Chemoenzymatic synthesis of spacer-linked oligosaccharides for the preparation of neoglycoproteins

Article abstract of DOI:10.1016/S0008-6215(99)00073-7

In the present work, the combination of chemical and enzymatic methods to obtain neoglycoproteins is described. Three bovine serum albumin (BSA)-conjugates, BSA-[GalNAcα-], BSA-[Gal(β1-3)GalNAc(α-], and BSA-[Neu5Ac(α2-3)Gal(β1-3)GalNAc(α-], were prepared.

Full text of DOI:10.1016/S0008-6215(99)00073-7

www.elsevier.nl/locate/carres
Carbohydrate Research 317 (1999) 180–190
Chemoenzymatic synthesis of spacer-linked
oligosaccharides for the preparation of neoglycoproteins
Raul Gonzalez Lio, Joachim Thiem *
Institut fu¨r Organische Chemie, Uni6ersita¨t Hamburg, Martin-Luther-King-Platz 6, D-20146 Hamburg, Germany
Received 7 December 1998; accepted 15 March 1999
Abstract
In the present work, the combination of chemical and enzymatic methods to obtain neoglycoproteins is described.
Three bovine serum albumin (BSA)-conjugates, BSA-[GalNAca-], BSA-[Gal(b1-3)GalNAc(a-], and BSA-
[Neu5Ac(a2-3)Gal(b1-3)GalNAc(a-], were prepared. aGalNAc derivatives were galactosylated employing crude
b-galactosidase from bovine testes. The use of oversaturated donor solutions (pNPbGal) enhanced the yields up to
60%. This method was verified using divalent structures as acceptors, that rendered di- and tri-galactosylated
products. Further treatment of the disaccharides with CMP-Neu5Ac and a2-3 sialyltransferase from pork liver led
to formation of trisaccharides. Finally, mono-, di-, and trisaccharides were coupled to BSA employing a thiolic group
introduced into the protein for Michael addition to a maleinimide group in the spacer-arm of the saccharide
components. The results were monitored by HPLC and MALDI-TOF. © 1999 Elsevier Science Ltd. All rights
reserved.
Keywords: Chemoenzymatic synthesis; Transglycosylation; b-Galactosidase; a(2“3)-Sialyltransferase; Neoglycoproteins
1. Introduction
synthesized and exploited by Verez-Bencomo
et al. [5]. Recently, we have reported syntheses
of analogues of the T-antigen determinant in
In recent years the use of neoglycoproteins
has expanded enormously [1,2]. The foremost
advantage of neoglycoconjugates is that they
contain carbohydrates of known structures and
assured purity. Galactose-, mannose-, and N-
which 2-acetamido-2-deoxy-3-O-(b-
D-galac-
topyranosyl)-a- -galactopyranose is a-glyco-
D
sidically linked to different diethylene glycol
derivatives, using crude extracts of b-galactosi-
dase (EC 3.2.1.23) from bovine testes [6].
Since a general and wider use of glycosidases
for the synthesis of biologically active oligosac-
charides is limited due to lower yields, we
sought to establish an improved method by
using b-galactosidase from bovine testes for the
galactosylation of the spacer-linked GalNAc-
derivative 1. In this paper, the synthesis of
mono-, di-, and trisaccharides by combination
of chemical and enzymatic methods and the
procedure for their coupling to bovine serum
albumin (BSA) is described to give neoglyco-
proteins.
acetylgalactosamine-containing
neoglyco-
proteins are used in different fields, for example
in the therapeutic area and in cancer research
[3,4].
For synthesis of neoglycoproteins, many dif-
ferent spacer molecules and coupling methods
have been employed. Oligosaccharides with an
ethylene glycol spacer have been classically
* Corresponding author. Tel.: +49-40-4123-4241; fax: +
49-40-4123-4325.
E-mail address: thiem@chemie.uni-hamburg.de (J. Thiem)
0008-6215/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(99)00073-7

R. Gonzalez Lio, J. Thiem / Carbohydrate Research 317 (1999) 180–190
181
transglycosylation products. Previously, the
use of excess of acceptor was described, and
proposed to shift the equilibrium of the reac-
tion to the right side (Scheme 1, right) [7–9].
Since the acceptor molecule 1 is very expen-
sive, it cannot be used in high excess. Alterna-
tively, we developed another strategy to shift
this equilibrium to the right by employing an
oversaturated solution of the donor molecule
pNPbGal (GX). Thus, a higher concentration
of the glycosyl enzyme intermediate (EG)
could be achieved and this enhanced the yield
of the enzymatic galactosylation considerably.
In contrast to previous preparations [6], the
transgalactosylation of 1 could be increased to
give the disaccharide 2 in 60% yield (Scheme
2).
The HPLC chromatogram of this reaction
is shown in Fig. 1. Apparently, due to the
high concentration of glycosyl enzyme inter-
mediate (EG), the donor (pNPbGal) can also
act as an acceptor for b-galactosidase. Thus,
by autogalactosylation of pNPbGal, the for-
mation of the b-(1“3)-galactobioside 4 was
observed in 6% yield. For NMR characteriza-
tion, both these compounds were acetylated to
give the corresponding heptaacetates 3 and 5.
Bis(galactosylation).—The use of oversatu-
rated donor solutions (pNPbGal) as an im-
proved method for transgalactosylation was
further checked and verified using divalent
acceptor structures. By reaction of the glyco-
2. Results and discussion
Use of o6ersaturated solutions of p-nitro-
phenyl i-
sylation.—Most
D
-galactopyranoside for transgalacto-
syntheses employing
glycosidases use the kinetic approach by trans-
glycosylation. Thus, the initial formation of a
glycosyl enzyme intermediate (EG) involves a
nucleophilic displacement of the aglycon of
the donor molecule GX.
The yield and optimal time of enzymic
transglycosylation are a function of the initial
concentration of reactants, and of various dis-
sociation and catalytic rate constants for the
different steps (Scheme 1, left). Thus, a high
yield may be expected if the rate constant (k1)
for the formation of the glycosyl enzyme inter-
mediate (EG) from the donor is high com-
pared with that of the product glycoside
(k−2). In cases of b-galactosidase from bovine
testes it was possible to use p- or o-nitro-
phenyl b- -galactopyranosides, because the
D
affinity for these donors to the active center of
the enzyme is higher than the affinity for the
Scheme 1. E, enzyme; GX, glycosyl donor; EG, glycosyl
enzyme intermediate; A, acceptor; GA, transglycosylation
product; GOH, hydrolysis product.
Scheme 2.

182
R. Gonzalez Lio, J. Thiem / Carbohydrate Research 317 (1999) 180–190
Fig. 1. HPLC of transgalactosylations of 1 with b-galactosidase employing pNPbGal in saturated (upper line) and oversaturated
(lower line) solutions. The relative absorptions of the acceptor 1, the product disaccharide 2, and the side product disaccharide
4, do not correlate to the yield.
Scheme 3. (a) Hg(CN)2, CH2Cl2; (b) CH3ONa–CH3OH; (c) PhCH(OCH3)2, pTsOH; (d) BnBr, BaO–Ba(OH)2, DMF; (e)
pTsNH2, Cs2CO3, DMF, 80 °C, 3 days; (f) H2, 10% Pd/C.
sylchloride 6 [10] (Scheme 3) with 1-chloro-3-
oxa-5-pentanol in the presence of Hg(CN)2, the
corresponding b-galactopyranoside 7 was ob-
tained in 60% yield. After deacetylation to
compound 8, benzylidenation gave derivative 9,
and the subsequent benzylation led to the fully
protected N-acetylglucosamine derivative 10.
Employing Richman–Atkins conditions [11]
to 10 and p-toluenesulfonamide gave the
bridged component 11, which, after hy-
drogenolysis with 10% Pd/C, led to the spacer-
linked N-acetyl glucosamine-derived dimeric
glycoside 12. Its enzymatic galactosylation was
performed using saturated and oversaturated

R. Gonzalez Lio, J. Thiem / Carbohydrate Research 317 (1999) 180–190
183
Fig. 2. MALDI-TOF spectra of mono- (13), bis- (14), and tris-galactosylated (15) spacer-linked dimeric GlcNAc acceptors. Upper
insert: saturated conditions, lower line: oversaturated donor solutions.
solutions of pNPbGal and the results were
monitored by MALDI-TOF MS as shown in
Fig. 2.
was done by incubation with stoichiometric
amounts of CMP-Neu5Ac in the presence of
sialyltransferase from pork liver for 24 h in
MES-buffer pH 6.8. After HPLC purification
on an RP-18 column and desalting, the trisac-
charide glycoside 16 was obtained in 50%
isolated yield (Scheme 4).
The subsequent reaction steps [15,16] re-
quired reduction of the azido groups in com-
pounds 1, 2 and 16. With hydrogen in the
presence of 5% Pd/C the corresponding mono-,
di-, and trisaccharide glycosides 17, 18, and 19
with terminal amino groups were obtained.
Further reaction of the free amines with N-suc-
cinimidyl-3-maleimidopropionate (NSMP) in
specifically deaminated DMF afforded
smoothly the activated derivatives 20, 21 and
22 carrying the desired thiophilic substituent.
For coupling to BSA with an average of 23
amino groups per mol, the protein 23 was
modified as outlined in Scheme 5. By reaction
with the N-hydroxysuccinimide dithiopropi-
onate (DTSP), compound 24 was obtained,
which was followed by treatment with dithio-
The peaks with molecular weights of 776, 938
and 1100 corresponded to the starting acceptor
material 12, the monogalactosylated com-
pounds 13 and the bisgalactosylated derivative
14, respectively. In fact, the use of oversatu-
rated solutions of pNPbGal led to an increased
formation of transgalactosylation products 13
and 14. In addition to these the MALDI-TOF
spectrum showed another peak at molecular
weight 1229 that corresponds to a trisgalactosy-
lated product 15. As there were only small
quantities of isolated 15, the structure of this
compound could not yet be determined un-
equivocally, since the third galactose residue
could be interglycosidically linked to either the
inner GlcNAc or the terminal Gal units.
3. Epitope synthesis and coupling to BSA
According to previous findings [12–14], dis-
accharide 2 could be selectively sialylated. This

184
R. Gonzalez Lio, J. Thiem / Carbohydrate Research 317 (1999) 180–190
Scheme 4. (g) H2, 5% Pd/C; (h) NSMP, DMF.
threitol (DTT) to allow the introduction of 20
thiol groups per mol of protein to give com-
pound 25.
(1-3)GalNAc per mol protein, 23 could be
measured by the TBA assay [18].
As shown in Fig. 3, MALDI-TOF spectra
of BSA 23, the two functionalized BSA com-
ponents 24 and 25, and two sugar-modified
neoglycoconjugates 26 and 28, were com-
pared. The molecular weights nicely reflected
the progress of the functionalization process
and the formation of neoglycoproteins. The
novel neoglycoproteins of the Tn, T, and sT
epitope type are now being subjected to for-
mation of monoclonal antibodies for further
investigations.
The final coupling reaction was performed
between derivative 25 and the corresponding
sugar epitopes carrying the maleinimide func-
tion 20, 21 and 22 in PBS-buffer at pH 7.2. By
this Michael addition of the thiol groups in
the protein to a maleimide group in the
spacer-armed sugar derivatives, the corre-
sponding neoglycoproteins 26, 27 and 28 were
obtained in almost quantitative yields.
For closer determination of the structures
of these neoglycoproteins, several different
methods had to be applied. As observed by
MALDI-TOF spectra, the neoglycoprotein
having the GalNAc monosaccharide epitope
26 contained 18 mol of GalNAc per mol
protein 23. Again 18 mol of Galb(1-3)GalNAc
disaccharide epitope per mol protein 23 were
determined for the conjugate 27 by use of the
phenol–sulfuric acid method [17]. Finally,
for compound 28 carrying the trisaccharide
epitope 15 mol of Neu5Aca(2-3)Galb-
4. Experimental
General procedures.—Specific rotations
were determined with a Perkin–Elmer polar-
imeter, 1H and 13C NMR spectra were
recorded at 400 or 500 MHz (100.6 or 125.7
MHz) with a Bruker AMX-400 or DRX-500
spectrometer. FAB spectra were recorded with
VG 70-250S. MALDI-TOF spectra were
recorded with a Bruker Biflex. Analytical and

R. Gonzalez Lio, J. Thiem / Carbohydrate Research 317 (1999) 180–190
185
Scheme 5. (i) DTSP, buffer pH 8; (j) DTT, buffer pH 8; (k) 20, 21, or 22, buffer pH 7.2.
5-Azido-3-oxapentyl 2-acetamido-2-deoxy-
3-O-(i- -galactopyranosyl)-h- -galactopyran-
oside (2).—(a) For preparation of 2 using a
saturated solution of pNPbGal, see Ref. [6].
(b) 5-Azido-3-oxapentyl 2-acetamido-2-de-
preparative HPLC was performed on silica gel
LiChrosorb RP-18 columns. Protein concen-
trations were determined by the method of
Bradford [19]. Thiol group concentrations
were determined by the method of Ellman
[20]. Sialic acid concentrations in glycocon-
jugtgates were determined by the TBA assay
[18]. Galactose concentrations in glycoconju-
gates were determined using the phenol–sulfu-
ric acid method [17].
D
D
oxy-a- -galactopyranoside [6] (1) (25 mg,
D
0.074 mmol) was dissolved in sodium phos-
phate–citrate buffer (50 mM, pH 4.3, 0.5
mL). The reaction mixture was incubated with
b-galactosidase (crude extract) from bovine

186
R. Gonzalez Lio, J. Thiem / Carbohydrate Research 317 (1999) 180–190
Fig. 3. MALDI-TOF spectra of bovine serum albumin (BSA-23 NH2, 23); the masked thiol-functionalized BSA compound 24; the
thiol-functionalized BSA 25, and the neoglycoproteins, with GalNAc monosaccharide epitopes 26 or with Neu5Aca2-3Galb-1-
3GalNAc trisaccharide epitopes 28.
3.0, H-4%), 5.03 (dd, 1 H, J1,2 7.6, H-2%), 4.87
(dd, 1 H, J2,3 10.1, H-3%), 4.82 (d, 1 H, J1,2 3.5,
H-1), 4.48 (d, 1 H, J1,2 7.6, H-1%), 4.43 (ddd, 1
H, J1,2 3.5, H-2), 4.11–4.01 (m, 1 H, H-5),
3.88 (dd, 1 H, J2,3 11.1, H-3), 3.72–3.68 (m, 9
H, H-5%), 3.66–3.59 (m, 6 H, H-spacer), 3.30
(t, 2 H, H-spacer), 2.22–1.81 (m, 21 H, 6
CH3CO, CH3CONH); 13C NMR (250 MHz,
CDCl3): l 100.4 (C-1%), 97.8 (C-1), 72.5 (C-3),
70.3 (C-3%), 70.2 (C-5%), 68.4 (C-4), 68.2 (C-2%),
67.0 (C-5), 66.3 (C-4%), 62.2, 60.4 (C-6, C-6%),
48.6 (C-2), 50.1 (CH2-N3). Anal. Calcd for
C30H44N4O18: 748.52. Found: m/z 749 [M+
1]+ (FABMS).
testes (10 mg, 0.5 U) at 37 °C. An oversatu-
rated solution of pNPbGal (1.5–2 equiv) in
phosphate–citrate–buffer was injected, and
the reaction mixture was further incubated for
60 h. The reaction was terminated by heating
for 5 min at 90 °C. The desired product was
purified by HPLC on an RP-18 column with
water/acetonitrile to give 2 (22.6 mg, 61%);
[h]D+143° (c 1.0, water); Rf 0.35 2:1 EtOAc–
1
MeOH, H NMR (400 MHz, D2O): l 4.93 (d,
1 H, J1,2 3.5, H-1), 4.46 (d, 1 H, J1,2 8.1, H-1%);
13C NMR (250 MHz, D2O): l 105.1 (C-1%),
97.8 (C-1), 48.9 (C-2), 50.6 (CH2–N3). Anal.
Calcd for C18H32N4O12: 496.48. Found: m/z
497 [M+1]+ (FABMS).
5-Azido-3-oxapentyl 2-acetamido-4,6-di-O-
acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-i-
p-Nitrophenyl 3-O-(i-D-galactopyranosyl)-
i- -galactopyranoside (4).—This compound
D
was isolated as a side-product during forma-
tion of 2. Based on the amount of pNPbGal,
the yield was 6%; [h]D +166° (c 1.0, H2O); Rf
D
- galactopyranosyl) - h -
D
- galactopyranoside
1
(3).—Treatment of 2 (100 mg, 0.22 mmol)
0.37 2:1 EtOAc–MeOH; H NMR (400 MHz,
with pyridine and Ac2O gave 3 (145 mg, 96%);
D2O): l 8.13 and 7.16 (d, 2 H, J 9.6, H-arom),
5.07 (d, 1 H, J1,2 7.6, H-1), 4.27 (d, 1 H, J1%,2%
1
[h]D+178° (c 1.0, CHCl3); H NMR (400
13
MHz, CDCl3): l 5.73 (d, 1 H, J2,NH 9.6, NH),
5.30 (d, 1 H, J3,4 3.0, H-4), 5.28 (d, 1 H, J3,4
7.1, H-1%), 3.98 (m, 1 H, H-3); C NMR (250
MHz, D2O): l 161.9 (OPh), 142.8 (C–NO2),

R. Gonzalez Lio, J. Thiem / Carbohydrate Research 317 (1999) 180–190
187
6.17; N, 3.08. Found: C, 47.75; H, 6.26; N,
3.02.
126.5, 116.5 (C-arom), 106.5 (C-1%), 100.2 (C-
1). Anal. Calcd for C18H25NO13: 463.25.
Found: m/z 464 [M+1]+ (FABMS).
5-Chloro-3-oxapentyl 2-acetamido-4,6-O-
benzylidene-2-deoxy-i- -glucopyranoside (9).
D
p-Nitrophenyl 2,4,6-tri-O-acetyl-3-O-(2,3,4,-
—Deacetylation of 7 (100 mg, 0.33 mmol) in
6-tetra-O-acetyl-i-
D
-galactopyranosyl)-i- -
D
NaOCH3–CH3OH at pH 9 gave 5-chloro-3-
galactopyranoside (5).—Treatment of 4 (100
mg, 0.21 mmol) with pyridine and acetic anhy-
dride gave 5 (155 mg, 95%); [h]D +177° (c 1.0,
oxapentyl 2-acetamido-2-deoxy-b- -glucopyr-
D
anoside (8). (70 mg, 98%); [h]D +55° (c 1.0,
1
1
chloroform); H NMR (400 MHz, CDCl3): l
water); H NMR (400 MHz, D2O): l 4.52 (d,
8.05 and 7.01 (d, 2 H, J 9.6, H-arom), 5.34 (d,
1 H, J3%,4% 3.5, H-4), 5.29 (d, 1 H, J3,4 3.5, H-4),
5.22 (dd, 1 H, J1,2 8.1, J2,3 10.2, H-2), 5.02 (dd,
1 H, J1%,2% 8.1, J2%,3% 10.1, H-2%), 4.92 (d, 1 H, J1,2
8.2, H-1), 4.58 (d, 1 H, J1%,2% 8.1, H-1%), 4.40
(dd, 1 H, J3,4 3.5, J2,3 10.2, H-3), 4.22 (dd, 1
H, Jgem 12.2, J5,6 4.8, H-6a), 4.11–4.02 (m, 3
H, H-6b, H-6%a/b), 3.84 (m, 2 H, H-5, H-5%),
2.16–1.92 (m, 21 H, 7 CH3CO); 13C NMR
(250 MHz, CDCl3): l 161.7 (OPh), 142.5 (C–
NO2), 128.9, 116.4 (C-arom), 101.6 (C-1%),
100.6 (C-1). Anal. Calcd for C32H39NO20:
757.23. Found: m/z 758 [M+1]+ (FABMS).
3-Oxa-5-chloro-pentyl 2-acetamido-3,4,6-
1 H, J1,2 8.6, H-1), 3.95 (m, 1 H, H-spacer),
3.88 (dd, 1 H, Jgem 12.2, J5,6a 1.0, H-6a),
3.79–3.63 (m, 9 H, H-spacer, H-5, H-2), 3.50
(t, 1 H, J 9.1, H-3), 3.41 (m, 2 H, H-4, H-6b),
2.00 (s, 3 H, CH3CONH); 13C NMR (250
MHz, D2O): l 174.9 (CONH), 101.3 (C-1),
76.2, 74.2 (C-3, C-4), 71.2, 69.2, 69.9 (C-
spacer), 70.2 (C-5), 61.1 (C-6), 55.8 (C-2), 43.6
(CH2–Cl), 22.6 (CH3CONH).
To a solution of 8 (1.0 g, 3.0 mmol) in
DMF (20 mL) were added benzaldehyde
dimethylacetal (1.85 mL, 12.2 mmol) and p-
toluenesulfonic acid (catalytic amount). The
mixture was stirred overnight and terminated
with a satd solution of NaHCO3. After work-
up and concentration, the material was crys-
tallized from ethyl acetate to give 9 (900 mg,
71%); [h]D+44° (c 1.0, CHCl3); Rf 0.42 4:1
tri-O-acetyl-2-deoxy-i- -glucopyranoside (7).
D
—To a solution of 6 [10] (1.88 g, 5.7 mmol)
and 5-chloro-3-oxapentanol (1.40 g, 5.7
mmol) in CH2Cl2 (40 mL) was added CaSO4
(4.2 g, 0.03 mol), and the mixture was stirred
for 30 min. Hg(CN)2 (1.44 g, 5.7 mmol) was
then added and stirring was continued for 48
h. The mixture was diluted with CH2Cl2,
washed with 10% KI solution, with satd
NaHCO3 solution and water. The organic
phase was separated and concentrated. The
residual syrup was chromatographed on a
column of silica gel with 4:1 CH2Cl2–acetone
to afford 7 (1.8 g, 60%); [h]D +82° (c 1.0,
CHCl3); Rf 0.52 3:2 petroleum ether–EtOAc;
1H NMR (400 MHz, CDCl3): l 6.30 (d, 1 H,
J2,NH 9.2, NH), 5.05 (t, 1 H, J 9.5, H-3), 4.80
(t, 1 H, J 9.5, H-4), 4.60 (d, 1 H, J1,2 9.1, H-1),
4.00 (dd, 1 H, Jgem 12.2, J5,6 7.0, H-6a), 3.88
(dd, 1 H, Jgem 12.2, J5,6 2.05, H-6b), 3.70–3.41
(m, 10 H, H-2, H-spacer, H-5), 1.81, 1.75, 1.68
(s, 12 H, CH3CONH, 3 CH3CO); 13C NMR
(250 MHz, CDCl3) l 171.0, 170.9, 170.8, 169.7
(3 CO, CONH), 101.2 (C-1), 73.0 (C-3), 72.0
(C-4), 69.0 (C-5), 70.8, 70.0, 68.8 (C-spacer),
62.6 (C-6), 54.5 (C-2), 43.6 (CH2–Cl), 23.5
(CH3CONH), 21.2, 21.0, 20.9 (CH3CO); Anal.
Calcd for C18H28ClNO10 (453.87): C, 47.63; H,
1
CH2Cl2–acetone; mp: 239–240 °C; H NMR
(400 MHz, CDCl3): l 8.03 (d, 1 H, J2,NH 8.8,
NH), 7.7–7.6 (m, 5 H, H-arom), 5.82 (s, 1 H,
H-benzylidene), 5.51 (d, 1 H, J 5.6, OH), 4.76
(d, 1 H, J1,2 8.6, H-1), 4.43 (dd, 1 H, J5,6 5.0
Jgem 10.1, H-6a), 4.01 (m, 1 H, H-spacer),
3.97–3.86 (m, 7 H, H-6b, H-spacer), 3.86–
3.71 (m, 3 H, H-3, H-2, H-spacer), 3.65 (t, 1
13
H, J 9.1, H-4), 2.05 (s, 3 H, CH3CONH); C
NMR (250 MHz, CDCl3): l 169.4 (CONH),
138.1 (C–CHO2), 129.2–126.7 (CH-arom),
101.8 (C-benzylidene), 101.0 (C-1), 81.6 (C-4),
70.8 (C-3), 70.9, 69.7, 68.5 (C-spacer), 68.2
(C-6), 66.3 (C-5), 56.4 (C-2), 43.9 (CH2–Cl),
23.4
(CH3CONH);
Anal.
Calcd
for
C19H26ClNO7 (415.5): C, 54.87; H, 6.25; N,
3.36. Found: C, 54.91; H, 6.32; N, 3.15.
5-Chloro-3-oxapentyl 2-acetamido-3-O-ben-
zyl-4,6-O-benzylidene-2-deoxy-i- -glucopyr-
D
anoside (10).—To a solution of 9 (900 mg, 2.1
mmol) in DMF (20 mL) were added BaO (900
mg, 5.8 mmol), Ba(OH)2×8H2O (290 mg, 0.9
mmol) and benzyl bromide (0.6 mL, 4.9
mmol). The reaction mixture was stirred for 2

188
R. Gonzalez Lio, J. Thiem / Carbohydrate Research 317 (1999) 180–190
82.5 (C-4), 77.5 (C-3), 74.7 (CH2Ph), 70.3,
70.0 (C-spacer), 68.7 (C-6), 68.6 (C-spacer),
65.9 (C-5), 56.8 (C-2), 49.3(CH2 –NTs), 23.4
(CH3CONH), 21.5 (CH3Ph); Anal. Calcd for
C59H71N3O16S (1109.87): C, 63.84; H, 6.40; N,
3.78. Found: C, 63.32; H, 6.51; N, 3.64.
h at rt, then filtered through Celite and the
filtrate washed with 2% aq HCl solution, satd
NaHCO3 solution, and water. The organic
phase was separated and concentrated, diethyl
ether added, and compound 10 (980 mg, 92%)
was isolated as white solid; [h]D +32° (c 1.0,
CHCl3); Rf 0.75 4:1 CH2Cl2–acetone; mp
Bis-1,11-[2-acetamido-2-deoxy-i- -gluco-
D
1
pyranosyl]-(6-tosyl-3,9-dioxa-6-aza-undecane)
(12).—Compound 11 (200 mg, 0.19 mmol)
was hydrogenolyzed for 2 days in methanol
(10 mL) in the presence of 10% Pd/C (cata-
lytic amount). The catalyst was filtered off, the
filtrate was concentrated, and the residue
chromatographed on a column of silica gel
with CH2Cl2–MeOH to give 12 (110 mg,
85%); [h]D +22° (c 1.0, water); Rf 0.15
10:10:2:1 EtOAc–MeOH–water–AcOH; 1H
NMR (400 MHz, D2O): l 7.48 and 7.19 (d, 2
H, H-arom), 4.26 (d, 2 H, J1,2 8.6, 2×H-1),
3.64 (m, 4 H, 2×H-3, 2×H-6a), 3.50–3.20
(m, 2×(H-2, H-4, H-6b, and H-spacer)), 3.17
(m, H-spacer, 2×H-5), 2.16 (s, 3 H, CH3Ph),
225–228 °C; H NMR (400 MHz, CDCl3): l
7.55–7.30 (m, 10 H, H-arom), 5.67 (d, 1 H,
J2,NH 7.1, NH), 5.62 (s, 1 H, H-benzylidene),
5.02 (d, 1 H, J1,2 8.6, H-1), 4.79 (AB, 2 H, J
11.7, PhCH2), 4.39 (dd, 1 H, J5,6 5.0 Jgem 10.6,
H-6a), 4.18 (t, 1 H, J 9.1, H-3), 3.95 (m, 1 H,
H-spacer), 3.85–3.65 (m, 9 H, H-5, H-4, H-
spacer), 3.52 (m, 2 H, H-6b, H-2), 1.95 (s, 3 H,
13
CH3CONH); C NMR (250 MHz, CDCl3): l
170.7 (CONH), 138.8 (C–CHO2), 137.7 (C–
CH2O), 129.3–126.4 (CH-arom), 101.6 (C-
benzylidene), 101.3 (C-1), 83.0 (C-4), 77.1
(C-3), 74.8 (CH2Ph), 71.6, 71.1, 69.2 (C-
spacer), 69.1 (C-6), 66.4 (C-5), 57.6 (C-2), 43.5
(CH2–Cl), 23.9 (CH3CONH); Anal. Calcd for
C26H32ClNO7 (505.5): C, 61.72; H, 6.33; N,
2.77. Found: C, 61.62; H, 6.44; N, 2.69.
13
1.79 (s, 6 H, 2×CH3CONH); C NMR (250
MHz, CDCl3): l 177.3 (CONH), 148.0 (C-
SO2), 137.9 (C–CH3), 133.0, 129.9 (C-arom),
104.0 (C-1), 78.8 (C-3), 76.8 (C-4), 74.5 (C-5),
72.9, 72.5, 71.4 (C-spacer), 63.7 (C-6), 58.2
(C-2), 50.6 (CH2–NTs), 25.2 (CH3CONH),
23.7 (CH3Ph).
Bis-1,11-[2-acetamido-3-O-benzyl-4,6-O-ben-
zylidene-2-deoxy-i- -glucopyranosyl]-(6-tosyl-
D
3,9-dioxa-6-aza-undecane) (11).—To a solu-
tion of 10 (1.0 g, 1.9 mmol) and p-toluene-sul-
fonamide (0.95 mmol) in DMF (10 mL) was
added Cs2CO3 (10 mmol). The reaction mix-
ture was stirred for 3 days at 80 °C, then
filtered through Celite/silica gel and concen-
trated. The residual syrup was chro-
matographed on a column of silica gel with
EtOAc to afford 11 (1.8 g, 85%); [h]D+56° (c
1.0, CHCl3); Rf 0.55 10:1 EtOAc–MeOH; mp
Bis-1-[2-acetamido-2-deoxy-3-O-(i-
actopyranosyl)-i- -glucopyranosyl]-11-[2-ace-
tamido-2-deoxy-i- -glucopyranosyl]-(6-tosyl-
3,9-dioxa-6-aza-undecane) (13) and bis-1,11-
[2-acetamido-2-deoxy-3-O-(i- -galactopyran-
osyl)-i- -glucopyranosyl]-(6-tosyl-3,9-dioxa-
D
-gal-
D
D
D
D
6-azaundecane) (14).—Compound 12 (25 mg,
0.033 mmol) was dissolved in sodium phos-
phate–citrate buffer, (50 mM, pH 4.3, 0.5
mL). The reaction mixture was incubated with
b-galactosidase (crude extract) from bovine
testes (10 mg, 0.5 U) at 37 °C. An oversatu-
rated solution of pNPbGal (1.5–2 equiv) in
phosphate–citrate buffer was injected and the
reaction mixture was further incubated for 5
days. The reaction was terminated by heating
for 5 min at 90 °C. The desired products were
purified by HPLC on a RP-18 column with
water–acetonitrile to give 13 (10 mg, 30%)
and 14 (5 mg, 15%).
1
214–216 °C; H NMR (400 MHz, CDCl3): l
7.70–7.20 (m, 20 H, H-arom), 6.44 (d, 2 H,
J2,NH 8.1, 2×NH), 5.47 (s, 2 H, 2×H-ben-
zylidene), 4.94 (d, 2 H, J1,2 8.6, 2×H-1), 4.74
(AB, 4 H, J 11.7, 2×PhCH2), 4.31 (dd, 2 H,
J5,6 4.5 Jgem 10.1, 2×H-6a), 4.14 (t, 2 H, J 9.6,
2×H-3), 3.86 (m, 4 H, H-spacer), 3.73 (t, 2
H, J 10.1, 2×H-6b), 3.66 (m, 2 H, 2×H-4),
3.52 (m, 2 H, 2×H-2), 3.50 (m, 2 H, 2×H-
5), 3.32 (t, 4 H, J 5.5, 2×CH2NTs), 2.42 (s, 3
13
H, CH3Ph), 1.89 (s, 6 H, 2×CH3CONH); C
NMR (250 MHz, CDCl3): l 170.6 (CONH),
143.5 (C–SO2), 138.5 (C–CHO2), 137.4 (C–
CH2O), 136.2 (C–CH3), 129.7–126.0 (C-
arom), 101.0 (C-benzylidene), 100.9 (C-1),
Compound 13: [h]D +46° (c 1.0, H2O);
Anal. Calcd for C37H61N3O21S: 915.38. Found
m/z 938 [M+Na]+ (MALDI-TOF).

R. Gonzalez Lio, J. Thiem / Carbohydrate Research 317 (1999) 180–190
189
1
The catalyst was filtered off and the filtrate
was concentrated to afford 17, 18 or 19 (0.048
mmol, 95%).
Compound 14: [h]D +165° (c 1.0, H2O); H
NMR (400 MHz, D2O): l 7.73 and 7.42 (d, 2
H, H-arom), 4.58 (d, 2 H, J1%,2% 8.3, 2×H-1%),
4.38 (d, 2 H, J1,2 8.0, 2×H-1), 3.79 (m, 2 H,
H-2%), 3.47 (m, 2 H, H-2), 2.39 (s, 3 H,
CH3Ph), 1.96 (s, 6 H, 2×CH3CONH); 13C
NMR (250 MHz, CDCl3): l 175.0 (CONH),
147.5 (C–SO2), 137.6 (C–CH3), 133.4, 129.0
(C-arom), 103.9 (C-1), 101.2 (C-1%), 50.3
(CH2–NTs), 25.0 (CH3CONH), 23.2 (CH3Ph).
Anal. Calcd for C43H71N3O26S: 1077.43.
Found m/z 1100 [M+Na]+ (MALDI-TOF).
5-Azido-3-oxapentyl 3-O-{(5-acetamido-3,5-
Introduction of the maleimide group.—To a
solution of the saccharides 17, 18 or 19 (4
mmol) in DMF (distilled in vacuo and pumped
for 30 min before use, 0.5 mL), was added
NSMP (1.3 mg, 5 mmol). After 2 h, the solu-
tion was concentrated and dried in vacuo. The
residue was resuspended in D2O (0.5 mL) and
centrifuged to remove the excess of reagent.
The maleimide derivatives 20, 21 and 22 were
obtained and could be stored in a lyophilized
form at 0 °C, or directly used in the coupling
dideoxy-h-
osonate)-3-O-(i-
amido-2-deoxy-h-
D
-glycero-
-galactopyranosyl)}-2-acet-
-galactopyranoside (16).—
D-galacto-2-nonulopyran-
1
reaction; H NMR (D2O): l 6.89 (s, 2 H,
D
HCꢀCH), 3.42 (t, 2 H, CH2N), 2.52 (t, 2 H,
CH2CO) [16].
D
A solution of 2 (50 mg, 0.10 mmol) and
CMP-Neu5Ac (45 mg, 0.075 mmol) in MES-
buffer (pH 6.8, 2.5 mL) was incubated at
37 °C with a-(2“3) sialyltransferase from
pork liver (crude extract, 30–50 mU) for 12 h.
At this time, more CMP-Neu5Ac (45 mg) was
added and incubation was continued for an-
other 12 h. HPLC then showed the formation
of 98% of 16. The mixture was centrifuged
and freeze-dried. The desired product was
purified by HPLC on a preparative RP-18
column with phosphate buffer acetonitrile. Af-
ter evaporation of solvents the residue was
applied to a G-15 column, equilibrated and
eluted with water, to afford 16 (40 mg, 50%);
[h]D+32° (c 1.0, in water); Rf 0.12; 7:3 1-
Preparation of neoglycoproteins.—Com-
pounds 20, 21 and 22 were added to a solution
of 25 [15] in PBS buffer (pH 7.2, 0.4 mL).
After 2 h, the process was complete as evi-
denced by a negative Ellman test [20]. The
resulting solution was diafiltrated against PBS
buffer (pH 7.2) to afford neoglycoproteins 26,
27 or 28. The carbohydrate content was deter-
mined by the Bradford method [19], phenol–
sulfuric acid method [17], TBA assay, and
MALDI-TOF analysis.
Acknowledgements
Support of this work by the Deutscher
Akademischer Austauschdienst (stipend to
R.G.L.), the Deutsche Forschungsgemein-
schaft (SFB 470) and the Fonds der Chemis-
chen Industrie is gratefully acknowledged.
1
propanol–water; H NMR (500 MHz, D2O):
l 4.83 (d, 1 H, J1,2 3.6, H-1), 4.43 (d, 1 H, J1,2
7.6, H-1%), 4.25 (dd, 1 H, J2,3 10.6, H-2), 4.14
(d, 1 H, J3,4 3.1, H-4), 3.97 (dd, 2 H, J2,3 10.6,
J3,4 3.1, H-3, H-3%), 3.90 (t, 2 H, H-5, H-5%),
3.84 (d, 1 H, J3,4 3.05, H-4%), 2.66 (dd, 1 H, J3,4
4.5, J3,3 12.2, H-3%%eq), 1.69 (t, 1 H, J3,3 12.2,
H-3%%ax), 1.93 (s, 6 H, 2 CH3CONH); 13C
NMR (250 MHz, D2O): l 174.0, 175.0 (CO,
CONH), 104.9 (C-1%), 100.0 (C-2%%), 97.7 (C-1),
77.8 (C-3), 76.0 (C-3%), 62.8 (C-9%%), 61.5, 61.3
(C-6, C-6%), 52.0 (C-5%%), 50.5 (CH2 –N3), 48.8
(C-2), 40.1 (C-3%%), 22.4 (2 CH3CONH). Anal.
Calcd for C29H49N5O20: 787.24. Found m/z
786 [M−1]− (MALDI-TOF).
References
[1] C.P. Stowell, Y.C. Lee, Ad6. Carbohydr. Chem. Biochem.,
37 (1980) 225–281.
[2] H.J. Gabius, A. Bardosi, Prog. Histochem. Cytochem., 22
(1991) 1–66.
[3] R. Koganty, M.A. Reddish, B.M. Longenecker, Drug
Disco6ery Today, No. 5, May, (1996) 190–198.
[4] T. Toyokuni, A.K. Singhal, Chem. Soc. Re6. (1995)
231–242.
[5] V. Verez-Bencomo, M.T. Campos-Valdes, J.R. Marino-
Albernas, V. Fernandez-Santana, Carbohydr. Res., 271
(1991) 263–267.
Hydrogenolysis of the azido group.—Com-
pounds 1, 2 or 16 (0.05 mmol) were hy-
drogenolyzed for 12 h in MeOH (5 mL) in the
presence of 5% Pd/C (catalytic amount) [16].
[6] U. Gambert, R. Gonzalez Lio, E. Farkas, J. Thiem, V.
Verez-Bencomo, A. Lipta´k, Bioorg. Med. Chem., 5
(1997) 1285–1291.

190
R. Gonzalez Lio, J. Thiem / Carbohydrate Research 317 (1999) 180–190
[7] L. Hedbys, E. Johansson, P.-O. Larsson, Carbohydr.
Res., 186 (1989) 217–223.
[8] K.G.I. Nilsson, Carbohydr. Res., 167 (1987) 95–103.
[9] K.G.I. Nilsson, Carbohydr. Res., 180 (1988) 53–59.
[10] D. Horton, Methods Carbohydr. Chem., 6 (1972) 282–
299.
[11] A. Rathjens, Dissertation, Hamburg, 1996.
[12] A. Lubineau, C. Auge´, P. Francois, Carbohydr. Res., 228
(1992) 137–144.
V. Kren, J. Thiem, Angew. Chem., Int. Ed. Engl., 34
(1995) 893–895.
[15] V. Fernandez Santana, R. Gonzalez Lio, V. Verez-Ben-
como, Glycoconjugate J., 15 (1998) 549–553.
[16] S. Figueroa-Perez, R. Gonzalez Lio, V. Fernandez San-
tana, V. Verez-Bencomo, J. Carbohydr. Chem., 17 (1998)
835–850.
[17] M. Dubois, K.H. Gilles, J.K. Hamilton, F. Smidt, Anal.
Biochem., 28 (1956) 350–356.
[13] S. David, C. Auge´, C. Gautheron, Ad6. Carbohydr.
Chem. Biochem., 49 (1991) 175–237.
[14] V. Kren, J. Thiem, Angew. Chem., 107 (1995) 979–981;
[18] L. Warren, J. Biol. Chem., 234 (1959) 1971–1975.
[19] M. Bradford, Anal. Biochem., 72 (1976) 248–254.
[20] G.L. Ellman, Arch. Biochem. Biophys., 74 (1958) 443–451.
.