Enzymatic glycosidation of sugar oxazolines having a carboxylate group catalyzed by chitinase

Article abstract of DOI:10.1016/j.carres.2004.08.016

Enzymatic glycosidation using sugar oxazolines 1-3 having a carboxylate group as glycosyl donors and compounds 4-6 as glycosyl acceptors was performed by employing a chitinase from Bacillus sp. as catalyst. All the glycosidations proceeded with full control in stereochemistry at the anomeric carbon of the donor and regio-selectivity of the acceptor. The N,N′-diacetyl-6′-O- carboxymethylchitobiose oxazoline derivative 1 was effectively glycosidated, under catalysis by the enzyme, with methyl N,N′-diacetyl-β- chitobioside (4), pent-4-enyl N-acetyl-β-d-glucosaminide (5), and methyl N-acetyl-β-d-glucosaminide (6), affording in good yields the corresponding oligosaccharide derivatives having 6-O-carboxymethyl group at the nonreducing GlcNAc residue. The N,N′-diacetyl-6-O-carboxymethylchitobiose oxazoline derivative 2 was subjected to catalysis by the enzyme catalysis; however, no glycosidated products were produced through the reactions with 4, 5, and 6. Glycosidation reactions of the β-d-glucosyluronic-(1→4)-N-acetyl-d- glucosamine oxazoline derivative 3 proceeded with each of the glycosyl acceptors, giving rise to the corresponding oligosaccharide derivative having a GlcA residue at their nonreducing termini in good yields.

Full text of DOI:10.1016/j.carres.2004.08.016

Carbohydrate
RESEARCH
Carbohydrate Research 339 (2004) 2769–2788
Enzymatic glycosidation of sugar oxazolines having a
carboxylate group catalyzed by chitinase
Hirofumi Ochiai, Masashi Ohmae and Shiro Kobayashi*
Department of Materials Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan
Received 7May 2004; accepted 20 August 2004
Available online 28 October 2004
Abstract—Enzymatic glycosidation using sugar oxazolines 1–3 having a carboxylate group as glycosyl donors and compounds 4–6
as glycosyl acceptors was performed by employing a chitinase from Bacillus sp. as catalyst. All the glycosidations proceeded with full
control in stereochemistry at the anomeric carbon of the donor and regio-selectivity of the acceptor. The N,N0-diacetyl-60-O-carb-
oxymethylchitobiose oxazoline derivative 1 was effectively glycosidated, under catalysis by the enzyme, with methyl N,N0-diacetyl-b-
chitobioside (4), pent-4-enyl N-acetyl-b-D-glucosaminide (5), and methyl N-acetyl-b-D-glucosaminide (6), affording in good yields
the corresponding oligosaccharide derivatives having 6-O-carboxymethyl group at the nonreducing GlcNAc residue. The N,N0-
diacetyl-6-O-carboxymethylchitobiose oxazoline derivative 2 was subjected to catalysis by the enzyme catalysis; however, no
glycosidated products were produced through the reactions with 4, 5, and 6. Glycosidation reactions of the b-D-glucosyluronic-
(1!4)-N-acetyl-D-glucosamine oxazoline derivative 3 proceeded with each of the glycosyl acceptors, giving rise to the corresponding
oligosaccharide derivative having a GlcA residue at their nonreducing termini in good yields.
Ó 2004 Published by Elsevier Ltd.
Keywords: Enzymatic glycosidation; Functionalized sugar oxazolines; Chitinase from Bacillus sp.; End functionalization
1. Introduction
acceptor with catalysis by a glycosyltransferase or glyco-
side hydrolase.12 The former enzyme catalyzes glycosidic
bond formations exclusively, whereas the latter, catalyz-
ing hydrolytic bond-cleavage, permits bond formation
under selected reaction conditions in vitro. Such in vitro
catalyses may be utilized for constructing structurally
complex carbohydrates, because of the high regio- and
stereo-selectivity of the reactions. Glycoside hydrolases
are readily available, are more stable, and less expensive
than glycosyltransferases, and have frequently been used
for glycosidation reactions, including polymerizations
requiring a repeated glycosidation.13,14
We previously reported the synthesis of N,N0-diacetyl-
chitobiose [b-GlcNAc-(1!4)-GlcNAc]; via enzymatic
glycosidation of an activated form of an N-acetyl-
D-glucosamine (GlcNAc) oxazoline derivative as the
glycosyl donor, with GlcNAc as the glycosyl acceptor.15
The reaction was catalyzed by a chitinase from Bacillus
sp. (EC 3.2.1.14), one of the endo-glycanases responsible
for chitin hydrolysis, belonging to the glycoside hydrol-
ase family 18.16 In this glycosidation, the oxazoline part
Carbohydrates exist in all living cells as components of
such glycoconjugates as glycoproteins, glycolipids, and
proteoglycans. They play critical roles for vital activities
of living cells; adhesion,1 development and proliferation
of cells,2 transformation and metastasis of tumors,3
infection of microorganisms,4 and parasites,5 and such
autoimmune diseases as rheumatoid arthritis.6 Chemical
synthesis provides structurally well-defined carbo-
hydrate samples for studying these processes, via
organo-chemical or enzyme-catalyzed methods. These
methodologies require a glycosyl donor and a glycosyl
acceptor. Various glycosyl donors have been found
effective;7–9 but, control of anomeric stereo- and regio-
chemistry is often difficult.10,11
Enzyme-catalyzed synthesis of carbohydrates may be
performed with a nonprotected glycosyl donor and
*
Corresponding author. Fax: +81 75 383 2461; e-mail: kobayasi@
mat.polym.kyoto-u.ac.jp
0008-6215/$ - see front matter Ó 2004 Published by Elsevier Ltd.
doi:10.1016/j.carres.2004.08.016

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H. Ochiai et al. / Carbohydrate Research 339 (2004) 2769–2788
Glycosyl donors
H3C
O
O
H3C
OH
O
CO2Na
OH
OH
O
NH
NH
O
HO
HO
O
HO
O
O
HO
HO
HO
HO
O
HO
O
HO
O
O
O
NaO2C
O
OH
O
NaO2C
O
N
N
N
CH3
CH3
CH3
3
1
2
Glycosyl acceptors
O
H3C
OH
OH
OH
O
NH
O
O
HO
HO
HO
HO
HO
HO
O
HO
O
OCH3
OCH3
O
OH
NH
O
NH
O
NH
O
H3C
H3C
H3C
4
5
6
Figure 1. Sugar oxazoline derivatives having a carboxylate group, designed as glycosyl donors (1–3), and glycosyl acceptors (4–6).
on the glycosyl donor is key for the reaction; the transi-
tion state of the enzymatic hydrolysis involves an oxazo-
linium ion species as a high-energy form structurally
analogous to the oxazoline.17 The GlcNAc oxazoline
derivative is, therefore, immediately recognized and cat-
alyzed by the chitinase as a Ôtransition-state analogue
substrateÕ, serving as an activated glycosyl donor. This
concept has been applied to the synthesis of natural
heteropolysaccharides of hyaluronan18 and chondroitin.19
Few reports describe the synthesis of functionalized
oligosaccharides via enzymatic glycosidation using func-
tionalized glycosyl donors.20 The functionalization of
oligosaccharides through enzymatic glycosidation is use-
ful for facile and rapid production of such artificial glyco-
conjugates as neoglycoconjugates,21 which are frequently
used as tools for investigating carbohydrate functions
in vivo. Here we report glycosidation reactions catalyzed
by a chitinase from Bacillus sp., using new sugar oxazoline
derivatives (1–3) having a carboxylate functional group
as glycosyl donors and GlcNAc derivatives (4–6) as
glycosyl acceptors. The functional group may serve for
further chemical modifications of the products (Fig. 1).
acetylation. The O-acetyl groups of 13 were all removed
by sodium methoxide, followed by selective protection of
the 60-hydroxyl group by the 4-methoxytrityl (MMTr)
group to provide 14. All of the hydroxyl groups of 14
were protected by 4-methoxybenzyl (MPM) groups and
then MMTr group was hydrolyzed by aqueous acetic
acid, giving rise to 15. Allyl bromide was allowed to react
with the 60-hydroxyl group of 15, converting it into 16.
The MPM groups of 16 were all removed by the oxida-
tion with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone
(DDQ), with subsequent acetylation of the hydroxyl
groups to produce 17. The alkenyl group of 17 was
oxidized to the carboxylic acid group by ozonolysis
and then methylated by Dowex 50W-X4 (H+ form) in
methanol to give 18. Compound 18 was treated with tri-
methylsilyl triflate (Me3SiOTf) to form the oxazoline
derivative 19. The O-acetyl groups of 19 were removed
by sodium methoxide, and the methyl ester was hydro-
lyzed in a carbonate buffer to provide the 60-O-carboxy-
methylated oxazoline 1.
The novel oxazoline derivative 2 was also synthesized
chemically (Scheme 2). Compound 11 was glycosidated
with 21 prepared via two-step reactions from 9 to afford
22. The phthalimido group of 22 was converted to an
amino group by treatment with hydrazine monohydrate,
followed by acetylation to give 23. All of the O-acetyl
groups of 23 were removed by the action of sodium
methoxide in methanol, and the resulting hydroxyl
groups were protected by the MPM group to provide
24. The tert-butyldimethylsilyl (TBDMS) group of 24
was removed by tetra-n-butylammonium fluoride in
tetrahydrofuran (THF), giving rise to 25. The hydroxyl
group of 25 reacted with allyl bromide in N,N-dimethyl-
formamide (DMF) to produce 26. Compound 27 was
obtained through the complete removal of the MPM
groups from 26 by DDQ oxidation followed by acetyl-
2. Results and discussion
2.1. Synthesis of disaccharide oxazoline derivatives having
a carboxylate group
The newly designed oxazoline derivative (1) was pre-
pared as outlined in Scheme 1. Compound 10, synthe-
sized via
a
four-step reactions from 722 was
glycosylated with a readily accessible glycosyl trichloro-
acetimidate 11 to afford the disaccharide derivative 12.
Compound 13 was prepared by removal of the phthalim-
ido group of 12 by hydrazine monohydrate followed by

H. Ochiai et al. / Carbohydrate Research 339 (2004) 2769–2788
2771
OAc
O
MPh
O
MPh
O
O
O
a
O
O
AcO
AcO
b
OMPM
NPhth
OMPM
NPhth
HO
OMPM
NPhth
MPMO
9
8
7
NH
CCl3
NPhth
AcO
AcO
O
O
OAc
11
OMPM
O
OR1
R2
O
d
c
O
R3O
R3O
HO
MPMO
O
9
OR1
OMPM
NPhth
R1O
R2
OR4
10
12: R1=MPM, R2=NPhth, R3=R4=Ac
13: R1=MPM, R2=NHAc, R3=R4=Ac
14: R1=MPM, R2=NHAc, R3=H, R4=MMTr
15: R1=R3=MPM, R2=NHAc, R4=H
16: R1=R3=MPM, R2=NHAc, R4=-CH2CH=CH2
17: R1=R3=Ac, R2=NHAc, R4=-CH2CH=CH2
18: R1=R3=Ac, R2=NHAc, R4=-CH2CO2Me
e
f
g
h
i
j
OAc
O
OH
NHAc
NHAc
l
k
O
AcO
AcO
O
HO
HO
O
HO
18
AcO
O
O
O
NPhth=phthalimido
O
O
CO2Me
O
CO2Na
N
N
MPh=4-methoxyphenyl
MPM=4-methoxylbenzyl
MMTr=4-methoxytrityl
1
19
CH3
CH3
Scheme 1. Reagents and conditions: (a) (i) NaOMe/MeOH, rt, 2h, (ii) 4-MeOC6H4CH(OMe)2, CSA/DMF, 30°C, 4h, 80% (two steps); (b) MPMCl,
NaH, n-Bu4NI/DMF, rt, overnight, 51%; (c) NaCNBH3, CF3CO2H, MS3A/DMF, rt, 48h, quant.; (d) Me3SiOSO2CF3, MS4A/CH2Cl2, À20°C, 1h,
91%; (e) (i) NH2NH2 monohydrate/EtOH, 90°C, 5h, (ii) Ac2O-pyridine, rt, 3h, 88% (two steps); (f) (i) NaOMe/MeOH, rt, 1h, quant, (ii) MMTrCl/
pyridine, rt, 7h, 96%; (g) MPMCl, NaOH/DMF, rt, overnight, then 70% aq AcOH, 50 °C, 4h, 71% (two steps); (h) CH2@CHCH2Br, NaOH/DMF,
rt, 3h, 92%; (i) (i) DDQ/CH2Cl2–H2O, rt, 4 h, (ii) Ac2O–pyridine, rt, 4h, 90% (two steps); (j) (i) O3/CH2Cl2–MeOH, À78°C, 0.5h, (ii) NaClO2,
NaH2PO4, Me2C@CHMe/tert-BuOH–H2O, rt, 2h, (iii) Dowex 50W-X4 (H+ form)/MeOH, 40°C, 4.5h, 74% (three steps); (k) Me3SiOSO2CF3/
CH2ClCH2Cl,40°C, 12h, 87%; (l) NaOMe/MeOH, rt, 2h, then carbonate buffer (25mM, pH12), rt, 2h.
ation. The alkenyl group of 27 was oxidized to the carb-
oxylic acid form of 28 by ozonolysis followed by methyl-
ation in methanol with Dowex 50W-X4 (H+ form) as
catalyst. Formation of the oxazoline ring to furnish 29
was performed in 1,2-dichloroethane by using Me3-
SiOTf. The O-acetyl groups of 29 were all removed by
sodium methoxide in methanol, followed by hydrolysis
of the methyl ester in a carbonate buffer, giving rise to
the 6-O-carboxymethylated oxazoline derivative 2.
The new oxazoline derivative 3 was synthesized as a
potential glycosyl donor for the chitinase (Scheme 3).
The acetylated methyl glucuronate trichloroacetimidate
3023 was glycosidated with the readily accessible com-
pound 3124 to give the disaccharide 32. The oxazoline
derivative 33 was produced by the treatment of 32 with
Me3SiOTf in 1,2-dichloroethane. Compound 33 was O-
deacylated by sodium methoxide in methanol, and the
methyl ester was hydrolyzed in carbonate buffer to yield
the target compound 3.
Bacillus sp. without any glycosyl acceptor in a carbonate
buffered D2O (50mM, pD9.0) at 30°C. The initial con-
centration of the substrate was 0.1M and the amount of
enzyme was 10wt% for the substrate. The reaction was
1
monitored by H NMR spectroscopy (Fig. 2). Without
the enzyme the substrates were not consumed, whereas
with addition of the enzyme the substrates were com-
pletely consumed after 4, 6, and 15h for 1, 3, and 2,
respectively. These observations indicate that these sub-
strates were recognized and activated by enzyme–sub-
strate complex formation. The reactivity order in
hydrolysis, 1 > 3 > 2, reflects the ability of these sub-
strates to be recognized at the donor site of the enzyme.
The products were confirmed as being 34, 35, and 36, de-
rived by hydrolysis via oxazoline ring opening from 1, 2,
and 3, respectively (Scheme 4). Analytical data for 34,
35, and 36 are given in Section 4.
2.3. Glycosidation reactions of 1 catalyzed by chitinase
2.2. Chitinase-catalyzed hydrolysis of the oxazoline
derivatives
The oxazoline derivative 1 as a glycosyl donor was
allowed to react with each glycosyl acceptor, methyl-
N,N0-diacetyl-b-chitobioside (4), pent-4-enyl N-acetyl-
b-D-glucosaminide (5), or methyl N-acetyl-b-D-glucos-
aminide (6). Compound 5 was designed as glycosyl
Compounds 1, 2, and 3 were subjected as substrates to
the enzymatic hydrolysis catalyzed by the chitinase from

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H. Ochiai et al. / Carbohydrate Research 339 (2004) 2769–2788
OH
OTBDMS
O
MPh
O
O
O
a
O
HO
MPMO
b
HO
MPMO
OMPM
NPhth
OMPM
NPhth
OMPM
NPhth
MPMO
20
21
9
NH
CCl3
NPhth
AcO
AcO
O
O
OAc
11
OR3
O
CO2Me
O
R2
NHAc
O
R4O
AcO
AcO
j
O
O
O
c
R4O
OR1
R1O
AcO
21
O
OR4
O
R2
OAc
N
22: R1=MPM, R2=NPhth, R3=TBDMS, R4=Ac
23: R1=MPM, R2=NHAc, R3=TBDMS, R4=Ac
24: R1=R4=MPM, R2=NHAc, R3=TBDMS
25: R1=R4=MPM, R2=NHAc, R3=H
26: R1=R4=MPM, R2=NHAc, R3=-CH2CH=CH2
27: R1=R4=Ac, R2=NHAc, R3=-CH2CH=CH2
28: R1=R4=Ac, R2=NHAc, R3=-CH2CO2Me
29
CH3
d
e
k
f
g
h
i
O
CO2Na
NHAc
O
O
HO
HO
O
HO
TBDMS=tert-butyldimethylsilyl
O
OH
N
2
CH3
Scheme 2. Reagents and conditions: (a) 70% aqAcOH, 70°C, 1h, 83%; (b) TBDMSCl/pyridine, rt, 1h, 97%; (c) BF3 etherate, MS4A/CH2Cl2,
À20°C, 1h, 87%; (d) (i) NH2NH2 monohydrate/EtOH, 90°C, 5h, (ii) Ac2O–pyridine, rt, 5h, 78% (two steps); (e) (i) NaOMe/MeOH, rt, 3h, quant,
(ii) NaOH, MPMCl/DMF, rt, overnight, 47%; (f) n-Bu4F/THF, rt, 1h, 99%; (g) NaOH, CH2@CHCH2Br/DMF, rt, 5h, 81%; (h) (i) DDQ/CH2Cl2–
H2O, rt, overnight, (ii) Ac2O–pyridine, 0°C, 5h, 87% (two steps); (i) (i) O3/CH2Cl2–MeOH, À78°C, 0.5h, (ii) NaClO2, NaH2PO4, (CH3)2C@CHCH3/
CH2Cl2-tert-BuOH–H2O, rt, 2h, (iii) Dowex 50W-X4 (H+ form)/CH2Cl2–MeOH, 40°C, 5h, 91% (three steps); (j) Me4SiOSO2CF3/CH2ClCH2Cl,
40°C, 5h, 56%; (k) (i) NaOMe/MeOH, rt, 2h, (ii) carbonate buffer (20mM, pH12), rt, 2h.
NH
O
CCl3
AcO
AcO
AcO
O
MeO2C
30
OAc
OAc
OAc
O
AcO
AcO
O
b
a
O
AcO
AcO
AcO
AcO
HO
AcO
O
AcO
O
AcO
O
O
AcHN
AcHN
MeO2C
MeO2C
O
OAc
OAc
N
33
32
31
CH3
c
OH
O
OH
O
HO
HO
O
HO
NaO2C
O
N
3
CH3
Scheme 3. Reagents and conditions: (a) BF3 etherate, MS4A/CH2Cl2, À20°C, 5h, 51%; (b) Me3SiOSO2CF3/CH2ClCH2Cl, 50°C, 5h, 77%; (c)
NaOMe/MeOH, rt, 1.5h, then carbonate buffer (25mM, pH12), rt, 1.5h.
acceptor because the pentenyl group serves as a reactive
alkene or a good leaving group for further reactions of
the glycosylated product.25
Enzymatic glycosidation of 1 with 4 was per-
formed under various reaction conditions (Scheme 5,
Table 1).

H. Ochiai et al. / Carbohydrate Research 339 (2004) 2769–2788
2773
40°C (entries 5, 8, and 9). The yield of 37 increased with
increasing concentration of 4, and reached a maximum
of 74% in 0.03M of 4 (entries 5, 10, and 11). The glyco-
sidation of 1 with an increased amount of 4 (10 or
30molequiv) gave somewhat lower yields (entries 12
and 13). A small amount of the enzyme was sufficient
for the reaction (entries 14–17and also entries 18–20
in large amount for comparison). The reaction in entry
5 gave 37 in 45% yield.
Enzymatic glycosidation of 1 with 5 was carried out
under the optimal conditions established for the reaction
(Scheme 6). The reaction was complete after 12h, giving
the product trisaccharide 38 in 37% yield. Compound 1
also reacted with 6 by enzymatic catalysis under similar
conditions (Scheme 7); 1 was completely consumed after
9h, affording the trisaccharide 39 as the sole glycosi-
dated product in 45% isolated yield.
0.1
0.08
0.06
0.04
0.02
0
0
5
10
Time (h)
15
20
Figure 2. Reaction–time courses of 1 (–m–), 2 (–j–), and 3 (–d–). The
arrow shows the time of enzyme addition.
In the reaction of 1 (0.01M) and 4 (0.03M) carried
out at a pHlower than 9.0 and at 30°C, 1 was rapidly
consumed within 1.5h, affording the product tetrasac-
charide 37 in lower yields (entries 1–3). The other com-
pounds detected in the mixture were hydrolysis product
34 and unchanged 4. These observations show that the
enzymatic and nonenzymatic hydrolysis of 1 is faster
than the enzymatic glycosidation of 1 with 4, although
the consumption of 1 was fast under these reaction con-
ditions. The reaction at pH9.5 progressed more slowly,
yet the yield of 37 was much improved (to 57%, entry 4).
The optimal yield was obtained at pH10.0 (74%, entry
5). At pH10.5, the yield was slightly decreased to 61%
(entry 6). It was drastically decreased (to 12%) at the
higher pHof 11.0 (entry 7). Thus, the reactions at a
pHranging from 9.5 to 10.5 gave the product tetrasac-
charide 37 in high yields. The reaction temperature
had little effect on the yield of 37, at 30, 20, and
All three acceptors can thus be glycosylated regio- and
stereo-specifically by donor 1 to give the corresponding
tetra- and trisaccharides in good yields. Other com-
pounds in the reaction mixture were the remaining
acceptor and 34.
2.4. Enzymatic reactions of 2 catalyzed by chitinase
The oxazoline derivative 2 was subjected to the chitinase
enzymatic reaction with glycosyl acceptor 4, 5, or 6, but
it did not react with these glycosyl acceptors, whereas it
was effectively hydrolyzed by the enzyme (Fig. 2), and
the compounds detected after the reaction were 35 (the
hydrolyzed product from 2), together with the un-
changed acceptors 4, 5, or 6. These observations can
be explained as illustrated in Figure 3. Compound 2 is
readily recognized and protonated at the donor site to
form the oxazolinium ion. Because of the steric
O
O
H3C
H3C
OH
O
O
CO2Na
OH
OH
NH
OH
O
NH
O
O
HO
HO
HO
HO
NaO2C
O
O
HO
HO
HO
O
HO
HO
O
OH
OH
OH
O
NH
O
NH
O
NH
O
O
CO2Na
H3C
H3C
H3C
35
36
34
Scheme 4.
O
H3C
OH
O
NH
chitinase
buffer
HO
HO
O
+
1
4
OCH3
HO
O
NH
O
NaO2C
O
H3C
3
37
Scheme 5.

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H. Ochiai et al. / Carbohydrate Research 339 (2004) 2769–2788
Table 1. Results of the enzymatic glycosidation of 1 with 4 under various conditionsa
Entry
[1]/M
[4]/M
Amount of
enzyme/wt% for 1
pH
Temperature/°C
Time/h
Yield of 37d/%
1
0.01
0.01
0.01
0.01
0.01
0.01
0.03
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.03
0.01
0.05
0.10
0.03
0.03
0.03
0.03
0.03
0.03
10
10
10
10
10
10
10
11.0
10
10
10
10
10
10
1
7.0
30
30
30
30
30
30
24
20
40
30
30
30
30
30
30
30
12
20
30
30
0.67b
1.0b
1.5b
12b
10
21
23
57
74
61
12
70
60
48
58
68
66
30
45
56
62
73
72
69
2
8.0
9.0
3
4
9.5
5
10.0
10.5
30
12b
6
18b
b
70.01
8
0.03
0.03
0.01
0.02
0.10
0.30
0.03
0.03
0.03
5
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
30
12c
12c
12c
12c
12c
12c
12c
12c
9
10
11
12
13
14
15
16
170.01
18
19
20
2
3
12c
c
10.0
20
10
20
0.03
0.15
0.30
10.0
10.0
10.0
12c
12c
12c
a In a carbonate buffer (50mM, 100lL).
b The time for complete consumption of 1.
c The reaction was terminated at the indicated time.
d Determined by HPLC based on 1.
O
H3C
OH
O
NH
chitinase
buffer
HO
HO
O
HO
+
1
5
O
O
NH
NaO2C
O
H3C
O
2
38
Scheme 6.
O
H3C
OH
O
NH
chitinase
buffer
HO
HO
O
+
1
6
OCH3
HO
NH
O
NaO2C
O
H3C
O
2
39
Scheme 7.
hindrance of the 6-O-CM group at the donor site, as
shown in A, the glycosyl acceptor cannot access the
acceptor site and even if it would do so, nucleophilic
attack of the 4-hydroxyl group of the GlcNAc residue
situated in the acceptor site cannot occur. The water
molecule is smaller than the CM group, and it can easily
locate at the acceptor site and attack the anomeric car-
bon of the oxazolinium intermediate in preference to
the 4-hydroxyl group of the glycosyl acceptor, as illus-
trated in B and C. Therefore, only hydrolysis of 2
occurred under enzymatic catalysis, that is, 2 is able to
act as a good donor molecule but reacts with difficulty
with the glycosyl acceptor.
2.5. Glycosidation reaction of 3 catalyzed by chitinase
The oxazoline derivative 3 was allowed to react enzy-
matically with 6 (Scheme 8).
Table 2 shows the results of two series of the reactions
(entries 1–7and 8–18). The enzymes used for these two

H. Ochiai et al. / Carbohydrate Research 339 (2004) 2769–2788
2775
Figure 3. Illustration of the reaction mechanism of 2.
In the second series of enzymatic reactions (entries 8–
18), the effects of enzyme amount (entries 8–11), the
reaction temperature (entries 12 and 13), and concentra-
tion of the acceptor and donor (entries 14–18) were
investigated. The yield of 40 increased with increasing
amount of enzyme (entries 8–10), and the maximum
yield was 56% on using 20wt% of the enzyme for 3 (en-
try 10). When the enzyme was used in 30wt% for 3, the
yield decreased a little (entry 11), and therefore, the en-
zyme amount was fixed to 20wt% for 3 in the following
reactions. The reactions at 20°C gave 40 in 33% yield
(entry 12), increasing at 40°C to 42% (entry 13). Next,
the concentration of donor 3 and acceptor 6 in a fixed
ratio (1:3) was varied to 0.10 and 0.30M (entry 14),
and 0.01 and 0.03 M (entry 15), respectively. Higher
concentrations of the substrates gave results similar to
entry 10, showing a little concentration effects on the
yield of 40 (entry 14). At 0.05M concentration of 3,
the reaction took place most efficiently. Effects of the
concentration of 6 were also investigated (entries 16–
18); but, the yield of 40 was decreased as compared with
that of the reaction using 0.15M of 6 (entry 10). These
OH
O
OH
chitinase
buffer
HO
HO
NaO2C
O
HO
+
3
6
OCH3
O
NH
O
H3C
2
40
Scheme 8.
series were from the same lot number but in the different
bottles. In the first series, the product 40 was obtained
under the pHrange from 7.0 to 10.5 (entries 1–6). In
the reactions at pH7.0 and 8.0, 3 was rapidly consumed
within 7h, giving 40 in slightly lower yields (entries 1
and 2). Reactions at pH 9.0–10.0 yielded 40 in good
yields with longer reaction time for the disappearance
of 3 (entries 3–5). At pH10.5, yield decreased drastically
to 18% (entry 6), and at pH11.0 the reaction did not
take place (entry 7). The best yield of 40 (58%) was
obtained at pH10.0. Other compounds detected after
the reaction were 36, the hydrolysis product from 3,
and the unchanged remaining 6.

2776
H. Ochiai et al. / Carbohydrate Research 339 (2004) 2769–2788
Table 2. Results of the enzymatic glycosidation of 3 with 6 under various conditionsa
Entry
[3]/M
[6]/M
Amount of
enzyme/wt% for 3
pH
Temperature/°C
Time/h
Yield of 40d/%
1
0.05
0.05
0.05
0.05
0.05
0.05
0.15
0.05
0.05
0.05
0.05
0.05
0.05
0.10
0.01
0.05
0.50
0.05
0.15
0.15
0.15
0.15
0.15
0.15
10
10
10
10
10
10
10
11.0
1
7.0
30
30
30
30
30
30
192
30
30
30
30
20
40
30
30
30
5.5b
7.0b
18b
56b
78b
24
32
38
41
58
18
0
2
8.0
9.0
3
4
9.5
5
10.0
10.5
30
6
168b
b
70.05
8
0.15
0.15
0.15
0.15
0.15
0.15
0.30
0.03
0.05
20
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
10.0
30
78c
78c
78c
72b
78c
60b
78c
60b
31
42
56
24
33
42
52
37
24
44
35
9
10
20
30
20
20
20
20
20
10.0
20
10
11
12
13
14
15
16
170.05
18
78c
c
8
7
1.50
10.0
30
78c
a In a carbonate buffer (50mM, 100lL).
b The time for complete consumption of 3.
c The reaction was terminated at the indicated time.
d Determined by HPLC based on 3.
results imply that 3 reacted with water rather than with 6
at lower concentration, and a higher concentration of 6
inhibited reaction, resulting in lower yields.
Enzymatic glycosidation of 3 with 5 was carried out
under the optimal conditions of the foregoing reaction
(Scheme 9), and was complete after 168h. The isolated
yield of 41 was 38%. The glycosyl donor 3 was also re-
acted with 4 under catalysis by the enzyme under similar
conditions (Scheme 10). The reaction was slowed, giving
rise to 42 in 36% yields after 168h.
OH
O
OH
chitinase
buffer
HO
HO
NaO2C
O
HO
OCH3
+
3
4
O
NH
O
H3C
3
42
Scheme 10.
Thus, 3 reacted with all of the acceptors, affording the
corresponding glycosidated products in satisfactory
yields. Other compounds in the reaction mixtures were
the hydrolyzed compound 36 and the remaining
acceptor.
the glycosidated products, 37–42, were formed with
complete stereo-chemical control (b configuration) at
C-1 carbon of donors 1 and 3, and regio-selectivity at
4-OH of acceptors 4–6, during the reaction. This is the
first example of chitinase-catalyzed terminal functional-
ization of chitooligosaccharide derivatives by using
glycosyl donors having a carboxylate group. These
combinations of enzyme and the glycosyl donors should
permit synthesis of carboxylate end-functionalized car-
bohydrate derivatives, providing tools for synthesis of
neoglycoconjugates and for other modifications of car-
bohydrate-based materials.
3. Conclusion
This study shows that chitinase from Bacillus sp. catal-
yzes the glycosidation reactions of carboxylate-function-
alized sugar oxazolines serving as glycosyl donors. All
OH
OH
O
chitinase
HO
HO
NaO2C
O
O
+
3
5
HO
O
buffer
NH
H3C
O
2
41
Scheme 9.

H. Ochiai et al. / Carbohydrate Research 339 (2004) 2769–2788
2777
4. Experimental
4.1. General methods
NMR (CDCl3): d 8.00–6.52 (m, 12H, Ar), 5.54 (s, 1H,
CHPh of benzylidene), 5.25 (d, 1H, J1,2 8.54Hz, H-1),
4.77 (d, 1H, J 12.05Hz, CH2Ph), 4.63 (m, 1H, H-3),
4.45 (d, 1H, J 12.05Hz, CH2Ph), 4.41 (dd, 1H, J6a,6b
10.29, J5,6a 4.77Hz, H-6a), 4.26 (dd, 1H, J2,3 10.54Hz,
H-2), 3.87–3.80 (m, 4H, H-6b, OCH3), 3.68 (s, 3H,
OCH3), 3.66–3.58 (m, 2H, H-4, H-5), 2.44 (d, 1H,
J3,3-OH 3.51Hz, 3-OH); 13C NMR (CDCl3): d 168.30,
163.01 (CO of phthalimido), 160.20–113.49 (CAr),
101.79 (CHPh of benzylidene), 97.63 (C-1), 82.03 (C-4),
71.04 (CH2Ph), 68.61 (C-6), 68.35 (C-3), 66.02 (C-5),
56.59 (C-2), 55.25, 54.98 (OCH3); HRMS (FAB+) calcd
for C30H30NO9 [M+H]+ 548.1921, found 548.1931.
NMR spectra were recorded with a Bruker DPX400
spectrometer. For solutions in D2O, acetone served as
the reference 2.24 (1H) and 30.91ppm (13C). All assign-
ments were based on COSY, DEPT, and HMQC exper-
iments. High-resolution fast-atom-bombardment mass
(HRFAB/MS) spectra were recorded on a Jeol XPS
spectrometer using 2,4-dinitrobenzyl alcohol or dithio-
threitol–thioglycerol (1:1, v/v) as the matrix. Optical
rotations were determined with a Jasco P-1010 polari-
meter. Melting points were determined by a Yamato
MP-21 apparatus. High-performance liquid chromato-
graphy (HPLC) was performed on a Tosoh LC-8020
system with a Shodex Asahipak NH2P-50 4E column.
Size-exclusion chromatography (SEC) was carried out
on a Tosoh GPC-8020 system with a Shodex Ohpak
SB-802HQ column, eluting with 0.1M aqueous sodium
nitrate (flow rate: 0.5mL/min). MALDI-TOF/MS spec-
tra were recorded on a Jeol JMS-ELITE spectrometer
by using 2,5-dihydroxybenzoic acid as the matrix on
Nafion-coated plate in the negative ion mode.26
4.2.2. 4-Methoxybenzyl 2-deoxy-3-O-(4-methoxybenzyl)-
4,6-O-(4-methoxybenzylidene)-2-phthalimido-b-D-gluco-
pyranoside (9). To a solution of compound 8 (300mg,
0.55mmol) in dry DMF (1mL) was added NaH (60%
oil dispersion, 32mg, 0.80mmol). The reaction mixture
was stirred at room temperature for 15min followed
by the addition of 4-methoxybenzyl chloride (0.11mL,
0.82mmol). The mixture was stirred at room tempera-
ture for 1h, and then tetrabutylammonium iodide
(100mg, 0.27mmol) was added. After stirring overnight,
additional NaH (60% oil dispersion, 21mg, 0.53mmol)
and 4-methoxybenzyl chloride (73lL, 0.54mmol) was
added to the mixture. Methanol (0.5mL) was added
after 4h to terminate the reaction. The mixture was con-
centrated under diminished pressure, diluted with
EtOAc, and washed with satd aq NaHCO3 and brine.
The organic layer was dried (MgSO4), and concentrated.
The residue was purified by silica gel column chroma-
1,2-Dichloroethane was distilled from diphosphorus
˚
pentoxide and stored over activated 4A molecular sieves
(MS4A) under argon before use. Other chemicals were
of reagent grade and used without further purification.
Chitinase from Bacillus sp. (Lot No LDH7064, EC
3.2.1.14, 0.04unit/mg) was purchased from Wako Pure
Chemicals, Inc. (Tokyo, Japan), and used without fur-
ther purification.
tography (5:1–2:1 n-hexane–EtOAc) to afford
(185mg, 51%) as a white amorphous powder: Rf 0.47
9
4.2. Synthesis
25
D
(1:1 n-hexane–EtOAc); ½aꢀ +7.0 (c 1.0, CHCl3); 1H
4.2.1. 4-Methoxybenzyl 2-deoxy-4,6-O-(4-methoxybenzyl-
idene)-2-phthalimido-b-D-glucopyranoside (8). 4-Meth-
oxybenzyl 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-
D-glucopyranoside (7)25 (2.20g, 4.0mmol) was dissolved
in 4:1 MeOH–CH2Cl2 (25mL), and then NaOMe in
MeOH (28wt%, 0.1mL) was added dropwise. After stir-
ring for 1h, additional NaOMe in MeOH (28wt%,
0.1mL) was added. The mixture was stirred for 1h at
room temperature, neutralized with Dowex 50W-X4
(H+ form), filtered through cotton and the filtrate con-
centrated to dryness under diminished pressure. The res-
idue was dissolved in DMF (10mL) followed by the
addition of 4-methoxybenzaldehyde dimethyl acetal
(1.5mL, 8.7mmol) and ( )-camphor-10-sulfonic acid
(134mg, 0.58mmol). The reaction mixture was stirred
at room temperature for 3h, and then at 30°C under
diminished pressure for 1h. The mixture was concen-
trated, and the residue was purified by silica gel column
chromatography (5:1–1:1 n-hexane–EtOAc) to afford 8
(2.52g, 80%) as a white amorphous powder: Rf 0.39
NMR (CDCl3): d 7.79–6.33 (m, 16H, Ar), 5.58 (s, 1H,
CHPh of benzylidene), 5.16 (d, 1H, J1,2 8.53Hz, H-1),
4.71 (d, 1H, J 12.55Hz, CH2Ph), 4.67(d, 1H,
J
12.55Hz, CH2Ph), 4.42–4.34 (m, 4H, H-3, H-6a,
CH2Ph), 4.17(dd, 1H, J2,3 10.29Hz, H-2), 3.88–3.75
(m, 5H, H-4, H-6b, OCH3), 3.65–3.59 (m, 7H, H-5,
OCH3); 13C NMR (CDCl3): d 167.38 (CO of phthalim-
ido), 159.97–113.21 (CAr), 101.22 (CHPh of benzylid-
ene), 97.55 (C-1), 82.85 (C-4), 73.87 (C-3), 73.52
(CH2Ph), 70.94 (CH2Ph), 68.70 (C-6), 65.97 (C-5),
55.80 (C-2), 55.24–54.80 (OCH3); HRMS (FAB+) calcd
for C38H38NO10 [M+H]+ 668.2496, found 668.2495.
4.2.3. 4-Methoxybenzyl 2-deoxy-3,6-di-O-(4-methoxy-
benzyl)-2-phthalimido-b-D-glucopyranoside (10). Com-
pound 9 (400mg, 0.60mmol) was added to a solution
of sodium cyanoborohydride (197mg, 3.2mmol) and
˚
activated 3A molecular sieves (MS3A, 5.0g) in DMF
(35mL). The mixture was stirred at 0°C under argon,
and then trifluoroacetic acid (TFA, 0.47mL, 6.0mmol)
in DMF (1.8mL) was added dropwise. The mixture
25
1
(1:1 n-hexane–EtOAc); ½aꢀ À68.0 (c 1.0, CHCl3); H
D

2778
H. Ochiai et al. / Carbohydrate Research 339 (2004) 2769–2788
was stirred at room temperature for 24h under argon
followed by further addition of TFA (0.45mL) in
DMF (1.8mL) at 0°C. After stirring for 24h at room
temperature, the mixture was filtered through Celite,
poured into satd aq NaHCO3, and extracted with
CHCl3. The organic layer was washed with brine, dried
(MgSO4) and concentrated. The residue was subjected
to silica gel column chromatography (5:1–1:1 n-hexane–
76.39 (C-3), 74.72 (C-5), 74.00 (CH2Ph), 72.84 (CH2Ph),
0
71.82 (C-50), 71.00 (C-30), 70.71 (CH2Ph), 69.27(C-4 ),
0
68.32 (C-6), 61.96 (C-60), 56.06 (C-2), 55.67(C-2
,
OCH3), 55.42, 55.16 (OCH3), 20.64, 20.59, 20.42
(COCH3); HRMS (FAB+) calcd for C58H58N2O19Na
[M+Na]+ 1109.3532, found 1109.3542.
4.2.5. 4-Methoxybenzyl 2-acetamido-3,4,6-tri-O-acetyl-2-
deoxy-b-D-glucopyranosyl-(1!4)-2-acetamido-2-deoxy-
EtOAc), affording 10 (400mg, quant.) as a colorless syr-
27
D
up: Rf 0.32 (1:1 n-hexane–EtOAc); ½aꢀ À10.0 (c 1.0,
3,6-di-O-(4-methoxybenzyl)-b-D-glucopyranoside
(13).
1
CHCl3); H NMR (CDCl3): d 7.77–6.42 (m, 16H, Ar),
Compound 12 (2.00g, 1.8mmol) was dissolved in a
10:1 mixture (v/v) of EtOH and H2O (22mL), and
hydrazine monohydrate (0.5mL, 10mmol) was added.
The mixture was stirred at 90°C for 2h followed by
the further addition of hydrazine monohydrate
(0.5mL, 10mmol). After stirring for 3h at 90°C, the
mixture was evaporated under diminished pressure,
and Ac2O (10mL) was added to the residue suspended
in pyridine (30mL) under a dry atmosphere. The mix-
ture was stirred at room temperature for 3h. After the
reaction was complete, MeOH (10mL) was added to
quench the excess of reagent, and the mixture was con-
centrated under diminished pressure. The residue was
diluted with CHCl3, washed with 1M aq HCl, satd aq
NaHCO3, and brine. The organic layer was dried
(MgSO4) and concentrated. Silica gel column chroma-
tography (2:1–1:1 toluene–EtOAc, then 3:1–2:1
CHCl3–acetone) purified the residue, giving rise to 13
(1.47g, 88%) as a white solid: Rf 0.30 (3:1 CHCl3–ace-
5.12 (d, 1H, J1,2 8.03Hz, H-1), 4.69 (d, 1H, J 12.05Hz,
CH2Ph), 4.64–4.58 (m, 2H, CH2Ph), 4.53 (d, 1H, J
11.54Hz, CH2Ph), 4.43 (d, 1H, J 12.04Hz, CH2Ph),
4.39 (d, 1H, J 12.04Hz, CH2Ph), 4.21–4.10 (m, 2H, H-
2, H-3), 3.82–3.72 (m, 6H, H-4, H-6a, H-6b, OCH3),
3.68 (s, 3H, OCH3), 3.65–3.60 (m, 4H, H-5, OCH3),
2.94 (s, 1H, 4-OH); 13C NMR (CDCl3): d 168.01 (CO
of phthalimido), 159.35–113.38 (CAr), 97.11 (C-1),
78.04 (C-3), 74.59 (C-4), 73.79 (CH2Ph), 73.41 (CH2Ph),
73.31 (C-5), 70.61 (CH2Ph), 70.48 (C-6), 55.40 (C-2),
55.27, 55.01, 54.83 (OCH3); HRMS (FAB+) calcd for
C38H40NO10 [M+H]+ 670.2652, found 670.2647.
4.2.4. 4-Methoxybenzyl 3,4,6-tri-O-acetyl-2-deoxy-2-
phthalimido-b-D-glucopyranosyl-(1!4)-2-deoxy-3,6-di-
O-(4-methoxybenzyl)-2-phthalimido-b-D-glucopyranoside
(12). To a solution of 3,4,6-tri-O-acetyl-2-deoxy-2-
phthalimido-b-D-glucopyranosyl trichloroacetimidate
(11, 172mg, 0.30mmol) and compound 10 (107mg,
0.16mmol) in dry CH2Cl2 (2.1mL) over activated
MS4A (326mg) was added trimethylsilyl triflate (Me3-
SiOTf; 5lL, 28lmol) in dry CH2Cl2 (0.2mL). The reac-
tion mixture was stirred at À20°C for 1h under argon
and then filtered through Celite, and diluted with
CHCl3. The solution was washed with satd aq NaHCO3
and brine. The organic layer was dried (MgSO4) and
concentrated. The residual mixture was purified by silica
gel column chromatography (3:1–1:1 n-hexane–EtOAc)
25
1
tone); mp 204–205°C; ½aꢀ À95.0 (c 0.1, CHCl3); H
D
NMR (CDCl3): d 7.25–6.79 (m, 12H, Ar), 6.10 (d, 1H,
J2,NH 9.03Hz, NH), 5.36 (d, 1H, J2 ,N H 9.54Hz, N0H),
0
0
5.06 (t, 1H, J4 ,5 9.54Hz, H-40), 4.92 (t, 1H, J3 ,4
0
0
0
0
9.54Hz, H-30), 4.76 (d, 1H, J 11.55Hz, CH2Ph), 4.63–
4.61 (m, 3H, H-1, CH2Ph), 4.55 (d, 1H, J 11.55Hz,
CH2Ph), 4.45 (d, 1H, J 11.54Hz, CH2Ph), 4.38 (d, 1H,
J1 ,2 8.54Hz, H-10), 4.37(d, 1H, J 11.55Hz, CH2Ph),
0
0
4.24 (dd, 1H, J6a ,6b 12.55, J5 ,6a 4.51Hz, H-6a0),
4.08–3.96 (m, 3H, H-2, H-20, H-6b0), 3.93 (t, 1H, J3.4
5.52Hz, H-3), 3.82–3.72 (m, 3H, H-4, H-6a, H-6b),
3.82, 3.80, 3.78 (3s, 9H, OCH3), 3.60 (m, 1H, H-5),
0
0
0
0
to provide 12 (159mg, 91%) as a white amorphous pow-
25
D
der: Rf 0.27(1:1 n-hexane–EtOAc); ½aꢀ À8.0 (c 1.0,
1
0
0
CHCl3); H NMR (CDCl3): d 7.93–6.30 (m, 20H, Ar),
3.45 (ddd, 1H, J5 ,6b 2.01Hz, H-50), 2.03, 2.03, 2.02,
1.93, 1.78 (5s, 15H, COCH3); 13C NMR (CDCl3): d
171.28, 170.69, 170.57, 170.190, 169.27 (COCH3),
159.53–113.58 (CAr), 100.17(C-1 ), 99.45 (C-1), 77.20
(C-4), 75.21 (C-3), 74.36 (C-5), 73.32 (CH2Ph), 72.56
(C-30), 72.26 (CH2Ph), 71.67 (C-50), 70.01 (CH2Ph),
69.24 (C-6), 68.09 (C-40), 61.76 (C-60), 55.27, 55.23,
55.20 (OCH3), 54.14 (C-2), 51.66 (C-20) 23.22, 23.19,
20.71, 20.65, 20.59 (COCH3); HRMS (FAB+) calcd for
C46H59N2O17 [M+H]+ 911.3814, found 911.3818.
5.80 (t, 1H, J3 ,4 9.79Hz, H-30), 5.48 (d, 1H, J1 ,2
0
0
0
0
8.03Hz, H-10), 5.13 (t, 1H, J4 ,5 9.54Hz, H-40), 4.91
(d, 1H, J1,2 8.03Hz, H-1), 4.69 (d, 1H, J 12.54Hz,
CH2Ph), 4.61 (d, 1H, J 12.04Hz, CH2Ph), 4.50 (s, 2H,
CH2Ph), 4.41 (d, 1H, J 12.55Hz, CH2Ph), 4.34–4.19
(m, 3H, H-4, H-20, H-6a0), 4.29 (d, 1H, J 12.55Hz,
CH2Ph), 4.10–4.07(m, 2H, H-2, H-3), 3.97(d, 1H,
0
0
J6a ,6b 10.04Hz, H-6b0), 3.84 (s, 3H, OCH3), 3.65 (s,
3H, OCH3), 3.57(s, 3H, OC H3), 3.57(m, 1H, H-6a),
3.47(dd, 1H, J6a,6b 11.29, J5,6b 3.76Hz, H-6b), 3.42
(m, 1H, H-50), 3.30 (m, 1H, H-5), 2.02, 2.00, 1.85 (3s,
9H, COCH3); 13C NMR (CDCl3): d 170.68, 170.08,
169.52 (COCH3), 167.46 (CO of phthalimido), 159.09–
113.12 (CAr), 96.74 (C-1), 96.62 (C-10), 76.48 (C-4),
0
0
4.2.6. 4-Methoxybenzyl 2-acetamido-2-deoxy-6-O-(4-
methoxytrityl)-b-D-glucopyranosyl-(1!4)-2-acetamido-2-
deoxy-3,6-di-O-(4-methoxybenzyl)-b-D-glucopyranoside
(14). Compound 13 (520mg, 0.57mmol) was dissolved

H. Ochiai et al. / Carbohydrate Research 339 (2004) 2769–2788
2779
in 7:15 (v/v) CHCl3–MeOH (22mL), and NaOMe in
6-di-O-(4-methoxybenzyl)-b-D-glucopyranoside as
yellowish syrup.
a
MeOH (28wt%, 0.2mL) was added dropwise. The reac-
tion mixture was stirred at room temperature for 1h.
The mixture was neutralized with Dowex 50W-X4 (H+
form), filtered through cotton, and evaporated. To the
residue dissolved in pyridine (10mL) was added 4-meth-
oxytrityl chloride (350mg, 1.1mmol), and the reaction
mixture was stirred at room temperature for 7h. Meth-
anol (5mL) was then added to quench the excess re-
agent. The mixture was then concentrated, poured into
satd aq NaHCO3 and extracted with CHCl3. The
organic layer was washed with brine, dried (MgSO4)
and concentrated. The residue was purified by silica
gel column chromatography (10:1 EtOAc–MeOH) to
To the crude product was added 70% aq AcOH
(40mL), and the mixture was stirred at 50°C for 4h.
The reaction mixture was then poured into satd aq
NaHCO3 and extracted with CHCl3. The extract was
washed with satd aq NaHCO3, brine, dried (MgSO4)
and concentrated. The residue was purified by silica
gel column chromatography (1:1 CHCl3–EtOAc, then
100:1–30:1 CHCl3–MeOH) to afford 15 (2.96g, 71%
from 14) as a white solid: Rf 0.44 (50:3 CHCl3–MeOH);
26
D
1
mp 208-209°C; ½aꢀ À17.3 (c 1.0, CHCl3); H NMR
0
0
(CDCl3): d 7.26–6.79 (m, 20H, Ar), 6.14 (d, 1H, J2 ,N H
8.53Hz, N0H), 4.77–4.69 (m, 5H, NH, CH2Ph), 4.63
0
0
give 14 (579mg, 96%) as a white amorphous powder:
(d, 1H, J1 ,2 6.03Hz, H-10), 4.59–4.51 (m, 4H, CH2Ph),
4.43 (d, 1H, J 11.54Hz, CH2Ph), 4.34 (d, 1H, J 11.55Hz,
CH2Ph), 4.23 (d, 1H, J1,2 8.03Hz, H-1), 3.94 (m, 1H, H-
26
D
Rf 0.43 (8:1 CHCl3–MeOH); ½aꢀ À45.0 (c 1.0, CHCl3);
1H NMR (CD3OD): d 7.45–6.62 (m, 26H, Ar), 4.78 (d,
1H, J 11.54Hz, CH2Ph), 4.74 (d, 1H, J 11.55Hz,
20), 3.88–3.67(m, 6H, H-2, H-6a, H-6b, H-3 , H-50, H-
6a0), 3.81, 3.79, 3.78, 3.76 (4s, 15H, OCH3), 3.57(m,
1H, H-40), 3.54–3.47(m, 2H, H-4, H-6b ), 3.35 (t, 1H,
J3,4 9.54Hz, H-3), 3.19 (m, 1H, H-5), 2.00 (t, 1H,
J6 ,6 -OH 6.78Hz, 60-OH), 1.93, 1.71 (2s, 6H, COCH3);
13C NMR (CDCl3):
159.44–113.61 (CAr), 99.98 (C-10), 99.29 (C-1), 80.86
(C-3), 78.03 (C-4), 77.20 (C-30), 75.07 (C-5), 74.82 (C-
50), 74.54 (CH2Ph), 74.38 (C-40), 74.10, 73.19, 72.39,
0
CH2Ph), 4.65 (d, 1H, J1 ,2 8.53Hz, H-10), 4.60 (d, 1H,
J 12.05Hz, CH2Ph), 4.58 (d, 1H, J 11.04Hz, CH2Ph),
4.51–4.47(m, 3H, H-1, C H2Ph), 4.27(t, 1H, J4,5
8.29Hz, H-4), 3.95 (t, 1H, J2,3 8.54Hz, H-2), 3.82 (m,
2H, H-6a, H-6b), 3.75 (s, 3H, OCH3), 3.70 (m, 1H,
H-20), 3.70, 3.68, 3.65 (3s, 9H, OCH3), 3.60 (t, 1H, J3,4
8.79Hz, H-3), 3.51–3.39 (m, 3H, H-5, H-30, H-40), 3.35
(d, 1H, J 7.53Hz, H-6a0), 3.21 (m, 1H, H-50), 3.14
(m, 1H, H-6b0), 1.97, 1.81 (2s, 6H, COCH3); 13C
NMR (CD3OD): d 174.22, 173.43 (COCH3), 161.19–
114.46 (CAr), 101.89 (C-1), 101.56 (C-10), 87.73
(C(Ph)3), 81.45 (C-3), 77.29 (C-50), 76.94 (C-5),
76.41 (C-30), 75.94 (C-4), 74.59 (CH2Ph), 74.44
(CH2Ph), 72.82 (C-40), 71.72 (CH2Ph), 70.26 (C-6),
64.33 (C-60), 58.68 (C-20), 56.10, 56.06, 56.04 (CH3O),
55.99 (C-2), 23.55, 23.43 (COCH3); HRMS (FAB+)
calcd for C60H69N2O15 [M+H]+ 1057.4698, found
1057.4664.
0
0
0
0
0
d 170.31, 170.25 (COCH3),
0
70.01 (CH2Ph), 69.17(C-6), 61.84 (C-6 ), 55.28, 55.26,
55.24, 55.22, 55.20 (OCH3), 55.12 (C-2), 52.06 (C-20),
23.45, 23.31 (COCH3); HRMS (FAB+) calcd for
C56H69N2O16 [M+H]+ 1025.4647, found 1025.4657.
4.2.8. 4-Methoxybenzyl 2-acetamido-6-O-allyl-2-deoxy-
3,4-di-O-(4-methoxybenzyl)-b-D-glucopyranosyl-(1!4)-
2-acetamido-2-deoxy-3,6-di-O-(4-methoxybenzyl)-b-D-
glucopyranoside (16). Compound 15 (2.96g, 2.9mmol)
was dissolved in dry DMF (20mL), and powdered
NaOH (173mg, 4.33mmol) was added under a dry
atmosphere. The mixture was stirred at room tempera-
ture and after 20min, allyl bromide (0.37mL, 4.3mmol)
was added, followed by powdered NaOH (58mg,
1.5mmol) after 12h during the reaction for 17h. Meth-
anol (10mL) was added to quench the excess of reagent
and the mixture was concentrated, diluted with CHCl3
and washed with satd aq NaHCO3, and brine. The or-
ganic layer was dried (MgSO4) and concentrated. Silica
gel column chromatography (3:1 CHCl3–EtOAc, then
3:1 CHCl3–acetone) of the residue afforded 16 (2.82g,
92%) as a white solid: Rf 0.35 (3:1 CHCl3–acetone);
4.2.7. 4-Methoxybenzyl 2-acetamido-2-deoxy-3,4-di-O-
(4-methoxybenzyl)-b-D-glucopyranosyl-(1!4)-2-acetam-
ido-2-deoxy-3,6-di-O-(4-methoxybenzyl)-b-D-glucopyrano-
side (15). To a solution of compound 14 (4.31g,
4.1mmol) in dry DMF (20mL) was added powdered
NaOH (490mg, 12mmol), then the mixture was stirred
at room temperature. After 20min, 4-methoxybenzyl
chloride (1.7mL, 12mmol) was added. To the mixture
was added powdered NaOH (160mg, 4.0mmol) twice
at 4.5 and 16.5h during the reaction for 20.5h. Metha-
nol (10mL) was added to quench the excess of reagent,
and the reaction mixture was concentrated, poured into
satd aq NaHCO3, extracted with CHCl3, and washed
with brine. The dried (MgSO4) extract was concentrated
and the residue roughly purified by silica gel column
chromatography (2:1–1:2 toluene–EtOAc, then EtOAc)
to provide crude 4-methoxybenzyl 2-acetamido-2-
deoxy-3,4-di-O-(4-methoxybenzyl)-6-O-(4-methoxytri-
tyl)-b-D-glucopyranosyl-(1!4)-2-acetamido-2-deoxy-3,
25
1
mp 174–175°C; ½aꢀ +42.0 (c 1.0, CHCl3); H NMR
D
0
0
(CDCl3): d 7.24–6.76 (m, 20H, Ar), 6.49 (d, 1H, J2 ,N H
9.54Hz, N0H), 5.87(m, 1H, C H@CH2), 5.27(dd, 1H,
J 17.32, 1.76Hz, CH@CH2), 5.16 (dd, 1H, J 10.29,
1.26Hz, CH@CH2), 4.78 (d, 1H, J2,NH 9.03Hz, NH),
4.75 (m, 3H, CH2Ph), 4.65 (d, 1H, J 11.05Hz, CH2Ph),
0
0
4.59 (d, 1H, J 10.54Hz, CH2Ph), 4.58 (d, 1H, J1 ,2
4.52Hz, H-10), 4.53 (d, 1H, J 11.54Hz, CH2Ph), 4.52

2780
H. Ochiai et al. / Carbohydrate Research 339 (2004) 2769–2788
(d, 1H, J 11.54Hz, CH2Ph), 4.42 (d, 2H, J 11.04Hz,
CH2Ph), 4.34 (d, 1H, J 12.05Hz, CH2Ph), 4.21–4.18
(m, 2H, H-1, H-20), 4.01–3.99 (m, 2H, CH2CH@CH2),
3.91 (t, 1H, J 4.02Hz, H-40), 3.82–3.61 (m, 8H, H-2,
H-4, H-6a, H-6b, H-30, H-50, H-6a0, H-6b0), 3.81, 3.79,
3.78, 3.76 (4s, 15H, OCH3), 3.38 (t, 1H, J3,4 9.54Hz,
H-3), 3.28 (m, 1H, H-5), 1.97, 1.69 (2s, 6H, COCH3);
170.13, 169.40, 168.92 (COCH3), 133.91 (CH@CH2),
117.74 (CH@CH2), 101.55 (C-10), 90.44 (C-1), 75.67
(C-4), 73.01 (C-50), 72.69 (C-30), 72.33 (CH2CH@CH2),
70.68 (C-5), 70.57 (C-3), 69.11 (C-40), 68.76 (C-60), 61.49
(C-6), 54.51 (C-20), 51.10 (C-2), 23.17, 23.02, 20.99,
20.96, 20.71, 20.68, 20.62 (COCH3); HRMS (FAB+)
calcd for C29H43N2O16 [M+H]+ 675.2613, found
675.2631.
13C NMR (CDCl3):
d 170.55, 170.19 (COCH3),
159.34–158.84 (CAr), 134.53 (CH@CH2), 130.61–129.02
(CAr), 117.04 (CH@CH2), 113.90–113.47(C Ar), 99.96
(C-1), 99.49 (C-10), 80.40 (C-3), 78.02 (C-4), 76.41 (C-
30), 74.90 (C-5), 74.49 (C-50), 74.44, 73.78 (CH2Ph),
73.53 (C-40), 73.07 (CH2Ph), 72.36 (CH2CH@CH2),
71.47 (CH2Ph), 69.79 (C-6), 69.74 (CH2Ph), 68.54 (C-
60), 52.22, 55.16, 55.12 (OCH3), 54.94 (C-2), 49.93 (C-
20), 23.40, 23.12 (COCH3); HRMS (FAB+) calcd for
C59H73N2O16 [M+H]+ 1065.4960, found 1065.4960.
4.2.10. 2-Acetamido-3,4-di-O-acetyl-2-deoxy-6-O-meth-
oxycarbonylmethyl-b-D-glucopyranosyl-(1!4)-2-acetam-
ido-1,3,6-tri-O-acetyl-2-deoxy-a-D-glucopyranose (18).
A solution of compound 17 (119mg, 0.18mmol) in 4:1
(v/v) CH2Cl2–MeOH (25mL) was cooled to À78°C,
purged with oxygen for 15min, and treated with O3 by
bubbling for 30min. The O3 was evacuated with oxygen
for 10min, and then triphenylphosphine (120mg,
0.46mmol) was added. The mixture was stirred at room
temperature overnight, evaporated, and the residue was
subjected to silica gel column chromatography (1:3 tolu-
ene–EtOAc, then 2:1–1:1 CHCl3–acetone) to provide
crude 2-acetamido-3,4-di-O-acetyl-2-deoxy-6-O-formyl-
methyl-b-D-glucopyranosyl-(1!4)-2-acetamido-1,3,6-
tri-O-acetyl-2-deoxy-a-D-glucopyranose as a white solid.
The crude product was dissolved in 2:12:3 (v/v/v)
CH2Cl2–tert-BuOH–H2O (17mL) followed by the addi-
tion of NaH2PO4 (31mg, 0.26mmol), NaClO2 (58mg,
0.64mmol) and 2-methyl-2-butene (83lL, 0.78mmol).
The mixture was stirred at room temperature for 2h,
and 1M aq HCl was added to adjust the pH to 1.0.
The mixture was diluted with brine and extracted with
CHCl3. The organic layer was dried (MgSO4) and evap-
orated. The residue was dissolved in 10:3 (v/v) MeOH–
CH2Cl2 (13mL) followed by the addition of Dowex
50W-X4 (H+ form) (100mg). The mixture was stirred
at 40°C for 4.5h, then filtered and evaporated. The resi-
due was dissolved in pyridine (10mL) and Ac2O (5mL)
added. After stirring for 5h, MeOH (5mL) was added to
terminate the reaction. The mixture was then concen-
trated, diluted with CHCl3, washed with 1M aq HCl,
satd aq NaHCO3, and brine. The organic layer was
dried (MgSO4) and evaporated. The residue was purified
by silica gel column chromatography (2:1 toluene–
EtOAc, then 1:1 CHCl3–acetone) to afford 18 (92mg,
4.2.9. 2-Acetamido-3,4-di-O-acetyl-6-O-allyl-2-deoxy-b-
D-glucopyranosyl-(1!4)-2-acetamido-3,6-di-O-acetyl-2-
deoxy-a-D-glucopyranosyl acetate (17). Compound 16
(300mg, 0.28mmol) was suspended in 18:1 (v/v)
CH2Cl2–H2O (19mL) and then 2,3-dichloro-5,6-dicy-
ano-1,4-benzoquinone (DDQ; 350mg, 1.5mmol) was
added. The mixture was stirred at room temperature
overnight followed by evaporation. The residue was sus-
pended in pyridine (10mL) at 0°C, and Ac2O (5mL)
was added. After stirring at 0°C for 5h, MeOH
(5mL) was added to quench the excess of reagent. The
mixture was concentrated, diluted with CHCl3, and
washed with 1M aq HCl, satd aq NaHCO3 and brine.
The organic layer was dried (MgSO4)and evaporated,
and the residue was re-suspended in 18:1 CH2Cl2–H2O
(19mL) and DDQ (190mg, 0.837mmol) was added.
The mixture was stirred at room temperature overnight,
and then evaporated. The residue was suspended in pyri-
dine (10mL) and Ac2O (5mL) was added at 0°C. After
stirring at 0°C for 5h, MeOH (5mL) was added to
terminate the reaction. The mixture was concentrated,
diluted with CHCl3, and washed with 1M aq HCl, satd
aq NaHCO3, and brine. The organic layer was dried
(MgSO4), evaporated, and the residue purified by silica
gel column chromatography (2:1 toluene–EtOAc, then
2:1 CHCl3–acetone) to give 17 (171mg, 90%) as a white
solid: Rf 0.19 (2:1 CHCl3–acetone); mp 279–280°C
74%) as a white solid: Rf 0.24 (1:1 CHCl3–acetone);
24
24
D
1
(dec.); ½aꢀ +28.0 (c 1.0, CHCl3); H NMR (CDCl3): d
mp 249–250°C (dec.); ½aꢀ +26.0 (c 1.0, CHCl3); 1H
D
0
0
6.10 (d, 1H, J1,2 3.51Hz, H-1), 5.87(d, 1H, J2 ,N H
9.53Hz, N0H), 5.84 (m, 1H, CH@CH2), 5.61 (d, 1H,
J2,NH 9.04Hz, NH), 5.28–5.18 (m, 3H, H-3, CH@CH2),
NMR (CDCl3): d 6.10 (d, 1H, J1,2 3.51Hz, H-1), 5.97
(d, 1H, J2 ,N H 9.54Hz, N0H), 5.69 (d, 1H, J2,NH
9.03Hz, NH), 5.24 (dd, 1H, J2,3 10.54, J3,4 9.04Hz, H-
0
0
5.12 (t, 1H, J3 ,4 9.79Hz, H-30), 5.05 (t, 1H, J4 ,5
3), 5.14 (t, 1H, J3 ,4 9.03Hz, H-30), 5.37(t, 1H, J4 ,5
0
0
0
0
0
0
0
0
9.54Hz, H-40), 4.47–4.44 (m, 2H, H-6a, H-10), 4.36 (m,
1H, H-2), 4.18 (d, 1H, J6a,6b 12.05Hz, H-6b), 3.95-3.88
(m, 4H, H-5, H-20, CH2CH@CH2), 3.76 (t, 1H, J4,5
9.53Hz, H-4), 3.56–3.46 (m, 3H, H-50, H-6a0, H-6b0),
2.19, 2.15, 2.09, 2.02, 2.01, 1.96, 1.93 (7s, 21H, COCH3);
13C NMR (CDCl3): d 171.68, 171.25, 170.89, 170.33,
9.04Hz, H-40), 4.50 (d, 1H, J1 ,2 8.54Hz, H-10), 4.43
(dd, 1H, J6a,6b 12.05, J5,6a 3.51Hz, H-6a), 4.35 (m, 1H,
H-2), 4.20 (m, 1H, H-6b), 4.08 (d, 1H, J 1.01Hz,
OCH2), 3.96–3.89 (m, 2H, H-5, H-20), 3.77 (m, 1H, H-
4), 3.74 (s, 3H, OCH3), 3.67–3.61 (m, 3H, H-50, H-6a0,
H-6b0), 2.19, 2.16, 2.10, 2.05, 2.02, 1.96, 1.93 (7s, 21H,
0
0

H. Ochiai et al. / Carbohydrate Research 339 (2004) 2769–2788
2781
COCH3); 13C NMR (CDCl3): d 171.59, 171.22, 170.83,
170.39, 170.28, 170.19, 169.58, 168.93 (COOCH3,
COCH3), 101.30 (C-10), 90.38 (C-1), 75.45 (C-4), 73.12
(C-50), 72.64 (C-30), 70.71 (C-3), 70.63 (C-5), 70.21 (C-
60), 68.78 (C-40), 68.39 (OCH2), 61.59 (C-6), 54.47(C-
20), 51.75 (OCH3), 51.13 (C-2), 23.11, 22.96, 20.93,
20.91, 20.78, 20.65, 20.59 (COCH3); HRMS (FAB+)
calcd for C29H43O18N2 [M+H]+ = 707.2511, found
707.2541.
4.41 (s, 1H, H-3), 4.21 (m, 1H, H-2), 4.05 (m, 1H,
OCH2), 4.00 (m, 1H, OCH2), 3.91 (d, 1H, J 11.54Hz,
H-6a0), 3.79 (m, 1H, H-6b0), 3.74–3.53 (m, 7H, H-4,
H-6a, H-6b, H-20, H-30, H-40, H-50), 3.30 (m, 1H, H-
5), 2.07, 2.05 (2s, 6H, CH3C of oxazoline, COCH3);
13C NMR (D2O): d 178.79 (COONa), 175.04 (COCH3),
168.94 (OC@N), 103.86 (C-10), 100.39 (C-1), 79.48
(C-4), 75.50 (C-50), 73.90 (C-30), 71.63 (C-5), 70.77
(OCH2), 70.47 (C-40), 70.23 (C-60), 69.46 (C-3), 65.80
(C-2), 62.42 (C-6), 56.41 (C-20), 22.70 (COCH3), 13.51
(CH3C of oxazoline); HRMS (FAB+) calcd for
C18H27O12N2Na2 [M+Na]+ 509.1359, found 509.1364.
4.2.11. 2-Methyl-[2-acetamido-3,4-di-O-acetyl-2-deoxy-
6-O-methoxycarbonylmethyl-b-D-glucopyranosyl-(1!4)-
3,6-di-O-acetyl-1,2-di-deoxy-a-D-glucopyrano]-[2,1-d]-2-
oxazoline (19). To a solution of compound 18 (90mg,
0.13mmol) in dry 1,2-dichloroethane (0.4mL) was
added Me3SiOTf (50lL, 0.28mmol) in dry MeCN
(0.2mL) under argon. The reaction mixture was stirred
at 40°C for 12h and cooled down to 0°C. Triethylamine
(0.1mL) was then added at 0°C to terminate the reac-
tion. The mixture was concentrated, and the residue
was purified by silica gel column chromatography (2:1
CHCl3–EtOAc) and size-exclusion chromatography on
a Sephadex LH-20 column using MeOH as eluent to af-
4.2.13.
4-Methoxybenzyl
2-deoxy-3-O-(4-methoxy-
benzyl)-2-phthalimido-b-D-glucopyranoside (20). Com-
pound 9 (185mg, 0.28mmol) was dissolved in 70% aq
AcOH (20mL), and the mixture stirred at 70°C for
1h, and then concentrated, diluted with CHCl3, washed
with satd aq NaHCO3, and brine. The organic layer was
dried (MgSO4) and evaporated. The residue was purified
by silica gel column chromatography (2:1–1:5 n-hexane–
EtOAc) to afford 20 (127mg, 83%) as a white amor-
25
phous powder: Rf 0.36 (1:5 n-hexane–EtOAc); ½aꢀ
D
ford 19 (72mg, 87%) as a white amorphous powder: Rf
+1.9 (c 1.0, CHCl3); 1H NMR (CDCl3): d 7.80–6.50
(m, 16H, Ar), 5.16 (d, 1H, J1,2 8.54Hz, H-1), 4.69 (d,
1H, J 12.04Hz, CH2Ph), 4.54 (d, 1H, J 12.05Hz,
CH2Ph), 4.43 (d, 1H, J 12.05Hz, CH2Ph), 4.43 (d, 1H,
J 12.05Hz, CH2Ph), 4.23 (dd, 1H, J3,4 8.54Hz, H-3),
4.13 (dd, 1H, J2,3 10.79Hz, H-2), 3.96 (m, 1H, H-6a),
3.85 (m, 1H, H-6b), 3.74 (m, 1H, H-4), 3.69 (s, 3H,
OCH3), 3.63 (s, 3H, OCH3), 3.52 (m, 1H, H-5), 2.40
(d, 1H, J4,4-OH 3.48Hz, 4-OH), 2.06 (t, 1H, J6,6ÀOH
6.78Hz, 6-OH); 13C NMR (CDCl3): d 167.62 (CO of
phthalimido) 158.81–113.16 (CAr), 97.11 (C-1), 78.23
(C-3), 75.35 (C-5), 73.92 (CH2Ph), 71.76 (C-6), 70.78
(CH2Ph), 61.81 (C-6), 55.55 (C-2), 54.85 (OCH3),
54.62 (OCH3); HRMS (FAB+) calcd for C30H31NNaO9
[M+Na]+ 572.1897, found 572.1871.
24
D
0.49 (10:1 CHCl3–MeOH); ½aꢀ +4.0 (c 1.0, CHCl3); 1H
NMR (CDCl3): d 5.91 (d, 1H, J2,NH 9.04Hz, NH), 5.90
(d, 1H, J1,2 7.53Hz, H-1), 5.63 (d, 1H, J2,3 1.51Hz, H-3),
5.18 (dd, 1H, J2,3 10.54, J3,4 9.53Hz, H-30), 5.02 (t, 1H,
H-40), 4.71 (d, 1H, J1 ,2 8.53Hz, H-10), 4.35 (dd, 1H,
J6a,6b 12.05, J5,6a 4.52Hz, H-6a), 4.18–4.08 (m, 4H, H-
2, H-6b, OCH2), 3.96 (m, 1H, H-20), 3.74 (s, 3H,
OCH3), 3.73 (m, 1H, H-50), 3.66–3.60 (m, 3H, H-4, H-
6a0, H-6b0), 3.48 (ddd, 1H, J4,5 9.87, J5,6b 1.89Hz, H-
5), 2.14 (s, 3H, COCH3), 2.09 (d, 3H, J 2.01Hz, CH3C
of oxazoline), 2.08, 2.03, 2.02, 1.94 (4s, 12H, COCH3);
13C NMR (CDCl3): d 171.15, 170.92, 170.56, 170.37,
169.61, 169.30 (COOCH3, COCH3), 166.79 (OC@N),
102.00 (C-10), 99.11 (C-1), 77.25 (C-4), 73.28 (C-50),
72.88 (C-30), 70.58 (C-3, C-60), 69.10 (C-40), 68.74
(OCH2), 67.62 (C-5), 65.03 (C-2), 62.96 (C-6), 54.29
(C-20), 51.74 (OCH3), 23.15, 22.62, 21.00, 20.87, 20.68
(COCH3), 13.94 (CH3 of oxazoline); HRMS (FAB+)
calcd for C27H39O16N2 [M+H]+ 647.2300, found
647.2297.
0
0
4.2.14. 4-Methoxybenzyl 6-O-(tert-butyldimethylsilyl)-2-
deoxy-3-O-(4-methoxybenzyl)-2-phthalimido-b-D-gluco-
pyranoside (21). Compound 20 (7.43g, 13.5mmol) was
dissolved in pyridine (50mL), and tert-butyldimethyl-
chlorosilane (4.07g, 27.0mmol) was added. The mixture
stirred at room temperature for 1h, then poured into
satd aq NaHCO3, extracted with CHCl3, and washed
with brine. The organic layer was dried (MgSO4) and
evaporated. The residue was chromatographed on silica
gel (5:1–2:1 n-hexane–EtOAc) to give 21 (8.72g, 97%) as
a white amorphous powder: Rf 0.55 (1:1 n-hexane–
4.2.12.
deoxy-b-D-glucopyranosyl-(1!4)-1,2-di-deoxy-a-D-gluco-
pyrano]-[2,1-d]-2-oxazoline sodium salt (1). To
2-Methyl-[2-acetamido-6-O-carboxymethyl-2-
a
MeOH solution (10mL) of compound 19 (40mg,
62lmol) was added NaOMe in MeOH (28wt%,
20lL). After stirring at room temperature for 2h, the
mixture was evaporated and the residue dissolved in a
carbonate buffer (25mM, pH12.0, 1238lL), and stirred
at room temperature for 2h. The mixture was then
28
D
1
EtOAc); ½aꢀ +1.9 (c 1.0, CHCl3); H NMR (CDCl3):
d 7.77–6.39 (m, 12H, Ar), 5.13 (d, 1H, J1,2 8.54Hz, H-
1), 4.70 (d, 1H, J 12.04Hz, CH2Ph), 4.68 (d, 1H, J
12.05Hz, CH2Ph), 4.45 (d, 1H, J 12.05Hz, CH2Ph),
4.38 (d, 1H, J 12.05Hz, CH2Ph), 4.20 (dd, 1H, J3,4
8.53Hz, H-3), 4.10 (dd, 1H, J2,3 10.54Hz, H-2), 4.01
1
lyophilized to give 1: H NMR (D2O): d 6.09 (d, 1H,
J1,2 7.03Hz, H-1), 4.56 (d, 1H, J1 ,2 8.03Hz, H-10),
0
0

2782
H. Ochiai et al. / Carbohydrate Research 339 (2004) 2769–2788
(dd, 1H, J6a,6b 10.29, J5,6a 4.77Hz, H-6a), 3.90 (dd, 1H,
J5,6b 6.52Hz, H-6b), 3.81 (t, 1H, J4,5 8.79Hz, H-4), 3.67,
3.59 (2s, 6H, OCH3), 3.41 (s, 1H, 4-OH), 0.94 (s, 9H,
C(CH3)3), 0.15 (s, 6H, SiCH3); 13C NMR (CDCl3): d
167.72, 167.56 (CO of phthalimido), 158.92–113.25
(CAr), 96.86 (C-1), 77.97 (C-3), 75.66 (C-4), 73.77
(CH2Ph), 73.49 (C-5), 70.44 (CH2Ph), 65.19 (C-6),
55.28 (C-2), 54.98 (OCH3), 54.76 (OCH3), 25.82
(C(CH3)3), 18.21 (C(CH3)3), À5.51 (SiCH3); HRMS
(FAB+) calcd for C36H46NO9Si [M+H]+ 664.2942,
found 664.2940.
acetamido group by treatment with hydrazine monohy-
drate (1.00mL, 20.6mmol) in 10:1 (v/v) EtOH–H2O
(55mL) followed by acetylation with Ac2O (10mL) in
pyridine (30mL) as described in the preparation of com-
pound 13. The mixture was subjected to silica gel col-
umn chromatography (2:1 toluene–EtOAc, then
EtOAc) to isolate 23 (2.78g, 78%) as a white amorphous
26
D
powder: Rf 0.48 (EtOAc); ½aꢀ À73.3 (c 1.0, CHCl3); 1H
NMR (CDCl3): d 7.27–6.75 (m, 8H, Ar), 6.44 (d, 1H,
J2,NH 9.54Hz, NH), 5.65 (d, 1H, J2 ,N H 9.04Hz, N0H),
0
0
5.12 (t, 1H, J4 ,5 9.54Hz, H-40), 5.02 (t, 1H, J3 ,4
0
0
0
0
10.04Hz, H-30), 4.73 (d, 1H, J 11.55Hz, CH2Ph), 4.68
(d, 1H, J 11.05Hz, CH2Ph), 4.51 (d, 1H, J1,2 4.51Hz,
H-1), 4.52 (d, 1H, J 11.04Hz, CH2Ph), 4.43 (d, 1H, J
4.2.15. 4-Methoxybenzyl 3,4,6-tri-O-acetyl-2-deoxy-2-
phthalimido-b-D-glucopyranosyl-(1!4)-6-O-tert-butyldi-
methylsilyl-2-deoxy-3-O-(4-methoxybenzyl)-2-phthalim-
ido-b-D-glucopyranoside (22). Compounds 11 (5.30g,
9.1mmol) and 21 (3.03g, 4.5mmol) were dissolved
together in dry CH2Cl2 (83mL) with activated MS4A
(10.0g). The mixture was cooled to À20°C and then
BF3 etherate (0.23mL, 1.8mmol) in dry CH2Cl2 (1mL)
was added. After stirring at À20°C for 1h, the mixture
was filtered through Celite, diluted with CHCl3, washed
with satd aq NaHCO3, and brine. The organic layer was
dried (MgSO4) and evaporated, and the residue was
purified by silica gel column chromatography (3:1–2:1
11.54Hz, CH2Ph), 4.41 (d, 1H, J1 ,2 8.03Hz, H-10),
0
0
4.29(dd, 1H, J6a ,6b 12.05, J5 ,6a 4.52Hz, H-6a0), 4.24
(m, 1H, H-2), 4.15–3.98 (m, 4H, H-3, H-6a, H-20, H-
6b0), 3.80 (s, 3H, OCH3), 3.77 (s, 3H, OCH3), 3.74–
3.70 (m, 2H, H-4, H-6b), 3.58 (m, 1H, H-5), 3.53 (ddd,
0
0
0
0
1H, J5 ,6b 2.00Hz, H-50), 2.06, 2.04, 2.03, 2.02, 1.89
(5s, 15H, COCH3), 0.87(s, 9H, C(C H3)3), 0.00, À0.04
(2s, 6H, SiCH3); 13C NMR (CDCl3): d 171.47, 170.84,
170.59, 170.31, 169.19 (COCH3), 158.91–113.43 (CAr),
99.99 (C-10), 99.14 (C-1), 76.47 (C-4), 75.81 (C-5),
73.02 (C-3), 72.27 (C-30), 71.94 (C-50), 71.40, 69.73
(CH2Ph), 67.92 (C-40), 62.51 (C-6), 61.79 (C-60), 55.17,
55.14 (OCH3), 54.11 (C-20), 49.53 (C-2), 25.82
(C(CH3)3), 23.27, 23.07, 20.67, 20.61, 20.53 (COCH3),
18.07( C(CH3)3), À5.35, À5.41 (SiCH3); HRMS
(FAB+) calcd for C44H65N2O16Si [M+H]+ 905.4103,
found 905.4110.
0
0
n-hexane–EtOAc) to provide 22 (4.32g, 87%) as a white
amorphous powder: Rf 0.31 (1:1 n-hexane–EtOAc); ½aꢀ
27
D
+25.1 (c 1.0, CHCl3); 1H NMR (CDCl3): d 7.91–6.30 (m,
16H, Ar), 5.88 (dd, 1H, J2 ,3 10.54, J3 ,4 9.04Hz,0 H-30),
0
0
0
0
5.58 (d, 1H, J1 ,2 8.03Hz, H-10), 5.17(t, 1H, H-4 ), 4.93
(d, 1H, J1,2 8.53Hz, H-1), 4.71 (d, 1H, J 12.55Hz,
CH2Ph), 4.57(d, 1H, J 11.55Hz, CH2Ph), 4.43 (d, 1H,
0
0
0
0
0
J 12.55Hz, CH2Ph), 4.42 (dd, 1H, J6a ,6b 12.05, J5,6a
4.2.17. 4-Methoxybenzyl 2-acetamido-2-deoxy-3,4,6-tri-
O-(4-methoxybenzyl)-b-D-glucopyranosyl-(1!4)-2-acet-
amido-6-O-tert-butyldimethylsilyl-2-deoxy-3-O-(4-meth-
oxybenzyl)-b-D-glucopyranoside (24). Sodium metho-
xide in MeOH (28wt%, 0.2mL) was added dropwise
to compound 23 (1.22g, 1.3mmol) in MeOH (10mL).
After stirring at room temperature for 3h, the mixture
was evaporated, and the residue was dissolved in dry
DMF (10mL) and then powdered NaOH (330mg,
8.3mmol) and 4-methoxybenzyl chloride (1.1mL,
8.2mmol) were added. The mixture stirred at room tem-
perature overnight and then MeOH (5mL) was added to
quench the excess of the reagent, and the mixture was
evaporated. The residue was diluted with CHCl3,
washed with satd aq NaHCO3, and brine. The organic
layer was dried (MgSO4), evaporated, and the residue
purified by silica gel column chromatography (2:1
4.52Hz, H-6a0), 4.34 (dd, 1H, H-20), 4.28 (d, 1H, J
12.04Hz, CH2Ph), 4.13–4.10 (m, 3H, H-3, H-4, H-6b0),
4.00 (t, 1H, J2,3 9.54Hz, H-2), 3.84 (m, 1H, H-50),
3.69–3.66 (m, 4H, H-6a, OCH3), 3.57(s, 3H, OC H3),
0
3.46 (dd, 1H, J6a,6b 12.04, J5,6b 3.51Hz, H-6b), 3.14
(d, 1H, H-5), 2.03, 2.03, 1.85 (3s, 9H, COCH3), 0.96
(s, 9H, C(CH3)3), 0.13, 0.10 (2s, 6H, SiCH3); 13C
NMR (CDCl3): d 170.14, 170.48, 170.00(COCH3),
167.94 (CO of phthalimido), 159.36–113.54 (CAr),
97.28 (C-10), 96.70 (C-1), 76.49 (C-4), 76.24 (C-3),
75.36 (C-5), 74.12 (CH2Ph), 71.94 (C-50), 71.04 (C-30),
70.10 (CH2Ph), 69.47(C-4 ), 62.24 (C-60), 61.68 (C-6),
0
56.14 (C-2), 55.74 (C-20), 55.45 (OCH3), 55.14 (OCH3),
26.30 (C(CH3)3), 21.09, 21.04, 20.85 (COCH3), 18.75
(C(CH3)3), À4.68, À4.90 (SiCH3); HRMS (FAB+) calcd
for C56H64N2O18SiNa [M+Na]+ 1103.3821, found
1103.3839.
CHCl3–EtOAc) to give 24 (715mg, 47%) as a white
amorphous powder: Rf 0.41 (3:1 CHCl3–acetone); ½aꢀ
28
D
1
4.2.16. 4-Methoxybenzyl 2-acetamido-3,4,6-tri-O-acetyl-
2-deoxy-b-D-glucopyranosyl-(1!4)-2-acetamido-6-O-
tert-butyldimethylsilyl-2-deoxy-3-O-(4-methoxybenzyl)-
b-D-glucopyranoside (23). The phthalimido group of
compound 22 (4.24g, 3.9mmol) was converted into the
À58.0 (c 1.0, CHCl3); H NMR (CDCl3): d 7.25–6.72
(m, 20H, Ar), 6.62 (d, 1H, J2 ,N H 9.04Hz, N0H), 4.87
(d, 1H, J2,NH 8.53Hz, NH), 4.79–4.68 (m, 4H, CH2Ph),
4.48 (m, 1H, H-10, CH2Ph), 4.29 (m, 1H, H-20), 4.22 (d,
1H, J1,2 8.03Hz, H-1), 4.02–3.98 (m, 2H, H-6a, H-40),
0
0

H. Ochiai et al. / Carbohydrate Research 339 (2004) 2769–2788
2783
27
D
1
3.86–3.54 (m, 7H, H-2, H-4, H-6b, H-30, H-50, H-6a0, H-
6b0), 3.81, 3.79, 3.79, 3.76, 3.75 (5s, 15H, OCH3), 3.43 (t,
1H, J3,4 9.53Hz, H-3), 3.30 (d, 1H, J4,5 9.54Hz H-5),
2.01, 1.71 (2s, 6H, COCH3), 0.82 (s, 9H, C(CH3)3),
À0.04, À0.08 (2s, 6H, SiCH3); 13C NMR (CDCl3): d
170.57, 170.27 (COCH3), 159.45–113.42 (CAr), 99.93
(C-1), 99.41 (C-10), 80.05 (C-3), 78.05 (C-4), 76.91 (C-
30), 76.24 (C-50), 74.96 (C-5), 74.53, 73.56, 73.12
(CH2Ph), 72.12 (C-40), 71.18, 69.73 (CH2Ph), 67.78 (C-
60), 62.80 (C-6), 55.28, 55.24, 55.51, 55.17, 55.14
(OCH3), 54.75 (C-2), 49.29(C-20), 25.85 (C(CH3)3),
23.48, 23.14 (COCH3), 18.12 (C(CH3)3), À5.40 (SiCH3);
HRMS (FAB+) calcd for C62H83N2O16Si [M+H]+
1139.5512, found 1139.5511.
202°C; ½aꢀ À51.0 (c 0.1, CHCl3); H NMR (CDCl3):
0
0
d 7.24–6.73 (m, 20H, Ar), 6.60 (d, 1H, J2 ,N H 9.04Hz,
N0H), 5.81 (m, 1H, CH@CH2), 5.16 (dd, 1H, J 17.56,
1.50Hz, CH@CH2), 5.09 (d, 1H, J 10.04Hz, CH@CH2),
4.88 (d, 1H, J2,NH 8.54Hz, NH), 4.78–4.68 (m, 4H,
CH2Ph), 4.59–4.41 (m, 7H, H-10, CH2Ph), 4.29–4.25
(m, 2H, H-1, H-20), 3.93 (m, 1H, H-40), 3.89 (d, 2H, J
5.52Hz, CH2CH@CH2), 3.81–3.68 (m, 5H, H-2, H-6a,
H-6b, H-30, H-50), 3.81, 3.80, 3.79, 3.76 (4s, 15H,
OCH3), 3.66–3.64 (m, 3H, H-4, H-6a0, H-6b0), 3.47
(dd, 1H, J2,3 10.04, J3,4 9.03Hz, H-3), 3.35 (m, 1H, H-
5), 1.99, 1.72 (2s, 6H, COCH3); 13C NMR (CDCl3): d
170.70, 170.24 (COCH3), 159.43–158.84 (CAr), 134.49
(CH@CH2), 130.55–129.04 (CAr), 117.19 (CH@CH2),
113.96–113.45 (CAr), 99.96 (C-1), 99.46 (C-10), 79.85
(C-3), 78.17 (C-4), 76.31 (C-30), 74.99 (C-5), 74.47 (C-
50, CH2Ph), 73.67 (CH2Ph), 73.24 (C-40), 73.10
(CH2Ph), 72.19 (CH2CH@CH2), 71.35 (CH2Ph), 70.18
(C-6), 69.72 (CH2Ph), 68.07(C-6 0), 55.25, 55.22,
55.18, 55.15, 55.13 (OCH3), 55.02 (C-2), 49.33
(C-20), 23.49, 23.14 (COCH3); HRMS (FAB+)
calcd for C59H73N2O16 [M+H]+ 1065.4960, found
1065.4984.
4.2.18. 4-Methoxybenzyl 2-acetamido-2-deoxy-3,4,6-tri-
O-(4-methoxybenzyl)-b-D-glucopyranosyl-(1!4)-2-acet-
amido-2-deoxy-3-O-(4-methoxybenzyl)-b-D-glucopyrano-
side (25). To a solution of compound 24 (1.61g,
1.4mmol) in dry THF (20mL) was added tetrabutylam-
monium fluoride (1.0M in THF, 2.8mL). The mixture
stirred at room temperature for 1h, then poured into
satd aq (NH4)2SO4, extracted with CHCl3, and washed
with brine. The organic layer was dried (MgSO4), evap-
orated, and the residue purified by silica gel column
chromatography (1:1 CHCl3–EtOAc, 5:1–3:1 CHCl3–
acetone, then 10:1 CHCl3–MeOH) to provide 25
4.2.20. 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-b-D-glu-
copyranosyl-(1!4)-2-acetamido-1,3-di-O-acetyl-6-O-
allyl-2-deoxy-a-D-glucopyranose (27). The 4-meth-
oxybenzyl groups of compound 26 (57.0mg, 54lmol)
were removed by treatment with DDQ (67.0mg,
0.30mmol) in 18:1 (v/v) CH2Cl2–H2O (3.8mL) followed
by acetylation with Ac2O (2mL) in pyridine (4mL) as
described in the preparation of compound 17. The resi-
due was purified by silica gel column chromatography
(3:1 CHCl3–acetone) to provide 27 (32.0mg, 87%) as a
(1.43g, 99%) as a white solid: Rf 0.29 (10:1 CHCl3–
28
D
MeOH); mp 212–213°C; ½aꢀ À35.6 (c 0.1, CHCl3);
1H NMR (CDCl3): d 7.22–6.76 (m, 20H, Ar), 6.15 (d,
1H, J2 ,N H 8.54Hz, N0H), 5.15 (d, 1H, J2,NH 8.03Hz,
NH), 4.76–4.39 (m, 12H, H-1, H-10, CH2Ph), 4.02 (m,
0
0
1H, H-20), 3.89 (t, 1H, J3 ,4 5.52Hz, H-40), 3.82–3.69
(m, 4H, H-2, H-6a, H-6b, H-30), 3.81, 3.79, 3.79, 3.75
(15H, 4s, OCH3), 3.65–3.55 (m, 5H, H-3, H-4, H-50,
H-6a0, H-6b0), 3.37(m, 1H, H-5), 2.34 (t, 1H, J6,6ÀOH
6.52Hz, 6-OH), 1.91, 1.73 (2s, 6H, COCH3); 13C
NMR (CDCl3): d 170.75, 170.27 (COCH3), 159.34–
113.63 (CAr), 100.37 (C-1), 97.74 (C-10), 80.27(C-3),
78.01 (C-4), 77.82 (C-30), 76.06 (C-50), 74.79 (C-5),
74.49 (C-40), 74.33, 73.86, 73.03, 72.14, 70.30 (CH2Ph),
68.20 (C-60), 62.29 (C-6), 55.57(C-2), 55.25, 55.21,
55.16 (OCH3), 52.34 (C-20), 23.44, 23.23 (COCH3);
HRMS (FAB+) calcd for C56H69N2O16 [M+H]+
1025.4647, found 1025.4640.
0
0
white solid: Rf 0.31 (1:1 CHCl3–acetone); mp 255–
28
D
256°C (dec.); ½aꢀ +36.0 (c 1.0, CHCl3); 1H NMR
(CDCl3): d 6.11 (d, 1H, J1,2 4.02Hz, H-1), 5.95 (m,
1H, CH@CH2), 5.68 (d, 1H, J2 ,N H 9.04Hz, N0H),
5.63 (d, 1H, J2,NH 9.03Hz, NH), 5.34 (dd, 1H, J 17.07,
1.51Hz, CH@CH2), 5.29 (dd, 1H, J 10.54, 1.50Hz,
CH@CH2), 5.21 (dd, 1H, J2,3 9.54, J3,4 4.01Hz, H-3),
0
0
5.18 (dd, 1H, J2 ,3 4.52, J3 ,4 9.54Hz, H-30), 5.06 (t,
0
0
0
0
1H, J4 ,5 9.79Hz, H-40), 4.73 (d, 1H, J1 ,2 8.53Hz, H-
10), 4.41–4.33 (m, 2H, H-2, H-6a0), 4.18 (m, 1H,
CH2CH@CH2), 4.06–3.99 (m, 3H, H-4, H-6b0,
CH2CH@CH2), 3.86–3.71 (m, 3H, H-5, H-6a, H-20),
0
0
0
0
3.65 (ddd, 1H, J5 ,6a 4.39, J5 ,6b 2.51Hz, H-50), 3.57
(dd, 1H, J6a,6b 11.55, J5,6b 2.01Hz, H-6b), 2.18, 2.09,
2.07, 2.03, 2.02, 1.93 (6s, 21H, COCH3); 13C NMR
(CDCl3): d 171.66, 170.91, 170.54, 170.09, 170.07,
169.35, 168.99 (COCH3), 134.36 (CH@CH2), 117.85
(CH@CH2), 100.62 (C-10), 90.67(C-1), 47.84 (C-4),
72.60 (CH2CH@CH2), 72.48 (C-30), 72.36 (C-5), 71.82
(C-50), 70.73 (C-3), 67.99 (C-40), 67.49 (C-6), 61.76 (C-
60), 54.94 (C-20), 51.17(C-2), 23.29, 23.02,
21.00, 20.65, 20.61, 20.57(CO CH3); HRMS (FAB+)
0
0
0
0
4.2.19. 4-Methoxybenzyl 2-acetamido-2-deoxy-3,4,6-tri-
O-(4-methoxybenzyl)-b-D-glucopyranosyl-(1!4)-2-acet-
amido-6-O-allyl-2-deoxy-3-O-(4-methoxybenzyl)-b-D-glu-
copyranoside (26). Compound 25 (1.43g, 1.4mmol) in
dry DMF (20.0mL) was treated with allyl bromide
(0.180mL, 2.13mmol) and powdered NaOH (84.0mg,
2.10mmol) as described in the preparation of compound
16. Silica gel column chromatography (3:1–2:1 CHCl3–
acetone) of the residue afforded 26 (1.22g, 81%) as a
white solid: Rf 0.47(2:1 CHCl 3–acetone); mp 201–

2784
H. Ochiai et al. / Carbohydrate Research 339 (2004) 2769–2788
calcd for C29H43N2O16 [M+H]+ 675.2613, found
675.2638.
5.10 (m, 2H, H-30, H-40), 4.79 (d, 1H, J1 ,2 8.54Hz, H-
0
0
10), 4.35 (d, 1H, J 17.07Hz, OCH2), 4.29 (dd, 1H, J6a ,6b
0
0
12.05, J5,6a 4.01Hz, H-6a0), 4.19–4.03 (m, 4H, H-2, H-
6a, H-20, H-6b0), 4.05 (d, 1H, J 17.07Hz, OCH2),
3.88–3.74 (m, 2H, H-4, H-50), 3.79 (s, 3H, OCH3), 3.56
(dd, 1H, J4,5 10.04Hz, J5,6a 2.51Hz, H-5), 3.41 (d, 1H,
J6a,6b 9.03Hz, H-6b), 2.09, 2.08, 2.03, 2.01, 1.88 (5s,
18H, CH3C of oxazoline, COCH3); 13C NMR (CDCl3):
0
4.2.21. 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-b-D-glu-
copyranosyl-(1!4)-2-acetamido-1,3-di-O-acetyl-2-deoxy-
6-O-methoxycarbonylmethyl-a-D-glucopyranose
(28).
Compound 27 (271mg, 0.40mmol) in 4:1 (v/v)
CH2Cl2–MeOH (30mL) was ozonized as described in
the preparation of compound 18. Silica gel column chro-
matography (2:1 n-hexane–EtOAc, then 1:1 CHCl3–ace-
tone) of the residue afforded crude 2-acetamido-3,4,
6-tri-O-acetyl-2-deoxy-b-D-glucopyranosyl-(1!4)-2-acet-
amido-1,3-di-O-acetyl-2-deoxy-6-O-formylmethyl-a-D-
glucopyranose as a white solid.
The crude product was oxidized with NaH2PO4
(31mg, 0.26mmol)–NaClO2 (58mg, 0.64mmol) in
2:12:3 (v/v/v) CH2Cl2–tert-BuOH–H2O (51mL) contain-
ing 2-methyl-2-butene (0.24mL, 2.3mmol) followed by
treatment with Dowex 50W-X4 (H+ form) (300mg) in
4:1 (v/v) MeOH–CH2Cl2 (25mL). Silica gel column
chromatography (2:1 n-hexane–EtOAc, then 2:1–1:1
d
171.67, 171.00, 170.90, 170.01, 169.42, 169.31
(COOCH3, COCH3), 166.61 (OC@N), 102.74 (C-10),
99.74 (C-1), 77.20 (C-4), 73.57 (C-30), 71.90 (C-50),
71.35 (C-3), 69.71 (C-6), 68.69 (C-5), 68.26 (C-40),
67.81 (OCH2), 65.09 (C-2), 61.95 (C-60), 53.72 (C-20),
52.17(O CH3), 23.03, 22.06, 21.01, 20.73, 20.70, 20.61
(COCH3), 14.00 (CH3 of oxazoline); HRMS (FAB+)
calcd for C27H39O16N2 [M+H]+ 647.2300, found
647.2309.
4.2.23. 2-Methyl-[2-acetamido-2-deoxy-b-D-glucopyrano-
syl-(1!4)-6-O-carboxymethyl-1,2-di-deoxy-a-D-glucopyr-
ano]-[2,1-d]-2-oxazoline sodium salt (2). Compound 29
(13mg, 20lmol) was treated with NaOMe in MeOH
(0.1M, 80lL) as described in the synthesis of compound
1. The remaining methyl ester was hydrolyzed in a car-
CHCl3–acetone) gave 28 (259mg, 91%) as a white solid:
Rf 0.29 (1:1 CHCl3–acetone); mp 250–251°C (dec.); ½aꢀ
28
D
1
+44.8 (c 1.0, CHCl3); H NMR (CDCl3): d 6.89 (d, 1H,
1
J2 ,N H 10.03Hz, N0H), 6.10 (d, 1H, J1,2 3.51Hz, H-1),
5.66 (d, 1H, J2,NH 9.04Hz, NH), 5.17(dd, 1H, J2,3
bonate buffer (25mM, pH12.0, 402lL) to afford 2: H
0
0
NMR (D2O): d 6.11 (d, 1H, J1,2 7.02Hz, H-1), 4.62 (d,
0
0
0
10.79, J3,4 9.28Hz, H-3), 5.09–5.07(m, 2H, H-3 , H-
1H, J1 ,2 8.53Hz, H-10), 4.43 (s, 1H, H-3), 4.22 (m,
1H, H-2), 4.00–3.93 (m, 3H, H-6a0, H-6b0, OCH2),
3.80–3.69 (m, 3H, H-4, H-20, OCH2), 3.64–3.44 (m,
6H, H-5, H-6a, H-6b, H-30, H-40, H-50), 2.09, 2.07(2s,
6H, CH3C of oxazoline, COCH3); 13C NMR (D2O): d
178.12 (COONa), 174.93 (COCH3), 168.76 (OC@N),
103.36 (C-1), 100.23 (C-10), 79.09 (C-4), 76.36 (C-50),
74.16 (C-30), 70.96 (C-6), 70.62 (OCH2), 70.23 (C-40),
70.00 (C-5), 69.22 (C-3), 65.51 (C-2), 61.10 (C-60),
56.22 (C-20), 22.67(CO CH3), 13.37( CH3 of oxazoline);
HRMS (FAB+) calcd for C18H27O12N2Na2 [M+Na]+
509.1359, found 509.1373.
40), 4.86 (d, 1H, J1 ,2 9.04Hz, H-10), 4.49 (d, 1H, J
17.57Hz, OCH2), 4.38 (dd, 1H, J 12.55, 4.01Hz, H-
6a0), 4.34 (m, 1H, H-2), 4.29 (dd, 1H, J6a,6b 10.29, J5,6a
2.26Hz, H-6a), 4.22 (m, 1H, H-20), 4.06–3.98 (m, 3H,
H-4, H-6b0, OCH2), 3.84 (s, 3H, OCH3), 3.77 (d, 1H,
H-5), 3.68 (m, 1H, H-50), 3.38 (d, 1H, H-6b), 2.18,
2.09, 2.06, 2.02, 2.01, 1.92 (6s, 21H, COCH3); 13C
NMR (CDCl3): d 172.43, 171.77, 170.89, 170.66,
170.10, 170.06, 169.34, 169.14 (COOCH3, COCH3),
101.44 (C-10), 90.75 (C-1), 74.93 (C-4), 73.43 (C-30),
71.65 (C-5, C-50), 0 70.34 (C-3), 68.97 (C-6), 68.45
(OCH2), 68.07(C-4 ), 61.88 (C-60), 53.41 (C-20), 52.38
(OCH3), 51.30 (C-2), 23.13, 22.97, 21.01, 20.68, 20.62,
20.58, 20.50 (COCH3); HRMS (FAB+) calcd for
C29H43O18N2 [M+H]+ 707.2511, found 707.2479.
0
0
4.2.24. (Methyl-2,3,4-tri-O-acetyl-b-D-glucopyranosyluro-
nate)-(1!4)-2-acetamido-1,3,6-tri-O-acetyl-2-deoxy-a-D-
glucopyranose (32). Methyl (2,3,4-tri-O-acetyl-a-D-glu-
copyranosyl trichloroacetimidate)uronate 3023 (570mg,
1.2mmol) and 2-acetamido-1,3,6-tri-O-acetyl-2-deoxy-
a-D-glucopyranose (31,24 276mg, 0.80mmol) were dis-
solved together in dry CH2Cl2 (8mL), and BF3 etherate
(0.15mL, 1.2mmol) in dry CH2Cl2 (0.35mL) was added
in the presence of activated MS4A (1.0g) at À20°C un-
der argon. After stirring at À20°C for 5h, the mixture
filtered through Celite, diluted with CHCl3, washed with
satd aq NaHCO3, and brine. The organic layer was
dried (MgSO4) and evaporated, and the residue purified
by silica gel column chromatography (2:1 n-hexane–
EtOAc, then EtOAc) and then size-exclusion chroma-
tography on a Sephadex LH-20 column eluted with
MeOH to provide 32 (268mg, 51%) as a white solid:
4.2.22.
2-Methyl-[2-acetamido-3,4,6-tri-O-acetyl-2-
deoxy-b-D-glucopyranosyl-(1!4)-3-O-acetyl-1,2-di-deoxy-
6-O-methoxycarbonylmethyl-a-D-glucopyrano]-[2,1-d]-2-
oxazoline (29). Compound 28 (118mg, 0.17mmol) in
dry 1,2-dichloroethane (10mL) was treated with Me3-
SiOTf (33lL, 0.18mmol) as described in the production
of compound 19. Compound 29 (60mg, 56%) was iso-
lated as a white amorphous powder through column
chromatography on silica gel (2:1 CHCl3–acetone) and
then a Sephadex LH-20 column (MeOH as eluent): Rf
28
D
0.53 (10:1 CHCl3–MeOH); ½aꢀ +8.3 (c 1.0, CHCl3);
1H NMR (CDCl3): d 6.48 (d, 1H, J2,NH 9.04Hz, NH),
5.93 (d, 1H, J1,2 7.53Hz, H-1), 5.59 (s, 1H, H-3), 5.13–

H. Ochiai et al. / Carbohydrate Research 339 (2004) 2769–2788
2785
3.34 (t, 1H, J2 ,3 7.28Hz, H-20), 2.08 (s, 3H, CH3C of
oxazoline); 13C NMR (D2O): d 176.53 (COONa),
168.88 (OC@N), 104.48 (C-10), 100.43 (C-1), 78.90 (C-
4), 76.60 (C-50), 75.94 (C-30), 73.59 (C-20), 72.38 (C-40),
71.55 (C-5), 69.70 (C-3), 65.79 (C-2), 62.23 (C-6),
13.58 (CH3 of oxazoline); HRMS (FAB+) calcd for
C14H20O11NNa2 [M+Na]+ 424.0832, found 424.0851.
0
0
Rf 0.38 (10:1 CHCl3–MeOH); mp 249–250°C (dec.);
25
1
½aꢀ +43.0 (c 1.0, CHCl3); H NMR (CDCl3): d 6.09
D
(d, 1H, J1,2 3.52Hz, H-1), 5.69 (d, 1H, J2,NH 9.04Hz,
NH), 5.26–5.14 (m, 3H, H-3, H-30, H-40), 4.96 (t, 1H,
J2 ,3 8.29Hz, H-20), 4.60 (d, 1H, J1 ,2 8.03Hz, H-10),
4.42 (d, 1H, J6a,6b 12.04Hz, H-6a), 4.38 (m, 1H, H-2),
0
0
0
0
0
4.10 (dd, 1H, J5,6b 2.77Hz, H-6b), 3.99 (d, 1H, J4 ,5
9.54Hz, H-50), 3.88–3.87(m, 2H, H-4, H-5), 3.75 (s,
0
3H, CH3O), 2.19, 2.13, 2.08, 2.05, 2.02, 2.01, 1.93 (7s,
21H, COCH3); 13C NMR (CDCl3): d 171.64, 170.31,
170.10, 169.98, 169.29, 169.17, 168.78, 166.66 (COCH3,
COOCH3), 100.85 (C-1), 90.43 (C-10), 76.11 (C-4), 72.69
(C-50), 72.00 (C-30), 71.33 (C-20), 70.51 (C-3), 70.24 (C-
5), 69.21 (C-40), 61.36 (C-6), 52.83 (CH3O), 51.03 (C-
2), 22.99, 20.94, 20.79, 20.63, 20.53, 20.50, 20.40
(CH3CO); HRMS (FAB+) calcd for C27H38NO18
[M+H]+ 664.2089, found 664.2086.
4.3. Chitinase-catalyzed hydrolysis of the oxazoline
derivatives
4.3.1. Hydrolysis of compound 1. A solution of com-
pound 1 (12.8mg, 26.3lmol) in carbonate buffered deu-
terium oxide solution (50mM, pD9.0, 263lL) was kept
in an NMR probe tube at 30°C for 5h. Chitinase from
Bacillus sp. (1.3mg) was added, and the mixture was
kept standing at 30°C. The concentration of 1 was cal-
1
culated from the integrated values of the H NMR sig-
4.2.25.
2-Methyl-[3,6-di-O-acetyl-1,2-di-deoxy-4-O-
nals from H-1 proton and the methyl protons as
internal standard. After complete consumption of 1,
the enzyme was thermally inactivated at 90°C for
20min. An aliquot of the mixture was subjected to
HPLC and MALDI-TOF/MS. The mixture was purified
through a Sephadex G-10 column running with distilled
H2O to give 2-acetamido-6-O-carboxymethyl-2-deoxy-
b-D-glucopyranosyl-(1!4)-2-acetamido-2-deoxy-D-glu-
copyranose sodium salt (34) (9.0mg, 68%) as a white
amorphous powder: 1H NMR (D2O): d 5.20 (d,
0.57H, J1a,2a 3.01Hz, H-1a), 4.72–4.70 (m, 0.43H, H-
(methyl-2,3,4-tri-O-acetyl-b-D-glucopyranosyluronate)-a-
D-glucopyrano]-[2,1-d]-2-oxazoline (33). A solution of
compound 32 (33.0mg, 50lmol) in dry 1,2-dichloro-
ethane (500lL) was treated with Me3SiOTf (18lL,
99lmol) as described in the preparation of compound
19. The residue was purified on a silica gel column (1:2
n-hexane–EtOAc) and then a Sephadex LH-20 column
(MeOH as eluent) to give 33 (23.0mg, 77%) as a white
amorphous powder: Rf 0.45 (15:1 CHCl3–MeOH); ½aꢀ
27
D
1
+6.0 (c 1.0, CHCl3); H NMR (CDCl3): d 5.91 (d, 1H,
J1,2 7.53Hz, H-1), 5.61 (m, 1H, H-3), 5.27–5.21 (m,
1b), 4.61 (d, 1H, J1 ,2 8.54Hz, H-10), 2.08, 2.05 (2s,
6H, COCH3); 13C NMR (D2O): d 178.73 (COONa),
175.39, 175.21, 175.12 (COCH3), 102.22, 102.18 (C-
10a, C-10b), 95.47(C-1 b), 91.02 (C-1a), 22.84–22.53
(COCH3); HRMS (FAB+) calcd for C18H29O13N2Na2
[M+Na]+, 527.1465, found 527.1453.
0
0
2H, H-30, H-40), 5.01 (m, 1H, H-20), 4.79 (d, 1H, J1 ,2
0
0
8.03Hz, H-10), 4.22 (dd, 1H, J6a,6b 12.05Hz, J5,6a
2.51Hz, H-6a), 4.13–4.11 (m, 2H, H-2, H-50), 4.04 (dd,
1H, J5,6b 5.52Hz, H-6b), 3.76 (s, 3H, COOCH3), 3.68
(d, 1H, J4,5 9.54Hz, H-4), 3.50 (m, 1H, H-5), 2.11,
2.09 (2s, 6H, COCH3), 2.09 (d, 3H, J 1.51Hz, CH3C
of oxazoline), 2.04, 2.02, 2.01 (3s, 9H, COCH3); 13C
NMR (CDCl3): d 170.56, 170.15, 169.40, 169.32,
169.28, 166.78, 166.67 (COCH3, COOCH3, OC@N),
101.65 (C-10), 99.02 (C-1), 78.12 (C-4), 72.55 (C-50),
72.30 (C-30), 70.96 (C-20), 70.51 (C-3), 69.08 (C-40),
67.35 (C-5), 65.09 (C-2), 63.32 (C-6), 52.88 (OCH3),
20.97, 20.79, 20.58, 20.50 (COCH3), 13.93 (CH3 of oxaz-
oline); HRMS (FAB+) calcd for C25H34O16N [M+H]+
604.1878, found 604.1862.
4.3.2. Hydrolysis of compound 2. The hydrolysis reac-
tion of 2 (22.0mg, 45.2lmol) was performed with
chitinase from Bacillus sp. (2.2mg) in a carbonate-
buffered deuterium oxide solution (50mM, pD9.0,
452lL) according to the procedure for hydrolysis of 1.
Isolation of the product was also carried out as already
described to provide 2-acetamido-2-deoxy-b-D-glucopyr-
anosyl-(1!4)-2-acetamido-2-deoxy-6-O-carboxymethyl-
D-glucopyranose sodium salt (35) (11.2mg, 49%) as a
1
white amorphous powder: H NMR (D2O): d 5.20 (d,
4.2.26. 2-Methyl-[1,2-di-deoxy-4-O-(sodium b-D-gluco-
pyranosyluronate)-a-D-glucopyrano]-[2,1-d]-2-oxazoline
(3). Compound 33 (23.0mg, 38lmol) was treated with
NaOMe in MeOH (28wt%, 20lL) as described for the
synthesis of compound 1. The methyl ester was hydro-
lyzed in a carbonate buffer (25mM, pH12.0, 600lL)
to provide 3: 1H NMR (D2O): d 6.10 (d, 1H, J1,2
0.5H, J1a,2a 3.01Hz, H-1a), 4.73–4.68 (m, 1.5H, H-1b,
H-10a, H-10b), 2.07, 2.05 (2s, 6H, COCH3); 13C NMR
(D2O): d 178.37 (COONa), 175.36, 175.13, 175.05
(COCH3), 101.81 (C-10), 95.39 (C-1b), 91.01 (C-1a),
22.89–22.54 (COCH3); HRMS (FAB+) calcd for
C18H29O13N2Na2 [M+Na]+, 527.1465, found 527.1484.
7.03Hz, H-1), 4.50 (d, 1H, J1 ,2 8.03Hz, H-10), 4.39 (s,
1H, H-3), 4.19 (m, 1H, H-2), 3.87–3.61 (m, 4H, H-4,
H-6a, H-6b, H-50), 3.46–3.39 (m, 3H, H-5, H-30, H-40),
4.3.3. Hydrolysis of compound 3. Compound 3 (10.9
mg, 27.2lmol) was enzymatically hydrolyzed with chiti-
nase from Bacillus sp. (1.1mg) in a carbonate-buffered
0
0

2786
H. Ochiai et al. / Carbohydrate Research 339 (2004) 2769–2788
deuterium oxide solution (50mM, pD9.0, 272lL)
according to the procedure for hydrolysis of 1. The mix-
ture was similarly purified through a Sephadex G-10
column to afford (sodium b-D-glucopyranosyluronate)-
(1!4)-2-acetamido-2-deoxy-4-O-D-glucopyranose (36)
1 (39.0mg, 80lmol) with 5 (69.0mg, 240lmol) in a car-
bonate buffer (50mM, pH10.0, 1.6mL) was incubated
with chitinase from Bacillus sp. (3.9mg) at 30°C as de-
scribed in the preparation of 37. The residue was puri-
fied by HPLC through a Shodex Asahipak NH2P-50
4E column eluting with 17:33 (v/v) phosphate buffer
(10mM, pH7.0)–MeCN. The combined fractions were
lyophilized and then desalted through a BioGel P-2 col-
1
(9.7mg, 85%) as a white amorphous powder: H NMR
(D2O): d 5.22 (d, 0.65H, J1a,2a 3.01Hz, H-1a), 4.74–
0
0
4.72 (m, 0.35H, H-1b), 4.55 (d, 1H, J1 ,2 8.03Hz, H-
10), 2.05 (s, 3H, COCH3); 13C NMR (D2O): d 176.20
(COONa), 175.37, 175.09 (COCH3), 102.91, 102.86 (C-
10a, C-10b), 95.49 (C-1b), 91.09 (C-1a), 22.84, 22.54
(COCH3); HRMS (FAB+) calcd for C14H23O12NNa
[M+H]+, 420.1118, found 420.1122.
umn eluting with distilled H2O to afford (38) (23.0mg,
26
D
37%) as a white amorphous powder: ½aꢀ À19.0 (c 0.1,
H2O); 1H NMR (D2O): d 5.88 (m, 1H, CH@CH2),
0
5.09–5.01 (m, 2H, CH@CH2), 4.61–4.57(m, 2H, H-1 ,
H-100), 4.49 (d, 1H, J1,2 8.03Hz, H-1), 3.98–3.97(d,
2H, J 4.02Hz, CH2COONa), 3.93–3.47(m, 20H, H-2,
H-3, H-4, H-5, H-6a, H-6b, H-20, H-30, H-40, H-50, H-
6a0, H-6b0, H-200, H-300, H-400, H-500, H-6a00, H-6b00,
OCH2CH2), 2.11–2.04 (m, 2H, CH2CH@CH2), 2.07,
2.07, 2.04 (3s, 9H, COCH3), 1.68–1.61 (m, 2H,
OCH2CH2); 13C NMR (D2O): d 178.42 (COONa),
174.99, 174.81 (COCH3), 139.10 (CH@CH2), 115.52
(CH@CH2), 102.00, 101.72 (C-10, C-100), 101.45 (C-1),
79.82, 79.68 (C-4, C-40), 75.02, 74.93 (C-5, C-50, C-500),
73.60, 72.83, 72.60 (C-3, C-30, C-300), 70.62 (CH2COO-
Na), 70.18 (C-400), 70.13 (OCH2CH2), 69.95 (C-600),
60.49 (C-6, C-60), 56.09, 55.54, 55.48 (C-2, C-20, C-200),
29.77 (CH2CH@CH2), 28.32 (OCH2CH2), 22.82, 22.74
(COCH3); HRMS (FAB+) calcd for C31H51O18N3Na
[M+H]+ 776.3065, found 776.3049.
4.4. Enzymatic glycosidation
4.4.1. Methyl 2-acetamido-6-O-carboxymethyl-2-deoxy-
b-D-glucopyranosyl-(1!4)-2-acetamido-2-deoxy-b-D-glu-
copyranosyl-(1!4)-2-acetamido-2-deoxy-b-D-glucopy-
ranosyl-(1!4)-2-acetamido-2-deoxy-b-D-glucopyranoside
sodium salt (37). Compound 4 (52.0mg, 120lmol) in a
carbonate buffer (50mM, pH10.0, 400lL) containing
chitinase from Bacillus sp. (2.0mg) was added to a solu-
tion of 1 (19.5mg, 40lmol) in a carbonate buffer
(50mM, pH10.0, 400lL). The mixture was kept at
30°C in an NMR probe tube. The concentration of 1
1
was calculated from the integrated H NMR values of
the H-1 signal and the methyl protons as the internal
standard. After complete consumption of 1, the reaction
was terminated by thermal inactivation of the enzyme at
90°C for 20min. The mixture was lyophilized and the
residue purified through a BioGel P-2 column using
0.1M aq NaCl as eluent. The combined fractions were
lyophilized and then desalted through a BioGel P-2 col-
4.4.3. Methyl 2-acetamido-6-O-carboxymethyl-2-deoxy-
b-D-glucopyranosyl-(1!4)-2-acetamido-2-deoxy-b-D-glu-
copyranosyl-(1!4)-2-acetamido-2-deoxy-b-D-glucopyr-
anoside sodium salt (39). A mixture of compounds 1
(19.5mg, 40lmol) and 6 (28.0mg, 120lmol) in a car-
bonate buffer (50mM, pH10.0, 800lL) was incubated
with chitinase from Bacillus sp. (2.0mg) at 30°C as de-
scribed in the production of 37. The residue was purified
by HPLC through a Shodex Asahipak NH2P-50 4E col-
umn using 17:33 (v/v) phosphate buffer (10mM,
pH7.0)–MeCN as eluent. The combined fractions were
lyophilized and then desalted through a BioGel P-2 col-
umn eluting with distilled H2O to give 37 (18.1mg, 49%)
26
D
as a white amorphous powder: ½aꢀ À53.0 (c 0.1, H2O);
1
H NMR (D2O): d 4.61–4.57(m, 2H, H-1 , H-100, H-1000),
0
4.44 (d, 1H, J1,2 7.53Hz, H-1), 3.98 (d, 2H, J 4.52Hz,
CH2COONa), 3.88–3.50 (m, 24H, H-2, H-3, H-4, H-5,
H-6a, H-6b, H-20, H-30, H-40, H-50, H-6a0, H-6b0, H-
200, H-300, H-400, H-500, H-6a00, H-6b00, H-2000, H-3000, H-
4000, H-5000, H-6a000, H-6b000), 3.50 (s, 3H, OCH3), 2.07,
2.07, 2.06, 2.04 (4s, 12H, COCH3); 13C NMR (D2O): d
178.41 (COONa), 174.96 (COCH3), 102.28 (C-1),
101.96, 101.65 (C-10, C-100, C-1000), 79.78, 79.67, 79.43
(C-4, C-40, C-400), 74.95 (C-5, C-50, C-500), 73.63, 72.96,
72.59, 72.48 (C-3, C-30, C-300, C-3000), 70.60 (CH2COO-
Na), 70.15 (C-4000), 69.90 (C-6000), 60.50 (C-6, C-60, C-
600), 57.67 (OCH3), 56.10, 55.53, 55.31 (C-2, C-20, C-200,
C-2000), 22.73 (COCH3); HRMS (FAB+) calcd for
C35H57O23N4Na2 [M+Na]+ 947.3209, found 947.3212.
umn eluting with distilled H2O to provide 39 (12.6mg,
26
D
45%) as a white amorphous powder: ½aꢀ À17.0 (c 0.1,
1
H2O); H NMR (D2O): d 4.65–4.56 (m, 2H, H-10, H-
100), 4.43 (d, 1H, J1,2 7.53Hz, H-1), 4.06–3.95 (m, 2H,
CH2COONa), 3.92–3.45 (m, 18H, H-2, H-3, H-4, H-5,
H-6a, H-6b, H-20, H-30, H-40, H-50, H-6a0, H-6b0, H-
200, H-300, H-400, H-500, H-6a00, H-6b00), 3.50 (s, 3H,
OCH3), 2.07, 2.04 (2s, 9H, COCH3); 13C NMR (D2O):
d 178.67 (COONa), 175.27, 175.21, 175.18 (COCH3),
102.48 (C-1), 102.18, 101.88 (C-10, C-100), 80.01, 79.81
(C-4, C-40), 75.21, 75.16, 75.09 (C-5, C-50, C-500),
73.83, 73.14, 72.80 (C-3, C-30, C-300), 70.71 (CH2COO-
Na), 70.33 (C-400), 70.08 (C-600), 60.68, 60.63 (C-6,
C-60), 57.76 (OCH3), 56.27, 55.69, 55.48 (C-2,
C-20, C-200), 22.81, 22.77 (COCH3); HRMS (FAB+)
4.4.2. Pent-4-enyl 2-acetamido-6-O-carboxymethyl-2-
deoxy-b-D-glucopyranosyl-(1!4)-2-acetamido-2-deoxy-
b-D-glucopyranosyl-(1!4)-2-acetamido-2-deoxy-b-D-glu-
copyranoside sodium salt (38). A solution of compound

H. Ochiai et al. / Carbohydrate Research 339 (2004) 2769–2788
2787
calcd for C27H45O18N3Na [M+H]+ 722.2596, found
722.2596.
(C-4, C-40), 76.36 (C-500), 75.83 (C-300), 75.33, 75.05 (C-
5, C-50), 73.49 (C-200), 72.96, 72.54 (C-3, C-30), 72.27
(C-400), 70.27 (OCH2CH2), 60.61, 60.44 (C-6, C-60),
55.75 (C-20), 55.58 (C-2), 29.85 (CH2CH@CH2), 28.41
(OCH2CH2), 22.84, 22.78 (COCH3); HRMS (FAB+)
calcd for C27H43O17N2Na2 [M+Na]+ 713.2357, found
713.2371.
4.4.4. Methyl (sodium b-D-glucopyranosyluronate)-
(1!4)-(2-acetamido-2-deoxy-b-D-glucopyranosyl)-(1!4)-
2-acetamido-2-deoxy-b-D-glucopyranoside (40).
Com-
pound 3 (28.0mg, 70lmol) was incubated with 6
(47.3mg, 209lmol) in a carbonate buffer (50mM,
pH10.0, 1.4mL) containing chitinase from Bacillus sp.
(2.0mg) at 30°C as described in the preparation of 37.
The residue was purified through a Shodex Asahipak
NH2P-50 4E column eluting with 3:2 (v/v) phosphate
buffer (10mM, pH7.0)–MeCN. The fractions were com-
bined, lyophilized and desalted through a BioGel P-2
4.4.6. Methyl (sodium b-D-glucopyranosyluronate)-
(1!4)-(2-acetamido-2-deoxy-b-D-glucopyranosyl)-(1!4)-
(2-acetamido-2-deoxy-b-D-glucopyranosyl)-(1!4)-2-ace-
tamido-2-deoxy-b-D-glucopyranoside
(42). Glycosyl
donor 3 (8.0mg, 20lmol) and glycosyl acceptor 4
(26.0mg, 60lmol) in a carbonate buffer (50mM,
pH10.0, 400lL) were incubated with chitinase from
Bacillus sp. (0.8mg) at 30°C as described for the prepa-
ration of compound 37. The residue was purified by
HPLC through a Shodex Asahipak NH2P-50 4E col-
umn eluting with 2:3 (v/v) phosphate buffer (10mM,
pH7.0)–MeCN. The combined fractions were lyophi-
lized and desalted through a Bio Gel P-2 column run-
ning with distilled H2O to give 42 (6.0mg, 36%) as a
column running with distilled H2O to give 40 (16.0mg,
26
D
36%) as a white amorphous powder: ½aꢀ À15.0 (c 0.1,
1
0
0
H2O); H NMR (D2O): d 4.61 (d, 1H, J1 ,2 7.53Hz,
H-10), 4.54 (d, 1H, J1 ,2 7.53Hz, H-100), 4.44 (d, 1H,
00 00
J1,2 8.03Hz, H-1), 3.99–3.51 (m, 15H, H-2, H-3, H-4,
H-5, H-6a, H-6b, H-20, H-30, H-40, H-50, H-6a0, H-6b0,
H-300, H-400, H-500), 3.51 (s, 3H, OCH3), 3.37(m, 1H,
H-200), 2.08, 2.04 (2s, 6H, COCH3); 13C NMR (D2O):
d 176.22 (COONa), 175.33, 175.24 (COCH3), 102.86
(C-100), 102.48 (C-1), 101.90 (C-10), 79.83, 79.17 (C-4,
C-40), 76.44 (C-500), 75.86 (C-300), 75.37, 75.12 (C-5, C-
50), 73.52 (C-200), 73.11, 72.56 (C-3, C-30), 72.32 (C-400),
60.66, 60.46 (C-6, C-60), 57.79 (OCH3), 55.80 (C-
2), 55.48 (C-20), 22.82 (COCH3); HRMS (FAB+)
calcd for C23H38O17N2Na [M+H]+ 637.2068, found
637.2062.
26
1
white amorphous powder: ½aꢀ À27.0 (c 0.1, H2O); H
D
NMR (D2O): d 4.61–4.57(m, 2H, H-1 , H-100), 4.54 (d,
0
1H, J 8.03Hz, H-1000), 4.44 (d, 1H, J1,2 8.03Hz, H-1),
4.00–3.50 (m, 21H, H-2, H-3, H-4, H-5, H-6a, H-6b,
H-20, H-30, H-40, H-50, H-6a0, H-6b0, H-200, H-300, H-
400, H-500, H-6a00, H-6b00, H-3000, H-4000, H-5000), 3.50 (s,
3H, OCH3), 3.36 (m, 1H, H-2000), 2.07, 2.04 (2s, 9H,
COCH3); 13C NMR (D2O): d 176.11 (COONa),
175.19, 175.12 (COCH3), 102.83 (C-1000), 102.40 (C-1),
101.85, 101.81 (C-10, C-100), 79.76, 79.54, 79.14 (C-4,
C-40, C-400), 76.32 (C-5000), 75.83 (C-3000), 75.32, 75.07
(C-5, C-50, C-500), 73.48 (C-2000), 73.06, 72.59, 72.51 (C-
3, C-30, C-300), 72.26 (C-4000), 60.60, 60.48 (C-6, C-60, C-
600), 57.73 (OCH3), 55.74, 55.60 (C-20, C-200), 55.43
(C-2), 22.79, 22.76 (COCH3); HRMS (FAB+)
calcd for C31H51O22N3Na [M+H]+ 840.2862, found
840.2864.
4.4.5. Pent-4-enyl (sodium b-D-glucopyranosyluronate)-
(1!4)-(2-acetamido-2-deoxy-b-D-glucopyranosyl)-(1!4)-
2-acetamido-2-deoxy-b-D-glucopyranoside (41). Com-
pound 3 (24.1mg, 60lmol) was dissolved together with
5 (54.0mg, 190lmol) in a carbonate buffer (50mM,
pH10.0, 1.2mL) containing chitinase from Bacillus sp.
(2.4mg) at 30°C. The mixture was treated according
to the procedure for the production of 37. The residue
was purified by HPLC through a Shodex Asahipak
NH2P-50 4E column using 1:1 (v/v) phosphate buffer
(10mM, pH7.0)–MeCN as eluent. The combined frac-
tions were lyophilized and desalted through a BioGel
Acknowledgements
The authors thank Professor Tamejiro Hiyama in Kyo-
to University for supplying an ozone generator and for
technical advice. This work was partially supported by
the Mitsubishi Foundation and by the 21st COE pro-
gram for a United Approach to New Materials Science
in Kyoto University.
P-2 column running with distilled H2O to afford 41
(15.8mg, 38%) as a white amorphous powder: ½aꢀ
26
D
1
À16.0 (c 0.1, H2O); H NMR (D2O): d 5.89 (m, 1H,
CH@CH2), 5.09–5.01 (m, 2H, CH@CH2), 4.61 (d, 1H,
J1 ,2 7.53Hz, H-10), 4.54 (d, 1H, J1 ,2 8.03Hz, H-100),
4.50 (d, 1H, J1,2 7.53Hz, H-1), 3.99–3.49 (m, 17H, H-
2, H-3, H-4, H-5, H-6a, H-6b, H-20, H-30, H-40, H-50,
H-6a0, H-6b0, H-300, H-400, H-500, OCH2CH2), 3.36 (m,
1H, H-200), 2.11–2.04 (m, 2H, CH2CH@CH2), 2.07,
2.04 (2s, 6H, COCH3), 1.68–1.62 (m, 2H, OCH2CH2);
13C NMR (D2O): d 176.16 (COONa), 175.17, 175.03
(COCH3), 139.23 (CH@CH2), 115.52 (CH@CH2),
102.83 (C-100), 101.88 (C-10), 101.58 (C-1), 79.83, 79.13
0
0
00 00
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