Synthesis of the Lewis b pentasaccharide and a HSA-conjugate thereof

Article abstract of DOI:10.1016/j.tet.2010.07.036

Helicobacter pylori, a gastric pathogen, binds to various blood group antigens, including the Lewis types, present in the gastric tissue and a relation between the presentation of the ligands and the overall strength of binding has been assumed. Synthetic Lewis b tetra- and hexasaccharide conjugates are available but not the analogous pentasaccharide. An efficient synthesis of the amino spacer equipped Lewis b pentasaccharide, 3-aminopropyl α-l-fucopyranosyl-(1→2)-β-d-galactopyranosyl-(1→3) -[α-l-fucopyranosyl-(1→4)]-2-acetamido-2-deoxy-β-d- glucopyranosyl-(1→3)-β-d-galactopyranoside, is presented to enable further investigation of the carbohydrate recognition process of H. pylori.

Full text of DOI:10.1016/j.tet.2010.07.036

Tetrahedron 66 (2010) 7850e7855
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of the Lewis b pentasaccharide and a HSA-conjugate thereof
,
a,
Viviane Fournière a, Linnéa Skantz b, Ferenc Sajtos b, Stefan Oscarson c , Martina Lahmann
*
*
a The School of Chemistry, University of Bangor, Alun Roberts Building, Deiniol Road, Bangor, Gwynedd LL57 2UW, UK
b Department of Organic Chemistry, Arrhenius Laboratory, University of Stockholm, S-106 91 Stockholm, Sweden
c Centre for Synthesis and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
Helicobacter pylori, a gastric pathogen, binds to various blood group antigens, including the Lewis
types, present in the gastric tissue and a relation between the presentation of the ligands and the
overall strength of binding has been assumed. Synthetic Lewis b tetra- and hexasaccharide conjugates
are available but not the analogous pentasaccharide. An efficient synthesis of the amino spacer
Received 4 February 2010
Received in revised form
29 June 2010
Accepted 15 July 2010
Available online 22 July 2010
equipped Lewis
b
pentasaccharide, 3-aminopropyl
a-
L-fucopyranosyl-(1/2)-
b
-D
-galactopyranosyl-
-galactopyr-
(1/3)-[ -fucopyranosyl-(1/4)]-2-acetamido-2-deoxy-
a
-
L
b
-
D
-glucopyranosyl-(1/3)-b-D
anoside, is presented to enable further investigation of the carbohydrate recognition process of
H. pylori.
Keywords:
Glycosylation
Regioselective 3,4-benzylidene opening
Glycoconjugates
Ó 2010 Elsevier Ltd. All rights reserved.
Helicobacter pylori
Carbohydrate synthesis
1. Introduction
palmitoylphosphatidylethanolamine (DPPE), concluding that the
recognition is affected by the carrier. The DPPE conjugate was
The spiral-shaped Gram-negative bacterium Helicobacter pylori,
a gastric pathogen, binds to various blood group antigens, including
the Lewis types, present in the gastric tissue.1,2 In developed
countries, approximately 50% of the population over the age of 50
are infected with this bacterium, while, in contrast, such infection is
uncommon in children. In developing nations, 70e90% of the
population are carrying H. pylori and virtually everyone gets
infected during childhood.3,4
The adhesion process of H. pylori bacteria to Lewis b structures is
mediated by a membrane lectin, the blood group antigen binding
adhesion protein (BabA).5 Synthetic glycoconjugates containing the
Lewis b hexasaccharide ligand are used in affinity chromatography
for purification of the BabA, but analogous Lewis b tetrasaccharide
conjugates are not effective for this purpose, although the free
tetrasaccharide can inhibit the adhesion process. Reductive ami-
nation, the conjugation technique preferably used for preparing
glycoconjugates from native Lewis b hexasaccharide, destroys the
reducing end glucose unit. These glycoconjugates are feasible for
purification purposes, although only a pentasaccharide unit is
available for recognition. Kojima et al.6 investigated the adhesion
properties of Lewis b oligosaccharide conjugates linked to Bovine
serum albumin (BSA), and polyacrylamide (PAA) and
synthesised via reductive amination from the hexasaccharide, but
no further information was given for the purchased conjugates
(Lewis b hexasaccharide BSA-conjugate, Funakoshi, Tokyo, Japan;
Lewis b tetrasaccharide PAA-conjugate, Seikagaku Kogyuo Co.,
Tokyo, Japan). All conjugates were recognised by H. pylori, but the
BSA-conjugate was clearly the best recognised conjugate.
Syntheses of both the Lewis b tetrasaccharide and the hex-
asaccharide have been published. The synthesis of the Lewis
b tetrasaccharide has been elaborated many times, e.g., as the
free reducing oligosaccharide by Matta et al.7 and as methyl
glycoside, first by Lemieux and Spohr8 and later by Kahne and
Yan.9 Solid phase synthesis has been employed by Danishefsky’s
group10 and a large scale approach, reducing chromatography to
a bare minimum, was achieved by Norberg et al.11 Recently an
enzymatic approach has been established.12 Also for the Lewis
b hexasaccharide several approaches have been investigated and
human serum albumin (HSA) conjugates have been prepared.13e16
The pentasaccharide structure (Fig. 1) has been prepared earlier
as a protected intermediate, e.g., in Danishefsky’s glycal approach,
but has never been deprotected and used for synthetic conju-
gates. To further investigate the binding specificity of BabA, we
envisaged using synthetic Lewis pentasaccharide glycoconjugates.
To obtain comparable parameters, we intended to use the same
spacer and conjugation techniques as in earlier studies with the
hexa- and tetrasaccharides.
* Corresponding authors. E-mail addresses: stefan.oscarson@ucd.ie (S. Oscarson),
m.lahmann@bangor.ac.uk (M. Lahmann).
0040-4020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.07.036

V. Fournière et al. / Tetrahedron 66 (2010) 7850e7855
7851
in a maximum yield of 66% after 24 h. Since large amounts of
compound 4 were required, we decided to employ a longer but
more reproducible route. The key step in this approach is the for-
mation of the endo-3,4-benzylidene acetal, which under reductive
conditions can be opened regioselectively to give the unprotected
3-OH product as major regioisomer.18 However, the 3,4-benzyli-
dene acetal cannot be introduced directly into tetraol 2, therefore
the spacer galactoside 2 was first treated with 2,2-dimethoxy-
propane and a catalytic amount of camphorsulfonic acid (CSA) to
form the interim 3,4-acetonide, followed by benzylation and
cleavage of the isopropylidene group, a procedure adapted from the
known 5-azidopentyl galactoside.19 Formation of the endo-3,4-
benzylidene acetal using benzaldehyde dimethyl acetal (PhCH
(OMe)2) and p-toluenesulfonic acid (pTSA) in THF was followed by
reductive opening using standard conditions to give the desired
acceptor 4 as major regioisomer.20,21
Figure 1. The target Lewis b pentasaccharide.
2. Results and discussion
Coupling of acceptor 4 with the donor ethyl 3-O-acetyl-4,6-O-
Attempts to adapt the synthetic approach used for our pre-
vious synthesis of the Lewis b hexasaccharide,16 where a suitable
protected lacto-N-tetraose unit was assembled by a 2þ2 approach
to gain quick access to the desired target pentasaccharide 1 were
hampered by the loss of regioselectivity during the coupling of
the disaccharide donor to the analogous 3-azidopropyl 2,6-di-O-
benzyl galactoside acceptor. Glycosylations, using the 4-O-benzyl
protected derivative 4 as glycosyl acceptor instead, produced
unsatisfactory yields. Therefore, we decided to trial a stepwise
synthesis starting from the reducing-end with acceptor galacto-
side 4.
benzylidene-2-deoxy-2-phthalimido-1-thio-b-D
-glucopyranoside 6,22
using N-iodosuccinimide (NIS)/silver trifluoromethanesulfonate
(AgOTf) as promoter system in CH2Cl2, gave disaccharide 7 in excellent
(93%) yield (Scheme 2). The phthaloyl protecting group ensured good
solubility of the donor and complete b-selectivity during the reaction.
Upon removal of the acetyl group, this disaccharide could serve as
glycosyl acceptor, but to avoid problems with late removal of the
phthaloyl group, experienced in the deprotection sequence of the
hexasaccharide,15 we decided to exchange it for the target acetyl amido
group already at this stage. Treating disaccharide 7 first with ethyl-
enediamine in ethanol at reflux temperature, followed by addition of
sodium methoxide to ensure complete removal of the 3-acetyl group,
At first, galactoside 4 (Scheme 1) was prepared from 3-azido-
propyl
b-D
-galactopyranoside 2.17 Regioselective allylation was
achieved by activating the OH-3 group using dibutyltin oxide fol-
lowed by treatment with allyl bromide and CsF in DMF to give 3-
azidopropyl 3-O-allyl-b-D-galactopyranoside. This material was
Ph
O
O
O
AcO
SEt
benzylated in the same reaction vessel using sodium hydride and
benzyl bromide to give 3 in 74% yield. When we tried to reproduce
this sequence on a multi-gram scale, substantially lower yields
were obtained. Also, the 3,6-di-O-allylated galactoside was pro-
duced as major product when the reaction mixture was co-evap-
orated with toluene after stannylidene formation in MeOH.
Furthermore, removal of the allyl group was not straightforward.
The use of Wilkinson’s catalyst for the isomerisation step produced
only base-line material on the TLC-plates, while PdCl2 in THF or
methanol was very slow. Using a methanol/ethanol (1:1) mixture
gave better results but unidentified side products appeared during
prolonged reaction times. Eventually, acceptor 4 could be isolated
NPhth
6
i
OBn
O
BnO
O
Ph
O
O
R
O
R'O
O(CH2)3N3
OBn
7 R = NPhth, R' = Ac
ii
8 R = NHAc, R' = H
BnO
OBn
O
BnO
SEt
OAc
9
iii
Ph
BnO
BnO
BnO
O
O
OBn
O
OBn
O
O
O
O
O(CH2)3N3
OAc
NHAc
OBn
10
iv, v
BnO
BnO
BnO
OBn
O
OBn
O
OBn
O
HO
O
O
NHAc
O(CH2)3N3
OH
OBn
11
Scheme 1. (i) (1) Bu2SnO, MeOH, reflux, 4 h; (2) AllBr, CsF, DMF, 30 ꢀC, overnight;
(3) BnBr, NaH, DMF, rt, 3 h, 74%; (ii) PdCl2, MeOH/EtOH (1:1), rt, 24 h, 15e66%; (iii) 2,
2-dimethoxypropane, CSA, 56%; (iv) BnBr, NaH, DMF, 66%; (v) TFA (90% aq), CH2Cl2,
95%; (vi) PhCH(OMe)2, pTSA, THF, 95%; (vii)NaBH3CN, HCl/Et2O, THF, 90%.
Scheme 2. i) 4, NIS, AgOTf, 4 A MS, CH2Cl2, ꢁ20 ꢀC, 30 min, 93%; (ii) (1) EtOH/ethyl-
enediamine (10:1), reflux, 2 h; (2) NaOMe/MeOH, reflux, 2 h; (3) Ac2O, MeOH, 0 ꢀC, 1 h,
ꢀ
83% over sequence; (iii) NIS, AgOTf, 4 A MS, CH2Cl2, 0 ꢀC, 30 min, 84%; (iv) (1)
NaBH3CN, HCl/Et2O, 3 A MS, THF, rt, 2 h; (2) NaOMe, MeOH, rt, 74% over two steps.
ꢀ
ꢀ

7852
V. Fournière et al. / Tetrahedron 66 (2010) 7850e7855
and subsequent N-acetylation with acetic anhydride in methanol at
0 ꢀC produced 8 (83%). Coupling between the disaccharide acceptor 8
and the suitably protected thioethyl galactoside donor 9 afforded the
stated. No reference was used for the spectrum in D2O. TLC was
performed on Silica Gel F254 (E. Merck) with detection by UV light
and/or charring with 8% sulfuric acid. Silica gel (0.041e0.063 mm,
Amicon) was used for column chromatography. Mass spectra were
recorded on a Bruker Daltonics MicrOTOF using electrospray
technique. MALDI-TOF spectra were recorded on a Bruker Reflex IV
using 20,40,60-trihydroxy-acetophenone monohydrate (THAP) as
matrix. For conjugation reactions a borate buffer was prepared
from 0.0125 M Na2B4O7$10H2O solution that was adjusted to pH 10
with 0.10 M NaOH.
trisaccharide 10 (84%). All three b-anomeric linkages were confirmed
by the coupling constants between the C-10s and H-10s (162.1 Hz,
162.5 Hz and 158.0 Hz). Regioselective opening of the benzylidene
acetal with NaBH3CN and HCl/Et2O in THF followed by Zemplén
deacetylation provided diol 11 (74% over two steps).
The subsequent coupling (Scheme 3) under halide-assisted
coupling conditions between the diol trisaccharide acceptor 11
and the halide donor 2,3,4-tri-O-benzyl-a-L-fucopyranosyl bro-
mide, prepared in situ by addition of bromine to the glycosylation
mixture already containing acceptor 11, the thioethyl fucoside 12
and tetraethyl ammonium bromide, furnished the protected
pentasaccharide 13 in 67% yield. Catalytic hydrogenolysis under
atmospheric hydrogen pressure, using Pd/C in the presence of
1 equiv of 1 M HCl in EtOAc/EtOH/H2O (2:2:1), gave complete
removal of the benzyl ethers and concomitant reduction of the
azido group in 1 day as verified by MALDI-TOF spectrometry
(1-HCl, 82%).
3.2. Synthetic procedures
3.2.1. 3-Azidopropyl 2,4,6-tri-O-benzyl-
b-D-galactopyranoside (4).
3-Azidopropyl -galactopyranoside 2 (2.58 g, 9.8 mmol) and
b-D
dibutyltin oxide (4.88 g, 19.6 mmol) were refluxed in dry MeOH
(50 mL) until the mixture became clear (e4 h). The solvent was
evaporated and the residue was dissolved in dry DMF (50 mL). Allyl
bromide (1.02 mL, 11.8 mmol) and CsF (1.93 g, 12.7 mmol) were
added to this solution and the mixture was stirred overnight. After
ꢀ
Scheme 3. i) 1. Et4NBr, CH2Cl2/DMF (10:1), 4 A MS, rt; (2) Br2, rt, 72 h, 67%; (ii) H2 (1 atm), 10% Pd/C, HCl, EtOAc/EtOH/H2O, rt, 24 h, 82%.
Since disuccinimidyl suberate (DSS) has been successfully
employed for the conjugation of the Lewis b hexasaccharide to HSA
in our group,16 we decided to follow the same two-step methodol-
ogy. An eightfold excess, to prevent dimerisation, of DSS, prepared
from suberic acid and N-hydroxy succinimide was reacted with
pentasaccharide 1 in DMSO, in the presence of triethylamine. After
complete conversion (30 mine1 h), the crude product was applied
onto a C18 column, pre-treated with DCM. Residual reagents and
DMSO were removed by elution with DCM, while the DSS/active-
ester conjugate was retained due to its poor solubility in the eluting
solvent. Elutionwith water provided eventually the DSS/active-ester
conjugate. This material was conjugateddmonitored by MALDI-
TOFdwith HSA in a buffer (pH 10) to give the Lewis b penta-
saccharide/HSA conjugate, with an average loading of 10 sugar res-
idues per protein molecule, ready for biological investigation.
In conclusion, a short and efficient way was developed for the
synthesis of the Lewis b pentasaccharide. DSS methodology was
successful to provide the Lewis b pentasaccharide/HSA conjugate.
that, NaH (60%; 2.35 g, 58.8 mmol) and BnBr (5.2 mL, 44.1 mmol)
were added and the reaction mixture was again left overnight. After
concentration and co-evaporation with toluene (3ꢂ15 mL), the res-
idue was dissolved in CH2Cl2 (300 mL), washed with 10% KI
(2ꢂ50 mL) andwater (2ꢂ50 mL), dried, concentrated andpurified by
silica gel flash chromatography (95:5 toluene/EtOAc) to produce 3-
azidopropyl 3-O-allyl-2,4,6-tri-O-benzyl-b-D-galactopyranoside (3)
22
(4.16 g, 74%); [
a
]
ꢁ10 (c 1, CHCl3); NMR (CDCl3): 13C,
d 29.4
D
(OCH2CH2CH2N3), 48.5 (OCH2CH2CH2N3), 66.6 (C-6), 69.0
(OCH2CH2CH2N3), 72.0 (OCH2CH]CH2), 73.4 (C-4), 73.6 (C-5), 73.7,
74.6 and 75.4 (3OCH2Ph), 79.6 (C-2), 82.2 (C-3), 104.0 (C-1), 116.8
(OCH2CH]CH2), 127.7e138.9 (aromatic C), 135.1 (OCH2CH]CH2);
1H,
d 1.86 (m, 2H, OCH2CH2CH2N3), 3.38 (t, 2H, OCH2CH2CH2N3), 3.41
(dd, 1H, J3,4¼2.8 Hz, H-3), 3.51e3.61 (m, 4H, H-5,60 and
OCH2CH2CH2N3), 3.74 (dd, 1H, J2,3¼8.0 Hz, H-2), 3.85 (d, 1H, H-4),
3.95 (m,1H, H-6), 4.18 (m, 2H, OCH2CH]CH2), 4.31 (d,1H, J1,2¼7.6 Hz,
H-1), 4.40 and 4.35 (2ꢂd, 2H, OCH2Ph), 4.60 and 4.92 (2ꢂd, 2H,
OCH2Ph), 4.77 and 4.83 (2ꢂd, 2H, OCH2Ph), 5.18 and 5.32 (2ꢂdq, 2H,
OCH2CH]CH2), 5.93 (m, 1H, OCH2CH]CH2), 7.25e7.39 (m, 15H, ar-
omatic H). Compound 3 (3.90 g, 6.80 mmol) was then dissolved in
MeOH/EtOH (1:1, 40 mL) and a catalytic amount of PdCl2 was added
to the solution. The reaction mixture was stirred for 24 h at room
temperature. After removal of the catalyst by filtration, the mixture
3. Experimental
3.1. General methods
Organic solutions were dried over Na2SO4 before concentration,
which was performed under reduced pressure at <40 ꢀC (bath).
NMR spectra were recorded at 25 ꢀC at 300 or 400 MHz (Varian) or
500 MHz (Bruker) (1H) or 75, 100 or 125 MHz (13C), respectively,
CDCl3, D2O or at ambient temperature if not otherwise stated. All
proton and carbon NMR spectra in CDCl3 were referenced to the
was concentrated and purified by silica gel flash chromatography
22
(9:1 toluene/EtOAc) to yield
4
d
(2.39 g, 66%);
29.4 (OCH2CH2CH2N3), 48.5
[
a
]
þ4
D
(c 1, CHCl3); NMR (CDCl3): 13C,
(OCH2CH2CH2N3), 66.6 (C-6), 68.8 (OCH2CH2CH2N3), 73.6, 74.9 and
75.1 (3OCH2Ph), 73.8, 74.2 and 79.8 (C-2,3,5), 75.6 (C-4), 103.9 (C-1),
127.9e138.5 (aromatic C); 1H,
d
1.84e1.97 (m, 2H, OCH2CH2CH2N3),
chloroform signal (1H
d C d 77.17 ppm) if not otherwise
7.27 ppm, 13
2.26 (d,1H, J3,OH¼5.0 Hz, OH), 3.41 (t, 2H, J¼6.8 Hz, OCH2CH2CH2N3),

V. Fournière et al. / Tetrahedron 66 (2010) 7850e7855
7853
3.562e3.68 (m, 6H, H-2,3,5,60 and OCH2CH2CH2N3), 3.88 (d, 1H,
J3,4¼3.2 Hz, H-4), 3.98e4.02 (m, 1H, H-6), 4.35 (d, 1H, J1,2¼7.6 Hz,
H-1), 4.47 (d, 2H,J¼11.8 Hz, OCH2Ph), 4.51 (d, 2H, J¼11.8 Hz, OCH2Ph),
4.66 (d, 2H, J¼11.4 Hz, OCH2Ph), 4.79 (d, 2H, J¼11.7 Hz, OCH2Ph), 4.93
(d, 2H, J¼11.7 Hz, OCH2Ph), 7.27e7.36 (m, 15H, aromatic H); HR-ESI
calcd for C30H35N3O6Na [MþNa]þ 556.2418. Found 556.2419.
(8). Disaccharide 7 (550 mg, 576
m
mol) in EtOH/ethylenediamine
(10:1, 18 mL) was stirred at reflux temperature for 2 h and then
treated with NaOCH3/MeOH (1 M, 1 mL) for an additional 2 h. The
reaction mixture was concentrated, then the residue was dissolved
in MeOH (20 mL), cooled to 0 ꢀC and acetic anhydride (2 mL) was
added. After 1 h, the solution was concentrated and purified by
silica gel flash chromatography (1:1 toluene/EtOAc) to give 8
22
(394 mg, 83%); [
a
]
ꢁ32 (c 1, CHCl3); NMR (CDCl3): 13C,
d 22.9
3.2.1.1. Alternative preparation. 3-Azidopropyl 2,4,6-tri-O-ben-
D
zyl-
O-benzylidene-
2,6-di-O-benzyl-3,4-dihydroxy-
b
-d-galactopyranoside (4) via 3-azidopropyl 2,6-di-O-benzyl-3,4-
-d-galactopyranoside (5). A mixture of 3-azidopropyl
-galactopyranoside (1.33 g,
(NHCOCH3), 29.3 (OCH2CH2CH2N3), 48.4 (OCH2CH2CH2N3), 59.4 (C-
2I), 66.6 (2C), 68.6, 68.8, 72.8, 73.7 (2C), 74.1, 74.7, 75.7, 79.3, 81.6
and 81.7 (C-2,3,4,5,6, C-3I,4I,5I,6I, OCH2CH2CH2N3 and 3OCH2Ph),
102.1, 102.8 and 104.0 (C-1, C-1I and CHPh), 126.5e138.7 (aromatic
b
b-D
3.01 mmol), benzaldehyde dimethyl acetal (2.3 mL, 15 mmol) and
pTSA (172 mg, 0.9 mmol) in dry THF (30 mL) was stirred at room
temperature. After 2 h, TLC (toluene/EtOAc 6:1) showed complete
conversion and the reaction was quenched with Et3N (1 mL). After
concentration, the crude mixture was purified by silica gel chroma-
tography (9:1/6:1 toluene/EtOAc) to give 3-azidopropyl 2,6-di-O-
C), 172.7 (NHCOCH3); 1H,
d 1.54 (s, 3H, NCOCH3), 1.80e1.93 (m, 2H,
OCH2CH2CH2N3), 3.35 (t, 2H, J¼6.7 Hz, OCH2CH2CH2N3), 3.43e3.50
(m,1H), 3.52 (dd,1H, J¼8.6, 6.2 Hz), 3.56e3.65 (m, 5H), 3.74 (dd,1H,
J¼9.9, 3.0 Hz), 3.77e3.86 (m, 3H), 3.93 (d, 1H, J¼2.8 Hz), 3.95e4.01
(m, 1H), 4.37 (d, 1H, J1,2¼7.5 Hz, H-1), 4.38 (dd, 1H, J¼10.3, 5.0 Hz),
4.41 (d, 1H, J¼11.8 Hz, OCH2Ph), 4.45 (d, 1H, J¼11.8 Hz, OCH2Ph),
4.61 (d, 1H, J¼11.7 Hz, OCH2Ph), 4.65 (d, 1H, J¼12.6 Hz, OCH2Ph),
4.76 (d, 1H, J1,2¼8.3 Hz, H-1I), 4.85 (d, 1H, J¼11.7 Hz, OCH2Ph), 5.02
(sb, 1H), 5.12 (d, 1H, J¼12.6 Hz, OCH2Ph), 5.53 (d, 1H, J¼5.0 Hz), 5.59
(s, 1H, CHPh), 7.28e7.43 (m, 2H, aromatic H), 7.50e7.54 (m, 18H,
aromatic H); HR-ESI calcd for C45H52N4O11Na [MþNa]þ 847.3525.
Found 847.3300.
benzyl-3,4-O-benzylidene-b-D-galactopyranoside (5) (1.526 g, 95%);
[
a
]
22 þ24 (c 1, CH2Cl2); NMR (CDCl3): 13C,
d 29.5 (OCH2CH2CH2N3), 48.6
D
(OCH2CH2CH2N3), 66.6 (C-6), 69.6 (OCH2CH2CH2N3), 73.2.3, 73.7, 73.9,
76.4, 79.0 and 80.4 (2OCH2Ph and C-2,3,4,5), 103.1 and 104.8 (C-1 and
CHPh), 125.6e138.4 (aromatic C); 1H,
d 1.91e2.03 (m, 2H,
OCH2CH2CH2N3), 3.47 (t, 2H, J¼6.8 Hz, OCH2CH2CH2N3), 3.57 (dd, 1H,
J¼7.7, 6.5 Hz), 3.71 (ddd, 1H, J¼10.1, 6.9, 5.5 Hz, H-5), 3.92 (dd, 1H,
J¼10.1, 6.9 Hz), 3.97 (dd,1H, J¼10.1, 5.5 Hz), 4.03e4.12(m, 2H), 4.31(dd,
1H, J¼6.2, 2.1 Hz), 4.42 (t, 1H, J¼6.3 Hz), 4.47 (d, 1H, J1,2¼7.8 Hz, H-1),
4.66 (d, 2H, J¼12.0 Hz, OCH2Ph), 4.72 (d, 2H, J¼12.0 Hz, OCH2Ph), 4.84
(s, 2H, OCH2Ph), 5.98 (s,1H, CHPh), 7.23e7.52 (m,15H, aromatic H); HR-
ESI calcd for C30H33N3O6Na [MþNa]þ 554.2263. Found 554.2472. A
3.2.4. 3-Azidopropyl (2-O-acetyl-3,4,6-tri-O-benzyl-
anosyl)-(1/3)-(2-acetamido-4,6-O-benzylidene-2-deoxy-
pyranosyl)-(1/3)-2,4,6-tri-O-benzyl- -galactopyranoside (10). A
solution of 8 (272 mg, 330 mol) and ethyl 2-O-acetyl-3,4,6-tri-O-
benzyl-1-thio- -galactopyranoside 9 (247 mg, 460 mol) in dry
CH2Cl2 (1.5 mL) was stirred with powdered 4 A molecular sieves
under argon for 1 h. After cooling it to 0 ꢀC, AgOTf (8 mg, 33
mol)
and NIS (148 mg, 660 mol) were added and the mixture was
stirred for an additional 15 min. The reaction was quenched with
Et3N (250 L) and concentrated. The residue was purified by col-
b
-D
-galactopyr-
b-D-gluco-
b-D
m
solution of 5 (509 mg, 960 mmol) in THF (5 mL) was stirred at room
b
-D
m
ꢀ
temperature with powdered 3 A molecular sieves. After 2 h, NaBH3CN
(362 mg, 5.76 mmol) and more molecular sieves were added and the
stirring continued for a further hour. HCl/Et2O was added dropwise
until the evolution of gas ceased. The reaction was monitored by TLC
(toluene/EtOAc 6:1) and quenched with Et3N after completion. The
mixture was concentrated and purified by flash column chromatog-
raphy (toluene/toluene/EtOAc 6:1) to give 4 (459 mg, 90%).
ꢀ
m
m
m
umn chromatography (toluene/6:1 toluene/EtOAc) to yield 10
22
(362 mg, 84%); [
a
]
ꢁ7 (c 1, CHCl3); NMR (CDCl3): 13C,
d 21.1
D
(OCOCH3), 23.3 (NHCOCH3), 29.3 (OCH2CH2CH2N3), 48.4
(OCH2CH2CH2N3), 58.7 (C-2I), 65.9, 66.6, 68.5, 68.9, 69.0, 71.9, 72.2,
72.3, 73.1, 73.4, 73.5 73.6, 74.5, 74.6, 74.8, 75.8, 76.3, 79.2, 80.5, 81.1
and 81.9 (C-2,3,4,5,6, C-3I,4I,5I,6I, C-2II,3II,4II,5II,6II, OCH2CH2CH2N3
and 6OCH2Ph), 100.7 (JC1,H1¼162.1 Hz), 101.3 (JC1,H1¼162.5 Hz) and
104.0 (JC1,H1¼158.0 Hz) (C-1, C-1I and C-1II), 101.0 (CHPh),
126.3e139.1 (aromatic C),169.6 and 170.6 (OCOCH3 and NHCOCH3);
3.2.2. 3-Azidopropyl
phthalimido- -glucopyranosyl)-(1/3)-2,4,6-tri-O-benzyl-
actopyranoside (7). A mixture of ethyl 3-O-acetyl-4,6-O-benzylidene-
2-deoxy-2-phthalimido-1-thio- -glucopyranoside (508 mg,
1.05 mmol), acceptor 4 (400 mg, 750 mol) and powdered molecular
(3-O-acetyl-4,6-O-benzylidene-2-deoxy-2-
b
-
D
b-D
-gal-
b-D
6
m
ꢀ
sieves (4 A, 2 g) in CH2Cl2 (15 mL) was stirred under argon at room
temperature. After 1 h, the reaction mixture was cooled to ꢁ20 ꢀC,
1H,
d 1.58 (s, 3H, NCOCH3), 1.78e1.90 (m, 2H, OCH2CH2CH2N3), 1.96
AgOTf (29 mg, 113 mmol) and NIS (337 mg, 1.50 mmol) were added.
(s, 3H, OCOCH3), 3.14e3.23 (m, 2H, OCH2CH2CH2N3), 3.30e3.80 (m,
15H), 3.90e4.00 (m, 4H), 4.27 (d, 1H, J¼5.0 Hz), 4.32 (d, 1H,
J1,2¼7.3 Hz, H-1), 4.38e4.71 (m, 10H), 4.87 (d, 1H, J¼12.1 Hz,
OCH2Ph) 4.89 (d, 1H, J¼12.0 Hz, OCH2Ph), 4.90 (d, 1H, J¼11.8 Hz,
OCH2Ph), 5.25 (dd, 1H, J¼10.0, 8.0 Hz), 5.31 (d, 1H, J1,2¼8.3 Hz, H-1),
5.59 (s, 1H, CHPh), 7.24e7.38 (m, 40H, aromatic H); HR-ESI calcd for
C74H82N4O17Na [MþNa]þ 1321.5567. Found 1321.5567.
After a further 30 min, the reaction was quenched with Et3N (50 mL).
The mixture was filtered through Celite, which was washed with
CH2Cl2 (3ꢂ10 mL). The combined filtratewas washed with 10% Na2S2O3
(2ꢂ10 mL) and water (2ꢂ10 mL), dried, concentrated and purified by
silica gel chromatography (9:1 toluene/EtOAc) to produce 7 (663 mg,
93%); [
a
]
22 ꢁ27 (c 1, CHCl3); NMR (CDCl3): 13C,
d 20.7 (OCOCH3), 29.2
D
(OCH2CH2CH2N3), 48.3 (OCH2CH2CH2N3), 55.9 (C-2I), 66.2, 66.6, 68.8,
68.9, 69.7, 73.4, 73.6, 74.0, 74.8, 76.0, 78.6, 79.5 and 81.7 (C-2,3,4,5,6, C-
3I,4I,5I,6I, OCH2CH2CH2N3 and 3OCH2Ph), 100.0, 101.8 and 104.0 (C-1, C-
3.2.5. 3-Azidopropyl
(1/3)-(2-acetamido-6-O-benzyl-2-deoxy-
(1/3)-2,4,6-tri-O-benzyl- -galactopyranoside (11). Compound
10 (50 mg, 38 mol) in dry THF (1 mL), NaBH3CN (17 mg, 269 mol)
(3,4,6-tri-O-benzyl-
b-
D-galactopyranosyl)-
b-D-glucopyranosyl)-
1I and CHPh),126.4e138.9 (aromatic C), 170.2 (OCOCH3).1H,
d 1.61e1.72
b-D
(m, 2H, OCH2CH2CH2N3), 1.88 (s, 3H, OCOCH3), 3.05e3.15 (m, 2H,
OCH2CH2CH2N3), 3.39 (m, 1H), 3.50e3.59 (m, 4H), 3.63e3.86 (m, 4H),
3.92 (d,1H, J¼2.9 Hz), 4.16 (d,1H, J¼11.8 Hz), 4.23 (d,1H, J¼7.6 Hz), 4.37
(dd, 1H, J¼10.2, 8.3 Hz), 4.38e4.48 (m, 4H), 4.60 (d, 1H, J¼11.6 Hz,
OCH2Ph), 4.91 (d,1H, J¼11.6 Hz, OCH2Ph), 5.55 (s,1H, CHPh), 5.77 (d,1H,
J1,2¼8.3 Hz, H-1II), 5.93 (dd, 1H, J¼10.2, 9.1 Hz, H-3II), 7.30e7.41 and
7.45e7.52 (m, 24H, aromatic H), 7.56 (sb,1H), 7.69 (sb,1H); HR-ESI calcd
for C53H54N4O13Na [MþNa]þ 977.3580. Found 977.3513.
m
m
ꢀ
and crushed molecular sieves (3 A) were added and stirred at room
temperature. After 2 h, HCl/Et2O was added dropwise until the
evolution of gas ceased. The reaction was stirred at room temper-
ature and followed by TLC (toluene/ethyl acetate 3:2). At comple-
tion, the reaction was quenched with Et3N (100
mL), evaporated and
purified on a silica gel column (9:1/3:1 toluene/EtOAc) to give
3-azidopropyl (2-O-acetyl-3,4,6-tri-O-benzyl-
(1/3)-(2-acetamido-6-O-benzyl-2-deoxy-
b
b
-
-
D
-galactopyranosyl)-
3.2.3. 3-Azidopropyl (2-acetamido-4,6-O-benzylidene-2-deoxy-
b-D-
D
-glucopyranosyl)-
22
glucopyranosyl)-(1/3)-2,4,6-tri-O-benzyl- -galactopyranoside
b-D
(1/3)-2,4,6-tri-O-benzyl-
b
-D-galactopyranoside
[a
]
þ4 (c 1,
D

7854
V. Fournière et al. / Tetrahedron 66 (2010) 7850e7855
CH2Cl2); NMR (CDCl3): 13C,
d
21.3 (OCOCH3), 23.5 (NHCOCH3), 29.4
residue purified on a Biogel P2 column eluting with H2O (1% n-
22
(OCH2CH2CH2N3), 48.4 (OCH2CH2CH2N3), 57.5, 66.8, 68.0, 69.5, 70.1,
70.4, 71.7, 72.7, 72.8, 73.8, 73.8, 73.9, 74.0, 74.0, 74.1, 74.9, 75.0, 75.4,
76.3, 79.5, 80.3, 81.7 and 83.6(C-2,3,4,5,6, C-3I,4I,5I,6I, C-
2II,3II,4II,5II,6II, OCH2CH2CH2N3 and 7OCH2Ph), 101.1, 101.3 and 103.9
(C-1, C-1I and C-1II), 127.2e139.5 (aromatic C), 170.2 and 171.1
(OCOCH3 and NHCOCH3); HR-ESI calcd for C74H84N4O17Na [MþNa]þ
1323.5724. Found 1323.6362. The obtained compound was then
stirred at room temperature in methanol (5 ml) with NaOMe (1 M,
1 mL) for 6 h. After neutralization with Dowex Hþ ion-exchange
BuOH) to give 1 (36 mg, 82%) after lyophilization; [
a
]
ꢁ6 (c 0.1,
D
H2O); NMR (D2O): 1H, 1.25e1.28 (m, 6H, H-6III and H-6IV),
d
1.99e2.04 (m, 2H, OCH2CH2CH2NH2), 2.06 (s, 3H, NHCOCH3),
3.14e3.19 (m, 2H, OCH2CH2CH2NH2), 3.51e3.63, 3.68e3.95, (m,
22H), 4.02e4.06 (m, 2H), 4.12e4.16 (m, 2H), 4.34 (dd, 1H, J¼13.5,
6.7 Hz), 4.38 (d, 1H, J1,2¼8.0 Hz), 4.61 (d, 1H, J1,2¼8.5 Hz), and 4.66
(d, 1H, J1,2¼7.7 Hz) (H-1, H-1I and H-1II), 4.88 (t, 1H, J¼6.4 Hz), 5.03
(d, 1H, J1,2¼3.8 Hz), and 5.16 (d, 1H, J1,2¼4.0 Hz) (H-1III and H-1IV);
13C,
d
15.4 and 15.4 (C-6III and C-6IV), 22.3 (NHCOCH3), 26.8
resin, the reaction mixture was concentrated to give 11 (36 mg, 74%,
(OCH2CH2CH2N3), 37.7 (OCH2CH2CH2NH2), 55.8 (C-2I), 59.5, 61.0,
61.6, 66.3, 67.1, 67.8, 67.9, 68.3, 68.6, 68.8, 69.1, 69.5, 70.0, 71.8, 72.0,
72.1, 73.7, 74.5, 74.7, 75.2, 76.5, and 81.8 (C-2,3,4,5,6, C-3I,4I,5I,6I, C-
2II,3II,4II,5II,6II, C-2III,3III,4III,5III, C-2IV,3IV,4IV,5IV, OCH2CH2CH2N3),
97.8, 99.6, 100.7, 103.1 and 103.2 (C-1, C-1I, C-1II C-1III and C-1IV),
174.3 (NHCOCH3); HR-ESI calcd for C35H63N2O24Na [MþH]þ
895.3765. Found 895.3750.
22
over two steps); [
a
]
D
þ3 (c 1, CH2Cl2); NMR (CDCl3): 13C,
d 23.0
(NHCOCH3), 29.0 (OCH2CH2CH2N3), 48.2 (OCH2CH2CH2N3), 55.6 (C-
2I), 66.3, 68.7, 69.1, 69.4, 69.9, 71.1, 72.8, 73.2, 73.3, 73.4, 73.5, 73.9,
74.2, 74.4, 74.5, 75.1, 75.7, 77.2, 79.1, 81.3, 81.7 and 86.8 (C-2,3,4,5,6,
C-3I,4I,5I,6I, C-2II,3II,4II,5II,6II, OCH2CH2CH2N3 and 7OCH2Ph), 101.6,
103.5 and 104.7 (C-1, C-1I and C-1II),126.6e139.0 (aromatic C), 171.8
(NHCOCH3); 1H,
d 1.62 (s, 3H, NCOCH3), 1.78e1.86 (m, 2H,
OCH2CH2CH2N3), 3.22 (sb, 1H), 3.32 (t. 2H, J¼6.8 Hz), 3.39e3.45 (m,
2H, OCH2CH2CH2N3), 3.48e3.68 (m, 10H), 3.75e3.79 (m, 4H),
3.87e3.99 (m, 4H), 4.09 (d, 1H, J1,2¼7.7 Hz, H-1), 4.31e4.33 (m, 1H),
4.37e4.47 (m, 5H), 4.54 (s,1H), 4.56 (d,1H, J¼11.8 Hz, OCH2Ph), 4.60
(d, 1H, J¼11.7 Hz, OCH2Ph), 4.66 (d, 1H, J¼12.3 Hz, OCH2Ph), 4.72 (d,
1H, J¼12.2 Hz, OCH2Ph), 4.79e4.82 (m, 2H), 4.90 (d, 1H, J¼11.5 Hz,
OCH2Ph), 4.91 (d, 1H, J¼11.7 Hz, OCH2Ph), 4.97 (d, 1H, J¼12.1 Hz,
OCH2Ph), 5.49 (d, 1H, J¼8.2 Hz), 7.25e7.39 (m, 35H, aromatic H);
HR-ESI calcd for C72H82N4O16Na [MþNa]þ 1281.5618. Found
1281.5574.
3.2.8. 3-Aminopropyl
anosyl)-(1/3)-[( -fucopyranosyl)-(1/4)]-(
(1/3)- -galactopyranoside conjugates. 3.2.8.1. General pro-
cedure for conjugation with DSS. Compound 1 (2 mg, 2.2 mol) and
DSS (6.5 mg, 17.6 mol) were dissolved in dry DMSO (100 L) and
Et3N (1 L) was added. The mixture was gently swirled and moni-
tored by MALDI-TOF spectrometry. When all 1 was converted into
the active-ester (e1 h), the reaction mixture was transferred onto
a C18 column (2 g) that had been pre-washed with CH2Cl2 (8 mL).
DMSO was washed off with CH2Cl2 (8 mL) and then the CH2Cl2 was
removed from the column with a stream of nitrogen. The active
ester was released from the column with double-distilled cold
water and product-containing fractions were pooled and freeze-
dried to produce the ester (2.1 mg, 82%).
(
a
-
L
-fucopyranosyl)-(1/2)-(
b-D-galactopyr-
a
-
L
b-D
-glucopyranosyl)-
b-D
m
m
m
m
3.2.6. 3-Azidopropyl (2,3,4-tri-O-benzyl-
(3,4,6-tri-O-benzyl- -galactopyranosyl)-(1/3)-[(2,3,4-tri-O-ben-
zyl- -fucopyranosyl)-(1/4)]-(2-acetamido-6-O-benzyl-2-deoxy-
-glucopyranosyl)-(1/3)-2,4,6-tri-O-benzyl- -galactopyrano-
side (13). A mixture of 10 (100 mg, 77 mol), ethyl 2,3,4-tri-O-
benzyl-1-thio- -fucopyranoside 12 (111 mg, 231 mol) and
Et4NBr (24 mg, 116 mol) in dry CH2Cl2/DMF (10:1, 1.5 mL) was
stirred with 4 A molecular sieves for 16 h. Bromine (20 L, 39 mol)
a-L-fucopyranosyl)-(1/2)-
b-D
a-L
b
-
D
b-D
m
3.2.9. General procedure for coupling to HSA. HSA (4.1 mg,
a
-L
m
0.06
and added to a solution of the DSS/active-ester conjugate (2.1 mg,
1.8 mol) in double-distilled water (1 mL). The mixture was stirred
mmol, Sigma) was treated with a borate buffer (pH 10, 200 ml)
m
ꢀ
m
m
m
was then added and the reaction stirred at room temperature. After
48 h, TLC (toluene/ethyl acetate 6:1) and Maldi-Tof showed com-
plete conversion of the starting trisaccharide. The crude mixture
for 24 h at room temperature and then transferred to a centrifugal
tube (30 kD, omega membrane, MicrosepÔ, Pall) and centrifuged
(3ꢂ1.5 h) after addition of double-distilled water (2ꢂ1 mL). The
was concentrated and purified on a silica gel column (toluene/6:1
filter residue was dissolved in water (3ꢂ400
mL) and freeze-dried to
22
toluene/EtOAc) to give 13 (112 mg, 67%); [
a
]
D
ꢁ44 (c 1, CHCl3);
yield the conjugate (4.0 mg, 85% calcd with respect to the protein).
MALDI-TOF: 78,570, corresponds to 10 incorporated receptor
saccharides.
NMR (CDCl3): 1H,
d
1.10e1.19 (m, 6H, H-6III and H-6IV) 1.55 (s, 3H,
NCOCH3), 1.69e1.82 (m, 2H, OCH2CH2CH2N3), 3.15 (s, 1H), 3.25 (d,
2H, J¼6.8 Hz), 3.31 (db, 1H J¼6.8 Hz), 3.39e4.02 (m, 26H), 4.23 (d,
1H, J¼7.3 Hz), 4.26e4.79 (m, 28H), 4.87 (d, 1H, J¼11.4 Hz, OCH2Ph),
4.89e4.95 (m, 1H), 5.48 (d, 1H, J¼3.7 Hz, NH), 6.88e7.33 (m, 65H,
Acknowledgements
aromatic H); 13C, 16.3 and 16.5 (C-6III and C-6IV) 23.6 (NHCOCH3),
d
Financial support from the Swedish Research Council (Veten-
skapsrådet), The Royal Society (RG081255), the European Com-
mission (MRTN-CT-2004-005645 GlycoGold), and the Science
Foundation Ireland (Grant Number 08/IN.1/B2067) are gratefully
acknowledged.
29.4 (OCH2CH2CH2N3), 48.5 (OCH2CH2CH2N3), 53.6 (C-2I), 66.6,
66.7, 67.1, 67.5, 68.7, 69.2, 71.3, 71.8, 71.9, 72.8, 73.0, 73.1, 73.3, 73.4,
73.7, 73.8, 74.0, 74.2, 74.6, 74.7, 74.9 (2C), 75.0, 75.2, 75.6, 75.8 (3C),
77.4, 78.1, 78.2, 79.3, 79.5, 80.5, 80.9 and 83.9 (C-2,3,4,5,6, C-
3I,4I,5I,6I,
C-2II,3II,4II,5II,6II,
C-2III,3III,4III,5III,
C-2IV,3IV,4IV,5IV,
OCH2CH2CH2N3 and 13OCH2Ph), 98.1, 98.7, 101.6, 102.0 and 103.9
(C-1, C-1I, C-1II C-1III and C-1IV), 126.3e139.7 (aromatic C), 169.4
(NHCOCH3); HR-ESI calcd for C126H138N4O24Na [MþNa]þ
2114.9632. Found 2114.9703.
Supplementary data
Supplementary data associated with this article can be found in
online version, at doi:10.1016/j.tet.2010.07.036. These data include
MOL files and InChIKeys of the most important compounds
described in this article.
3.2.7. 3-Aminopropyl
anosyl)-(1/3)-[( -fucopyranosyl)-(1/4)]-(
(1/3)- -galactopyranoside (1-HCl). Pentasaccharide 13 (102 mg,
10.0 mol) was dissolved in EtOAc/EtOH (95%)/H2O (2:2:1, 10 mL),
then HCl (1 M, 10 L) and Pd/C (10%, 100 mg) were added. Hydro-
genolysis was carried out under H2 at atmospheric pressure for 1
day. The suspension was filtered through a filter sandwich (5 m/
10 m/20 m pore size), then the filtrate was concentrated and the
(a-
L
-fucopyranosyl)-(1/2)-(b-D-galactopyr-
a
-
L
b-D
-glucopyranosyl)-
b-D
m
References and notes
m
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M.; Alarcon, T.; Lopez-Brea, M.; Nakazawa, T.; Fox, J. G.; Correa, P.; Dominguez-
m
m
m

V. Fournière et al. / Tetrahedron 66 (2010) 7850e7855
7855
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21. An analytical sample of 4 was benzoylated to prove the position of the hydroxyl
group by its significant downfield shift. 4OBz (1H NMR, CDCl3,
J2,3¼10.2 Hz, J3,4¼3.1 Hz, H-3)).
d 5.05 ppm (dd,
22. Kihlberg, J. O.; Leigh, D. A.; Bundle, D. R. J. Org. Chem. 1990, 55, 2860e2863.