Enzymatic synthesis of galactose-containing disaccharides employing β- galactosidase from Bacillus circulans

Article abstract of DOI:10.1002/(sici)1099-0690(199911)1999:11<3073::aid-ejoc3073>3.0.co;2-5

Galβ1→4 disaccharide structures are vital core units of the oligosaccharide components of glycoconjugates. β-Galactosidase from Bacillus circulans (E.C.3.2.1.23) catalyses the transfer of galactose from a donor structure such as GalβOpNP to various GlcNAc

Full text of DOI:10.1002/(sici)1099-0690(199911)1999:11<3073::aid-ejoc3073>3.0.co;2-5

FULL PAPER
Enzymatic Synthesis of Galactose-Containing Disaccharides Employing
β-Galactosidase from Bacillus circulans
[a]
[a]
´
Erzsebet Farkas and Joachim Thiem*
Keywords: Carbohydrates / β-Galactosidase / Bacillus circulans / Galβ1Ǟ4GlcNAc / Galβ1Ǟ4Gal / Enzyme catalysis
Galβ1Ǟ4 disaccharide structures are vital core units of the
oligosaccharide components of glycoconjugates. β-Galactosi-
rides {Galβ1Ǟ4GlcNAcαOAll (3), Galβ1Ǟ4GlcNAcβOAll (5),
Galβ1Ǟ4GalαOAll (10), Galβ1Ǟ4GalβOAll (12), Galβ1Ǟ
dase from Bacillus circulans (E.C.3.2.1.23) catalyses the transfer 4GalβSPh (14), Galβ1Ǟ4GalβOpNP (16) and the trisaccharide
of galactose from a donor structure such as GalβOpNP to Galβ1Ǟ4Galβ1Ǟ4GalβOpNP (18)} has been achieved in 30–66
various GlcNAc and galactose derivatives, forming β1Ǟ4 lin- % yield by application of this enzyme.
kages. The synthesis of several biologically relevant disaccha-
ases to oligosaccharide synthesis has been restricted by their
Introduction
sensitivity, rarity, the requirement for expensive nucleotide
donors, and cofactors, including cofactor regeneration.
In nature, glycosylhydrolases (glycosidases) usually cleave
oligosaccharides to yield monosaccharides, but under un-
natural conditions the equilibrium of the reversible cleavage
can be shifted.[10Ϫ13] Thus, glycosylhydrolases can be used
to form interglycosidic linkages under appropriate reaction
conditions (transglycosylation). Glycosidase-catalysed reac-
tions have the advantage of high enzyme stability and sim-
ple reaction conditions, and are relatively low-cost.
The oligosaccharide components of glycoproteins and
glycolipids play key roles in many vital biological processes,
such as cellϪcell recognition and interaction, cell communi-
cation and growth regulation.[1] Oligosaccharides with
Galβ1Ǟ4 linkages are fundamental constituents of various
biologically important glycoconjugates. For example, N-
acetyllactosamine (Galβ1Ǟ4GlcNAc) is a typical terminal
sequence of N-linked oligosaccharides and a core unit of
carbohydrates isolated from human milk. It has been found
in sialooligosaccharides, such as sialyl Lewisx, as well as in
ligands of various lectins.[2][3] Biologically active
Galβ1Ǟ4Gal structures[4] can be isolated by partial hy-
drolysis of several of the polysaccharides obtained from the
white birch (Betula papyrifera).[5][6] The need to investigate
such compounds further is a powerful incentive to develop
a synthesis of such oligosaccharides, and their analogues,
inexpensively and in sufficient quantities.
β-Galactosidase from Bacillus circulans[14Ϫ23] catalyses
the transfer of galactose from GalβOpNP or lactose pre-
dominantly to the OH-4 position of Glc, GlcNAc, Gal, and
GalNAc. We report here the enzymatic synthesis of the
following di- and trisaccharides using β-galactosidase from
Bacillus
Galβ1Ǟ4GlcNAcβOAll (5); Galβ1Ǟ4GalαOAll (10);
Galβ1Ǟ4GalβOAll (12); Galβ1Ǟ4GalβSPh (14);
circulans:
Galβ1Ǟ4GlcNAcαOAll
(3);
Galβ1Ǟ4GalβOpNP (16); Galβ1Ǟ4Galβ1Ǟ4GalβOpNP
(18).
The isolation of Galβ1Ǟ4-linked oligosaccharides from
natural sources is both labour-intensive and difficult. Be-
cause of their low concentrations in complex natural prod-
uct mixtures, such an approach is uneconomic on a large
scale. Access to the desired Galβ1Ǟ4-linked structures by
classical organic synthetic methodologies is often restricted
by the need to perform laborious protective-group chemis-
try, which requires multistep syntheses resulting in low over-
all yields. In recent years, the use of enzymes has been inves-
tigated as an alternative to classical synthesis, following the
biochemical pathways of carbohydrate metabolism.[7Ϫ9]
Two groups of enzymes can be used for the synthesis of
glycosidic bonds: glycosyltransferases and glycosylhydrol-
ases. Glycosyltransferases[10Ϫ12] act as in vivo biocatalysts
and attach monosaccharide units to sugar chains, via the
activated nucleotide components, with high stereo- and re-
giospecificity. To date, the application of glycosyl transfer-
Results and Discussion
The chemoenzymatic synthesis of Galβ1Ǟ4 structures
was achieved by transglycosylation using β--galactosidase
from Bacillus circulans (E.C.3.2.1.23). The donor p-nitro-
phenyl β--galactopyranoside (GalβOpNP) was transferred
to the 4-position of the appropriate glycosyl acceptor unit,
resulting in the required β1Ǟ4 linkage. The enzymatic gly-
cosylation was successfully applied to the following ac-
ceptors: GlcNAcαOAll[24] (1), GlcNAcβOAll[24] (2), Ga-
lαOAll[24] (7), GalβOAll[24] (8) and GalβSPh[25] (9).
In order to optimise the reaction conditions, the effect
of the donor-acceptor ratio, buffer pH, reaction time and
temperature were examined in a comparison with the yield
of the desired disaccharide product. The optimised reaction
conditions were found to involve a 1:7.5 donor/acceptor
mixture which was incubated in a 1:1 sodium phosphate
buffer/acetonitrile mixture with 0.7Ϫ1.0 U β--galactosid-
[a]
Institut für Organische Chemie, Universität Hamburg,
Martin-Luther-King-Platz 6, D-20146 Hamburg, Germany
Fax: (internat.) ϩ 49-(0)40/42838-4325
E-mail: thiem@chemie.uni-hamburg.de
Eur. J. Org. Chem. 1999, 3073Ϫ3077
WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999
1434Ϫ193X/99/1111Ϫ3073 $ 17.50ϩ.50/0
3073

E. Farkas, J. Thiem
FULL PAPER
Scheme 1. Galactosylation of GlcNAc derivatives with β-galactosidase from Bacillus circulans
Scheme 2. Galactosylation of galactose derivatives with β-galactosidase from Bacillus circulans
ase (E.C.3.2.1.23) for 72 h at 30°C. The reaction was ter- (664 m) and an increased amount of enzyme (25 U), for-
minated by heating to 90°C for 10 min, and worked up by mation of the disaccharide (16) and trisaccharide
extraction with ethyl acetate to remove p-nitrophenol fol- Galβ1Ǟ4Galβ1Ǟ4GalβOpNP (18) was observed in 23%
lowed by lyophilisation and purification by column chroma- and 10% yields, respectively. The reaction was stopped after
tography (Biogel P2). After lyophilisation the residue was 2 h and the products were isolated and purified as above.
acetylated, the product was isolated and purified by silica
gel chromatography. The disaccharides 3, 5, 10, 12 and 14
were isolated in 66, 30, 55, 49, and 63% yields, respectively.
These yields are more than satisfactory, especially in light
Conclusion
of the simplicity of the method.
β--galactosidase from Bacillus circulans (E.C.3.2.1.23)
In the course of optimising the reaction, other donor- has proved to be an excellent catalyst for the formation of
acceptor ratios were examined. When a 3:2 donor/acceptor Galβ1Ǟ4-linked structures. An efficient method has been
system was employed, the disaccharide Galβ1Ǟ4Galβ- developed for the one-step synthesis of N-acetyllactosamine
OpNP (16) was formed (< 4% yield). This self-galactosyl- and analogous Galβ1Ǟ4Gal oligosaccharides in excellent
ation is attributed to the high donor concentration, and was (30Ϫ66%) yields. The allyl glycosides thus synthesised (3,
investigated further by using GalβOpNP both as donor and 5, 10, 12) can easily be coupled to BSA or other proteins
acceptor. Employing a high GalβOpNP concentration to form the corresponding glycoprotein. The thiophenyl ga-
3074
Eur. J. Org. Chem. 1999, 3073Ϫ3077

Enzymatic Synthesis of Galactose-Containing Disaccharides
FULL PAPER
Scheme 3. Galactosylation with β-galactosidase from Bacillus circulans using GalβOpNP as both donor and acceptor substrate
Allyl β-D-galactopyranosyl-(1Ǟ4)-2-acetamido-2-deoxy-␣-D-glucop-
lactoside 14 and p-nitrophenyl galactosides 16 and 18 can
be used for the construction of larger oligosaccharides. It is
to be expected that the methodology developed herein can
be applied to the synthesis of any number of analogous
Galβ1Ǟ4GlcNAc and Galβ1Ǟ4Gal structures.
yranoside (3): To compound 1[24] (81 mg, 0.31 mmol) in a 1:1 solu-
tion of 50 m sodium phosphate buffer (pH ϭ 7.0) and acetonitrile
(0.8 mL) was added p-nitrophenyl β--galactopyranoside (14 mg,
0.046 mmol). The mixture was incubated with β-galactosidase from
Bacillus circulans (0.7 U) at 30°C. After 72 h the reaction was
stopped by heating at 90°C for 10 min. The solution was extracted
with ethyl acetate to remove p-nitrophenol and lyophilised. The
residue was applied to a Biogel P2 column and eluted with water
to give compound 3 (13 mg, 66%). Ϫ TLC (ethyl acetate/methanol/
Experimental Section
General Remarks: The reactions were monitored by TLC analysis
with silica gel plates (Kieselgel 60 F254, Merck). Compounds were
visualised by spraying with 20% sulfuric acid in ethanol, followed
by charring at 150°C and/or UV irradiation. Ϫ Column chroma-
tography was performed on silica gel 60 M (0.040Ϫ0.063 mm,
Merck) or Biogel P2. Ϫ Optical rotations were determined at room
temperature with a PerkinϪElmer 241 and 341 polarimeter. Ϫ
NMR spectra were accumulated with a Bruker AMX 400 spec-
trometer. Chemical shifts are given in ppm (δ). Ϫ Mass spectra
were recorded with a Bruker MALDI-TOF mass spectrometer
(with N2 laser operating at 337 nm and 5 µL of 2,5-dihydroxyben-
zoic acid as matrix) and FAB with a VG 7035 mass spectrometer.
Ϫ β-Galactosidase from Bacillus circulans (E. C. 3. 2. 1. 23) was
purchased from ”Biolacta” Daiwa Kasei Co., Ltd., Osaka, Japan,
and p-nitrophenyl β--galactopyranoside from Sigma, Germany.
20
water, 7:3:1): Rf ϭ 0.59. Ϫ [α]D ϭ ϩ 93.3 (c ϭ 0.15, H2O). Ϫ 1H
NMR (400 MHz, D2O): δ ϭ 5.75 (m, 1 H, CHϭ), 5.10 (dd, 2 H,
CH2ϭ), 4.74 (d, 1 H, J1,2 ϭ 3.1 Hz, 1-H), 4.28 (d, 1 H, J1Ј,2Ј
ϭ
7.6 Hz, 1Ј-H), 4.03 (dd, 1 H, CH2), 3.84 (dd, 1 H, CH2), 3.48 (dd,
1 H, 3Ј-H), 3.35 (dd, 1 H, 2Ј-H), 1.84 (s, 3 H, CH3CONH). Ϫ
MALDI-TOF MS: C17H29NO11 (423.42): m/z 424 [M ϩ H]ϩ.
Allyl (2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-(1Ǟ4)-2-aceta-
mido-3,6-di-O-acetyl-2-deoxy-␣-D-glucopyranoside (4): Compound
3 (13 mg, 0.03 mmol) was dissolved in pyridine (1 mL), and acetic
anhydride (1 mL) and DMAP (10 mg) were added. The reaction
mixture was stirred at room temperature for 12 h and dried in va-
cuo. The residue was co-evaporated with toluene, concentrated, and
purified by column chromatography (dichloromethane/acetone,
20
85:15) to yield compound 4 (13 mg, 63%). Ϫ [α]D ϭ ϩ 33.3 (c ϭ
1
Enzyme Assay: Enzyme solution (0.2 mL, 0.12 g enzyme powder/
0.13, CHCl3). Ϫ H NMR (400 MHz, CDCl3): δ ϭ 5.80 (m, 1 H,
mL) was added to p-nitrophenyl β--galactopyranoside {0.8 mL of CHϭ), 5.62 (d, 1 H, NH), 5.28Ϫ5.15 (m, 4 H, 4Ј-H, CH2ϭ, 3-H),
a 12.2 m solution in 2  sodium acetate buffer (pH ϭ 6.0)} and
the mixture was incubated at 37°C for 15 min. The reaction was
stopped by adding 1 mL of 0.1  Na2CO3 solution and the liber-
5.06 (dd, 1 H, J2Ј,3Ј ϭ 7.6 Hz, 2Ј-H), 4.89 (dd, 1 H, J3Ј,4Ј ϭ 3.6 Hz,
3Ј-H), 4.74 (d, 1 H, J1,2 ϭ 3.6 Hz, 1-H), 4.45 (d, 1 H, J1Ј,2Ј
8.1 Hz, 1Ј-H), 4.39Ϫ4.36 (dd, 1 H, 6aЈ-H,), 4.17 (m, 1 H, J2,3
ϭ
ϭ
ated p-nitrophenol was determined spectrophotometrically. The ab- 10.7 Hz, 2-H), 4.10 (m, 1 H, CH2), 4.11Ϫ3.99 (m, 3 H, 6a,b-H,
sorbance was measured in cells with 1 cm light path at 420 nm.
One unit of activity was defined as the amount of the enzyme re-
leasing 1µmol p-nitrophenol per min.
6bЈ-H), 3.90 (m, 1 H, CH2), 3.83Ϫ3.78 (m, 2 H, 5-H, 5Ј-H), 3.70
(m, 1 H, 4-H), 2.08Ϫ1.88 (m, 21 H, 6 CH3CO, CH3CONH). Ϫ
13C NMR (100.62 MHz, CDCl3): δ ϭ 169.9Ϫ168.1 (CϭO), 132.04
Eur. J. Org. Chem. 1999, 3073Ϫ3077
3075

E. Farkas, J. Thiem
FULL PAPER
(CHϭ), 117.48 (CH2ϭ), 100.20 (1Ј-C), 95.19 (1-C), 75.34 (4-C), H,
7
CH3CO).
Ϫ δ ϭ
13C NMR (100.62 MHz, CDCl3):
70.61 (3-C), 70.00 (3Ј-C), 69.57 (5-C), 68.16 (2Ј-C), 67.71 (CH2),
168.8Ϫ168.3 (CϭO), 132.3 (CHϭ), 116.8 (CH2ϭ), 100.8 (1Ј-C),
67.49 (5Ј-C), 65.55 (4Ј-C), 60.96 (6-C), 59.71 (6Ј-C), 51.00 (2-C), 94.3 (1-C), 74.1 (4-C), 69.7 (3Ј-C), 69.7 (5Ј-C), 69.3 (3-C), 67.6 (2Ј-
22.2Ϫ19.5 (CH3).
C), 67.4 (CH2), 66.7 (5-C), 66.6 (2-C), 65.9 (4Ј-C), 62.7 (6-C), 60.4
(6Ј-C), 19.8Ϫ19.5 (CH3). FAB-MS: C29H40O18 (676.63): m/z 678
[M ϩ H]ϩ.
Allyl (2,3,4,6-tetra-O-acetyl-β-
mido-3,6-di-O-acetyl-2-deoxy-β-
D
-galactopyranosyl)-(1Ǟ4)-2-aceta-
-glucopyranoside (6): To com-
D
pound 2[24] (120 mg, 0.46 mmol) in a 1:1 solution of 50 m sodium
phosphate buffer (pH ϭ 7.0) and acetonitrile (0.6 mL) was added
p-nitrophenyl β--galactopyranoside (21 mg, 0.07 mmol). The mix-
ture was incubated with β-galactosidase from Bacillus circulans
(0.9 U) at 30°C. After 72 h the reaction was stopped by heating at
90°C for 10 min. The solution was extracted with ethyl acetate to
remove p-nitrophenol and lyophilised. The residue was applied to
a Biogel P2 column and eluted with water to give allyl β--galacto-
Allyl β-D-galactopyranosyl-(1Ǟ4)-β-D-galactopyranoside (12): To
compound 8[24] (42 mg. 0.19 mmol) in a 1:1 solution of 50 m so-
dium phosphate buffer (pH ϭ 7.0) and acetonitrile (0.6 mL) was
added p-nitrophenyl β--galactopyranoside (8 mg, 0.026 mmol).
The solution was incubated with β-galactosidase from Bacillus cir-
culans (0.7 U) at 30°C. After 72 h the reaction was stopped by heat-
ing at 90°C for 10 min. The solution was extracted with ethyl acet-
ate to remove p-nitrophenol and lyophilised. The residue was ap-
plied to a Biogel P2 column and eluted with water to give com-
pound 12 (5 mg, 49%). Ϫ TLC (ethyl acetate/methanol/water,
pyranosyl-(1Ǟ4)-2-acetamido-2-deoxy-β--glucopyranoside
5
(9 mg, 30%). Ϫ TLC (ethyl acetate/methanol/water, 7:3:1): Rf ϭ
0.58. Ϫ Compound 5 (9 mg, 0.02 mmol) was dissolved in pyridine
(1 mL), and acetic anhydride (1 mL) and DMAP (10 mg) were ad-
ded. The reaction mixture was stirred at room temperature for 12 h
and dried in vacuo. The residue was co-evaporated with toluene,
concentrated, and purified by column chromatography (dichloro-
methane/acetone, 85:15) to yield compound 6 (9 mg, 63%). Ϫ
1
7:3:1): Rf ϭ 0.50. Ϫ H NMR (400 MHz, D2O): δ ϭ 5.82 (m, 1 H,
CHϭ), 5.17 (m, 2 H, CH2ϭ), 4.42 (d, 1 H, J1,2 ϭ 7.6 Hz, 1-H),
4.30 (d, 1 H, J1Ј,2Ј ϭ 7.6 Hz, 1Ј-H), 4.23Ϫ4.06 (m, 2 H, CH2).
Allyl (2,3,4,6-tetra-O-acetyl-β-
D-galactopyranosyl)-(1Ǟ4)-2,3,6-tri-
O-acetyl-β- -galactopyranoside (13): Compound 12 (5 mg,
D
0.013 mmol) was dissolved in pyridine (1 mL), and acetic anhydride
(1 mL) and DMAP (10 mg) were added. The reaction mixture was
stirred at room temperature for 12 h and dried in vacuo. The resi-
due was co-evaporated with toluene, concentrated, and purified by
column chromatography (dichloromethane/acetone, 94:6) to yield
20
1
[α]D ϭ Ϫ 25 (c ϭ 0.5, CHCl3). Ϫ H NMR (400 MHz, CDCl3):
δ ϭ 5.80 (m, 1 H, CHϭ), 5.55 (d, 1 H, NH), 5.29 (d, 1 H, 4Ј-H),
5.22Ϫ5.10 (m, 2 H, CH2ϭ), 5.08Ϫ4.98 (m, 2 H, 2Ј-H, 3-H), 4.91
α-(dd, 1 H, J3Ј,4Ј ϭ 3.6 Hz, 3Ј-H), 4.45 (m, 1 H, 6b-H), 4.43 (d, 1
H, J1Ј,2Ј ϭ 8.1 Hz, 1Ј-H), 4.42 (d, 1 H, J1,2 ϭ 7.1 Hz, 1-H), 4.24
(m, 1 H, CH2), 4.09Ϫ3.94 (m, 5 H, 2-H, 6a-H, 6Јa,b-H, CH2), 3.81
(m, 1 H, 5Ј-H), 3.72 (t, 1 H, 4-H), 3.55 (m, 1 H, 5-H), 2.08Ϫ1.90
(m, 21 H, 6 CH3CO, CH3CONH). Ϫ 13C NMR (100.62 MHz,
CDCl3): δ ϭ 169.2Ϫ168.3 (CϭO), 132.48 (CHϭ), 116.59 (CH2ϭ),
99.94 (1Ј-C), 98.70 (1-C), 74.53 (4-C), 71.57 (5-C), 71.15 (3-C),
69.78 (3Ј-C), 69.73 (5Ј-C), 69.59 (CH2), 68.05 (2Ј-C), 65.60 (4Ј-C),
61.37 (6Ј-C), 59.80 (6-C), 51.96 (2-C), 22.2Ϫ19.5 (CH3). Ϫ
MALDI-TOF MS: C29H41NO17 (675.64): m/z 677 [M ϩ H]ϩ.
20
compound 13 (5 mg, 57%). Ϫ [α]D ϭ Ϫ 8.1 (c ϭ 0.3, CHCl3). Ϫ
1H NMR (400 MHz, CDCl3): δ ϭ 5.79 (m, 1 H, CHϭ), 5.30 (d, 1
H, 4Ј-H), 5.23Ϫ5.09 (m, 3 H, J2Ј,3Ј ϭ 10.7 Hz, 2Ј-H, CH2ϭ), 5.04
(dd, 1 H, J2,3 ϭ 10.2 Hz, 2-H), 4.94 (dd, 1 H, 3Ј-H), 4.82 (dd, 1 H,
3-H), 4.40 (d, 1 H, J1,2 ϭ 8.1 Hz, 1-H), 4.37 (d, 1 H, J1Ј,2Ј ϭ 7.6 Hz,
1Ј-H), 4.30 (dd, 1 H, 6Јa-H), 4.25 (m, 1 H, CH2), 4.18 (dd, 1 H,
6Јb-H), 4.07Ϫ4.00 (m, 4 H, 4-H, 6a,b-H, CH2), 3.77 (m, 1 H, 5-
H), 3.62 (m, 1 H, 5Ј-H), 2.10Ϫ1.92 (m, 21 H, 7 CH3CO). Ϫ 13C
NMR (100.62 MHz, CDCl3): δ ϭ 169.6Ϫ168.3 (CϭO), 132.6
(CHϭ), 116.4 (CH2ϭ), 100.8 (1Ј-C), 98.5 (1-C), 73.3 (4-C), 72.3 (3-
C), 70.8 (5Ј-C), 69.7 (3Ј-C), 69.6 (5-C), 68.3 (CH2), 68.1 (2-C), 67.5
(2Ј-C), 65.8 (4Ј-C), 62.3 (6-C), 60.3 (6Ј-C), 19.9Ϫ19.5 (CH3). Ϫ
FAB-MS:C29H40O18 (676.63): m/z 678 [M ϩ H]ϩ, 700 [M ϩ Na]ϩ.
Allyl β-D-galactopyranosyl-(1Ǟ4)-␣-D-galactopyranoside (10): To
compound 7[24] (55 mg, 0.25 mmol) in a 1:1 solution of 50 m so-
dium phosphate buffer (pH ϭ 7.0) and acetonitrile (0.6 mL) was
added p-nitrophenyl β--galactopyranoside (10 mg, 0.033 mmol).
The solution was incubated with β-galactosidase from Bacillus cir-
culans (0.7 U) at 30°C. After 72 h the reaction was stopped by heat-
ing at 90°C for 10 min. The solution was extracted with ethyl acet-
ate to remove p-nitrophenol and lyophilised. The residue was ap-
plied to a Biogel P2 column and eluted with water to give com-
pound 10 (7 mg, 55%). Ϫ TLC (ethyl acetate/methanol/water,
Phenyl β-D-galactopyranosyl-(1Ǟ4)-1-thio-β-D-galactopyranoside
(14): To compound 9[25] (68 mg, 0.25 mmol) in a 1:1 solution of
50 m sodium phosphate buffer (pH ϭ 7.0) and acetonitrile
(0.6 mL) was added p-nitrophenyl β--galactopyranoside (10 mg,
0.033 mmol). The solution was incubated with β-galactosidase
from Bacillus circulans (0.7 U) at 30°C. After 72 h the reaction was
stopped by heating at 90°C for 10 min. The solution was extracted
with ethyl acetate to remove p-nitrophenol and lyophilised. The
residue was applied to a Biogel P2 column and eluted with water
to give compound 14 (9 mg, 63%). Ϫ TLC (ethyl acetate/methanol/
water, 7:3:1): Rf ϭ 0.52. Ϫ 1H NMR (400 MHz, D2O): δ ϭ 7.60
(m, 2 H, Ph), 7.35 (m, 3 H, Ph), 4.65 (d, 1 H, J1,2 ϭ 9.7 Hz, 1-H),
4.52 (d, 1 H, J1Ј,2Ј ϭ 7.6 Hz, 1Ј-H).
1
7:3:1): Rf ϭ 0.50. Ϫ H NMR (400 MHz, D2O): δ ϭ 6.03 (m, 1 H,
CHϭ), 5.40 (m, 2 H, CH2ϭ), 5.08 (d, 1 H, J1,2 ϭ 3.6 Hz, 1-H),
4.65 (d, 1 H, J1Ј,2Ј ϭ 7.6 Hz, 1Ј-H).
Allyl (2,3,4,6-tetra-O-acetyl-β-
D-galactopyranosyl)-(1Ǟ4)-2,3,6-tri-
O-acetyl-␣- -galactopyranoside (11): Compound 10 (7 mg,
D
0.018 mmol) was dissolved in pyridine (1 mL), and acetic anhydride
(1 mL) and DMAP (10 mg) were added. The reaction mixture was
stirred at room temperature for 12 h and dried in vacuo. The resi-
due was co-evaporated with toluene, concentrated, and purified by
Phenyl (2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-(1Ǟ4)-2,3,6-
tri-O-acetyl-1-thio-β- -galactopyranoside (15): Compound 14
D
column chromatography (dichloromethane/acetone, 94:6) to yield (9 mg, 0.021 mmol) was dissolved in pyridine (1 mL), and acetic
20
compound 11 (7 mg, 56%). Ϫ [α]D ϭ ϩ 54.1 (c ϭ 0.4, CHCl3).
anhydride (1 mL) and DMAP (10 mg) were added. The reaction
mixture was stirred at room temperature for 12 h and dried in va-
Ϫ 1H NMR (400 MHz, CDCl3): δ ϭ 5.86 (m, 1 H, CHϭ), 5.35 (d,
1 H, 4Ј-H), 5.28 (m, 1 H, CH2ϭ), 5.25Ϫ5.13 (m, 4 H, 3-H, 2Ј-H, cuo. The residue was co-evaporated with toluene, concentrated, and
2-H, CH2ϭ), 5.00 (d, 1 H, J1,2 ϭ 3.6 Hz, 1-H), 4.96 (dd, 1 H, 3Ј- purified by column chromatography (dichloromethane/acetone,
20
H), 4.38 (d, 1 H, J1Ј,2Ј ϭ 8.1 Hz, 1Ј-H), 4.32 (dd, 1 H, 6a-H),
95:5) to yield compound 15 (9 mg, 60%). Ϫ [α]D ϭ ϩ 2.7 (c ϭ
4.18Ϫ4.13 (m, 3 H, 4-H, 6b-H, CH2), 4.09Ϫ4.07 (m, 3 H, 6Јa,b-H, 0.5, CHCl3). Ϫ 1H NMR (400 MHz, CDCl3): δ ϭ 7.42 (m, 2 H,
5-H), 4.00 (m, 1 H, CH2), 3.82 (m, 1 H, 5Ј-H), 2.15Ϫ1.97 (m, 21 Ph), 7.23 (m, 3 H, Ph), 5.29 (d, 1 H, 4Ј-H), 5.15 (dd, 1 H, J2Ј,3Ј
ϭ
3076
Eur. J. Org. Chem. 1999, 3073Ϫ3077

Enzymatic Synthesis of Galactose-Containing Disaccharides
FULL PAPER
10.7 Hz, 2Ј-H), 5.06 (dd, 1 H, J2,3 ϭ 9.7 Hz, 2-H), 4.93 (dd, 1 H, (d, 1 H, J1Ј,2Ј ϭ 8.1 Hz, 1Ј-H), 4.31Ϫ4.26 (dd, 1 H, 6a-H),
3Ј-H), 4.86 (dd, 1 H, 3-H), 4.57 (d, 1 H, J1,2 ϭ 10.2 Hz, 1-H), 4.36 4.22Ϫ4.17 (dd, 1 H, 6Јb-H), 4.13Ϫ4.07 (m, 2 H, 4-H, 6b-H),
(d, 1 H, J1Ј,2Ј ϭ 8.1 Hz, 1Ј-H), 4.29Ϫ4.18 (m, 2 H, 6a,b-H), 4.08 4.05Ϫ3.97 (m, 3 H, 4Ј-H, 6ЈЈa,b-H), 3.84 (m, 1 H, 5Ј-H), 3.76 (m,
(d, 1 H, 4-H), 4.02 (d, 2 H, 6Јa,b-H), 3.77 (m, 1 H, 5Ј-H), 3.68 1 H, 5ЈЈ-H), 3.61 (m, 1 H, 5-H), 2.13Ϫ1.91 (m, 30 H, 10 CH3CO).
(m, 1 H, 5Ј-H), 2.10Ϫ1.93 (m, 21 H, 7 CH3CO). Ϫ 13C NMR
Ϫ
13C NMR (100.62 MHz, CDCl3): δ ϭ 169.70Ϫ168.05 (Cϭ
(100.62 MHz, CDCl3): δ ϭ 169.6Ϫ168.2 (CϭO), 131.8, 131.07, O),160.20 (Ph), 142.07 (p-Ph), 124.70, (2 C, m-Ph), 118.26,115.88
127.9, 126.8 (Ph), 100.81 (1Ј-C), 85.3 (1-C), 74.8 (5-C), 73.3 (4-C), (o-Ph), 100.76 (1ЈЈ-C), 100.61 (1Ј-C), 97.3 (1-C), 72.79 (4-C), 72.73
73.2 (3-C), 69.7 (5Ј-C), 69.7 (3Ј-C), 67.5 (2Ј-C), 66.5 (2-C), 65.9 (4Ј-C), 71.87 (3-C, 3Ј-C, 5-C), 71.27 (5ЈЈ-C), 69.79 (5Ј-C), 69.63
(4Ј-C), 62.7 (6-C), 60.3 (6Ј-C), 19.85Ϫ19.51 (CH3). Ϫ FAB-MS: (3ЈЈ-C), 67.84 (2Ј-C), 67.53 (2ЈЈ-C), 67.37 (2-C), 65.84 (4ЈЈ-C), 62.46
C32H40O17S (728.72): m/z 730 [M ϩ H]ϩ, 745 [M ϩ Na]ϩ.
p-Nitrophenyl β- -galactopyranosyl-(1Ǟ4)-β- -galactopyranoside
(16) and p-Nitro-phenyl β- -galactopyranosyl-(1Ǟ4)-β- -galactopy-
ranosyl-(1Ǟ4)-β- -galactopyranoside (18): p-Nitrophenyl β--gal-
actopyranoside (200 mg, 0.66 mmol) was incubated with β-galacto-
sidase from Bacillus circulans (25 U) in 1 mL of 50 m sodium
acetate buffer (pH ϭ 5.0) at 30°C. After 2 h the reaction was
stopped by heating at 90°C for 10 min. The reaction mixture was
lyophilised, the residue was applied to a Biogel P2 column and
eluted with water to give compounds 16 (36 mg, 23%) and 18
(15 mg, 10%).
(6ЈЈ-C, 6Ј-C), 60.27 (6-C), 19.8Ϫ1955 (CH3).
Ϫ FAB-MS:
C44H55NO28 (1045.91): m/z 1069 [M ϩ Na]ϩ.
D
D
D
D
D
Acknowledgments
Support of this study by the European Comission, FAIR CT
97Ϫ3142, programme NOFA, and the Fonds der Chemischen In-
dustrie is gratefully acknowledged.
[1]
A. Varki, Glycobiology 1993, 3, 97Ϫ130.
[2]
M. L. Phillips, E. Nudelman, F. C. A. Gaeta, M. Perez, A. K.
20
16: [α]D ϭ Ϫ 24.5 (c ϭ 0.1, H2O); 1H NMR (400 MHz, D2O):
Singhal, S. Hakomori, J. C. Paulson, Science 1990, 250,
1130Ϫ1131.
δ ϭ 8.11 (d, 2 H, Ph), 7.13 (d, 2 H, Ph), 4.94 (d, 1 H, J1,2 ϭ 7.6 Hz,
1-H), 4.40 (d, 1 H, J1Ј,2Ј ϭ 7.6 Hz, 1Ј-H).
[3]
G. Walz, A. Aruffo, W. Kolanus, M. Bevilacqua, B. Seed, Sci-
ence 1990, 1132Ϫ1135.
Compound 18 was identified and characterised only after acety-
lation.
[4]
C. T. M. Fransen, K. M. J. Van Laere, A. A. C. van Wijk, L.
P. Brüll, M. Dignum, J. E. Thomas-Oates, J. Haverkamp, A.
Schols, A. G. J. Voragen, J. P. Kamerling, J. F. G. Vliegenthart,
Carbohydr. Res. 1998, 314, 101Ϫ104.
p-Nitrophenyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-(1Ǟ4)-
[5]
2,3,6-tri-O-acetyl-β- -galactopyranoside (17): Compound 16
D
G. K. Gillham, A. S. Perlin, T. E. Timell, Can. J. Chem. 1958,
36, 1741Ϫ1743.
(30 mg, 0.065 mmol) was dissolved in pyridine (1 mL), and acetic
anhydride (1 mL) and DMAP (10 mg) were added. The reaction
mixture was stirred at room temperature for 12 h and dried in va-
cuo. The residue was co-evaporated with toluene, concentrated, and
purified by column chromatography (dichloromethane/acetone,
[6]
H. A. El-Shenawy, C. Schuerch, Carbohydr. Res. 1984, 131,
227Ϫ238.
[7]
J. Thiem, FEMS Microbiol. Rev. 1995, 16, 193Ϫ211.
[8]
D. G. Drueckhammer, W. J. Hennen, R. L. Pederson, C. F.
Barbas, C. M. Gautheron, T. Krach, C.-H. Wong, Synthesis
1991, 499Ϫ525.
20
95:5) to yield compound 17 (40 mg, 82%). Ϫ [α]D ϭ ϩ 9.1 (c ϭ
[9]
G. J. McGarvey, C.-H. Wong, Liebigs. Ann./Recueil 1997,
0.4, CHCl3). Ϫ 1H NMR (400 MHz, CDCl3): δ ϭ 8.15 (d, 2 H,
Ph), 7.00 (d, 2 H, Ph), 5.31 (d, 1 H, 4Ј-H), 5.32 (dd, 1 H, 2-H),
5.20 (dd, 1 H, 2Ј-H), 5.04 (d, 1 H, J1,2 ϭ 7.6 Hz, 1-H), 4.95 (m, 2
H, 3-H, 3Ј-H), 4.40 (d, 1 H, J1Ј,2Ј ϭ 8.1 Hz, 1Ј-H), 4.32 (dd, 1 H,
6a-H), 4.22 (dd, 1 H, 6b-H), 4.12 (d, 1 H, 4-H), 4.04 (m, 2 H, 6Јa,b-
H), 3.86 (m, 1 H, 5-H), 3.80 (m, 1 H, 5Ј-H), 2.13Ϫ1.94 (m, 21 H,
1059Ϫ1074.
[10]
G. L. Cote, B. Y. Tao, Glycoconjugate J 1990, 7, 145Ϫ162.
[11]
T. Murata, T. Usui, Biosci. Biotech. Biochem. 1997, 61,
1059Ϫ1066.
[12]
V. Kren, J. Thiem, Chem. Soc. Rev. 1997, 26, 463Ϫ473.
[13]
C. Bucke, J. Chem. Tech. Biotechnol. 1996, 67, 217Ϫ220.
[14]
S. Singh, M. Scigelova, G. Vic, D. H. G. Crout, J. Chem. Soc.,
13C NMR (100.62 MHz, CDCl3):
δ
ϭ
Perkin Trans. 1 1996, 1921Ϫ1926.
7
CH3CO).
Ϫ
[15]
T. Usui, S. Kubota, H. Ohi, Carbohydr. Res. 1993, 244,
169.53Ϫ168.04 (CϭO), 160.30 (Ph) 142.10 (p-Ph), 124.69, 124.35
(m-Ph), 118.53, 115.71 (o-Ph), 100.97 (1Ј-C), 97.27 (1-C), 72.92 (4-
C), 71.87 (3-C), 71.67 (5-C), 69.79 (5Ј-C), 69.60 (3Ј-C), 67.59 (2-
C), 67.54 (2Ј-C), 65.84 (4Ј-C), 62.31 (6-C), 60.31 (6Ј-C), 19.8Ϫ19.6
(CH3). Ϫ FAB-MS: C32H39NO20 (757.66): m/z 759 [M ϩ H]ϩ.
315Ϫ323.
[16]
A. Vetere, S. Paoletti, Biochem. Biophys. Res. Commun. 1996,
219, 6Ϫ13.
[17]
K. Sakai, R. Katsumi, H. Ohi, T. Usui, Y. Ishido, J. Carbohydr.
Chem. 1992, 11, 553Ϫ565.
[18]
G. F. Herrmann, Y. Ichikawa, C. Wandrey, F. C. A. Gaeta, J. C.
p-Nitrophenyl 2,3,4,6-tetra-O-acetyl-β-
2,3,6-tri-O-acetyl-β- -galactopyranosyl-(1Ǟ4)-2,3,6-tri-O-acetyl-
β- -galactopyranoside (19): Compound 18 (15 mg, 0.024 mmol)
D-galactopyranosyl-(1Ǟ4)-
Paulson, C.-H. Wong, Tetrahedron Lett. 1993, 34, 3091Ϫ3094.
[19]
T. Usui, S. Morimoto, Y. Hayakawa, M. Kawaguchi, T. Murata,
D
Y. Matahira, Y. Nishida, Carbohydr. Res. 1996, 285, 29Ϫ39.
D
[20]
T. Kimura, S. Takayama, H. Huang, C.-W. Wong, Angew.
was dissolved in pyridine (1 mL), and acetic anhydride (1 mL) and
DMAP (10 mg) were added. The reaction mixture was stirred at
room temperature for 12 h and dried in vacuo. The residue was
co-evaporated with toluene, concentrated, and purified by column
chromatography (dichloromethane/acetone, 9:1) to yield compound
Chem. Int. Ed. Engl. 1996, 35, 2348Ϫ2350.
[21]
G. Vic, J. J. Hastings, O. W. Howarth, D. H. G. Crout, Tetra-
hedron: Asymmetry 1996, 7, 709Ϫ720.
[22]
S. Yanahira, Y. Yabe, M. Nakakoshi, S. Miura, N. Matsubara,
H. Ishikawa, Biosci. Biotechnol. Biochem. 1998, 62, 1791Ϫ1794.
[23]
S. Yanahira, T. Kobayashi, T. Suguri, M. Nakakoshi, S. Miura,
20
1
19 (21 mg, 84%). Ϫ [α]D ϭ ϩ 25 (c ϭ 0.5, CHCl3). Ϫ H NMR
(400 MHz, CDCl3): δ ϭ 8.25 (d, 2 H, Ph), 7.05 (d, 2 H, Ph), 5.29
(m, 2 H, 2-H, 4ЈЈ-H), 5.14Ϫ5.04 (m, 2 H, 2Ј-H, 2ЈЈ-H), 5.02 (d, 1
H, J1,2 ϭ 8.1 Hz, 1-H), 4.96Ϫ4.90 (m, 2 H, 3-H, 3ЈЈ-H), 4.83 (dd,
1 H, 3Ј-H), 4.43Ϫ4.38 (m, 2 H, J1ЈЈ,2ЈЈ ϭ 8.1 Hz, 1ЈЈ-H, 6Јa-H), 4.33
H. Ishikawa, I. Nakajima, Biosci. Biotechnol. Biochem. 1995,
59, 1021Ϫ1026.
[24]
R. T. Lee, Y. C. Lee, Carbohydr. Res. 1974, 37, 193Ϫ201.
[25]
V. Pozsgay, H. J. Jennings, J. Org. Chem. 1988, 53, 4042Ϫ4055.
Received April 21 1999
[O99225]
Eur. J. Org. Chem. 1999, 3073Ϫ3077
3077