Glycosidase-catalyzed synthesis of fucosyl di- and trisaccharide derivatives using α-L-fucosidase from Alcaligenes sp.

Article abstract of DOI:10.1081/CAR-120023474

p-Nitrophenyl glycosides of di-and trisaccharides containing a (1→2)-, (1→3)-, (1→4)-, or (1→6)-linked α-L-fucosyl group were synthesized using the transglycosylation reaction mediated by α-L-fucosidase from Alcaligenes sp. with p-nitrophenyl glycosides of N-acetyllactosamine, lactose, D-GlcNAc, and D-Glc as acceptors. The enzymatic process for preparing these compounds is simple, and the yield is sufficiently high to make the method practical.

Full text of DOI:10.1081/CAR-120023474

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Glycosidase‐Catalyzed Synthesis of Fucosyl Di‐ and
Trisaccharide Derivatives Using α‐l‐Fucosidase from
Alcaligenes sp
Xiaoxiong Zeng a b , Takeomi Murata a & Taichi Usui a
a Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University,
Ohya 836, Shizuoka, 422‐8529, Japan
b Laboratory of Advanced Bioelectronics, National Institute of Advanced Industrial Science
and Technology, Higashi 1‐1‐1, Tsukuba Central 5, Tsukuba, 305‐8565, Japan
Available online: 16 Aug 2006
To cite this article: Xiaoxiong Zeng, Takeomi Murata & Taichi Usui (2003): Glycosidase‐Catalyzed Synthesis of Fucosyl Di‐ and
Trisaccharide Derivatives Using α‐l‐Fucosidase from Alcaligenes sp , Journal of Carbohydrate Chemistry, 22:5, 309-316
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JOURNAL OF CARBOHYDRATE CHEMISTRY
Vol. 22, No. 5, pp. 309–316, 2003
Glycosidase-Catalyzed Synthesis of Fucosyl Di- and
Trisaccharide Derivatives Using a-L-Fucosidase
from Alcaligenes sp.
*
Xiaoxiong Zeng, Takeomi Murata, and Taichi Usui
Department of Applied Biological Chemistry, Faculty of Agriculture,
Shizuoka University, Shizuoka, Japan
ABSTRACT
p-Nitrophenyl glycosides of di-and trisaccharides containing a (1!2)-, (1!3)-,
(1!4)-, or (1!6)-linked a-L-fucosyl group were synthesized using the transglyco-
sylation reaction mediated by a-L-fucosidase from Alcaligenes sp. with p-nitrophenyl
glycosides of N-acetyllactosamine, lactose, D-GlcNAc, and D-Glc as acceptors. The
enzymatic process for preparing these compounds is simple, and the yield is
sufficiently high to make the method practical.
Key Words: Transglycosylation; Regioselectivity; a-L-Fucosidase; Fucosyl
oligosaccharide.
*
Correspondence: Xiaoxiong Zeng, Laboratory of Advanced Bioelectronics, National Institute of
Advanced Industrial Science and Technology, Higashi 1-1-1, Tsukuba Central 5, Tsukuba 305-
8565, Ibaraki, Japan; Fax: 81-298-61-4680; E-mail: zeng.x@aist.go.jp.
309
DOI: 10.1081/CAR-120023474
0732-8303 (Print); 1532-2327 (Online)
www.dekker.com
Copyright D 2003 by Marcel Dekker, Inc.

310
Zeng, Murata, and Usui
INTRODUCTION
The L-sugar fucose is an important recognition component of cell-surface glycans.
A number of L-fucose-containing glycoconjugates have been reported to be involved in
a variety of biological functions, such as growth regulation, receptor function, cell-cell
interaction, and antigenicity.[1] To elucidate the biological functions of L-fucose-
containing glycoconjugates, great attention have been paid to the chemical and
enzymatic synthesis of fucosyl oligosaccharides. Chemical procedures have been
extensively developed,[2–4] but they are frequently complicated due to multiple steps of
protection and deprotection. In contrast, protection and deprotection are not required for
enzymatic procedures. The enzymatic approach has been done in part with glycosyl-
transferases and glycosidases.[5] From a practical viewpoint, the use of glycosidases is
attractive for oligosaccharides synthesis because glycosidases are inexpensive, stable,
and readily available. In the case of a-L-fucosidase, various sources of a-L-fucosidase
have been investigated for the ability to transfer a fucose residue to D-Gal, D-Glc, and
D-GlcNAc.[6–11] In our previous works, we synthesized a-L-Fuc-(1!3)-b-D-Gal-(1!4)-
D-GlcNAc, a-L-Fuc-(1!3)-b-D-Gal-(1!4)-D-Glc, and their analogues through the trans-
glycosylation reaction mediated by a-L-fucosidase from Alcaligenes sp.[8] Here, we
describe the transglycosylation mediated by a-L-fucosidase from Alcaligenes sp. for
the synthesis of p-nitrophenyl glycosides of fucosyl di- and trisaccharides that could
be useful as starting substances for glycopolymers[12,13] or as intermediates for further
sugar elongation.
RESULTS AND DISCUSSIONS
It has been reported that a-L-fucosidase from Alcaligenes sp. mediates the
regioselective transglycosylation from a-L-Fuc-OC6H4NO2-p 1 to the 3-position of the
Gal moiety of b-D-Gal-(1!4)-b-D-GlcNAc (acetyllactosamine), lactose, b-D-Gal-
(1!4)-b-D-GlcNAc-OMe, b-D-Gal-(1!4)-b-D-Glc-OMe, and b-D-Gal-(1!3)-b-D-Glc-
OMe as the acceptors. The enzyme, however, shows hydrolytic activity to a-L-Fuc-
(1!2)-b-D-Gal-(1!4)-D-Glc (2’-fucosyllactose), and the relative hydrolysis rate of 2’-
fucosyllactose is 77.5 as compared to that of a-L-Fuc-(1!3)-b-D-Gal-(1!4)-D-Glc
whose hydrolytic rate is set arbitrarily at 100.[8] Thus, it should form a(1!2)-linked
transfer product rather than the a(1!3)-linked product by transglycosylation. Therefore,
we chose the p-nitrophenyl derivatives of N-acetyllactosamine, lactose, D-Glc, and D-
GlcNAc as acceptors in order to investigate the regioselectivity of the enzyme. The
rational choice of p-nitrophenyl derivative as an acceptor was advantageous for detecting
and separating transfer products by chromatography due to the presence of the p-
nitrophenyl group.
At first, 1 and b-D-Gal-(1!4)-b-D-GlcNAc-OC6H4NO2-p 2, which was prepared
as described previously,[14] were incubated with a-L-fucosidase from Alcaligenes sp. at
50°C. The extent of enzymatic conversion was monitored in the course of reaction by
HPLC of aliquots (10 mL) taken from the incubation mixture. The chromatogram
(Figure 1) showed that two transfer products (26.091 min and 30.809 min, respec-
tively) were formed during the incubation. The reaction mixture was separated by ODS
and Toyopearl HW-40S, giving two products in a total yield of 38% based on 1 added.

Synthesis of Fucosyl Di- and Trisaccharide Derivatives
311
Figure 1. HPLC chromatogram of Alcaligenes sp. a-L-fucosidase-mediated transglycosylation
reaction mixture with 1 and 2 as substrates. 1: compound 2, 2: compound 4, 3: compound 3, 4:
p-nitrophenol, 5: compound 1.

312
Zeng, Murata, and Usui
Scheme 1. Formation of fucosyl trisaccharides 3 and 4 by Alcaligenes sp. a-L-fucosidase-
mediated transglycosylation.
The a(1!3)-linked product 4 as a main transfer product along with a(1!2)-linked
isomer 3 was formed in a ratio of 97:3 (Scheme 1, Table 1). By replacing b-D-Gal-
(1!4)-b-D-Glc-OC6H4NO2-p 5[15] with 2 as acceptor, the a(1!2)-linked transfer
product was also detected. However in this case, the a(1!6)-linked isomer was
formed in addition to the a(1!2)-and a(1!3)-linked products. The three transfer
products 6–8 were obtained in an overall yield of 38% and in a molar ratio of 8:88:4
(Scheme 2, Table 1). In both cases, the fucosylation favored the 3-position of the Gal
moiety rather than the 2-position.
When b-D-Glc-OC6H4NO2-p 9 was used as the acceptor instead of 2, the a(1!3)-
linked product 10 was the main product along with the a(1!4)-linked isomer 11. The
two products were obtained in 53% overall yield and in a molar ratio of 85:15. When
b-D-GlcNAc-OC6H4NO2-p 12 was used as acceptor, the a(1!3)-(13) and a(1!4)-
linked (14) transfer products were obtained in a ratio of 91:9, and the yield (13%
overall yield) was much lower than that obtained with 9 as the acceptor (Scheme 3,
Table 1). It appears that the regioselectivity of fucosyl transfer to the D-GlcNAc moiety
is not influenced by the presence of the acetyl group. The low yield may be due to the
low solubility of 12 (<1%) in aqueous solution as compared to the higher aqueous
Table 1. Transfer products and yields mediated by a-L-fucosidase from Alcaligenes sp.
Acceptor
Product
Yield* (%)
2
3
a-L-Fuc-(1!2)-b-D-Gal-(1!4)-D-GlcNAc-OC6H4NO2-p
a-L-Fuc-(1!3)-b-D-Gal-(1!4)-D-GlcNAc-OC6H4NO2-p
a-L-Fuc-(1!2)-b-D-Gal-(1!4)-D-Glc-OC6H4NO2-p
a-L-Fuc-(1!3)-b-D-Gal-(1!4)-D-Glc-OC6H4NO2-p
a-L-Fuc-(1!6)-b-D-Gal-(1!4)-D-Glc-OC6H4NO2-p
a-L-Fuc-(1!3)-b-D-Glc-OC6H4NO2-p
1
37
3
4
5
6
7
34
1
8
9
10
11
13
14
45
8
a-L-Fuc-(1!4)-b-D-Glc-OC6H4NO2-p
12
a-L-Fuc-(1!3)-b-D-GlcNAc-OC6H4NO2-p
12
1
a-L-Fuc-(1!4)-b-D-GlcNAc-OC6H4NO2-p
*Isolated yield based on the donor substrate added.

Synthesis of Fucosyl Di- and Trisaccharide Derivatives
313
Scheme 2. Formation of fucosyl trisaccharides 6–8 by Alcaligenes sp. a-L-fucosidase-mediated
transglycosylation.
solubility of 9 (>10%), because it is known that the efficiency of the transglycosylation
process is dependent on an excess of substrate.
With p-nitrophenyl glycoside as acceptor, a-L-fucosidase from Alcaligenes sp.
catalyzed the transfer of the a-L-fucosyl group to the 2-, 3-, 6-positions of the D-Gal
residue, and to the 3- and 4-positions of the D-Glc or D-GlcNAc residues. The transfer
products were detected and separated easily by chromatography due to the presence of
Scheme 3. Formation of fucosyl disaccharides 10, 11, 13, 14 by Alcaligenes sp. a-L-fucosidase-
mediated transglycosylation.

314
Zeng, Murata, and Usui
the p-nitrophenyl group. The enzymatic process for preparation of fucosyl di- and
trisaccharide derivatives is simple and the yield is sufficiently high to make the method
practical. These synthesized compounds will be useful as starting substances for
glycopolymers that are valuable for investigation of their biological recognition phe-
nomena by lectins or as intermediates for further sugar elongation.
EXPERIMENTAL SECTION
Analytical methods. HPLC was done with a Jasco Gulliver series liquid
chromatography with a column of Mightsyl RP-18 GP (5ꢀ150 mm, Kanto Chemical
1
Co., Inc., Tokyo, Japan) eluted with 10% MeOH at a flow rate of 1.0 mL/min. H and
13C NMR spectra were recorded with a Jeol JNM-EX 270 Fourier transform NMR
spectrometer at 30°C. Chemical shifts are expressed in d units relative to sodium 4,4-
dimethyl-4-silapentanoate (TPS) as the external standard in D2O. Specific rotation was
determined with a Digital Automatic Polarimeter DIP-1000 apparatus (Jasco, Japan).
Enzyme assay. The activity of a-L-fucosidase from Alcaligenes sp. (kindly
supplied by Kumiai Chemical Industry Co., Ltd., Shizuoka, Japan) was assayed as
follows. A mixture containing 0.4 mM 1 in 0.9 mL of 50 mM potassium phosphate
buffer (pH 7.0) and an appropriate amount of enzyme in a total volume of 1.0 mL was
incubated for 10 min at 50°C. Adding 0.5 mL of 1.0 M Na2CO3 solution stopped the
reaction, and the liberated p-nitrophenol was determined spectrophotometrically at 410
nm. One unit of enzyme activity was defined as the amount of enzyme that hydrolyzes
1 mmol of 1 per min.
-L-Fuc-(1 2)-b-D-Gal-(1 4)-b-D-GlcNAc-OC H NO -p(3) and -L-Fuc-(1
!
!
!
6
4
2
3)-b-D-Gal-(1 4)-b-D-GlcNAc-OC H NO -p (4). To a solution of 1 (50 mg) and 2
!
6
4
2
(200 mg) in 12 mL of 0.1 M potassium phosphate buffer (pH 7.0) containing 0.8 mL of
dimethyl sulfoxide (DMSO) was added a-L-fucosidase from Alcaligenes sp. (0.47 U).
The reaction mixture was incubated for 80 h at 50°C, and heating in a boiling water
bath for 5 min terminated the reaction. The resulting insoluble material was filtered off.
The solution was directly loaded onto a Chromatorex-ODS DM 1020T column (3ꢀ50
cm) equilibrated with 10% MeOH in aqueous solution. The column was eluted with the
same solution. The fractions (tubes 41–75, 60 mL/tube) were collected, concentrated,
and loaded onto a column of Toyopearl HW-40S (4.5ꢀ90 cm) equilibrated with 25%
MeOH in water. The column was eluted with the same solution. F-1 (tubes 89–93, 20
mL/tube) and F-2 (tubes 95–101) were collected, concentrated, and lyophilized to
afford compounds 3 (1.2 mg) and 4 (40.8 mg), respectively. NMR data for compound
1
3: H NMR d 8.30 (d, 2H, J 9.2 Hz, m-Ph), 7.24 (d, 2H, J 9.2 Hz, o-Ph), 5.40 (d, 1H, J
4.3 Hz, H-1@), 4.63 (d, 1H, J 7.3 Hz, H-1), 2.09 (s, 3H, Me of Ac), and 1.31 (3H, Me
of fucosyl residue). 13C NMR d 177.50 (C O of Ac), 164.58 (Ph carbon attached to
the phenolic oxygen), 145.69 (p-Ph carbon), 129.05 (m-Ph carbon), 119.53 (o-Ph
carbon), 103.27 (C-1’), 102.33 (C-1), 101.54 (C-1@), 79.33 (C-4), 78.79 (C-2’), 78.63
(C-5’), 78.24 (C-5), 76.49 (C-3@), 74.63 (C-4@, C-3), 72.59 (C-3@), 72.06 (C-4@), 71.17
(C-2@), 69.92 (C-5@), 64.04 (C-6’), 62.93 (C-6), 57.74 (C-2), 25.03 (Me of Ac), and
1
18.29 (C-6@). Compound 4 had [a]D25-75.6 (c 0.7, water). NMR data: H NMR d 8.24

Synthesis of Fucosyl Di- and Trisaccharide Derivatives
315
(d, 2H, J 9.2 Hz, m-Ph), 7.18 (d, 2H, J 9.2 Hz, o-Ph), 5.33 (d, 1H, J 8.4 Hz, H-1’), 5.17
(d, 1H, J 3.8 Hz, H-1@), 4.57 (d, 1H, H-1), 2.02 (s, 3H, Me of Ac), and 1.20 (3H, Me of
fucosyl residue). 13C NMR d 177.79 (C O of Ac), 164.54 (Ph carbon attached to the
phenolic oxygen), 145.57 (p-Ph), 128.97 (m-Ph), 119.39 (o-Ph), 105.55 (C-1’), 103.79
(C-1), 101.38 (C-1@), 83.18 (C-3’), 80.86 (C-4), 78.17 (C-5’), 78.00 (C-5), 74.91 (C-3),
73.29 (C-2’), 72.27 (C-3@), 71.50 (C-4’), 71.28 (C-2@), 70.04 (C-5@), 63.83 (C-6’), 62.66
(C-6), 57.72 (C-2), 24.94 (Me of Ac), and 18.20 (C-6@).
-L-Fuc-(1 2)-b-D-Gal-(1 4)-b-D-Glc-OC H NO -p (6), -L-Fuc-(1 3)-b-D-
!
!
!
6
4
2
Gal-(1 4)-b-D-Glc-OC H NO -p (7), and -L-Fuc-(1 6)-b-D-Gal-(1 4)-b-D-Glc-
!
!
!
6
4
2
OC6H4NO2-p (8). To a solution of 1 (100 mg) and 5 (500 mg) in 24 mL of 0.1 M
potassiumphosphate buffer (pH7.0) containing1.0mL of DMSO was added a-L-fucosidase
from Alcaligenes sp. (1.0 U). The mixture solution was incubated for 68 h at 50°C;
heating in a boiling water bath for 5 min terminated the reaction. The reaction mixture
was treated with Chromatorex-ODS DM 1020T and Toyopearl HW-40S columns as
described as above to afford 6 (6.5 mg), 7 (69.6 mg), and 8 (2.7 mg). The NMR data of
6–8 were identical to those corresponding compounds reported previously.[16]
-L-Fuc-(1 3)-b-D-Glc-OC H NO -p (10), -L-Fuc-(1 4)-b-D-Glc-OC H NO -
!
!
6
4
2
6
4
2
p (11), -L-Fuc-(1 3)-b-D-GlcNAc-OC H NO -p (13), and -L-Fuc-(1 4)-b-D-
!
!
6
4
2
GlcNAc-OC6H4NO2-p (14). In a similar manner, compounds 10, 11, 13, and 14 were
prepared from 9 and 12 as acceptors, respectively, by use of the transglycosylation of
a-L-fucosidase from Alcaligenes sp. with 1 as the donor. The NMR data of these
compounds were identical to those corresponding compounds reported previously.[16]
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Received May 31, 2002
Accepted March 13, 2003