Synthesis of the spacer-containing β-D-GalpNAc-(1 →4)-β-D-GlcpNAc-(1→3)-α-D-Galp moiety, representing the non-fucosylated backbone trisaccharide of the glycocalyx glycan of the parasite Schistosoma mansoni

Article abstract of DOI:10.1016/S0008-6215(98)00087-1

The chemical synthesis of β-D-GalpNAc-(1→4)-β-D-GlcpNAc-(1→3)-α-D-Galp-(1→O)-(CH₂)₅NH₂ is described. This structure represents the nonfucosylated backbone trisaccharide of the glycocalyx glycan of the cercarial stage of the parasite Schistosoma mansoni. Synthesis of the trisaccharide was achieved via a stepwise coupling approach. 5-Azidopentyl 4-O-acetyl-2,6-di-O-benzyl-α-D-galactopyranoside was condensed with ethyl 6-O-benzyl-2-deoxy-3,4-di-O-dimethylisopropylsilyl-2-phthalimido- 1-thio-β-D-glucopyranoside, using N-iodosuccinimide and silver trifluoromethanesulfonate as a catalyst system, followed by the removal of the silyl ether groups to afford a disaccharide acceptor. Coupling of ethyl 4,6-di-O-acetyl-3-O-allyloxycarbonyl-2-deoxy-2- phthalimido-1-thio-β-D-galactopyranoside to the disaccharide acceptor, using methylsulfenyl bromide and silver trifluoromethanesulfonate as a catalyst system, gave a protected trisaccharide. Deprotection of this compound yielded the target structure.

Full text of DOI:10.1016/S0008-6215(98)00087-1

Carbohydrate Research 308 (1998) 329±338
Synthesis of the spacer-containing ꢀ-d-
GalpNAc-(1!4)-ꢀ-d-GlcpNAc-(1!3)-ꢁ-d-Galp
moiety, representing the non-fucosylated
backbone trisaccharide of the glycocalyx glycan
of the parasite Schistosoma mansoni
Koen M. Halkes, Dirk J. Lefeber, Carolus T.M. Fransen,
Johannis P. Kamerling *, Johannes F.G. Vliegenthart
BijvoetCenter,DepartmentofBio-OrganicChemistry,UtrechtUniversity,POBox80.075,NL-3508TB
Utrecht, The Netherlands
Received 22 January 1998; accepted 30 March 1998
Abstract
The chemical synthesis of ꢀ-d-GalpNAc-(1!4)-ꢀ-d-GlcpNAc-(1!3)-ꢁ-d-Galp-(1!O)-
(CH2)5NH2 is described. This structure represents the nonfucosylated backbone trisaccharide of
the glycocalyx glycan of the cercarial stage of the parasite Schistosoma mansoni. Synthesis of the
trisaccharide was achieved via a stepwise coupling approach. 5-Azidopentyl 4-O-acetyl-2,6-di-O-
benzyl-ꢁ-d-galactopyranoside was condensed with ethyl 6-O-benzyl-2-deoxy-3,4-di-O-dimethyli-
sopropylsilyl-2-phthalimido-1-thio-ꢀ-d-glucopyranoside, using N-iodosuccinimide and silver tri-
¯uoromethanesulfonate as a catalyst system, followed by the removal of the silyl ether groups to
a€ord a disaccharide acceptor. Coupling of ethyl 4,6-di-O-acetyl-3-O-allyloxycarbonyl-2-deoxy-2-
phthalimido-1-thio-ꢀ-d-galactopyranoside to the disaccharide acceptor, using methylsulfenyl bro-
mide and silver tri¯uoromethanesulfonate as a catalyst system, gave a protected trisaccharide.
Deprotection of this compound yielded the target structure. # 1998 Elsevier Science Ltd. All
rights reserved
Keywords: Oligosaccharide synthesis; Glycocalyx; Schistosoma mansoni
1. Introduction
human schistosomiasis are Schistosoma mansoni,
Schistosoma japonicum, and Schistosoma haemato-
bium [1]. The infective parasitic stage, the cercaria,
enters the host through the skin, evoking an
in¯ammatory response. From this stage to about 3
weeks after infection the parasite, present as a
young schistosomulum, is most susceptible to
immune damage [2]. During the cercarial stage of
Schistosomes, which are blood-dwelling ¯ukes
belonging to the class Trematode, cause in human
beings the infectional disease schistosomiasis.
Schistosoma species that are most important in
* Corresponding author. Fax: 00-31-30-2540980.
0008-6215/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved
PII S0008-6215(98)00087-1

330
K.M. Halkes et al./Carbohydrate Research 308 (1998) 329±338
the life cycle of the parasite, the entire surface of
the parasite is covered by a 1 ꢂm-thick highly
immunogenic, fucose-rich glycocalyx. Recently, the
nonreducing terminal sequences of the O-linked
carbohydrate chain as part of the glycocalyx
(GCX) were elucidated by Khoo et al. [3] (Fig. 1).
These epitopes are carried on type 1 or type 2
core structures via units of {!3)-ꢀ-d-GalpNAc-
(1!4)-[ꢁ-l-Fucp-(1!2)-ꢁ-l-Fucp-(1!2)-ꢁ-l-Fucp-
(1!3)-]ꢀ-d-GlcpNAc-(1!3)-ꢁ-d-Galp-(1!}n,
galactose residue are likely to be involved in
GCX's action as a very potent immunological
modulator [3]. However, the exact role of the var-
ious domains of the GCX in immunological sti-
mulation can only be assessed with testing with
neoglycoconjugates prepared with fragments of the
GCX.
The availability of well-de®ned oligosaccharides
of the GCX from biological sources in sucient
amounts is limited. In order to replace isolated
material in both immunological studies and diag-
nostic purposes, a synthetic program for the pre-
paration of several oligosaccharide fragments
present in the GCX was initiated. Here, we report
the chemical synthesis of the nonfucosylated
backbone trisaccharide ꢀ-d-GalpNAc-(1!4)-ꢀ-d-
GlcpNAc-(1!3)-ꢁ-d-Galp 1, having an amino-
pentyl spacer for conjugation to carrier molecules.
In the course of our studies, a Note appeared
reporting the synthesis of ꢁ-l-Fucp-(1!2)-ꢁ-l-
Fucp-(1!3)-ꢀ-d-GalpNAc-(1!O)(CH2)8COOMe
[12].
Treating trisaccharide 1 with fucosyltransferases
present in di€erent schistosoma species [13], will
give partially fucosylated structures which can be
used in the above described biological tests. On the
other hand, one of the intermediates generated
along the route towards the preparation of 1, can
be coupled with chemically synthesised di- and tri-
saccharide fucosyl molecules which are prepared as
described elsewhere [14], to achieve the total
synthesis of the octasaccharide structure (Fig. 1).
where n is mainly 0 or 1.
After the cercarial stage the developing worm
becomes resistant to, or even invisible to, certain
parts of the host's defence system by several eva-
sion mechanisms. Some of the evasion techniques
employed by schistosomula and adult worms are
camou¯age by acquisition of host antigens [4],
reduction of surface antigenicity by tegument anti-
gen shedding [5], modulation of the host's immune
response by eliciting blocking antibodies [6],
induction of immunosuppressive mechanisms by
excretion of speci®c T cell suppressor factors [7],
and clearance from the surface by sloughing of
antibody-antigen complexes or of molecules which
are potentially harmful to the parasite [8]. Due to
all these evasion mechanisms, the parasite can per-
sist in the host for approximately 3±5 years. The
most severe pathology of the infection is caused by
the eggs of the parasite which 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 needed. Serological detection of
antibodies, raised against the glycocalyx of the
cercarial stage of the parasite S. mansoni, could be
such an early diagnostic method.
2. Results and discussion
A vaccine against schistosomiasis would be even
more desirable. Although much research has been
directed to this ®eld, no vaccine has yet been
developed to the stage of application in man
[10,11]. It is known that man once infected seldom
become re-infected, probably due to the generation
of protective antibodies against the cercarial stage
of the parasite S. mansoni. It is speculated that
both the fucosyl appendages and the ꢁ-linked
Trisaccharide 1 can be synthesised via a stepwise
approach using the monosaccharide building
blocks 5-azidopentyl 4-O-acetyl-2,6-di-O-benzyl-
ꢁ-d-galactopyranoside (7) (Scheme 1), ethyl 6-O-
benzyl-2-deoxy-3,4-di-O-dimethylisopropylsilyl-2-
phthalimido-1-thio-ꢀ-d-glucopyranoside (10), and
ethyl
4,6-di-O-acetyl-3-O-allyloxycarbonyl-2-
deoxy-2-phthalimido-1-thio-ꢀ-d-galactopyranoside
(13) (Scheme 2). In order to be able to conjugate
Fig. 1. Nonreducing terminus of the glycocalyx glycan of the cercarial stage of Schistosoma mansoni.

K.M. Halkes et al./Carbohydrate Research 308 (1998) 329±338
331
Scheme 1. Synthesis of spacer-containing galactose acceptor 7.
Scheme 2. Synthesis of glucosamine donor 10 and galactosamine donor 13; DMIPS, ((CH3)2C)(CH3)2Si; AOC,
CH2CHCH2OC(O).
deprotected oligosaccharides to a suitable carrier
molecule for immunological testing, a spacer
molecule is needed. As indicated by 7, in our
strategy an azidopentyl spacer has been chosen,
since it is stable to all the required reaction condi-
tions [15]. This spacer is introduced at an early
stage of the synthetic route by coupling ethyl
2,6-di-O-benzyl-3,4-O-isopropylidene-1-thio-ꢀ-d-
galactopyranoside 4 (Scheme 1) to 5-azidopentanol.
The obtained product can easily be transformed
into acceptor 7.
For the synthesis of acceptor 7 (Scheme 1),
donor molecule 4 was prepared by deacetylation of
ethyl 2,3,4,6-tetra-O-acetyl-1-thio-ꢀ-d-galactopyr-
anoside (2) using the Zemplen procedure, followed
by the introduction of an isopropylidene function
at HO-3,4 using 2,2-dimethoxypropane in the pre-
sence of p-toluenesulfonic acid (!3, 67%), and

332
K.M. Halkes et al./Carbohydrate Research 308 (1998) 329±338
benzylation of the remaining hydroxyl functions
using benzyl bromide in the presence of sodium
hydride (!4, 73%). Condensation of 4 with 5-azi-
dopentanol [15], in the presence of methyl tri-
¯uoromethanesulfonate, yielded a mixture of the
1,2-cis and 1,2-trans glycosides, from which the
desired compound 5 could be isolated in a yield of
43%. The presence of the azido group in 5 was
Diol 15 can be used directly as an acceptor for
coupling with 13. A similar condensation of a
galactosamine donor to the 4-position of a gluco-
samine acceptor having both the HO-3 and HO-4
functions available, and the amine groups of both
the donor and acceptor protected with a phthali-
mido function, has been described earlier [22].
Condensation of 15 with 13 (Scheme 3), using
methylsulfenyl bromide and silver tri¯uoro-
1
established by FT-IR (ꢃ 2096 cm ), and the 1,2-cis
1
ꢀ
glycosidic linkage by H NMR (J1,2 3.5 Hz). After
methanesulfonate [23], at 50 C a€orded in 79%
removal under acidic conditions of the isopropyl-
idene function (!6, quantitative), an orthoester
function was introduced at HO-3,4, using trimethyl
orthoacetate and p-toluenesulfonic acid in N,N-
dimethylformamide, and subsequently selectively
[16] opened towards the 4-position of the galactose
derivative to yield acceptor molecule 7 (97%).
yield a 1:4 mixture of the (1!3) and (1!4) cou-
pling products, which could be separated by col-
umn chromatography, to give 16 in 63% yield. For
product identi®cation, two-dimensional rotating
frame nuclear Overhauser enhancement NMR
spectroscopy of the products and their acetylated
derivatives was performed.
The starting compound for both the glucosa-
mine donor 10 (Scheme 2) and the galactosamine
donor 13 (Scheme 2) was ethyl 4,6-O-benzylidene-
2-deoxy-2-phthalimido-1-thio-ꢀ-d-glucopyranoside
[17] (8). Synthesis of 10 was achieved by the
reductive opening of the benzylidene ring of 8
towards the primary hydroxyl function using
sodium cyanoborohydride and anhydrous HCl in
diethyl ether [18] (!9, 60%), followed by silylation
of the hydroxyl groups at C-3 and C-4 with dime-
thylisopropylchlorosilane in pyridine to a€ord the
desired glucosamine donor (66%).
For the preparation of the galactosamine donor
13 (Scheme 2), compound 8 was ®rst allyloxy-
carbonylated [19] using allyl chloroformate in
pyridine, to a€ord intermediate 11 (97%). Acidic
removal of the benzylidene function, followed by
selective acetylation of the primary hydroxyl group
using acetyl chloride in pyridine, gave 12 in an
overall yield of 70%. For the conversion of 12 into
13 (epimerization at C-4) use was made of an SN2
displacement reaction of O-tri¯ate by O-acetate
[20]. To this end, 12 was treated with tri¯ic anhy-
dride in dichloromethane in the presence of pyr-
idine, and then the 4-O-tri¯ated intermediate was
treated with tetrabutylammonium acetate in N,N-
dimethylformamide, to yield 13 (90%).
A strategy, which is under current investigation
involves the chemical fucosylation of intermediate
16 at the glucosamine residue, and after deallyl-
oxycarbonylation at the galactosamine residue, to
a€ord the total octasaccharide structure, as depic-
ted in Fig. 1.
In order to obtain a suitable substrate for fuco-
syltransferases, 16 was deprotected to yield target
molecule 1. To this end a deacylation was achieved
using ethylenediamine in 1-butanol [24], and after
conventional N,O-acetylation followed by de-O-
acetylation, a catalytic hydrogenolysis, using pal-
ladium on carbon was carried out, to a€ord 1 in an
overall yield of 70%. In Table 1 the 1H NMR data
of trisaccharide 1 are depicted.
3. Experimental
General methods.ÐReactions were monitored by
TLC on Kieselgel 60 F254 (E. Merck); compounds
were visualised by charring with aq 50% H2SO4,
after examination under UV light. In the work-up
procedures of reaction mixtures, organic solns were
washed with appropriate amounts of the indicated
aq solns, then dried (MgSO4), and concentrated
ꢀ
under reduced pressure at 20±40 C (water-bath).
Condensation of 7 with 10 (Scheme 3) in dichloro-
methane in the presence of the catalyst system N-
iodosuccinimide and silver tri¯uoromethane-
Column chromatography was performed on Kie-
selgel 60 F254 (E. Merck, 70-230 mesh). Size-exclu-
sion chromatography was performed on Sephadex
LH-20 or on Hi-Trap.
ꢀ
sulfonate [21] at 60 C a€orded stereoselectively
ꢀ
disaccharide derivative 14 (84%). Removal of
both silyl ether groups under acidic conditions,
using p-toluenesulfonic acid in acetonitrile and
water, gave disaccharide acceptor 15 (93%).
Optical rotations were measured at 20 C for
solns in CHCl3, unless otherwise stated, with a
Perkin±Elmer 241 polarimeter, using a 10 cꢀm 1 mL
1
cell. H NMR spectra were recorded at 27 C with

K.M. Halkes et al./Carbohydrate Research 308 (1998) 329±338
333
Scheme 3. Synthesis of the protected trisaccharide 16.
Bruker AC 300 or Bruker AMX 500 spectrometers;
the values of ꢄH are given in ppm relative to the
signal for internal Me4Si (ꢄ 0) for solns in CDCl3,
or by reference to acetone (ꢄ 2.225) for solns in
D2O. 13C (APT, 75 MHz) NMR spectra were
recorded at 27 ꢀC with a Bruker AC 300 or a Varian
Gemini-300 instrument; indicated ppm values for
ꢄC are relative to the signal of CDCl3 (ꢄ 76.9) for
solns in CDCl3. Two-dimensional double-quantum
®ltered 1H±1H correlation spectra (2D DQF 1H±1H
COSY) were recorded usingꢀa Bruker AMX 500
apparatus (500 MHz) at 27 C. Two-dimensional
Table 1
500-MHz H NMR data of ꢀ-d-GalpNAc-(1!4)-ꢀ-d-GlcpNAc-(1!3)-ꢁ-d-Galp-(1!O)(CH2)5NH2 (1). Assignment was achieved
1
1
1
using two-dimensional double-quantum ®ltered H±1H correlation spectroscopy (2D DQF H±1H COSY) and two-dimensional
rotating frame nuclear Overhauser enhancement spectroscopy (2D ROESY)
Proton
ꢄ (ppm)/J (Hz)
ꢀ-d-GlcpNAc
ꢁ-d-Galp
ꢀ-d-GalpNAc
H-1
H-2
H-3
H-4
H-5
NAc
4.903 (2.5)
3.85
3.90
4.703 (7.9)
3.78
3.74
3.66
3.51
4.522 (8.5)
3.93
3.76
3.92
3.73
4.18
Ð
2.029
2.068
OCHaHb(CH2)3CH2NH2
OCHaHb(CH2)3CH2NH2
OCHaHb(CH2)3CH2NH2
3.722,3.532
2.995
1.49±1.44, 1.72±1.64

334
K.M. Halkes et al./Carbohydrate Research 308 (1998) 329±338
rotating frame nuclear Overhauser enhancement
spectroscopy (2D ROESY) of 1, 16, and the acety-
lated derivative of 16 was carried out using a mix-
ing time of 250 ms at a spin±lock ®eld strength
corresponding to a 90ꢀ pulse-width between 100±
120 ꢂs. The carrier frequency was placed at the left
side of the spectrum at ꢄ 7.3 ppm in order to mini-
mise HOHAHA-type magnetisation transfer. The
spectral width was 6580 Hz in each dimension, and
400 experiments of 2K data points were recorded.
(15 mL). After stirring for 1.5 h, the reaction was
quenched by addition of MeOH, and the mixture
was diluted with EtOAc (300 mL) and washed with
aq 10% NaCl. The organic layer was dried, ®l-
tered, concentrated, and the residue was subjected
to column chromatography (95:5 CH2Cl2±EtOAc)
to a€ord 4, isolated as a syrup (2.44 g, 73%); (Rf
0.80; 95:5 CH2Cl2±acetone); [ꢁ]d 15.8ꢀ (c 1);
NMR (CDCl3): 1H, ꢄ 7.44±7.21 (m, 10 H, 2
CH2C6H5), 4.835, 4.751, 4.627, and 4.542 (4 d,
each 1 H, 2 CH2C6H5), 4.435 (d, 1 H, J1,2 9.6 Hz,
H-1), 3.451 (dd, 1 H, J2,3 6.2 Hz, H-2), 2.79±2.65
(m, 2 H, SCH2CH3), 1.428 and 1.348 (2 s, each 3
H, C(CH3)2), 1.304 (t, 3 H, SCH2CH3); 13C, ꢄ
138.1, 137.7, 128.2 (5 C), and 127.5 (5 C) (2
CH2C6H5), 109.8 [(CH3)2C], 83.6 (C-1), 73.3 (2 C)
(2 CH2C6H5), 69.5 (C-6), 27.7 and 26.2 [(CH3)2C],
24.6 (SCH2CH3), 14.8 (SCH2CH3). FABMS (posi-
tive-ion mode; C25H32O5S): m/z 467 [M+Na]+.
5-Azidopentyl 2,6-di-O-benzyl-3,4-O-isopropy-
lidene-a-d-galactopyranoside (5).ÐTo a mixture of
4 (1.03 g, 2.31 mmol), 5-azidopentanol [15] (0.39 g,
3.02 mmol) and 4 A molecular sieves in Et2O
(30 mL) was added at 0 ꢀC MeOTf (0.63 mL,
5.78 mmol). After stirring for 6 h, TLC (7:3 hex-
ane±EtOAc) showed the formation of a new spot
(Rf 0.54). Then, Et3N (0.2 mL) was added and the
mixture was diluted with CH2Cl2 (200 mL), washed
with water, and the organic layer was dried, ®l-
tered, and concentrated. Column chromatography
(7:3 hexane±EtOAc) of the residue yielded 5, iso-
lated as a glass (0.51 g, 43%); [ꢁ]d +31.8ꢀ (c 1);
NMR (CDCl3): 1H, ꢄ 7.35±7.25 (m, 10 H, 2
CH2C6H5), 4.773 (d, 1 H, J1,2 3.5 Hz, H-1), 4.800,
4.679, 4.640, and 4.528 (4 d, each 1 H, 2
CH2C6H5), 4.165 (dd, 1 H, J2,3 7.8, J3,4 5.3 Hz, H-3),
3.511 (dd, 1 H, H-2), 3.228 (t, 2 H, O(CH2)4-
CH2N3), 1.72±1.54 (m, 4 H, OCH2CH2CH2CH2-
CH2N3), 1.46±1.37 (m, 2 H, O(CH2)2CH2(CH2)2-
N3), 1.393 and 1.328 (2 s, each 3 H, C(CH3)2); 13C,
ꢄ 137.5, 128.2, 127.7, and 127.5 (CH2C6H5), 109.0
[(CH3)2C], 96.5 (C-1), 73.3 and 72.2 (2 CH2C6H5),
69.5 (C-6), 67.9 and 51.2 (OCH2(CH2)3CH2N3),
28.8, 28.5, and 23.3 (OCH2(CH2)3CH2N3), 28.0
and 26.3 [(CH3)2C]. FABMS (positive-ion mode;
C28H37N3O6): m/z 534 [M+Na]+. The ꢀ anomer
was isolated in a yield of 17%.
Fast-atom-bombardment
mass
spectrometry
(FABMS) was performed on a JEOL JMS SX/SX
102A four-sector mass spectrometer, operated at
10 kV accelerating voltage, equipped with a JEOL
MS-FAB 10 D FAB gun, operated at 10 mA emis-
sion current, producing a beam of 6 keV Xe atoms.
Ethyl
3,4-O-isopropylidene-1-thio-b-d-galacto-
pyranoside (3).ÐTo a soln of ethyl 2,3,4,6-tetra-O-
acetyl-1-thio-ꢀ-d-galactopyranoside (2; 3.65 g,
9.30 mmol) in MeOH was added NaOMe (pH 10).
After stirring for 1 h, the soln was neutralised by
the addition of Dowex-50 (H+). Then, the suspen-
sion was ®ltered, concentrated and twice co-con-
centrated with CH2Cl2. To a soln of the residue
and 2,2-dimethoxypropane (1.37 mL, 11.2 mmol)
in DMF (20 mL) was added p-toluenesulfonic acid
(pH 2±3). The mixture was stirred overnight and
then neutralised by the addition of Amberlyst A-
21, ®ltered, and co-concentrated with toluene. In
order to remove the hemiacetal at C-6, a soln of
the residue in CH2Cl2 (40 mL) was treated with aq
50% CF3CO2H for 10 min, when TLC (9:1
CH2Cl2-MeOH) showed the formation of one
lower moving spot (Rf 0.39). Et3N was added to
quench the reaction, and the mixture was con-
centrated. Column chromatography (9:1 CH2Cl2±
acetone) of the residue gave 3, isolated as a syrup
(1.65 g, 67%); [ꢁ]d +19.7ꢀ (c 1); NMR (CDCl3):
1H, ꢄ 4.270 (d, 1 H, J1,2 10.2 Hz, H-1), 4.207 (dd, 1
H, J3,4 5.5, J4,5 2.2 Hz, H-4), 4.092 (dd, 1 H, J2,3
7.0 Hz, H-3), 3.560 (ddd, 1 H, J2,OH 1.6 Hz, H-2),
2.82±2.64 (m, 2 H, SCH2CH3), 1.519 and 1.356 (2
s, each 3 H, C(CH3)2), 1.324 (t, 3 H, SCH2CH3);
13C, ꢄ 110.1 [(CH3)2C], 85.2 (C-1), 62.2 (C-6), 27.9
and 26.1 [(CH3)2C], 24.3 (SCH2CH3), 15.1
(SCH2CH3).
FABMS
(positive-ion
mode;
C11H20O5S): m/z 287 [M+Na]+.
Ethyl 2,6-di-O-benzyl-3,4-O-isopropylidene-1-
thio-b-d-galactopyranoside (4).ÐTo a suspension
of NaH (1.63 g, 41.33 mmol) in DMF (10 mL) was
added slowly a soln of 3 (2.3 g, 7.56 mmol) and
benzyl bromide (5.1 mL, 42.64 mmol) in DMF
5-Azidopentyl 2,6-di-O-benzyl-a-d-galactopyr-
anoside (6).ÐTo a soln of 5 (0.24 g, 0.47 mmol) in
CH2Cl2 (15 mL) were added CF3CO2H (0.41 mL,
5.17 mmol) and water (32 mL). After stirring for 1 h,
TLC (55:45 hexane±EtOAc) showed a complete

K.M. Halkes et al./Carbohydrate Research 308 (1998) 329±338
335
conversion of 5 into 6 (Rf 0.15). The mixture was
diluted with CH2Cl2 (100 mL), washed with saturated
aq NaHCO3, and the organic layer was dried, ®ltered,
and concentrated. Column chromatography (55:45
hexane±EtOAc) of the residue gave 6, isolated as a
glass (0.22 g, quant.); 1H NMR (CDCl3): ꢄ 7.36±7.26
(m, 10 H, 2 CH2C6H5), 4.842 (d, 1 H, J1,2 3.6 Hz, H-
1), 4.683 and 4.614 (2 d, each 1 H, CH2C6H5),
4.577 (bs, 2 H, CH2C6H5), 4.076 (dd, 1 H, J3,4 3.4,
J4,5 1.1 Hz, H-4), 3.980 (dd, 1 H, J2,3 9.6, H-3),
3.750 (dd, 1 H, H-2), 3.656 and 3.343 (2 dt, each 1
H, OCH2(CH2)4N3), 3.230 (t, 2 H, O(CH2)4CH2N3),
2.50 and 2.83 (2 bs, 2 H, 2 OH), 1.66±1.55 (m, 4 H,
OCH2CH2CH2CH2CH2N3), 1.46±1.33 (m, 2 H,
O(CH2)2CH2(CH2)2N3). FABMS (positive-ion
mode; C25H33N3O6): m/z 494 [M+Na]+.
1 M HCl/Et2O soln until the evolution of gas
ceased (38 mL). After stirring for 2.5 h, TLC (9:1
CH2Cl2±MeOH) showed the disappearance of 8
and the formation of a new spot (Rf 0.33). Then,
Et3N (2.5 mL) was added and the mixture was ®l-
tered through Celite, diluted with CH2Cl2
(300 mL), washed with aq 10% NaHCO3, and the
organic layer was dried, ®ltered, and concentrated.
Column chromatography (9:1 CH2Cl2±acetone) of
the residue yieldeꢀd 9, isolated as a syrup (1.16 g,
1
60%); [ꢁ]d 16.3 (c 0.8); NMR (CDCl3): H, ꢄ
7.30±7.20 (m, 5 H, CH2C6H5), 5.322 (d, 1 H, J1,2
10.4 Hz, H-1), 4.623 and 4.569 (2 d, each 1 H,
CH2C6H5), 4.152 (t, 1 H, J2,3 10.4 Hz, H-2), 2.72±
2.58 (m, 2 H, SCH2CH3), 1.181 (t, 3 H, SCH2CH3);
13C, ꢄ 168.1, 134.1, 131.4 (Phth), 137.4, 128.3 (2 C),
127.7 (2 C), and 127.6 (CH2C6H5), 81.0 (C-1), 73.5
and 70.2 (C-6 and CH2C6H5), 24.0 (SCH2CH3),
14.8 (SCH2CH3).
5-Azidopentyl
4-O-acetyl-2,6-di-O-benzyl-a-d-
galactopyranoside (7).ÐA mixture of 6 (0.09 g,
0.19 mmol), trimethyl orthoacetate (49 ꢂL) and a
catalytic amount of p-toluenesulfonic acid in DMF
(3.5 mL) was stirred for 30 min, when TLC (45:55
EtOAc±hexane) showed the formation of the
orthoester (Rf 0.75). Upon addition of aq 80%
HOAc (3 mL), the orthoester was opened within
10 min to the 4-position. The mixture was diluted
with EtOAc (100 mL), washed with aq 10%
NaHCO3 and aq 10% NaCl, and the organic layer
was dried, ®ltered, and concentrated. Column
chromatography (45:55 EtOAc±hexane) of the
residue a€orded 7, isolated as a glass (96 mg, 97%);
Ethyl 6-O-benzyl-2-deoxy-3,4-di-O-dimethyliso-
propylsilyl-2-phthalimido-1-thio-b-d-glucopyrano-
side (10).ÐTo a soln of 9 (0.372 g, 0.82 mmol) in
dry pyridine (8 mL) were added in two portions
dimethylisopropylchlorosilane (0.9 mL, 6.0 mmol)
and a catalytic amount of 4-dimethylaminopyr-
idine. The mixture was stirred for 48 h, then diluted
with CH2Cl2 (200 mL), washed with aq 10%
NaHCO3 and aq 10% NaCl, and the organic layer
was dried, ®ltered, and concentrated. Column
chromatography (98:2 CH2Cl2±EtOAc) of the
residue gave 10, isolated as a syrup (0.35 g, 66%);
Rf 0.84 (95:5 CH2Cl2±acetone); [ꢁ]d +62.6ꢀ (c 0.8);
NMR (CDCl3): 1H, ꢄ 7.35±7.25 (m, 5 H,
CH2C6H5), 5.243 (d, 1 H, J1,2 10.5 Hz, H-1), 4.652
and 4.583 (2 d, each 1 H, CH2C6H5), 4.234 (dd, 1
H, J2,3 10.3 Hz, H-2), 2.76±2.54 (m, 2 H, SCH2-
CH3), 1.182 (t, 3 H, SCH2CH3), 0.75 and 0.70 (2 d,
each 6 H, 2 (CH3)2CHSi), 0.59±0.50 (m, 2 H, 2
(CH3)2CHSi), 0.094, 0.065, 0.045, and 0.285 (4
s, each 3 H, 2 Si(CH3)2); 13C, ꢄ 138.2, 128.1 (3 C),
and 127.3 (2 C) (CH2C6H5), 170.2, 134.0, 131.8,
and 123.0 (Phth), 80.7 (C-1), 73.1 (CH2C6H5), 69.2
(C-6), 23.7 (SCH2CH3), 17.0, 16.9, 16.7, and 16.6
(2 (CH3)2CHSi), 14.9 (SCH2CH3), 14.8 and 14.4 (2
(CH3)2CHSi). FABMS (positive-ion mode;
C33H49NO6SSi2): m/z 666 [M+Na]+.
ꢀ
1
[ꢁ]d +75.5 (c 1); NMR (CDCl3): H, ꢄ 7.34±7.26
(m, 10 H, 2 CH2C6H5), 5.434 (dd, 1 H, J3,4 3.5, J4,5
1.3 Hz, H-4), 4.843 (d, 1 H, J1,2 3.6 Hz, H-1), 4.709,
4.623, 4.543, and 4.438 (4 d, each 1 H, 2
CH2C6H5), 4.161 (ddd, 1 H, J2,3 10.0, J3,OH 2.7 Hz,
H-3), 3.693 (dd, 1 H, H-2), 3.660 and 3.337 (2 dt,
each 1 H, OCH2(CH2)4N3), 3.224 (t, 2 H,
O(CH2)4CH2N3), 2.272 (d, 1 H, OH), 2.070 (s, 3 H,
Ac), 1.64±1.52 (m, 4 H, OCH2CH2CH2CH2CH2-
N3), 1.46±1.37 (m, 2 H, O(CH2)2CH2(CH2)2N3);
13C, ꢄ 170.7 (COCH3), 137.7 (2 C), 128.4 (5 C), and
127.6 (5 C) (2 CH2C6H5), 96.8 (C-1), 73.4 and 72.6
(2 CH2C6H5), 68.6 (C-6), 67.8 and 51.2 (OCH2-
(CH2)3CH2N3), 28.8, 28.5, and 23.3 (OCH2(CH2)3-
CH2N3), 20.7 (COCH3). FABMS (positive-ion
mode; C27H35N3O7): m/z 536 [M+Na]+.
Ethyl 6-O-benzyl-2-deoxy-2-phthalimido-1-thio-
b-d-glucopyranoside (9).ÐTo a soln of ethyl 4,6-
O-benzylidene-2-deoxy-2-phthalimido-1-thio-ꢀ-d-
glucopyranoside [17] (8; 1.93 g, 4.37 mmol) and
NaCNBH3 (3.77 g, 60 mmol) in dry THF (75 mL),
containing 4 A molecular sieves (1 g), was added a
Ethyl 3-O-allyloxycarbonyl-4,6-O-benzylidene-
2-deoxy-2-phthalimido-1-thio-b-d-glucopyranoside
(11).ÐTo a stirred soln of ethyl 4,6-O-benzylidene-
2-deoxy-2-phthalimido-1-thio-ꢀ-d-glucopyranoside
[17] (8; 1.16 g, 2.63 mmol) in 1:1 dry pyridine±
CH2Cl2 (24 mL) was added at
30 ꢀC allyl

336
K.M. Halkes et al./Carbohydrate Research 308 (1998) 329±338
chloroformate (0.41 mL, 3.87 mmol). After stirring
for 30 min, another portion of allyl chloroformate
(0.41 mL, 3.87 mmol) was added. After stirring for
1 h, TLC (95:5 CH2Cl2±acetone) showed a com-
plete conversion of 8 into 11 (Rf 0.67). Then, the
mixture was diluted with CH2Cl2 (350 mL), washed
with aq 10% NaHCO3 and aq 10% NaCl, and the
organic layer was dried, ®ltered, concentrated and
co-concentrated with toluene. The residue was
subjected to column chromatography (97.5:2.5
CH2Cl2±acetone) to give 11, isolated as a syrup
(1.31 g, 97%); [ꢁ]d 6.7 (c 1); NMR (CDCl3): H,
ꢄ 7.48±7.26 (m, 5 H, CH2C6H5), 5.789 (dd, 1 H, J2,3
9.8, J3,4 7.9 Hz, H-3), 5.67±5.57 (m, 1 H,
COOCH2CH=CH2), 5.551 (s, 1 H, CHC6H5),
5.533 (d, 1 H, J1,2 10.5 Hz, H-1), 5.12±4.94 (m, 2 H,
COOCH2CH=CH2), 4.445 (dd, 1 H, H-2), 4.38±
4.36 (m, 2 H, COOCH2CH=CH2), 2.75±2.62 (m, 2
H, SCH2CH3), 1.201 (t, 3 H, SCH2CH3); 13C, ꢄ
173.8, 134.0, 131.5, and 123.4 (Phth), 154.0
(COOCH2CH=CH2), 130.7 (COOCH2CH=CH2),
136.6, 128.9 (2 C), 128.0 (2 C), 126.0 (CHC6H5),
118.4 (COOCH2CH=CH2), 101.4 (CHC6H5), 81.6
(C-1), 68.3 (C-6 and COOCH2CH=CH2), 24.1
(SCH2CH3), 14.7 (SCH2CH3). FABMS (positive-
ion mode; C27H27NO8S): m/z 548 [M+Na]+.
Ethyl 6-O-acetyl-3-O-allyloxycarbonyl-2-deoxy-
2-phthalimido-1-thio-b-d-glucopyranoside (12).ÐTo
a stirred soln of 11 (1.31 g, 2.51 mmol) in CH2Cl2
(40 mL) was added water (0.2 mL) and CF3CO2H
(2 mL). After stirring for 1.5 h, the mixture was
diluted with CH2Cl2 (300 mL), washed with aq
10% NaHCO3 and aq 10% NaCl, and the organic
layer was dried, ®ltered, and concentrated. The
residue was dissolved in dry CH2Cl2 (15 mL) and
dry pyridine (0.1ꢀ6 mL, 2.79 mmol), and the mixture
was stirred at 0 C under Ar, then a soln of acetyl
chloride (71 ꢂL, 3.2 mmol) in dry CH2Cl2 (8 mL)
was added dropwise. After stirring for 1 h, TLC
(95:5 CH2Cl2±acetone) showed a new spot (Rf
0.30), and the mixture was diluted with CH2Cl2
(150 mL) and washed with aq 10% NaHCO3. The
organic layer was dried, ®ltered, and co-con-
centrated with toluene. Column chromatography
(95:5 CH2Cl2±acetone) of the residue a€orded 12,
isolated as a glass (0.30 g, 70%); [ꢁ]d +0.9ꢀ (c 1);
NMR (CDCl3): 1H, ꢄ 5.73±5.60 (m, 1 H, COOCH2-
CH=CH2), 5.558 (dd, 1 H, J2,3 10.3, J3,4 8.5 Hz,
H-3), 5.449 (d, 1 H, J1,2 10.5 Hz, H-1), 5.17±5.00
(m, 2 H, COOCH2CH=CH2), 4.43±4.41 (m, 2 H,
COOCH2CH=CH2), 4.361 (dd, 1 H, H-2), 3.050
(d, 1 H, J4,OH 1.2 Hz, OH), 2.76±2.58 (m, 2 H,
SCH2CH3), 2.144 (s, 3 H, Ac), 1.208 (t, 3 H,
SCH2CH3); 13C, ꢄ 171.4 (COCH3), 167.0, 167.7,
134.1, 134.0, 131.4, 131.1, and 123.4 (2 C) (Phth),
154.6 (COOCH2CH=CH2), 130.6 (COOCH2-
CH=CH2), 118.6 (COOCH2CH=CH2), 80.9 (C-
1), 68.5 and 63.2 (C-6 and COOCH2CH=CH2),
24.2 (SCH2CH3), 20.6 (COCH3), 14.7 (SCH2CH3).
FABMS (positive-ion mode; C22H25NO9S): m/z
502 [M+Na]+.
Ethyl 4,6-di-O-acetyl-3-O-allyloxycarbonyl-2-
deoxy-2-phthalimido-1-thio-b-d-galactopyranoside
(13).ÐTo a soln of 12 (30 mg, 60 mmol) in dry
CH2Cl2 (3 mL) and pyridine (61 ꢂL) was added at
ꢀ
1
ꢀ
0 C tri¯ic anhydride (63 ꢂL, 0.38 mmol). After
45 min, TLC (95:5 CH2Cl2±acetone) showed the
formation of a new spot (Rf 0.65). The mixture was
diluted with CH2Cl2 (50 mL), washed with cold aq
10% NaHCO3 and aq 5% NaCl, and the organic
layer was dried, ®ltered, and concentrated
ꢀ
(T<40 C). The yellow residue was dissolved in
dry DMF (1.5 mL) and tetrabutylammonium
acetate (200 mg, 0.60 mmol) was added in two
portions. The mixture was stirred overnight, when
TLC (95:5 CH2Cl2±acetone) showed a complete
conversion of 12 into 13 (Rf 0.61), then diluted
with CH2Cl2 and washed with aq 10% NaCl, and
the organic layer was dried, ®ltered, and con-
centrated. The residue was subjected to column
chromatography (97:3 CH2Cl2±acetone) to yield
13, isolated as a glass (29 mg, 90%); [ꢁ]d +60.1ꢀ (c
1); NMR (CDCl3): 1H, ꢄ 5.81±5.68 (m, 1 H,
COOCH2CH=CH2), 5.716 (dd, 1 H, J2,3 10.7, J3,4
3.4 Hz, H-3), 5.639 (dd, 1 H, J4,5<1 Hz, H-4),
5.421 (d, 1 H, J1,2 10.5 Hz, H-1), 5.21±5.07 (m, 2 H,
COOCH2CH=CH2), 4.645 (dd, 1 H, H-2), 4.55±
4.41 (m, 2 H, COOCH2CH=CH2), 2.77±2.63 (m, 2
H, SCH2CH3), 2.203 and 2.069 (2 s, each 3 H, 2
Ac), 1.224 (t, 3 H, SCH2CH3); 13C, ꢄ 170.1 and
169.9 (2 COCH3), 167.8, 166.9, 134.1 (2 C), 131.3,
131.2, 123.5, and 123.3 (Phth), 153.4 (COOCH2-
CH=CH2), 130.8 (COOCH2CH=CH2), 118.5
(COOCH2CH=CH2), 81.5 (C-1), 68.6 and 61.5 (C-
6 and COOCH2CH=CH2), 24.3 (SCH2CH3), 20.4
(COCH3), 14.7 (SCH2CH3). FABMS (positive-ion
mode; C24H27NO10S): m/z 544 [M+Na]+.
5-Azidopentyl (6-O-benzyl-2-deoxy-3,4-di-O-di-
methylisopropylsilyl-2-phthalimido-b-d-glucopyrano-
syl)-(1!3)-4-O-acetyl-2,6-di-O-benzyl-a-d-galacto-
pyranoside (14).ÐTo
a
soln of 10 (0.17 g,
0.26 mmol) and 7 (0.11 g, 0.21 mmol) in dry
CH2Cl2 (20 mL), containingꢀ4 A molecular sieves
(0.3 g), was added at 60 C N-iodosuccinimide

K.M. Halkes et al./Carbohydrate Research 308 (1998) 329±338
337
(0.11 g) and a catalytic amount of AgOTf. The
mixture was allowed to warm to 50 ꢀC, and after
30 min, when TLC (Rf 0.67; 4:6 EtOAc±hexane)
showed a complete reaction, it was neutralised with
Et3N. The mixture was diluted with CH2Cl2
(150 mL), washed with aq 10% NaHSO3, aq 10%
KI, aq 10% NaHCO3, and aq 10% NaCl, and the
organic layer was dried, ®ltered, and concentrated.
Column chromatography (95:5 CH2Cl2±acetone)
of the residue gave 14, isolated as a glass (0.19 g,
3.496 and 3.196 (2 dt, each 1 H, OCH2(CH2)4N3),
3.164 (t, 2 H, O(CH2)4CH2N3), 2.066 (s, 3 H, Ac),
1.58±1.26 (m, 6 H, OCH2(CH2)3CH2N3); 13C, ꢄ
170.2 (COCH3), 167.9, 133.6, 131.4, and 123.0
(Phth), 98.6 and 97.1 (C-1,10), 73.2, 73.1, and 72.8
(3 CH2C6H5), 69.8 and 69.1 (C-6,60), 67.6 and 50.9
(OCH2(CH2)3CH2N3), 28.4, 28.2, and 23.0 (OCH2-
(CH2)3CH2N3), 20.5 (COCH3). FABMS (positive-
ion mode; C48H54N4O13): m/z 917 [M+Na]+.
5-Azidopentyl (4,6-di-O-acetyl-3-O-allyloxy-
carbonyl-2-deoxy-2-phthalimido-1-thio-b-d-galacto-
pyranosyl)-(1!4)-(6-O-benzyl-2-deoxy-2-phthal-
imido-b-d-glucopyranosyl)-(1!3)-4-O-acetyl-2,6-
di-O-benzyl-a-d-galactopyranoside (16).ÐTo a soln
of 15 (60 mg, 0.070 mmol) and 13 (78 mg,
0.15 mmol) in dry CH2Cl2 (1.7 mL), containing 3 A
powdered molecular sieves (0.15 g), was added a
soln of AgOTf (82 mg) in dry CH3CN (2.5 mL). At
50 ꢀC, a 1 M methylsulfenyl bromide soln
(137 ꢂL) in 1,2-dichloroethane was added in two
portions with an interval of 20 min. After stirring
for 1 h, TLC (35:65 EtOAc±hexane) showed the
disappearance of 15 and the formation of a new
spot (Rf 0.70). Then the reaction was quenched by
stirring for 30 min with N-diisopropylethylamine
(137 ꢂL), and the mixture was diluted with CH2Cl2
(100 mL) and ®ltered through Celite. The organic
phase was washed with aq 10% NaHSO3, aq 10%
KI, aq 10% NaHCO3, and aq 10% NaCl, and the
organic layer was dried, ®ltered, and concentrated.
Puri®cation of the residue by gel ®ltration over
LH-20 (1:1 CH2Cl2±MeOH) gave a 1:4 mixture
(79%) of the (1!3) and (1!4) coupling products.
Column chromatography (45:55 hexane±EtOAc)
of the mixture gave 16, isolated as a glass (57 mg,
ꢀ
1
84%); [ꢁ]d +30.0 (c 0.8); NMR (CDCl3): H, ꢄ
7.45±7.20 (m, 15 H, 3 CH2C6H5), 5.471 (dd, 1 H,
0
0
J3,4 3.6, J4,5<1 Hz, H-4), 5.276 (d, 1 H, J1 ,2
8.4 Hz, H-10), 4.684, 4.620, 4.467, 4.389, 4.101, and
3.766 (6 d, each 1 H, 3 CH2C6H5), 4.501 (dd, 1 H,
J2 ,3 10.4, J3 ,4 8.0 Hz, H-30), 4.394 (d, 1 H, J1,2
3.8 Hz, H-1), 4.105 (dd, 1 H, H-20), 4.018 (dd, 1 H,
J2,3 10.1 Hz, H-3), 3.448 (dd, 1 H, H-2), 3.136 (t, 2
H, O(CH2)4CH2N3), 2.080 (s, 3 H, Ac), 1.54±1.24
(m, 6 H, OCH2(CH2)3CH2N3), 0.725 and 0.675 (2
d, each 6 H, 2 (CH3)2CHSi), 0.55±0.45 (m, 2 H, 2
(CH3)2CHSi), 0.089, 0.074, 0.054, and 0.284 (4
s, each 3 H, 2 Si(CH3)2); 13C, ꢄ 169.9 (COCH3),
138.7, 138.4, 137.8, 128.0 (5 C), 127.3 (5 C), and
127.1 (5 C) (3 CH2C6H5), 167.2, 133.8, 131.8, and
122.9 (Phth), 98.6 and 97.1 (C-1,10), 73.1 and 72.8
(2 C) (3 CH2C6H5), 69.4 and 69.1 (C-6,60), 67.7 and
51.0 (OCH2(CH2)3CH2N3), 28.5, 28.3, and 23.1
(OCH2(CH2)3CH2N3), 20.6 (COCH3), 17.0, 16.9,
16.7, and 16.5 (2 (CH3)2CHSi), 14.9 and 14.5
(2 (CH3)2CHSi). FABMS (positive-ion mode;
C58H87N4O13Si2): m/z 1117 [M+Na]+.
0
0
0
0
5-Azidopentyl (6-O-benzyl-2-deoxy-2-phthal-
imido-b-d-glucopyranosyl)-(1!3)-4-O-acetyl-2,6-
di-O-benzyl-a-d-galactopyranoside (15).ÐTo a soln
of 14 (0.19 g, 0.17 mmol) in CH3CN (30 mL) were
added water (4.5 mL) and p-toluenesulfonic acid
(56 mg). After stirring for 2 h, TLC (8:2 CH2Cl2±
acetone) showed the complete conversion of 14
into 15 (Rf 0.67). The mixture was diluted with
CH2Cl2 (100 mL), washed with aq 10% NaHCO3
and aq 10% NaCl, and the organic layer was dried,
®ltered, and concentrated. The residue was sub-
jected to column chromatography (9:1 CH2Cl2±
acetone) to give 15, isolated as a glass (0.14 g, 93%);
[ꢁ]d +14.9ꢀ (c 1.2); NMR (CDCl3): 1H, ꢄ 7.36±7.18
(m, 15 H, 3 CH2C6H5), 5.417 (dd, 1 H, J3,4 3.6, J4,5
ꢀ
1
63%); [ꢁ]d +18.5 (c 0.4); NMR (CDCl3): H, ꢄ
7.30±7.07 (m, 15 H, 3 CH2C6H5), 5.80±5.67 (m, 1
00 00
H, COOCH2CH =CH2), 5.733 (dd, 1 H, J2 ,3 11.5,
00 00
J3 ,4 3.4 Hz, H-300), 5.558 (bd, 1 H, J4 ,5 <1 Hz,
00 00
H-400), 5.397 (d, 1 H, J1 ,2 8.5 Hz, H-100), 5.346 (bd,
00 00
0
0
1 H, J3,4 3.7, J4,5 <1 Hz, H-4), 5.298 (d, 1 H, J1 ,2
8.4 Hz, H-10), 5.21±5.07 (m, 2 H, COOCH2-
CH=CH2), 4.596 (dd, 1 H, H-200), 4.456, 4.362,
4.227, 4.158, 4.084, and 3.923 (6 d, each 1 H, 3
CH2C6H5), 4.283 (d, 1 H, J1,2 1.8 Hz, H-1), 3.941
(dd, 1 H, J2,3 9.9 Hz, H-3), 3.132 (t, 2 H,
O(CH2)4CH2N3), 2.176, 1.982, and 1.863 (3 s, each
3 H, 3 Ac), 1.53±1.30 (m, 6 H, OCH2(CH2)3-
CH2N3); 13C, ꢄ 170.3 (2 C) and 170.1 (3 COCH3),
153.4 (COOCH2CH=CH2), 130.9 (COOCH2-
CH=CH2), 167.8 (2 C), 167.1 (2 C), 134.3, 134.0,
133.8 (2 C), 131.6 (2 C), 131.2 (2 C), 123.6 (2 C),
<1 Hz, H-4), 5.393 (d, 1 H, J1 ,2 8.3 Hz, H-10),
4.662, 4.592, 4.478, 4.399, 4.253, and 3.906 (6 d,
each 1 H, 3 CH2C6H5), 4.449 (d, 1 H, J1,2 3.6 Hz,
0
0
H-1), 4.123 (dd, 1 H, J2 ,3 11.0 Hz, H-20), 4.036
(dd, 1 H, J2,3 10.0 Hz, H-3), 3.503 (dd, 1 H, H-2),
0
0

338
K.M. Halkes et al./Carbohydrate Research 308 (1998) 329±338
and 123.0 (2 C) (2 Phth), 118.8 (COOCH2-
CH=CH2), 99.3, 99.0, and 97.3 (C-1,10,100), 73.3,
73.0, and 72.7 (3 CH2C6H5), 69.3, 68.9, and 68.0
(C-6,60,600), 61.8 (COOCH2CH=CH2), 67.8 and
51.2 (OCH2(CH2)3CH2N3), 28.6, 28.4, and 23.2
(OCH2(CH2)3CH2N3), 20.7, 20.5, and 20.1 (3
[2] E.J. Pearce and S.L. James, Parasite Immunol., 8
(1993) 513±527.
[3] K.-H. Khoo, S. Sarda, X. Xu, J.P. Caul®eld, M.R.
McNeil, S.W. Homans, H.R. Morris, and A. Dell,
J. Biol. Chem., 270 (1995) 17114±17123.
[4] J.A. Clegg, S.R. Smithers, and R.J. Terry, Nature,
232 (1971) 653±654.
COCH3).
FABMS
(positive-ion
mode;
[5] E.J. Pearce, P.F. Basch, and A. Sher, Parasite
Immunol., 8 (1986) 79±94.
C70H75N5O23): m/z 1376 [M+Na]+.
5-Aminopentyl (2-deoxy-2-acetamido-b-d-galacto-
pyranosyl)-(1!4)-(2-deoxy-2-acetamido-b-d-gluco-
pyranosyl)-(1!3)-a-d-galactopyranoside (1).ÐTo
a soln of 16 (80 mg, 60 mmol) in 1-butanol (8 mL)
was added ethylenediamine (2 mL). The mixture
[6] A.E. Butterworth, R. Bensted-Smith, A. Capron,
M. Capron, P.R. Dalton, D.W. Dunne, J.-M.
Grzych, H.C. Kariuki, J. Khalife, D.K. Koech,
M. Mugambi, J.H. Ouma, T.K. Arab Siongok,
and R.F. Sturrock, Parasitology, 94 (1987) 281±
300.
ꢀ
was stirred overnight under Ar at 90 C, and then
co-concentrated with toluene and dried under high
vacuo for 1 h. A soln of 1:1 dry pyridine±Ac2O
(10 mL) was added and the mixture was stirred
overnight, and then co-concentrated with toluene.
The residue was subjected to column chromato-
graphy (94:4 CH2Cl2±MeOH) to obtain 46 mg of
the acetylated intermediate. The obtained com-
pound was dissolved in dry MeOH (10 mL) and
solid NaOMe was added (pH 10). The mixture was
stirred overnight under Ar, neutralised with
Dowex-50 (H+), ®ltered, and concentrated. A soln
of the residue in tert-butanol (6 mL) and water
(2.3 mL), containing 10% Pd-C (100 mg) and two
drops of aq 25% NH3 (pH 9) was hydrogenated
for 1 h. By ¯ushing with N2 for 1 h the pH of the
solution decreased to 7, then HOAc was added (pH
5), and the mixture was hydrogenated for another
1.5 h. The mixture was ®ltered and concentrated to
obtain 24 mg of the crude product 1. HiTrap gel
®ltration (aq 5 mM NH4HCO3) a€orded pure 1
[7] M.W. Lightowers and M.D. Rickard, Parasitology,
96 (1988) 123±166.
[8] F.J. Kruger and P.H. Joubert, Int. J. Parasitol., 20
(1990) 965±967.
[9] K.S. Warren, Nature, 273 (1978) 609±612.
[10] R.E. Blanton, Y. Matsumoto, P.A. Peters, S. El
Ibiary, C.H. King, A.A. Mahmoud, and Aikawa,
Am. J. Trop. Med. Hyg., 45 (1991) 112±120.
[11] A. Sher, S.L. James, R. Correa-Oliveira, S. Hieny,
and E. Pearce, Parasitology, 98 (1989) S61±S68.
[12] V.P. Kamath and O. Hindsgaul, Carbohydr. Res.,
280 (1996) 323±330.
[13] A.P. Neeleman, Ph. D. Thesis, Free University of
Amsterdam, The Netherlands, 1996.
[14] K.M. Halkes, Ph. D. Thesis, Utrecht University,
The Netherlands, 1997.
[15] P.B. van Seeventer, J.P. Kamerling, and J.F.G.
Vliegenthart, Carbohydr. Res., 299 (1997) 181±195.
[16] R.U. Lemieux and H.J. Driguez, J. Am. Chem.
Soc., 97 (1975) 4069±4075.
[17] H. Lohn, Carbohydr. Res., 139 (1985) 105±113.
[18] P.J. Garegg, H. Hultberg, and S.W. Wallin, Car-
bohydr. Res., 108 (1982) 97±101.
(15 mg, 70%); [ꢁ]d 108ꢀ (c 0.5, H2O); for H
1
NMR data, see Table 1; FABMS (positive-ion
mode; C27H49N3O16): m/z 672 [M+H]+.
[19] P. Boullanger, P. Chaterlard, G. Descotes, M.
Kloosterman, and J.H. van Boom, J. Carbohydr.
Chem., 5 (1986) 541±559.
Acknowledgements
[20] R.W. Binkley and M.G. Ambrose, J. Carbohydr.
Chem., 3 (1984) 1±49.
The authors thank Mrs. A.C.H.T.M. van der
Kerk-van Hoof for recording FAB mass spectra.
[21] P. Konradsson, U.E. Udodong, and B. Fraser-
Reid, Tetrahedron Lett., 31 (1990) 4313±4316.
[22] U. Ellervik and G. Magnusson, Carbohydr. Res.,
280 (1996) 251±260.
References
[23] F. Dasgupta and P.J. Garegg, Carbohydr. Res., 177
(1988) C13±C17.
[1] M. Katz, D.D. Despommier, and R. Gwadz,
Parasitic Diseases, 1st ed., Springer±Verlag, New
York, 1982.
[24] O. Kanie, S.C. Crawley, M.M. Palcic, and O.
Hindsgaul, Carbohydr. Res., 243 (1993) 139±164.