A new glycosylation method part 3: study of microwave effects at low temperatures to control reaction pathways and reduce byproducts

Article abstract of DOI:10.1016/j.tet.2008.08.011

The efficiency of microwave irradiation at low temperature for glycosylations is described. Although oligosaccharide synthesis usually requires reactive donors for glycosylations, which have leaving groups on the anomer positions, i.e., trichloroacetoimidates, halogenates, thioalkyl glycosides, etc., the suitable donors in our microwave supported synthesis of Lewis X oligosaccharide were very stable acetate derivatives. Regarding glycosylation with a fucosyl acetate donor and a glucosamine acceptor, microwave irradiation with simultaneous cooling improved yields. Moreover, further synthesis to Lewis X derivatives was achieved only with microwave irradiation at low temperatures. Without microwave irradiation, we could only obtain byproducts and none of the designed product at any reaction temperature.

Full text of DOI:10.1016/j.tet.2008.08.011

Tetrahedron 64 (2008) 10091–10096
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
A new glycosylation method part 3: study of microwave effects at low
temperatures to control reaction pathways and reduce byproductsq
,
b
b
a,b,
*
Hiroki Shimizu a , Yayoi Yoshimura , Hiroshi Hinou , Shin-Ichiro Nishimura
*
a Drug-Seeds Discovery Research Laboratory, Hokkaido Center, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo 062-8517, Japan
b Division of Advanced Chemical Biology, Graduate School of Advanced Life Science, Frontier Research Center for Post-Genomic Science and Technology, Hokkaido University,
Sapporo 011-0021, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 April 2008
Received in revised form 2 August 2008
Accepted 6 August 2008
Available online 9 August 2008
The efficiency of microwave irradiation at low temperature for glycosylations is described. Although
oligosaccharide synthesis usually requires reactive donors for glycosylations, which have leaving groups
on the anomer positions, i.e., trichloroacetoimidates, halogenates, thioalkyl glycosides, etc., the suitable
donors in our microwave supported synthesis of Lewis X oligosaccharide were very stable acetate
derivatives. Regarding glycosylation with a fucosyl acetate donor and a glucosamine acceptor, microwave
irradiation with simultaneous cooling improved yields. Moreover, further synthesis to Lewis X de-
rivatives was achieved only with microwave irradiation at low temperatures. Without microwave irra-
diation, we could only obtain byproducts and none of the designed product at any reaction temperature.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Microwave
Nonthermal effect
Glycosylation
Carbohydrate
1. Introduction
carbohydrate chemistry, including Fischer glycosylation,14 solvent
free glycosylation,15 Ferrier rearrangement,16,17 glycosylation with
With the development of carbohydrate research, the biological
importance of oligosaccharides has been revealed.1–3 For example,
one of its important roles is cell to cell adhesion4 and in-
flammation.5 These phenomena are the result of molecular binding
systems, typically carbohydrate–protein and carbohydrate–carbo-
hydrate. We are interested in studying these carbohydrate recog-
nition systems at the molecular level and believe that these studies
will open a door to a new drug discovery system. We have pre-
viously reported the verotoxin-oligosaccharide binding conforma-
tion6–9 and the dynamics of carbohydrate binding protein.10
Since 1986, microwave irradiation has been applied widely in the
synthetic research field,11,12 especially as an effective heating tool in
chemical reactions.13 Although many glycosylations are performed
under Lewis acidic conditions and usually proceed at low tempera-
tures, microwave irradiation has been well studied in for
oxazoline donor,18 glycosylation with pentenyl orthoesters,19 glyco-
sylationwithtrichloroacetoimidatesandDISAL(3,5-dinitrosalicylate)
donors,20,21 synthesis of glycosyl amino acids,22 etc. These reactions
use microwave irradiation as an effective heating tool, but we
revealed another factor, which can control chemical reactions and
was very effective for the glycosylation23,24 and glycopeptides syn-
theses.25,26 The technique of using microwave with simultaneous
cooling is reported27 and an established machine is now commer-
cially available.28 In this report, we demonstrate the effectiveness of
microwave irradiation at low temperatures, i.e., non-heating for
glycosylations through syntheses of Lewis X derivatives (Fig. 1).
2. Results and discussion
2.1. Strategy
Lewis X (Lex) antigen (1) is the terminal oligosaccharide in nu-
merous cell surface glycolipids and glycoproteins that playa key role
in selectin-mediated cell–cell adhesion.29 On a molecular level, this
process is dominated by Lex–Lex recognition and we plan to study
conformations of this weak homo-recognition by NMR. For this
purpose, we set thiomethyl Lex derivative (2) as a synthetic target
because the thiomethyl group can behave either as a protecting
group at the reducingend or as a leaving groupto introduce avariety
of ligands. Thus, 2 could be converted to a variety of Lex derivatives.
q
Part 1: Y. Yoshimura, H. Shimizu, H. Hinou, S.-I. Nishimura ‘A novel glycosyla-
tion concept; microwave-assisted acetal-exchange type glycosylations from methyl
glycosides as donors’, Tetrahedron Lett., 2005, 46, 4701–4705.
Part 2: H. Shimizu, M. Sakamoto, N. Nagahori, S.-I. Nishimura ‘A new glyco-
sylation method part 2: study of carbohydrate elongation onto the gold nano-
particles in a colloidal phase’, Tetrahedron, 2007, 63, 2418–2425.
* Corresponding authors. Fax: þ81 11 857 8435 (H.S.); fax: þ81 11 706 9042
(S.-I.N.).
E-mail addresses: hiroki.shimizu@aist.go.jp (H. Shimizu), shin@glyco.sci.
hokudai.ac.jp, tiger.nishimura@aist.go.jp (S.-I. Nishimura).
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.08.011

10092
H. Shimizu et al. / Tetrahedron 64 (2008) 10091–10096
OH
O
Ph
O
HO
OBn
O
OH
O
AcO
O
O
SMe
O
HO
HO
O
BnO
BnO
SMe
O
O
O
NDCPhth
OAc
OBn
OH
O
4
N
HNAc
H3C
O
OH
OBn
O
5
H3C
O
H3C
O
OBn
BnO
OH
OBn
(2 eq.)
OH
HO
OBn
Cl
BnO
Cl
TMSOTf,
2
conditions as below
Lewis X oligosacchride: 1
Ph
O
O
O
SMe
O
Figure 1. Lewis X oligosaccharide (1) and the synthetic target 2.
NDCPhth
H3C
O
OBn
NDCPhth =
In addition, we introduced the fucosyl moiety first to the glucos-
amine acceptor followed by the introduction of the galactose unit.
Although the Lex analogues are important targets in the history of
carbohydrate synthetic research field and have been well studied,30
the synthesis of thiomethyl Lex (2) in the order described above has
several difficulties. The 3-OH and the 4-OH of the glucosamine ac-
ceptor are potentially less reactive, the glucosamine acceptor (3),
which had a fucose unit on the 3-OH produced steric hindrance for
the 4-OH, the thiomethyl group had good nucleophilicity, which
lead to byproducts, and generally the fucosyl bond was relatively
unstable in acidic conditions like glycosylation (Fig. 2). Therefore, for
the construction of Lex building blocks, it has been recommended to
first glycosylate the galactose donor to the 4-OH of the glucosamine
acceptor followed by the introduction of the fucosyl moiety at 3-OH.
There have been some reports of this reverse synthesis strategy.31
Stahl et al. successfully fucosylated andgalactosylated ontothe 3-OH
and 4-OH of glucosamine acceptor in 95% and 75% yields, re-
spectively,32 and de la Fuente et al. fucosylated and galactosylated to
a phenylthio protected glucosamine acceptor in 83% and 80% yields,
respectively.33 Our synthesis of 2 was more difficult. Because of the
thiomethyl groups’ nucleophilicity and low reactivity of the 4-OH,
the activated oxocarbenium ion from galactosyl donors was more
likely to be attacked at S rather than O, and as a result we only
achieved byproducts instead of the designed product.
OBn
BnO
4,5-dichlorophthalimido
6
Conditions:
Entry
Temp.a) Timeb) Yield
Solv.
ether
Microwave
1
2
none
none
-10 °C
-45 °C
1 h 27 %
6 h 71 %
ether/THF (5/1)
3
4
ether
ether
300W
100W
-10 °C
-18 °C
1 h
2.5 h
90 %
83 %
5
6
300W
300W
-20 °C
-20 °C
1 h
2.5 h
21 %
9 %
CH3CN
CH2Cl2
Scheme 1. Synthesis of disaccharide 6. Microwave irradiation in low temperatures
gave fruitful results. (a) Observed temperatures by the CoolMate equipped sensor with
the use of microwave irradiation, within ꢂ5 ꢁC. (b) The reactions were monitored by
TLC and stopped when acceptor (6) was consumed.
yield improved to 71% in mixed solvents such as ether/THF (5:1).
On the other hand, in the same conditions with additional micro-
wave irradiation, the yield increased to 90%. In addition, byprod-
ucts, which were hardly purified by column chromatography, were
not observed by NMR (Fig. S1 in Supplementary data). We would
like to note here that the reaction temperature was ꢀ10 ꢁC, which
meant that microwave was effective even at low temperatures and
played a more significant role than heating. It was also interesting
to note that microwave supported glycosylation in a solvent de-
pendent manner as the yields became 21% and 9% in cases using
acetonitrile or dichrolomethane, respectively, although we do not
know the underlying reason for this yet.
Despite of these findings, we found that microwave irradiation
could achieve the synthesis of thiomethyl Lex (2) in high yield in the
syntheses order described above. At the same time, we found that
microwave irradiation was effective even at low temperatures,
which indicates that microwave provides not only a heating effect
in chemical reactions but also plays a role at the molecular level.
2.3. Trisaccharide synthesis
2.2. Disaccharide synthesis
After selective reduction of the 4,6-benzylidene group of 6,37 the
galactose unit was introduced for the synthesis of Lex analogue 2.
As we already discussed, galactosylation to 3 required ‘paradoxical’
conditions, namely strong glycosylation conditions, i.e., high tem-
peratures, due to the poor reactivity of the hydroxyl group of 3, but
the fucosyl bond was generally weak in acidic conditions. Thus,
carrying out the reaction in low temperatures was favorable.
Moreover, as a potential nucleophilic group, the thiomethyl group
would be more reactive than the hydroxyl group on 3, which meant
the reaction of the oxocarbenium ion would be more favored with
1-SMe than 4-OH, and this synthetic strategy may not result in the
production of 2. In fact, we could not obtain trisaccharide 2 despite
many attempts in any temperatures between ꢀ45 ꢁC to room
temperature (we have shown one typical example in Scheme 2).
Despite these attempts and unlikely results, microwave irradia-
tion turned the ‘impossible’ into a ‘possible’. Microwave irradiation
with Lewis acid, trimethylsilyl trifluoromethanesulfonate (TMSOTf),
to glycosyl donor 7 gave successfully designed Lex analogues (2) in
70% and 82% yield when 15% of recovered disaccharide acceptor 3
was considered (entry 2 in Scheme 2). This reaction was performed
at less than zero degrees for 5 h but when the reaction temperature
was raised to 30 ꢁC, the yield became 37% and 43% considered 14% of
recovered 3 (entry 3 in Scheme 2). In this case, we isolated the lacto-
samine derivative (9)38 as a byproduct, which would result in the
After syntheses of monosaccharide building blocks by common
procedures,34–36 fucosyl donors, fluoride, trichloroacetoimidate,
and acetate have been used for glycosylations to a glucosamine
acceptor (4) without microwave irradiation. We have tried to syn-
thesize 6 under a variety of conditions, promoters, solvents, and
temperatures. (Selected attempts are shown in Scheme S1 in Sup-
plementary data.) Although monitoring by TLC showed that the
donors were successfully activated in all cases, only limited at-
tempts resulted in disaccharide 6. As shown in Scheme 1, glyco-
sylation between 4 and 5 in ether gave 6 in only 27% yield but the
AcO
BnO
X
O
neucleophilicity
BnO
hinder
OBn
BnO
O
HO
H3C
SMe
O
O
N
O
O
Bn
O
BnO
BnO
weak
Cl
3
Cl
Figure 2. Problems in the glycosylation between oxocarbenium ion from the galactose
donor and the fucosyl-glucosamine acceptor 3.

H. Shimizu et al. / Tetrahedron 64 (2008) 10091–10096
10093
R2O
it is. Deprotection of the benzyl groups by Pd(OH)2 under H2 and of
the acetyl groups by saponification yielded 1. NMR and MS data
were compared with commercially available one to confirm its
structure (Scheme 3).
R1O
O
O
SMe
NDCPhth
H3C
O
OBn
OBn
BnO
6: R1, R2 = CHPh
3: R1 = H, R2 = Bn
TES, TFA, CH2Cl2,
0 °C - RT, 18 h 88 %
OBn
AcO
O
O
1) H2NCH2CH2NH2
EtOH, 70 °C, 2h
BnO
BnO
SMe
O
O
OBn
NHAc
2
7 (R = Bn, α:β = 1:2)
RO
RO
OR
O
2) Py, Ac2O, CH2Cl2
RT, 3h
or
H3C
O
OBn
8 (R = Ac, β only )
OBn
BnO
95 % (2 steps)
AcO
OAc
11
TMSOTf,
(2 eq.)
conditions as below
OBn
AcO
O
O
BnO
BnO
NIS, TMSOTf
OH
O
O
2
wet CH2Cl2
0 °C, 10 min
HNAc
OBn
Conditions:
Timeb) Yield
Temp.a)
H3C
O
Entry Donor
Solv.
ether
MW
OBn
56 %
OBn
BnO
24 h
c)
1
7
none
-20 °C to RT
12
1) Pd(OH)2, H2
5 h
6 h
82%
43%d)
-20 to -5 °C
-20 to 30 °C
-20 to 0 °C
300W
300W
300W
2
3
4
7
7
8
ether
ether
ether
MeOH, RT, 18h
1
2) NaOH, H2O
RT, 2h
quant. (2 steps)
6 h none
6 h
2 h
6 h
e)
f)
(CH2Cl)2
CH2Cl2
0 to 50 °C
-20 to 5 °C
-12 to 5 °C
50W
300W
300W
8
7
5
6
7
Scheme 3. Further conversion to Lewis X oligosaccharide (1).
f)
7
CH3CN
BnO
3. Conclusion
OBn
O
Byproducts:
OBn
Although there are some reports of chemical reactions using
microwave irradiation with simultaneous cooling, many succeeded
only in the improvement of product yields,39–41 although Horikoshi
et al. found a case of changing reaction pathways.42 Earlier this year,
Kappe et al. reported inaccuracies in the case of heating by mi-
crowave irradiation. They found that the reaction temperature
might not be homogeneous in the reaction vessel despite stirring,
resulting in partial heating areas, heat-spots or self-heating of the
vessels. They were not convinced of the existence of microwave
nonthermal effects.43 However, our results indicated that micro-
wave irradiation worked not only to improve glycosylation yield
(Scheme 1) but also to allow for the completion of previously im-
possible reactions (Scheme 2). Temperature control without mi-
crowave irradiation gave only byproducts and microwave
irradiation at low temperatures successfully produced the designed
product 2 in the reaction showed in Scheme 2. It would be hard to
explain our results based on temperature inhomogeneities.
It is also hard to imagine that a microwave can activate mole-
cules directly based on potential energy. We conjecture that one of
the efficiencies of microwave irradiation at low temperatures may
be the destruction of a cluster. It is reasonable to think that syn-
thons, especially the glycosyl acceptor, might exist as clusters, such
as micelles, and the reaction points, hydroxyl groups, may be hid-
den inside the cluster. As a result, under normal conditions, the
hydroxyl groups could not be attacked by the oxocarbenium ion,
but microwave irradiation will be able to break the cluster, thus
freeing the hydroxyl groups to react. Next, we are planning to study
not only the utility of microwave irradiation but also the microwave
effect itself in chemical reactions. By revealing the reason for the
dependency of the solvents in Schemes 1 and 2, we will determine
the true microwave effect itself.
O
O
9
BnO
SMe
HO
AcO
NDCPhth
AcO
AcO
AcO
OBn
O
O
O
10
OH
O
AcO
H3C
NDCPhth
O
OBn
OBn
BnO
Scheme 2. Synthesis of trisaccharide 2. Microwave irradiation at low temperatures
made this synthesis possible. (a) Observed temperatures by CoolMate equipped sensor
with the use of microwave irradiation. (b) The reactions were monitored by TLC and
stopped when acceptor (3) was consumed. (c) TLC monitoring showed that 7 was
activated but 2 was not observed. (d) Byproduct 9 was also observed. (e) Only 30% of
de-thiomethyl trisaccharide (10) was isolated. (f) We could not detect 2.
galactosylation of 3 but the fucose unit was detached. MALDI-TOF
MS monitoring showed that the byproduct was observed when the
reaction temperature increased to over ꢀ5 ꢁC (Fig. S2 in Supple-
mentary data). In the case of using galactose pentaacetate (8) as
a donor, the reaction did not proceed as 8 was not activated under
0 ꢁC evenwith microwave irradiation (entry 4 in Scheme 2), thenwe
treated 8 at higher temperatures. In this case, we changed the
solvent to dichloroethane due to the low boiling point of ether.
Although entry 5 in Scheme 2 gave a trisaccharide, it was 10,38 the
thiomethyl group detached from the Lex analogue. As we have de-
scribed in the synthesis of disaccharide (6), solvents’ dependency
was remarkable in this case as acetonitrile and dichrolomethane led
to low yields even in similar conditions, entry 2 in Scheme 2.
2.4. Further conversion to Lewis X oligosaccharide (1)
Lex analogues (2) will be a key compound for further research as
it can be converted to a variety of Lex derivatives. At the same time,
2 was deprotected to become a Lex oligosaccharide (1). Treatment
with ethylenediamine, followed by acetylation for 2 gave 11. The
thiomethyl group on anomer was removed by NIS/TMSOTf in wet
4. Experimental
4.1. General
Microwave supported syntheses at low temperatures were
performed in the CEM Discover microwave system and the
dichloromethane, and resulting 12 was only b-hemiacetal although

10094
H. Shimizu et al. / Tetrahedron 64 (2008) 10091–10096
CoolMate cooling unit. The CoolMate equipped reaction flasks were
jacked by a cooling medium, perfluorinated fluid GALDEN HT55.
Reaction temperatures were monitored by an equipped fiber optic
probe, which was inserted into the reaction medium. A silicone cap
with two holes was placed on the reaction flask. One hole was for
the temperature probe and another was for a nitrogen balloon. All
reagents were purchased from Wako Pure Chemical Industries Ltd.
(Osaka, Japan), Tokyo Kasei Kogyo Co. (Tokyo, Japan), or Aldrich
Chemical Co. (Milwaukee, WI, USA) and used as purchased without
further purification. All dry solvents were purchased from Wako
Pure Chemical Industries Ltd. and stored with activated molecular
sieves 4A. Reactions were monitored by TLC, which was performed
with 0.25 mm precoated silica gel 60F254 on glass from Merck
(Darmstadt, Germany). Compounds were detected by dipping the
TLC plates in an ethanolic solution of sulfuric acid (5% v/v) and
heating. Silica gel N60 (40–50 nm) from Kanto Chemical (Tokyo,
Japan) was used for silica gel chromatography. Optical rotations
were recorded on a Perkin Elmer Polarimeter 343 at room tem-
perature (approximately 18–23 ꢁC). All NMR data are reported in
parts per million downfield shift from tetramethylsilane. 1H NMR
spectra were routinely recorded at 400 MHz on a BRUKER AVANCE
400 spectrometer and 500 MHz on a VARIAN Unity INOVA 500
spectrometer at 300 K, and chemical shifts were expressed relative
(1H, br, H-40), 2.17 (3H, s, CH3S–), 0.90 (3H, d, J¼6.5 Hz, H-60); 13C
NMR (125 MHz, CDCl3):
d 138.5, 138.5, 138.2, 137.0, 128.9, 128.4,
128.2, 128.2, 128.1, 127.6, 127.5, 127.5, 127.3, 127.3, 126.1, 125.4, 125.1,
101.3, 99.3, 82.1, 81.3, 79.7, 77.9, 77.9, 73.4, 73.2, 70.7, 68.6, 67.5,
54.4,16.6,14.1; HRMS FAB m/z for C49H47O10NCl2SþH (MþH)þ calcd
912.2376, found 912.2382.
4.3. Methyl O-(2,3,4-tri-O-benzyl-a-L-fucopyranosyl)-(1/3)-
6-O-benzyl-2-deoxy-2-(4,5-dichlorophthalimido)-1-thio-b-D-
glucopyranoside (3)
Trifluoroacetic acid (282
a stirred solution of 6 (562 mg, 616
3.87 mmol) in dichloromethane (5 ml) at 0 ꢁC. The mixture was
stirred for 4 h at 0 ꢁC and additional triethylsilane (617
l,
3.87 mmol) and trifluoroacetic acid (282 l, 3.71 mmol) were
ml, 3.71 mmol) was added dropwise to
m
mol) and triethylsilane (617 l,
m
m
m
added. The reaction mixture was stirred overnight as it was allowed
to slowly warm to room temperature. The mixture was then diluted
with EtOAc and neutralized with satd NaHCO3 aq. The aqueous
layer was extracted with EtOAc three times, combined extracts
were dried over MgSO4 and concentrated in vacuo. The resulting
residue was purified by column chromatography [silica gel:
n-hexane/EtOAc (3:1)] to give 3 (496 mg, 88%) as a white solid.
16
to methyl proton of tetramethylsilane (
d
0.00 ppm). In disaccharide
[
a
]
þ12.1 (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3):
d 7.78 (1H,
D
or trisaccharide cases, proton positions of fucose and galactose
parts were shown by using single prime (0) and double prime (00),
respectively. 13C NMR spectra were routinely recorded at 100 MHz
on a BRUKER AVANCE 400 spectrometer at 300 K and chemical
s, Aromatic-H), 7.48 (1H, s, Aromatic-H), 7.38–6.97 (20H, m, Aro-
matic-H), 5.27 (1H, d, J¼10.3 Hz, H-1), 4.90 (1H, d, J¼11.3 Hz,
PhCH2–), 4.84 (1H, d, J¼3.3 Hz, H-10), 4.63 (1H, d, J¼11.5 Hz, PhCH2–),
4.61 (1H, d, J¼12.0 Hz, PhCH2–), 4.60 (1H, d, J¼11.3 Hz, PhCH2–), 4.58
(1H, d, J¼11.5 Hz, PhCH2–), 4.56 (1H, d, J¼12.0 Hz, PhCH2–), 4.31
(1H, t, J¼10.3 Hz, H-2), 4.30 (1H, d, J¼12.0 Hz, PhCH2–), 4.29 (1H, d,
J¼12.0 Hz, PhCH2–), 4.26 (1H, t, J¼10.3 Hz, H-3), 4.21 (1H, dd,
J¼10.3, 7.8 Hz, H-4), 4.12 (1H, q, J¼6.5 Hz, H-50), 3.90–3.76 (2H, m,
H-6), 3.89–3.85 (1H, m, 4-OH), 3.84 (1H, dd, J¼10.3, 3.3 Hz, H-20),
3.79 (1H, dd, J¼2.7, 10.3 Hz, H-30), 3.62–3.59 (1H, m, H-5), 3.57 (1H,
br, H-40), 2.15 (3H, s, SCH3), 1.11 (3H, d, J¼6.5 Hz, H-60); 13C NMR
shifts were expressed relative to that of the deuterated solvents (
d
77.0 ppm for CDCl3). MALDI-TOF MS analyses were performed on
a BRUKER REFLEX III with DHB (2,5-dihydroxybenzoic acid). Ions
generated by a pulsed UV laser beam (nitrogen laser,
l
¼337 nm,
5 Hz) were accelerated to a kinetic energy of 23.5 kV. High-reso-
lution FAB-mass data were recorded by the in-house mass spec-
trometry services at the Center for Instrumental Analysis, Hokkaido
University.
(100 MHz, CDCl3):
d 166.5, 166.0, 138.6, 138.6, 138.5, 138.2, 137.9,
131.1, 130.8, 128.5, 128.3, 128.3, 128.2, 127.7, 127.7, 127.6, 127.6, 127.5,
126.7, 125.2, 124.9, 100.1, 83.8, 80.4, 79.4, 78.8, 77.7, 75.8, 74.9, 73.5,
73.0, 71.0, 69.2, 68.6, 53.5, 16.5, 11.5; HRMS FAB m/z for
4.2. Methyl O-(2,3,4-tri-O-benzyl-
4,6-O-benzylidene-2-deoxy-2-(4,5-dichlorophthalimido)-1-
thio- -glucopyranoside (6)
a-L-fucopyranosyl)-(1/3)-
b-D
C
49H49O10NCl2SþH (MþH)þ calcd 914.2533, found 914.2534.
4.2.1. Typical method with microwave irradiation: entry 3 in
Scheme 1
4.4. Methyl O-(2-O-acetyl-3,4,6-tri-O-benzyl-
galactopyranosyl)-(1/4)-[O-(2,3,4-tri-O-benzyl-
b
-
D
-
a
-L-
A solution of TMSOTf (73
m
l, 0.403 mmol) in diethyl ether (1 ml)
fucopyranosyl)-(1/3)]-6-O-benzyl-2-deoxy-2-(4,5-
dichlorophthalimido)-1-thio- -glucopyranoside (2)
was added dropwise to a stirred ꢀ30 ꢁC solution, cooled by the
CoolMate, of pre-dried glucosamine acceptor (4) (100 mg,
0.201 mmol) and acetate donor (5) (190 mg, 0.399 mmol) in diethyl
ether (2 ml) in a CEM equipped vessel under nitrogen. The reaction
mixture was stirred for 1 h with microwave irradiation (300 W)
and the reaction temperature was monitored every second auto-
matically. The mixture was then diluted with chloroform and
neutralized with satd NaHCO3 aq. The aqueous layer was extracted
with chloroform three times, combined extracts were dried over
MgSO4 and concentrated in vacuo. The resulting residue was pu-
rified by column chromatography [silica gel: n-hexane/EtOAc (4:1)]
b-D
4.4.1. Typical method with microwave irradiation: entry 2 in
Scheme 2
A solution of TMSOTf (20 ml, 0.110 mmol) in diethyl ether
(1.5 ml) was added dropwise to a stirred ꢀ30 ꢁC solution, cooled by
the CoolMate, of pre-dried disaccharide acceptor (3) (51.5 mg,
0.0563 mmol) and galactosyl donor (7) (59.7 mg, 0.112 mmol) in
diethyl ether (3 ml) in a CEM equipped vessel under nitrogen at-
mosphere. The reaction mixture was stirred for 5 h with microwave
irradiation (300 W) and the reaction temperature was monitored
every second automatically. The mixture was then diluted with
chloroform and neutralized with satd NaHCO3 aq. The aqueous
layer was extracted with chloroform four times, and combined
extracts were dried over MgSO4 and concentrated in vacuo. The
resulting residue was purified by column chromatography
[Sephadex LH-20: chloroform/methanol (1:1)] to give 2 (54.4 mg,
70%) as a white solid and recovered as 3 (7.6 mg, with a calculated
yield of 2 of 82%).
to give 6 (165 mg, 90%) as a white solid.
16
[a
]
ꢀ45.6 (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3):
d 7.68 (1H,
D
s, Aromatic-H), 7.43 (1H, s, Aromatic-H), 7.37–6.99 (20H, m, Aro-
matic-H), 5.57 (1H, s, PhCH–), 5.32 (1H, d, J¼10.5 Hz, H-1), 4.94 (1H,
d, J¼3.5 Hz, H-10), 4.86 (1H, d, J¼11.8 Hz, PhCH2–), 4.64 (1H, t,
J¼10.5 Hz, H-3), 4.55 (1H, d, J¼11.8 Hz, PhCH2–), 4.51 (1H, d,
J¼11.8 Hz, PhCH2–), 4.45 (1H, dd J¼4.5, 9.5 Hz, H-5), 4.40 (1H, d, J¼
11.8 Hz, PhCH2–), 4.40 (1H, t, J¼10.5 Hz, H-2), 4.33 (1H, d, J¼12.0 Hz,
PhCH2–), 4.13 (1H, d, J¼12.0 Hz, PhCH2–), 4.08 (1H, q, J¼6.5 Hz,
H-50), 3.82 (1H, dd, J¼9.5, 10.5 Hz, H-4), 3.79 (1H, dd, J¼3.5, 10.5, H-
20), 3.75 (1H, dd, J¼2.5, 10.0 Hz, H-30), 3.74–3.69 (2H, m, H-6), 3.56
1H NMR (400 MHz, CDCl3):
d 7.80 (1H, s, Aromatic-H), 7.38–6.97
(36H, m, Aromatic-H), 5.27 (1H, dd, J¼9.8, 8.3 Hz, H-200), 4.99 (1H, d,
J¼10.4 Hz, H-1), 4.87 (1H, d, J¼10.6 Hz, PhCH2–), 4.76 (1H, d,

H. Shimizu et al. / Tetrahedron 64 (2008) 10091–10096
10095
J¼12.0 Hz, PhCH2–), 4.67 (1H, d, J¼12.2 Hz, PhCH2–), 4.61 (1H, t,
J¼9.6 Hz, H-3), 4.61 (1H, m, H-50), 4.60 (1H, d, J¼8.3 Hz, H-100), 4.59
(1H, d, J¼4.6 Hz, H-10), 4.52 (1H, d, J¼12.2 Hz, PhCH2–), 4.50 (1H, d,
J¼11.2 Hz, PhCH2–), 4.45 (1H, d, J¼12.0 Hz, PhCH2–), 4.44 (1H, d,
J¼12.4 Hz, PhCH2–), 4.43–4.27 (6H, m, PhCH2–), 4.40 (1H, m, H-2),
4.29 (1H, d, J¼12.4 Hz, PhCH2–), 4.11 (1H, t, J¼9.6 Hz, H-4), 3.99 (1H,
d, J¼2.6 Hz, H-400), 3.89–3.79 (2H, m, H-6), 3.85 (1H, dd, J¼10.3,
2.6 Hz, H-30), 3.76–3.67 (2H, m, H-600), 3.71 (1H, m, H-20), 3.55 (1H,
d, J¼9.6 Hz, H-5), 3.34 (1H, dd, J¼9.8, 2.6 Hz, H-300), 3.33 (1H, m, H-
500), 3.18 (1H, br, H-40), 2.07 (3H, s, SCH3), 2.02 (3H, s, COCH3), 1.13
reaction mixture was stirred for 10 min. The mixture was diluted
with chloroform and washed with satd Na2S2O3 aq and satd
NaHCO3 aq successively. The combined aqueous layer was extrac-
ted with chloroform three times, and combined extracts were dried
over MgSO4 and concentrated in vacuo. The resulting residue was
purified by column chromatography [silica gel: n-hexane/EtOAc
(2:1)] to give
b
-12 (6.4 mg, 56%) as a white solid.
1H NMR (400 MHz, CDCl3):
d
7.36–7.21 (35H, m, Aromatic-H),
5.90 (1H, d, J¼7.5 Hz, H-1), 5.22 (1H, dd, J¼10.1, 8.0 Hz, H-200), 5.19
(1H, d, J¼3.7 Hz, H-10), 4.87 (1H, d, J¼11.2 Hz, PhCH2–), 4.77 (2H, d,
J¼11.5 Hz, PhCH2–), 4.76 (1H, d, J¼11.7 Hz, PhCH2–), 4.70–4.61 (5H,
m, PhCH2–), 4.50 (1H, d, J¼11.2 Hz, PhCH2–), 4.45 (1H, d, J¼12.1 Hz,
PhCH2–), 4.45 (1H, d, J¼11.7 Hz, PhCH2–), 4.44 (1H, d, J¼8.0 Hz, H00-
1), 4.34 (2H, J¼11.5 Hz, PhCH2–), 4.32 (1H, d, J¼12.0 Hz, PhCH2–),
4.30 (1H, d, J¼12.0 Hz, PhCH2–), 4.19 (1H, q, J¼6.4 Hz, H-50), 4.14
(1H, t, J¼4.4 Hz, H-3), 4.05–4.02 (1H, m, H-2), 3.99 (1H, m, H-4),
3.96 (1H, dd, J¼10.1, 3.7 Hz, H-20), 3.94 (1H, m, H-400), 3.82 (1H, dd,
J¼10.1, 2.7 Hz, H-30), 3.66–3.61 (2H, m, H-6), 3.63–3.61 (1H, m,
H-500), 3.55 (1H, dd, J¼8.6, 5.0 Hz, H-600), 3.43–3.38 (1H, m, H-5),
3.41–3.37 (1H, m, H-600), 3.38–3.36 (1H, m, H-40), 3.33 (1H, dd,
J¼10.1, 2.8 Hz, H-300), 2.75 (1H, s, OH), 1.99 (3H, s, NHCOCH3), 1,93
(3H, s, COCH3), 1.04 (3H, d, J¼6.4 Hz, H-60); 13C NMR (100 MHz,
(3H, d, J¼6.5 Hz, H-60); 13C NMR (100 MHz, CDCl3):
d 169.0, 166.4,
165.2, 139.2, 138.9, 138.7, 138.3, 138.1, 138.0, 137.8, 128.8, 128.6,
128.4,128.4, 128.3, 128.1, 127.9, 127.9, 127.9, 127.8, 127.7, 127.7, 127.6,
127.5, 127.4, 127.3, 127.2, 127.1, 127.1, 127.0, 125.8, 99.8, 97.7, 80.8,
80.2, 80.0, 79.9, 78.4, 77.2, 75.5, 74.9, 74.4, 73.8, 73.4, 73.3, 73.2, 73.1,
72.8, 72.7, 72.0, 71.8, 71.5, 67.9, 67.7, 66.8, 55.1, 16.2, 10.7; MALDI-
TOF MS m/z for C78H79Cl21NO18SþNa [MþNa]þ calcd 1410.4, found
1410.5; HRMS ESI m/z for C78H79Cl21NO18SþNa [MþNa]þ calcd
1410.4394, found 1410.4374.
4.5. Methyl O-(2-O-acetyl-3,4,6-tri-O-benzyl-
b-D
-
galactopyranosyl)-(1/4)-[O-(2,3,4-tri-O-benzyl-
a-L-
fucopyranosyl)-(1/3)]-6-O-benzyl-2-acetoamide-2-deoxy-1-
thio- -glucopyranoside (11)
CDCl3): d 169.1, 165.4, 139.1, 139.0, 138.6, 138.5, 138.0, 137.9, 137.8,
b-D
101.3, 102.3, 97.7, 80.3, 79.3, 78.2, 77.8, 76.3, 74.9, 74.7, 73.4, 73.3,
73.0, 72.9, 72.8, 72.6, 71.8, 71.6, 70.9, 68.6, 67.9, 67.16, 66.6, 21.0,
16.4, 14.1; MALDI-TOF MS m/z for C71H79NO16þNa [MþNa]þ calcd
1224.5, found 1224.6; HRMS ESI m/z for C71H79NO16þNa [MþNa]þ
calcd 1224.5297, found 1224.5300.
Ethylenediamine (30 ml, 0.449 mmol) was added dropwise to
a stirred solution of 2 (16.3 mg, 0.0117 mmol) in ethanol (1.5 ml) at
room temperature. The reaction mixture was stirred for 2 h at 70 ꢁC
and concentrated in vacuo. The resulting residue was solved with
dichloromethane (0.5 ml), and pyridine (1 ml) and acetic anhydride
(1 ml) were added successively at 0 ꢁC. The reaction mixture was
stirred for 3 h at room temperature and concentrated in vacuo. The
resulting residue was purified by column chromatography [silica
gel: n-hexane/EtOAc (2:1)] to give 11 (13.8 mg, 95%) as a colorless oil.
4.7. O-(b-
D
-Galactopyranosyl)-(1/4)-[O-(
a
-L
-fucopyranosyl)-
(1/3)]-2-acetoamide-2-deoxy-a,b-D-gluco-pyranose; Lewis X
oligosaccharide (1)44
Pd(OH)2/C (cat.) was added to a stirred solution of 10 (6.4 mg,
5.32 mol) in methanol (1 ml) at room temperature and the mix-
1H NMR (400 MHz, CDCl3):
d 7.34–7.14 (35H, m, Aromatic-H),
m
5.53 (1H, d, J¼8.4 Hz, –NHAc), 5.24 (1H, dd, J¼10.0, 8.2 Hz, H-200),
5.06 (1H, br, H-10), 4.88 (1H, d, J¼10.8 Hz, PhCH2–), 4.73 (1H, d,
J¼12.7 Hz, PhCH2–), 4.70 (1H, d, J¼12.4 Hz, PhCH2–), 4.68 (1H, d,
J¼12.4 Hz, PhCH2–), 4.66 (1H, d, J¼12.4 Hz, PhCH2–), 4.57 (2H, d, J¼
11.4 Hz, PhCH2–), 4.52 (1H, d, J¼12.2 Hz, PhCH2–), 4.50 (1H, d,
J¼8.2 Hz, H-100), 4.49 (1H, q, J¼6.5 Hz, H-50), 4.47 (1H, d, J¼10.7 Hz,
PhCH2–), 4.42 (1H, d, J¼12.2 Hz, PhCH2–), 4.35 (2H, s, PhCH2–),
4.15–4.05 (1H, m, H-2 or H-3), 4.09 (2H, d, J¼11.4 Hz, PhCH2–), 4.00
(1H, m, H-4), 3.99 (1H, m, H-20), 3.98 (1H, m, H-400), 3.83 (1H, dd,
J¼7.5, 3.1 Hz, H-30), 3.78 (1H, dd, J¼11.0, 3.3 Hz, H-6), 3.75–3.70 (1H,
m, H-500), 3.75–3.70 (1H, m, H-2 or H-3), 3.73 (1H, dd, J¼11.0, 2.8 Hz,
H-6), 3.62 (1H, dd, J¼8.5, 4.7 Hz, H-600), 3.45 (1H, m, H-5), 3.37 (1H,
dd, J¼10.0, 2.7 Hz, H-300), 3.32 (1H, dd, J¼8.5, 4.9 Hz, H-600), 3.26 (1H,
br, H-40), 2.11 (3H, s, SCH3), 1.99 (3H, s, COCH3 or NHCOCH3), 1.73
(3H, s, COCH3 or NHCOCH3), 1.11 (3H, d, J¼6.5 Hz, H-60); 13C NMR
ture was treated under hydrogen. The reaction mixture was stirred
overnight at room temperature. The mixture was filtered through
Celite. The filtrate was concentrated in vacuo. The resulting residue
was solved with water (1 ml), and 15% NaOH aq (one drop) was
added. The reaction mixture was stirred for 2 h at room tempera-
ture, neutralized with Dowex 500WX8-400 and filtered. The filtrate
was concentrated in vacuo. The resulting residue was purified by
column chromatography [Sephadex G-10: water] to give Lewis X
oligosaccharide (1)44 (2.8 mg, 99%).
Acknowledgements
This research was partially supported by the Ministry of Edu-
cation, Science, Sports and Culture, Grant-in-Aid for Scientific Re-
search (C), 1858009, 2006 and by a fund from Tokyo Rikakikai Co.,
Ltd (EYELA). The authors thank members of the Center for In-
strumental Analysis, Hokkaido University, for their high-resolution
FAB-MS analysis, Ms. T. Yamada at Tohoku University for supporting
normal-resolution FAB-MS analysis, and Ms. I. Nagashima and Dr. K.
Shimizu in our group (AIST) for rearrangement experimental data
and technical support.
(100 MHz, CDCl3):
d 170.0, 169.2, 139.2, 139.1, 138.8, 138.6, 138.2,
137.9, 137.8, 128.7, 128.5, 128.5, 128.4, 128.4, 128.3, 126.3, 128.2,
128.0, 127.9, 127.8, 127.7, 127.7, 127.6, 127.6, 127.5, 127.4, 127.3, 127.2,
99.8, 97.7, 80.5, 79.6, 79.4, 78.3, 75.3, 74.9, 73.4, 73.3, 73.2, 73.0,
72.9, 72.2, 72.1, 71.6, 68.4, 67.7, 66.7, 23.4, 21.0, 16.3, 11.9; MALDI-
TOF MS m/z for C72H81Cl21NO15SþNa [MþNa]þ calcd 1254.5, found
1254.8; HRMS ESI m/z for C72H81Cl21NO15SþNa [MþNa]þ calcd
1254.5224, found 1254.5222.
Supplementary data
4.6. O-(3,4,6-Tri-O-benzyl-2-O-acetyl-
(1/4)-[O-(2,3,4-tri-O-benzyl- -fucopyranosyl)-(1/3)]-6-O-
benzyl-2-acetoamide-2-deoxy- -glucopyranose (12)
b-D-galactopyranosyl)-
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.tet.2008.08.011.
a-L
b-D
References and notes
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m
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mol) in aqueous dichloromethane (1 ml) at 0 ꢁC, and the
ml,
55.5
95.7
m
m
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