Convenient enzymatic synthesis of a p-nitrophenyl oligosaccharide series of sialyl N-acetyllactosamine, sialyl Lex and relevant compounds

Article abstract of DOI:10.1016/j.carres.2005.08.019

From the β-d-Gal-(1→4)-β-d-GlcNAc-OC₆H ₄NO₂-p (1) prepared by the transglycosylation of β-galactosidase from Bacillus circulans, α-d-Neu5Ac-(2→3)- β-d-Gal-(1→4)-β-d-GlcNAc-OC₆H₄NO ₂-p (9) a

Full text of DOI:10.1016/j.carres.2005.08.019

Carbohydrate
RESEARCH
Carbohydrate Research 340 (2005) 2469–2475
Convenient enzymatic synthesis of a p-nitrophenyl
oligosaccharide series of sialyl N-acetyllactosamine, sialyl Lex
and relevant compounds
Xiaoxiong Zenga,b and Hirotaka Uzawaa,*
aResearch Center of Advanced Bionics, National Institute of Advanced Industrial Science and Technology (AIST),
Tsukuba Central 5, 1-1-1 Higashi, Tsukuba 305-8565, Japan
bDepartment of Biotechnology, College of Food Science and Technology, Nanjing Agricultural University, Weigang, Nanjing 210095,
Jiangsu, PR China
Received 8 February 2005; accepted 22 August 2005
Available online 5 October 2005
Abstract—From the b-D-Gal-(1!4)-b-D-GlcNAc-OC6H4NO2-p (1) prepared by the transglycosylation of b-galactosidase from
Bacillus circulans, a-D-Neu5Ac-(2!3)-b-D-Gal-(1!4)-b-D-GlcNAc-OC6H4NO2-p (9) and a-D-Neu5Ac-(2!6)-b-D-Gal-(1!4)-b-
D-GlcNAc-OC6H4NO2-p (10) were effectively synthesized with an equimolar ratio of CMP-Neu5Ac by recombinant rat a-(2!3)-
N-sialyltransferase and rat liver a-(2!6)-N-sialyltransferase, respectively. The former enzyme also transferred effectively the
Neu5Ac residue from CMP-Neu5Ac to the location of OH-3 in the non-reducing terminal of b-D-Gal-(1!4)-b-D-Gal-
OC6H4NO2-p or b-D-Gal-(1!4)-b-D-Gal-(1!4)-b-D-GlcNAc-OC6H4NO2-p, while the latter enzyme did not. In the case of equimo-
lar ratio of GDP-Fuc/acceptor, 1 and 9 were further fucosylated quantitatively to form b-D-Gal-(1!4)-b-D-(a-L-Fuc-(1!3)-)-Glc-
NAc-OC6H4NO2-p (14) and a-D-Neu5Ac-(2!3)-b-D-Gal-(1!4)-b-D-(a-L-Fuc-(1!3)-)-GlcNAc-OC6H4NO2-p (13) by recombinant
human a-(1!3)-fucosyltransferase VII, respectively.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Keywords: Enzymatic synthesis; Sialyl Lewis X; Lewis X
1. Introduction
biological functions increases, the need for practical syn-
thetic procedures of oligosaccharides and their analogs
in large quantities has become a major subject. Practical
syntheses potentially contribute to studies on the molec-
ular mechanisms of carbohydrate-mediated biological
processes. In the literature, several synthetic approaches
have been proposed to sialyl-N-acetyllactosamine, sLex
and their related derivatives.8–12 Among them, enzy-
matic and chemo-enzymatic methods seem to have re-
ceived increasing attention from the viewpoint of green
chemistry.13 They are advantageous over their chemical
counterparts also in terms of efficiency in obtaining the
desired oligosaccharides without using protecting
groups. Moreover, the problem of controlling the ano-
meric configuration is eliminated because the enzymatic
glycosylation is usually stereospecific to provide a well-
defined glycosidic linkage.
The oligosaccharides found in glycoconjugates and gly-
coproteins play crucial roles in a variety of biological
events, for example, immunological responses, cellular
recognition and host–toxin interactions.1,2 Sialyl Lewisx
(sLex) tetrasaccharide is considered to serve as a com-
mon ligand of the three members of selectins, namely,
E-, P- and L-selectins, which are involved in the biolog-
ical events of inflammation and metastasis.3,4 Sialyloli-
gosaccharides and sialylglycoproteins also serve as the
cell-surface receptor determinants of pathogenic bacte-
ria, viruses, bacterial toxins and carbohydrate-recogniz-
ing proteins like lectins.5–7 As the understanding of these
*
Corresponding author. Tel.: +81 29 861 4431; fax: +81 29 861
4680; e-mail: h.uzawa@aist.go.jp
0008-6215/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2005.08.019

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X. Zeng, H. Uzawa / Carbohydrate Research 340 (2005) 2469–2475
Recently, we reported the chemo-enzymatic synthesis
phobic interaction chromatography on a column of
highly substituted Phenyl Sepharose 6 Fast Flow. This
simple and convenient purification enabled us to synthe-
size a key substrate 1 in large scale. To the solution of
b-D-GlcNAc-OC6H4NO2-p (1.0 g) and lactose (4.0 g)
dissolved in 35 mL of sodium phosphate buffer (20
mM, pH 6.8) containing 50% acetonitrile was added
the partially purified b-galactosidase (3.2 U). The pro-
gress of the reaction at 30 ꢁC was monitored by an
HPLC fitted with an Amide-80 column and a UV detec-
tor, showing that four new peaks appeared. The reaction
mixture was stopped by heating at 95 ꢁC for 2 min and
each product was purified with ODS (C-18), Toyopearl
HW-40S and Bio-Gel P-2, successively, to yield transfer
products 1–4 in the order of 1 > 3 > 4 ꢀ 2. Apparently, 3
and 4 are produced by further regioselective galactosyla-
tion from 1 formed initially. The structure of galactosyl
of sialyl-a-(2!3)-LacNAc by using a-(2!3)-(N)-sialyl-
transferase.14 In our chemo-enzymatic synthesis, a 3-
aminopropyl group is introduced persistently at the
reducing terminal sugar to give the corresponding b-
glycosides. These functional groups are valuable for
immobilization onto sensor chip surfaces and fabrica-
tion of glycomaterials. For instance, the p-aminophenyl
group can be converted to thiolated compounds for this
purpose.15
In this paper, we describe a convenient synthesis of
pNP sLex and Lex, which utilizes pNP monosaccharides
as the starting sugars and the multiple enzymes of b-
galactosidase, sialyltransferases and fucosyltransferase
for their assembly. Obviously, these pNP-based oligo-
saccharides are designed for the study on specific
carbohydrate–carbohydrate and carbohydrate–protein
interactions with a quartz crystal microbalance or sur-
face plasmon resonance methods.16–19
1
trisaccharide 3 was easily confirmed by H NMR spec-
00 00
troscopy [dH 5.30 (J1,2 7.8 Hz), 4.57 (J1 ,2 7.8 Hz),
4.51 (J1 ,2 7.8 Hz); dC 79.9 (C-4) and 78.9 (C-40)], which
was accorded well with the data of the previous report.20
Similarly, the structure of tetrasaccharide 4 was also
confirmed by 1H NMR [d 5.31 (J1,2 8.4 Hz), 4.63
0
0
2. Results and discussion
000 000
00 00
0
0
2.1. Preparation of pNP galactosylated substrates for
sialyltransferases
(J1 ,2 7.8 Hz), 4.57 (J1 ,2 8.4 Hz), 4.52 (J1 ,2 7.8 Hz)]
13
and C NMR spectra [d 79.9 (C-4), 79.3 (C-40) and
78.9 (C-400)]. These 13C NMR signals were shown to be
clearly shifted downfield compared to other carbons (d
56.6–76.8 ppm). FABMS analysis supports the struc-
tures. In a similar analysis, self-condensed galactosyl
oligosaccharides (6 and 7) were formed by successive
regioselective galactosylation through the initially
formed major intermediate 5 from b-D-Gal-OC6H4-
NO2-p which was applied as a common glycosyl donor
and acceptor (Scheme 2). The structure of pNP galac-
tosylated trisaccharide 6 was confirmed compared to
the previous data.23 The peaks appearing at d 5.22
The glycosyl acceptors for sialylation were prepared by
use of the transglycosylation of b-galactosidase from
Bacillus circulans in a similar way to the previous re-
ports.20–22 As our initial approach, the commercially
available b-galactosidase used here was partially purified
because it contained high b-N-acetylhexosaminidase
(NAHase) activity. When b-D-GlcNAc-OC6H4NO2-p is
applied as a glycosyl acceptor as shown in Scheme 1,
the enzyme preparation should be free of the b-N-acetyl-
hexosaminidase (NAHase) activity that might hydrolyze
the acceptor used. Therefore, the crude enzyme was par-
tially purified to remove the NAHase activity by hydro-
R2O
R1O
OH
O
OR2
R1O
HO
O
O
O
OH
NHAc
NO2
NO2
R2
R1
R1
R2
H
H
H
H
H
H
1
Galβ1
Galβ1
5
i
i
Galβ1
H
H
H
2
3
4
H
H
Galβ1-4Galβ1
Galβ1-4Galβ1-4Galβ1
6
7
8
Galβ1-4Galβ1
Galβ1-4Galβ1-4Galβ1
Galβ1
H
Scheme 1. Reagents and conditions: (i) Partially purified B. circulans
b-galactosidase and lactose in 20 mM sodium phosphate buffer
(pH 6.8) containing 50% acetonitrile.
Scheme 2. Reagents and conditions: (i) Partially purified B. circulans
b-galactosidase and b-D-Gal-OC6H4NO2-p in 20 mM sodium phos-
phate buffer (pH 6.8) containing 50% acetonitrile.

X. Zeng, H. Uzawa / Carbohydrate Research 340 (2005) 2469–2475
2471
000 000
00 00
(J1,2 7.2 Hz), 4.66 (J1 ,2 8.4 Hz), 4.62 (J1 ,2 7.8 Hz)
(10.5 mg, 16.0 lmol) in 40 mM MES (2-morpholinoe-
thanesulfonic acid) buffer (pH 6.3, 0.8 mL) containing
BSA (1.2 mg/mL) and 20 mM MnCl2, 25 U alkaline
phosphatase, and a-2,3-NST (20 mU) was incubated at
30 ꢁC. The reaction was monitored by HPLC. After
48 h of incubation, HPLC analysis revealed that most
of 1 was sialylated. The reaction mixture was purified
by chromatography on ODS (15% acetonitrile contain-
ing 0.1% TFA as the eluent) and Bio-Gel P-2 columns
to give 9 in 91% yield based on the donor. Similarly,
10 was obtained in excellent yield (93% based on
donor) with a-2,6-NST as catalyst. In both cases, 1
1
0
0
and 4.56 (J1 ,2 7.8 Hz) in the H NMR spectrum of tetra-
saccharide 7 were shown to be all b-linkage. The 13C
NMR signals also revealed the structure 7 [d 79.3
(C-4), 79.0 (C-40) and 78.8 (C-400)], which was down-
shifted compared to other carbons.
2.2. Substrate specificity in sialyltransferase reaction for
galactosylated oligosaccharides
The synthetic galactosylated acceptors 1–3, 5, 8, Galb-
(1!4)-Glcb-OC6H4NO2-p24 and Galb-(1!3)Glcb-
OC6H4NO2-p24 were then subjected to enzyme-cata-
lyzed sialylation with two different enzymes, recombi-
nant rat a-(2!3)-N-sialyltransferase (a-2,3-NST) and
rat liver a-(2!6)-(N)-sialyltransferase (a-2,6-NST).
The relative activities are summarized as shown in Table
1. The results are in good agreement with the specificity
reported for both enzymes (entries 1, 2, 6 and 7).25–28 a-
2,6-NST shows limited acceptor specificity to type 2
structure (Galb(1!4)GlcNAc) (entries 2 and 6), while
a-2,3-NST shows a relatively broader specificity as com-
pared to a-2,6-NST (entries 1, 2, 6 and 7). It has been
reported that a-2,3-NST is capable of using terminal
Galb(1!4)Glc and both type 2 and type 1 (Galb(1!3)
GlcNAc) structures as acceptors. The present results
demonstrated that even the GlcNAc residues in type 2
and type 1 could be replaced by the Gal residue (entries
4 and 5) although the replacement resulted in a decrease
of activity. The relative rate for 3 was 29 when the rate
of 1 was arbitrarily set at 100 for a-2,3-NST. It indicates
that trisaccharide 3 further galactosylated at O-4 in 1
could be acceptable for a-2,3-NST (entry 3).
Table 1. Relative ratesa of transfer Neu5Ac to synthetic acceptors with
CMP-Neu5Ac as donor by recombinant rat a-2,3-NST and rat liver a-
2,6-NST
Entry
Acceptor
Relative rate
a-2,3-NST a-2,6-NST
1
2
3
4
5
6
7
1
100
0
100
0
0
0
0
2
3
29
48
10
45
97
5
8
Galb(1!4)Glcb-ORb
3
0
Galb(1!3)Glcb-ORb
a The activities were assayed with synthetic acceptors in the following
way: a solution (200 lL) of 1 mM acceptor, 1 mM CMP-Neu5Ac,
and a-2,3-NST (400 lU) or a-2,6-NST (400 lU) in 40 mM MES
buffer (pH 6.3) containing 1.0 mg/mL BSA and 20 mM MnCl2 was
incubated at 30 ꢁC. Samples (20 lL) were taken out at appropriate
time intervals (0, 15, 30, 60 and 90 min) during the incubation, and
were heated over water bath at 95 ꢁC for 2 min to stop each enzyme
reaction. Every sample was then analyzed by HPLC with a column of
TSKgel ODS-80Ts (4.6 · 250 mm) eluted with 15% acetonitrile in
10 mM sodium phosphate buffer (pH 7.1) at a flowrate of 1.0 mL/
min and with a UV detector monitored at 300 nm. The relative rates
on 1 for both enzymes are arbitrarily set at 100.
Sialylation of 1 was carried out with a-2,3-NST and a-
2,6-NST to afford 9 and 10, respectively (Scheme 3).
Enzymatic synthesis was performed in the following
way: a solution of 1 (8 mg, 15.8 lmol), CMP-Neu5Ac
b R=C6H4NO2-p. Galb(1!4)Glc-OR and Galb(1!3)Glc-OR were
prepared by b-galactosidase from B. circulans according to the pre-
viously reported method.24
OR3
OH
HO
R2O
R1O
OH
O
O
O
OH
O
O
RO
O
HO
OH
HO
O
OH
NHAc
O
NO2
OH
R2
R1
H
R3
H
H
R
NO2
1
9
10
3
H
H
H
5
i
i
i
Neu5Acα2
ii
12
Neu5Acα2
Neu5Acα2
H
H
H
H
H
H
Galβ1
Neu5Acα2-3Galβ1
11
Scheme 3. Reagents and conditions: (i) Recombinant rat a-(2!3)-(N)-sialyltransferase, CMP-Neu5Ac, alkaline phosphatase in 40 mM MES buffer
(pH 6.3) containing BSA and 20 mM MnCl2 at 30 ꢁC. (ii) Rat liver a-(2!6)-(N)-sialyltransferase, and other reagents and conditions are the same
as (i).

2472
X. Zeng, H. Uzawa / Carbohydrate Research 340 (2005) 2469–2475
was effectively sialylated although the donor CMP-
Neu5Ac was used in an equimolar ratio to the acceptor.
When 3 was used as the acceptor instead of 1, HPLC
chromatogram showed that one new peak with UV
absorbance at 300 nm appeared in the reaction mixture
with a-2,6-NST as catalyst. The newcompound was
purified and determined by NMR spectroscopy to be
tetrasaccharide 11. The downfield shift for galactose
When using an equimolar ratio of acceptor and donor,
9 was fucosylated at O-3 of the GlcNAc residue almost
quantitatively. The structure of 13 was determined by
NMR spectroscopy. The characteristic peaks at d
5.12 ppm (J 4.2 Hz) for the fucose H-1, 4.82 ppm for
the fucose H-5, and 1.16 ppm (J 6.6 Hz) for the fucose
H-6 in the 1H NMR spectrum and peaks at d
100.4 ppm for the fucose C-1 and 17.0 ppm for the fu-
cose C-6 in the 13C NMR spectrum evidenced the pres-
ence of the fucose residue to 9 through an a linkage. In
the 13C NMR spectrum, the signal of GlcNAc C-3
appearing at 76.6 ppm shifted down-field from
73.5 ppm in that of 9, while the signal of GlcNAc C-4
appearing at 74.6 ppm shifted up-field from 79.5 ppm
in that of 9. These results indicate that the fucose has
been transferred regioselectively onto GlcNAc OH-3
of 9. Surprisingly, 1 was also fucosylated by human re-
combinant Fuc-T VII to give 14 in an excellent yield
(91%), while 14 is obtained in a yield of 20% based on
1 (14% based on donor GDP-Fuc) by partially purified
chicken serum a-(1!3)-fucosyltransferase.31 Appar-
ently, this Fuc-T VII in the present study is a valuable
enzyme for the efficient synthesis of both sLex-OC6H4-
NO2-p and Lex-OC6H4NO2-p.
1
H-300 at d 4.09 ppm in the H NMR spectrum and the
characteristic peaks (d 2.74 and 1.77 ppm for Neu5Ac
H-3, d 41.4 ppm for Neu5Ac C-3) of Neu5Ac residue
in the 1H and 13C NMR spectra confirmed the structure
of 11. Similarly, 5 could be sialylated by a-2,3-NST to
afford 12. In the case of an equimolar ratio of accep-
tor/donor, 11 and 12 were obtained in yield of 72%
and 76%, respectively. In contrast, none of the transfer
products was detected when applying 3 and 5 with the
a-2,6-NST as catalyst. These results indicate that the
a-2,3-NST has broader specificity to 1, 3, 5 and 8, while
the a-2,6-NST could accept only 1, not 2–5.
2.3. Synthesis of sLex-OC6H4NO2-p (13) and Lex-
OC6H4NO2-p (14) oligosaccharides
Recombinant human a-(1!3)-fucosyltransferase VII
(Fuc-T VII) has an extremely limited acceptor specificity
restricted to 30-sialylated type 2 oligosaccharides.29,30
We examined the substrate specificity of this enzyme to-
wards sialyl lactosamine 9 and lactosamine 1 (Scheme
4). The trisaccharide 9 was successfully fucosylated by
incubation with GDP-Fuc, the a-(1!3)-fucosyltransfer-
ase and alkaline phosphatase in HEPES buffer (50 mM,
pH 7.4) containing BSA (1.5 mg/mL) and MnCl2
(15 mM) at 30 ꢁC, producing 13 in a quite good yield.
In conclusion, a series of pNP sialyl oligosaccharides
(9–14) were synthesized efficiently by combination with
b-galactosidase, sialyltransferases and/or fucosyl-
transferase. These synthetic compounds will be very
useful for enzyme kinetic analysis or activity assay,
synthesis of artificial glycopolymers and investigation
of carbohydrate-mediated biological functions with
a surface plasmon resonance technique, a quartz
crystal microbalance and other cell-free assay
systems.
OH
HO
OH
O
OH
O
OH
i
HOOC
O
9
O
O
HO
AcNH
O
O
OH
NHAc
NO2
O
H3C
HO
OH
OH
HO
13
OH
O
OH
OH
HO
H3C
HO
i
O
O
NHAc
O
1
O
OH
NO2
O
OH
OH
14
Scheme 4. Reagents and conditions: (i) Recombinant human a-(1!3)-fucosyltransferase VII, GDP-Fuc, and alkaline phosphatase in HEPES buffer
(50 mM, pH 7.4) containing BSA and 15 mM MnCl2 at 30 ꢁC.

X. Zeng, H. Uzawa / Carbohydrate Research 340 (2005) 2469–2475
2473
3. Experimental
3.1. General methods
with 12% MeOH in water, and the column was eluted
with the same solution. The fractions containing the
transfer products were collected and concentrated to a
small volume for further purification. The concentrated
solution was loaded onto a column (4 · 50 cm) of Toyo-
pearl HW-40S equilibrated with 25% methanol in water,
and the column was eluted with the same solution. The
fractions (tubes 46–50, 20 mL/tube; tubes 52–59) were
Recombinant rat a-(2!3)-N-sialyltransferase and re-
combinant human a(1!3)-fucosyltransferase VII were
purchased from Calbiochem-Novabiochem Corp. (La
Jolla, CA) and used without further purification. Rat
liver a-(2!6)-N-sialyltransferase was purchased from
Wako Pure Chemical Industries, Ltd. (Tokyo, Japan).
CMP-Neu5Ac sodium salt and GDP-Fuc were obtained
from Kyowa Hakko Kogyo Co., Ltd. (Tokyo, Japan).
b-Galactosidase from B. circulans (Biolacta) was a gift
from Daiwa Kasei Co., Ltd. (Osaka, Japan). Optical
rotations were measured with a JASCO DIP-1000
digital polarimeter at ambient temperature, using a
10-cm micro cell. The 1H (600 MHz) and 13C NMR
(150 MHz) spectra were recorded on a JEOL LA-600
spectrometer for solutions in D2O. Chemical shifts are
given in ppm and referenced to internal tert-butyl alco-
hol (d 1.23 in D2O or d 31.2 in D2O). All data are as-
sumed to be first order with apparent doublet and
triplets reported as d and t, respectively. Resonances
that appear broad are designated b. FAB mass spectra
(FABMS) were recorded using a JEOL DX 303 mass
spectrometer.
collected, concentrated and dried to afford
3
(103.7 mg) and 1 (265.1 mg), respectively. Other frac-
tions (tubes 41–44) were further purified, then the frac-
tions were collected, concentrated and loaded in a
column (2.0 · 100 cm) of Bio-Gel P-2, and the column
was eluted with water at a flow rate of 0.8 mL/min.
The transfer products 2 (11.2 mg) and 4 (24.5 mg) were
obtained. The physical and NMR data for compounds
1–3 were identical to those of b-D-Gal-(1!4)-b-D-
GlcNAc-OC6H4NO2-p, b-D-Gal-(1!6)-b-D-GlcNAc-
OC6H4NO2-p and b-D-Gal-(1!4)-b-D-Gal-(1!4)-b-D-
GlcNAc-OC6H4NO2-p, respectively.20 Data for 4: 1H
NMR, d 8.21 (d, J 9.0 Hz, 2H, o-Ph), 7.16 (d, J
9.0 Hz, 2H, m-Ph), 5.31 (d, J 8.4 Hz, 1H, H-1), 4.63
(d, J 7.8 Hz, 1H, H000-1), 4.57 (d, J 8.4 Hz, 1H, H00-1),
4.52 (d, J 7.8 Hz, 1H, H0-1) and 1.99 (s, 3H, NHAc);
13C NMR, d 79.9, 79.3 and 78.9 (C-4, C-40 and
C-400); [a]D ꢁ86.5 (0.53, H2O); FABMS 829 m/z for
[M+H]+.
3.2. Purification of b-galactosidase from B. circulans
3.4. b-D-Gal-(1!4)-b-D-Gal-OC6H4NO2-p (5), b-D-Gal-
(1!4)-b-D-Gal-(1!4)-b-D-Gal-OC6H4NO2-p (6), b-D-
Gal-(1!4)-b-D-Gal-(1!4)-b-D-Gal-(1!4)-b-D-Gal-
OC6H4NO2-p (7) and b-D-Gal-(1!3)-b-D-Gal-
OC6H4NO2-p (8)
The crude b-galactosidase from B. circulans (Biolacta,
50 mg) was dissolved in sodium phosphate buffer
(20 mM, pH 6.8) containing 0.4 M ammonium sulfate.
The supernatant was loaded onto a phenyl-Sepharose
6FF column equilibrated with the same buffer as de-
scribed above. The column was developed with a linear
decrease of ammonium sulfate concentration (0.4–0 M)
in sodium phosphate buffer. The b-galactosidase was
eluted at the beginning of the gradient. The fractions
containing b-galactosidase activity without NAHase
activity were collected and concentrated to a small vol-
ume by Amico Diaflo Unit with a PM10 membrane.
b-D-Gal-OC6H4NO2-p (500 mg) was incubated with
partially purified b-galactosidase (5 U) in 1.8 mL of so-
dium phosphate buffer (20 mM, pH 6.8) containing
50% acetonitrile at 30 ꢁC. After 5 h incubation, the reac-
tion mixture was stopped and passed through columns
of ODS, Toyopearl HW-40S and Bio-Gel P-2 as
described above, affording 5 (162.7 mg), 6 (145.4 mg),
7 (45.7 mg) and 8 (3.9 mg) as transfer products. The
physical and NMR data for compounds 5 and 6 were
identical to those of b-D-Gal-(1!4)-b-D-Gal-OC6H4-
NO2-p and b-D-Gal-(1!4)-b-D-Gal-(1!4)-b-D-Gal-
3.3. b-D-Gal-(1!4)-b-D-GlcNAc-OC6H4NO2-p (1), b-D-
Gal-(1!6)-b-D-GlcNAc-OC6H4NO2-p (2), b-D-Gal-
(1!4)-b-D-Gal-(1!4)-b-D-GlcNAc-OC6H4NO2-p (3)
and b-D-Gal-(1!4)-b-D-Gal-(1!4)-b-D-Gal-(1!4)-b-D-
GlcNAc-OC6H4NO2-p (4)
1
OC6H4NO2-p, respectively.23 Data for 7: H NMR, d
8.24 (d, J 9.6 Hz, 2H, o-Ph), 7.23 (d, J 9.6 Hz, 2H, m-
Ph), 5.22 (d, J 7.2 Hz, 1H, H-1), 4.66 (d, J 8.4 Hz, 1H,
H000-1), 4.62 (d, J 7.8 Hz, 1H, H00-1) and 4.56 (d, J
7.8 Hz, 1H, H0-1); 13C NMR, d 79.3, 79.0 and 78.8
(C-4, C-40 and C-400); [a]D ꢁ58.8 (0.96, H2O); FABMS
A reaction mixture (35 mL) of b-D-GlcNAc-OC6H4-
NO2-p (1.0 g), lactose (4.0 g) and partially purified b-
galactosidase (3.2 U) in sodium phosphate buffer
(20 mM, pH6.8) containing 50% acetonitrile was incu-
bated at 30 ꢁC. After 24 h incubation, the reaction mix-
ture was stopped by heating in a boiling water bath for
5 min. The supernatant from the centrifugation was
applied to a column (2.5 · 45 cm) of ODS equilibrated
1
788 m/z for [M+H]+. Data for 8: H NMR, d 8.25 (d,
J 9.6 Hz, 2H, o-Ph), 7.24 (d, J 9.6 Hz, 2H, m-Ph), 5.25
(d, J 7.8 Hz, 1H, H-1), 4.64 (d, J 7.8 Hz, 1H,
H0-1); 13C NMR, d 83.5 (C-3); FABMS 486 m/z for
[M+H]+.

2474
X. Zeng, H. Uzawa / Carbohydrate Research 340 (2005) 2469–2475
3.5. a-D-Neu5Ac-(2!3)-b-D-Gal-(1!4)-b-D-GlcNAc-
OC6H4NO2-p (9), a-D-Neu5Ac-(2!6)-b-D-Gal-(1!4)-b-
D-GlcNAc-OC6H4NO2-p (10), a-D-Neu5Ac-(2!3)-b-D-
Gal-(1!4)-b-D-Gal-(1!4)-b-D-GlcNAc-OC6H4NO2-p
(11) and a-D-Neu5Ac-(2!3)-b-D-Gal-(1!4)-b-D-Gal-
OC6H4NO2-p (12)
(1.5 mg/mL) and MnCl2 (15 mM) were incubated for
20 h at 30 ꢁC. The reaction mixture was heated in boil-
ing water for 5 min, and the reaction solution was
passed through columns of ODS and Bio-Gel P-2 to af-
1
ford 13 (8.9 mg). Data for 13: H NMR, d 8.23 (d, J
9.0 Hz, 2H, o-Ph), 7.17 (d, J 9.0 Hz, 2H, m-Ph), 5.32
(d, J 8.4 Hz, 1H, H-1), 5.12 (d, J 4.2 Hz, 1H, H-1Fuc),
4.82 (dd, 1H, H-5Fuc), 4.54 (d, 1H, 8.4 Hz, H-10), 4.22
(t, 1H, H-2), 2.74 (dd, J 12.0, 4.8 Hz, 1H, H ꢁ 30e0),
2.00, 1.97 (2s, 6H, 2 NHAc), 1.77 (t, J 12.0 Hz, 1H,
H ꢁ 30a0) and 1.16 (d, 3H, 6.6 Hz, H-6Fuc); 13C NMR, d
101.4 (C-1Fuc), 76.7 (C-3), 74.6 (C-4) and 17.0
(C-6Fuc). [a]D ꢁ9.2 (0.86, H2O); FABMS 942 m/z for
[M+H]+.
In a similar manner, 14 was prepared from 1 with re-
combinant a-(1!3)-fucosyltransferase VII as catalyst
under the following conditions: 1 (8 mg), GDP-Fuc
disodium salt (10.5 mg), recombinant a-(1!3)-fucosyl-
transferase VII (30 mU), and alkaline phosphatase
(25 U) in HEPES buffer (50 mM, pH 7.4) containing
BSA (1.5 mg/mL) and MnCl2 (15 mM), incubation for
20 h at 30 ꢁC. The reaction mixture was then processed
in the same way as described for compound 13 to give 14
(9.9 mg, 91%). The NMR data for 14 were identical to
that of b-D-Gal-(1!4)-b-D-(a-L-Fuc-(1!3)-)-GlcNAc-
OC6H4NO2-p.31 Data for 14: [a]D ꢁ85.8 (0.46, H2O);
FABMS 651 m/z for [M+H]+.
A reaction mixture of 1 (8 mg), CMP-Neu5Ac disodium
salt (10.5 mg) in 0.8 mL MES (2-morpholinoethanesulf-
onic acid) buffer (40 mM, pH 6.3) containing BSA
(1.2 mg/mL) and 20 mM MnCl2, 25 U alkaline phos-
phatase, and a-2,3-NST (20 mU) was incubated at
30 ꢁC. After 48 h incubation, the reaction solution was
passed through columns of ODS (15% acetonitrile con-
taining 0.1% TFA as eluent) and Bio-Gel P-2 to afford 9
(14.8 mg). Data for 9: 1H NMR, d 8.18 (d, J 9.0 Hz, 2H,
o-Ph), 7.14 (d, J 9.0 Hz, 2H, m-Ph), 5.31 (d, J 7.8 Hz,
1H, H-1), 4.57 (d, J 7.8 Hz, 1H, H0-1), 273 (dd, J 12.0,
4.8 Hz, 1H, H ꢁ 30e0), 2.01, 1.99, (2s, 6H, 2 NHAc) and
1.78 (t, J 12.0 Hz, 1H, H ꢁ 30a0); 13C NMR, d 77.1
(C-3); [a]D ꢁ46.7 (1.20, H2O); FABMS 796 m/z for
[M+H]+.
In a similar manner, 10 (15.2 mg) was obtained from
compound 1 in the case with rat liver a-2,6-NST as cat-
alyst. Also in a similar manner, 11 (8.6 mg) and 12
(9.9 mg) were obtained from 3 and 5, respectively, with
1
recombinant rat a-2,3-NST as catalyst. Data for 10: H
NMR, d 8.23 (d, J 9.6 Hz, 2H, o-Ph), 7.17 (d, J 9.6 Hz,
2H, m-Ph), 5.35 (d, J 8.4 Hz, 1H, H-1), 4.46 (d, J 7.2 Hz,
1H, H0-1), 2.67 (dd, J 12.0, 4.8 Hz, 1H, H ꢁ 30e0), 2.01,
1.99, (2s, 6H, 2 NHAc) and 1.72 (t, J 12.0 Hz, 1H,
H ꢁ 30a0); 13C NMR, d 65.1 (C-6); [a]D ꢁ132.7 (0.43,
Acknowledgements
A part of this work was financially supported by a
Grant-in-Aid for Scientific Research (No.17380202)
from the Ministry of Education, Culture, Sports, Sci-
ence and Technology of Japan and also by the special
Coordination Fund from the Science and Technology
Agency of the Japanese Government.
1
H2O); FABMS 796 m/z for [M+H]+. Data for 11: H
NMR, d 8.23 (d, J 9.0 Hz, 2H, o-Ph), 7.17 (d, J
9.0 Hz, 2H, m-Ph), 5.32 (d, J 9.0 Hz, 1H, H-1), 4.65
(d, J 8.4 Hz, 1H, H00-1), 4.51 (d, J 8.4 Hz, 1H, H0-1),
4.09 (dd, J 10.2 Hz, 3.0 Hz, 1H, H-300), 2.74 (dd, J
12.0, 4.8 Hz, 1H, H ꢁ 30e0), 2.00, 1.98 (2s, 6H, 2 NHAc)
and 1.77 (t, J 12.0 Hz, 1H, H ꢁ 30a0); 13C NMR, d 74.5
(C-300) and 41.5 (C-3000); FABMS 1002 m/z for
1
[M+H]+. Data for 12: H NMR d 8.25 (d, J 9.0 Hz,
Supplementary data
2H, o-Ph), 7.23 (d, J 9.0 Hz, 2H, m-Ph), 5.23 (d, J
7.8 Hz, 1H, H-1), 4.67 (d, J 7.8 Hz, 1H, H0-1),
4.11 (dd, J 3.0 Hz, 10.2 Hz, 1H, H-30), 2.75 (dd, J
12.0, 4.8 Hz, 1H, H ꢁ 30e0), 1.99 (s, 3H, NHAc) and
1.78 (t, J 12.0 Hz, 1H, H ꢁ 30a0); FABMS 777 m/z for
[M+H]+.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.carres.
2005.08.019.
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