The design and synthesis of a selective inhibitor of fucosyltransferase VI.

Article abstract of DOI:10.1039/b317067e

Inversion of configuration of the C-2[prime or minute] hydroxyl of methyl N-acetyllactosamine was accomplished by a two-step procedure involving oxidation to a ketone followed by reduction with NaBH(4). After deprotection, the resulting derivative was examined as a substrate for [small alpha]-(2,6)- and [small alpha]-(2,3)-sialyltransferase and fucosyltransferase III, IV, V and VI. It was found that none of these enzymes could glycosylate. However, it showed exquisite selectivity for inhibition of fucosyltransferase VI. The kinetic data support an unusual mechanism in which the inhibitor can bind to the GDP-fucose complex as well as another enzyme form.

Full text of DOI:10.1039/b317067e

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The design and synthesis of a selective inhibitor of
fucosyltransferase VI
M. Carmen Galan,a Andre P. Venot,a Robert S. Phillipsb and Geert-Jan Boons*a,b
a Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens,
GA 30602, USA
b Department of Chemistry, University of Georgia, Athens, GA 30602, USA.
E-mail: gjboons@ccrc.uga.edu; Fax: ϩ1 706-5424412; Tel: ϩ1 706 5429161
Received 5th January 2004, Accepted 12th March 2004
First published as an Advance Article on the web 1st April 2004
Inversion of configuration of the C-2Ј hydroxyl of methyl N-acetyllactosamine was accomplished by a two-step
procedure involving oxidation to a ketone followed by reduction with NaBH4. After deprotection, the resulting
derivative 2 was examined as a substrate for α-(2,6)- and α-(2,3)-sialyltransferase and fucosyltransferase III, IV, V
and VI. It was found that none of these enzymes could glycosylate 2. However, it showed exquisite selectivity for
inhibition of fucosyltransferase VI. The kinetic data support an unusual mechanism in which the inhibitor can bind
to the GDP-fucose complex as well as another enzyme form.
accepts replacements and modification of the N-acetyl group of
Introduction
the GlcNAc unit.16–18
Protein- and lipid-bound oligosaccharides play critical roles in
a diverse range of biological processes such as embryogenesis,
Recently, we found that rat liver α-(2,6)- and α-(2,3)-sialyl-
transferase and fucosyltransferase III, V and VI allow modifi-
fertilization, neuronal development, hormonal activities, cell
cations at the C-2Ј and C-6 hydroxyl of LacNAc, however,
proliferation and cells’ organization into specific tissues.1,2 They
different enzymes responded differently.19 For example, methyl-
are also important in health science and are involved in the
invasion and attachment of pathogens, inflammation, meta-
ation of the C-6 and C-2Ј hydroxyls of LacNAc had only a
minimal effect on α-2,3-sialyltransferase, thus indicating that
stasis, and xenotransplantation. Not surprisingly, consider-
these hydroxyls make marginal interactions with the binding
able effort has been dedicated to the design and synthesis of
site. On the other hand, a significant loss of catalytic efficiency
inhibitors of carbohydrate processing enzymes.3,4 Remarkable
progress has been made in the development of glycosidase
was observed when the same substrate was employed for rat
liver α-2,6-sialyltransferase. Fucosyltransferase IV was not
affected by methylation of the C-6 and C-2 hydroxyls, whereas
the catalytic efficiency for α-1,3-fucosyltransferases VI was
slightly increased. These findings indicate that in some cases,
functionalities at C-6 and C-2Ј hydroxyl can make interactions
inhibitors and some of these compounds have entered advance
clinical trials.5 On the other hand, the search for glyco-
syltransferase inhibitors has been slow. This problem is mainly
due to intrinsic features of these enzymes such as a complex
four-partner transition state (sugar donor, acceptor, metal,
with the periphery of the binding site. It is to be expected that
nucleotide), weak binding of the enzyme with their natural sub-
the improved selectivities may be exploited in the design of
strates (usual Km values are in the mM or high µM range) and
limited structural data. Moreover, many aspects of the catalytic
mechanisms of glycosyltransferases are still unknown, thus
complicating the rational design of inhibitors. Nevertheless,
selective inhibitors for particular transferases.
To further explore the effect of modifying the C-2Ј hydroxyl
of LacNAc, we synthesized derivative 2, in which the C-2Ј
hydroxyl of LacNAc was epimerized (Fig. 1). It was found that
this modification abolished catalytic activity for α-(2,6)- and
α-(2,3)-sialyltransferase and fucosyltransferase III and V. Inter-
estingly, compound 2 is a remarkably selective inhibitor for the
α-1,3-fucosyltransferase VI. Detailed analysis of inhibition
several strategies for inhibitor design have been pursued with
some success. The most important approaches involve acceptor
and donor analogues, and transition state mimetics.6–10
N-Acetyllactosamine is a substrate for a wide range of glyco-
syltransferases. For example fucosyltransferase III, IV, V and
VI catalyze the transfer of a fucosyl residue from GDP-fucose
data revealed that 2 can bind to the GDP-fucose complex as
well as to another enzyme form.
to the C-3 hydroxyl of N-acetyllactosamine,11 whereas α-(2,6)-
sialyltransferase and α-(2,3)-sialyltransferase transfer an
N-acetyl neuraminic acid (Neu5Ac) moiety from CMP-
Neu5Ac to the C-6Ј and C-3Ј hydroxyl of LacNAc, respect-
ively.12 The substrate specificity of these enzymes has been
probed by using modified LacNAc derivatives. These studies
have revealed that the C-6Ј hydroxyl and acetamido group of
Fig. 1 Compounds 1 and 2.
LacNAc are essential for sialylation by rat liver α-(2,6)-sialyl-
transferase.13 Furthermore, glycosylation of the C-3Ј, C-4Ј and
C-3 hydroxyl lead to inactivation of the substrate. Other trans-
ferases have different acceptor requirements. For example,
Results and discussion
α-(2,3)-sialyltransferase requires the 3-OH, 4-OH and 6-OH of
Gal for recognition13 whereas all three fucosyltransferases
(FucTs III, IV and V) had an absolute requirement for a
hydroxyl at C-6 of galactose in addition to the accepting
hydroxyl at C-3 or C-4 of GlcNAc.14,15 Furthermore, FucT VI
Epimerization of the C-2Ј hydroxyl of 320 was achieved through
a two-step procedure involving oxidation to a ketone followed
by a stereoselective reduction. Thus, the acetyl group of 3 was
removed by treatment with sodium methoxide and methanol to
give compound 4 in an almost quantitative yield (Scheme 1).
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 3 7 6 – 1 3 8 0
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
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rate of transfer indicating that only this enzyme can recognize
compound 2.
To explore in detail the mode of inhibition, kinetic param-
eters were determined at different concentrations of 2 with
respect to both methyl LacNAc (1) and GDP-fucose. Concen-
trations of 2 were chosen in such a manner that the inhibition
was between 15–75% for accurate data collection. With respect
to methyl LacNAc (1), data were collected at 0, 0.2, and 0.4
mM, and fitted to the equations for competitive and mixed
inhibition with COMPO and NCOMP.24 The data were fitted
all at once without inhibitor present and with all inhibitor con-
centrations. As can be seen in Fig. 2, a mixed inhibition model
gave the best fit for the data of methyl LacNAc. From the data
shown in Fig. 2A, the Kis value was determined to be 0.193
0.056 mM and the Kii value was 0.382 0.116 mM. Compound
2 showed a weaker inhibitory effect with respect to GDP-fucose
(Fig. 3). The data do not easily distinguish between competitive
(dashed line) and mixed inhibition (solid line). However, the
best fit was for a mixed inhibition model (Fig. 3A). The double
reciprocal plot also showed better statistics for this mode of
inhibition (Fig. 3B). Fitting the data to COMPO24 gave a Ki
value of 0.475 0.096 mM.
Scheme 1 Reagents and conditions: i) NaOMe, MeOH; ii) Ac2O,
DMSO; iii) NaHB4, DCM/MeOH; iv) HBF4, MeCN; v) Pd(OH)2, H2,
EtOH.
The C-2Ј hydroxyl of 4 was oxidized using the procedure of
Albright and Goldman using DMSO and Ac2O.21 The resulting
ketone 5 was immediately reduced with sodium borohydride in
a mixture of dichloromethane and methanol to give, after puri-
fication by silica gel column chromatography, compound 6 in
an overall yield of 60%. Finally, removal of the TBDMS pro-
tecting group, using standard conditions followed by catalytic
hydrogenation over Pd(OH)2, gave the target compound 2. The
appearance of H-1Ј (4.58 ppm) and H-3Ј (3.74 ppm) as singlets
1
in the H spectra of compound 2 confirmed the axial orien-
tation of the C-2Ј hydroxyl.
Next, the apparent kinetic parameters for the α-2,3- and
α-2,6-sialyltransferase and fucosyltransferases III, IV, V and VI
catalyzed transfer of CMP-[14C]-Neu5Ac or GDP-[14C]-fucose
Fig. 2 Michaelis–Menten plot (A) and Lineweaver–Burk plot (B) for
LacNAc-OMe at different inhibitor concentrations. The lines are the
result of fitting the data to NCOMP. Competitive inhibition (dashed
line) and mixed inhibition (solid line) fits.
to acceptors
1
and
2
were determined using reported
Fucosyltransferases have been shown to obey an ordered,
sequential BiBi mechanism,25 with GDP-fucose being the first
substrate to bind to the free enzyme and GDP the last product
released. Normally, the acceptor sugar binds as the second
substrate exclusively to the GDP-fucose–enzyme complex.
However, the mixed inhibition observed for 2 when the con-
centration of methyl LacNAc was varied indicates that this
compound can bind to the GDP-fucose complex as well as to
another enzyme form. There is reasonable agreement in the
values of Ki (0.475 mM) and Kis (0.193 mM), which are the
apparent dissociation constants of 2 for binding to the free
enzyme. With respect to GDP-fucose, the best fit was for mixed
inhibition, which would indicate that 2 can also bind to the
E-GDP-Fuc complex substrate as well as the E-GDP product
assays.13,15,22,23 In each case, the Km for 1 was in close agreement
with previously reported data. Surprisingly, none of the
enzymes showed any activity when the modified compound 2
was used.
It may be possible that 2 can be recognized by the glyco-
syltransferases, but is unable to accept the monosaccharide
moiety from the sugar nucleotide. Thus, to investigate whether
2 exhibits inhibitory activity, relative rates of transfer were
measured at different concentrations of 2 (0 to 1.5 mM) while a
saturating concentration of GDP-fucose was used in combin-
ation with a fixed concentration of methyl LacNAc (1) that
corresponded to the Km value for each of the six enzymes.
Surprisingly, only FucT VI showed a decrease in the relative
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 3 7 6 – 1 3 8 0
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α-1,3-fucosyltransferase III, IV,
V
and VI. Surprisingly,
fucosyltransferase VI was the only enzyme that was inhibited
by 2. Detailed kinetic analysis revealed that the inhibition was
of a mixed type with respect to LacNAc. The data fit a model in
which compound 2 can bind the GDP-fucose complex as well
as another enzyme form. Compound 2 is the first inhibitor that
displays exquisite selectivity for a particular fucosyltransferase.
Furthermore, the fact that FucT VI was inhibited is highly
relevant given that this enzyme is involved in the biosynthesis of
sialyl Lewisx. The over-expression of this oligosaccharide has
been implicated in numerous diseases, such as cancer and
inflammation.29
Experimental
General methodology for synthesis
Chemicals were purchased from Aldrich and Fluka and used
without further purification. Molecular sieves were activated at
350 ЊC for 3 h in vacuo. Dichloromethane was distilled from
CaH2 and stored over 4 Å molecular sieves. All reactions were
performed under anhydrous conditions and monitored by TLC
on Kieselgel 60 F254 (Merck). Detection was by examination
under UV light (254 nm) and by charring with 10% sulfuric acid
in methanol. Flash chromatography was performed on silica gel
(Merck, mesh 70–230). Extracts were concentrated under
Fig. 3 Michaelis–Menten plot (A) and Lineweaver–Burk plot (B) for
GDP-fucose at different inhibitor concentrations. The lines are the
result of fitting the data to COMPO. Competitive inhibition (dashed
line) and mixed inhibition (solid line) fits.
reduced pressure at <40 ЊC (bath). 1H NMR (1D, 2D) and 13
C
NMR spectra were recorded on a Varian Merc300 spectrometer
and Varian 500, 600 and 800 MHz spectrometers equipped with
1
Sun workstations. For H and 13C NMR spectra recorded in
CDCl3, chemical shifts (δ) are given in ppm relative to solvent
peaks (1H, δ = 7.26; 13C, δ = 77.3) as an internal standard for the
protected compounds and using NH proton (1.91 ppm) and
OMe carbon (56.83 ppm) as internal standard for deprotected
molecules. Negative ion matrix assisted laser desorption ioniz-
ation time of flight (MALDI-TOF) mass spectra were recorded
using an HP-MALDI instrument using gentisic acid matrix.
High-resolution mass spectra were obtained using a Voyager
delayed extraction STR with 2,5-dihydroxybenzoic acid as an
internal calibration matrix. Optical rotations were measured on
a Jasco P-1020 polarimeter, and [α]D values are given in units of
10Ϫ1 deg cm3 gϪ1 at 26 ЊC, 50 mm cell. Human recombinant
α-1,3-fucosyltransferases V and VI, rat liver α-2,6-sialyltrans-
ferase, CTP, CMP-Neu5Ac and calf alkaline phosphatase
were purchased from Calbiochem. α-2,3-Sialyltransferase was
obtained from Sigma. Human recombinant α-1,3-fucosyl-
transferases III and IV were a generous gift from Dr. Theodora
de Vries. ACS liquid scintillation cocktail was obtained from
Fisher Scientific.
complex. However, a competitive inhibition model could not be
excluded and in this case the inhibitor can also bind to the free
enzyme.
Observations of structural studies provide a possible ration-
ale for the sequential BiBi mechanism. A disordered loop in the
vicinity of the donor/acceptor-binding site is a common feature
of glycosyltransferases. The binding of a nucleotide sugar
structures the loop by making a direct hydrogen bond with the
phosphate groups. It has been suggested that the ordered loop
provides a monosaccharide-sized pocket centered directly over
the catalytic base and the C-1 of the nucleotide. Furthermore,
ordering of the loop may also be a means of protecting the
bound nucleotide sugar from hydrolysis and, thus, be important
for product release. The fact that compound 2 can be bound
to FucT VI in the absence of bound GDP-Fuc shows that
ordering of the loop is not important for the binding of this
compound. The analysis of the inhibitory data suggests that
compound 2 can be bound to FucT VI without prior binding to
GDP-Fuc. Furthermore, the compound is probably complexed
in a different fashion than the natural substrate LacNAc since it
is unable to accept the fucosyl moiety.
Methyl
2-acetamido-2-deoxy-3-O-benzyl-6-O-tert-butyl-
Recombinant Fuc-Ts share significant amino acid sequence
homology,26 despite differences in acceptor specificity. DNA
sequence analysis of α-1,3-fucosyltransferase III, V and VI
revealed greater than 85% homology, suggesting that these
enzymes evolved from common ancestor genes.27 The hyper-
variable region containing 85–90% of the amino acid differ-
ences between FucT III, V and VI28 and approximately 60% of
the amino acid differences for FucT-IV14 is confined to the
N-terminal part of the proteins.26 It has been suggested that this
domain controls the acceptor specificity and the differences in
this region may be important for selective inhibition of FucT
VI by compound 2.
dimethylsilyl-4-O-(3,4,6-tri-O-benzyl-ꢀ-D-galactopyranosyl)-ꢀ-
D-glucopyranoside 4. Sodium methoxide (0.5 mg, 0.01 mmol)
was added to a stirred solution of 3 (50 mg, 0.055 mmol) in
methanol (7 mL). The mixture was left stirring at room temper-
ature for 48 hours. TLC (chloroform/methanol, 9/1, v/v) indi-
cated completion of the reaction. The mixture was neutralized
with Dowex 50Hϩ resin, and then filtered and concentrated
in vacuo. The residue was purified by flash chromatography
(flash silica gel, gradient hexane/acetone, 3/1 to 1/1, v/v) to
1
afford compound 4 (42.5 mg, 86%) as a white solid. H NMR
(CDCl3, 300 MHz); δ 7.40–7.10 (m, 20H, arom), 5.58 (d, 1H,
JNH,2 8.0), 4.83, 4.80 (AB q, 2H, JAB 11.5, OCH2Ph), 4.68, 4.60
(AB q, 2H, JAB 12.1, OCH2Ph), 4.63, 4.46 (AB q, 2H, JAB 12.3,
OCH2Ph), 4.54 (d, 1H, J1,2 7.4, H-1), 4.41 (d, 1H, J1,2 7.4, H-1Ј),
4.15, 4.10 (AB q, 2H, JAB 11.8, OCH2Ph), 3.95 (d, 1H, J3Ј-4Ј 2.75,
H-4Ј), 3.98 (dd, 1H, J2Ј3Ј 11.8 H-3Ј), 3.90–3.80( m, 4H, H-2Ј,
H-5Ј, H-6bЈ, H6b), 3.50 (t, 1-H, J3,4 8.0, H-3), 3.49–3.40 (m,
4H, H-6a, H-6aЈ, H-5, H-2), 3.43 (s, 3H, OCH3), 1.97 (s,
3H, AcNH), 0.81 (s, 9H, Si(CH3)3), Ϫ0.03, Ϫ0.05 (2s, 6H,
Conclusion
Although the C-2Ј hydroxyl of LacNAc is not essential for
sialyl- and fucosyltransferase mediated glycosylations, inver-
sion of the configuration led to a compound that was not
accepted as a substrate by α-2,3- and α-2,6-sialyltransferase and
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 3 7 6 – 1 3 8 0
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Si(CH3)2). MALDI-TOF: m/z 896.4 [M ϩ Na]ϩ. Anal. calcd for
C49H65NO11Si: C, 67.48; H, 7.51; N, 1.61; O, 20.18; Si, 3.22;
found: C, 67.56; H, 7.49; N, 1.69%. [α]D = Ϫ8.5 (c 0.32, DCM).
Methyl
2-acetamido-2-deoxy-4-O-(ꢀ-D-talopyranosyl)-ꢀ-D-
glucopyranoside 2. Palladium hydroxide (4 mg) was added to a
solution of 7 (3.5 mg, 0.0046 mmol) in ethanol (2 mL). The
mixture was vigorously stirred under an atmosphere of hydro-
gen for 3 hours. TLC (chloroform/methanol, 9/1, v/v) indicated
the completion of the reaction. After filtration on Celite and
concentration, the crude material was purified by chromato-
graphy (Iatrobeads, chloroform/methanol/water, 74/24/2, v/v/v)
to afford compound 2 (1.8 mg, 98%) as an amorphous white
solid. 1H NMR (D2O, 300 MHz); δ 4.58 (s, 1H, H-1Ј), 4.35 (d,
1H, J1,2 5.9, H-1), 3.91 (d, 1H, J3Ј,4Ј < 0.2, H-4Ј), 3.80 (dd, 1H,
J6a-6b 12.2, H-6a), 3.74 (s, 1H, H-3Ј), 3.64 (m, 1H, H-6b), 3.70–
3.58( m, 3-H, H-4, H-3, H-2Ј), 3.54 (m, 1-H, H-5), 3.46 (m, 1-H,
H-5Ј), 3.38 (s, 3H, OCH3), 1.91 (s, 3H, AcNH). 13C NMR (D2O,
125 MHz); δ 101.70 (C-1), 100.65 (C-1Ј), 79.01 (C-4), 76.00
(C-5), 74.49 (C-5Ј), 72.25 (C-3), 70.55 (C-4Ј), 68.43 (C-3Ј),
68.02 (C-2Ј), 60.86 (C-6), 60.12 (C-6Ј), 56.83 (OCH3), 54.64
(C-2), 21.92 (NHCOCH3). HRMS: calculated m/z 397.1584
[M ϩ Na]ϩ; observed 397.1569.
Methyl
2-acetamido-2-deoxy-3-O-benzyl-6-O-tert-butyl-
dimethylsilyl-4-O-(3,4,6-tri-O-benzyl-ꢀ-D-talopyranosyl)-ꢀ-D-
glucopyranoside 6. A solution of 4 (8.72 mg, 0.01 mmol) in
acetic anhydride/dimethyl sulfoxide (1/2, v/v, 12 ml) was left
stirring at room temperature overnight. TLC (chloroform/
methanol, 9/1, v/v) indicated completion of the reaction. The
mixture was concentrated in vacuo to give compound 5 as a
white solid. Without further purification, sodium borohydride
(3.8 mg, 0.1 mmol) was added to a stirred solution of 5 in
DCM/MeOH (6 ml, 1/1, v/v). The mixture was left stirring at
room temperature for 4 h. After completion of the reaction, the
mixture was diluted with dichloromethane (20 mL), washed
successively with water, citric acid (5% in water), a saturated
solution of NaHCO3 (5 mL), water (2 × 5 mL) and brine
(5 mL), followed by drying over MgSO4. After evaporation of
the solvent, the residue was purified by flash chromatography
(flash silica gel, gradient hexane/ethyl acetate, 3/1 to 1/1, v/v) to
afford compound 6 (5.2 mg, 60% over 2 steps) as a white foam.
1H NMR (CDCl3, 300 MHz); δ 7.40–7.10 (m, 20H, arom), 6.10
(d, 1H, JNH,2 7.8), 4.98, 4.58 (AB q, 2H, JAB 10.7, OCH2Ph),
4.81, 4.57 (AB q, 2H, JAB 11.8, OCH2Ph), 4.78, 4.50 (AB q,
2H, JAB 11.7, OCH2Ph), 4.52 (s, 1H, H-1Ј), 4.51 (d, 1H, J1,2 7.2,
H-1), 4.44, 4.38 (AB q, 2H, JAB 11.5, OCH2Ph), 4.08 (m, 1H,
H-2Ј), 3.96 (d, 1H, J3Ј-4Ј < 2, H-4Ј), 3.87 (m, 1H, H-4), 4.00–3.80
(m, 3H, H-6b, H-6bЈ–H-5Ј), 3.79 (m, 1H, H-2), 3.60–3.50 (m,
2H, H-3, H-5), 3.51 (m, 1H, H-6aЈ), 3.46 (m, 1H, H-6a), 3.45–
3.34 (m, 2H, H-3, H-3Ј), 3.39 (s, 3H, OCH3), 1.96 (s, 3H,
AcNH), 0.82 (s, 9H, Si(CH3)3), Ϫ0.02, Ϫ0.04 (2s, 6H,
Si(CH3)2). 13C NMR (CDCl3, 125 MHz); δ 170.26, 139.03,
138.04, 137.91, 128.76, 128.68, 128.62, 128.45, 128.37, 128.20,
128.10, 128.05, 127.85, 127.56, 101.63, 101.32. 78.25, 76.79,
75.88, 75.66, 74.98, 74.09, 73.75, 73.32, 69.94, 69.55, 68.06,
62.55, 56.34, 53.34, 29.91, 26.10, 26.06, 23.57, 18.45, 0.20,
Ϫ4.90. HRMS: calculated m/z 894.4225 [M ϩ Na]ϩ; observed
894.4247. [α]D = Ϫ31 (c 0.09, DCM).
ꢁ-2,6- and ꢁ-2,3-Sialyltransferase assays
Reported methods13,22,23,30 were employed for assaying sialyl-
transferase activity at different inhibitor concentrations. For
studies of the relative rates of transfer, incubation mixtures
contained saturating concentration of CMP[14C]Neu5Ac
(200 µM, 1655 cpm nmolϪ1), substrate (1.6 mM for α-2,6-ST
and 4 mM for α-2,3-ST) and different inhibitor concen-
tration (0–1.5 mM), bovine serum albumin (1 mg mlϪ1), 57 µU
of α-2,6-Sialyltransferase and 370 µU of α-2,3-sialyltransferase
in sodium cacodylate (50 mM, pH 6.5) containing 0.1%
Triton X100 in a total volume of 60 µL were incubated at
37 ЊC for a period of 30 min. The radiolabeled product
was isolated using a procedure modified by Horenstein et al.30
based on Paulson’s ion-exchange chromatography on a Dowex
1X8-200 (PO42Ϫ, 100–200 mesh) Pasteur pipette column.22
2Ϫ
Columns (5 cm high) were eluted twice with 1 mM PO4
(4 mL) buffer to ensure that no radiolabeled product was left
on the column.
Fucosyltransferase assays
Methyl
2-acetamido-2-deoxy-3-O-benzyl-4-O-(3,4,6-tri-O-
Reported methods15,23were employed for assaying fucosyl-
transferase activity. For studies of the relative rates, incubation
mixtures contained a saturating concentration of GDP[14C]-
fucose (45 µM, 6532 cpm nmolϪ1), substrate (100 nmol), inhibi-
tor (0–1.5 mM) and an amount of enzyme corresponding
to initial velocity for each fucosyltransferase (56 µU of FucT
VI, 4 µL of FucT IV (6 µg protein µLϪ1), 100 µU of FucT V and
10 µU of FucT III) assayed in sodium cacodylate (25 mM,
pH 6.5) containing MnCl2 (8 mM), ATP (1.6 mM) and NaN3
(1.6 mM) in a total volume of 50 µL were incubated at 37 ЊC for
a period of 60 min. The radiolabeled product was isolated using
ion-exchange chromatography on a Dowex 1X8-200 (ClϪ, 100–
200 mesh) Pasteur pipette column.15 Columns (2.5 cm high)
were eluted twice with ice cold water (1.5 mL) to ensure that no
radiolabeled product was left on the column.
benzyl-ꢀ-D-talopyranosyl)-ꢀ-D-glucopyranoside 7. Tetrafluoro-
boric acid (48% in water, 2 µL, 0.015 mmol) was added to the
stirred solution of 6 (4.1 mg, 0.005 mmol). The mixture was left
stirring at room temperature for 5 minutes. TLC analysis (tolu-
ene/ethyl acetate, 1/1, v/v) indicated completion of the reaction.
The mixture was neutralized with triethylamine (4 µL, 0.03
mmol) and concentrated in vacuo. The residue was then diluted
in dichloromethane (5 mL), washed successively with a satur-
ated solution of NaHCO3 (2 mL), water (2 × 2 mL) and brine
(2 mL), followed by drying over MgSO4. After evaporation of
the solvent, the residue was purified by flash chromatography
(flash silica gel, gradient hexane/ethyl acetate, 3/1 to 1/1, v/v) to
1
afford compound 7 (3.7 mg, 94%) as a white foam. H NMR
(CDCl3, 300 MHz); δ 7.40–7.10 (m, 20H, arom), 5.85 (d, 1H,
JNH,2 7.9), 4.98, 4.58 (AB q, 2H, JAB 10.7, OCH2Ph), 4.81, 4.55
(AB q, 2H, JAB 11.7, OCH2Ph), 4.80, 4.51 (AB q, 2H, JAB 11.7,
OCH2Ph), 4.63 (d, 1H, J1,2 7.8, H-1), 4.51 (s, 1H, H-1Ј), 4.44,
4.35 (AB q, 2H, JAB 11.9, OCH2Ph), 4.14 (m, 1H, H-2Ј), 3.98 (t,
1-H, J3-4 7.3, H-3), 3.94 (d, 1H, J3Ј-4Ј < 2, H-4Ј), 3.88 (m, 1H,
H-4), 3.88–3.80 (m, 3H, H-6b, H-6bЈ- H-5Ј), 3.70 (dd, 1H, H-2),
3.62 (m, 1H, H-5), 3.57 (m, 1H, H-6aЈ), 3.47 (s, 3H, OCH3),
3.43 (ddd, 1H, J6a-5 3.41, H-6a), 3.39 (s, 1-H, H-3Ј), 1.96 (s, 3H,
AcNH). 13C NMR (CDCl3, 125 MHz); δ 170.42, 138.74, 137.99,
137.85, 137.80, 128.78, 128.69, 128.65, 128.52, 128.50, 128.24,
128.20, 128.13, 128.06, 127.85, 127.87, 101.99, 101.37, 78.91,
76.00, 79.00, 75.93, 75.02, 74.21, 73.77, 69.99, 68.91, 68.17,
62.54, 56.98, 54.74, 29.92, 16.44, 6.01. HRMS: calculated m/z
780.3360 [M ϩ Na]ϩ; observed 780.3387. [α]D = ϩ4.3 (c 0.12,
DCM).
Inhibition studies
With respect to methyl LacNAc. Apparent kinetic parameters of
the human recombinant FucT VI for synthetic acceptors were
determined under the above standard conditions using a satur-
ating concentration of GDP-fucose and different inhibitor con-
centrations (0, 0.2, 0.3 and 0.4 mM). Assays were performed in
duplicate using the appropriate amount of enzyme. The con-
centration of oligosaccharide acceptor was varied around the
Km value (0.1–1.0 mM), whereas the concentration of GDP-
fucose was kept constant at 45 µM. The time of incubation at
37 ЊC was varied to 15 min. to limit the GDP-[14C]fucose
consumption to 10–15 % to ensure initial rate conditions. The
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 3 7 6 – 1 3 8 0
1379

View Article Online
8 S. J. Chung, S. Takayama and C. H. Wong, Bioorg.Med. Chem. Lett.,
1998, 8(23), 359–3364.
9 J. M. Elhalabi and K. G. Rice, Curr. Med. Chem., 1999, 6, 93.
10 X. Qian and M. M. Palcic, Carbohydrates in Chemistry and Biology,
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13 K. B. Wlasichuk, M. Kashem, P. V. Nikrad, P. Bird, C. Jiang and
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kinetic parameters were determined by fitting the data to the
Fortran programs of Cleland, COMPO and NCOMP.24
With respect to GDP-Fuc. Apparent kinetic parameters of
the human recombinant FucT VI for GDP-Fuc were deter-
mined under the above standard conditions using a saturating
concentration of LacNAc-OMe. Assays were performed in
duplicate using the appropriate amount of enzyme (56 µU).
The concentration of GDP-[14C]fucose was varied around the
Km value (0.4–6.6 µM), whereas the concentration of LacNAc-
OMe was kept constant at 1.5 mM and different inhibitor
concentrations (0, 0.2 and 0.4 mM) were used. The time
of incubation at 37 ЊC was varied to 15 min. The kinetic
parameters were determined by fitting the data to the Fortran
programs of Cleland, COMPO and NCOMP.24
17 G. Baisch, R. Ohrlein and A. Katopodis, Bioorg. Med. Chem. Lett.,
1997, 7, 2431.
18 G. Baisch, R. Ohrlein and M. Streiff, Bioorg. Med. Chem. Lett.,
1998, 8, 161.
Acknowledgements
19 M. C. Galan, A. P. Venot and G. J. Boons, Biochemistry, 2003, 42,
8522–8529.
20 M. C. Galan, A. P. Venot, J. Glushka, A. Imberty and G. J. Boons,
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22 J. C. Paulson, J. I. Rearick and R. L. Hill, J. Biol. Chem., 1977,
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This research was supported by the NIH Research Resource
Center for Biomedical Complex Carbohydrates (P41-RR-
5351). We are grateful to Dr. Theodora de Vries for the kind gift
of the FucT IV CHO cells and the FucT III cell extracts. We are
also thankful to Dr. Margreet Wolfert for assistance with the
extraction of CHO cells to provide sufficient amounts of FucT
IV.
23 M. M. Palcic, A. P. Venot, R. M. Ratcliffe and O. Hindsgaul,
Carbohydr. Res., 1989, 190, 1–11.
24 W. W. Cleland, Methods Enzymol., 1979, 63, 103.
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26 B. W. Weston, P. L. Smith, R. J. Kellyand and J. B. Lowe, J. Biol.
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27 I. Reguigne-Arnould, P. Coullin, R. Mollicone, S. Faure,
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