Non-isosteric C-glycosyl analogues of natural nucleotide diphosphate sugars as glycosyltransferase inhibitors

Article abstract of DOI:10.1016/j.bmc.2006.06.057

A series of C-glycosyl ethylphosphonophosphate analogues of UDP-Glc, UDP-Gal, UDP-GlcNAc and GDP-Fuc were synthesized from the corresponding C-glycosyl ethylphosphonic acids. Analogues were obtained as α-anomers through either diastereoselective photo-induced radical addition of glycosyl bromides (d-Glc, d-Gal and l-Fuc) to diethyl vinylphosphonate, or a multi-step sequence (d-GlcNAc), with subsequent coupling with morpholidate-activated nucleotide monophosphates. The in vitro inhibitory activity of UDP-Gal, GDP-Fuc and UDP-GlcNAc analogues towards glycosyltransferases (β-1,4-GalT, FUT3 and LgtA) was evaluated through a competition fluorescence assay and IC₅₀ values of 40 μM, 2 mM and 3.5 mM were obtained, respectively.

Full text of DOI:10.1016/j.bmc.2006.06.057

Bioorganic & Medicinal Chemistry 14 (2006) 7293–7301
Non-isosteric C-glycosyl analogues of natural nucleotide
diphosphate sugars as glycosyltransferase inhibitors
a
a
b
´
`
Sebastien Vidal, Isabelle Bruyere, Annie Malleron,
b
a,
*
´
Claudine Auge and Jean-Pierre Praly
aLaboratoire de Chimie Organique 2, UMR-CNRS 5181, Universite´ Claude Bernard Lyon 1,
ˆ
CPE-Lyon Batiment 308, 43 Boulevard du 11 Novembre 1918, F-69622 Villeurbanne, France
bLaboratoire de Chimie Organique Multifonctionnelle, UMR-CNRS 8614, GDR 2590,
ˆ
Institut de Chimie Mole´culaire et des Mate´riaux d’Orsay, Universite´ Paris-Sud, Batiment 420, F-91405 Orsay, France
Received 21 December 2005; revised 13 June 2006; accepted 23 June 2006
Available online 14 July 2006
Abstract—A series of C-glycosyl ethylphosphonophosphate analogues of UDP-Glc, UDP-Gal, UDP-GlcNAc and GDP-Fuc were
synthesized from the corresponding C-glycosyl ethylphosphonic acids. Analogues were obtained as a-anomers through either dia-
stereoselective photo-induced radical addition of glycosyl bromides (D-Glc, D-Gal and L-Fuc) to diethyl vinylphosphonate, or a mul-
ti-step sequence (D-GlcNAc), with subsequent coupling with morpholidate-activated nucleotide monophosphates. The in vitro
inhibitory activity of UDP-Gal, GDP-Fuc and UDP-GlcNAc analogues towards glycosyltransferases (b-1,4-GalT, FUT3 and
LgtA) was evaluated through a competition fluorescence assay and IC50 values of 40 lM, 2 mM and 3.5 mM were obtained,
respectively.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
Since glycoconjugates are involved in numerous cell–cell
recognition and communication processes, the rational
design of glycosyltransferase inhibitors has been inten-
sively investigated9–33 for a better understanding of bio-
chemical pathways implicating these enzymes and also
for potential therapeutical applications. Glycosyltrans-
ferase inhibitors are generally designed based on analo-
gies between the three different moieties composing the
NDP sugar natural substrates, mimicking either the car-
bohydrate part,9–12,31 the diphosphate linkage,13–20 the
nucleoside moiety or combinations of these.21–30 The
diphosphate linkage, essential for most glycosyltransfe-
rases’ activity, interacts via its dianionic charge with
metallic cations, for example, Mn2+, coordinated with
two aspartate residues within the active site (Fig. 1). Sev-
eral mimics of the dianionic charge of NDP sugars have
been designed ranging from phosphonates,32 methylpyro-
phosphates,19,33 methylenediphosphonates,14 to malo-
nates, tartrates or even monosaccharidic structures.13–20
Even though several syntheses of glycoconjugates mim-
icking the diphosphate linkage have been described in
the literature, the biological evaluation of these analogues
towards glycosyltransferases is rather limited.13–15,32,33
Formation of glycosidic linkages in the biosynthesis of
oligosaccharides, glycolipids and glycoproteins usually
occurs through the Leloir pathway1 under the control
of glycosyltransferases,2–5 by transfer of a carbohydrate
from a nucleotide diphosphate sugar (NDP sugar) as
donor to the acceptor substrate, releasing the corre-
sponding nucleotide diphosphate. Glycosyltransferases
catalyze the transfer of sugar units to acceptors with
either inversion or retention of the anomeric configura-
tion with respect to the NDP sugar donor. The proposed
mechanism6–8 of glycosyl transfer with inversion of con-
figuration is occurring through weakening of the donor
glycosidic bond implicating an acidic residue in the en-
zyme active site, followed by a SN2-type transition state
involving an oxonium ion. Final attack of the acceptor’s
hydroxyl group with assistance of a basic group in the
enzyme catalytic site provides the glycosylated acceptor
(Fig. 1).
Keywords: Glycosyl radical; C-Glycoside; Inhibitor; Glycosyltransfer-
ase; NDP sugar analogue.
*
C-Glycosyl analogues are useful mimetics due to their
higher stability towards acid- or enzyme-catalyzed
Corresponding author. Tel.: +33 4 72 43 11 61; fax: +33 4 72 44 83
49; e-mail: jean-pierre.praly@univ-lyon1.fr
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.06.057

7294
S. Vidal et al. / Bioorg. Med. Chem. 14 (2006) 7293–7301
H
B
H
B
H
B
O
O
Acceptor
O
Acceptor
O
Acceptor
O
N
O
N
O
N
O
O
NH
NH
NH
O
P
O
P
O
P
O
P
O
P
O
P
H
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
A
H
A
H
A
Mn2+
OH OH
Mn2+
OH OH
Mn2+
OH OH
O
O
O
O
O
O
O
O
O
O
O
Figure 1. Proposed mechanism for inverting glycosyltransferases.26
hydrolysis compared to O-glycosides and several
approaches have been developed for their stereocon-
trolled synthesis.34–39 Being involved in a continuing
programme focusing on a series of glycosyltransferases
with mostly a-configured natural substrates, we wanted
to prepare analogues with the same a-configuration. The
use of radical chemistry,40 for example, addition of gly-
cosyl radicals to suitable alkenes, is probably the most
efficient method41–48 due to mild conditions, atom econ-
omy and high stereocontrol. Although this approach is
leading predominantly to a-configured C-glycosyl deriv-
atives, we could achieve the synthesis of the b-config-
ured analogues41 under very mild conditions using
photo-induced activation.
diethyl vinylphosphonate,41 the GlcNAc derivative
being obtained by a multi-step route, to afford C-glyco-
syl ethylphosphonates. The corresponding phosphonic
acids were then coupled to activated nucleotide mono-
phosphates in order to obtain the desired analogues
for enzymatic studies.
2.1. Synthesis of UDP-Glc and UDP-Gal analogues
(4a,b)
Reaction of commercially available acetobromoglycoses
1a,b with diethyl vinylphosphonate under photo-
induced conditions (Scheme 1) yielded the desired a-
configured C-glycosyl ethylphosphonates 2a,b.41 Depro-
tection49 of the diethylphosphonates upon treatment
with Me3SiBr in MeCN followed by deacetylation
(H2O/MeOH/Et3N) and C18-reverse phase column chro-
matography afforded the fully deprotected C-glycosyl
ethylphosphonic acids 3a,b in high yields. Final coupling
of 3a,b and UMP-morpholidate50,51 provided the de-
sired analogues 4a,b after purification52 by ion exchange
resin chromatography using DEAE-Sephadex A-25
Based on our previously developed radical chemistry,41
we are now reporting on the design of donor substrate
analogues, with a modification at the diphosphate link-
age (Fig. 2). These compounds are not isosteric to natu-
ral substrates due to the replacement of the oxygen atom
by an ethylene moiety, resulting in an increased distance
between the sugar anomeric centre and the phosphorus
atom. Therefore, it was assumed that these structures
would mimic the lengthening of the anomeric C–O bond
which occurs in the SN2-type transition state (Fig. 1) in-
volved for the natural substrate according to the pro-
posed mechanism.26
ꢀ
(HCO3 form).
2.2. Synthesis of GDP-L-Fuc analogue (8)
Preparation of the GDP-L-fucose analogue 8 was also
achieved through a similar procedure (Scheme 2). Acet-
ylation of L-fucose followed by conversion of 5 into L-
fucosyl bromide53 and subsequent treatment under pho-
tochemical conditions41 in the presence of diethyl vinyl-
phosphonate afforded the L-fucosyl ethylphosphonates
in good yield, as a mixture of anomers predominantly
a (a/b, 9:1).47 The a-anomer 6 could be easily purified
2. Results and discussion
The preparation of three C-glycosyl analogues (D-Glc,
D-Gal and L-Fuc) of NDP sugar was achieved through
photo-induced radical addition of glycosyl bromides to
O
O
O
O
NH
NH
O
P
O
P
O
P
O
P
N
O
N
O
O
O
O
O
O
O
O
O
O
O
O
Mn2+
Mn2+
OH OH
OH OH
C-glycosyl ethylphosphonophosphates
(analogues)
NDP sugar
(natural substrates)
Figure 2. Glycosyltransferase natural substrates and C-glycosyl ethylphosphonophosphate analogues.

S. Vidal et al. / Bioorg. Med. Chem. 14 (2006) 7293–7301
7295
R2
OAc
O
R2
OAc
O
R1
AcO
R1
AcO
b
a
O
P
AcO
AcO
OEt
OEt
Br
1a R1 = OAc, R2 = H
1b R1 = H, R2 = OAc
2a R1 = OAc, R2 = H
2b R1 = H, R2 = OAc
R2
OH
O
O
R1
NH
OH
O
O
P
R2
HO
OH
P
2 Et3NH+
O
R1
HO
N
O
O
O
O
c
O
O
O
O
P
OH
2 Et3NH+
O
OH OH
4a R1 = OH, R2 = H
4b R1 = H, R2 = OH
3a R1 = OH, R2 = H
3b R1 = H, R2 = OH
Scheme 1. Synthesis of C-glycosyl ethylphosphonophosphate analogues of UDP-Glc 4a and UDP-Gal 4b. Reagents: (a) diethyl vinylphosphonate,
NaBH3CN, n-Bu3SnCl, AIBN, t-BuOH, hm, 72–75%; (b) Me3SiBr, C5H5N, MeCN then MeOH, H2O, Et3N, 92–94%; (c) 4-morpholine-N,N0-
dicyclohexylcarboxamidinium uridine 50-monophosphomorpholidate, tetrazole, C5H5N, 10–24%.
O
OEt
P
OEt
OH
OH
OAc
OAc
a
b
Me
Me
Me
O
O
O
OAc
HO
OAc
OAc
c
HO
AcO
AcO
5
6
O
N
O
P
O
O
N
NH
O
P
O
P
2Et3NH+
N
NH2
O
O
Me
d
O
O
O
O
OH
Me
O
OH
2Et3NH+
OH
OH OH
HO
7
OH
8
HO
Scheme 2. Synthesis of GDP-L-fucose analogue 8. Reagents and conditions: (a) Ac2O, C5H5N, quantitative; (b) HBr/AcOH, CH2Cl2 then diethyl
vinylphosphonate, NaBH3CN, n-Bu3SnCl, AIBN, t-BuOH, hm, 59%, a/b 9:1; (c) Me3SiBr, C5H5N, MeCN then MeOH, H2O, Et3N, 89%; (d) 4-
morpholine-N,N0-dicyclohexylcarboxamidinium guanosine 50-monophosphomorpholidate, tetrazole, C5H5N, 24%.
by silica gel chromatography and its stereochemistry
was assigned by a detailed analysis of the 1H NMR data.
The observed coupling constant between H-1 and H-2
(3J1,2 = 5.6 Hz) is in agreement with the a-configuration
at the anomeric centre as noted for similar C-glycosyl
derivatives.54 Deprotection49 of the diethylphosphonate
followed by deacetylation provided the fully deprotected
phosphonic acid 7. Coupling with GMP-morpholi-
date50,51 afforded the desired analogue 8. As compared
to uracil derivatives 4a,b and 10, we noticed a relatively
poor stability of analogue 8 resulting in partial decom-
position into GMP and 7 when stored for 2 weeks at
ꢁ+4 ꢁC. Slow decomposition is probably due to the sen-
sitivity of the P–O–P–O linkage and the increased basici-
ty and nucleophilicity of the guanine moiety, in
comparison to uracil.
ro- or bromo-activated N-acetylglucosamine derivative
A (Fig. 3) were subjected to coupling under our standard
conditions41 in the presence of a large excess of diethyl
vinylphosphonate (up to 15 equiv), the major compound
isolated after irradiation was the oxazoline B in >80%
yield. Despite several attempts using acetamide or triflu-
oroacetamide derivatives, we were not able to obtain the
expected C-glycosyl ethylphosphonate C, even though
Junker and Fessner47 previously reported the radical
coupling of the glucosamine trifluoroacetamide deriva-
tive with diethyl vinylphosphonate. Radical reactions
in the GlcNAc series being highly sensitive to different
parameters,42,46 one can therefore assume that using dif-
ferent conditions (Et2O/5 · 50 W halogen lamps under
Fessner’s conditions47 and t-BuOH/450 W UV lamp in
our case) would greatly alter the outcome of the
reaction.
2.3. Synthesis of UDP-GlcNAc analogue (10)
Nevertheless, the protected GlcNAc analogue could be
prepared through a multi-step approach55,56 and cou-
pling of the corresponding phosphonic acid 9 with
Synthesis of the GlcNAc-based analogue 10 using the
radical approach was ineffective. When either the chlo-

7296
S. Vidal et al. / Bioorg. Med. Chem. 14 (2006) 7293–7301
OAc
O
AcO
AcO
N
OAc
O
O
Activation
AcO
AcO
B
Me
(X = Cl, Br)
AcHN
X
OAc
A
O
AcO
AcO
O
P
O
P
OEt
OEt
AcHN
OEt
OEt
C
Figure 3. Attempted syntheses of GlcNAc-based C-glycosyl ethylphosphonates using photo-induced radical conditions.
UMP-morpholidate afforded the desired GlcNAc C-gly-
cosyl ethylphosphonophosphate analogue 10 (Scheme
3).
two other enzymes studied (vide infra), and compared
to the corresponding Km values reported in the literature
(Table 1). In each case, a fairly good agreement was ob-
served, thus proving the validity of the applied method.
The IC50 value of 40 lM observed for 4b is similar to the
Km value58 for the natural donor substrate, UDP-Gal
(Table 1). This compound is therefore a good inhibitor
of b-1,4-GalT. It is noteworthy that non-isosteric a-
and b-configured analogues of UDP-Gal displaying an
extra methylene between the sugar and UDP have no
inhibitory activity against recombinant pig a(1–3)-galac-
tosyltransferase (pa(1–3)GalT).59 On the other hand, the
C-glycosyl ethylphosphonic acid 3b, evaluated in the
same conditions up to 16 mM concentration, did not
display any inhibitory activity towards b-1,4-GalT, thus
The formation of the P–O–P bond in C-glycosyl ethyl-
phosphonophosphate analogues 4a,b, 8 and 10 could
be easily evidenced by 31P NMR spectroscopy display-
ing two doublets resonating at d ꢁ ꢀ10 (phosphate)
2
and d ꢁ 19 ppm (phosphonate) with a JP,P = 26 Hz.
The presence of the desired phosphonophosphates in
the crude reaction mixtures was checked by 31P · 31P
2D COSY NMR analysis with observation of the
expected correlation between these two doublets.
3. Biological evaluations
3.1. Inhibitory activity of UDP-Gal analogue (4b)
Table 1. Inhibitory activities (IC50) of NDP-sugar analogues 4b, 8 and
10
Compound 4b is a non-isosteric a-configured analogue
of the sugar nucleotide donor UDP-Gal, the natural
substrate of bovine milk b-1,4-galactosyltransferase (b-
1,4-GalT, EC 2.4.1.22). This inverting enzyme catalyzes
the transfer, with the b-configuration, of a galactose unit
from UDP-Gal to the 4-position of a GlcNAc residue.
Human b-1,4-GalT plays a role in the biosynthesis of
blood group antigens and sialyl Lewis x (sLex) but is
also involved in some pathologies such as arthritis and
cancer.31 The biological evaluation of 4b towards bovine
milk b-1,4-GalT was carried out through a competition
assay using a fluorescent acceptor substrate, GlcNAc-b-
O-(CH2)6-NH-dansyl,57 where the reaction product was
quantified by RP-HPLC. Km constants were measured
for bovine milk b-1,4-galactosyltransferase and the
a
b
Glycosyltransferases
Km
Km
Analogues
IC50
(lM)
(lM)
b-1,4-GalT
FUT3
LgtA
4458
3361
22062
51
43
4b
8
40 lMc
2 mMd
3.5 mMe
540
10
a Km values previously reported in the literature.
b Km values measured with our enzymatic assay using fluorescent
acceptor substrates, under similar experimental conditions as
described for the measurement of the IC50 values of 4b, 8 and 10.
c Measured at 0.2 mM dansylated acceptor substrate and 0.04 mM
UDP-Gal.
d Measured at 0.2 mM dansylated acceptor substrate and 0.05 mM
GDP-Fuc.
e Measured at 0.8 mM dansylated acceptor substrate and 0.25 mM
UDP-GlcNAc.
OH
O
OH
O
O
HO
HO
HO
HO
a
NH
O
P
O
P
O
P
AcHN
O
O
AcHN
N
O
O
O
O
O
O
10
9
2Et3NH+
2Et3NH+
OH OH
Scheme 3. Synthesis of UDP-GlcNAc analogue 10. Reagents: (a) 4-morpholine-N,N0-dicyclohexylcarboxamidinium uridine 50-monophosphomor-
pholidate, tetrazole, C5H5N, 13%.

S. Vidal et al. / Bioorg. Med. Chem. 14 (2006) 7293–7301
7297
indicating that the nucleotide moiety of UDP-Gal ana-
logues cannot be omitted without altering the inhibitory
activity.
afforded three C-glycosyl ethylphosphonophosphate
analogues of glycosyltransferase donor substrates (4a
for UDP-Glc, 4b for UDP-Gal and 8 for GDP-Fuc),
the UDP-GlcNAc analogue 10 being obtained through
a multi-step sequence. The radical-based approach al-
lowed for the syntheses of C-glycosyl ethylphosphonates
with high diastereoselectivity in favour of the a-anomer.
Biological evaluations of UDP-Gal, GDP-Fuc and
UDP-GlcNAc analogues (4b, 8 and 10, respectively)
were achieved using a fluorescence assay and IC50 values
of 40 lM, 2 mM and 3.5 mM were determined for b-1,4-
GalT, FUT3 and LgtA, respectively. Based on these re-
sults, the replacement of the O–P oxygen–phosphorus
bond in the natural donor substrates by C–C–P car-
bon–carbon–phosphorus bonds led to non-isosteric ana-
logues displaying weak inhibitory activities, except for
UDP-Gal analogue 4b which turned out to be an inter-
esting candidate for b-1,4-galactosyltransferase inhibi-
tion, at least in vitro. Further investigations of the
biological properties of these glycoconjugates are being
pursued in our efforts to better understand the mecha-
nism of action of glycosyltransferases and their biologi-
cal implications.
3.2. Inhibitory activity of GDP-Fuc analogue (8)
The inhibitory activity of GDP-Fuc analogue 8 towards
recombinant human Galb(1 ! 3/4)GlcNAc-a-1,4/3-
fucosyltransferase60 (FUT3, EC 2.4.1.65) was deter-
mined. This enzyme catalyzes the transfer of a fucose
unit from GDP-Fuc, with the a-configuration, to the
3- or 4-position of a GlcNAc residue belonging either
to disaccharide acceptors Galb(1 ! 3)GlcNAc or
Galb(1 ! 4)GlcNAc. Inhibition tests were similarly car-
ried out through a competition assay using a fluorescent
acceptor substrate, Galb(1 ! 3)GlcNAc-b-O-(CH2)6-
NH-dansyl,57 in which the reaction product was quanti-
fied by RP-HPLC. An IC50 value of 2 mM, about 2 or-
ders of magnitude higher than the Km for GDP-Fuc,61
was found for GDP-Fuc analogue 8 (Table 1). The weak
inhibitory activity of 8 against FUT3 could be due to the
a-anomeric configuration of this C-glycosyl analogue,
opposite to the b-configuration of the natural donor
substrate GDP-Fuc, although an a-anomer analogue
of GDP-Fuc has been reported to inhibit significantly
human fucosyltransferases,31 even though their natural
substrates possess the opposite b-configuration. The
elongation of the distance between the guanosine and
the sugar moiety in 8 can also account for the lower inhi-
bition observed.12
5. Materials and methods
5.1. General methods
tert-Butanol was distilled over CaH2 and under argon
atmosphere. Diethyl vinylphosphonate (97% grade)
and NaBH3CN (95% grade) were purchased from Al-
drich and used as received. Air was removed from the
reaction media by bubbling argon into organic solu-
tions. Thin-layer chromatography (TLC) was carried
out on aluminium sheets coated with silica gel 60 F254
(Merck). TLC plates were inspected under UV light
and developed by charring after spraying with 5%
H2SO4 in EtOH. Preparative C18-reversed phase chro-
3.3. Inhibitory activity of UDP-GlcNAc analogue (10)
The inhibitory activity of UDP-GlcNAc analogue
10 against recombinant Neisseria meningitidis
Galb(1 ! 4)Glc-b-1,3-N-acetyl-glucosaminyltransferase57
(LgtA, EC 2.4.1.56) was measured according to the same
procedure with Galb(1 ! 4)Glcb-O-(CH2)6-NH-dan-
syl57 as the fluorescent acceptor substrate. This enzyme
catalyzes the transfer of a GlcNAc residue from the su-
gar nucleotide donor UDP-GlcNAc to the 3-position of
the terminal galactose of a lactose unit. With a IC50 val-
ue of 3.5 mM that is about 1 order of magnitude higher
than the Km value for UDP-GlcNAc,62 the analogue 10
turned out to be only a poor inhibitor towards the bac-
terial enzyme LgtA (Table 1), unlike the analogue 4b
that exhibited a significant inhibitory activity against
the bovine b-1,4-GalT. Considering that both glyco-
syltransferases are inverting enzymes that require man-
ganese ions for proper activity, a different spatial
arrangement of the amino acid residues involved in the
enzyme active sites may account for this discrepancy.
Other elongated non-isosteric compounds have been
shown to be weaker inhibitors of UDP-GlcNAc 2-epi-
merase, as compared to analogues which closely resem-
ble UDP-GlcNAc.29,33
matography (RP-18) was performed using
a
15 · 15 mm column of fully endcapped silica gel 100
C18 (>400 mesh, Fluka). Ion exchange chromatography
was performed using a 25 · 200 mm column of DEAE-
Sephadex A25 resin (HCO3 form, Pharmacia). 1H
ꢀ
and 13C NMR spectra were recorded using Bruker
AC200, DRX300 or DRX500 spectrometers with the
residual solvent as the internal standard.63 The chemical
shifts are expressed on the d scale in parts per million
(ppm). 31P NMR spectra were recorded with a Bruker
spectrometer at 80 MHz (AC200) or 120 MHz
(DRX300) from D2O or CDCl3 solutions with H3PO4
as the external reference. The following abbreviations
are used to explain the observed multiplicities: s, singlet;
d, doublet; dd, doublet of doublets; ddd, doublet of dou-
blet of doublets; t, triplet; q, quadruplet; m, multiplet;
br, broad. NMR solvents were purchased from Euriso-
top (Saint Aubin, F-91194, France). Mass spectra were
measured with a Finnigan Mat95XL spectrometer.
Optical rotations were measured using a Perkin-Elmer
polarimeter. Reversed phase HPLC (RP-HPLC) analy-
ses were performed using a Waters 600 pump equipped
with a reversed phase column (C18, Nucleosil, 5 lm,
3.9 mm id · 300 mm; mobile phase, H2O–MeCN:
4. Conclusion
A photo-induced addition of glycosyl radicals to diethyl
vinylphosphonate followed by deprotection and
coupling with activated nucleotide monophosphates

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S. Vidal et al. / Bioorg. Med. Chem. 14 (2006) 7293–7301
73:27; flow rate, 1 mL/min), with a Luminescence Spec-
trometer LS50B (Perkin-Elmer) as the detection device.
Fluorescence of substrates and products was read at 385
nm excitation/540 nm emission. b-1,4-GalT was pur-
chased from Sigma. LgtA64 and FUT360 were prepared
according to previously described procedures.52 Struc-
ture elucidation was deduced from 1D and 2D NMR
spectroscopy which allowed, in most cases, complete sig-
nal assignments based on COSY, HSQC and HMBC
correlations. The glycopyranosyl rings existed in all
NCH2CH3), 1.30–1.85 (m, 4H, CH2CH2P), 3.08 (q, 6H,
J = 7.3 Hz, NCH2CH3), 3.57–3.65 (m, 3H, H-5, H-6, H-
60), 3.70 (dd, 1H, J2,1 = 3.4 Hz, J2,3 = 9.3 Hz, H-2),
3.83–3.94 (m, 3H, H-1, H-3, H-4). 13C NMR (D2O,
75 MHz): d 8.6 (NCH2CH3), 18.8 (d, JC,P = 2.8 Hz,
CH2CH2P), 24.3 (d, JC,P = 135.0 Hz, CH2CH2P), 47.0
(NCH2CH3), 61.6 (C-6), 68.7 (C-2), 69.5, 70.0 (2s, C-3,
C-4), 72.0 (C-5), 76.2 (d, JC,P = 16.6 Hz, C-1). 31P NMR
(D2O, 80 MHz): d 26.2. LSIMS (negative mode, glycerol)
m/z: 271 [Mꢀ2HNEt3+H]ꢀ. HRLSIMS (negative mode,
glycerol) m/z: calcd for C8H16O8P1 [Mꢀ2HNEt3+H]ꢀ,
271.0583; found, 271.0584.
1
cases under a chair conformation (4C1 or C4), as de-
duced from the observed values of the vicinal H–H cou-
pling constants. Abbreviations Glc, Gal, Fuc, Rib and
Ura were used for the NMR assignments of D-glucose,
D-galactose, L-fucose, D-ribose and uracil protons and
carbons, respectively.
5.4. General procedure for the synthesis of 4a,b, 8 and 10
A water solution of 3a,b, 7 or 9 was evaporated to dryness
then dried under high vacuum over P2O5 for 24 h. The
morpholidate-activated nucleotide monophosphate and
tetrazole were added. The solvent was evaporated off
and the mixture dried under high vacuum over P2O5 for
24 h. The solid residue was dissolved in anhydrous
C5H5N (10 mL) and stirred under argon at room temper-
ature for 10 days. The reaction mixture was diluted with
H2O (100 mL) then evaporated to dryness and the yellow
powder purified (Sephadex DEAE-A25 HCO3ꢀ form/0 to
1 M TEAB) to obtain 4a,b, 8 or 10 as white foams.
5.2. Bis(triethylammonium) 2-(a-D-glucopyranosyl)-eth-
ylphosphonate (3a)
To a solution of 2a41 (1.854 g, 3.73 mmol) in MeCN
(40 mL) was added C5H5N (3.12 mL, 37.88 mmol) at
0 ꢁC followed by dropwise addition of Me3SiBr (5 mL,
37.88 mmol). The solution was stirred at 0 ꢁC for 2 h then
quenched with H2O/C5H5N (9:1/30 mL). The mixture
was evaporated to dryness and the white powder purified
(RP-18/H2O then H2O/MeOH, 1:1) to obtain the acety-
lated phosphonic acid intermediate. The product was stir-
red in H2O/MeOH/Et3N (7:3:1/30 mL) for 16 h then
evaporated to dryness and the white powder purified
(RP-18/H2O) to afford 3a (933 mg, 92%) as a colourless
foam. [a]D +37 (c 1, H2O). 1H NMR (D2O, 300 MHz): d
1.23 (t, 18H, J = 7.3 Hz, NCH2CH3), 1.38–1.92 (m, 4H,
CH2CH2P), 3.15 (q, 12H, J = 7.3 Hz, NCH2CH3), 3.28
(t, 1H, J4,3 = J4,5 = 9.8 Hz, H-4), 3.46 (ddd, 1H,
5.4.1. Bis(triethylammonium) [2-(a-D-glucopyranosyl)-
ethylphosphono]uridin-50-yl phosphate (4a). A mixture of
3a (509 mg, 1.87 mmol), 4-morpholine-N,N0-dic-
yclohexylcarboxamidinium uridine 50-monophospho-
morpholidate (470 mg, 0.68 mmol) and tetrazole
(16.2 mL, 7.27 mmol, 0.45 M in MeCN) was treated
according to the general procedure described above to ob-
tain 4a (95 mg, 24%). [a]D +18 (c 1, H2O). 1H NMR (D2O,
300 MHz): d 1.24 (t, 18H, J = 7.3 Hz, NCH2CH3), 1.58–
2.00 (m, 4H, CH2CH2P), 3.13 (q, 12H, J = 7.3 Hz,
NCH2CH3), 3.28–3.36 (m, 1H, H-4Glc), 3.52 (ddd, 1H,
0
J5;6 ¼ 2:2 Hz, J5,6 = 5.9 Hz, J5,4 = 9.8 Hz, H-5), 3.58–
3.72 (m, 3H, H-2, H-3, H-6), 3.82 (dd, 1H,
0
J6 ;6 ¼ 12:3 Hz, H-60), 3.94 (m, 1H, H-1). 13C NMR
0
(D2O, 75 MHz): d 9.1 (NCH2CH3), 19.4 (d, JC,P = 3.4 Hz,
CH2CH2P), 24.4 (d, JC,P = 174.2 Hz, CH2CH2P), 47.5
(NCH2CH3), 62.0 (C-6), 71.3 (C-4), 72.1, 74.0 (2s, C-2,
C-3), 73.2 (C-5), 77.0 (d, JC,P = 16.5 Hz, C-1). 31P NMR
(D2O, 80 MHz): d 26.2. LSIMS (negative mode, glycerol)
m/z: 271 [Mꢀ2HNEt3+H]ꢀ. HRLSIMS (negative mode,
glycerol) m/z: calcd for C8H16O8P1 [Mꢀ2HNEt3+H]ꢀ,
271.0583; found, 271.0584.
J5,4 = 9.7 Hz, J5,6 = 5.7 Hz, J5;6 ¼ 2:2 Hz, H-5Glc),
3.62–3.75 (m, 3H, H-2Glc, H-3Glc, H-6Glc), 3.84 (dd, 1H,
0
0
J6 ,6 = 12.3 Hz, H-6 Glc), 3.92–4.03 (m, 1H, H-1Glc),
4.15–4.21 (m, 2H, H-5Rib, H-50Rib), 4.21–4.28 (m, 1H, H-
4Rib), 4.30–4.39 (m, 2H, H-2Rib, H-3Rib), 5.88 (d, 1H,
J = 7.8 Hz, H-5Ura), 6.00 (d, 1H, J1,2 = 4.6 Hz, H-1Rib),
7.83 (d, 1H, J = 7.7 Hz, H-6Ura). 13C NMR (D2O,
125 MHz): d 8.7 (NCH2CH3), 18.9 (d, JC,P = 3.0 Hz,
CH2CH2P), 24.1 (d, JC,P = 139.5 Hz, CH2CH2P), 47.0
(NCH2CH3), 61.6 (C-6Glc), 65.1 (d, JC,P = 5.3 Hz, C-
5Rib), 70.0, 74.1 (C-2Rib, C-3Rib), 70.9 (C-4Glc), 71.7 (C-
2Glc), 72.7 (C-5Glc), 73.6 (C-3Glc), 76.5 (d, JC,P = 17.5 Hz,
C-1Glc), 83.6 (d, JC,P = 9.1 Hz, C-4Rib), 88.8 (C-1Rib),
103.0 (C-5Ura), 142.1 (C-6Ura), 152.1 (C-2Ura), 166.5 (C-
5.3. Bis(triethylammonium) 2-(a-D-galactopyranosyl)-
ethylphosphonate (3b)
To a solution of 2b41 (668 mg, 1.34 mmol) in MeCN
(20 mL) was added C5H5N (1.11 mL, 13.4 mmol) at
0 ꢁC followed by Me3SiBr (1.8 mL, 13.4 mmol). The solu-
tion was stirred at 0 ꢁC for 1 h then quenched with H2O/
C5H5N (9:1/10 mL). The mixture was evaporated to dry-
ness and the white powder purified (RP-18/H2O then
H2O/MeOH, 1:1) to obtain the acetylated phosphonic
acid intermediate. The product was stirred in H2O/
MeOH/Et3N (7:3:1/30 mL) for 16 h then evaporated to
dryness and the white powder purified (RP-18/H2O) to af-
ford 3b (342 mg, 94%) as a colourless foam and as its
4Ura). 31P NMR (D2O, 80 MHz):
d
ꢀ10.8 (d,
JP,P = 26.3 Hz, CPOPO), 19.6 (d, JP,P = 26.3 Hz, CPO-
PO). LSIMS (negative mode, glycerol) m/z: 577
[Mꢀ2HNEt3+H]ꢀ. HRLSIMS (negative mode, glycerol)
m/z: calcd for C17H27N2O16P2 [Mꢀ2HNEt3+H]ꢀ,
577.0836; found, 577.0834.
5.4.2. Bis(triethylammonium) [2-(a-D-galactopyranosyl)-
ethylphosphono]uridin-50-yl phosphate (4b). A mixture of
3b (177 mg, 0.65 mmol), 4-morpholine-N,N0-dicyclo-
hexylcarboxamidinium uridine 50-monophosphomor-
1
mono-triethylammonium salt. [a]D +53 (c 1, H2O). H
NMR (D2O, 300 MHz): d 1.17 (t, 9H, J = 7.3 Hz,

S. Vidal et al. / Bioorg. Med. Chem. 14 (2006) 7293–7301
7299
pholidate (250 mg, 0.36 mmol) and tetrazole (5.6 mL,
2.53 mmol, 0.45 M in MeCN) was treated according to
the general procedure described above to obtain 4b
J4,5 = 1.5 Hz, H-4), 5.14 (dd, 1H, J1,2 = 5.6 Hz, H-2).
13C NMR (CDCl3, 75 MHz): d 15.7 (C-6), 16.1 (d,
JC,P = 5.8 Hz, POCH2CH3), 18.3 (d, JC,P = 3.8 Hz,
CH2CH2P), 20.3, 20.4 (2s, CH3CO), 21.3 (d,
JC,P = 136.6 H z, CH2CH2P), 61.3, 61.4 (2d,
JC,P = 6.3 Hz, POCH2CH3), 65.1 (C-5), 67.6 (C-2),
68.0 (C-3), 70.2 (C-4), 72.1 (d, JC,P = 16.9 Hz, C-1),
169.4, 169.6, 170.0 (3s, CH3CO). 31P NMR (CDCl3,
80 MHz): d 31.8. LSIMS (positive mode, glycerol) m/z:
439 [M+H]+, 461 [M+Na]+. HRLSIMS (positive mode,
glycerol) m/z: calcd for C18H32O10P1 [M+H]+, 439.1733;
found, 439.1732.
1
(21 mg, 10%). [a]D +39 (c 1.05, H2O). H NMR (D2O,
300 MHz): d 1.23 (t, 18H, J = 7.3 Hz, NCH2CH3),
1.30–2.00 (m, 4H, CH2CH2P), 3.10 (q, 12H,
J = 7.3 Hz, NCH2CH3), 3.66–3.75 (m, 3H, H-5Gal, H-
6Gal, H-60Gal), 3.80 (dd, 1H, J3,4 = 3.4 Hz, J3,2 = 9.4 Hz,
H-3Gal), 3.90–3.93 (m, 1H, H-4Gal), 3.94–4.04 (m, 2H,
H-1Gal H-2Gal), 4.10–4.20 (m, 2H, H-5Rib, H-50Rib),
4.20–4.26 (m, 1H, H-4Rib), 4.29–4.38 (m, 2H, H-2Rib
,
H-3Rib), 5.87 (d, 1H, J = 7.7 Hz, H-5Ura), 6.00 (d, 1H,
J1,2 = 4.6 Hz, H-1Rib), 7.82 (d, 1H, J = 7.7 Hz, H-6Ura).
13C NMR (D2O, 125 MHz): d 8.6 (NCH2CH3), 18.7
(d, JC,P = 2.4 Hz, CH2CH2P), 24.3 (d, JC,P = 139.4 Hz,
CH2CH2P), 47.1 (NCH2CH3), 61.6 (C-6Gal), 65.1 (d,
JC,P = 5.2 Hz, C-5Rib), 68.7 (C-2Gal), 69.5 (C-4Gal),
69.99 (C-3Gal), 70.02, 74.2 (C-2Rib, C-3Rib), 72.0 (C-
5Gal), 76.1 (d, JC,P = 18.1 Hz, C-1Gal), 83.7 (d,
JC,P = 9.0 Hz, C-4Rib), 88.8 (C-1Rib), 103.0 (C-5Ura),
142.1 (C-6Ura), 152.2 (C-2Ura), 166.6 (C-4Ura). 31P
NMR (D2O, 120 MHz): d ꢀ10.3 (d, JP,P = 26.8 Hz,
CPOPO), 20.3 (d, JP,P = 26.8 Hz, CPOPO). LSIMS
(negative mode, glycerol) m/z: 577 [Mꢀ2HNEt3+H]ꢀ.
HRLSIMS (negative mode, glycerol) m/z: calcd for
C17H27N2O16P2 [Mꢀ2HNEt3+H]ꢀ, 577.0836; found,
577.0832.
5.4.4. Bis(triethylammonium) 2-(a-L-fucopyranosyl)-eth-
ylphosphonate (7). To
a solution of 6 (630 mg,
1.44 mmol) in MeCN (20 mL) was added C5H5N
(1.18 mL, 14.42 mmol) at 0 ꢁC followed by Me3SiBr
(1.90 mL, 14.42 mmol). The solution was stirred at
0 ꢁC for 1 h then quenched with H2O/C5H5N (9:1/
15 mL). The mixture was evaporated to dryness and
the white powder purified (RP-18/H2O then H2O/
MeOH, 1:1) to obtain the acetylated phosphonic acid
intermediate. The product was stirred in H2O/MeOH/
Et3N (7:3:1/25 mL) for 16 h then evaporated to dryness
and the white powder purified (RP-18/H2O) to afford 7
(459 mg, 89%) as a colourless foam and as its mono-tri-
ethylammonium salt. [a]D ꢀ62 (c 1, H2O). 1H NMR
(D2O, 300 MHz): d 1.03 (d, 3H, J6,5 = 6.4 Hz, H-6),
1.12 (t, 9H, J = 7.3 Hz, NCH2CH3), 1.20–1.82 (m, 4H,
CH2CH2P), 3.03 (q, 6H, J = 7.3 Hz, NCH2CH3), 3.59–
3.84 (m, 5H, H-1 to H-5). 13C NMR (D2O, 75 MHz):
d 8.6 (NCH2CH3), 16.2 (C-6), 18.8 (d, JC,P = 2.1 Hz,
CH2CH2P), 24.6 (d, JC,P = 134.8 Hz, CH2CH2P), 46.9
(NCH2CH3), 67.2, 68.3, 70.1, 72.1 (4s, C-2 to C-5),
76.6 (d, JC,P = 17.1 Hz, C-1). 31P NMR (D2O,
80 MHz): d 25.9. LSIMS (negative mode, glycerol)
m/z: 255 [Mꢀ2HNEt3+H]ꢀ. HRLSIMS (negative mode,
glycerol) m/z: calcd for C8H16O7P1 [Mꢀ2HNEt3+H]ꢀ,
255.0634; found, 255.0631.
5.4.3. Diethyl 2-(2,3,4-tri-O-acetyl-a-L-fucopyranosyl)-
ethylphosphonate (6).
A
solution of 553 (1.5 g,
4.52 mmol) and HBr (5 mL, 33% wt in AcOH) in
CH2Cl2 (5 mL) was stirred at 0 ꢁC for 10 min, then
warmed to rt and stirred for an additional 45 min. The
mixture was poured into saturated NaHCO3 (100 mL)
and extracted with CH2Cl2 (2 · 100 mL). The organic
layers were combined, dried (Na2SO4), filtered and
evaporated to dryness. The crude orange gum was used
for the next step without further purification. A solution
of the crude fucosyl bromide (1.6 g, 4.52 mmol),
NaBH3CN (426 mg, 6.77 mmol), AIBN (445 mg,
2.71 mmol), n-Bu3SnCl (370 lL, 1.35 mmol) and diethyl
vinylphosphonate (3.5 mL, 22.6 mmol) in t-BuOH
(80 mL) was introduced into a quartz tube, then de-
gassed (argon) for 15 min and finally irradiated for 5 h
with a medium pressure mercury lamp (Hanovia/
450 W) equipped with a Vycor filter (k > 254 nm) and
inserted in a two-wall jacket made of quartz with water
flow for cooling. The mixture was evaporated to dryness
and purified (SiO2/petroleum ether then EtOAc, then
EtOAc/MeOH, 95:5) to afford the pure a-anomer
(718 mg) and a second crop (450 mg) of a 77:23 mixture
of a/b-anomers, respectively, as determined by 1H
NMR. Compound 6 (1.17 g) was obtained in 59% total
yield with an a/b global ratio of 9:1. The a-anomer was
used for characterization and the next synthetic steps.
5.4.5. Bis(triethylammonium) [2-(a-L-fucopyranosyl)-eth-
ylphosphono]guanosin-50-yl phosphate (8). A mixture of 7
(114 mg, 0.44 mmol), 4-morpholine-N,N0-dicyclohexyl-
carboxamidinium guanosine 50-monophosphomorpholi-
date (90 mg, 0.12 mmol) and tetrazole (2.8 mL,
1.24 mmol, 0.45 M in MeCN) was treated according to
the general procedure described above to obtain 8
(17.6 mg, 24%) as 1:3 mixture with excess TEAB salts.
1H NMR (D2O, 300 MHz): d 1.14 (d, 3H, J6,5 = 6.4 Hz,
H-6Fuc), 1.31 (t, 18H, J = 7.3 Hz, NCH2CH3), 1.50–2.00
(m, 4H, CH2CH2P), 3.50 (q, 12H, J = 7.3 Hz,
NCH2CH3), 3.68–3.83 (m, 4H, H-2Fuc to H-5Fuc), 3.90–
3.99 (m, 2H, H-1Fuc, H-5Rib), 4.16–4.23 (m, 2H, H-4Rib
H-50Rib), 4.30–4.38 (m, 1H, H-3Rib), 4.52 (dd, 1H,
J2,1 = 6.0 Hz, J2,3 = 3.3 Hz, H-2Rib), (d, 1H,
,
1
[a]D ꢀ70 (c 1, CHCl3). H NMR (CDCl3, 300 MHz): d
J1,2 = 6.0 Hz, H-1Rib), 8.13 (s, 1H, H-8Gua). 13C NMR
(D2O, 125 MHz): d 15.8 (C-6), 18.4 (d, JC,P = 3.4 Hz,
CH2CH2P), 24.1 (d, JC,P = 140.0 Hz, CH2CH2P), 65.3
(d, JC,P = 5.2 Hz, C-5Rib), 66.9 (C-5Fuc), 70.4 (C-3Rib),
68.2, 69.9, 71.9 (C-2Fuc to C-4Fuc), 73.9 (C-2Rib), 76.3 (d,
JC,P = 17.2 Hz, C-1Fuc), 83.7 (d, JC,P = 8.6 Hz, C-4Rib),
87.3 (C-1Rib), 137.4 (C-8Gua), 151.4, 153.8, 157.8, 158.5
(C-2Gua, C-4Gua, C-5Gua, C-6Gua). 31P NMR (D2O,
0.98 (d, 3H, J6,5 = 6.4 Hz, H-6), 1.17 (dt, 6H,
JCH ;CH ¼ 7:3 Hz, JCH ;P ¼ 1:5 Hz, POCH2CH3), 1.40–
2
3
3
1.91 (m, 4H, CH2CH2P), 1.82, 1.89, 1.98 (3s, 3· 3H,
CH3CO), 3.74 (dq, 1H, J5,4 = 1.5 Hz, J5,6 = 6.4 Hz, H-
5), 3.94 (qd, 4H, JCH ;CH ¼ 7:3 Hz, JCH ;P ¼ 2:7 Hz,
2
3
3
POCH2CH3), 3.90–4.00 (m, 1H, H-1), 5.01 (dd, 1H,
J2,3 = 10.1 Hz, J3,4 = 3.3 Hz, H-3), 5.08 (dd, 1H,

7300
S. Vidal et al. / Bioorg. Med. Chem. 14 (2006) 7293–7301
80 MHz): d ꢀ10.7 (d, JP,P = 26.2 Hz, CPOPO), 19.7 (d,
JP,P = 26.2 CPOPO). LSIMS (negative mode, glycerol)
m/z: 600 [Mꢀ2HNEt3+H]ꢀ. HRLSIMS (negative
mode, glycerol) m/z: calcd for C18H28N5O14P2
[Mꢀ2HNEt3+H]ꢀ, 600.1108; found, 600.1104.
and inhibitor 4b (1, 5, 10, 25, 50 and 100 lM) in 50 lL to-
tal volume. Assays were incubated at 30 ꢁC for 14 min.
5.7. Inhibition of Galb(1 ! 3/4)GlcNAc-a-1,4/3-fucosyl-
transferase (EC 2.4.1.65, FUT3)
5.4.6. Bis(triethylammonium) [2-(2-acetamido-2-deoxy-a-
D-glucopyranosyl)-ethylphosphono]uridin-50-yl phosphate
(10). A mixture of 956 (214 mg, 0.68 mmol), 4-morpho-
line-N,N0-dicyclohexylcarboxamidinium uridine 50-
monophosphomorpholidate (440 mg, 0.64 mmol) and
tetrazole (14.2 mL, 6.41 mmol, 0.45 M in MeCN) was
treated according to the general procedure described
above to obtain 10 (50 mg, 13%). [a]D +35 (c 1, H2O).
1H NMR (D2O, 300 MHz): d 1.22 (t, 18H, J = 7.3 Hz,
NCH2CH3), 1.47–1.94 (m, 4H, CH2CH2P), 1.99 (s, 3H,
CH3CONH), 3.11 (q, 12H, J = 7.3 Hz, NCH2CH3), 3.35
(t, 1H, J4,3 = J4,5 = 9.5 Hz, H-4Glc), 3.42–3.50 (m, 1H,
Enzyme assay conditions: 10 mM MnCl2, 25 mM caco-
dylate buffer (pH 6), 200 lM Galb(1 ! 3)GlcNAcb-O-
(CH2)6-NH-dansyl,57 50 lM GDP-Fuc, 0.2 mU FUT3
and inhibitor 8 (0.5, 1, 2, 4 and 8 mM) in 50 lL total
volume. Assays were incubated at 37 ꢁC for 9 min.
5.8. Inhibition of Galb(1 ! 4)Glc-b-1,3-N-acetyl-glucos-
aminyltransferase (EC 2.4.1.56, LgtA)
Enzyme assay conditions: 15 mM MnCl2, 50 mM caco-
dylate buffer (pH 7.2), 800 lM Galb(1 ! 4)Glcb-O-
(CH2)6-NH-dansyl,57 250 lM UDP-GlcNAc, 0.4 mU
LgtA and inhibitor 10 (1, 2, 4, 8 and 16 mM) in 50 lL
total volume. Assays were incubated at 37 ꢁC for 6 min.
0
H-5Glc), 3.67 (dd, 1H, J6,5 = 5.5 Hz, J6;6 ¼ 12:4 Hz, H-
6Glc), 3.73 (t, 1H, J3,4 = J3,2 = 9.5 Hz, H-3Glc), 3.83 (dd,
0
1H, J6 ;5 ¼ 1:5 Hz, H-60 ), 3.75–3.80 (m, 2H, H-1Glc, H-
Glc
2Glc), 4.05–4.15 (m, 2H, H-5Rib, H-50Rib), 4.15–4.20 (m,
1H, H-4Rib), 4.25–4.30 (m, 2H, H-2Rib, H-3Rib), 5.86 (d,
1H, J = 7.7 Hz, H-5Ura), 5.98 (d, 1H, J1,2 = 4.4 Hz, H-
1Rib), 7.81 (d, 1H, J = 7.7 Hz, H-6Ura). 13C NMR (D2O,
125 MHz): d 8.7 (NCH2CH3), 19.6 (d, JC,P = 3.0 Hz,
CH2CH2P), 22.3 (CH3CONH), 24.3 (d, JC,P = 139.6 Hz,
CH2CH2P), 47.0 (NCH2CH3), 53.8 (C-2Glc), 61.5 (C-
Acknowledgments
This research was supported by the University Claude-
Bernard Lyon 1, University Paris-Sud XI, CNRS (Cen-
tre National de la Recherche Scientifique) and the
French Ministry of Education and Research (stipend
to I.B.).
6Glc), 65.1 (d, JC,P = 5.3 Hz, C-5Rib), 70.0, 74.1 (C-2Rib
,
C-3Rib), 71.0 (C-3Glc), 71.4 (C-4Glc), 72.7 (C-5Glc), 74.5
(d, JC,P = 18.3 Hz, C-1Glc), 83.6 (d, JC,P = 9.1 Hz, C-
4Rib), 88.8 (C-1Rib), 103.0 (C-5Ura), 142.1 (C-6Ura), 152.1
(C-2Ura), 166.5 (C-4Ura), 174.8 (CH3CONH). 31P NMR
(D2O, 80 MHz): d ꢀ10.7 (d, JP,P = 26.4 Hz, CPOPO),
19.2 (d, JP,P = 26.4 Hz, CPOPO). LSIMS (positive
mode, glycerol) m/z: 620 [Mꢀ2HNEt3+3H]+, 721
[MꢀHNEt3+2H]+, 823 [M+H]+. LSIMS (negative mode,
glycerol) m/z: 618 [Mꢀ2HNEt3+H]ꢀ. HRLSIMS (nega-
tive mode, glycerol) m/z: calcd for C19H30N3O16P2
[Mꢀ2HNEt3+H]ꢀ, 618.1101; found, 618.1108.
References and notes
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¨
5.5. General procedure for the determination of IC50
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Solutions were prepared and incubated as indicated be-
low. Incubation was halted by immersion for 2 min in a
boiling water bath. The solution was diluted with water
(100 lL) and filtered. The samples were analyzed by RP-
HPLC using fluorescence detection. The percentage of
conversion was quantified from the fluorescence intensi-
ty of the peaks corresponding, respectively, to acceptor
substrate and product. A control reaction without inhib-
itor was run simultaneously and measurements for each
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