5-Amino-2-pyridyl 1-thioglycosides in synthesis of analogs of glycosyltransferases substrates

Article abstract of DOI:10.1016/j.bioorg.2009.04.002

We present the synthesis of 1-thioglycosyl derivatives of uridine, which were designed to act as potential donor substrates for glycosyltransferases. We constructed such analogs using 5-amino-2-pyridyl 1-thioglycosides as glycosyl units which were connect

Full text of DOI:10.1016/j.bioorg.2009.04.002

Bioorganic Chemistry 37 (2009) 77–83
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
5-Amino-2-pyridyl 1-thioglycosides in synthesis of analogs
of glycosyltransferases substrates
*
Gabriela Pastuch-Gawolek , Tadeusz Bieg, Wieslaw Szeja, Jakub Flasz
Silesian University of Technology, Faculty of Chemistry, Department of Organic Chemistry, Bioorganic Chemistry and Biotechnology, 44-100 Gliwice, Krzywoustego 4, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 December 2008
Available online 19 April 2009
We present the synthesis of 1-thioglycosyl derivatives of uridine, which were designed to act as potential
donor substrates for glycosyltransferases. We constructed such analogs using 5-amino-2-pyridyl 1-thio-
glycosides as glycosyl units which were connected to uridine via succinic linker. For preparation of the
amide bonds we applied different condensation procedures.
Keywords:
Ó 2009 Elsevier Inc. All rights reserved.
GTs substrate analogs
1-Thioglycosides
Amide bond formation
1. Introduction
Affecting glycosylation processes might bring about various
changes within the biological processes utilizing glycoproteins
Modulation of biological adhesion processes has been a long
standing goal of pharmaceutical invention in pathological pro-
cesses ranging from inflammation, arteriosclerosis to viral infec-
tion and tumor metastasis [1]. In fact, cellular or cell-surface-
associated cell adhesion molecules responsible for diseases are
attractive drug targets because of their accessibility, which is
evoked by the fact that the drug molecules no longer need to trans-
fer through cell membranes [2]. Alternatively, enzymes utilizing
such molecules or responsible for the biosynthesis of the recogni-
tion epitopes may also be targeted in order to alter the process of
cell adhesion [3]. Actually, they should be chemically more tracta-
ble than the adhesion molecules that function in the targeted
pathway.
Glycosylation catalyzed by a family of glycosyltransferases
(GTs) is one of post-translational modification of peptides impor-
tant in cell-adhesion [4]. GTs of the Leloir pathway [5] which cat-
alyze the transfer of a sugar moiety from an activated nucleotide
sugar to a hydroxyl group of an acceptor (a growing oligosaccha-
ride, a lipid or a protein [6]) are responsible for synthesizing most
of glycoproteins and other glycoconjugates in mammalian systems.
Glycosyltransferases are usually metal ion dependent, where mag-
nesium or manganese are the most typical metals found in the ac-
tive site. The metal ion is essential for catalytic properties of GTs
since it interacts with pyrophosphate group of the UDP-sugar do-
nor in enzyme active site, thus binding it and forcing its correct ori-
entation in space [7].
(e.g. enzyme activity or ligand binding). In some reported cases,
absence of particular glycans or introduction of other particular
glycans within the glycoprotein structure has been found to influ-
ence its biological activity [8]. Therefore, search for potent inhibi-
tors of such processes is of grave importance.
Among known GT inhibitors there are such natural compounds
as tunicamycin (a GlcNAc phosphotransferase inhibitor which may
also act as GT inhibitor) [9], Nikkomycin Z and Polyoxin D (a chitin
synthetase inhibitor) [10] or moenomycin A (a bacterial transgly-
cosylase inhibitor) [11]. However, tunicamycin exhibits high cyto-
toxicity which prevents from its use in clinical therapy. Therefore,
more selective inhibitors are needed.
Searching for carbohydrate mimetics-based GT inhibitors ap-
pears to be a task of substantial and still growing interest. Different
strategies for designing new GTs inhibitors were applied [12].
Structures of proposed inhibitors are based on their structural
analogies between donor substrates, acceptor substrates and tran-
sition state, respectively. In case of donor substrate analogs some
structural changes concerned the carbohydrate part or pyrophos-
phate linkage [13].
Numerous analogs of pyrophosphate linker have been pro-
posed, most of which would theoretically come into interactions
with Mn2+ similarly to their natural counterparts. Some of the pro-
posed structures bear carbon or sulfur atoms in the phosphoester
structures instead of oxygen, which is expected to increase the
overall stability against enzymatic hydrolysis [14]. Malonates, tart-
arates and even monosaccharide moieties have also been proposed
as replacements for pyrophosphate linkage [15]. However, almost
none of the proposed potential inhibitors exhibited significant
activity.
* Corresponding author.
E-mail address: gabriela.pastuch@polsl.pl (G. Pastuch-Gawolek).
0045-2068/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2009.04.002

78
G. Pastuch-Gawolek et al. / Bioorganic Chemistry 37 (2009) 77–83
In our study, we focused on simple and efficient synthesis of
was synthesised according to
Kunishima [27].
Other chemicals were purchased from Aldrich, Fluka and Acros
Chemical Companies and were used without purification. Solvents
were dried and stored over molecular sieves (4 Å).
a
procedure described by
carbohydrate derivatives conjoined with uridine part, which is
responsible for the recognition and proper binding within the ac-
tive site. The structures we proposed are believed to exhibit im-
proved stability against hydrolytic cleavage.
Stability of the structure would be increased by replacing the
oxygen atom between the linker and sugar moiety with sulfur.
Thioglycosides are well tolerated by most of biological systems
[16,17]. They are also known for their increased resistance against
enzymatic degradation [18,19]. Stability of (5-nitro-2-pyridyl) 1-
thioglycosides has been shown while utilizing their selectively
protected derivatives as glycosyl acceptors in the presence of var-
ious acidic promoters [20]. That is the main reason for choosing
such structures to be the sugar fragment analogs joined with uri-
dine. Although all of the proposed structures have b-configuration
which stays in contradiction to the naturally occurring GT
substrates, there are reports stating that such sugar donor analog
as 50-O-b-lactosyl-uridine [15] exhibited biological activity despite
having b-configuration at the anomeric carbon. Disodium
uridin-50-yl (2-acetamido-2-deoxy-b-D-glucopyranosylthiometh-
ylphosphono) phosphate was also tested as inhibitor of UDP-
GlcNAc 2-epimerase [19].
Within the structures we proposed, the pyrophosphate was re-
placed with succinic linkage. Choice of this particular fragment was
based on the reports in which succinic derivatives were effectively
employed to coordinate divalent metal ions [21,22]. The aforemen-
tioned studies proved that carbonyl oxygen atoms of the succinic
fragment are responsible for the complexation process. Linker sta-
bility was additionally enhanced by introduction of amide bond
between the linker and sugar in the reaction between the succinic
carboxyl groups with primary amine present within the structure
of sugar aglycone.
2.2. General procedures for condensation of amines and uridine
derivatives
2.2.1. Procedure A using DCC as a coupling agent
To a solution of amine 1 (0.25 mmol), DCC (0.25 mmol) and
DMAP (0.25 mmol) in dry CHCl3 (5 mL), an equimolar amount of
uridine derivative 8 (0.25 mmol) in CHCl3 (2 mL) was added. The
mixture was stirred at room temperature for 48 h. The reaction
mixture was concentrated to give a mixture of crude products
which was purified directly by column chromatography with
CHCl3/MeOH 100:1 ? 20:1 solvent system.
2.2.2. Procedure B using DCC as a coupling agent
To a solution of amine 1 or 2 (0.25 mmol) and uridine derivative
7, 8 or 9 (0.25 mmol) in dry pyridine (3 mL), DCC (51 mg,
0.25 mmol) and DMAP (31 mg, 0.25 mmol) were added. The mix-
ture was stirred at room temperature for 17–24 h (appropriate
reaction times are given in Table 1). After completion (TLC, 10:1
CHCl3–MeOH or 1:1 toluene–AcOEt) the reaction mixture was con-
centrated with toluene (3 ꢀ 10 mL) in order to remove the whole
amount of pyridine. The crude product was purified directly by col-
umn chromatography with an appropriate solvent system as
indicated.
2.2.3. Procedure C using ethyl chloroformate as coupling agent
To a solution of ethyl chloroformate (0.25 mmol) in dry CH2Cl2
(4 mL) with an addition of Et3N (0.25 mmol), a solution of uridine
derivative 8 or 9 in CH2Cl2 (4 mL) was added in portions. Resulting
reaction mixture was stirred at room temperature for 1.5 h and
then the amine 1 (0.25 mmol) was added. The mixture was stirred
at room temperature for 48 h. Finally, the reaction mixture was
concentrated to give a crude product that was purified directly
by column chromatography with an appropriate solvent system
as indicated.
2. Experimental
2.1. General methods
NMR spectra were recorded for solutions in CDCl3 or DMSO-d6
using Varian spectrometer at a frequency of 300 MHz with TMS
as internal reference. NMR solvents were purchased from ACROS
Organics (Geel, Belgium). Chemical shifts (d) are expressed in
ppm and coupling constants (J) in Hz. Optical rotations were mea-
sured on Perkin–Elmer 141 polarimeter using a sodium lamp
(589.3 nm) at room temperature. Mass spectra were measured in
the positive mode with a Mariner (Perspective Biosystem) detector
using the electrospray-ionization (ESI) technique.
2.2.4. Procedure D using DMT-MM as coupling agent
To a solution of amine 1 or 2 (0.25 mmol) and uridine derivative
7, 8 or 9 (0.25 mmol) in dry THF (5 mL) with addition of MeOH
(0.3 mL), an equimolar amount of DMT-MM (69 mg, 0.25 mmol)
was added (Table 1). The mixture was stirred at room temperature
for 24–48 h (appropriate reaction times are given in Table 1). After
Reactions were monitored by TLC on precoated plates of silica
gel 60 F254 (Merck). TLC plates were inspected under UV light
and developed by charring after spraying with 10% sulphuric acid
in ethanol. Column chromatography was performed on Silica Gel
60 (70–230 mesh, Fluka) developed with either toluene–EtOAc or
CHCl3–MeOH solvent systems. Organic solvents were evaporated
on a rotary evaporator under reduced pressure at 50 °C.
Table 1
Condensation of amines 1–2 with uridine derivatives 7–9.
Entry Amine Uridine
derivative
Product Reaction time Procedure Yield
(h)
(%)
1
1
8
15
11
10
10
14
12
11
14
10
11
14
12
13
48
A
34
5
20,30-O-isopropylideneuridine (3) [23], 3-N-benzoyl-20,30-O-iso-
propylideneuridine (4) [24], 20,30-di-O-tert-butyldimethylsilyl-uri-
dine (5) [25] were prepared according to the published
procedures. (5-amino-2-pyridyl) 2,3,4,6-tetra-O-acetyl-1-thio-b-
D-glucopyranoside (1) and (5-amino-2-pyridyl) 2,3,4,6-tetra-O-
acetyl-1-thio-b-D-galactopyranoside (2) were synthesized by
reduction of the corresponding (5-nitro-2-pyridyl) 2,3,4,6-tetra-
O-acetyl-1-thio-b-D-glycopyranosides [26] with zinc powder/
acetic acid system in CH2Cl2; 3-N-benzoyl-20,30-di-O-tert-butyldi-
methylsilyl-uridine (6) was synthesized by benzoylation of 5 with
benzoyl chloride/Et3N system in CH2Cl2. 4-(4,6-Dimethoxy-
(1,3,5)triazin-2-yl)-4-methyl-morpholinium chloride (DMT-MM)
2
3
4
5
6
7
8
9
1
1
1
2
1
1
1
1
1
2
2
7
8
9
8
8
9
7
8
9
7
8
24
17
20
19
48
48
48
48
48
24
48
B
B
B
B
C
Tracesa
23
14
10
40
27
53
42
51
39
C
D
D
D
D
D
10
11
12
37
a
Only TLC analysis, product was not isolated by column chromatography.

G. Pastuch-Gawolek et al. / Bioorganic Chemistry 37 (2009) 77–83
79
completion (TLC, 10:1 CHCl3–MeOH or 1:1 toluene–AcOEt) the
reaction mixture was concentrated to give a crude product which
was purified directly by column chromatography with an appropri-
ate solvent system as indicated.
2.3.5. Procedure D using uridine derivative 7 for preparation of 10
Compound 1 (114 mg, 0.25 mmol) and uridine derivative 7
(96 mg, 0.25 mmol) were submitted to general procedure D de-
scribed above. The resulting crude product 10 was purified by col-
umn chromatography to yield 10 as a white solid (109 mg, 53%):
2.3. Compound (10)
mp 145–148 °C; ½a D25
ꢁ
ꢂ5.4 (c 1.6, CHCl3); 1H NMR (CDCl3): d
10.10 (bs, 1H, NH), 8.72 (s, 1H, NH), 8.52 (d, 1H, J 2.4 Hz, H-6pyr),
7.98 (dd, 1H, J 2.4, 8.6 Hz, H-4pyr), 7.35 (d, 1H, J 8.0 Hz, H-6ur),
7.20 (d, 1H, J 8.6 Hz, H-3pyr), 5.70 (d, 1H, J 8.0 Hz, H-5ur), 5.65 (d,
2.3.1. Succinic acid mono-20,30-isopropylidene-uridin-50-yl ester (7)
To
a 3 (500 mg,
solution of 20,30-isopropylidene-uridine
1.76 mmol) in a mixture of CH2Cl2 (7 mL) and pyridine (3 mL) suc-
cinic anhydride (176 mg, 1.76 mmol) and DMAP (215 mg,
1.76 mmol) were added. The resulting mixture was stirred at room
temperature for 2 h. After completion (TLC, 10:1 CHCl3–MeOH) the
reaction mixture was concentrated with toluene (3 ꢀ 10 mL) in or-
der to remove the whole amount of pyridine. The crude product
was purified on a column of silica gel using CHCl3–MeOH 10:1 sol-
vent system as the eluent to yield 7 as a white powder (490 mg,
1H, J 10.4 Hz, H-1glu), 5.62 (d, 1H, J 1.5 Hz, H-10 ), 5.35 (dd ꢃ t,
ur
1H, J 9.3 Hz, H-3glu), 5.18 (dd ꢃ t, 1H, J 9.8 Hz, H-2glu), 5.15 (dd ꢃ t,
1H, J 9.5 Hz, H-4glu), 5.05 (dd, 1H, J 1.6, 6.4 0Hz, H-20 ), 4.85 (dd, 1H, J
ur
3.3, 6.4 Hz, H-3u0 r), 4.40–4.32 (m, 3H, H-4ur, H-50aur, H-50bur), 4.26
(dd, 1H, J 4.4, 12.4 Hz, H-6aglu), 4.10 (dd, 1H, J 3.3, 12.3 Hz, H-6bglu),
3.88 (ddd, 1H, J 2.4, 4.2, 10.0 Hz, H-5glu), 2.78–2.65 (m, 4H,
2 ꢀ CH2), 2.04, 2.03, 2.01 (3s, 12H, 4 ꢀ CH3CO), 1.55, 1.32 (2s, 6H,
C(CH3)2); 13C NMR (CDCl3): d 172.86, 171.02, 170.68, 170.41,
169.80, 169.71 (CO), 162.11 C-4ur), 150.38 (C-2ur), 149.43 (C-2pir),
143.09 (C-6ur), 141.27 (C-6pyr), 133.55 (C-5pyr),128.33 (C-4pyr),
72%): mp 101–102 °C; ½a D25
ꢁ
+3.1 (c 1.0, MeOH); 1H NMR (DMSO-
d6): d 12.24 (s, 1H, COOH), 11.44 (s, 1H, NH), 7.69 (d, 1H, J
8.1 Hz, H-6ur), 5.81 (d, 1H, J 2.0 Hz, H-10 ), 5.65 (dd, 1H, J 2.0,
124.03 (C-3pyr), 114.70 (C(CH3)2), 102.57 (C-5ur), 95.45 ðC-10 Þ,
ur
ur
8.1 Hz, H-5ur), 5.03 (dd, 1H, J 2.2, 6.4 Hz, H-20 ), 4.78 (dd, 1H, J
85.56 ðC-40 Þ, 84.67 ðC-20 Þ, 82.70 (C-1glu), 81.17 ðC-30 Þ, 75.97 (C-
ur
ur
ur
ur
3.1, 6.4 Hz, H-3u0 r), 4.31–4.13 (m, 3H, H-40ur, H-50aur, H-50bur),
2.56–2.45 (m, 4H, 2 ꢀ CH2), 1.49, 1.29 (2s, 6H, C(CH3)2); 13C NMR
(DMSO-d6): d 174.07 (COOH), 172.66 (CO), 163.91 (C-4ur), 150.98
(C-2ur), 143.30 (C-6ur), 114.01 (C(CH3)2), 102.53 (C-5ur), 92.97
ðC-10urÞ, 84.80 ðC-2u0 rÞ, 84.31 ðC-3u0 rÞ, 81.27 ðC-40urÞ, 64.65 ðC-5u0 rÞ,
29.29, 29.27 (2 ꢀ CH2CO), 27.65, 25.85 (C(CH3)2).
5glu), 74.25 (C-3glu), 69.85 (C-2glu), 68.47 (C-4glu), 64.51 ðC-50 Þ,
ur
62.20 (C-6glu), 31.71 (CH2COO), 29.33 (CH2CONH), 27.31, 25.44
(C(CH3)2), 20.95, 20.89, 20.82, 20.80 (4 ꢀ CH3CO). ESI-MS: Calcd
for C35H42N4O17SNa ([M+Na]+): m/z 845.2. Found: m/z 845.2.
2.4. Compound (11)
2.3.2. Succinic acid mono-3-N-benzoyl-20,30-isopropylidene-uridin-50-
yl ester (8)
2.4.1. Procedure A using uridine derivative 8 for preparation of 11 and
(15)
To a solution of 3-N-benzoyl-20,30-isopropylidene-uridine 4
(500 mg, 1.24 mmol) in CH2Cl2 (5 mL) succinic anhydride
(124 mg, 1.24 mmol) and DMAP (152 mg, 1.24 mmol) were
added. The resulting mixture was stirred at room temperature
for 6 h. After completion (TLC, 10:1 CHCl3–MeOH) the reaction
mixture was concentrated. The crude product was purified on a
column packed with silica gel using CHCl3–MeOH 100:1 ? 20:1
solvent systems as the eluent to yield 8 as a white powder
(5-Amino-2-pyridyl)
tetra-O-acetyl-1-thio-b-D-glucopyrano-
side 1 (114 mg, 0.25 mmol) and uridine derivative 8 (122 mg,
0.25 mmol) were submitted to general procedure A described
above. Two compounds were isolated: product 11 (12 mg, 5%) as
a white foam and byproduct 15 (41 mg, 34%) as a colorless oil;
½
a 2D5 +7.4 (c 0.4, CHCl3); 1H NMR (CDCl3): d 7.98–7.91 (m, 4H,
ꢁ
Ph(o)), 7.70–7.62 (m, 2H, Ph(p)), 7.55–7.46 (m, 4H, Ph(m)), 7.43 (d,
2H, J 8.1 Hz, H-6ur), 5.85 (d, 2H, J 8.1 Hz, H-5ur), 5.68 (d, 2H, J
1.8 Hz, H-10ur), 5.04 (dd, 2H, J 1.8, 6.1 Hz, H-2u0 r), 4.79 (dd, 2H, J
3.3, 6.1 Hz, H-3u0 r), 4.40–4.24 (m, 6H, H-40ur, H-50aur, H-50bur),
2.68-2.56 (m, 8H, 4 ꢀ CH2), 1.54, 1.33 (2s, 12H, C(CH3)2).
(425 mg, 48%): mp 146–147 °C; ½a D25
ꢁ
+6.3 (c 2.0, CHCl3); 1H
NMR (CDCl3): d 8.5 (bs, 1H, COOH), 7.98–7.93 (m, 2H, Ph(o)),
7.67 (m, 1H, Ph(p)), 7.55–7.45 (m, 2H, Ph(m)), 7.42 (d, 1H, J
8.1 Hz, H-6ur), 5.87 (d, 1H, J 8.4 Hz, H-5ur), 5.67 (d, 1H, J 1.8 Hz,
H-10ur), 5.04 (dd, 1H, J 1.8, 6.1 Hz, H0-2u0 r), 4.79 (dd, 1H, J 3.3,
6.1 Hz, H-30ur), 4.40–4.28 (m, 3H, H-4ur, H-50aur, H-50bur), 2.70–
2.55 (m, 4H, CH2), 1.55, 1.33 (2s, 6H, C(CH3)2); 13C NMR (CDCl3):
d 172.62, 171.76, 168.31 (CO), 161.94 (C-4ur), 149.07 (C-2ur),
141.89 (C-6ur), 135.22, 131.16, 130.49, 129.16 (PhCO), 114.48
(C(CH3)2), 102.41 (C-5ur), 95.08 ðC-10urÞ, 85.35 ðC-2u0 rÞ, 84.43
ðC-30 Þ, 80.77 ðC-40 Þ, 63.91 ðC-50 Þ, 28.81, 28.65 (2 ꢀ CH2CO),
2.4.2. Procedure C using uridine derivative 8 for preparation of 11
Compound 1 (114 mg, 0.25 mmol) and uridine derivative 8
(122 mg, 0.25 mmol) were submitted to general procedure C de-
scribed above. The resulting crude product 11 was purified on a
column packed with silica gel using CHCl3/MeOH 30:1 solvent sys-
tem as the eluent to yield 11 as a white solid (92 mg, 40%).
ur
ur
ur
27.01, 25.14 (C(CH3)2).
2.4.3. Procedure D using uridine derivatives 8 for preparation of 11
Compound 1 (114 mg, 0.25 mmol) and uridine derivative 8
(122 mg, 0.25 mmol) were submitted to general procedure D de-
scribed above. The resulting crude product 11 was purified by col-
umn chromatography to yield 11 as a white solid (97 mg, 42%): mp
2.3.3. Procedure B using uridine derivative 7 for preparation of 10
(5-Amino-2-pyridyl) tetra-O-acetyl-1-thio-b-D-glucopyrano-
side 1 (114 mg, 0.25 mmol) and uridine derivative 7 (96 mg,
0.25 mmol) were submitted to general procedure B described
above. After 24 h TLC analysis indicated presence of substrates 1
and 7 and only traces of 10. So, product 10 was not isolated from
the reaction mixture.
142–143 °C; ½a 2D5
ꢁ
+2.3 (c 1.3, CHCl3); 1H NMR (CDCl3): d 8.49 (d, 1H,
J 2.4 Hz, H-6pyr), 8.02 (s, 1H, NH), 7.98–7.85 (m, 3H, Ph(o), H-4pyr),
7.66 (m, 1H, Ph(p)), 7.55–7.45 (m, 2H, Ph(m)), 7.43 (d, 1H, J 8.1 Hz,
H-6ur), 7.18 (d, 1H, J 8.8 Hz, H-3pyr), 5.84 (d, 1H, J 8.2 Hz, H-5ur),
5.66 (d, 1H, J 10.4 Hz, H-1glu), 5.62 (d, 1H, J 1.6 Hz, H-10 ), 5.33
ur
2.3.4. Procedure B using uridine derivative 8 for preparation of 10
(dd ꢃ t, 1H, J 9.3 Hz, H-3glu), 5.19 (dd ꢃ t, 1H, J 10.4 Hz, H-2glu),
5.15 (dd ꢃ t, 1H, J 9.8 Hz, H-4glu), 5.05 (dd, 1H, J 1.6, 6.2 Hz,
H-20ur), 4.85 (dd, 1H, J 3.3, 6.2 Hz, H-3u0 r), 4.40–4.28 (m, 3H, H-4u0 r,
H-50aur, H-50bur), 4.26 (dd, 1H, J 4.4, 12.4 Hz, H-6aglu), 4.09 (dd,
1H, J 2.2, 12.4 Hz, H-6bglu), 3.85 (ddd, 1H, J 2.2, 4.4, 10.1 Hz, H-
5glu), 2.70 (m, 2H, NHCOCH2), 2.60 (m, 2H, CH2COO), 2.04, 2.03,
2.02, 2.01 (4s, 12H, 4 ꢀ CH3CO), 1.53, 1.28 (2s, 6H, C(CH3)2); 13C
(5-Amino-2-pyridyl)
tetra-O-acetyl-1-thio-b-D-glucopyrano-
side 1 (114 mg, 0.25 mmol) and uridine derivative 8 (112 mg,
0.25 mmol) were submitted to general procedure B described
above. The crude product was purified on a column packed with
silica gel using CHCl3/MeOH 30:1 solvent system as the eluent to
yield 10 as a white solid (47 mg, 23%).

80
G. Pastuch-Gawolek et al. / Bioorganic Chemistry 37 (2009) 77–83
NMR (CDCl3): d 172.26, 170.75, 170.18, 170.11, 169.57, 169.48,
168.69 (CO), 162.08, (C-4ur), 149.44, 149.16 (C-2pyr, C-2ur), 142.52
(C-6ur), 141.01 (C-6pyr), 135.54 (Ph(p)CO), 132.69 (C-5pyr), 131.11
(PhCO), 130.57 (Ph(o)CO), 129.35 (Ph(m)CO), 128.11 (C-4pyr),
170.19, 169.93, 169.61, 168.51 (CO), 162.05 (C-4ur), 159.42 (CO),
149.31 (C-2pyr), 149.05 (C-2ur), 142.33 (C-6ur), 140.93 (C-6pyr),
135.35 (Ph(p)CO), 133.09 (C-5pyr), 130.99 (PhCO), 130.41 (Ph(o)CO),
129.19 (Ph(m)CO), 127.94 (C-4pyr), 123.53 (C-3pyr), 114.40
(C(CH3)2), 102.20 (C-5ur), 95.27 ðC-10urÞ, 85.22 ðC-4u0 rÞ, 84.30
ðC-20 Þ, 82.85 (C-1gal), 80.47 ðC-30 Þ, 74.31 (C-5gal), 71.91 (C-3gal),
123.66 (C-3pyr), 114.58 (C(CH3)2), 102.38 (C-5ur), 95.72 ðC-10 Þ,
ur
85.38 ðC-40 Þ, 84.39 ðC-20 Þ, 82.37 (C-1glu), 80.50 ðC-30 Þ, 75.83 (C-
ur
ur
ur
ur
ur
5glu), 74.07 (C-3glu), 69.53 (C-2glu), 68.20 (C-4glu), 63.85 ðC-50 Þ,
67.18, 66.78 (C-2gal, C-4gal), 63.78 ðC-50 Þ, 61.15 (C-6gal), 31.17,
ur
ur
61.92 (C-6glu), 31.42 (CH2COO), 29.12 (CH2CONH), 27.10, 25.17
(C(CH3)2), 20.76, 20.70, 20.64, 20.61 (4 ꢀ CH3CO). ESI-HRMS: Calcd
for C42H46N4O18SNa ([M+Na]+): m/z 949.2420. Found: m/z
949.2447.
28.92 (2 ꢀ CH2CO), 26.94, 25.03 (C(CH3)2), 20.64, 20.55, 20.52,
20.46 (4 ꢀ CH3CO). ESI-HRMS: Calcd for C42H46N4O18SNa
([M+Na]+): m/z 949.2420. Found: m/z 949.2438.
2.7. Compound (14)
2.5. Compound (12)
2.7.1. Succinic acid 3-N-benzoyl-20,30-di-O-tert-butyldimethylsilyl-
uridin-5’-yl ester (9)
2.5.1. Procedure B using uridine derivative 8 for preparation of 12
(5-Amino-2-pyridyl) tetra-O-acetyl-1-thio-b-D-galactopyrano-
side 2 (114 mg, 0.25 mmol) and uridine derivative 8 (122 mg,
0.25 mmol) were submitted to general procedure B described
above. The crude product was purified on a column packed with
silica gel using CHCl3/MeOH 60:1 solvent system as the eluent to
yield 12 as a white solid (21 mg, 10%).
To a solution of 20,30-di-O-tert-butyldimethylsilyl-uridine 6
(1.015 g, 1.76 mmol) in a mixture of CH2Cl2 (10 mL) and pyridine
(3 mL) succinic anhydride (176 mg, 1.76 mmol) and DMAP
(215 mg, 1.76 mmol) were added. The resulting mixture was stir-
red at room temperature for 6 h. After completion (TLC, 10:1
CHCl3–MeOH) the reaction mixture was concentrated with toluene
(3 ꢀ 10 mL) in order to remove the whole amount of pyridine. The
crude product was purified on a column packed with silica gel
using CHCl3–MeOH 30:1 solvent system as the eluent to yield 9
2.5.2. Procedure D using uridine derivative 7 for preparation of 12
Compound 2 (114 mg, 0.25 mmol) and uridine derivative 7
(96 mg, 0.25 mmol) were submitted to general procedure D de-
scribed above. The resulting crude product 12 was purified by col-
umn chromatography to yield 12 as a white solid (81 mg, 39%): mp
as a white powder (1.048 g, 88%): mp 144–145 °C; ½a D25
ꢁ
+38.9 (c
1.2, CDCl3); 1H NMR (CDCl3): d 10.38 (bs, 1H, COOH), 7.96–7.87
(m, 3H, Ph(o), H-6ur), 7.64 (m, 1H, Ph(p)), 7.52–7.44 (m, 2H, Ph(m)),
115–118 °C; ½a 2D5
ꢁ
+8.9 (c 1.5, CHCl3); 1H NMR (CDCl3): d 9.95 (s, 1H,
NH), 8.63 (s, 1H, NH), 8.49 (d, 1H, J 2.4 Hz, H-6pyr), 8.01 (dd, 1H, J
2.4, 8.6 Hz, H-4pyr), 7.34 (d, 1H, J 8.1 Hz, H-6ur), 7.23 (d, 1H, J
8.6 Hz, H-3pyr), 5.72 (dd, 1H, J 1.7, 8.1 Hz, H-5ur), 5.64 (d, 1H, J
5.97 (d, 1H, J 8.1 Hz, H-5ur), 5.81 (d, 1H, J 3.9 Hz, H-10 ), 4.47 (m,
ur
1H, H-50aur), 4.31–4.18 (m, 3H, H-2u0 r, H-30ur, H-50bur), 4.08 (m, 1H,
H-30 ), 2.82–2.50 (m, 4H, CH2), 0.89, 0.88 (2s, 18H, 2 ꢀ (CH3)3CSi),
ur
0.06, 0.07, 0.09 (3s, 12H, CH3Si); 13C NMR (CDCl3): d 172.56
(COOH), 168.74 (CO), 162.18 (C-4ur), 156.69 (CO), 149.20 (C-2ur),
139.87 (C-6ur), 134.99 (Ph(p)CO), 131.42 (PhCO), 130.45(Ph(o)CO),
129.03 (Ph(m)CO), 102.15 (C-5ur), 89.59 ðC-10 Þ, 81.42 ðC-40 Þ,
10.2 Hz, H-1gal), 5.60 (d, 1H, J 1.5 Hz, H-10 ), 5.48 (d, 1H, J 3.3 Hz,
ur
H-4gal), 5.38 (dd, 1H, J 10.2, J 9.9 Hz, H-2gal), 5.19 (dd, 1H, J 3.3,
9.9 Hz, H-3gal), 5.05 (dd, 1H, J 1.8, 6.3 Hz, H-20 ), 4.85 (dd, 1H, J
ur
ur
ur
3.3, 6.3 Hz, H-3u0 r), 4.42–4.30 (m, 3H, H-30ur, H-50aur, H-50bur),
4.18–4.02 (m, 3H, H-5gal, H-6agal, H-6agal), 2.81–2.64 (m, 4H,
2 ꢀ CH2), 2.17, 2.03, 2.00 (3s, 12H, 4 ꢀ CH3CO), 1.55, 1.32 (2s, 6H,
C(CH3)2); 13C NMR (CDCl3): d 172.70, 170.51, 170.44, 170.33,
170.10, 169.77 (CO), 163.83 (C-4ur), 150.12 (C-2ur), 149.58 (C-
2pyr), 142.92 (C-6ur), 141.04 (C-6pyr), 133.24 (C-5pyr), 128.17 (C-
4pyr), 123.83 (C-3pyr), 114.54 (C(CH3)2), 102.41 (C-5ur), 95.42
ðC-10 Þ, 85.40 ðC-40 Þ, 84.45 ðC-20 Þ, 83.01 (C-1gal), 80.97 ðC-30 Þ,
75.40 ðC-20 Þ, 70.86 ðC-30 Þ, 62.48 ðC-50 Þ, 28.85, 28.70 (2 ꢀ CH2CO),
ur
ur
ur
25.70 (2 ꢀ (CH3)3CSi), 17.92, 17.88 (2 ꢀ (CH3)3CSi), ꢂ4.41, ꢂ4.53,
ꢂ4.87, ꢂ5.07 (4 ꢀ CH3Si).
2.7.2. Procedure B using uridine derivative 9 for preparation of 14
Compound 1 (114 mg, 0.25 mmol) and uridine derivative 9
(169 mg, 0.25 mmol) were submitted to general procedure B de-
scribed above. The crude product was purified on a column of silica
gel using CHCl3–MeOH 60:1 solvent system as the eluent to yield
14 as a white solid (39 mg, 14%).
ur
ur
ur
ur
74.43 (C-5gal), 72.05 (C-3gal), 67.38 (C-4gal), 67.02 (C-2gal), 64.33
ðC-50urÞ, 61.34 (C-6gal), 31.58, 29.18 (CH2CO), 27.11, 25.24
(C(CH3)2), 20.79, 20.68, 20.62 (4 ꢀ CH3CO). ESI-MS: Calcd for
C35H42N4O17SNa ([M+Na]+): m/z 845.2. Found: m/z 845.2.
2.7.3. Procedure C using uridine derivative 9 for preparation of 14
Compound 1 (114 mg, 0.25 mmol) and uridine derivative 9
(169 mg, 0.25 mmol) were submitted to general procedure C de-
scribed above. The resulting crude product 14 was purified by col-
umn chromatography to yield 14 as a white solid (75 mg, 27%).
2.6. Compound (13)
2.6.1. Procedure D using uridine derivatives 8 for preparation of 13
Compound 2 (114 mg, 0.25 mmol) and uridine derivative 8
(122 mg, 0.25 mmol) were submitted to general procedure D de-
scribed above. The resulting crude product 13 was purified on a
column packed with silica gel using CHCl3/MeOH 60:1 solvent sys-
tem as the eluent to yield 13 as a white solid (86 mg, 37%): mp
2.7.4. Procedure D using uridine derivatives 9 for preparation of 14
Compound 1 (114 mg, 0.25 mmol) and uridine derivative 9
(169 mg, 0.25 mmol) were submitted to general procedure D de-
scribed above. The resulting crude product 14 was purified by col-
umn chromatography to yield 14 as a white solid (142 mg, 51%):
122–124 °C; ½a 2D5
ꢁ
+6.8 (c 1.1, CHCl3); 1H NMR (CDCl3): d 8.57 (s,
1H, NH), 8.53 (d, 1H, J 2.4 Hz, H-6pyr), 8.01–7.90 (m, 3H, Ph(o), H-
4pyr), 7.65 (m, 1H, Ph(p)), 7.54–7.45 (m, 2H, Ph(m)), 7.44 (d, 1H, J
8.1 Hz, H-6ur), 7.21 (d, 1H, J 8.8 Hz, H-3pyr), 5.86 (d, 1H, J 8.1 Hz,
mp 143–144 °C; ½a D25
ꢁ
+5.6 (c 0.9, CHCl3); 1H NMR (CDCl3): d 8.49
(d, 1H, J 2.6 Hz, H-6pyr), 7.97–7.88 (m, 3H, Ph(o), H-4pyr), 7.82 (s,
1H, NH), 7.77(d, 1H, J 8.2 Hz, H-6ur), 7.66 (m, 1H, Ph(p)), 7.55–
7.45 (m, 2H, Ph(m)), 7.19 (d, 1H, J 8.6 Hz, H-3pyr), 5.90 (d, 1H, J
8.2 Hz, H-5ur), 5.68 (d, 1H, J 10.2 Hz, H-1glu), 5.68 (d, 1H, J 3.3 Hz,
H-5ur), 5.66 (d, 1H, J 1.7 Hz, H-10 ), 5.62 (d, 1H, J 10.2 Hz, H-1gal),
ur
5.48 (d, 1H, J 3.2 Hz, H-4gal), 5.39 (dd ꢃ t, 1H, J 10.1 Hz, H-2gal),
5.17 (dd, 1H, J 3.4, 9.8 Hz, H-3gal), 5.05 (dd, 1H, J 1.7, 6.3 Hz,
H-20ur), 4.78 (dd, 1H, J 3.4, 6.3 Hz, H-3u0 r), 4.39–4.25 (m, 3H, H-3u0 r,
H-5’aur, H-5’bur), 4.15–4.04 (m, 3H, H-6agal, H-6bgal, H-5gal), 2.74-
2.59 (m, 4H, CH2), 2.17, 2.02, 2.00, 1.99 (4s, 12H, 4 ꢀ CH3CO),
1.52, 1.28 (2s, 6H, C(CH3)2); 13C NMR (CDCl3): d 172.22, 170.31,
H-10 ), 5.34 (dd ꢃ t, 1H, J 9.2 Hz, H-3glu), 5.18 (dd, 1H, J 9.3,
ur
10.2 Hz, H-2glu), 5.15 (dd ꢃ t, 1H, J 9.6 Hz, H-4glu), 4.47 (dd, 1H, J
3.9, 13.4 Hz, H-50aur), 4.36–4.18 (m, 4H, H-20 ; H-30 , H-6aglu, H-
ur
ur
50bur), 4.14–4.04 (m, 2H, H-30 , H-6bglu), 3.86 (ddd, 1H, J 2.1, 4.3,
ur
9.9 Hz, H-5glu), 2.84–2.59 (m, 4H, 2 ꢀ CH2), 2.04, 2.03, 2.02 (3s,

G. Pastuch-Gawolek et al. / Bioorganic Chemistry 37 (2009) 77–83
81
12H, 4 ꢀ CH3CO), 0.89, 0.88 (2s, 18H, 2 ꢀ (CH3)3CSi), 0.10, 0.08,
0.07, 0.06 (4s, 12H, 4 ꢀ CH3Si); 13C NMR (CDCl3): d 172.22,
170.65, 170.12, 169.69, 169.50, 169.41, 168.72 (CO), 162.16 (C-
4ur), 149.67 (C-2pyr), 149.04 (C-2ur), 140.97 (C-6pyr), 140.37 (C-
6ur), 135.23 (Ph(p)CO), 132.69 (C-5pyr), 131.28 (PhCO), 130.49 (Ph(o)-
CO), 129.16 (Ph(m)CO), 128.11 (C-4pyr), 123.61 (C-3pyr), 102.03 (C-
5ur), 91.42 ðC-10 Þ, 82.31 (C-1glu), 81.31 ðC-40 Þ, 75.82 (C-5glu),
1-thio-b-D-glycosides 1 or 2 (Fig. 1) with a succinic linker, which
together with the pyridyl ring is expected to mimic the pyrophos-
phate bridge (Scheme 1).
For the preparation of amine 1 or 2 (5-nitro-2-pyridyl) 1-thio-
glycosides were used as starting compounds and nitro group
reduction procedure with zinc powder/acetic acid system in CH2Cl2
described by Roy and co-workers for 4-nitrophenyl 1-thioglyco-
sides was applied [28]. (5-Amino-2-pyridyl) 2,3,4,6-tetra-O-acet-
ur
ur
74.91 ðC-20 Þ, 74.06 (C-3glu), 70.87 ðC-30 Þ, 69.50 (C-2glu), 68.20
ur
ur
(C-4glu), 62.86 ðC-5u0 rÞ, 61.89 (C-6glu), 31.35 (CH2COO), 28.96
(CH2CONH), 25.72 ((CH3)3CSi), 20.70, 20.65, 20.58 (4 ꢀ CH3CO),
17.96, 17.92 (2 ꢀ (CH3)3CSi), ꢂ4.34, ꢂ4.46, ꢂ4.81, ꢂ5.05
(4 ꢀ CH3Si). ESI-MS: Calcd for C51H70N4O18SSi2Na ([M+Na]+): m/z
1137.4. Found: m/z 1137.3.
yl-1-thio-b-D-glycoside derivatives of D-glucose and D-galactose
were obtained in a good yields (86% and 73%, respectively).
Uridine derivatives 7–9 served as the second structural compo-
nent of glycoconjugates 10–14. They were first selectively pro-
tected at 20 and 30-positions in ribose and at N3-position in uracil
ring. For protection of hydroxyl groups isopropylidene or tert-
butyldimethylsilyl (TBDMS) groups were chosen, while for protec-
tion of uracil nitrogen benzoyl group was used. Having selectively
protected uridine derivatives 3–6, we converted them into 5-succi-
nic acid monoesters by acylation reaction with succinic anhydride
in dry pyridine or in CH2Cl2 (choice of solvent was dependent of
substrate solubility). Reaction proceeded at room temperature in
3. Results and discussion
Taking into account previously mentioned structural and stabil-
ity requirements for glycosyltransferases sugar donor analogs we
would like to present here compounds consisting of variously pro-
tected 5’-uridine derivatives connected with (5-amino-2-pyridyl)
Amines:
AcO
AcO
AcO
AcO
S
NH2
S
O
NH2
O
OAc
OAc
N
N
OAc
OAc
2
1
Uridine derivatives:
O
N
O
N
O
O
N
NR
NR
NR
O
HO
NR
O
O
O
N
O
HO
O
HO
HO
O
O
O
O
O
O
O
O
O
O
O
O
TBDMSO
OTBDMS
TBDMSO
OTBDMS
3: R = H
4: R = Bz
5: R = H
7: R = H
9: R = Bz
6: R = Bz
8: R = Bz
Main products:
O
N
NR
AcO
R2
O
S
NH
O
O
OAc
N
O
O
O
R3
OAc
R1O
OR1
10: R = H, R1 = _CMe2_ , R2 = H, R3 = OAc
11: R = Bz, R1 = _CMe2_ , R2 = H, R3 = OAc
12: R = H, R1 = _CMe2_ , R2 = OAc, R3 = H
13: R = Bz, R1 = _CMe2_ , R2 = OAc, R3 = H
14: R = Bz, R1 = TBDMS , R2 = H, R3 = OAc
Byproduct:
O
N
O
NBz
BzN
O
O
O
O
N
O
O
O
O
O
O
O
O
O
O
O
15
Fig. 1. Structures of reagents and products.

82
G. Pastuch-Gawolek et al. / Bioorganic Chemistry 37 (2009) 77–83
AcO
S
NH2
O
OAc
N
AcO
O
N
OAc
+
NR
AcO
O
1 or 2
S
NH
O
A, B, C or D
O
O
OAc
N
AcO
O
N
O
O
NR
OAc
O
R1O
OR1
HO
O
O
10 -14
O
O
R1O
OR1
R = H or Bz
_
R1
=
_CMe2 or TBDMS
7-9
(A) CHCl 3, DCC/DMAP, r.t., (B) pyridine, DCC/DMAP, r.t., (C) DCM, ethyl chloroformate/Et3 N, r.t.,
(D) THF, DMT-MM, r.t..
Scheme 1. Synthesis of compounds 11–14.
the presence of DMAP [29]. 1H NMR and 13C NMR spectra proved
the structures of uridine derivatives 7–9. Succinyl methylene pro-
tons gave multiplets at approximately 2.50–2.80 ppm. Appearance
of a broad signal corresponding to carboxylic proton also con-
firmed products structure. Desired products of esterification reac-
tions 7-9 were obtained in satisfactory yields of 72%, 48% and
88%, respectively.
9 with 1 equivalent each of ethyl chloroformate and triethylamine
in CH2Cl2 at room temperature gave the mixed anhydrides, which
were condensed, without isolation, with amine 1 to give the corre-
sponding amides 11 or 14 as white solids in a 40% and 27% yields,
respectively (Table 1, entries 6 and 7).
Kaminski and co-workers showed the efficiency of 2-chloro-
4,6-disubstituted-1,3,5-triazines in formation of the peptide bond
[35]. Activated ester resulting from reaction of carboxylic acid with
triazine derivative (‘‘superactive ester”) contains an excellent leav-
ing group which is displaced by the amine. Such reaction requires
the presence of a tertiary amine in the reaction medium. A range of
tertiary amines were tested and the best results were obtained
when N-methylmorpholine (NMM) was applied [36]. Kunishima
and co-workers found that 4-(4,6-dimethoxy-(1,3,5)triazin-2-yl)-
4-methyl-morpholinium chloride (DMT-MM) can be formed and
isolated in THF and then used as an efficient condensing agent
facilitating formation amides and esters. They tested stability of
this compound in different solvents because DMT-MM is suscepti-
ble to demethylation at the morpholinium nitrogen. They showed
that only small amount of demethylation product was formed
when THF or MeOH was used as a solvent [37]. Taking advantage
of these information we performed condensation reactions amine
1 or 2 with uridine derivatives 7–9 using equimolar amount of
DMT-MM as condensing agent. Reactions were carried out in THF
at room temperature for 24–48 h. We obtained expected products
10–14 in yields superior to those obtained in case of using other
condensing procedures (Table 1, entries 8-12). Additionally, no
debenzoylation products were observed, so a mild and effective
method for amide bond preparation in desired glycoconjugates
was found. All glycoconjugates were purified by column chroma-
tography and their structures were elucidated by 1H and 13C
NMR data (including 1H, 13C HETCOR experiments and simulations
analysis) and mass spectrometry analysis.
With the amines and uridine derivatives of succinic acid in
hand, different methods for construction of an amide bond be-
tween these building blocks were tested next. Although amide
bond formation between an acid and amine is formally a conden-
sation, yet mixing an amine with a carboxylic acid results in an
acid–base reaction and formation of a stable salt. The direct con-
densation becomes feasible at high temperature (above 150 °C),
which may be troublesome when other sensitive functionalities
are present within coupled compounds. Therefore, activation of
carboxylic acid seems to be necessary [30]. There are numerous
commercially available coupling reagents for construction of
amide bond including carbodiimides alone [31] or plus additives
such as HOBt or DMAP [32,33]. The carbodiimide reacts with the
carboxylic acid to form O-acylisourea mixed anhydride which
can react directly with amine to yield the desired amide. Unfortu-
nately, isomerisation of reactive O-acylisourea into unreactive N-
acylurea may also be observed. Addition of nucleophiles such as
DMAP or HOBt hampers the side reaction [28]. We applied DCC/
DMAP condensing system for coupling amine 1 with uridine deriv-
ative 8 with CHCl3 as a solvent. Reaction was carried out at room
temperature for 48 h (Table 1, procedure A). The desired product
11 was isolated in only 5% yield. We also isolated symmetrical
anhydride 15 in a 34% yield. When CHCl3 was replaced with pyri-
dine (procedure B) the reaction yield increased but N-benzoyl pro-
tection in uracil ring was not stable enough under such conditions.
Glycoconjugates 10 and 12 were isolated in a 23% and 10% yields,
respectively (Table 1, entries 3 and 5). When uridine derivative of
succinic acid 9 with silyl protective groups was used, debenzoyla-
tion of uracil nitrogen was no longer observed but the yield of gly-
coconjugate 14 was far from satisfactory (Table 1, entry 4).
Interestingly, when uridine derivative 7 with unprotected uracil
nitrogen was applied any significant amount of product could
not be isolated. Only TLC analysis showed traces of product 10 (Ta-
ble 1, entry 2) and finally, this experiment proved the necessity of
uracil nitrogen protection.
Biological evaluations of obtained glycoconjugates 10, 12 and
14 were performed by Szewczyk’s team [38,39]. These compounds
were tested for the ability to inhibit the propagation of classical
swine fever virus (CSFV). After adsorption with CSFV virus swine
kidney cells SK6 were washed, replenished with medium contain-
ing varying amounts of glycoconjugates 10, 12 or 14 (10–140 lg/
ml) and incubated for 2 days. The extent of CSFV infection in these
cells was measured by immunoperoxidase Monolayer Assay. It was
observed that compounds 10, 12 and 14 reduced the number of
CSFV-infected cells in dose-dependent manner without significant
toxicity for mammalian cells. The best results were obtained for
compound 14 (reduction of CSFV-infected cells was observed for
Low yield of glycoconjugates induced us to search for another
method of amide bond formation. We tried to apply uridine eth-
oxycarbonyl anhydride which can be generated using ethyl chloro-
formate [34]. Treatment of uridine derivatives of succinic acid 8 or
dose 30 lg/ml of compound 14 at 82% survival of mammalian

G. Pastuch-Gawolek et al. / Bioorganic Chemistry 37 (2009) 77–83
83
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cells) [40]. Detailed results of these investigations will be pub-
lished after patent proceedings are completed.
Acknowledgment
Financial support from the Polish State Committee for Scientific
Research (Grant No. 1T09 A 08630) is gratefully acknowledged.
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