Synthesis of galactose-linked uridine derivatives with simple linkers as potential galactosyltransferase inhibitors

Article abstract of DOI:10.1016/j.tet.2005.04.019

Galactose-linked uridine derivatives without charge or dipole contributions in the linker were designed and synthesized via cross metathesis (CM). This strategy would provide a ready access to a range of hybrid compounds linking uridine and galactose deri

Full text of DOI:10.1016/j.tet.2005.04.019

Tetrahedron 61 (2005) 5837–5842
Synthesis of galactose-linked uridine derivatives with simple
linkers as potential galactosyltransferase inhibitors*
*
Shunpei Murata, Satoshi Ichikawa and Akira Matsuda
Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan
Received 16 February 2005; accepted 7 April 2005
Available online 11 May 2005
Abstract—Galactose-linked uridine derivatives without charge or dipole contributions in the linker were designed and synthesized via cross
metathesis (CM). This strategy would provide a ready access to a range of hybrid compounds linking uridine and galactose derivatives.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
charge is disfavored in terms of cell permeability. Some
glycosyltransferases are known to require a divalent metal
for their activity and form a salt bridge with the NDP-sugar
through the metal in their active site called a DXD motif.
Thus, modification of a diphosphate found in an NDP-sugar
has been conducted primarily with respect to its ability to
interact with the divalent metal ion in the acceptor analogue.
In addition to the charge or dipole interaction mentioned
above, the relative position and orientation of nucleoside
and sugar moiety within the enzyme upon binding are
likewise important concerns. Here we describe in an initial
study the design and synthesis of galactose-linked uridine
derivatives without charge or dipole contributions in the
linker, which would be useful for the development of novel
NDP-sugar analogues. Bovine b1,4-galactosyltransferase
(b4GalTI) was chosen as the target enzyme because it is the
most well-studied enzyme among the glycosyltransferases,
and because X-ray crystal structure analysis data are
available.6–8 b4GalT catalyzes the transfer of galactose
from UDP-galactose (Fig. 1, 1) to the 4-hydroxyl group
of N-acetylglucosamine found in oligosaccharides. We
designed two sets of galactose-linked uridine derivatives,
one of which possesses the simple alkane linker (Fig. 1, 2),
the other the alkene (Fig. 1, 3), with the same number of
atoms between the 50-position of uridine and the anomeric
position of galactose. Each set of compounds also consists
of diastereomers at the 50-position of uridine. With the
introduction of an olefin and a hydroxyl group, a systematic
structure-activity relationship could be conducted in terms
of the distance between the uridine and galactose moiety
and the relative orientation toward each other. Compounds 3
are also versatile intermediates because a variety of
functional groups can be introduced at the olefinic position,
which corresponds to the diphosphate in the natural
substrate.
Oligosaccharides on cell surfaces and other glycoconjugates
are responsible for several intercellular and intracellular
events, including intercellular adhesion in inflammation,
tumor metastasis, bacterial or viral infection, or activation
of the innate immune system.1–5 Since diseases such as
rheumatoid arthritis, for example, are caused by malfunc-
tion of glycoconjugate biosynthesis, the challenge to
modulate glycoconjugate biosynthesis is compelling and is
being actively undertaken. Glycosyltransferases are key
enzymes responsible for oligosaccharide biosynthesis and
for catalysis of the sugar-transfer reaction in a stereo- and
regiospecific manner. Each individual glycosyltransferase is
responsible for each glycosylation step in oligosaccharide
biosynthesis. Thus, inhibition of glycosyltransferases
leads to the modulation of oligosaccharide biosynthesis,
providing us with an opportunity to study their biological
functions and to develop new therapeutic agents. Generally,
a nucleoside diphosphate sugar (an NDP-sugar) is utilized
as the sugar donor and the sugar moiety found in an
NDP-sugar is transferred to the hydroxyl group of the
acceptor molecule such as a growing oligosaccharide, a
protein, or a lipid. Extensive efforts have been devoted to
developing inhibitors for these glycosyltransferases with
donor analogues.9–20 However, only limited success has
been achieved.9–11 The diphosphate linkage found in an
NDP-sugar is prone to hydrolysis, and its inherent negative
* This paper constitutes Part 233 of Nucleosides and Nucleotides: for part
232 in this series, Kaga, D.; Minakawa, N.; Matsuda, A. Nucleoside
Nucleotide Nucl. Acids, 2005, in press.
Keywords: Glycosyltransferase inhibitor; b1,4-Galactosyltransferase;
Cross metathesis.
Corresponding author. Tel.: C81 11 706 3228; fax: C81 11 706 4980;
e-mail: matuda@pharm.hokudai.ac.jp
*
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.04.019

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S. Murata et al. / Tetrahedron 61 (2005) 5837–5842
Figure 1. Structures of UDP-Gal (1) and designed compounds (2, 3).
Scheme 1. Preparation of 6 and 8. Reagents and conditions: (a) TBDPSCl, imidazole, DMF, room temperature; (b) 2,2-dimethoxypropane, TsOH, acetone,
room temperature (90%, two steps); (c) MPMCl, NaH, DMF, room temperature (87%); (d) IBX, MeCN, 80 8C; (e) allylmagnesium bromide, THF, K78 8C
(89%, two steps).
Table 1
Entry
Catalyst
Solvent
Temp. (8C)
Yield (%)
9a
10
6a (recovered)
1
2
A
B
B
B
CH2Cl2
CH2Cl2
toluene
CH2Cl2
40
40
110
40
trace
35
15
89
trace
38
54
6
-
43
40
62
3
4b
a Yield was based on the sugar derivative 6.
b The reaction was carried out using 5 equiv of 6.
2. Results and discussion
and Scheme 2. When 6 (1 equiv) and 8a (1 equiv) in a 0.1 M
CH2Cl2 solution was treated with catalyst B33,34 (Fig. 2) at
reflux for 12 h, the desired 9a was obtained in 35% yield
(E/ZZ5/1) as well as the homodimer of the uridine
derivative 10 (Fig. 3) in 38% yield (entry 2). The use of
catalyst A35,36 gave only a trace amount of 9a and was not
effective for CM, as reported (entry 1). A trace amount of the
homodimer of the allyl galactoside 11 was observed in
the reaction mixture. Changing the solvent to toluene and
the reaction temperature decreased the yield of 9a (entry 3).
Our strategy for the synthesis of compounds 2 and 3
consisted of connecting the suitably protected allyl galacto-
side and the 50-C-allyluridine derivative via olefin cross
metathesis (CM).21–29
The CM precursors were prepared as shown in Scheme 1.
Allyl a-galactopyranoside 430 was sequentially protected with
TBDPS, isopropylidene, and MPM groups to give 6. 20,30-O-
Isopropylideneuridine (7) was oxidized with IBX31 to give the
50-aldehyde, which was allylated upon treatment with
allylmagnesium bromide to provide the 50-C-allyl uridine32
8 in 89% yield (8a/8bZ5/1) for two steps. Each diastereomer
was separated by HPLC to give the pure material.
With these compounds in hand, we next examined the key
cross metathesis, and the results are summarized in Table 1
Figure 2. Structure of ruthenium catalysts.

S. Murata et al. / Tetrahedron 61 (2005) 5837–5842
5839
It is known that the ruthenium carbene complex coordinates
with the neighboring oxygen, and this coordination affects
the selectivity and reactivity.21 It is suggested that an initial
[2C2] cycloaddition of the ruthenium carbene in the
catalytic process might selectively0proceed with 8a rather
than 6 via coordination with the 5 -hydroxyl group in 8a.
This result prompted us to use an excess of 6 to increase the
ratio of the desired cross metathesis product 9a to the
homodimer 10. Thus, when 6 (1 equiv) and 8a (5 equiv) in
0.1 M CH2Cl2 solution was treated with catalyst B at reflux
for 12 h, the yield of the desired 9a was increased to 89%
with the decreased amount of homodimer 10 (6% yield,
entry 4). A limited amount of dimerized product 11 was also
obtained under these conditions and the unreacted 6 was
recovered and recycled. The CM with the diastereomer 8b
gave similar results to provide 9b.
All the synthesized target compounds 2a, 2b, 3a and 3b
were tested as inhibitors of bovine b1,4-galactosyltransfer-
ase I, according to the previously reported procedure18,19
using UDP-[3H]Gal as the glycosyl donor. Compounds 2a
and 2b were tested as a geometric mixture because the
geometrical isomers were inseparable. However, none of
them exhibited inhibitory activities at concentrations up to
1 mM. Compounds 3 possess neutral and hydrophobic liker
between uridine and galactose moieties. Thus, these results
suggest that charge and/or dipole interaction with DXD
motif found in b4GalTI is important for binding. However,
compounds 3 also versatile intermediates because a variety
of functional groups can be introduced at the olefinic
position, which can interact with DXD motif.
3. Conclusion
Deprotection of 9a and 9b by a two-step sequence
afforded the target compounds 3a and 3b, respectively.
Hydrogenation of 3a and 3b gave 2a and 2b, respectively
(Scheme 3).
Galactose-linked uridine derivatives without charge or
dipole contributions in the linker were designed and
synthesized via CM. This strategy would provide a ready
access to a range of hybrid compounds linking uridine and
galactose derivatives. Further transformation of the olefin
found in compounds 3 could provide additional function-
ality, and these studies are in progress.
4. Experimental
4.1. General
Scheme 2. Cross metathesis of 6 and 8a.
NMR spectra were obtained on a JEOL EX270, JEOL
Figure 3. Structures of compound 10 and 11.
Scheme 3. Synthesis of compound 2 and 3. Reagents and conditions: (a) TBAF, THF, room temperature (91% for 12a, 93% for 12b); (b) 80% aq. TFA, room
temperature (quant. for 3a and 3c); (c) H2, Pd(OH)2/C, MeOH, room temperature (quant. for 2a and 2c).

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S. Murata et al. / Tetrahedron 61 (2005) 5837–5842
GX270, JEOL AL400 or Bruker ARX-500 and were
reported in parts per million (d) relative to tetramethylsilane
(0.00 ppm) as an internal standard otherwise noted.
Coupling constant (J) was reported in herz (Hz). Abbrevi-
ations of multiplicity were as follows; s: singlet, d; doublet,
t: triplet, q: quartet, m: multiplet, br: broad. Data were
presented as follows; chemical shift (multiplicity, inte-
gration, coupling constant). Assignment was based on
1H–1H COSY, HMBC and HMQC NMR spectra. Optical
rotations were recorded on JASCO DIP-370 digital
polarimeter or JASCO P-1030 polarimeter. FAB-MS were
obtained on a JEOL JMS-HX101 or JEOL JMS-700TZ.
Analytical thin layer chromatography (TLC) was performed
on Merck silica gel 60F254 plates. Normal-phase column
chromatography was performed on Merck silica gel 5715 or
Kanto Chemical silica gel 60N (neutral). Flash column
chromatography was performed on Merck silica gel 60.
Reverse-phase column chromatography was performed on
3H), 1.07 (s, 9H); 13C NMR (100 MHz, CDCl3) d 135.6,
133.6, 133.4, 133.3, 129.6, 127.6, 117.7, 109.3, 96.4, 76.2,
72.9, 69.7, 68.8, 68.4, 63.0, 27.8, 26.8, 26.0, 19.3; FAB-
LRMS m/z 499 (MHC); FAB-HRMS calcd for C28H39O6Si
499.2516, found 499.2520 (MH).
4.1.2. Allyl 6-O-(tert-butyldiphenylsilyl)-3,4-O-isopropyl-
idene-2-O-(4-methoxybenzyl)-a-D-galactopyranoside
(6). To a solution of 5 (4.56 mmol, 2.27 g) in DMF (40 ml),
NaH (5.47 mmol, 131 mg) was added and the mixture was
stirred for 1 h. After 1 h stirring, MPMCl (5.47 mmol,
742 ml) was added and the mixture was stirred for further
10 h. The reaction was quenched with MeOH and the
solvent was removed in vacuo. The residue was diluted with
AcOEt (300 ml), and the organic layer was washed with
H2O (100 ml!4) and brine (70 ml), dried over Na2SO4,
filtered and concentrated. The resulting residue was purified
by column chromatography (SiO2, 10/1-8/1-7/1; hexane/
AcOEt) to give 6 (3.97 mmol, 2.45 g, 87%) as a colorless
˚
Waters Preparative C18 125 A (55–105 mm). HPLC sys-
1
syrup. [a]1D9 C52.3 (c 1.44, CDCl3); H NMR (500 MHz,
tems were Shimadzu LC-8A, Shimadzu SPD-6A, Shimadzu
C-R6A. Dichloromethane and acetonitorile were distilled
from P2O5 and then CaH2. Methanol was distilled from
sodium metal or directly used HPLC grade solvent
from Kanto Chemical Co., Inc. Toluene was distilled from
sodium metal/benzophenone ketyl. N,N-Dimethylforma-
mide and dimethylsulfoxide were distilled from CaH2
under reduced pressure or purchased dehydrated solvent
from Kishida Chemical Co., Ltd. Tetrahydrofuran was
purchased dehydrated stabilizer free solvent from Kanto
Chemical Co., Inc. Bovine b1,4-galactosyltransferase I
(b4GalTI) was purchased from TOYOBO Co. Ltd. Uridine
diphosphate-[3H]-galactose was purchased from American
Radiolabeled Chemical Inc. Ovalbmin was purchased from
nacalai tesque. Scintillation count was performed on
PACKARD 1600 TR and Aloka LSC-120.
CDCl3) d 7.68 (m, 4H), 7.41 (m, 6H), 7.27 (m, 2H), 6.85 (m,
2H), 5.91 (m, 1H), 5.30 (dd, 1H, JZ1.4, 17.3 Hz), 5.20 (dd,
1H, JZ1.4, 10.6 Hz), 4.76 (d, 1H, JZ3.5 Hz), 4.72 (d, 1H,
JZ12.3 Hz), 4.62 (d, 1H, JZ12.3 Hz), 4.33 (m, 1H), 4.25
(dd, 1H, JZ2.4, 5.4 Hz), 4.14 (dd, 1H, JZ5.1, 13.0 Hz),
4.08 (m, 1H), 3.96 (dd, 1H, JZ13.0, 6.4 Hz), 3.90 (m, 1H),
3.83 (m, 1H), 3.79 (s, 3H), 3.49 (dd, 1H, JZ3.6, 7.9 Hz),
1.38, 1.35 (each s, each 3H), 1.05 (s, 9H); 13C NMR
(100 MHz, CDCl3) d 159.1, 135.5, 133.7, 133.5, 133.3,
130.4, 129.7, 129.4, 127.6, 117.8, 113.7, 108.9, 95.8, 76.1,
76.0, 73.3, 71.9, 68.2, 68.0, 63.0, 55.3, 28.3, 26.8, 26.4,
19.3; FAB-LRMS m/z 619 (MHC); FAB-HRMS calcd for
C36H47O7Si 619.3091, found 619.3088 (MHC).
4.1.3. 1-(20,30-O-Isopropylidene-60,70,80-trideoxy-a-L-
talo-oct-70-enofranosyl)uracil (8a) and 1-(20,30-O-isopro-
pylidene-60,70,80-trideoxy-b-D-allo-oct-70-enofranosyl)-
uracil (8b). To a solution of 7 (4.32 mmol, 1.23 g) in MeCN
(40 ml), IBX (12.9 mmol, 3.60 g) was added and the
mixture was refluxed for 1.5 h. The reaction mixture was
cooled to 0 8C and the precipitate was filtered off. The
filtrate was concentrated and the residue was coevaporated
with toluene (5 ml!2). A solution of the residue in THF
(40 ml) was cooled to K78 8C. To the solution, allylmag-
nesium bromide (1.0 M solution in Et2O; 13.0 mmol,
13.0 ml) was added dropwise over 10 min and the mixture
was stirred for further 10 min. The reaction was quenched
with saturated aqueous NH4Cl and the precipitate was
filtered off. The filtrate was diluted with AcOEt (200 ml)
and the organic layers were washed with H2O (70 ml) and
brine (70 ml). The aqueous layer was extracted with CHCl3
(70 ml) and combined organic layers were dried over
Na2SO4, filtered and concentrated. The resulting residue
was purified by column chromatography (SiO2, 0–2%
ethanol in CHCl3) to give 8 (3.84 mmol, 1.24 g, 89% for
two steps; diastereomixture of 50R/S-isomer) as a white
foam. Separation of 50-stereoisomer was accomplished by
HPLC (YMC-PACK SIL-06; 1/3; hexane/AcOEt). Physical
data for 50R-isomer 8a were identical with the properties of
the previously reported.32 For 50R-isomer 8a; 1H NMR
(500 MHz, CDCl3) d 8.43 (br s, 1H, exchangeable with
D2O), 7.42 (d, 1H, JZ8.0 Hz), 5.84 (m, 1H), 5.72 (d, 1H,
JZ8.0 Hz), 5.62 (d, 1H, JZ2.7 Hz), 5.20 (m, 2H), 4.97 (m,
4.1.1. Allyl 6-O-(tert-butyldiphenylsilyl)-3,4-O-isopropyl-
idene-a-D-galactopyranoside (5). To a solution of 430
(5.00 mmol, 1.10 g) in DMF (40 ml), imidazole
(12.0 mmol, 817 mg) and TBDPSCl (6.0 mmol, 1.56 ml)
were added and the mixture was stirred for 12 h. The
reaction was quenched with MeOH, and the solvent was
removed in vacuo. The residue was partitioned between
AcOEt (300 ml) and H2O (100 ml) and the organic layer
was washed with H2O (100 ml!2) and brine (70 ml), dried
over Na2SO4, filtered and concentrated. To the residue in
acetone (100 ml), 2,2-dimethoxypropane (50 mmol, 6.1 ml)
and TsOH (0.5 mmol, 86 mg) were added and the mixture
was stirred for 12 h. The reaction mixture was neutralized
with saturated aqueous NaHCO3 and the solvent was
removed in vacuo. The residue was partitioned between
AcOEt (200 ml) and H2O (100 ml) and the organic layer
was washed with H2O (100 ml) and brine (70 ml), dried
over Na2SO4, filtered and concentrated. The residue was
purified by column chromatography (SiO2, 7/1-6/1-5/1;
hexane/AcOEt) to give 5 (4.50 mmol, 2.24 g, 90% for two
steps) as a colorless syrup. [a]2D0 C62.6 (c 1.00, CDCl3); 1H
NMR (400 MHz, CDCl3) d 7.70 (m, 4H), 7.38 (m, 6H), 5.90
(m, 1H), 5.29 (ddd, 1H, JZ1.7, 17.2 Hz), 5.21 (ddd, 1H, JZ
1.7, 10.4 Hz), 4.89 (d, 1H, JZ3.9 Hz), 4.26 (m, 3H), 4.11
(ddd, 1H, JZ2.3, 6.5, 6.6 Hz), 4.03 (ddd, 1H, JZ6.3,
12.8 Hz), 3.94 (m, 2H), 3.80 (m, 1H), 2.30 (d, 1H, JZ
7.1 Hz, exchangeable with D2O), 1.48, 1.34 (each s, each

S. Murata et al. / Tetrahedron 61 (2005) 5837–5842
5841
2H), 4.13 (m, 1H), 3.94 (m, 1H), 2.93 (br s, 1H, exchange-
able with D2O), 2.32 (m, 2H), 1.57, 1.37 (each s, each 3H).
added and stirred for 8 h. The solvent was removed in
vacuo, and the residue was purified by flash column
chromatography (SiO2, 0–1–2–3–4% ethanol in CHCl3) to
1
For 50S-isomer 8b; [a]D21 K7.15 (c 0.91, CDCl3); H NMR
1
(400 MHz, CDCl3) d 8.09 (br s, 1H, exchangeable with
D2O), 7.44 (d, 1H, JZ8.3 Hz), 5.83 (m, 1H), 5.73 (d, 1H,
JZ8.3 Hz), 5.67 (d, 1H, JZ3.4 Hz), 5.19 (d, 1H, JZ
5.8 Hz), 5.15 (s, 1H), 4.96 (dd, 1H, JZ3.4, 6.9 Hz), 4.92
(dd, 1H, JZ3.4, 6.9 Hz), 4.15 (dd, 1H, JZ3.0, 3.4 Hz), 3.83
(m, 1H), 2.65 (d, 1H, JZ6.4 Hz, exchangeable with D2O),
2.36 (m, 2H), 1.55, 1.36 (each s, each 3H); 13C NMR
(100 MHz, CDCl3) d 162.3, 142.5, 133.8, 118.8, 114.5,
102.8, 95.0, 87.4, 83.2, 81.3, 71.1, 38.5, 27.4, 25.3; FAB-
LRMS m/z 325 (MHC); FAB-HRMS calcd for C15H21N2O6
325.1400, found 325.1399 (MHC).
give 12a (127 mmol, 86 mg, 91%) as a colorless glass. H
NMR (500 MHz, CDCl3) for E geometrical isomer d 8.21
(br s, 1H, exchangeable with D2O), 7.38 (d, 1H, JZ8.0 Hz),
7.27 (m, 2H), 6.87 (m, 2H), 5.74 (m, 3H), 5.58 (d, 1H, JZ
1.5 Hz), 4.97 (m, 2H), 4.83 (d, 1H, JZ3.4 Hz), 4.74 (d, 1H,
JZ12.2 Hz), 4.62 (d, 1H, JZ12.2 Hz), 4.35 (dd, 1H, JZ
5.5, 7.7 Hz), 4.21 (dd, 1H, JZ2.7, 5.5 Hz), 4.06 (m, 4H),
3.91 (m, 2H), 3.80 (s, 3H), 3.49 (m, 2H), 3.13 (d, 1H, JZ
2.5 Hz, exchangeable with D2O), 2.31 (m, 2H), 1.57, 1.41,
1.36, 1.34 (each s, each 3H); FAB-LRMS m/z 677 (MHC);
FAB-HRMS calcd for C33H45N2O13 677.2922, found
677.2918 (MHC).
4.1.4. 1-{90-O-[600-O-(tert-Butyldiphenylsilyl)-300,400-O-
isopropylidene-200-O-(4-methoxybenzyl)-a-D-galactopyr-
anosyl]-20,30-O-isopropylidene-60,70,80-trideoxy-a-L-talo-
non-70-enofranosyl}uracil (9a). To a solution of 8a
(59 mmol, 19 mg) and 6 (295 mmol, 182 mg) in CH2Cl2
(2 ml), Grubbs second catalyst B (10 mol%, 6.0 mg) was
added and the mixture was refluxed for 12 h. The solvent
was removed in vacuo, and the residue was purified by
column chromatography (SiO2, 0–2% ethanol in CHCl3) to
give 9a (52 mmol, 48 mg, 89%) as a brown foam. 1H NMR
(500 MHz, CDCl3) for E geometrical isomer d 8.13 (br s,
1H, exchangeable with D2O), 7.67 (m, 3H), 7.41 (m, 7H),
7.36 (m, 3H), 6.85 (m, 2H), 5.71 (m, 3H), 5.58 (d, 1H, JZ
2.7 Hz), 4.97 (m, 2H), 4.76 (d, 1H, JZ3.6 Hz), 4.73 (d, 1H,
JZ12.1 Hz), 4.61 (d, 1H, JZ12.1 Hz), 4.32 (m, 1H), 4.26
(m, 1H), 4.12 (m, 1H), 4.07 (m, 2H), 3.91 (m, 3H), 3.86 (m,
1H), 3.79 (s, 3H), 3.49 (m, 1H), 2.90 (br s, 1H, exchangeable
with D2O), 2.29 (m, 2H), 1.53, 1.37, 1.34, 1.31 (each s, each
3H), 1.05 (s, 9H); FAB-LRMS m/z 915 (MHC); FAB-
HRMS calcd for C49H63N2O13Si 915.4099, found 915.4095
(MHC).
4.1.7. 1-{90-O-[300,400-O-Isopropylidene-200-O-(4-methoxy-
benzyl)-a-D-galactopyranosyl]-20,30-O-isopropylidene-
60,70,80-trideoxy-b-D-allo-non-70-enofranosyl}uracil
(12b). Compound 12b (0.10 mmol, 68 mg, 93%) was
obtained as a colorless glass from 9b (0.11 mmol,
100 mg) as described above for the synthesis of 12a, after
purification by flash column chromatography (SiO2, 0–1–2–
3–4% ethanol in CHCl3). 1H NMR (500 MHz, CDCl3) for E
geometrical isomer d 7.40 (d, 1H, JZ8.1 Hz), 7.29 (d, 2H,
JZ8.5 Hz), 6.87 (d, 2H, JZ8.5 Hz), 5.72 (m, 3H), 5.61
(d, 1H, JZ3.0 Hz), 4.97 (m, 1H), 4.91 (m, 1H), 4.86 (d, 1H,
JZ3.5 Hz), 4.72 (d, 1H, JZ12.1 Hz), 4.62 (d, 1H, JZ
12.1 Hz), 4.34 (dd, 1H, JZ5.5, 7.7 Hz), 4.19 (dd, 1H, JZ
2.5, 5.5 Hz), 4.13 (m, 2H), 4.09 (m, 1H), 3.90 (m, 1H), 3.82
(m, 2H), 3.80 (s, 3H), 3.49 (dd, 1H, JZ3.6, 8.0 Hz), 3.24 (br
s, 1H, exchangeable with D2O), 1.57, 1.39, 1.35, 1.33 (each
s, each 3H); FAB-LRMS m/z 677 (MHC); FAB-HRMS
calcd for C33H45N2O13 677.2922, found 677.2930 (MHC).
4.1.8. 1-(90-O-a-D-Galactopyranosyl-60,70,80-trideoxy-a-
L-talo-non-70-enofranosyl)uracil (3a). 12a (30 mmol,
20 mg) was dissolved in 80% aqueous TFA (200 ml) and
the mixture was stirred for 5 min. The solvent was removed
in vacuo and the residue was coevaporated with EtOH
(1 ml!6). The resulting residue was purified by column
chromatography (C18, 5–10–15% MeOH in H2O) to give
4.1.5. 1-{90-O-[600-O-(tert-Butyldiphenylsilyl)-300,400-O-
isopropylidene-200-O-(4-methoxybenzyl)-a-D-galactopyr-
anosyl]-20,30-O-isopropylidene-60,70,80-trideoxy-b-D-allo-
non-70-enofranosyl}uracil (9b).
Compound 9b
(0.14 mmol, 125 mg, 80%) was obtained as a pale brown
foam from 8a (0.17 mmol, 54 mg) and 6 (0.85 mmol,
556 mg) as described above for the synthesis of 9b, after
purification by flash column chromatography (SiO2, 3/1-2/
1-1/1-1/2; hexane/AcOEt). 1H NMR (500 MHz, CDCl3) for
E geometrical isomer d 8.27 (br s, 1H, exchangeable with
D2O), 7.68 (m, 3H), 7.41 (m, 7H), 7.28 (m, 3H), 6.86 (m,
2H), 5.71 (m, 3H), 5.60 (d, 1H, JZ3.3 Hz), 4.95 (m, 1H),
4.89 (m, 1H), 4.76 (d, 1H, JZ3.3 Hz), 4.71 (d, 1H, JZ
12.2 Hz), 4.60 (d, 1H, JZ12.2 Hz), 4.31 (m, 1H), 4.26 (m,
1H), 4.12 (m, 3H), 4.07 (m, 1H), 3.92 (m, 2H), 3.84 (m, 1H),
3.81 (s, 3H), 3.49 (dd, 1H, JZ3.6, 7.9 Hz), 2.87 (d, 1H, JZ
6.0 Hz, exchangeable with D2O), 2.33 (m, 2H), 1.56, 1.40,
1.37, 1.35 (each s, each 3H), 1.05 (s, 9H); FAB-LRMS m/z
915 (MHC); FAB-HRMS calcd for C49H63N2O13Si
915.4099, found 915.4103 (MHC).
1
3a (30 mmol, 14 mg, quant.) as a colorless glass. H NMR
(500 MHz, DMSO-d6, D2O) for E geometrical isomer d 7.82
(d, 1H, JZ8.0 Hz), 5.89 (m, 1H), 5.87 (m, 1H), 5.75 (m,
2H), 4.97 (d, 1H, JZ2.3 Hz), 4.18 (m, 2H), 4.20 (dd, 1H,
JZ5.4, 12.1 Hz), 4.05 (m, 2H), 3.92 (m, 3H), 3.78 (m, 2H),
3.71 (m, 2H), 2.42 (m, 1H), 2.29 (m, 2H); FAB-LRMS m/z
477 (MHC); FAB-HRMS calcd for C19H29N2O12 477.1720,
found 477.1731 (MHC).
4.1.9. 1-(90-O-a-D-Galactopyranosyl-60,70,80-trideoxy-b-
D-allo-non-70-enofranosyl)uracil (3b). Compound 3b
(74 mmol, 35 mg, quant.) was obtained as a colorless glass
from 12b (74 mmol, 50 mg) as described above for the
synthesis of 3a, after purification by column chroma-
tography (C18, 5–10% methanol in H2O). 1H NMR
(400 MHz, DMSO-d6, D2O) d 7.97 (d, 1H, JZ7.8 Hz),
5.77 (d, 1H, JZ5.4 Hz), 5.62 (d, 1H, JZ5.62 Hz), 4.62 (d,
1H, JZ3.4 Hz), 4.00 (m, 3H), 3.53 (m, 7H), 3.44 (m, 2H),
1.48 (m, 6H); FAB-LRMS m/z 477 (MHC); FAB-HRMS
calcd for C19H29N2O12 477.1720, found 477.1735 (MHC).
4.1.6. 1-{90-O-[300,400-O-Isopropylidene-200-O-(4-methoxy-
benzyl)-a-D-galactopyranosyl]-20,30-O-isopropylidene-
60,70,80-trideoxy-a-L-talo-non-70-enofranosyl}uracil
(12a). To a solution of 9a (140 mmol, 125 mg) in THF
(2 ml), TBAF (1 M solution in THF; 210 mmol, 210 ml) was

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S. Murata et al. / Tetrahedron 61 (2005) 5837–5842
4.1.10. 1-(90-O-a-D-Galactopyranosyl-60,70,80-trideoxy-a-
L-talo-nonofranosyl)uracil (2a). To a solution of 3a
(15 mmol, 7.0 mg) in MeOH (2 ml), palladium hydroxide
on carbon (wet. 10%; 5 mg) was added and the mixture was
vigorously stirred under H2 atmosphere for 10 min. The
catalyst was filtered off through Celite pad and the filtrate
was concentrated. The resulting residue was purified by
column chromatography (C18, 5–10–15% methanol in
H2O) to give 2a (15 mmol, 7.0 mg, quant.) as a colorless
glass. [a]2D1 C21.6 (c 1.09, H2O); 1H NMR (500 MHz,
DMSO-d6, D2O) d 7.82 (d, 1H, JZ8.1 Hz), 5.77 (d, 1H, JZ
6.6 Hz), 5.62 (d, 1H, JZ8.1 Hz), 4.61 (d, 1H, JZ3.4 Hz),
3.68 (m, 2H), 3.57 (m, 4H), 3.53 (m, 3H), 3.42 (m, 2H), 1.47
(m, 6H); 13C NMR (100 MHz, DMSO-d6, D2O) d 163.2,
151.0, 140.9, 101.9, 98.8, 87.8, 86.7, 73.3, 71.1, 70.2, 69.6,
69.0, 68.8, 68.4, 66.9, 60.6, 32.8, 29.1, 22.1; FAB-LRMS
m/z 479 (MHC); FAB-HRMS calcd for C19H31N2O12
479.1877, found 479.1868 (MHC).
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4.1.11. 1-(90-O-a-D-Galactopyranosyl-60,70,80-trideoxy-
b-D-allo-nonofranosyl)uracil (2b). Compound 2b
(10 mmol, 7.0 mg, quant.) was obtained as a colorless
glass from 3b (10 mmol, 7.0 mg) as described above for the
synthesis of 2a, after purification by column chroma-
tography (C18, 5–10% methanol in H2O). [a]2D1 C22.2
(c 1.02, H2O); 1H NMR (400 MHz, DMSO-d6, D2O) d 7.97
(d, 1H, JZ7.8 Hz), 5.77 (d, 1H, JZ5.4 Hz), 5.63 (d, 1H,
JZ7.8 Hz), 4.62 (d, 1H, JZ3.4 Hz), 4.01 (m, 2H), 3.97 (m,
1H), 3.76 (m, 1H), 3.69 (br s, 1H), 3.50 (m, 7H), 1.46 (m,
6H); 13C NMR (100 MHz, DMSO-d6, D2O) d 140.5, 101.7,
98.8, 87.6, 86.6, 73.7, 71.1, 70.7, 69.6, 69.3, 68.9, 68.4,
66.9, 60.6, 33.3, 29.1, 22.2; FAB-LRMS m/z 479 (MHC);
FAB-HRMS calcd for C19H31N2O12 479.1877, found
479.1875 (MHC); FAB-HRMS calcd for C19H31N2O12
479.1877, found 479.1878 (MHC).
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4.2. b1,4-Galactosyltransferase assay
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Assays were performed in a total volume of 100 ml. The
assay medium contained ovalbmin (1 mg/ml), 0.1 M
sodium cacodylate buffer, 10 mM MnCl2, 2 mM AMP,
inhibitor (varying conc.), 1 mCi of UDP-[3H]-galactose, and
b1,4-galactosyltransferase (4 mU). The enzyme assay was
incubated for 3 h at 37 8C and the reaction stopped with 1 ml
of 10% TCA. The precipitate was washed with 10% TCA
and once with ethanol/ether (2/1). The precipitate was
dissolved in 200 ml of 2 N NaOH and the solution was
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Acknowledgements
This work was supported financially by a Grant-in-Aid from
the Ministry of Education, Science, Sports, and Culture.
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