Synthesis of 2-[(2-pyridyl)amino]ethyl β-D-lactosaminide and evaluation of its acceptor ability for sialyltransferase: A comparison with 4-methylumbelliferyl and dansyl β-D-lactosaminide

Article abstract of DOI:10.1016/j.carres.2004.03.015

We reported the synthesis of β-D-lactosaminide with a 2-aminopyridyl group that is linked to a glycosyl tether at the reducing end. This fluorescent disaccharide acts as an acceptor for both α-(2→6)- and α-(2→3)-sialyltransferases. In addition, the acceptor ability of this disaccharide was evaluated and compared with that of β-D-lactosaminide having a dansyl or a 4-methylumbelliferyl group.

Full text of DOI:10.1016/j.carres.2004.03.015

Carbohydrate
RESEARCH
Carbohydrate Research 339 (2004) 1545–1550
Synthesis of 2-[(2-pyridyl)amino]ethyl b-
evaluation of its acceptor ability for sialyltransferase: a comparison
with 4-methylumbelliferyl and dansyl b- -lactosaminide
D
-lactosaminide and
D
Yasuhiro Kajihara,a,* Daisuke Kamiyama,a Naoki Yamamoto,a Tohru Sakakibara,a
Masayuki Izumib and Hironobu Hashimotob
aGraduate School of Integrated Science, Yokohama City University, 22-2, Seto, Kanazawa-ku, Yokohama 236-0027, Japan
bDepartment of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta,
Midori-ku, Yokohama 226-8501, Japan
Received 15 December 2003; accepted 9 March 2004
Abstract—We report the synthesis of b-D-lactosaminide with a 2-aminopyridyl group that is linked to a glycosyl tether at the
reducing end. This fluorescent disaccharide acts as an acceptor for both a-(2 fi 6)- and a-(2 fi 3)-sialyltransferases. In addition, the
acceptor ability of this disaccharide was evaluated and compared with that of b-
4-methylumbelliferyl group.
D-lactosaminide having a dansyl or a
Ó 2004 Elsevier Ltd. All rights reserved.
Keywords: Sialyltransferase; Enzyme assay; 2-Aminopyridyl-tethered oligosaccharide; Fluorescence assay; N-Acetyl-D-lactosaminide
1. Introduction
assay method for sialyltransferases is essential in order
to estimate the quantity of sialyltransferase expressed in
a target cell.
Sialic acid residues at the nonreducing end of oligosac-
charides are responsible for several biological events,
such as binding with CD22 (Siglec), the lifetime of gly-
coproteins in plasma, and viral invasion.1;2 Biosynthesis
of the sialyl linkages are performed by the action of
several sialyltransferases that catalyze transfer of a sialic
acid residue to the nonreducing end of the oligosac-
charide from cytidine-50-monophospho-neuraminic acid
(CMP-NeuAc). Increasing or decreasing the quantity of
sialyltransferases in the cell may be related to pathogens,
proliferation, and differentiation. Indeed, a deficiency of
a-(2 fi 6)-sialyltransferase causes a fatal decrease in IgM
production due to a loss of ligand, NeuAc-a-(2 fi 6)-
Gal-b-(1 fi 4)-GlcNAc, of the CD22 (Siglec) on the B-
cell.3 Therefore a convenient and sensitive diagnostic
The most sensitive assay method is one using radio-
labeled sugar nucleotides, such as CMP-[U-14C]-sialic
acid.4 We have also examined fluorometric enzyme
assay methods for sialyltransferases. However, although
our methods are highly sensitive, these methods are time
consuming5 and the 4-methylumbelliferyl b-
D-lactoside
acceptor is only slightly soluble in buffer solution.6
Palcic and co-workers have reported a fluorescence-
labeled acceptor in order to assay several enzymes
including sialyltransferases.7;8 This superb substrate
based on the LacNAc skeleton can be detected with as
few as 100 molecules.
On the other hand, pyridyamination9 is now widely
used as a labeling method for oligosaccharides, and its
high sensitivity makes it potentially useful for oligosac-
charide analysis. This method, however, causes loss of
the characteristic nature of the sugar ring at the reducing
end, because pyridylamino labeling by reductive ami-
nation requires an aldehyde at the anomeric position.
* Corresponding author. Tel.: +81-45-787-2210; fax: +81-45-787-2413;
e-mail: kajihara@yokohama-cu.ac.jp
0008-6215/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2004.03.015

1546
Y. Kajihara et al. / Carbohydrate Research 339 (2004) 1545–1550
Although b-
D
-LacNAc can be easily prepared from
phosphorane group, which did not hydrolyze at room
temperature. Therefore, this reaction was performed at
40 °C to afford the 2-amino derivative (Scheme 1).
The 2-amino group was then acetylated using acetic
anhydride, after which all the hydroxyl groups were
acetylated for isolation as 3. To attach the 2-amino-
pyridyl group by reductive amination, the allyl group at
the anomeric position was converted into an aldehyde
group by ozonolysis in 70% yield. This aldehyde was
fluorescence labeled with 2-aminopyridine. After con-
firmation of imine formation between this aldehyde and
the 2-aminopyridine by TLC, the imino group was
reduced by acidic NaBH3CN in quantitative yield. After
O-deacetylation by NaOMe, the PA-LacNAc was ana-
lyzed by HPLC using a fluorescence detector. However,
although fluorescence labeling was quantitatively per-
formed, small amounts of impurities were detected due
to the high sensitivity of detection. Therefore an aliquot
of PA-LacNAc was isolated by HPLC and used for the
sialyltransferase assays.
lactose,10 pyridylaminated LacNAc thus obtained by
reductive amination does not act as an acceptor for
a-(2 fi 6)- and a-(2 fi 3)-sialyltransferase due to its strict
acceptor specificity requiring a complete LacNAc skele-
ton.11 Although lactose having a pyridylamino group
that is linked to the glycosyl tether derived from 2,3-
dihydroxypropyl glycoside at the reducing end, has been
synthesized,12 the synthesis afforded several byproducts.
In addition, the b-D-LacNAc derivative, which is a
common acceptor for both a-(2 fi 6)- and a-(2 fi 3)-
sialyltransferases, has not yet been synthesized. There-
fore, we examined the synthesis of 2-aminopyridyl
b-D-LacNAc (PA-LacNAc) as a new type of acceptor by
a convenient synthetic route. In this paper, we describe
the synthesis of the PA-LacNAc from lactose and the
evaluation of its reactivity toward sialyltransferases in
comparison with other acceptors.
2. Results and discussion
In order to evaluate the acceptor ability for the sialyl-
transferase reaction, other acceptors having 4-methyl-
umbelliferyl or dansyl groups were also prepared and
then compared for their acceptor ability toward PA-
LacNAc.
For the synthesis of allyl O-(LacN3), LacNAc chloride
(the glycosyl chloride)13 was first prepared from lactose
by use of azidonitration10 and then conversion to the
glycosyl chloride 1. Sodium allyloxide was added to
form the b-allyl glycoside 2. This reaction proceeded
For the synthesis of 4-methylumbelliferyl b-D-Lac-
NAc (UM-LacNAc),14 a galactosyltransferase-mediated
reaction with commercially available 4-methylumbel-
smoothly and afforded the desired All b-D-LacN3 (3), as
the sole product.
liferyl b-D-GlcNAc (UM-GlcNAc) was performed
In order to reduce the azide group to an amino group
in the presence of the allyl glycosyl group, triphenyl-
phosphine was used in dioxane–MeOH–H2O. However,
this reduction stopped after the formation of the imino-
(Scheme 2). Although the solubility of UM-GlcNAc in
the buffer solution was extremely poor, the galactosyl-
transferase reaction occurred in 90% yield. We assumed
that the small amount of UM-GlcNAc dissolved was
1) PPh3,
1) NaOAll, AllOH-CH2Cl2,
r.t.
Dioxane-MeOH-H2O,40 o
2) Ac2O, pyridine
C
AcO OAc
Cl
N3
AcO OAc
AcO
2) Ac2O, Pyridine
N3
O
AcO
O
AcO
O
O
O
AcO
O
O
OAc
71%
OAc
OAc
OAc
1
2
1) 2-aminopyridine, AcOH
NaBH3CN
2) NaOMe, MeOH
AcO OAc
AcO OAc
O3, MeOH,
-78 o
NHAc
NHAc
O
C
O
O
AcO
AcO
O
O
AcO
O
AcO
O
O
O
OAc
OAc
OAc
OAc
4
3
OH OH
NHAc
H
N
N
O
AcO
O
O
HO
O
OH
OH
5
Scheme 1.

Y. Kajihara et al. / Carbohydrate Research 339 (2004) 1545–1550
1547
HEPES buffer. With the rat recombinant a-(2 fi 3)-
sialyltransferase, adequate transfer velocity was only
observed at pH 5 in cacodylate buffer. Therefore, HE-
PES buffer (pH 7.0) and sodium cacodylate buffer
(pH 5.0) were chosen for use of a-(2 fi 6)- and a-(2 fi 3)-
sialyltransferase assays, respectively. In order to esti-
mate Km and Vmax values, an assay solution containing
CMP-NeuAc (300 lM), acceptor (various concentra-
tions), sialyltransferase, and bovine serum albumin were
used, and the formation of sialyl-LacNAc was estimated
by HPLC.6 Although a solution of PA-LacNAc can be
prepared as a 100 mM solution, MU-LacNAc, and
dansyl-LacNAc are troublesome to prepare over 20 and
2 mM, respectively.
Lineweaver–Burk plots of MU-, PA-, and dansyl-
LacNAc toward a-(2 fi 6)- and a-(2 fi 3)-sialyltransfe-
rases are shown in Figure 3, and Km and Vmax values thus
obtained are also summarized in Tables 1 and 2.
As shown in Figure 3, these three LacNAc derivatives
act as acceptors for both a-(2 fi 6) and a-(2 fi 3)-sial-
yltransferases. In the case of a-(2 fi 6)-sialyltransferase,
the dansyl-LacNAc shows the smallest Km value
(56.0 lM) compared to MU-LacNAc (634 lM) and PA-
LacNAc (442 lM). This phenomenon may be a cluster
effect of the dansyl group or a hydrophobic interaction
between the enzyme and the dansyl group. For MU- and
PA-LacNAc, both acceptors show same Km and Vmax
OH OH
O
NHAc
O(CH2)6
H
N
O
HO
O
S
HO
O
O
OH
OH
NMe2
8 : Dansyl-LacNAc
Figure 1.
galactosylated, after which UM-LacNAc (7) easily dis-
solved.
For the synthesis of the dansyl-b-D-LacNAc (8) (Fig.
1), reported procedure was used.15
In order to evaluate acceptor ability of these three
fluorescence-labeled-LacNAcs, we measured their
transfer rates by HPLC equipped with a fluorescence
detector. For the estimation of sialyl-LacNAc forma-
tion, a new fluorescence peak by the sialyltransfer
reaction on HPLC analysis was estimated by use of a
calibration curve made from the corresponding fluo-
rescence-labeled acceptor.
For the assay toward PA-LacNAc, we also optimized
assay conditions by use of rat a-(2 fi 6) and rat
recombinant a-(2 fi 3) sialyltransferases. As shown in
Figure 2, in the case of sodium cacodylate buffer, a
higher transfer velocity by a-(2 fi 6)-sialyltransferase at
pH 6 was observed, but only half the transfer velocities
were observed at pH 5 and 7 compared to those in
OH
O
CH3
O
HO
HO
O
O
NHAc
OH OH
NHAc
β-(1
4)-GalTase
O
O
O
HO
6
O
HO
O
O
OH
OH
Alkaline phosphatase,
MnCl2, BSA
CH3
+
OH OH
O
NH2
N
O
7 : 4MU-LacNAc
HO
OH OH
HO
N
O
O
O
O
P
O
P
O
HO OH
UDP-galactose
Scheme 2.
Figure 2. Transfer rate is dependent on buffer conditions. Rat-liver a-(2 fi 6)-sialyltransferase (left), rat-recombinant a-(2 fi 3)-sialyltransferase
(right).

1548
Y. Kajihara et al. / Carbohydrate Research 339 (2004) 1545–1550
Figure 3. Kinetics of rat-liver a-(2 fi 6)-(A), (B), and (C), and rat recombinant a-(2 fi 3)-sialyltransferases (D), (E), and (F).
Table 1. Kinetic parameters toward rat-liver a-(2 fi 6)-sialyltransfer-
ase
observed. PA-LacNAc showed a moderate Km value,
but the lowest Vmax value was observed (Table 2).
For the synthesis of these acceptors, MU-LacNAc
was easily prepared by use of commercially available
enzyme and substrates. However, the solubilities of both
MU- and dansyl-LacNAc are remarkably poor com-
pared to that of PA-LacNAc. Therefore preparation of
relatively dilute solution by use of MU-LacNAc or
dansyl-LacNAc in an assay mixture is troublesome. In
addition, dansyl-LacNAc is the most insensitive during
fluorescence detection, but PA-LacNAc is highly sensi-
tive to fluorescence detection.
Substrate
Km (lM)
Vmax
(pmol/min)
Vmax=Km
(relative)
MU-LacNAc
PA-LacNAc
Dansyl-LacNAc
634 99
442 104
56.0 12
58.7 5.0
43.9 6.3
23.2 2.7
22.3
24.0
100
Table 2. Kinetic parameters toward rat recombinant a-(2 fi 3)-sialyl-
transferase
Substrate
Km
(mM)
Vmax
(pmol/min)
Vmax=Km
(relative)
In summary, we synthesized PA-LacNAc and evalu-
ated its ability to act as an acceptor. We found that
solubility, sensitivity, and affinity toward both sial-
yltransferases make it suitable as a new probe for sial-
yltransferase assays.
MU-LacNAc
PA-LacNAc
3.37 0.93
1.78 0.34
Dansyl-LacNAc 0.354 0.091 200.1 41.2
1613 406
62.05 9.20
84.8
6.17
100
values. However, the Vmax=Km value of dansyl-LacNAc
is four-fold higher than that of MU- and PA-LacNAc
(Table 1).
3. Experimental
For a-(2 fi 3)-sialyltransferase, dansyl-LacNAc shows
the smaller Km (354 lM) value than that of the other
acceptors. In contrast, although MU-LacNAc showed a
large Km value, a remarkable increase in the Vmax value
compared to that of the other acceptors was also
3.1. General procedure
1H NMR spectra were recorded with Bruker Avance-
400 instrument. Optical rotations were measured with a
JASCO DIP-4 polarimeter. High-resolution mass spec-

Y. Kajihara et al. / Carbohydrate Research 339 (2004) 1545–1550
1549
0
0
0
0
0
0
tra were recorded using a Shimazu/Kratos concept-IIH
under FAB conditions. The rat-liver a-(2 fi 6)-sial-
yltransferases [EC 2.4.99.1] and rat-liver-recombinant
a-(2 fi 3)-sialyltransferases [EC 2.4.99.6] were purchased
from Calbiochem.
J4 ;3 3.4 Hz, J4 ;5 1.0 Hz, H-40) 5.12 (dd, 1H, J2 ;1 7.8 Hz,
J2 ;3 10.5 Hz, H-20) 5.08 (dd, 1H, J3;2 9.9 Hz, J3;4 8.4 Hz,
0
0
H-3) 4.97 (dd, 1H, J3 ;2 10.5 Hz, J3 ;4 3.4 Hz, H-30) 4.51
0
0
0
0
(d, 1H, J1 ;2 7.9 Hz, H-10) 4.50 (d, 1H, J1;2 7.9 Hz, H-1)
4.47 (dd, 1H, J6a;5 2.5 Hz, Jgem 11.9 Hz, H-6a) 4.31 (d,
1H, Jgem 17.9 Hz, –OCH2CHO) 4.21 (dd, 1H, J 1.2 Hz,
Jgem 17.9 Hz, –OCH2CHO) 4.15 (ddd, 1H, J2;1 7.9 Hz,
0
0
3.2. Allyl 2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl-
0
0
(1 ! 4)-3,6-di-O-acetyl-2-azido-2-deoxy-b-
D
-gluco-
J2;3 9.9 Hz, J2;NH 9.3 Hz, H-2) 4.14 (dd, 1H, J6 a;5 6.6 Hz,
pyranoside (2)
Jgem 11.2 Hz, H-60a) 4.11 (dd, 1H, J6b;5 5.7 Hz, Jgem
0
0
11.9 Hz, H-6b) 4.08 (dd, 1H, J6 b;5 7.2 Hz, Jgem 11.2 Hz,
0
0
0
0
0
0
A solution of 1 (2.01 g, 3.15 mmol) and AllONa
(8.0 mmol) in 1:2 CH2Cl2–AllOH (18 mL) was stirred at
room temperature. After 10 min, the mixture was neu-
tralized with AcOH and then concentrated. The residue
was treated with pyridine–Ac2O in the presence of 4-
dimethylaminopyridine. After 12 h, the mixture was
concentrated. Purification of the residue with a column
of charcoal (3.5 i.d. · 10 cm, water to 80% EtOH)
afforded allyl glycoside 2 (908 mg, 71%), which was
identical to the reported data.16
H-60b) 3.89 (ddd, 1H, J5 ;4 1.0 Hz, J5 ;6 a 6.6 Hz,J5 ;6 b
7.2 Hz, H-50) 3.79 (dd, 1H, J4;3 ¼ J4;5 8.4 Hz, H-4) 3.61
(ddd, 1H, J5;4 8.4 Hz, J5;6a 2.5 Hz, J5;6b 5.7 Hz, H-5) 2.15,
2.11, 2.09, 2.07, 2.05, 2.00, 1.97 (7s, 21H, Ac);
HRFABMS: Calcd for C28H40N1O18 (M+H)þ m=z
678.2245; found m=z 678.2182.
3.5. 2-[(2-Pyridyl)amino]ethyl b-
(1 ! 4)-2-acetamido-2-deoxy-b-
D
-galactopyranosyl-
-glucopyranoside (5)
D
To a solution of aldehyde 4 (255 mg, 0.376 mmol) in
MeOH (4.0 mL) was added 2-aminopyridine (178 mg,
1.89 mmol) and HOAc (55 lL), and the mixture was
stirred at room temperature. After 30 min NaBH3CN
(48 mg, 0.76 mmol) was added, and the mixture was
stirred for 18 h at room temperature. The mixture was
diluted with EtOAc and washed with brine. The organic
phase was dried (MgSO4) and then concentrated in
vacuo to give a residue that was dried in vacuo. To a
solution of this residue in dry MeOH (5.0 mL) was
added a catalytic amount of NaOMe. After 4 h the
mixture was neutralized by addition of Dowex 50Wx8
(Hþ form) and then filtered. The filtrate was then con-
centrated in vacuo. Purification of this residue by HPLC
(column: YMC-Pack ODS-A SH-343-5 S-5 120 A 2.0
i.d. · 25 cm, 10:1 50 mM ammonium acetate–MeCN,
monitoring by 210 nm or Ex 320 nm/Em 400 nm) and
desalting by same HPLC system (H2O for 30 min and
3.3. Allyl 2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl-
(1 ! 4)-2-acetamido-3,6-di-O-acetyl-2-deoxy-b-
D-gluco-
pyranoside (3)
To a solution of allyl glycoside 2 (859 mg, 2.03 mmol) in
10:3:1 dioxane–MeOH–H2O (26 mL) was added Ph3P
(705 mg, 2.69 mmol), and this mixture was stirred at
40 °C. After 20 h, Ac2O (1.0 mL) was added, and the
mixture was stirred for 1 h. The mixture was evaporated,
and the residue was suspended into water. The water
phase was washed with CH2Cl2, and then the water
phase was concentrated in vacuo. The residue was dis-
solved in a solution of 1:1 Ac2O–pyridine (42 mL) con-
taining 4-dimethylaminopyridine (77 mg, 0.63 mmol),
and this mixture was stirred for 12 h. After concentra-
tion of this mixture, purification of the residue by silica
gel column chromatography (1:4 hexane–EtOAc) affor-
ded lactosaminide 3 (687 mg, 50%), which was identical
to that reported.17
then 1:1 H2O–MeCN gradient system) afforded pure
21
D
PA-lactosaminide 5 (55 mg, 29%): ½aŠ À1:57 (c 0.57,
H2O); 1H NMR (400 MHz, D2O): d 8.04, 7.67, 6.81, 6.76
0
0
3.4. 2-Oxoethyl 2,3,4,6-tetra-O-acetyl-b-
D
-galactopyran-
D-
(each m, each 1H, pyridylamino group) 4.64 (d, 1H, J1 ;2
osyl-(1 ! 4)-2-acetamido-3,6-di-O-acetyl-2-deoxy-b-
7.7 Hz, H-10) 4.55 (d, 1H, J1;2 7.8 Hz, H-1) 4.01 (bd, 1H,
J4 ;3 3.3 Hz, H-40) 3.75 (dd, 1H, J3 ;2 10.0 Hz, J3 ;4 3.3 Hz,
0
0
0
0
0
0
glucopyranoside (4)
H-30) 3.62 (dd, 1H, J2 ;1 7.8 Hz, J2 ;3 10.0 Hz, H-20) 1.96
(s, 3H, Ac); HRFABMS: Calcd for C21H34N3O11
(M+H)þ m=z 504.2193; found m=z 504.2213.
0
0
0
0
Ozone was passed through a solution of lactosaminide 3
(130 mg, 0.19 mmol) in CH2Cl2 (9.6 mL) for 15 min at
)78 °C until the solution became slightly blue. Excess
ozone was displaced by an argon stream for 5 min, and
then the ozonide was reduced by addition of an excess of
Me2S at )78 °C. The cooling bath was removed, and the
mixture was concentrated in vacuo. Purification of the
3.6. 4-Methylumbelliferyl b-
D
-galactopyranosyl-(1 ! 4)-
2-acetamido-2-deoxy-b- -glucopyranoside (7)
D
To a solution of 4-methylumbelliferyl 2-acetamido-2-
deoxy-b- -glucopyranoside (10.0 mg, 0.026 mmol), BSA
(5.0 mg), UDP-galactose (16.0 mg, 0.025 mmol), and
alkaline phosphatase (50 U) in HEPES buffer (100 mM,
pH 7.0 containing 30 mM MgCl2) was added bovine
residue by silica gel column (20:1 EtOAc–MeOH)
D
21
D
afforded aldehyde 4 (91 mg, 70%): ½aŠ À10:0 (c 0.6,
1
EtOH); H NMR (400 MHz, CDCl3): d 9.65 (br s, 1H,
aldehyde) 5.77 (d, 1H, JNH;2 9.3 Hz, NH) 5.36 (dd, 1H,

1550
Y. Kajihara et al. / Carbohydrate Research 339 (2004) 1545–1550
D
-LacNAc (50, 100, 200, 300, and 500 lM), and dansyl-
b-(1 ! 4)-galactosyltransferase (12 mU), and the mixture
was incubated at 37 °C. After 48 h, purification with an
ODS column (1.5 i.d. · 22 cm, elution by H2O (100 mL),
then 9:1 H2O–MeCN) afforded 4-methylumbelliferyl-
LacNAc 714 (10.8 mg, 0.019 mmol) in 74% yield. 1H
NMR (400 MHz, D2O): d 7.58 (d, 1H, J 8.8 Hz, MU
group) 6.98 (dd, 1H, J 2.3 Hz, 8.8 Hz, MU group) 6.90
(d, 1H, J 2.3 Hz, MU group) 7.58 (s, 1H, MU group)
b-
D
-LacNAc (17, 23, 33, 67, and 100 lM) were used for
the assays. PA-LacNAc, MU-LacNAc, and dansyl-
LacNAc were detected by following excitation and
emission values: PA:Ex 320 nm, Em 400 nm; MU: Ex
325 nm, Em 372 nm; dansyl: Ex 340 nm, Em 540 nm.
Rat-liver-recombinant
assays were performed with 50 mM HEPES (30 lL;
pH 5.0) containing CMP-b- -NeuAc (300 lM), fluores-
cence labeled-b- -LacNAc (varied concentrations),
BSA (10 lg), rat-liver-recombinant a-(2 fi 3)-sial-
yltransferases (0.1–0.2 mU). Estimation of Km and Vmax
values were same as in the case of rat liver a-(2 fi 6)-
sialyltransferases. MU-b-
and 1000 lM), PA-b- -LacNAc (100, 200, 300, 500, and
1000 lM), and dansyl-b- -LacNAc (25, 35, 50, 100, and
a-(2 fi 3)-sialyltransferase
D
0
0
5.27 (d, 1H, J1;2 8.4 Hz, H-1) 4.54 (d, 1H, J1 ;2 7.8 Hz, H-
10) 4.10 (dd, 1H, J2;1 8.4 Hz, J2;3 9.6 Hz, H-2) 3.95 (bd,
D
1H, J4;3 3.2 Hz, J4 ;5 ꢀ 0 Hz, H-40) 4.08–3.76 (m, 8H, H-
0
0
3, H-4, H-5, H-6a, H-6b, H-50, H-60a, H-60b) 3.70 (dd,
1H, J3 ;2 9.9 Hz, J3 ;4 3.2 Hz, H-30) 3.59 (dd, 1H, J2 ;1
0
0
0
0
0
0
7.8 Hz, J2 ;3 9.9 Hz, H-20) 2.35, 2.08 (2s, 6H, CH3).
D-LacNAc (100, 200, 300, 500,
0
0
D
D
3.7. Sialyltransferase assay with varying pH and buffer
conditions
150 lM) were used for the assays.
Assays6 were performed with 50 mM HEPES or 50 mM
cacodylate buffer solution (30 lL; pH was varied:
References
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D-NeuAc
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(300 lM), PA-b- -LacNAc (1 mM), BSA (10 lg), and
D
rat-liver-recombinant a-(2 fi 3)- (1 mU) or rat-liver
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up to 15% consumption of donor substrate. After
incubation, the mixture was diluted iced H2O (170 lL),
and then NeuAc transfer was estimated with HPLC
(C18-column: i.d. 0.46 · 25 cm, 15:1 25 mM ammonium
acetate–MeCN) equipped with fluorescence detector (Ex
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Rat-liver a-(2 fi 6)-sialyltransferase assays6 were per-
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CMP-b-
D
-NeuAc (300 lM), fluorescence labeled-b-D-
LacNAc (varied concentrations), BSA (10 lg), rat-liver
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to 15% consumption of donor substrate. After incuba-
tion, the mixture was diluted with iced H2O (170 lL),
and then NeuAc transfer was estimated by HPLC (C18-
column: i.d. 0.46 · 25 cm, 15:1 25 mM ammonium acet-
ate–MeCN) equipped with fluorescence detector. Km
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ꢀ
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b-
D
-LacNAc (100, 200, 300, 500, and 1000 lM), PA-b-