New derivatives of reducing oligosaccharides and their use in enzymatic reactions: Efficient synthesis of sialyl Lewis a and sialyl dimeric Lewis x glycoconjugates

Article abstract of DOI:10.1016/S0008-6215(00)00128-2

The reducing oligosaccharides lactose and lacto-N-tetraose were reductively aminated with benzyloxycarbonylaminoaniline and sodium cyanoborohydride, followed by treatment of the resulting secondary amines with acetic anhydride. The resulting N-acetyl-N-(4-benzyloxycarbonylaminophenyl)-1-amino-1-deoxyalditol oligosaccharide derivatives were subjected to stepwise enzymatic elongation with various glycosyltransferases/nucleotide sugars. Purification of the products after each enzymatic step was conveniently performed by solid-phase extraction on silica gel C-18 cartridges. Two oligosaccharide derivatives (with sialyl Lewis a and sialyl dimeric Lewis x structures) were prepared. Conversion of the obtained derivatives into neoglycoproteins by the sequence hydrogenolysis, thiophosgene treatment, and protein coupling was carried out. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0008-6215(00)00128-2

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Carbohydrate Research 328 (2000) 525–531
New derivatives of reducing oligosaccharides and their
use in enzymatic reactions: efficient synthesis of sialyl
Lewis a and sialyl dimeric Lewis x glycoconjugates
Mattias Ba˚rstro¨m, Mia Bengtsson, Ola Blixt, Thomas Norberg *
Department of Chemistry, Swedish Uni6ersity of Agricultural Sciences, PO Box 7015, S-750 07 Uppsala, Sweden
Received 19 October 1999; accepted 19 April 2000
Abstract
The reducing oligosaccharides lactose and lacto-N-tetraose were reductively aminated with benzyloxycarbony-
laminoaniline and sodium cyanoborohydride, followed by treatment of the resulting secondary amines with acetic
anhydride. The resulting N-acetyl-N-(4-benzyloxycarbonylaminophenyl)-1-amino-1-deoxyalditol oligosaccharide
derivatives were subjected to stepwise enzymatic elongation with various glycosyltransferases/nucleotide sugars.
Purification of the products after each enzymatic step was conveniently performed by solid-phase extraction on silica
gel C-18 cartridges. Two oligosaccharide derivatives (with sialyl Lewis a and sialyl dimeric Lewis x structures) were
prepared. Conversion of the obtained derivatives into neoglycoproteins by the sequence hydrogenolysis, thiophosgene
treatment, and protein coupling was carried out. © 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Derivatives; Oligosaccharides; Enzymatic reactions
aqueous solutions containing considerable
amounts of buffers and metal salts, and it is
from this mixture that the oligosaccharide
product (usually a minor component com-
pared with the salts) has to be isolated. One
approach to the product purification problem
has been to attach the oligosaccharide accep-
tor to a solid phase and conduct the enzymatic
steps in a solid-phase synthesis fashion [2].
Another approach has been to attach the
oligosaccharide acceptor to a lipophilic moiety
before the enzymatic steps. This enables facile
product isolation from the reaction mixture by
solid-phase extraction using C-18 silica car-
tridges (Sep-Pak, Bond-Elut or similar). The
lipophilic moiety most commonly used for this
purpose has been the methoxycarbonyloctyl
glycosidic group [3,4]. However, this group
has to be attached to the oligosaccharide ac-
ceptor by a multi-step organic synthetic se-
1. Introduction
The use of glycosyltransferases in prepara-
tive oligosaccharide synthesis is an alternative
to traditional chemical synthesis [1]. Increased
availability of nucleotide sugars and glycosyl-
transferases from commercial sources has to-
day made enzymatic synthesis possible in
laboratories that do not have cloning or en-
zyme purification capabilities. Some practical
difficulties in the preparative use of glycosyl-
transferase enzymes remain, however. For ex-
ample, purification of the oligosaccharide
product after each enzymatic steps is not triv-
ial. Enzymatic reactions are carried out in
* Corresponding author. Tel.: +46-18-671578; fax: +46-
18-673477.
E-mail address: thomas.norberg@kemi.slu.se (T. Norberg).
0008-6215/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(00)00128-2

526
M. Ba˚rstro¨m et al. / Carbohydrate Research 328 (2000) 525–531
2. Results and discussion
Reaction of p-benzyloxycarbonylaminoani-
line (1) [6,7] and sodium cyanoborohydride
with lactose (2) or lacto-N-tetraose (3) gave
the reductive amination products 4 and 5,
respectively. Treatment in situ of 4 or 5 with
acetic anhydride produced the corresponding
N-acetylated derivatives 6 and 7, which were
isolated (60 and 80% yields, respectively) and
characterized. The N-acetylations were per-
formed in order to stabilize the derivatives
towards air oxidation, which is otherwise
known [8] to be rather rapid with p-
phenylenediamine derivatives like 4 or 5
(Scheme 1).
Scheme 1.
quence, and it might be difficult to realize this
attachment onto larger oligosaccharides.
Therefore, alternative oligosaccharide deriva-
tization procedures are interesting, especially
for work with complex reducing oligosaccha-
rides of natural origin. We have previously
reported [5] the use of glycosylamine deriva-
tization/squaric acid coupling to long-chain
aliphatic amines for this purpose.
The lacto-N-tetraose derivative 7 was sub-
jected to enzymatic elongation first using re-
combinant
a-(2“3)-(N)-sialyltransferase/
CMP-Neu5Ac, then human milk a-(1“3/4)-
fucosyltransferase/GDP-fucose (Scheme 2).
Purification by solid-phase extraction with C-
18 silica cartridges after each step was per-
formed, as well as a final gel filtration. The
resulting sialyl Lewis a (sialyl LNF II) hexas-
accharide derivative 8 was isolated in a 67%
yield. The 1H NMR data of 8 agreed well with
those for a commercial sample of sialyl-Lea
hexasaccharide (which has the same oligosac-
charide structure as 8 except for the reducing
glucosyl unit).
We now report an alternative procedure for
attachment, through reductive amination, of a
lipophilic
benzyloxycarbonylaminoaniline
group to the reducing end of oligosaccharides.
Oligosaccharides thus derivatized were enzy-
matically elongated and the products conve-
niently isolated from the reaction mixture
after each step by solid-phase extraction with
C-18 silica cartridges. The benzyloxycarbonyl-
aminoaniline group of the synthesized
oligosaccharides could be converted, by cata-
lytic hydrogenation, followed by treatment
with thiophosgene, into an isothiocyanate,
which coupled well to proteins giving glyco-
conjugates. Two biologically interesting
oligosaccharide derivatives, with sialyl Lewis a
and sialyl dimeric Lewis x carbohydrate struc-
tures, were synthesized in good yield from
lacto-N-tetraose and lactose, respectively, and
were subsequently conjugated to human
serum albumin (HSA).
It should be noted here that earlier attempts
at enzymatic elongation of an oligosaccharide
derivative carrying a p-trifluoroacetamidoaryl
group instead of
a
benzyloxycarbonyl-
aminoaryl group resulted [9] in partial loss of
the trifluoroacetamido group. Other unpub-
lished experiments have given the same result.
Presumably, enzyme preparations often con-
tain a de-trifluoroacetylating activity. Because
of this sensitivity to deacylation, the use of
p-trifluoroacetamidoaryl derivatized oligosac-
charides in enzymatic synthesis is less practi-
cal, even though the p-trifluoroacetamidoaryl
group is also lipophilic and the oligosaccha-
ride is therefore easy to isolate by solid-phase
extraction. No corresponding cleavage was
observed with the present benzyloxycarbonyl-
aminoaniline derivatives.
Scheme 2.

M. Ba˚rstro¨m et al. / Carbohydrate Research 328 (2000) 525–531
527
Scheme 3.
The derivative 8 could be converted into an
isothiocyanate by catalytic hydrogenation, fol-
lowed by in situ treatment of the product
amine with thiophosgene. Coupling of the
isothiocyanate to HSA gave the sialyl Lewis a
neoglycoconjugate 9 (Scheme 4). The spacer in
this conjugate is the same [8] as has been
published previously.
the sluggish reaction with the first enzyme.
Extended oligosaccharides like 10 are known
[18] to be poor acceptors for N. meningitidis
b - (1“3) - N - acetylglucosaminyltransferase.
The hexasaccharide was purified by gel filtra-
tion prior to treatment first with recombinant
a - (2“3) - (N) - sialyltransferase/CMP-NeuAc,
then recombinant human a-(1“3)-fucosyl-
transferase V/GDP-Fuc. A final gel filtration
purification gave 11 in 21% yield, calculated
To further demonstrate the usefulness of
oligosaccharide benzyloxycarbonylaminoani-
line derivatives in enzymatic synthesis, the lac-
tose derivative 6 was subjected to a six-step
enzymatic elongation sequence. The product
nonasaccharide derivative 11 has an oligosac-
charide part (‘sialyl dimeric Lewis x’) that is
implicated as a high-affinity selectin ligand
[10–12] or as a tumor marker [13]. The fol-
lowing steps were carried out (Scheme 3). The
lactose derivative 6 was treated with first re-
combinant Neisseria meningitidis b-(1“3)-N-
acetylglucosaminyltransferase/UDP - GlcNAc,
then recombinant bovine milk b-(1“4)-galac-
tosyltransferase/UDP-Gal. Purification after
each step by solid-phase extraction with C-18
silica cartridges was performed here as well as
in all subsequent steps. After gel filtration, the
lacto-N-neotetraose derivative 10 was ob-
tained in 53% yield from 6. Further treatment
of 10 first with recombinant Neisseria menin-
1
from 10. The H NMR data of 11 agreed well
with data [15,16] for similar structures
(Scheme 3). Conversion of 11 into the neogly-
coconjugate 12 was carried out as for 8
(Scheme 4).
It should be noted that nonasaccharide
derivatives containing the same sugar se-
quence as 11 have been prepared previously
gitidis
ferase/UDP-GlcNAc,
bovine milk b-(1“4)-galactosyltransferase/
UDP-Gal gave hexasaccharide major
b-(1“3)-N-acetylglucosaminyltrans-
then recombinant
a
product (approximately 60% yield), contami-
nated with residual tetrasaccharide 10 (ap-
proximately 40%). The reason for the
incomplete conversion to hexasaccharide is
Scheme 4.

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M. Ba˚rstro¨m et al. / Carbohydrate Research 328 (2000) 525–531
[14–17], in these cases by multi-step chemical
synthesis. The total yields (from component
protected monosaccharides) were in the 1%
range. The total yield (from the lactose deriva-
tive 2) in the present enzymatic synthesis of 11
was 11%. This illustrates that preparative en-
zymatic synthesis of oligosaccharides is in cer-
tain cases an attractive alternative to chemical
synthesis, provided that enzymes and nucleo-
tide sugars are available and that purification
after each step can be carried out in a simple
way.
Gel filtrations were performed on Bio-Gel P2
(fine) columns using MilliQ-purified water
with added n-butanol (5%, to prevent mi-
croorganism contamination) as eluant.
Materials.—Uridine 5%-diphosphogalactose
(UDP-Gal), uridine 5%-diphospho-N-acetylglu-
cosamine (UDP-GlcNAc), cytidine mono-
phospho-N-acetylneuraminic acid (CMP-
NeuAc), bovine serum albumin (BSA) and
human serum albumin (HSA) were from
Sigma Chemical Co., (St. Louis, IL, USA).
Sialyl-Lea hexasaccharide and lacto-N-tetra-
ose were from IsoSep AB (Tullinge, Sweden).
Guanosine
5%-(b- -fucopyranosyl)-diphos-
L
phate (GDP-Fuc) was prepared by chemical
synthesis as described previously [2]. Recom-
binant bovine milk b-(1“4)-galactosyltrans-
ferase (Gal-T), rat a-(2“3)-(N)-sialyltrans-
ferase (NeuAc-T), and human a-(1“3)-fuco-
syltransferase V (Fuc-T-V) were from Cal-
biochem/Novabiochem Corp. (La Jolla, CA,
USA), the manufacturers unit definition was
used. Recombinant N. meningitidis b-(1“3)-
N-acetylglucosaminyltransferase (GlcNAc-T)
was prepared as described [18], unit definition
was based on p-nitrophenyl b-lactoside as
substrate. Human milk a-(1“3/4)-fucosyl-
transferase (3/4-Fuc-T) was prepared from hu-
man milk as described [9], unit definition was
based on compound 5 as the substrate.
3. Experimental
General methods.—Concentrations were
performed at reduced pressure at B40 °C
bath temperature. Proton NMR spectra were
recorded at 25, 30 or 60 °C in D2O using
Bruker 400 and 600 MHz instruments. The
benzyl methylene signal (at l 5.244 against
external TMS in compound 10 at 30 °C)
present in all conjugates was used as internal
1H NMR reference signal. For 13C NMR
spectra, dioxane (l 67.4) was used as refer-
ence. The FABMS spectra were recorded with
a JEOL JMS-SX/SX-102A instrument. Ions
were produced by a beam of Xe atoms (6
keV), using a glycerol matrix. MALDI MS
spectra were recorded in the positive ion mode
with a Bruker reflex 3 instrument, using a
2,5-dihydroxybenzoic acid matrix. Thin-layer
chromatography (TLC) was performed on
Kieselgel 60 F254 Fertigplatten (E. Merck,
Darmstadt, Germany). The plates were eluted
with X:3:3:2 EtOAc–MeOH–AcOH–water
mixtures, where X was varied between 4 and
12, depending on the polarity of the analytes.
After elution, spots were visualized by UV
light and by dipping in 5% H2SO4, followed
by charring. For solid-phase extractions, Iso-
lute C-18 EC silica cartridges (International
Sorbent Technology, Mid-Glamorgan, UK)
were used. The cartridges were wetted thor-
oughly with several void volumes of MeOH
and then washed with at least 20 void volumes
of water before use. Water for all solutions
was from a MilliQ water purification system
(Millipore Corp., Bedford, MA, USA), and
was degassed by vacuum treatment before use.
i-
D
-Galactopyranosyl-(1“4)- -[N-acetyl-
D
N - (4 - benzyloxycarbonylaminophenyl) - 1-
amino-1-deoxy]-glucitol (6).—Acetic acid (100
mL) was added to a solution of lactose (2, 33
mg, 0.1 mmol) in 2:1 EtOH–water (6 mL),
then p-benzyloxycarbonylaminoaniline [6,7]
(1, 100 mg, 0.41 mmol) was added. When all
had dissolved, sodium cyanoborohydride (50
mg) was added, and the mixture was stirred
for 48 h, after which TLC indicated no more
change. Acetic anhydride (1.0 mL) was added,
and the mixture was stirred for 5 h, then
partitioned between EtOAc and water. The
organic layer was washed with water, and the
combined aqueous layers were concentrated to
2 mL and then applied onto an Isolute car-
tridge (1.0 g), which was then eluted first with
water and then with MeOH. Appropriate frac-
tions were concentrated and lyophilized to
1
give 6 as a white powder (36 mg, 60%). H

M. Ba˚rstro¨m et al. / Carbohydrate Research 328 (2000) 525–531
529
mixture was kept at 37 °C for 24 h after which
TLC indicated complete reaction. The mixture
was diluted with water (1 mL), then slowly
applied to an Isolute cartridge (1.0 g). Elution
first with water and then with 20, 40, and 60%
aq MeOH gave, after concentration and
lyophilization of appropriate fractions, sialy-
lated 7 (7.9 mg, homogenous on TLC), which
was dissolved in buffer (0.05 M sodium ca-
codylate, 0.005 M MnCl2, pH 7.2, 2.5 mL)
containing GDP-Fuc (6.1 mg, 13 mmol) and
human milk 3/4-Fuc-T (100 mU). After 24 h
at 37 °C, TLC indicated complete reaction,
and the mixture was purified on an Isolute
cartridge as described above. The obtained
lyophilized material (9.2 mg) was further
purified by gel filtration to give pure 8 (5.8
NME (25 °C): l 4.459 (d, J1,2 7.8 Hz, H-1%),
3.541 (dd, H-2%), 1.893 (s, NAc); 13C NMR
(25 °C): l 104.0 (C-1%), 81.4, 75.9, 73.4, 72.2,
71.9, 70.7, 70.1, 69.3 (C-2, 3, 4, 5, 2%, 3%, 4%, 5%),
68.0 (OCH2Ph), 62.8, 61.5 (C-6, 6%), 52.2 (C-
1), 22.8 (NAc). FABMS showed an [M+H]
molecular ion at m/e=611.
i-
2 - deoxy - i -
galactopyranosyl-(1“4)-
D
-Galactopyranosyl-(1“3)-2-acetamido-
- glucopyranosyl - (1“3) - i -
-[N-acetyl-N-(4-
D
D-
D
benzyloxycarbonylaminophenyl) - 1 - amino - 1-
deoxy]-glucitol (7).—Acetic acid (50 mL) was
added to a solution of lacto-N-tetraose (3, 30
mg, 0.042 mmol) in 2:1 EtOH–water (3.0
mL), then p-benzyloxycarbonylaminoaniline
[6,7] (1, 60 mg, 0.25 mmol) was added. When
all had dissolved, sodium cyanoborohydride
(25 mg) was added, and the mixture was
stirred for 48 h, after which TLC indicated no
more change. Acetic anhydride (0.6 mL) was
added, and the mixture was stirred for 3 h,
then partitioned between EtOAc (20 mL) and
water (20 mL). The organic layer was washed
with 2:1 water–brine, and the combined
aqueous layers were concentrated to 10 mL
and then applied onto an Isolute cartridge (1.0
g), which was then eluted first with water and
then with 20, 40, and 60% aq MeOH. Appro-
priate fractions were concentrated and
lyophilized to give 7 as a white powder (33
1
mg, 67%); H NMR (25 °C): l 5.017 (d, J1,2
3.9 Hz, Fuc H-1), 4.700 (d, J1,2 8.6 Hz, Glc-
NAc H-1), 4.557 (d, J1,2 7.6 Hz, Gal H-1),
4.421 (d, J1,2 7.8 Hz, Gal H-1), 2.775 (dd, J2a,3
4.5, J2a,2b 12.3 Hz, NeuAc H-3a), 2.036, 2.033
(2 s, 2 NAc), 1.882 (s, aromatic NAc), 1.776
(t, NeuAc H-3b), 1.179 (d, J5,6 6.6 Hz, H-6
Fuc). FABMS showed an [M+Na] molecular
ion at m/z=1436.
O-(Sodium 5-acetamido-3,5-dideoxy-
ero - h - - galacto - 2 - nonulpyranosyluronate)-
-galactopyranosyl-(1“3)-O-[h-
D-glyc-
D
(2“3)-O-i-
D
L
-fucopyranosyl-(1“4)]-O-(2-acetamido-2-
1
mg, 80%). H NMR (25 °C): l 4.739 (d, J1,2
deoxy-i-
D
-glucopyranosyl)-(1“3)-O-i-
D
-
7.8 Hz, H-1¦), 4.459 (d, J1,2 7.3 Hz, H-1%),
galactopyranosyl - (1“4) -
D
- [N - acetyl - N-
4.441 (d, J1,2 7.3 Hz, H-1§), 2.038 (s, NAc),
(4 - isothiocyanatophenyl) - 1 - amino - 1 - deoxy]-
glucitol and its HSA conjugate (9).—A solu-
tion of 8 (5.5 mg, 3.9 mmol) in water (0.50
mL) was stirred in a hydrogen atmosphere in
the presence of 10% palladium on charcoal (5
mg) until TLC indicated complete conversion
into a slower-migrating (and ninhydrin-posi-
tive) compound. Acetic acid (15 mL) and
sodium acetate (20 mg) was then added, fol-
lowed by EtOH (0.5 mL), then thiophosgene
(5 mL) was added with stirring. Rapid (1 min)
conversion into a faster-migrating compound
was indicated by TLC, and the mixture was
filtered (through glass wool), the filtrate was
partitioned between water (1 mL) and diethyl
ether (1 mL), the aqueous layer was, if neces-
sary, pH-adjusted to 6–8, then concentrated
to 0.3 mL and added to a solution of HSA (10
mg, 0.16 mmol, 1/24 equiv) in phosphate
13
1.885 (s, aromatic NAc); C NMR (25 °C): l
104.2 (C-1%), 103.7 (C-1§), 103.3 (C-1¦), 68.0
(OCH2Ph), 62.8, 61.5 (C-6, 6%), 55.4 (C-2¦),
52.2 (C-1), 23.0, 22.8 (NAc). FABMS showed
an [M+H] molecular ion at m/z=976.
O-(Sodium 5-acetamido-3,5-dideoxy-
ero - h - - galacto - 2 - nonulpyranosyluronate)-
(2“3) - O - i - - galactopyranosyl - (1“3) - O-
[h- -fucopyranosyl-(1“4)]-O-(2-acetamido-2-
deoxy-i- -glucopyranosyl)-(1“3)-O-i-
galactopyranosyl-(1“4)- -[N-acetyl-N-(4-
D
-glyc-
D
D
L
D
D-
D
benzyloxycarbonylaminophenyl) - 1 - amino - 1-
deoxy]-glucitol (8).—A solution of 7 (6.0 mg,
6.15 mmol) and CMP-NeuAc (6.5 mg, 9.9
mmol) in buffer (0.05 M sodium cacodylate,
0.015 M MnCl2, 0.2% Triton-X, pH 7.4, 1.0
mL) was mixed with NeuAc-T (100 mU, 0.1
mL of a 1.0 U/mL stock solution), and the

530
M. Ba˚rstro¨m et al. / Carbohydrate Research 328 (2000) 525–531
buffer (1.5 mL, pH 9.0). The pH was adjusted
to 9.5 with aq NaOH (0.5 M), then the mixture
was gently stirred overnight at room tempera-
ture (rt). Low-molecular compounds were re-
moved from the mixture by repeating (×5) a
water dilution–ultrafiltration cycle (10 K cut-
off filter, 10 mL). The resulting solution was
lyophilized to give conjugate (12.9 mg), which
in MALDI showed a broad molecular ion
centered around m/z 83743, which corre-
sponds to an average sugar incorporation of
13 haptens/HSA molecule.
glucopyranosyl)-(1“3)-O-i-
D
-galactopyra-
nosyl-(1“4)- -[N-acetyl-N-(4-benzyloxycar-
D
bonylaminophenyl)-1-amino-1-deoxy]-glucitol
(11).—A solution of 10 (26 mg, 26.6 mmol),
UDP-GlcNAc (265 mg, 407 mmol, and BSA
(26 mg) in buffer (0.5 M sodium cacodylate,
0.015 M MnCl2, pH 7.3, 1.8 mL) was mixed
with GlcNAc-T (1.5 U, 3.6 mL of a 0.41
U/mL stock solution), and the mixture was
stirred for 5 days at rt. Then TLC analysis
indicated 60% conversion to product (lower
Rf). The mixture was diluted with water (20
mL) and was then purified on an Isolute car-
tridge (1.0 g) as described for 3 and 8, to give
material (33 mg, a mixture of 10 and pen-
tasaccharide product), which was taken up in
buffer (0.5 M sodium cacodylate, 0.02 M
MnCl2, pH 7.3, 8.0 mL) and mixed with
UDP-Gal (33 mg, 54 mmol) and Gal-T (0.75
U, 150 mL of a 5 U/mL stock solution). After
2 days at rt, the mixture was diluted with
water and purified on an Isolute cartridge (1.0
g) as described for 3 and 8, to give material,
which was purified by gel filtration to give a
i-
2 - deoxy - i -
galactopyranosyl-(1“4)-
D
-Galactopyranosyl-(1“4)-2-acetamido-
- glucopyranosyl - (1“3) - i -
-[N-acetyl-N-(4-
D
D-
D
benzyloxycarbonylaminophenyl) - 1 - amino - 1-
deoxy]-glucitol (10).—A solution of 6 (44 mg,
72 mmol), UDP-GlcNAc (100 mg, 154 mmol,
and BSA (60 mg) in buffer (0.5 M sodium
cacodylate, 0.015 M MnCl2, pH 7.3, 8.5 mL)
was mixed with GlcNAc-T (1.4 U, 3.5 mL of
a 0.41 U/mL stock solution), and the mixture
was stirred for 3 days at rt. Then TLC analysis
indicated 50% conversion to product (lower
Rf). More UDP-GlcNAc (75 mg, 115 mmol)
and GlcNAc-T (0.82 U, 2.0 mL) was added,
and the mixture was stirred for another 48 h.
The mixture was diluted with water (33 mL)
and was then purified on an Isolute cartridge
(4.0 g) as described for 3 and 8, to give
material (38 mg), which was taken up in buffer
(0.5 M sodium cacodylate, 0.015 M MnCl2,
pH 7.3, 16 mL) and mixed with UDP-Gal (44
mg, 72 mmol) and Gal-T (1.5 U, 300 mL of a
5 U/mL stock solution). After 2 days at rt, the
mixture was diluted with water and purified on
an Isolute cartridge (4.0 g) as described for 3
and 8 to give material, which was further
purified by gel filtration to give pure 10 (37
1
hexasaccharide material (14.7 mg); H NMR
(40 °C): l 4.718 (d, J1,2 7.8 Hz, GlcNAc H-1),
4.710 (d, J1,2 7.8 Hz, GlcNAc H-1), 4.480 (d,
J1,2 7.6 Hz, Gal H-1), 4.467 (d, J1,2 7.7 Hz, Gal
H-1), 4.426 (d, J1,2 7.7 Hz, Gal H-1), 4.151 (d,
Gal H-4), 4.102 (d, Gal H-4), 2.034, 2.020 (2 s,
2 NAc), 1.877 (s, aromatic NAc). Part of this
material (9.7 mg) and CMP-NeuAc (8.9 mg,
14.4 mmol) in buffer (0.05 M sodium cacody-
late, 0.015 M MnCl2, 0.2% Triton-X, pH 7.0,
1.1 mL) was mixed with NeuAc-T (250 mU,
0.25 mL of a 1.0 U/mL stock solution), and
the mixture was kept at 37 °C for 4 days.
Dilution with water (5 mL) and purification
on an Isolute cartridge (0.5 g) gave material
(11.8 mg), which was taken up in buffer (0.10
M sodium cacodylate, 0.020 M MnCl2, pH
7.3, 1.3 mL) and mixed with GDP-Fuc (19
mg, 23 mmol) and Fuc-T-V (0.21 U, 0.42 mL
of a 0.5 U/mL stock solution). After 4 days at
37 °C, the mixture was diluted with water (8
mL) and purified on an Isolute cartridge (0.5
g) as described for 3 and 8, to give material,
which was further purified by gel filtration to
give pure 11 (7.0 mg, 3.6 mmol, 21% from 10);
1H NMR (25 °C): l 5.112, 5.108 (2 d, 2 H, J1,2
3.3 Hz, 2 H-1 Fuc), 4.809 (d, GlcNAc H-1),
4.797 (d, GlcNAc H-1), 4.524 (d, J1,2 7.7 Hz,
Gal H-1), 4.439 (d, J1,2 7.7 Hz, Gal H-1),
1
mg, 38 mmol, 53%); H NMR (60 °C): l 4.731
(d, J1,2 7.6 Hz, GlcNAc H-1), 4.475 (d, J1,2 7.6
Hz, Gal H-1), 4.433 (d, J1,2 7.6 Hz, Gal H-1),
4.097 (d, Gal H-4), 2.022 (s, NAc), 1.871 (s,
aromatic NAc). FABMS showed an [M+H]
molecular ion at m/z=976.
O-(Sodium 5-acetamido-3,5-dideoxy-
ero - h - - galacto - 2 - nonulpyranosyluronate)-
-galactopyranosyl-(1“4)-O-[h-
D
-glyc-
D
(2“3)-O-i-
D
L
-fucopyranosyl-(1“3)]-O-(2-acetamido-2-
deoxy-i-
D
-glucopyranosyl)-(1“3)-O-i-
D
-
galactopyranosyl - (1“4) - O - [h -
L
- fucopyran-
osyl-(1“3)]-O-(2-acetamido-2-deoxy-i-
D-

M. Ba˚rstro¨m et al. / Carbohydrate Research 328 (2000) 525–531
531
4.418 (d, J1,2 7.7 Hz, Gal H-1), 2.763 (dd, J2a,3
References
4.0, J2a,2b 12.3 Hz, NeuAc H-3a), 2.029, 1.999
(2 s, 2 NAc), 1.878 (s, aromatic NAc), 1.788
(t, NeuAc H-3b), 1.166, 1.145 (2 d, J5,6 6.6 Hz,
2 H-6 Fuc). MALDI-MS showed an [M+Na]
molecular ion at m/z=1946.
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-galactopyranosyl-(1“4)-O-[h-
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We thank the Swedish Natural Science Re-
search Council for financial support. We also
thank Isosep AB (Tullinge, Sweden) for gifts
of oligosaccharides, and the Tejbrant family
for supplying the human milk.
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