Chemo-enzymatic synthesis of C-9 acetylated sialosides

Article abstract of DOI:10.1016/j.carres.2008.05.002

A chemo-enzymatic synthesis of [(5-acetamido-9-O-acetyl-3,5-dideoxy-d-glycero-α-d-galacto-2-nonulopyranosylonic acid)-(2→3)-O-(β-d-galactopyranosyl)-(1→3)-O-(2-acetamido-2-deoxy-α-d-galactopyranosyl)]-l-serine acetate (1) has been accomplished by a regioselective chemical acetylation of Neu5Ac (2) to give 9-O-acetylated sialic acid 3, which was enzymatically converted into CMP-Neu5,9Ac₂ (4) employing a recombinant CMP-sialic acid synthetase from Neisseria meningitis [EC 2.7.7.43]. The resulting compound was then employed for the enzymatic glycosylation of the C-3′ hydroxyl of chemically prepared glycosylated amino acid 10 using recombinant rat α-(2→3)-O-sialyltransferase expressed in Spodooptera frugiperda [EC 2.4.99.4] to give, after deprotection of the N^α-benzyloxycarbonyl (CBz)-protecting group of serine, target compound 1. The N^α-CBz-protected intermediate 11 can be employed for the synthesis of glycolipopeptides for immunization purposes.

Full text of DOI:10.1016/j.carres.2008.05.002

Available online at www.sciencedirect.com
Carbohydrate Research 343 (2008) 1605–1611
Chemo-enzymatic synthesis of C-9 acetylated sialosides
Jana Rauvolfova, Andre Venot and Geert-Jan Boons*
Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, GA 30602, USA
Received 28 March 2008; received in revised form 1 May 2008; accepted 4 May 2008
Available online 8 May 2008
Abstract—A chemo-enzymatic synthesis of [(5-acetamido-9-O-acetyl-3,5-dideoxy-D-glycero-a-D-galacto-2-nonulopyranosylonic
acid)-(2?3)-O-(b-D-galactopyranosyl)-(1?3)-O-(2-acetamido-2-deoxy-a-D-galactopyranosyl)]-L-serine acetate (1) has been accom-
plished by a regioselective chemical acetylation of Neu5Ac (2) to give 9-O-acetylated sialic acid 3, which was enzymatically con-
verted into CMP-Neu5,9Ac2 (4) employing a recombinant CMP-sialic acid synthetase from Neisseria meningitis [EC 2.7.7.43].
The resulting compound was then employed for the enzymatic glycosylation of the C-30 hydroxyl of chemically prepared glycosyl-
ated amino acid 10 using recombinant rat a-(2?3)-O-sialyltransferase expressed in Spodooptera frugiperda [EC 2.4.99.4] to give,
after deprotection of the Na-benzyloxycarbonyl (CBz)-protecting group of serine, target compound 1. The Na-CBz-protected inter-
mediate 11 can be employed for the synthesis of glycolipopeptides for immunization purposes.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Chemo-enzymatic synthesis; Sialic acid; Acetylation; CMP-Neu5,9Ac2; Immunogens
1. Introduction
strate specificity. Initially, acetyl transferases introduce
an acetyl ester at C-7 or C-9 of sialic acid and under
physiological conditions, the C-7 acetyl ester can mi-
grate to C-8 or C-9 making C-9 acetylation (Neu5,9Ac2)
the most prevalent sialic acid modification of cell surface
glycoconjugates.2,7 Acetylation of the C-4 hydroxyl of
sialosides has also been observed.9
It has been shown that 9-O-acetylation of sialic acids
can dramatically influence biological properties of gly-
coconjugates.2,7,10 For example, O-acetylation of sialo-
sides on the surface of thymocytes results in loss of
binding to Siglec-2 (CD22b) on B-cells.11 Abrogation
of 9-O-acetylated deleteriously affects development of
transgenic mice. This modification is also involved in
cell specific recognition by influenza C and corona
viruses.12,13 It has also been demonstrated that sialogly-
coproteins having 9-O-acetylation are expressed on lym-
phoblasts of children with acute lymphoblastic
leukemia.14
Sialic acids are a family of approximately 50 naturally
occurring 2-keto-3-deoxy-nononic acids that are
involved in a wide range of biological processes.1–3 The
C-5-amino derivative represents the long-known
neuraminic acid and its amino function can either be
acetylated (Neu5Ac) or glycolylated (Neu5Gc). As termi-
nal components of glycoproteins and glycolipids, neu-
raminic acids are a-(2?3)- or a-(2?6)-linked to
galactosides or a-(2?6)-linked to 2-acetamido-galacto-
sides.4–6
Enzymatic acetylation, lactylation, sulfation, methyl-
ation and phosphorylation of free hydroxyls give a large
structural diversity of sialic acids.3,7 O-Acetylation of
sialic acids takes place in the Golgi apparatus by mem-
brane bound O-acetyltransferases. Interestingly, differ-
ent cell types show different substrate selectivity for
acetylation of sialic acid indicating that there are differ-
ent classes of acetyltransfereases.2,3,8 Alternatively, it
may be possible that adaptor proteins control the sub-
Glycoproteins and gangliosides from rat, mouse and
chicken erythrocytes express Neu5,9Ac2-a-(2?3)-Gal-
b-(1?3)-GalNAc, which serves as targets for the bind-
ing of naturally occurring antibodies.12,15,16 In microor-
ganisms, such as the trypanosoma parasite Crithidia
fasciculata and the pathogenic fungus Cryptococcus
*
Corresponding author. Tel.: +1 706 542 9161; fax: +1 706 542
4412; e-mail: gjboons@ccrc.uga.edu
0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2008.05.002

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J. Rauvolfova et al. / Carbohydrate Research 343 (2008) 1605–1611
neoformans, Neu5,9Ac2 can be glycosidically linked to
Gal-b-(1?3)-GalNAc.17,18 The potential impact of 9-
O-acetylation of the GD1a ganglioside on the overall
conformation and recognition by a natural human anti-
body has been investigated by molecular dynamics sim-
ulations and NMR. It was found that acetylation did
not affect the conformational properties of the oligosac-
charide. However, it was shown that an antibody spe-
cific for a 9-O-acetylated ganglioside can make direct
interactions with the acetyl group rationalizing the selec-
tive recognition of acetylated derivatives.16,19
As part of a program to develop monoclonal antibod-
ies against sialo-oligosaccharides bearing 9-O-acetyl es-
ters, we report here an efficient chemo-enzymatic
synthesis of Neu5,9Ac2-a-(2?3)-Gal-b-(1?3)-Gal-
NAc-O-Ser (1). It is to be expected that this compound
can be employed for the preparation of fully synthetic
immunogens for the development of monoclonal anti-
bodies.20–22
fer modified sialic acids from corresponding CMP-deriv-
atives.24,25,27 For example,
rat liver a-(2?3)-
a
sialyltransferase has been used to transfer Neu5,9Ac2
from the corresponding CMP-donor to various glyco-
proteins,28 and a porcine liver a(2?6)-sialyltransferase
has been employed for the chemo-enzymatic synthesis
of Neu5,9Ac2-a-(2?6)-Gal-b-(1?4)-GlcNAc-a-OCH3.24
Recently, a library of (2?6)-linked modified sialosides
of Gal-b-(1?4)-GlcNAc-a-O-(CH2)3N3, which includes
four 9-O-acetylated sialoside derivatives, has been
prepared using the flexible recombinant Photobacterium
damsela [EC 2.4.99.1] a-(2?6)-sialyltransferase by a
one-pot three-enzyme system.29 The enzymatic synthesis
of the Neu5,9Ac2-a-(2?3)-Gal-O-p-nitrophenyl glyco-
side has also been reported.30
It was anticipated that C-9 derivatives of sialic acid
(Neu5Ac) would be readily available from Neu5Ac by
a chemical regioselective acetylation.23 Thus, treatment
of Neu5Ac (2) with trimethylorthoacetate in DMSO in
the presence of a catalytic amount of p-TsOHꢀH2O gave,
after purification by an ion exchange column chro-
ꢁ
matography (Dowex 50W-X2, HCO2 form, eluent: a
2. Results and discussion
mixture of H2O and formic acid) followed by a size
exclusion chromatography (Biogel P-2; eluent: H2O)
Neu5,9Ac2 (3) in an excellent yield of 92% (Scheme 1).
Condensation of Neu5,9Ac2 (3) with CTP in the pres-
ence of the commercial recombinant CMP-sialic acid
synthetase from N. meningitis [EC 2.7.7.43] and the inor-
ganic pyrophosphatase from Thermus thermophilus [EC
3.1.3.1] afforded CMP-Neu5,9Ac2 (4). The reported
purification strategy25 gave a compound 4 contaminated
with phosphorylated nucleotides (CTP, CDP and CMP)
and a small amount of CMP-Neu5Ac and Neu5Ac.
However, it was found that highly pure 4 could be
obtained by removal of inorganic salts by selective
precipitation31 in an ethanol–H2O mixture (9:1, v:v,
twice) followed by size exclusion chromatography over
extra fine Biogel P-2 using ammonium bicarbonate
buffer as eluent at 4 °C.
While relatively efficient methods have been developed
for the introduction of sialosides, the preparation of
acetylated analogues is less well developed. It was
anticipated that compound 1 could be prepared by a
regioselective chemical acetylation23 of Neu5Ac (2) to
give 9-O-acetylated sialic acid 3, which can then be
enzymatically converted into CMP-Neu5,9Ac2 (4)
employing the recombinant CMP-sialic acid synthetase
from Neisseria meningitis. This enzyme possesses a flexi-
ble substrate specificity and has been employed for the
synthesis of CMP-sialic acid analogues such as CMP-
Neu5,9Ac2.24,25,30 The resulting compound can then be
used for the enzymatic glycosylation26 of the C-30 hydro-
xyl of chemically prepared glycopeptide 10 using the
recombinant rat a-(2?3)-sialyltransferase [EC 2.4.99.4]
expressed in Spodooptera frugiperda to give, after
removal of the Na-CBz protecting group of serine, target
compound 1.
Glycosylation of thioglycoside 532 with properly pro-
tected serine derivative 626 in the presence of Ph2SO–
Tf2O and DTBMP provided the a-linked glycopeptide
7 in 83% yield with excellent a-anomeric selectivity
Sialyltransferases exhibit some flexibility with regard
to the structure of their acceptors and are able to trans-
NH2
OH
HO
R1O
OH
HO
O
O-
AcO
O
N
-
P
CO2
O
O
O
(ii)
O
-
O
OH
AcHN
CO2
AcHN
HO
HO
HO
OH
2. R1 = H
3. R1 = Ac
4
(i)
Scheme 1. Reagents and conditions: (i) CH3(OCH3)3, DMSO, p-TsOHꢀH2O, 92%; (ii) CTP, 3, Tris buffer (0.1 M, pH 7.5), CMP-sialic acid
synthetase, inorganic pyrophosphatase.

J. Rauvolfova et al. / Carbohydrate Research 343 (2008) 1605–1611
Ph
1607
Ph
NHCbz
O
O
OAc
O
AcO
AcO
O
OAc
O
HO
AcO
AcO
O
CO2tBu
O
O
6
SPh
O
O
NHCbz
CO2tBu
dFBzO
(i)
N3
dFBzO
N
3O
7
5
OAc
OAc
O
AcO
AcO
OH
OH
O
HO
HO
HO
O
(iv)
O
O
(ii)
O
AcO
NHCbz
NHCbz
CO2H
dFBzO
8 R = N3
9 R = NHAc
R
HO
AcNH
O
O
CO2tBu
10
(iii)
Scheme 2. Reagents and conditions: (i) Ph2SO, DTBMP, CH2Cl2, ꢁ70 °C, Tf2O then 6, 83%; (ii) (a) CH3CO2H–H2O, 80 °C, 1 h; (b) Ac2O, Py,
DMAP, 92%; (iii) CH3COSH, Py, 16 h, 67%; (iv) (a) TFA, H2O, 1 h; (b) CH3ONa, CH3OH, pH < 10, 86%.
OH
HO
AcO
-
CO2
OH
OH
O
HO
HO
O
(i)
O
O
4
+
10
AcHN
HO
O
NHR
CO2H
HO
AcHN
O
11 R = Cbz
R = H
1
Scheme 3. Reagents and condition: (i): a-(2?3)-sialyltransferase, cacodylate buffer (50 mM, pH 6.2), alkaline phosphatase; Pd–C, CH3OH–H2O–
AcOH.
(Scheme 2). Treatment of 7 with aqueous acetic acid
removed the benzylidene acetal and acetylation of the
resulting free hydroxyls gave compound 8 in excellent
overall yield. Next, the azido moiety of 8 was converted
into an acetamido derivative by reaction with thioacetic
acid in pyridine33–35 and the resulting compound 9 was
treated with a mixture of TFA and water to remove
the t-butyl ester, and then with NaOMe in methanol
to cleave the acetyl esters to give glycopeptide 10.36 At
this stage of the synthesis, the Cbz group was kept intact
because protection of the amino group of serine is
required for glycopeptide synthesis.
Next, attention was focused on the enzymatic sialyl-
ation of 10 using a CMP-Neu5,9Ac2 donor (4) (Scheme
3). Thus, the recombinant rat a-(2?3)-sialyltransferase
[EC 2.4.99.4] expressed in S. frugiperda and a calf intes-
tine alkaline phosphatase [EC 3.6.1.1] were added to a
mixture of 4 and 10 dissolved in cacodylate buffer
(50 mM; pH 6.2) containing Triton X-100 and BSA.37
After incubation at 37 °C for 2 h, another portion of
alkaline phosphatase and CMP-Neu5,9Ac2 (4) was
added and after occasional stirring for an additional
3.5 h, TLC analysis showed complete consumption of
the starting material 4 and the formation of a new prod-
uct. Purification of the crude reaction mixture by a
reverse-phase C18 column chromatography followed by
an ion exchange chromatography using Biorad AG
50W-X8 (100–200 mesh, Na+ form) at 4 °C, gave a pure
trisaccharide 11 in a yield of 69%. In addition, a small
amount (<5%) of 9-O-deacetylated product was also
isolated.
Compound 11 is appropriately protected for glyco-
peptide synthesis. However, fully unprotected 1 could
be obtained by catalytic hydrogenation over Pd–C to
remove the Na-Cbz group followed by purification by
a Biogel P-2 size exclusion column chromatography.
The chemical shift of the C-9 protons clearly demon-
strated the presence of the C-9 acetyl ester: 4.37 (dd,
1H, J8 ;9 a ¼ 2:2 Hz, J9 a;9 b ¼ 12:0 Hz, H-900a) and 4.19
00 00
00
00
(dd, 1H, J8 ;9 b ¼ 5:8 Hz, J9 a;9 b ¼ 12:0 Hz, H-900b).
In conclusion, we have developed an efficient chemo-
enzymatic synthesis of the trisaccharide 11, which is
appropriately protected for glycopeptide synthesis. It
has been shown that the rat recombinant a-(2?3)-sialyl-
transferase expressed in S. frugiperda can accept CMP-
Neu5,9Ac2 as a donor. The improved purification proto-
col for CMP-Neu5,9Ac2 was essential for the successful
transfer of CMP-Neu5,9Ac2 to glycosyl acceptor Gal-b-
(1?3)-GalNAc-O-Ser.
00 00
00
00
3. Experimental
3.1. General methods
The recombinant N. meningitis CMP-sialic acid synthe-
tase expressed in Escherichia coli [EC 2.7.7.43], the
recombinant rat a-(2?3)-sialyltransferase [EC 2.4.99.4]

1608
J. Rauvolfova et al. / Carbohydrate Research 343 (2008) 1605–1611
expressed in S. frugiperda and calf intestine alkaline
phosphatase [EC 3.6.1.1] were purchased from
Calbiochem. The inorganic pyrophosphatase from T.
thermophilus [EC 3.1.3.1], bovine serum albumin
(BSA), cytidine 50-monophosphate (CMP), cytidine
50-triphosphate (CTP), 1,4-dithioerythritol (DET),
manganese(II) chloride (MnCl2) and Triton X-100 were
purchased from Sigma. All other chemicals were pur-
chased from Aldrich. Solvents were dried using standard
procedures, and all chemical reactions were performed
under an atmosphere of Argon. Column chromato-
graphy was performed on F60 Silica Gel (60–200 lm)
from Silicycle. Bio-Gel P-2 Gel fine (200–3000 Da; 45–
90 lm), Bio-Gel P-2 extra fine (200–3000 Da, <45 lm)
and AG 50W-X8 (100–200 mesh) ion exchange resin
were obtained from Bio-Rad; preparative C18 silica gel
indicated completion of the reaction, which was
quenched by the addition of solid NaHCO3. After
addition of CH2Cl2, the mixture was filtered, washed
with H2O, aqueous NaHCO3 and brine, dried (MgSO4),
filtered and concentration in vacuo. The residue was
purified by silica gel column chromatography (hex-
anes–EtOAc 65:35, v:v) to provide 7 (122 mg, 83%);
½aꢂD +22 (c 0.2; CHCl3); H NMR (CDCl3): d 7.30–
7.00 (m, 13H, aromatics), 5.66 (d, 1H, JNH,Ha = 8.0 Hz,
NH), 5.51 (m, 2H, H-20, CH, benzylidene), 5.43 (d, 1H,
25
1
J3 ;4 ¼ 3:0 Hz, H-40), 5.16 (dd, J3 ;4 ¼ 3:0 Hz,
0
0
0
0
J2 ;3 ¼ 10:3 Hz, H-30), 5.10 (s, 2H, PhCH2), 4.91 (d,
0
0
0
0
1H, J1,2 = 3.0 Hz, H-1), 4.86 (d, 1H, J1 ;2 ¼ 7:9 Hz, H-
10), 4.40–3.66 (m, 12H), 2.16, 2.03, 1.93 (3s, 3H each,
3 ꢃ CH3, OAc), 1.44 (s, 9H, CH3 t-Bu); 13C NMR: d
170.5, 170.3, 137.7, 129.1 128.8, 128.6, 128.4, 128.3,
126.3, 118.4, 118.1, 102.4 (C-1), 100.8 (PhCh), 99.9
(C-1), 83.1, 75.8, 75.7, 71.3, 71.1, 70.0, 69.9, 69.2, 67.3,
63.7, 61.6, 58.9, 55.1, 28.1, 20.9, 20.7; HRMALDI-
ToF-MS: calcd for C47H52F2N4O18 [M+Na]+: m/z:
1021.3231; found m/z: 1021.3224.
˚
(125 A, 55–105 lm) was obtained from Waters and
Iatrobeads 6RS-8060 (60 lm) were purchased from Bio-
scan. Thin layer chromatography (TLC) was performed
using Kieselgel 60 F254 (Merck) plates with detection by
UV and/or by charring with 5% sulfuric acid in ethanol,
cerium ammonium nitrate or p-anisaldehyde reagents.
Solvent evaporations were done in vacuo with a bath
temperature <25 °C. Optical rotations were measured
with a Jasco P-1020 polarimeter. Both positive and neg-
ative ion matrix-assisted laser desorption ionization time
of flight (MALDI-TOF) mass spectra were recorded
using an Applied Biosystems 4700 instrument with gen-
tisic acid as matrix. 1D and 2D 1H NMR and 13C NMR
spectra were recorded at 25 °C on Varian Inova300,
Varian Inova500, Varian Inova600 or Varian Inova900
spectrometers. Assignments were obtained from COSY
3.3. N-(Benzyloxycarbonyl) [4,6-di-O-acetyl-2-azido-2-
deoxy-3-O-(3,4,6-tri-O-acetyl-2-O-(2,5-difluorobenzoyl)-
b-D-galactopyranosyl)-(1?3)-a-D-galactopyranosyl]-L-
serine t-butyl ester (8)
A solution of disaccharide 7 (143 mg, 0.142 mmol) in a
mixture of CH3CO2H (4 mL) and H2O (1 mL) was kept
at 80 °C for 1 h after which TLC analysis (hexanes–
EtOAc 1:1, v:v) indicated completion of the reaction.
The mixture was co-evaporated with toluene and the
residue dried in vacuo. The recovered material was acet-
ylated with pyridine (2 mL) and Ac2O (0.2 mL) in the
presence of a catalytic amount of dimethylaminopyr-
idine (DMAP) for 12 h. After the addition of CH3OH,
the mixture was co-evaporated with toluene, the residue
dissolved in CH2Cl2 and washed with aqueous NaH-
CO3, H2O and brine. The organic solvents were dried
(MgSO4), filtered and the filtrate concentrated in vacuo.
The residue was purified by silica gel column chromato-
graphy (hexanes–acetone 70:30, v:v) to afford 8 (129 mg,
1
and HSQC spectra. For H spectra recorded in CDCl3,
D2O and CH3OD and 13C spectra recorded in CDCl3,
chemical shifts are given in ppm relative to solvents
peaks (CDCl3, 1H 7.24; 13C, 77.00; D2O, 1H, 4.76;
1
CH3OD, H, 3.31, 4.78). 13C Spectra recorded in D2O
were indirectly referenced.
3.2. N-(Benzyloxycarbonyl) [2-azido-4,6-O-benzylidene-2-
deoxy-3-O-(3,4,6-tri-O-acetyl-2-O-(2,5-difluorobenzoyl)-
b-D-galactopyranosyl)-(1?3)-a-D-galactopyranosyl]-L-
serine t-butyl ester (7)
25
1
92%); ½aꢂD +52.8 (c 0.2; CHCl3); H NMR (CDCl3): d
7.3–7.0 (m, 8H, aromatics), 5.65 (d, 1H, JNH,Ha
=
Ph2SO (83 mg, 0.411 mmol), di-t-butyl-3-methyl pyr-
8.0 Hz, NH), 5.45–5.35 (m, 3H, H-4, H-20, H-40), 5.16–
idine (90.4 mg, 0.441 mmol) and crushed activated
5.08 (m, 3H, H-30, PhCH2), 4.85 (d, J1,2 = 3,3 Hz, H-
molecular sieves 4 A (500 mg) were added to a cooled
1), 4.77 (d, 1H, J1 ;2 ¼ 7:6 Hz, H-10), 4.40 (m, 1H, Ha-
Ser), 4.18–3.84 (m, 10H), 3.58 (dd, 1H, J1,2 = 3.3 Hz,
J2,3 = 10.7 Hz, H-2), 2.16, 2.10, 2.03, 2.02, 1.92 (5s,
15H, 5 ꢃ CH3, OAc), 1.42 (s, 9H, CH3, t-Bu); 13C
NMR: d 170.7, 170.6, 170.4, 170.2, 169.8 168.6, 136.3,
129.2, 128.8, 128.6, 128.5, 128.4, 101.5, 98.9, 83.1,
74.6, 71.2, 70.8, 70.1, 69.6, 69.4, 68.1, 67.3, 67.0, 62.8,
61.2, 59.6, 55.0, 28.0, 20.9, 20.8, 20.7; HRMALDI-
ToF-MS: calcd for C44H52F2N4O20 [M+Na]+: m/z:
1017.3124; found m/z: 1017.3141.
˚
0
0
solution (ꢁ70 °C) of disaccharide 5, (120 mg,
0.147 mmol) in CH2Cl2 (6 mL) prior to the addition of
trifluoromethanesulfonic anhydride (58 mg, 36 lL,
0.205 mmol). After stirring the mixture at ꢁ70 °C for
5 min, a solution of 6 (85.5 mg, 0.29 mmol) in CH2Cl2
(0.5 mL) was added dropwise by syringe. The resulting
reaction mixture was stirred at ꢁ60 °C for 1 h and then
allowed to warmed to room temperature over a period
of 3 h. TLC analysis (hexanes–acetone 70:30, v:v)

J. Rauvolfova et al. / Carbohydrate Research 343 (2008) 1605–1611
1609
0
0
3.4. N-(Benzyloxycarbonyl) [-2-acetamido-4,6-di-O-
acetyl-2-deoxy-3-O-(3,4,6-tri-O-acetyl-2-O-(2,5-di-
fluorobenzoyl)-b-D-galactopyranosyl)-(1?3)-a-D-galacto-
pyranosyl]-L-serine t-butyl ester (9)
Hb0Ser), 3.72 (d, 1H, J3 ;4 ¼ 2:7 Hz, H-40), 3.68–3.57
(m, 4H, H6a, H6b, H60a, H60b), 3.47–3.38 (m, 2H, H-
20, H-50), 3.34 (dd, 1H, J2 ;3 ¼ 9:8 Hz, J3 ;4 ¼ 2:7 Hz,
H-30), 1.86 (s, 3H, CH3, NHAc); 13C NMR (D2O): d
174.8, 174.7, 136.4, 129.9, 128.9, 128.6, 127.9, 104.7,
98.2, 77.0, 75.1, 72.7, 71.0, 70.8, 68.7, 68.2, 67.3, 61.2,
61.1, 55.3, 48.7, 22.16; HRMALDI-ToF-MS: calcd for
C25H36N2O15 [M+Na+]: m/z: 627.2147; found m/z:
627.2156.
0
0
0
0
Freshly distilled thioacetic acid (0.6 mL) was added
by syringe to a solution of disaccharide 8 (128 mg,
0.128 mmol) in pyridine. The resulting reaction mixture
was stirred under an atmosphere of argon for 18 h after
which TLC (hexanes–EtOAc 1:1, v:v) indicated comple-
tion of the reaction. The mixture was diluted with
CH2Cl2, successively washed with H2O, aqueous NaH-
CO3 and brine, dried (MgSO4), filtered and the filtrate
concentrated in vacuo. The residue was purified by silica
gel column chromatography (hexanes–EtOAc 55:45,
3.6. 5-Acetamido-9-O-acetyl-3,5-dideoxy-D-glycero-b-D-
galacto-2-nonulopyranosylonic acid (3)
Trimethyl orthoacetate23 (2.0 mL, 16.0 mmol) and p-
TsOHꢀH2O (20 mg) were added to a solution of Neu5Ac
2 (0.5 g, 1.6 mmol) in DMSO (5.0 mL) under an atmo-
sphere of argon. The reaction mixture was stirred for
1.5 h and then purified by anion exchange resin ꢁcolumn
chromatography using Dowex 50W-X2 (HCO2 form,
40 ꢃ 1 cm). Elution with H2O (110 mL), followed by
formic acid (1 N, 75 mL) provided appropriate fractions
(detected by TLC), which were combined and first evap-
orated under vacuum (T < 25 °C) and then lyophilized
to afford crude Neu5,9Ac2 3 (0.54 g, 1.54 mmol, 96%).
The resulting material was dissolved in H2O (1 mL)
25
v:v) to give 9 (86 mg, 67%); ½aꢂD +66 (c 0.1; CHCl3);
1H NMR (CDCl3): d 7.3–7.0 (m, 8H, aromatics), 5.55
(d, 1H, JNH,Ha = 8.0 Hz, NH), 5.45–5.35 (m, 3H, H-4,
H-20, H-40), 5.16–5.30 (m, 3H, H-30, PhCH2), 4.78 (d,
J1,2 = 3,3 Hz, H-1), 4.68 (d, 1H, J1 ;2 ¼ 7:7 Hz, H-10),
0
0
4.50 (m, 1H, H-2), 3.38 (m, 1H, HaSer), 4.20–3.80 (m,
0
8H), 3.70 (dd, 1H, JHa,Hb = 3.0 Hz, JHb;Hb ¼ 10:4 Hz,
HbSer), 2.14, 2.10, 2.03, 2.02, 1.92 (6s, 18H, 5 ꢃ CH3,
OAc and NHAc), 1.39 (s, 9H, CH3, t-Bu); 13C NMR:
d 170.5, 170.4, 170.3, 170.1, 170.0, 169.5, 168.9, 135.9,
128.6, 128.5, 128.3, 118.4, 118.0, 100.5 (C-10), 98.2
(C-1), 82.8, 74.8, 70.9, 70.7, 70.2, 68.6, 68.2, 67.8, 67.2,
66.7, 62.8, 60.8, 54.5, 48.2, 22.6, 20.7, 20.6, 20.5; HRM-
ALDI-ToF-MS: calcd for C46H56F2N2O4 [M+Na]+:
m/z: 1033.3362; found m/z: 1033.3341.
and then loaded on
a Biogel P-2 fine column
(75 ꢃ 2.5 cm, flow rate 20.5 mL/h, H2O) to give pure 3
1
(0.52 g, 92.5%); [a]D ꢁ8.6 (c 1; H2O); H NMR (D2O,
300 MHz): d 4.36 (dd, 1H, J8,9a = 2.4 Hz, J9a,9b
11.6 Hz, H-9a), 4.17 (dd, 1H, J8,9b = 5.5 Hz, J9a,9b
=
=
11.6 Hz, H-9b), 4.10–3.80 (m, 4H), 3.60 (d, 1H,
J7,8 = 9.4 Hz, H-7), 2.30 (dd, 1H, J3eq,4 = 4.9 Hz,
J3eq,3ax = 13.0 Hz, H-3eq), 2.10 and 2.04 (2s, 6H,
2 ꢃ CH3, OAc, NHAc), 1.86 (dd, J3ax,4 = 12.2 Hz,
J3eq,3ax = 13.0 Hz, H-3ax); HRMALDI-ToF-MS: calcd
for C13H21NO10 [M+Na]+: m/z: 374.1118; found m/z:
374.1134.
3.5. N-(Benzyloxycarbonyl)-[-2-acetamido-2-deoxy-3-O-
(b-D-galactopyranosyl)-a-D-galactopyranosyl]-L-serine
(10)
H2O (50 lL) was added to a solution of the disaccharide
9 (83 mg, 0.082 mmol) in trifluoroacetic acid (0.95 mL).
The mixture was stirred at 22 °C for 1 h until TLC (hex-
anes–acetone 1:1, v:v) indicated completion of the reac-
tion. The mixture was co-evaporated with toluene and
the residue dried in vacuo. The crude material was
dissolved in dry CH3OH (3 mL), and a solution of
NaOCH3 in CH3OH (0.5 M) was carefully added while
keeping the pH < 10 and the reaction monitored by
TLC (CHCl3–CH3OH–H2O 65:35:5, v:v:v). The
reaction was neutralized by the addition of Dowex
50W-X2 (H+ form). After filtration, the product was
dry-loaded on Iatrobeads (200 mg) and purified by
3.7. Cytidine 50-(5-acetamido-9-O-acetyl-3,5-dideoxy-b-
D-glycero-D-galacto-2-nonulopyranosylonic acid mono-
phosphate) ammonium salt (4)
CTP (126 mg, 0.239 mmol) was added to a solution of
Neu5,9Ac2 3 (50 mg, 0.142 mmol) in a Tris–HCl buffer
(0.1 M, 9 mL, pH 7.5) containing DET (4.75 mM) and
MnCl2 (13.80 mM). Addition of CTP resulted in a
change of pH from 7.5 to 6.5, which was re-adjusted
to pH 7.5 using aqueous NaOH (0.3 M). The recombi-
nant CMP-sialic acid synthetase from N. meningitis
(9.2 U, 27.0 lL) and the inorganic pyrophosphatase
from T. thermophilus (4.6 U, 60.0 lL) were added, and
the reaction mixture was incubated at 37 °C for 1 h with
occasional shaking. Another portion of CMP-sialic acid
synthetase and inorganic pyrophosphatase (1.5 U,
19.5 lL) was added after 2.5 h. The formation of a white
precipitate (presumably manganese–ammonium phos-
chromatography on
a Iatrobeads column (1.8 g)
(CHCl3–CH3OH–H2O 65:35:5, v:v:v) to afford di-
25
saccharide 10 (43 mg, 83%); ½aꢂD +85 (c 0.09; H2O);
1H NMR (CD3OD, 500 MHz): d 7.30 (m, 5H, aro-
matics), 5.04 (m, 2H, PhCH2), 4.73 (J1,2 = 3.2 Hz,
H-1), 4.33–4.25 (m, 3H, H-10, H-2, HaSer), 4.06 (br d,
J3,4 = 2.6 Hz, H-4), 3.87 (dd, 1H, JHa,Hb = 3.3 Hz,
0
JHb;Hb ¼ 11:0 Hz, HbSer), 3.83–3.75 (m 3H, H-3, H-5,

1610
J. Rauvolfova et al. / Carbohydrate Research 343 (2008) 1605–1611
phate) was observed after 1.5 h. The progress of the
reaction was monitored by TLC (EtOH–1 M
NH4HCO3, 4:1, v:v), which after 5 h indicated comple-
tion of the reaction. Ethanol (80 mL) was added and
the mixture was kept on ice for 2 h prior to centrifu-
gation. The supernatant was decanted and the pellet
(mostly inorganic salts) was re-suspended in EtOH
(30 mL), cooled on ice for 1 h and centrifuged. The com-
bined ethanol extracts were concentrated in vacuo pro-
viding crude material (230 mg). Ethanol (1.8 mL) was
slowly added to the material dissolved in H2O
(0.2 mL) and precipitation occurred immediately. The
mixture was kept on ice for 2 h, and the supernatant
was removed after centrifugation. After drying, NMR
(D2O) of the first pellet (114 mg) indicated the presence
of the expected product, some Tris, ethanol and a small
amount of Neu5,9Ac2. The recovered material was
again dissolved in H2O (0.2 mL) and re-precipitated by
the addition of ethanol (1.80 mL) as described above
providing 75 mg of material, which was loaded on a col-
umn of extra-fine Biogel P-2 (75 ꢃ 1.5 cm) and eluted
with 0.1 M NH4HCO3 at 4 °C. The appropriate frac-
tions were detected by UV and TLC (as above), col-
lected, concentrated in vacuo (bath temperature
<25 °C) and lyophilized from H2O (three times to com-
pletely remove NH4HCO3) to afford 3 (60 mg, 61%,
yltransferase (10 mU), calf intestine alkaline phospha-
tase and CMP-Neu5,9Ac2 (7.0 mg) was added to each
of the five tubes. After 5.5 h, TLC indicated the disap-
pearance of disaccharide 10. Each reaction was
quenched by the addition of CH3OH (10 lL), combined
and freeze dried to provide a crude material (200 mg),
which was applied on a Biogel fine P-2 column
(75 ꢃ 1.5 cm, using H2O as eluent at 8 mL/h) at 4 °C.
The product was detected by TLC, and appropriate
fractions were combined and freeze dried to provide
11 (37 mg). This material was passed through a short
column of BioRad AG 50W-X8 (100–200 mesh, Na+)
resin at 4 °C, and the appropriate fractions freeze dried.
The recovered material was purified by C-18 silica gel (5
g) column chromatography using a gradient of CH3CN
in H2O (95:5?90:10, v:v, at 4 °C), which removed the
9-O-deacetylated trisaccharide (1.0 mg) from the prod-
25
uct 11 (22.5 mg, 69%); ½aꢂD +12 (c 0.05; H2O); 1H
NMR (600 MHz): d 7.40 (m, 5H, aromatics), 5.18–
5.08 (m, 2H, PhCH2), 4.85 (d, 1H, J1,2 = 3.3 Hz, H-1),
4.50 (d, 1H, J1 ;2 ¼ 7:6 Hz, H-10), 4.35 (br d, 1H,
0
0
J9 a;00b ¼ 10:7 Hz, H-900a), 4.28 (dd, 1H, J1,2 = 3.3 Hz,
00
J2,3 = 11.0 Hz, H-2), 4.20–4.15 (m, 3H, H-4, H-900b,
HaSer), 4.08–4.00 (m, 3H, H-3, H-30, H-800), 3.95–3.90
(m, 3H), 3.83 (t, 1H, J4 ;5 ¼ J5 ;6 ¼ 10:1 Hz, H-500),
3.78 (m, 1H, Hb0Ser), 3.73–3.55 (m, 8H), 3.50 (dd, 1H,
00 00
00 00
25
ammonium salt). ½aꢂD ꢁ4 (c 1.5; H2O); 1H NMR
J1 ;2 ¼ 7:6 Hz, J2 ;3 ¼ 9:4 Hz, H-20), 2.74 (dd, 1H,
0
0
0
0
J3 eq;4 ¼ 4:6 Hz, J3 eq;3 ax ¼ 12:5 Hz, H-300eq), 2.10 (s,
00
00
00
00
(D2O, 500 MHz): d 7.94 (d, 1H, J5,6 = 7.4 Hz, H-5,
cyt.), 6.12 (d, 1H, J5,6 = 7.4 Hz, H-5, cyt.), 6.00 (d,
1H, J1,2 = 4.9 Hz, H-1 rib.), 4.40–4.10 (m, 8H), 4.03
(ddd, 1H, J8,9b = 5.1 Hz, J7,8 = 9.8 Hz, J8,9a = 11.0 Hz,
H-8), 3.95 (t, 1H, J4,5 = J5,6 = 10.4 Hz, H-5), 3.49 (d,
1H, J7,8 = 9.8 Hz, H-7), 2.47 (dd, 1H, J3eq,4 = 4.9 Hz,
J3eq,3ax = 13.4 Hz, H-3eq), 2.08 and 2.04 (2s, 6H,
2 ꢃ CH3, OAc, NHAc), 1.64 (ddd, 1H, J3ax,P = 5.8 Hz,
J3ax,3eq = 13.4 Hz, J3ax,4 = 12.5 Hz, H-3ax) in agreement
with reported data;24 HRESIMS: calcd for C23H24N3-
O17P [MꢁH]ꢁ: m/z: 655.1626; found 654.1469.
3H, OAc), 2.01, 1.95 (2s, 6H, NHAc), 1.77 (t, 1H,
J3 eq;3 ax ¼ J3 ax;4 12.5 Hz, H-300ax). HRMALDI-ToF-
MS: calcd for C38H55N3O24 [M+Na]+: m/z: 960.3342;
m/z: found 960.3351.
00
00
00
00
3.9. [(5-Acetamido-9-O-acetyl-3,5-dideoxy-D-glycero-
a-D-galacto-2-nonulopyranosylonic acid)-(2?3)-O-
(b-D-galactopyranosyl)-(1?3)-O-(2-acetamido-2-deoxy-
a-D-galacto-pyranosyl)]-L-serine acetate (1)
Pd–C (10%, 18 mg) was added to trisaccharide 11
(20.0 mg, 0.021 mmol) in a mixture of H2O and CH3OH
(1:1, 3.5 mL) containing CH3CO2H (0.14 mL). The mix-
ture was hydrogenated at 1 atm for 30 min after which
TLC (C2H5OH–1 M NH4CH3CO2 8:2, v:v) indicated
completion of the reaction. The mixture was filtered
through a 0.2 lM PTFE syringe filter, the solvents were
evaporated and the residue was freeze dried from H2O
3.8. Sodium N-(benzyloxycarbonyl)-[(5-acetamido-9-O-
acetyl-3,5-dideoxy-D-glycero-a-D-galacto-2-nonulopyr-
anosylonic acid)-(2?3)-O-(b-D-galactopyranosyl)-
(1?3)-O-(2-acetamido-2-deoxy-a-D-galactopyranosyl)]-
L-serine sodium salt (11)
Recombinant rat a-(2?3)-sialyltransferase from S. fru-
giperda [EC 2.4.99.4] (10 mU, 7 lL) and calf intestine
alkaline phosphatase (15 U, 15 lL) were added to a mix-
ture of disaccharide 10 (4.6 mg, 7.1 lmol) in cacodylate
buffer (50 mM, pH 6.2; 600 lL) containing 0.1% Triton
X-100, 0.1% BSA and CMP-Neu5,9Ac2 4 (6.7 mg,
9.5 lmol) in an Eppendorf tube, and five similar reac-
tions were run in parallel. The tubes were incubated at
37 °C, and progress of the reactions were monitored
by TLC (CHCl3–CH3OH–H2O–AcOH 60:40:10:0.5,
v:v:v:v). After 2 h, an additional amount of a(2?3)-sial-
25
to provide trisaccharide 1 (18.1 mg, 100%). ½aꢂD +8.4
(c 0.1; H2O); 1H NMR (900 MHz): d 4.91 (d, 1H,
J1,2 = 3.0 Hz, H-1), 4.52 (d, 1H, J1 ;2 ¼ 7:7 Hz, H-10),
0
0
00 00
00
00
4.37 (dd, 1H, J8 ;9 a ¼ 2:2 Hz, J9 a;9 b ¼ 12:0 Hz,
H-900a), 4.35 (dd, 1H, J1,2 = 3.2 Hz, J2,3 = 11.0 Hz,
H-2). 4.23 (d, 1H, J3,4 = 2.5 Hz, H-4), 4.19 (dd, 1H,
J8 ;9 b ¼ 5:8 Hz, J9 a;9 b ¼ 12:0 Hz, H-900b), 4.11 (dd,
00 00
00
00
0
1H, JHa,Hb = 2.8 Hz, JHa;Hb ¼ 11:2 Hz, HaSer), 4.09–
4.03 (m, 3H, H-3, H-30, H-800), 3.97 (m, 2H, Hb-Ser,
H-5), 3.92 (d, 1H, J3 ;4 ¼ 3:0 Hz H-40), 3.88 (dd, 1H,
0
0

J. Rauvolfova et al. / Carbohydrate Research 343 (2008) 1605–1611
13. Kunkel, F.; Herrler, G. Virology 1993, 195, 195–202.
1611
0
J
Ha,Hb = 5.2 Hz,
JHb;Hb
Hb-Ser),
3.85
(t,
J4 ;5 ¼ J5 ;6 ¼ 11:1 Hz, H-500), 3.80–3.64 (m, 4H, H6a,
14. Mandal, C.; Chatterjee, M.; Sinha, D. Br. J. Haematol.
2000, 110, 801–812.
00 00
00 00
H6b, H60a, H60b), 3.61 (m, 2H, H-50, H-700), 3.52 (dd,
15. Gowda, D. C.; Reuter, G.; Shukla, A. K.; Schauer, R.
Hoppe-Seylers Z. Physiol. Chem. 1984, 365, 1247–1253.
16. Siebert, H. C.; von der Lieth, C. W.; Dong, X.; Reuter, G.;
Schauer, R.; Gabius, H. J.; Vliegenthart, J. F. Glycobiol-
ogy 1996, 6, 561–572.
1H, J1 ;2 ¼ 7:7 Hz, J2 ;3 ¼ 9:8 Hz, H-20), 2.74 (dd, 1H,
0
0
0
0
J3 eq;4 ¼ 2:6 Hz, J3 eq;3 ax ¼ 12:2 Hz, H-300eq), 2.12 (s,
00
00
00
00
3H, OAc), 2.02, 1.98 (2s, 6H, NHAc), 1.97 (s,
00
00
00
00
CH3CO2H), 1.78 (t, 1H, J3 ax;3 eq ¼ J3 ax;4 ¼ 12:2 Hz,
H-300ax); HRMALDI-ToF-MS: calcd for C30H49N3O22
[M+Na]+: m/z: 826.3025; found: m/z: 826.3033.
17. Matta, M. A. D.; Alviano, D. S.; Couceiro, J. N. D. S.;
Nazareth, M.; Meirelles, L.; Alviano, C. S.; Angluster, J.
Parasitol. Res. 1999, 85, 293–299.
18. Rodrigues, M. L.; Rozental, S.; Couceiro, J. N. S. S.;
Angluster, J.; Alviano, C. S.; Travassos, L. R. Infect.
Immun. 1997, 65, 4937–4942.
19. Pal, S.; Chatterjee, M.; Bhattacharya, D. K.; Bandhyo-
padhyay, S.; Mandal, C. Glycobiology 2000, 10, 539–
549.
Acknowledgement
This research was supported by the National Cancer
Institute of the National Institute of Health (Grant
No. RO1 CA88986).
20. Buskas, T.; Ingale, S.; Boons, G. J. Angew. Chem., Int. Ed.
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Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.carres.
2008.05.002.
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