FRET-based direct and continuous monitoring of human fucosyltransferases activity: An efficient synthesis of versatile GDP-L-fucose derivatives from abundant D-galactose

Article abstract of DOI:10.1002/chem.200700760

We have developed a facile and versatile protocol for the continuous monitoring of human fucosyltransferases activity by using fluorescence energy resonance transfer (FRET), and have explored the feasibility of its use in an inhibitor screening assay. A convenient sugar nucleotide with a fluorogenic probe, 6-deoxy-6-N-(2-naphalene-2-yl-acetamide)-β-L-galactopyranos-1-yl- guanosine 5′-diphosphate disodium salt (1), was efficiently synthesized from naturally abundant D-galactopyranose via a key intermediate, 6-azide-1,2,3,4-tetra-O-benzoyl-6-deoxy-β-L-galactopyranose (10). It was demonstrated that the combined use of the glycosyl donor 1 and a dansylated acceptor substrate, sialyl-α2,3-LacNAc derivative (2) allowed us to carry out highly sensitive, direct, and continuous in vitro monitoring of the generation of sialyl Lewis X (SLe^x), which is catalyzed by human α1-,3-fucosyltransferase VI (FUT-VI). A kinetic analysis revealed that compound 1 was an excellent donor substrate (K_M = 0.94 μM and K_max = 0.14 μM min^-1) for detecting human FUT-VI activity. To the best of our knowledge, this synthetic fluorogenic probe is the most sensitive and selective donor substrate for FUT-VI among all of the known GDP-Fuc analogues, including the parent GDP-Fuc. When a dansylated asparagine-linked glycopeptide 20, which is derived from egg yolk was employed as an alternate acceptor substrate, a FRET-based assay with compound 1 could be used to directly monitor the α1,6-fucosylation at the reducing terminal GlcNAc residue by human FUT-VIII (K_M = 175 μM and V_max = 0.06 μM/min); this indicates that the present method might become a general protocol for the characterization of various mammalian fucosyltransferases in the presence of designated fluorogenic acceptor substrates. The present protocol revealed that compound 23, which was obtained by a 1,3-dipolar cycloaddition between the disodium salt 16 and 1-ethynylnaphthalene exhibits highly potent inhibitory effects against the FUT-VI-mediated sialyl Lewis X synthesis (IC ₅₀ = 5.4 μM).

Full text of DOI:10.1002/chem.200700760

DOI: 10.1002/chem.200700760
FRET-Based Direct and Continuous Monitoring of Human
Fucosyltransferases Activity: An Efficient synthesis of Versatile GDP-l-
Fucose Derivatives from Abundant d-Galactose**
[
a]
[a, b]
Takahiro Maeda and Shin-Ichiro Nishimura*
Abstract: We have developed a facile
and versatile protocol for the continu-
ous monitoring of human fucosyltrans-
ferases activity by using fluorescence
energy resonance transfer (FRET), and
have explored the feasibility of its use
in an inhibitor screening assay. A con-
venient sugar nucleotide with a fluoro-
genic probe, 6-deoxy-6-N-(2-napha-
lene-2-yl-acetamide)-b-l-galactopyra-
nos-1-yl-guanosine 5’-diphosphate di-
in vitro monitoring of the generation of
sialyl Lewis X (SLe ), which is cata-
lyzed by human a-1,3-fucosyltransfera-
se VI (FUT-VI). A kinetic analysis re-
vealed that compound 1 was an excel-
egg yolk was employed as an alternate
acceptor substrate, FRET-based
assay with compound 1 could be used
to directly monitor the a1,6-fucosyla-
tion at the reducing terminal GlcNAc
x
a
lent donor substrate (K =0.94 mm and
residue by human FUT-VIII (K =
M
M
À1
Vmax =0.14 mmmin )
for
detecting
175 mm and Vmax =0.06 mm/min); this in-
dicates that the present method might
become a general protocol for the char-
acterization of various mammalian
human FUT-VI activity. To the best of
our knowledge, this synthetic fluoro-
genic probe is the most sensitive and
selective donor substrate for FUT-VI
among all of the known GDP-Fuc ana-
logues, including the parent GDP-Fuc.
When a dansylated asparagine-linked
glycopeptide 20, which is derived from
A
H
R
U
G
A
C
H
T
R
E
U
N
G
sodium salt (1), was efficiently synthe-
designated fluorogenic acceptor sub-
strates. The present protocol revealed
that compound 23, which was obtained
by a 1,3-dipolar cycloaddition between
the disodium salt 16 and 1-ethynyl-
naphthalene exhibits highly potent in-
hibitory effects against the FUT-VI-
mediated sialyl Lewis X synthesis
(IC =5.4 mm).
sized from naturally abundant d-
galactopyranose via a key intermediate,
6-azide-1,2,3,4-tetra-O-benzoyl-6-
deoxy-b-l-galactopyranose (10). It was
demonstrated that the combined use of
the glycosyl donor 1 and a dansylated
acceptor substrate, sialyl-a2,3-LacNAc
derivative (2) allowed us to carry out
highly sensitive, direct, and continuous
Keywords: carbohydrates · FRET
(
fluorescence
resonant
energy
transfer) · fucose · fucosyltransfer-
ase · galactose
5
0
Introduction
[
a] T. Maeda, Dr. S.-I. Nishimura
Glycosylation, which is conducted by various glycosyltrans-
ferases, is one of the most important biological processes in
the posttranslational modification of protein/lipid structures
and functions. This modification greatly influences various
molecular recognition processes, including bacterial/viral in-
fections and cell adhesion in inflammation, as well as basic
biological phenomena such as immune response, cellular dif-
ferentiation, development, regulation, and many other inter-
Laboratory of Advanced Chemical Biology
Graduate School of Advanced Life Science, and
Frontier Research Center for Post-Genome Science and Technology
Hokkaido University
N21, W11, Kita-ku, Sapporo 001-0021 (Japan)
Fax : (+81)11-706-9042
E-mail: shin@glyco.sci.hokudai.ac.jp
[
b] Dr. S.-I. Nishimura
Drug-Seeds Discovery Research Laboratory
National Institute of Advanced Industrial Science and Technology
[1]
cellular communication and signal transductions. It is evi-
(
AIST)
dent that synthetic substrates and inhibitors of glycosyltrans-
ferases have become powerful tools, and they might lead to
the development of novel therapeutic reagents, as well as
specific molecular probes for characterizing enzymatic gly-
Toyohira-ku, Sapporo 062-8517 (Japan)
[
**] A part of this work was presented at XXIIIth International
Carbohydrate Symposium held in Whistler, Canada on July 25, 2006.
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
[2–11]
cosylation processes.
478
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 478 – 487

FULL PAPER
Fucosyltransferases (FucTs) are enzymes that catalyze the
transfer of the l-fucose moiety from guanosine diphosphate
b-l-fucose (GDP-Fuc) to various acceptors in an a1,2-,
[12]
a1,3-, a1,4-, or a1,6-linkage. It has been well documented
that fucosylation conducted by mammalian FucTs is often
the final glycosylation step in a range of biologically signifi-
x
cant glycans such as sialyl Lewis X (sLe ), sialyl Lewis A
a
b
[13]
(
sLe ), and sialyl Lewis B (sLe ) structures.
It was also
suggested that the attachment of a l-fucose residue to the
core chitobiose unit of asparagine-linked glycans reduces
[14]
the antibody-dependent cellular cytotoxicity drastically.
Given that fucosylation is essential for basic biological
events, especially fertilization, embryogenesis, lymphocyte
[15]
trafficking, immune responses, and various diseases, it is
not surprising that extensive efforts have been made toward
[2,9,16–29]
the synthesis of potential inhibitors of FucTs
and
toward specific tagging methods to visualize fucosylated gly-
[30]
cans in vivo.
Our attention is now directed toward the need for a new
method that allows for the direct, continuous, and quantita-
tive monitoring of the FucTs reactions which yield fucosylat-
ed glycoconjugates of interest. The advent of facile and reli-
able screening methods should facilitate the process of
searching for specific inhibitors against various FucTs. We
hypothesized that an approach that uses fluorescent reso-
nance energy transfer (FRET) might become a key to ad-
Scheme 1. Concept of FRET-based detection of sialyl Lewis X synthe-
sized by FucTs. The increase of the dansyl emission at 540 nm under an
excitation at 290 nm will be observed only when sialyl Lewis X tetrasac-
charide is formed by glycosylation catalyzed by FucTs.
[31,32]
dress this issue,
although modern mass spectrometry
and surface plasmon resonance (SPR) approaches have
been feasible in combination with the use of gold nanoparti-
cles or high-performance microarrays to display glycosyl ac-
intermediate, 6-azide-1,2,3,4-tetra-O-benzoyl-6-deoxy-b-l-
galactopyranose (10), by using d-galactose as a starting ma-
A
H
R
U
G
[33–36]
À1
ceptor substrates or glycosyltransferases.
As illustrated
terial. It seems likely that the use of d-galactose ($1.6 g )
would make the large-scale synthesis of various l-fucose de-
rivatives possible through a series of established chemical
manipulations; most C6-modified l-fucose derivatives have
in Scheme 1, we imagined that a FRET-based assay would
allow a quite simple and specific sensing of the generation
of the desired product (structures) as an enhanced fluores-
cence due to dansyl emission at 540 nm under an excitation
of naphtylmethyl group at 290 nm, only when the FucT-
mediated glycosylation occurs between the designated fluo-
rescent donor 1 and acceptor 2 substrates. Its feasibility in
continuous and quantitative kinetic analysis is evident be-
cause the fluorescence intensity at 540 nm directly repre-
been prepared by starting from l-galactose, which is very
À1 [30,38]
expensive ($945 g ).
Conversion of the d-galacto into
the l-galacto configuration was carried out by chemical
modifications of compound 5, which is derived from a
[37]
known d-galactose diethyl dithioacetal 4,
as shown in
Scheme 2. Selective liberation of the thioacetal 5 yielded an
alcohol 6, which was tosylated and subjected to a subse-
quent substitution reaction to afford the fully protected
azide 8 in high yield. After de-O-silylation, the primary hy-
droxyl group was oxidized by treatment with oxalyl chloride
to afford a hemiacetal 9, which was subjected to de-O-iso-
propylidenation. Subsequent O-benzoylation gave 6-azide-l-
fucose as per-O-benzoate 10 in 73% overall yield from com-
pound 9 (three steps). With key intermediate 10 in hand, we
prepared compound 1 and another 6-azide analogue of
x
sents the concentration of the sLe derivative generated by
glycosylation. In the present study, we describe an efficient
synthesis of versatile GDP-fucose analogues for direct, con-
tinuous, and quantitative monitoring of glycosylation cata-
lyzed by human FucTs.
Results and Discussion
[
30]
Synthesis of versatile GDP-l-fucose analogues from d-galac-
tose: Because synthetic studies on GDP-l-fucose analogues
had revealed a tolerance at the C6 position for FucTs-cata-
GDP-l-fucose (16)
dure.
according to the published proce-
[39]
As anticipated, it was demonstrated that our ap-
proach permits an efficient and large-scale synthesis of ver-
satile 6-azide-l-fucose analogues; these have become key in-
termediates for a further derivatization study to develop
potent inhibitors and novel molecular probes. We are cur-
rently investigating the feasibility of using compound 16 for
the construction of potent inhibitor libraries.
[30,37,38]
lyzed glycosylations,
we selected compound 1 as an
ideal fluorogenic donor (naphthylmethyl) substrate for use
with acceptor substrate 2 (dansyl) as shown in Scheme 1.
We established an efficient synthetic route to a novel
fluorescence-labeled GDP-l-fucose derivative 1 via a key
Chem. Eur. J. 2008, 14, 478 – 487
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
479

S.-I. Nishimura and T. Maeda
chemical and enzymatic proce-
dure
Scheme 3.
as
indicated
in
[
32]
A coupling reaction between
compounds 1 and 2 in the pres-
ence of human recombinant
FucT VI was monitored under
excitation at 290 nm, and the
emission spectra were taken at
appropriate intervals as shown
in Figure 1. The results indicat-
ed that the fucosylation caused
increases in the dansyl emis-
sion at 540 nm by FRET from
the naphtylmethyl group in ac-
cordance with the progress of
the reaction. In
a
similar
manner, decreases in the naph-
tylmethyl emission at 340 nm,
due to quenching by the dansyl
group were also observed. In
addition, the existence of the
isofluorometric
point
at
4
80 nm clearly indicates that
the attachment of the l-fucose
residue to sialyl-LacNAc tri-
saccharide induces a drastic
and convergent structural alter-
ation. This result implies that
the formation of a stable con-
x
formation of the sLe structure
might be necessary for the spe-
cific interaction with partner
carbohydrate-binding proteins
such as P-selectin, E-selectin,
and L-selectin. Figure 2 shows
a typical FRET spectra, which
was observed in the FucT VI-
mediated reactions. The kinetic
constants were determined to
Scheme 2. Synthetic route of the GDP-l-fucose derivatives. a) ethanethiol, conc. HCl, 64%; b) i) t-butyldime-
thylsilyl chloride, pyridine, 08C to RT; ii) 2,2-dimethoxypropane, pyridinium p-toluenesulfonic acid, CH CN,
RT, 86 %; c) i) N-bromosuccimide, 95% aq. acetone, 08C; ii) NaBH , EtOH, RT, 79% (2 steps), d) p-toluene-
sulfonyl chloride, pyridine, RT, 97%; e) NaN , DMF, 608C, 87%; f) (tBu) NF, THF, RT, 87%; g) i) (COCl)
DMSO, Et N, CH Cl
, À788C to 08C; ii) 60% aq. AcOH, MeOH, 608C; iii) benzoyl chloride, dimethylamino-
pyridine, pyridine, RT, 73% (3 steps); h) i) TiBr , EtOAc, CH Cl , RT; ii) (BnO) PO H, Ag CO , CH Cl
Et O, CH CN, RT, 70% (2 steps), i) i) H , Pd/C, MeOH, RT; ii) naphthyl acetic acid succinimidyl ester, diiso-
propyl ethyl amine, THF, RT, 64% (2 steps); j) Et N/MeOH, 608C, 87%; k) GMP-morpholidate, 1H-tetrazole,
pyridine, RT, 39%; l) i) NaOMe, MeOH, rt; ii) Ac O, pyridine, RT, quantitative yield; m) conditions described
3
4
be K =0.94 mm and Vmax
=
M
3
4
2
,
À1
0
.14 mmmin , which indicates
3
2
2
that compound 1 is a much
better donor substrate than the
4
2
2
2
2
2
3
2
2
,
2
3
2
3
parent GDP-l-fucose (K =
M
[9]
2
5
.5 mm).
The HPLC and
in reference [38] n) GMP-morpholidate, 1H-tetrazole, pyridine, RT, 3 days, 11%.
MALDI analyses also suggest-
ed that the above assay condi-
x
tion generates only sLe deriv-
Direct monitoring of human FucTs-mediated glycan synthe-
ative 19 from two fluorogenic substrates; the kinetic con-
sis by FRET: Fucosylation is often the final step in the bio-
stants that were determined by HPLC methods were esti-
À1
synthetic pathways of many biologically significant glycan
mated to be K =3.5 mm and V =0.46 mmmin (Support-
M
max
x
structures, such as sLe tetrasaccharides, which are critical
selectin ligands that modulate cellular adhesion and lympho-
ing Information).
Next, our interest was directed to the feasibility of using
compound 1 for the characterization of FucT VIII. For this
purpose, we prepared the designated glycosyl acceptor sub-
[13]
cyte recirculation. To test the feasibility of 1 in the FRET-
based direct monitoring of this unique glycan synthesis, a
dansylated acceptor substrate 2 was readily synthesized by a
[40]
strate 20 according to the published procedure. As expect-
480
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Chem. Eur. J. 2008, 14, 478 – 487

Carbohydrate Chemistry
FULL PAPER
Scheme 3. Preparation of a glycosyl acceptor substrate 2. a) conditions described in reference [32]. b) CMP-Neu-5-Ac, rat recombinant a2,3-sialyltrans-
ferase, CIAP, pH 7.4, 378C, 2 days, 97%.
Figure 1. The FRET that was observed in FucT VI mediated sialyl Lewis X synthesis. Condition: a) 1 (20 mM), 2 (20 mM), FucT VI (3.5 mU), 20 mm
HEPES buffer that contained 20 mm MnCl , pH 7.5, at 258C, total volume 2.5 mL; A) The time course of the relative fluorescence emission of 1 and 2
2
during sugar transferring with FucT VI. The slit width of excitation was 5 nm and emission was 20 nm, and emission spectra were scanned at 300 nm/min
from 300 nm to 650 nm; B) Fluorescence change of naphthylmethyl (square), naphthalene excimer (triangle), and dansyl (circle).
ed, the combined use of 1 and 20 allowed the FucT VIII-
mediated synthesis of glycopeptide 21 to be monitored, as
shown in Figure 3. The increase of the dansyl emission at
5
3
25 nm and the decrease of the naphtylmethyl emission at
40 nm were observed, and the spectra could be used for
further kinetic studies. Interestingly, a typical isofluoromet-
ric point was not detected in the conversion of 20 into 21;
however one was observed in the case of the construction of
x
sLe tetrasaccharide 19 from sialyl-LacNAc derivative 2 by
fucosylation with FucT VI. Kinetic constants of 1 for human
A
H
R
U
G
recombinant FucT VIII were estimated to be K =175 mm
M
À1
and Vmax =0.06 mmmin (Figure 4). This means that modifi-
cation at the C6 position of GDP-fucose with a sterically
hindered aromatic group might slightly reduce the affinity
of the parent GDP-fucose for this enzyme, because the KM
value of the GDP-fucose for recombinant FucT VIII was re-
[41]
ported to be 19.3 mm by conventional HPLC analysis.
These results imply that the 3D structures of the donor-
binding pockets of FucT VI and FucT VIII seem to be dif-
ferent. To achieve a desired FRET assay for FucT VIII-se-
lective inhibitors, it is likely that an appropriate structural
optimization might be required for enhancing the affinity
and specificity of fluorogenic GDP-l-fucose donor sub-
strates toward FucT VIII. However, it must be noted that
the present strategy might not be feasible for discriminating
Figure 2. Determination of kinetic constants of 1 for FucT VI. Condi-
tions: 1, 2, 5, 10, 15, and 20 mm of 1, 2 (20 mm), and 3.5 mU FucT VI were
incubated in 20 mm HEPES buffer that contained 20 mm MnCl , pH 7.5,
2
at 258C for 10 min. A) Changes in the dansyl emission at 540 nm.
B) Lineweaver–Burk plot of a1,3-fucosylation reactions measured as a
function of the concentration of glycosyl donor substrate.
Chem. Eur. J. 2008, 14, 478 – 487
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www.chemeurj.org
481

S.-I. Nishimura and T. Maeda
Figure 3. FRET analysis of the generation of glycopeptide 21 by FucT VIII in the presence of compound 1 and 20. Conditions: a) 1 (50 mm), 20 (100 mm),
8
0 mU a1,6-FucT (FucT VIII), 50 mm sodium cacodyrate buffer, pH 7.5, total volume: 200 mL, 258C. A) The changes in the fluorescence emission spectra
during the sugar-transfer reaction conducted by FucT VIII; B) Time course of the fluorescence intensity due to the dansyl emission at 525 nm.
FRET under an excitation of naphtylmethyl group at
2
90 nm. For the proof of this principle, we tested preliminar-
x
ily the effect of compound 23 on the FucT VI-mediated sLe
tetrasaccharide (19) synthesis from 1 and 2. Here, com-
pound 23 is a GDP-l-fucose analogue that was obtained
[42]
I
by means of Cu -catalyzed [3+2] cycloaddition of 6-azide-
GDP-l-fucose 16 (the kinetic constants of 16 were estimated
À1
as follows: K =19.9 mm and Vmax =0.073 mmmin
for
M
FucT VI, Supporting Information) with 1-ethynyl-naphtha-
lene (Scheme 4). The IC50 value of 23 was determined by
measuring the decrease of the fluorescence intensity at
5
40 nm that was caused through FRET as shown in Figure 5.
When the inhibition assay was carried out on the basis of
the present FRET strategy, FucT VI activity was determined
with a fixed concentration of 1 (20 mm, 2 (20 mm), and
FucT VI (1.4 mU) in the presence of various concentrations
of 23 (5, 10, and 15 mm). The estimated inhibitory effect by
2
3 (IC =5.4 mm) was in good quite agreement with the
50
Figure 4. Determination of kinetic constants of 1 for FucT VIII. Condi-
tions: 10, 25, 50, 75, 100 mm of 1, 20 (100 mm), and 80 mU a1,6-FucT VIII
were incubated in 50 mm sodium cacodylate buffer, pH 7.5, at 258C for
result that was obtained by the HPLC-based inhibition
assay (IC =3.6 mm). Judging from the difference in the
5
0
1
0 min. A) Changes in the dansyl emission at 525 nm. B) Lineweaver–
chemical structures of the very similar GDP-l-fucose ana-
logues 1 and 23, it is evident that the triazole ring that con-
nects the naphtylmethyl group and GDP-l-fucose moiety is
an important motif for this compound to function as a
potent inhibitor for FucT VI. Interestingly, the naphtylmeth-
yl group that was combined simply by an amide bond did
not exhibit any inhibitory effect; this suggests that the tri-
Burk plot of a1,6-fucosylation reactions measured as a function of the
concentration of glycosyl donor substrate.
the functions among several FucTs that exhibit very similar
substrate specificity.
Feasibility of the FRET protocol for inhibitor screening:
The versatility of the present protocol in the inhibitor
screening of FucTs is evident, because the inhibitory effect
can be detected simply as the decrease of the fluorescence
intensity at 540 nm, which is due to dansyl emission by
A
T
E
N
azole ring might bind tightly with this binding pocket by
forming multiple hydrogen bonds rather than by contribu-
ting to hydrophobic interactions. The relative inhibitory ef-
fects of related compounds such as 2-naphtylphosphate,
GMP, and CMP (all at 20 mm) were preliminarily compared
482
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Chem. Eur. J. 2008, 14, 478 – 487

Carbohydrate Chemistry
FULL PAPER
[
9]
Scheme 4. Synthesis of a potential inhibitor 23 by means of the Crick reaction of 6-azide-GDP-l-fucose (16) with 1-ethynyl-naphthalene. a) 6-azide-
GDP-l-fucose 16 (2 mm), 1-ethynyl-naphthalene (2 mm), CuSO (0.2 mm), and sodium ascorbate (2 mm) in H O/methanol (75:25 v/v, the total volume
4
2
was adjusted to 50 mL) were incubated at room temperature for 7 h. The completion of the coupling reaction was monitored and judged by mass spec-
trometry as described in the Supporting Information. (50 mL) was added to the mixture to adjust the concentration of product to be 1 mm, and this solu-
tion was used directly for the subsequent inhibition assay.
In conclusion, we have established an efficient synthetic
route to highly useful GDP-l-fucose derivatives by starting
from inexpensive d-galactose, through a key intermediate
1
0. The use of these compounds in the FRET-based assay
systems for the characterization of human recombinant
FucT VI and FucT VIII has also been explored. This
method allowed us to rapidly and easily evaluate the inhibi-
tory effect of synthetic compounds on the FucT-mediated
x
sLe synthesis, and we found that compound 23, which is de-
rived by click chemistry by using 6-azide GDP-fucose 16 is a
potent inhibitor for FucT VI. It should also be noted that
the present protocol might be suitable not only for investi-
gating mammalian FucTs, but also for bacterial FucTs when
a designated glycosyl acceptor substrate is optimized.
Experimental Section
Reagents and general methods: Recombinant human a1,3-fucosyltrans-
ferase VI (FucT VI, one unit of this enzyme is defined as the amount of
enzyme that will transfer 1.0 mmol of fucose from GDP-Fuc to N-acetyl-
lactosamine per minute at 378C, pH 7.5) and recombinant rat a2,3-sialyl-
transferase (a2,3-siaT, one unit of this enzyme is defined as the amount
of enzyme that will transfer 1.0 mmol of sialic acid from CMP-Neu5 Ac to
N-acetyllactosamine per minute at 378C, pH 7.4) were purchased from
Calbiochem (San Diego, CA). Recombinant human a1,6-fucosyltransfer-
ase (FucT VIII, one unit of this enzyme is defined as the amount of
enzyme that will transfer 1.0 mmol of fucose from GDP-Fuc to aspara-
gine-linked N-acetyl-b-d-glucosamine of the chitobiose moiety per
minute at 378C, pH 7.5) was purchased from Toyobo Co. Ltd.(Osaka,
Japan). b-Galactosidase was purchased from Seikagaku Co. Ltd. (Tokyo,
Japan). Solvents and other reagents for chemical synthesis were pur-
chased from Aldrich Chemical Co. (Milwaukee, WI), Tokyo Kasei
(Tokyo, Japan), and Wako Pure Chemical Industries Ltd. (Osaka, Japan)
x
Figure 5. Inhibitory effect of compound 23 on the synthesis of sLe tetra-
saccharide 19 by FucT VI in the presence of 1 and 2. The IC50 value of 23
was determined by measuring the decrease of the fluorescence intensity
at 540 nm (square) and by
FucT VI activity was determined with a fixed concentration of 1 (20 mm),
(20 mm) and FucT VI (1.4 mU) in the presence of various concentra-
tions of 23 (5, 10, and 15 mm) in 20 mm HEPES buffer (pH 7.2) that con-
tained 20 mm MnCl (total volume 250 mL
a conventional HPLC assay (triangle).
2
2
by using 23 (20 mm) as a positive control, and the results are
summarized in Table 1.
1
13
and used without further purification. H and C NMR was recorded
with Bruker lambda 600 MHz(Bruker BioSpin, Rheinstten Germany).
Chemical shifts are given in ppm and referenced to internal TMS (d
H
=
Table 1. The relative inhibitory effects determined by FRET assay. The
effects of 2-naphtylphosphate, GMP, and CMP on the FucT VI-mediated
sLe synthesis were evaluated by employing the following conditions: 1
0.00 ppm in CDCl
), CHCl
(d
H
=7.26 ppm in CDCl
), tert-BuOH (d
=
3
3
3
H
1.24 ppm in O), or CDCl
D
2
3
C
(d =77.00 ppm). Assignments of the
x
1
H NMR spectra were made by first-order analysis, and were verified by
H,H COSY, TOCSY, and HMQC experiments. Elemental analyses were
performed with MT-6 CHN CORDER (Yanako, Japan). Thin-layer chro-
matography (TLC) was performed on Merck precoated plates (20”
20 cm; layer thickness: 0.25 mm; silica gel 60F254); spots were visualized
by spraying a solution of 90:5:5 (v/v/v) MeOH/p-anisaldehyde/concen-
trated sulfuric acid and by heating at 1808C for about 30 s, and by UV
light (256 or 365 nm), when applicable. Flash column chromatography
was performed on silica gel 60 (spherical type, particle size 40–50 mm;
Wako Pure Chemical, Osaka, Japan) with the solvent systems specified,
and the ratio of solvent systems was given in (v/v). Organic extracts were
(
(
20 mm), 2 (20 mm), and FucT VI (0.7 mU) in 25 mm HEPES buffer
250 mL, pH 7.2) that contained 20 mm MnCl at 258C for 1 h. The inhibi-
2
tion (%) was determined by comparing this result with the result that
was obtained by using compound 23 as a positive control.
[
a]
[a]
Compound
-naphtylphosphate
CMP
Inhibition [%]
Compound
Inhibition [%]
2
26
35
GMP
23
56
100
[
a] The concentration of each compound was 20 mm.
Chem. Eur. J. 2008, 14, 478 – 487
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
483

S.-I. Nishimura and T. Maeda
dried over anhydrous MgSO
4
, and solutions were concentrated under di-
88.19 (C5), 88.27 ppm (C2); HR-MS (FAB): m/z calcd for C18
377.2359; found: 377.2364 [M+H] .
H
36
O
6
Si:
+
minished pressure below 508C. Column chromatography was performed
on silica gel (Kanto Kagaku, Japan), Iatrobeads (6RS-8060, Jatron Labo-
ratories, Mitsubishi Kagaku Iatron, Tokyo, Japan), Sephadex G-15 (GE
Healthcare Bio-Sciences,Buckinghamshire England), Dowex 50W X8
6
-O-tert-Butyldimethylsilyl-2,3:4,5-di-O-isopropylidene-1-O-p-toluenesul-
fonyl-d-galactitol (7): A cold solution of 6 (2.0 g, 5.31 mmol), 4-(dimethy-
lamino)pyridine (1.30 g, 10.62 mmol) in pyridine (10 mL) was treated
with p-toluenesulfonyl chloride (1.52 g, 7.97 mmol) in CH Cl (2 mL) and
(
Muromachi Chemicals, Shizuoka, Japan), DEAE-Cellulose, or Wako-
2
2
gel C-18 (Wako Pure Chemical Industries Ltd., Osaka, Japan). Fluores-
cence measurement of emission spectra and excitation spectra was car-
ried out by using a Hitachi F2500 fluorescence spectrometer. The excita-
tion wavelength was 290 nm and the emission spectrum was scanned
the mixture was stirred at room temperature. After 15 h, the mixture was
concentrated and the residue was dissolved in CHCl . The solution was
3
washed with 1n H SO , aq. sat. NaHCO , and brine. The organic layer
2
4
3
was dried and concentrated. The residue was subjected to the purification
by chromatography on silica gel (hexane/EtOAc 8:1), and the fractions
that contained the product were collected and concentrated. The syrupy
product was dissolved in EtOH and stored in a refrigerator to yield 7
À1
from 300 nm to 650 nm at 300 nmmin . The data were processed with
FL-solution data manager software (Hitachi), Microsoft Excel, and
GraphPad Prism. High-performance liquid chromatography (HPLC) was
conducted on an Hitachi L-7100 HPLC system equipped with a Inertsil-
ODS column (4.6 mm”250 mm, GL Science Inc.) and Hitachi L-7485
fluorescence detector. Samples for MALDI-TOFMS were desalted and
(2.73 g, 97%) as white powdery precipitate. R
f
=0.64 (toluene/EtOAc
), 3.89–3.96 (m, 9H;
tBu), 1.34, 1.35, 1.36. 1.39 (each s, 12H; iPr), 2.48 (s, 3H; CH ), 3.73 (dd,
6a,6b =11.3 Hz, J5,6a =4.0 Hz, 1H; H6a), 3.80 (t, J2,3 =7.7 Hz, 1H; H2),
1
3:1); H NMR (CDCl
3
A
H
R
U
G
3 2
): d=0.09 (m, 6H; Si(CH )
3
TM
concentrated by using 10 mL C18 ZipTips (Millipore) according to the
J
manufacturerꢁs instructions. Typically, samples were dissolved in 1 mL of
3.87 (d, 1H; H3), 3.89 (dd, J5,6b =2.8 Hz, 1H; H6b), 3.99 (m, 1H; H4),
4.13 (t, J1b,2 =5.1 Hz, 1H; H1b), 4.15–4.17 (m, 1H; H5), 4.34 (dd, 1H;
1
0% trifluoroacetic acid in acetonitrile (v/v) and mixed with the same
1
3
volume of a saturated solution of 2,5-dihydroxybenzoic acid in 33% ace-
tonitrile that contained 0.1% trifluoroacetic acid. The above mixtures
H1), 7.37, 7.84 ppm (each d, J=8.1 Hz4H; Ar); C NMR (CDCl
3
): d=
0.59, 0.76 (Si(CH ), 24.44 (tBu), 27.65 (CH ), 31.96, 32.81, 33.03, 33.11
A
H
R
U
G
3
)
2
3
(
(
iPr), 68.94 (C6), 75.38 (C1), 83.40 (C3), 83.84 (C2), 84.20 (C5), 87.65
C4), 134.11, 135.79 ppm (Ar); elemental analysis calcd (%) for
(
1 mL) were applied to a stainless steel target MALDI plate and air-dried
before analysis in the mass spectrometer. All measurements were per-
formed by using an Ultraflex TOF/TOF mass spectrometer equipped
with a reflector and controlled by the Flexcontrol 1.2 software package
C H O SSi: C 56.57, H 7.98, S 6.04; found: C 56.43, H 7.93, S 6.24; HR-
MS (FAB): m/z calcd: 530.2370; found: 530.7467 [M+H] .
25 42 8
+
(
Bruker Daltonics GmbsH, Bremen, Germany). Ions were generated by
1-Azide-1-deoxy-6-O-tert-butyldimethylsilyl-2,3:4,5-di-O-isopropylidene-
d-galactitol (8): A mixture of tosylate 7 (2.73 g, 5.14 mmol) and sodium
azide (1.34 g, 20.56 mmol) in dimethylfolmamide (20 mL) was stirred at
608C for 24 h. The mixture was concentrated and the residue was dis-
solved in EtOAc. The solution was washed with brine, dried, and evapo-
rated. The residue was purified by chromatography on silica gel (hexane/
EtOAc 45:1), and the fractions that contained the crude product were
collected and concentrated. The residual syrupy material was dissolved in
EtOH and the solution was stored in a refrigerator to yield a precipitate.
a pulsed-UV laser beam (nitrogen laser, l=337 nm) and were accelerat-
ed to a kinetic energy of 23.5 kV. External calibration of MALDI mass
spectra was carried out by using singly charged monoisotopic peaks of a
mixture of human angiotensin II (m/z: 1046.542), bombesin (m/z:
1
619.823), ACTH (m/z: 2465.199), and somatostatin 28 (m/z: 3147.472).
The mixture of these peptides was measured on the central spot of a 3”3
square with external calibration. To achieve mass accuracy better than
6
tion with a mixture of the calibration peptides. The calibration of these
mass spectra was performed automatically by using a customized macro
command of the XMASS 5.1.2 NT software package. The macro com-
mand was used for the calibration of the monoisotopic singly charged
peaks of the above-mentioned peptides.
0 ppm, internal calibration was carried out by doping the matrix solu-
The white amorphous powder was obtained by filtration to give the azide
1
8
0
1
(1.80 g, 87%). R
.08 (m, 6H; Si(CH
2H; iPr), 3.31 (dd, J5,6a =5.9 Hz, J6a,6b =14.1 Hz, 1H; H6a), 3.60 (dd,
5,6b =2.8 Hz, 1H; H6b), 3.72 (dd, J=9.5, J=11.3 Hz, 1H; H1a), 3.84–
.90 (m, 3H; H1b, H3, H4), 4.00 (m, 1H; H2), 4.13 ppm (m, 1H; H5);
f
=0.68 (toluene/EtOAc 7:1); H NMR (CDCl
3
): d=
A
H
R
U
G
3 2
) ), 0.91 (m, 9H; tBu), 1.34, 1.35, 1.36, 1.42 (each s,
J
3
Synthesis of novel GDP-fucose derivatives
1
3
C NMR (CDCl
iPr), 59.5 (C6), 70.69 (C1), 85.11 (C3), 86.28 (C4), 87.73 (C5), 89.52 ppm
C2); elemental analysis calcd (%) for C18 Si: C 53.84, H 8.78, N
3
): d=1.21, 1.34 (Si
A
H
R
U
G
3 2
(CH ) ), 32.85 (tBu), 33.65–33.83
6
-O-tert-Butyldimethylsilyl-2,3:4,5-di-O-isopropylidene-d-galactose
di-
(
(
methyl dithioacetal (5): Compound 5 was synthesized from compound
35 3 5
H N O
[
37]
[43]
4
according to a previously reported procedure, and the details are
described in the Supporting Information.
-O-tert-Butyldimethylsilyl-2,3:4,5-di-O-isopropylidene-d-galactitol (6):
A solution of compound 5 (3.6 g, 7.49 mmol) in 95% aq. acetone
30 mL) was treated with N-bromosuccinimide (2.93 g, 16.5 mmol). The
mixture was stirred at 08C for 5 min, and then Na (5 g) and
NaHCO (5 g) were added. The reaction mixture was evaporated to
remove acetone. The residue was dissolved in CHCl , and the solution
1
4
0.46; found: C 53.75, H 8.74, N, 10.39; HR-MS (FAB): m/z calcd:
02.2424; found: 402.2444 [M+H] .
+
6
1
8
-Azide-1-deoxy-2,3:4,5-di-O-isopropylidene-d-galactitol (9): Compound
(1.80 g, 4.48 mmol) was dissolved in THF (10 mL) at 08C. Then, a 1m
(
THF solution of tetrabutyl ammonium fluoride (TBAF; 6.17 mL,
5.28 mmol) was added to this solution. The mixture was stirred at room
temperature for 1 h. The mixture was concentrated and the residue was
subjected to chromatography on silica gel (hexane/EtOAc 4:1). The frac-
tions that contained product were collected and concentrated. The
syrupy crude material was dissolved in EtOH and the solution was stored
2 2 3
S O
3
3
was washed with brine, dried, and concentrated. The residual syrup was
subjected to chromatography on silica gel (hexane/EtOAc 5:1). The frac-
tions that contained the aldehyde were collected, concentrated, and the
in refrigerator to give white powdery 9 (1.80 g, 87%). R =0.21 (toluene/
f
1
residue was dissolved in EtOH (20 mL). NaBH
4
(85 mg, 2.25 mmol) was
EtOAc 7:1); H NMR (CDCl ): d=1.34, 1.36, 1.38, 1.42 (each s, 12H;
3
added to this solution and the mixture was stirred at room temperature.
After 40 min, the mixture was concentrated to a syrup and subjected to
chromatography on silica gel (hexane/EtOAc 6:1) to afford the crude al-
cohol 6. The syrupy 6 was dissolved in EtOH and the solution was stored
iPr), 3.32 (dd, J6a,6b =13.2 Hz, J5,6a =5.1 Hz, 1H; H6a), 3.60 (dd, J1a,1b
12.0 Hz, J1a,2 =5.6 Hz, 1H; H1a), 3.61 (dd, J5,6b =2.8 Hz, 1H; H6b), 3.77
(t, J3,4 =7.5 Hz, 1H; H3), 3.79 (dd, J1b,2 =2.4 Hz, 1H; H1b), 3.85 (t, J4,5
=
=
1
3
8.0 Hz, 1H; H4), 4.03 (m, 1H; H2), 4.13 ppm (m, 1H; H5); C NMR
(CDCl ): d=28.90–29.80 (iPr), 54.76 (C6), 64.04 (C1), 80.81 (C3), 81.50
(C4), 83.09 (C5), 84.82 ppm (C2); elemental analysis calcd (%) for
12 21 3 5
C H N O : C 50.16, H 7.37, N 14.63; found: C 50.30, H 7.18, N 14.52;
in a refrigerator to yield pure compound 6 (2.22 g, 79%) as a white amor-
3
1
phous powder. R
f
=0.53 (hexane/EtOAc 2:1); H NMR (CDCl
3
): d=
0
.07–0.08 (m, 6H; Si
A
C
H
T
R
E
U
N
G
(CH
3
)
2
), 0.87–0.94 (m, 9H; tBu), 1.35, 1.36, 1.37, 1.38
+
(
(
each s, 12H; iPr), 3.59 (dd, J5,6 =12.0 Hz, J6a,6b =5.6 Hz, 1H; H6a), 3.72
dd, J1,2 =11.3 Hz, J1a,1b =4.6 Hz, 1H; H1a), 3.79 (dd, J5,6b =2.2 Hz, 1H;
HR-MS (FAB): m/z calcd: 288.1559; found: 288.1556 [M+H] .
6-Azide-1,2,3,4-tetra-O-benzoyl-6-deoxy-b-l-galactopyranose (10): Com-
pound 9 (1.20 g, 4.18 mmol) was added to a cooled solution (À788C) of
oxalyl chloride (0.73 mL, 8.35 mmol) and DMSO (1.19 mL, 16.7 mmol) in
CH Cl (5 mL). After 15 min, Et N (2.91 mL, 20.88 mmol) was added and
H6b), 3.80 (dd, J4,5 =5.3 Hz, J3,4 =2.4 Hz, 1H; H4), 3.88 (t, J1b,2 =2.6 Hz,
H; H1b), 3.90 (t, J2,3 =1.3 Hz, 1H; H3), 3.99–4.03 ppm (m, 2H; H2,
1
1
3
H5); C NMR (CDCl
3
): d=0.10, 0.19 (Si
A
H
R
U
G
3 2
(CH ) ), 24.57 (tBu), 31.73,
2
2
3
3
2.70, 32.73, 32.75 (iPr), 68.73 (C6), 69.69 (C1), 88.19 (C4), 84.73 (C3),
the mixture was stirred at 08C for 2 h. The mixture was then poured into
484
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 478 – 487

Carbohydrate Chemistry
FULL PAPER
CHCl
The residue was purified roughly by silica gel column chromatography
hexane/EtOAc 8:1). The fractions that contained the aldehyde were col-
3
and the solution was washed with brine, dried, and concentrated.
6-Deoxy-6-N-(2-naphthalen-2-yl-acetamide)-b-l-galactopyranos-1-yl-
phosphate triethylammonium salt (13): A mixture of tri-O-benzoate 12
(
(86.5 mg, 0.12 mmol) and Et N (1 mL) in MeOH (5 mL) was stirred at
3
lected and concentrated. The residue was dissolved in a mixture of meth-
608C for 4 h. The mixture then was concentrated and the residual syrup
anol (5 mL) and 60% aq. acetic acid (10 mL), and stirred at 608C. After
was purified by chromatography on Iatrobeads (0.5% w/w Et N in
3
4
h, the solution was concentrated and co-evaporated with toluene to
CHCl /MeOH 2:1). The fractions of the product were collected and con-
3
remove water, and the resulted syrup was dissolved in pyridine (30 mL).
Benzoyl chloride (2.33 mL, 20.05 mmol) was added to the solution and
the mixture was stirred at room temperature for 24 h. The mixture was
centrated and the residual syrup was lyophilized with water to afford a
white amorphous 13 (49.4 mg, 87%). R =0.52 (chloroform/MeOH/water
f
1
5:4:1); H NMR (MeOD): d=3.36 (dd, J =8.3 Hz, J6a,6b =14.1 Hz, 1H;
5,6a
concentrated and the residue was dissolved in CHCl
layer was washed with 1n H SO , aq. sat. NaHCO , and brine; dried; and
concentrated. The residue was purified by chromatography on silica gel
hexane/EtOAc 5:1) to afford compound 10 (1.90 g, 73%). R =0.61 (tol-
): d=3.42 (dd, J5,6a =5.6 Hz, J6a,6b
3.1 Hz, 1H; H6a), 3.61 (dd, J5,6b =7.2 Hz, 1H; H6b), 4.59 (m, 1H; H5),
.01–6.06 (m, 3H; H2, H3, H4), 6.94 (d, J1,2 =3.6 Hz, 1H; H1), 7.26–
3
and the organic
H6a), 3.55 (dd, J5,6b =4.6 Hz, 1H; H6b), 3.60 (dd, J2,3 =10.0 Hz, J1,2 =
2
4
3
7.6 Hz, 1H; H2), 3.66 (dd, J3,4 =3.4 Hz, 1H; H3), 3.70 (m, 1H; H5), 3.78
(s, 2H; CH ), 3.80 (d, J =3.2 Hz, 1H; H4), 4.90 (t, J =7.7 Hz, 1H;
2
4,5
1,P
1
3
(
f
H4), 7.55–7.57 (m, 3H; Ar), 7.90–7.92 (m, 4H; Ar); C NMR (MeOD):
1
uene/EtOAc 7:1); H NMR (CDCl
3
=
d=43.10 (C6), 45.14 (CH ), 71.94 (C4), 74.81 (C2), 75.25 (C3), 76.18
2
1
6
8
7
(
C
C5), 100.57 (C1), 129.00–131.25 (ar); HR-MS (FAB): m/z calcd for
À
18
H
22NO
9
P: 426.0959: found: 426.3349 [MÀH] .
1
3
.14 ppm (m, 20H; Ar); C NMR (CDCl
3.34 (C2, C3, C4), 94.90 ppm (C1); elemental analysis calcd (%) for
: C 65.70, H 4.38, N 6.76; found: C 65.66, H 4.43, N 6.89; HR-
3
): d=54.88 (C6), 71.90, 72.94,
6
-Deoxy-6-N-(2-naphthalen-2-yl-acetamide)-b-l-galactospyranos-1-yl-
guanosine 5’-diphosphate disodium salt (1): Compound 13 (40 mg,
.08 mmol) was co-evaporated with pyridine three times, and the residual
syrup was dissolved in dry pyridine (4 mL). GDP-morphoridate
114.2 mg, 0.16 mmol) and 1H-tetrazole (22.4 mg, 0.32 mmol) was added
34 27 3 9
C H N O
MS (FAB): m/z calcd for [M+Na]
0
+
34 27 3 9
C H N O Na: 644.1645; found:
6
44.1646.
(
6
-Azide-2,3,4-tri-O-benzoyl-6-deoxy-b-l-galactopyranos-1-yl-dibenzyl
to this solution and the mixture was stirred at room temperature under
nitrogen for 72 h. Then, the mixture was concentrated and the residue
was subjected to purification by chromatography on DEAE-Cellulose
phosphate (11): A solution of benzoate 10 (500 mg, 0.80 mmol) in
CH Cl (10 mL) and EtOAc (1 mL) was treated with TiBr (1.18 g,
.2 mmol) and the mixture was stirred at room temperature. After 24 h,
2
2
4
3
(
0.1m aq. ammonium bicarbonate). The fractions that contained the
sodium acetate (2.0 g) was added and the mixture was stirred for 10 min
at room temperature. The mixture was concentrated and the residue was
dissolved in CHCl . The organic layer was washed with water, dried, and
3
concentrated. The residual syrup was co-evaporated with toluene to
remove water. The dried material was then dissolved in a mixture of
target material were collected and directly subjected to treatment with
+
Dowex 50W”8 (Na form). The solution was then concentrated and the
residue was purified by chromatography on Wako-gel C18 (water only).
The obtained material was finally purified by chromatography on a Se-
phadex G15 column (water only) to yield white amorphous 1 (25.2 mg,
CH
2
Cl
2
(3 mL), acetonitrile (3 mL), and Et
2
O (3 mL). Crushed molecular
1
3
9%). H NMR (D
2
O): d=3.33 (dd, J= J5,6a =14.1, J6a,6b =9.8 Hz, 1H;
), 3.76 (dd,
4’’,5’’ =2.3 Hz, 1H; H5’’), 3.89 (d, J3’’,4’’ =3.0, 1H; H4’’), 4.30 (s, 2H; H5’),
sieves (3 Š, 2 g) were added, and the mixture was stirred under nitrogen
for 30 min. Dibenzyl phosphate (445.2 mg, 1.60 mmol) and silver carbon-
ate (441.2 mg, 1.60 mmol) were added to this solution, and the mixture
was stirred at room temperature under nitrogen. After 7 h, the molecular
sieves were removed by filtration, and the filtrate was concentrated. The
residue was subjected to the purification by chromatography on silica gel
H6’’a), 3.63 (m, 2H; H2’’, H6’’b), 3.71 (m, 3H; H3’’, CH
2
J
4
.35 (s, 1H; H4’), 4.49 (m, 2H; H2’, H3’), 5.00 (t, J1’’,2’’ =J1’’,P =7.9 Hz, 1H;
H4’’), 5.72 (d, J1’,2’ =4.4 Hz, 1H; H1’), 7.27–7.96 (m, 7H; naphthyl),
13
8
6
1
1
7
.59 ppm (s, 1H; H8); C NMR (D
2
O): d=43.14 (C6’’), 44.91 (CH
2
),
7.78 (C5’), 71.98 (C4’’), 75.01 (C3’’), 76.49 (C5’’), 86.18 (C4’), 89.84 (C1’),
01.26 (C1’’), 127.64, 128.58, 128.97, 129.76, 130.18, 130.29. 130.78,
31.261 ppm (naphthyl); HR-MS (FAB): m/z calcd for C28
(
3
0.5% w/w Et N in hexane/EtOAc 3:1). The fractions that contained the
product were collected and concentrated. The syrupy material was
35 6 16 2
H N O P :
lyophilized with benzene to afford a white amorphous powdery 11
+
73.1585: found: 773.5556 [M+H] .
1
(
350 mg, 70%). R
f
=0.25 (toluene/EtOAc 7:1); H NMR (CDCl
3
): d=
6
-Azide-6-deoxy-b-l-galactopyranos-1-yl-guanosine 5’-diphosphate diso-
3
(
.41 (m, 1H; H6a), 3.59 (m, 1H; H6b), 4.20 (m, 1H; H5), 4.85, 4.77
each m, 2H; CH ), 5.11–5.15 (m, 2H; CH ), 5.58 (d, J=9.8 Hz, 1H;
H3), 5.74 (t, J1,P =J1,2 =7.2 Hz, 1H; H1), 5.89–5.94 ppm (m, 2H; H2, H4);
[
30]
dium salt (16):
Compound 10 (820 mg, 1.32 mmol) and NaOMe
2
2
(
0.4 equiv, 17.28 mg) were dissolved in EtOH (5 mL) and the solution
was stirred for 1 h. Then the mixture was neutralized with Dowex 50W”
(H form) and filtered. The filtrate was concentrated and the residue
1
3
C NMR (CDCl
2.26 (CH ), 73.95 (C3), 76.38 (C5), 99.61 (C1), 129.08–136.38 ppm (Ar);
HR-MS (FAB): m/z calcd for C41 11P: 778.2166; found: 778.2147
3 2
): d=53.10 (C6), 70.86 (C4), 72.07 (C2), 72.22 (CH ),
+
8
7
2
was dissolved in pyridine (3 mL). Acetic anhydride (2 mL) was added to
this solution and the mixture was stirred at room temperature for 4 h.
36 3
H N O
+
[
M+H] .
The mixture was concentrated and the residue was dissolved in CHCl
3
.
6
-Deoxy-2,3,4-tri-O-benzoyl-6-N-(2-naphthalen-2-yl-acetamide)-b-l-gal-
The organic layer was washed with 1n HCl, sat. NaHCO , and brine;
3
actopyranos-1-yl-phosphate triethylammonium salt (12): A mixture of
compound 11 (145 mg, 0.19 mmol) and 10% palladium/charcoal (300 mg)
in methanol (5 mL) was stirred at room temperature under a hydrogen
atmosphere. After 1 h, the palladium/charcoal was removed by filtration,
and the filtrate was concentrated. Naphthyl acetic acid succinimidyl ester
dried; and concentrated. The residue was subjected to chromatography
on silica gel (hexane/EtOAc 3:1) to provide a known 6-azide-6-deoxy-
[
38]
1
,2,3,4-tetra-O-acetyl-a-l-galactopyranose (14)
(493 mg, quantitative
1
yield). H NMR (CDCl
3
): d=2.01, 2.03, 2.17, 2.18 (each s, 3H; Ac), 3.22
(
dd, J5,6a =7.5, J6a,6b =5.4, 1H; H6a) 3.45 (dd, J5,6b =7.5 1H; H6b), 4.23 (t,
(
80.7 mg, 0.29 mmol) and diisopropyl ethylamine (33.0 mL, 0.19 mmol)
J
5,6a =J5,6a =5.7 Hz, 1H; H5), 5.34 (m, 2H; H2, H3), 5.48 (s, 1H; H4),
.40 ppm (s, 1H; H1). This material 14 (373 mg, 1.0 mmol) was converted
directly into 6-azide-6-deoxy-b-l-galactopyranosyl phosphate n-tetrabutyl-
were added to a solution of this material in THF (5 mL), and the mixture
was stirred at room temperature for 24 h. The mixture was concentrated
6
and the residue was dissolved in CHCl
with water, dried, and concentrated. The residual syrup was purified by
chromatography on silica gel (0.5% w/w Et N in CHCl /MeOH 4:1) to
afford a white amorphous powdery 12 (92.2 mg, 64%). R =0.85 (CHCl
MeOH/water 5:4:1); H NMR (MeOD): d=3.34 (dd,
6a,6b =13.9 Hz, 1H; H6a), 3.69 (dd, J5,6b =4.6 Hz, 1H; H6b), 3.69 (s, 2H;
CH ), 4.27 (m, 1H; H5), 5.56 (t, J1,2 =J1,P =7.9 Hz, 1H; H1), 5.66 (dd,
2,3 =10.3 Hz, J3,4 =3.4 Hz, 1H; H3), 5.75 (dd, J1,2 =7.9, J2,3 =10.3 Hz, 1H;
3
. The organic layer was washed
[
38]
1
ammonium salt (15) (72 mg, overall yield 18%). H NMR (D
.31, 3.17 (Et N) 3.49 (dd, J5,6a =12.9, J6a,b =7.2 Hz, 1H; H6a), 3.53 (dd,
2,3 =9.7, J3,4 =3.4 Hz, 1H; H3), 3.54–3.57 (m, 2H; H5, H6b), 3.66 (t,
J1,2 =J2,3 =5.3 Hz, 1H; H2), 3.77 (d, J3,4 =4.0 Hz, 1H; H4), 4.89 ppm (t,
2
O): d=
1
J
3
3
3
f
3
/
1
J
5,6a =8.2 Hz,
J
1,2 =J1,P =7.6 Hz, 1H; H1) by phosphorylation and the subsequent de-
J
[
38]
O-acetylation according to the reported procedure.
2
J
Compound 15 (50 mg, 0.098 mmol) was co-evaporated with pyridine
three times to remove water and the residual syrup was dissolved in dry
pyridine (1 mL). GMP-morpholidate (142 mg, 2.0 equiv) and 1H-tetra-
zole (27.4 mg, 4.0 equiv) were added to this solution and the mixture was
stirred at room temperature under nitrogen for 48 h. The mixture was
1
3
H2), 5.84 (d, J=3.4 Hz, 1H; H4), 7.24–8.03 (m, 22H; Ar); C NMR
MeOD): d=22.52 (C6), 72.50 (C4), 73.83 (C2), 75.36 (C3), 76.31 (C5),
(
9
C
8.91 (C1), 130.20–132.59 (ar); HR-MS (FAB): m/z calcd for
À
39
H
34NO12P: 738.1746; found: 738.1747 [MÀH] .
Chem. Eur. J. 2008, 14, 478 – 487
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
485

S.-I. Nishimura and T. Maeda
concentrated and the residue was dissolved in water. The aqueous solu-
tion was washed with Et O and concentrated. The residue was subjected
to purification by a series of chromatography on Wako-gel C18 (water
In the case of FucT VIII, the general condition was as follows: glycosyl
donor 1 (10–100 mm), an acceptor 20 (100 mm), and FucT VIII (80 mU)
were incubated in 50 mm sodium cacodyrate buffer (200 mL, pH 7.5) by
using a 700 mL cuvette.
2
only) and DEAE-Cellulose (0.06 mm ammonium bicarbonate aq.), Se-
+
phadex G25 (water only), and Dowex 50W”8 (Na form, water only).
Inhibition assay by FRET method: The FRET-based inhibition assay was
performed by employing a fixed condition as follows: 1 (20 mm), 2
(20 mm), and FucT VI (1.4 mU) in 20 mm HEPES buffer (250 mL, pH 7.5)
that contained 20 mm MnCl at 258C for 2 h. To measure the IC of com-
The factions that contained the product were collected and lyophilized to
1
yield a white amorphous 16 (9.6 mg, 11%). H NMR (D
2
O): d=8.52 (s,
1
H; H8), 5.99 (d, J1,2 =5.1 Hz, 1H; H1’), 4.96 (t, J1’’,2’’ =J1’’,P’’ =7.8 Hz, 1H;
2
50
H1’’), 3.88 (d, J3,4 =3.2 Hz, 1H; H4’’), 3.79 (m, 1H; H5’’), 3.67 (dd, J2’’,3’’
=
pound 23, the inhibitory effect by compound 23 (5, 10, and 15 mm) was
evaluated by measuring the change (decrease) of the dansyl emission at
540 nm in comparison with the standard emission spectrum in the ab-
sence of the inhibitor. The glycosylation reactions were stopped by the
addition of 250 mL of acetonitrile. The reactions were monitored by
FRET and HPLC (Supporting Information). Relative inhibitory effects
by 2-naphtylphosphate, GMP, and CMP on FucT VI activity were deter-
mined by employing the following condition: 1 (20 mm), 2 (20 mm), each
compound (20 mm), and FucT VI (0.7 mU) in 25 mm HEPES buffer
9
.6, J3’’,4’’ =3.6 Hz, 1H; H3’’), 3.61–3.59 (m, 2H; H2’’, H6a’’), 3.46 ppm
1
3
(
dd, J5,6 =13.0 Hz, J6a,6b =5.5 Hz, 1H; H6b’’); C NMR (D
2
O): d=98.52
(
C1’’), 86.84 (C1’), 73.66 (C5’’), 72.16 (C2’’), 70.48 (C3’’), 68.57 (C4’’),
2
2.61 ppm (C6’’); HR-MS (FAB): m/z calcd for
C
16
H
22
N
8
O
15
P
2
Na:
+
6
51.0583; found 651.0587 [M+Na] . These physical data were found to
be in good agreement with the data that were reported recently by Wong
et al. (see reference [30]).
Preparation of dansylated sialyl-LacNAc (2): Dansyl-N-acethyllactosa-
mine (18), which was prepared from per-O-acetate 17 by the previously
(
2
250 mL, pH 7.2) that contained 20 mm MnCl at 258C for 1 h.
[
32]
reported procedure was subjected to the sialylation by using recombi-
nant rat a2,3-siaT as follows: a2,3-siaT (50 mU) was added to a solution
of 18 (11.0 mg, 0.16 mmol) in 50 mm sodium cacodyrate buffer (300 mL,
that contained 5 mm MnCl
2
, 1 mm NaN
3
, 0.05% v/v CF-54, 300 mg BSA,
Acknowledgement
pH 7.4) and CMP-Neu5 Ac (14.51 mg, 1.35 equiv) . The reaction mixture
was incubated at 378C for 2 d. After ultracentrifugation, the mixture was
concentrated and the residue was subjected to purification on DEAE-cel-
lulose column chromatography with 30 mm aq. ammonium bicarbonate.
The fractions that contained the product were collected and concentrat-
ed, and the residue was treated with Dowex 50W”8 (Na form). The so-
lution was concentrated and subjected to the chromatography on Sepha-
dex G25 (water only). The fractions that contained the product were col-
This work was partly supported by grants for the National Projects on
“Functional Glycoconjugate Research Aimed at Developing for New In-
dustry” and “Innovation COE Program for Future Drug Discovery and
Medical Care” from JST and the Ministry of Education, Culture, Science,
Sports, and Technology of Japan. The authors thank to Dr. H. Hinou, Dr.
J. Furukawa, Dr. D. Kumagai, Dr. T. Furuike, Dr. N. Miura, and Dr. K.
Takaya of Hokkaido University for their helpful suggestions and techni-
cal assistance.
+
lected and lyophilized to afford white amorphous 2 (15.7 mg, 97%).
1
H NMR (D
1.6 Hz, 1H; H3’’ax), 1.95, 2.03 (each s, 6H; Ac), 2.84–2.85 (m, 7H;
NMe”2, H3’’eq), 2.90–2.93 (m, 2H; CH ), 3.39–3.43 (m, 1H; CH ), 3.49
d, J=9.0 Hz, 1H; H8’’), 3.55–3.78 (m, 13H; H3, H5, H6a, H6b, H2’,
H5’, H6a’, H6b’, H4’’, H7’’, H9a’’, H9b’’, CH ), 3.83–3.91 (m, 5H; H2,
H4, H4’, H5’’, H6’’), 4.04 (dd, J2,3 =9.7 Hz, J3,4 =3.0 Hz, 1H; H3’), 4.28 (d,
1,2 =8.4 Hz, 1H; H1), 4.43 (d, J1’2 =7.8 Hz, 1H; H1), 7.28 (d, J=7.6 Hz,
H; Ar), 7.57–7.61 (m, 2H; Ar), 8.18 (dd, J=1.2, J=7.3 Hz, 1H; Ar),
2 2
O) d=1.57–1.62 (m, 2H; CH ), 1.72 (t, J3’’ax,3’’eq =J3’’ax,4 =
1
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[
(
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C H O N S: 1733.6098; found:
À
1
733.6233 [MÀH] .
6
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À1
emission spectra were scanned from 300 nm to 650 nm at 300 nmmin
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486
www.chemeurj.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 478 – 487

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Published online: October 11, 2007
Chem. Eur. J. 2008, 14, 478 – 487
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
487