Polymer-supported and chemoenzymatic synthesis of the Neisseria meningitidis pentasaccharide: A methodological comparison

Article abstract of DOI:10.1016/S0008-6215(00)00086-0

Neisseria meningitids trisaccharide [GlcNAcβ(1 → 3)Galβ(1 → 4)Glc-R], tetrasaccharide [Galβ(1 → 4)GlcNAcβ(1 → 3)Galβ(1 → 4)Glc-R], and a pentasaccharide [Neu5Acα(2 → 3)Galβ(1 → 4)GlcNAcβ(1 → 3)Galβ(1 → 4)Glc-SPh] were prepared via conventional chemical synthesis, polymer-supported synthesis, and chemoenzymatic methods, starting from D-lactose. The polymer polyethyleneglycol monomethylether (MPEG) and the linker dioxyxylene (DOX) were used with a lactose-bound acceptor to improve the purification process. Several enzymes (LgtA, GalE-LgtB fusion, and CMP-Neu5Ac synthetase/sialyltransferase fusion) were used for syntheses of these oligosaccharides. Excellent stereo- and regioselectivities as well high yield (> 90% from Galβ(1 → 4)Glc-SPh) of the pentasaccharide were obtained. Both of the convenient processes are suitable for efficient preparation of target oligosaccharides. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0008-6215(00)00086-0

www.elsevier.nl/locate/carres
Carbohydrate Research 328 (2000) 3–16
Polymer-supported and chemoenzymatic synthesis of the
Neisseria meningitidis pentasaccharide: a methodological
comparison
Fengyang Yan, Warren W. Wakarchuk, Michel Gilbert, James C. Richards,
Dennis M. Whitfield *
Institute for Biological Sciences, National Research Council of Canada, Room 3024, 100 Sussex Dri6e, Ottawa,
Ont., Canada K1A 0R6
Received 10 January 2000; accepted 6 March 2000
Abstract
Neisseria meningitidis trisaccharide [GlcNAcb(1“3)Galb(1“4)Glc-R], tetrasaccharide [Galb(1“4)GlcNAcb(1“
3)Galb(1“4)Glc-R], and a pentasaccharide [Neu5Aca(2“3)Galb(1“4)GlcNAcb(1“3)Galb(1“4)Glc-SPh] were
prepared via conventional chemical synthesis, polymer-supported synthesis, and chemoenzymatic methods, starting
from
D-lactose. The polymer polyethyleneglycol monomethylether (MPEG) and the linker dioxyxylene (DOX) were
used with a lactose-bound acceptor to improve the purification process. Several enzymes (LgtA, GalE-LgtB fusion,
and CMP-Neu5Ac synthetase/sialyltransferase fusion) were used for syntheses of these oligosaccharides. Excellent
stereo- and regioselectivities as well as high yield (\90% from Galb(1“4)Glc-SPh) of the pentasaccharide were
obtained. Both of the convenient processes are suitable for efficient preparation of target oligosaccharides. © 2000
Elsevier Science Ltd. All rights reserved.
Keywords: Polymer-supported synthesis; Chemoenzymatic synthesis; Glycosyltransferase; Neisseria meningitidis LOS
1. Introduction
transformations involving iterative protec-
tion–glycosylation–deprotection reaction se-
quences with chromatographic purification of
intermediates at each stage of the synthesis.
Such preparations would greatly benefit from
developments in polymer-supported and
chemoenzymatic oligosaccharide synthetic
strategies. Polymer-supported syntheses offer
many advantages over solution-phase reac-
tions including increased speed of synthesis,
due to the minimization of purification pro-
cesses [3]. Polymer-supported methods based
on a soluble polymer polyethyleneglycol
monomethylether [MeOꢀ(CH2CH2O)nH, aver-
age Mw 5000], MPEG, have been studied in
our group in order to synthesize oligosaccha-
ride fragments of the serotype specific capsu-
The emerging understanding of the critical
role of oligosaccharides in cellular biological
recognition events and the promise of thera-
peutics based on oligosaccharides creates an
urgent need for an efficient synthetic method-
ology for oligosaccharides [1]. Although the
progress made in chemical oligosaccharide
synthesis in recent years has been remarkable
[2], oligosaccharide synthesis is still difficult
and time-consuming. These problems are
mainly due to the requirement of multistep
* Corresponding author. Tel.: +1-613-9935265; fax: +1-
613-9529092.
E-mail address: dennis.whitfield@nrc.ca (D.M. Whitfield).
0008-6215/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(00)00086-0

4
F. Yan et al. / Carbohydrate Research 328 (2000) 3–16
lar polysaccharide as a part of the process
towards developing vaccines against Group B
Streptococcus bacteria [4]. On the other hand,
as more glycosyltransferases become commer-
cially available, enzymatic synthesis is becom-
ing an alternative for the preparation of
oligosaccharides [5]. By using enzymes, glyco-
sylations can be performed under mild reac-
tion conditions with high stereo- and
regioselectivity, without the need to protect
the functional groups of the sugars. Both
methods can facilitate large-scale synthesis of
oligosaccharides.
Fig. 1. Neisseria meningitidis pentasaccharide 1.
2. Results and discussion
For comparison, trisaccharide 13 and tetra-
saccharide 14 were prepared by conventional
chemical methods (Scheme 2). The key step
was the formation of the GlcNAcb(1“3)Gal
glycosidic linkage. For this purpose,
trichloroacetimidate 3 [11–13] (Scheme 1)
containing a 2-N-phthalimido group, was se-
lected as a glycosyl donor. The phthalimido
group aids the stereoselectivity, both by neigh-
boring group participation and by its bulki-
ness. Monosaccharide glycosyl donor 4 was
prepared similarly [14]. The diol acceptor 8
Our interest is directed toward an efficient
approach to the synthesis of Neisseria menin-
gitidis
pentasaccharide
[Neu5Aca(2“3)-
Galb(1“4)GlcNAcb(1“3)Galb(1“4)Glc-R]
(1) (Fig. 1) [6]. This pentasaccharide is the
upstream terminus of the N. meningitidis
lipooligosaccharide (LOS), and it has been
shown to play an important role in the patho-
genesis of disease caused by these organisms
[7]. A process for synthesizing these oligosac-
charides is clearly needed to enable wide-
spread use. Several papers have been
published describing the chemical syntheses of
lacto-N-neotetraose and similar structures [8].
Also, both glycosyltransferases and glycosy-
lases have been used, but the yields of the
enzymatic reaction were low (10–30%)
[9,10]. In this communication, polymer-
supported and chemoenzymatic methods for
the preparation of the N. meningitidis pen-
tasaccharide 1 are compared with classical
chemical methods.
[15] (50%) was prepared from -lactose via its
D
phenylthio derivative 5 [16a] and the 3,4-O-
isopropylidene derivative 6 [16b]. Benzoyla-
tion of the free OH groups in 6, followed by
deprotection of the isopropylidene group (1 M
HCl–AcOH–THF) gave the diol acceptor 8.
The donor (4 or 3) and the diol acceptor 8
were then coupled through a regioselective
Schmidt glycosylation [9] with BF3·Et2O to
give only b(1“3) trisaccharide 9 or b(1“3)
tetrasaccharide 10 in 40–45% isolated yields.
These low yields are the result of the losses
during repeated chromatography. The b(1“
3) glycosidic linkage was determined first by
1D and 2D NMR spectroscopy (gCOSY and
Scheme 1.

F. Yan et al. / Carbohydrate Research 328 (2000) 3–16
5
Scheme 2. Reagents: (a) BzCl, Py; (b) MeONa, MeOH; (c) NH2NH2; (d) Ac2O, Py.
III
gHSQC) (9: l , 5.48 ppm, J1,2 8.5 Hz and
dioxyxylene ꢀCH2C6H4CH2Oꢀ (DOX), intro-
duced by Krepinsky et al. [18], is a superior
linker because it can be bound via an ether
linkage, it is stable under most reaction condi-
tions, and is removable from final oligosac-
charides. Thus, the phenylthio derivative 7
was glycosylated with alcohol MPEG-DOX-
OH under NIS–AgOTf conditions, then the
3,4-O-isopropylidene group was cleaved (60%
AcOH) to give a nearly quantitative yield of
the polymer-bound diol acceptor 15 (Scheme
3). Glycosylation of 15 with 4 or 3, under the
same reaction conditions as the solution
method (BF3·Et2O, −20 °C), led to the b(1“
3) linked 16 or 17 polymer-bound tri- or tetra-
saccharide in a high yield (based on the
acceptor). Unreacted diol acceptor 15 (about
10%) was also present in each of the glycosy-
lation reactions, and it was isolated as per-
acetylated compound 21 after the cleavage
step (Scheme 3).
H-1
III
III
l
, 98.58 ppm; 10: l , 5.48 ppm, J1,2 8.5
C-1
H-1
III
C-1
Hz and l , 98.15 ppm) and confirmed by
1H–13C gHMBC NMR spectroscopy, which
clearly showed that GlcNAc C-1III is con-
nected to Gal H-3II in both 9 and 10. The
sequence deprotection–acetylation–deprotec-
tion of 9 and 10 afforded trisaccharide 13
1
(75%) and tetrasaccharide 14 (66%). The H
and 13C NMR spectra in D2O of 13 and 14
could be completely assigned (Tables 1 and 2).
Evidently, this is a quite laborious and time
consuming protocol involving extensive purifi-
cation by chromatography and is not suitable
for a large-scale synthesis.
As a solution to this problem, MPEG poly-
mer-supported strategy was examined. The
MPEG strategy is based on the carbohydrate-
linker–MPEG synthon that is soluble under
reaction conditions, but insoluble during work
up, while all impurities are soluble [17]. The

6
F. Yan et al. / Carbohydrate Research 328 (2000) 3–16
ing Sc(OTf)3–Ac2O [19]. The cleavage occurs
In order to characterize these compounds,
the products were cleaved from the polymer
using the recently developed protocol involv-
at the CꢀO bond between the benzylic carbon
of the DOX and the terminal oxygen of the
Table 1
1H NMR chemical shifts a in ppm for tri-, tetra-, and pentasaccharides 13, 14, 20, and 1
13
I
II
III
H-1
H-2
H-3
H-4
H-5
H-6
H-6%
4.86 (10.0)
3.45
3.70
3.72
3.67
4.47 (8.0)
3.61
3.75
4.18 (2.5)
3.76
3.81
4.71 (8.0)
3.78
3.60
3.50
3.48
4.00
3.83
3.93
3.79
3.77
Other resonances:
SPh 7.62 (d, 2 H, Ph), 7.48–7.41 (m, 3 H, Ph)
2-NHAc 2.06
14
I
II
III
IV
H-1
H-2
H-3
H-4
H-5
H-6
H-6%
4.86 (10.0)
3.44
3.71
3.69
3.66
4.47 (7.5)
3.61
3.75
4.18
3.74
3.83
3.76
4.73 (8.5)
3.83
3.75
3.77
3.61
4.51 (7.5)
3.57
3.70
3.96
3.73
3.99
3.82
3.98
3.90
3.83
3.76
Other resonances:
SPh 7.62 (d, 2 H, Ph), 7.48–7.41 (m, 3 H, Ph)
2-NHAc 2.06
20
I
II
III
IV
H-1
H-2
H-3
H-4
H-5
H-6
H-6%
4.58 (7.5)
3.38
3.65
3.66
3.61
4.01
3.83
4.47 (8.0)
3.61
3.75
4.18
3.75
3.84
3.76
4.74 (8.0)
3.84
3.76
3.77
3.62
4.51 (8.0)
3.57
3.70
3.96
3.73
3.99
3.88
3.84
3.76
Other resonances:
CH2PhOCH2OH 7.50, 7.46, 4.97 (d, CH2Ph), 4.80 (d, CH2Ph), 4.68 (s, CH2OH)
2-NHAc 2.07
1
I
II
III
IV
V
H-1
H-2
H-3
H-3%
H-4
H-5
H-6
H-6%
H-7
H-8
H-9
H-9%
4.84 (10.0)
3.42
3.70
4.45 (7.5)
3.60.
3.74
4.71 (8.0)
3.82
3.77
4.57 (7.5)
3.59
4.14
1.82 (13.0)
2.78 (12.5, 5.0)
3.71
3.87
3.65
3.68
3.65
3.98
3.81
4.17
3.74
3.76
3.65
3.74
3.59
3.98
3.89
3.98
3.79
3.65
3.76
3.60
3.90
3.89
3.65
Other resonances:
SPh 7.61 (d, 2 H, Ph), 7.46–7.41 (m, 3 H, Ph)
2-NHAc 2.06; 5-NHAc 2.05
a Recorded at 500 MHz in D2O at 300 K.

F. Yan et al. / Carbohydrate Research 328 (2000) 3–16
7
Table 2
saccharide 19 (53%). The stereo- and regio-
chemistry of b(1“3) linked 18 and 19 was
13C NMR chemical shifts a (l) in ppm for tri-, tetra-, and
pentasaccharides 13, 14, 20, 1
confirmed by 1D and 2D NMR spectroscopy
III
(gCOSY, gHSQC, and gHMBC) (18: l
,
H-1
13
I
II
III
III
5.01 ppm, J1,2 8.0 Hz and l , 99.49 ppm; 19:
C-1
C-2
C-3
C-4
C-5
C-6
88.14
72.45
78.91
76.80
79.73
61.14
103.86
71.03
75.92
69.38
82.95
62.00
103.86
56.70
74.59
70.71
76.68
61.53
C-1
III
H-1
III
C-1
l
, 5.19 ppm, J1,2 8.0 Hz and l , 100.19
ppm). Then, 19 was converted to the final
tetrasaccharide 20 with NaOMe–MeOH in an
1
87% yield. The assignment of the H and 13C
NMR spectra of 20 is shown in Tables 1 and
2. Since elaborate and extensive chromatogra-
phy was avoided in the purification process,
this polymer-supported method is suitable for
synthesis of large quantities of oligosaccha-
rides. Typical laboratory scales are 50 mg–50
g of MPEG. Larger scales are mainly limited
by apparatus size and not the chemistry [20].
The enzymatic method is a powerful
tool for oligosaccharide synthesis. Several
Neisserial glycosyltransferases such as b(1“
3)-N-acetylglucosaminyl transferase (LgtA),
b(1“4)-galactosyltransferase (LgtB), UDP-
galactose-4-epimerase (GalE), CMP-Neu5Ac-
synthetase, and a(2“3)-sialyltransferase from
N. meningitidis have been cloned and overex-
pressed in Escherichia coli in our institute [7].
These enzymes can catalyze the transfer of an
oligosaccharide from a sugar nucleotide donor
(e.g., UDP-GlcNAc, UDP-Glc, and CMP-
Neu5Ac) to an acceptor. The lactose deriva-
Other resonances:
SPh 132.84, 132.62, 130.30, 129.11
2-NHAc 175.88, 23.24
14
I
II
III
IV
C-1
C-2
C-3
C-4
C-5
C-6
88.12
72.42
73.50
78.88
79.70
61.08
103.86
70.95
76.34
69.34
83.02
61.96
103.74
56.18
73.17
79.54
75.54
60.86
103.86
71.96
76.76
69.54
75.87
61.96
Other resonances:
SPh 132.87, 132.65, 130.33, 129.14
2-NHAc 175.90, 23.16
20
I
II
III
IV
C-1
C-2
C-3
C-4
C-5
C-6
101.70
73.50
73.10
79.06
75.48
61.43
103.57
70.65
76.04
69.02
82.73
61.72
103.44
55.89
72.88
78.87
75.25
61.63
103.56
71.66
73.20
69.24
75.57
61.72
Other resonances:
CH2PhOCH2OH 141.05, 136.66, 129.72, 128.35, 71.94 (C,
CH2Ph), 64.29 (C, OCH2OH)
2-NHAc 175.61, 22.87
tive phenyl b-D-galactopyranosyl-(1“4)-
1-thio-b- -glucopyranoside (5) was chosen as
D
1
I
II
III
IV
V
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
88.24
72.55
76.90
79.03
79.84
61.23
104.00
71.09
76.22
69.74
83.14
62.13
103.93
56.30
73.26
76.30
75.68
60.97
103.69
70.51
76.62
68.60
79.15
62.13
174.98
100.93
40.78
69.74
52.82
74.02
69.22
72.89
63.70
an initial acceptor substrate for enzymatic
synthesis of trisaccharide 13. The phenylthio
group was purposefully introduced to aid in
monitoring of the separation of the target
product by C18 reversed-phase chromatogra-
phy and for further chemical or enzymatic
manipulation.
The
b(1“3)-N-acetylglu-
cosaminyltransferase (LgtA) catalyzed glyco-
sylation used UDP-GlcNAc as a donor and 5
as an acceptor to give stereo- and regioselec-
tively the b(1“3) linked trisaccharide 13 in an
95% yield (see Scheme 4). The pure compound
13 was obtained by a single C18 reversed-phase
chromatography using 17:3–4:1 water–
MeOH as eluent, followed by freeze-drying.
Subsequently, the tetrasaccharide Galb(1“
Other resonances:
SPh 133.00, 132.77, 130.44, 129.25
2-NHAc 175.98, 23.14; 5-NHAc 176.13, 23.28
a Recorded at 75 MHz in D2O at 300 K.
MPEG while all hydroxyls are acetylated.
First, the O- and N-acyl groups in 16 and 17
were removed by treatment with 1,8-diazabi-
cyclo[5,4,0]undec-7-ene (DBU), followed by
ethylenediamine. The product was cleaved
from the polymer with Sc(OTf)3–Ac2O to give
peracetylated trisaccharide 18 (52%) and tetra-
4)GlcNAcb(1“3)Galb(1“4)Glc-SPh
(14)
was constructed. A fusion protein GalE-LgtB
[21] was used as a catalyst with UDP-glucose
(UDP-Glc) as a glycosyl donor. This fusion

8
F. Yan et al. / Carbohydrate Research 328 (2000) 3–16
enzyme allows the relatively inexpensive
UDP-Glc to be converted to the more expen-
sive UDP-Gal under equilibrium conditions,
with the enzyme GalE as the catalyst. The
equilibrium is in favor of the formation of
UDP-Glc (7:1), but the equilibrium is driven
by the UDP-Gal being consumed by the trans-
ferase LgtB to give the b(1“4) linked tetra-
saccharide 14. The enzymes showed excellent
stereo- and regioselectivity (see Scheme 4).
The desired compound 14 was isolated and
purified in 96% yield using a C18 reversed-
phase column (9:1–17:3 water–MeOH).
Sialyltransferases transfer sialic acid from cy-
tidine 5%-monophospho N-acetylneuraminic
acid (CMP-Neu5Ac) onto the specific hy-
droxyl group of the acceptor sugar. CMP-
Neu5Ac is costly and unstable, and therefore
CMP-Neu5Ac was generated in situ by CMP-
Neu5Ac synthetase. Also, the a(2“3)-sialyl-
transferase from N. meningitidis as expressed
from E. coli is relatively insoluble and has a
tendency to precipitate with the loss of activity
during storage. Thus, a fused form of these
two enzymes has been constructed and ex-
pressed in E. coli [23]. tetrasaccharide 14 was
enzymatically glycosylated using in situ gener-
ation of CMP-Neu5Ac from CTP and sialic
acid by this fusion enzyme. The reaction was
very fast and complete in 2 h at 32 °C. The
Since the chemical sialylation is one of the
most difficult reactions in synthetic carbohy-
drate chemistry, sialylation with sialyltrans-
ferases is
a
valuable alternative [22].
Scheme 3. Reagents: (a) DBU, MeOH; (b) ethylenediamine; (c) Sc(OTf)3, Ac2O; (d) MeONa, MeOH.

F. Yan et al. / Carbohydrate Research 328 (2000) 3–16
9
Scheme 4. Enzymatic synthesis of N. meningitidis pentasaccharide 1.
oligosaccharide syntheses. Notable a yield of
final pentasaccharide 1 was successfully iso-
lated using C18 reversed-phase chromatogra-
phy by controlled elution with water and then
99:1 water–MeOH. The yield of 1 was nearly
nearly 80% of N. meningitidis pentasaccharide
1 from
D-lactose was obtained via the
chemoenzymatic method. Similar chemoenzy-
matic syntheses have been easily performed on
gram scales in our laboratory [24]. The synthe-
sized oligosaccharides are useful as starting
substances for more complex molecules and
will be used in the biophysical studies of the
transferases used for their synthesis.
1
quantitative. Complete H and 13C NMR as-
signments for 1 were achieved based on vari-
ous 1D and 2D NMR experiments (1D
TOCSY, gCOSY, gHSQC, gHMBC) (Tables
1 and 2). The H-3 resonance of GalIV in 1
(4.14 ppm) versus 14 (3.79 ppm) shows a large
downfield shift confirming the a(2“3) linkage
in compound 1.
3. Experimental
Thus, we developed three methodologies for
the syntheses of tri-, tetrasaccharides, and sia-
lylated pentasaccharide (13, 14, 20 and 1).
General methods.—Melting points were
measured on a Buchi 535 melting point ap-
paratus and were not corrected. Optical rota-
tions were obtained (u=589 nm) at 20 °C in a
Polymer-supported
and
chemoenzymatic
methodologies are powerful tools for these

10
F. Yan et al. / Carbohydrate Research 328 (2000) 3–16
atmosphere of argon, CH2Cl2 (10 mL) was
added, followed by BF3·Et2O (33 mL), and the
mixture was stirred for 4 h. The reaction was
quenched with several drops of Et3N, and the
mixture was concentrated under reduced pres-
sure. Chromatography thrice afforded 9 (0.16
g, 42%). Mp 104–106 °C; [h]D +56.6° (c 0.6,
10-cm, 1-mL cell using a Perkin–Elmer 243
polarimeter. NMR spectra were recorded on
INOVA-500 or INOVA-200 instrument at 300
K. Chemical shifts were given in ppm relative
to the signal of internal TMS or indirectly to
solvent signals 7.26 (CDCl3) or 4.81 (D2O) for
1H NMR spectra, and to the solvent signals
77.0 (CDCl3) or 49.15 (internal methyl alco-
hol) for 13C NMR spectra. All signal assign-
1
CHCl3); H NMR (CDCl3): l 8.10–7.02 (m,
34 H), 5.62 (t, 1 H, J 8.0 Hz, H-3I), 5.60 (t, 1
H, J 9.0 Hz, H-3III), 5.48 (d, 1 H, J 8.5 Hz,
H-1III), 5.33 (t, 1 H, J 10.0 Hz, H-2I), 5.25 (t,
1 H, J 9.5 Hz, H-2II), 5.06 (t, 1 H, J 9.5 Hz,
H-4III), 4.78 (d, 1 H, J 10.0 Hz, H-1I), 4.46 (d,
1 H, J 8.0 Hz, H-1II), 4.37 (d, 1 H, J 12.0 Hz,
H-6I), 4.28 (dd, 1 H, J 11.5 Hz, 5.5 Hz, H-6I),
4.25 (dd, 1 H, J 11.0 Hz, 9.5 Hz, H-2III),
4.20–4.13 (m, 2 H, H-6II and H-6III), 4.10 (d,
1 H, J 10.5 Hz, H-6III), 4.00 (s, 1 H, H-4II),
3.97 (t, 1 H, J 9.5 Hz, H-4I), 3.84–3.78 (m, 1
H, H-5III), 3.71 (dd, 1 H, J 10.0 Hz, 3.0 Hz,
H-3II), 3.68–3.63 (m, 1 H, H-5I), 3.63–3.58
(m, 1 H, H-6II), 3.56–3.49 (m, 1 H, H-5II),
2.00 (s, 3 H, CH3), 1.98 (s, 3 H, CH3), 1.74 (s,
1
ments were made by standard H–1H COSY
and 1H-decoupled 13C–1H COSY experi-
ments. MALDIMS spectra were taken on a
Voyager-De STR Biochemistry Workstation
(PerSeptive Biosystems, Framingham, MA,
USA). Thin-layer chromatography (TLC) was
performed on E. Merck Silica Gel 60 F254
plates. Silica gel (230–400 mesh) was used for
flash chromatography. C18 Silica gel (10%
capped with TMS, 35–70 mesh) was used for
reversed-phase chromatography.
Phenyl 2,6-di-O-benzoyl-i-
osyl-(1“4)-2,3,6-tri-O-benzoyl-1-thio-i-
glucopyranoside (8).—The acceptor 8 was pre-
pared in six steps starting from -lactose
D
-galactopyran-
D-
13
D
3 H, CH3); C NMR (CDCl3): l 170.47 (Ac),
[9,15,16]. Mp 117–119 °C; [h]D +39.0° (c 0.4,
CHCl3); 1H NMR (CDCl3): l 8.10–7.10 (m 30
H, 6Ph), 5.63 (t, 1 H, J 9.5 Hz, H-3I), 5.39 (t,
1 H, J 9.5 Hz, H-2I), 5.34 (t, 1 H, J 7.5 Hz,
H-2II), 4.83 (d, 1 H, J 10.0 Hz, H-1I), 4.60 (d,
1 H, J 11.5 Hz, H-6I), 4.59 (d, 1 H, J 8.0 Hz,
H-1II), 4.50 (dd, 1 H, J 11.5 Hz, 5.0 Hz, H-6I),
4.04 (t, 1 H, J 9.5 Hz, H-4I), 3.98 (dd, 1 H, J
11.0 Hz, 5.0 Hz, H-6II), 3.88–3.80 (m, 1 H,
H-5I), 3.83 (d, 1 H, J 4.0 Hz, H-4II), 3.71 (dd,
1 H, J 9.5 Hz, 3.0 Hz, H-3II), 3.58–3.48 (m, 2
H, H-5II and H-6II); 13C NMR (CDCl3): l
166.36 (Bz), 166.03 (Bz), 165.95 (Bz), 165.87
(Bz), 165.15 (Bz), 100.96 (C-1II), 85.78 (C-1I),
77.00 (C-5I), 76.20 (C-4I), 74.20 (C-3I), 73.65
(C-2II), 72.58 (C-5II) 72.58 (C-3II), 70.17 (C-2I),
68.56 (C-4II), 62.85 (C-6I), 61.75 (C-6II);
MALDIMS: Anal. Calcd for C53H46O15SNa:
977.24. Found: m/z 977.16 [M+Na].
169.89 (Ac), 169.26 (Ac), 167.60 (Phth),
166.50 (Phth), 165.98 (Bz), 165.71 (Bz), 165.39
(Bz), 165.11 (Bz), 163.95 (Bz), 100.52 (C-1II),
98.58 (C-1III), 85.73 (C-1I), 81.20 (C-3II), 76.86
(C-5I), 75.37 (C-4I), 73.72 (C-3I), 72.19 (C-5II),
72.02 (C-5III), 70.46 (C-2II), 70.32 (C-2I), 70.22
(C-3III), 68.60 (C-4III), 67.81 (C-4II), 62.64 (C-
6I), 62.63 (C-6II), 61.78 (C-6III), 54.17 (C-2III);
Anal. Calcd for C73H65NO24S (1372.38): C,
63.38; H, 4.77; N, 1.02. Found: C, 63.22; H,
4.43; N, 1.01.
Phenyl 2,3,4,6-tetra-O-acetyl-i-
pyranosyl)-(1“4)-3,6-di-O-acetyl-2-deoxy-2-
phthalimido-i- -glucopyranosyl-(1“3)-2,6-
di-O-benzoyl-i- -galactopyranosyl-(1“4)-
2,3,6-tri-O-benzoyl-1-thio-i- -glucopyrano-
D-galacto-
D
D
D
side (10).—Disaccharide donor 3 and receptor
8 were treated with BF3·Et2O, as described
above for 9, to yield tetrasaccharide 10 (0.26
g, 40%). Mp 149–151 °C; [h]D +48.5° (c 0.5,
Phenyl 3,4,6-tri-O-acetyl-2-deoxy-2-phthal-
1
imido - i -
benzoyl-i-
tri-O-benzoyl-1-thio-i-
D
- glucopyranosyl - (1“3) - 2,6-di-O-
-galactopyranosyl-(1“4)-2,3,6-
-glucopyranoside (9).
CHCl3); H NMR (CDCl3): l 8.09–6.99 (m,
D
34 H, Ph), 5.62 (t, 1 H, J 9.0 Hz, H-3I), 5.55
(dd, 1 H, J 10.0 Hz, 8.0 Hz, H-3III), 5.48 (d, 1
H, J 8.5 Hz, H-1III), 5.32 (t, 1 H, J 9.5 Hz,
H-2I), 5.30 (d, 1 H, J 3.0 Hz, H-4IV), 5.25 (t, 1
H, J 8.0 Hz, H-2II), 5.08 (dd, 1 H, J 10.5 Hz,
8.5 Hz, H-2IV), 4.94 (dd, 1 H, J 10.0 Hz, 3.0
D
—The mixture of monosaccharide donor 4
(0.25 g, 0.26 mmol) and acceptor 8 (0.18 g,
0.31 mmol) was dried under high vacuum
overnight. After cooling to −20 °C under an

F. Yan et al. / Carbohydrate Research 328 (2000) 3–16
11
NH), 5.46 (t, 1 H, J 11.5 Hz, H-3III), 5.31 (d,
1 H, J 3.5 Hz, H-4II), 5.18 (t, 1 H, J 9.0 Hz,
H-3I), 5.02 (t, 1 H, J 10.0 Hz, H-4III), 5.00 (d,
1 H, J 8.0 Hz, H-1III), 4.99 (t, 1 H, J 10.5 Hz,
H-2II), 4.89 (t, 1 H, J 9.5 Hz, H-2I), 4.66 (d, 1
H, J 10.5 Hz, H-1I), 4.48 (dd, 1 H, J 12.0, 2.0
Hz, H-6I), 4.36 (dd, 1 H, J 12.0, 2.5 Hz,
H-6III), 4.33 (d, 1 H, J 8.0 Hz, H-1II), 4.19 (dd,
1 H, J 11.5, 6.0 Hz, H-6I), 4.04 (dd, 1 H, J
11.0, 4.0 Hz, H-6III), 4.02 (m, 2 H, 2×H-6II),
3.82–3.73 (m, 2 H, H-3II and H-5II), 3.69 (t, 1
H, J 12.0 Hz, H-4I), 3.66 (m, 1 H, H-5III), 3.63
(m, 1 H, H-5I), 3.32 (m, 1 H, H-2III), 2.10 (s,
6 H, 2×CH3), 2.09 (s, 3 H, CH3), 2.07 (s, 3
H, CH3), 2.06 (s, 3 H, CH3), 2.05 (s, 3 H,
CH3), 2.04 (s, 3 H, CH3), 1.99 (s, 6 H, 2×
Hz, H-3IV), 4.78 (d, 1 H, J 10.0 Hz, H-1I), 4.51
(d, 1 H, J 8.0 Hz, H-1IV), 4.50 (d, 1 H, J 12.0
Hz, H-6III), 4.47 (d, 1 H, J 8.0 Hz, H-1II), 4.39
(d, 1 H, J 11.0 Hz, H-6I), 4.26 (dd, 1 H, J 11.5
Hz, 5.0 Hz, H-6I), 4.19 (dd, 1 H, J 11.0 Hz,
4.5 Hz, H-2II), 4.16 (t, 1 H, J 10.0 Hz, H-2III),
4.07–3.95 (m, 5 H, H-4I, H-4II, H-6III, and
2×H-6IV), 3.82 (t, 1 H, J 6.5 Hz, H-5IV),
3.78–3.72 (m, 2 H, H-4III and H-5III), 3.70–
3.64 (m, 2 H, H-3II and H-5I), 3.62 (dd, 1 H,
J 11.5, 7.5 Hz, H-6II), 3.52 (m, 1 H, H-5II),
2.10 (s, 3 H, CH3), 2.04 (s, 3 H, CH3), 2.01 (s,
3 H, CH3), 1.99 (s, 3 H, CH3), 1.94 (s, 3 H,
13
CH3), 1.78 (s, 3 H, CH3); C NMR (CDCl3):
l 170.24 (Ac), 170.19 (Ac), 170.00 (Ac),
169.95 (Ac), 169.43 (Ac), 196.04 (Ac), 167.75
(Phth), 166.71 (Phth), 165.94 (Bz), 165.65
(Bz), 165.35 (Bz), 165.08 (Bz), 163.92 (Bz),
100.98 (C-1IV), 100.47 (C-1II), 98.15 (C-1III),
85.66 (C-1I), 81.35 (C-5I), 76.85 (C-3II), 76.68
(C-5III), 75.39 (C-4I), 73.72 (C-3I), 72.74 (C-
4III), 72.24 (C-5II), 70.90 (C-3III) 70.80 (C-3IV),
70.53 (C-5IV), 70.35 (C-2II), 70.35 (C-2I), 69.02
(C-2IV), 67.53 (C-4II), 66.44 (C-4IV), 62.74 (C-
6II), 62.58 (C-6I), 61.69 (C-6III), 60.55 (C-6IV),
54.38 (C-2III), 20.58 (Ac), 20.51 (Ac), 20.49
(Ac), 20.42 (Ac), 20.28 (Ac); Anal. Calcd for
C85H81NO32S (1660.63): C, 61.47; H, 4.92; N,
0.84. Found: C, 61.73; H, 4.99; N, 0.83.
13
CH3), 1.89 (s, 3 H, CH3); C NMR (CDCl3):
l 170.69 (Ac), 170.49 (Ac), 169.74 (Ac),
169.56 (Ac), 169.50 (Ac), 169.43 (Ac), 169.07
(Ac), 132.84 (Ph), 131.95 (Ph), 128.85 (Ph),
128.21 (Ph), 100.62 (C-1II), 99.52 (C-1III),
85.57 (C-1I), 76.74 (C-5I), 75.92 (C-3II), 75.57
(C-4I), 73.57 (C-3I), 71.68 (C-5III), 71.18 (C-
5II), 71.13 (C-3III), 70.92 (C-2II), 70.20 (C-2I),
68.83 (C-4II), 68.66 (C-4III), 62.27 (C-6I), 61.51
(C-6II), 61.09 (C-6III), 56.12 (C-2III), 20.83
(Ac), 20.79 (Ac), 20.73 (Ac), 20.71 (Ac), 20.68
(Ac), 20.60 (Ac); Anal. Calcd for
C44H57NO24S (1015.96): C, 52.02; H, 5.66; N,
1.38. Found: C, 51.94; H, 5.70; N, 1.38.
Phenyl 2-acetamido-3,4,6-tri-O-acetyl-2-
deoxy-i-
acetyl-i-
O-acetyl-1-thio-i-
D
-glucopyranosyl-(1“3)-2,4,6-tri-O-
-galactopyranosyl-(1“4)-2,3,6-tri-
-glucopyranoside (11).—
Phenyl 2,3,4,6-tetra-O-acetyl-i-
pyranosyl-(1“4)-2-acetamido-3,6-di-O-acetyl-
2-deoxy-i- -glucopyranosyl-(1“3)-2,4,6-tri-
O - acetyl - i - - galactopyranosyl-(1“4)-2,-
3,6 - tri - O - acetyl - 1 - thio-i- -glucopyranoside
D-galacto-
D
D
D
To a solution of 9 (100 mg, 0.07 mmol) in
MeOH (10 mL) was added 1 M CH3ONa (15
mL). After 4 h, the reaction mixture was
neutralized with Rexyn 101 (H+) resin and
filtered, and the filtrate was concentrated. A
mixture of the residue and hydrazine hydrate
(1 mL) in EtOH (30 mL) was refluxed for 5 h.
Upon cooling and concentration, the residue
was treated with 1:1 Ac2O–pyridine (10 mL)
overnight. The reaction was quenched with
MeOH (0 °C), and the mixture was extracted
with EtOAc (3×30 mL). The organic layers
were combined and dried over Na2SO4. The
solvent was removed and the residue was
chromatographed (10:1 EtOAc–MeOH) to
give 64 mg (86%) of 11. Mp 89–92 °C; [h]D
D
D
(12).—The protected compound 10 (165 mg,
0.10 mmol) was treated with CH3ONa, hydra-
zine hydrate, and Ac2O, as described for 11, to
yield the peracylated tetrasaccharide 12 (110
mg, 86%). Mp 129–130 °C; [h]D +3.5° (c 0.5,
1
CHCl3); H NMR (CDCl3): l 7.48–7.28 (m 5
H, Ph), 5.34 (d, 1 H, J 3.0 Hz, H-4IV), 5.32 (d,
1 H, J 8.5 Hz, NH), 5.29 (d, 1 H, J 3.0 Hz,
H-4II), 5.18 (t, 2 H, J 9.0 Hz, H-3III and H-3I),
5.10 (dd, 1 H, J 11.0, 7.5 Hz, H-2IV), 4.99–
4.94 (m, 2 H, H-2II and H-3IV), 4.90 (t, 1 H, J
9.5 Hz, H-2I), 4.77 (dd, 1 H, J 11.5, 2.0 Hz,
H-6III), 4.66 (d, 2 H, J 9.0 Hz, H-1I and
H-1III), 4.54 (d, 1 H, J 8.0 Hz, H-1IV), 4.48 (d,
1 H, J 10.5 Hz, H-6I), 4.33 (d, 1 H, J 8.0 Hz,
1
+14.0° (c 0.9, CHCl3); H NMR (CDCl3): l
H-1II),
4.14–4.08
(m,
3
H,
H-6I
7.50–7.27 (m 5 H, Ph), 5.47 (d, 1 H, J 8.5 Hz,

12
F. Yan et al. / Carbohydrate Research 328 (2000) 3–16
and 2×H-6IV), 4.08–3.99 (m, 2 H, 2×H-6II),
3.96 (dd, 1 H, J 11.5, 3.0 Hz, H-6III), 3.86 (t,
1 H, J 7.0 Hz, H-5IV), 3.78 (t, 1 H, J 9.5 Hz,
H-4III), 3.76 (t, 1 H, J, 6.0 Hz, H-5II), 3.72 (m,
1 H, H-3II), 3.70 (t, 1 H, J 10.0 Hz, H-4I),
3.66–3.60 (m, 1 H, H-5I), 3.55–3.48 (m, 2 H,
H-2III and H-5III), 2.14 (s, 6 H, 2×CH3), 2.10
(s, 6 H, 2×CH3), 2.07 (s, 6 H, 2×CH3), 2.06
(s, 3 H, CH3), 2.05 (s, 6 H, 2×CH3), 2.04 (s,
3 H, CH3), 2.00 (s, 3 H, CH3), 1.96 (s, 3 H,
6.49; N, 1.64. Found: C, 44.66; H, 6.33; N,
1.74.
MPEGDOXyl 2,6-di-O-benzoyl-i- -galac-
D
topyranosyl-(1“4)-2,3,6-tri-O-benzoyl-i- -
D
glucopyranoside (15).—Compound 7 (300 mg,
0.30 mmol), HODOXMPEG (772 mg, 0.15
,
mmol) and activated 4 A molecular sieves (500
mg) were dried overnight at high vacuum in a
flask
covered
with
aluminum
foil.
Dichloromethane (5 mL) was added and the
mixture was cooled in an ice bath. After stir-
ring for 0.5 h, N-iodosuccinimide (NIS, 136
mg, 0.60 mmol) was added, followed by silver
trifluoromethanesulfonate (77.5 mg, 0.30
mmol), and the reaction was maintained at
0 °C for 3 h. The mixture was cooled to 0 °C,
neutralized with several drops of diisopropyl-
ethylamine, and the polymer was precipitated
with TBME (260 mL). The solids were recov-
ered by filtration and washed several times
with diethyl ether. The solids were dissolved
with a hot solution of 0.5% ethanolic imida-
zole (175 mL), and the resulting solution was
kept at −20 °C for 1 h to complete precipita-
tion. The precipitate was collected by filtration
and, after washing with cold EtOH and di-
ethyl ether, it was taken up in CH2Cl2 and
filtered, and the filtrate was evaporated to give
a polymer-bound product. A solution of the
foregoing product in 60% AcOH (120 mL)
was stirred at 60 °C for 2 h. The solvent was
evaporated under reduced pressure, the
residue was dissolved in CH2Cl2 (3 mL), and
TBME (250 mL) was added to precipitate the
polymer. The precipitate was recovered by
filtration and, after rinsing with TBME, re-
precipitated from absolute EtOH (150 mL).
The resulting solid was collected by filtration,
and after washing with cold EtOH and diethyl
ether it was dissolved in CH2Cl2, filtered, and
evaporated to yield the polymer-bound accep-
tor 15 (840 mg, 92%).
13
CH3), 1.89 (s, 3 H, CH3); C NMR (CDCl3):
l 170.56 (Ac), 170.42 (Ac), 170.38 (Ac),
170.35 (Ac), 170.25 (Ac), 170.08 (Ac), 170.03
(Ac), 169.75 (Ac), 169.72 (Ac), 169.50 (Ac),
169.06 (Ac), 168.82 (Ac), 132.83 (Ph), 131.97
(Ph), 128.85 (Ph), 128.21 (Ph), 101.06 (C-1IV),
100.61 (C-1II), 100.20 (C-1III), 85.58 (C-1I),
76.75 (C-5I), 75.74 (C-3II), 75.69 (C-4III), 75.60
(C-4I), 73.59 (C-3I), 72.66 (C-5III), 71.82 (C-
3III), 71.08 (C-5II), 70.99 (C-2II), 70.85 (C-3IV),
70.69 (C-5IV), 70.22 (C-2I), 69.15 (C-2IV), 68.86
(C-4II), 66.60 (C-4IV), 62.28 (C-6I), 61.51 (C-
6II), 60.75 (C-6IV), 60.03 (C-6III), 55.04 (C-2III),
20.83 (Ac), 20.82 (Ac), 20.80 (Ac), 20.73 (Ac),
20.71 (Ac), 20.68 (Ac), 20.63 (Ac), 20.62 (Ac),
20.59 (Ac), 20.48 (Ac); Anal. Calcd for
C56H73NO32S (1304.25): C, 51.57; H, 5.64; N,
1.07. Found: C, 51.30; H, 5.97; N, 1.10.
Phenyl 2-acetamido-2-deoxy-i-
anosyl-(1“3)-i- -galactopyranosyl-(1“4)-
1-thio-i- -glucopyranoside (13).—Compound
D-glucopyr-
D
D
11 (50 mg, 0.05 mmol) was deacetylated (Zem-
pen), and the reaction mixture was neutralized
with Rexyn 101 (H+) resin and filtered. The
filtrate was concentrated, and the residue was
dissolved in water and freeze-dried to give the
final trisaccharide 13 (27 mg, 87%). [h]D
+
26.7° (c 0.2, MeOH); Anal. Calcd for
C26H39NO15S (637.66): C, 48.97; H, 6.16; N,
2.20. Found: C, 48.96; H, 6.05; N, 2.09;
MALDIMS: Anal. Calcd for C26H39NO15SNa:
660.1938. Found: m/z 660.295 [M+Na].
MPEGDOXyl 3,4,6-tri-O-acetyl-2-deoxy-2-
phthalimido - i -
di-O-benzoyl-i-
2,3,6-tri-O-benzoyl-i-
D
- glucopyranosyl - (1“3)-2,6-
-galactopyranosyl-(1“4)-
-glucopyranoside (16).
Phenyl i -
acetamido - 2 - deoxy-i-
(1“3)-i- -galactopyranosyl-(1“4)-1-thio-
i - - glucopyranoside (14).—Protected tetra-
D
- galactopyranosyl) - (1“4) - 2-
D
D
-glucopyranosyl)-
D
D
—A mixture of monosaccharide donor 4 (50
mg, 0.09 mmol) and polymer-bound acceptor
15 (430 mg, 0.07 mmol) was dried under high
vacuum overnight. After cooling (−20 °C) in
an argon atmosphere CH2Cl2 (10 mL) was
added, followed by BF3·Et2O (20 mL), and the
D
saccharide 12 (60 mg, 0.05 mmol) was treated
as above for 13 to give 14 (30 mg, 77%). [h]D
−138.26° (c 0.07, H2O); Anal. Calcd for
C26H39NO15S·3 H2O (853.84): C, 45.06; H,

F. Yan et al. / Carbohydrate Research 328 (2000) 3–16
13
added, and the mixture was left to stir for 5 h.
Ammonium bicarbonate (15 mg) was added,
and the polymer was precipitated with TBME
(100 mL) and, after filtration, the residue was
reprecipitated from absolute EtOH (50 mL).
The combined filtrates were evaporated to
dryness, followed by co-evaporation with tolu-
ene (2×20 mL). The residue was purified by
chromatography (19:1 EtOAc–MeOH) to
yield 18 (35 mg, 52%) and the by-product 21
(4.6 mg, 10%). 18: [h]D −1.2° (c 0.8 CHCl3);
1H NMR (CDCl3): l 7.33 (d, 2 H, J 7.5 Hz,
Ph), 7.27 (d, 2 H, J 7.0 Hz, Ph), 5.48 (t, 1 H,
J 10.0 Hz, H-3III), 5.44 (d, 1 H, J 8.0 Hz,
NH), 5.32 (d, 1 H, J 3.0 Hz, H-4II), 5.13 (t, 1
H, J 9.0 Hz, H-3I), 5.09 (s, 2 H, CH2OAc),
5.04 (t, 1 H, J 9.5 Hz, H-4III), 5.01 (d, 1 H, J
8.0 Hz, H-1III), 4.98 (t, 1 H, J 7.5 Hz, H-2II),
4.96 (t, 1 H, J 9.5 Hz, H-2I), 4.85 (d, 1 H, J
12.0 Hz, CH2Ph), 4.59 (d, 1 H, J 12.0 Hz,
CH2Ph), 4.52–4.46 (m, 2 H, H-1I and H-6I),
4.38 (dd, 1 H, J 12.0, 1.5 Hz, H-6III), 4.35 (d,
1 H, J 7.5 Hz, H-1II), 4.13 (dd, 1 H, J 12.0, 5.5
Hz, H-6I), 4.09–4.01 (m, 3 H, H-6III and
2×H-6II), 3.81–3.74 (m, 3 H, H-3II, H-5II,
and H-4I), 3.66 (m, 1 H, H-5III), 3.60–3.56 (m,
1 H, H-5I), 3.28 (m, 1 H, H-2III), 2.14 (s, 3 H,
CH3), 2.10 (s, 9 H, 3×CH3), 2.08 (s, 6 H,
2×CH3), 2.02 (s, 3 H, CH3), 2.01 (s, 3 H,
CH3), 2.00 (s, 6 H, 2×CH3), 1.90 (s, 3 H,
CH3); 13C NMR (CDCl3): l 170.76 (Ac),
170.65 (Ac), 170.43 (Ac), 170.42 (2×Ac),
170.31 (Ac), 169.76 (Ac), 169.74 (2×Ac),
169.38 (Ac), 169.05 (Ac), 136.70 (Ph), 135.70
(Ph), 128.36 (Ph), 127.82 (Ph), 100.62 (C-1II),
99.49 (C-1III), 99.05 (C-1I), 75.64 (C-3II), 75.64
(C-4I), 72.79 (C-5I), 72.51 (C-3I), 71.72 (C-2I),
71.58 (C-5III), 71.19 (C-5II), 71.18 (C-3III),
70.99 (C-2II), 70.31 (C-CH2Ph), 68.87 (C-4II),
68.63 (C-4III), 65.94 (CꢀCH2OAc), 62.08 (C-
6I), 61.59 (C-6II), 61.12 (C-6III), 56.23 (C-2III),
23.36 (Ac), 21.06 (Ac), 20.95 (Ac), 20.78 (Ac),
20.72 (Ac); MALDIMS: Anal. Calcd for
C48H63NO27Na: 1108.35. Found: m/z 1108.12
[M+Na].
mixture was stirred for 4 h. The reaction was
quenched with several drops of diisopropyl-
ethylamine, and the polymer was precipitated
with TBME (150 mL). The solid was collected
by filtration and re-precipitated from absolute
EtOH (90 mL). The precipitate was filtered
and washed with cold EtOH and diethyl ether.
Then it was dissolved in CH2Cl2, filtered and
evaporated to yield polymer-bound trisaccha-
ride 16 (420 mg, 91%).
MPEGDOXyl 2,3,4,6-tetra-O-acetyl-i-
galactopyranosyl - (1“4) - 3,6 - di - O - acetyl - 2-
deoxy - 2 - phthalimido - i - - glucopyranosyl-
-galactopyrano-
-glucopyrano-
D
-
D
(1“3)-2,6-di-O-benzoyl-i-
D
syl(1“4)-2,3,6-tri-O-benzoyl-i-
D
side (17).—The mixture of disaccharide donor
3 (174 mg, 0.20 mmol) and polymer-bound
acceptor 15 (600 mg, 0.10 mmol) was treated
with BF3·Et2O (30 mL), as described above for
16 to give 17 (620 mg, 92%).
4-Acetoxymethylbenzyl 2-acetamido-3,4,6-
tri - O - acetyl - 2 - deoxy - i -
(1“3)-2,4,6-tri-O-acetyl-i-
nosyl-(1“4)-2,3,6-tri-O-acetyl-i-
D
- glucopyranosyl-
-galactopyra-
-glucopy-
D
D
ranoside (18).—The polymer-bound trisaccha-
ride 16 (400 mg, 0.06 mmol) was dissolved in
MeOH (10 mL), 1,8-diazabicyclo[5,4,0]undec-
7-ene (five drops) was added, and the mixture
was stirred under an argon atmosphere
overnight. After cooling in ice, the polymer
was precipitated with TBME (140 mL). The
solid was collected by filtration and reprecipi-
tated from absolute EtOH (80 mL), and the
solid was dissolved in CH2Cl2, filtered and
evaporated to yield O-de-acylated polymer-
bound trisaccharide. To this solid was added
absolute EtOH (6 mL) and ethylenediamine
(0.30 mL, 4.5 mmol), and the mixture was
heated at 70 °C under an atmosphere of argon
for 2 h. The reaction mixture was diluted with
absolute EtOH (10 mL) and tightly stoppered
and put in a freezer for about 1 h. The
resulting precipitate was recovered by filtra-
tion, washed with cold EtOH and diethyl
ether, and then dissolved in CH2Cl2, filtered
and evaporated to obtain the crude amine.
After drying under high vacuum, this solid
was dissolved in CH2Cl2 (3 mL), and Ac2O (3
mL) was added under an atmosphere of ar-
gon. After 15 min scandium(III) trifluoro-
methanesulfonate (15 mg, 0.03 mmol) was
4-Acetoxymethylbenzyl 2,3,4,6-tetra-O-ace-
tyl-i-
D
-galactopyranosyl - (1“4) - 2,3,6-tri-O-
acetyl-i-
D
-glucopyranoside (21).—[h]D −26.9°
1
(c 0.4 CHCl3); H NMR (CDCl3): l 7.33 (d, 2
H, J 8.0 Hz, Ph), 7.27 (d, 2 H, J 8.0 Hz, Ph),
5.34 (d, 1 H, J 3.0 Hz, H-4II), 5.16 (t, 1 H, J
10.0 Hz, H-3I), 5.10 (s, 2 H, CH2OAc),

14
F. Yan et al. / Carbohydrate Research 328 (2000) 3–16
5.10 (t, 1 H, J 7.5 Hz, H-2II), 4.96 (t, 1 H, J
7.5 Hz, H-2I), 4.95 (dd, 1 H, J 10.0, 4.0 Hz,
H-3II), 4.85 (d, 1 H, J 12.5 Hz, CH2Ph), 4.59
(d, 1 H, J 12.5 Hz, CH2Ph), 4.52 (d, 1 H, J 8.0
Hz, H-6I), 4.50 (d, 1 H, J 7.5 Hz, H-1I), 4.48
(d, 1 H, J 8.0 Hz, H-1II), 4.16–4.04 (m, 3 H,
H-6I and 2×H-6II), 3.86 (t, 1 H, J 7.0 Hz,
H-5II), 3.82 (t, 1 H, J 10.0 Hz, H-4I), 3.60–
3.55 (m, 1 H, H-5I), 2.14 (s, 3 H, CH3), 2.13 (s,
3 H, CH3), 2.10 (s, 3 H, CH3), 2.05 (s, 3 H,
CH3), 2.04 (s, 3 H, CH3), 2.03 (s, 3 H, CH3),
2.01 (s, 3 H, CH3), 1.96 (s, 3 H, CH3); 13C
NMR (CDCl3): l 170.84 (Ac), 170.35 (2×
Ac), 170.14 (Ac), 170.06 (Ac), 169.78 (Ac),
169.59 (Ac), 169.05 (Ac), 136.80 (Ph), 135.82
(Ph), 128.44 (Ph), 127.91 (Ph), 101.08 (C-1II),
99.09 (C-1I), 76.25 (C-4I), 72.82 (C-5I), 72.74
(C-3I), 71.69 (C-2I), 71.07 (C-3II), 70.73 (C-5II),
70.33 (CꢀCH2Ph), 69.16 (C-CH2OAc), 66.64
(C-4II), 65.94 (C-2II), 61.98 (C-6I), 60.83 (C-
6II), 21.01 (Ac), 20.88 (Ac), 20.81 (Ac), 20.70
(Ac), 20.63 (Ac), 20.51 (Ac); MALDIMS:
Anal. Calcd for C36H46O20Na: 821.25. Found:
m/z 821.43 [M+Na].
2×CH3), 2.09 (s, 3 H, CH3), 2.06 (s, 6 H,
2×CH3), 2.05 (s, 3 H, CH3), 2.04 (s, 3 H,
CH3), 2.01 (s, 3 H, CH3), 2.00 (s, 3 H, CH3),
1.97 (s, 3 H, CH3), 1.90 (s, 3 H, CH3); 13C
NMR (CDCl3): l 170.82 (Ac), 170.58 (Ac),
170.49 (Ac), 170.44 (Ac), 170.38 (Ac), 170.33
(Ac), 170.32 (Ac), 170.09 (Ac), 170.04 (Ac),
169.80 (Ac), 169.78 (Ac), 169.55 (Ac), 169.08
(Ac), 168.85 (Ac), 136.75 (Ph), 135.73 (Ph),
128.38 (Ph), 127.83 (Ph), 101.05 (C-1IV),
100.64 (C-1II), 100.19 (C-1III), 99.05 (C-1I),
76.76 (C-3II), 75.67 (C-4III), 75.63 (C-4I), 72.74
(C-5I), 72.66 (C-5III), 72.51 (C-3I), 71.83 (C-
3III), 71.51 (C-2I), 71.05 (C-5II), 71.01 (C-3IV),
70.83 (C-2II), 70.67 (C-5IV), 70.27 (C-CH2Ph),
69.13 (C-2IV), 68.85 (C-4II), 66.59 (C-4IV),
65.89 (C-CH2OAc), 62.05 (C-6I), 61.50 (C-6II),
60.73 (C-6IV), 60.28 (C-6III), 55.02 (C-2III),
23.13 (Ac), 20.96 (Ac), 20.86 (Ac), 20.83 (Ac),
20.77 (Ac), 20.71 (Ac), 20.69 (Ac), 20.65 (Ac),
20.60 (Ac), 20.48 (Ac); MALDIMS: Anal.
Calcd for C60H79NO35Na: 1396.43. Found:
m/z 1396.32 [M+Na].
4-Hydroxymethylbenzyl i-
nosyl-(1“4)-2-acetamido-2-deoxy-i-
pyranosyl - (1“3) - i - - galactopyranosyl-
(1“4)-i- -glucopyranoside (20).—To a solu-
D
-galactopyra-
4-Acetoxymethylbenzyl 2,3,4,6-tetra-O-ace-
D-gluco-
tyl-i-
do-3,6-di-O-acetyl-2-deoxy-i-
syl-(1“3)-2,4,6-tri-O-acetyl-i-
pyranosyl-(1“4)-2,3,6-tri-O-acetyl-i-
D
-galactopyranosyl-(1“4)-2-acetami-
-glucopyrano-
-galacto-
-gluco-
D
D
D
D
tion of 19 (60 mg, 0.05 mmol) in dry MeOH
(5 mL) was added 1 M CH3ONa in MeOH (5
mL). The mixture was stirred at room temper-
ature for 5 h, then diluted with MeOH, the
mixture was neutralized with Rexyn 101 (H+)
resin and filtered. The combined filtrates were
concentrated and dissolved in water for freeze-
drying to give tetrasaccharide 20 (27 mg,
87%). [h]D +15.4° (c 0.3, H2O); Anal. Calcd
for C34H53NO22 (827.79): C, 49.33; H, 6.45; N,
1.69. Found: C, 49.33; H, 6.81; N, 1.87.
D
pyranoside (19).—tetrasaccharide 19 (65 mg,
53%) was obtained from 17 as described
1
above for 18. [h]D −4.8° (c 2.6 CHCl3); H
NMR (CDCl3): l 7.33 (d, 2 H, J 7.5 Hz, Ph),
7.26 (d, 2 H, J 8.0 Hz, Ph), 5.39 (d, 1 H, J 9.0
Hz, NH), 5.34 (d, 1 H, J 3.0 Hz, H-4IV), 5.29
(d, 1 H, J 3.0 Hz, H-4II), 5.19 (t, 1 H, J 9.5
Hz, H-3III), 5.13 (t, 1 H, J 9.0 Hz, H-3I), 5.11
(t, 1 H, J 9.5 Hz, H-2IV), 5.09 (s, 2 H,
CH2OAc), 5.00–4.93 (m, 3 H, H-2I, H-3IV,
and H-2II), 4.85 (d, 1 H, J 11.5 Hz, CH2Ph),
4.77 (d, 1 H, J 10.5 Hz, H-6III), 4.68 (d, 1 H,
J 8.0 Hz, H-1III), 4.59 (d, 1 H, J 12.0 Hz,
CH2Ph), 4.54 (d, 1 H, J 8.0 Hz, H-1IV), 4.50
(d, 1 H, J 8.0 Hz, H-1I), 4.48 (d, 1 H, J 10.5
Hz, H-6I), 4.34 (d, 1 H, J 8.0 Hz, H-1II),
4.16–4.00 (m, 5 H, H-6I, 2×H-6IV, and 2×
H-6II), 3.96 (dd, 1 H, J 12.05, 2.5 Hz, H-6III),
3.87 (t, 1 H, J 7.0 Hz, H-5IV), 3.82–3.70 (m, 4
H, H-4III, H-5II, H-4I, and H-3II), 3.59–3.53
(m, 1 H, H-5I), 3.53–3.48 (m, 2 H, H-2III and
H-5III), 2.14 (s, 9 H, 3×CH3), 2.10 (s, 6 H,
Enzymatic synthesis of phenyl 2-acetamido-
2 - deoxy - i -
galactopyranosyl - (1“4) - 1 - thio - i -
pyranoside (13).—b(1“3)N-Acetylglucos-
D
- glucopyranosyl - (1“3) - i -
D
-
D
- glucoz
aminyltransferase (LgtA, 0.5 units, 9.2 mL)
was added to a solution of 2 mM acceptor 5
(20 mg, 460 mL of 100 mM), 10 mM MnCl2
(2300 mL of 100 mM), and 4 mM UDP-Glc-
NAc donor (4600 mL of 20 mM) in 50 mM
HEPES buffer (1150 mL of 1 M, pH 7.4). The
reaction was performed at 37 °C for 2 days.
The product formation (Rf 0.55) was followed
by TLC on silica (9:9:2 MeOH–CHCl3–0.5%

F. Yan et al. / Carbohydrate Research 328 (2000) 3–16
15
C, 46.40; H, 5.89; N, 2.52. Found: C, 46.23;
H, 5.86; N, 2.70.
CaCl2). The total volume was adjusted to 23
mL with water and applied to a C18 reversed-
phase column, eluting with 17:3–4:1 water–
MeOH, which afforded pure 13 (28.2 mg,
96%).
Acknowledgements
Enzymatic synthesis of phenyl i-
pyranosyl - (1“4) - 2 - acetamido - 2 - deoxy - i-
-glucopyranosyl-(1“3)-i- -galactopyrano-
syl-(1“4)-1-thio-i- -glucopyranoside (14).—
D
-galacto-
The authors would like to thank Ms
Melissa J. Schur for preparation of enzymes.
The authors are also grateful to Drs J.R.
Brisson and Y. Wu for excellent NMR techni-
cal help, Mr D. Krajcarski for mass spectral
analyses, and Ms A. Webb for elemental
analyses. The authors also thank Ms Arlene
Chan, an NRC summer student, who pre-
pared some of the intermediates. This work is
part of the Scientific Technical Cooperation
Program between NRC (Canada) and CNRS
(France).
D
D
D
A fusion enzyme UDP-galactose 4-epimerase/
b(1“4)galactosyltransferase (GalE-LgtB,
1.2 units, 6803 mL) was added to a solution of
2 mM acceptor 13 (20 mg, 313 mL of 20 mM),
10 mM MnCl2 (1565 mL of 100 mM), and 1
mM UDP-Glc (940 mL of 20 mM) in 50 mM
HEPES buffer (782 mL of 1 M, pH 7.4). The
reaction was performed at 37 °C for a total of
2 h, while two additional portions of 1 mM
UDP-Glc (940 mL of 20 mM) were added in
30 min intervals. The formation of 14 (Rf
0.40) was monitored by TLC (9:9:2 MeOH–
CHCl3–0.5% CaCl2). Chromatography of the
crude reaction mixture on a C18 reversed-
phase column (9:1–17:3 water–MeOH) af-
forded pure 14 (24 mg, 96%).
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