Chemoenzymatic synthesis of sialylated oligosaccharides for their evaluation in a polysialyltransferase assay

Article abstract of DOI:10.1016/S0040-4039(03)01509-0

A series of sialylated β-D-Gal-(1→3)-α-D-GalNAc-octyl containing oligosaccharides representative of those found on mucin type complex O-glycans were synthesized by a chemoenzymatic approach for use in the kinetic characterization of recently cloned polysialyltransferases. Enzymatic incorporation of N-acetylneuraminic acid (sialic acid) into the synthetic acceptors was accomplished by 2,3-(N) and (O)-sialyltransferases to give the target compounds 6-10 in a practical yield.

Full text of DOI:10.1016/S0040-4039(03)01509-0

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 6037–6042
Chemoenzymatic synthesis of sialylated oligosaccharides for their
evaluation in a polysialyltransferase assay
Prabal Sengupta, Anup Kumar Misra,* Misa Suzuki, Minoru Fukuda and Ole Hindsgaul
The Burnham Institute, 10901 N. Torrey Pines Road, La Jolla, CA 92037, USA
Received 4 April 2003; revised 29 May 2003; accepted 13 June 2003
Abstract—A series of sialylated b-
D
-Gal-(1“3)-a-D-GalNAc-octyl containing oligosaccharides representative of those found on
mucin type complex O-glycans were synthesized by a chemoenzymatic approach for use in the kinetic characterization of recently
cloned polysialyltransferases. Enzymatic incorporation of N-acetylneuraminic acid (sialic acid) into the synthetic acceptors was
accomplished by 2,3-(N) and (O)-sialyltransferases to give the target compounds 6–10 in a practical yield.
©
2003 Published by Elsevier Ltd.
Oligosaccharides on cell surfaces play an important role
in many biological recognition events including cell–cell
adhesion, bacterial attachment and viral infection. The
Polysialic acid is a unique carbohydrate of a linear
homopolymer of a-2,8-linked sialic acid, which may
1
7
contain as many as 55 sialic acid residues per polymer.
availability of oligosaccharides and their analogues as
inhibitors can provide insight into their biological roles
and might lead to the discovery of novel carbohydrate-
Neural Cell Adhesion Molecules (NCAM) are highly
polysialylated in embryonic tissues, although the major-
ity of NCAM in the adult olfactory bulb and the
hippocampus of adult brain contains polysialic acid
2
based therapeutics. Despite many advances in the
8
chemical synthesis of oligosaccharides, it is still time-
consuming and often difficult to synthesize these highly
where neuronal regeneration persists. There is increas-
ing evidence that polysialylated NCAM promotes cell
migration and enhances neurite outgrowth and branch-
3
asymmetric and densely functionalized molecules. Pro-
9
gress in recombinant DNA technology has made it
possible to express large quantities of enzymes, includ-
ing several glycosyltransferases that biosynthesize
oligosaccharides. Many glycosyltransferases have been
isolated and cloned are now readily available in quanti-
ing during development and neural regeneration.
Polysialic acid is thought to modulate the functional
properties of NCAM by rendering it less adhesive to
10
itself (homophilic binding) or to other cell surface
molecules (heterophilic binding). Taken together, these
results strongly suggest that polysialylated NCAM
plays a critical role in neural development and plastic-
ity. Polysialic acid is present in mucin type O-glycans as
4
ties sufficient for their use in synthesis. The uses of
glycosyltransferases to prepare oligosaccharides offer a
number of advantages: high regio- and stereoselectivity,
no requirement for chemical protection of functional
11
shown in trout eggs. Trisialylated core 1 disaccharide
-GalNac-Ser/Thr) was also found
5
groups, and very mild reaction conditions.
(b-
D
-Gal-(1“3)-a-
D
12
in the human erythrocyte membrane. However, it has
not been shown which core structure(s) is a preferable
acceptor for polysialyltransferases.
Numerous examples have been reported in which glyco-
syltransferases tolerate structural changes on both
donor and acceptor substrates, thus making enzymatic
synthesis a promising alternative for the preparation of
Herein, we describe the chemoenzymatic syntheses of
the sialylated oligosaccharides 6–10, which represent
biosynthetic intermediates in the synthesis of polysialyl-
ated mucin type O-glycans (Fig. 1). These compounds
are for use in kinetic studies of recently cloned polysial-
6
both natural and unnatural oligosaccharides.
13
yltransferase, ST8SiaIV.
Our synthetic approach
involves enzymatic incorporation of sialic acid residues
to the synthetic di-, tri- and tetrasaccharide acceptors
by recombinant rat liver Galb-1,4/3-GlcNAca-2,3-(N)-
sialyltransferase (EC 2.4.99.5) and recombinant rat liver
*
0
Corresponding author. Present address: Medicinal Chemistry Divi-
sion, Central Drug Research Institute, Lucknow-226 001, U.P.,
India.
Fax:
+91-522-223938/223405;
e-mail:
akmisra69@
rediffmail.com
040-4039/$ - see front matter © 2003 Published by Elsevier Ltd.
doi:10.1016/S0040-4039(03)01509-0

6
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P. Sengupta et al. / Tetrahedron Letters 44 (2003) 6037–6042
17
Galb-1,3-GalNAca-2,3-(O)-sialyltransferase
(EC
yield. The low yield in the glycosylation step may be
due to the lower reactivity of the sialic acid donor as
well as competition between glycosylation and elimina-
tion reactions. To increase the access of the donor to
the acceptor site, a 4,6-diol acceptor was used instead
of using only a 6-hydroxylated acceptor.
2
.4.99.4) (purchased from Calbiochem, USA) using
cytidine-5%-monophospho-N-acetylneuraminic
acid
sodium salt (CMP-NeuAc) as sialic acid donor. The
construction of branched Galb-(1“3)-[NeuAca-(2“6)]-
GalNAca-octyl trisaccharide 5b was achieved by a
chemical approach.
The enzymatic incorporations of sialic acid into com-
pounds 2, 4 and 5b were achieved through the initial
addition of the CMP-NeuAc donor along with rat liver
A few reports have appeared in the literature regarding
the enzymatic synthesis of sialylated oligosaccharides.
Suitably protected TF-disaccharide acceptor 1 and
deprotected mucin type core 2 tetrasaccharide acceptor
14
Galb-1,4-GlcNAc-a-2,3-(N)-sialyltransferase
2.4.99.5), which selectively adds sialic acid to the 3-OH
of the -galactose residue linked to a (b- -Gal-(1“4)-
a- -GlcNac) chain of the N-glycan structure, or
Galb-1,3-GalNAc-a-2,3-(O)-sialyltransferase (EC
2.4.99.4), which selectively adds sialic acid to the 3-OH
of the -galactose residue linked to a core-1 chain
(b- -Gal-(1“3)-a- -GalNac) of the O-glycan
(EC
4
were prepared following the procedure reported
D
D
1
5
18
earlier (Scheme 1). Condensation of the sialic acid
D
16
thioglycoside donor 3 with the disaccharide diol
acceptor using dimethyl (methylthio)trifluoro-
1
methanesulfonate (DMTST) as glycosyl promoter in
acetonitrile–dichloromethane (5:1) solvent at −10°C
over 12 h gave the (2“6)-a-sialylated trisaccharide 5a
in 43% yield together with a minor amount of a (2“6)-
b-sialylated trisaccharide (12%). On saponification of
D
D
D
19
structure at 37°C on a 1–2 mmol scale (Scheme 2). For
the 2,3-(O)-sialyltransferase, the reaction was complete
20
in 16–18 h, while in the case of the 2,3-(N)-sialyltrans-
ferase, the reaction required from 30 min to 18 h
depending upon the number of sialic acids incorpo-
5
a using wet sodium methoxide the deprotected (2“6)-
a-sialylated TF-disaccharide 5b was obtained in 62%
Figure 1. Structures of the compounds synthesized enzymatically.

P. Sengupta et al. / Tetrahedron Letters 44 (2003) 6037–6042
6039
Scheme 1. Reagents and conditions: (a) DMTST, CH CN–CH Cl (5:1), 3 A MS, −10°C, 12 h, 43% together with 40% of 12; (b)
,
3
2
2
0.5 M MeONa, MeOH, rt, 12 h, then a few drops of water, rt, 12 h, 62%.
Scheme 2. Reagents and conditions: Rat recombinant 2,3-a-(O)-sialyltransferase (0.25 mU), CMP-NeuAc (2.25 equiv.), sodium
cacodilate, Triton, calf intestine alkaline phosphatase, 37°C, 16 h, quantitative.
2
1
rated. An interesting point to note here is that, while
attempting to prepare 8 by using the 2,3-(N)-sialyl-
transferase, the reaction seemed to be completed in only
acid to the compound 10 (obtained from the reaction
of CMP-sialic acid and acceptor 4 in presence of 2,3-
(O)-sialyltransferase) was done by using 2,3-(N)-sialyl-
transferase and CMP-sialic acid, while in the other
case, the same reaction was performed using com-
pound 8 (obtained from the reaction of CMP-sialic
acid and acceptor 4 in the presence of 2,3-(N)-sialyl-
transferase), CMP-sialic acid and 2,3-(O)-sialyltrans-
ferase (Scheme 3). In both cases 9 was isolated as the
sole product (TLC, MS and NMR analyses). This
result can be explained based on the extremely high
3
0 min. When we allowed the reaction to proceed for a
longer time, disialylated product 9 was obtained in a
quantitative
yield,
2 4
as
confirmed
by
TLC
i
(
PrOH:H O:NH OH=8:2:0.2 as running solvent, R
f
values for compounds 8: 0.32, 10: 0.26 and 9: 0.5),
NMR and MS analyses. In order to confirm the for-
mation of product 9, two control experiments were
performed. For one, the further incorporation of sialic

6
040
P. Sengupta et al. / Tetrahedron Letters 44 (2003) 6037–6042
Scheme 3. Reagents and conditions: (a) Rat recombinant 2,3-a-(N)-sialyltransferase (0.025 mU), CMP-NeuAc (1.4 equiv.), MOPS
buffer, BSA, Triton, calf intestine alkaline phosphatase, 37°C, 30 min, 87%; (b) Rat recombinant 2,3-a-(N)-sialyltransferase (0.25
mU), CMP-NeuAc (2.25 equiv.), MOPS buffer, BSA, Triton, calf intestine alkaline phosphatase, 37°C, 16 h, quantitative; (c) Rat
recombinant 2,3-a-(O)-sialyltransferase (0.25 mU), CMP-NeuAc (2.25 equiv.), sodium cacodilate, Triton, calf intestine alkaline
phosphatase, 37°C, 18 h, quantitative.
reactivity of 2,3-(N)-sialyltransferase in comparison
with that of 2,3-(O)-sialyltransferase. All the sialylated
oligosaccharides 5b–10 were purified using C -SepPak
added to both compounds 5b and 10, which is consis-
12
tent with the results obtained in the previous study.
By contrast, in trout eggs, polysialic acid is added only
1
8
11
cartridges and LH-20 sephadex gel filtration. Their
to compound 5b. These results suggest either that
polysialylation in trout eggs is unique or polysialic acid
can be added to the a-(2,3)-sialylated core 1 if the core
2
2
NMR and MS spectra supported the structures of all
the products.
2
branch is also present as seen in compound 10.
These synthetic oligosaccharides and NeuAca-(2“3)-
2
3
Galb-(1“4)-GlcNAcb-1“octyl 11 were then incu-
In conclusion, chemoenzymatic syntheses of six mono-
and disialylated mucin type O-glycans (5b–10) were
performed by addition of sialic acid in a stepwise
manner or in a single step using the regio- and stereose-
lectivity of two sialyltransferase enzymes (Fig. 2). The
results obtained using these oligosaccharides indicate
that ST8Sia IV acts on mucin-type O-glycans as
efficiently as on N-glycans, and exhibits minimum dis-
crimination towards different linkages of sialic acids on
acceptors.
bated with a soluble form of ST8Sia IV and a donor
3
24
substrate CMP-[ H]-NeuAc. After the reaction, the
incubation mixture was directly applied to a column of
Mono-Q anion exchange chromatography and eluted
with a linear gradient of increasing concentrations of
25,26
NaCl as described previously.
Surprisingly, com-
pounds 5b (49%) and 10 (65%) served as better accep-
tors than NeuAca-(2“3)-Galb-(1“4)-GlcNAcb1-octyl
1
1 (both in the relative reaction rates). Interestingly, all
these acceptors produced polysialic acid with 35–45
sialic acid residues, which is similar to that obtained on
2
6
NCAM. These results indicate that both a-(2,3)-
linked and a-(2,6)-linked sialic acid on O-glycans serve
as efficient precursors. Similarly, we have previously
found that both a-(2,3)- and a-(2,6)-linked sialic acids
Acknowledgements
This work was supported by NIH grants POI CA71932
and RO1 CA33895. The authors would like to thank
the referee for valuable suggestions.
2
5
on N-glycans serve as efficient precursors. In the
human erythrocyte membrane, the second sialic acid is

P. Sengupta et al. / Tetrahedron Letters 44 (2003) 6037–6042
6041
Figure 2. Addition of polysialic acid to compounds 5b, 10 and NeuAc-a-(2-3)-Gal-b-(1-4)-GlcNAcb-octyl 11. Each compound (1.0
14
nmol each) was incubated with CMP-[ C]-NeuAc and the soluble form of ST8SiaIV for 20 h at 37°C. The reaction products were
directly subjected to Mono-Q anion exchange chromatography. The elution position for polysialic acid containing multiple
25,26
numbers of sialic acids are shown as numbers above the chromatogram as described previously.
molecules are underlined.
Sialic acid residues in acceptor
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6
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. Palcic, M. M.; Hindsgaul, O. Trends Glycosci. Glycotech-
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. Rutishauser, U.; Landmesser, L. Trends Neurosci. 1996,
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1
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2
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chromatography on SiO2 using toluene–acetone (5:1)

6
042
P. Sengupta et al. / Tetrahedron Letters 44 (2003) 6037–6042
1
gave pure 2,6-a-sialylated product 5a (194 mg, 43%)
along with some 2,6-b-sialylated product (12%). The pro-
tected trisaccharide (194 mg, 0.14 mmol) thus obtained
was dissolved in 50 mM NaOMe in MeOH (5 ml) and
stirred for 12 h at room temperature. A few drops of
water was added to the reaction mixture which was then
allowed to stir for another 12 h. The reaction mixture
was neutralized with Amberlite IR-120 (H ) resin, filtered
and purified on LH-20 sephadex gel using MeOH as
solvent to afford pure 5b (70 mg, 62%).
22. Partial H NMR (500 MHz, D O): The following com-
2
mon signals for the octyl aglycon were observed in D O
soln: l 1.60–1.40 (2H, m, OCH CH ), 1.40–1.10 (10H, m,
OCH CH (CH ) CH ), 0.85 (t, J=6.9 Hz, 3H, octyl
CH ). H-1 indicates the anomeric proton of the GalNAc
residue, H-1% the anomeric proton of the Gal residue
linked to O-3 of the GalNAc residue and H-1%% the
anomeric proton of the GlcNAc residue linked to O-6 of
the GalNAc residue and onwards. Compound 5b: l 4.43
(d, J=8.4 Hz, 1H, H-1%), 4.28 (d, J=3.6 Hz, 1H, H-1),
2
2
2
2
2
2 5
3
3
+
1
1
8. Williams, M. A.; Kitagawa, H.; Datta, A. K.; Paulson, J.
2.72 (dd, J=12.5, 4.8 Hz, 1H, H-3%%), 1.99 and 2.00 (2s,
e
C.; Jamieson, J. C. Glycoconjugate J. 1995, 12, 755.
9. Lee, Y.-C.; Kojima, N.; Wada, E.; Kurosawa, N.;
Nakaoka, T.; Hamamoto, T.; Tsuji, S. J. Biol. Chem.
6H, 2 NHAc), 1.64 (t, J=12.5 Hz, 1H, H-3%%); TOFMS
a
m/z for C H O N Na (found) 808.7 (calcd) 808.7.
33
57 19
2
Compound 6: l 4.92 (d, J=3.0 Hz, 1H, H-1), 4.57 (d,
J=8.0 Hz, 1H, H-1%), 2.77 (dd, J=12.5, 5.0 Hz, 1H,
1
994, 269, 10028.
20. Enzymatic glycosylation using 2,3-(O)-sialyltransferase
H-3%%), 2.01–2.02 (2s, 6H, 2 NHAc), 1.81 (t, J=12.0 Hz,
e
for the synthesis of 6, 7 or 10: To a solution of compound
1H, H-3%%); TOFMS m/z for C H O N Na (found)
a
33 57 19
2
2
(
1
, 4 or 5b (2.0 mmol) in 50 mM sodium cacodylate buffer
pH 6.0; 100 mL) was added recombinant rat liver Galb-
,3-GalNAca-2,3-(O)-sialyltransferase (0.25 mU), CMP-
808.6, (calcd) 808.7. Compound 7: l 4.90 (d, J=3.3 Hz,
1H, H-1), 4.56 (d, J=7.8 Hz, 1H, H-1%), 2.78–2.82 (m,
2H, H-3
1.68–1.82 (m, 2H, H-3
Na (found) 1122.0 (calcd) 1122.0. Com-
%% and H-3
e e
NeuAc (3.0 mg, 4.50 mmol), Triton CF-54 (0.1%), calf
intestinal alkaline phosphatase (5.0 U) and the solution
was kept at 37°C for 16 h. The reaction mixture was
passed through a QAE mini column to remove the side
products formed and the product was isolated on a
C -SepPak cartridge, which was pre-washed with water.
The product was eluted with methanol. The methanolic
fractions were concentrated and further purified on sep-
hadex LH-20 to give pure 6, 7 or 10.
a
a
C
44
H
O
27
N
73
3
2
pound 8: l 4.88 (d, J=4.0 Hz, 1H, H-1), 4.57 (d, J=7.5
Hz, 1H, H-1%%%), 4.55 (d, J=7.5 Hz, 1H, H-1%%), 4.48 (d,
J=7.5 Hz, 1H, H-1%), 2.77 (dd, J=12.0, 4.5 Hz, 1H,
H-3
1H, H-3
%%%%), 2.03–2.05 (3s, 9H, 3 NHAc), 1.82 (t, J=12.5 Hz,
%%%%); TOFMS m/z for C47 Na (found)
18
e
H O N
80 29 3
a
1174.1 (calcd) 1174.1. Compound 9: l 4.86 (d, J=3.9 Hz,
1H, H-1), 4.53 (d, J=7.5 Hz, 1H, H-1%%%), 4.52 (d, J=7.5
Hz, 1H, H-1%%), 4.46 (d, J=7.5 Hz, 1H, H-1%), 2.73–2.75
2
1. Enzymatic glycosylation using 2,3-(N)-sialyltransferase
for the synthesis of 8 and 9: To a solution of compound
(m, 2H, H-3
NHAc), 1.74–1.81 (m, 2H, H-3
m/z for C58 Na (found) 1487.4 (calcd) 1487.4.
Compound 10: l 4.87 (d, J=3.9 Hz, 1H, H-1), 4.54 (d,
J=7.5 Hz, 1H, H-1%%%), 4.52 (d, J=7.5 Hz, 1H, H-1%%),
4.45 (d, J=7.5 Hz, 1H, H-1%), 2.73 (dd, J=12.5, 5.0 Hz,
e e
%%%%%), 1.99–2.03 (4s, 12H, 4
4
(1.7 mg, 2.0 mmol) in 50 mM 3-[N-mor-
pholino]propanesulfonic acid (MOPS) buffer (pH 7.4;
00 mL) was added recombinant rat liver Galb-1,4-Glc-
a
a
H
O
N
96
37
4
2
1
NAca-2,3-(N)-sialyltransferase (0.025 mU for compound
8
and 0.25 mU for compound 9), CMP-NeuAc (3.0 mg,
4
.50 mmol for compound 9 and 1.8 mg, 2.70 mmol for
1H, H-3
Hz, 1H, H-3
(found) 1174.1 (calcd) 1174.1.
e
compound 8), Triton CF-54 (0.2%), calf intestinal alka-
line phosphatase (5.0 U) and bovine serum albumin (1.0
mg) and the solution was kept at 37°C for 30 min to give
a
H O N
80 29 3
23. Ding, Y.; Fukuda, M.; Hindsgaul, O. Bioorg. Med.
Chem. Lett. 1998, 8, 1903.
8
and for 16 h to give 9. The reaction mixture was passed
through a QAE mini column to remove the side products
24. Nakayama, J.; Fukuda, M. J. Biol. Chem. 1996, 271,
1829.
and the product was isolated on a C -SepPak cartridge,
18
which was pre-washed with water. The product was
eluted with methanol. The methanolic fractions were
concentrated and further purified on sephadex LH-20 to
give pure 8 or 9.
25. Angata, K.; Suzuki, M.; McAuliffe, J.; Ding, Y.; Hinds-
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