Accelerated glycosylation under frozen conditions

Article abstract of DOI:10.1016/j.tetlet.2004.03.100

O-Glycosylations using thiomethyl glycosides as donors were compared under both frozen and unfrozen conditions. In the presence of MeOTf as a promoter, enormous rate acceleration was observed when the glycosylation was conducted in p-xylene below its freezing point.

Full text of DOI:10.1016/j.tetlet.2004.03.100

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 3929–3932
Accelerated glycosylation under frozen conditions
Maki Takatani,a,b Jun Nakano,a,c Midori A. Arai,a Akihiro Ishiwata,a,b Hiromichi Ohtac
and Yukishige Itoa,b,
*
aRIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
bCREST (JST), Kawaguchi, Saitama 332-0012, Japan
cDepartment of Chemistry, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-0061, Japan
Received 26 February 2004; revised 16 March 2004; accepted 18 March 2004
Abstract—O-Glycosylations using thiomethyl glycosides as donors were compared under both frozen and unfrozen conditions. In
the presence of MeOTf as a promoter, enormous rate acceleration was observed when the glycosylation was conducted in p-xylene
below its freezing point.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Chemical reactions are believed to proceed most
smoothly under freely diffusing conditions, typically in
homogeneous solutions. Therefore, it is taken as granted
that the reaction temperature must be higher than the
melting point of the solvent. In frozen solutions, reac-
tions are considered to be extremely slow, because
diffusion of reactants would be severely restricted.
However, there have been several reports, which
revealed the dramatic rate acceleration of chemical1 and
enzymatic2 reactions under frozen conditions both in
aqueous and nonaqueous systems. These observations
were explained by the concentration effect.3 Below
freezing point, but above the eutectic temperature, fro-
zen solution contains a small volume of liquid phase
that is in an equilibrium with solid phase. According
to the freezing point–composition relationship, it is
expected that solutes (reactants and reagents) are highly
concentrated in liquid particles. If the effect of concen-
tration overrides the rate reduction caused by cooling,
bimolecular reaction would be accelerated in frozen
media. Our thought was that freezing would increase the
velocity of intermolecular coupling reaction like glyco-
sylation. In this paper, we wish to report the first dem-
onstration of accelerated O-glycosylation in frozen
medium.
As part of our effort to develop facile synthetic routes to
oligosaccharide molecular probes related to high-man-
nose type glycoproteins,4 we planned the preparation of
trimannoside 5, starting from the mannose derivative 15
having lipophilic tether (Scheme 1). Glycosylation of 1
with thioglycoside 25 was attempted in the presence of
methyl trifluoromethanesulfonate (MeOTf)6 and 2,6-
di-tert-butyl-4-methylpyridine (DTBMP). This glyco-
sylation turned out to be quite slow at room tempera-
ture. In toluene, it resulted in ca. 60% conversion after 2
days, and elevated temperature (50 ꢁC) was required for
completion (Table 1, entry 1). The use of 1,2-dichloro-
ethane facilitated the reaction to an extent that it com-
pletes at room temperature, albeit after 19 h (entry 2).
When the same glycosylation was conducted in p-xylene
(mp 12–13 ꢁC) below freezing temperature, marvelous
rate acceleration was observed (entry 3). For instance, at
4 ꢁC, the reaction was complete after 7 h to provide
disaccharide 37 in 93% yield (a:b ¼ 8:3:1). Since sol-
vating properties of p-xylene and toluene are likely to be
very similar, this result implied that the rate acceleration
originates from the unique nature of the frozen envi-
ronment. After chromatographic separation, anomeri-
cally pure 3a was desilylated (1.5 equiv Bu4NF, THF,
50 ꢁC, 4 h) to give 4 (quantitative). The latter was then
subjected to the second glycosylation, again using 2 and
MeOTf/DTBMP as a donor and a promoter, respec-
tively. The effect of freezing was even more prominent in
this case (entries 4–6). Namely, the smooth formation of
the trisaccharide 5 (77%, a:b ¼ 18:1) was observed
in frozen p-xylene (4 ꢁC), while the same reaction in
Keywords: Glycosylation; Frozen; Rate acceleration; High-mannose
type; Oligosaccharide.
* Corresponding author. Tel.: +81-48-462-1111; fax: +81-48-462-4680;
e-mail: yukito@riken.jp
0040-4039/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.03.100

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M. Takatani et al. / Tetrahedron Letters 45 (2004) 3929–3932
Scheme 1.
Table 1. Glycosylation with thioglycoside 2 or 7
Entry
Donor
(equiv)
Acceptor
Solvent
MeOTf/DTBMP
(equiv)
Temperature/
time (ꢁC/h)
Product
Yield (%)
Ratioa (a:b)
1
2
3
4
5
6
7
8
2 (2.5)
2 (2.5)
2 (2.0)
2 (2.0)
2 (1.5)
2 (1.5)
7 (1.3)
7 (1.3)
1
1
1
4
4
4
6
6
Toluene
(ClCH2)2
p-Xylene
(ClCH2)2
Toluene
p-Xylene
Toluene
p-Xylene
4.5/4.5
4.5/4.5
3.5/2.2
4.5/4.5
2.7/1.6
2.7/1.6
8.0/8.0
8.0/8.0
50/14
rt/19
4/7
3
3
3
5
5
5
8
8
88
91
93
65b
50c
77
6
ꢁ40:1
3.8:1
8.3:1
15:1
ꢁ50:1
18:1
ad
rt/19, 50/10
50/22
4/18
4/22
4/22
61
ad
a Based on the amounts of isolated products.
b 18% Recovery of 4.
c 43% Recovery of 4.
d Only a-isomer was isolated.
toluene (22 h) or 1,2-dichloroethane (19 h) was incom-
plete even at 50 ꢁC.
yield, while conversion was only marginal at room
temperature (entry 1). Remarkably, reaction at room
temperature (entry 1) was even slower than at ꢀ20 ꢁC
(entry 8) and comparable with conducted at ꢀ40 ꢁC
(entry 10). At ꢀ20 and ꢀ40 ꢁC, complete conversion was
observed after 24 h (entry 9) and 48 h (entry 11),
respectively, while reactions were extremely sluggish in
toluene at these temperatures (entries 3–6) (Scheme 2).
Block condensation of larger oligosaccharide fragments
(64 þ 74 fi 8) was also accelerated dramatically in frozen
p-xylene (entries 7, 8).
With these results in hand, systematic comparison was
made using 9 and 10 as an acceptor and donor pair, and
results are summarized in Table 2. In p-xylene, both
donor (9) and acceptor (10) were completely consumed
after 6 h at 4 ꢁC (entry 7) to give disaccharide 118 in high
Our results clearly demonstrated that freezing the mix-
ture increases the rate of O-glycosylation. The use of
frozen media is technically simple and does not require

M. Takatani et al. / Tetrahedron Letters 45 (2004) 3929–3932
Table 2. Glycosylation of 9 with 10; comparison of frozen and unfrozen systemsa
3931
Entryb
Solventc
Temperature (ꢁC)
Time (h)
Yield (%)
Ratio (a:b)
Recovery (%)
10
9
Unfrozen
1
X
4
rt
6
24
16:1
<1 6
5
46:1d
60
64
66
87
80
85
80
e
2T
3
6
11
73
89
78
88
80
T
T
T
T
ꢀ2
ꢀ20
ꢀ40
ꢀ40
0
af
16:1e
4
24
6
5
0g
––
10:1e
6
48
6
Frozen
7
8
X
X
X
X
X
4
ꢀ20
ꢀ20
ꢀ40
ꢀ40
6
6
86
66
3.4:1d
4.5:1d
4.0:1d
NDg
NDg
NDg
2
8
34
9
24
6
81
17
10
11
10:1d
7274
d
48
825.0:1
a Performed by using 0.07–0.12mmol of 9 (0.03 M in p-xylene or toluene), 1.35 equiv of 10, 2.5 equiv of MeOTf, 1.5 equiv of DTBMP, and molecular
ꢀ
sieves 4 A (ca. 0.3 g/0.1 mmol of 9).
b All reactions were performed in refrigerator (except entry 1) adjusted to the appropriate temperature.
c X: p-xylene, T: toluene.
d Based on the amounts of isolated products.
e Determined by 1H NMR (400 MHz, CDCl3).
f Only a-isomer could be detected.
g Not detectable by TLC.
2. Grant, N. H.; Alburn, H. E. Nature 1966, 212, 194;
Ullmann, G.; Haensler, M.; Gruender, W.; Wagner, M.;
Hofmann, H.-J.; Jakubke, H.-D. Biochim. Biophys. Acta
1997, 1338, 253–258; Wehofsky, N.; Kirbach, S. W.;
Haensler, M.; Wissmann, J.-D.; Bordusa, F. Org. Lett.
2000, 2, 2027–2030; Wehofsky, N.; Haensler, M.; Kirbach,
S. W.; Wissmann, J.-D.; Bordusa, F. Tetrahedron: Asym-
metry 2000, 11, 2421–2428; Schuster, M.; Ullmann, G.;
Jakubke, H.-D. Tetrahedron Lett. 1993, 34, 5701–5702;
Schuster, M.; Aaviksaar, A.; Jakubke, H.-D. Tetrahedron
1990, 46, 8093–8102.
3. Pincock, R. E.; Kiovsky, T. E. J. Chem. Educ. 1966, 43,
358–360.
Scheme 2.
4. Matsuo, I.; Wada, M.; Manabe, S.; Yamaguchi, Y.; Otake,
K.; Kato, K.; Ito, Y. J. Am. Chem. Soc. 2003, 125, 3402–
3403.
5. Preparation of these compounds will be reported in a
separate account.
any special devise. It may be suggested as a clue to
facilitate glycosylation under certain circumstances.
Somewhat unexpectedly, the stereoselectivity seems to
be attenuated under frozen conditions (e.g., Table 2,
entries 1, 2, 7, 8). In this context, more systematic
studies, with various types of glycosyl donors, promot-
ers, and solvents that can be frozen, would be required
to clarify the scope and advantage of frozen system in
oligosaccharide synthesis.
€
6. Lonn, H. Carbohydr. Res. 1985, 139, 105–113.
7. Typical experimental procedure (preparation of disaccha-
ride 3): a solution of compound 1 (21 mg, 0.039 mmol) and
2 (47 mg, 0.079 mmol) in p-xylene (3 mL) that contained
ꢀ
activated molecular sieves 4 A (200 mg) and DTBMP
(18 mg, 0.086 mmol) was stirred under Ar at room
temperature for 0.5 h to ensure dehydration. Subse-
quently, MeOTf (9.5 lL, 0.141 mmol) was added, and the
mixture was rapidly mixed and frozen by liq. N2. The
mixture was stored in refrigerator at 4 ꢁC for 7 h and was
defrosted at room temperature. Et3N (29 mL, 0.212 mmol)
was added to quench MeOTf and the mixture was diluted
with AcOEt, filtered through Cerite. The filtrate was
washed with brine, dried (MgSO4), and evaporated in
vacuo. The residue was chromatographed over silica gel
(hexane–AcOEt ¼ 3:1) to afford 34.8 mg (83%) of 3a
together with the stereoisomer 3b (4.2mg, 10%). Com-
pound 3a: 1H NMR (CDCl3) d 8.09 (d, 2H, J 7.8 Hz,
aromatic), 7.55–7.18 (m, 18H, aromatic), 5.67 (s, 1H, H-2I),
5.08 (s, 1H, H-1II), 4.79 (d, 1H, J 11.7 Hz, CHHPh), 4.67 (d,
Acknowledgements
Financial support from a Grant-in-Aid for Scientific
Research from the Japan Society for the Promotion of
Science (Grant no. 13480191) is acknowledged with
thanks. M.A.A. is a Basic Science Special Postdoctoral
Fellow of RIKEN. The authors thank Ms. Akemi
Takahashi for technical assistance.
1H, J 11.7 Hz, CHHPh), 4.38 (s, 1H, H-1I), 4.52(d, 2H,
J
References and notes
11.7 Hz, CH2Ph), 4.38 (s, 2H, CH2Ph), 4.07–4.01 (m, 3H,
H-3I, 4I, 5II), 3.96–3.87 (m, 4H, H-2II, 4II, 6II), 3.72–3.70 (m,
3H, H-6I, OCHH(CH2)7–CO2CH3), 3.65 (s, 3H, CO2CH3),
3.61 (dd, 1H, J 2.2, 9.3 Hz, H-3II), 3.45–3.40 (m, 1H,
1. Pincock, R. E.; Kiovsky, T. E. J. Am. Chem. Soc. 1965, 87,
4100–4107, 1965, 87, 2072–2073; 1966, 88, 51–55; Grant, N.
H.; Alburn, H. E. Science 1965, 150, 1589–1590.

3932
M. Takatani et al. / Tetrahedron Letters 45 (2004) 3929–3932
OCHH(CH2)7–CO2CH3), 3.30–3.24 (m, 1H, H-5I), 2.24 (t,
H-6I, 2II, 4II, 6II, OCHH(CH2)7CO2CH3), 3.64 (s, 3H,
2H, 7.6 Hz, CH2CO2CH3), 2.28–1.41 (m, 14 H,
OCH2CH2–(CH2)4CH2CH2CO2CH3, cyclohexyl), 1.18 (s,
H, O(CH2)2(CH2)4(CH2)2CO2CH3), 0.85 (s, H,
J
CO2CH3),
3.52–3.29 (m, 4H, H-5I, 3II, 5II,
OCHH(CH2)7CO2CH3), 2.39–2.31 (br m, 1H, cyclohexyl),
2.25 (t, 2H, J 7.6 Hz, CH2CO2CH3), 1.64–1.26 (m, 21H,
OCH2(CH2)6CH2CO2CH3, cyclohexyl), 0.78 (s, 9H,
C(CH3)3), ꢀ0.18 (s, 3H, SiCH3), ꢀ0.25 (s, 3H, SiCH3);
13C NMR (CDCl3) 173.98 (CO2CH3), 165.93 (OCOPh),
8
9
C(CH3)3), 0.00 (s, 3 H, SiCH3), ꢀ0.04 (s, 3 H, SiCH3);
13C NMR (CDCl3) 173.81 (C2CH3), 165.36 (OCOPh),
1
I
100.88 (C-1II, JCH 175.0 Hz), 99.53 ꢂ 2(C-1
,
1JCH
156.7 Hz, cyclohexylidene), 79.72(C-3 II), 73.91 (C-4II),
73.75, 72.71, 72.27 (3 ꢂ CH2Ph), 72.48 (C-5II), 72.16 (C-
3I), 71.44 (C-2I), 71.10 (C-4I), 69.88 (C-2II), 69.81
(OCH2(CH2)7CO2CH3), 69.39 (C-6II), 67.91 (C-5I), 61.33
(C-6I), 51.27 (CO2CH3), 37.87, 27.89, 25.57, 22.89,
99.97 (cyclohexylidene), 99.45 (C-1I, JCH 155.1 Hz), 96.69
1
1
(C-1II, JCH 153.4 Hz), 82.61 (C-3II), 76.61 (C-5II), 74.92,
73.31, 71.56 (3 ꢂ CH2Ph), 74.50 (C-3II), 74.29 (C-4II), 70.11
(OCH2(CH2)7CO2CH3), 69.34 (C-2II), 68.77 (C-5I), 68.66
(C-2I), 68.57 (C-6II), 68.48 (C-4I), 61.45 (C-6I), 51.46
(CO2CH3), 38.02, 27.61, 25.79, 22.79, 22.72 (cyclohexyl),
34.14 (CH2CO2CH3), 29.45 (OCH2CH2(CH2)6CO2CH3),
29.16, 29.07, 25.86 (O(CH2)2(CH2)4(CH2)2CO2CH3), 25.95
(C(CH3)3), 25.00 (CH2CH2CO2CH3), 18. 48 (C(CH3)3),
ꢀ4.20 (SiCH3), ꢀ5.21 (SiCH3).
22.62
(cyclohexyl),
33.96
(CH2CO2CH3),
29.31
(OCH2CH2(CH2)6CO2CH3), 29.00, 28.96, 28.88, 25.65
(O(CH2)2(CH2)4(CH2)2CO2CH3), 25.65 (C(CH3)3), 24.81
(CH2CH2CO2CH3), 18.04 (C(CH3)3), ꢀ4.51 (SiCH3),
ꢀ5.04 (SiCH3). Compound 3b: 1H NMR (CDCl3) d 8.09
(d, 2H, J 7.1 Hz, aromatic), 7.60–7.24 (m, 18H, aromatic),
5.71 (s, 1H, H-2I), 4.85 (d, 1H, J 11.1 Hz, CHHPh), 4.75 (d,
1H, J 11.5 Hz, CHHPh), 4.68 (d, 2H, J 11.5 Hz, CH2Ph),
4.62(s, 1H, H-1 I), 4.59 (d, 1H, J 11.5 Hz, CHHPh), 4.57 (d,
8. a-Isomer: 1H NMR (CDCl3) d 5.16 (d, 1H, J 1.5 Hz, H-1II),
4.95 (d, 1H, J 1.7 Hz, H-1I); 13C NMR (CDCl3) d 99.5 (JC–H
170 Hz), 98.2( JC–H 170 Hz). b-Isomer: 1H NMR (CDCl3) d
4.97 (d, 1H, J 1.7 Hz, H-1I), 4.56–4.30 (m, 11H, H-1II, H-2I,
1
1H,
J
11.1 Hz,
CHHPh),
4.50
(s,
1H,
PhCH2); 13C NMR (CDCl3) d 99.2( JC–H 155 Hz), 96.4
H-1II), 4.14–4.05 (m, 2H, H-3I, 4I), 3.98–3.74 (m, 7H,
(1JC–H 169 Hz).