Synthesis and conjugation of oligosaccharide fragments related to the immunologically reactive part of the circulating anodic antigen of the parasite Schistosoma mansoni

Article abstract of DOI:10.1039/b002083o

The immunoreactive part of the circulating anodic antigen (CAA) from the parasite Schistosoma mansoni is a threonine-linked polysaccharide consisting of →6)-[β-D-GlcpA-(1→3)]-β-D-GalpNAc-(1→ repeating disaccharides. In the framework of an immunochemical p

Full text of DOI:10.1039/b002083o

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Synthesis and conjugation of oligosaccharide fragments related to
the immunologically reactive part of the circulating anodic antigen
of the parasite Schistosoma mansoni
1
Henricus J. Vermeer, Koen M. Halkes, J. Albert van Kuik, Johannis P. Kamerling* and
Johannes F. G. Vliegenthart
Bijvoet Center, Department of Bio-Organic Chemistry, Utrecht University, P.O. Box 80.075,
NL-3508 TB Utrecht, The Netherlands
Received (in Cambridge, UK) 14th March 2000, Accepted 25th April 2000
Published on the Web 21st June 2000
The immunoreactive part of the circulating anodic antigen (CAA) from the parasite Schistosoma mansoni
is a threonine-linked polysaccharide consisting of →6)-[β--GlcpA-(1→3)]-β--GalpNAc-(1→ repeating
disaccharides. In the framework of an immunochemical project, as a follow-up of earlier synthesized di- to
tetrasaccharide CAA fragments, the synthesis of a spacer-containing pentasaccharide fragment, 3-(2-amino-
ethylthio)propyl (2-acetamido-2-deoxy-β--galactopyranosyl)-(1→6)-[(β--glucopyranosyluronic acid)-(1→3)]-
(2-acetamido-2-deoxy-β--galactopyranosyl)-(1→6)-[(β--glucopyranosyluronic acid)-(1→3)]-2-acetamido-2-
deoxy-β--galactopyranoside, is described. Moreover, 1-O-[3-(2-aminoethylthio)propyl]-N-acetyl-β--galactosamine
was synthesized. Oxidation steps in the synthesis of tri- to pentasaccharide CAA fragments were performed using
pyridinium dichromate and acetic anhydride. TEMPO-catalyzed oxidations were explored in the synthesis of
1-O-[6-aminohexyl]-β--glucuronicacidand3-aminopropyl(2-acetamido-2-deoxy-β--galactopyranosyl)-(1→6)-[(β--
glucopyranosyluronic acid)-(1→3)]-2-acetamido-2-deoxy-β--galactopyranoside, affording short reaction times
and high yields. All synthesized compounds, including the earlier described 3-(2-aminoethylthio)propyl-spacered
di-, tri-, and tetrasaccharide CAA fragments, were conjugated to BSA using squaric diester chemistry with coupling
efficiencies in the range of 30–90%. The efficiency decreased when larger oligosaccharides were coupled to BSA.
Finally, conformational analyses of the tri- and tetrasaccharide fragments were performed using Molecular
Mechanics (MM) and Molecular Dynamics (MD) calculations.
fragments as diagnostic markers for the detection of human
Introduction
schistosomiasis. As a first result the stereoselective synthesis of
Schistosomiasis (or bilharzia), caused by infection with worms
di-, tri-, and tetrasaccharide fragments of the CAA poly-
(blood-dwelling flukes) belonging to the class of Trematoda, is
saccharide (1, 2 and 4, Fig. 1) has been described.5 Here, we
one of the most important and widespread parasitic diseases.
describe the synthesis of other spacer-armed CAA glycan
The number of infected people is estimated to surpass 250
fragments, i.e. the tri- (3) and pentasaccharide fragment (5),
and the spacer-armed monosaccharide constituents (6 and 7).
million,1 and the main species of medical importance to man
are Schistosoma mansoni, S. haematobium, and S. japonicum.
Compounds 1–7 were conjugated to bovine serum albumin
The life cycle of the parasite involves several parasitic stages,
(BSA). Additionally, conformational studies were performed
alternated by free-living stages in either intermediate hosts
on the tri- and tetrasaccharide fragments using molecular
mechanics and molecular dynamics calculations. Results of the
(fresh-water snails) or definitive hosts.2 Human infection is
initiated during exposure to water containing cercariae, free-
immunological studies with the neoglycoconjugates will be
living mobile stages of the parasite. Since, for cure of the infec-
published elsewhere.
tion, chemotherapy is available, early diagnosis is important. In
this context, the development of diagnostic protocols based on
the detection of schistosome antigens in the circulatory system
of the host is receiving more and more attention. More specific-
ally, in recent years much research is focused on the use of
monoclonal antibody-based assays for the detection of highly
immunogenic schistosomal antigens in serum and urine.
One of the major antigens of S. mansoni is the gut-associated
circulating anodic antigen (CAA).3 The immunologically
dominant part of CAA is a threonine-linked polysaccharide
consisting of disaccharide repeating units, {→6)-[β--GlcpA-
(1→3)]-β--GalpNAc-(1→}n (n = 30), probably connected to
the protein via an as-yet-unknown, core saccharide with Glc-
NAc at the reducing end.4 Recently, we have started a pro-
gramme to synthesize well defined oligosaccharide fragments
of the polysaccharide in order to identify the immunologic
epitopes of CAA for the generated anti-CAA monoclonal anti-
bodies, and to investigate the potential of synthetic CAA glycan
Results and discussion
Synthesis of CAA fragments
The synthesized spacer-containing saccharide fragments of the
CAA glycan are shown in Fig. 1. The convergent synthesis of 1,
2, and 45 involved the preparation of the allyl glycoside pre-
cursors of 1, 2, and 4, followed by an elongation of the allyl
functions with 2-aminoethanethiol (cysteamine).6 Compounds
5 and 6 were synthesized using a similar strategy. In view of
the severe problems in realizing a general protocol for the
oxidation of Glc to GlcA during the preparation of 1 [oxalyl
dichloride–dimethyl sulfoxide (DMSO); NaClO2], 2 [pyridin-
ium dichromate(PDC)–dichloromethane–molecular sieves 4
Å], 4 and 5 (PDC–dichloromethane–acetic anhydride), and
structures larger than 5 (see below), an alternative approach
using 2,2,6,6-tetramethylpiperidine 1-oxide7–9 (TEMPO) was
DOI: 10.1039/b002083o
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This journal is © The Royal Society of Chemistry 2000
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Fig. 1 Structures of the synthesized oligosaccharides representing fragments of the circulating anodic antigen of Schistosoma mansoni.
explored. However, the application of TEMPO required the use
of other spacer systems than the 3-(2-aminoethylthio)propyl
function, as evidenced in the synthesis of compounds 3 and 7.
Compounds 1–7 were conjugated to BSA using diethyl squar-
ate; this reagent has been shown to be effective when small
quantities of oligosaccharides (≈1 mg) are coupled to carrier
proteins.10–12
toluene13,14 gave stereospecifically pentamer 11 (66%). Conden-
sation of 9 with 10 conducted in other solvent systems or in the
presence of NIS and triflic acid (cat.)15 did not result in higher
yields. Selective delevulinoylation of 11 was performed by
treatment with hydrazinium acetate16 in toluene–ethanol
(→ 12, 92%), followed by oxidation of HO-6Ј, -6ٞ of 12 using
PDC and acetic anhydride in dichloromethane17 (3.5 h) to give
1
13 (73%). The complete oxidation of 12 was confirmed by H
Synthesis of pentasaccharide 5
NMR analysis of a minor amount of methyl-esterified 13
(→ 13a, COOCH3: δ 3.64 and 3.67). After deacylation/
dephthaloylation using methylamine in ethanol18 (2 weeks at
room temperature), and subsequent N-acetylation with acetic
anhydride in methanol at 0 ЊC, compound 14 was isolated in
65% yield. Subsequently, allyl glycoside 14 was treated with
cysteamine hydrochloride under radical conditions (UV irradi-
ation) to afford 5 (80%), suitable for conjugation to BSA (vide
infra). The structures of 14 and 5 were unambiguously ascer-
For the synthesis of 5, allyl (6-O-levulinoyl-2,3,4-tri-O-p-
toluoyl-β--glucopyranosyl)-(1→3)-(4-O-acetyl-2-deoxy-2-
phthalimido-β--galactopyranosyl)-(1→6)-[(6-O-levulinoyl-
2,3,4-tri-O-p-toluoyl-β--glucopyranosyl)-(1→3)]-4-O-acetyl-
2-deoxy-2-phthalimido-β--galactopyranoside 9 was selected
as glycosyl acceptor and ethyl 3,4,6-tri-O-acetyl-2-deoxy-2-
phthalimido-1-thio-β--galactopyranoside5 10 as glycosyl
donor (Scheme 1). The levulinoyl groups at the C-6 positions
of the β--glucopyranosyl moieties were chosen in view of
the proposed oxidation of the primary hydroxy functions
after deprotection in the final stage of the synthesis. Compound
1
tained by mass spectrometry and H (1D and 2D TOCSY)
NMR analysis (Table 1).
Preparation of spacer containing monosaccharides 6 and 7
9
was prepared from allyl (6-O-levulinoyl-2,3,4-tri-O-p-
toluoyl-β--glucopyranosyl)-(1→3)-[4-O-acetyl-6-O-(tert-butyl-
dimethylsilyl)-2-deoxy-2-phthalimido-β--galactopyranosyl]-
(1→6)-[(6-O-levulinoyl-2,3,4-tri-O-p-toluoyl-β--glucopyrano-
syl)-(1→3)]-4-O-acetyl-2-deoxy-2-phthalimido-β--galacto-
pyranoside5 8 by desilylation using toluene-p-sulfonic acid in
acetonitrile–water (→ 9, 82%). Galactosylation of 9 with
donor 10 using N-iodosuccinimide (NIS) and silver triflate in
For immunological purposes, the preparation of neoglyco-
conjugates bearing the individual monosaccharide constituents
of the repeating disaccharide unit, i.e. N-acetyl-β--galactos-
amine and β--glucuronic acid, was also of interest. The syn-
thesis of 3-(2-aminoethylthio)propyl 2-acetamido-2-deoxy-β--
galactopyranoside 6 is depicted in Scheme 2. Reaction of
2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy--galactopyranose
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Table 1 500 MHz 1H NMR data (1D and 2D TOCSY) of compound 5
Proton (δH)
GalNAc/GalNAcЉ
GlcAЈ/GlcAٞ
GalNAc٣
H-1 (J1,2
H-2 (J2,3
H-3 (J3,4
H-4 (J4,5
H-5
)
)
)
)
4.46 and 4.50 (8.5)
3.97 and 4.01 (11.0)
3.83 and 3.84
4.13 and 4.15
n.d.a
4.48 and 4.51 (7.5)
3.33 (9.4)
3.45 (9.0)
3.47 (8.3)
3.67 and 3.70
4.47 (8.5)
3.91 (10.5)
3.75 (3.0)
3.94
n.d.a
OCH2CH2CH2S(CH2)2NH2
OCH2CH2CH2S(CH2)2NH2
O(CH2)3SCH2CH2NH2
OCH2(CH2)2SCH2CH2NH2
NHCOCH3
1.87
2.63
3.23 and 3.47
3.69 and 3.96
2.00, 2.03 and 2.04
a n.d. = not determined.
Scheme 1 Reagents and yields: i, p-TsOH (82%); ii, NIS, AgOTf (66%); iii, H2NNH2ؒHOAc (92%); iv, PDC, Ac2O (73%); v, CH2N2 (quant.);
vi, (a) MeNH2; (b) Ac2O, MeOH (65%); vii, HS(CH2)2NH2 (80%).
15 with hydrazinium acetate in dichloromethane for 1 h at 60 ЊC
furnished 16, which was imidoylated19 using trichloroaceto-
nitrile in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU) to yield 17 (56% over two steps). Condensation of 17
with allyl alcohol in dichloromethane in the presence of tri-
methylsilyl triflate (0.15 equiv. based on 17) gave 18 (80%).
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Table 2 500 MHz 1H NMR data (1D and 2D TOCSY) of compound
Although only a few methods can be found in the literature
describing successful glycoside bond formation with N-acetyl
glycosyl donors,20,21 glycosidation using the trichloroacetimid-
ate N-acetyl donor 17 proceeded in high yield and with the
exclusive formation of the β-glycosidic linkage. Zemplén
de-O-acetylation (→ 19, 88%), followed by reaction with
cysteamine hydrochloride under radical conditions in an anti-
Markownikoff way for 2 h yielded target compound 6 (81%).
As the removal of excess of reagent from 6 was rather compli-
cated, only 1 equiv. of cysteamine was used for the reaction.
For the preparation of 7 a TEMPO-mediated oxidation step
was used.7,9 This type of oxidation was explored for reasons
mentioned above. It should be noted that structures larger than
the precursor of 5 could not be oxidized using PDC, pyridinium
chlorochromate,22 Jones oxidation procedure,22 or Swern oxid-
ation procedure23 (data not shown). However, the application
of nitroxyl radicals for oxidation in the presence of an allyl
group is impossible as the double bond of this group is sensi-
tive to radicals. Furthermore, it was found that oxidation of
3-(2-aminoethylthio)propyl (2,3,4-tri-O-p-toluoyl-β--gluco-
pyranosyl)-(1→3)-4,6-di-O-acetyl-2-deoxy-2-phthalimido-β--
galactopyranoside (an alternative precursor of disaccharide 1)
resulted in the formation of by-products due to oxidation of the
thio function (results not shown). The latter finding is in agree-
ment with recent data indicating that oxidation of alkyl/aryl
thioglycosides with TEMPO and sodium hypochlorite leads
to mixtures of sulfoxides and sulfones in preference to the
oxidation of primary hydroxy groups.24 For this reason,
the anomeric allyl group was changed for a 6-aminohexyl
spacer moiety. The preparation of 6-aminohexyl β--
glucopyranosiduronic acid 7 is outlined in Scheme 2. Con-
densation of 6-O-levulinoyl-2,3,4-tri-O-p-toluoyl-α--gluco-
pyranosyl trichloroacetimidate 205 with 6-azidohexan-1-ol
21 in dichloromethane catalyzed by trimethylsilyl triflate
(0.05 equiv. based on 20) gave 22 (83%). Then, 22 was de-
levulinoylated with hydrazinium acetate in toluene–ethanol to
afford 23 (89%). Oxidation of the primary hydroxy function
in 23 in a two-phase system (dichloromethane–saturated aq.
sodium bicarbonate) with sodium hypochlorite in the presence
of a catalytic amount of TEMPO gave a rapid conversion of 23
into its uronic acid derivative (→ 24, 79%). As the oxidation
was performed under alkaline conditions, the reaction time was
kept to a minimum in order to avoid cleavage of the base-labile
acyl functions (15 min). The presence of a carboxylic function
3
Proton (δH)
GalNAc
GlcA
GalNAcЈ
H-1 (J1,2
H-2 (J2,3
H-3 (J3,4
H-4 (J4,5
)
)
)
)
4.46 (8.4)
4.03 (10.8)
3.84 (3.9)
4.15 (< 1)
4.53 (7.4)
3.34 (9.3)
3.48 (9.3)
3.48
4.48 (8.8)
3.89 (10.8)
3.73 (3.0)
3.94 (< 1)
OCH2CH2CH2NH2
O(CH2)2CH2NH2
OCH2(CH2)2NH2
NHCOCH3
1.93–2.00
3.10
3.76 and 3.98
2.03
methanol gave 31 (84%), which was oxidized to afford 32 using
the TEMPO-mediated protocol as described for the oxidation
of 23 to 24 (→ 32, 75%). Treatment of a small amount of acid
32 with diazomethane in diethyl ether gave ester 32a; 1H NMR
analysis showed the presence of the methyl ester at δ 3.69.
Dephthaloylation/deacylation of 32 using methylamine in
ethanol (14 days), and subsequent N-acetylation with acetic
anhydride in methanol at 0 ЊC, afforded target compound 33
(80%). Finally, hydrogenation of the azido group of 33 was
performed using sodium borohydride in the presence of Pd-C
(→ 3, 93%). For 1H NMR data of compound 3, see Table 2.
In conclusion, sodium hypochlorite oxidation catalyzed by
TEMPO is a promising alternative for oxidation steps in the
synthesis of larger CAA glycan fragments.
Conversion of 1–7 into neoglycoconjugates
Reaction of the amine functions of compounds 1–7 with
diethyl squarate in a phosphate-buffered mixture of water and
ethanol at pH 7 afforded the squarate adducts 34–40, respect-
ively (Scheme 4).26,27 Squarate reactions were performed start-
ing with 1.0–1.5 mg of carbohydrate, and TLC analysis showed
in most cases complete reactions without decomposition of the
starting compounds. Reactions with larger compounds (4 and
5) required relatively long reaction times (up to 1 day). Most
products were readily purified by passing the reaction mixtures
through a C-18 Sep-Pak cartridge. However, purification of 37
and 38 was complicated, and additional HiTrap gel filtration
was needed to isolate the adducts. Purification of 36 was very
complicated and isolation using a Sep-Pak cartridge was not
successful, most likely due to the shorter spacer-arm of 36
compared with that of other adducts; 36 could only be isolated
by HiTrap gel filtration. The isolated products 34–40 were
immediately used for the subsequent covalent coupling to BSA
via the ε-amino groups of the lysine residues of the protein. The
targeted incorporation onto BSA was in all cases 15 oligo-
saccharide units per BSA molecule, and 35–40 were added to a
solution of BSA in bicarbonate buffer (pH 9). After stirring for
3 days, the isolation of the neoglycoconjugates was performed
by HiTrap gel filtration. Lyophilization provided the neoglyco-
proteins 41–47 (Scheme 4). The average degree of incorpor-
ation was determined by MALDI-TOF MS (Table 3) by
determination of the centre of the distribution of the singly
charged molecular ion.
1
in 24 was confirmed by H NMR analysis of methyl-esterified
24 (→ 24a; COOCH3 singlet at δ 3.69). After detoluoylation
of 24 using methylamine in ethanol (7 days; → 25, 88%), fol-
lowed by hydrogenation25 in the presence of Pd-C, compound 7
was obtained in 75% yield.
Synthesis of trisaccharide 3
To explore if nitroxyl radical-catalyzed oxidations could be
implemented in synthetic strategies towards the preparation of
larger CAA glycan fragments, trisaccharide 3 was assembled
having a 3-aminopropyl spacer at its anomeric centre (Scheme
3). Condensation of disaccharide donor 26 with 3-azido-
propan-1-ol 27 in dichloromethane in the presence of trimeth-
ylsilyl triflate (0.05 equiv. based on 26) afforded disaccharide
derivative 28 in 81% yield. Removal of the silyl ether group
under acidic conditions (toluene-p-sulfonic acid in acetonitrile–
water) gave 29 (67%). During purification, some product
was lost due to migration of the acetyl function from O-4 to
O-6. Conventional NIS/silver triflate-assisted glycosylation of
acceptor 29 with donor 10 in toluene afforded trisaccharide
derivative 30 in 42% yield. This rather low yield can probably be
assigned to acceptor loss during coupling by in situ O-acetyl
migration from O-4 to O-6, as TLC analysis during the reaction
showed the presence of the 6-O-acetylated by-product. Dele-
vulinoylation of 30 using hydrazinium acetate in toluene–
It is evident from Table 3 that, in general, coupling efficien-
cies, based on two reaction steps, decreased when larger
oligosaccharides were conjugated to BSA. The low coupling
efficiency of the conjugation reaction with 36 is most likely
due to the difficult isolation procedure. Therefore, the use
of spacer systems longer than the 3-aminopropyl moiety is
recommended.
For uncharged carbohydrate structures, it was claimed11 that
the nature of the carbohydrate will not substantially affect the
efficiency of conjugations using squarate chemistry. However,
other authors found that the rate and level of incorporation of
charged glycosides, e.g. sialic acid derivatives,28 were lower than
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Scheme 2 Reagents and yields: i, H2NNH2ؒHOAc; ii, CCl3CN, DBU (56% over i and ii); iii, CH2᎐᎐CHCH2OH, TMSOTf (80%); iv, NaOMe (88%);
v, HS(CH2)2NH2 (81%); vi, TMSOTf (83%); vii, H2NNH2ؒHOAc (89%); viii, TEMPO, KBr, Bu4NCl, NaOCl (79%); ix, CH2N2 (quant.); x, MeNH2
(88%); xi, H2, Pd–C (75%).
in corresponding reactions with neutral glycosides. The results
Conformational studies on structures 2 and 4
presented here suggest an influence of size and charge on the
efficiency of the conjugation. In terms of charge, this effect can
probably be explained by a repulsive build-up of negative
charges on the acidic BSA protein.29
In order to get insight into the conformational effect of the
additional GalNAc residue in 2 and 5, compared with 1 and 4,
respectively, the preferred conformations of compounds 2 and 4
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Table 3 Degree of incorporation of 34–40 onto BSA
Targeted
incorporation
(n-value)
Achieved
incorporation
(n-value)
Oligosaccharide
used (mg)
Incorporation
efficiency (%)
Compound
Product
34
35
36
37
38
39
40
1.0
1.0
1.5
1.0
1.0
1.5
1.5
15
15
15
15
15
15
15
41
42
43
44
45
46
47
13.5
6.5
4.4
5.4
4.5
14.2
12.8
90
43
29
36
30
95
85
Scheme 3 Reagents and yields: i, TMSOTf (81%); ii, p-TsOH, water (67%); iii, NIS, AgOTf (42%); iv, H2NNH2ؒHOAc (84%); v, TEMPO, KBr,
Bu4NCl, NaOCl (75%); vi, CH2N2 (quant.); vii, (a) MeNH2; (b) Ac2O, MeOH (80%); viii, NaBH4, Pd–C (93%).
were investigated by performing molecular mechanics and
molecular dynamics calculations. First, relaxed energy maps
of the methyl glycosides of the constituting disaccharides,
β--GlcpA-(1→3)-β--GalpNAc (cf. structure 1) and β--
GalpNAc-(1→6)-β--GalpNAc, in vacuo were constructed with
the CHEAT30,31 force field. All maps were generated by chan-
ging the interresidual torsion angles in steps of 10Њ, resulting in
a 36 × 36 grid. Three dihedral angles per sugar ring and both
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Scheme 4 Reagents and conditions: i, diethyl squarate, pH 7.0; ii, BSA, pH 9.0 (conversion in Naϩ-form).
interglycosidic angles are kept fixed by setting constraints
clature G stands for gauche ( 60Њ) and T for trans ( 180Њ). The
during a first minimization at each grid point. Subsequently,
all constraints in the ring were removed and the molecule was
minimized again. Contour levels were plotted in steps of 1 kcal
molϪ1† from the global minimum. In this force field hydroxy
groups are treated as united atoms, which prevents the
formation of intramolecular hydrogen bonds, and whereby the
energies are independent of the hydroxy-group orientation. For
β--GlcpA-(1→3)-β--GalpNAc-(1→OMe) one energy map
was generated to study the preferred conformations for the
glycosidic linkages φ (O5Ј–C1Ј–O3–C3) and ψ (C1Ј–O3–C3–
C2) (Fig. 2a).32 For β--GalpNAc-(1→6)-β--GalpNAc-
(1→OMe), three φ/ψ energy maps were generated, correspond-
ing to the three staggered conformations around the C5–C6
bond, commonly denoted GG, GT and TG. In this nomen-
torsion angle of the O6–C6–C5–O5 moiety is indicated by the
first letter, and the torsion angle ω32 of the O6–C6–C5–C4
moiety by the second. The three isoenergy contour plots (Fig.
2b for the TG contour plot) show basically the same profile,
with the global energy minimum at φ/ψ Ϫ65Њ/Ϫ175Њ (Table 4).
A set of three second-lowest energy minima had an energy that
was at least 3 kcal molϪ1 higher than the global minima, and
were not further considered. For each energy map, the con-
formations with the lowest energy were minimized again, but
with no constraints on the interglycosidic dihedral angles. The
resulting disaccharides were used as building blocks to create
the tri- and tetrasaccharide starting structures (cf. 2 and 4) used
in the molecular dynamics (MD) calculations.
To consider the conformational aspects in solution, for the
methyl glycosides of the tri- and tetrasaccharide, MD runs
in water with a duration of 1 ns were performed with the
GROMOS force field. All runs were started from the global-
† 1 cal = 4.184 J.
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Table 4 Inter-glycosidic dihedral angles of the TG, GG and GT
lowest-energy conformations for β--GalpNAc-(1→6)-β--GalpNAc-
(1→OMe)
Table 5 Probability distributions of conformations of the β--galacto-
pyranoside hydroxymethyl group,34 and distributions around C5–C6
of the β--GalpNAc-(1→6)-β--GalpNAc part in 2 and 4
Dihedral anglesa
Trisaccharide
(2)
Tetrasaccharide
(4)
Gal34
Conformation
φ
ψ
ω
Energyb
TG
GG
GT
25
5
70
18
2
80
31
3
66
TG
GG
GT
TG
GG
GT
Ϫ63
Ϫ64
Ϫ66
51
36
40
Ϫ178
Ϫ179
Ϫ172
Ϫ172
164
Ϫ57
72
Ϫ175
Ϫ59
68
49.76
49.65
48.39
52.77
52.23
53.20
179
Ϫ173
a Angles are in degrees. b Energy is in kcal molϪ1
.
Fig. 3 Probability distribution profiles of the ω dihedral angle orient-
ation in the tri- and the tetrasaccharide.
Fig. 2 Selected trajectories of the GROMOS MD runs. Panel a shows
the results of the β--GlcpA-(1→3)-β--GalpNAc φ/ψ dihedral angles
for a 1 ns MD simulation of the trisaccharide. The trajectory is pro-
jected on top of the energy-contour map calculated for inter-glycosidic
linkages of the β--GlcpA-(1→3)-β--GalpNAc disaccharide, and the
arrow indicates the global energy minimum. Panel b displays the trajec-
tory of the β--GalpNAc-(1→6)-β--GalpNAc φ/ψ dihedral angles of
the trisaccharide displayed on top of the energy-contour plot of φ/ψ of
the β--GalpNAc-(1→6)-β--GalpNAc disaccharide in the GT con-
formation. Panels c and d show the MD trajectories of the ω dihedral
angle of the tetra- and trisaccharide, respectively.
minimum-energy conformation. For the simulations of both
structures, the φ dihedral angle of the glycosidic linkage(s)
between GlcA and GalNAc changed rapidly from 40Њ to some-
where in the region of Ϫ100Њ/Ϫ180Њ; see, for example, Fig. 2d.
Further investigation showed that for φ = 40Њ, a hydrogen bond
is formed between the COOϪ group of GlcA and the NH group
of GalNAc. During the grid search in vacuo, this hydrogen
bond is the cause of an unrealistically low energy for this con-
formation (note that NH is NOT a united atom in CHEAT and
that the carboxy group is implemented as a charged group). The
β--GalpNAc-(1→6)-β--GalpNAc interglycosidic dihedral
angle ω is shown in Fig. 2c and 2d, for the tetra- and trisacchar-
ide, respectively. The plots show some interchange between
the GT and TG conformation for the tetrasaccharide, the tri-
saccharide existing mostly in the GT conformation.
Fig. 4 GT Conformation of tetrasaccharide 4 as its methyl glycoside.
dihedral angle ω. The distribution profiles for the tri- and tetra-
saccharide (displayed in Fig. 3) show that the GT conformation
is predominant, but also that the TG conformation contributes
significantly to the overall conformations of both the tri- and
tetrasaccharide.
In summary, the results of the GG, GT and TG distributions
around C5–C6 of β--GalpNAc-(1→6)-β--GalpNAc corre-
spond with those found for the hydroxymethyl distribution
for Gal34 (for comparison see Table 5). However, no striking
differences are observed between the tri- and tetrasaccharide
To estimate the rotamer population distribution of ω, the
method of adaptive umbrella sampling of the potential of
mean force33 was used. With this method it is possible to obtain
free-energy differences between conformers, and as a con-
sequence from this information the distribution. Umbrella
sampling runs, with a duration of 15 ns, were performed for the
2256
J. Chem. Soc., Perkin Trans. 1, 2000, 2249–2263

View Article Online
fragments. Interestingly, the resulting conformations show no
interactions between the residues, except over the glycosidic
linkages. The low free-energy barrier between the GT and TG
conformations, which the umbrella sampling method estimated
at 2 kcal molϪ1 for both the tri- and tetrasaccharide, suggests a
flexible polysaccharide molecule. In Fig. 4 the tetrasaccharide 4
in the GT conformation is represented.
silyl)-2-deoxy-2-phthalimido-β--galactopyranosyl]-(1→6)-
[(6-O-levulinoyl-2,3,4-tri-O-p-toluoyl-β--glucopyranosyl)-
(1→3)]-4-O-acetyl-2-deoxy-2-phthalimido-β--galactopyran-
oside5 8 (272 mg, 0.13 mmol) in acetonitrile (12 ml) and water
(1.3 ml) was added p-TsOH monohydrate (83 mg, 0.44 mmol).
The mixture was stirred for 20 min at rt, then neutralized with
triethylamine, and the solution was diluted with CH2Cl2 (250
ml), washed successively with 10% aq. NaHCO3 (same vol., 1 ×)
and 5% aq. NaCl (half vol., 1 ×), dried, filtered, and concen-
trated. Column chromatography (CH2Cl2–acetone, 9:1; 0.1%
triethylamine) of the residue gave 9, isolated as a colourless
glass (208 mg, 82%); TLC (CH2Cl2–acetone, 9:1) Rf 0.94 (8),
0.22 (9); [α]D ϩ 1 (c 1, CHCl3); δH (300 MHz; CDCl3) 2.15, 2.20,
2.21 (2 ×), 2.29 (2 ×), 2.30 (2 ×) and 2.31 (2 ×) [30 H, 6 s,
6 × COC6H4CH3, 2 × CO(CH2)2COCH3 and 2 × COCH3], 2.68
[8 H, m, 2 × CO(CH2)2COCH3], 4.35 and 4.51 (2 H, 2 dd, J2,3
11.2, J2Љ,3Љ 11.2, 2- and 2Љ-H), 4.71 and 4.86 (2 H, 2 d, 1Ј- and 1ЉЈ-
H), 4.70 and 4.91 (2 H, 2 dd, J3,4 3.3, J3Љ,4Љ 3.3, 3- and 3Љ-H), 4.78
and 5.05 (2 H, 2 d, J1,2 8.5, J1Љ,2Љ 8.5, 1- and 1Љ-H), 5.20 and 5.29
(2 H, 2 dd, J1Ј,2Ј 7.8, J1ٞ,2ٞ 7.8, J2Ј,3Ј/2ٞ,3ٞ 9.8/10.0, 2Ј- and 2ٞ-H),
Experimental
General procedures
In the work-up procedures of reaction mixtures, organic solu-
tions were washed with appropriate amounts of indicated
aqueous solutions, then dried (MgSO4), and concentrated
under reduced pressure at 40 ЊC (water-bath). Reactions were
monitored by TLC on Silica Gel 60 F254 (Merck) with detection
by UV light, then charring with either 10% H2SO4 in EtOH or
0.2% orcinol in 20% methanolic H2SO4. Column chromato-
graphy was performed on Silica Gel 60 F254 (Merck, 0.063–
0.200 mm). Gel-permeation chromatography was carried out
on Sephadex LH-20 or Toyopearl HW-40S (Supelco)
5.29 (1 H, m, OCH2CH᎐CH2), 5.37 (2 H, br t, 3Ј- and 3ٞ-H),
᎐
5.43 and 5.62 (2 H, 2 d, 4- and 4Љ-H), 5.59 and 5.64 (2 H, 2 br t,
4Ј- and 4ٞ-H), 6.82, 6.84, 6.96 (2 ×), 7.11, 7.12, 7.29, 7.30, 7.53
(2 ×), 7.72 and 7.74 (24 H, 10 d, 6 × COC6H4CH3); δC (75.5
MHz; CDCl3) 20.6 and 20.7 (2 × COCH3), 21.3 (2 C) and 21.4
(4 C) (6 × COC6H4CH3), 27.6, 27.7 and 37.8 (2 C) [2 ×
CO(CH2)2COCH3], 29.5 and 29.6 [2 × CO(CH2)2COCH3], 52.1
and 52.2 (C-2, -2Љ), 60.0, 62.0, 62.2, 64.1 and 68.8 (C-6, -6Ј, -6Љ,
1
(2.0 × 60 cm). H NMR spectra (300 MHz) were recorded at
25 ЊC with a Bruker AC 300 spectrometer. Chemical shifts (δ)
are given in ppm relative to the signal for internal Me4Si for
solutions in CDCl3 (δ 0), or by reference to acetone (δ 2.225) for
solutions in D2O. J-Values are given in Hz. Two-dimensional
1
double-quantum filtered H–1H correlated spectra (2D DQF
1H–1H COSY spectra with various mixing times) were recorded
at 300 K using a Bruker AMX 500 spectrometer. 13C NMR
spectra (75.5 MHz) were recorded with a Bruker AC 300 spec-
trometer; δC-values are given in ppm relative to the signal for
CDCl3 (δC 76.9). Optical rotations were determined for solu-
tions in CHCl3, unless otherwise stated, at 20 ЊC with a Perkin-
Elmer 241 polarimeter, using a 10-cm 1-ml cell. [α]D-Values
are given in 10Ϫ1 deg cm2 gϪ1. UV irradiations for synthetic
purposes were performed in quartz vials at 254 nm using a
Cole-Parmer. Fast-atom-bombardment mass spectrometry
(FABMS) was performed on a JEOL JMS SX/SX 102A four-
sector mass spectrometer, operated at 10 kV accelerating volt-
age, equipped with a JEOL MS-FAB 10 D FAB gun operated at
10 mA emission current, producing a beam of 6 keV xenon
atoms. MALDI-TOF mass spectra were recorded on a Voyager-
DE (PerSeptive Biosystems) instrument using sinapinic acid
as the matrix; proteins were analyzed in the linear mode at an
acceleration voltage of 22.5 kV. Samples for MALDI-TOF
analysis were prepared as follows. First, an aliquot of 1 µl of
the matrix solution was placed on the target stage of the mass
spectrometer. After complete evaporation of the solvent, a 1-µl
droplet of a 7.5 pmol mlϪ1 sample solution in acetonitrile–water
(1:1) acidified with 0.1% trifluoroacetic acid was applied to the
thin matrix film. Spectra were obtained by summing positive-
ion signals of 183 to 188 laser shots. GLC was performed using
a CP-Sil5 CB WCOT fused-silica capillary column (25 m × 0.34
mm, Chrompack). Native SDS-PAGE (8% acrylamide) was run
on a Pharmacia Phast System according to the manufacturer’s
instructions. Bovine serum albumin (BSA) together with pre-
treated BSA were used as reference and the gel was stained by
a standard Coomassie technique. 3-Azidopropan-1-ol and
6-azidohexan-1-ol were prepared from the commercially avail-
able 3-bromopropan-1-ol and 6-bromohexan-1-ol, respectively.
Crystalline BSA was obtained from Bayer Corporation.
-6ٞ and OCH2CH᎐CH2), 96.7 and 98.4 (C-1, -1Љ), 101.0 and
᎐
101.4 (C-1Ј, -1ٞ), 117.4 (OCH CH᎐CH ), 164.0 (2 C), 164.8,
᎐
2
2
164.9 and 165.4 (2 C) (6 × COC6H4CH3), 166.6, 168.4 (2 C) and
170.1 (2 × COCH3 and 2 × COPhth), 172.2 and 172.3 [2 ×
CO(CH2)2COCH3]; FABMS of C105H104N2O35 (M, 1954.0) m/z
1976.8 (M ϩ Na)ϩ.
Allyl (3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-ꢀ-D-galacto-
pyranosyl)-(1→6)-[(6-O-levulinoyl-2,3,4-tri-O-p-toluoyl-ꢀ-D-
glucopyranosyl)-(1→3)]-(4-O-acetyl-2-deoxy-2-phthalimido-ꢀ-
D-galactopyranosyl)-(1→6)-[(6-O-levulinoyl-2,3,4-tri-O-p-
toluoyl-ꢀ-D-glucopyranosyl)-(1→3)]-4-O-acetyl-2-deoxy-2-
phthalimido-ꢀ-D-galactopyranoside 11
A mixture of ethyl 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-1-
thio-β--galactopyranoside5 10 (56 mg, 0.12 mmol) and 9 (115
mg, 59 µmol) in dry toluene (2 ml), containing molecular sieves
4 Å (0.2 g), was stirred under Ar for 2 h at rt. Then, NIS (16 mg,
72 µmol) and silver trifluoromethanesulfonate (15 mg, 58 µmol)
were added, and the resulting suspension was stirred for 2.5 h at
rt. After neutralization with pyridine, the mixture was diluted
with CH2Cl2 (200 ml), and filtered over Celite. The organic
phase was washed successively with 10% aq. NaHSO3 (half
vol., 2 ×), 10% aq. NaHCO3 (same vol., 1 ×) and 5% aq. NaCl
(half vol., 1 ×), dried, and concentrated. Sephadex LH-20
chromatography (CH2Cl2–MeOH, 1:1), followed by column
chromatography (CH2Cl2–acetone, 92:8) of the residue yielded
11, isolated as a colourless glass (92 mg, 66%); TLC (CH2Cl2–
acetone, 9:1) Rf 0.67 (10), 0.54 (11); [α]D ϩ80 (c 1, CHCl3); δH
(300 MHz; CDCl3) 1.84, 2.11, 2.15 (2 ×), 2.16, 2.17, 2.21 (3 ×)
and 2.31 (4 ×) [39 H, 7 s, 6 × COC6H4CH3, 2 × CO(CH2)2-
COCH3 and 5 × COCH3], 2.60 [8 H, m, 2 × CO(CH2)2COCH3],
3.28 and 3.50 (2 H, 2 m, OCH CH᎐CH ), 4.40, 4.41 and 4.47
᎐
2
2
(3 H, 3 dd, J1,2/1Љ,2Љ/1٣,2٣ 8.4/8.5/8.5, J2,3 11.3, J2Љ,3Љ 11.3, J2٣,3٣ 11.3,
2-, 2Љ- and 2٣-H), 4.64 and 4.79 (2 H, 2 d, 1Ј- and 1ٞ-H), 4.69
and 4.74 (2 H, 2 dd, J3,4 3.4, J3Љ,4Љ 3.4, 3- and 3Љ-H), 4.76 and 4.94
Allyl (6-O-levulinoyl-2,3,4-tri-O-p-toluoyl-ꢀ-D-gluco-
pyranosyl)-(1→3)-(4-O-acetyl-2-deoxy-2-phthalimido-ꢀ-D-
galactopyranosyl)-(1→6)-[(6-O-levulinoyl-2,3,4-tri-O-p-
toluoyl-ꢀ-D-glucopyranosyl)-(1→3)]-4-O-acetyl-2-deoxy-2-
phthalimido-ꢀ-D-galactopyranoside 9
(2 H, 2 d, 1- and 1Љ-H), 5.22 (1 H, m, OCH CH᎐CH ), 5.18 and
᎐
2
2
5.22 (2 H, 2 dd, J1Ј,2Ј 7.8, J1ٞ,2ٞ 7.8, J2Ј,3Ј 9.8, J2ٞ,3ٞ 9.8, 2Ј- and 2ٞ-
H), 5.36 and 5.40 (2 H, 2 br t, 3Ј- and 3ٞ-H), 5.46, 5.52 and 5.58
(3 H, 3 d, 4-, 4Љ- and 4٣-H), 5.55 and 5.60 (2 H, 2 t, J4Ј,5Ј 9.6,
J4ٞ,5ٞ 9.6, 4Ј- and 4ٞ-H), 5.64 (1 H, d, J1٣,2٣ 8.4, 1٣-H), 5.93
(1 H, dd, J3٣,4٣ 3.5, 3٣-H), 6.85 (2 ×), 6.95 (2 ×), 7.09, 7.11, 7.29
To a solution of allyl (6-O-levulinoyl-2,3,4-tri-O-p-toluoyl-β-
-glucopyranosyl)-(1→3)-[4-O-acetyl-6-O-(tert-butyldimethyl-
J. Chem. Soc., Perkin Trans. 1, 2000, 2249–2263
2257

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(2 ×), 7.51 (2 ×), 7.69 and 7.70 (24 H, 8 d, 6 × COC6H4CH3);
δC (75.5 MHz; CDCl3) 20.4 and 20.7 (4 C) (5 × COCH3), 21.4
(2 C) and 21.5 (4 C) (6 × COC6H4CH3), 27.7 (2 C) and 37.8
(2 C) [2 × CO(CH2)2COCH3], 29.5 and 29.6 [2 × CO(CH2)2-
COCH3], 52.1 (3 C) (C-2, -2Љ, -2٣), 96.7, 96.8 and 98.2 (C-1, -1Љ,
CDCl3) 1.84, 2.10, 2.15, 2.17, 2.20, 2.21, 2.23 (2 ×), 2.33 (2 ×)
and 2.34 (33 H, 9 s, 6 × COC6H4CH3 and 5 × COCH3), 3.64
and 3.67 (6 H, 2 s, 2 × COOCH3), 4.66, 4.80, 4.85 and 4.91 (4 H,
4 d, J1,2/1Љ,2Љ/1ٞ,2ٞ/1٣,2٣ 7.7/8.6/7.7/8.4, 1-, 1Љ-, 1ٞ- and 1٣-H), 5.24
(1 H, m, OCH CH᎐CH ), 5.42 and 5.55 (2 H, 2 br t, J
᎐
2
2
3Ј,4Ј/3ٞ,4ٞ
-1٣), 100.9 and 101.0 (C-1Ј, -1ٞ), 117.4 (OCH CH᎐CH ), 164.1
9.6, 3Ј- and 3ٞ-H), 5.44, 5.49 and 5.67 (3 H, 3 d, 4-, 4Љ- and 4٣-
H), 5.93 (1 H, dd, 3٣-H), 6.90, 6.98, 7.11, 7.29, 7.54 and 7.71
(24 H, 6 dd, 6 × COC6H4CH3); for acid 13: FABMS of
C115H109N3O42 (M, 2203.1) m/z 2225.8 (M ϩ Na)ϩ.
᎐
2
2
(2 C), 164.8 (2 C) and 165.4 (2 C) (6 × COC6H4CH3), 169.6,
169.8, 170.2, 170.5 and 170.7 (5 × COCH3), 172.1 and 172.2
[2 × CO(CH2)2COCH3], 192.9 and 193.0 [2 × CO(CH2)2-
COCH3]; FABMS of C125H123N3O44 (M, 2371.3) m/z 2394.0
(M ϩ Na)ϩ.
Allyl (2-acetamido-2-deoxy-ꢀ-D-galactopyranosyl)-(1→6)-[(ꢀ-D-
glucopyranosyluronic acid)-(1→3)]-(2-acetamido-2-deoxy-ꢀ-D-
galactopyranosyl)-(1→6)-[(ꢀ-D-glucopyranosyluronic acid)-
(1→3)]-2-acetamido-2-deoxy-ꢀ-D-galactopyranoside 14
Allyl (3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-ꢀ-D-galacto-
pyranosyl)-(1→6)-[(2,3,4-tri-O-p-toluoyl-ꢀ-D-glucopyranosyl)-
(1→3)]-(4-O-acetyl-2-deoxy-2-phthalimido-ꢀ-D-galacto-
pyranosyl)-(1→6)-[(2,3,4-tri-O-p-toluoyl-ꢀ-D-glucopyranosyl)-
(1→3)]-4-O-acetyl-2-deoxy-2-phthalimido-ꢀ-D-galactopyrano-
side 12
A solution of compound 13 (27 mg, 12 µmol) in ethanolic 33%
MeNH2 (15 ml) was stirred for 2 weeks at rt, during which the
mixture was concentrated repeatedly and new reagent (8 × 10
ml) added. After concentration, the residue was dissolved in dry
MeOH (3.6 ml), and acetic anhydride (100 µl) was added at
0 ЊC. The mixture was stirred for 2 h, then concentrated and co-
concentrated with 1:1 toluene–MeOH (3 × 10 ml). The residue
was purified on Toyopearl HW-40S (5 mM aq. NH4HCO3) to
give, after lyophilization, 14 (8 mg, 65%); TLC (butan-1-ol–
MeOH–water–HOAc, 8:4:4:1) Rf 0.30 (14); [α]D Ϫ14 (c 0.8,
water); δH (500 MHz; D2O; TOCSY) 2.00, 2.02 and 2.03 (9 H,
3 s, 3 × NHCOCH3), 3.33 and 3.34 (2 H, 2 dd, J1Ј,2Ј/1ٞ,2ٞ 8.0,
J2Ј,3Ј/2ٞ,3ٞ 9.2, 2Ј- and 2ٞ-H), 3.45 (2 H, br t, J3Ј,4Ј/3ٞ,4ٞ 9.2, 3Ј- and
3ٞ-H), 3.48 (2 H, t, J4Ј,5Ј/4ٞ,5ٞ 8.3, 4Ј- and 4ٞ-H), 3.69 (2 H, d,
5Ј- and 5ٞ-H), 3.76 (1 H, dd, J3٣,4٣ 3.0, 3٣-H), 3.82 and 3.86
(2 H, 2 dd, 3- and 3Љ-H), 3.93 (1 H, dd, 2٣-H), 3.95 (1 H, dd,
J4٣,5٣ < 1, 4٣-H), 3.98 and 4.01 (2 H, 2 dd, 2- and 2Љ-H), 4.13
and 4.15 (2 H, 2 d, 4- and 4Љ-H), 4.48 (1 H, d, 1٣-H), 4.50 and
4.51 (2 H, 2 d, 1Ј- and 1ٞ-H), 4.51 and 4.53 (2 H, 2 d, J1,2/1Љ,2Љ 8.6,
To a solution of compound 11 (39 mg, 16 µmol) in 2:1 EtOH–
toluene (4 ml) was added hydrazinium acetate (15 mg, 0.16
mmol). The mixture was stirred for 20 min at rt, then concen-
trated. Column chromatography (CH2Cl2–acetone, 9:1) of the
residue gave 12, isolated as a colourless glass (32 mg, 92%);
TLC (toluene–EtOAc, 1:1) Rf 0.47 (11), 0.46 (12); [α]D ϩ4 (c 1,
CHCl3); δH (300 MHz; CDCl3) 1.85, 2.12, 2.21 (3 ×), 2.22, 2.25,
2.27 (2 ×) and 2.32 (2 ×) (33 H, 7 s, 6 × COC6H4CH3 and
5 × COCH ), 3.17 and 3.37 (2 H, 2 m, OCH CH᎐CH ), 4.38,
᎐
3
2
2
4.42 and 4.47 (3 H, 3 dd, J1,2/1Љ,2Љ/1٣,2٣ 8.4, J2,3/2Љ,3Љ/2٣,3٣ 11.1/11.2/
11.3, 2-, 2Љ- and 2٣-H), 4.74 and 4.94 (2 H, 2 d, 1- and 1Љ-H),
4.74 and 4.85 (2 H, 2 d, J1Ј,2Ј/1ٞ,2ٞ 7.8, 1Ј- and 1ٞ-H), 5.18 (1 H, m,
OCH CH᎐CH ), 5.30 and 5.32 (2 H, 2 t, J 9.6, J3Ј,4Ј/3ٞ,4ٞ
᎐
2
2
2Ј,3Ј/2ٞ,3ٞ
9.6, 3Ј- and 3ٞ-H), 5.55, 5.60 and 5.70 (3 H, 3 d, J4,5/4Љ,5Љ/4٣,5٣ 3.4/
3.7, 4-, 4Љ- and 4٣-H), 5.59 and 5.63 (2 H, 2 t, 4Ј- and 4ٞ-H),
5.92 (1 H, dd, J3٣,4٣ 3.6, 3٣-H), 6.77 (2 ×), 6.94 (2 ×), 7.11 (2 ×),
7.24, 7.27, 7.49, 7.50, 7.72 and 7.73 (24 H, 9 d, 6 × COC6-
H4CH3); δC (75.5 MHz; CDCl3) 20.4, 20.7 (2 C) and 20.9 (2 C)
(5 × COCH3), 21.3 (2 C) and 21.5 (4 C) (6 × COC6H4CH3),
52.0 and 52.2 (2 C) (C-2, -2Љ, -2٣), 61.0 (2 C) (C-6, -6Љ), 62.0
(C-6٣), 65.7 and 66.0 (C-6Ј, -6ٞ), 96.2, 96.8 and 98.2 (C-1, -1Љ,
1- and 1Љ-H), 5.27–5.39 (2 H, m, OCH CH᎐CH ), 5.91 (1 H, m,
᎐
2
2
OCH CH᎐CH ); FABMS of C H N O (M, 1019.6) m/z
᎐
2
2
39 61
3
28
1042.4 (M ϩ Na)ϩ.
3-(2-Aminoethylthio)propyl (2-acetamido-2-deoxy-ꢀ-D-galacto-
pyranosyl)-(1→6)-[(ꢀ-D-glucopyranosyluronic acid)-(1→3)]-(2-
acetamido-2-deoxy-ꢀ-D-galactopyranosyl)-(1→6)-[(ꢀ-D-gluco-
pyranosyluronic acid)-(1→3)]-2-acetamido-2-deoxy-ꢀ-D-galacto-
pyranoside 5
-1٣), 101.7 (C-1Ј, -1ٞ), 117.5 (OCH CH᎐CH ), 163.9 (2 C),
᎐
2
2
165.0 (2 C), 166.5 (2 C) (6 × COC6H4CH3), 166.7, 168.3 and
168.5 (3 × COPhth), 169.6, 170.2, 170.8, 171.3 and 172.4
(5 × COCH3); FABMS of C115H111N3O40 (M, 2175.1) m/z
2197.8 (M ϩ Na)ϩ.
Allyl glycoside 14 (2.5 mg, 2.5 µmol) was dissolved in aq.
cysteamine hydrochloride (1.5 mg, 13.2 µmol in 250 µl), and the
solution was irradiated with UV light for 4.5 h at rt. The prod-
uct was purified by HiTrap gel filtration (5 mM aq. NH4HCO3)
to afford, after lyophilization, the title compound 5 (2.2 mg,
80%). TLC (butan-1-ol–water–HOAc, 2:1:1) Rf 0.53 (14), 0.10
(5); [α]D Ϫ15 (c 0.2, water); 1H NMR data are given in Table 1;
FABMS of C41H68N4O28S (M, 1096.1) m/z 1094.9 (M Ϫ H)Ϫ.
Allyl (3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-ꢀ-D-galacto-
pyranosyl)-(1→6)-[(2,3,4-tri-O-p-toluoyl-ꢀ-D-glucopyranos-
yluronic acid)-(1→3)]-(4-O-acetyl-2-deoxy-2-phthalimido-ꢀ-D-
galactopyranosyl)-(1→6)-[(2,3,4-tri-O-p-toluoyl-ꢀ-D-gluco-
pyranosyluronic acid)-(1→3)]-4-O-acetyl-2-deoxy-2-phthal-
imido-ꢀ-D-galactopyranoside 13
To a solution of compound 12 (30 mg, 14 µmol) in dry CH2Cl2
(1 ml) were added PDC (21 mg, 84 µmol), acetic anhydride
(14 µl, 0.15 mmol) and a catalytic amount of dry pyridine. The
reaction mixture was stirred for 3.5 h at rt, then EtOAc (1 ml)
was added. Column chromatography (EtOAc → EtOAc–
HOAc, 98:2) of the suspension yielded 13 (22 mg, 73%); TLC
(CH2Cl2–acetone–HOAc, 18:2:1) Rf 0.19 (13); [α]D ϩ3 (c 1,
CHCl3). A solution of 13 in 5:1 MeOH–CH2Cl2 (5 ml) was
stirred with Dowex-50 (Naϩ) for 30 min, filtered, concentrated,
and analyzed by 13C NMR: δC (75.5 MHz; CDCl3) 20.4 and
20.6 (4 C) (5 × COCH3), 21.3 (2 C) and 21.5 (4 C)
(6 × COC6H4CH3), 51.3 and 52.4 (2 C) (C-2, -2Љ, -2٣), 62.7
(C-6, -6Љ, -6٣), 97.1, 97.4 and 97.5 (C-1, -1Љ, -1٣), 100.9 and
Allyl 2-acetamido-2-deoxy-ꢀ-D-galactopyranoside 19
To a solution of 2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy--
galactopyranose 15 (85 mg, 0.22 mmol) in dry CH2Cl2 (1 ml)
was added NH2NH2ؒHOAc (26 mg, 0.28 mmol). The mixture
was stirred for 1 h at 60 ЊC, when TLC (CH2Cl2–acetone, 9:1)
showed the complete conversion of 15 into 16. The mixture was
concentrated, and the material was directly used for the next
reaction.
To a solution of the residue in dry CH2Cl2 (2.2 ml) and
trichloroacetonitrile (0.2 ml, 2.0 mmol) at 0 ЊC was added DBU
(10 µl). The mixture was stirred overnight, then concentrated.
Column chromatography (CH2Cl2–acetone, 9:1) of the residue
furnished 17, isolated as a syrup (61 mg, 56% over two steps).
A solution of 17 (50 mg, 0.10 mmol) and allyl alcohol (88 µl,
1.0 mmol) in dry CH2Cl2 (0.75 ml) containing molecular sieves
4 Å (10 mg) was stirred for 1 h under Ar. Then, Me3SiOTf (2.7
µl, 14 µmol) was added and the mixture was stirred for 30 min.
101.0 (C-1Ј, -1ٞ), 117.1 (OCH CH᎐CH ), 164.3, 165.4, 166.0
᎐
2
2
(2 C) and 166.5 (2 C) (6 × COC6H4CH3), 169.8 (3 C) and 170.4
(2 C) (5 × COCH3).
A small amount of acid 13 was esterified with diazomethane
in diethyl ether (13a), and analyzed by 1H NMR: δH (300 MHz;
2258
J. Chem. Soc., Perkin Trans. 1, 2000, 2249–2263

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After neutralization with pyridine, dilution with CH2Cl2
(80 ml), and filtration, the solution was washed with 5% aq.
NaCl (half vol., 1 ×), dried, filtered, and concentrated. Column
chromatography (CH2Cl2–acetone, 95:5) of the residue gave
18, isolated as a syrup (30 mg, 80%); TLC (CH2Cl2–acetone,
9:1) Rf 0.72 (15), 0.22 (16), 0.60 (17), 0.55 (18); [α]D Ϫ17 (c 1,
CHCl3) (Found: C, 52.58; H, 6.51. C17H25NO9 requires C,
52.71; H, 6.46%); δH (300 MHz; CDCl3) 1.95, 2.00, 2.05 and
2.15 (12 H, 4 s, 4 × COCH3), 3.99 (1 H, m, J1,2 8.5, J2,3 11.1,
2-H), 4.75 (1 H, d, J1,2 8.4, 1-H), 5.20 and 5.28 (2 H, 2 m,
29.0 [OCH2(CH2)4CH2N3], 27.7 and 37.7 [CO(CH2)2COCH3],
29.6 [CO(CH2)2COCH3], 51.0 [O(CH2)5CH2N3], 62.7 (C-6),
69.7 [OCH2(CH2)5N3], 101.1 (C-1), 164.8, 165.0 and 165.5
(3 × COC6H4CH3), 172.1 [CO(CH2)2COCH3]; FABMS of
C41H47N3O11 (M, 757.8) m/z 758.5 (M ϩ H)ϩ, 780.5 (M ϩ
Na)ϩ.
6-Azidohexyl 2,3,4-tri-O-p-toluoyl-ꢀ-D-glucopyranoside 23
To a solution of 22 (107 mg, 0.14 mmol) in 2:1 EtOH–toluene
(14 ml) was added NH2NH2ؒHOAc (60 mg, 0.65 mmol). The
mixture was stirred for 45 min, then concentrated. Column
chromatography (CH2Cl2–acetone, 95:5) of the residue fur-
nished 23, isolated as a colourless glass (82 mg, 89%); TLC
(CH2Cl2–acetone, 9:1) Rf 0.76 (22), 0.55 (23); [α]D Ϫ5 (c 0.7,
CHCl3) (Found: C, 65.63; H, 6.32. C36H41N3O9 requires C,
65.54; H, 6.26%); δH (300 MHz; CDCl3) 1.18–1.58 [8 H, m,
O(CH2)(CH2)4(CH2)N3], 2.27, 2.34 and 2.35 (9 H, 3 s, 3 ×
COC6H4CH3), 3.06 [2 H, t, O(CH2)5CH2N3], 3.53 and 3.95 [2 H,
2 m, OCH2(CH2)5N3], 4.79 (1 H, d, J1,2 7.9, 1-H), 5.47 (1 H, dd,
J2,3 9.9, 2-H), 5.44 (1 H, t, J3,4 9.7, 3-H), 5.90 (1 H, t, J4,5 9.7,
4-H), 7.06, 7.16, 7.17, 7.74, 7.83 and 7.85 (12 H, 6 d, 3 ×
COC6H4CH3); δC (75.5 MHz; CDCl3) 21.4 and 21.5 (2 C)
(3 × COC6H4CH3), 25.2, 26.1, 28.4 and 29.1 [OCH2(CH2)4-
CH2N3], 51.0 [O(CH2)5CH2N3], 61.3 (C-6), 69.7 [OCH2(CH2)5-
N3], 101.2 (C-1), 164.8, 165.7 and 166.0 (3 × COC6H4CH3);
FABMS of C36H41N3O9 (M, 659.7) m/z 660.5 (M ϩ H)ϩ, 682.4
(M ϩ Na)ϩ.
OCH CH᎐CH ), 5.31 (1 H, dd, J3,4 3.4, 3-H), 5.37 (1 H, dd, J4,5
᎐
2
2
1.0, 4-H), 5.88 (1 H, m, OCH CH᎐CH ); δ (75.5 MHz; CDCl )
᎐
2
2
C
3
20.6 (COCH3), 23.4 (NHCOCH3), 51.7 (C-2), 61.4 (C-6), 99.8
(C-1), 117.8 (OCH CH᎐CH ), 133.5 (OCH CH᎐CH ), 170.2
᎐
᎐
2
2
2
2
(2 C), 170.3 and 170.4 (4 × COCH3); FABMS of C17H25NO9
(M, 387.1) m/z 388.1 (M ϩ H)ϩ, 410.1 (M ϩ Na)ϩ.
To a solution of 18 (25 mg, 65 µmol) in 5:1 MeOH–CH2Cl2
(3 ml) was added NaOMe to pH 10, and the mixture was stirred
overnight. After neutralization with Dowex-50 (Hϩ), the mix-
ture was filtered and concentrated. Column chromatography
(EtOAc–MeOH, 3:1) of the residue gave 19, isolated as a
colourless glass (15 mg, 88%); TLC (EtOAc–MeOH, 3:1) Rf
0.29 (19); [α]D Ϫ45 (c 0.6, water); δH (300 MHz; D2O) 2.04 (3 H,
s, NHCOCH3), 3.72 (1 H, dd, J2,3 10.8, 3-H), 3.90 (1 H, dd,
2-H), 3.93 (1 H, d, J3,4 3.3, J4,5 < 1, 4-H), 4.50 (1 H, d, J1,2 8.5,
1-H), 5.26 and 5.31 (2 H, 2 m, OCH CH᎐CH ), 5.91 (1 H, m,
᎐
2
2
OCH CH᎐CH ); FABMS of C H NO (M, 261.3) m/z 262.1
᎐
2
2
11 19
6
(M ϩ H)ϩ.
6-Azidohexyl 2,3,4-tri-O-p-toluoyl-ꢀ-D-glucopyranosiduronic
acid 24
3-(2-Aminoethylthio)propyl 2-acetamido-2-deoxy-ꢀ-D-galacto-
pyranoside 6
To a solution of 23 (42 mg, 64 µmol) in CH2Cl2 (0.3 ml) were
added a catalytic amount of TEMPO, KBr (740 µg, 6.2 µmol),
Bu4NCl (1.7 mg, 6.2 µmol), and saturated aq. NaHCO3 (0.2
ml). The suspension was cooled to 0 ЊC and under vigorously
stirring a mixture of 0.35 M aq. NaOCl (84 µl, 79 µmol), satur-
ated aq. NaHCO3 (108 µl), and saturated aq. NaCl (138 µl)
were added dropwise. The mixture was stirred for 15 min, then
acidified (pH 2) by addition of 4 M aq. HCl and mixed with
CH2Cl2 (100 ml). The organic layer was washed with 10% aq.
NaCl (half vol., 1 ×), dried, filtered, and concentrated. Column
chromatography (CH2Cl2–acetone, 8:2 → CH2Cl2–acetone–
HOAc, 8:2:0.5) of the residue afforded 24 (34 mg, 79%); TLC
(CH2Cl2–acetone–HOAc, 9:1:0.5) Rf 0.37 (24); [α]D Ϫ1 (c 1,
CHCl3) (Found: C, 63.89; H, 6.08. C36H39N3O10 requires C,
64.08; H, 5.98%); δH (300 MHz; CDCl3) 1.18–1.29 [4 H, m,
O(CH2)2(CH2)2(CH2)2N3], 1.31–1.40 [2 H, m, O(CH2)4CH2-
CH2N3], 1.45–1.60 [2 H, m, OCH2CH2(CH2)4N3], 2.29, 2.34
and 2.37 (9 H, 3 s, 3 × COC6H4CH3), 3.07 [2 H, t, O(CH2)5-
CH2N3], 3.53 and 3.97 [2 H, 2 m, OCH2(CH2)5N3], 4.37 (1 H, d,
J4,5 9.2, 5-H), 4.85 (1 H, d, J1,2 7.3, 1-H), 5.48 (1 H, dd, J2,3 9.1,
2-H), 5.69 (1 H, t, J3,4 9.2, 3-H), 5.86 (1 H, t, 4-H), 7.08, 7.13,
7.17, 7.74, 7.80 and 7.84 (12 H, 6 d, 3 × COC6H4CH3); δC (75.5
MHz; CDCl3) 21.5 (COC6H4CH3), 25.2, 26.1, 28.5 and 29.0
[OCH2(CH2)4CH2N3], 51.1 [O(CH2)5CH2N3], 70.2 [OCH2-
(CH2)5N3], 101.0 (C-1), 164.9, 165.3 and 165.5 (3 × COC6H4CH3),
170.1 (COOH).
Allyl glycoside 19 (3 mg, 11 µmol) was dissolved in aq. cysteam-
ine hydrochloride (1.3 mg, 11 µmol in 130 µl), and the solution
was irradiated with UV light for 2 h at rt. After concentration
of the mixture, column chromatography (MeOH–water, 9:1) of
the residue afforded 6, isolated as a colourless glass (3 mg,
81%); TLC (butan-1-ol–MeOH–water–HOAc, 4:2:2:1) Rf
0.57 (19), 0.32 (6); [α]D Ϫ1 (c 0.2, water); δH (300 MHz; D2O)
1.81–1.90 [2 H, m, OCH2CH2CH2S(CH2)2NH2], 2.05 (3 H, s,
NHCOCH3), 2.63 and 3.03 [4 H, 2 t, O(CH2)2CH2SCH2-
CH2NH2], 3.41 (2 H, t, SCH2CH2NH2), 3.72 (1 H, dd, J2,3 10.9,
3-H), 3.87 (1 H, dd, 2-H), 3.94 (1 H, d, J3,4 3.3, J4,5 < 1, 4-H),
4.44 (1 H, d, J1,2 8.6, 1-H); FABMS of C13H26N2O6S (M, 338.4)
m/z 339.1 (M ϩ H)ϩ.
6-Azidohexyl 6-O-levulinoyl-2,3,4-tri-O-p-toluoyl-ꢀ-D-gluco-
pyranoside 22
A solution of 6-O-levulinoyl-2,3,4-tri-O-p-toluoyl-α--gluco-
pyranosyl trichloroacetimidate5 20 (135 mg, 0.19 mmol) and
6-azidohexan-1-ol 21 (140 mg, 0.97 mmol) in dry CH2Cl2
(2 ml), containing molecular sieves 4 Å (10 mg), was stirred for
1 h under Ar. Then, Me3SiOTf (1.7 µl, 8.8 µmol) was added and
the mixture was stirred for 15 min. After neutralization with
pyridine, dilution with CH2Cl2 (150 ml) and filtration, the solu-
tion was washed with 5% aq. NaCl (half vol., 1 ×), dried,
filtered, and concentrated. Column chromatography (CH2Cl2–
acetone, 95:5) of the residue gave 22, isolated as a syrup (119
mg, 83%); TLC (CH2Cl2–acetone, 9:1) Rf 0.59 (20), 0.65 (22);
[α]D Ϫ7 (c 1, CHCl3) (Found: C, 64.76; H, 6.22. C41H47N3O11
requires C, 64.98; H, 6.25%); δH (300 MHz; CDCl3) 1.18–1.26
[4 H, m, O(CH2)2(CH2)2(CH2)2N3], 1.32–1.38 [2 H, m,
O(CH2)4CH2CH2N3], 1.45–1.54 [2 H, m, OCH2CH2(CH2)4N3],
2.26, 2.32, 2.33 and 2.35 [12 H, 4 s, CO(CH2)2COCH3 and
3 × COC6H4CH3], 2.55–2.74 [4 H, m, CO(CH2)2COCH3], 3.06
[2 H, t, O(CH2)5CH2N3], 3.54 and 3.94 [2 H, 2 m, OCH2-
(CH2)5N3], 4.78 (1 H, d, J1,2 7.9, 1-H), 5.46 (1 H, dd, J2,3 9.8,
2-H), 5.51 (1 H, t, J3,4 9.7, 3-H), 5.84 (1 H, t, J4,5 9.7, 4-H), 7.05,
7.14, 7.17, 7.71, 7.80 and 7.84 (12 H, 6 d, 3 × COC6H4CH3);
δC (75.5 MHz; CDCl3) 21.4 (COC6H4CH3), 25.2, 26.1, 28.4 and
A small amount of acid 24 was esterified with diazomethane
in diethyl ether (24a), and analyzed by 1H NMR: δH (300 MHz;
CDCl3) 1.18–1.60 [8 H, m, OCH2(CH2)4(CH2)N3], 2.30, 2.36
and 2.37 (9 H, 3 s, 3 × COC6H4CH3), 3.08 [2 H, t, O(CH2)5-
CH2N3], 3.52 and 3.97 [2 H, 2 m, OCH2(CH2)5N3], 3.69 (3 H, s,
COOCH3), 4.31 (1 H, d, J4,5 9.6, 5-H), 4.81 (1 H, d, J1,2 7.5,
1-H), 5.49 (1 H, dd, J2,3 9.5, 2-H), 5.64 (1 H, t, J3,4 9.5, 3-H),
5.87 (1 H, t, 4-H), 7.09, 7.17, 7.18, 7.75, 7.81 and 7.84 (12 H,
6 d, 3 × COC6H4CH3); for acid 24: FABMS of C36H39N3O10
(M, 673.7) m/z 674.4 (M ϩ H)ϩ, 696.4 (M ϩ Na)ϩ.
6-Azidohexyl ꢀ-D-glucopyranosiduronic acid 25
A solution of compound 24 (22 mg, 32 µmol) in ethanolic 33%
J. Chem. Soc., Perkin Trans. 1, 2000, 2249–2263
2259

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MeNH2 (5 ml) was stirred for 7 days at rt, during which the
mixture was concentrated repeatedly and new reagent (3 × 5
ml) was added. Column chromatography (EtOAc–MeOH–H2O,
10:5:1) of the residue gave 25 (9 mg, 88%); TLC (butan-1-ol–
MeOH–water–HOAc, 8:4:4:1) Rf 0.65 (25); [α]D Ϫ30 (c 1,
water); δH (300 MHz; D2O) 1.35–1.44 and 1.56–1.69 [8 H,
2 m, OCH2(CH2)4CH2N3], 3.16 (1 H, dd, 2-H), 3.32 [2 H, t,
O(CH2)5CH2N3], 3.51 (1 H, d, J4,5 8.9, 5-H), 3.64 (1 H, t, J2,3 9.5,
J3,4 9.8, 3-H), 3.67 and 3.93 [2 H, 2 m, OCH2(CH2)5N3], 3.70
(1 H, t, 4-H), 4.45 (1 H, d, J1,2 8.0, 1-H); FABMS of C12H21-
N3O7 (M, 319.3) m/z 318.1 (M Ϫ H)Ϫ.
(30 mg, 0.16 mmol). The mixture was stirred for 45 min, then
diluted with CH2Cl2 (150 ml). The organic layer was washed
successively with 10% aq. NaHCO3 (same vol., 1 ×) and 5% aq.
NaCl (half vol., 1 ×), dried, filtered, and concentrated. Column
chromatography (CH2Cl2–acetone, 96:4; 0.1% triethylamine)
of the residue gave 29 (59 mg, 67%); TLC (CH2Cl2–acetone,
9:1) Rf 0.16 (29); [α]D ϩ4 (c 1, CHCl3) (Found: C, 61.20; H,
5.03. C54H56N4O18 requires C, 61.83; H, 5.34%); δH (300 MHz;
CDCl3) 1.63 (2 H, m, OCH2CH2CH2N3), 2.21 (3 H, s, COCH3),
2.23, 2.30, 2.31 and 2.32 [12 H, 4 s, CO(CH2)2COCH3 and
3 × COC6H4CH3], 2.51–2.87 [4 H, m, CO(CH2)2COCH3], 3.41
and 3.82 (2 H, 2 m, OCH2CH2CH2N3), 4.53 (1 H, dd, J1,2 8.5,
J2,3 11.2, 2-H), 4.88 (1 H, d, J1Ј,2Ј 7.9, 1Ј-H), 4.91 (1 H, dd, J3,4
3.4, 3-H), 5.02 (1 H, d, 1-H), 5.32 (1 H, dd, J2Ј,3Ј 9.9, 2Ј-H), 5.40
(1 H, t, J3Ј,4Ј 9.8, 3Ј-H), 5.60 (1 H, d, J4,5 <1, 4-H), 5.68 (1 H, t,
J4Ј,5Ј 9.7, 4Ј-H), 6.84, 6.98, 7.13, 7.33, 7.55 and 7.74 (12 H, 6 d,
3 × COC6H4CH3); δC (75.5 MHz; CDCl3) 20.4 (COCH3), 21.4
(COC6H4CH3), 27.7 and 37.8 [CO(CH2)2COCH3], 29.9
[CO(CH2)2COCH3], 28.6, 47.6 and 66.0 (OCH2CH2CH2N3),
52.3 (C-2), 59.9 and 62.2 (C-6, -6Ј), 98.6 (C-1), 101.5 (C-1Ј),
122.6, 123.1, 130.7 and 133.4 (Phth), 164.1, 165.0 and 165.4
(3 × COC6H4CH3), 172.4 [CO(CH2)2COCH3]; FABMS of
C54H56N4O18 (M, 1048.8) m/z 1071.4 (M ϩ Na)ϩ.
6-Aminohexyl ꢀ-D-glucopyranosiduronic acid 7
A solution of 25 (5.0 mg, 16 µmol) in MeOH (0.5 ml) and
HOAc (50 µl) was hydrogenolyzed in the presence of 10% Pd–C
(6.4 mg) under H2 for 2 h at rt. Then, the mixture was filtered
and concentrated. Column chromatography (EtOAc–MeOH–
water, 7:5:1) of the residue, followed by lyophilization from
water, afforded 7, isolated as a white powder (3.5 mg, 75%);
TLC (EtOAc–MeOH–H2O, 10:5:1) Rf 0.54 (25), 0.05 (7); [α]D
Ϫ17 (c 0.4, water); δH (300 MHz; D2O) 1.32–1.46 and 1.58–1.70
[8 H, 2 m, OCH2(CH2)4CH2NH2], 2.93 [2 H, t, O(CH2)5-
CH2NH2], 3.17 (1 H, dd, 2-H), 3.50 (1 H, d, J4,5 8.8, 5-H), 3.66
and 3.92 [2 H, 2 m, OCH2(CH2)5NH2], 3.66 (1 H, t, J2,3 9.2, J3,4
9.2, 3-H), 3.68 (1 H, t, 4-H), 4.45 (1 H, d, J1,2 7.9, 1-H); FABMS
of C12H23NO7 (M, 293.3) m/z 294.1 (M ϩ H)ϩ.
3-Azidopropyl (3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-ꢀ-D-
galactopyranosyl)-(1→6)-[(6-O-levulinoyl-2,3,4-tri-O-p-
toluoyl-ꢀ-D-glucopyranosyl)-(1→3)]-4-O-acetyl-2-deoxy-2-
phthalimido-ꢀ-D-galactopyranoside 30
3-Azidopropyl (6-O-levulinoyl-2,3,4-tri-O-p-toluoyl-ꢀ-D-gluco-
pyranosyl)-(1→3)-4-O-acetyl-6-O-(tert-butyldimethylsilyl)-2-
deoxy-2-phthalimido-ꢀ-D-galactopyranoside 28
A mixture of 10 (52 mg, 0.11 mmol) and 29 (54 mg, 51 µmol) in
dry toluene (1.5 ml) containing molecular sieves 4 Å (0.1 g) was
stirred under Ar for 2 h at rt. Then, NIS (15 mg, 67 µmol) and
silver trifluoromethanesulfonate (14 mg, 55 µmol) were added,
and the resulting suspension was stirred for 2 h at rt. After
neutralization with pyridine, the mixture was diluted with
CH2Cl2 (100 ml), filtered over Celite, washed successively with
10% aq. NaHSO3 (half vol., 3 ×), 10% aq. NaHCO3 (same vol.,
1 ×) and 5% aq. NaCl (half vol., 1 ×), dried, and concentrated.
The residue was purified by sequential Sephadex LH-20
chromatography (CH2Cl2–MeOH, 1:1) and Silica Gel column
chromatography (CH2Cl2 → CH2Cl2–acetone, 95:5) to yield
30, isolated as a colourless glass (33 mg, 42%); TLC (CH2Cl2–
acetone, 9:1) Rf 0.65 (10), 0.40 (30); [α]D ϩ4 (c 1, CHCl3);
δH (300 MHz; CDCl3) 1.29–1.48 (2 H, m, OCH2CH2CH2N3),
1.83, 2.09, 2.18 and 2.20 (12 H, 4 s, 4 × COCH3), 2.20, 2.23,
2.31 and 2.33 [12 H, 4 s, CO(CH2)2COCH3 and 3 ×
COC6H4CH3], 2.59 and 2.78 [4 H, 2 m, CO(CH2)2COCH3],
2.85–2.90 (2 H, m, OCH2CH2CH2N3), 3.12 and 3.42 (2 H, 2 m,
OCH2CH2CH2N3), 4.37 (1 H, dd, J1Љ,2Љ 8.5, J2Љ,3Љ 11.2, 2Љ-H), 4.52
(1 H, dd, J1,2 8.5, J2,3 11.5, 2-H), 4.72 (1 H, d, J1Ј,2Ј 7.7, 1Ј-H),
4.73 (1 H, dd, J3,4 3.4, 3-H), 4.82 (1 H, d, 1-H), 5.22 (1 H, dd,
J2Ј,3Ј 9.8, 2Ј-H), 5.36 (1 H, d, 1Љ-H), 5.39 (1 H, t, J3Ј,4Ј 9.8, 3Ј-H),
5.46 (1 H, d, J4,5 <1, 4-H), 5.48 (1 H, d, J4Љ,5Љ <1, 4Љ-H), 5.60
(1 H, t, J4Ј,5Ј 9.7, 4Ј-H), 5.80 (1 H, dd, J3Љ,4Љ 3.4, 3Љ-H), 6.86, 6.97,
7.12, 7.32, 7.54 and 7.73 (12 H, 6 d, 3 × COC6H4CH3); δC (75.5
MHz; CDCl3) 20.3, 20.5 (2 C) and 20.7 (4 × COCH3), 21.4
(COC6H4CH3), 27.7 and 37.8 [CO(CH2)2COCH3], 28.6, 47.6
and 66.5 (OCH2CH2CH2N3), 51.3 and 52.2 (C-2, -2Љ), 98.1
(2 C) (C-1, -1ٞ), 101.1 (C-1Ј), 123.3, 130.7 and 133.5 (Phth);
FABMS of C74H75N5O27 (M, 1465.0) m/z 1488.5 (M ϩ Na)ϩ.
To a solution of (6-O-levulinoyl-2,3,4-tri-O-p-toluoyl-β--
glucopyranosyl)-(1→3)-4-O-acetyl-6-O-(tert-butyldimethyl-
silyl)-2-deoxy-2-phthalimido-β--galactopyranosyl trichloro-
acetimidate5 26 (0.14 g, 0.12 mmol) and 3-azidopropan-1-ol 27
(53 mg, 0.52 mmol) in dry CH2Cl2 (3.2 ml), containing molec-
ular sieves 4 Å (0.1 g), was added at rt Me3SiOTf (1.1 µl, 5.7
µmol). After stirring for 15 min, the mixture was neutralized
with triethylamine, and CH2Cl2 (150 ml) was added. The
organic layer was washed with 5% aq. NaCl (half vol., 1 ×),
dried, filtered, and concentrated. Column chromatography
(CH2Cl2–acetone, 96:4) of the residue gave 28 (0.11 g, 81%);
TLC (CH2Cl2–acetone, 9:1) Rf 0.75 (26), 0.80 (28); [α]D ϩ7 (c 1,
CHCl3) (Found: C, 62.11; H, 6.15. C60H70N4O18Si requires C,
61.91; H, 6.02%); δH (300 MHz; CDCl3) 0.06 and 0.07 [6 H, 2 s,
Si(CH3)2C(CH3)3], 0.89 [9 H, s, Si(CH3)2C(CH3)3], 1.50–1.75 (2
H, m, OCH2CH2CH2N3), 2.20 (3 H, s, COCH3), 2.22 (2 ×), 2.32
and 2.33 [12 H, 3 s, CO(CH2)2COCH3 and 3 × COC6H4CH3],
2.60 and 2.76 [4 H, 2 m, CO(CH2)2COCH3], 2.98–3.09 (2 H, m,
OCH2CH2CH2N3), 3.43 and 3.79 (2 H, 2 m, OCH2CH2CH2N3),
4.48 (1 H, dd, J1,2 8.5, J2,3 11.2, 2-H), 4.78 (1 H, d, J1Ј,2Ј 7.8,
1Ј-H), 4.84 (1 H, dd, J3,4 3.3, 3-H), 5.00 (1 H, d, 1-H), 5.26 (1 H,
dd, J2Ј,3Ј 9.8, 2Ј-H), 5.44 (1 H, t, J3Ј,4Ј 9.7, 3Ј-H), 5.61 (1 H, d,
J4,5 <1, 4-H), 5.64 (1 H, t, 4Ј-H), 6.88, 6.98, 7.12, 7.37, 7.56
and 7.74 (12 H, 6 d, 3 × COC6H4CH3); δC (75.5 MHz; CDCl3)
18.0 [Si(CH3)2C(CH3)3], 20.7 (COCH3), 21.2 and 21.4 (2 C)
(3 × COC6H4CH3), 25.6 [Si(CH3)2C(CH3)3], 27.7 and 37.8
[CO(CH2)2COCH3], 29.6 [CO(CH2)2COCH3], 28.5, 47.7 and
65.8 (OCH2CH2CH2N3), 52.4 (C-2), 62.1 (2 C) (C-6, -6Ј), 98.4
and 101.0 (C-1, -1Ј), 122.7, 123.1, 130.7 and 133.5 (Phth), 164.2,
164.8 and 165.4 (3 × COC6H4CH3), 169.8 (COCH3), 172.1
[CO(CH2)2COCH3]; FABMS of C60H70N4O18Si (M, 1162.8)
m/z 1185.5 (M ϩ Na)ϩ.
3-Azidopropyl (3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-ꢀ-D-
galactopyranosyl)-(1→6)-[(2,3,4-tri-O-p-toluoyl-ꢀ-D-gluco-
pyranosyl)-(1→3)]-4-O-acetyl-2-deoxy-2-phthalimido-ꢀ-D-
galactopyranoside 31
3-Azidopropyl (6-O-levulinoyl-2,3,4-tri-O-p-toluoyl-ꢀ-D-gluco-
pyranosyl)-(1→3)-4-O-acetyl-2-deoxy-2-phthalimido-ꢀ-D-
galactopyranoside 29
To a solution of compound 30 (25 mg, 18 µmol) in 2:1 MeOH–
toluene (2.8 ml) was added hydrazinium acetate (17 mg, 0.18
mmol). The mixture was stirred for 20 min at rt, then concen-
trated. Column chromatography (CH2Cl2–acetone, 93:7) of the
residue gave 31 (20 mg, 84%); TLC (CH2Cl2–acetone, 9:1) Rf
To a solution of 28 (96 mg, 85 µmol) in acetonitrile (7.9 ml),
containing water (0.9 ml), was added p-TsOH monohydrate
2260
J. Chem. Soc., Perkin Trans. 1, 2000, 2249–2263

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0.48 (31); [α]D Ϫ1 (c 1, CHCl3) (Found: C, 60.18; H, 4.92.
C69H69N5O25 requires C, 60.55; H, 5.04); δH (300 MHz; CDCl3)
1.84, 2.09, 2.21 and 2.22 (12 H, 4 s, 4 × COCH3), 2.25, 2.28 and
2.33 (9 H, 3 s, 3 × COC6H4CH3), 2.84–2.90 (2 H, m, OCH2-
CH2CH2N3), 3.15 and 3.41 (2 H, 2 m, OCH2CH2CH2N3), 4.38
(1 H, dd, J1Љ,2Љ 8.4, J2Љ,3Љ 11.5, 2Љ-H), 4.53 (1 H, dd, J1,2 8.5, J2,3
11.4, 2-H), 4.73 (1 H, dd, J3,4 3.4, 3-H), 4.81 (1 H, d, J1Ј,2Ј 7.8,
1Ј-H), 4.82 (1 H, d, 1-H), 5.24 (1 H, dd, J2Ј,3Ј 9.9, 2Ј-H), 5.33
(1 H, t, J3Ј,4Ј 9.7, 3Ј-H), 5.36 (1 H, d, 1Љ-H), 5.48 (1 H, d, J4,5 <1,
4-H), 5.59 (1 H, d, J4Љ,5Љ <1, 4Љ-H), 5.64 (1 H, t, J4Ј,5Ј 9.8, 4Ј-H),
5.79 (1 H, dd, J3Љ,4Љ 3.3, 3Љ-H), 6.78, 6.96, 7.13, 7.29, 7.52 and
7.75 (12 H, 6 d, 3 × COC6H4CH3); δC (75.5 MHz; CDCl3) 20.4
and 20.6 (COCH3), 21.0, 21.3 and 21.4 (3 × COC6H4CH3),
28.4, 47.6 and 65.4 (OCH2CH2CH2N3), 51.2 and 52.2 (C-2,
-2Љ), 97.8 and 98.2 (C-1, -1ٞ), 101.8 (C-1Ј), 165.1, 165.4 and
166.7 (3 × COC6H4CH3), 169.6, 170.2, 170.3 and 171.5
(4 × COCH3); FABMS of C69H69N5O25 (M, 1367.3) m/z 1390.4
(M ϩ Na)ϩ.
water–HOAc, 2:1:0.5) Rf 0.35 (33); [α]D Ϫ10 (c 1, water);
δH (500 MHz; D2O; TOCSY) 1.82–1.89 (2 H, m, OCH2CH2-
CH2N3), 2.03 (6 H, s, 2 × NHCOCH3), 3.33 (1 H, dd, J1Ј,2Ј 8.0,
2Ј-H), 3.39 [2 H, t, O(CH2)2CH2N3], 3.47 (1 H, t, J2Ј,3Ј 9.3, J3Ј,4Ј
9.3, 3Ј-H), 3.50 (1 H, t, 4Ј-H), 3.68 (1 H, d, J4Ј,5Ј 8.3, 5Ј-H), 3.74
(1 H, dd, J3Љ,4Љ 3.1, 3Љ-H), 3.84 (1 H, dd, J3,4 3.1, 3-H), 3.89 (1 H,
dd, J1Љ,2Љ 8.6, J2Љ,3Љ 10.7, 2Љ-H), 3.94 (1 H, d, J4Љ,5Љ <1, 4Љ-H), 4.00
(1 H, dd, J1,2 8.6, J2,3 10.7, 2-H), 4.16 (1 H, d, J4,5 <1, 4-H), 4.47
(1 H, d, 1-H), 4.49 (1 H, d, 1Љ-H), 4.50 (1 H, d, 1Ј-H); FABMS
of C25H41N5O17 (M, 683.0) m/z 682.0 (M Ϫ H)Ϫ.
3-Aminopropyl (2-acetamido-2-deoxy-ꢀ-D-galactopyranosyl)-
(1→6)-[(ꢀ-D-glucopyranosyluronic acid)-(1→3)]-2-acetamido-2-
deoxy-ꢀ-D-galactopyranoside 3
To a solution of 33 (5 mg, 7.3 µl) in 0.05 M aq. NaOH were
added 10% Pd–C (2.5 mg) and a catalytic amount of NaBH4,
and the mixture was stirred for 2 h at rt. Then, the mixture was
filtered and concentrated. Column chromatography (MeOH–
H2O, 2:1) of the residue gave 3 (4.5 mg, 93%); TLC (MeOH–
1
3-Azidopropyl (3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-ꢀ-D-
galactopyranosyl)-(1→6)-[(2,3,4-tri-O-p-toluoyl-ꢀ-D-gluco-
pyranosyluronic acid)-(1→3)]-4-O-acetyl-2-deoxy-2-phthal-
imido-ꢀ-D-galactopyranoside 32
H2O, 2:1) Rf 0.56 (3); [α]D Ϫ130 (c 0.1, water); H NMR data
are given in Table 2; FABMS of C25H43N3O17 (M, 657.3) m/z
658.3 (M ϩ H)ϩ.
3-{2-[N-(2-Ethoxy-3,4-dioxocyclobut-1-enyl)amino]ethylthio}-
propyl (ꢀ-D-glucopyranosyluronic acid)-(1→3)-2-acetamido-2-
deoxy-ꢀ-D-galactopyranoside 34
To a solution of 31 (17 mg, 13 µmol) in CH2Cl2 (0.3 ml) were
added a catalytic amount of TEMPO, KBr (160 µg, 1.3 µmol),
Bu4NCl (181 µg, 0.66 µmol), and saturated aq. NaHCO3 (0.3
ml). The suspension was cooled to 0 ЊC and under vigorously
stirring a mixture of 0.35 M aq. NaOCl (17 µl, 16 µmol), satur-
ated aq. NaHCO3 (22 µl), and saturated aq. NaCl (28 µl) was
added dropwise. The mixture was stirred for 10 min, and there-
after the solution was acidified (pH 2) by adding 4 M aq. HCl.
Then, CH2Cl2 (50 ml) was added and the organic layer was
washed with 10% aq. NaCl (half vol., 1 ×), dried, filtered
and concentrated. Column chromatography (CH2Cl2–acetone,
8:2 → CH2Cl2–acetone–HOAc, 8:2:0.5) of the residue
furnished 32 (13 mg, 75%); TLC (CH2Cl2–acetone–HOAc,
9:1:0.5) Rf 0.37 (32); [α]D ϩ4 (c 1, CHCl3). A solution of 32
in 5:1 MeOH–CH2Cl2 (3 ml) was stirred with Dowex-50 (Naϩ)
for 30 min, filtered, concentrated and analyzed by 13C NMR:
δC (75.5 MHz; CDCl3) 20.4 and 20.6 (COCH3), 21.1, 21.4
and 21.5 (3 × COC6H4CH3), 29.6, 47.6 and 65.6 (OCH2CH2-
CH2N3), 51.2 and 52.2 (C-2, -2Љ), 97.6 and 98.3 (C-1, -1ٞ), 101.4
(C-1Ј), 164.0, 165.2 and 166.6 (3 × COC6H4CH3), 168.8, 169.7
and 170.3 (2 C) (4 × COCH3).
A small amount of acid 32 was esterified with diazomethane
in diethyl ether (32a), and analyzed by 1H NMR: δH (300 MHz;
CDCl3) 1.83, 2.09, 2.18 and 2.20 (12 H, 4 s, 4 × COCH3), 2.24,
2.33 and 2.34 (9 H, 3 s, 3 × COC6H4CH3), 2.85–2.91 (2 H, m,
OCH2CH2CH2N3), 3.69 (3 H, s, COOCH3), 4.39 and 4.52 (2 H,
2 dd, 2- and 2Љ-H), 4.72 (1 H, dd, 3-H), 4.75 (1 H, d, J1Ј,2Ј
7.8, 1Ј-H), 4.81 (1 H, d, J1,2 8.4, 1-H), 5.27 (1 H, dd, 2Ј-H),
5.35 (1 H, d, J1Љ,2Љ 8.6, 1Љ-H), 5.41 (1 H, d, J3,4 3.5, 4-H), 5.48
(1 H, d, 4Љ-H), 5.52 (1 H, t, J2Ј,3Ј/3Ј,4Ј 9.7, 3Ј-H), 5.65 (1 H, t,
4Ј-H), 5.79 (1 H, dd, J2Љ,3Љ 11.3, J3Љ,4Љ 3.3, 3Љ-H), 6.88, 7.00, 7.14,
7.31, 7.57 and 7.74 (12 H, 6 d, 3 × COC6H4CH3); FABMS of
C69H67N5O26 (M, 1381.4) m/z 1404.4 (M ϩ Na)ϩ.
To a solution of compound 15 (1.0 mg, 1.9 µmol) in 75 mM
sodium phosphate buffer (pH 7.0) (100 µl) was added a solution
of 3,4-diethoxycyclobut-3-ene-1,2-dione (diethyl squarate; 0.27
µl, 1.86 µmol) in EtOH (100 µl). The mixture was stirred for 4 h
at rt, when TLC (butan-1-ol–MeOH–water–HOAc, 4:2:2:0.5)
showed an incomplete conversion into a higher moving spot.
Again, diethyl squarate (0.1 µl, 0.69 µmol) was added and the
mixture was stirred overnight. After concentration, a solution
of the crude residue in water (1 ml) was loaded on a C-18 Sep-
Pak cartridge. The column was washed with water (2 ml, 3 ×),
then the product was eluted with MeOH (2 ml, 2 ×). The MeOH
phase was evaporated, and a solution of the residue in water
(2 ml) was concentrated to yield 34 as a colourless glass; TLC
(butan-1-ol–MeOH–water–HOAc, 4:2:2:0.5) Rf 0.22 (1), 0.46
(34). The material was directly used for the preparation of
neoglycoconjugate 41.
3-{2-[N-(2-Ethoxy-3,4-dioxocyclobut-1-enyl)amino]ethylthio}-
propyl (2-acetamido-2-deoxy-ꢀ-D-galactopyranosyl)-(1→6)-
[(ꢀ-D-glucopyranosyluronic acid)-(1→3)]-2-acetamido-2-deoxy-
ꢀ-D-galactopyranoside 35
A similar protocol as described for 34 was followed: compound
25 (1.0 mg, 1.4 µmol) in 75 mM sodium phosphate buffer (pH
7.0) (100 µl); diethyl squarate (0.18 µl, 1.26 µmol) in EtOH
(100 µl); reaction time, overnight at rt; TLC with butan-1-ol–
MeOH–water–HOAc, 4:2:2:0.5. Product 35 was isolated
as a colourless glass; TLC (butan-1-ol–MeOH–water–HOAc,
4:2:2:0.5) Rf 0.16 (2), 0.35 (34). The material was directly used
for the preparation of neoglycoconjugate 42.
3-[N-(2-Ethoxy-3,4-dioxocyclobut-1-enyl)amino]propyl (2-acet-
amido-2-deoxy-ꢀ-D-galactopyranosyl)-(1→6)-[(ꢀ-D-gluco-
pyranosyluronic acid)-(1→3)]-2-acetamido-2-deoxy-ꢀ-D-
galactopyranoside 36
3-Azidopropyl (2-acetamido-2-deoxy-ꢀ-D-galactopyranosyl)-
(1→6)-[(ꢀ-D-glucopyranosyluronic acid)-(1→3)]-2-acetamido-2-
deoxy-ꢀ-D-galactopyranoside 33
A solution of compound 32 (24 mg, 18 µmol) in ethanolic 33%
MeNH2 (5 ml) was stirred for 2 weeks at rt, during which the
mixture was concentrated repeatedly and new reagent (3 × 5
ml) added. After concentration, the residue was dissolved in dry
MeOH (3 ml) and acetic anhydride (100 µmol) was added at
0 ЊC. The mixture was stirred for 2 h, then concentrated and co-
concentrated with 1:1 toluene–MeOH (3 × 10 ml). Purification
of the residue on Toyopearl HW-40S with 5 mM aq. NH4HCO3
gave, after lyophilization, 33 (9.9 mg, 80%); TLC (butan-1-ol–
A similar protocol as described for 34 was followed: compound
3 (1.0 mg, 1.5 µmol) in 75 mM sodium phosphate buffer (pH
7.0) (100 µl); diethyl squarate (0.19 µl, 1.35 µmol) in EtOH
(100 µl); reaction time, overnight at rt; TLC with butan-1-ol–
MeOH–water–HOAc, 4:2:2:0.5. Product 36 was purified by
HiTrap gel filtration, and isolated as a colourless glass; TLC
(butan-1-ol–MeOH–water–HOAc, 3:3:3:1) Rf 0.10 (3), 0.23
(36). The material was directly used for the preparation of
neoglycoconjugate 43.
J. Chem. Soc., Perkin Trans. 1, 2000, 2249–2263
2261

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3-{2-[N-(2-Ethoxy-3,4-dioxocyclobut-1-enyl)amino]ethylthio}-
propyl (ꢀ-D-glucopyranosyluronic acid)-(1→3)-(2-acetamido-2-
deoxy-ꢀ-D-galactopyranosyl)-(1→6)-[(ꢀ-D-glucopyranosyluronic
acid)-(1→3)]-2-acetamido-2-deoxy-ꢀ-D-galactopyranoside 37
Preparation of BSA-glycoconjugates 41–47
Pretreated BSA (25 mg mlϪ1) was dissolved in 0.1 M NaHCO3
buffer (pH 9.0) and the solution was stirred for 30 min. Then,
the oligosaccharide–squarate adduct (34–40) in water (0.5 mg
mlϪ1) was added to the pretreated-BSA solution (15 mequiv.
based on BSA) and the resulting mixture was stirred for 3 days
at rt. The mixture was purified by HiTrap gel filtration (5%
aq. NH4HCO3) to afford, after lyophilization from water, the
neoglycoconjugate (41–47). The degree of incorporation of 34–
40 onto BSA was determined by MALDI-TOF MS (Table 3).
A similar protocol as described for 34 was followed: compound
45 (1.0 mg, 1.1 µmol) in 75 mM sodium phosphate buffer (pH
7.0) (100 µl); diethyl squarate (0.15 µl, 1.0 µmol) in EtOH (80
µl); reaction time, 4.5 h at rt; further diethyl squarate (0.07 µl,
0.5 µmol); reaction time, overnight at rt; TLC with butan-1-ol–
MeOH–water–HOAc, 4:2:2:0.5. Product 37 was isolated,
after an additional chromatographic purification on HiTrap
(5% aq. NH4HCO3), as a colourless glass; TLC (butan-1-ol–
MeOH–water–HOAc, 3:3:3:1) Rf 0.34 (4), 0.47 (37). The
material was directly used for the preparation of neoglycocon-
jugate 44.
Molecular mechanics calculations
Minimum-energy calculations in vacuo were performed with
CHEAT,30,31 a CHARMm-based force field for carbohydrates
wherein OH groups are represented by extended atoms to pre-
vent intramolecular hydrogen-bond formation. Carboxylic and
N-acetyl parameters were taken from the CHARMm force
field.
3-{2-[N-(2-Ethoxy-3,4-dioxocyclobut-1-enyl)amino]ethylthio}-
propyl (2-acetamido-2-deoxy-ꢀ-D-galactopyranosyl)-(1→6)-
[(ꢀ-D-glucopyranosyluronic acid)-(1→3)]-(2-acetamido-2-deoxy-
ꢀ-D-galactopyranosyl)-(1→6)-[(ꢀ-D-glucopyranosyluronic acid)-
(1→3)]-2-acetamido-2-deoxy-ꢀ-D-galactopyranoside 38
Molecular dynamics calculations
Molecular dynamics simulations were carried out using the
GROMOS program package and the updated carbohydrate
force field for GROMOS34 on Silicon Graphics O2 computers.
Each molecule was surrounded by SPC/E37 water molecules
and placed in a truncated octahedral periodic box. All
A similar protocol as described for 34 was followed: compound
5 (0.78 mg, 0.71 µmol) in 75 mM sodium phosphate buffer (pH
7.0) (50 µl); diethyl squarate (0.1 µl, 0.7 µmol) in EtOH (60 µl);
reaction time, 16 h at rt; TLC with butan-1-ol–MeOH–water–
HOAc, 4:2:2:0.5. Product 38 was isolated, after an additional
chromatographic purification on HiTrap (5% aq. NH4HCO3),
as a colourless glass; TLC (butan-1-ol–MeOH–water–HOAc,
4:2:2:0.5) Rf 0.09 (5), 0.20 (38). The material was directly used
for the preparation of neoglycoconjugate 45.
bond lenghts were kept fixed using the SHAKE procedure.38
A
cut-off radius of 0.8 nm and a time step of 2 fs was used.
Simulations were performed with loose coupling to a pressure
bath at 1 atm and a temperature bath at 300 K39 with time
constants of 0.5 and 0.1 ps, respectively.
3-{2-[N-(2-Ethoxy-3,4-dioxocyclobut-1-enyl)amino]ethylthio}-
propyl 2-acetamido-2-deoxy-ꢀ-D-galactopyranoside 39
Potential of mean force calculations on the ꢀ-D-GalpNAc-(1→6)-
ꢀ-D-GalpNAc rotamer distribution
A similar protocol as described for 34 was followed: compound
6 (3.0 mg, 8.9 µmol) in 75 mM sodium phosphate buffer (pH
7.0) (150 µl); diethyl squarate (1.3 µl, 8.9 µmol) in EtOH (150
µl); reaction time, overnight at rt; TLC with EtOAc–MeOH–
water, 10:5:1. Product 39 was isolated as a colourless glass;
TLC (MeOH–water, 9:1) Rf 0.19 (6), 0.72 (39). The material
was directly used for the preparation of neoglycoconjugate 46.
To calculate the free-energy differences of the GG, GT and TG
conformations around the GalNAc–GalNAc glycosidic ω
angle, potential-of-mean-force calculations33 were run with the
GROMOS force field for the methyl glycoside analogues of
compounds 2 and 4. All simulations were divided into jobs of
10 ps. The φ-values were collected into 72 classes, each with
a width of ∆φ = 5Њ. The derivatives were fitted to a 12-term
Fourier series. The first 0.2 ps of each job was discarded.
6-[N-(2-Ethoxy-3,4-dioxocyclobut-1-enyl)amino]hexyl-
ꢀ-D-glucopyranosiduronic acid 40
A similar protocol as described for 34 was followed: compound
7 (1.5 mg, 34.5 µmol) in 75 mM sodium phosphate buffer
(pH 7.0) (130 µl); diethyl squarate (0.63 µl, 4.28 µmol) in EtOH
(100 µl); reaction time, 2.5 h at rt; TLC with EtOAc–MeOH–
water, 10:5:1. Product 40 was isolated as a colourless glass;
TLC (EtOAc–MeOH–water, 10:5:1) Rf 0.46. The material was
directly used for the preparation of neoglycoconjugate 47.
Acknowledgements
We thank Dr C. T. M. Fransen for recording the NMR spec-
trum of compound 5 and 14, Mr R. Gutiérrez Gallego for
recording the NMR spectra of compound 33, Dr J. L. Jimenez
Blanco for recording the NMR spectrum of compound 3, and
Mr C. H. Grün for recording the FAB mass spectra.
Pretreatment of bovine serum albumin (BSA)
References
BSA (40 mg mlϪ1) was stirred in 0.1 M NaOAc buffer (pH 4.5)
containing 10 mM NaIO4 for 1.5 h at rt to oxidize carbohydrate
of glycoprotein contaminants.35 Excess of periodate was des-
troyed by adding glycerol to a final concentration of 10 mM.
The solution was dialyzed against water (three changes; Milli
Q), followed by lyophilization. After treatment of the material
with aq. NaBH4 (catalytic amount) for 1 h at rt, the solution
was diluted with water (1 ml), and neutralized with 4 M HOAc.
After lyophilization, the quality of pretreated BSA was checked
by sodium dodecyl sulfate (SDS)-polyacrylamide gel electro-
phoresis. To verify the complete removal of carbohydrate con-
taminants with GLC, a small amount of the protein material
(1 mg) was subjected to methanolysis (1.0 M methanolic HCl,
24 h, 85 ЊC) followed by re-N-acetylation and trimethylsilyl-
ation.36
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