Chemoenzymatic synthesis of L-galactosylated dimeric Sialyl Lewis X structures employing α-1,3-fucosyltransferase V

Article abstract of DOI:10.1016/S0968-0896(00)00187-5

L-Galactosylated dimeric sialyl Lewis X (SLe(x)) has been prepared employing a combination of chemical and enzymatic synthetic methods. GDP-L-galactose has been chemically synthesised. Enzymatic transfer of L-galactose onto the acceptor (Sia-α2,3-Gal-β1,4-GlcNAc-β1,3/6)₂-Man-α1-OMe was achieved using the human α-1,3-fucosyltransferase V. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00187-5

Bioorganic & Medicinal Chemistry 8 (2000) 2519±2525
Chemoenzymatic Synthesis of L-Galactosylated Dimeric Sialyl
Lewis X Structures Employing ꢀ-1,3-Fucosyltransferase V
Arno Du€els,a Luke G. Green,a Roman Lenz,a Steven V. Ley,a,*
Stephane P. Vincentb and Chi-Huey Wongb
aDepartment of Chemistry, University of Cambridge, Lens®eld Road, Cambridge CB2 1EW, UK
bThe Scripps Research Institute, Arnold and Mable Beckman Center, 10550 North Torrey Pines Road,
La Jolla, CA 92037, USA
Received 18 April 2000; accepted 3 July 2000
AbstractÐl-Galactosylated dimeric sialyl Lewis X (SLex) has been prepared employing a combination of chemical and enzymatic
synthetic methods. GDP-l-galactose has been chemically synthesised. Enzymatic transfer of l-galactose onto the acceptor (Sia-
a2,3-Gal-b1,4-GlcNAc-b1,3/6)2-Man-a1-OMe was achieved using the human a-1,3-fucosyltransferase V. # 2000 Elsevier Science
Ltd. All rights reserved.
Introduction
be very attractive for the synthesis of natural and
unnatural oligosaccharides.2 4 The most important
strategy for in vitro enzymatic oligosaccharide synthesis
is the use of the glycosyltransferases of the Leloir path-
way, which require sugar nucleotides as donors.5,6
White blood cells, or leukocytes, are important species
for the repair of tissue damage and defense against
microbial infection. Leukocytes are brought to the site
of injury by a complex series of steps, referred to as the
in¯ammatory cascade. The initial events in this cell±cell
recognition process, the tethering and rolling of leuko-
cytes, are mediated by selectins, a family of calcium
dependent carbohydrate binding cell adhesion mole-
cules.1 SLex, among others, has been recognised as a
small molecule ligand for the selectins.1 An attractive
strategy for treating in¯ammation-related diseases, such
as rheumatoid arthritis, is to inhibit the selectin±ligand
interaction that initiates these processes. Consequently,
attempts have been made to develop SLex inhibitors and
much recent e€ort has gone into preparing multimers of
SLex in hope of mimicking nature's use of polyvalency.1
The use of these `Leloir' glycosyltransferases in pre-
parative enzymatic glycosylation to date has mainly
focused on the transferases involved in the biosynthesis
of sialyl Lewis X (SLex), namely b-1,4-galactosyltrans-
ferase (b-1,4-GalT), a-2,3-sialyltransferase (a-2,3-SiaT)
and a-1,3-fucosyltransferase (a-1,3-FucT).7 9 In addition
a-2,6-sialyltransferase (a-2,6-SiaT) has been frequently
used.10,11
In order to extend the synthetic applicability of the
limited number of glycosyltransferases available, inten-
sive studies towards the substrate speci®city of these
enzymes have been carried out. A number of unnatural
acceptor and donor substrates have been employed,
disproving the original `one enzyme±one linkage' con-
ception and broadening the scope of glycosyltransfera-
ses in synthetic chemistry. In this context derivatives of
GDP-l-fucose have been chemically synthesised and
examined as donor substrates for various fucosyl-
transferases.12
Since Whitesides' pioneering work 20 years ago, chemo-
enzymatic methods in carbohydrate chemistry have
undergone extensive development and have proven to
Abbreviations: ATP, adenosine 50-triphosphate; BSA, bovine serum
albumin; CMP-NeuAc, cytidine 50-monophospho-N-acetylneuraminic
acid; GDP, guanidine 50-diphosphate; HEPES, N-2-hydroxyethyl-
piperazine-N0-2-ethane sulfonic acid; MES, 2-(N-morpholine)ethane
sulfonic acid; MK, myokinase; NMK, nucleoside monophosphate
kinase; PEP, phosphoenol pyruvate; PK, pyruvate kinase; PPase,
inorganic pyrophosphorylase.
In earlier work concerning the N-glycans from the Gly-
codelins, human glycoproteins with immunosuppressive
and contraceptive activity,13,14 we reported the chemo-
enzymatic synthesis of monomeric SLex structures
*Corresponding author. Tel.: +44-1-223-336-398; fax: +44-1-223-
336-442; e-mail: svl1000@cam.ac.uk
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00187-5

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A. Du€els et al. / Bioorg. Med. Chem. 8 (2000) 2519±2525
È
containing the l-fucose and the l-galactose motif.15
Additionally the preparation of dimeric SLex was pre-
sented.15 Encouraged by these results, we attempted the
synthesis of the dimeric SLex undecasaccharide 1 (Fig. 1)
using a-1,3-FucT V to achieve enzymatic bis-l-galacto-
sylation of a suitable precursor.
to keep the product in solution. Subsequent debenzoy-
lation was achieved by re¯uxing a solution of 4 in
methanol and cyclohexylamine (1:1) giving access to the
desired b-l-fucopyranosyl dicyclohexylammonium phos-
phate 5 in crystalline form. The best results for sub-
sequent coupling of 5 with GDP-morpholidate are
usually obtained if the reaction is carried out in pyri-
dine.18,19 In order to make the b-l-galactopyranosyl
phosphate soluble in pyridine, the counterion has to be
changed from cyclohexylammonium to triethylammo-
nium. The ®nal coupling reaction was carried out under
strictly anhydrous conditions using 1H-tetrazole as a
catalyst.16 GDP-l-galactose 6 was isolated in 74% yield
after puri®cation by size exclusion chromatography.
Results and Discussion
Synthesis of GDP-L-galactose
The synthesis of GDP-l-galactose was achieved in six
steps and 57% overall yield starting from l-galactose
employing an improved procedure for the coupling of
sugar diphosphate nucleosides (Scheme 1).16 Glycosyla-
tion of the galactosyl bromide 217 with dibenzyl phos-
phate in the presence of silver carbonate gave the
protected b-l-galactopyranosyl phosphate 3 as the sole
product. The phosphate group was deprotected by
hydrogenation in a mixture of toluene, pyridine and
triethylamine using Pd/C as a catalyst to give galacto-
pyranosyl phosphate 4. The solvent system was chosen
Synthesis of the undecasaccharide 1
Our synthesis of undecasaccharide 1 is based on the
enzymatic decoration of pentasaccharide precursor 7
(Fig. 2). A similar strategy has been employed by
Unverzagt in their synthesis of complex N-glycans20 and
presented an alternative to our route in the synthesis
of complex N-glycans from the human glycoproteins
Glycodelin A and Glycodelin S reported earlier.15
Double glycosylation of the readily available diol
acceptor 915 with three equivalents of the b-thioglyco-
side 821 in the presence of silver(I) tri¯ate/NIS as acti-
vator system gave the symmetrical pentasaccharide 10
in 85% yield (Scheme 2). A four step deprotection
sequence ®nally gave pentasaccharide 7 as a colourless
amorphous powder after size exclusion chromatography.
1H NMR analysis revealed that 7 existed as a 40:1 b:a
mixture of anomers. This result re¯ects a slight
improvement over a 30:1 b:a ratio for the double glyco-
sylation of the diol acceptor 9 with a fully acetylated
lactosamine bromide donor reported earlier.15
With pentasaccharide 7 in hand, selective galactosyla-
tion was achieved in a single step using b-1,4-GalT and
the donor substrate UDP-d-galactose in the presence of
alkaline phosphatase.10 Heptasaccharide 11 was isolated
in 69% yield. In our hands the puri®cation of 11 by size
exclusion chromatography proved dicult, restricting
this synthesis to a small preparative scale (10 mg).
Incubation of 11 with a-2,3-sialyltransferase and CMP-
NeuAc gave the bis-sialylated nonasaccharide 12 in
nearly quantitative yield (Scheme 3). When nonasac-
charide 12 was incubated with GDP-l-galactose and
a-1,3-FucT V the bivalent SLex analogue 1 was isolated
Figure 1. l-Galactosylated SLex undecasaccharide.
Scheme 1. (i) Ag2CO3, (Bn)2P(O)OH, CH2Cl2, Et2O, CH3CN, 93%
over two steps; (ii) Pd/C, H2, toluene, pyridine, NEt3, 97%; (iii) MeOH,
cyclohexylamine, 90%; (iv) BioRad AG 50W-X2 (NEt3-form), quant,
then guanosinemorpholidate, tetrazole, pyridine, 3 days, 74%.
Figure 2. Pentasaccharide precursor for enzymatic decoration.

A. Du€els et al. / Bioorg. Med. Chem. 8 (2000) 2519±2525
È
2521
Scheme 2. (i) NIS, AgOTf, 4 A ms, CH2Cl2, toluene, 50 ꢀC to rt,
85%; (ii) (a) NH2NH2, EtOH; (b) Ac2O, pyridine; (c) K2CO3, MeOH;
(d) Pd/C, CH2Cl2:EtOH:H2O 3:3:1, 79% over four steps.
Figure 3. Monomeric and dimeric SLex analogues undergoing inhibi-
tion assays.
galactose as an unnatural substrate for a-1,3-fucosyl-
transferase III have been reported by Ohrlein et al.23
Conclusion
The high yielding synthesis of the l-galactosylated
undecasaccharide 1 gives testimony to the broad
applicability of glycosyltransferases and the power of
the chemoenzymatic approach chosen.
On a small preparative scale the chemoenzymatic syn-
thesis of heptasaccharide 11 presents a viable alternative
to the purely chemical synthesis reported earlier.15
Also noteworthy is the high yield observed in the enzy-
matic sialylation. These results are of particular interest
as the chemical glycosylation of N-acetylneuraminic
acid su€ers from complex problems such as the stereo-
chemical outcome of the reaction and the formation of
by-products by elimination.24,25
Although in the ®nal step GDP-l-galactose showed a
decrease in donor eciency when compared with the
natural substrate GDP-l-fucose, l-galactosylation of
the acceptor saccharide 12 was achieved in good yield.
Scheme 3. (i) UDP-galactose, b-1,4-galactosyltransferase, alkaline
phosphatase, BSA, HEPES (50 mM, pH 7.2), 69%; (ii) CMP-NeuAc,
a-2,3-sialyltransferase, MnCl2, MgCl2, alkaline phosphatase, BSA,
HEPES (200 mM, pH 7.5), 99%; (iii) GDP-l-galactose, a-1,3-fuco-
syltransferase V, MnCl2, alkaline phosphatase, BSA, MES (50 mM,
pH 6.0), 56%.
The dimeric SLex analogue 1 and the related structures
13±1515 (Fig. 3) are currently undergoing tests concern-
ing their binding anity towards E-selectin.
as a colourless solid after size exclusion chromato-
graphy in 56% yield.
Experimental
A prolonged reaction time in comparison with the
synthesis of the l-fucosylated analogue of 115 indicated
the decreased donor eciency of GDP-l-galactose
compared to GDP-l-fucose. This observation is con-
sistent with kinetic data reported by Hindsgaul et al. for
the enzymatic transfer of l-galactose using an a-1,3/
4 FucT.22 Similar experiments employing GDP-l-
1H NMR spectra were recorded in CDCl3 or D2O on
Bruker DRX-600, DRX-500 and DPX-200 spectrom-
eters at 300 K. Residual protic solvent CHCl3 (dH=
7.26 ppm) was used as the external reference. 13C NMR
spectra were recorded in CDCl3 or D2O at 150 or 100
MHz on Bruker DRX-600 and AC400 spectrometers,
respectively, using the central resonance of CDCl3

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A. Du€els et al. / Bioorg. Med. Chem. 8 (2000) 2519±2525
È
(dC=77.0 ppm) as the external reference. DQF-COSY,
HMQC, decoupled-HMQC, HMBC, TOCSY and 1-D
TOCSY experiments were used to assist assignment of
the products. NMR assignments are as indicated in
Figure 3. Infra-red spectra were recorded as thin ®lms
between sodium chloride plates, deposited from chloro-
form solution on an FTIR 1620 spectrometer. Mass
spectra were obtained on Micromass Platform LC±MS
and Q-Tof, Kratos MS890MS and Kompact 4, Brucker
Daltonics Bio-Apex II (FTICR) spectrometers at the
Department of Chemistry, University of Cambridge and
on a Voyager STR spectrometer at M-Scan, Silwood
Park, Ascot.
(1.80 mL) and pyridine (2.5 mL). The mixture was stir-
red under an H2 atmosphere with 10% activated Pd/C
(90 mg) as catalyst. After 36 h the mixture was ®ltered and
the ®ltrate was concentrated to give 4 as a colourless foam
i
(1.40 g, 97%). Rf=0.52 (silica gel, PrOH:NH3: dH (400
MHz, CDCl3) 1.23 (3H, t, J=7.3 Hz, N(CH2CH3)3), 3.03
(2H, q, J=7.3 Hz, N(CH2CH3)3), 4.45 (1H, dd, J=7.3
and 10.8 Hz, Ha-6), 4.57±4.63 (1H, m, H-1), 4.69 (1H,
dd, J=5.4 and 10.8 Hz, Hb-6), 5.63±5.68 (1H, m, H-3),
5.75±5.84 (2H, m, H-2, H-5), 6.04 (1H, dd, J=0.8 and
3.0 Hz, H-4), 7.20±8.06 (20H, m, Harom); dC (100 MHz,
CDCl3) 20.8, 42.8, 43.4, 60.5, 67.5, 69.6 (d, J=8.7 Hz),
70.1, 94.8 (d, J=4.4 Hz), 126.8, 126.9, 127.0, 127.2, 127.3,
127.9, 128.8, 128.9, 131.7, 131.8, 131.9, 132.2, 164.0,
164.6, 164.7, 164.8; dP (162 MHz, CDCl3) 2.06; m/z
(FAB) found: [M + Cs]+, 809.0432. C34H29O13P requires
M+Cs, 809.0400.
Flash column chromatography was carried out using
Merck Kieselgel (230±400 mesh). Analytical thin layer
chromatography (tlc) and preparative tlc were performed
using precoated glass-backed plates (Merck Kieselgel 60
F254) and visualised by uv and acidic ammonium
molybdate (IV). Petrol refers to petroleum ether bp 40±
60 ꢀC, which was distilled prior to use.
Di(cyclohexylammonium)phosphoryl-ꢁ-L-galactopyrano-
side (5). Cyclohexylamine (5 mL) was added to a solu-
tion of compound 4 (1.30 g, 1.48 mmol) in dry MeOH
(10 mL). The mixture was heated at re¯ux for 4 h after
which the mixture was concentrated, partitioned
between water and CHCl3, and the aqueous phase was
washed three times with CHCl3 and then concentrated
to dryness by coevaporating with MeOH. The product
was dissolved in hot EtOH and triturated with acetone.
Filtration gave a colourless solid (610 mg, 90%). Rf=0.14
All reactions were carried out under an argon atmos-
phere in oven-dried glassware unless otherwise stated.
Diethyl ether was distilled from sodium benzophenone
ketyl, dichloromethane and toluene from calcium
hydride. Other reagents and solvents were puri®ed using
standard procedures. Aqueous solutions are saturated
unless otherwise speci®ed.
i
(silica gel, PrOH:NH3:H2O, 6:3:1); dH (400 MHz, D2O)
1.06±1.16 (2H, m, C6H11NH2), 1.20±1.34 (8H, m,
C6H11NH2), 1.55±1.62 (2H, m, C6H11NH2), 1.69±1.77
(4H, m, C6H11NH2), 1.90±1.96 (4H, m, C6H11NH2), 3.03±
3.12 (2H, m, C6H11NH2), 3.48 (1H, dd, J=7.6 and 9.7
Hz), 3.62 (1H, dd, J=3.5 and 10.3 Hz), 3.65±3.68 (2H, m),
3.75 (1H, dd, J=9.4 and 12.4 Hz), 3.83 (1H, d, J=3.5
Hz), 4.77 (1H, t, J=7.6 Hz, H-1); dC (100 MHz, D2O)
26.2, 26.7, 32.8, 52.7, 63.9, 71.3, 74.5 (d, J=5.8 Hz), 75.0,
78.0, 100.0 (d, J=4.4 Hz); m/z (FAB) found: [M + Na]+,
283.0187. C6H13O9P requires M + Na, 283.0195.
a-1,3-Fucosyltransferase V and a-2,3-sialyltransferase
were purchased from Calbiochem1. CMP-Sialic acid
and GDP-galactose were purchased from Calbiochem1
or synthesised using published protocols.16
Dibenzylphosphoryl 2,3,4,6-tetra-O-benzoyl-ꢁ-L-galacto-
pyranoside (3). Ag2CO3 (1.08 g 3.94 mmol) was added
in one portion to a cooled mixture of 217 (1.30 g, 1.97
mmol), dibenzylphosphate (1.65 g, 5.91 mmol) and 3 A
ms (3.50 g) in CH2Cl2, Et2O, CH3CN (11 mL each). The
mixture was stirred for 24 h at room temperature with
exclusion of light, ®ltered through Celite1 and the ®l-
trate was concentrated. The residue was puri®ed by
column chromatography (toluene:EtOAc, 2:1) to give
3 as a colourless foam (1.59 g, 95%). Rf=0.48 (silica gel,
1:2 EtOAc:hexane); dH (400 MHz, CDCl3) 4.41±4.52 (3H,
m, CH2Ph), 4.62 (1H, dd, J=6.2 and 10.8 Hz, CH2Ph),
4.76 (1H, dd, J=7.0 and 11.6 Hz, Ha-6), 4.86 (1H, dd,
J=6.8 and 11.6 Hz, Hb-6), 5.07 (1H, d, J=7.9 Hz, H-1),
5.65 (1H, dd, J=3.5 and 10.5 Hz, H-3), 5.76 (1H, m, H-5),
5.95 (1H, dd, J=7.9 and 10.5 Hz, H-2), 6.05 (1H, d,
J=3.5 Hz, H-4), 6.98±8.12 (30H, m, Harom); dC (100
MHz, CDCl3) 62.0, 67.8, 69.4 (d, J=5.8 Hz), 69.5 (d,
J=5.8 Hz), 69.6 (d, J=7.2 Hz), 71.4, 72.4, 97.0 (d, J=4.4
Hz), 127.4, 127.9, 128.3, 128.4, 128.5, 128.7, 129.7, 129.8,
129.9, 130.0, 133.3, 133.4, 133.5, 133.7, 165.2, 165.4, 165.9;
dP (162 MHz, CDCl3) 2.22; m/z (FAB) found: [M +
Cs]+, 989.1306. C48H41O13P requires M + Cs, 989.1339.
Guanosine 50-diphospho-ꢁ-L-galactopyranoside (6). Com-
pound 15 (184 mg, 401 mmol) was dissolved in water (3
mL), applied to a Bio-Rad AG 50 W-X2 cation-
exchange column (Et3N+-form, 2.5Â6 cm) and eluted
with water (150 mL). The eluent was evaporated, co-
evaporated with MeOH (2Â5 mL) and dried for 3 days
under vacuum to give triethylammonium b-l-galactopyr-
anosyl phosphate. After addition of 4-morpholino-N,N0-
dicyclohexylcarboxamidinium guanosine 50-monophos-
phomorpholidate (319 mg, 440 mmol) the mixture was
coevaporated with dry pyridine (3Â5 mL). 1H-Tetrazole
(56.0 mg, 800 mmol) and dry pyridine (3 mL) were
added and the solution was stirred at room temperature.
After 2 days, the mixture was diluted with water (3 mL)
to become a clear solution, and it was extracted with
CH2Cl2 (3Â5 mL). The aqueous phase was concentrated
and applied to a Bio-Gel1 P-2 column (2.5Â70 cm, eluent:
50 mM NH4HCO3) to give the desired product as a col-
ourless solid after lyophilization (180 mg, 74%). Rf=0.26
(silica gel, 2:1 iPrOH:1 M NH4OAc); dH (400 MHz, D2O)
1.18 (1H, d, J=6.7 Hz, H-600), 3.56±3.82 (5H, m), 3.88
(1H, d, J=3.2 Hz), 4.18±4.22 (1H, m), 4.31±4.34 (1H, m),
4.50 (1H, t, J=4.2 Hz), 4.71 (1H, t, J=5.4 Hz, H-20), 4.93
Di(triethylammonium)phosphoryl 2,3,4,6-tetra-O-benzoyl-
ꢁ-L-galactopyranoside (4). Compound 3 (1.40 g, 1.64
mmol) was dissolved in toluene (12 mL), triethylamine

A. Du€els et al. / Bioorg. Med. Chem. 8 (2000) 2519±2525
È
2523
(1H, t, J=7.6 Hz, H-100), 5.87 (1H, d, J=5.7 Hz, H-10), 8.05
(1H, s, H-8); dC (100 MHz, D2O) 63.6, 67.7 (d, J=5.8 Hz),
71.1, 72.8, 73.7 (d, J=7.3 Hz), 74.7, 76.2, 78.3, 86.1 (d,
J=8.7 Hz), 89.3, 100.9 (d, J=5.8 Hz), 118.5, 139.9, 154.1,
156.3, 161.2; dP (162 MHz, D2O) 12.1 (d, J=18.3), 10.4
(d, J=18.3 Hz); m/z (ESI) found: [M H] , 604.
C16H25N5O16P2 requires M H, 604.
protected pentasaccharide 10 was suspended in EtOH
.
(10 mL) and NH2NH2 H2O (1 mL) was added. The
reaction mixture was heated under re¯ux for 18 h. The
solvent was removed in vacuo and the residue co-
evaporated with toluene (3Â5 mL). Ac2O (2 mL) and
pyridine (4 mL) were added and the solution was stirred
for 18 h. The reaction mixture was concentrated and co-
evaporated with toluene to remove traces of pyridine and
the residue was puri®ed by size exclusion chromatography
on Sephadex1 LH-20 (DCM/MeOH 1:1).
Methyl 3-O-benzyl-4,6-O-benzylidene-2-deoxy-2-phthali-
mido-ꢁ-D-glucopyranosyl-(1!2)-3,4,6-tri-O-benzyl-ꢀ-D-
mannopyranosyl-(1!3)-[3-O-benzyl-4,6-O-benzylidene-
2-deoxy-2-phthalimido-ꢁ-D-glucopyranosyl-(1!2)-3,4,6-
tri-O-benzyl-ꢀ-D-mannopyranosyl-(1!6)]-2,4-di-O-ben-
zyl-ꢀ-D-mannopyranoside (10). A mixture of glycosyl
donor 8 (51.3 mg, 96.6 mmol) and glycosyl acceptor 9
(40.0 mg, 32.2 mmol) was dried by azeotropic distillation
with toluene and left under vacuum for 18 h. After
addition of NIS (29.0 mg, 129 mmol) and 4 A molecular
sieves (400 mg) the mixture was suspended in CH2Cl2 (1
mL) and cooled to 50 ꢀC. AgOTf (8.3 mg, 32.2 mmol)
in toluene (0.4 mL) was added over 20 min and the
reaction mixture was warmed slowly to 30 ꢀC. After
12 h at 20 ꢀC it was diluted with ether, ®ltered through
Celite1, washed with Na2S2O3, concentrated and pur-
i®ed by size exclusion chromatography on Sephadex1
LH-20 (CH2Cl2:MeOH 1:1). Pentasaccharide 10 was
obtained in 85% yield (60.1 mg, 27.4 mmol). Rf=0.33
(silica gel, PE:Et2O, 1:2); dH (600 MHz, CDCl3) 2.74±
2.78 (1H, m, H-5c), 2.80 (1H, dd, J=7.4 and 10.7 Hz,
The resulting oil was dissolved in CH2Cl2 (1 mL) and
MeOH (2 mL) and a catalytic amount of K2CO3 was
added. The mixture was stirred overnight and Amberlite
IR-120 was added to neutralise the reaction mixture.
After ®ltration and concentration the resulting material
was dissolved in CH2Cl2 (6 mL), MeOH (6 mL) and
H2O (2 mL), and Pd(OH)2/C was added. The reaction
mixture was stirred under an atmosphere of H2 for 24 h
and ®ltered through Celite1. Size exclusion chromatog-
raphy on Sephadex1 G-15 gave pentasaccharide 7 in
79% (19.5 mg, 21.0 mmol) over four steps. dH (600 MHz,
D2O) 1.98 (3H, s, NHC(O)CH3), 1.99 (3H, s,
NHC(O)CH3), 3.35 (3H, s, OCH3), 3.36±3.41 (4H, m, H-
0
4c, H-5c, H-4c , H-5c ), 3.43±3.51 (4H, m, H-4b, H-3c, H-
0
0
4b , H-3c ), 3.55±3.60 (3H, m, Ha-6b, H-5b , Ha-6b ), 3.61±
0
0
0
0
3.66 (4H, m, Ha-6a, H-5b, H-2c, H-2c ), 3.67±3.74 (3H, m,
0
H-5a, Ha-6c, Ha-6c ), 3.77±3.87 (8H, m, H-3a, H-4a, H-3b,
0
0
0
Hb-6b, Hb-6c, H-3b , Hb-6b , Hb-6c ), 3.98 (1H, dd, J=4.0
and 11.4 Hz, Hb-6a), 4.02 (1H, dd, J=1.7 and 3.1 Hz, H-
0
Ha-6b), 2.99 (1H, dd, J=6.3 and 11.0 Hz, Ha-6b ), 3.21
0
(3H, s, OCH3), 3.27±3.32 (2H, m, Ha-6a, Hb-6b ), 3.39
(1H, t, J=9.7 Hz, H-4b), 3.43±3.47 (2H, m, Hb-6b, H-
0
2a), 4.06 (1H, dd, J=1.6 and 3.1 Hz, H-2b ), 4.11 (1H, dd,
0
J=1.6 and 3.2, H-2b), 4.48 (1H, d, J=8.4 Hz, H-1c ),
0
5b ), 3.48±3.53 (2H, m, H-5a, H-4b ), 3.55±3.60 (1H, m,
0
H-5c ), 3.63±3.67 (3H, m, H-2a, H-4a, Hb-6a), 3.69±3.73
(3H, m, H-5b, H-4c, Ha-6c), 3.75 (2H, s, H-2b), 3.76±3.82
0
4.50 (1H, d, J=8.4 Hz, H-1c), 4.67 (1H, s, H-1a), 4.85
0
(1H, s, H-1b ), 5.05 (1H, s, H-1b); dC (125 MHz, D2O)
0
22.3 (NHC(O)CH3), 54.9 (OCH3), 55.3 (C-2c ), 55.4 (C-
0
(3H, m, H-3a, H-3b, H-3b ), 3.84±3.89 (3H, m, H-4c , Ha-
0
0
2c), 60.6 (C-6c, C-6c ), 61.6 (C-6b, C-6b ), 65.4 (C-6a),
0
0
6c , CH2Ph), 3.92±3.97 (2H, m, CH2Ph), 4.01 (1H, d, J=
11.8, CH2Ph), 4.05 (1H, dd, J=4.9 and 10.1 Hz, Hb-6c),
0
65.6 (C-4a), (67.2, 67.3 (C-4b, C-4b )), (69.4, 69.5, 69.6
0 0
(C-2a, C-3b, C-3b )), 69.9 (C-4c, C-4c ), 71.0 (C-5a), 71.9
0
4.14 (1H, s, H-2b ), 4.22±4.29 (2H, m, H-2c, H-3c), 4.31±
0 0
(C-5b ), (73.3, 73.4 (C-3c, C-3c )), 73.5 (C-5b), (75.8, 75.9
0
4.34 (1H, m, Hb-6c ), 4.37±4.52 (11H, m, H-2c , H-3c ,
0
0
0 0
(C-5c, C-5c )), 76.4, (C-2b ), 76.6 (C-2b), 78.6 (C-3a), 96.8
0
CH2Ph), 4.53±4.55 (2H, m, H-1b , CH2Ph), 4.59 (1H, s,
0
0
(C-1b ), 99.5 (C-1b), 99.6 (C-1c ), 99.7 (C-1c), 101.4 (C-
1a); m/z (FAB) found: [M
Na]+, 947.3319.
C35H60N2O26 requires M + Na, 947.3326.
H-1a), 4.65 (1H, d, J=11.6 Hz, CH2Ph), 4.71 (1H, s, H-1b),
4.75 (1H, d, J=12.3, CH2Ph), 4.79 (1H, d, J=12.3 Hz,
CH2Ph), 4.83±4.86 (2H, m, CH2Ph), 4.91 (1H, d, J=8.1
+
0
Hz, H-1c), 5.28 (1H, s, H-1c ), 5.53 (1H, s, CHPh), 5.62
(1H, s, CHPh), 6.84±7.71 (68H, m, Harom); dC (125 MHz,
0
CDCl3) 54.7 (OCH3), 55.5 (C-2c), 55.6 (C-2c ), 65.6 (C-5c),
Methyl ꢁ-D-galactopyranosyl-(1!4)-ꢁ-D-2-acetamido-2-
deoxy-glucopyranosyl-(1!2)-ꢀ-D-mannopyranosyl-(1!3)-
[ꢁ-D-galactopyranosyl-(1!4)]-ꢁ-D-2-acetamido-2-deoxy-
glucopyranosyl-(1!2)-ꢀ-D-mannopyranosyl-(1!6)]-ꢀ-D-
mannopyranoside (11). b-1,4-Galactosyltransferase (300
mU, 438 mL) was added to a solution of pentasacchar-
ide 7 (5.0 mg, 5.4 mmol) and UDP-d-galactose (8.2 mg,
13.5 mmol) in HEPES bu€er (0.5 mL, 50 mM, pH 7.2)
containing 1 mg BSA and 7 U alkaline phosphatase. The
mixture was shaken at 37 ꢀC for 5 days. The reaction
mixture was centrifuged, the supernatant was con-
centrated and puri®ed on Sephadex1 G-15 (100 mM
NH4HCO3) to give 11 as a white solid after lyophiliza-
tion (4.6 mg, 5.0 mmol, 69%). dH (600 MHz, D2O) 1.98
(3H, s, NHC(O)CH3), 1.99 (6H, s, NHC(O)CH3), 3.34
(3H, s OCH3), 3.42±3.87 (33H, m), 3.90 (2H, d, J=11.7
0
66.1 (C-6a), 66.2 (C-5c ), 68.5 (C-6c), 68.6 (C-6c ), 69.8 (C-
0
0
6b ), 70.5 (C-6b), 70.8 (C-5a), (70.9, 71.6, 72.2, 72.5, 72.9,
0
73.8, 74.0, 74.7, 75.1 (CH2Ph)), 71.7 (C-5b ), 72.6 (C-5b),
0
73.7 (C-2b, C-2b ), 73.9 (C-4a), 74.1 (C-3c, C-3c ), 74.6 (C-
0
0
0
4b, C-4b ), 77.6 (C-3b ), 77.7 (C-2a), 78.1 (C-3b), 80.2 (C-
0
3a), 82.8 (C-4c), 82.9 (C-4c ), 96.8 (C-1c), 97.4 (C-1b ), 97.6
0
0
(C-1c ), 98.3 (C-1a), 99.2 (C-1b), 101.3 (CHPh), (123.1,
126.1, 127.3, 127.4, 127.6, 128.0, 128.1, 128.2, 128.3, 129.0,
131.8, 133.5, 133.6, 137.4, 137.5, 138.0, 138.1, 138.2, 138.5,
138.6, 138.8 (Carom)); m/z (ESI) found: [M + Na]+,
2199.8504. C131H128N2O28 requires M + Na, 2199.8546.
Methyl 2-acetamido-2-deoxy-ꢁ-D-glucopyranosyl-(1!2)-
ꢀ-D-mannopyranosyl-(1!3)-[2-acetamido-2-deoxy-ꢁ-D-
glucopyranosyl-(1!2)-3,4,6-ꢀ-D-mannopyranosyl-(1!6)]-
ꢀ-D-mannopyranoside (7). 58 mg (26.6 mmol) of fully
0
Hz, Hb-6c, Hb-6c ), 3.98 (1H, d, J=11.1 Hz, Hb-6a), 4.02
0
(1H, s, H-2a), 4.06 (1H, s, H-2b ), 4.11 (1H, s, H-2b),
0
4.40 (2H, d, J=7.8 Hz, H-1d, 1d ), 4.51 (1H, d, J=7.5 Hz,

2524
A. Du€els et al. / Bioorg. Med. Chem. 8 (2000) 2519±2525
È
0
H-1c), 4.53 (1H, d, J=7.7 Hz, H-1c ), 4.66 (s, 1H, H-1a),
The reaction mixture was centrifuged, the supernatant
was concentrated and puri®ed with Sephadex1 G-15
(150 mM NH4HCO3) to give 1 as a colourless solid after
lyophilization in 56% yield (3.3 mg, 1.5 mmol). dH (600
MHz, D2O) 1.70 (2H, t, J 12.2 Hz, Hax-3e, Hax-3e ), 1.92
(3H, s, HNC(O)CH3), 1.94±1.97 (9H, m, HNC(O)CH3),
0
4.86 (1H, s, H-1b ) 5.04 (1H, s, H-1b); dC (125 MHz, D2O,
selected data) 22.3 (NHC(O)CH3), 54.9 (OCH3), 60.0 (C-
0
6c, 6c ), 65.4 (C-6a), 69.5 (C-2a), 76.3 (C-2b ), 76.5 (C-2b),
0
0
96.8 (C-1b ), 99.4 (C-1b, C-1c ), 99.5 (C-1c), 101.0 (C-1a),
0
0
+
0
102.9 (C-1d, 1d ); m/z (ESI) found: [M + Na] ,
1271.4355. C47H80 N2O26 requires M + Na, 1271.4388.
0
2.64 (2H, dd, J=4.5 and 12.2 Hz, Heq-3e, Heq-3e ), 3.32
(3H, s, OCH3), 3.38±3.96 (60H, m), 3.98±4.01 (3H, m,
0
H-2a, H-3d, H-3d ), 4.04 (1H, s, H-2b ), 4.08 (1H, m, H-
0
2b), 4.34±4.38 (1H, m, H-1d/d ), 4.40±4.44 (1H, m, H-1d/
0
Methyl ꢀ-D-5-acetamido-3,5-dideoxy-D-glycero-ꢀ-D-ga-
lacto-2-nonulo-pyranuronic acid-(2!3)-ꢁ-D-galactopyra-
nosyl-(1!4)-ꢁ-D-2-acetamido-2-deoxy-glucopyranosyl-
(1!2)-ꢀ-D-mannopyranosyl-(1!3)-[ꢀ-D-5-acetamido-3,5-
dideoxy-D-glycero-ꢀ-D-galacto-2-nonulo-pyranuronic acid-
(2!3)-ꢁ-D-galactopyranosyl-(1!4)-ꢁ-D-2-acetamido-2-
deoxy-glucopyranosyl-(1!2)-ꢀ-D-mannopyranosyl-(1!
6)] - ꢀ - D - mannopyranoside (12). a-2,3-Sialyltransferase
(60 mU, 60 mL) was added to a solution of the hepta-
saccharide 11 (10.0 mg, 8.0 mmol) and CMP-sialic acid
(10.0 mg, 16.0 mmol) in HEPES bu€er (2.0 mL, 100
mM, pH 7.5) containing 4 mg BSA, 14 U alkaline
0
0
d ), 4.47±4.52 (2H, m, H-1c, H-1c ), 4.64 (1H, s, H-1a),
0
4.82 (1H, s, H-1b ), 5.01 (1H, s, H-1b), 5.09±5.12 (2H, m,
H-1f, H-1f'); dC (125 MHz, D2O, selected data according
to HMQC) 22.0 (NHC(O)CH3), 22.5 (NHC(O)CH3),
0
39.7 (C-3e, C-3e ), 54.8 (OCH3), [69.4, 75.7, 75.8, 76.4
0
(C-2a, C-2b, C-2b , C-3d, C-3d )], 96.7 (C-1b ), 98.2 (C-1f,
0
0
0
0
C-1f ), 99.2 (C-1c, C-1c ), 99.4 (C-1b), 101.1 (C-1a), 102.0
0
(C-1d, C-1d ).
.
phosphatase, MnCl2 4H2O (10 mL of a 1 M solution)
Acknowledgements
and MgCl2 (40 mL of a 1 M solution). The mixture was
shaken at 37 ꢀC for 9 days (additions of identical
amounts of CMP-sialic acid, a-2,3-sialyltransferase and
alkaline phosphatase were repeated after 2, 4, and 7
days). The reaction mixture was centrifuged, the super-
natant was concentrated and puri®ed with Bio Gel1 P-4
(150 mM NH4HCO3) to give 12 as a colourless solid
after lyophilization (14.7 mg, 7.8 mmol, 98%). dH (600
This work was supported by the BP Endowment, the
Novartis Research Fellowship, the Zeneca Strategic
Research Fund (SVL), the Ernst Schering Research
Foundation (AD), the Swiss National Science Founda-
tion, the Novartis-Stiftung (RL), and BBSRC (for
NMR facilities, ROPA, LGG). We are especially grate-
ful to Dr. Andrew Reason (M-Scan, Silwood Park,
Ascot) for the mass measurements of the deprotected
oligosaccharides.
0
MHz, D2O) 1.73 (2H, t, J 12.2 Hz, Hax-3e, Hax-3e ), 1.96
(6H, s, HNC(O)CH3), 1.98 (3H, s, HNC(O)CH3), 1.99
(3H, s, HNC(O)CH3), 2.69 (2H, dd, J=4.4 and 12.2 Hz,
0
Heq-3e, Heq-3e ), 3.34 (3H, s, OCH3), 3.43±3.96 (50H,
m), 3.97±3.99 (1H, m, H-6a), 4.02±4.08 (4H, m, H-2a, H-
References and Notes
0
2b , H-3d, H-3d ), 4.11 (1H, s, H-2b), 4.46±4.49 (2H, m, H-
0
0
1d, H-1d ), 4.50 (1H, d, J=7.9 Hz, H-1c), 4.52 (1H, d,
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J=7.9 Hz, H-1c ), 4.67 (1H, s, H-1a), 4.86 (1H, s, H-1b ),
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0
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0
75.5, 75.6, 76.4 (C-2a, C-2b, C-3d, C-3d )), 76.6 (C-2b),
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96.7 (C-1b ), 99.3 (C-1b), 99.4 (C-1c ), 99.6 (C-1c), 101.0
0
0
0
(C-1a), 102.6 (C-1d/d ), 102.7 (C-1d/d ); m/z (ESI after
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Methyl ꢀ-L-galactopyranosyl-(1!3)-[ꢀ-D-5-acetamido-
3,5-dideoxy-D-glycero-ꢀ-D-galacto-2-nonulo-pyranuronic
acid-(2!3)-ꢁ-D-galactopyranosyl-(1!4)]-ꢁ-D-2-aceta-
mido-2-deoxy-glucopyranosyl-(1!2)-ꢀ-D-mannopyranosyl-
(1!3)-{ꢀ-L-galactopyranosyl-(1!3)-[ꢀ-D-5-acetamido-
3,5-dideoxy-D-glycero-ꢀ-D-galacto-2-nonulo-pyranuronic
acid-(2!3)-ꢁ-D-galactopyranosyl-(1!4)]-ꢁ-D-2-aceta-
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(5.0 mg, 2.7 mmol) and GDP-l-galactose (4.0 mg, 6.5
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