Synthesis of 2-deoxy-hexopyranosyl derivatives of uridine as donor substrate analogues for glycosyltransferases

Article abstract of DOI:10.1016/j.bioorg.2009.08.001

A series of 2-deoxy-hexopyranosyl derivatives of uridine have been synthesized as analogues of UDP-sugar. These compounds were tested as inhibitors against bovine β-1,4-galactosyltransferase I in fluorescent assays and showed no significant inhibition.

Full text of DOI:10.1016/j.bioorg.2009.08.001

Bioorganic Chemistry 37 (2009) 211–216
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis of 2-deoxy-hexopyranosyl derivatives of uridine as donor substrate
analogues for glycosyltransferases
a
b
Ilona Wandzik a, , Tadeusz Bieg , Marianna Czaplicka
*
a Silesian University of Technology, Faculty of Chemistry, Department of Organic Chemistry, Bioorganic Chemistry and Biotechnology, 44-100 Gliwice, Krzywoustego 4, Poland
b Institute of Non-Ferrous Metals, 44-10 Gliwice, Sowin´skiego 5, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 July 2009
Available online 15 August 2009
A series of 2-deoxy-hexopyranosyl derivatives of uridine have been synthesized as analogues of UDP-
sugar. These compounds were tested as inhibitors against bovine b-1,4-galactosyltransferase I in fluores-
cent assays and showed no significant inhibition.
Ó 2009 Elsevier Inc. All rights reserved.
Keywords:
Glycosyltransferases
Donor substrate analogues
Glycals
1. Introduction
[11a]) with modification of the carbohydrate part are: carbacyclic
analogue of UDP-galactose (Ki 58
galactose (Ki 149
M) [11c] or 200-, 300-, 400- and 600-methyl derivatives
of UDP-galactose (Ki 44–270 M) [11d]. Interestingly, UDP-fucose
and UDP-mannose are also powerful inhibitors of b4GalT I (Ki 10.0
and 8.8 M, respectively) [11d]. The pyrophoshpate group is known
lM) [11b], UDP-2-deoxy-2-fluoro-
Oligosaccharide chains in glycoconjugates play important roles
in molecular recognition processes including adhesion in inflamma-
tion, bacterial or viral infection, tumor metastasis and immune re-
sponse [1]. Construction of glycosidic linkages in the biosynthesis
of oligosaccharides, glycolipids, glycoproteins and other glucocon-
jugates is catalysed by glycosyltransferases (GTs), usually through
the Leloir pathway [2]. These enzymes catalyse the transfer of a
monosaccharide unit from an activated nucleotide sugar donor to
the hydroxyl group of an acceptor, which is a growing oligosaccha-
ride, a lipid or a protein. Since GTs are responsible for the synthesis
of glycoconjugates, modulation of their activities has potential for
the control of certain cellular functions and make them desirable
targets for inhibition. Identification of potent inhibitors has been
developing very rapidly during the last two decades since the 3D
structure of several GTs were determined [3–5] and catalytic mech-
anism proposed [6]. Different approaches based on analogies with
donor substrates, acceptor substrates and transition state, respec-
tively, have been used to design potent inhibitors of GTs [7–10].
Although many compounds have been designed and synthesized,
only few of them exhibited significant activity against GTs. Among
them the development of donor substrate analogues has received
considerable attention. These compounds must display Kis value of
the same order of magnitude as that of KM. They feature some struc-
tural changes at the carbohydrate part or at the pyrophosphate
linkage. Examples of inhibitors of one of the most extensively stud-
l
l
l
to bind strongly to a divalent cation, most commonly Mn2+, in the
enzyme active site. Some pyrophosphate analogues were obtained
by varying the diphosphate moiety, resulting in higher stability
towards enzymatic hydrolysis. The reported inhibitors of b4GalT I
of that type are: glycosyl phosphonate [12a], methylenediphospho-
nate [12b] or C-glycosyl ethyl phosphonophosphate [13]. It is
believed that replacement of pyrophosphate group is possible, for
example natural glycosyltransferase inhibitor, such as tunicamycin
is thought to use a sugar ring to mimic the pyrophosphate group
[14]. Several synthetic analogues containing monosaccharide moie-
ties acting as pyrophosphate-metal ion complex mimics were
synthesized as GTs inhibitors [15–17]. Some examples with biolog-
ical data are presented in Fig. 1.
Encouraged by the reports mentioned above, we made another
attempt at using monosaccharide ring as pyrophosphate mimic in
the synthesis of donor substrate analogues. In our study we report
on efficient and stereoselective synthesis of 2-deoxy monosaccha-
rides and disaccharides coupled with the uridine part, which
according to our idea is treated as a necessary moiety for the rec-
ognition and proper binding in the active site. This concept is in
accordance with saturation transfer difference (STD) NMR spec-
troscopy experiments applied to map the binding epitope of
UDP-Gal and UDP-Glc bound to b4GalT I with atomic resolution
[18]. These experiments revealed that binding of a donor substrate
is essentially controlled by the uridine part. In both cases, the
ied b-1,4-galactosyltransferase I (b4GalT I, KM (UDP-Gal) = 44 lM
* Corresponding author.
E-mail address: ilona.wandzik@polsl.pl (I. Wandzik).
0045-2068/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2009.08.001

212
I. Wandzik et al. / Bioorganic Chemistry 37 (2009) 211–216
pyrophosphate mimic
OH
O
pyrophosphate group
OH
HO
O
N
O
OH
O
N
HO
NH
O
O
P
O-
N
H
NHAc
OH
O
OH
NH
9
O
O
P
O-
O
O
O
O
O
O
O
OH
Mn++
OH OH
OH OH
HO
natural donor substrate
UDP-α-D-galactopyranose
OH
tunicamycin
IC50 7 nM (GlcNAc phosphotransferase) 14
O
N
O
OH OH
OH
O
OH
OH
NH
O
NH
O
O
O
HO
HO
HO
O
O
O
HO
O
O
N
OH
O
HO
O
OH
OH
OH
OH OH
OH
Ki 119.6 μM (β4GalT I) 15
inhibition <10% at 0.5 mM (β4GalT I) 17
O
O
O
N
O
HO
NH
N
OH
O
OH
O
NH
NH
O
OH
O
N
O
HO
O
HO
O
N
O
N
O
O
HO
O
O
O
O
N
OH
OH
OH OH
OH OH
OH OH
IC50 3.2 mM (chitin synthase) 16
IC50 0.8 mM (chitin synthase) 16
IC50 2.0 mM (chitin synthase) 16
Fig. 1. Examples of glycosyltransferase inhibitors containing monosaccharide ring as a pyrophosphate mimic.
anomeric proton H-1 of the ribose ring and H-5 of the uracil
received the largest amount of saturation transfer. This indicates
that the uridine residue is in tight contact with the active site of
the protein. The galactose protons show smaller STD effects, which
suggests a minor role of this residue in the recognition step. For
UDP-Glc in the presence of b4GalT I, STD effect was also observed,
although only the uridine part received saturation transfer, the
glucose protons give no STD responses. This is in accordance with
the fact that b4GalT I does not process UDP-Glc at a significant rate
[19]. Now, we propose compounds presented in Fig. 2 as potential
inhibitors containing the uridine moiety, which is supposed to
exhibit significant binding affinity for the enzyme. We hope that
2. Experimental
2.1. General
Melting points were determined on a Boetius apparatus and are
uncorrected. Optical rotations were measured with a Perkin–Elmer
141 polarimeter using a sodium lamp (589 nm) at room tempera-
ture. NMR spectra were recorded with a Varian spectrometer at a
frequency of 600 MHz with Me4Si as internal reference in CD3OD
as a solvent. Mass spectra were recorded in the positive mode on
a Mariner (Perseptive Biosystem) detector using the electrospray-
ionisation (ESI) technique.
2-deoxy-D-gluco- and 2-deoxy-D-galactopyranosyl residues can
provide additional binding with protein, not necessary specific
for glycosyl transfer, which is not expected.
Reactions were monitored by TLC on precoated plates of silica
gel 60 F254 (Merck), and visualising using UV light (254 nm or
366 nm) and/or by charring with 10% sulphuric acid in ethanol.
OH
Y
OH
O
X
HO
O
HO
HO
O
OH
O
N
OH
O
N
O
O
NH
O
NH
O
O
NH
HO
O
HO
HO
HO
O
N
O
O
O
O
O
HO
HO
O
O
O
O
OH
O
O
O
O
O
O
4
3
1 X=OH, Y=H
2 X=H, Y=OH
H3C
CH3
H3C
CH3
H3C
CH3
OH
X"
O
X'
OR2
O
N
"Y
HO
NR3
O
O
OH
R1O
R1O
Y
O
N
O
O
NH
O
X
HO
O
NH
O
Y'
HO
N
O
O
O
O
O
O
O
O
H3C
O
OH
OH
CH3
OH
OH
5 R1=Bn, R2=TBDMS, R3=Bz
6 R1=Bn, R2=H, R3=Bz
7 R1=H, R2=H, R3=H
10 X'=H, Y'=OH, X"=H, Y"=OH
11 X'=H, Y'=OH, X"=OH, Y"=H
12 X'=OH, Y'=H, X"=H, Y"=OH
13 X'=OH, Y'=H, X"=OH, Y"=H
8 X=OH, Y=H,
9 X=H, Y=OH
Fig. 2. 2-Deoxy-hexopyranosyl derivatives of uridine as donor substrate analogues for glycosyltransferases.

I. Wandzik et al. / Bioorganic Chemistry 37 (2009) 211–216
213
Column chromatography was performed on silica gel 60 (70–230
mesh, Merck) developed with one of the hexane/EtOAc or CHCl3/
MeOH solvent systems. All evaporations were performed under
diminished pressure at 50 °C. Reversed phase HPLC analyses were
performed using Perkin–Elmer Series 200 apparatus equipped with
for
413.1151.
C
15H22N2O10Na ([M + Na]+): m/z 413.1167, found: m/z
2.4. 2-deoxy-a-D-glucopyranosyl-(1 ? 6)-2-deoxy-a-D-glucopyrano-
syl-(1 ? 5)- uridine (10)
a reverse phase column (Aquapore OD-300, 7
l
m, 4.6 ꢀ 250 mm;
mobile phase: H2O/MeCN, 73:27, flow rate 1 ml/min) with a fluo-
rescence detector. Fluorescence of acceptor substrate and product
was read at 385 nm excitation/540 nm emission.
Glycal 14 (132 mg, 0.30 mmol) and uridine derivative 18
(232 mg, 0.30 mmol) were submitted to general procedure A (reac-
tion time 1 h). The resulting adduct 19 (273 mg, 0.24 mmol) was
desilylating according to the general procedure B and submitted
to the next addition step with glycal 16 (85 mg, 0.20 mmol)
according to the general procedure A (reaction time 3 h). Deprotec-
tion of the resulting adduct 25 (165 mg, 0.12 mmol) according to
the procedure C yielded 10 as a white solid (59 mg, 35% overall
N3,20,30-O-tris-(benzyloxycarbonyl)uridine (18) [20], 3,4-di-O-
benzyl-6-O-tert-butyldimethylsilyl-
D
-glucal (14) and 3,4-di-O-
-galactal (15) [21], 3,4,6-tri-
benzyl-6-O-tert-butyldimethylsilyl-
D
O-benzyl-D-glucal (16) and 3,4,6-tri-O-benzyl-D-galactal (17) [22]
were prepared according to the published procedures. Uridine 50-
diphosphogalactose disodium salt (UDP-Gal) and other chemicals
were purchased from Aldrich and Fluka Chemical Companies and
were used without purification. Bovine milk b-1,4-galactosyltrans-
ferase I (b4GalT I) was purchased from Sigma at 1 U/mg. Solvents
were dried and stored over molecular sieves (4 Å) under an inert
atmosphere.
yield): mp 132–135 °C; ½a D20
ꢂ
+ 58.0 (MeOH, c 0.5); 1H NMR: d
1.61 (ddd, 1H, J 3.6, 11.7, 13.0 Hz, H-2000ax), 1.71 (ddd, 1H, J 3.7,
11.8, 13.4 Hz, H-200ax), 2.09 (ddd, 1H, J ꢃ0, 5.1, 13.4 Hz, H-200eq),
2.11 (ddd, 1H, J ꢃ0, 5.1, 13.0 Hz, H-2000eq), 3.27 (t, 1H, J 9.3 Hz,
H-4000), 3.33 (dd, 1H, J 9.7, 9.2 Hz, H-400), 3.60–3.74 (m, 5H, H-50a,
H-500, H-5000, H-600a, H-6000a), 3.76–3.88 (m, 4H, H-300, H-3000, H-600b,
H-6000b), 3.73 (dd, 1H, J 2.4, 11.1 Hz, H-50b), 4.14 (dd, 1H, J 3.7,
4.4 Hz, H-20), 4.15–4.19 (m, 2H, H-30, H-40), 4.95, 4.97 (2 dd, 2H,
H-100, H-1000), 5.76 (d, 1H, J 8.1 Hz, H-5), 5.89 (d, 1H, J 3.7, Hz, H-
2.2. General synthetic procedures
13
2.2.1. Procedure A
10), 7.98 (d, 1H, J 8.1 Hz, H-6); C NMR: d 38.82, 38.82 (C-200, C-
To a solution of glycal 14, 15, 16 or 17 (0.30 mmol) and uridine
derivative 18, 23 or 24 (0.30 mmol) in dry CH2Cl2 (5 mL) a catalytic
amount of TPHB (10 mg, 0.03 mmol) was added. The mixture was
kept at room temperature for 1–20 h. Then the reaction mixture
was concentrated to give crude product purified directly by col-
umn chromatography with hexane/AcOEt 2:1 solvent system to
yield adducts 19, 20, 21, 22, 25, 26, 27, 28 as oils.
2000), 62.82 (C-6000), 66.98, 66.98 (C-50, C-600), 71.28 (C-30), 70.01,
70.16, 72.99, 73.08, 73.28, 73.88 (C-300, C-400, C-500, C-3000, C-4000, C-
500), 76.05 (C-20), 84.31 (C-40), 91.03 (C-10), 98.49, 98.75 (C-100, C-
1000), 102.59 (C-5), 142.05 (C-6), 152.24 (C-2), 166.17(C-4); ESI-
HRMS: Calcd for C21H32N2O14Na ([M + Na]+): m/z 559.1746, found:
m/z 559.1720.
2.5. 2-deoxy-a-D-galactopyranosyl-(1 ? 6)-2-deoxy-a-D-glucopyra-
2.2.2. Procedure B
nosyl-(1 ? 5)- uridine (11)
Desilylation of adduct 19 or 20 (0.20 mmol) was achieved with
AcCl (16
l
L, 0.20 mmol) in a MeOH/CHCl3 3:1 solvent system
Glycal 14 (132 mg, 0.30 mmol) and uridine derivative 18
(232 mg, 0.30 mmol) were submitted to general procedure A (reac-
tion time 1 h). The resulting adduct 19 (273 mg, 0.24 mmol) was
desilylating according to the general procedure B and submitted
to the next addition step with glycal 17 (85 mg, 0.20 mmol)
according to the general procedure A (reaction time 20 h). Depro-
tection of the resulting adduct 26 (141 mg, 0.10 mmol) according
to the procedure C yielded 11 as a white solid (40 mg, 25% overall
(8 mL) at room temperature within 15 min. The reaction mixture
was neutralized with a basic resin, Amberlyst 21 (OHꢁ), filtered
and concentrated to give crude product 23 or 24 as an oil.
2.2.3. Procedure C
A solution of 21, 22, 25, 26, 27 or 28 (0.10 mmol) in a 1:6:2 mix-
ture of cyclohexene/EtOH/THF (10 mL) was heated under reflux in
the presence of Pd(OH)2/C (75 mg) for 30 min. After removal of the
catalyst by filtration the reaction mixture was concentrated and
crude product was purified by column chromatography with
CHCl3/MeOH 5:1 ? 2:1 solvent system to yield final products 8–
13 as white solids.
yield): mp 142–145 °C; ½a D20
ꢂ
+ 67.5 (MeOH, c 0.5); 1H NMR: d 1.71
(ddd, 1H, J 3.7, 11.8, 13.4 Hz, H-200ax), 1.80 (ddd, 1H, J 0.8, 4.9,
12.7 Hz, H-2000eq), 1.94 (ddd, 1H, J 3.7, 11.9, 12.7 Hz, H-2000ax),
2.10 (ddd, 1H, J 1.0, 5.1, 13.4 Hz, H-200eq), 3.32 (t, 1H, J 9.8 Hz, H-
400), 3.64 (ddd, 1H, J 2.0, 5.4 9.8 Hz, H-500), 3.66–3.74 (m, 4H, H-
50a, H-600a, H-6000a, H-6000b), 3.76–3.82 (m, 2H, H-300, H-4000), 3.83–
3.88 (m, 2H, H-5000, H-600b), 3.96 (ddd, 1H, J 3.0, 4.9, 11.9 Hz, H-
3000), 3.90 (dd, 1H, J 2.5, 11.2, Hz, H-50b), 4.14 (dd, 1H, J 3.7,
4.4 Hz, H-20), 4.15–4.19 (m, 2H, H-30, H-40), 4.97, 4.98 (2 dd, 2H,
H-100, H-1000), 5.75 (d, 1H, J 8.1 Hz, H-5), 5.88 (d, 1H, J 3.7, Hz, H-
Compound 8 was synthesized in 60% overall yield and identified
in our recent report [20].
2.3. 2-deoxy-a-D-galactopyranosyl – (1 ? 5)- uridine (9)
13
Glycal 17 (125 mg, 0.30 mmol) and uridine derivative 18
(232 mg, 0.30 mmol) were submitted to general procedure A (reac-
tion time 1 h). The resulting adduct 22 (230 mg, 0.20 mmol) was
deprotected according to the general procedure C to yield 9 as a
white solid (68 mg, 58% overall yield): mp 130–132 °C;
10), 7.98 (d, 1H, J 8.1 Hz, H-6); C NMR: d 33.63 (C-2000), 38.86 (C-
200), 63.19 (C-6000), 66.58 (C-50), 66.96 (C-3000), 67.02 (C-600), 69.66
(C-4000), 70.12 (C-300), 71.26 (C-30), 72.33 (C-5000), 73.04 (C-500),
73.07 (C-400), 76.06 (C-20), 84.30 (C-40), 91.06 (C-10), 98.78, 98.81
(C-100, C-1000), 102.56 (C-5), 142.07 (C-6), 152.33 (C-2), 166.19 (C-
4); ESI-HRMS: Calcd for C21H32N2O14Na ([M + Na]+): m/z
559.1746, found: m/z 559.1731.
½
a 2D0 + 34.5 (MeOH, c 0.5); 1H NMR: d 1.79 (ddd, 1H, J 1.0, 13.1,
ꢂ
5.0 Hz, H-200eq), 2.04 (ddd, 1H, J 3.7, 12.2, 13.1 Hz, H-200ax), 3.66–
3.80 (m, 5H, H-50a, H-400, H-500, H-600a,b), 3.89–3.95 (m, 2H, H-50b,
H-300), 4.11–4.16 (m, 3H, H-20, H-30, H-40), 5.01 (dd, 1H, J 1.1,
3.7 Hz, H-100), 5.74 (d, 1H, J 8.1 Hz, H-5), 5.90 (d, 1H, J 3.9 Hz,
2.6. 2-deoxy-a-D-glucopyranosyl-(1 ? 6)-2-deoxy-a-D-
galactopyranosyl-(1 ? 5)-uridine (12)
13
H-10), 7.98 (d, 1H, J 8.1 Hz, H-6); C NMR: d 33.72 (C-200), 63.36
(C-600), 66.74 (C-300), 67.25 (C-50), 69.57 (C-400), 71.38 (C-30), 73.00
(C-500), 76.10 (C-20), 84.47 (C-40), 90.88 (C-10), 99.15 (C-100), 102.57
(C-5), 142.04 (C-6), 152.38 (C-2), 166.18 (C-4); ESI-HRMS: Calcd
Glycal 15 (132 mg, 0.30 mmol) and uridine derivative 18
(232 mg, 0.30 mmol) were submitted to general procedure A (reac-
tion time 1 h). The resulting adduct 20 (217 mg, 0.20 mmol) was

214
I. Wandzik et al. / Bioorganic Chemistry 37 (2009) 211–216
desilylating according to the general procedure B and submitted to
the next addition step with glycal 16 (70 mg, 0.17 mmol) according
to the general procedure A (reaction time 20 h). Deprotection of
the resulting adduct 27 (143 mg, 0.10 mmol) according to the pro-
cedure C yielded 12 as a white solid (48 mg, 30% overall yield): mp
(10 mL). The resin was washed with methanol (180 mL) and drips
containing product were concentrated. TLC analysis was performed
in CHCl3/MeOH 4:1 solvent system and product was visualised
using UV light (366 nm): RF = 0.4. The crude product was purified
by column chromatography with CHCl3/MeOH 25:1 ? 10:1 sol-
vent system to yield final product as a yellowish solid (116 mg,
134–136 °C; ½a 2D0
ꢂ
+ 52.7 (MeOH, c 0.5); 1H NMR: d 1.61 (ddd, 1H, J
3.7, 11.6, 13.0 Hz, H-2000ax), 1.80 (ddd, 1H, J 1.0, 5.0, 13.0 Hz, H-
200eq), 2.03 (ddd, 1H, J 3.6, 12.2, 13.0 Hz, H-200ax), 2.07 (ddd, 1H, J
1.3, 5.2, 13.0 Hz, H-2000eq), 3.25 (dd, 1H, J 9.0, 9.8 Hz, H-4000), 3.58–
3.63 (m, 2H, H-5000, H-600a), 3.67–3.74 (m, 2H, H-50a, H-6000a), 3.77
(bd, 1H, J 3.0 Hz, H-400), 3.79–3.85 (m, 3H, H-3000, H-600b, H-6000b),
3.87–3.96 (m, 3H, H-300, H-50b, H-500), 4.12–4.19 (m, 3H, H-20, H-
30, H-40), 4.94 (dd, 1H, J 1.3, 3.7 Hz, H-1000), 4.99 (dd, 1H, J 1.0,
3.6 Hz, H-100), 5.76 (d, 1H, J 8.1 Hz, H-5), 5.89 (d, 1H, J 4.0 Hz, H-
52%): mp 86–89 °C; ½a D20
ꢂ
ꢁ 3.1 (MeOH, c 0.5); 1H NMR: d 1.05–
1.20 (m, 4H, (CH2)2); 1.23–1.39 (m, 4H, (CH2)2), 1.93 (s, 3H, Ac);
2.82 (t, 2H, J 6.8 Hz, CH2NH); 2.85 (s, 6H, N(CH3)2); 3.25 (ddd,
1H, J 2.3, 5.7, 9.6 Hz, H-5); 3.31 (m, 2H, H-4, OCH2a); 3.45 (dd,
1H, J 8.7, 10.2 Hz, H-3); 3.61 (dd, 1H, J 8.4, 10.2 Hz, H-2); 3.68
(dd, 1H, J 5.7, 11.9 Hz, H-6a); 3.75 (m, 1H, OCH2b); 3.87 (dd, 1H,
J 2.3, 11.9 Hz, H-6b); 4.34 (d, 1H, J 8.4 Hz, H-1); 7.25 (dd, 1H, J
0.7, 7.6 Hz, H-dansyl); 7.55 (dd, 1H, J 7.3, 8.5 Hz, H-dansyl); 7.57
(dd, 1H, J 7.6, 8.6 Hz, H-dansyl); 8.18 (dd, 1H, J 1.2, 7.3 Hz, H-dan-
syl); 8.36 (ddd, 1H, J 0.7, 1.2, 8.6 Hz, H-dansyl); 8.54 (d, 1H, J 8.5 Hz,
H-dansyl); 13C NMR: d 23.08 (CH3CO); 26.53, 27.19, 30,33, 30,57
(C-hexyl); 43.86 (CH2NH); 45.85 (N(CH3)2); 57.89 (C-2); 62.81
(C-6); 70.34 (OCH2); 72.16 (C-4); 76.06 (C-3); 77.87 (C-5); 102.65
(C-1); 116.42, 120.61, 124.31, 129.08, 130.11, 131,09 (C-dansyl),
130.99, 131.20, 137.20, 153.167 (Cq-dansyl), 173.62 CH3CO); ESI-
HRMS: Calcd for C26H40N3O8S ([M + H]+): m/z 554.2531, found:
m/z 554.2545.
13
10), 7.98 (d, 1H, J 8.1 Hz, H-6); C NMR: d 33.68 (C-200), 38.86 (C-
2000), 62.85 (C-6000), 66.67 (C-300), 67.15 (C-50), 67.88 (C-600), 69.50
(C-400), 69.95 (C-3000), 70.89 (C-500), 71.36 (C-30), 73.27 (C-4000),
74.05 (C-5000), 76.04 (C-20), 84.34 (C-40), 90.97 (C-10), 98.69 (C-1000),
98.99 (C-100) 102.59 (C-5), 142.10 (C-6), 152.34 (C-2), 166.16 (C-
4); ESI-HRMS: Calcd for C21H32N2O14Na ([M + Na]+): m/z
559.1746, found: m/z 559.1734.
2.7. 2-deoxy-a-D-galactopyranosyl-(1 ? 6)-2-deoxy-a-D-galactopyra-
nosyl-(1 ? 5)-uridine (13)
2.9. Bovine milk b-1,4-galactosyltransferase I assay
Glycal 15 (132 mg, 0.30 mmol) and uridine derivative 18
(232 mg, 0.30 mmol) were submitted to general procedure A (reac-
tion time 1 h). The resulting adduct 20 (217 mg, 0.20 mmol) was
desilylating according to the general procedure B and submitted
to the next addition step with glycal 17 (70 mg, 0.17 mmol)
according to the general procedure A (reaction time 20 h). Depro-
tection of the resulting adduct 28 (108 mg, 0.08 mmol) according
to the procedure C yielded 13 as a white solid (36 mg, 22% overall
b4GalT I activity was assayed using UDP-Gal as glycosyl donor
and b-GlcNAc-O-(CH2)6-dansyl as glycosyl acceptor as described
previously [13]. Assays were performed in a total volume of
100
final concentrations: 50 mM Hepes buffer (pH 7.4), 10 mM MnCl2,
0.2 mg/ml BSA, 200 V b-GlcNAc-O-(CH2)6-dansyl, 40 V UDP-Gal
ll. The reaction mixtures contained reagents in the following
l
l
and potential inhibitors 1–13 at a range of concentrations from
0 mM (control) to 2.4 mM. The enzymatic reactions were started
by the addition of 0.2 mU b4GalT I and incubated at 30 °C for
14 min. Inactivation was quickly done by immersion of the reac-
tion solutions for 2 min in a boiling water bath. The solutions were
yield): mp 146–149 °C; ½a D20
ꢂ
+ 23.2 (MeOH, c 0.25); 1H NMR: d
1.75–1.82 (2 ddd, 2H, H-200eq, H-2000eq), 1.94 (ddd, 1H, J 3.7, 11.9,
12.7 Hz, H-2000ax), 2.03 (ddd, 1H, J 3.6, 12.2, 13.0 Hz, H-200ax),
3.57–3.63 (2 dd, 2H, H-600a, H-6000a), 3.66–3.76 (m, 2H, H-50a, H-
500), 3.76–3.84 (m, 4H, H-400, H-4000, H-5000, H-600b), 3.87–3.96 (m,
4H, H-300, H-3000, H-50b, H-6000b), 4.12–4.19 (m, 3H, H-20, H-30, H-40),
4.97 (bd, 1H, J 3.5 Hz, H-100), 5.00 (bd, 1H, J 3.5 Hz, H-1000), 5.75 (d,
1H, J 8.1 Hz, H-5), 5.89 (d, 1H, J 3.8 Hz, H-10), 7.98 (d, 1H, J
diluted with water (200 ll) and centrifuged for 10 min, and the
supernatant was injected into RP-HPLC system. The percentage of
inhibition was evaluated from the fluorescence intensity of the
peaks referring to product (Galb-1,4-GlcNAcb-O-(CH2)6-dansyl).
13
8.1 Hz, H-6); C NMR: d 33.69, 33.69 (C-200, C-2000), 63.24 (C-6000),
66.66, 66.69, 67.10, 67.88, 69.48, 69.67, 70.90, 71.37, 72.49 (C-30,
C-50, C-300, C-400, C-500, C-600, C-3000, C-4000, C-5000), 76.04 (C-20), 84.35
(C-40), 91.03 (C-10), 99.01, 99,05 (C-10, C-1000), 102.58 (C-5), 142.08
(C-6), 152.34 (C-2), 166.15 (C-4); ESI-HRMS: Calcd for
C21H32N2O14Na ([M + Na]+): m/z 559.1746, found: m/z 559.1737.
3. Results and discussion
Recently, we have synthesized 2-deoxy-hexopyranosyl deriva-
tives of 2,3-O-isopropylidene-uridine as donor substrate analogues
of glycosyltransferases [24] (compounds 1–7, Fig. 2). These
compounds were composed of 2,3-O-isopropylidene-uridine and
2.8. 6-N-(dansylamino)hexyl 2-acetamido-2-deoxy-b-
side (b-GlcNAc-O-(CH2)6-dansyl)
D-glucopyrano-
one or two residues of 2-deoxy-
-galactopyranose. The central 2-deoxy-
replacing the key pyrophosphate group was linked with terminal
2-deoxy-O-glycosyl moiety through -(1 ? 3)-, -(1 ? 4)- or
-(1 ? 6)-linked glycosidic linkage. Compounds 1–4 were synthe-
sized in two sequential addition reactions of 2,3-O-isopropyli-
dene-uridine acceptor to the double bond of glycal in
a-D
-glucopyranose or 2-deoxy-
a-
D
a-D-glucopyranose moiety
6-N-(Benzyloxycarbonylamino)hexyl 2-acetamido-3,4,6-tri-O-
acetyl-2-deoxy-b- -glucopyranoside [23] (232 mg, 0.40 mmol))
D
a
a
was deacetylated with 0.033 M solution of MeONa in methanol
(30 mL) at room temperature within 20 h. The reaction mixture
was neutralized with an acidic resin, Dowex 50WX8 (H+), filtered
and concentrated. The residue was dissolved in EtOH (10 mL) then
cyclohexene (2 mL) and Pd(OH)2/C (150 mg) were added. The solu-
tion was heated under reflux for 5 min. After removal of the cata-
lyst by filtration the reaction mixture was concentrated and the
residue was dissolved in 0.033 M solution of Na2CO3 in water
(25 mL). To the resulting solution suspension of dansyl chloride
(108 mg, 0.40 mmol) in aceton (8 mL) was added dropwise. The
reaction mixture was stirred at room temperature for 1 h. Then
the reaction mixture was passed through the column with mac-
roreticular strong base anion exchange resin Amberlyst A-26
a
a
stereoselective manner using the Falck–Mioskowski protocol [25].
Synthesized compounds 1–7 feature an acetonide moiety as a
protection of two hydroxyl groups on the ribose part. We origi-
nally attempted to synthesize totally deblocked structures.
Unfortunately, acidic removal of the acetonide group occurred in
low yield due to the poor stability of 2-deoxyglycosidic linkage
in acid medium. There was a concern that acetonide derivatives
will not fit into the active site of the enzyme owing to the steric
hindrances created by the isopropylidene group. Therefore, we
decided to improve our methodology by applying the another

I. Wandzik et al. / Bioorganic Chemistry 37 (2009) 211–216
215
way of protecting the uridine moiety. The second synthetic ques-
tion was a kind of glycosidic linkage between two glycosyl units.
Recently, we reported on molecular docking simulations aiming
at investigating interactions between 2-deoxy-hexopyranosyl
derivatives of uridine and the active site of b-1,4-galactosyltrans-
ferase I [26]. Structures composed of uridine and two glycosyl
we used N3,20,30-O-tris-(benzyloxycarbonyl)uridine (18) [20] as a
substrate. Uridine derivative 18 enabled the addition reaction to
3,4-di-O-benzyl-6-O-tert-butyldimethylsilyl-
D
-glucal (14) and
-galactal (15) to be
3,4-di-O-benzyl-6-O-tert-butyldimethylsilyl-
D
performed at 5-OH in the presence of TPHB as a catalyst in CH2Cl2
as a solvent (Scheme 1).
residues: 2-deoxy-
D
-glucopyranose and 2-deoxy-
D
-galactopyra-
Removal of the orthogonal TBDMS protecting group in 19 and
20 was effected by treatment of AcCl in methanol [28]. Compounds
23 and 24, which possessed free hydroxyl at C-6, were submitted
without purification to the second addition step. In addition reac-
tion to glycals 16 and 17, respectively, we have synthesized four
nose connected by different glycosidic linkages were analysed.
Simulated binding modes of the top ranked ligands suggested that
two hydroxyl groups: 2-OH and 3-OH in the ribose moiety as well
as the uracil nitrogen exhibited interactions with the key amino
acids residues similar to that of the natural substrate. Simulations
isomers as
a-1,6-linked derivatives 25–28. Simultaneous removal
showed that binding affinity of structures featuring
sidic linkage between glycosyl units compared better with
or -1,4 analogues. Therefore, we wish to describe supplementary
synthesis of 2-deoxy-hexopyranosyl derivatives of uridine 10–13
as totally deblocked compounds, which contained two residues
a
-1,6 glyco-
of benzyl and benzyloxycarbonyl groups in compounds 21, 22,
25–28 was accomplished by catalytic transfer hydrogenolysis on
Pearlman’s catalyst (palladium(II) hydroxide) [29] in the presence
of cyclohexene [30] in reflux of ethanol affording the unprotected,
new compounds 9–13 without side reactions. All new compounds
were purified by column chromatography and their structures
were elucidated with the aid of 1H and 13C NMR spectroscopy data
(including two-dimensional DQCOSY, HMQC, HMBC experiments
and simulation analysis) and mass spectrometry analysis (for de-
tails see Sections 2.3–2.8).
The main goal of this work was to evaluate synthesized com-
pounds 1–13 as galactosyltransferase inhibitors. Compounds 1–4
and 10–13 can be treated as UDP-sugar analogues, while com-
pounds 5–9 as UDP analogues. Compounds 5 and 6 were included
in examination due to their antiviral activity against classical
swine fever virus, which can be associated with the inhibition of
a
-1,3
a
of 2-deoxy sugars linked through
a-1,6 glycosidic linkage
(Fig. 2). In order to synthesize the target compounds we applied
glycal derivatives 14–17 with benzyl protection of hydroxyl
groups (Scheme 1). Unfortunately, N3,20,30-O-tri-benzyl-uridine
could not be applied due to a problem in the final N-debenzylation
[27]. Our goal was to apply a protective group that could be
removed in neutral conditions, preferably by catalytic hydrogenol-
ysis, together with O-benzyl protection in 2-deoxy-hexopyranose
part. Our previous experiments revealed beneficial influence of
using a large group as uridine N-imide protection on
a-selectivity
in the addition of uridine derivatives to
D-glucal [24]. Therefore,
O
N
OR
O
N
OH
O
N
X'
X'
NCbz
O
O
O
NCbz
O
NCbz
O
Y'
BnO
Y'
BnO
OR
X
HO
O
O
O
Y
i
ii
O
O
O
BnO
+
OCbz
OCbz
OCbz
OCbz
OCbz
OCbz
18
14 R=TBDMS, X=H, Y=OBn
15 R=TBDMS, X=OBn, Y=H
16 R=Bn, X=H, Y=OBn
19 R=TBDMS, X'=H, Y'=OBn
20 R=TBDMS, X'=OBn, Y'=H
23 X'=H, Y'=OBn
24 X'=OBn, Y'=H
iii
iii
21 R=Bn, X'=H, Y'=OBn
8
9
17 R=Bn, X=OBn, Y=H
22 R=Bn, X'=OBn, Y'=H
OBn
X"
"Y
OH
O
N
X'
O
O
NCbz
O
BnO
Y'
BnO
OBn
X
O
O
X'
i
O
Y
O
O
NCbz
Y'
BnO
BnO
+
O
N
O
O
OCbz
OCbz
O
16 X=H, Y=OBn
17 X=OBn, Y=H
23 X'=H, Y'=OBn
24 X'=OBn, Y'=H
25 X'=H, Y'=OBn, X"=H, Y"=OBn
26 X'=H, Y'=OBn, X"=OBn, Y"=H
27 X'=OBn, Y'=H, X"=H, Y"=OBn
28 X'=OBn, Y'=H, X"=OBn, Y"=H
OCbz
OCbz
iii
OH
X"
O
"Y
HO
Bn - benzyl
Bz - benzoyl
O
N
O
X'
Cbz - benzyloxycarbonyl
O
NH
Y'
HO
TBDMS - tert-butyldimethylsilyl
O
O
O
10 X'=H, Y'=OH, X"=H, Y"=OH
11 X'=H, Y'=OH, X"=OH, Y"=H
12 X'=OH, Y'=H, X"=H, Y"=OH
13 X'=OH, Y'=H, X"=OH, Y"=H
OH
OH
Scheme 1. (i): TPHB (0.1 equiv.), CH2Cl2, rt., 1–20 h; (ii): AcCl (1.0 equiv.), MeOH, rt., 15 min.; and (iii): Pd(OH)2/C, cyclohexene, EtOH/THF, reflux, 30 min.

216
I. Wandzik et al. / Bioorganic Chemistry 37 (2009) 211–216
[7] K.-H. Jung, R.R. Schmidt, in: C.-H. Wong (Ed.), Carbohydrate-Based Drug
Discovery, vol. 2, Wiley-VCH Verlag, Weinheim, 2003, pp. 609–659.
[8] P. Compain, O.R. Martin, Bioorg. Med. Chem. 9 (2001) 3077–3092.
[9] W. Zou, Curr. Top. Med. Chem. 5 (2005) 1363–1391.
[10] T. Kajimoto, M. Node, J. Synth. Org. Chem. Jpn. 64 (2006) 894–912.
[11] (a) G. Srivastava, O. Hindsgaul, M.M. Palcic, Carbohydr. Res. 245(1993)137–144;
(b) H. Yuasa, M.M. Palcic, O. Hindsgaul, Can. J. Chem. 73 (1995) 2190–2195;
(c) T. Hayashi, B.W. Murray, R. Wang, C.-H. Wong, Bioorg. Med. Chem. 5 (1997)
497–500;
(d) T. Ende, Y. Kajihara, H. Kodana, H. Hashimoto, Bioorg. Med. Chem. Lett. 4
(1996) 1939–1948.
[12] (a) M.M. Vaghefi, R.J. Bernacki, N.K. Dalley, B.E. Wilson, R.K. Robins, J. Med.
Chem. 30 (1987) 1383–1391;
(b) M.M. Vaghefi, R.J. Bernacki, W.J. Hennen, R.K. Robins, J. Med. Chem. 30
(1987) 1391–1399.
[13] S. Vidal, I. Bruyère, A. Malleron, C. Augé, J.-P. Praly, Bioorg. Med. Chem. 14
(2006) 7293–7301.
[14] A. Heifetz, R.W. Keenan, A.D. Elbein, Biochemistry 18 (1979) 2186–2192.
[15] R. Wang, D.H. Steensma, Y. Takaoka, J.W. Yun, T. Kajimoto, C-H. Wong, Bioorg.
Med. Chem. 5 (1997) 661–672.
[16] J-B. Behr, T. Gourlain, A. Helimi, G. Guillerm, Bioorg. Med. Chem. Lett. 13
(2003) 1713–1716.
[17] L. Ballell, R.J. Young, R.A. Field, Org. Biomol. Chem. 3 (2005) 1109–1115.
[18] T. Biet, T. Peters, Angew. Chem. Int. Ed. 40 (2001) 4189–4192.
[19] M.M. Palcic, O. Hindsgaul, Glycobiology 1 (1991) 205–209.
[20] I. Wandzik, T. Bieg, M. Kadela, Nucleos. Nucleot. Nucl. 27 (2008) 1250–1256.
[21] J-P. Lellouche, S. Koeller, J. Org. Chem. 66 (2001) 693–696.
[22] S.K. Madhusudan, G. Agnihotri, D.S. Negi, A.K. Misra, Carbohydr. Res. 340
(2005) 1373–1377.
[23] A.-M.M. van Roon, B. Aguilera, F. Cuenca, A. van Remoortere, G.A. van der
Marel, A.M. Deelder, H.S. Overkleeft, C.H. Hokke, Bioorg. Med. Chem. 13 (2005)
3553–3564.
[24] I. Wandzik, T. Bieg, Bioorg. Chem. 35 (2007) 401–416.
[25] V. Bolitt, Ch. Mioskowski, S.-G. Lee, J.R. Falck, J. Org. Chem. 55 (1990) 5812–5813.
[26] I. Wandzik, Acta Pol. Pharm. 65 (2008) 735–741.
[27] D.C. Johnson II, T.S. Widlanski, Org. Lett. 6 (2004) 4643–4646.
[28] A.T. Khan, E. Mondal, Synlett 5 (2003) 694–698.
glycosylation at the stage of glycan modification characteristic for
mammalian cells [31].
Bovine milk b4GalT I was chosen as the target enzyme. It is one
of the most well-studied enzyme among glycosyltransferases with
available X-ray crystal structure analysis data [32] and commercial
availability. D4GalT I is responsible for biosynthesizing N-acetyllac-
tosamine by the transfer of galactose from UDP-Gal to the 4-OH
group of N-acetylglucosamine of an acceptor sugar in glycopro-
teins or glycolipids with b-1,4-linkage.
Radioassays [12,33] and fluorescence assays [13,34] are
routinely used to measure GTs activity. We have applied
the fluorescence assay developed by Praly and co-workers [13].
b-GlcNAc-O-(CH2)6-dansyl was used instead of the natural accep-
tor substrate. Firstly, compounds 1–13 were tested as possible
substrates. 1 mM sample of investigated compound and fluores-
cent acceptor substrate were incubated at 37 °C for 30 min in the
presence of bovine milk b4GalT I. Reactions were monitored by
TLC on Silica Gel 60F254 with UV detection. TLC analysis revealed
that acceptor substrate was the only one bearing fluorescent label,
this indicated that no reaction occurred between acceptor
substrate and synthesized donor analogues.
All the synthesized compounds 1–13 were then tested as poten-
tial inhibitors in a competition assay against bovine milk b4GalT I
using fluorescent acceptor b-GlcNAc-O-(CH2)6-dansyl as
a
substrate. None of the compounds presented in Fig. 2 displayed
significant inhibitory activity against bovine milk b4GalT I at con-
centrations up to 2.4 mM. Very poor inhibition was observed for
compound 9 (IC30 2.4 mM). Our results suggest that derivatives
of uridine connected with one or two 2-deoxy-hexopyranose rings
[29] W.M. Pearlman, Tetrahedron Lett. 17 (1967) 1663–1664.
[30] S. Hanessian, T.J. Liak, B. Vanasse, Synthesis (1980) 396–398.
[31] [a] I. Wandzik, G. Pastuch-Gawołek, W. Szeja, B. Szewczyk, E. Król, G.
Grynkiewicz, Polish Patent Application No P 381955, 13.03. 2007.;
(b) E. Król, I. Wandzik, B. Szewczyk, G. Grynkiewicz, W. Szeja, Antivir. Res. 82
(2009) A78.
in
a-configuration of glycosidic linkages are not able to ensure
binding into the enzyme active site with strength comparable to
the natural donor substrate.
[32] (a) L.N. Gastinel, C. Cambillau, Y. Bourne, EMBO J. 18 (1999) 3546–3557;
(b) B. Ramakrishnan, P.V. Balaji, P.K. Qasba, J. Mol. Biol. 318 (2002) 491–502.
[33] (a) M.M. Palcic, L.D. Hoerze, M. Pierce, O. Hindsgaul, Glycoconjugate J. 5 (1988)
49–63;
Acknowledgment
Financial support from Rector of The Silesian University of
(b) Y. Ichikawa, Y.C. Lin, D.P. Dumas, G.J. Shen, E. Garcia-Lunceda, M.A.
Williams, R. Bayer, C. Ketcham, L.E. Walker, J. Am. Chem. Soc. 114 (1992)
9283–9298;
Technology
(Grant BW/RGW-7/RCh-0/2009) is gratefully
acknowledged.
(c) I. Brockhausen, M. Benn, S. Bhat, S. Marone, J.G. Riley, P. Montoya-Peleaz,
J.Z. Vlahakis, H. Paulsen, J.S. Schutzbach, W.A. Szarek, Glycoconjugate J. 23
(2006) 123–525.
References
[34] (a) G. Limberg, G.C. Slim, C.A. Compston, P. Stangier, M.M. Palcic, Liebigs Ann.
(1996) 1773–1784;
[1] A. Varki, R.D. Cummings, J.D. Esco, H.H. Freeze, P. Stanley, C.R. Bertozzi, G.W.
Hart, M.E. Etzler (Eds.), Essentials of Glycobiology, second ed., CSHL Press, 2009.
[2] L.F. Leloir, Science 172 (1971) 1299–1303.
[3] N. Kikuchi, H. Narimatsu, Biochim. Biophys. Acta 1760 (2006) 578–583.
[4] U.M. Ünligil, J.M. Rini, Curr. Opin. Struct. Biol. 10 (2000) 510–517.
(b) Y. Kanie, A. Kirsch, O. Kanie, C-H. Wong, Anal. Biochem. 263 (1998) 240–245;
(c) T. Ivannikova, F. Bintein, A. Malleron, S. Juliant, M. Cerutti, A. Harduin-Lepers,
P. Delannoy, C. Augé, A. Lubineau, Carbohydr. Res. 338 (2003) 1153–1161;
(d) K. Takaya, N. Nagahori, M. Kurogochi, T. Furuike, N. Miura, K. Monde, Y.C. Lee,
S-I. Nishimura, J. Med. Chem. 48 (2005) 6054–6065.
ˇ
[5] C. Breton, L. Šnajdrowa, C. Jeanneau, J. Koca, A. Imberty, Glycobiology 16 (2006)
29R–37R.
[6] I. Tvoroška, I. André, J.P. Carver, J. Am. Chem. Soc. 122 (2000) 8762–8776.