Recombinant (2-->3)-alpha-sialyltransferase immobilized on nickel-agarose for preparative synthesis of sialyl Lewis(x) and Lewis(a) precursor oligosaccharides.

Article abstract of DOI:10.1016/S0008-6215(03)00130-7

The specificity of recombinant (2-->3)-alpha-sialyltransferase (ST3Gal-III), expressed in baculovirus-infected insect cells, has been determined with various oligosaccharide acceptors and sugar-nucleotide donors using a fluorescence based assay. Recombina

Full text of DOI:10.1016/S0008-6215(03)00130-7

Carbohydrate Research 338 (2003) 1153–1161
www.elsevier.com/locate/carres
Recombinant (2“3)-a-sialyltransferase immobilized on
nickel-Agarose for preparative synthesis of sialyl Lewisx and
Lewisa precursor oligosaccharides
Tatiana Ivannikova,a Fabrice Bintein,a Annie Malleron,a Sylvie Juliant,b
Martine Cerutti,b Anne Harduin-Lepers,c Philippe Delannoy,c Claudine Auge´,a,*
Andre´ Lubineaua
aLaboratoire de Chimie Organique Multifonctionnelle, UMR 8614, Uni6ersite´ de Paris-Sud, F-91405 Orsay, France
bStation de Recherche de Pathologie Compare´e, Unite´ de Biologie Cellulaire et Mole´culaire, UMR 5087,
F-30380 Saint Christol lez Ale`s, France
cUnite´ de Glycobiologie Structurale et Fonctionnelle, UMR CNRS-USTL 8576 Uni6ersite´ des Sciences et Technologies de Lille, F-59655,
Villeneu6e d’Ascq, France
Received 23 December 2002; accepted 21 February 2003
Abstract
The specificity of recombinant (2“3)-a-sialyltransferase (ST3Gal-III), expressed in baculovirus-infected insect cells, has been
determined with various oligosaccharide acceptors and sugar-nucleotide donors using a fluorescence based assay. Recombinant
ST3Gal-III tagged with a polyhistidine tail was immobilized on Ni2+-NTA-Agarose as an active enzyme for use in the synthesis
of three sialylated oligosaccharides: (i) the divalent molecule [a-Neu5Ac-(2“3)-
(CH2OBn)2 (12); (ii) the dansylated derivative, a-Neu5Ac-(2“3)- -Galp-(1“3)-b- -GlcpNAc-O-(CH2)6-NH-dansyl and; (iii) the
tetrasacharide a-Neu5Ac-(2“3)-b- -Galp-(1“4)-b- -GlcpNAc-(1“2)-a- -Manp-O-CH3. Compound 12 was itself prepared
D
-Galp-(1“4)-b-D-GlcpNAc-O-CH2]2-C-
D
D
D
D
D
from the divalent N-acetyllactosamine molecule built on pentaerythritol by a chemo-enzymatic route. © 2003 Elsevier Science
Ltd. All rights reserved.
Keywords: Chemo-enzymatic synthesis; Recombinant sialyltransferase; Baculovirus-infected insect cells; Enzyme immobilization
sialyltransferase (ST3Gal-III according to Tsuji’s
nomenclature1), first purified from rat liver,2 was cloned
10 years ago3 and the recombinant enzyme expressed in
insect cells allowed the enzymatic synthesis of sialyl
Lewisx tetrasaccharide with in situ regeneration of
CMP-Neu5Ac.4 Another construct of recombinant
ST3Gal-III was used for the efficient production of
sialyl Lewisx and Lewisa libraries5–7 and saccharopep-
tides.8,9
In this report, the cDNA coding for the rat liver
ST3Gal-III starting at amino acid 34 with a hexahis-
tidine tag added to the N-terminus, was expressed in
baculovirus-infected insect cells. Here we wish to report
on: (i) the specificity of the recombinant ST3Gal-III
based on a novel fluorescent assay; (ii) enzyme immobi-
lization on Nickel charged beads via the His6 tag; (iii)
the use of the immobilized enzyme in the synthesis of
1. Introduction
Sialyltransferases catalyzing the transfer of N-acetyl-
neuraminic acid at the nonreducing end of oligosaccha-
rides with complete regio- and stereoselectivity have
been widely used over the past decade in the synthesis
of bioactive oligosaccharides related to selectin-medi-
ated cell adhesion. These enzymes have become increas-
ing available by genetic engineering, and soluble forms
of glycosyltransferases, originally membrane-bound
proteins, have been generated by removal of the mem-
brane spanning region. A soluble form of CMP-
Neu5Ac:b-
D
-Galp-(1“3/4)-b- -GlcpNAc-(2“3%)-a-
D
* Corresponding author
E-mail address: clauauge@icmo.u-psud.fr (C. Auge´).
0008-6215/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0008-6215(03)00130-7

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T. I6anniko6a et al. / Carbohydrate Research 338 (2003) 1153–1161
Scheme 1. Fluorescent substrates for ST3Gal-III.
new sialylated oligosaccharides belonging to Type 2
(b- -Galp-(1“4)-b- -GlcpNAc) or Type 1 (b- -Galp-
(1“3)-b- -GlcpNAc) series.
sodium carbonate (Scheme 1). In evaluation as sub-
strates, acceptor 1 exhibited the highest activity,
whereas the relative rate with acceptors 2 and 3 were,
64 and 30%, respectively of the rate observed with
acceptor 1 (Table 1). These results are in agreement
with the specificity studies reported both for the
natural10 and recombinant enzyme.11 The cation Mn2+
has no effect on enzyme activity. From 1 L of insect
cells culture, about 5 U of ST3Gal-III could be repro-
ducibly obtained (1 U of enzyme activity is defined as
the amount of enzyme that catalyzes the transfer of 1
D
D
D
D
2. Results and discussion
2.1. Expression of soluble ST3Gal-III in Sf9
Ten recombinant viral clones were used to infect Sf9
cell cultures and the conditioned culture media were
tested for (2“3)-a-sialyltransferase activity 5 days after
mmol of Neu5Ac from CMP-Neu5Ac to b- -Galp-(1“
D
infection using b-
D-Galp-(1“3)-b-D-GlcpNAc-O-
(CH2)7-CH3 as an acceptor substrate. For two indepen-
dent clones (c6703 and c6708), a functional soluble
form of ST3Gal-III (EC 2.4.99.5) was produced based
on enzymatic assay of the culture supernatant and by
anti-His mAb detection (Qiagen, France) (data not
shown). Clone c6703 was chosen for large scale pro-
duction of ST3Gal-III in 400 mL roller bottles. The
medium was collected and the recombinant His6-tagged
ST3Gal-III was visualized by Western blotting as a
single 60 kDa band using anti-His mAb that is in
agreement with the theoretical molecular weight of the
His6-tagged ST3Gal-III of 39 kDa (data not shown).
Table 1
Activity of recombinant ST3Gal-III expressed in baculovirus-
infected insect cells on various oligosaccharide acceptors and
sugar-nucleotide donors, both tested at 0.8 mM concentration
Donor
Acceptor
Relative
velocities a
CMP-Neu5Ac
CMP-Neu5Ac
CMP-Neu5Ac
CMP-Neu5Gc
CMP-KDN
b-
-GlcNAcp-OR b (1)
b- -Galp-(1“4)-b-
-GlcNAcp-OR (4)
b- -Galp-(1“4)-b-
-Glcp-OR (6)
b- -Galp-(1“3)-b-
-GlcNAcp-OR (1)
b- -Galp-(1“3)-b-
-GlcNAcp-OR (1)
D
-Galp-(1“3)-b-
100
64
30
28
D
D
D
D
D
2.2. Substrate specificity of ST3Gal-III
D
D
As an alternative to radioactive assays routinely used
for glycosyltransferases, a novel fluorescent assay was
developed. Novel disaccharide derivatives 1, 2, 3, bear-
ing an aminohexyl chain substituted by a dansyl group
at the anomeric center were prepared, from glycosides
4, 5, 6, respectively by reaction with dansyl chloride in
D
26
D
a Relative velocities with acceptor and donor substrates
tested at 0.8 mM.
b R=(CH2)6-NH-dansyl.

T. I6anniko6a et al. / Carbohydrate Research 338 (2003) 1153–1161
1155
Table 2
Immobilization of His-tagged recombinant ST3Gal-III on Ni2+-NTA-Agarose
Initial soluble
Vol of culture
supernatant
(mL)
Vol of
Gel-bound
enzyme activity
(mU)
Remaining soluble
enzyme activity
(mU)
Immobilization
yield c (%)
enzyme activity
Ni2+-NTA-Agarose
(mL)
(mU mL−1
)
6 a
5
2
15
6
1
1
6
2
2
1
38
66
44
58
22.5
18.5
66
12
32
B5
59
44
54
40
32 a
10 a
13.5 b
29 b
5
a Culture supernatant free of fetal calf serum.
b Culture supernatant containing 5% fetal calf serum.
c Immobilization yield is expressed as the ratio of immobilized activity to that initially present in solution.
3)-b-
D
-GlcpNAc-O-(CH2)6-NH-dansyl). The fluores-
probably due to the loss of the His6 tag owing to
enzyme proteolysis in the course of concentration and
immobilization steps. No significant difference in im-
mobilization yields was observed between culture media
with or without fetal calf serum. The enzymatic activity
of the gel stored at 4 °C was stable for at least 5
months, whereas crude culture supernatants were inac-
tivated in 2 weeks of storage at 4 °C. By avoiding the
lengthy procedures of enzyme purification, immobiliza-
tion on Ni2+-Agarose appears to be an ideal method
for general use of recombinant glycosyltransferases in
synthesis. In addition to the earliest report on immobi-
lization of recombinant mannosyltransferase,14 there is
a recent example describing the use of immobilized
enzymes for the efficient production of UDP-Gal from
UDP.15
cent assay was used to measure enzyme activity with
CMP-sialic acids other than CMP-Neu5Ac, the donor
commonly used in sialyltransferase-catalyzed reactions,
and the only radiolabeled CMP-donor that is commer-
cially available. Both donors CMP-Neu5Gc and CMP-
KDN were recognized by ST3Gal-III but with a lower
rate of transfer, 28 and 26%, respectively, as compared
to CMP-Neu5Ac (Table 1).
2.3. Enzyme immobilization
Recombinant ST3Gal-III was tagged with a polyhis-
tidine tail. A stretch of His6 is now commonly added to
the primary sequence of recombinant proteins in order
to facilitate their purification by Ni2+ affinity chro-
matography, based on strong interactions between
Ni2+ immobilized on Agarose through nitriloacetic
acid (NTA) and the polyhistidine tag.12 We previously
reported that the His6 tag of recombinant FucT-III
could be exploited for enzyme immobilization and that
FucT-III immobilized onto nickel-nitriloacetate beads
exhibited enhanced stability compared to the soluble
enzyme.13 This result prompted us to examine immobi-
lization for ST3Gal-III secreted in the culture superna-
tant of baculovirus-infected insect cells. Prior to the
binding step, the culture media must be ultrafiltrated to
adjust the pH to 8 and to remove any interfering
components. Then the His6 tagged ST3Gal-III was
immobilized on Ni2+-NTA-Agarose under native con-
ditions at 4 °C using a batch procedure. In order to
optimise the immobilization yield, several parameters
were tested (Table 2). For optimal immobilization, the
culture supernatant must be concentrated with a ratio
of the volume of the soluble enzyme to that of the gel
of :2/1. ST3Gal-III was immobilized, in an active
form, in 40–59% yield depending on the experiments.
The soluble enzymatic activity remaining after the im-
mobilization step could not be re-immobilized when
again treated with a new batch of nickel-Agarose,
2.4. Sialylation with immobilized ST3Gal-III
The immobilized enzyme preparation was first used to
sialylate compound 10, comprised of two N-acetyllac-
tosamine residues built on the pentaerythritol molecule
according to previous work on the preparation of diva-
lent selectin ligands.16 Compound 10 was prepared by a
chemo-enzymatic route. First, condensation of the chlo-
ride 7 with the dibenzyl ether of pentaerythritol 817 in
toluene–nitromethane at 45 °C in the presence of mer-
curic cyanide as the promotor, afforded after deacetyla-
tion the starting divalent molecule 9 in 68% yield
(Scheme 2). Then, enzymatic galactosylation was
achieved with b-(1“4)-galactosyltransferase (EC
2.4.1.22), together with UDP-glucose, UDP-glucose
epimerase, manganese chloride and alkaline phos-
phatase, leading to the di-galactosylated derivative 10
in 65% isolated yield, in addition to the mono-galacto-
sylated derivative 11 obtained in 24% yield. Compound
10 was sialylated by incubation with recombinant
ST3Gal-III adsorbed on Ni2+-Agarose in 25 mM
sodium cacodylate buffer pH 7.1 and CMP-Neu5Ac
added in three portions during the 40-h incubation time
(Scheme 3). Unlike CDP and CTP, CMP released in the

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T. I6anniko6a et al. / Carbohydrate Research 338 (2003) 1153–1161
reaction was not a strong inhibitor, therefore the addi-
tion of alkaline phosphatase was unnecessary. The bis-
sialylated lactosamine derivative 12 was obtained in
73% yield by methanol elution from C18 Sep-Pak car-
tridges and gel permeation on Biogel P-2. This deriva-
tive has been further used for the synthesis of a divalent
sialyl Lewisx molecule, required as a reference com-
pound in the study of low entropy ligands.18 At the end
of incubation, about 75% of the enzymatic activity still
remained on the gel. The same immobilized enzyme
preparation could then be used for sialylation of the
dansylated disaccharide 1. This substrate was incubated
like compound 10 with CMP-Neu5Ac and immobilized
ST3Gal-III, and gave trisaccharide 13 in 65% isolated
yield. This trisaccharide may serve as a substrate for
testing endogenous sialidase activity present in culture
of Chinese hamster ovary (CHO) cells.19 The prepara-
tion of immobilized sialyltransferase was also utilized
3. Experimental
3.1. General
NMR spectra were recorded with a Bruker AC-200 or
AC-250 spectrometer; chemical shifts are given relative
to the signal of tetramethylsilane in CDCl3; for H and
1
13C NMR spectra in D2O, acetone (l 2.22 and 30.5
ppm) was used as an internal reference. Optical rota-
tions were measured with a JASCO digital micropolar-
imeter. Mass spectra were performed either on a
Finigan MATT 95 apparatus using the ESI technique,
or on a VOYAGER DE STR Pro instrument (Persep-
tive Biosystem) using MALDI-TOF-MS with 2,5-dihy-
droxybenzoic acid as the matrix. Reactions were
monitored by TLC on Silica Gel 60F254 with detection
by charring with 10% H2SO4 in EtOH or 2% orcinol in
10% H2SO4. C18 Sep-Pak cartridges were from Mil-
lipore-Waters. Fluorescence measurements were per-
for sialylation of trisaccharide b-
D
-Galp-(1“4)-b-D-
GlcpNAc-(1“2)-a-
D
-Manp-O-CH3 (14), previously
formed with a Perkin–Elmer LS50B fluorimeter,
synthesized by a chemo-enzymatic method.20 The sialy-
controlled by a PC and equipped with a plate reader
allowing TLC plates scanning. Fluorescence was read
at 385 nM excitation/540 nM emission. CMP-Neu5Ac
was purchased from Kyowa Hakko; CMP-Neu5Gc and
CMP-KDN were synthesized according to Lubineau
lated tetrasaccharide 15, together with its regio-isomer
a-Neu5Ac-(2“6)-b-
D
-Galp-(1“4)-b-D-GlcpNAc-
(1“2)-a-
D
-Manp-O-CH320, are expected to be useful
substrates to define the specificity of new (2“8)-a-sia-
lyltransferases.
and co-workers21,22 Bovine milk
D
-GlcNAc-b-(1“4)-
Scheme 2. Reagents and conditions: (a) HgCN2, CaSO4, 1:1 PhCH3–CH3NO2, 45 °C, 16 h; (b) 8:1:1 MeOH–water–Et3N, rt, 16
h, 68% for two steps; (c) GalT, UDPGE, UDP-Glc 2 equiv, AP, 15 mM MnCl2, 25 mM sodium cacodylate buffer pH 7.4, 37 °C,
3 days, 65%.

T. I6anniko6a et al. / Carbohydrate Research 338 (2003) 1153–1161
1157
Scheme 3. Reagents and conditions: (a) ST3Gal-III immobilized on Ni2+-NTA-Agarose, CMP-Neu5Ac 1.7 equiv, 25 mM sodium
cacodylate buffer pH 7.1, 37 °C, 4 days.
galactosyltransferase and UDP-glucose 4-epimerase
were from Calbiochem, UDP-Glc was from Sigma,
protease inhibitor cocktail and calf intestine alkaline
phosphatase from Roche Molecular Biochemicals.
peptide sequence of the EGT gene was obtained after
the annealing of the following two synthetic oligonucle-
otides: EGT 5%-GA-TCC-GCC-ACC-ATG-ACC-ATC-
TTA-TGT-TGG-CTC-GCT-CTC-CTG-AGC-ACA-CT
C-ACA-GCT-GTT-AAC-GCT-GAC-ATC-A-3%, and
Back EGT 5%-GA-TCT-GAT-GTC-AGC-GTT-AAC-
AGC-TGT-GAG-TGT-GCT-CAG-GAG-AGC-GAG-
CCA-ACA-TAA-GAT-GGT-CAT-GGT-GGC-G-3% to
generate pUC-PS-EGT. A 108 bp DNA fragment con-
taining an HpaI site, the last codon of the EGT signal
peptide sequence, 6 histidine codons and 19 codons
corresponding to aa 34–52 of the N-terminal domain
of the rat ST3Gal-III was reconstituted using a
3.2. Construction of a recombinant baculovirus and ex-
pression of a soluble ST3Gal III in Sf9
To express a soluble and His6-tagged form of the rat
ST3Gal-III, the N-terminal sequence (aa 9–33), includ-
ing the transmembrane domain, was eliminated and
replaced by the signal peptide sequence of the viral
EGT gene. 23A 78 bp fragment encoding the signal

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T. I6anniko6a et al. / Carbohydrate Research 338 (2003) 1153–1161
set of nine overlapping synthetic oligonucleotides. This
DNA fragment was subcloned and inserted into the
HpaI–SacI site of pUC-PS-EGT.23 In order to avoid
homologous recombination between virus and vector
encoded EGT, codons were made degenerate. The re-
sulting construct was digested with A6rII–MunI to
receive the 1900 bp A6rII–EcoRI fragment prepared
from pBS-SK-ST3Gal-III (clone ST3N-13). The full-
length modified rat ST3Gal-III gene was then excised
by digestion with BamHI and HindIII and inserted into
the BglII–HindIII site of the p119 transfer vector24
designed for recombination into the p10 locus of the
baculovirus, giving p119-PS-ST3Gal-III. Sf9 Cells
(ATCC CRL1711) were cotransfected by lipofection25
using DOTAP (Roche Applied Science, Germany) with
p119-PS-ST3Gal-III and purified viral DNA. The
ST3Gal-III recombinant baculoviruses were purified by
plaque assay and viral clones were tested in vitro by
assaying for a a-(2“3)-sialyltransferase activity using 2
added, and the solution was ultrafiltered 10-fold. The
concentrated solution was diluted in 9 vol of 20 mM
Tris pH 8 containing 0.2 M NaCl, 5 mM imidazole, 1
mM mercaptoethanol, 0.01% NaN3, and again ultrafil-
tered. The resulting solution was incubated with Ni2+
-
NTA-Agarose (Qiagen, France) at 4 °C on a rotary
shaker overnight. Then the gel was centrifuged at 3000
rpm for 10 min, the supernatant was removed and the
gel washed with 0.25 mM sodium cacodylate buffer pH
6.6 containing 0.01% NaN3 and again centrifuged. The
gel was resuspended in the same buffer (5 mL); the
enzymatic activity was measured in the supernatant and
on aliquots of the gel suspension.
3.5. General procedure for dansylation
The 6-aminohexyl b-D
-glycoside27 (0.5 mmol) was dis-
solved in water (32 mL) containing sodium carbonate
(0.11 g, 1.03 mmol). A solution of dansyl chloride (148
mg, 0.55 mmol) in acetone was added dropwise and the
mixture was stirred at room temperature (rt) for 1 h.
Solvents were then evaporated and the residual mixture
was purified by HPLC on a Waters 600 equipped with
a preparative reversed-phase column (Nucleosil C-18)
using elution with a solvent gradient of water–MeOH.
mM b-D-Galp-(1“3)-b-D-GlcpNAc-O-(CH2)7-CH3 as
acceptor and 50 mM CMP-[14C]-Neu5Ac (1.85 KBq) as
donor.26 Large scale production of ST3Gal-III was
performed in Sf9 cells (growing temperature 28 °C),
infected with the recombinant baculovirus clone 6703 at
a multiplicity of infection of five PFU/cell. This produc-
tion was undertaken under two different conditions.
The first was done in a 400 mL roller bottle using
EXTRA-1X medium (Eurobio, France) supplemented
with 5% heat-inactivated fetal calf serum (Life Tech-
nologies, France). The second one was performed in
4× 400 mL roller bottles using only serum-free
medium EXTRA-SB5 (Eurobio, France).
3.6. 6-(Dansyl)-aminohexyl O-b-
(1“3)-(2-acetamido-2-deoxy-b-
D
-galactopyranosyl-
D-glucopyranoside) (1)
Disaccharide 4 was treated as described above afford-
ing disaccharide 1 (0.22 g, 64%); [h]2D9 −19° (c 0.98,
MeOH);1H NMR (250 MHz, CD3OD): l 8.55 (d, J 7
Hz 1 H, dansyl), 8.30 (d, 1 H, dansyl), 8.19 (d, 1 H,
dansyl), 7.62 (d, 1 H, dansyl), 7.55 (d, 1 H, dansyl), 7.25
(d, 1 H, dansyl), 4.45 (d, 1 H, J1,2 7 Hz, H-1), 4.25 (d,
1 H, J1%,2% 6 Hz, H-1%), 2.89 (s, 6 H, N(CH3)2), 1.92 (s, 3
H, NAc) and 1.35–0.90 (m, 8 H, 4 CH2); 13C NMR
(62.9 MHz, CD3OD) l: 173.9 (CO), 153.0, 137.0, 131.0,
130.0, 128.9, 124.1, 120.4, 116.3 (CꢀAr), 105.4 (C-1%),
102.1 (C-1), 84.8 (C-3) 77.3, 76.9, 74.5, 72.2, 70.4, 70.3,
70.0 (C-5, C-5%, C-4%, C-4, C-3%, C-2%, OCH2), 62.6, 62.3
(C-6, C-6%), 56.2 (C-2), 45.8 (N(CH3)2), 43.7 (NCH2),
30.4, 30.1, 27.0, 26.3 (CH2), 23.1 (CH3CO); HRMS
Calcd for C32H49N3NaO13S [M+Na]+: 738.2883.
Found: m/z 738.2882.
3.3. Enzymatic assay
Enzyme assay with fluorescent acceptors: 50 mM
sodium cacodylate buffer pH 6.6, 5 mg bovine serum
albumin, 0.8 mM CMP-Neu5Ac (or CMP-Neu5Gc or
CMP-KDN when other donors were tested), 0.8 mM
fluorescent disaccharide acceptors. The mixture was
incubated at 37 °C under stirring for 15–60 min with
10–20 mL of 10-fold concentrated culture supernatant
or a Nickel bead suspension containing ST3Gal-III.
Incubation mixtures were analyzed by TLC on silica gel
(3:3:2 EtOAc–2-propanol–water) and the percentage
of conversion was evaluated from the fluorescence in-
tensity of the spots referring to substrate and product,
which was directly quantified on the plate.
3.7. 6-(Dansyl)-aminohexyl O-b-
D
-galactopyranosyl-
(1“4)-(2-acetamido-2-deoxy-b-
D
-glucopyranoside) (2)
3.4. Immobilization of ST3Gal-III
Disaccharide 5 was treated as described above afford-
ing disaccharide 2 (0.148 g, 43%); [h]2D9 −11° (c 1.01,
MeOH); H NMR (200 MHz, CD3OD): l 8.56 (d, J 7
Hz 1 H, dansyl), 8.35 (d, 1 H, dansyl), 8.19 (d, 1 H,
dansyl), 7.61 (d, 1 H, dansyl), 7.54 (d, 1 H, dansyl), 7.25
(d, 1 H, dansyl), 4.38 (d, 1 H, J1,2 7.7 Hz, H-1), 4.36 (d,
1 H, J1%,2% 8 Hz, H-1%), 2.90 (s, 6 H, N(CH3)2), 2.84 (t, 2
1
To 10 vol of culture medium containing the His6-tagged
soluble form of ST3Gal-III from baculovirus-infected
insect cells, 1 vol of 0.2 M Tris pH 8 containing 2 M
NaCl, 50 mM imidazole, 10 mM mercaptoethanol,
0.1% NaN3 and a protease inhibitor cocktail was

T. I6anniko6a et al. / Carbohydrate Research 338 (2003) 1153–1161
1159
H, NCH2),1.92 (s, 3 H, NAc) and 1.5–1.1 (m, 8 H, 4
CH2); 13C NMR (50.2 MHz, CD3OD) l: 173.2
(CO),152.97, 136.9, 131.0, 130.0, 128.9, 124.1, 120.4,
116.3 (C-Ar), 104.9 (C-1%), 102.5 (C-1), 81.0 (C-4) 76.8,
76.2, 73.9, 72.3, 70.2, 70.1 (C-5, C-5%, C-4%, C-3, C-3%,
C-2%, OCH2), 62.3, 61.9 (C-6, C-6%), 56.5 (C-2), 45.7
(N(CH3)2), 43.6 (NCH2), 30.3, 30.1, 26.9, 26.2 (CH2))
and 22.9 (CH3CO); HRMS Calcd for C32H49N3
NaO13S [M+Na]+: 738.2883. Found: m/z 738.2882.
2 H-4), 3.24–3.07 (m, 4 H, J1,2 8.3 Hz, 2 H-2, 2 H-5)
and 1.8 (s, 6 H, 2 OAc); 13C NMR (62.9 MHz,
CD3OD): l 173.5 (CO), 140.1, 129.3, 128.5 (ArꢀC),
103.5 (C-1), 77.9, 75.7, 72.1 (C-3, C-4 and C-5), 74.5
(CH2ꢀPh), 70.3, 69.8 (CꢀCH2), 62.7 (C-6), 57.4 (C-2),
46.5 (C(CH2)4) and 23.3 (CH3CO); ES+MS m/z 745.5
[M+Na]+; Anal. Calcd for C35H50N2O14·0.5 H2O: C,
57.44; H, 7.02; N, 3.83. Found: C, 57.76; H, 7.32; N,
3.51.
3.8. 6-(Dansyl)-aminohexyl O-b-
(1“4)-b- -glucopyranoside (3)
D
-galactopyranosyl-
3.10. 2,2-Bis(benzyloxymethyl)-1,3-bis[O-b-
ranosyl-(1“4)-(2-acetamido-2-deoxy-b- -glucopyran-
osyl-oxy)]-propane (10) and 2,2-bis(benzyloxymethyl)-1-
(2-acetamido-2-deoxy-b- -glucopyranosyl-oxy)-3-[O-b-
-galactopyranosyl-(1“4)-(2-acetamido-2-deoxy-b-
glucopyranosyl-oxy)]-propane (11)
D-galactopy-
D
D
Disaccharide 6 was treated as described above afford-
ing disaccharide 3 (0.141 g, 42%); [h]2D9 −10° (c 0.99,
D
D
D-
1
MeOH); H NMR (250 MHz, CDCl3–CD3OD): l 8.54
(d, 1 H, J 7 Hz, dansyl), 8.32 (d, 1 H, dansyl), 8.21 (d,
1 H, dansyl), 7.60 (d, 1 H, dansyl), 7.53 (d, 1 H,
dansyl), 7.22 (d, 2 H, dansyl), 4.37 (d, 1 H, J1%,2% 7.7 Hz,
H-1%), 4.26 (d, 1 H, J1,2 8 Hz, H-1), 2.90 (s, 6 H,
N(CH3)2), 2.86 (t, 2 H, J 7 Hz, NCH2) and 1.54–1.15
(m, 8 H, 4 CH2); 13C NMR (62.9 MHz, CD3OD): l
153.1, 137.2, 131.0, 130.1, 129.0, 124.3, 120.6, 116.4
(CꢀAr), 105.0 (C-1%), 104.1 (C-1), 80.7 (C-4) 77.0, 76.4,
74.7, 72.5, 70.6, 70.3 (C-5, C-5%, C-4%, C-3, C-3%, C-2,
C-2%, OCH2), 62.5, 62.0 (C-6, C-6%), 45.8 (N(CH3)2),
43.7 (NCH2) and 30.4, 27.2, 26.3 (CH2); HRMS Calcd
for C30H46N2NaO13S [M+Na]+: 697.2618. Found: m/z
697.2619.
Compound 9 (50 mg, 0.07 mmol), UDP-Glc (45 mg,
0.07 mmol), bovine milk D-GlcNAc b-(1“4)-galacto-
syltransferase (0.3 U) and UDP-glucose-4-epimerase
(1.5 U), were incubated at 37 °C in 25 mM sodium
cacodylate buffer pH 7.4 (5 mL) containing 15 mM
MnCl2. The reaction was monitored by TLC on silica
gel (3:3:1 EtOAc–2-propanol–water). After 48 h UDP-
Glc (45 mg, 0.07 mmol), bovine milk -GlcNAc b-(1“
D
4)-galactosyltransferase (0.2 U), UDP-glucose-4-
epimerase (1 U) and MnCl2 (10 mg, 0.05 mmol) were
added again together with alkaline phosphatase (2 U)
and incubation was continued for 24 h. The reaction
mixture was divided into four portions and applied to
Sep-Pak C18 cartridges; the combined MeOH eluates
were evaporated to dryness and the residue was purified
by flash chromatography on silica gel (6:4:1 EtOAc–2-
propanol–water) to give the di-galactosylated com-
pound 10 (45 mg, 65%) and the mono-galactosylated
compound 11 (15 mg, 24%).
3.9. 2,2-Bis-(benzyloxymethyl)-1,3-bis(2-acetamido-2-de-
oxy-b-D-glucopyranosyl-oxy)-propane (9)
To a mixture of diol 8 (0.438 mg, 1.39 mmol), chloride
7 (1.52 g, 4.16 mmol), calcium sulfate (0.566 g, 4.16
mmol) suspended in 1:1 toluene–nitromethane (10 mL)
was added mercuric cyanide (1.05 g, 4.16 mmol). The
mixture was stirred for 16 h at 45 °C under Ag, then
solvents were evaporated and the residue dissolved in
CH2Cl2 and filtered through Celite. The filtrate was
washed with saturated aq NaHCO3, water, 20% aq
potassium iodide and water again, dried (MgSO4) and
concentrated. Flash chromatography (1:3 petroleum
ether-EtOAc) of the residue afforded the diglycosyla-
tion product slightly contaminated by the monoglyco-
sylation product. This mixture (1.04 g), dissolved in
8:1:1 MeOH–water–Et3N (100 mL), was stirred
overnight at rt, then concentrated to dryness and co-
evaporated several times with toluene. The residue,
chromatographed on silica gel (4:1 EtOAc–MeOH),
afforded 9 (0.683 g, 68%); [h]2D7 −12.6° (c 1, CH3OH);
1H NMR (250 MHz, CD3OD): l 7.30–7.10 (m, 10 H,
Ph), 4.31 (s, 4 H, 2 PhCH2), 4.19 (d, 2 H, J1,2 8.3 Hz,
2 H-1), 3.84 (d, 2 H, Jgem 9.8 Hz, 2 CH), 3.72 (d, 2 H,
J6,6% 11.7 Hz, 2 H-6), 3.6–3.45 (m, J6,6% 11.7, J6%,5 4.9 Hz,
4 H, 2 H-6%, 2 CH), 3.40–3.27 (m, 8 H, 2 CH2, 2 H-3,
1
Compound 10: [h]2D7 −30° (c 0.5, water); H NMR
(200 MHz, D2O): l 7.5–7.3 (m, 10 H, Ph), 4.45 (m, 6
H, J1%,2% 8.8 Hz, 2 H-1%, 2 CH2Ph), 4.34 (d, 2 H, J1,2 7.8
Hz, 2 H-1) and 1.95 (s, 6 H, 2 OAc); 13C NMR (50.3
MHz, D2O): l 174.9 (CO), 138.7, 129.7, 129.1 (ArꢀC),
103.9 (C-1%), 102.8 (C-1), 79.6, 76.3, 75.7, 74.3, 73.5,
73.2, 69.6 (C-2%, C-3%, C-4%, C-5%, C-3, C-4 and C-5),
74.4 (PhCH2), 69.5 (CH2), 62.0, 61.1 (C-6, C-6%), 56.1
(C-2), 45.7 (C(CH2)4 and 23.2 (CH3CO); HRMS: Calcd
for C47H70N2NaO24 [M+Na]+: 1069.4216. Found: m/z
1069.4239.
1
Compound 11: [h]2D7 −29° (c 0.5, MeOH); H NMR
(200 MHz, D2O): l 7.5–7.3 (m, 10 H, Ph), 4.50–4.41
(m, 5 H, J1%,2% 8.8 Hz, H-1%, 2 CH2Ph), 4.34 (d, 2 H, J1,2
7.8 Hz, 2 H-1) and 1.95 (s, 6 H, 2 OAc); 13C NMR
(50.3 MHz, D2O): l 173.3 (CO), 140.1, 129.3, 128.6
(ArꢀC), 105.1 (C-1%), 103.5 (C-1), 81.1, 78.0, 77.1, 76.5,
75.8, 74.9, 73.9, 72.6, 72.2, 70.4 (C-2%, C-3%, C-4%, C-5%, 2
C-3, 2 C-4, 2 C-5), 74.5 (PhCH2), 70.3, 68.8, 68.7
(CH2), 62.8, 62.5, 61.9 (2 C-6, C-6%), 57.4, 56.8 (2 C-2),

1160
T. I6anniko6a et al. / Carbohydrate Research 338 (2003) 1153–1161
46.6 (C(CH2)4 and 23.2 (CH3CO); HRMS Calcd for
C41H60N2NaO19 [M+Na]+: 907.3688. Found: m/z
907.3684.
purified by chromatography on DEAE-Sephadex A-25
(HCO−3 form); elution with a gradient of 0–0.4 M
triethylammonium hydrogen carbonate (pH 8.0) gave
13 as its triethylammonium salt (14 mg, 65%); [h]D27
1
3.11. 2,2-Bis(benzyloxymethyl)-1,3-bis-[5-acetamido-3,5-
−19° (c 0.44, H2O); H NMR (250 MHz, D2O) l: 8.45
dideoxy-a-
(2“3)-O-b-
deoxy-b- -glucopyranosyloxy]-propane (12)
D
-glycero-
D
-galacto-2-nonulopyranosylonate-
(d, 1 H, J 7 Hz, dansyl), 8.29 (d, 1 H, dansyl), 8.21 (d,
1 H, dansyl), 7.68 (d, 1 H, dansyl), 7.61 (d, 1 H,
dansyl), 7.39 (d, 1 H, dansyl), 4.47 ( d, 1 H, J1%,2% 7.8 Hz,
H-1%), 4.37 (d, 1 H, broad, H-1), 4.09 (dd, 1 H, J3%,4% 3,
J3%,2% 10 Hz, H-3%), 3.26 (q, 6 H, 3 CH2CH3), 2.84 (m, 10
H, 2 NCH3, OCH2, NCH2), 2.76 (dd, 1 H, J3¦e,3¦a 12.5,
J3¦e,4¦ 4 Hz, H-3¦e), 2.02 (s, 3 H, NAc), 1.92 (s, 3 H,
NAc), 1.79 (t, 1 H, H-3¦a), 1.24 (t, 9 H, 3 CH2CH3) and
1.15–0.7 (m, 8 H, 4 CH2); 13C NMR (62.9 MHz, D2O)
l: 177.4, 176.5, 176.3 (CO), 156.0, 153.1, 136.5, 132.4,
132.04, 131.3, 131.1, 118.4 (CꢀAr), 105.8 (C-1%), 103.1
(C-1), 102.0 (C-2¦), 84.9 (C-3), 78.0, 77.7, 77.4 (C-5,
C-5%, C-3%), 75.2 (C-6¦), 74.2 (C-8¦), 72.4, 71.45, 71.1,
70.7, 70.4, 69.6 (C-2%, C-4, C-4%, C-4¦, C-7¦, CH2), 64.8
(C-9¦), 63.4, 63.10 (C-6, C-6%), 61.3 (CH2), 56.8 (C-2),
54.1 (C-5¦), 47.3 (N(CH2CH3)3), 44.7 (N(CH3)), 42.1
(C-3¦, N(CH2)), 30.5, 27.3, 26.5 (CH2), 24.6, 24.4
(CH3CO) and 9.8 (N(CH2CH3)3); MALDI-HRMS
Calcd for C43H65N4Na2O21S [M+2 Na−H]+:
1051.366. Found: m/z 1051.365.
D-galactopyranosyl-(1“4)-2-acetamido-2-
D
Compound 10 (20 mg, 0.019 mmol), CMP-NeuAc (12.6
mg, 0.02 mmol) and ST3Gal-III adsorbed on Ni2+
-
Agarose (45 mU) were incubated in 25 mM sodium
cacodylate buffer pH 7.1 (6 mL) at 30 °C for 4 days
with gentle stirring. Additional CMP-NeuAc (15.1 mg,
0.022 mmol) was added twice, after 24 and 48 h. At the
end of incubation the gel was filtered off, washed with
10 mM sodium cacodylate buffer pH 7.1 and filtrate
and washings were combined, divided into two portions
and applied to Sep-Pak C18 cartridges; the combined
methanol eluates were evaporated to dryness; then the
residue dissolved in water was purified on Bio-Gel P2
and passed through a small column of AG 50W-X8
ion-exchange resin (Na+ form) to give 12 as its sodium
1
salt (23 mg, 73%); [h]2D7 −18° (c 1, water); H NMR
(D2O, 250 MHz): l 7.5–7.3 (m, 10 H, Ph), 4.5 (d, 2 H,
J1%,2% 7.8 Hz, 2 H-1%), 4.46 (s, 4 H, 2 CH2Ph), 4.32 (d, 2
H, J1,2 7.3 Hz, 2 H-1), 4.08 (dd, 2 H, J3%,4% 3, J3%,2% 8.8
Hz, 2 H-3%), 2.72 (dd, 2 H, J3¦e,3¦a 11.7, J3¦e,4¦ 4 Hz, 2
H-3¦e), 2.02 (s, 6 H, 2 OAc), 1.90 (s, 6H, 2 OAc) and
1.75 (t, 2 H, J3¦a,3¦e 11.7 Hz, 2 H-3¦a); 13C NMR (D2O,
62.9 MHz): l 175.9 (CO2Na), 175.0, 174.9 (CO), 138.6,
129.7, 129.2 (Ph), 103.6 (C-1%), 102.9 (C-1), 100.8 (C-
2¦), 79.4 (C-4), 76.5, 76.1, 75.6 (C-5, C-5%, C-3%), 74.4,
73.8, 73.1, 72.7 (PhCH2, C-3, C-6¦, C-8¦), 70.7, 69.7,
69.3, 69.1, 68.5 (C-2%, C-4%, C-4¦, C-7¦, CH2), 63.6
(C-9¦), 62.0, 61.0 (C-6, C-6%), 56.1 (C-2), 52.7 (C-5¦),
45.7 (Cq), 40.6 (C-3¦), 23.2 and 23.0 (CH3CO); HRMS
(negative mode): Calcd for C69H102N4O40·Na2 [(M−2
Na+)/2]− 813.3035. Found: m/z 813.3031.
3.13. Methyl (5-acetamido-3,5-dideoxy-a-
galacto-2-nonulopyranosylonate)-(2“3)-(O-b-
pyranosyl)-(1“4)-O-(2-acetamido-2-deoxy-b-
pyranosyl)-(1“2)-a- -mannopyranoside (15)
D
-glycero-
-galacto-
-gluco-
D-
D
D
D
Trisaccharide 14 (8.5 mg, 0.015 mol) and CMP-NeuAc
(6.9 mg, 0.011 mmol) were incubated in 25 mM sodium
cacodylate buffer pH 7.1 (5 mL) at 30 °C for 4 days
with gentle stirring in the presence of ST3Gal-III ad-
sorbed on Ni2+-Agarose (25 mU). Additional CMP-
NeuAc (6.9 mg) was added twice, after 24 and 48 h. At
the end of incubation the gel was filtered off, washed
with 10 mM sodium cacodylate buffer pH 7.1 and
filtrate and washings were evaporated to dryness; then
the residue taken up in water was purified on Bio-Gel
P2 and passed through a small column of AG 50W-X8
ion exchange resin (Na+ form) to give 15 as its sodium
3.12. 6-(Dansyl)-aminohexyl (5-acetamido-3,5-dideoxy-
a-
3)-O-b-
deoxy-b-
D
-glycero-
-galactopyranosyl-(1“3)-2-acetamido-2-
-glucopyranoside (13)
D-galacto-2-nonulopyranosylonic acid)-(2“
D
1
D
salt (9 mg, 69%); [h]D27 +0.5° (c 0.3, water); H NMR
(D2O, 250 MHz): l 4.88 (d, 1 H, J1,2 1.5 Hz, H-1), 4.75
(d, 1 H, J1¦,2¦ 8.5 Hz), 4.54 (d, 1 H, J1%,2% 8 Hz, H-1%),
4.11 (dd, J3¦,4¦ 3, J3¦,2¦ 10 Hz, H-3¦), 3.40 (s, 3 H, CH3),
2.75 (dd, 1 H, J3§e,3§a 12.5, J3¦e,4¦ 4 Hz, H-3§e), 2.05 (s,
3 H, NAc), 2.03 (s, 3 H, NAc) and 1.80 (t, 1 H, H-3§a);
13C RMN (D2O, 62.9 MHz): l 175.3 (CO2Na), 175.0,
174.2 (CO), 102.9 (C-1¦), 100.1 (C-2§), 99.7 ( C-1%), 98.2
(C-1), 78.5 (C-4%), 76.4, 75.7, 75.4, 75.0 (C-2, C-3¦, C-5%,
C-5¦), 73.2, 72.9, 72.2, 72.0 (C-3%, C-5, C-6§, C-8§),
69.8, 69.7, 68.7, 68.4, 67.7, 67.7 (C-4, C-4%, C-2¦, C-4¦,
C-4§, C-7§), 62.8 (C-9§), 61.9, 61.3, 60.2 (C-6, C-6%,
C-6¦, 55.1 (C-2%, OCH3), 51.9 (C-5§), 39.9 (C-3§), 22.6
The dansylated disaccharide 1 (14 mg, 0.0195 mmol)
and CMP-NeuAc (6.9 mg, 0.011 mmol) were incubated
in 25 mM sodium cacodylate buffer pH 7.1 (5 mL) at
30 °C for 4 days with gentle stirring in the presence of
ST3Gal-III adsorbed on Ni2+-Agarose (35 mU). Addi-
tional CMP-NeuAc (6.9 mg) was added twice, after 24
and 48 h. At the end of incubation, the gel was filtered
off, washed with 10 mM sodium cacodylate buffer pH
7.1 and filtrate and washings were applied onto a
Sep-Pak C18 cartridge; the methanol eluate was evapo-
rated to dryness and the residue dissolved in water was

T. I6anniko6a et al. / Carbohydrate Research 338 (2003) 1153–1161
1161
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and 22.3 (CH3CO); MALDI-HRMS: Calcd for
C32H53N2Na2O24 [M+2 Na−H]+: 895.278. Found:
m/z 895.279.
Acknowledgements
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This work has been performed in the frame of the
French GTrec Network, supported by MENRT (ACC
SV no. 951411) and CNRS (Programme Interdisci-
plinaire Physique et Chimie du Vivant and Programme
Interdisciplinaire en Ge´nie des Proce´de´s). Since January
2003 this network has been going on with GDR 2590
supported by CNRS. We thank Willy Morelle for
performing MALDI-TOF MS analyses.
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