Synthesis of fragments of the glycocalyx glycan of the parasite Schistosoma mansoni

Article abstract of DOI:10.1002/1521-3765(20020104)8:1<151::AID-CHEM151>3.0.CO;2-C

The chemical synthesis of α-L-Fucp-(1→3)-β-D-GalpNAc-(1→4)-β-D-GlcpNAc- (1→3)-α-D-GalpO(CH₂)₅NH₂, β-D-GalpNAc-(1→4)-[α-L-Fucp-(1→3)-]β-D-GlcpNAc- (1→3)-α-D-GalpO(CH₂)₅NH₂, and α-L-Fucp-(1→3

Full text of DOI:10.1002/1521-3765(20020104)8:1<151::AID-CHEM151>3.0.CO;2-C

FULL PAPER
Synthesis of Fragments of the Glycocalyx Glycan of
the Parasite Schistosoma mansoni
[a]
[a]
[a]
[b]
[c]
¬
Ka¬roly Agoston, Ja¬nos Kere¬kgya¬rto¬,* Ja¬nos Hajko¬, Gyula Batta, Dirk J. Lefeber,
Johannis P. Kamerling,*[c] and Johannes F. G. Vliegenthart[c]
Abstract: The chemical synthesis of a-l-Fucp-(1 ! 3)-b-d-GalpNAc-(1 ! 4)-b-d-
GlcpNAc-(1 ! 3)-a-d-GalpO(CH2)5NH2, b-d-GalpNAc-(1 ! 4)-[a-l-Fucp-(1 ! 3)-
]b-d-GlcpNAc-(1 ! 3)-a-d-GalpO(CH2)5NH2, and a-l-Fucp-(1 ! 3)-b-d-GalpNAc-
Keywords: carbohydrates
calyx ¥ glycosylation ¥ oligosacchar-
ides ¥ Schistosoma mansoni
¥ glyco-
(1 ! 4)-[a-l-Fucp-(1 ! 3)-]b-d-GlcpNAc-(1 ! 3)-a-d-GalpO(CH2)5NH2 is
de-
scribed. These structures represent fucosylated oligosaccharide fragments of the
glycocalyx glycan of the cercarial stage of the parasite Schistosoma mansoni, and in
protein-conjugated form they are potential diagnostics in the search for antibodies
raised against the glycan in the serum of infected humans.
resistant to, or even invisible to, certain parts of the host×s
Introduction
8]
defence system by several evasion mechanisms.[4
The
Schistosomiasis, a parasitic infection affecting more than 200
million people in tropical and subtropical regions, is caused by
blood-dwelling flukes of the genus Schistosoma. The most
important species of this genus are S. mansoni, S. haematobi-
um, and S. japonicum.[1] The infective parasitic stage, the
cercaria, enters the host through the skin, evoking an
inflammatory response. From this stage, until approximately
three weeks after infection, the parasite, present as a young
schistosomulum, is most susceptible to immune damage.[2] In
the cercarial stage of the life cycle of the parasite, the entire
surface of the parasite is covered by a 1 mm thick, highly
immunogenic, fucose-rich glycocalyx. Recently, the nonre-
ducing terminal sequences of the O-linked carbohydrate
chain as part of the glycocalyx (GCX) were elucidated as
follows:[3]
severest pathology of the infection is caused by the eggs of
the parasite that get stuck in the human body.[9] In order to be
able to treat the infection with chemotherapeutics before the
onset of egg production, an early diagnostic method for
schistosomiasis is required. Serological detection of antibod-
ies, raised against the glycocalyx of the cercarial stage of the
parasite S. mansoni, could certainly be such an early
diagnostic method.
It is speculated that both the fucosyl appendages and the a-
linked galactose residue are likely to be involved in GCX×s
action as a potent immunological modulator.[3] The exact role
of the various domains of the GCX in immunological
stimulation can only be assessed with neoglycoconjugates
prepared from fragments of the GCX. However, the avail-
ability of well-defined oligosaccharides of the GCX from
biological sources in sufficient amounts is limited. To replace
isolated material in both immunological studies and for
diagnostic purposes, a synthet-
An important feature of the Schistosoma infection is that,
after the cercarial stage, the developing worm becomes
ic program for the preparation
of several oligosaccharide frag-
ments present in the GCX was
initiated. In the first part of this project the chemical synthesis
of the nonfucosylated backbone trisaccharide b-d-GalpNAc-
¬
¬
¬
¬
¬
[a] Dr. J. Kerekgyarto, K. Agoston, Dr. J. Hajko
Institute of Biochemistry, Faculty of Sciences
University of Debrecen
(1 ! 4)-b-d-GlcpNAc-(1 ! 3)-a-d-GalpO(CH2)5NH2
has
been undertaken.[10] Here, we report the synthesis of fucosy-
P.O. Box 55, 4010 Debrecen (Hungary)
Fax :(‡36)52-512-913
E-mail: kerek@tigris.klte.hu
[c] Prof. J. P. Kamerling, D. J. Lefeber, Prof. J. F. G. Vliegenthart
Bijvoet Center, Department of Bio-Organic Chemistry
Utrecht University, P.O. Box 80.075
[b] Dr. G. Batta
Research Group of Antibiotics of the Hungarian Academy of Sciences
University of Debrecen
3508 TB Utrecht (The Netherlands)
P.O. Box 70, 4010 Debrecen (Hungary)
Fax :(‡36)52-512-914
Fax : (‡31)30-2540980
E-mail: kame@boc.chem.uu.nl
Chem. Eur. J. 2002, 8, No. 1
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0947-6539/02/0801-0151 $ 17.50+.50/0
151

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J. Kerekgyarto, J. P. Kamerling et al.
FULL PAPER
lated oligosaccharide fragments a-l-Fucp-(1 ! 3)-b-d-
GalpNAc-(1 ! 4)-b-d-GlcpNAc-(1 ! 3)-a-d-Galp (1), b-d-
GalpNAc-(1 ! 4)-[a-l-Fucp-(1 ! 3)]-b-d-GlcpNAc-(1 ! 3)-
a-d-Galp (2) and a-l-Fucp-(1 ! 3)-b-d-GalpNAc-(1 ! 4)-
[a-l-Fucp-(1 ! 3)]-b-d-GlcpNAc-(1 ! 3)-a-d-Galp (3) of
the GCX glycan of the cercarial stage of the parasite S.
mansoni bearing an aminopentyl spacer for conjugation to
carrier molecules (Figure 1).
Results and Discussion
Our initial synthetic strategy was based on the preparation of
a suitably protected backbone trisaccharide b-d-GalpNPhth-
(1 ! 4)-b-d-GlcpNPhth-(1 ! 3)-a-d-Galp (23) carrying an
azidopentyl spacer. Temporary protection of the positions to
be fucosylated with an O-acetyl group for the GalpNPhth
residue and an O-allyl group for the GlcpNPhth moiety
provides a possibility to prepare both tetrasaccharides 1 and 2
as well as the pentasaccharide 3. The remaining hydroxyl
groups are protected with benzyl groups. Relevant mono-
saccharide building blocks for the stepwise synthesis of 23 are
5-azidopentyl 2,4,6-tri-O-benzyl-a-d-galactopyranoside (11),
ethyl 4-O-acetyl-3-O-allyl-6-O-benzyl-2-deoxy-2-phthalimi-
do-1-thio-b-d-glucopyranoside (15), and ethyl 3-O-acetyl-
4,6-di-O-benzyl-2-deoxy-2-phthalimido-1-thio-b-d-galacto-
pyranoside (20) (Scheme 1).
Scheme 1. a) 60% aq HOAc, 608C; b) a,a-dimethoxytoluene, pTsOH;
c) LiAlH4, AlCl3, DCM, Et2O; d) pyridine, Ac2O; e) 5-azidopentanol,
MeOTf, Et2O, 0 8C; f) NaOMe, MeOH, DCM; then TiCl4, 4 ä, DCM;
g) AllBr, NaH, THF; h) Me3NBH3, AlCl3, 4 ä, THF; i) Tf2O, pyridine,
DCM, 08C, then TBAA, DMF; j) K2CO3, MeOH, THF; k) BzlBr, Ag2O,
KI, 4 ä, DMF; l) (Ph3P)3RhCl, EtOH, HCl, acetone; m) Br2, DCM, 08C.
For the synthesis of acceptor 11 the isopropylidene group of
ethyl 2,6-di-O-benzyl-3,4-O-isopropylidene-1-thio-b-d-galac-
topyranoside[10] (4) was removed (!5, quantitative), and a
dioxolane-type endo-3,4-O-benzylidene acetal was stereo-
selectively introduced by a kinetically controlled reaction
(!6, 76%).[11] The reductive opening of the benzylidene ring
with lithium aluminium hydride/aluminium(iii) chloride[12]
resulted in derivative 7 (77%). Conventional acetylation of
7 afforded thioglycoside 8 (96%). Condensation of 8 with
5-azidopentanol[13] in diethyl ether in the presence of methyl
triflate[14] as a promoter gave an inseparable mixture of the
1,2-cis- (9) and 1,2-trans-glycosides (10) in approximately a 1:1
molar ratio (1H NMR data) in a yield of 89%. After removal
of the acetyl function, the anomeric mixture could be
separated by means of column chromatography to give pure
11 (32%) and a mixture of the a- (11) and b- (11b) anomers
(59%). The rapid anomerization of the anomeric mixture
1
(a:b-ratio 3:7; H NMR data) with titanium tetrachloride[15]
resulted in a mixture of 11 and 11b in a 9:1 molar ratio
(1H NMR data). Separation of the mixture by column
chromatography afforded pure 11 in 30% yield (total yield
62%). The presence of the azido group in 11 was established
by IR analysis (nmax 2098 cmÀ1), and the 1,2-cis glycosidic
linkage by 1H NMR analysis (J(H1,H2) ˆ 4.1 Hz).
Ethyl 4,6-O-benzylidene-2-deoxy-2-phthalimido-1-thio-b-
d-glucopyranoside[14] (12) (Scheme 1) was the starting com-
pound for both the glucosamine donor 15 and the galactos-
amine donor 20. For the synthesis of 15, compound 12 was
treated with allyl bromide in the presence of sodium hydride
to give crystalline 13 in a yield of 80%. Regioselective
opening of the 4,6-O-benzylidene ring in 13 with borane-
trimethylamine complex and aluminium(iii) chloride[16] in
tetrahydrofuran yielded 14 (85%). Conventional acetylation
of 14 resulted in the desired
glucosamine donor 15. For the
preparation of the galactos-
amine donor 20, glucosamine
derivative 14 was converted in-
to the corresponding galactos-
amine derivative 16 (epimeriza-
tion at C-4) by an SN2 displace-
ment reaction of O-triflate by
O-acetate. For this, 14 was
treated with triflic anhydride
in dichloromethane in the pres-
ence of pyridine, and then the
4-O-triflated intermediate was
Figure 1. Synthesized oligosaccharide fragments of the glycocalyx glycan.
treated with tetrabutylammoni-
152
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Chem. Eur. J. 2002, 8, No. 1

Glycocalyx Glycan
151 161
um acetate[17] in N,N-dimethyl-
formamide to produce 16 (78%).
The removal of the acetyl func-
tion with potassium carbonate in
tetrahydrofuran/methanol
1:1
(!17, 73%), followed by benzy-
lation using benzyl bromide in
the presence of potassium iodide
and silver(i) oxide in N,N-di-
methylformamide afforded de-
rivative 18 (82%). Compound
18 was O-deallylated with tris-
(triphenylphosphine)rhodium(i)
chloride[18] as catalyst in ethanol
to yield 19 (72%). Conventional
acetylation of 19 resulted in the
desired galactosamine donor 20.
Condensation of 15 with 11 in
diethyl ether in the presence of
methyl triflate as a promoter
afforded disaccharide 21 in a
yield of 78% (Scheme 2). After
removal of the acetyl group with
potassium carbonate in tetrahydrofuran/methanol 1:1 (!22,
89%), acceptor 22 was coupled with galactosamine donor 20
in diethyl ether in the presence of methyl triflate as a
promoter to furnish the aimed backbone trisaccharide 23
(82%), carrying O-benzyl-persistent, and O-allyl and O-
acetyl temporary protecting groups. O-Deacetylation of 23
with potassium carbonate in tetrahydrofuran/methanol 1:1
gave the trisaccharide acceptor 24 (85%).
Scheme 3. a) Br2, DCM, 08C; b) CuBr2, TBABr, 4 ä, DCM, DMF; c) TBABr, 4 ä, DCM, DMF; d) MeOTf,
4 ä, Et2O, 0 8C.
tetrabutylammonium bromide[19] to give tetrasaccharide 27 in
a yield of 32%. Condensation of 24 with 2,3,4-tri-O-benzyl-a-
l-fucopyranosyl bromide (26) under Lemieux conditions[20]
yielded tetrasaccharide 27 in a yield of 42%. Because
fucosylations of 24 could only be achieved with moderate
yields, the introduction of the a-fucosyl linkage at an earlier
stage of the synthesis was also investigated. Thus, tetrasac-
charide 27 was prepared by a 2‡2 block synthesis as follows:
Galactosamine derivative 19 was fucosylated with donor 26
under Lemieux conditions to yield disaccharide donor 28
(35%). Condensation of thioglycoside 28 with disaccharide
acceptor 22 in the presence of methyl triflate resulted in
tetrasaccharide 27 in a yield of 65%. For the deprotection of
tetrasaccharide 27 as well as for the preparation of oligosac-
charides bearing a fucose residue attached to the 3-position of
the glucosamine moiety of the backbone trisaccharide, the
removal of the allyl group in the presence of an azido function
was required. However, under the tested conditions (tris-
(triphenylphosphine)rhodium(i) chloride in ethanol, palladi-
um(ii) chloride/copper(i) chloride, sodium borohydride/iodine,
palladium on carbon/acetic acid), complex reaction mixtures
and low yields were obtained. To overcome this difficulty, our
preliminary synthetic strategy had to be modified. For the
preparation of the tetrasaccharide a-l-Fucp-(1 ! 3)-b-d-
GalpNAc-(1 ! 4)-b-d-GlcpNAc-(1 ! 3)-a-d-GalpO(CH2)5NH2
(1), ethyl 4-O-acetyl-3,6-di-O-benzyl-2-deoxy-2-phthalimido-
1-thio-b-d-glucopyranoside (29)[21] was used, bearing a 3-O-
benzyl instead of a 3-O-allyl group (Scheme 4). Condensation
of 29 with acceptor 11 in the presence of N-iodosuccinimide/
silver triflate[22] afforded disaccharide 30 (83%), which was
O-deacetyled to give 31 (81%). Glycosylation of 31 with
bromosugar 32, obtained from thioglycoside 28, in the
presence of silver triflate afforded tetrasaccharide 33 in
84% yield.
Scheme 2. a) MeOTf, Et2O, 0 8C; b) K2CO3, MeOH, THF.
For the fucosylation of the backbone trisaccharide at the
3-position of the galactosamine residue, both ethyl 2,3,4-tri-O-
benzyl-1-thio-b-l-fucopyranoside (25)[14] and the correspond-
ing bromo sugar 26 were used (Scheme 3). The reaction of
acceptor 24 with 25 was promoted by copper(ii) bromide/
For the preparation of the tetrasaccharide b-d-GalpNAc-
1 ! 4)-[a-l-Fucp-(1 ! 3)-]b-d-GlcpNAc-(1 ! 3)-a-d-GalpO-
Chem. Eur. J. 2002, 8, No. 1
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153

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J. Kerekgyarto, J. P. Kamerling et al.
FULL PAPER
silver triflate afforded 39
(Scheme 6). Disaccharide 39
turned out to be a suitably
protected key intermediate for
both the preparation of tetra-
saccharide 2 and pentasacchar-
ide 3. O-Deallylation of com-
pound 39 using tris(triphenyl-
phosphine)rhodium(i) chloride
as catalyst gave disaccharide
acceptor 40 in 71% yield. At-
tempted fucosylation of accept-
or 40 with donor 26 in the
presence of tetrabutylammoni-
um bromide failed to give ac-
ceptable yields. However, con-
densation of disaccharide 40
with 26 in the presence of silver
triflate resulted in trisaccharide
Scheme 4. a) NIS, AgOTf, 4 ä, DCM, acetonitrile, À158C; b) K2CO3, MeOH, THF; c) AgOTf, 4 ä, DCM,
toluene, À508C.
41 in
a
yield of 48%
(Scheme 7). Coupling of thio-
(CH2)5NH2 (2), disaccharide 34[23] was applied as a fucosylated
glucosamine building block (Scheme 5). Chloroacetylation[24]
of disaccharide 34 gave glycosyl donor 35 in 85% yield.
Condensation of 35 with acceptor 11 in diethyl ether in the
presence of methyl triflate as a promoter yielded trisaccharide
36 (65%). Surprisingly, the removal of the chloroacetyl group
with thiourea failed. However, the chloroacetyl function of 36
could be removed by treatment with potassium carbonate in
tetrahydrofuran/methanol 1:1 to furnish trisaccharide accept-
or 37 in 90% yield. Attempted glycosylation of 37 by using
galactosamine donor 20 with various promoters (methyl
triflate, N-iodosuccinimide silver triflate) failed. Condensa-
tion of 37 with the corresponding bromosugar and trichloro-
acetimidate galactosamine donors (both prepared from 20)
promoted by silver triflate and trimethylsilyl triflate, respec-
tively, also failed (data not shown). The low reactivity of HO-
4' in 37 may be due to its sterically hindered position; this
might explain both the unsuccessful removal of the chloro-
acetyl group of 36 with thiourea and results of attempted
glycosylations under various conditions. To overcome this
difficulty, the order of glycosylation reactions had to be
changed. Condensation of bromosugar 38 (prepared from 20,
Scheme 1) with glucosamine derivative 14 in the presence of
Scheme 6. a) AgOTf, 4 ä, DCM, toluene, À408C; b) [Rh(Ph3P)3Cl],
EtOH, HCl, acetone; c) AcCl, MeOH, DCM, 08C/RT.
glycoside 41 with acceptor 11 in diethyl ether in the presence
of methyl triflate as a promoter yielded tetrasaccharide 42
(64%).
For the preparation of pentasaccharide 3, disaccharide 40
was O-deacetylated with acetyl chloride[25] in methanol/
dichloromethane (3:1) to give disaccharide 43 (Scheme 6).
Condensation of acceptor 43 with fucosyl donor 26
in the presence of silver triflate afforded tetrasaccharide
44 in
a
yield of 75%
(Scheme 8). Finally, coupling
of tetrasaccharide thioglycoside
44 with acceptor 11 af-
forded pentasaccharide 45 in
54% yield.
To furnish target compounds
1, 2, and 3, the phthalimido
functions of tetrasaccharides
33 and 42, and pentasaccharide
45, respectively, were removed
by treatment with ethylenedi-
amine in 1-butanol,[26] and the
resulting products N-acetylat-
ed, then hydrogenolyzed in or-
Scheme 5. a) Chloroacetylchloride, DCM, pyridine, À408C/ À 208C; b) MeOTf, 4 ä, Et2O, 0 8C; c) K2CO3,
MeOH, THF.
154
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Glycocalyx Glycan
151 161
200SY (300 K), Bruker AM-300 (300 K), Bruker DRX-500, and Bruker
AMX-500 spectrometers. Internal references: TMS (d ˆ 0.00 for 1H in
CDCl3), CDCl3 (d ˆ 77.00 for 13C in CDCl3), and acetone (d ˆ 2.225 for 1H
in D2O). Matrix-assisted laser desorption ionisation time-of-flight (MAL-
DI-TOF) spectra were obtained on a Voyager-DETM mass spectrometer.
Samples were dissolved in doubly distilled water (2 mgmLÀ1) and mixed on
the sample plate with the matrix 2,4-dihydroxybenzoic acid (DHB) in
doubly distilled water (10 mgmLÀ1) in a ratio of 1:1.
Ethyl 2,6-di-O-benzyl-1-thio-b-d-galactopyranoside (5): A solution of 4[10]
(11.3 g, 25.4 mmol) in acetic acid/water 6:4 (200 mL) was kept for 1 h at
608C, and was then concentrated and co-concentrated with toluene (3 Â
15 mL). Purification of the residue by column chromatography (CH2Cl2/
EtOAc 95:5) gave 5 (9.87 g, 96%), isolated as a syrup. [a]2D0 ˆ À6.1 (c ˆ 0.8
1
in CHCl3); H NMR (300 MHz, CDCl3): d ˆ 1.32 (t, 3H; SCH2CH3), 2.18
and 2.34 (2 brs, 2H; 2OH, can be deuterated), 2.68 2.83 (m, 2H;
SCH2CH3), 4.02 (dd, 3J(H3,H4) ˆ 3.3 Hz, 3J(H4,H5) < 1 Hz, 1H; H4), 4.42
(d, 3J(H1,H2) ˆ 9.4 Hz, 1H; H1), 4.70 and 4.83 (2 ABq, each 2H; 2PhCH2),
7.11 7.39 (m, 10H; aromatic); elemental analysis calcd (%) for C22H28O5S
(404.16): C 65.32, H 6.98; found: C 65.36, H 6.97.
Scheme 7. a) AgOTf, 4 ä, DCM, sym-collidine, toluene, À308C;
b) MeOTf, 4 ä, Et2O, 0 8C.
Ethyl 2,6-di-O-benzyl-endo-3,4-O-benzylidene-1-thio-b-d-galactopyrano-
side (6): p-Toluenesulfonic acid monohydrate (50 mg) was added to a
stirred solution of 5 (1.00 g, 2.47 mmol) in a,a-dimethoxytoluene (8 mL).
After 6 min, NaHCO3 (100 mg) was added, and the mixture diluted with
CH2Cl2 (200 mL). The organic layer was then washed with water (3 Â
50 mL), dried (MgSO4), filtered, and concentrated. Purification of the
residue by column chromatography (hexane/EtOAc 8:2) gave 6 (925 mg,
76%), isolated as a syrup. [a]2D0 ˆ À18.4 (c ˆ 1.3 in CHCl3); 1H NMR
(300 MHz, CDCl3): d ˆ 1.29 (t, 3H; SCH2CH3), 2.62 2.85 (m, 2H;
SCH2CH3), 4.52 (d, 3J(H1,H2) ˆ 9.4 Hz, 1H; H1), 4.55 4.80 (m, 4H;
2PhCH2), 5.91 (s, 1H; PhCH), 7.23 7.37 (m, 15H; aromatic); elemental
analysis calcd (%) for C29H32O5S (492.10): C 70.70, H 6.55; found: C 70.65,
H 6.58.
Ethyl 2,4,6-tri-O-benzyl-1-thio-b-d-galactopyranoside (7): A mixture of
LiAlH4 (250 mg) and AlCl3 (250 mg) in Et2O (6 mL) was added to a
solution of 6 (750 mg, 1.52 mmol) in CH2Cl2 (6 mL). After 20 min, the
excess of reagent was decomposed with EtOAc (5 mL), and Al(OH)3 was
precipitated with water. The organic layer was decanted, and the residue
washed with EtOAc (2 Â 50 mL). The combined organic solutions were
washed with water (3 Â 50 mL), dried (MgSO4), filtered, and concentrated.
Purification of the residue by column chromatography (hexane/EtOAc 7:3)
gave 7 (580 mg, 77%), isolated as a syrup. [a]2D0 ˆ À2.2 (c ˆ 0.7 in CHCl3);
1H NMR (200 MHz, CDCl3): d ˆ 1.25 (t, 3H; SCH2CH3), 2.19 (brs, 1H;
OH, can be deuterated), 2.68 2.85 (m, 2H; SCH2CH3), 4.41 (d,
3J(H1,H2) ˆ 9.5 Hz, 1H; H1), 4.45 4.90 (m, 6H; 3 PhCH2), 7.25 7.38
(m, 15H; aromatic); elemental analysis calcd (%) for C29H34O5S (494.21):
C 70.42, H 6.93; found: C 70.37, H 6.90.
Scheme 8. a) AgOTf, 4 ä, DCM, sym-collidine, toluene, À308C;
b) MeOTf, 4 ä, Et2O, 0 8C.
Ethyl 3-O-acetyl-2,4,6-tri-O-benzyl-1-thio-b-d-galactopyranoside (8):
der to convert the azido function into an amino group and to
remove the benzyl functions. The identity of the deprotected
oligosaccharides was established by H NMR spectroscopy.
The synthesized compounds in protein-conjugated form are
potential diagnostics in the search for antibodies raised
against the glycan in the serum of infected humans. Results
of interaction studies using surface plasmon resonance
between panels of GCX-specific monoclonal antibodies and
protein-conjugated 1, 2, and 3 will be published elsewhere.
Compound
7 (450 mg, 0.91 mmol) was treated with pyridine/acetic
anhydride 1:1 (20 mL) for 2 h. The mixture was concentrated, and toluene
(3 Â 15 mL) was evaporated from the residue. Purification of the residue by
column chromatography (hexane/EtOAc 8:2) gave 8 (468 mg, 96%),
1
1
isolated as a syrup. [a]2D0 ˆ À28.4 (c ˆ 0.7 in CHCl3); H NMR (300 MHz,
CDCl3): d ˆ 1.30 (t, 3H; SCH2CH3), 1.88 (s, 3H; OAc), 2.75 2.85 (m, 2H;
3
3
SCH2CH3), 3.81 (dd, J(H2,H3) ˆ 9.8 Hz, 1H; H2), 4.01 (dd, J(H4,H5) <
3
1 Hz, 1H; H4), 4.40 4.88 (m, 6H; 3 PhCH2), 4.48 (d, J(H1,H2) ˆ 9.8 Hz,
1H; H1), 4.93 (dd, 3J(H3,H4) ˆ 3.1 Hz, 1H; H3), 7.20 7.40 (m, 15H;
aromatic); elemental analysis calcd (%) for C31H36O6S (536.22): C 69.37, H
6.77; found: C 69.29, H 6.70.
5-Azidopentyl 2,4,6-tri-O-benzyl-a-d-galactopyranoside (11): MeOTf
(310 mL, 2.75 mmol) was added to a mixture of 8 (590 mg, 1.1 mmol),
5-azidopentanol[13] (213 mg, 1.65 mmol), and 4 ä molecular sieves in Et2O
(15 mL) at 08C. After stirring for 5 h, TLC (hexane/EtOAc 8:2) showed the
formation of a new spot (Rf ˆ 0.23). Et3N was added, and the mixture
diluted with CH2Cl2 (100 mL), washed with water, dried, filtered, and
concentrated. Column chromatography of the residue gave an inseparable
mixture of the 1,2-cis- (9) and 1,2-trans-glycosides (10) (590 mg, 89%) in
approximately a 1:1 molar ratio (1H NMR data). The mixture of 9 and 10
(560 mg, 0.93 mmol) was dissolved in MeOH/CH2Cl2 9:1 (15 mL), and
NaOMe was added. After 1 h, the solution was neutralised with DOWEX-
Experimental Section
General methods: Optical rotations were measured at room temperature
with a Perkin Elmer 241 automatic polarimeter. Melting points were
determined on
a Kofler apparatus and are uncorrected. TLC was
performed on Kieselgel 60F254 (Merck) with detection by charring with
50% aqueous sulfuric acid. Column chromatography was performed on
Silica gel 60 (Merck 63 200 mesh). 1H (200, 300, and 500 MHz) and 13C
(50.3 and 125.76 MHz) NMR spectra were recorded with Bruker WP-
‡
50 (H ) resin, filtered, and concentrated. Purification of the residue by
Chem. Eur. J. 2002, 8, No. 1
¹ WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002
0947-6539/02/0801-0155 $ 17.50+.50/0
155

¬
¬ ¬
J. Kerekgyarto, J. P. Kamerling et al.
FULL PAPER
column chromatography (CH2Cl2/EtOAc 97:3) gave a mixture of the a,b-
anomers 11/11b (308 mg, 59%) and pure 11 (179 mg, 32%). A solution of
the mixture of 11 and 11b (308 mg, 0.55 mmol) in CH2Cl2 (5 mL)
containing molecular sieves (4 ä) was stirred for 30 min under Ar. Then,
a solution of TiCl4 (64 mL, 0.58 mmol) in CH2Cl2 (2 mL) was added, and
after 10 min solid NaHCO3 (50 mg) was also added. The mixture was
diluted with CH2Cl2 (100 mL), washed with water (2 Â 20 mL), dried
(MgSO4), filtered, and concentrated. Purification of the residue by column
chromatography (CH2Cl2/EtOAc 95:5) gave 11 (168 mg, 30%; total yield
62%), isolated as a syrup. [a]2D0 ˆ ‡46.0 (c ˆ 0.3 in CHCl3); 1H NMR
(300 MHz, CDCl3): d ˆ 1.37 1.72 (m, 6H; OCH2(CH2)3CH2N3), 2.29 (brs,
new spot. Then, tetrabutylammonium acetate (1.95 g, 6.48 mmol) and
DMF (5 mL) were added at 08C. After 5 h, an additional amount of
tetrabutylammonium acetate (1.95 g, 6.48 mmol) was added, and the
reaction was stirred overnight at room temperature. Then, the mixture was
diluted with EtOAc (200 mL), washed with 10% aq NaCl (3 Â 30 mL),
dried (MgSO4), filtered, concentrated, and co-concentrated with toluene.
Purification of the residue by column chromatography (hexane/EtOAc 7:3)
afforded 16 (1.08 g, 78%), isolated as a syrup. [a]2D0 ˆ ‡24.1 (c ˆ 0.6 in
1
CHCl3); H NMR (200 MHz, CDCl3): d ˆ 1.22 (t, 3H; SCH2CH3), 2.14 (s,
3H; OAc), 2.60 2.79 (m, 2H; SCH2CH3), 4.39 (dd, 3J(H3,H4) ˆ 3.0 Hz,
1H; H3), 4.51 (dd, 3J(H2,H3) ˆ 10.0 Hz, 1H; H2), 4.55 (ABq, 2H; PhCH2),
3
ˆ
1H; OH, can be deuterated), 3.21 (t, 2H; CH2N3), 3.81 (dd, J(H2,H3) ˆ
4.94 5.12 (m, 2H; H2C CHCH2O), 5.34 (d, 3J(H1,H2) ˆ 10.0 Hz, 1H;
10.0 Hz, 1H; H2), 4.40 4.85 (m, 6H; 3 PhCH2), 4.81 (d, 3J(H1,H2) ˆ
H1), 5.45 5.61 (m, 1H; H2C CHCH2O), 5.61 (dd, 3J(H4,H5) < 1 Hz,
ˆ
4.1 Hz, 1H; H1), 7.25 7.40 (m, 15H; aromatic); IR (KBr): nÄmax
ˆ
1H; H4), 7.29 7.48 and 7.73 7.91 (m, 9H; aromatic); elemental analysis
calcd (%) for C28H31NO7S (525.18): C 63.98, H 5.94; found: C 63.96,
H 5.92.
2098 cmÀ1 (N3); elemental analysis calcd (%) for C32H39N3O6 (561.28): C
69.41, H 7.00; found: C 69.44, H 7.05.
For analytical purposes 15 mg of 11b was also collected. [a]2D0 ˆ ‡2.3 (c ˆ
0.6 in CHCl3); 1H NMR (300 MHz, CDCl3): d ˆ 1.35 1.75 (m, 6H;
OCH2(CH2)3CH2N3), 2.29 (brs, 1H; OH, can be deuterated), 3.20 (t, 2H;
CH2N3), 4.33 (d, 3J(H1,H2) ˆ 7.1 Hz, 1H; H1), 4.48, 4.69, and 4.82 (3ABq,
each 2H; 3PhCH2), 7.20 7.40 (m, 15H; aromatic).
Ethyl 3-O-allyl-6-O-benzyl-2-deoxy-2-phthalimido-1-thio-b-d-galactopyr-
anoside (17): Potassium carbonate (521 mg, 3.77 mmol) was added to a
solution of 16 (990 mg, 1.88 mmol) in MeOH/THF 1:1 (10 mL). After
stirring for 4 h, the mixture was diluted with CH2Cl2 (100 mL), washed with
10% aq NaCl (3 Â 10 mL), dried (MgSO4), filtered, and concentrated.
Purification of the residue by column chromatography (CH2Cl2/EtOAc
95:5) gave 17 (664 mg, 73%), isolated as a syrup. [a]2D0 ˆ ‡26.5 (c ˆ 0.4 in
CHCl3); 1H NMR (200 MHz, CDCl3): d ˆ 1.19 (t, 3H; SCH2CH3), 2.55
2.81 (m, 3H; SCH2CH3 and OH, the OH can be deuterated), 4.21 (dd,
3J(H4,H5) < 1 Hz, 1H; H4), 4.32 (dd, 3J(H3,H4) ˆ 3.0 Hz, 1H; H3), 4.59
Ethyl 3-O-allyl-4,6-O-benzylidene-2-deoxy-2-phthalimido-1-thio-b-d-glu-
copyranoside (13): A solution of 12[14] (3.74 g, 8.47 mmol) and allyl
bromide (3.58 mL, 41.3 mmol) in THF (30 mL) was added dropwise to
sodium hydride (634 mg, 26.41 mmol), and the mixture stirred overnight.
When TLC (hexane/EtOAc 7:3) indicated the reaction was complete, the
mixture was diluted with EtOAc, filtered through Celite, and washed with
water (3 Â 25 mL), dried (MgSO4), filtered, and concentrated. Purification
of the residue by column chromatography (CH2Cl2 ! CH2Cl2/EtOAc 97:3)
(dd, 3J(H2,H3) ˆ 10.0 Hz, 1H; H2), 4.95 5.14 (m, 2H; H2C CHCH2O),
ˆ
5.27 (d, 3J(H1,H2) ˆ 10.0 Hz, 1H; H1), 5.53 5.73 (m, 1H;
ˆ
H2C CHCH2O), 7.26 7.95 (m, 9H; aromatic); elemental analysis calcd
(%) for C26H29NO6S (483.17): C 64.57, H 6.05; found: C 64.61, H 6.01.
gave crystalline 13 (3.26 g, 80%). M.p. 130 1328C (from EtOH); [a]D20
ˆ
Ethyl 3-O-allyl-4,6-di-O-benzyl-2-deoxy-2-phthalimido-1-thio-b-d-galacto-
pyranoside (18): Ag2O (786 mg, 3.39 mmol) and benzyl bromide (300 mL,
2.52 mmol) were added to a mixture of 17 (410 mg, 0.85 mmol), potassium
iodide (307 mg, 1.85 mmol), and 4 ä molecular sieves in DMF (5 mL) at
08C. The mixture was stirred for 5 h, then diluted with CH2Cl2 (100 mL),
filtered through Celite, washed with 10% aq Na2S2O3 (3 Â 25 mL) and
water (2 Â 25 mL), dried (MgSO4), filtered, and concentrated. Purification
of the residue by column chromatography (CH2Cl2 ! CH2Cl2/EtOAc 98:2)
gave 18 (400 mg, 82%), isolated as a syrup. [a]2D0 ˆ ‡29.8 (c ˆ 0.6 in
CHCl3); 1H NMR (300 MHz, CDCl3): d ˆ 1.17 (t, 3H; SCH2CH3), 2.58
2.80 (m, 2H; SCH2CH3), 3.77 3.86 and 4.00 4.06 (m, 2H;
‡9.6 (c ˆ 1 in CHCl3); 1H NMR (200 MHz, CDCl3): d ˆ 1.21 (t, 3H;
3
SCH2CH3), 2.62 2.78 (m, 2H; SCH2CH3), 4.38 (dd, J(H2,H3) ˆ 10.0 Hz,
1H; H2), 4.83 5.06 (m, 2H; H2C CHCH2O), 5.41 (d, 3J(H1,H2) ˆ
ˆ
ˆ
10.0 Hz, 1H; H1), 5.45 5.65 (m, 1H; H2C CHCH2O), 5.59 (s, 1H;
PhCH), 7.35 7.85 (m, 9H; aromatic); elemental analysis calcd (%) for
C26H27NO6S (481.16): C 64.84, H 5.66; found: C 64.87, H 5.61.
Ethyl 3-O-allyl-6-O-benzyl-2-deoxy-2-phthalimido-1-thio-b-d-glucopyra-
noside (14):
A mixture of borane-trimethylamine complex (2.78 g,
38.1 mmol), powdered 4 ä molecular sieves (3 g), 13 (2.50 g, 5.20 mmol),
and THF (50 mL) was stirred for 1 h at room temperature. Then, AlCl3
(5.12 g, 38.4 mmol) was added, and the mixture stirred for 5 h in the dark,
when TLC (CH2Cl2/EtOAc 95:5) showed the conversion of 13 into 14. The
mixture was diluted with CH2Cl2 (250 mL), filtered through Celite, washed
with cold 0.5m H2SO4, water, 5% aq NaHCO3, and water, dried (MgSO4),
filtered, and concentrated. Purification of the residue by column chroma-
tography (CH2Cl2/EtOAc 95:5) gave 14 (2.13 g, 85%), isolated as a syrup.
[a]2D0 ˆ ‡5.5 (c ˆ 1.2 in CHCl3); 1H NMR (200 MHz, CDCl3): d ˆ 1.19 (t,
3H; SCH2CH3), 2.53 2.78 (m, 2H; SCH2CH3), 2.94 (brs, 1H; OH, can be
H2C CHCH2O), 4.04 (dd, 3J(H4,H5) < 1 Hz, 1H; H4), 4.33 (dd,
ˆ
3J(H3,H4) ˆ 2.6 Hz, 1H; H3), 4.47 (ABq, 2H; PhCH2), 4.76 (ABq, 2H;
PhCH2), 4.79 (dd, 3J(H2,H3) ˆ 10.4 Hz, 1H; H2), 4.92 5.14 (m, 2H;
H2C CHCH2O), 5.26 (d, 3J(H1,H2) ˆ 10.4 Hz, 1H; H1), 5.55 5.68 (m,
ˆ
ˆ
1H; H2C CHCH2O), 7.25 7.35 (m, 10H; 2 Ph), 7.60 7.85 (m, 4H; Phth);
elemental analysis calcd (%) for C33H35NO6S (573.22): C 69.08, H 6.15;
found: C 69.12, H 6.09.
Ethyl 4,6-di-O-benzyl-2-deoxy-2-phthalimido-1-thio-b-d-galactopyrano-
deuterated), 4.27 (dd, 3J(H2,H3) ˆ 9.0 Hz, 1H; H2), 4.61 (ABq, 2H;
side (19):
A solution of 18 (384 mg, 0.67 mmol) in EtOH (30 mL)
3
ˆ
PhCH2), 4.85 5.10 (m, 2H; H2C CHCH2O), 5.32 (d, J(H1,H2) ˆ 9.1 Hz,
containing tris(triphenylphosphine)rhodium(i) chloride (284 mg, 307 mmol)
was boiled under reflux for 3 h, then cooled, and concentrated. A solution
of the residue in acetone/1m hydrochloric acid 9:1 (20 mL) was boiled for
1 h, when TLC (CH2Cl2/EtOAc 95:5) showed the complete conversion of
the prop-1-enyl ether into 19 (Rf ˆ 0.46). The mixture was concentrated,
and purification of the residue by column chromatography (CH2Cl2/EtOAc
95:5) gave 19 (257 mg, 72%), isolated as a syrup. [a]2D0 ˆ ‡20.2 (c ˆ 0.8 in
CHCl3); 1H NMR (200 MHz, CDCl3): d ˆ 1.22 (t, 3H; SCH2CH3), 1.77 (brs,
1H; OH, can be deuterated), 2.57 2.77 (m, 2H; SCH2CH3), 4.03 (dd,
3J(H3,H4) ˆ 2.5 Hz, 3J(H4,H5) < 1 Hz, 1H; H4), 5.31 (d, 3J(H1,H2) ˆ
10.0 Hz, 1H; H1), 7.19 7.38 and 7.58 7.80 (m, 14H; aromatic); elemental
analysis calcd (%) for C30H31NO6S (533.19): C 67.52, H 5.86; found: C 67.47,
H 5.91.
ˆ
1H; H1), 5.50 5.70 (m, 1H; H2C CHCH2O), 7.26 7.91 (m, 9H; aromat-
ic); elemental analysis calcd (%) for C26H29NO6S (483.17): C 64.57, H 6.05;
found: C 64.61, H 6.08.
Ethyl 4-O-acetyl-3-O-allyl-6-O-benzyl-2-deoxy-2-phthalimido-1-thio-b-d-
glucopyranoside (15): Compound 14 (1.5 g, 3.1 mmol) was treated with
pyridine/acetic anhydride 1:1 (10 mL), as described for 8. Purification of
the residue by column chromatography (CH2Cl2/EtOAc 98:2) gave 15
(1.55 g, 95%), isolated as a syrup. [a]2D0 ˆ ‡61.4 (c ˆ 0.7 in CHCl3); 1H NMR
(300 MHz, CDCl3): d ˆ 1.25 (t, 3H; SCH2CH3), 2.05 (s, 3H; OAc), 2.62
2.78 (m, 2H; SCH2CH3), 4.39 (dd, 3J(H2,H3) ˆ 9.0 Hz, 1H; H2), 4.58 (brs,
2H; PhCH2), 4.82 5.15 (m, 2H; H2C CHCH2O), 5.10 (dd, 3J(H3,H4) ˆ
ˆ
8.7 Hz, 3J(H4,H5) ˆ 9.9 Hz, 1H; H4), 5.32 (d, 3J(H1,H2) ˆ 9.2 Hz, 1H;
ˆ
H1), 5.44 5.63 (m, 1H; H2C CHCH2O), 7.28 7.81 (m, 9H; aromatic);
Ethyl 3-O-acetyl-4,6-di-O-benzyl-2-deoxy-2-phthalimido-1-thio-b-d-galac-
topyranoside (20): Compound 19 (200 mg, 0.37 mmol) was treated with
pyridine/acetic anhydride 1:1 (10 mL), as described for 8, to give 20
(210 mg, 97%), isolated as a syrup. [a]2D0 ˆ ‡25.4 (c ˆ 0.4 in CHCl3);
1H NMR (300 MHz, CDCl3): d ˆ 1.18 (t, 3H; SCH2CH3), 1.79 (s, 3H; OAc),
elemental analysis calcd (%) for C28H31NO7S (525.18): C 63.98, H 5.95;
found: C 63.94, H 5.89.
Ethyl 4-O-acetyl-3-O-allyl-6-O-benzyl-2-deoxy-2-phthalimido-1-thio-b-d-
galactopyranoside (16): A solution of trifluoromethanesulfonic anhydride
(654 mL, 3.89 mmol) in CH2Cl2 (4 mL) was added to a solution of 14 (1.27 g,
2.63 mmol) in CH2Cl2 (10 mL) and pyridine (485 mL, 6.0 mmol) at 08C.
After stirring for 2 h, TLC (CH2Cl2/EtOAc 95:5) showed the formation of a
3
2.59 2.78 (m, 2H; SCH2CH3), 4.12 (dd, J(H4,H5) < 1 Hz, 1H; H4), 4.48
(ABq, 2H; PhCH2), 4.64 (ABq, 2H; PhCH2), 4.81 (dd, 3J(H2,H3) ˆ
10.7 Hz, 1H; H2), 5.41 (d, 3J(H1,H2) ˆ 10.0 Hz, 1H; H1), 5.73 (dd,
156
¹ WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002
0947-6539/02/0801-0156 $ 17.50+.50/0
Chem. Eur. J. 2002, 8, No. 1

Glycocalyx Glycan
151 161
3J(H3,H4) ˆ 3.1 Hz, 1H; H3), 7.20 7.40 and 7.60 7.90 (m, 14H; aromatic);
elemental analysis calcd (%) for C32H33NO7S (575.20): C 66.77, H 5.78;
found: C 66.71, H 5.79.
5-Azidopentyl (2,3,4-tri-O-benzyl-a-l-fucopyranosyl)-(1 ! 3)-(4,6-di-O-
benzyl-2-deoxy-2-phthalimido-b-d-galactopyranosyl)-(1 ! 4)-(3-O-allyl-6-
O-benzyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl)-(1 ! 3)-2,4,6-tri-O-
benzyl-a-d-galactopyranoside (27)
5-Azidopentyl (4-O-acetyl-3-O-allyl-6-O-benzyl-2-deoxy-2-phthalimido-b-
d-glucopyranosyl)-(1 ! 3)-2,4,6-tri-O-benzyl-a-d-galactopyranoside (21):
A solution of 11 (320 mg, 0.57 mmol) and 15 (390 mg, 0.74 mmol) in
Et2O (15 mL) containing 4 ä molecular sieves was stirred for 30 min under
Ar, then methyl triflate (500 mL, 4.44 mmol) was added at 08C. After
stirring for 12 h, Et3N was added, and the mixture diluted with CH2Cl2
(100 mL), washed with water, dried (MgSO4), filtered, and concentrated.
Purification of the residue by column chromatography (CH2Cl2/EtOAc
97:3) gave 21 (456 mg, 78%), isolated as a glass. [a]2D0 ˆ ‡7.1 (c ˆ 2.5 in
CHCl3); 1H NMR (200 MHz, CDCl3): d ˆ 1.27 1.59 (m, 6H;
OCH2(CH2)3CH2N3), 1.99 (s, 3H; OAc), 3.14 (t, 2H; CH2N3), 4.78 5.15
Procedure A: Compound 23 (120 mg, 79 mmol) was treated with potassium
carbonate (11 mg, 79 mmol) in MeOH/THF 1:1 (6 mL), as described for 17,
to afford 24 (100 mg, 85%; [a]D20 ˆ À13.2 (c ˆ 0.61 in CHCl3)). Bromine
(10 mL, 0.19 mmol) was added to a solution of 25 (60 mg, 125 mmol) in dry
CH2Cl2 (3 mL) at 08C. After 20 min, when TLC showed the disappearance
of the starting compound, the mixture was concentrated and co-concen-
trated with dry CH2Cl2 (2 Â 3 mL). A solution of the residue (26) in dry
CH2Cl2 (2 mL) was added to a stirred mixture of 24 (88 mg, 59 mmol),
tetrabutylammonium bromide (60 mg, 188 mmol), and 4 ä molecular sieves
in DMF (2 mL). After stirring for 24 h, the mixture was diluted with CH2Cl2
(50 mL), filtered through Celite, washed with water (3 Â 20 mL), dried
(MgSO4), filtered, and concentrated. Purification of the residue by column
chromatography (hexane/EtOAc 7:3) gave 27 (47 mg, 42%), isolated as a
syrup.
(m, 2H; H2C CHCH2O), 5.05 (dd, 3J(H4',H5') ˆ 10.5 Hz, 1H; H4'), 5.46
ˆ
(d, 3J(H1',H2') ˆ 8.1 Hz, 1H; H1'), 5.45 5.67 (m, 1H; H2C CHCH2O),
ˆ
7.14 7.95 (m, 24H; aromatic); elemental analysis calcd (%) for
C58H64N4O13 (1024.45): C 67.94, H 6.30; found: C 67.97, H 6.35.
5-Azidopentyl
(3-O-allyl-6-O-benzyl-2-deoxy-2-phthalimido-b-d-gluco-
Procedure B: A mixture of 24 (70 mg, 47 mmol), 25 (60 mg, 124 mmol),
CuBr2 (63 mg, 284 mmol), tetrabutylammonium bromide (68 mg,
213 mmol), and 4 ä molecular sieves was stirred in CH2Cl2/DMF 3:1
(4 mL) for 24 h under Ar. Then, the mixture was diluted with CH2Cl2
(50 mL), filtered through Celite, washed with water (2 Â 10 mL), dried
(MgSO4), filtered, and concentrated. Purification of the residue by column
chromatography (hexane/EtOAc 7:3) gave 27 (29 mg, 32%), isolated as a
syrup.
pyranosyl)-(1 ! 3)-2,4,6-tri-O-benzyl-a-d-galactopyranoside (22): Com-
pound 21 (500 mg, 0.49 mmol) was treated with potassium carbonate
(140 mg, 1.01 mmol) in MeOH/THF 1:1 (8 mL), as described for 17, to
afford 22 (425 mg, 89%), isolated as a syrup. [a]2D0 ˆ À10.9 (c ˆ 0.9 in
CHCl3); 1H NMR (200 MHz, CDCl3): d ˆ 1.25 1.59 (m, 6H;
OCH2(CH2)3CH2N3), 2.90 (brs, 1H; OH, can be deuterated), 3.14 (t, 2H;
3
ˆ
CH2N3), 4.81 5.12 (m, 2H; H2C CHCH2O), 5.47 (d, J(H1',H2') ˆ 8.1 Hz,
ˆ
1H; H1'), 5.52 5.78 (m, 1H; H2C CHCH2O), 7.15 7.94 (m, 24H;
Procedure C: A solution of 22 (44 mg, 44 mmol) and 28 (62 mg, 65 mmol) in
Et2O (2 mL) containing 4 ä molecular sieves was stirred for 30 min under
Ar, then methyl triflate (44 mL, 0.39 mmol) was added at 08C. After 12 h,
pyridine (0.2 mL) was injected and the mixture filtered through Celite,
concentrated, and co-concentrated with toluene. Purification of the residue
by gel filtration over LH-20 (CH2Cl2/MeOH 1:1), followed by column
chromatography (hexane/EtOAc 7:3) yielded 27 (50 mg, 65%), isolated as
a syrup. [a]2D0 ˆ À6.6 (c ˆ 0.1 in CHCl3). For 1H and 13C NMR data, see
Tables 1 and 2, respectively; elemental analysis calcd (%) for C111H115N5O22
(1869.80): C 71.24, H 6.20; found: C 71.20, H 6.21.
aromatic); elemental analysis calcd (%) for C56H62N4O12 (982.44): C
68.40, H 6.36; found: C 68.37, H 6.34.
5-Azidopentyl
(3-O-acetyl-4,6-di-O-benzyl-2-deoxy-2-phthalimido-b-d-
galactopyranosyl)-(1 ! 4)-(3-O-allyl-6-O-benzyl-2-deoxy-2-phthalimido-
b-d-glucopyranosyl)-(1 ! 3)-2,4,6-tri-O-benzyl-a-d-galactopyranoside
(23): A solution of 22 (580 mg, 0.59 mmol) and 20 (466 mg, 0.81 mmol) in
Et2O (20 mL) was stirred in the presence of 4 ä molecular sieves for 20 min
under Ar, then methyl triflate (550 mL, 4.86 mmol) was added at 08C. After
stirring for 12 h, pyridine (1 mL) was added, and the mixture was
concentrated. Purification of the residue by column chromatography
(CH2Cl2/EtOAc 96:4) gave 23 (724 mg, 82%), isolated as a syrup. [a]D20
ˆ
5-Azidopentyl
(4-O-acetyl-3,6-di-O-benzyl-2-deoxy-2-phthalimido-b-d-
1
glucopyranosyl)-(1 ! 3)-2,4,6-tri-O-benzyl-a-d-galactopyranoside (30): A
mixture of AgOTf (20 mg, 80 mmol) and N-iodosuccinimide (180 mg,
798 mmol) in acetonitrile (2 mL) was added to a solution of 11 (100 mg,
177 mmol) and 29[21] (153 mg, 266 mmol) in dry CH2Cl2 (3 mL) containing
4 ä molecular sieves at À158C. When TLC showed the disappearance of
11 and the formation of a new spot (Rf ˆ 0.45 hexane/EtOAc 6:4), pyridine
(0.1 mL) was added. The mixture was diluted with CH2Cl2 (100 mL),
filtered through Celite, washed with 10% aq Na2S2O3 (3 Â 20 mL), 10% aq
NaHCO3 (3 Â 20 mL), and water (2 Â 20 mL), dried (MgSO4), filtered, and
concentrated. Purification of the residue by column chromatography
(hexane/CH2Cl2/EtOAc 6:3:1) gave 30 (160 mg, 83%), isolated as a glass.
[a]2D0 ˆ ‡16.5 (c ˆ 0.6 in CHCl3); 1H NMR (200 MHz, CDCl3): d ˆ 1.25
1.54 (m, 6H; OCH2(CH2)3CH2N3), 1.94 (s, 3H; OAc), 3.14 (t, 2H; CH2N3),
5.16 (dd, 3J(H3',H4') ˆ 9.1 Hz, 1H; H4'), 5.42 (d, 3J(H1',H2') ˆ 8.2 Hz, 1H;
H1'), 6.87 7.74 (m, 29H; aromatic); 13C NMR (50.3 MHz, CDCl3): d ˆ
20.91 (COCH3), 23.24, 28.47, and 28.74 (OCH2(CH2)3CH2N3), 51.21
À18.6 (c ˆ 0.2 in CHCl3); H NMR (200 MHz, CDCl3): d ˆ 1.25 1.59 (m,
6H; OCH2(CH2)3CH2N3), 1.77 (s, 3H; OAc), 3.06 (t, 2H; CH2N3), 4.56
5.05 (m, 2H; H2C CHCH2O), 5.28 (d, 3J(H1'',H2'') ˆ 7.7 Hz, 1H; H1''),
ˆ
5.47 (d, 3J(H1',H2') ˆ 8.1 Hz, 1H; H1'), 5.41 5.61 (m, 1H;
H2C CHCH2O), 5.67 (dd, 3J(H2'',H3'') ˆ 11.4 Hz, 3J(H3'',H4'') ˆ 3.1 Hz,
ˆ
1H; H3''), 7.01 7.95 (m, 38H; aromatic); 13C NMR (50.3 MHz, CDCl3):
d ˆ 20.56 (COCH3), 23.24, 28.47, and 28.74 (OCH2(CH2)3CH2N3), 51.17
(CH2N3), 52.57 and 56.49 (C2', C2''), 97.39, 97.14, and 99.66 (C1, C1', C1''),
ˆ
À
ˆ
ˆ
115.84 (H2C ), 135.00 ( CH ), 167.52, 168.48, and 169.96 (C O);
elemental analysis calcd (%) for C86H89N5O19 (1495.62): C 69.00, H 6.00;
found: C 69.08, H 5.97.
Ethyl
(2,3,4-tri-O-benzyl-a-l-fucopyranosyl)-(1 ! 3)-4,6-di-O-benzyl-2-
deoxy-2-phthalimido-1-thio-b-d-galactopyranoside (28): Bromine (40 mL,
0.79 mmol) was added to a solution of 25 (250 mg, 0.52 mmol) in dry
CH2Cl2 (5 mL) at 08C. After 20 min, when TLC showed the disappearance
of the starting compound, a few drops of cyclohexene followed by 4 ä
molecular sieves were added. After stirring the mixture for 30 min under
Ar, a solution of 19 (140 mg, 0.26 mmol) in DMF (3 mL) and tetrabuty-
lammonium bromide (250 mg, 0.79 mmol) were added. The reaction
mixture was stirred for 3 d, diluted with CH2Cl2 (100 mL), filtered through
Celite, washed with 5% aq NaHCO3 (3 Â 20 mL) and water (3 Â 20 mL),
dried (MgSO4), filtered, and concentrated. Purification of the residue by
column chromatography (hexane/EtOAc 8:2 ! 7:3) afforded 28 (87 mg,
35%), isolated as a syrup. [a]2D0 ˆ ‡22.5 (c ˆ 0.2 in CHCl3); 1H NMR
(200 MHz, CDCl3): d ˆ 0.86 (d, 3H; CMe), 1.22 (t, 3H; SCH2CH3), 2.55
2.85 (m, 2H; SCH2CH3), 4.66 (d, 3J(H1',H2') ˆ 3.1 Hz, 1H; H1'), 4.85 (dd,
3J(H2,H3) ˆ 10.5 Hz, 1H; H2), 5.50 (d, 3J(H1,H2) ˆ 10.5 Hz, 1H; H1),
7.13 7.85 (m, 29H; aromatic); 13C NMR (50.3 MHz, CDCl3): d ˆ 14.96
(CMe), 16.33 (SCH2CH3), 23.72 (SCH2CH3), 51.27 (C2), 67.77 (C5'), 72.81,
72.99, 73.41, 74.17, and 74.59 (OCH2Ph), 80.99 (C1), 99.46 (C1'), 168.76
ˆ
(CH2N3), 56.14 (C2'), 97.38 (C1), 99.67 (C1'), 169.77 (C O); elemental
analysis calcd (%) for C62H66N4O13 (1074.46): C 69.24, H 6.19; found: C
69.20, H 6.22.
5-Azidopentyl (3,6-di-O-benzyl-2-deoxy-2-phthalimido-b-d-glucopyrano-
syl)-(1 ! 3)-2,4,6-tri-O-benzyl-a-d-galactopyranoside (31): Compound 30
(140 mg, 0.13 mmol) was treated with potassium carbonate (36 mg,
0.26 mmol) in MeOH/THF 1:1 (4 mL) as described for 17, to afford 31
(110 mg, 81%), isolated as a syrup. [a]2D0 ˆ À5.1 (c ˆ 0.2 in CHCl3);
1H NMR (200 MHz, CDCl3): d ˆ 1.25 1.57 (m, 6H; OCH2(CH2)3CH2N3),
3.07 3.17 (m, 3H; CH2N3 and OH, can be deuterated), 5.44 (d,
3J(H1',H2') ˆ 8.1 Hz, 1H; H1'), 6.95 7.77 (m, 29H; aromatic); 13C NMR
(50.3 MHz, CDCl3): d ˆ 23.24, 28.47, and 28.74 (OCH2(CH2)3CH2N3), 51.17
(CH2N3), 55.91 (C2'), 67.57 (OCH2(CH2)4N3), 72.91, 73.24, 73.56, 74.33, and
ˆ
74.71 (OCH2Ph), 97.30 (C1), 99.57 (C1'), 167.57 and 167.79 (C O);
ˆ
(C O); elemental analysis calcd (%) for C57H59NO10S (949.39): C 72.05, H
elemental analysis calcd (%) for C60H64N4O12 (1032.45): C 69.75, H 6.24;
found: C 69.68, H 6.28.
6.26; found: C 72.02, H 6.29.
Chem. Eur. J. 2002, 8, No. 1
¹ WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002
0947-6539/02/0801-0157 $ 17.50+.50/0
157

¬
¬ ¬
J. Kerekgyarto, J. P. Kamerling et al.
FULL PAPER
Table 1. 500 MHz 1H NMR data (300 K) of the oligosaccharides 27, 33, 36, 42, and 45. For the assignments, two-dimensional COSY, HSQC, TOCSY,
HMBC, and ROESY experiments were applied.
GalNPhth
GlcNPhth
Gal
Fuc (GalNPhth)
Fuc (GlcNPhth)
27
H1 (J(H1,H2) [Hz])
H2
5.58 (8.4)
4.72
5.33 (8.1)
4.28
4.37 (ꢀ5.0)
3.62
4.66 (3.6)
3.75
H3
4.40
4.36
3.95
3.57
H4
3.96
4.12
3.94
3.41
H5
3.72
3.50
3.81
3.93
H6
3.61, 3.61
3.57, 3.55
3.42, 3.32
0.89
33
H1 (J(H1,H2) [Hz])
H2
5.58 (8.4)
4.82
5.31 (8.1)
4.29
4.36 (3.6)
3.59
4.66 (3.6)
3.75
H3
4.43
4.43
3.93
3.58
H4
H5
3.94
n.d.[a]
4.25
3.50
3.94
3.79
3.41
3.90
H6
3.61, 3.59
3.55, 3.47
3.41, 3.33
0.86
36
H1 (J(H1,H2) [Hz])
H2
5.70 (8.4)
4.44
4.46 (5.3)
3.74
4.52 (3.3)
3.61
H3
4.56
4.15
3.73
H4
5.14
4.06
3.44
H5
3.85
3.93
3.92
H6
3.64, 3.64
3.48, 3.42
0.99
42
H1 (J(H1,H2) [Hz])
H2
5.52 (8.5)
4.69
5.23 (8.7)
4.56
4.32 (3.5)
3.58
4.79 (2.9)
3.70
H3
H4
H5
5.78
4.13
3.70
4.27
3.88
3.95
3.77
3.30
n.d.[a]
3.66
n.d.[a]
3.43
H6
3.68, 3.76
3.08, 3.40
3.28, 3.46
1.36
45
H1 (J(H1,H2) [Hz])
5.58 (8.4)
4.72
5.24 (7.9)
4.55
4.27
4.38
n.d.[a]
4.32 (4.2)
3.58
3.92
n.d.[a]
3.79
3.29, 3.41
4.67 (3.2)
3.78
3.58
3.42
3.94
4.80 (3.7)
3.69
3.31
n.d.[a]
3.70
H2
H3
H4
H5
H6
4.42
n.d.[a]
3.62
n.d.[a]
3.09, 3.40
0.69
1.38
[a] n.d.: not determined.
1
5-Azidopentyl (2,3,4-tri-O-benzyl-a-l-fucopyranosyl)-(1 ! 3)-(4,6-di-O-
benzyl-2-deoxy-2-phthalimido-b-d-galactopyranosyl)-(1 ! 4)-(3,6-di-O-
benzyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl)-(1 ! 3)-2,4,6-tri-O-
benzyl-a-d-galactopyranoside (33): Bromine (8 mL, 0.15 mmol) was added
to a solution of 28 (114 mg, 120 mmol) in dry CH2Cl2 (4 mL) at 08C. After
20 min, when TLC showed the disappearance of the starting compound and
the formation of a new spot (32), the mixture was concentrated and co-
concentrated with dry CH2Cl2 (2 Â 3 mL). A solution of the residue (32) in
dry CH2Cl2 (2 mL) was added to a stirred mixture of 31 (77 mg, 75 mmol)
and 4 ä molecular sieves in dry CH2Cl2 (2 mL). The mixture was stirred for
30 min under Ar, and then a solution of AgOTf (54 mg, 215 mmol) in
toluene (2 mL) was added at À508C. After 1 h, pyridine (0.1 mL) was
added and the mixture diluted with CH2Cl2 (50 mL), filtered through
Celite, washed with 10% aq Na2S2O3 (3 Â 10 mL), 5% aq NaHCO3 (3 Â
10 mL), and water (2 Â 10 mL), dried (MgSO4), filtered, and concentrated.
Purification of the residue by column chromatography (hexane/CH2Cl2/
EtOAc 6:3:1) gave 33 (113 mg, 84%), isolated as a glass. [a]2D0 ˆ ‡7.6 (c ˆ
0.3 in CHCl3). For 1H and 13C NMR data, see Tables 1 and 2, respectively;
elemental analysis calcd (%) for C115H117N5O22 (1919.82): C 71.88, H 6.14;
found: C 71.92, H 6.11.
syrup. [a]2D0 ˆ ‡38.0 (c ˆ 0.3 in CHCl3); H NMR (200 MHz, CDCl3): d ˆ
0.95 (d, 3H; CMe), 1.24 (t, 3H; SCH2CH3), 2.61 2.81 (m, 2H; SCH2CH3),
4.51 (d, 3J(H1',H2') ˆ 2.0 Hz, 1H; H1'), 5.08 (dd, 3J(H3,H4) ˆ 3J(H4,H5) ˆ
9.7 Hz, 1H; H4), 5.51 (d, 3J(H1,H2) ˆ 10.0 Hz, 1H; H1), 6.99 7.85
(m, 24H; aromatic); 13C NMR (50.3 MHz, CDCl3): d ˆ 14.98 (SCH2CH3),
À
16.04 (CMe), 24.03 (SCH2CH3), 41.18 (ClCH2 ), 54.17 (C2), 68.37
(C5'), 69.85 (C6), 72.55, 73.12, 73.47, and 73.68 (OCH2Ph), 80.97
(C1), 101.96 (C1'), 166.71, 167.80, and 168.69 (C O); elemental analysis
calcd (%) for C52H54ClNO11S (935.31): C 66.72, H 5.82; found: C 66.75, H
5.78.
ˆ
5-Azidopentyl (2,3,4-tri-O-benzyl-a-l-fucopyranosyl)-(1 ! 3)-(6-O-ben-
zyl-4-O-chloroacetyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl)-(1 ! 3)-
2,4,6-tri-O-benzyl-a-d-galactopyranoside (36): Methyl triflate (180 mL,
1.6 mmol) was added to a mixture of 11 (100 mg, 177 mmol), 35 (236 mg,
248 mmol) and 4 ä molecular sieves in Et2O (7 mL) at 08C. After stirring
for 24 h, Et3N (0.2 mL) was added, and the mixture diluted with CH2Cl2
(50 mL), washed with 5% aq NaHCO3 (2 Â 10 mL) and water (2 Â 10 mL),
dried (MgSO4), filtered, and concentrated. Purification of the residue by
column chromatography (CH2Cl2 ! CH2Cl2/EtOAc 98:2) gave 36 (166 mg,
65%), isolated as a syrup. [a]2D0 ˆ ‡27.5 (c ˆ 0.3 in CHCl3). For 1H and
13C NMR data, see Tables 1 and 2, respectively; elemental analysis
calcd (%) for C82H87ClN4O17 (1434.58): C 68.59, H 6.11; found: C 68.62,
H 6.13.
Ethyl
(2,3,4-tri-O-benzyl-a-l-fucopyranosyl)-(1 ! 3)-6-O-benzyl-4-O-
(35):
chloroacetyl-2-deoxy-2-phthalimido-1-thio-b-d-glucopyranoside
Chloroacetylchloride (50 mL, 0.63 mmol) in CH2Cl2 (1 mL) was added to
a solution of 34[23] (270 mg, 314 mmol) and pyridine (0.25 mL) in CH2Cl2
(2.5 mL) at À408C, and the mixture was kept for 24 h at À208C. Then, the
mixture was diluted with CH2Cl2 (50 mL), washed with 5% aq NaHCO3
(3 Â 10 mL) and water (2 Â 10 mL), dried (MgSO4), filtered, and concen-
trated. Purification of the residue by column chromatography (CH2Cl2 !
CH2Cl2/EtOAc 98:2) gave 35 (250 mg, 85%), isolated as a pale yellow
5-Azidopentyl (2,3,4-tri-O-benzyl-a-l-fucopyranosyl)-(1 ! 3)-(6-O-ben-
zyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl)-(1 ! 3)-2,4,6-tri-O-ben-
zyl-a-d-galactopyranoside (37): Potassium carbonate (20 mg, 146 mmol)
was added to a solution of 36 (140 mg, 97 mmol) in MeOH/THF 1:1 (4 mL),
and the mixture stirred for 2 h. The mixture was then diluted with CH2Cl2
(50 mL), washed with water (3 Â 10 mL), and the combined washings were
158
¹ WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002
0947-6539/02/0801-0158 $ 17.50+.50/0
Chem. Eur. J. 2002, 8, No. 1

Glycocalyx Glycan
151 161
Table 2. 125.76 MHz 13C NMR data (300 K) of the oligosaccharides 27, 33,
36, 42, and 45. For the assignments, HSQC and HMBC experiments were
applied.
chromatography (CH2Cl2/EtOAc 98:2 ! 96:4) gave 39 (330 mg, 45%),
1
isolated as a syrup. [a]2D0 ˆ ‡10.9 (c ˆ 1.8 in CHCl3); H NMR (200 MHz,
CDCl3): d ˆ 1.11 (t, 3H; SCH2CH3), 1.77 (s, 3H; OAc), 2.47 2.66
ˆ
(m, 2H; SCH2CH3), 4.85 5.19 (m, 2H; H2C CHCH2O), 5.12 (d,
GalNPhth
GlcNPhth
Gal
Fuc
Fuc
3J(H1,H2) ˆ 10.5 Hz, 1H; H1), 5.49 (d, 3J(H1',H2') ˆ 8.3 Hz, 1H; H1'),
(GalNPhth)
(GlcNPhth)
5.38 5.58 (m, 1H; H2C CHCH2O), 5.65 (dd, 3J(H2',H3') ˆ 11.5 Hz,
ˆ
3J(H3',H4') ˆ 3.0 Hz, 1H; H3'), 7.19 7.89 (m, 23H; aromatic); 13C NMR
(50.3 MHz, CDCl3): d ˆ 14.90 (SCH2CH3), 20.62 (COCH3), 23.66
(SCH2CH3), 52.61 and 54.94 (C2, C2'), 80.91 (C1), 97.26 (C1'), 116.11
27
C1
C2
C3
C4
C5
C6
33
97.68
53.83
78.28
74.51
73.39
68.44
100.13
56.90
77.71
76.32
74.64
68.86
97.80
76.23
78.91
77.79
69.63
69.95
99.88
75.45
80.00
78.18
68.00
16.77
ˆ
À
ˆ
ˆ
(H2C ), 134.04 ( CH ), 167.63, 168.47, and 170.02 (C O); elemental
analysis calcd (%) for C56H56N2O13S (996.35): C 67.45, H 5.66; found: C
67.51, H 5.64.
Ethyl (3-O-acetyl-4,6-di-O-benzyl-2-deoxy-2-phthalimido-b-d-galactopyr-
anosyl)-(1 ! 4)-(6-O-benzyl-2-deoxy-2-phthalimido-1-thio-b-d-glucopyra-
noside (40): A solution of 39 (610 mg, 0.61 mmol) and tris(triphenylphos-
phine)rhodium(i) chloride (276 mg, 299 mmol) in EtOH (40 mL) was boiled
under reflux for 5 h, then cooled and concentrated. A solution of the
residue in acetone/1m hydrochloric acid 9:1 (20 mL) was boiled for 30 min,
when TLC (CH2Cl2/EtOAc 98:2) showed a complete conversion of the
prop-1-enyl ether into 40 (Rf ˆ 0.43). Then, the mixture was concentrated,
and a solution of the residue in CH2Cl2 (100 mL) was washed with 10% aq
NaCl (3 Â 20 mL) and water (2 Â 20 mL), dried (MgSO4), filtered, and
concentrated. Purification of the residue by column chromatography
C1
C2
C3
C4
C5
C6
36
97.51
53.86
78.09
74.66
73.39
68.90
100.06
56.90
77.39
76.45
74.70
68.35
97.70
76.18
79.05
77.80
69.64
69.94
99.92
75.48
80.12
78.15
68.06
16.73
C1
C2
C3
C4
C5
C6
42
99.90
56.19
79.16
74.93
72.59
70.21
97.84
76.11
79.47
77.75
68.65
69.79
102.29
75.16
79.90
78.94
69.55
16.36
(CH2Cl2/EtOAc 98:2) gave 40 (415 mg, 71%), isolated as a syrup. [a]D20
ˆ
‡24.3 (c ˆ 0.6 in CHCl3); 1H NMR (200 MHz, CDCl3): d ˆ 1.13 (t, 3H;
SCH2CH3), 1.58 (brs, 1H; OH, can be deuterated), 1.81 (s, 3H; OAc),
2.51 2.65 (m, 2H; SCH2CH3), 5.20 (d, 3J(H1,H2) ˆ 10.5 Hz, 1H; H1), 5.45
(d, 3J(H1',H2') ˆ 8.4 Hz, 1H; H1'), 5.68 (dd, 3J(H2',H3') ˆ 11.5 Hz,
3J(H3',H4') ˆ 3.0 Hz, 1H; H3'), 7.00 7.88 (m, 23H; aromatic); elemental
analysis calcd (%) for C53H52N2O13S (956.32): C 66.51, H 5.48; found: C
66.61, H 5.50.
C1
C2
C3
C4
C5
C6
45
97.28
52.87
71.54
74.39
74.43
67.69
100.01
57.79
73.69
n.d.[a]
74.70
67.98
97.74
76.66
78.57
77.54
69.65
70.06
97.20
74.50
79.01
n.d.[a]
68.77
16.98
Ethyl (3-O-acetyl-4,6-di-O-benzyl-2-deoxy-2-phthalimido-b-d-galactopyr-
anosyl)-(1 ! 4)-[(2,3,4-tri-O-benzyl-a-l-fucopyranosyl)-(1 ! 3)]-(6-O-
benzyl-2-deoxy-2-phthalimido-1-thio-b-d-glucopyranoside (41): Bromine
(46 mL, 0.89 mmol) was added to a solution of 25 (610 mg, 1.27 mmol) in
dry CH2Cl2 (10 mL) at 08C. After 20 min, when TLC showed the
disappearance of the starting compound, the mixture was concentrated
and co-concentrated with dry CH2Cl2 (2 Â 5 mL). Then, a solution of 40
(344 mg, 0.36 mmol) and sym-collidine (300 mL) in dry CH2Cl2 (10 mL) and
4 ä molecular sieves were added to the residue. The mixture was stirred for
30 min under Ar, then AgOTf (514 mg, 2.0 mmol) in toluene (10 mL) was
added at À308C. After 2 h, pyridine (0.5 mL) was added, and the mixture
diluted with CH2Cl2 (200 mL), filtered through Celite, washed with 10% aq
Na2S2O3 (3 Â 20 mL), 5% aq NaHCO3 (3 Â 20 mL), and water (2 Â 20 mL),
dried (MgSO4), filtered, and concentrated. Purification of the residue by
column chromatography (hexane/CH2Cl2/EtOAc 4:2:1) gave 41 (237 mg,
48%), isolated as a glass. [a]2D0 ˆ À44.5 (c ˆ 0.2 in CHCl3); 1H NMR
(500 MHz, CDCl3, 300 K) (for the assignments, two-dimensional COSY
and HSQC experiments were applied): d ˆ 1.09 (t, 3H; SCH2CH3), 1.32 (d,
3H; CMe), 1.84 (s, 3H; OAc), 2.50 2.64 (m, 2H; SCH2CH3), 5.01 (d,
3J(H1,H2) ˆ 10.5 Hz, 1H; H1), 5.47 (d, 3J(H1',H2') ˆ 8.5 Hz, 1H; H1'), 5.72
(dd, 3J(H2',H3') ˆ 11.5 Hz, 3J(H3',H4') ˆ 3.0 Hz, 1H; H3'), 7.00 7.91 (m,
38H; aromatic); 13C NMR (125.76 MHz, CDCl3): d ˆ 14.75 (SCH2CH3),
16.56 (CMe), 20.59 (COCH3), 29.65 (SCH2CH3), 52.37 and 55.56 (C2, C2'),
C1
C2
C3
C4
C5
C6
97.70
53.91
72.31
n.d.[a]
73.39
n.d.[a]
100.01
57.90
73.12
73.45
n.d.[a]
68.05
97.80
76.61
78.80
n.d.[a]
69.70
70.10
99.88
75.54
80.05
78.35
68.42
16.62
97.00
74.80
79.24
n.d.[a]
68.82
17.45
[a] n.d.: not determined.
extracted with CH2Cl2 (3 Â 10 mL). The combined organic phases were
dried (MgSO4), filtered, and concentrated. Purification of the residue by
column chromatography (hexane/CH2Cl2/EtOAc 6:3:1) gave 37 (120 mg,
90%), isolated as a syrup. [a]2D0 ˆ ‡4.9 (c ˆ 0.6 in CHCl3); 1H NMR
(200 MHz, CDCl3): d ˆ 1.06 (d, 3H; CMe), 1.35 1.62 (m, 6H;
OCH2(CH2)3CH2N3), 3.21 (brs, 1H; OH, can be deuterated), 3.13 (t, 2H;
CH2N3), 4.52 (d, 3J(H1'',H2'') ˆ 2.7 Hz, 1H; H1''), 5.59 (d, 3J(H1',H2') ˆ
7.8 Hz, 1H; H1'), 6.93 7.82 (m, 39H; aromatic); 13C NMR (50.3 MHz,
CDCl3): d ˆ 16.41 (CMe), 23.30, 28.53, and 28.80 (OCH2(CH2)3CH2N3),
51.64 (CH2N3), 60.36 (C2'), 67.71 (OCH2(CH2)4N3), 97.56 (C1), 99.82 (C1'),
ˆ
80.86 (C1), 96.84 and 96.99 (C1', C1''), 167.41, 168.61, and 169.91 (C O);
ˆ
100.79 (C1''), 168.08 and 168.36 (C O); elemental analysis calcd (%) for
elemental analysis calcd (%) for C80H80N2O17S (1372.52): C 69.94, H 5.87;
found: C 69.90, H 5.89.
C80H86N4O16 (1358.60): C 70.66, H 6.38; found: C 70.62, H 6.41.
Ethyl (3-O-acetyl-4,6-di-O-benzyl-2-deoxy-2-phthalimido-b-d-galactopyr-
anosyl)-(1 ! 4)-(3-O-allyl-6-O-benzyl-2-deoxy-2-phthalimido-1-thio-b-d-
glucopyranoside (39): Bromine (55 mL, 1.0 mmol) was added to a solution
of 20 (470 mg, 0.82 mmol) in dry CH2Cl2 (10 mL) at 08C. After 20 min,
when TLC showed the disappearance of the starting compound and the
formation of a new spot (38), the mixture was concentrated and co-
concentrated with dry CH2Cl2 (2 Â 5 mL). A solution of 14 (352 mg,
0.73 mmol) in CH2Cl2 (4 mL) and 4 ä molecular sieves were added to the
residue (38). After stirring the mixture for 20 min under Ar, a solution of
AgOTf (320 mg, 1.25 mmol) in toluene (7 mL) was added at À408C. After
1.5 h, pyridine (0.5 mL) was added, and the mixture diluted with CH2Cl2
(200 mL), filtered through Celite, washed with 10% aq Na2S2O3 (3 Â
50 mL), 5% aq NaHCO3 (3 Â 50 mL), and water (2 Â 50 mL), dried
(MgSO4), filtered, and concentrated. Purification of the residue by column
5-Azidopentyl
(3-O-acetyl-4,6-di-O-benzyl-2-deoxy-2-phthalimido-b-d-
galactopyranosyl)-(1 ! 4)-[(2,3,4-tri-O-benzyl-a-l-fucopyranosyl)-(1! 3)]-
(6-O-benzyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl)-(1 ! 3)-2,4,6-tri-
O-benzyl-a-d-galactopyranoside (42): A solution of 11 (22 mg, 40 mmol)
and 41 (55 mg, 39 mmol) in dry Et2O (2 mL) containing 4 ä molecular
sieves was stirred for 30 min under Ar, then methyl triflate (45 mL,
0.4 mmol) was added at 08C. After 20 h, pyridine was injected, and the
mixture diluted with CH2Cl2 (50 mL), washed with water (2 Â 10 mL),
dried (MgSO4), filtered, and concentrated. Gel filtration of the residue over
LH-20 (CH2Cl2/MeOH 1:1), followed by purification by silica column
chromatography (hexane/EtOAc 7:3) afforded 42 (46 mg, 64%), isolated
as a syrup. [a]2D0 ˆ À35.5 (c ˆ 2.1 in CHCl3). For 1H and 13C NMR data, see
Tables 1 and 2, respectively; elemental analysis calcd (%) for C110H113N5O23
(1871.78): C 70.52, H 6.08; found: C 70.49, H 6.12.
Chem. Eur. J. 2002, 8, No. 1
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159

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¬ ¬
J. Kerekgyarto, J. P. Kamerling et al.
FULL PAPER
Ethyl
(4,6-di-O-benzyl-2-deoxy-2-phthalimido-b-d-galactopyranosyl)- Table 3. 500 MHz 1H NMR data of the oligosaccharides 1 (295 K), 2 (280 K), and
3 (300 K). For the assignments, two-dimensional TOCSY experiments with short
(1 ! 4)-(6-O-benzyl-2-deoxy-2-phthalimido-1-thio-b-d-glucopyranoside
(43): Acetyl chloride (300 mL) was added to a solution of 40 (287 mg, and long mixing times were applied.
100 mmol) in MeOH/CH2Cl2 3:1 (15 mL) at 08C, and the mixture stirred for
GalNAc
GlcNAc
Gal
Fuc
Fuc
3d at room temperature, then concentrated. Purification of the residue by
(GalNAc) (GlcNAc)
column chromatography (hexane/EtOAc 6:4) gave 43 (184 mg, 67%),
isolated as a syrup. [a]2D0 ˆ ‡11.9 (c ˆ 0.3 in CHCl3); H NMR (200 MHz,
1
1
CDCl3): d ˆ 1.13 (t, 3H; SCH2CH3), 1.43 and 2.03 (2brs, 2H; 2OH, can be
H1 (J(H1,H2) [Hz]) 4.59 (8.4) 4.70 (7.3) 4.90 (2.0) 5.00 (3.4)
3
deuterated), 2.50 2.70 (m, 2H; SCH2CH3), 5.20 (d, J(H1,H2) ˆ 10.5 Hz,
H2
H3
H4
H5
4.06
3.79
3.98
n.d.[a]
3.77
3.74
n.d.[a]
n.d.[a]
n.d.[a]
3.87
3.88
4.17
n.d.[a]
n.d.[a]
3.70
3.89
3.81
4.12
3
1H; H1), 5.33 (d, J(H1',H2') ˆ 8.0 Hz, 1H; H1'), 7.22 7.41 and 7.65 7.90
(m, 23H; aromatic); elemental analysis calcd (%) for C51H50N2O12S
(914.31): C 66.94, H 5.51; found: C 66.88, H 5.53.
H6 (J(H5,H6) [Hz]) n.d.[a]
1.20 (6.8)
Ethyl (2,3,4-tri-O-benzyl-a-l-fucopyranosyl)-(1 ! 3)-(4,6-di-O-benzyl-2-
deoxy-2-phthalimido-b-d-galactopyranosyl)-(1 ! 4)-[(2,3,4-tri-O-benzyl-
a-l-fucopyranosyl)-(1 ! 3)]-(6-O-benzyl-2-deoxy-2-phthalimido-1-thio-b-
d-glucopyranoside (44): Bromine (110 mL, 2.0 mmol) was added to a
solution of 25 (747 mg, 1.56 mmol) in dry CH2Cl2 (10 mL) at 08C. After
20 min, when TLC showed the disappearance of the starting compound, the
mixture was concentrated and co-concentrated with dry CH2Cl2 (2 Â 5 mL).
Then, a solution of 43 (155 mg, 170 mmol) and sym-collidine (316 mL) in
CH2Cl2 (10 mL) and 4 ä molecular sieves were added to the residue. The
mixture was stirred for 30 min under Ar, then a solution of AgOTf (616 mg,
2.4 mmol) in toluene (10 mL) was added at À308C. After 2 h, pyridine
(0.5 mL) was added, and the mixture diluted with CH2Cl2 (100 mL), filtered
through Celite, washed with 10% aq Na2S2O3 (3 Â 20 mL), 5% aq NaHCO3
(3 Â 20 mL), and water (2 Â 20 mL), dried (MgSO4), filtered, and concen-
trated. Purification of the residue by column chromatography (hexane/
EtOAc 7:3) yielded 44 (224 mg, 75%), isolated as a glass. [a]2D0 ˆ À20.5
(c ˆ 1.1 in CHCl3); 1H NMR (500 MHz, CDCl3, 300 K) (for the assignments
two-dimensional COSY and HSQC experiments were applied): d ˆ 0.72 (d,
3H; CMe), 1.15 (t, 3H; SCH2CH3), 1.40 (d, 3H; CMe), 2.55 2.75 (m, 2H;
SCH2CH3), 4.63 (d, 3J(H1'',H2'') ˆ 3.6 Hz, 1H; H1''), 4.79 (d,
3J(H1''',H2''') ˆ 3.6 Hz, 1H; H1'''), 5.07 (d, 3J(H1,H2) ˆ 8.1 Hz, 1H; H1),
5.58 (d, 3J(H1',H2') ˆ 8.5 Hz, 1H; H1'), 7.08 7.90 (m, 53H; aromatic);
13C NMR (125.76 MHz, CDCl3): d ˆ 16.49 (CMe), 17.17 (CMe), 53.53 and
56.13 (C2, C2''), 81.35 (C1), 97.20 (C1'''), 97.30 (C1'), 99.61 (C1''); elemental
analysis calcd (%) for C105H106N2O20S (1746.71): C 72.14, H 6.12; found: C
72.21, H 6.15.
NC(O)CH3
2
2.05, 2.03
H1 (J(H1,H2) [Hz]) 4.46 (8.4) 4.67 (7.8) 4.89 (ꢀ1)
5.13 (br s)
3.69
3.96
3.84
4.89
H2
H3
H4
H5
3.99
3.72
3.90
n.d.[a]
3.97
3.86
3.90
3.51
n.d.[a]
3.83
3.88
4.18
n.d.[a]
n.d.[a]
H6 (J(H5,H6) [Hz]) n.d.[a]
NC(O)CH3
3
1.27 (5.9)
2.04, 2.02
H1 (J(H1,H2) [Hz]) 4.51 (7.9) 4.70 (7.9) 4.90 (ꢀ1) 4.98 (4.3) 5.13 (3.7)
H2
H3
H4
H5
4.08
3.73
3.95
n.d.[a]
3.95
3.87
3.90
n.d.[a]
n.d.[a]
3.86
3.86
4.17
n.d.[a]
n.d.[a]
3.69
3.92
3.82
4.11
3.69
3.96
3.83
4.85
H6 (J(H5,H6) [Hz]) n.d.[a]
NC(O)CH3
1.20 (6.7) 1.27 (6.7)
2.03, 2.02
[a] n.d.: not determined.
converted into 2, as described for 1, to afford 2 (5.1 mg, 57%). [a]2D0 ˆ À2.1
(c ˆ 0.7 in water); for 1H NMR data, see Table 3; MS (MALDI-TOF): m/z
‡
calcd for C33H59N3O20 (817.37); found: 840.32 [M‡Na] .
5-Aminopentyl
(a-l-fucopyranosyl)-(1 ! 3)-(2-deoxy-2-acetamido-b-d-
galactopyranosyl)-(1 ! 4)-[(a-l-fucopyranosyl)-(1 ! 3)]-(2-deoxy-2-acet-
amido-b-d-glucopyranosyl)-(1 ! 3)-a-d-galactopyranoside (3): Compound
45 (7.2 mg, 3.17 mmol) was converted into 3, as described for 1, to give 3
(1.8 mg, 54%). [a]2D0 ˆ À34.7 (c ˆ 0.1 in water); for 1H NMR data, see
Table 3; MS (MALDI-TOF): m/z calcd for C39H69N3O24 (963.43); found:
5-Azidopentyl (2,3,4-tri-O-benzyl-a-l-fucopyranosyl)-(1 ! 3)-(4,6-di-O-
benzyl-2-deoxy-2-phthalimido-b-d-galactopyranosyl)-(1 ! 4)-[(2,3,4-tri-
O-benzyl-a-l-fucopyranosyl)-(1 ! 3)]-(6-O-benzyl-2-deoxy-2-phthalimi-
do-b-d-glucopyranosyl)-(1 ! 3)-2,4,6-tri-O-benzyl-a-d-galactopyranoside
(45): A solution of 11 (19 mg, 34 mmol) and 44 (51 mg, 29 mmol) in Et2O
(2 mL), containing 4 ä molecular sieves was stirred for 30 min under Ar,
then methyl triflate (24 mL, 214 mmol) was added at 08C. After 20 h,
pyridine (0.2 mL) was injected, and the mixture filtered through Celite,
concentrated and co-concentrated with toluene. Gel filtration of the
residue over LH-20 (CH2Cl2/MeOH 1:1), followed by purification by silica
column chromatography (hexane/EtOAc 8:2) afforded 45 (41 mg, 54%),
‡
986.37 [M‡Na] .
Acknowledgements
1
isolated as a syrup. [a]D20 ˆ À22.8 (c ˆ 1.2 in CHCl3); for H and 13C NMR
This work was supported by the Hungarian National Science Foundation
(OTKA-T-019404, OTKA-T-029089), and drx500 purchase grants
(OMFBMEC-93-0098, Phare-Accord-H-9112-0198, OTKAA084). Gener-
ous support (to J.H.) from a Bolyai Research Scholarship is also acknowl-
edged.
data, see Tables 1 and 2, respectively; elemental analysis calcd (%) for
C135H139N5O26 (2245.97): C 72.13, H 6.24; found: C 72.21, H 6.20.
5-Aminopentyl
(a-l-fucopyranosyl)-(1 ! 3)-(2-deoxy-2-acetamido-b-d-
galactopyranosyl)-(1 ! 4)-(2-deoxy-2-acetamido-b-d-glucopyranosyl)-
(1 ! 3)-a-d-galactopyranoside (1): Ethylenediamine (1 mL) was added to
a solution of 33 (18.7 mg, 9.8 mmol) in 1-butanol (1 mL). The mixture was
stirred overnight under Ar at 908C, then co-concentrated with toluene and
dried under high vacuum. The residue was dissolved in MeOH (1 mL), and
Ac2O (1 mL) added at 08C. The mixture was kept for 1 h at 08C, then
concentrated and co-concentrated with toluene. A solution of the residue in
1-butanol (1.6 mL) and water (0.6 mL), containing 10% Pd C (10 mg) and
a few drops of 25% aq NH3 (pH 9), was hydrogenated for 1 h. By flushing
with Ar for 1 h, the pH of the solution decreased to 7, then HOAc was
added (pH 5), and the mixture hydrogenated for another two days. After
filtration and concentration, the crude product was purified by HiTrap gel
filtration (aq 5 mm NH4HCO3) to give 1 (5.3 mg, 61%). [a]2D0 ˆ À1.6 (c ˆ 1
in water); for 1H NMR data, see Table 3; MS (MALDI-TOF): m/z calcd for
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5-Aminopentyl
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[(a-l-fucopyranosyl)-(1 ! 3)]-(2-deoxy-2-acetamido-b-d-glucopyranosyl)-
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Received: April 30, 2001 [F3226]
Chem. Eur. J. 2002, 8, No. 1
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0947-6539/02/0801-0161 $ 17.50+.50/0
161