Synthesis of the Lewis b hexasaccharide and squarate acid-HSA conjugates thereof with various saccharide loadings

Article abstract of DOI:10.1016/S0008-6215(00)00189-0

The Lewis b hexasaccharide, α-Ll-Fucp-(1 → 2)-β-D-Galp-(1 → 3)-[α-L-Fucp-(1 → 4)]-β-D-GlcpNAc-(1 → 3)-β-D-Galp-(1 → 4)-β-D-Glcp, has been synthesised using a convergent synthesis. Starting from ethyl 4,6-O-benzylidene-2-deoxy-2-phthalimido-1-thio-β-D-gluc

Full text of DOI:10.1016/S0008-6215(00)00189-0

www.elsevier.nl/locate/carres
Carbohydrate Research 329 (2000) 309–316
Synthesis of the Lewis b hexasaccharide and squarate
acid–HSA conjugates thereof with various saccharide
loadings
Anatoly Chernyak 1, Stefan Oscarson *, Dominika Turek
Department of Organic Chemistry, Arrhenius Laboratory, Uni6ersity of Stockholm, S-106 91 Stockholm, Sweden
Received 8 March 2000; accepted 20 June 2000
Abstract
The Lewis b hexasaccharide, a-
Galp-(1“4)-b- -Glcp, has been synthesised using a convergent synthesis. Starting from ethyl 4,6-O-benzylidene-2-
deoxy-2-phthalimido-1-thio-b- -glucopyranoside, a thioglycoside tetrasaccharide donor block, was constructed
L
-Fucp-(1“2)-b-
D
-Galp-(1“3)-[a-
L
-Fucp-(1“4)]-b-
D
-GlcpNAc-(1“3)-b-
D-
D
D
through two orthogonal glycosylations with glycosyl bromide donors. First, the galactose moiety was introduced
using silver triflate as a promoter and then the two fucose residues under halide-assisted conditions. Finally, this
tetrasaccharide was linked to a spacer-equipped 3I,4I-diol lactose acceptor in a DMTST-promoted coupling to give,
after deprotection, the Lewis b hexasaccharide as its 2-aminoethyl spacer-equipped derivative. This was coupled to
human serum albumin (HSA), using the squarate ester methodology, in various saccharide–protein ratios, to give
neoglycoconjugates with different saccharide loadings in about 50% efficiency. © 2000 Elsevier Science Ltd. All
rights reserved.
Keywords: Oligosaccharide synthesis; Glycoconjugates; Helicobacter pylori; Blood group antigens
1. Introduction
[4]. To investigate this binding, we synthesised
the tetrasaccharide with the 8-methoxycar-
bonyloctyl spacer as described by Lemieux [5]
and made BSA-conjugates of this. However,
experiments showed that these conjugates did
not bind to the bacteria and the free spacer
tetrasaccharide was not an inhibitor to the
binding [6]. Results that the free reducing
tetrasaccharide did function as an inhibitor
indicated that this might be due to a spacer
effect, but the idea of an extended binding
epitope could not be excluded. To allow inves-
tigation of multivalency effects [7], the
aminoethanol glycoside of hexasaccharide Leb
structure was synthesised, and human serum
albumin (HSA) conjugates thereof with differ-
ent loadings were made using the squarate
acid coupling chemistry [8,9].
The Lewis b (Leb) blood group antigen
structure is known to have a number of im-
portant biological functions. Its effective bind-
ing to the Griffonia simplicifolia lectin [1,2]
and interest in studying carbohydrate–protein
interactions in general, prompted Lemieux
and co-workers to synthesise the tetra-
saccharide structure and numerous analogues
thereof [2,3]. Rather recently it has been found
that the Leb structure is also a human receptor
for the bacteria Helicobacter pylori, a binding
event that causes gastritic and duodenal ulcers
* Corresponding author. Fax: +46-8-154908.
E-mail address: s.oscarson@organ.su.se (S. Oscarson).
1 On leave from N.D. Zelinsky Institute of Organic Chem-
istry, Russian Academy of Sciences, Moscow, Russia.
0008-6215/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(00)00189-0

310
A. Chernyak et al. / Carbohydrate Research 329 (2000) 309–316
thioglycoside disaccharide 3 in 84% yield. Re-
2. Results and discussion
ductive opening of the benzylidene acetal with
NaCNBH3–HCl in THF (“4, 77%) [15] fol-
lowed by deacetylation gave the 4,2I-diol 5.
The normally straightforward Ze´mplen
deacetylation was accompanied by extensive
opening of the phthalimido ring. Reclosing
the ring by treatment with trifluoroacetic an-
hydride followed by de-O-trifluoroacetylation
(Mg(OMe)2) was necessary to obtain 5 in an
overall 91% yield. The subsequent coupling,
between the thioglycoside acceptor 5 and the
The Leb hexasaccharide has been synthe-
sised earlier by Danishefsky et al. using glycals
as glycosyl donors [10,11]. In our approach,
bromide and thioglycoside donors were
utilised. Since the tetrasaccharide structure
with other spacer molecules might be of inter-
est, we synthesised the hexasaccharide through
a 4+2 block approach (Scheme 3), in which
the formed tetrasaccharide thioglycoside
donor block would be suitable also for the
synthesis of various spacer tetrasaccharide
derivatives. The required lactose acceptor
block 7 could easily be prepared from lactose.
The tetrasaccharide thioglycoside 6 was
synthesised using a strategy similar to that of
Lemieux et al. in the synthesis of the corre-
sponding methyl glycoside [2] (Scheme 1).
Thus, ethyl 4,6-O-benzylidene-2-deoxy-2-ph-
halide donor 2,3,4-tri-O-benzyl-a- -fucopyran-
L
osyl bromide [16], using halide-assisted cou-
pling conditions [17], afforded the desired tet-
rasaccharide thioglycoside block 6 in 94%
yield. Although efficiently prepared, the tetra-
saccharide was found to be quite unstable.
Even when kept in the freezer, substantial
elimination of ethyl mercaptan was observed
to produce the corresponding glucal deriva-
tive. Consequently, compound 6 had to be
prepared just before use.
thalimido-1-thio-b-
was coupled with 2-O-acetyl-3,4,6-tri-O-ben-
-galactopyranosyl bromide (2) [13],
D-glucopyranoside (1) [12]
zyl-b-D
[14], using silver trifluoromethanesulfonate
(silver triflate) as a promoter, to give the
The lactoside acceptor 10 was synthesised
according to Scheme 2. The 2-azidoethyl lacto-
Scheme 1. (i) AgOTf, 2,6-di-tert-butyl pyridine, CH2Cl2, toluene, −74°C; (ii) NaCNBH3, HCl–Et2O, THF; (iii) NaOMe,
MeOH–CH2Cl2; (iv) TFAA, pyridine; (v) Mg(OMe)2, MeOH–CH2Cl2; (vi) Et4NBr, CH2Cl2.
Scheme 2. (i) NaOMe–MeOH; (ii) acetone, PTS; (iii) BnBr, NaH, DMF; (iv) 70% TFA, CH2Cl2.

A. Chernyak et al. / Carbohydrate Research 329 (2000) 309–316
311
ing in a low yield of the desired product.
However, with 20 molar equivalents of the
promoter DMTST, a 72% yield of the hexa-
saccharide 11 could be obtained.
Deprotection of hexasaccharide 11 was
rather complicated, mainly due to problems
with the removal of the phthalimido group.
When 11 was heated with hydrazine hydrate
for 5 days, the corresponding free amine was
obtained in 59% yield. Subsequent selective
N-acetylation (acetic anhydride in methanol/
CH2Cl2, 73%) and simultaneous catalytic
debenzylation and reduction of the azido
group gave the aminoethyl glycoside hexasac-
charide 12 (67%) ready for conjugation to a
protein.
The squaric acid diester chemistry seems to
be one of the best choices for preparation of
neo-glycoconjugates [8,9]. Recently, this ap-
proach has been successfully used for conjuga-
tion of various oligosaccharide haptens to
proteins [18–20]. The effect of some parame-
ters (reaction time, concentration, molar ex-
cess of hapten) upon the efficiency of
conjugation was also described.
Scheme 3. (i) DMTST, CH2Cl2, −78°C“r.t.; (ii) NH2NH2–
H2O, EtOH–dioxane, D; (iii) Ac2O–MeOH–CH2Cl2; (iv)
10% Pd/C, H2 (120 psi), EtOH–EtOAc, 60% HOAc.
We used this approach, with some slight
modifications, for the preparation of Leb-
tetra- and hexasaccharide conjugates with
HSA. In order to avoid complications on the
stage of derivatisation with diethyl squarate
(caused by the ability of the amino group in a
spacer to form salts) up to one equivalent of
Et3N was added to complete the reaction in a
quantitative yield. To elute the oligosaccha-
ride-squarate derivative from a C-18 Sep-Pak
cartridge a step-wise gradient of aqueous
methanol (usually 10–40%) was used. The
effect of pH upon the efficiency of the cou-
pling step was also investigated. We found
that at pH 10 the coupling was more efficient
than at pH 9.0, in contrast to Pozsgay et al.,
who obtained slightly higher incorporation at
pH 9 [19]. In a model experiment with an
appropriate globotrioside at a 20:1 hapten–
protein ratio, the average incorporation was
found to be 8 (MALDI-TOF data) at pH 9.0
and 10.5 at pH 10.0 [21]. In case of Leb-tetra-
saccharide (15:1 hapten–HSA), we have
reached loading of 6 at pH 9.0 and 8 at pH
10.0. In our hands, in order to reach the
targeted incorporation of hapten, a 2:1 hap-
ten–protein ratio was needed (Table 1).
Table 1
Degree of incorporation of Leb-hexaoligosaccharide–HSA
conjugates
Hapten–protein ratio
Incorporation achieved a
2:1
10:1
20:1
30:1
1.5
6
11
14
a Confirmed by MALDI-TOF.
side 7 was deacetylated (Zemple´n, 95%) and
the product 8 was treated with acetone and
p-TsOH to give the 3I,4I-O-isopropylidene
acetal derivative, which was directly benzy-
lated to give 9 (55%). It was de-isopropyli-
denated by treatment with aqueous tri-
fluoroacetic acid to give the 3I,4I diol accep-
tor 10 in 92% yield.
In the coupling between tetrasaccharide
donor 6 and disaccharide acceptor 10 (Scheme
3), it was important to use a large excess of
promoter to effectuate the glycosylation reac-
tion, otherwise the elimination reaction of 6,
known to occur in similar couplings [12], be-
came an important competing reaction result-

312
A. Chernyak et al. / Carbohydrate Research 329 (2000) 309–316
In conclusion, an efficient way to Leb-hexa-
71.6, 72.0, 72.8, 73.3, 74.2, 75.9, 80.3, 80.9,
81.5 (C-1, 3- 6, 2I-6I, 3 CH2Ph), 100.5, 101.0
(C-1I, PhCH), 122.4–138.2 (aromatic C),
166.5, 168.1 (PhthCO) and 168.5 (CH3CO);
1H, l 5.13 (dd, J=8 and 10 Hz, H-2I). Anal.
Calcd for C52H53NO12S: C, 68.18; H, 5.83.
Found: C, 68.28; H, 5.97.
saccharide–HSA-conjugates based on syn-
thetic oligosaccharide structures and with dif-
ferent carbohydrate loading is described. The
synthetic strategy allows the formation of Leb
tetrasaccharide structures with various spac-
ers. The conjugates are presently being evalu-
ated for their binding to Helicobacter pylori
bacteria.
Ethyl 3,4,6-tri-O-benzyl-i-
osyl-(1“3)-6-O-benzyl-2-deoxy-2-phthal-
imido - 1 - thio - i - - glucopyranoside (5).—
D
-galactopyran-
D
NaCNBH3 (2.0 g, 31.8 mmol) and crushed
,
3 A MS (3.2 g) were added to a stirred solution
3. Experimental
of 3 (2.0 g, 2.2 mmol) in THF (50 mL) under
Ar. After 5 min, HCl–Et2O was added drop-
wise until the evolution of gas ceased. After an
additional 20 min, the mixture was filtered
through Celite, concentrated and purified on a
silica gel column (5:1 toluene–EtOAc) to give
General methods.—Concentration of solu-
tions was performed under reduced pressure
at B40 °C (bath). NMR spectra were
recorded at 25 °C at 300 or 400 MHz (1H) or
75 or 100 MHz (13C) in CDCl3 with Me4Si as
internal standard (l=0 ppm) or in D2O with
acetone as an internal standard (13C: l=31.0
ppm; 1H: l=2.225 ppm). TLC was performed
on Silica Gel F254 (E. Merck) with detection
by UV light and/or by charring with 8% sulfu-
ric acid. Silica Gel (0.040–0.063 mm, Amicon)
was used for column chromatography.
ethyl (2 - O - acetyl - 3,4,6 - tri - O - benzyl - b -
galactopyranosyl)-(1“3)-6-O-benzyl-2-deoxy-
2-phthalimido-1-thio-b- -glucopyranoside (4,
D
-
D
1.55 g, 77%); [h]D +16.4° (c 1.0, CHCl3);
NMR (CDCl3): 13C, l 14.9 (SCH2CH3), 20.0
(CH3CO), 23.9 (SCH2CH3), 53.9 (C-2), 68.6,
69.8, 69.9, 70.9, 72.1, 72.2, 73.4, 73.6, 73.8,
74.4, 79.9, 80.4, 81.0, 82.7 (C-1, 3–6, 2I –6I, 4
CH2Ph), 101.2 (C-1I), 123.3–138.6 (aromatic
C), 167.0, 168.4 (PhthCO) and 168.9
(CH3CO). 1 M NaOMe (15 drops) was added
to a solution of 4 (1.21 g, 1.3 mmol) in
MeOH–CH2Cl2 (2:1, 30 mL), and the reaction
was stirred for 72 h, during which time more 1
M NaOMe (15 drops) was added every 24 h.
The mixture was neutralised with Dowex H+
ion-exchange resin and filtered. The Dowex
resin was washed thoroughly with CH2Cl2 to
minimize losses. After concentration of the
filtrate, the residue was dissolved in pyridine
(30 mL) and TFAA (10 mL) was added at
0 °C. After 45 min the reaction mixture was
diluted with CH2Cl2 and washed with water,
10% CuSO4, 1 M HCl, NaHCO3 and water,
dried (MgSO4), filtered and concentrated. The
residue was dissolved in 2:1 MeOH–CH2Cl2
(60 mL) and the trifluoroacetyl groups were
removed by the addition of 1 M Mg(OMe)2
(20 drops). After 80 min, Dowex (H+) ion-
exchange resin was added, whereafter the mix-
ture was filtered, concentrated and chro-
matographed (8:1“6:1 toluene–EtOAc) to
yield 5 (1.05 g, 91%); [h]D +5.1° (c 0.97,
Ethyl (2-O-acetyl-3,4,6-tri-O-benzyl-i-
galactopyranosyl)-(1“3)-4,6-O-benzylidene-
2-deoxy-2-phthalimido-1-thio-i- -glucopyran-
oside (3).—A solution of ethyl 2-O-acetyl-
3,4,6-tri-O-benzyl-1-thio-b- -galactopyran-
D-
D
D
oside [22](4.194 g, 7.8 mmol) and bromine
(459 mL, 9.0 mmol) in CH2Cl2 (30 mL) was
stirred under argon for 40 min at rt, where-
after the mixture was concentrated and co-
evaporated with CH2Cl2 twice. The residue,
crude 2 (7.8 mmol) [13,14], 1 (2.0 g, 4.7 mmol)
[12], and 2,6-di-tert-butylpyridine (1.89 mL,
8.4 mmol) was dissolved in CH2Cl2 (20 mL)
,
and crushed 4 A MS (3 g) was added. The
stirred mixture was cooled to −74 °C under
argon. After 1 h at this temperature, AgOTf
(2.705 g, 10.5 mmol) dissolved in toluene (10
mL) was added. After another hour the reac-
tion was left to attain rt over night. The
reaction mixture was filtered through Celite,
concd and purified by silica gel chromatogra-
phy (6:1“2:1 light petroleum bp 45–60 °C–
EtOAc) to produce 3 (3.58 g, 84%); [h]D
+3.3° (c 1.0, CHCl3); NMR (CDCl3): 13C, l
14.7 (SCH2CH3), 20.3 (CH3CO), 23.6
(SCH2CH3), 54.1 (C-2), 68.0, 68.5, 70.6, 71.4,
CHCl3); NMR (CDCl3): 13C,
l
15.0

A. Chernyak et al. / Carbohydrate Research 329 (2000) 309–316
313
,
mmol) and crushed 4 A MS (1.2 g) in MeCN
(10 mL), which had been stirred at ambient
temperature under argon for 1 h. After 48 h
the reaction mixture was diluted with CHCl3,
filtered through Celite and the filtrate was
washed with water and NaI (aq 1 M), dried
(MgSO4), filtered and concd. The residue was
chromatographed (6:1“2:1 toluene–EtOAc)
to yield 7 (2.285 g, 44%); [h]D −22° (c 1.1,
CHCl3); NMR (CDCl3): 13C, l 20.4, 20.6,
20.75 and 20.8 (CH3CO), 50.4 (OCH2CH2N3),
60.7, 61.7, 66.5, 68.5, 69.0, 70.6, 70.8, 71.4,
72.6, 72.7, 76.0 (C-2–6, 2I –6I, OCH2CH2N3),
100.3, 100.9 (C-1, 1I), 168.8, 169.4, 169.5,
169.7, 169.8, 170.0 (MeCO). Anal. Calcd for
C28H39N3O18: C, 47.66; H, 5.57. Found: C,
47.76; H, 5.54.
(SCH2CH3), 24.1 (SCH2CH3), 53.9 (C-2),
68.2, 69.9, 70.1, 70.2, 72.0, 72.2, 73.3, 73.4,
73.6, 74.3, 79.3, 80.9, 81.6, 83.4 (C-1, 3–6,
2I –6I, 4 CH2Ph), 103.9 (C-1I), 122.9–138.2
(aromatic C), 167.6 and 168.2 (PhthCO).
Anal. Calcd for C50H53NO11S: C, 68.55; H,
6.10. Found: C, 68.27; H, 6.17.
Ethyl (2,3,4-tri-O-benzyl-h-
(1“2)-(3,4,6-tri-O-benzyl-i-
osyl)-(1“3)-[(2,3,4-tri-O-benzyl-h-
ranosyl)-(1“4)]-6-O-benzyl-2-deoxy-2-phthal-
imido-1-thio-i- -glucopyranoside (6).—Ethyl
2,3,4-tri-O-benzyl-1-thio-a- -fucopyranoside
L
-fucopyranosyl)-
-galactopyran-
-fucopy-
D
L
D
L
(1.191 g, 2.5 mmol) [12] in CH2Cl2 (7 mL) was
treated with bromine (150 mL, 3.0 mmol) at
0 °C for 10 min and then concentrated and
co-evaporated twice with CH2Cl2. The residue,
2-Azidoethyl (2,6-di-O-benzyl-i-
pyranosyl)-(1“4)-2,3,6-tri-O-benzyl-i-
D-galacto-
crude
2,3,4-tri-O-benzyl-a- -fucopyranosyl
L
D
-
bromide (2.5 mmol) [16], was dissolved in
CH2Cl2 (2.5 mL) and added to a mixture of 5
(433 mg, 494 mmol), Et4NBr (107 mg, 510
glucopyranoside (10).—A solution of 7 (7.63
g, 10.8 mmol) in MeOH (100 mL) was made
basic by the addition of 1 M NaOMe (4 mL).
After 20 h the reaction was neutralised with
Dowex (H+) ion-exchange resin, filtered and
,
mmol) and crushed 4 A MS (1.67 g) in
CH2Cl2 –DMF (2.5/1, 7 mL), which had been
stirred under argon for 50 min. After 72 h
MeOH (2 mL) was added. The mixture was
then filtered through Celite, concentrated, co-
concentrated to give crude 2-azidoethyl b- -
D
galactopyranosyl-(1“4)-b- -glucopyranoside
D
(8, 4.214 g, 95%); [h]D +4.8° (c 1.0, water);
evaporated
with
toluene
and
chro-
13
NMR (D2O): C, l 51.3 (OCH2CH2N3), 60.8,
matographed (two columns: 6:1“2:1 light
petroleum bp 45–60 °C–EtOAc and 15:1“
12:1 toluene–EtOAc) to yield 6 (791 mg,
61.7, 69.2, 71.6, 73.2, 73.5, 75.0, 75.5, 76.0,
79.0, 85.5 (C-2–6, 2I –6I, OCH2CH2N3), 102.8
and 103.6 (C-1, 1I); 1 H, l 4.36 (d, J=8 Hz,
1 H) and 4.28 (d, J=8 Hz, 1 H) (H-1 and
H-1I). A solution of 8 (4.2 g, 10.2 mmol) in
acetone (200 mL) was acidified by the addi-
tion of p-TsOH. After 24 h the amount of
solvent and p-TsOH was doubled and, after
another 72 h, the solution was neutralised by
the addition of Et3N, and concd. The residue
was dissolved in DMF (30 mL), benzyl bro-
mide (7.3 mL, 61.5 mmol) was added and the
mixture was added dropwise to a cooled
(0 °C) slurry of 60% NaH (3.06 g, 76.5 mmol)
in DMF (4 mL). After 1 h, the mixture was
allowed to attain room temperature and, after
another 3 h, the reaction was quenched by the
successive addition of MeOH and ice–water.
The mixture was extracted with toluene, the
organic phase was washed with water, dried
(MgSO4), filtered, concd and the residue was
chromatographed (20:1 toluene–EtOAc) to
afford 9 (5.044 g, 55%); [h]D +19.1° (c 1.0,
CHCl3); NMR (CDCl3): 13C, l 26.4 and 28.0
13
94%); NMR (CDCl3): C, l 14.9 (SCH2CH3),
16.3 (C-6II, 6III), 24.3 (SCH2CH3), 55.8 (C-2),
66.4, 66.9, 67.3, 67.8, 70.6, 71.4, 71.8, 72.5,
72.6, 72.7, 72.8, 73.4, 73.5, 73.8, 74.5, 74.6,
74.8, 75.0, 75.2, 75.6, 77.7, 77.9, 79.4, 80.0,
80.3, 81.5, 83.4 (C-1, 3–6, 2I –6I, 2II–5II, 2III–
5III, 10 CH2Ph), 97.8, 98.3, 100.4 (C-1I, 1II,
1III), 123.3–139.3 (aromatic C), 167.3 and
168.4 (PhthCO).
2-Azidoethyl
galactopyranosyl)-(1“4)-2,3,6-tri-O-acetyl-
i- -glucopyranoside (7).—Peracetylated lac-
(2,3,4,6-tetra-O-acetyl-i-
D-
D
tose (5.0 g, 7.4 mmol) was treated with HBr–
HOAc (20 mL) and after 1 h, the reaction
mixture was concentrated. The residue was
dissolved in CHCl3, washed with NaHCO3 (aq
sat) and water, dried (MgSO4), filtered and
concd. A soln of the residue in MeCN (10
mL) was added dropwise to a mixture of
2-azidoethanol (650 mg, 7.4 mmol), HgCN2
(938 mg, 3.7 mmol), HgBr2 (1.340 g, 3.7

314
A. Chernyak et al. / Carbohydrate Research 329 (2000) 309–316
168.4 (PhthCO). FABMS Calcd for C151H158-
N4O30+Na: 2530.09. Found: 2530.17.
(Me2CO2), 51.0 (OCH2CH2N3), 68.0, 68.2,
68.9, 71.9, 73.1, 73.3, 73.5, 74.97, 75.04, 75.3,
76.2, 79.3, 80.6, 81.7, 82.8 (C-2–6, 2I –6I,
OCH2CH2N3, 5 CH2Ph), 101.7, 103.5 (C-1,
1I), 109.6 (Me2CO2), 127.1–138.7 (aromatic
C). 70% TFA (1 mL) was added to a solution
of 9 (600 mg, 665 mmol) in CH2Cl2 (5 mL).
After stirring at room temperature for 1 h
more CH2Cl2 was added and the reaction
mixture was washed with water, NaHCO3 (aq
sat), and water, dried (MgSO4), filtered, concd
and the residue was chromatographed (6:1“
2:1 toluene-EtOAc) to give 10 (527 mg, 92%);
[h]D +16.8° (c 1.1 CHCl3); NMR (CDCl3):
13C, l 50.8 (OCH2CH2N3), 67.9, 68.1, 68.5,
68.6, 72.8, 73.0, 73.3, 73.4, 74.7, 74.8, 74.95,
75.0, 76.3, 79.9, 81.5, 82.5 (C-2–6, 2I –6I,
OCH2CH2N3, 5 CH2Ph), 102.4, 103.4 (C-1, 1I)
and 127.0–38.8 (aromatic C). Anal. Calcd for
C49H55N3O11: C, 68.28; H, 6.43. Found: C,
68.20; H, 6.56.
An aliquote of 11 was benzoylated to give
1H NMR: l 5,67 (d, J=3.7 Hz, 1 H, H-4I).
2-Aminoethyl (h-
-galactopyranosyl)-(1“3)-[h-
osyl-(1“4)]-(2-acetamido-2-deoxy-i-
copyranosyl)-(1“3)-(i- -galactopyranosyl)-
(1“4)-i- -glucopyranoside (12).—Hexasac-
L
-fucopyranosyl)-(1“2)-(i-
-fucopyran-
-glu-
D
L
D
D
D
charide 11 (203 mg, 81 mmol) in 3:1 dioxane–
EtOH 95% (12 mL) was treated with
hydrazine hydrate (1 mL) and the mixture
heated at 90 °C for 5 days, whereafter concen-
tration, co-evaporation with toluene and chro-
matography (2:1 toluene–EtOAc) gave
2-azidoethyl(2,3,4-tri-O-benzyl-a-
osyl)-(1“2)-(3,4,6-tri-O-benzyl-b-
pyranosyl)-(1“3)-[(2,3,4-tri-O-benzyl-a-
fucopyranosyl)-(1“4)]-(6-O-benzyl-2-deoxy-
L
-fucopyran-
D
-galacto-
L
-
2-amino-b-
D
-glucopyranosyl)-(1“3)-(2,6-di-
-galactopyranosyl) -(1“4)-2,3,
O-benzyl-b-
D
6-tri-O-benzyl-b- -glucopyranoside (114 mg,
D
2-Azidoethyl (2,3,4-tri-O-benzyl-h-
pyranosyl)-(1“2)-(3,4,6-tri-O-benzyl-i-
galactopyranosyl)-(1“3)-[2,3,4-tri-O-benzyl-
h - - fucopyranosyl - (1“4)] - (6 - O - benzyl - 2-
deoxy - 2 - phthalimido - i - - glucopyranosyl)-
(1“3)-(2,6-di-O-benzyl-i- -galactopyran-
osyl)-(1“4)-2,3,6-tri-O-benzyl-i- -gluco-
L
-fuco-
13
59%); NMR (CDCl3): C, l 16.3, 16.5 (C-6IV,
6V), 50.9 (OCH2CH2N3), 59.0 (C-2II), 66.6,
66.8, 67.4, 67.9, 68.2, 68.6, 71.0, 71.5, 72.4,
72.6, 72.8, 73.0, 73.3, 73.4, 73.6, 74.5, 74.6,
74.7, 74.8, 74.9, 75.3, 75.4, 75.8, 75.9, 77.1,
77.8, 78.1, 78.9, 79.5, 79.6, 80.2, 81.5, 82.1,
82.6, 83.8 (C-2–6, 2I –6I, 3II–6II, 2III–6III, 2IV –
5IV, 2V–5V, OCH2CH2N3, 15 CH2Ph), 97.8
and 98.1 (C-1IV, 1V), 101.5, 102.1, 103.4, 103.8
(C-1, 1I, 1II, 1III), 126.0–139.0 (aromatic C).
Ac2O (800 mL) was added to the above free
amine (114 mg, 50 mmol) in 4:1 MeOH–
CH2Cl2 (10 mL). After 3.5 h the reaction
mixture was concentrated and co-evaporated
with toluene. Purification by chromatography
(3:1“2:1 toluene–EtOAc) yielded 2-azi-
D-
L
D
D
D
pyranoside (11).—Tetrasaccharide 6 (791 mg,
463 mmol), diol 10 (559 mg, 649 mmol) and 4
,
A MS (2 g) in CH2Cl2 (12 mL) were stirred
under argon at −65 °C. After 40 min, a
slurry of DMTST (2.388 g, 9.3 mmol) in
CH2Cl2 (10 mL) was added via a syringe. The
reaction was left to reach −25 °C, at which it
was kept overnight, then stirred at room tem-
perature for 1 h, when Et3N (4 mL) was
added, and the mixture was filtered through
Celite, concd and chromatographed (4:1“2:1
light petroleum bp 45–60 °C–EtOAc) to give
11 (836 mg, 72%); [h]D −30° (c 1.0 CHCl3);
NMR (CDCl3): 13C, l 16.2, 16.4 (C-6IV, 6V),
50.7 (OCH2CH2N3), 56.3 (C-2II), 66.4, 66.9,
67.4, 67.6, 67.7, 67.8, 68.5, 70.6, 71.0, 71.3,
71.5, 71.8, 72.6, 72.6, 72.8, 72.9, 73.2, 73.5,
73.8, 74.5, 74.6, 74.7, 74.8, 75.1, 75.2, 75.3,
75.5, 77.6, 77.8, 78.0, 79.3, 80.2, 81.4, 82.6,
82.9, 83.4 (C-2–6, 2I–6I, 3II –6II, 2III–6III, 2IV–
5IV, 2V–5V, OCH2CH2N3, 15 CH2Ph), 98.0,
98.2, 98.6, 100.4, 101.6, 103.3 (C-1, 1I, 1II, 1III,
1IV, 1V), 122.9–139.3 (aromatic C), 167.0 and
doethyl
osyl)-(1“2)-(3,4,6-tri-O-benzyl-b-
pyranosyl)-(1“3)-[(2,3,4-tri-O-benzyl-a-
fucopyranosyl)-(1“4)]-(6-O-benzyl-2-deoxy-
(2,3,4-tri-O-benzyl-a-
L
-fucopyran-
D
-galacto-
L
-
2 - acetamido - b -
D
- glucopyranosyl) - (1“3)-
-galactopyranosyl)-(1“4)-
(2,6-di-O-benzyl-b-
D
2,3,6-tri-O-benzyl-b- -glucopyranoside (85
D
mg, 73%); NMR (CDCl3): 13C, l 16.3, 16.4
(C-6IV, 6V), 23.3 (NHCOCH3), 50.9 (OCH2-
CH2N3), 56.6 (C-2II), 66.7, 67.4, 67.9, 68.1,
68.2, 68.5, 71.3, 71.5, 72.0, 72.6, 72.9, 73.1,
73.3, 73.4, 73.6, 74.2, 74.5, 74.6, 74.8, 74.9,
75.1, 75.3, 75.4, 76.1, 77.5, 77.9, 79.3, 79.6,
80.2, 81.5, 81.6, 82.5, 83.7 (C-2–6, 2I –6I, 3II –

A. Chernyak et al. / Carbohydrate Research 329 (2000) 309–316
315
6II, 2III–6III, 2IV –5IV, 2V–5V, OCH2CH2N3, 15
CH2Ph), 97.7, 97.9 (C-1IV, 1V) 101.2, 101.8,
102.0, 103.4 (C-1, 1I, 1II, 1III), 126.1–139.0
(aromatic C) and 169.6 (NHCOCH3). This
compound (85 mg, 35 mmol) was dissolved in
10:15:2 EtOH (95%)–EtOAc–HOAc (60% aq)
(17 mL) and 10% Pd–C (150 mg) was added.
Hydrogenolysis was carried out under H2 at
120 psi for 2 days. The suspension was filtered
through silanated Celite, which was washed
with EtOAc, EtOH and water. The combined
filtrate was concd and co-evaporated with
EtOH–toluene and toluene. The residue was
purified on a Biogel P2 column eluted with
water (1% n-BuOH)) to give 12 (24 mg, 67%)
after lyophilization; [h]D −49° (c 1.0 water);
NMR (D2O): 13C (acetone, l 31.0), l 16.1,
16.2 (C-6IV, 6V), 23.0 (NHCOCH3), 40.2
(OCH2CH2N3), 56.5 (C-2II), 60.2, 60.6, 61.7,
62.3, 66.5, 66.9, 67.7, 68.5, 69.0, 69.3, 69.4,
69.8, 70.1, 70.8, 72.5, 72.7, 73.4, 74.3, 74.9,
75.1, 75.4, 75.5, 75.5, 75.5, 75.8, 77.2, 78.8,
82.2 (C-2–6, 2I –6I, 3II –6II, 2III–6III, 2IV –5IV,
2V–5V, OCH2CH2N3), 98.4, 100.2, 101.2,
102.6, 103.6, 103.8 (C-1, 1I, 1II, 1III, 1IV, 1V),
was subsequently washed with water (20 mL).
The product was eluted with a step-wise gradi-
ent of MeOH in water (10%, 1 mL of eluent
each step) collecting fractions of 1 mL. The
product was usually eluted with 10–40% aq
MeOH. The pooled fractions were concd and
dried to yield 90–100% of the oligosaccharide-
squarate derivative.
General procedure for coupling to HSA.—
The required amount of HSA (Sigma) was
dissolved in a Labassco buffer (1 mL, pH
10.0). Then, with stirring, the oligosaccharide-
squarate derivative was added as a soln in the
same buffer (1 mL). The mixture was stirred
for 16 h at rt and then dialyzed against 15
changes of deionized water in an Amicon
ultrafiltration cell (10 mL) with PM-10 mem-
brane, filtered through Millex-GS 0.22 mm
filter (Millipore), and lyophilized to give the
glycoprotein.
Acknowledgements
Financial support from EU (Contract Num-
ber BIO 4 CT 960158), from the Swedish
Foundation for Strategic research (the Glyco-
conjugates in Biological Systems programme)
and from the Swedish Natural Science Re-
search Council are gratefully acknowledged.
We thank Professor Per-Erik Jansson at the
Clinical Research Center, University College
of South Stockholm, Sweden, for running the
MALDI-TOF spectra.
1
and 174.6 (CO); H (25 °C, acetone, l 2.225),
l 1.26 (t×2, 6 H, H-6IV, 6V), 2.06 (s, 3 H,
NHCOCH3), 3.27 (t, 2 H), 3.37 (m, 1 H),
3.52–3.99 (m, 32 H), 4.13 (m, 3 H), 4.34 (q, 1
H), 4.42 (d, 1 H, J1,2 =7.8 Hz), 4.54 (d, 1 H,
J1,2 =8.3 Hz), 4.60 (d, 1 H, J1,2 =8.8 Hz), 4.66
(d, 1 H, J1,2=7.8 Hz) (H-1, 1I, 1II, 1III), 4.87
(q, 1 H), 5.02 (d, 1 H, J1,2=3.9 Hz), 5.15 (d,
1 H, J1,2 =3.9 Hz) (H-1IV, 1V). HRMS Calcd
for C40H71O29N2 [M+H]+: 1043.4143. Found
1043.4159.
General procedure for deri6atization of
spacer glycoside with diethyl squarate.—Di-
ethyl squarate (0.95 equiv) as a solution in
MeOH (10/50 mL of MeOH) was added with
stirring to a solution of the spacer glycoside
(1–7 mg) in MeOH (in some cases in aq
MeOH, 1 mL). After 12 h, TLC on silica
gel (4:3:3:2 CHCl3–MeOH–AcOH–water)
showed complete conversion to a faster mov-
ing squarate derivative. In some cases addition
of Et3N (up to 1 equiv) was required to com-
plete the conversion. The mixture was concd,
dissolved in Me2SO (0.4 mL), and diluted with
water (4 mL). The soln was passed through a
Sep-Pak C18 Plus (Waters) cartridge which
References
[1] U. Spohr, O. Hindsgaul, R.U. Lemieux, Can. J. Chem.,
63 (1985) 2644–2652.
[2] U. Spohr, R.U. Lemieux, Carbohydr. Res., 174 (1988)
211–237.
[3] P.V. Nikrad, H. Beierbeck, R.U. Lemieux, Can. J.
Chem., 70 (1992) 241–253.
[4] T. Bore´n, P. Falk, K.A. Roth, G. Larsson, S. Normark,
Science, 262 (1993) 1892–1895.
[5] R.U. Lemieux, D.A. Baker, D.R. Bundle, US Patent
4,137,401, 1979.
[6] T. Bore´n, personal communication.
[7] M. Mammen, S.-K. Choi, G.M. Whitesides, Angew.
Chem., Int. Ed. Engl., 37 (1998) 2754–2794.
[8] L.F. Tietze, C. Schro¨ter, S. Gabius, U. Brink, A. Goer-
lach-Graw, H.-J. Gabius, Bioconjug. Chem., 2 (1991)
148–153.

316
A. Chernyak et al. / Carbohydrate Research 329 (2000) 309–316
[9] L.F. Tietze, M. Arlt, M. Beller, K.-H. Gluesenkamp, E.
[16] M. Dejter-Juszynski, H.M. Flowers, Carbohydr. Res., 18
(1971) 219–226.
[17] R.U. Lemieux, K.B. Hendriks, R.V. Stick, K. James, J.
Am. Chem. Soc., 97 (1975) 4056–4062.
[18] V.P. Kamath, P. Diedrich, O. Hindsgaul, Glycoconjugate
J., 13 (1996) 315–319.
[19] V. Pozsgay, E.P. Dubois, L. Panell, J. Org. Chem., 62
(1997) 2832–2846.
[20] J. Zhang, A. Yergey, J. Kowalak, P. Kovac, Carbohydr.
Res., 313 (1998) 15–20.
Ja¨hde, M.F. Rajewsky, Chem. Ber., 124 (1991) 1215–1221.
[10] J.T. Randolph, S. Danishefsky, Angew. Chem., Int. Ed.
Engl., 33 (1994) 1470–1473.
[11] J.T. Randolph, K.F. McClure, S. Danishefsky, J. Am.
Chem. Soc., 117 (1995) 5701–5711, 5712–5719.
[12] H. Lo¨nn, Carbohydr. Res., 139 (1985) 105–113.
[13] N.K. Kotchetkov, B.A. Dmitriev, O.S. Chizhov, E.M.
Klimov, N.N. Malysheva, A.Y. Chernyak, N.E. Bayra-
mova, V.I. Torgov, Carbohydr. Res., 33 (1974) C5–C7.
[14] O. Hindsgaul, T. Norberg, J.L. Pendu, R.U. Lemieux,
Carbohydr. Res., 109 (1982) 109–142.
[21] A. Chernyak, unpublished results.
[22] S.-B. Nilsson, H. Lo¨nn, T. Norberg, Glycoconjugate J., 6
(1989) 21–34.
[15] P.J. Garegg, H. Hultberg, S. Wallin, Carbohydr. Res., 108
(1982) 97–101.
.