Synthesis of unnatural sugar nucleotides and their evaluation as donor substrates in glycosyltransferase-catalyzed reactions

Article abstract of DOI:10.1016/j.carres.2004.09.002

New unnatural sugar nucleotides, UDP-Fuc and CDP-Fuc were synthesized from fucose-β-1-phosphate and nucleotide monophosphates activated as morpholidates. Furthermore, a nucleotide analogue was prepared by phosphorylation of 1-(β-D-ribofuranosyl)cyanuric acid, itself obtained as a protected derivative by condensation of the persilylated derivative of cyanuric acid with 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose in 74% yield. This phosphate activated according to the same procedure was condensed with fucose-β-1-phosphate, affording a new sugar nucleotide conjugate (NDP-Fuc) which was evaluated together with UDP-Fuc, CDP-Fuc and ADP-Fuc, as fucose donors in α-(1→4/3)-fucosyltransferase (FucT-III) catalyzed reaction. Fucose transfer could be observed with each of the donors and kinetic parameters were determined using a fluorescent acceptor substrate. Efficiency of the four analogues towards FucT-III was in the following order: UDP-Fuc = ADP-Fuc > NDP-Fuc > CDP-Fuc. According to the same strategy ADP-GlcNAc was prepared from AMP-morpholidate and N-acetylglucosamine-α-1-phosphate; tested as a glucosaminyl donor towards Neisseria meningitidis N-acetylglucosaminyl transferase (LgtA), ADP-GlcNAc was recognized with 0.1% efficiency as compared with UDP-GlcNAc, the natural donor substrate.

Full text of DOI:10.1016/j.carres.2004.09.002

Carbohydrate
RESEARCH
Carbohydrate Research 339 (2004) 2641–2649
Synthesis of unnatural sugar nucleotides and their evaluation
as donor substrates in glycosyltransferase-catalyzed reactions
*
´
Amira Khaled, Tatiana Ivannikova and Claudine Auge
´ ´
Laboratoire de Chimie Organique Multifonctionnelle, UMR 8614, GDR 2590, Institut de Chimie Moleculaire et des Materiaux
´ ˆ
d’Orsay (ICMMO), Universite Paris-Sud, Bat 420, F-91405Orsay, France
Received 7 July 2004; accepted 1 September 2004
Available online 23 September 2004
Abstract—New unnatural sugar nucleotides, UDP-Fuc and CDP-Fuc were synthesized from fucose-b-1-phosphate and nucleotide
monophosphates activated as morpholidates. Furthermore, a nucleotide analogue was prepared by phosphorylation of 1-(b-D-
ribofuranosyl)cyanuric acid, itself obtained as a protected derivative by condensation of the persilylated derivative of cyanuric acid
with 1-O-acetyl-2,3,5-tri-O-benzoyl-b-D-ribofuranose in 74% yield. This phosphate activated according to the same procedure was
condensed with fucose-b-1-phosphate, affording a new sugar nucleotide conjugate (NDP-Fuc) which was evaluated together with
UDP-Fuc, CDP-Fuc and ADP-Fuc, as fucose donors in a-(1!4/3)-fucosyltransferase (FucT-III) catalyzed reaction. Fucose transfer
could be observed with each of the donors and kinetic parameters were determined using a fluorescent acceptor substrate. Efficiency
of the four analogues towards FucT-III was in the following order: UDP-Fuc = ADP-Fuc > NDP-Fuc > CDP-Fuc.
According to the same strategy ADP-GlcNAc was prepared from AMP-morpholidate and N-acetylglucosamine-a-1-phosphate;
tested as a glucosaminyl donor towards Neisseria meningitidis N-acetylglucosaminyl transferase (LgtA), ADP-GlcNAc was recog-
nized with 0.1% efficiency as compared with UDP-GlcNAc, the natural donor substrate.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Keywords: Sugar nucleotides; Donor substrate; Glycosyltransferases; Specificity
1. Introduction
requirement for stoichiometric amounts of sugar nucleo-
tides. In situ regeneration of the sugar nucleotides is
the most elegant way of using these cofactors, but it calls
for additional enzymes and complicates the process.4
For preparative purposes donor substrates with a sim-
pler structure than sugar nucleotides, prepared at low
cost, would be highly desirable. The pyrophosphate
bridge plays a key role in enzyme mechanism: as a leav-
ing group it interacts with Mn2+ when this cation is re-
quired for enzyme activity, which, according to the
limited number of known glycosyltransferase crystal
structures, is coordinated to two aspartate residues of
the enzyme.5 Mimics of the diphosphate bond that
might act as alternate donor substrates would be of
great interest, but searching for such analogues is some-
what hazardous. Therefore, we decided to first look at
the possible changes, that glycosyltransferases are able
to tolerate with respect to the nucleoside part of the
sugar nucleotide. Thus, we report here the synthesis of
Glycosyltransferases of Leloir-type that typically catal-
yze sugar unit transfer from a sugar nucleotide donor
to an unprotected saccharide acceptor with complete
regio- and stereoselectivity, have proved to be precious
tools in preparative oligosaccharide synthesis.1 Nowa-
days the number of glycosyltransferases cloned in
research laboratories is increasing very rapidly and
much improvement has been achieved in this area: these
enzymes, originally membrane-associated proteins are
now produced in soluble form, and mostly tagged with
a six histidine tail, which turned out to be of benefit
for synthetic purposes.2 Supported synthesis with glycos-
yltransferases is also expected to develop.3 However the
major drawback to the enzymatic approach remains the
*
Corresponding author. Tel.: +33 1 69 15 47 18; fax: +33 1 69 15 47
15; e-mail: clauauge@icmo.u-psud.fr
0008-6215/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2004.09.002

2642
A. Khaled et al. / Carbohydrate Research 339 (2004) 2641–2649
NH
N
2
O
HN
N
NH
NH
-
-
O
O- O-
O
O
P
O
P
N
O
OH
O
O
O
N
O
2
H C
O
P
P
O
H C
O
O
3
3
OH
OH
O
O
O
O
OH
HO
HO
HO OH
3
HO OH
1
O
O
NH
NH
HN
-
-
O
-
-
O
O
N
O
O
O
P
O
P
O
N
O
O
O
O
OH
O
O
H C
3
P
P
O
O
H C
O
OH
3
O
O
OH
HO
OH
HO
O
O
4
HO OH
HO OH
2
NH
2
NH
N
N
O
-
-
O
O
P
N
O
O
O
O
H C
3
P
OH
O
O
OH
HO
HO
OH
5
O
NH
OH
2
OH
HN
N
O
NH
O
HO
HO
HO
HO
-
-
-
-
O
N
O
P
O
P
O
P
O
P
N
O
O
O
O
AcHN
AcHN
N
O
O
O
O
O
O
O
O
7
6
HO OH
HO OH
Chart 1. Unnatural sugar nucleotides UDP-Fuc (2), CDP-Fuc (3), NDP-Fuc (4), ADP-Fuc (5), ADP-GlcNAc (6) to be tested as donor substrates
and natural donor substrates GDP-Fuc (1) and UDP-GlcNAc (7).
three new unnatural sugar nucleotide analogues of
GDP-Fuc (1): UDP-Fuc (2), CDP-Fuc (3) and the so
called NDP-Fuc (4) derived from 1-(b-D-ribofuranos-
yl)cyanuric acid (Chart 1). These analogues were evalu-
ated along with the previously described ADP-Fuc (5),6
as fucose donors in the reaction catalyzed by GDP-Fuc:
b-D-Galp-(1!3/4)-b-D-GlcpNAc-(1!4/3)-a-fucosyltrans-
ferase (EC 2.4.1.65, FucT-III). Furthermore, we de-
scribe the synthesis of another new sugar nucleotide
ADP-GlcNAc (6), an analogue of UDP-GlcNAc (7),
which was tested as an N-acetylglucosaminyl donor in
the reaction catalyzed by Neisseria meningitidis UDP-
GlcNAc: b-D-Galp-(1!4)-b-D-Glcp-(1!30)-b-N-acetyl-
glucosaminyltransferase (EC 2.4.1.56, LgtA).
nucleotide. To our knowledge there is only one
¨
report by Ohrlein et al. who have shown that two rec-
ombinant fucosyltransferases FucT-III and FucT-VI
were able to accept unnatural donor substrates in which
the guanine of the natural donor GDP-Fuc (1) has been
replaced by other purine bases.6,7 Following this work
we decided to examine the base specificity in more
details, and we prepared various fucose nucleotide con-
jugates, analogues to 1, which have the purine base
replaced either by the pyrimidine bases, like uracil
(UDP-Fuc 2) or cytosine (CDP-Fuc 3), or cyanuric acid
(NDP-Fuc 4). Their syntheses rely on the coupling of
fucose-b-1-phosphate and the activated form of the
nucleotide monophosphate. We chose the morpholidate
first introduced by Moffatt and co-workers8 and com-
monly used in the synthesis of GDP-Fuc,9–11 instead
of the imidazolide alternatively described in the synthe-
sis of ADP-Fuc.12 These morpholidates, commercially
available at a very high price, were synthesized in the
laboratory.13
2. Results and discussion
Glycosyltransferases are known to exhibit strict donor
specificity with a high affinity constant for the sugar

A. Khaled et al. / Carbohydrate Research 339 (2004) 2641–2649
2643
Reaction of the triethylammonium salt of b-L-fucosyl-
1-phosphate11 8 with uridine 50-monophosphoromor-
pholidate 913 was carried out in pyridine. Work-up
and purification with anionic exchange chromatography
on DEAE-Sephadex afforded UDP-Fuc (2) in 33% yield
(Scheme 1). In view of the modest yield, condensation of
phosphate 8 with cytidine 50-monophosphoromorpholi-
date 10 was performed in pyridine in the presence of 1H-
tetrazole14 reported as an activator for this coupling, but
no increased yield was observed and CDP-Fuc (3) was
obtained in the same yield.
phate was unsuitable due to low solubility of starting
material in this solvent, phosphorylation of compound
14 performed in trimethyl phosphate, afforded the
nucleotide analogue 15 as its ammonium salt in 48%
yield after purification by chromatography on silica
gel. To prepare the corresponding fucose phosphate
conjugate, we again applied the procedure relying on
nucleotide activation as the morpholidate. To this end
compound 15, as its acidic form, was heated under
reflux with morpholine in the presence of dicyclohexyl-
carbodiimide in a mixture of water and tert-butyl
alcohol.13 After removing dicyclohexylurea, the phos-
phoromorpholidate could be isolated as a precipitate
identified by 1H NMRspectroscopy as its 4-morpholine
N,N0,-dicyclohexylcarboxamidine salt 16. The coupling
reaction of compound 16 with fucose phosphate 8 was
performed in the presence of 1H-tetrazole; contrary to
the previous coupling reactions, an excess of sugar phos-
phate was used. The sugar nucleotide analogue (NDP-
Fuc) 4 was isolated in poor yield (20%) in addition to
73% of the starting fucose 8; the reaction was not optim-
ized, but compound 4 was directly tested as a donor sub-
strate with FucT-III. ADP-Fuc (5) known to serve as a
donor substrate for FucT-III and FucT-VI was also syn-
thesized in 54% yield by condensation of phosphate 8
and AMP-morpholidate 17. This analogue was needed
for comparison with the other unnatural substrates.
Finally, in order to extend this study to another gly-
cosyltransferase, we prepared ADP-GlcNAc 6 as an
analogue of UDP-GlcNAc 7. The coupling reaction of
N-acetylglucosamine a-1-phosphate19 18 and AMP-mor-
pholidate 17 performed in the presence of 1H-tetrazole,
provided after anionic chromatography, ADP-GlcNAc
6 (Scheme 1). This remained contaminated by traces of
As a substitute for the nucleotide base to be tested
with glycosyltransferases we next selected cyanuric acid
for three reasons. Firstly it is a very cheap starting mate-
rial, secondly it is easy to handle in coupling reactions
because of its C-3 symmetry, and finally it is a compo-
nent of dye reagents recognized as good ligands for
enzymes exhibiting high affinity with nucleotide diphos-
phates, such as glycosyltransferases.15 Cyanuric acid 11
was persilylated by refluxing with trimethylchlorosilane
and hexamethyldisilazane, and the tris(trimethylsilyl)-
derivative was treated with 1-O-acetyl-2,3,5-tri-O-benz-
oyl-b-D-ribofuranose 12 in the presence of SnCl4,
according to Vorbruggen conditions (Scheme 2).16 We
¨
used an excess of cyanuric acid since stoichiometric
amounts led to an equimolecular mixture of mono-
and di-substituted cyanuric acid. In this way, the cou-
pling compound could be isolated after chromatography
on silica gel as the pure b-anomer 13 in 74% yield.
Saponification with methanolic ammonia afforded the
crystalline deprotected nucleoside analogue 14 (53%).17
Compound 14 was then phosphorylated according to
the phosphorylation procedure with phosphorus oxy-
chloride in trialkyl phosphate.18 Whereas triethyl phos-
O
P
O
B
O
-
-
O
O
P
O
O
N
N
+
O
O
-
-
O
B
O
NHEt
O
O
P
3
H C
O
P
O
3
-
OH
+
O
+
NHEt
3
NHC H
6
H C
3
O
11
+
i
OH
O
OH
HO
HO OH
O
O
OH
HO
HO OH
NHC H
6
11
8
B = uracil, 9
B = uracil, 2
B = cytosine, 3
B = adenine , 5
B = cytosine, 10
B = adenine, 17
NH
2
OH
N
NH
OH
NH
2
O
O
N
+
3
HO
HO
O
NH
NHEt
N
HO
-
N
+
O
-
O
P
O
P
-
HO
+
O
P
O
O
P
i
-
NHEt
O
N
AcHN
O
N
3
O
O
O
O
-
AcHN
N
O
O
O
+
NHC H
6
11
O
18
HO OH
O
N
HO OH
NHC H
6
11
17
6
Scheme 1. Reagents and conditions: (i) 9 1.6equiv, pyridine, 7days, rt, 2: 33%; 10 1.6equiv, 1H-tetrazole 3equiv, pyridine, 6days, rt, 3: 32%; 17
1.6equiv, 1H-tetrazole 3equiv, pyridine, 2days, rt, 5: 54%; 17 1.1equiv, 1H-tetrazole 3.3equiv, pyridine, 3days, rt, 6: 58%.

2644
A. Khaled et al. / Carbohydrate Research 339 (2004) 2641–2649
O
N
OSi(CH3)3
HN
NH
O
BzO
OAc
O
N
N
ii
+
O
RO
O
N
(H3C)3SiO
OSi(CH3)3
BzO OBz
i
12
RO OR
OH
13 R = Bz
14 R = H
iii
N
N
11
N
HO
OH
iv
O
O
HN
NH
HN
NH
O
P
O-
N
O
O
N
O
P
O-
O
vii
O
O
-O
O
O
O
N
4
O
v, vi
+NHC6H11
NHC6H11
HO OH
HO OH
O
N
15
16
Scheme 2. Reagents and conditions: (i) TMSi-Cl 0.003equiv, HMDS 4.2equiv, 6h, reflux; (ii) 12 0.59equiv, SnCl4 0.38equiv, CH3CN, 1h, rt, 13:
74%; (iii) NH3 in MeOH, 4days, 0ꢁC, 14: 53%; (iv) POCl3 3.3equiv, PO(OCH3)3, 10h, 0ꢁC, 15: 48%; (v) Bio-Rad AG 50 H+ form; (vi)
H11C6N@C@NC6H11 4equiv, tert-butyl alcohol–water, morpholine 5equiv, 4h, reflux, 16: 65%; (vii) 8 1.7equiv, 1H-tetrazole 2equiv, pyridine,
2days, rt, 6: 20%.
100
75
50
25
0
1H-tetrazole and further purification with Bio-Gel P2
was required to obtain the pure compound 6 in 58%
yield.
All synthesized sugar nucleotide analogues 2, 3, 4, 5
were tested as fucose donors towards recombinant a-
(1!3/4)-fucosyltransferase, which was produced in
Baculovirus-infected insect cells.2 This enzyme is able
to catalyze transfer of a fucose residue to position-4 of
the N-acetylglucosamine unit belonging to the disacchar-
ide b-D-Galp-(1!3)-b-D-GlcpNAc or position-3 of the
N-acetylglucosamine unit belonging to the disaccharide
b-D-Galp-(1!4)-b-D-GlcpNAc. For evaluation of the
analogues, we applied a fluorescent assay utilising as
an acceptor substrate b-D-Galp-(1!3)-b-D-GlcpNAc-
O-(CH2)6-NH-dansyl.20 This disaccharide was incubated
with each of the analogues 2, 3, 4 or 5 at a concentration
of 20mM. The kinetics of the conversion are shown in
Figure 1.
0
5
10
15
20
time (hours)
Figure 1. Kinetics of fucose transfer reaction catalyzed by FucT-III
Ç
using analogues 2, 3, 4, 5 as donor substrates; UDP-Fuc 2 ( ), CDP-
Fuc 3 (d), NDP-Fuc 4 (m), ADP-Fuc 5 (.).
Surprisingly total conversion was observed after 3h
for UDP-Fuc (2) as well as for ADP-Fuc (5), after
12h for NDP-Fuc (4), whereas for CDP-Fuc (3) conver-
sion was only 70% after 18h. It is worthy of mention
that under these conditions, only 10min were required
for total conversion with the natural substrate, GDP-
Fuc (1). Unexpectedly, FucT-III does not only tolerate
the exchange of guanosine for adenine but may also ac-
cept a pyrimidine base. NDP-Fuc and CDP-Fuc were
utilized with lower efficiency than UDP-Fuc, but never-
theless they could act as fucose donors. In order to get
further insights into the specificity of the transferase,
we determined the kinetic parameters of FucT-III for
each donor analogue and compared their values with
those for the natural substrate 1. The Lineweaver–Burk
plots for analogues 2, 3, 4 and 5 are reported in Figure 2.
Kinetic parameters of FucT-III for GDP-Fuc(1) and its
analogues 2, 3, 4 and 5 are gathered in Table 1. The
affinity constant we measured for 1 (43lM) is in good

A. Khaled et al. / Carbohydrate Research 339 (2004) 2641–2649
2645
3
2.5
2
80
60
40
20
0
1.5
1
0.5
0
0
10
20
30
40
50
0
0.1
0.2
0.3
1/[S] mM-1
0.4
0.5
0.6
time (hours)
Figure 3. Kinetics of N-acetylglucosamine transfer reaction catalyzed
by LgtA using analogue 6 as a donor substrate.
Figure 2. The Lineweaver–Burk plots for 2, 3, 4, 5; UDP-Fuc 2 (j),
Ç
CDP-Fuc 3 (m), NDP-Fuc 4 (d), ADP-Fuc 5 ( ).
5
4
3
2
1
0
Table 1. Comparison of the kinetic parameters of FucT-III for GDP-
Fuc (1) and analogues 2, 3, 4, 5
Donor substrates Vmax [nmolminÀ1 mLÀ1
]
Km mM Vmax/Km
GDP-Fuc 1
UDP-Fuc 2
ADP-Fuc 5
NDP-Fuc 4
CDP-Fuc 3
148
52
40
29
3
0.043 3440
13
13
4
3
140
38
0.2
0.1
agreement with the literature value (33lM).21 The data
corroborate the results observed in the kinetics experi-
ments: UDP-Fuc 2 turns out to be as efficient a substrate
as ADP-Fuc 5. The Km values are much higher for 2 and
3 than for 1 (13mM vs 43lM) and the Vmax/Km ratios
are three orders of magnitude lower for these analogues
than for 1. NDP-Fuc 4 exhibits a lower affinity but a
higher Vmax than CDP-Fuc 3, the poorest substrate
out of the four analogues.
0
1
2
3
4
5
6
1/ [ADP-GlcNAc] 10-1 mM-1
ADP-GlcNAc 6 was also tested as an analogue of
UDP-GlcNAc 7 with N. meningitidis UDP-GlcNAc: b-
D-Galp-(1!4)-b-D-Glcp-(1!3)-b-N-acetylglucosaminyl-
transferase (lgtA) cloned and expressed in E. coli.22,23
This enzyme catalyzes transfer of N-acetylglucosamine
onto position-3 of the terminal galactose of lactose unit.
Thus b-D-Gal p-(1!4)-b-D-Glcp O-(CH2)6-NH-dansyl
disaccharide20 was incubated with analogue 6 at a
concentration of 20mM in the presence of the enzyme.
Kinetics for the conversion are reported in Figure 3.
Again the donor specificity proved not to be as strict
as was supposed, since ADP-GlcNAc turned out to
serve as a donor substrate for LgtA. The kinetic
parameters for ADP-GlcNAc (6) were measured and
compared with those for the natural substrate, UDP-
GlcNAc (7). The Lineweaver–Burk plot is reported in
Figure 4. Data are similar to those previously observed
Figure 4. The Lineweaver–Burk plot for ADP-GlcNAc (6).
with fucose donor analogues and FucT-III: the Vmax/Km
ratio is three orders of magnitude lower for analogue 6
than for 7, with a Km value multiplied by a factor of
135 for ADP-GlcNAc as compared to UDP-GlcNAc
(Table 2). Nevertheless, working at high donor substrate
concentrations allowed sugar transfer onto the acceptor
substrate.
Table 2. Kinetic parameters of LgtA for ADP-GlcNAc (6) as
compared to those for UDP-GlNAc (7)
Donor substrates Vmax [nmolminÀ1 mLÀ1
]
Km mM Vmax/Km
0.64 1000
88 1.5
UDP-GlcNAc 7
ADP-GlcNAc 6
640
110

2646
A. Khaled et al. / Carbohydrate Research 339 (2004) 2641–2649
These kinetics studies with donor substrate analogues
72.1, 70.8, (C-20, C-30, C-2, C-3, C-4, C-5), 65.9 (d,
JC,P 4Hz, C-50), 47.5 (CH2–CH3)3, 16.5 (C-6) and 9.2
(CH2–CH3)3; 31P NMR(D 2O, 101MHz): d À10.55
and À12.28 (JP,P 20.2Hz); ESMS (negative mode): m/z
274 [(M À 2 NHEt3)/2]À, 549 [(M À 2NHEt3 + 1]À.
HRMS (negative mode): Calcd for C15H23N2O16P2
[(M À 2NHEt3 + 1]À 549.0517. Found: m/z 549.0527.
provide new and useful information on structure–activ-
ity relationships. Together with molecular modeling
studies they may be helpful for the rational design of
enzyme specificity.
3. Experimental
3.3. Cytidine-50-(b-L-fucopyranosyl)-diphosphate, bis(tri-
ethylammonium) salt (CDP-Fuc) (3)
3.1. General methods
NMRspectra were recorded with a Bruker AC-200 or
AC-250 spectrometers; the chemical shifts are given rel-
ative to the signal of tetramethylsilane in CDCl3; for 1H
To a soln of b-L-fucose-1-phosphate bis(triethylammo-
nium) salt 8 (100mg, 0.225mmol) and CMP-morpholi-
date 10 (246mg, 0.36mmol) in dry pyridine (5mL) was
added 1H-tetrazole (47mg, 0.67mmol) and the soln
was stirred for 6d at rt under argon. Then the mixture
was concentrated under diminished pressure to dryness
and the residue dissolved in water was purified by chro-
matography on a DEAE-Sephadex A-25 (HCOÀ3 form)
column. Elution with a gradient of 0 to M triethylam-
NMRand 13C NMRspectra in D O, acetone (d 2.22
2
and 30.5ppm) was used as an internal reference.
H3PO4 in D2O was used as an external reference for
31P NMR. Optical rotations were measured with a Jasco
digital micropolarimeter. Mass spectra were performed
either on a Finigan MATT 95 apparatus using the ESI
technique. Reactions were monitored by TLC on Silica
Gel 60F254 with detection by charring with 10%
H2SO4 in EtOH or 2% orcinol in 10% H2SO4. HPLC
measurements were performed using the Waters 600
pump equipped with a reverse-phase column (C18
Nucleosil, 5lm, 4.6mm id · 200mm; mobile phase,
water–MeCN: 65:35; flow rate 1mL/min). Detection
was done with a Luminescence Spectrometer LS50B
from Perkin–Elmer. Fluorescence of substrate and prod-
uct was read at 385nm excitation/540nm emission.
monium hydrogenocarbonate buffer (pH7.8) afforded
30
D
3 as its triethylammonium salt (56mg, 32%); ½aŠ þ2
1
(c 1, water); H NMR(D O, 250MHz): d 7.95 (d, 1H,
2
J5,6 8Hz, H-6 cytosine), 6.10 (d, 1H, H-5 cytosine),
5.95 (d, 1H, J1 ,2 4Hz, H-10), 4.88 (t, 1H, J1,2 = J1,P
8Hz, H-1), 4.40–4.15 (m, 5H, H-20, H-30, H-40, H-50),
3.75 (q, 1H, J5,6 6.6Hz, H-5), 3.65 (d, 1H, J3,4 3Hz,
H-4), 3.60 (dd, 1H, J2,3 9.5Hz, H-3), 3.50 (dd, 1H, H-
2), 3.07 (q, 12H, 2(CH2–CH3)3 and 1.20 (t, 18H,
2(CH2–CH3)3, H-6); 13C NMR(D 2O, 62.9MHz): d
166.9, 158.4, 156.4, 142.6 (4C-cytidine), 99.6 (d, JC,P
5.5Hz, C-1), 90.1 (C-10), 83.8 (d, JC,P 9.5Hz, C-40),
75.3, 73.6, 72.4, 72.2, 72.1, 70.3 (C-20, C-30, C-2, C-3,
C-4, C-5), 65.6 (C-50), 47.5 (CH2–CH3)3, 16.5 (C-6)
and 9.2 (CH2–CH3)3; 31P NMR(D 2O, 101MHz): d
À10.51 and À12.28 (JP,P 21.5Hz); ESMS (negative
mode): m/z 273.5 [(M À 2NHEt3)/2]À; HRMS (negative
mode): Calcd for C15H23N3O15P2 [(M À 2NHEt3)/2]À
273.5302. Found: m/z 273.5298.
0
0
3.2. Uridine-50-(b-L-fucopyranosyl)-diphosphate, bis(tri-
ethylammonium) salt (UDP-Fuc) (2)
To a soln of fucose-b-1-phosphate bis(triethylammo-
nium) salt 8 (222mg, 0.5mmol) in dry pyridine (5mL)
was added UMP-morpholidate 9 (549mg, 0.8mmol)
and the soln was stirred for 7d at rt under argon. Then
the mixture was concentrated in vacuo to dryness and
the residue dissolved in water was purified by chroma-
tography on a DEAE-Sephadex A-25 (HCOÀ3 form)
column. Elution with a gradient of 0 to M triethyl-
ammonium hydrogenocarbonate buffer (pH7.8) first
afforded remaining 8 (88mg, 40%) and then 2 as its
triethylammonium salt (125mg, 33%, 55% based on
3.4. 1-(b-D-Ribofuranosyl)cyanuric acid (14)
Cyanuric acid (636mg, 4.93mmol) and trimethylchloro-
silane (0.1mL, 0.013mmol) were refluxed for 6h with
hexamethyldisilazane (4.4mL, 20.9mmol). Then sol-
vents were evaporated to dryness (1.609g, 95%). To
the residue were added 1-O-acetyl-2,3,5-tri-O-benzoyl-
b-D-ribofuranose24 (1.458g, 2.9mmol), SnCl4 (0.225
mL, 1.9mmol) and dry MeCN (60mL); the mixture
was stirred at rt for 1h. Then solvents were evaporated
and the residue was extracted with hot toluene. The or-
ganic phase was concentrated and purified by flash col-
umn chromatography (4:1, toluene–EtOAc), affording
13 (1.24g, 74%), which crystallized from MeOH–
EtOAc–petroleum ether; mp 211ꢁC (lit.16 211–213ꢁC);
29
1
recovered fucose phosphate); ½aŠ À5 (c 1, water); H
D
NMR(D 2O, 250MHz): d 7.90 (d, 1H, J5,6 8Hz, H-6 uri-
0
0
0
dine), 5.95 (d, 1H, J1 ,2 4Hz, H-1 ), 5.93 (d, 1H, H-5 uri-
dine), 4.88 (t, 1H, J1,2 = J1,P 8Hz, H-1), 4.33 (m, 2H,
H-20, H-30), 4.27–4.15 (m, 3H, H-40, H-50), 3.78 (q,
1H, J5,6 6.6Hz, H-5), 3.70 (d, 1H, J3,4 3Hz, H-4), 3.64
(dd, 1H, J2,3 9.5Hz, H-3), 3.51 (dd, 1H, H-2), 3.16 (q,
12H, 2(CH2–CH3)3) and 1.22 (t, 21H, 2 (CH2–CH3)3,
H-6); 13C NMR(D 2O, 50.3MHz): d 167.8, 153.3,
142.6, 103.6 (4C-uridine), 99.5 (d, JC,P 5Hz, C-1), 89.2
(C-10), 84.3 (d, JC,P 9Hz, C-40), 74.7, 73.6, 72.4, 72.2,
1H NMR(CDCl , 250MHz): d 9.7 (s, 2H, NH), 8.06–
3

A. Khaled et al. / Carbohydrate Research 339 (2004) 2641–2649
2647
7.84 (3d, 6H, Bz), 7.60–7.20 (m, 9H, Bz), 6.45 (d, 1H,
J1 ,2 2Hz, H-10), 6.17 (dd, 1H, J2 ,3 6.5Hz, H-2), 6.08
reaction. Conversion of 15 into the nucleoside morphol-
idate 16 was evidenced by TLC on silica gel (6:3:2 2-pro-
panol–NH4OH–water). The mixture was then cooled to
rt, the crystalline dicyclohexylurea was filtered and
washed with water. The filtrate was concentrated and
then extracted with ether to remove excess dicyclohexyl-
carbodiimide. The aqueous soln was evaporated to dry-
ness. The residue was taken up in MeOH (1mL);
addition of dry ether (8mL) to this soln gave a precipi-
tate as a gum, which upon trituration with fresh ether
gave the salt of 4-morpholine N,N0-dicyclohexylcarbox-
0
0
0
0
(t, 1H, H-3) and 4.86–4.56 (m, 3H, H-4, H-50); 13C
NMR(CDCl , 62.9MHz): d 166.3, 165.6, 165.2 (CO),
3
148.2 (CO), 129.4, 128.8, 128.6, 128.5, 128.2, 128.0 (C–
Ar), 87.1 (C-1), 79.1 (C-4), 74.0 (C-2), 71.0 (C-3) and
63.8 (C-5); ESMS (positive mode): m/z 596.1
[M + Na]+ C29H23N3O10.
Compound 13 (1.24g, 2.16mmol) was dissolved in
MeOH (20mL), saturated at 0ꢁC with ammonia and left
at rt for 4d. Then the methanolic ammonia was evapo-
rated, the residue was taken up in water and extracted
four times with ether. The aqueous phase was then evap-
orated to dryness and the residue crystallized from
1
amidine 16 as a white powder (106mg, 65%); H NMR
0
(D2O, 200MHz): d 6.13 (d, 1H, J1 ,2 3Hz, H-10), 4.73
0
(dd, 1H, J2 ,3 6.3Hz, H-20), 4.50 (t, 1H, J3 ,4 6.3Hz, H-
30) and 4.10–3.95 (m, 3H, H-40, H-50), 3.83–3.06 (m,
18H, 8CH2 morpholidate, 2CH–N), 2.10–1.25 (m, 20H,
cyclohexyl).
0
0
0
0
MeOH to give compound 14 (299mg, 53%); mp 224–
29
D
225ꢁC (lit.16 229–230ꢁC); ½aŠ À24 (c 1, water); 1H
0
0
NMR(D O, 250MHz): d 6.10 (d, 1H, J1 ,2 3.5Hz, H-
2
10), 4.74 (dd, 1H, J2 ,3 6Hz, H-20), 4.41 (t, 1H, H-30),
To a soln of fucose-b-1-phosphate bis(triethylammo-
nium) salt 8 (120mg, 0.26mmol) and morpholidate 16
(105mg, 0.15mmol) in dry pyridine (1mL) was added
1H-tetrazole (19mg, 0.28mmol). Reaction was moni-
tored by TLC on silica gel (6:3:2 2-propanol–NH4OH–
water). The soln was stirred for 2d at rt under argon.
Then the mixture was concentrated to dryness and the
residue dissolved in water was purified by chromatogra-
phy on a DEAE-Sephadex A-25 (HCOÀ3 form) column.
Elution with a gradient of 0 to M triethylammonium
hydrogenocarbonate buffer (pH7.8) first afforded
0
0
3.99 (dt, 1H, J4 ,5 a 6Hz, J4 ,5 b 3Hz, H-40), 3.90 (dd,
0
0
0
0
1H, J5 a,5 b 12Hz, H-50a) and 3.76 (dd, 1H, H-5b0 ); 13C
0
0
NMR(D O, 50.3MHz) d 155.1 (CO), 89.2 (C-1), 84.5
2
(C-4), 72.5 (C-2), 70.5 (C-3) and 62.6 (C-5).
3.5. 1-(b-D-Ribofuranosyl)cyanuric acid-50-phosphate,
ammonium salt (15)
To a soln of POCl3 (0.172mM, 2mmol) in trimethyl
phosphate (1mL) cooled at 0ꢁC was added compound
14 (0.162mg, 0.6mmol) and water (0.004mL, 0.2mmol).
The mixture was stirred at 0ꢁC for 10h, then acidified to
pH1.5 by adding M NaOH and heated at 70ꢁC for
30min. The solution was evaporated to dryness and
the residue was purified by flash chromatography on sil-
remaining 8 (84mg, 73%) and then 4 as its triethylam-
29
D
monium salt (23mg, 20%); ½aŠ À11 (c 1.18, water);
1H NMR (D2O, 200MHz): d 6.10 (d, 1H, J1 ,2 3.5Hz,
H-10), 4.92 (t, 1H, J1,2 = J1,P 8Hz, H-1), 4.74 (dd, 1H,
0
0
J2 ,3 6.5Hz, H-20), 4.48 (t, 1H, H-30), 4.24 (t, 1H, J3 ,4
0
0
0
0
ica gel (6:3:2 2-propanol–NH4OH–water) to afford the
6.5Hz, H-40), 4.30 (m, 2H, H-50), 3.84 (q, 1H, J5,6
6.6Hz, H-5), 3.75 (d, 1H, J3,4 3Hz, H-4), 3.69 (dd,
1H, J2,3 10Hz, H-3), 3.57 (dd, 1H, J1,2 8Hz, J2,3
10Hz, H-2), 3.21 (q, 12H, 2(CH2–CH3)3) and 1.28 (t,
21H, J 7Hz, 2(CH2–CH3)3, H-6); 13C NMR(D 2O,
50.3MHz): d 151.2 (CO), 98.7 (d, JC,P 5.5Hz, C-1),
88.6 (C-10), 82.1 (d, JC,P 9Hz, C-40), 72.8, 71.7, 71.5,
71.3, 72.1, 69.7 (C-20, C-30, C-2, C-3, C-4, C-5), 65.8
(d, JC,P 6Hz, C-50), 46.8 (CH2–CH3)3, 15.8 (C-6) and
27
D
phosphate 15 (129mg, 48%); ½aŠ À20 (c 0.5, water);
1H NMR(D O, 200MHz): d 6.07 (d, 1H, J1 ,2 3.2Hz,
0
0
2
H-10), 4.70 (dd, 1H, J2 ,3 6.5Hz, H-20), 4.40 (t, 1H, H-
30) and 4.05–3.80 (m, 3H, H-40, H-50); 13C NMR
(D2O, 62.9MHz): d 155.1 (CO), 90.4 (C-1), 84.1 (d,
JC,P 8Hz, C-4), 73.7 (C-2), 71.6 (C-3) and 65.3 (d, JC,P
0
0
6Hz, C-5); 31P NMR(D O, 101MHz): d 4.34; HRMS
2
(negative mode): Calcd for C8H11N3O10P [M–NHþ4 ]À
340.0182. Found: m/z 340.0185.
8.5 (CH2–CH3)3; 31P NMR(D O, 101MHz): d À10.42
2
and À12.37 (JP,P 20.3Hz); HRMS (negative mode):
3.6. 1-(b-D-Ribofuranosyl)cyanuric acid-50-(b-L-fucopyr-
anosyl)-diphosphate, bis(triethylammonium) salt (4)
Calcd
282.5173. Found: m/z 282.5175.
for
C14H21N3O17P2
[(M À 2NHEt3)/2]À
A soln of the ammonium salt 15 (82mg, 0.23mmol) was
passed through a column of Bio-Rad AG 50W-X8 resin
(H+ form); the soln was freeeze-dried and the residue
was dissolved in a mixture of water (2.3mL), tert-butyl
alcohol (2.3mL) and distilled morpholine (0.078mL,
1.14mmol). To this soln heated under reflux was added
dropwise dicyclohexylcarbodiimide (190mg, 0.92mmol)
in tert-butyl alcohol (3.5mL) and the reaction mixture
was maintained under reflux for 3–4h until complete
3.7. Adenosine-50-(b-L-fucopyranosyl)-diphosphate,
bis(triethylammonium) salt (ADP-Fuc) (5)
To a soln of b-L-fucose-1-phosphate bis(triethylammo-
nium) salt 8 (222mg, 0.5mmol) and AMP-morpholidate
17 (565mg, 0.8mmol) in dry pyridine (7.5mL) was
added 1H-tetrazole (105mg, 1.5mmol). The reaction
was monitored by TLC on silica gel (6:3:2 2-propanol–
NH4OH–water). The soln was stirred for 2d at rt under

2648
A. Khaled et al. / Carbohydrate Research 339 (2004) 2641–2649
argon, then concentrated to dryness and the residue dis-
solved in water was purified by chromatography on a
DEAE-Sephadex A-25 (HCOÀ3 form) column. Elution
with a gradient of 0 to M triethylammonium hydrogeno-
tive mode): Calcd for C18H26N6O15P2 [(M À 2NHEt3)/
2]À 314.0465. Found: m/z 314.04656.
3.9. GDP-Fuc: b-D-Galp-(1!3/4)-b-D-GlcpNAc-(1!4/
3)-a-fucosyltransferase kinetics studies using donor
analogues
carbonate buffer (pH7.8) afforded 5 as its triethylammo-
30
D
nium salt (209mg, 54%); ½aŠ À17 (c 1, water); 1H NMR
(D2O, 250MHz): d 8.50 (s, 1H adenosine), 8.25 (s, 1H
adenosine), 6.12 (d, 1H, J1 ,2 5.5Hz, H-10), 4.88 (t,
FucT-III was a 20-fold concentrated culture supernatant
of recombinant enzyme produced in baculovirus-in-
fected insect cells.2 Each GDP-Fuc analogue (2 or 3 or
4 or 5, 2lmol) and b-D-Galp-(1!3)-b-D-GlcNAcp-O-
(CH2)6-NH-dansyl (40nmol) were incubated at 37ꢁC
with FucT-III (20lL, 0.02U) in 50mM sodium cacodyl-
ate buffer pH6 containing 10mM MnCl2 in a total vol-
ume of 100lL. Aliquots of the reaction mixture were
withdrawn at different incubation times and analyzed
by HPLC to determine the percentage of conversion.
0
0
0
0
1H, J1,2 = J1,P 8Hz, H-1), 4.74 (t, 1H, J2 ,3 5.5Hz H-
20), 4.53 (dd, J3 ,4 4Hz, 1H, H-30), 4.38 (m, 1H, H-40),
4.2 (m, 2H, 2H-50), 3.72 (q, 1H, J5,6 6.6Hz, H-5),
3.69–3.58 (m, 2H, H-3, H-4), 3.55 (dd, 1H, J2,3 9.5Hz,
H-2), 3.16 (q, 12H, J 7Hz, 2(CH2–CH3)3), 1.24 (t,
18H, J 7Hz, 2(CH2–CH3)3) and 1.18 (d, 3H, J5,6
6.6Hz, H-6); 13C NMR(D 2O, 62.5MHz): d 158.6,
153.6, 149.7, 140.6, 119.3 (5C-adenosine), 99.5 (d, JC,P
6Hz, C-1), 87.9 (C-10), 84.8 (d, JC,P 10Hz, C-40), 75.3,
73.5, 72.4, 72.1, 71.4, (C-20, C-30, C-2, C-3, C-4, C-5),
66.3 (C-50), 47.6 (CH2–CH3)3, 16.4 (C-6) and 9.2
(CH2–CH3)3; 31P NMR (D2O, 101MHz): d À10.85
and À12.71 (JP,P 20.4Hz); ESMS (negative mode): m/z
285.5 [(M À 2NHEt3)/2]ÀC16H23N5O14P2.
0
0
3.10. Determination of kinetic parameters of FucT-III for
GDP-Fuc, UDP-Fuc, CDP-Fuc, NDP-Fuc, ADP-Fuc
Enzyme assay conditions: 50mM sodium cacodylate
buffer pH6.0, 15mM MnCl2, 0.4mM Gal-b-(1!3)-Glc-
NAc-b-O-(CH2)6-NH-dansyl disaccharide, sugar-nucleo-
tide analogue concentrations varying from 2 to 30mM;
FucT-III, 5lL (0.4mU); for GDP-Fuc, concentrations
were 20, 40, 80, 160lM. Total volume incubation was
50lL. Assays were incubated at 37ꢁC for 15–60min.
Incubation was stopped by immersion in a boiling
water bath for 1min; after centrifugation and filtration,
samples were analyzed by HPLC.
3.8. Adenosine-50-(2-acetamido-2-deoxy-a-D-glucopyran-
osyl)-diphosphate, bis(triethylammonium) salt (ADP-
GlcNAc) (6)
To a solution of 2-acetamido-2-deoxy-a-D-glucopyrano-
syl phosphate bis(triethylammonium) salt 18 (87mg,
0.173mmol) in dry pyridine (5mL) was added AMP-
morpholidate 17 (135mg, 0.191mmol) and 1H-tetrazole
(40mg, 0.573mmol) and the soln was stirred for 3d at rt
under argon. The reaction mixture was then concen-
trated to dryness and the residue dissolved in water
was purified by anionic exchange chromatography on
DEAE-Sephadex A-25 (HCOÀ3 form) column eluted
with a gradient of triethylammonium hydrogenocarbo-
nate. Fractions containing compound 6 were still con-
taminated with traces of 1H-tetrazole; they were
3.11. Preparation of N. meningitidis UDP-GlcNAc: b-D-
Galp-(1!4)-b-D-Glcp-(1!30)-b-N-acetyl-glucosaminyl-
transferase (LgtA)
LgtA was produced in E. coli transfected with the plas-
mid carrying the LgtA gene.22,23 The crude enzyme was
prepared according to the procedure of Blixt et al.25 with
some modifications. At the end of the fermentation, the
cells were harvested and frozen. After thawing, the cells
were suspended in 0.05M sodium cacodylate buffer
pH7.2 containing protease inhibitor cocktail (from
Roche Diagnostics) and lysed with (106 microns size)
glass beads using the mixer mill MM 200 from Retsch
at the maximum frequency for 2 · 10min; the lysate
was then centrifuged at 20,000g, at 4ꢁC for 10min and
the supernatant was ready to be used for enzymatic
assays.
freeze-dried, and the residue taken up in water was puri-
29
D
fied on Bio-Gel P2 to afford pure 6 (84mg, 58%); ½aŠ 21
(c 0.45, water); 1H NMR(D 2O, 250MHz): d 8.45 (s, 1H
0
0
adenosine), 8.19 (s, 1H adenosine), 6.08 (d, 1H, J1 ,2
5.5Hz, H-10), 5.45 (dd, 1H, J1,2 3.5, JP,1 7.5Hz, H-1),
4.70 (dd, J2,3 5Hz, 1H, H-20), 4.46 (dd, J3,4 4Hz, 1H,
H-30), 4.32 (m, 1H, H-40), 4.15 (m, 2H, 2 H-50), 3.96–
3.65 (m, 5H, H-2, H-4, H-5, 2H-6), 3.45 (t J2,3 = J3,4
9.5Hz, H-3), 3.14 (q, J 7Hz, 12H, 2(CH2–CH3)3), 1.98
(s, 3H, NHAc) and 1.22 (t, J 7Hz, 18H, 2 (CH2–
CH3)3); 13C NMR(D 2O, 50.3MHz): d 174.9 (CO),
154.9, 152.0, 149.1, 146.9, 140.3 (5C-adenosine) 94.8
(d, JC,P 4.5Hz, C-1), 87.3 (C-10), 84.1 (d, JC,P 9Hz, C-
40), 74.6, 73.4, 71.2, 70.7, 69.9 (C-20, C-30, C-3, C-4, C-
5), 65.5 (C-50), 60.7 (C-6), 53.9 (d, JC,P 9Hz, C-2) 46.8
(C2–CH3)3 22.4 (CH3) and 8.5 (CH2–CH3)3 31P NMR
(D2O, 101MHz): d À10.77 and À12.40; HRMS (nega-
3.12. LgtA kinetics studies using ADP-GlcNAc as a donor
analogue
ADP-GlcNAc (6, 2lmol) and b-D-Galp-(1!4)-b-D-
Glcp-O-(CH2)6-NH-dansyl (80nmol) were incubated at
37ꢁC with LgtA (30lL, 0.01U) in 50mM sodium caco-

A. Khaled et al. / Carbohydrate Research 339 (2004) 2641–2649
2649
4. Koeller, K. M.; Wong, C.-H. In Carbohydrates in Chem-
istry and Biochemistry, Part I: Chemistry of Saccharides;
dylate buffer pH7.4 containing 15mM MnCl2 in a total
volume of 100lL. Aliquots of the reaction mixture were
withdrawn at different incubation times and analyzed by
HPLC to determine the percentage of conversion.
€
Ernst, B., Hart, G. W., Sinay, P., Eds.; Wiley-VCH:
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7. Ohrlein, R. Top. Curr. Chem. 1999, 200, 227–254.
3.13. Determination of the kinetic parameters of LgtA for
ADP-GlcNAc and UDP-GlNAc
8. Roseman, S.; Distler, J. J.; Moffatt, J. G.; Khorana, H. G.
J. Am. Chem. Soc. 1961, 82, 659–663.
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Enzyme assay conditions: 50mM sodium cacodylate
buffer pH7.4, 15mM MnCl2, 1mM Gal-b-(1!4)-Glc-
b-O-(CH2)6-NH-dansyl disaccharide, ADP-GlcNAc
concentration: 32, 16, 8, 4, 2mM; LgtA, 9lL (3mU);
for UDP-GlcNAc, concentrations were 0.05, 0.1, 0.2,
0.5, 0.9mM. Total volume incubation was 30lL. Assays
were incubated at 37ꢁC for 20–100min. Incubation was
stopped by immersion in a boiling water bath for 1min;
after centrifugation and filtration, samples were ana-
lyzed by HPLC.
15. Kaminska, J.; Dzieciol, J.; Koscielak, J. Glycoconjugate J.
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Acknowledgements
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3654–3660.
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18. Yoshikawa, T.; Kato, T.; Takenishi, T. Bull. Chem. Soc.
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¨
The present study was funded in part by the interre-
gional network (MIIAT) project Ôglycosylation mutantsÕ
and A.K. was recipient of a fellowship from Region Ile
de France. CNRS and the University of Paris-Sud are
also gratefully acknowledged. We thank Professor
´
Andre Lubineau for helpful discussions and Dr. Eric
Samain for providing the recombinant E. coli strain pro-
ducing LgtA.
´
Cerutti, M.; Harduin-Lepers, A.; Delannoy, P.; Auge, C.;
Lubineau, A. Carbohydr. Res. 2003, 338, 1153–1161.
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