
3.99 (dd, J = 9.2, 3.7 Hz, 3-HMan2, 1H), 4.11 (dd, J = 10.1, 9.6 Hz,
4-HMan1, 1H), 4.17 (s, 2-HMan2, 1H), 4.18 (d, J = 11.0 Hz, –CH2Ph,
1H), 4.36–4.44 (m, –CH2Ph, 3H), 4.44 (s, 1-HMan1, 1H), 4.50
(d, J = 12.4 Hz, –CH2Ph, 1H), 4.64 (d, J = 11.9 Hz, –CH2Ph, 1H), 4.67
(d, J = 11.5 Hz, –CH2Ph, 1H), 4.74 (d, J = 11.4 Hz, –CH2Ph, 1H), 4.90
(d, J = 2.3 Hz, 1-HMan2, 1H), 6.97–7.01 (m, Ar, 2H), 7.10–7.27 (m,
Ar, 16H), 7.35–7.37 (m, Ar, 2H); 13C NMR (100 MHz, CDCl3): d
22.6, 23.0, 25.6, 28.0, 38.1, 61.5, 68.8, 68.9, 69.2, 70.5, 70.7, 71.6,
19. Shimizu, H.; Ito, Y.; Ogawa, T. Synlett 1994, 535.
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Wiley-VHC: Weinheim, 2003.
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S.; Hayashi, M.; Noyori, R. Tetrahedron Lett. 1984, 25, 1379; (c) Fukase, K.;
Hasuoka, A.; Kinoshita, I.; Aoki, Y.; Kusumoto, S. Tetrahedron 1995, 51, 4923; (d)
Fukase, K.; Kinoshita, I.; Kanoh, T.; Nakai, Y.; Hasuoka, A.; Kusumoto, S.
Tetrahedron 1996, 52, 3897; (e) Uchiro, H.; Mukaiyama, T. Chem. Lett. 1996,
271; (f) Demchenko, A.; Stauch, T.; Boons, G. J. Synlett 1997, 818; (g) Fukase, K.;
Nakai, Y.; Kanoh, T.; Kusumoto, S. Synlett 1998, 84; (h) Manabe, S.; Ito, Y.;
Ogawa, T. Synlett 1998, 628; (i) Fraser-reid, B.; Udodong, U. E.; Wu, Z. F.;
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72.9, 73.3, 74.9, 76.3, 77.6, 78.1, 96.7, 99.9 (C1Man2
, JC–H =
169.8 Hz), 100.4 (C1Man1, JC–H = 157.4 Hz), 127.3, 127.5, 127.8,
127.9, 128.0, 128.07, 128.12, 128.2, 128.4, 137.1, 138.1, 138.4,
138.6, 138.8; MALDI-TOF MS: [M+Na]+ calcd for C60H52D14O11Na1,
999.54, found 999.51; ESI-TOF MS: [M+Na]+ calcd for C60H52D14
-
24. Yamasaki, F.; Sato, S.; Nukada, T.; Ogawwa, T. Carbohydr. Res. 1990, 201, 31.

A. Ishiwata et al. / Bioorg. Med. Chem. 18 (2010) 3687–3695
3695
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