α-(2,6)-sialyltransferase-catalyzed sialylations of conformationally constrained oligosaccharides

Article abstract of DOI:10.1021/ja0169771

It is demonstrated that conformationally restricted oligosaccharides can act as acceptors for glycosyltransferases. Correlation of the conformational properties of N-acetyl lactosamine (Galβ/(1-4)GlcNAc, LacNAc) and several preorganized derivatives with t

Full text of DOI:10.1021/ja0169771

Published on Web 05/04/2002
r-(2,6)-Sialyltransferase-Catalyzed Sialylations of
Conformationally Constrained Oligosaccharides
M. Carmen Galan,† Andre P. Venot,† John Glushka,† Anne Imberty,‡ and
Geert-Jan Boons*,†
Contribution from the Complex Carbohydrate Research Center, UniVersity of Georgia,
220 RiVerbend Road, Athens, Georgia 30602, and CERMAV-CNRS (affiliated to
UniVersite´ Joseph Fourier), BP 53, 38041 Grenoble, Cedex 9, France
Received August 30, 2001
Abstract: It is demonstrated that conformationally restricted oligosaccharides can act as acceptors for
glycosyltransferases. Correlation of the conformational properties of N-acetyl lactosamine (Galâ(1-4)GlcNAc,
LacNAc) and several preorganized derivatives with the corresponding apparent kinetic parameters of rat
liver R-(2,6)-sialyltransferase-catalyzed sialylations revealed that this enzyme recognizes LacNAc in a low
energy conformation. Furthermore, small variations in the conformational properties of the acceptors resulted
in large differences in catalytic efficiency. Collectively, our data suggest that preorganization of acceptors
in conformations that are favorable for recognition by a transferase may improve catalytic efficiencies.
Introduction
properties of a wide range of glycoproteins has revealed that
glycosidic torsional angles may deviate as much as 20-30° from
Protein- and lipid-bound oligosaccharides play critical roles
in a diverse range of biological processes such as protein folding,
cell-cell communication, bacterial adhesion, viral infection, and
masking of immunological epitopes.1,2 They are also important
in health science and are involved in the attachment and invasion
of pathogens, inflammation, metastasis, and xenotransplantation.
Unlike nucleic acid and protein biosynthesis in which the order
of attachment of nucleotides and amino acids is read from a
template, glycosylation is a nontemplated process. Oligosac-
charides are assembled by glycosyltransferases, which transfer
monosaccharide residues from nucleoside mono- or diphosphate
sugars to growing oligosaccharide chains.3,4 The level of
expression of glycosyltransferases and their donor and acceptor
specificities determine the structure of a biosynthesized oli-
gosaccharide. These parameters may differ between cell type
and development stage leading to cell-specific glycosylation.
The mechanism of this metabolic control is complex, and, in
particular, the origin of acceptor specificity is not well under-
stood. Several studies have indicated that not only primary
structures but also conformational properties of oligosaccharides
are important for acceptor specificities. Most oligosaccharides
have some degree of flexibility around their glycosidic linkages
and, as a result, can adopt several distinct low energy minima
on their conformational surface.5,6 Examination of the structural
minimum energy conformations.7 Furthermore, in the binding
site of proteins, not only global but also secondary minima with
very different conformations can be complexed.6 In addition,
there is evidence that the conformational properties of a
glycosidic linkage can be modulated by the macromolecular
structure it is part of.8 In particular, addition or removal of
flanking oligosaccharide residues or attachment of oligosac-
charides to proteins and lipids may result in conformational
changes. This modulation of properties may have important
implications in the control of glycan biosynthesis.
Little is known about the effect of interresidual flexibility of
glycosyl acceptors on kinetic parameters of glycosyl transfer.9
Furthermore, structural studies have provided little information
about the conformation of a glycosyl acceptor that is recognized
by a glycosyltransferase.10 To address these issues, we have
designed and synthesized several conformationally con-
strained11-18 N-acetyl lactosamine (LacNAc) derivatives for
(6) Imberty, A.; Perez, S. Chem. ReV. 2000, 100, 4567-4588.
(7) Petrescu, A. J.; Petrescu, S. M.; Dwek, R. A.; Wormald, M. R. Glycobiology
1999, 9, 343-352.
(8) Carver, J. P.; Cumming, D. A. Pure Appl. Chem. 1987, 59, 1465-1476.
(9) Lindh, I.; Hindsgaul, O. J. Am. Chem. Soc. 1991, 113, 216-223.
(10) Rini, J. M.; Sharon, N. Curr. Opin. Struct. Biol. 2000, 10, 507-509.
(11) Bundle, D. R.; Alibes, R.; Nilar, S.; Otter, A.; Warwas, M.; Zhang, P. J.
Am. Chem. Soc. 1998, 120, 5317-5318.
(12) Wilstermann, M.; Balogh, J.; Magnusson, G. J. Org. Chem. 1997, 62, 3659-
3665.
* To whom correspondence should be addressed. Telephone: (706)-
5429161. Fax: (706)5424412. E-mail: gjboons@ccrc.uga.edu.
† CCRC, University of Georgia.
(13) Navarre, N.; Amiot, N.; van Oijen, A.; Imberty, A.; Poveda, A.; Jimenez-
Barbero, J.; Cooper, A.; Nutley, M. A.; Boons, G. J. Chem.-Eur. J. 1999,
5, 2281-2294.
‡ CERMAV-CNRS.
(14) Milton, M. J.; Bundle, D. R. J. Am. Chem. Soc. 1998, 120, 10547-10548.
(15) Alibes, R.; Bundle, D. R. J. Org. Chem. 1998, 63, 6288-6301.
(16) Wacowich-Sgarbi, S. A.; Bundle, D. R. J. Org. Chem. 1999, 64, 9080-
9089.
(1) Varki, A. Glycobiology 1993, 3, 97-130.
(2) Dwek, R. A. Chem. ReV. 1996, 96, 683-720.
(3) Wong, C. H.; Halcomb, R. L.; Ichikawa, Y.; Kajimoto, T. Angew. Chem.,
Int. Ed. Engl. 1995, 34, 521-546.
(17) Koviach, J. L.; Chapel, M. D.; Halcomb, R. L. J. Org. Chem. 2001, 66,
2318-2326.
(18) Thoma, G.; Magnani, J. L.; Patton, J. T.; Ernst, B.; Jahnke, W. Angew.
Chem., Int. Ed. 2001, 40, 1941-1945.
(4) Palcic, M. M.; Hindsgaul, O. Trends Glycosci. Glycotechnol. 1996, 8, 37-
49.
(5) Homans, S. W. Glycobiology 1993, 3, 551-555.
9
5964
J. AM. CHEM. SOC. 2002, 124, 5964-5973
10.1021/ja0169771 CCC: $22.00 © 2002 American Chemical Society

R-(2,6)-Sialyltransferase-Catalyzed Sialylations
A R T I C L E S
Figure 2. Disaccharide derivatives 1-5.
showed that in the bound state, LacNAc is mainly observed in
conf A, some in conf B, and one case in conf D.7,22
In conformations A and B, the C-6′ hydroxyl and the
acetamido moiety are at the same face of the molecule, and
both of these residues serve critical roles in substrate recognition
by rat liver R-(2,6)-sialyltransferase.20 The C-6 and C-2′
hydroxyls, which are not critical for transferase activity, are at
the other face of the molecule, and these functionalities were
selected for incorporation of tethers (Figure 2). Compound 2 is
a reference compound in which the C-2′ and C-6 hydroxyls of
LacNAc are methylated. Compound 3 is a 2′,6-anhydro-
derivative, whereas 4 and 5 contain methylene and ethylene
tethers, respectively.
Figure 1. Energy map of LacNAc glycosidic linkage21 calculated with
MM3. Conformations accessible to compounds 3-5 have been superim-
posed as 9 ) 3, 2 ) 4, b ) 5.
which apparent kinetic parameters of rat liver R-(2,6)-sialyl-
transferase-catalyzed sialylations have been determined. These
data have been correlated with conformational properties of
these acceptors obtained by computer modeling and NMR
spectroscopy.
Results and Discussion
Synthesis. The target compounds 2, 3, and 5 were prepared
from orthogonally protected LacNAc 8, which has a tert-butyl
dimethyl silyl ether at C-6 and an acetyl ester at C-2′ (Scheme
1). This disaccharide could be converted into alcohol 9 which
was the precursor of 3 and into diol 10 which was used for the
preparation of 2 and 5. Compound 4 was obtained from the
corresponding disaccharide analogue 21 (Scheme 2).
The key disaccharide 8 was obtained in good yield of 69%
by coupling the known trichloroacetimidate donor 623 with
glycosyl acceptor 724 in dichloromethane at -40 °C using BF3‚
OEt2 as the promoter. Treatment of 8 with HBF4 in H2O/
acetonitrile resulted in removal of the TBDMS ether to afford
9 in almost quantitative yield. Deacetylation of 9 under standard
conditions gave the desired diol 10.
The reference compound 2 was easily obtained by O-
methylation of 10 using methyl iodide, BaO, and Ba(OH)2 in
DMF25 to give 11 followed by debenzylation by catalytic
hydrogenation over Pd/C. Other methylation methods such as
treatment with methyl triflate in the presence of lutidine or
methyl iodide in combination with silver oxide26 were unsuc-
cessful.
The protected 2′,6 anhydro-derivative 327 was prepared by a
three-step procedure starting from 9. Thus, mesylation of 9 using
mesyl chloride (MsCl) in pyridine gave 12, which was deacety-
lated using sodium methoxide in methanol to afford 13.
Cyclization of 13 was accomplished using NaH, and although
Sialyltransferases are an important class of glycosyltrans-
ferases that catalyze the transfer of the N-acetyl neuraminic acid
(Neu5Ac) moiety of CMP-Neu5Ac to the carbohydrate moiety
of glycolipids and proteins. All known sialyltransferases have
two conserved regions in their catalytic domain, which are
proposed to be important for binding the common substrate
CMP-Neu5Ac and catalysis.19 To date, no NMR or X-ray crystal
structures of sialyltransferases have been reported, and informa-
tion about acceptor specificities has mainly been obtained from
studies with chemically modified acceptors. These studies have
revealed the C-6′ hydroxyl and acetamido group of LacNAc
are essential for sialylation by rat liver R-(2,6)-sialyltransferase.20
Furthermore, glycosylation of the C-3′ and C-4′ and C-3
hydroxyl lead to inactivation of the substrate, whereas removal
of other hydroxyls such as those at C-2′ and C-6 leads to some
reduction of acceptor activity.
Previously, the conformational properties of LacNAc have
been investigated by molecular mechanics calculations (Figure
1).21 This disaccharide is highly flexible with a global minimum
energy conformation around Φ ) 50° (Φ ) H1′-C1′-O1′-
C4) and Ψ ) 0° (Ψ ) C1′-O1′-C4-H4) (conf A) and a second
low minimum around Φ ) 30° and Ψ ) -60° (conf B).
Regions C and D correspond to remote parts of the main low
energy region and should be considered as secondary minima.
An analysis of reported X-ray crystal structures of oligosac-
charides/protein complexes that contain a LacNAc moiety
(22) Imberty, A.; Bourne, Y.; Cambillau, C.; Rouge, P.; Perez, S. AdV. Biophys.
Chem. 1993, 3, 71-118.
(23) Schmidt, R. R.; Michel, J. J. Angew. Chem. 1980, 92, 763-765.
(24) Sakai, K.; Nakahara, Y.; Ogawa, T. Tetrahedron Lett. 1990, 31, 3035-
(19) Datta, A. K.; Paulson, J. C. Indian J. Biochem. Biophys. 1997, 34, 157-
165.
3038.
(25) Petrakova, E.; Spohr, U.; Lemieux, R. U. Can. J. Chem. 1992, 70, 233-
(20) Wlasichuk, K. B.; Kashem, M.; Nikrad, P. V.; Bird, P.; Jiang, C.; Venot,
A. P. J. Biol. Chem. 1993, 268, 13971-13977.
240.
(26) Matta, K. L.; Rana, S. S.; Abbas, S. A. Carbohydr. Res. 1984, 131, 265-
(21) Imberty, A.; Mikros, E.; Koca, J.; Mollicone, R.; Oriol, R.; Pe´rez, S.
Glycoconjugate J. 1995, 12, 331-349.
272.
(27) Spohr, U., private communication.
9
J. AM. CHEM. SOC. VOL. 124, NO. 21, 2002 5965

A R T I C L E S
Galan et al.
Scheme 1a
The ethylene-bridged derivative 5 was obtained by a multistep
procedure, whereby 10 was first regioselectively condensed with
mesylate 15 followed by removal of the TBDMS ether;
conversion of the resulting alcohol into a good leaving group
allowed macrocyclization. The ethylene linker 15 was prepared
by a one-pot two-step procedure,28 whereby ethylene glycol was
monosilylated with sodium hydride and TBDMSCl in DMF
followed by sulfonylation by addition of methanesulfonyl
chloride and triethylamine. Condensation of 10 with 15 in the
presence of NaH gave 16, and although a relatively large excess
of 15 was required to drive the reaction to completion, very
little substitution at C-2′ was observed. The TBDMS group of
16 was removed by treatment with HBF4 in H2O/acetonitrile to
give alcohol 17 which was mesylated using standard conditions
to give methyl sulfonate ester 18 in a yield of 47% over three
steps. Ethylene-bridged 19 was obtained in a yield of 60% by
NaH-mediated cyclization of 18 in DMF. Catalytic hydrogena-
tion over Pd/C of compounds 14 and 19 gave 3 and 5,
respectively.
Attempts to prepare methylene-bridged LacNAc 4 by reaction
12
of diol 10 with (PhS)2CH2 in the presence of NIS/TMSOTf
resulted only in the formation of trace amounts of product. The
failure of this reaction was attributed to the presence of the
acetamido group, which interfered unfavorably with the reagents.
To address this problem, the analogous disaccharide 21 was
prepared, which instead of the acetamido has an azido moiety
at C-2 (Scheme 2). This disaccharide was obtained in a yield
of 80% by a BF3‚OEt2-promoted glycosylation of 6 with 20 at
-40 °C in dichloromethane. Treatment of 21 with tetrafluo-
roboric acid in dichloromethane gave 22, which was deacety-
lated under standard conditions to afford 23. In this case, the
diol 23 could be cyclized by intramolecular methylene acetal
formation by reaction with (PhS)2CH2 in the presence of NIS/
TMSOTf12 to give 24 in a acceptable yield of 60%. Reduction
of the azido group of 24 with propanedithiol29 followed by
N-acetylation with acetic anhydride in pyridine gave 25. Finally,
debenzylation of 25 by catalytic hydrogenation over Pd/C gave
the 2′6-methylene acetal 4.
a Reagents and conditions: (i) BF3‚Et2O, CH2Cl2, -40 °C; (ii) 48% HBF4
in H2O, MeCN; (iii) NaOMe, MeOH; (iv) MeI, BaO, Ba(OH)2, DMF; (v)
MsCl, pyridine; (vi) NaH, DMF; (vii) Pd/C, H2; (viii) NaH, DMF, 50 °C.
The presence of the linkers of 3, 4, and 5 was confirmed by
HMBC experiments which allows the correlation between
carbons and protons linked over more than one bond and
supported by NOE data. Cross-peaks in the HMBC spectra were
observed corresponding to the connectivities between the two
sugars other than the glycosidic linkage, that is, H-2′ of
galactoside and C-6 of the glucoside of compound 3, C-7 from
the tether of compound 4, and C-8 from the tether of compound
5, demonstrating that the disaccharides were connected.
Conformational Analysis. The conformational properties of
compounds 1-5 were probed by a combination of molecular
mechanics simulations and NMR spectroscopy. A systematic
conformational search was performed on Ψ and Φ torsional
angles of 3-5 using the SEARCH procedure of the SYBYL
software package (Tripos Inc., St Louis, MO). For the cyclic
compounds, conformational families were determined using a
clustering procedure. Compound 3 appears to be the most
restricted compound with only 14 possible conformers in two
conformational families. Compound 4 can adopt 242 conforma-
Scheme 2a
a Reagents and conditions: (i) BF3‚Et2O, CH2Cl2, -40 °C; (ii) 48% HBF4
in H2O, MeCN; (iii) NaOMe, MeOH; (iv) (PhS)2CH2, NIS, TfOH, CH2Cl2;
(v) HS(CH2)3SH then Ac2O, pyridine; (vi) Pd/C, H2.
(28) McDougal, P. G.; Rico, J. G.; Oh, Y.-I; Condon, B. D. J. Org. Chem.
1986, 51, 3388-3390.
(29) Dullenkopf, W.; Ritter, G.; Fortunato, S.; Old, L. J.; Schmidt, R. Chem.-
Eur. J. 1999, 5, 2432-2438.
this reaction involved the formation of a rigid eight-membered
ring, compound 14 was obtained in a yield of 70%.
9
5966 J. AM. CHEM. SOC. VOL. 124, NO. 21, 2002

R-(2,6)-Sialyltransferase-Catalyzed Sialylations
A R T I C L E S
Figure 3. Low energy conformations of compounds 3-5. For each compound, the lowest energy conformation has been colored according to atom types.
Conformer A of LacNAc is also displayed for comparison.
tions scattered in seven families, and compound 5 is the most
flexible of the cyclic compounds with 2989 conformers in 14
different families. The lowest energy conformer of each family
was fully optimized using the MM3 program.30 Figure 3 displays
the obtained low energy conformations, and their complete
geometrical characterizations are listed in Supporting Informa-
tion. To compare the conformational properties of the confor-
mationally constrained compounds with the properties of
LacNAc, the low energy conformations of 3-5 were superim-
posed on the energy map of LacNAc (Figure 1). Derivative 3
is highly constrained and can adopt only two conformations,
both with similar but unusual conformations at the linkage.
Compound 4 can exist in a larger range of conformations;
however, the Φ dihedral angle is restricted to values smaller
than 30° which are not entirely similar to minimum energy
conformations of LacNAc. Compound 5 can attain conforma-
tions in the relatively large energy plateau of LacNAc that
include syn conformations A and B. However, the anti- (conf
C) and gauche-gauche conformer (conf D) cannot be adopted.
In all derivatives, the ω torsion angle that is involved in the
bridge cannot adopt its lowest energy orientation since such
orientation directs the O-6 atom far from the galactose residue
and is not compatible with the establishment of the bridge.
NMR Data and Comparison with Molecular Modeling.
The 1H and 13C spectra of compounds 1-5 were assigned by a
combination of one-dimensional proton, and two-dimensional
HSQC and HSQC-TOCSY experiments. Assignment of pro-R
and pro-S hydroxymethylene protons of the GlcNAc moiety of
1 and 2 was based on relative 3JHH coupling constants assuming
predominantly gg and gt rotamer populations.31 The stereospe-
Trans-glycosidic heteronuclear coupling constants, which are
sensitive to orientations around glycosidic linkages, were
measured using a quantitative HMBC experiment.32 The result-
ing values are listed in Table 1 together with the calculated
values for each low energy conformer using the empirical
Karplus-type equation proposed by Tvaroska et al.33 This
approach allowed a direct comparison of the experimental
coupling constants with the theoretical ones obtained by
averaging the populations of conformers using a Boltzmann
equation for energy-weighting. For LacNAc 1, the 3JH1′-C4 and
3JH4-C1′ values of 2.9 and 2.5 Hz, respectively, support the
mixture of conformers determined by the modeling studies. To
obtain a good agreement with experimental data, the computed
population of conformer B should be higher. As expected,
compound 2 gave similar values to 1 (3.3, 2.6 Hz). Compound
3 provided a small value of 3JH1′-C4 (1.2 Hz) corresponding to
Φ of approximately -60°, and due to spectral overlap, 3JH4-C1′
could not be measured. Compound 3 provided additional NOE
and J coupling data that was most consistent with the lowest
energy conformer. A 3JH5-H6R value of 7.3 Hz, a 3JH6S-C2′ value
3
3
of 7 Hz, small JH6R-C4 and JH6S-C4 values (<1.5 Hz), and a
large H2′-H6R NOE (2.4 Å) are in agreement with a very
unusual eclipsed orientation of the ω torsion angle. In the lowest
energy conformer, ω adopts a value of -102° (see Supporting
Information) with a small H2′-H6R distance (2.1 Å vs
experimentally derived 2.4 Å). Experiments and modeling
(32) Additional measurements were made using two-dimensional HSQC type
experiments such as “Exside” (Krishnamurthy, V. J. Magn. Reson., Ser. A
1996, 121, 33-41) and HSQCMBC (Williamson, R.; Marquez, B.;
Gerwick, W.; Kover, K. Magn. Reson. Chem. 2000, 38, 265-273). In all
the compounds, significant signal overlap and strong coupling restricted
their application, and only a few values (3JH1′-C4 for compounds 1, 2, and
3) could be obtained which agreed within experimental error with those
obtained via the HMBC experiment. The application of other methods that
depend on carbon selective pulses (for example, Nishida, T.; Widmalm,
G.; Sandor, P. Magn. Reson. Chem. 1996, 34, 377-382) did not provide
useful data.
3
cific assignments for compounds 3-5 were based on JH5,H6
3
and JC4,H6 values and interresidual NOE’s assuming tg or
eclipsed Gln-H5/H6 rotamer populations.
(30) Allinger, N. L.; Yuh, Y. H.; Lii, J. H. J. Am. Chem. Soc. 1989, 111, 8551-
8566.
(31) Hounsell, E. Prog. Nucl. Magn. Reson. Spectrosc. 1995, 27, 445-474.
(33) Tvaroska, I.; Hricovini, M.; Petrakova, E. Carbohydr. Res. 1989, 189, 359-
362.
9
J. AM. CHEM. SOC. VOL. 124, NO. 21, 2002 5967

A R T I C L E S
Galan et al.
Table 1. Comparison of Experimental and Theoretical NMR
Table 2. Apparent Kinetic Parameters of Rat Liver
Values of LacNAc and LacNAc Derivativesa
R-(2,6)-Sialyltranferase-Catalyzed Sialylation of Compounds 1-5a
ΦΗ
(deg)
JH1′-C4
(Hz)
ΨH
(deg)
JH4-C1′ H1′−H4 H1′−H6R H1′−H6S
(rel Vmax)/
Km (mM-1
)
(Hz)
(Å)
(Å)
(Å)
acceptor
Km (mM)
rel Vmax
compd 1
conf•A
conf•B
conf•C
conf•D
av conf
exp
1
2
3
4
5
1.7 ( 0.2
11.2 ( 0.8
n.a.
11.6 ( 1.9
1.0 ( 0.1
1
0.6
0.04
n.a
0.09
1.2
45
28
43
2.9
4.4
3.1
6.8
3.7
2.9
-5
-54
5.5
2.1
6.7
2.2
2.4
3.7
3.6
2.3
2.4
2.8
4.1
5.2
4.5
3.1
2.9
3.2
4.5
4.1
5.4
3.5
3.0
0.5
n.a.
1.0
1.3
-174
176
-2.6 5.6
3.9
2.5
a n.a. compound not active up to 2.0 mM of substrate.
compd 2
exp
3.3
2.6
2.5
2.9
n.m.
assay34,35 (Table 2). The Km for 1 was in close agreement with
previous data, and its Vmax was set at 1. Compound 2, containing
methoxy groups at C-6 and C-2′, has a significantly elevated
Km and a somewhat smaller Vmax, indicating that these hydroxyls
are of some importance for the transferase. The anhydro-
derivative 3 is not a substrate, whereas the methylene-bridged
compound 4 has similar kinetic parameters as compared to that
of 2. Importantly, the ethylene-tethered derivative 5 has the
highest catalytic efficiency of all compounds tested, suggesting
that it is preorganized in a favorable conformation.
compd 3
conf•1
conf•2
av conf
exp
compd 4
conf•1
conf•2
conf•3
conf•4
conf•5
av conf
exp
compd 5
conf•1
conf•2
conf•3
conf•4
conf•5
conf•6
conf•7
conf•8
conf•9
av conf
exp
-47
-29
2.7
4.3
3.1
1.2
25
49
4.6
2.6
4.2
n.m.
2.1
2.2
2.1
2.3
4.5
2.1
2.7
4.1
4.2
3.7
4.1
4.0
-0
-42
13
24
12
5.6
3.2
5.3
4.7
5.4
5.5
4.2
-14
-13
17
5.3
5.3
5.1
5.6
5.6
5.3
5.9
2.0
2.3
2.1
2.2
2.0
2.0
2.1
3.5
5.5
2.2
3.7
4.3
3.3
n.o.
4.9
4.2
3.9
4.4
3.8
4.8
n.o
0
3
A preparative enzymatic reaction was performed to ensure
that the sialylation had occurred at C-6 and that the tether of
compound 5 was still intact. The structure of the resulting
-5
-1
3
24
13
-18
46
15
39
5.6
5.6
5.6
4.7
5.3
5.1
2.9
5.2
3.4
5.3
4.5
-32
-14
-18
-48
-9
-22
6
-59
13
4.0
5.3
5.1
2.7
5.5
4.8
5.5
3.4
5.3
4.2
4.6
2.15
2.01
2.02
2.31
2.05
2.16
2.36
2.28
2.25
2.13
2.3
5.40
3.47
5.01
4.39
2.80
4.06
3.37
4.10
2.46
4.42
n.o.
4.38
4.94
3.83
5.41
4.41
5.36
4.12
5.34
3.91
4.38
n.o.
1
tethered trisaccharide 6 was confirmed by H NMR and MS
analyses. The spectroscopic data were in agreement with that
of the methyl glycoside of the corresponding linear trisaccha-
ride.36
The observation that compound 5 is the best acceptor tested
indicates that it is preorganized in a conformation that is
favorable for the transferase. The low energy conformations of
this compound are centered about the A- and B-conformers of
LacNAc with A being the most populated one. Thus, these
combined findings indicate that the enzyme recognizes LacNAc
in one of its main low energy conformations with conf A being
the most probable one. Correlating the properties of compounds
4 and 5 reveals that small differences in conformational behavior
may result in significant differences in apparent kinetic param-
eters. In the case of 4, a small shift outside the main minimum
resulted in a much larger Km, but, importantly, the compound
can still act as an acceptor. When larger conformational changes
are induced, such as in compound 3, glycosyl-accepting
properties are lost. The similarity of apparent kinetic parameters
for compounds 2 and 4 was surprising. Probably, the unfavorable
conformation of 4 is compensated by less reduction of flexibility
upon binding. The Km of 5 is approximately 10 times smaller
than that of the dimethoxy analogue 2 with a 3-fold increase in
a n.m. not measured, n.o. not observed; exp. J values (15%; exp. NOE
values (0.3 Å.
therefore indicate that the 6-hydroxymethyl-C2′ bridge rotates
H6R to eclipse H5, and thus rotates the Φ angle into the unusual
-60° value. The proposed conformation moves the C-2 acet-
amido group away from the face containing the C-6′ hydroxyl.
Compound 4 has values of both JH1′-C4 (4.2 Hz) and JH4-C1′
(5.9 Hz) significantly larger than those of 1, consistent with
the major cluster of conformers proposed by the modeling
studies. Compound 5 also has coupling values larger than that
3
3
3
of 1 (4.5, 4.6 Hz), and the JH4-C1′ is consistent with the
predicted high population of Ψ torsional angle close to 0°, and
therefore similar to conformer A of compound 1.
Distance constraints were obtained from relative NOE
intensities using the isolated spin-pair approximation. In Table
1, the measured distances are compared to the ones obtained
from each low energy conformer. The theoretical averaged
distances have been calculated from an r-6 1/6 distance using
a Boltzmann population. All compounds showed a strong NOE
between H1′ and H4, which was expected for compounds
restricted to syn conformations. Compounds 1 and 2 showed
an NOE between H1′-H6R and H6S, which is indicative of
favored gt orientation of the ω torsion angle for the GlcNAc
residue in LacNAc. This NOE was very weak for compound 3
and was not observed for compounds 4 and 5, which is in
agreement with the computer modeling data.
V
max. Both compounds essentially have the same hydrogen-
bonding potential and hydrophobic and hydrophilic surface but
differ in conformational properties. Therefore, the significantly
higher catalytic efficiency (Vmax/Km) of 5 probably results from
a more favorable enzyme-substrate association (approximately
1.5-2.0 kcal/mol) due to preorganization of the acceptor in a
conformation that is recognized by the enzyme. It is important
to note that apart from preorganization of the glycosidic linkages
of 2 and 5, their C-6 and C-2′ substitutions (ethyl bridge vs
two methyl groups) are also conformationally different. These
Enzyme Kinetics. The apparent kinetic parameters for the
R-(2,6)-sialyltransferase-catalyzed reaction of CMP-(14C)-
Neu5Ac with acceptors 1-5 were determined using a reported
(34) Paulson, J. C.; Rearic, J. I.; Hill, R. L. J. Biol. Chem. 1977, 252, 2363-
2371.
(35) Horenstein, B. A.; Bruner, M. J. Am. Chem. Soc. 1998, 120, 1357-1362.
(36) Sabesan, S.; Paulson J. C. J. Am. Chem. Soc. 1986, 108, 2068-2080.
9
5968 J. AM. CHEM. SOC. VOL. 124, NO. 21, 2002

R-(2,6)-Sialyltransferase-Catalyzed Sialylations
A R T I C L E S
presented in the Supporting Information. Standard Varian gradient
HSQC and HSQC-TOCSY pulse sequences were used for the assign-
ment data.
positions of the disaccharide are thought to be at the periphery
of the enzyme-binding site, and it is unlikely that these
conformational modifications can account for the observed
differences in catalytic efficiency. Thus, we propose that the
glycosidic linkage of 5 will lose less conformational flexibility
upon binding than 2, resulting in a more favorable association.
This result is highly significant because most of the previously
reported studies with conformational constrained oligosaccha-
rides failed to produce more favorable free energies of bind-
ing.11,13,18
Heteronuclear Coupling Constants. The three-bond proton-carbon
coupling constants (3JCH) were measured according to Zhu et al.40
A
typical data set was 600 × 64 complex points, covering a 1H range of
3.5 ppm and 13C range of 60 ppm. Sufficient transients were collected
to obtain a signal-to-noise of ∼20:1 for long-range cross-peaks. Data
were processed using Gaussian apodization functions and zero-filling
in both dimensions. No special noise reduction procedures were used.
The cross-peak intensities were measured by integrating the peak
volumes in the two-dimensional spectra, or by extracting one-
dimensional slices from these cross-peaks. Ratios of the long-range
cross-peak and the one-bond reference volumes were used to calculate
J values, according to Zhu et al.40 In cases where neighboring cross-
peaks distorted the baseline significantly, making volume integration
unreliable, the one-dimensional slices of these volumes were matched,
and a scaling factor was extracted. The corrections due to differences
in T1 and T2 for 13C- and 12C-bonded protons canceled within the
experimental error for the protons that were measured. The values
reported thus have an error of (15% primarily due to signal-to-noise
and uncertainties in integration due to baseline distortions.
Experimental Section
Molecular Modeling. Nomenclature. Torsion angles at the glyco-
sidic linkage have been defined with reference to the hydrogen atom:
ΦH ) H1′-C1′-O1′-C4 and ΨH ) C1′-O1′-C4-H4 (see Supporting
Information).
Conformational Analysis of the Cyclic Derivatives. All energy
calculations were performed using the TRIPOS force field37 comple-
mented by carbohydrate energy parameters.38 First, it was checked that
the relaxed (Φ,Ψ) energy map of LacNAc calculated with the TRIPOS
force field was similar to the MM3 energy map, previously published.21
Starting conformations of compounds 3, 4, and 5 were built using low
energy conformation of LacNAc and editing and optimizing the
molecule while checking that no distortion of pyranose rings occurred.
The possible conformations of compounds 3, 4, and 5 were determined
using the SEARCH procedure of SYBYL. The ring closure bond was
chosen to be µ6 in compound 3, ν6 in compound 4, and ν2 in compound
5. Tolerance for ring closure was 0.3 Å for bond length and 20° for
bond angles. Several conditions were tested on compound 3 and 4. A
search step of 10° with van der Waals cutoff of 0.6 and energy window
of 20 kcal/mol was appropriate for the conformational studies. The
hydroxylic hydrogen atoms were not taken into account during this
rigid search. The energy was evaluated for each conformation, but the
electrostatic contribution was not included. These conditions were
applied for the three compounds. The resulting conformations were
then analyzed by a homemade clustering program to determine
conformational families. The lowest energy conformer of each family
was then fully optimized with the use of the MM3 force field,30 using
a dielectric constant of 80 to simulate water environment. Some of the
families converged to the same conformation. Low energy conforma-
tions are displayed, and their characteristics are listed in Supporting
Information.
NOE Experimental Procedures. For each compound, selective one-
dimensional NOESY experiments41 were used to obtain buildup curves
for the anomeric and other resolved protons. For compound 1, the
anomeric protons were overlapped, and therefore a one-dimensional-
TOCSY-NOESY sequence was employed.42 In this experiment, H4′
was selectively irradiated, followed by TOCSY transfer to H1′, which
then was selectively irradiated for the subsequent NOESY step.
Similarly, H-5 was selectively irradiated prior to the TOCSY transfer
to H-1. For each compound, good linearity of the NOE buildup curves
was observed up to a mixing time of 300 ms. For compounds 2, 3, and
5, the interproton distances were determined by using the intraring
H-1′-H-2′ NOE as an internal reference (3.09 Å).43 For compounds 1
and 4, the NOE between H-1′ and H-2′ could not be accurately
measured; therefore, H-1-H-5 (2.37 Å)43 was employed as internal
reference.
General Methodology. Chemicals were purchased from Aldrich and
Fluka and used without further purification. Molecular sieves were
activated at 350 °C for 3 h in vacuo. Dichloromethane was distilled
from CaH2 and stored over 4 Å molecular sieves. All of the reactions
were performed under anhydrous conditions and monitored by TLC
on Kieselgel 60 F254 (Merck). Detection was by examination under UV
light (254 nm) and by charring with 10% sulfuric acid in methanol.
Flash chromatography was performed on silica gel (Merck, mesh 70-
230). Extracts were concentrated under reduced pressure at <40 °C
(bath). 1H NMR (1-D, 2-D) and 13C NMR spectra were recorded on a
Varian Merc300 spectrometer and Varian 500, 600, and 800 MHz
Heteronuclear 3JC-H coupling constants across the glycosidic linkages
were calculated using the Karplus-type equation proposed by Tvaro-
ska.33
1
spectrometers equipped with Sun workstations. For H and 13C NMR
3JC-H ) 5.7 cos2 Φ - 0.6 cos Φ + 0.5
spectra recorded in CDCl3, chemical shifts (δ) are given in ppm relative
to solvent peaks (1H, δ ) 7.26; 13C, δ ) 77.3) as internal standard.
Negative ion matrix assisted laser desorption ionization time-of-flight
(MALDI-TOF) mass spectra were recorded using an HP-MALDI
instrument using gentisic acid matrix. Optical rotations were measured
on a Jasco P-1020 polarimeter, and [R]D values are given in units of
10-1 deg cm3 g-1 at 25 °C, 50 mm cell.
The rat liver R-(2,6)-sialyltransferase was purchased from Sigma.
CMP-[14C]Neu5Ac was obtained from Amersham Corp. CTP, CMP-
Neu5Ac, and calf alkaline phosphatase were purchased from Calbio-
chem. ACS liquid scintillation cocktail was obtained from Fisher
3
Averaging of NMR parameters JC-H and r-6 was performed on
the population of conformers, taking into account the probability of
existence (Pi) of each conformer with energy Ei at a given temperature
T as governed by a Boltzmann distribution. This procedure has been
detailed in the conformational analysis of sucrose.39
NMR Spectroscopy. The disaccharides (2-4 mg) were dissolved
in 99.96% D2O. Data were collected on Varian 500, 600, and 800 MHz
spectrometers at 25 °C, except for some data on compound 4, which
were collected at 21 °C. Complete proton and carbon assignments are
(37) Clark, M.; Cramer, R. D. I.; van den Opdenbosch, N. J. Comput. Chem.
1989, 10, 982-1012.
(38) Imberty, A.; Bettler, E.; Karababa, M.; Mazeau, K.; Petrova, P.; Pe´rez, S.
Building Sugars: The Sweet Part of Structural Biology; Indian Academy
of Sciences and Universities Press: Hyderabad, 1999; pp 392-409.
(39) Herve´ du Pehnoat, C.; Imberty, A.; Roques, N.; Michon, V.; Mentech, J.;
Descotes, G.; Pe´rez, S. J. Am. Chem. Soc. 1991, 113, 3720-3727.
(40) Zhu, G.; Renwick, A.; Bax, A. J. Magn. Reson., Ser. A 1994, 110, 257-
261.
(41) Stott, K.; Stonehous, J.; Keeler, J.; Hwang, T.-L.; Shaka, A. J. J. Am. Chem.
Soc. 1995, 117, 4199-4200.
(42) Uhrin, D.; Barlow, P. J. Magn. Reson. 1997, 126, 248-255.
(43) Perez, S.; Mazeau, K.; Imberty, A.; Karababa, M.; Bettler, E., http://
webenligne.cermav.cnrs.fr/databank/monosaccharides/index.html.
9
J. AM. CHEM. SOC. VOL. 124, NO. 21, 2002 5969

A R T I C L E S
Galan et al.
Scientific. All other chemicals were of analytical grade. Compounds
1-5 were purified by chromatography on Iatrobeads (Iatron laborato-
ries, 6RS-8060), followed by chromatography on biogel P2 (Biorad).
Methyl 2-Acetamido-2-deoxy-3-O-benzyl-4-O-(3,4,6-tri-O-benzyl-
D-galactopyranosyl)-â-D-glucopyranoside (10). Sodium methoxide
â-
(5.4 mg, 0.1 mmol) was added to a stirred solution of 9 (450 mg, 0.56
mmol) in methanol (20 mL). The mixture was left stirring at room
temperature for 48 h. TLC (hexane/acetone, 1/1, v/v) indicated
completion of the reaction. The mixture was neutralized with Dowex
50H+ resin until pH ) 7, filtered, and concentrated in vacuo. The
residue was purified by flash silica gel chromatography (gradient
hexane/acetone, 3/1 to 1/1, v/v) to afford compound 10 (418 mg, 98%)
as a white foam. 1H NMR (CDCl3, 300 MHz): δ 7.40-7.10 (m, 20H,
arom), 5.42 (d, 1H, JNH,2 7.9, NH), 4.92, 4.59 (AB q, 2H, JAB 11.4,
OCH2Ph), 4.80, 4.48 (AB q, 2H, JAB 11.4, OCH2Ph), 4.59, 4.23 (AB
q, 2H, JAB 11.4, OCH2Ph), 4.50, 4.18 (AB q, 2H, JAB 11.4, OCH2Ph),
4.6 (d, 1H, J1,2 7.9, H-1), 4.52 (d, 1H, J1′,2′ 8.3, H-1′), 4.04 (t, 1H, J3,4
9.2, H-3), 3.95 (dd, 1H, J6a-5 < 2, J6a-6b 12.1, H-6a), 3.90 (m, 1H,
H-4), 3.85 (m, 1H, J2′,3′ 9.7, H-2′), 3.82 (dd, 1H, J3′-4′ 2.6, H-4′), 3.81
(dd, 1H, H-6b), 3.5 (m, 2H, H-6a′, H6b′), 3.40 (s, 3H, OCH3), 3.30
(dd, 1H, J3′,4′ 2.6, H-3′), 3.29 (m, 1H, H-2), 3.25 (m, 1H, H-5), 3.22
(dt, 1H, J5′-6a′ 7.9, J5′-6b′ 7.9, H-5′), 1.79 (s, 3H, CH3C(O)NH). 13C
NMR (CDCl3, 125 MHz): δ 170.90, 139.13, 138.78, 138.13, 137.93,
128.68, 128.54, 128.43, 128.33, 128.03, 127.99, 127.92, 127.82, 127.65,
127.52, 104.03, 101.41, 82.15, 79.86, 76.803, 75.29, 74.71, 74.11, 73.71,
73.58, 72.87, 72.50, 72.14, 71.00, 68.36, 56.96, 56.83, 23.63. MALDI-
TOF: m/z 780.4 [M + Na]+. Anal. Calcd for C43H51NO11: C, 68.15;
H, 6.78; N, 1.85; O, 23.22. Found: C, 68.28; H, 6.91; N, 1.90. [R]D )
5.6 (c 0.43, CHCl3).
Methyl 2-Acetamido-2-deoxy-3-O-benzyl-6-O-tert-butyldimeth-
ylsilyl-4-O-(2-O-acetyl-3,4,6-tri-O-benzyl-â-D-galactopyranosyl)-â-
D-glucopyranoside (8). Boron trifluoride diethyl etherate (0.9 mL, 7.1
mmol) was added to a stirred suspension of 6 (4.5 g, 7.1 mmol), 7 (2.0
g, 4.7 mmol), and powdered molecular sieves 4 Å (4.0 g) in dry
dichloromethane (50 mL). The mixture was left stirring at -40 °C for
3 h. TLC (toluene/ethyl acetate, 1/1, v/v) indicated completion of the
reaction. The mixture was neutralized with triethylamine (1.4 mL, 10.0
mmol) and then filtered over Celite. The filtrate washings were
combined and concentrated under reduced pressure. The residue was
then diluted in dichloromethane (100 mL), washed successively with
a saturated solution of NaHCO3 (20 mL), water (2 × 20 mL), and
brine (20 mL), followed by drying over MgSO4. After evaporation of
the solvent, the residue was purified by flash silica gel chromatography
(gradient hexane/ethyl acetate, 3/1 to 1/1, v/v). The yellow solid was
crystallized from diethyl ether/ethanol, and the product was washed
with cold diethyl ether to give compound 8 (2.97 g, 69%) as a white
solid. 1H NMR (CDCl3, 300 MHz): δ 7.60-7.15 (m, 20H, arom), 6.1
(d, 1H, JNH,2 8.8, NH), 5.30 (dd, 1H, J2′,3′ 9.7, H-2′), 4.73 (AB q, 2H,
JAB 11.4, OCH2Ph), 4.46 (AB q, 2H, JAB 12.3, OCH2Ph), 4.73-4.20
(m, 4H, 2 x OCH2Ph), 4.40 (d, 1H, J1,2 5.7, H-1), 4.38 (d, 1H, J1′,2′ 7.9,
H-1′), 4.0-3.88 (m, 2H, H-6b, H-6b′), 3.96 (d, 1H, H-4′, J3′-4′ 2.74),
3.90 (m, 1H, H-2), 3.72-3.68 (m, 2H, H-5′, H-6a′), 3.61-3.38 (m,
4H, H-3, H-4, H-5, H-6a), 3.50 (m, 1H, H-3′), 3.35 (s, 3H, OCH3),
2.02 (s, 3H, CH3C(O)NH), 1.90 (s, 3H, CH3C(O)O), 0.81 (s, 9H, Si-
(CH3)3), -0.03, -0.05 (2s, 6H, Si(CH3)2). 13C NMR (CDCl3, 125
MHz): δ 169.94, 138.25, 137.74, 137.61, 128.30, 128.27, 128.07,
127.95, 127.78, 127.77, 127.70, 127.62, 127.46, 127.27, 127.19, 101.17,
99.72, 79.99, 75.96, 74.63, 73.47, 73.32, 72.53, 72.42, 72.06, 71.80,
67.79, 62.09, 56.16, 51.01, 25.87, 23.34, 21.16, 18.32, 18.20, -5.21,
-5.30. FAB-MS: m/z 936.5 [M + Na]+. Anal. Calcd for C51H67NO12-
Si: C, 67.01; H, 7.39; N, 1.53; O, 21.00; Si, 3.07. Found: C, 67.18;
H, 7.55; N, 1.49. [R]D ) -17.6 (c 0.59, CHCl3). mp ) 124.9 °C.
Methyl 2-Acetamido-2-deoxy-6-O-methyl-4-O-(2-O-methyl-â-D-
galactopyranosyl)-â-D-glucopyranoside (2). Methyl iodide (0.86 mL,
9.23 mmol), anhydrous barium oxide (778 mg, 5.0 mmol), and barium
hydroxide octahydrate (750 mg, 2.5 mmol) were added to a stirred
solution of 10 (318 mg, 0.42 mmol) in dry N,N-dimethylformamide (3
mL). The mixture was left stirring at room temperature for 24 h. TLC
(chloroform/methanol, 9/1, v/v) indicated completion of the reaction.
The mixture was diluted with ethyl acetate (20 mL), washed succes-
sively with a saturated solution of NaHCO3 (10 mL), water (5 × 10
mL), and brine (10 mL), followed by drying over MgSO4. After
evaporation of the solvent, the residue was purified by flash chroma-
tography (gradient hexane/acetone, 3/1 to 1/1, v/v) to afford compound
11 (198 mg, 60%). 10% palladium on charcoal (125.0 mg) was added
to a solution of 11 (100 mg, 0.13 mmol) in ethanol (10 mL). The
mixture was vigorously stirred under an atmosphere of hydrogen for
16 h. TLC (chloroform/methanol, 9/1, v/v) indicated the completion
of the reaction. After filtration on Celite and concentration, the crude
material was purified by chromatography (Iatrobeads, chloroform/
methanol/water, 74/24/2, v/v/v) to afford compound 2 (52 mg, 94%)
as an amorphous white solid. MALDI-TOF: m/z 448.3 [M + Na]+.
Methyl 2-Acetamido-2-deoxy-3-O-benzyl-4-O-(2-O-acetyl-3,4,6-
tri-O-benzyl-â-D-galactopyranosyl)-â-D-glucopyranoside (9). Tet-
rafluoroboric acid (48% in water, 0.31 mL, 2.33 mmol) was added to
a stirred solution of 8 (2.0 g, 2.2 mmol) in acetonitrile (50 mL). The
mixture was left stirring at room temperature for 5 min. TLC (toluene/
ethyl acetate, 1/1, v/v) indicated completion of the reaction. The mixture
was neutralized with triethylamine (0.33 mL, 2.3 mmol) and concen-
trated in vacuo. The residue was then dissolved in dichloromethane
(100 mL), washed successively with a saturated solution of NaHCO3
(20 mL), water (2 × 20 mL), and brine (20 mL), followed by drying
over MgSO4. After evaporation of the solvent, the residue was purified
by flash silica gel chromatography (gradient hexane/ethyl acetate, 3/1
1
[R]D ) -20.6 (c 0.30, H2O). For H NMR and 13C NMR data, see
Supporting Information.
Methyl 2-Acetamido-2-deoxy-3-O-benzyl-6-O-methanesulfonyl-
4-O-(2-O-acetyl-3,4,6-tri-O-benzyl-â-D-galactopyranosyl)-â-D-glu-
copyranoside (12). Methanesulfonyl chloride (70 µL, 0.9 mmol) was
added to a cooled (-20 °C) and stirred solution of 9 (479 mg, 0.6
mmol) in dry pyridine (20 mL). After stirring for 6 h at -20 °C, TLC
(toluene/ethyl acetate, 1/1, v/v) indicated completion of the reaction.
After coevaporation with toluene, the reaction mixture was diluted with
dichloromethane (100 mL), washed successively with a saturated
solution of NaHCO3 (20 mL), water (2 × 20 mL), and brine (20 mL),
followed by drying over MgSO4. After evaporation of the solvent, the
residue was purified by flash silica gel chromatography (hexane/ethyl
acetate, gradient 3/1, 3/2 followed by 1/1, v/v) to afford compound 12
(463 mg, 88%) as a white foam. 1H NMR (CDCl3, 300 MHz): δ 7.40-
7.10 (m, 20H, arom), 6.40 (d, 1H, JNH,2 9.7, NH), 5.34 (dd, 1H, J1′,2′
7.9 J2′3′ 10.1, H-2′), 4.94, 4.51 (AB q, 2H, JAB 11.4, OCH2Ph), 4.74,
4.44 (AB q, 2H, JAB 11.4, OCH2Ph), 4.69, 4.54 (AB q, 2H, JAB 11.9
OCH2Ph), 4.63 (dd, 1H, J6b,5 4.0, H-6b), 4.60, 4.40 (AB q, 2H, JAB
12.3, OCH2Ph), 4.55 (d, 1H, J1,2 5.7, H-1), 4.47 (dd, 1H, J6a,5 5.3, J6a,6b
1
to 1/1, v/v) to afford compound 9 (1.7 g, 99%) as a white foam. H
NMR (CDCl3, 300 MHz): δ 7.40-7.10 (m, 20H, arom), 5.82 (d, 1H,
JNH,2 8.3, NH), 5.26 (dd, 1H, J2′,3′ 10.1, H-2′), 4.91, 4.50 (AB q, 2H,
JAB 11.4, OCH2Ph), 4.76, 4.60 (AB q, 2H, JAB 11.4, OCH2Ph), 4.63,
4.46 (AB q, 2H, JAB 12.3, OCH2Ph), 4.54 (d, 1H, J1,2 7.9, H-1), 4.43
(d, 1H, J1′,2′ 8.4, H-1′), 4.35, 4.22 (AB q, 2H, JAB 11.9, OCH2Ph), 3.91
(d, 1H, J3′-4′ 2.64, H-4′), 3.88-3.35 (m, 5H, H-4, H-5, H-5′, H-6a′,
H6b′), 3.86 (dd, 1H, J6a-5 3.86, J6a-6b 11.6, H-6a), 3.82 (m, 1-H, H-3),
3.80 (dd, 1H, H-6b), 3.70 (m, 1H, H-2), 3.50 (m, 1H, H-3′), 3.41 (s,
3H, OCH3), 1.97 (s, 3H, CH3C(O)NH), 1.84 (s, 3H, CH3C(O)O). 13C
NMR (CDCl3, 125 MHz): δ 170.50, 170.08, 138.76, 138.69, 138.05,
137.98, 128.62, 128.38, 128.23, 128.10, 128.06, 127.99, 127.97, 127.72,
127.62, 127.59, 101.75, 100.81, 80.34, 80.26, 75.86, 75.52, 74.81, 73.70,
73.55, 72.90, 72.51, 72.23, 72.13, 68.30, 61.90, 56.94, 53.85, 23.56,
21.22. FAB-MS: m/z 821.3 [M + Na]+. Anal. Calcd for C45H53NO12:
C, 67.57; H, 6.68; N, 1.75; O, 24.00. Found: C, 67.69; H, 6.80; N,
1.88. [R]D ) -9.6 (c 0.73, CHCl3).
9
5970 J. AM. CHEM. SOC. VOL. 124, NO. 21, 2002

R-(2,6)-Sialyltransferase-Catalyzed Sialylations
A R T I C L E S
10.1, H-6a), 4.39 (d, 1H, J1′,2′ 7.9, H-1′), 4.23 (dd, 1H, J2,3 8.4, H-2),
3.99-3.79 (m, 1H, H-4), 3.97 (d, 1H, J3′-4′ 2.6, H-4′), 3.93 (m, 1H,
H-5), 3.82 (m, 1H, H-3), 3.61-3.46 (m, 3H, H-5′, H-6a′, H-6b′), 3.53
(dd, 1H, J2′-3′ 10.1, H-3′), 3.44 (s, 3H, OCH3), 2.82 (s, 3H, CH3SO2),
2.07 (s, 3H, CH3C(O)O), 1.99 (s, 3H, CH3C(O)NH). 13C NMR (CDCl3,
125 MHz): δ 170.78, 170.51, 138.47, 138.10, 137.99, 128.84, 128.70,
128.58, 128.55, 128.48, 128.28, 128.15, 128.12, 127.95, 127.73, 101.77,
100.19, 80.00, 77.66, 77.43, 77.23, 76.81, 75.50, 74.93, 74.08, 73.78,
72.89, 72.63, 72.43, 71.90, 69.42, 68.39, 56.92, 48.41, 37.26, 29.92,
23.34, 21.27. MALDI-TOF: m/z 900.5 [M + Na]+. Anal. Calcd for
C46H55NO14: C, 62.93; H, 6.31; N, 1.60; O, 25.51; S, 3.65. Found: C,
63.05; H, 6.36; N, 1.75. [R]D ) -24.8 (c 0.13, CHCl3).
Methyl 2-Acetamido-2-deoxy-3-O-benzyl-6-O-methanesulfonyl-
4-O-(3,4,6-tri-O-benzyl-â-D-galactopyranosyl)-â-D-glucopyrano-
side (13). Sodium methoxide (5.4 mg, 0.1 mmol) was added to a
solution of 12 (447 mg, 0.51 mmol) in methanol (10 mL). After stirring
at room temperature for 48 h, TLC (hexane/acetone, 1/1, v/v) indicated
completion of the reaction; the mixture was then neutralized with
Dowex 50H+ until pH ) 7.0, filtered, and concentrated in vacuo. The
residue was purified by flash chromatography (gradient hexane/acetone,
3/1 to 1/1, v/v) to afford compound 13 (418 mg, 98%) as a white foam.
1H NMR (CDCl3, 300 MHz): δ 7.40-7.10 (m, 20H, arom), 5.88 (d,
1H, JNH,2 7.7, NH), 4.86, 4.72 (AB q, 2H, JAB 11.5, OCH2Ph), 4.81,
4.54 (AB q, 2H, JAB 11.3, OCH2Ph), 4.72, 4.57 (AB q, 2H, JAB 11.8,
OCH2Ph), 4.60, 4.40 (AB q, 2H, JAB 12.3, OCH2Ph), 4.62 (d, 1H, J1,2
7.4, H-1), 4.60 (m, 1H, H-6b), 4.47 (m, 1H, H-6a), 4.44 (d, 1H, J1′,2′
7.7, H-1′), 4.00 (dd, 1H, J3,4 8.5, H-3), 3.99 (dd, 1H, J3′,4′ 3.8, H-3′),
3.96 (dd, 1H, J2′,3′ 9.6, H-2′), 3.86 (m, 2H, H-4, H-5), 3.80 (m, 1H,
H-4′), 3.64 (m, 1H, H-2), 3.60 (dd, 1H, J6a′-6b′ 11.8, H-6b′), 3.57 (dd,
1H, J6a′-5 2.2, H-6a′), 3.48-3.30 (m, 1H, H-5′), 3.46 (s, 3H, OCH3),
2.96 (s, 3H, CH3SO2), 1.85 (s, 3H, CH3C(O)NH). MALDI-TOF: m/z
858.0 [M + Na]+. Analytical data are in agreement with Lemieux et
al.25
tion of the filtrate, the crude material was purified by column
chromatography (Iatrobeads, chloroform/methanol/water, 74/24/2, v/v/
v) to afford compound 3 (21 mg, 95%) as a white amorphous solid.
MALDI-TOF: m/z 402.3 [M + Na]+. [R]D ) -15.6 (c 0.79, D2O).
1
For H NMR and 13C NMR data, see Supporting Information.
1-O-tert-Butyldimethylsilyl-2-O-methanesulfonyloxy-ethane (15).
Sodium hydride (60% in oil suspension, 28.2 mg, 0.71 mmol) was
added to a solution of ethylene glycol (2.05 g, 33 mmol) in dry N,N-
dimethylformamide (25 mL). After stirring at room temperature for
30 min, tert-butyldimethylsilyl chloride (5.0 g, 33 mmol) was added
dropwise. The mixture was left stirring at room temperature for 16 h.
Triethylamine (6.91 mL, 50 mmol) was then added, followed by slow
addition of methanesulfonyl chloride (3.6 mL, 46 mmol). TLC (hexane/
ethyl acetate, 3/1, v/v) indicated completion of the reaction. The reaction
mixture was quenched with methanol, diluted with ethyl acetate (100
mL), and washed successively with water (5 × 20 mL), brine (20 mL),
followed by drying over MgSO4. After evaporation of the solvent, the
residue was then purified by flash chromatography (gradient hexane/
ethyl acetate, 5/1 to 3/2, v/v) to afford compound 15 (5.9 g, 70%) as
1
a white foam. H NMR (CDCl3, 300 MHz): 4.58 (dd, 2H, J 4.7, 9.1,
MsOCH2), 3.88 (dd, 2H, J 4.7, 9.1, TBDMSOCH2), 3.02 (s, 3H, CH3-
SO2), 0.90 (s, 9H, Si(CH3)3), 0.09 (2s, 6H, Si(CH3)2). 13C NMR (CDCl3,
125 MHz): δ 71.22, 61.28, 37.60, 25.96, 25.87, 18.37, -5.27. MALDI-
TOF: m/z 277.1 [M + Na]+. Anal. Calcd for C9H22O4SSi: C, 42.49;
H, 8.72; O, 25.15; S, 12.60; Si, 11.04. Found: C, 42.65; H, 8.76; O,
25.26.
Methyl 2-Acetamido-2-deoxy-3-O-benzyl-4-O-(3,4,6-tri-O-benzyl-
â-D-galactopyranosyl)-6-di-O-(2-O-methanesulfonyl-ethane)-â-D-glu-
copyranoside (18). Sodium hydride (60% in oil suspension, 28.2 mg,
0.71 mmol) was added to a solution of 10 (67 mg, 0.09 mmol) in dry
N,N-dimethylformamide (10 mL). After stirring for 30 min at 50 °C,
15 (336 mg, 1.32 mmol) was added dropwise. The mixture was stirred
at 50 °C for 16 h. TLC (chloroform/methanol, 9/1, v/v) indicated
completion of the reaction. The reaction mixture was quenched with
methanol, diluted in ethyl acetate (30 mL), washed successively with
water (5 × 10 mL), brine (10 mL), followed by drying over MgSO4.
After evaporation of the solvent, the residue was dissolved in acetonitrile
(20 mL), and tetrafluoroboric acid (48% in water, 21.3 µL, 0.16 mmol)
was added. The reaction mixture was stirred at room temperature for
5 min after which TLC (chloroform/methanol, 9/1, v/v) indicated
completion of the reaction. The mixture was neutralized with triethyl-
amine (23.0 µL, 0.16 mmol) and concentrated in vacuo. The residue
was then diluted in dichloromethane (10 mL), successively washed with
a saturated solution of NaHCO3 (5 mL), water (2 × 5 mL), brine (5
mL), followed by drying over MgSO4. After concentration of the filtrate,
the crude mixture was purified by flash chromatography (gradient
hexane/acetone, 3/1 to 1/1, v/v). The dried product was then dissolved
in pyridine (5 mL), and methanesulfonyl chloride (7.0 µL, 0.09 mmol)
was added to the stirred mixture at -20 °C. The reaction mixture was
then left stirring at room temperature for 2 h. TLC (chloroform/
methanol, 9/1, v/v) indicated completion of the reaction. The reaction
mixture was quenched with methanol. Toluene was then added to the
crude mixture which was concentrated in vacuo. The reaction mixture
was diluted with dichloromethane (10 mL) and then washed succes-
sively with water (2 × 5 mL), brine (5 mL), followed by drying over
MgSO4. After evaporation of the solvent, the residue was purified by
flash silica gel chromatography (gradient hexane/acetone, 3/1 to 1/1,
v/v) to afford compound 18 (37 mg, 47%) as a white amorphous solid.
1H NMR (CDCl3, 300 MHz): δ 7.40-7.10 (m, 20H, arom), 5.64 (d,
1H, JNH,2 8.3, NH), 4.92, 4.38 (AB q, 2H, JAB 12.3, OCH2Ph), 4.88,
4.56 (AB q, 2H, JAB 12.3, OCH2Ph), 4.75, 4.61 (AB q, 2H, JAB 11.8,
OCH2Ph), 4.67 (d, 1H, J1,2 7.4, H-1), 4.50 (m, 2H, H-2, H-5′), 4.36,
4.28 (AB q, 2H, JAB 11.4, OCH2Ph), 4.33 (d, 1H, J1′,2′ 8.0, H-1′), 4.1-
4.0 (m, 1H, H-4), 4.0-3.88 (m, 1H, H-5), 3.92 (dd, 1H, J3,4 8.4 H-3),
3.84-3.44 (m, 2H, H-6b, H-6a), 3.80 (d, 1H, J3′,4′ 3.0, H-4′), 3.72 (m,
1H, H-3′), 3.58 (m, 1H, H-6b′), 3.45 (s, 3H, OCH3), 3.42 (dd, 1H, J2,3
Methyl 2-Acetamido-2-deoxy-3-O-benzyl-2′,6-anhydro-4-O-(3,4,6-
tri-O-benzyl-â-D-galactopyranosyl)-â-D-glucopyranoside (14). So-
dium hydride (60% in oil suspension, 19.3 mg, 0.48 mmol) was added
to a solution of 13 (134 mg, 0.16 mmol) in dry N,N-dimethylformamide
(20 mL). After stirring at room temperature for 16 h, TLC (chloroform/
methanol, 9/1, v/v) indicated completion of the reaction. The reaction
mixture was quenched with methanol, diluted in ethyl acetate (100 mL),
and successively washed with water (2 × 20 mL) and brine (20 mL),
followed by drying over MgSO4. After evaporation of the solvent, the
residue was then purified by flash silica gel chromatography (hexane/
acetone, gradient 3/1, 3/2 followed by 1/1, v/v) to afford compound
1
14 (83 mg, 70%) as a white foam. H NMR (CDCl3, 300 MHz): δ
7.40-7.10 (m, 20H, arom), 5.40 (d, 1H, JNH,2 7.4, NH), 4.96, 4.62
(AB q, 2H, JAB 11.3, OCH2Ph), 4.93, 4.60 (AB q, 2H, JAB 11.6, OCH2-
Ph), 4.81, 4.70 (AB q, 2H, JAB 12.1, OCH2Ph), 4.70 (d, 1H, J1,2 7.2,
H-1), 4.48, 4.42 (AB q, 2H, JAB 11.8, OCH2Ph), 4.37 (d, 1H, J1′,2′ 7.4,
H-1′), 4.20 (dd, 1H, J6b,5 6.9, J6b,6a 13.5, H-6b), 4.09 (m, 1H, H-3),
4.02 (dd, 1H, J6a,5 3.3, H-6a), 3.89 (d, 1H, J3′,4′ 2.5, H-4′), 3.82 (dd,
1H, J3,4 8.5, J4,5 9.6, H-4), 3.75 (m, 1H, H-5), 3.68-3.62 (m, 3H, H6b′,
H-6a′, H-5′), 3.61 (m, 1H, H-2′), 3.49 (m, 1H, H-3′), 3.46 (s, 3H,
OCH3), 3.28 (m, 1H, H-2), 1.82 (s, 3H, CH3C(O)NH). 13C NMR
(CDCl3, 125 MHz): δ 170.39, 138.79, 138.63, 137.92, 128.47, 128.23,
127.88, 128.68, 127.59, 127.50, 103.75, 101.03, 85.83, 80.19, 78.83,
78.66, 75.27, 75.08, 74.98, 74.78, 74.31, 73.70, 73.49, 68.74, 57.17,
56.86, 29.95, 23.86. MALDI-TOF: m/z 762.2 [M + Na]+. Anal. Calcd
for C43H49NO10: C, 69.81; H, 6.68; N, 1.89; O, 21.63. Found: C, 69.95;
H, 6.76; N, 1.92. [R]D ) -20.8 (c 0.27, CH2Cl2).
Methyl 2-Acetamido-2-deoxy-2′,6-anhydro-4-O-(â-D-galactopy-
ranosyl)-â-D-glucopyranoside (3). 10% palladium on charcoal (55.0
mg) was added to a solution of 14 (43 mg, 0.058 mmol) in ethanol (3
mL). The mixture was vigorously stirred under an atmosphere of
hydrogen for 16 h. TLC (chloroform/methanol, 9/1, v/v) indicated
completion of the reaction. After filtration using Celite and concentra-
9
J. AM. CHEM. SOC. VOL. 124, NO. 21, 2002 5971

A R T I C L E S
Galan et al.
6.3, H-2), 3.38 (m, 1H, H-6a′), 2.98 (s, 3H, CH3SO2), 1.84 (s, 3H,
CH3C(O)NH). 13C NMR (CDCl3, 125 MHz): δ 170.42, 138.87, 138.21,
132.51, 128.70, 128.55, 128.41, 128.55, 128.4, 128.35, 127.96, 127.63,
118.28, 112.42, 103.72, 103.53, 101.46, 100.26, 82.32, 78.85, 74.81,
74.70, 74.07, 73.82, 73.70, 73.07, 72.47, 71.91, 69.88, 69.51, 69.40,
68.69, 57.65, 57.05, 56.20, 37.85, 23.89. MALDI-TOF: m/z 902.6 [M
+ Na]+. Anal. Calcd for C46H57NO14S: C, 62.78; H, 6.53; N, 1.59; O,
25.45; S, 3.64. Found: C, 62.89; H, 6.68; N, 1.72. [R]D ) 4.0 (c 0.07,
CH2Cl2).
room temperature for 5 min. TLC (hexane/ethyl acetate, 7/3, v/v)
indicated completion of the reaction. The mixture was neutralized with
triethylamine (0.325 mL, 2.3 mmol) and concentrated in vacuo. The
residue was then diluted in dichloromethane (50 mL) and successively
washed with a saturated solution of NaHCO3 (10 mL), water (2 × 10
mL), and brine (10 mL), followed by drying over MgSO4. After
evaporation of the solvent, the residue was purified by flash silica gel
chromatography (gradient hexane/ethyl acetate, 9.5/0.5 to 8/2, v/v) to
give compound 22 (297.67 mg, 95%) as an amorphous white solid. 1H
NMR (CDCl3, 300 MHz): δ 7.40-7.10 (m, 20H, arom), 5.42 (dd, 1H,
J2′3′ 9.9, H-2′), 5.06, 4.74 (AB q, 2H, JAB 10.7, OCH2Ph), 5.00, 4.56
(AB q, 2H, JAB 11.5, OCH2Ph), 4.69, 4.51 (AB q, 2H, JAB 12.4, OCH2-
Ph), 4.61 (d, 1H, J1′,2′ 8.0, H-1′), 4.33, 4.23 (AB q, 2H, JAB 11.8, OCH2-
Ph), 4.18 (d, 1H, J1,2 8.0, H-1), 3.99 (d, 1H, J3′-4′ 2.5, H-4′), 3.87 (dd,
1H, J6b,5 2.5, J6b,6a 12.1, H-6b), 3.76 (dd, 1H, J6a,5 3.3, H-6a), 3.62-
3.44 (m, 3H, H-3, H-4, H5′), 3.54 (s, 3H, OCH3), 3.52 (m, 1H, H-6b′),
3.51 (dd, 1H, H-3′), 3.33 (m, 1H, H-6a′), 3.32 (m, 1H, H-2), 3.30 (m,
1H, H-5), 2.10 (s, 3H, CH3C(O)O). 13C NMR (CDCl3, 125 MHz): δ
169.61, 138.83, 138.55, 138.14, 138.10, 128.63, 128.58, 128.39, 128.25,
128.17, 128.02, 127.94, 127.70, 127.58, 127.51, 103.10, 101.36, 81.38,
80.68, 76.28, 75.52, 75.49, 74.99, 73.79, 73.73, 73.02, 72.37, 72.16,
68.28, 66.25, 60.90, 57.60, 21.37. MALDI-TOF: m/z 806.3 [M + Na]+.
Anal. Calcd for C43H49N3O11: C, 65.89; H, 6.30; N, 5.36; O, 22.45.
Found: C, 65.96; H, 6.43; N, 5.41. [R]D ) -17.3 (c 0.40, CH2Cl2).
Methyl 2-Azido-2-deoxy-3-O-benzyl-4-O-(3,4,6-tri-O-benzyl-â-D-
galactopyranosyl)-â-D-glucopyranoside (23). Sodium methoxide (5.4
mg, 0.1 mmol) was added to a solution of 22 (223 mg, 0.29 mmol) in
dry methanol (30 mL). The mixture was stirred at room temperature
for 48 h. TLC (hexane/ethyl acetate, 7/3, v/v) indicated completion of
the reaction. The mixture was neutralized with Dowex 50H+ resin,
filtered, and the filtrate was concentrated in vacuo. The residue was
purified by flash silica gel chromatography (gradient hexane/acetone,
9/1 to 7/3, v/v) to afford compound 23 (210 mg, 98%) as an amorphous
white solid. 1H NMR (CDCl3, 300 MHz): δ 7.40-7.10 (m, 20H, arom),
4.97, 4.82 (AB q, 2H, JAB 11.0, OCH2Ph), 4.83, 4.55 (AB q, 2H, JAB
11.5, OCH2Ph), 4.68, 4.53 (AB q, 2H, JAB 11.9, OCH2Ph), 4.61 (d,
1H, J1′,2′ 7.5, H-1′), 4.37 (d, 1H, J1,2 7.9, H-1), 4.29, 4.21 (AB q, 2H,
Methyl 2-Acetamido-2-deoxy-3-O-benzyl-4-O-(3,4,6-tri-O-benzyl-
â-D-galactopyranosyl)-2′,6-di-O-(ethane-1,2-diyl)-â-D-glucopyrano-
side (19). A suspension of sodium hydride (60% in oil suspension, 9.6
mg, 0.24 mmol) in N,N-dimethylformamide (2 mL) was added over a
period of 2 h to a solution of 18 (26.4 mg, 0.03 mmol) in N,N-
dimethylformamide (10 mL). The reaction mixture was then stirred at
50 °C for 16 h. TLC (chloroform/methanol, 9/1, v/v) indicated
completion of the reaction. The reaction mixture was quenched with
methanol, and then diluted in ethyl acetate (30 mL), successively
washed with water (5 × 10 mL), brine (10 mL), followed by drying
over MgSO4. After evaporation of the solvent, the residue was purified
by flash silica gel chromatography (hexane/acetone, gradient 3/1, 3/2
followed by 1/1, v/v) to afford compound 19 (14.1 mg, 60%) as an
1
amorphous white solid. H NMR (CDCl3, 300 MHz): δ 7.40-7.10
(m, 20H, arom), 5.48 (d, 1H, JNH,2 7.5, NH), 5.01, 4.66 (AB q, 2H, JAB
12.3, OCH2Ph), 4.88, 4.64 (AB q, 2H, JAB 11.9, OCH2Ph), 4.81 (d,
1H, J1,2 7.8, H-1), 4.80, 4.12 (AB q, 2H, JAB 11.9, OCH2Ph), 4.77 (d,
1H, J1′,2′ 7.5, H-1′), 4.70, 4.54 (AB q, 2H, JAB 11.4, OCH2Ph), 4.29
(dd, 1H, J3,4 8.8, H-3), 4.20 (m, 1H, H-4), 4.16, 3.84 (m, 2H, H6b′,
H6a′), 4.10-3.87 (m, 2H, H5′, H6a), 3.93 (m, 1H, H6b), 3.85 (dd,
1H, J3′-4′ 2.2, H-4′), 3.60 (dd, 1H, J2′,3′ 9.4, H-2′), 3.51 (m, 2H, OCH2-
CH2O), 3.50 (m, 1H, H-5), 3.46 (s, 3H, OCH3), 3.45 (m, 1H, H-3′),
3.27 (dd, 1H, J 5.27, 8.79, OCH2CH2O), 3.18 (m, 1H, H-2), 2.99 (dd,
1H, OCH2CH2O), 1.80 (s, 3H, CH3C(O)NH). 13C NMR (CDCl3, 125
MHz): δ 170.53, 139.46, 138.71, 138.40, 137.45, 128.38, 128.30,
128.05, 127.90, 127.83, 127.68, 127.58, 127.36, 127.12, 126.24, 104.84,
100.65, 83.27, 82.08, 81.10, 78.67, 76.57, 74.46, 74.27, 74.18, 73.43,
73.32, 72.99, 72.42, 70.35, 68.11, 64.78, 58.61, 56.71, 30.90, 23.62.
MALDI-TOF: m/z 806.2 [M + Na]+. [R]D ) 3.5 (c 0.25, CH2Cl2).
JAB 11.4, OCH2Ph), 4.20 (dd, 1H, J6b,5 2.7, J6b,6a 12.6, H-6b), 3.93 (m,
1H, H-5′), 3.91 (d, 1H, J3′-4′ 1.9, H-4′), 3.90 (m, 1H, H-2′), 3.84 (dd,
1H, J6a,5 2.2, H-6a), 3.56 (m, 1H, H-6b′), 3.55 (s, 3H, OCH3), 3.49-
3.20 (m, 3H, H-3, H-4, H-5), 3.36 (m, 1H, H-2), 3.35 (m, 1H, H-3′),
3.27 (dd, 1H, J6a′,5′ 2.7, J6b′,6a′ 12.4, H-6a′). 13C NMR (CDCl3, 125
MHz): δ 138.85-128.58 (arom), 104.08, 103.33, 82.21, 75.38, 75.03,
74.80, 76.75, 75.52, 75.16, 73.67, 73.50, 72.35, 72.17, 72.01, 68.21,
66.60, 66.31, 61.22, 57.32. MALDI-TOF: m/z 764.3 [M + Na]+. Anal.
Calcd for C41H47N3O10: C, 66.38; H, 6.39; N, 5.66; O, 21.57. Found:
C, 66.48; H, 6.47; N, 5.78. [R]D ) -12.4 (c 0.35, CH2Cl2).
Methyl 2-Azido-2-deoxy-3-O-benzyl-4-O-(3,4,6-tri-O-benzyl-â-D-
galactopyranosyl)-2′,6-di-O-(methane-1,2-diyl)-â-D-glucopyrano-
side (24). Formaldehyde diphenylmercaptal (58.0 mg, 0.25 mmol) and
powdered molecular sieves 4 Å (50 mg) were added to a stirred solution
of 23 (156 mg, 0.21 mmol) in a mixture of acetonitrile and dichlo-
romethane (34 mL, 7/3, v/v). The mixture was stirred at room
temperature for 1 h, then cooled to -35 °C, and a solution of
N-iodosuccinimide (234 mg, 1.04 mmol) and trifluoromethanesulfonic
acid (5.5 µL, 0.063 mmol) in dry acetonitrile (3 mL) was added to the
reaction mixture. The mixture was left stirring at -35 °C for 2 h. TLC
(hexane/ethyl acetate, 3/1, v/v) indicated completion of the reaction.
The mixture was neutralized with triethylamine (50 µL) and filtered
over Celite. The filtrate and washings were combined and concentrated.
The residue was then diluted in dichloromethane (30 mL), successively
washed with a saturated solution of Na2S2O3 (10 mL), a saturated
solution of NaHCO3, water (2 × 10 mL), brine (10 mL), followed by
drying over MgSO4. After evaporation of the solvents, the residue was
purified by flash silica gel chromatography (gradient hexane/acetone,
9/0.5 to 8/2, v/v) to afford compound 24 (95 mg, 60%) as an amorphous
Methyl 2-Acetamido-2-deoxy-4-O-(â-D-galactopyranosyl)-2′,6-di-
O-(ethane-1,2-diyl)-â-D-glucopyranoside (5). 10% palladium on
charcoal (14.0 mg) was added to a solution of 19 (9.0 mg, 0.012 mmol)
in ethanol (3 mL). The mixture was vigorously stirred under an
atmosphere of hydrogen for 16 h. TLC (chloroform/methanol, 9/1, v/v)
indicated completion of the reaction. After filtration using Celite and
concentration, the crude material was purified by chromatography
(Iatrobeads, chloroform/methanol/water, 74/24/2, v/v/v) to afford
compound 5 (4.9 mg, 96%) as an amorphous white solid. MALDI-
1
TOF: m/z 446.0 [M + Na]+. [R]D ) -14.6 (c 0.56, D2O). For H
NMR and 13C NMR data, see Supporting Information.
Methyl 2-Azido-2-deoxy-3-O-benzyl-4-O-(2-O-acetyl-3,4,6-tri-O-
benzyl-â-D-galactopyranosyl)-â-D-glucopyranoside (22). Boron tri-
fluoride diethyl etherate (87.5 µL, 0.7 mmol) was added to a stirred
solution of 6 (438.2 mg, 0.70 mmol), 20 (194.8 mg, 0.50 mmol), and
powdered molecular sieves 4 Å (200 mg) in dichloromethane (20 mL).
The mixture was stirred at -40 °C for 3 h. TLC (hexane/ethyl acetate,
7/3, v/v) indicated completion of the reaction. The mixture was
neutralized with triethylamine (97.6 µL, 0.70 mmol) and filtered over
Celite. The filtrate washings were combined and concentrated in vacuo.
The residue was then diluted in dichloromethane (50 mL) and
successively washed with a saturated solution of NaHCO3 (10 mL),
water (2 × 10 mL), and brine (10 mL), followed by drying over MgSO4.
After evaporation of the solvent, the residue was purified by flash silica
gel chromatography (gradient hexane/ethyl acetate, 9.5/0.5 to 8/2, v/v)
to give compound 21 (359 mg, 80%). Tetrafluoroboric acid (48% in
water, 129 µL, 0.70 mmol) was added to a stirred solution of 21 (359
mg, 0.40 mmol) in acetonitrile (50 mL). The mixture was stirred at
9
5972 J. AM. CHEM. SOC. VOL. 124, NO. 21, 2002

R-(2,6)-Sialyltransferase-Catalyzed Sialylations
A R T I C L E S
white solid. 1H NMR (CDCl3, 300 MHz): δ 7.40-7.10 (m, 20H, arom),
5.10, 4.71 (AB q, 2H, JAB 7.4, OCH2O), 4.98, 4.86 (AB q, 2H, JAB
10.7, OCH2Ph), 4.90, 4.56 (AB q, 2H, JAB 11.3, OCH2Ph), 4.69, 4.64
(AB q, 2H, JAB 12.4, OCH2Ph), 4.68 (d, 1H, J1′,2′ 7.4, H-1′), 4.38, 4.30
(AB q, 2H, JAB 11.8, OCH2Ph), 4.18 (d, 1H, J1,2 7.7, H-1), 4.08 (dd,
1H, J6b′,5′ 2.8, J6b′,6a′ 12.1, H-6b′), 4.02 (m, 1H, H-5′), 3.98 (dd, 1H,
J6a′,5′ 4.7, H-6a′), 3.88 (d, 1H, J3′-4′ 2.8, H-4′), 3.78 (dd, 1H, J2′3′9.6,
H-2′), 3.60 (dd, 1H, J6b,6a 8.5, H-6b), 3.49 (m, 1H, H-3), 3.46 (m, 1H,
H-5), 3.44 (t, 1H, J3,4˜J4,5 9.1, H-4), 3.41 (dd, 1H, H-3′), 3.38 (m, 1H,
H-2), 3.32 (m, 1H, J6a,5 4.9, J6a,6b 8.5 H-6a), 3.54 (s, 3H, OCH3). 13C
NMR (CDCl3, 125 MHz): δ 138.83, 138.68, 138.46, 137.97, 128.66,
128.64, 128.53, 128.37, 128.18, 128.13, 128.05, 127.96, 127.86, 127.80,
127.70, 127.62, 104.54, 103.25, 100.29, 84.51, 83.33, 81.41, 75.76,
74.79, 74.14, 73.73, 73.26, 73.06, 72.81, 70.71, 68.41, 66.31, 57.32.
MALDI-TOF: m/z 776.3 [M + Na]+. Anal. Calcd for C42H47N3O10:
C, 66.92; H, 6.28; N, 5.57; O, 21.22. Found: C, 66.96; H, 6.43; N,
5.66. [R]D ) -57.7 (c 0.53, CH2Cl2).
compound 4 (28.7 mg, 90%) as a white amorphous solid. MALDI-
1
TOF: m/z 432.2 [M + Na]+. [R]D ) -11.0 (c 0.57, D2O). For H
NMR and 13C NMR data, see Supporting Information.
Enzyme Studies. Reported methods20,34,35,44 were employed for
assaying sialyltransferase activity. For studies of the relative rates,
incubation mixtures contained CMP[14C]NeuAc (9 nmol, 6180 cpm/
nmol) and substrate (120 nmol), bovine serum albumin (1 mg/ml), 0.2
mU of enzyme in sodium cacodylate (50 mM, pH 6.5) containing 0.1%
Triton ×100 in a total volume of 60 µL were incubated at 37 °C for a
period of 30 min. The radiolabeled product was isolated using a
procedure modified by Horenstein et al.35 based on Paulson’s ion-
exchange chromatography on a Dowex 1 × 8-200 (PO42-, 100-200
mesh) Pasteur pipet column.34 Columns (5 cm high) were eluted twice
with 1 mM PO42- (4 mL) buffer to ensure that no radiolabeled product
was left on the column.
Kinetic Studies. Apparent kinetic parameters of the R-2,6-ST for
synthetic acceptors were determined under the above standard conditions
using a saturating concentration of CMP-[14C]Neu5Ac.43 Assays were
performed in duplicate using 50 µU of enzyme. The concentration of
oligosaccharide acceptor was varied around the Km value (six different
concentrations, ranging from 0.5 to 3.6 mM for acceptors 1 and 5, and
0.5-6.0 mM for acceptors 2 and 4), whereas the concentration of CMP-
[14C]Neu5Ac was kept constant at 200 µM (1655 cpm/nmol). The time
of incubation at 37 °C was varied to 15 min to limit the CMP-[14C]-
Neu5Ac consumption to 10-15% to ensure initial rate conditions. The
kinetic parameters Vmax and Km were determined using the GraphPad
computer program obtained from Prism.
Methyl 2-Acetamido-2-deoxy-3-O-benzyl-4-O-(3,4,6-tri-O-benzyl-
â-D-galactopyranosyl)-2′,6-di-O-(methane-1,2-diyl)-â-D-glucopyra-
noside (25). 1,3-Propanedithiol (43.5 µL, 0.43 mmol) and triethylamine
(42 µL, 0.43 mmol) were added to a stirred solution of 24 (83 mg,
0.11 mmol) in a mixture of pyridine and water (10 mL, 4/1, v/v). The
mixture was stirred at room temperature for 6 h. TLC (hexane/acetone,
7/3, v/v) indicated completion of the reaction. The mixture was
coevaporated with toluene, concentrated in vacuo, and then chromato-
graphed over silica gel (gradient DCM/methanol, 1/0 to 9/1, v/v) to
afford compound 25 (73 mg, 89%). Acetic anhydride (5 mL) was added
to a solution of the above crude product (70 mg, 0.096 mmol) in
pyridine (10 mL). The mixture was stirred at room temperature for 16
h. TLC (chloroform/methanol, 9/1, v/v) indicated completion of the
reaction. Toluene was added to the mixture which was concentrated in
vacuo. The residue was purified by flash chromatography (gradient
hexane/acetone, 3/1 to 1/1, v/v) to afford compound 25 (72.4 mg, 98%)
Preparative Sialylation. Incubations were carried out in 150 µL of
sodium cacodylate buffer (25 mM, pH 7.2) containing 0.5% Triton
×100 and bovine serum albumin (1 mg/mL). Acceptor 4 (1.0 mg),
CMP-Neu5Ac (0.9 mg), 3 U of alkaline phosphatase, and 6.9 mU of
sialyltranferase are incubated at 37 °C for 36 h. Every 12 h, 0.9 mg of
CMP-Neu5Ac was added to the incubation mixture to compensate for
any hydrolysis of the donor. The reaction was monitored by TLC on
silica gel plates using CHCl3-MeOH-H2O (60:35:6, v/v). The acceptor
Galâ1 f 4GlcNAcâ-OMe has an Rf of 0.63, and the product NeuAcR2
f 6Galâ1f4GlcNAcâ-OMe has an Rf of 0.33. At the completion of
the reaction, the sample was purified on Iatrobeads followed by biogel
1
as an amorphous white solid. H NMR (CDCl3, 300 MHz): δ 7.40-
7.10 (m, 20H, arom), 5.36 (d, 1H, JNH,2 8.0, NH), 5.07, 4.69 (AB q,
2H, JAB 7.4, OCH2O), 4.98, 4.64 (AB q, 2H, JAB 11.8, OCH2Ph), 4.86,
4.54 (AB q, 2H, JAB 11.5, OCH2Ph), 4.68, 4.60 (AB q, 2H, JAB 12.1,
OCH2Ph), 4.70 (d, 1H, J1,2 7.2, H-1), 4.68 (d, 1H, J1′,2′ 7.4, H-1′), 4.32,
4.25 (AB q, 2H, JAB 11.8, OCH2Ph), 4.12-3.92 (m, 1H, H-5′), 4.07
(dd, 1H, J2,3 7.8, H-3), 4.04 (dd, 1H, J6b′,5 2.7, J6b′,6a′ 12.3, H-6b′), 3.96
(m, 1H, 3.3, H-6a′), 3.86 (d, 1H, J3′,4′ 2.2, H-4′), 3.76 (dd, 1H, J2,′3′
9.6, H-2′), 3.60-3.40 (m, 1H, H-5), 3.56 (m, 1H, H-6b), 3.43 (s, 3H,
OCH3), 3.33 (dd, 1H, J6b,5 2.7, H-6a), 3.42 (m, 1H, H-4), 3.41 (dd,
1H, H-3′), 3.28 (m, 1H, H-2), 1.80 (s, 3H, CH3C(O)NH). 13C NMR
(CDCl3, 125 MHz): 170.6, 139.81, 139.23, 138.81, 138.49, 138.06,
137.95, 128.63, 128.59, 128.57, 128.35, 128.17, 128.03, 127.96, 127.91,
127.84, 127.74, 127.66, 127.61, 104.84, 101.26, 100.06, 84.32, 81.53,
80.94, 75.31, 74.72, 74.15, 73.68, 73.13, 73.00, 72.87, 70.90, 68.64,
57.55, 56.90, 23.84. MALDI-TOF: m/z 792.4 [M + Na]+. Anal. Calcd
for C44H51NO11: C, 68.64; H, 6.68; N, 1.82; O, 22.86. Found: C, 68.78;
H, 6.67; N, 1.90. [R]D ) -19.2 (c 0.68, CH2Cl2).
Methyl 2-Acetamido-2-deoxy-4-O-(â-D-galactopyranosyl)-2′,6-di-
O-(methane-1,2-diyl)-â-D-glucopyranoside (4). 10% palladium on
charcoal (74.0 mg) was added to a solution of 25 (60.0 mg, 0.078 mmol)
in ethanol (3 mL). The mixture was vigorously stirred under an
atmosphere of hydrogen for 16 h. TLC (chloroform/methanol, 9/1, v/v)
indicated completion of the reaction. After filtration using Celite and
concentration, the crude material was purified by chromatography
(Iatrobeads, chloroform/methanol/water, 74/24/2, v/v/v) to afford
1
P2. H NMR (D2O, 500 MHz, DOH set at 4.76): δ 4.55 (d, 1H, J1′2′
) 7.8 Hz, H-1′), 4.43 (d, 1H, J1,2 ) 8.8 Hz, H-1), 3.48 (s, 3H, OCH3),
3.34 (m, 1H, H-2′), 2.67 (dd, 1H, J3e′′,3a′′ ) 12.7, J3e′′,4′′ ) 4.9 Hz, H-3e′′),
2.04, 2.02 (2s, 6H, 2 CH3C(O) NH), 1.68 (t, 1H, J3a′′,4′′ ) 11.7 Hz,
H-3a′′). MALDI-TOF: m/z ) 737.3 [M+ + Na].
Acknowledgment. This research was supported by the Office
of the Vice President of Research of the University of Georgia.
Supporting Information Available: Synthesis of monosac-
1
charide 20, enzyme kinetics plots, H NMR and 13C NMR
spectra of compounds 1-5, NOE experiments figure of
compounds 1, 3, and 5, showing NOEs from Gal-H-1, region
of quantitative HMBC for compound 4, nomenclature, and
detailed geometrical description of all optimized conformers of
1, 3, 4, and 5 (PDF). This material is available free of charge
via the Internet at http://pubs.acs.org.
JA0169771
(44) Palcic, M. M.; Venot, A. P.; Ratcliffe, R. M.; Hindsgaul, O. Carbohydr.
Res. 1989, 190, 1-11.
9
J. AM. CHEM. SOC. VOL. 124, NO. 21, 2002 5973