Adducts of uridine and glycals as potential substrates for glycosyltransferases

Article abstract of DOI:10.1016/j.bioorg.2007.07.001

We report on the synthesis of 2-deoxyglycosyl derivatives of uridine as potential donor substrates for glycosyltransferases. The totally stereoselective synthesis is accomplished by two sequential addition reactions of uridine derivatives to glycals promo

Full text of DOI:10.1016/j.bioorg.2007.07.001

Bioorganic Chemistry 35 (2007) 401–416
www.elsevier.com/locate/bioorg
Adducts of uridine and glycals as potential
substrates for glycosyltransferases
*
Ilona Wandzik , Tadeusz Bieg
Silesian University of Technology, Faculty of Chemistry, Department of Organic Chemistry,
Bioorganic Chemistry and Biotechnology, 44-100 Gliwice, Krzywoustego 4, Poland
Received 14 June 2007
Abstract
We report on the synthesis of 2-deoxyglycosyl derivatives of uridine as potential donor substrates
for glycosyltransferases. The totally stereoselective synthesis is accomplished by two sequential addi-
tion reactions of uridine derivatives to glycals promoted by triphenylphosphine-hydrogen bromide.
ꢀ 2007 Elsevier Inc. All rights reserved.
Keywords: GTs substrate analogues; Glycals; Stereoselective synthesis
1. Introduction
Glycosyltransferases (GTs) of the Leloir pathway [1] catalyse the transfer of a monosac-
charide unit from an activated nucleotide sugar donor to the hydroxyl group of an accep-
tor with complete regio- and stereoselectivity. The molecules to which GTs transfer
monosaccharide units include oligosaccharides, proteins or lipids. GTs are involved in sev-
eral metabolic pathways and modulation of their activities by efficient inhibitors has
potential for the control of certain cellular functions [2]. Compounds that can modulate
the biosynthesis of glycoconjugates are of great interest as novel therapeutic agents. They
may also find applications in the study of biological pathways and can be valuable tools in
the preparative stereoselective synthesis of oligosaccharides [3,4]. The majority of GTs uti-
lise donors containing uridine pyrophosphate leaving group (UDP). These enzymes can be
*
Corresponding author.
E-mail address: ilona.wandzik@polsl.pl (I. Wandzik).
0045-2068/$ - see front matter ꢀ 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2007.07.001

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I. Wandzik, T. Bieg / Bioorganic Chemistry 35 (2007) 401–416
grouped into two different structural superfamilies: GT-A and GT-B. Enzymes belonging
to the GT-A superfamily employ a DxD motif, which is present on the N-terminal
domain, to bind a divalent metal, most commonly Mn2+ cation. The metal ion is essential
for catalysis since it interacts with the pyrophosphate group of the UDP-sugar donor in
the enzyme active site.
Different strategies have been used in order to design potent inhibitors of GTs [5–8].
Identification of potent inhibitors has been developing very rapidly during the last two
decades since the 3D structures of several GTs were found [9–11] and catalytic mech-
anism proposed [12]. Structures of GTs’ inhibitors are based on analogies between
donor substrates, acceptor substrates and transition state, respectively. Several inhibi-
tors were designed as donor substrate analogues. They feature some structural changes
at the carbohydrate part or at the pyrophosphate linkage. Among mimetics of carbo-
hydrates, iminosugars and carbasugars are typical examples [6]. A variety of pyrophos-
phate analogues that are capable of mimicking pyrophosphate–metal interactions have
been developed. A common approach is the substitution of a phosphoester oxygen
atom by a carbon or sulphur atom, resulting in higher stability towards enzymatic
hydrolysis [13]. It has been reported that monosaccharide units might act as pyrophos-
phate–metal ion complex mimetics [14–16]. It is likely that complexation with Mn2+ is
feasible with two hydroxyl group of the sugar. Wang and co-workers reported on 50-O-
b-lactosyl-uridine which proved to be an inhibitor of the b-1,4-galactosyltransferase
present in L1210 leukaemia ascites fluid (Ki 119.6 lM) [14]. Behr and co-workers pre-
pared 3-, 4- and 6-hetaryl-glucose derivatives of uridine which were tested against chi-
tin synthase (IC50 0.8–3.2 mM) [15]. Ballell and co-workers demonstrated sceptical
attitude in application of glycosyl units as pyrophosphate linkage mimetics [16]. Series
of 50-O-b-glycosyl-uridine and thymidine derivatives showed moderate inhibition of
Salmonella dTDP-a-D-glucose 4,6-dehydratase and no significant inhibition of the
bovine b-1,4-galactosyltransferase.
Being inspired by the reports mentioned above, we have synthesised 2-deoxy-hexo-
pyranosyl derivatives of uridine as donor substrate analogues of gluco- and galactosyl-
transferases (Fig. 1). According to our knowledge, there is no report on application of
2-deoxy-a-D-glucosyl unit as a surrogate of the pyrophosphate linkage. Compounds 1–5
are composed of uridine and one or two residues of 2-deoxy-hexopyranose and can be syn-
thesised in a totally stereoselective manner using the Falck–Mioskowski protocol [17]. In
order to construct target compounds 1–5 we used orthogonally protected glycal substrates
and uridine. In our study, the central 2-deoxy-a-D-glucopyranose moiety replacing the key
pyrophosphate unit can be linked with terminal 2-deoxyglycosyl moiety through a-
(1 fi 3)-, a-(1 fi 4)-and a-(1 fi 6)-linked glycosidic linkages.
2. Experimental
2.1. General
NMR spectra were recorded with a Varian spectrometer at a frequency of 300 MHz
with Me4Si as internal reference. Optical rotations were measured with a Perkin–Elmer
141 polarimeter using a sodium lamp (589 nm) at room temperature. Mass spectra were
recorded in the positive mode on a Mariner (Perseptive Biosystem) detector using the elec-
trospray-ionisation (ESI) technique.

I. Wandzik, T. Bieg / Bioorganic Chemistry 35 (2007) 401–416
403
pyrophosphate
linkage
OH
O
N
O
HO
HO
O
P
O
P
NH
HO
O
O
O
O
O
O
O
-
-
Mn++
natural donor substrate
OH OH
UDP-α-D-glucose
OH
HO
HO
O
pyrophosphate
linkage mimetics
Precursors of pyrophosphate
linkage
O
O
N
HO
TBDMSO
BnO
O
HO
O
NH
BnO
O
α -(1,6) 2
O
O
8
HO
BnO
BnO
TBDMSO
HO
O
O
O
O
O
O
H3C
CH3
O
α -(1,3) 3
9
HO
TBDMSO
AcO
TBDMSO
O
O
HO
α -(1,4) 4
10
O
OH
OH
O
OH
HO
OH
O
N
O
O
O
HO
HO
O
NH
NH
HO
O
O
N
O
O
O
O
1
5
O
O
O
O
H3C
CH3
H3C
CH3
Fig. 1. 2-Deoxy-O-glycosyl rings as pyrophosphate linkage mimetics.
Reactions were monitored by TLC on precoated plates of silica gel G (Merck), compo-
nents were detected under UV light or by charring with 10% sulphuric acid in ethanol.
Column chromatography was performed on silica gel 60 (70–230 mesh, E. Merck) devel-
oped with one of the hexane/EtOAc or CHCl3/MeOH solvent systems. All evaporations
were performed under diminished pressure at 50 ꢁC.
20,30-Isopropylidene-uridine (13) [18], 3-N-benzyl-20,30-isopropylidene-uridine (14) [19]
and 3-N-benzoyl-20,30-isopropylidene-uridine (15) [20] were prepared according to the

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I. Wandzik, T. Bieg / Bioorganic Chemistry 35 (2007) 401–416
published procedures. 4-O-acetyl-3,6-di-O-benzyl-D-glucal (11) was prepared by acetyla-
tion of 3,6-di-O-benzyl-D-glucal [21]. 3,4,6-Tri-O-benzyl-D-galactal (6) and 3,4,6-tri-O-ben-
zyl-D-glucal (7) were prepared by benzylation of corresponding per-O-acetylated
derivatives with benzyl bromide in NaOH/DMSO system. Pearlman catalyst Pd(OH)2/
C and other chemicals were purchased from Aldrich and Fluka Chemical Companies
and were used without purification. Solvents were dried and stored over molecular sieves
˚
(4 A) under an inert atmosphere.
2.2. 3,4-Di-O-benzyl-6-O-tert-butyldimethylsilyl-D-glucal (8), 4,6-di-O-benzyl-3-O-tert-
butyldimethylsilyl-D-glucal (9) and 4-O-benzyl-6-O-tert-butyldimethylsilyl-D-glucal (10)
To a solution of D-glucal (15.0 g, 0.103 mol) in DMF (45 mL), imidazole (15.4 g,
0.227 mol) and TBDMSCl (16.9 g, 0.113 mol) were added. Reaction mixture was stirred
at room temperature for 20 min and after this time was diluted with CH2Cl2 (400 mL),
washed with water (2 · 150 mL), dried over anhydrous MgSO4 and concentrated to give
crude product. Crude product was subsequently purified by column chromatography with
hexane/AcOEt 15:1 fi 4:1 solvent system to yield 3,6-di-O-tert-butyldimethylsilyl-D-glucal
(7.3 g, 19%) as white crystals and 6-O-tert-butyldimethylsilyl-D-glucal (14.5 g, 54%) as a
colourless oil. 6-O-tert-butyldimethylsilyl-D-glucal (7.0 g, 0.028 mol) was dissolved in
DMF (70 mL) and the solution was cooled to ꢀ5 ꢁC. Then sodium hydride, 60% disper-
sion in oil (2.0 g) and benzyl bromide (6.6 mL, 0.056 mol) were added. After stirring for
4 h at room temperature, the solution was neutralized with 1 M HCl aqueous solution,
diluted with hexane (500 mL) and washed with water (3 · 150 mL). The organic layer
was then dried (MgSO4) and the solvent removed under reduced pressure. The resulting
crude product mixture was purified by column chromatography with hexane/AcOEt
20:1 fi 10:1 solvent system to yield 8 (6.5 g, 53%) as a colourless oil, 9 (0.9 g, 8%) as a col-
ourless oil and 10 (1.7 g, 17%) as white crystals. Glycal 8 was identified by comparison
with literature data [22].
20
1
Compound 9: ½aꢁD +13.0 (CHCl3, c 1.0); H NMR (CDCl3): d 0.08, 0.09 (2s, 6H,
Si[C(CH3)3(CH3)2]), 0.89 (s, 9H, Si[C(CH3)3(CH3)2]), 3.66 (dd, 1H, J 5.9, 8.4 Hz, H-4),
3.70 (dd, 1H, J 2.6, 11.0 Hz, H-6a), 3.78 (dd, 1H, J 5.5, 11.0 Hz, H-6b), 4.07 (ddd, 1H,
J 2.6, 5.5, 8.4 Hz, H-5), 4.34 (m, 1H, H-3), 4.56 (s, 2H, PhCH2), 4.62, 4.82 (qAB, 2H, J
11.3 Hz, PhCH2), 4.64 (dd, 1H, J 2.7, 6.1 Hz, H-2), 6.34 (dd, 1H, J 1.2, 6.1 Hz, H-1),
7.23–7.33 (m, 10H, PhCH2); 13C NMR (CDCl3): d ꢀ4.64, ꢀ4.36 (Si[C(CH3)3(CH3)2]),
17.98 (Si[C(CH3)3(CH3)2]), 25.82 (Si[C(CH3)3(CH3)2], 68.72, 69.07, 73.49, 73.96, 76.61,
76.81 (C-3, C-4, C-5, C-6, 2 PhCH2), 103.52 (C-2), 125.32–128.40 (PhCH2), 138.04,
138.19 (Cq, PhCH2), 143.39 (C-1); ESI-HRMS: Calcd for C26H36O4SiNa ([M+Na]+):
m/z 463.2277. Found: m/z 463.2269.
20
1
Compound 10: ½aꢁD +27.2 (CHCl3, c 1.0); mp. 55–57 ꢁC; H NMR (CDCl3): d 0.09 (s,
6H, Si[C(CH3)3(CH3)2]), 0.92 (s, 9H, Si[C(CH3)3(CH3)2]), 2.31 (d, 1H, J 6.3 Hz, C-3OH),
3.68 (dd, 1H, J 6.2, 8.2 Hz, H-4), 3.86 (ddd, 1H, J 2.6, 2.6, 8.2 Hz, H-5), 3.95 (dd, 1H, J
2.9, 11.9 Hz, H-6a), 3.96 (dd, 1H, J 2.5, 11.9 Hz, H-6b), 4.29 (dddd, 1H, J 1.5, 2.8, 6.2,
6.3 Hz, H-3), 4.72 (dd, 1H, J 2.8, 6.1 Hz, H-2), 4.79 (s, 2H, PhCH2), 6.36 (dd, 1H, J
1.5, 6.1 Hz, H-1), 7.26–7.37 (m, 5H, PhCH2); 13C NMR (CDCl3): d ꢀ5.11, ꢀ5.42
(Si[C(CH3)3(CH3)2]), 18.37 (Si[C(CH3)3(CH3)2]), 25.91 (Si[C(CH3)3(CH3)2], 62.41 (C-6),
68.07 (C-3), 73.68 (PhCH2), 77.14 (C-4), 77.57 (C-5), 102.20 (C-2), 127.93, 127.95,

I. Wandzik, T. Bieg / Bioorganic Chemistry 35 (2007) 401–416
405
128.59 (PhCH2), 138.48 (Cq, PhCH2), 144.54 (C-1); ESI-HRMS: Calcd for C19H30O4SiNa
([M+Na]+): m/z 373.1806. Found: m/z 373.1809.
2.3. 3,4-Di-O-benzyl-6-O-tert-butyldimethylsilyl-2-deoxy-a-D-glucopyranosyl-(1 fi 5)-2,3-
O-isopropylideneuridine (16)
To a solution of glucal 8 (0.30 mmol) and uridine derivative 13 (85 mg, 0.30 mmol) in
dry CH2Cl2 (15 mL), a catalytic amount of TPHB (10 mg, 0.03 mmol) was added. The
mixture was stirred at room temperature for 24 h. Then the reaction mixture was concen-
trated to give crude product mixture purified directly by column chromatography with
20
hexane/AcOEt 1:1 solvent system to yield 16 (54 mg, 25%) as a white foam; ½aꢁD +16.1
1
(CHCl3, c 1.0); H NMR (CDCl3): d 0.06, 0.07 (2s, 6H, Si[C(CH3)3(CH3)2]), 0.90 (s,
9H, (Si[C(CH3)3(CH3)2]), 1.35, 1.57 (2s, 6H, C(CH3)2), 1.69 (ddd, 1H, J 3.0, 11.4,
12.6 Hz, H-200ax), 2.13 (ddd, 1H, J ꢂ 0, 4.5, 12.6 Hz, H-200eq), 3.49–3.91 (m, 7H,
H-50a,b, H-300, H-400, H-500, H-600a,b), 4.34–4.38 (m, 1H, H-40), 4.61–4.96 (m, 6H, H-20,
H-30, 2 Æ PhCH2), 4.92 (m, 1H, H-100), 5.66 (dd, 1H, J 8.1, 2.0 Hz, H-5), 5.84 (d, 1H, J
2.0 Hz, H-10), 7.22–7.38 (m, 10H, 2PhCH2), 7.41 (d, 1H, J 8.1 Hz, H-6), 9.77 (bs, 1H,
NH); 13C NMR (CDCl3): d ꢀ5.35, ꢀ5.15 (Si[C(CH3)3(CH3)2]), 18.29 (Si[C(CH3)3(CH3)2]),
25.41, 27.25 (C(CH3)2), 25.89 (Si[C(CH3)3(CH3)2]), 35.33 (C-200), 62.32 (C-50), 66.83, 72.84,
76.83, 78.02 (C-300, C-400, C-500, C-600), 71.96, 75.12 (2PhCH2), 80.68 (C-30), 85.10 (C-20),
85.35 (C-40), 92.94 (C-10), 97.43 (C-100), 101.95 (C-5), 114.30 (C(CH3)2), 127.75, 128.08,
128.45 (PhCH2), 138.23, 138.34 (Cq, PhCH2), 140.60 (C-6), 149.80 (C-2), 162.76 (C-4);
ESI-HRMS: Calcd for C38H52N2O10SiNa ([M+Na]+): m/z 747.3283. Found: m/z
747.3319.
2.4. 3,4-Di-O-benzyl-6-O-tert-butyldimethylsilyl-2-deoxy-a-D-glucopyranosyl (1 fi 5)-3-N-
benzyl-2,3-O-isopropylideneuridine (17)
Procedure A. To a solution of glucal 8 (132 mg, 0.30 mmol) and uridine derivative 14
(112 mg, 0.30 mmol, 1 equiv.) in dry CH2Cl2 (2 mL), a catalytic amount of TPHB
(10 mg, 0.03 mmol, 0.1 equiv.) was added. The mixture was kept at room temperature
for 1 h. Then the reaction mixture was concentrated to give crude product mixture purified
directly by column chromatography with hexane/AcOEt 2:1 solvent system to yield 17
20
1
(161 mg, 66%) as a white foam; ½aꢁD +20.4 (CHCl3, c 1.0); H NMR (CDCl3): d 0.01,
0.05 (2s, 6H, Si[C(CH3)3(CH3)2]), 0.89 (s, 9H, (Si[C(CH3)3(CH3)2]), 1.35, 1.57 (2s, 6H,
C(CH3)2), 1.68 (ddd, 1H, J 3.5, 11.5, 13.2 Hz, H-200ax), 2.10 (ddd, 1H, J ꢂ 0, 4.9,
13.2 Hz, H-200eq), 3.49–3.86 (m, 7H, H-50a,b, H-300, H-400, H-500, H-600a,b), 4.36–4.41 (m,
1H, H-40), 4.52–4.94 (m, 6H, H-20, H-30, 2 Æ PhCH2), 4.90 (bd, 1H, J 3.5 Hz, H-100),
5.04, 5.16 (qAB, 2H, J 12.6 Hz, PhCH2), 5.73 (d, 1H, J 8.1 Hz, H-5), 5.81 (d, 1H, J
2.4 Hz, H-10), 7.21–7.50 (m, 15H, 3PhCH2), 7.42 (d, 1H, J 8.1 Hz, H-6); 13C NMR
(CDCl3): d ꢀ5.34, ꢀ5.15 (Si[C(CH3)3(CH3)2]), 18.29 (Si[C(CH3)3(CH3)2]), 25.42, 27.25
(C(CH3)2), 25.89 (Si[C(CH3)3(CH3)2]), 35.19 (C-200), 44.11 (N-CH2Ph), 62.34, 66.83,
71.83, 72.91, 75.11, 76.81, 77.93 (C-500, C-300, C-400, C-500, C-600, 2PhCH2), 80.68 (C-30),
85.47 (C-20), 85.55 (C-40), 93.75 (C-10), 97.45 (C-100), 101.44 (C-5), 114.18 (C(CH3)2),
127.66–129.13 (PhCH2), 136.52 (Cq, PhCH2N), 138.15, 138.22 (Cq, PhCH2), 138.34 (C-6),
150.76 (C-2), 162.48 (C-4); ESI-HRMS: Calcd for C45H58N2O10SiNa ([M+Na]+): m/z
837.3753. Found: m/z 837.3745.

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I. Wandzik, T. Bieg / Bioorganic Chemistry 35 (2007) 401–416
2.5. 3,4-Di-O-benzyl-6-O-tert-butyldimethylsilyl-2-deoxy-a-D-glucopyranosyl (1 fi 5)-3-N-
benzoyl-2,3-O-isopropylideneuridine (18)
Glucal 8 (528 mg, 1.20 mmol) and uridine derivative 15 (233 mg, 1.20 mmol) were
submitted to general procedure A described above for the preparation of 17. The
resulting crude product was purified by column chromatography with hexane/AcOEt
20
2:1 solvent system to yield 18 (706 mg, 71%) as a white foam; ½aꢁD +18.8 (CHCl3, c
2.0); 1H NMR (CDCl3): d 0.01, 0.07 (2s, 6H, Si[C(CH3)3(CH3)2]), 0.90 (s, 9H,
(Si[C(CH3)3(CH3)2]), 1.34, 1.54 (2s, 6H, C(CH3)2), 1.69 (ddd, 1H, J 3.7, 11.4,
12.5 Hz, H-200ax), 2.14 (ddd, 1H, J ꢂ 0, 4.0, 12.5 Hz, H-200eq), 3.47–3.92 (m, 7H,
H-50a,b, H-300, H-400, H-500, H-600a,b), 4.37–4.42 (m, 1H, H-40), 4.68, 4.94 (qAB, 2H, J
10.7 Hz, PhCH2), 4.66 (s, 2H, PhCH2), 4.74 (m, 1H, H-30), 4.74 (dd, 1H, J 2.4,
6.1 Hz, H-20), 4.90 (bd, 1H, J 3.7 Hz, H-100), 5.77 (d, 1H, J 8.3 Hz, H-5), 5.82 (d,
1H, J 2.4 Hz, H-10), 7.27–7.39 (m, 10H, 2PhCH2), 7.43–7.50 (m, 2H, Ph(m)), 7.54
(d, 1H, J 8.1 Hz, H-6), 7.59–7.65 (m, 1H, Ph(p)), 7.89–7.95 (m, 1H, Ph(o)); 13C
NMR (CDCl3): d ꢀ5.31, ꢀ5.11 (Si[C(CH3)3(CH3)2]), 18.33 (Si[C(CH3)3(CH3)2]),
25.44, 27.27 (C(CH3)2), 25.94 (Si[C(CH3)3(CH3)2]), 35.51 (C-200), 62.36 (C-50), 66.86,
72.92, 76.81, 78.04 (C-300, C-400, C-500, C-600), 71.92, 75.17 (2PhCH2), 80.76 (C-30),
85.18 (C-20), 85.68 (C-40), 93.79 (C-10), 97.53 (C-100), 101.92 (C-5), 114.30 (C(CH3)2),
127.77, 127.79, 127.83, 128.13, 128.47, (PhCH2), 129.19, 130.50, 136.13, (PhCO),
131.40, (Cq, PhCO), 138.33, 138.43, (Cq, PhCH2), 140.48 (C-6), 149.10 (C-2), 161.94
(C-4), 168.32 (PhCO); ESI-HRMS: Calcd for C45H56N2O11SiNa ([M+Na]+): m/z
851.3546. Found: m/z 851.3527.
2.6. 3,4-Di-O-benzyl-2-deoxy-a-D-glucopyranosyl-(1 fi 5)-3-N-benzoyl-2,3-O-isopropyl-
ideneuridine (19)
Procedure B. Desilylation of adduct 18 (650 mg, 0.79 mmol) was achieved with
AcCl (62 lL, 0.79 mmol, 1 equiv.) in a solution of MeOH (15 mL) within 10 min.
The reaction mixture was neutralized with a basic resin, Amberlyst 21 (OHꢀ), filtered
and concentrated to give crude product mixture, which was subsequently purified by
column chromatography with hexane/AcOEt 1:1 solvent system to yield 19 (465 mg,
20
83%) as a white foam; ½aꢁD +26.8 (CHCl3, c 1.0); 1H NMR (CDCl3): d 1.33, 1.54
(2s, 6H, C(CH3)2), 1.67 (ddd, 1H, J 3.3, 11.3, 13.2 Hz, H-200ax), 2.03 (d, 1H, J
2.9 Hz, C-6OH), 2.18 (ddd, 1H, J ꢂ 0, 5.1, 13.2 Hz, H-200eq), 3.45–3.82 (m, 6H, H-
50a,b, H-400, H-500, H-600a,b), 3.90 (ddd, 1H, J 5.1, 8.3, 11.3 Hz, H-300), 4.36 (m, 1H,
H-40), 4.61–4.97 (m, 4H, PhCH2), 4.70 (dd, 1H, J 3.6, 6.1 Hz, H-30), 4.80 (dd, 1H,
J 2.2, 6.1 Hz, H-20), 4.90 (bd, 1H, J 3.3 Hz, H-100), 5.74 (d, 1H, J 2.2 Hz, H-10),
5.78 (d, 1H, J 8.1 Hz, H-5), 7.24–7.39 (m, 10H, 2PhCH2), 7.42–7.50 (m, 2H,
Ph(m)), 7.47 (d, 1H, J 8.1 Hz, H-6), 7.58–7.65 (m, 1H, Ph(p)), 7.89–7.95 (m, 1H,
Ph(o)); 13C NMR (CDCl3): 25.39, 27.22 (C(CH3)2), 35.21 (C-200), 62.08, 67.28,
71.71, 71.91, 75.15, 76.69, 77.99 (C-50, C-300, C-400, C-500, C-600, 2PhCH2), 80.79 (C-
30), 84.91 (C-20), 85.89 (C-40), 94.44 (C-10), 97.71 (C-100), 102.01 (C-5), 114.35
(C(CH3)2), 127.69–128.53 (PhCH2), 129.20, 130.50, 135.21, (PhCO), 131.33, (Cq,
PhCO), 138.10, 138.30, (Cq, PhCH2), 141.08 (C-6), 149.06 (C-2), 161.96 (C-4),
168.37 (PhCO); ESI-HRMS: Calcd for C39H42N2O11Na ([M+Na]+): m/z 737.2681.
Found: m/z 737.2680.

I. Wandzik, T. Bieg / Bioorganic Chemistry 35 (2007) 401–416
407
2.7. 4,6-Di-O-benzyl-2-deoxy-a-D-glucopyranosyl-(1 fi 5)-3-N-benzoyl-2,3-O-isopropyl-
ideneuridine (21)
Glucal 9 (300 mg, 0.68 mmol) and uridine derivative 15 (265 mg, 0.68 mmol) were
submitted to general procedure A described above for the preparation of 17. The
resulting crude product was purified by column chromatography with hexane/AcOEt
3:1 solvent system to yield 20 (230 mg, 41%) as a white foam. Desilylation of adduct
20 according to the procedure B described above for the preparation of 18 yielded 21
20
(130 mg, 65%) in 1.5 h as a white foam; ½aꢁD +28.0 (CHCl3, c 1.0) 1H NMR
(CDCl3): d 1.31, 1.53 (2s, 6H, C(CH3)2), 1.75 (ddd, 1H, J 3.7, 11.5, 13.1 Hz,
H-200ax), 2.02 (ddd, 1H, J ꢂ 0, 5.1, 13.1 Hz, H-200eq), 2.62 (s, 1H, C-3OH), 3.46 (t,
1H, J 9.3 Hz, H-400) 3.64 (m, 1H, H-500), 3.76 (dd, 1H, J 3.8, 10.9 Hz, H-600a), 3.83
(dd, 1H, J 3.7, 10.9 Hz, H-600b), 3.95 (ddd, 1H, J 5.1, 9.3, 11.5 Hz, H-300), 4.39 (m,
1H, H-40), 4.49–4.72 (2qAB, 4H, PhCH2), 4.72 (dd, 1H, J 2.8, 6.1 Hz, H-30), 4.82
(dd, 1H, J 2.2, 6.1 Hz, H-20), 4.92 (bd, 1H, J 3.7 Hz, H-100), 5.81 (d, 1H, J 2.2 Hz,
H-10), 5.81 (d, 1H, J 8.1 Hz, H-5), 7.21–7.35 (m, 10H, 2CH2Ph), 7.42–7.50 (m, 2H,
Ph(m)), 7.59 (d, 1H, J 8.1 Hz, H-6), 7.58–7.65 (m, 1H, Ph(p)), 7.89–7.95 (m, 1H,
Ph(o)); 13C NMR (CDCl3): 25.43, 27.27 (C(CH3)2), 37.32 (C-200), 67.22, 68.73,
68.93, 71.38, 73.65, 74.97, 79.65 (C-50, C-300, C-400, C-500, C-600, 2 PhCH2), 80.75 (C-
30), 85.30 (C-20), 85.97 (C-40), 93.77 (C-10), 97.82 (C-100), 101.87 (C-5), 114.31
(C(CH3)2), 127.85–128.47, (PhCH2), 129.27, 130.60, 135.30, (PhCO), 131.32 (Cq,
PhCO), 137.87, 138.18, (Cq, PhCH2), 140.88 (C-6), 149.12 (C-2), 162.16 (C-4),
168.67 (PhCO); ESI-HRMS: Calcd for C39H42N2O11Na ([M+Na]+): m/z 737.2681.
Found: m/z 737.2667.
2.8. 3,6-Di-O-benzyl-2-deoxy-D-glucopyranosyl-(1 fi 5)-2,3-O-isopropylideneuridine (23)
Glucal 11 (250 mg, 0.68 mmol) and uridine derivative 15 (265 mg, 0.68 mmol) were
submitted to general procedure A described above for the preparation of 17. The
resulting crude product 22 was purified by column chromatography with hexane/
AcOEt 2:1 solvent system to yield 22 (298 mg, 58%) as an inseparable a,b-anomeric
(6:1) mixture. Deacylation of adduct 22 was achieved with a 0.1 M solution of sodium
methoxide in methanol (4 mL) in 4 days. The reaction mixture was neutralized with an
acidic resin, Dowex 50WX8 (H+), filtered, concentrated and purified by column chro-
matography with hexane/AcOEt 1:1 solvent system to yield 23 (180 mg, 75%) as a
white foam; a:b = 7:1; 1H NMR (CD3OD) a anomer: d 1.34, 1.54 (2s, 6H,
C(CH3)2), 1.50–1.62 (m, 1H, H-200ax), 2.06 (ddd, 1H, J ꢂ 0, 4.8, 12.9 Hz, H-200eq),
3.36–3.86 (m, 6H, H-50, H-300, H-400, H-500, H-600a,b), 4.44 (m, 1H, H-40), 4.54–4.68
(m, 4H, PhCH2), 4.68 (dd, 1H, J 2.4, 6.1 Hz, H-30), 4.74 (dd, 1H, J 2.4, 6.1 Hz, H-
20), 4.91 (bd, 1H, J 2.5 Hz, H-100), 5.66 (d, 1H, J 8.1 Hz, H-5), 5.77 (d, 1H, J
2.4 Hz, H-10), 7.21–7.35 (m, 10H, 2CH2Ph), 7.63 (d, 1H, J 8.1 Hz, H-6), 7.90 (s, 1H,
13
NH); C NMR (CD3OD) a anomer: 25.51, 27.53 (C(CH3)2), 36.01 (C-200), 68.45,
70.95, 72.03, 72.77, 73.69, 74.56, 77.22 (C-50, C-300, C-400, C-500, C-600, 2PhCH2), 82.81
(C-30), 86.88 (C-20), 87.20 (C-40), 95.29 (C-10), 98.81 (C-100), 102.08 (C-5), 114.79
(C(CH3)2), 128.69–129.41, (PhCH2), 139.70, 139.87, (Cq, PhCH2), 143.06 (C-6),
152.03 (C-2), 166.31 (C-4). ESI-HRMS: Calcd for C32H38N2O10Na ([M+Na]+): m/z
633.2419. Found: m/z 633.2438.

408
I. Wandzik, T. Bieg / Bioorganic Chemistry 35 (2007) 401–416
2.9. 3,4,6-Tri-O-benzyl-2-deoxy-a-D-galactopyranosyl-(1 fi 6)-3,4-di-O-benzyl-2-deoxy-a-
D-glucopyranosyl-(1 fi 5)-3-N-benzoyl-2,3-O-isopropylideneuridine (24)
Glucal 6 (116 mg, 0.28 mmol) and uridine derivative 19 (200 mg, 0.28 mmol) were
submitted to general procedure A described above for the preparation of 17. Crude
product 24 was purified by column chromatography with hexane/AcOEt 2:1 solvent
20
system to yield 24 (155 mg, 49%) as a white foam; ½aꢁD +40.5 (CDCl3, c 1.0); 1H
NMR (CDCl3): d 1.32, 1.53 (2s, 6H, C(CH3)2), 1.61–1.75 (m, 2H, H-200ax, H-2000eq),
2.12–2.27 (m, 2H, H-200eq, H-2000ax), 3.43–3.94 (m, 12 H, H-50a,b, H-300, H-400, H-500,
H-600a,b, H-3000, H-4000, H-5000, H-6000a,b), 4.35, 4.44 (qAB, 2H, J 11.8 Hz, PhCH2),
4.35 (m, 1H, H-40), 4.51–4.95 (m, 8H, 4PhCH2), 4.68 (dd, 1H, J 3.4, 6.1 Hz, H-30),
4.77 (dd, 1H, J 2.2, 6.1 Hz, H-20), 4.88 (bd, 1H, J 2.7 Hz, H-100), 5.02 (bd, 1H, J
2.7 Hz, H-1000), 5.75 (d, 1H, J 8.1 Hz, H-5), 5.75 (d, 1H, J 2.2 Hz, H-10), 7.20–7.40
(m, 25H, 5CH2Ph), 7.42–7.50 (m, 2H, Ph(m)), 7.45 (d, 1H, J 8.1 Hz, H-6), 7.55–
7.65 (m, 1H, Ph(p)), 7.86–7.94 (m, 1H, Ph(o)); 13C NMR (CDCl3): d 25.42, 27.24
(C(CH3)2), 30.99 (C-2000), 35.08 (C-200), 66.09, 67.02, 69.43, 70.10, 70.27, 71.17,
71.73, 72.92, 73.36, 74.28, 74.41, 75.11, 76.89, 78.09 (C-50, C-300, C-400, C-500, C-600,
C-3000, C-4000, C-5000, C-6000, 5PhCH2), 80.74 (C-30), 85.07 (C-20), 85.78 (C-40), 94.21
(C-10), 97.54 (C-100), 98.30 (C-1000), 101.90 (C-5), 114.31 (C(CH3)2), 127.34–128.47
(PhCH2), 129.19, 130.48, 135.18, (PhCO), 131.32 (Cq, PhCO), 138.11, 138.15,
138.23, 138.39, 138.83 (Cq, PhCH2), 140.79 (C-6), 149.02 (C-2), 161.92 (C-4), 168.30
(PhCO); ESI-MS: Calcd for C66H70N2O15Na ([M+Na]+): m/z 1153.5. Found: m/z
1153.4.
2.10. 3,4,6-Tri-O-benzyl-2-deoxy-a-D-glucopyranosyl-(1 fi 6)-3,4-di-O-benzyl-2-deoxy-a-D-
glucopyranosyl-(1 fi 5)-3-N-benzoyl-2,3-O-isopropylideneuridine (25)
Glucal 7 (116 mg, 0.28 mmol) and uridine derivative 19 (200 mg, 0.28 mmol) were
submitted to general procedure A described above for the preparation of 17. Crude
product 25 was purified by column chromatography with hexane/AcOEt 2:1 solvent sys-
20
1
tem to yield 25 (193 mg, 61%) as a white foam; ½aꢁD +43.7 (CDCl3, c 1.0); H NMR
(CDCl3): d 1.32, 1.53 (2s, 6H, C(CH3)2), 1.61–1.75 (m, 2H, H-200ax, H-2000ax), 2.17,
2.30 (2dd, 2H, J ꢂ 0, 5.1, 12.5 Hz, H-20eq, H-2000eq), 3.43–3.94 (m, 12 H, H-50a,b,
H-300, H-400, H-500, H-600a,b, H-3000, H-4000, H-5000, H-6000a,b), 4.35 (m, 1H, H-40), 4.38–
5.00 (m, 10H, 5PhCH2), 4.70 (dd, 1H, J 3.0, 6.1 Hz, H-30), 4.78 (dd, 1H, J 2.1,
6.1 Hz, H-20), 4.89 (bd, 1H, J 3.3 Hz, H-100), 4.98 (bs, 1H, H-1000), 5.76 (d, 1H, J
2.1 Hz, H-10), 5.75 (d, 1H, J 8.1 Hz, H-5), 7.20–7.40 (m, 25H, 5PhCH2), 7.42–7.50
(m, 2H, Ph(m)), 7.45 (d, 1H, J 8.1 Hz, H-6), 7.55–7.65 (m, 1H, Ph(p)), 7.86–7.94 (m,
13
1H, Ph(o)); C NMR (CDCl3): d 25.44, 27.25 (C(CH3)2), 35.06, 35.25 (C-200, C-2000),
65.80, 67.12, 68.76, 69.95, 70.89, 71.15, 71.66, 73.06, 73.42, 74.40, 74.88, 75.04, 76.01,
78.12 (C-50, C-300, C-400, C-500, C-600, C-3000, C-4000, C-5000, C-6000, 5PhCH2), 80.76 (C-30),
85.10 (C-20), 85.86 (C-40), 94.28 (C-10), 97.57 (C-100), 97.78 (C-1000), 101.90 (C-5),
114.31 (C(CH3)2), 127.50–128.48 (PhCH2), 129.20, 130.49, 135.17 (PhCO), 131.36 (Cq,
PhCO), 138.13, 138.21, 138.23, 138.39, 138.83 (Cq, PhCH2), 140.81 (C-6), 149.90 (C-
2), 161.93 (C-4), 168.30 (PhCO); ESI-MS: Calcd for C66H70N2O15Na ([M+Na]+): m/z
1153.5. Found: m/z 1153.4.

I. Wandzik, T. Bieg / Bioorganic Chemistry 35 (2007) 401–416
409
2.11. 3,4,6-Tri-O-benzyl-2-deoxy-a-D-glucopyranosyl-(1 fi 3)-4,6-di-O-benzyl-2-deoxy-a-D-
glucopyranosyl-(1 fi 5)-3-N-benzoyl-2,3-O-isopropylideneuridine (26)
Glucal 7 (116 mg, 0.28 mmol) and uridine derivative 21 (200 mg, 0.28 mmol) were
submitted to general procedure A described above for the preparation of 17. Crude
product 26 was purified by column chromatography with hexane/AcOEt 2:1 solvent
20
system to yield 26 (152 mg, 48%) as a white foam; ½aꢁD +48.1 (CDCl3, c 0.5); 1H
NMR (CDCl3): d 1.31, 1.53 (2s, 6H, C(CH3)2), 1.64–1.81 (m, 2H, H-200ax, H-2000ax),
2.17, 2.39 (2dd, 2H, J ꢂ 0, 5.1, 12.3 Hz, H-20eq, H-2000eq), 3.44–4.05 (m, 12 H, H-
50a,b, H-300, H-400, H-500, H-600a,b, H-3000, H-4000, H-5000, H-6000a,b), 4.29 (m, 1H, H-40),
4.41–4.73 (m, 9H, PhCH2), 4.69 (dd, 1H, J 2.2, 6.0 Hz, H-30), 4.76 (dd, 1H, J 2.1,
6.2 Hz, H-20), 4.79 (bd, 1H, J 2.7 Hz, H-100), 4.86–4.92 (m, 1H, PhCH2), 5.16 (bd,
1H, J 2.6 Hz, H-1000), 5.89 (d, 1H, J 2.1 Hz, H-10), 6.03 (d, 1H, J 8.2 Hz, H-5),
7.10–7.38 (m, 25H, 5 PhCH2), 7.40–7.48 (m, 2H, Ph(m)), 7.54 (d, 1H, J 8.2 Hz,
H-6), 7.56–7.64 (m, 1H, Ph(p)), 7.86–7.94 (m, 1H, Ph(o)); 13C NMR (CDCl3): d
25.49, 27.28 (C(CH3)2), 36.11, 36.74 (C-200, C-2000), 66.98, 68.64, 69.46, 71.18, 71.39,
71.78, 73.20, 73.54, 75.05, 75.19, 77.16, 77.25, 77.64, 78.43 (C-50, C-300, C-400, C-500,
C-600, C-3000, C-4000, C-5000, C-6000, 5PhCH2), 80.22 (C-30), 84.82 (C-20), 85.36 (C-40),
92.30 (C-10), 97.76 (C-100), 99.77 (C-1000), 102.82 (C-5), 114.52 (C(CH3)2), 127.47-
128.51 (PhCH2), 129.15, 130.48, 135.05, (PhCO), 131.40 (Cq, PhCO), 137.96, 138.04,
138.25, 138.29, 138.53 (Cq, PhCH2), 140.41 (C-6), 149.09 (C-2), 161.98 (C-4), 168.47
(PhCO); ESI-MS: Calcd for C66H70N2O15Na ([M+Na]+): m/z 1153.5. Found: m/z
1153.7.
2.12. 3,4,6-Tri-O-benzyl-2-deoxy-a-D-glucopyranosyl-(1 fi 4)-3,6-di-O-benzyl-2-deoxy-D-
glucopyranosyl-(1 fi 5)-2,3-O-isopropylideneuridine (27)
Glucal 7 (123 mg, 0.30 mmol) and uridine derivative 23 (180 mg, 0.30 mmol) were
submitted to general procedure A described above for the preparation of 17, reaction
time 4.5 h. Crude product 27 was purified by column chromatography with hexane/
20
AcOEt 1:1 solvent system to yield 27 (170 mg, 55%) as a white foam; ½aꢁD +26.0
(CDCl3, c 0.5); 1H NMR (CDCl3)a,a-anomer : d 1.37, 1.58 (2s, 6H, C(CH3)2),
1.58–1.76 (m, 2H, H-200ax, H-2000ax), 2.15, 2.22 (2dd, 2H, J ꢂ 0, 5.1, 13.2 Hz, H-
20eq, H-2000eq), 3.40–3.92 (m, 12 H, H-50a,b, H-300, H-400, H-500, H-600a,b, H-3000, H-
4000, H-5000, H-6000a,b), 4.33-4.64 (m, 10H, H-40, PhCH2), 4.73 (dd, 1H, J 3.0, 6.2 Hz,
H-30), 4.77 (dd, 1H, J 2.2, 6.2 Hz, H-20), 4.82–4.88 (m, 1H, PhCH2), 4.94 (bd, 1H,
J 2.5 Hz, H-100), 5.47 (bd, 1H, J 2.4 Hz, H-1000), 5.65 (dd, 1H, J 2.2, 8.1 Hz, H-5),
5.78 (d, 1H, J 2.2 Hz, H-10), 7.13–7.36 (m, 25H, 5 PhCH2), 7.38 (d, 1H, J 8.1 Hz,
H-6), 8.93 (bd, 1H, J 1.9 Hz); 13C NMR (CDCl3)a,a-anomer: d 25.47, 27.26
(C(CH3)2), 34.40, 35.57 (C-200, C-2000), 67.22, 68.66, 69.61, 71.05, 71.15, 71.67, 71.80,
73.29, 73.46, 74.88, 75.64, 77.19, 77.33, 78.07 (C-50, C-300, C-400, C-500, C-600, C-3000,
C-4000, C-5000, C-6000, 5PhCH2), 81.03 (C-30), 85.14 (C-20), 85.54 (C-40), 93.59 (C-10),
97.32 (C-100), 99.10 (C-1000), 101.96 (C-5), 114.26 (C(CH3)2), 127.42-128.47 (PhCH2),
137.92, 138.11, 138.27, 138.56, 138.73 (Cq, PhCH2), 140.88 (C-6), 149.88 (C-2),
162.89 (C-4); ESI-MS: Calcd for C59H66N2O14Na ([M+Na]+): m/z 1049.4. Found:
m/z 1049.7.

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I. Wandzik, T. Bieg / Bioorganic Chemistry 35 (2007) 401–416
2.13. 2-Deoxy-a-D-galactopyranosyl-(1 fi 6)-2- deoxy-a-D-glucopyranosyl-(1 fi 5)-2,3-O-
isopropylideneuridine (1)
Procedure C. To a solution of 24 (115 mg, 0.10 mmol) in MeOH (5 mL) 25% aqueous
NH4OH (0.8 mL) was added and the mixture was stirred for 2.5 h at room temperature.
The solution was concentrated under reduced pressure and the residue was dissolved in a
1:10:2 mixture of cyclohexene/EtOH/THF (6 mL). The resulting solution was heated
under reflux in the presence of Pd(OH)2/C (75 mg) for 1 h. After removal of the catalyst
by filtration, crude product was purified by column chromatography with CHCl3/MeOH
20
10:1 fi 5:1 solvent system to yield 1 (32 mg, 55%) as a white foam; ½aꢁD +67.3 (MeOH, c
1
0.5); H NMR (CD3OD): d 1.36, 1.55 (2s, 6H, C(CH3)2), 1.62 (ddd, 1H, J 3.6, 11.7,
13.2 Hz, H-200ax), 1.79 (ddd, 1H, J ꢂ 0, 5.1, 12.7 Hz, H-2000eq), 1.95 (ddd, 1H, J 3.4,
12.0, 12.7 Hz, H-2000ax), 2.01 (ddd, 1H, J ꢂ 0, 5.1, 13.2 Hz, H-200eq), 3.28 (t, 1H, J
9.3 Hz, H-400), 3.59–3.90 (m, 9H, H-50a,b, H-500, H-600a,b, H-300, H-5000, H-6000a,b), 3.64
(bd, 1H, J 2.9 Hz, H-4000), 3.96 (ddd, 1H, J 2.9, 5.1, 12.0 Hz, H-300), 4.40 (m, 1H, H-40),
4.82 (dd, 1H, J 3.0, 6.2 Hz, H-30), 4.91 (dd, 1H, J 2.2, 6.2 Hz, H-20) 4.92 (bd, 1H, J
3.6 Hz, H-100), 4.95 (bd, 1H, J 3.4 Hz, H-1000), 5.72 (d, 1H, J 8.2 Hz, H-5), 5.84 (d, 1H, J
2.2 Hz, H-10), 7.76 (d, 1H, J 8.2 Hz, H-5), 7.90 (s, 1H, NH); 13C NMR (CD3OD): d
25.58, 27.57 (C(CH3)2), 33.62 (C-2000), 38.70 (C-200), 63.16, 66.96, 68.37 (C-50, C-600, C-
6000), 66.74 (C-3000), 69.62, 69.99, 72.31 (C-300, C-500, C-5000), 73.01, 73.09 (C-400, C-4000),
82.68 (C-30), 86.50 (C-20), 87.17 (C-40), 94.85 (C-10), 98.77 (C-1000), 98.92 (C-100), 102.38
(C-5), 115.06 (C(CH3)2), 143.48 (C-6), 152.00 (C-2), 166.27 (C-4); ESI-HRMS: Calcd
for C24H36N2O14Na ([M+Na]+): m/z 599.2059. Found: m/z 599.2073.
2.14. 2-Deoxy-a-D-glucopyranosyl-(1 fi 6)-2-deoxy-a-D-glucopyranosyl-(1 fi 5)-2,3-O-
isopropylideneuridine (2)
Deprotection of adduct 25 (115 mg, 0.10 mmol) according to the procedure C described
above for the preparation of 1 yielded 2 (45 mg, 78%) as a white foam; ½aꢁ2D0 +50.4 (MeOH,
1
c 0.5); H NMR (CD3OD): d 1.36, 1.55 (2s, 6H, C(CH3)2), 1.60, 1.65 (2ddd, 2H, H-200ax,
H-2000ax), 2.01, 2.09 (2ddd, 2H, H-200eq, H-2000eq), 3.23–3.31 (2t, 2H, H-400, H-4000), 3.59–3.90
(m, 10 H, H-50a,b, H-300, H-500, H-600a,b, H-3000, H-5000, H-6000a,b), 4.40 (m, 1H, H-40), 4.82
(dd, 1H, J 3.1, 6.1 Hz, H-30), 4.88–4.94 (m, 3H, H-20, H-100, H-1000), 5.72 (d, 1H, J
8.0 Hz, H-5), 5.84 (d, 1H, J 2.4 Hz, H-10) ), 7.75 (d, 1H, J 8.0 Hz, H-5), 7.90 (s, 1H,
13
NH); C NMR (CD3OD): d 25.60, 27.58 (C(CH3)2), 38.65, 38.79 (C-200, C-2000), 62.78
(C-6000), 66.92 (C-600), 68.37 (C-50), 69.98, 70.04 (C-300, C-3000), 72.99, 73.08, 73.26, 73.82
(C-400, C-500, C-4000, C-5000), 82.63 (C-30), 86.46 (C-20), 87.12 (C-40), 94.69 (C-10), 98.50,
98.91 (C-100, C-1000), 102.44 (C-5), 115.07 (C(CH3)2), 143.40 (C-6), 152.01 (C-2), 166.23
(C-4); ESI-HRMS: Calcd for C24H36N2O14Na ([M+Na]+): m/z 599.2059. Found: m/z
599.2065.
2.15. 2-Deoxy-a-D-glucopyranosyl-(1 fi 3)-2-deoxy-a-D-glucopyranosyl-(1 fi 5)-2,3-O-
isopropylideneuridine (3)
Deprotection of adduct 26 (115 mg, 0.10 mmol) according to the procedure C described
20
above for the preparation of 1 yielded 3 (32 mg, 55%) as a white foam; ½aꢁD +122.3
1
(MeOH, c 0.5); H NMR (CD3OD): d 1.36, 1.54 (2s, 6H, C(CH3)2), 1.58 (ddd, 1H, J

I. Wandzik, T. Bieg / Bioorganic Chemistry 35 (2007) 401–416
411
2.9, 11.7, 12.4 Hz, H-2000ax), 1.65 (ddd, 1H, J 3.3, 11.7, 12.4 Hz, H-200 ax), 2.17 (ddd, 1H, J
0.9, 5.1, 12.4 Hz, H-2000eq), 2.21 (ddd, 1H, J 0.9, 5.1, 12.4 Hz, H-200 eq), 3.27 (dd, 1H, J 9.0,
9.8 Hz, H-400), 3.36 (dd, 1H, J 9.0, 9.9 Hz, H-4000), 3.51 (ddd, J 2.2, 5.6, 9.9 Hz, H-5000), 3.55
(ddd, J 2.4, 5.0, 9.8 Hz, H-500) 3.69 (dd, J 3.0, 11.2 Hz, H-50a), 3.56–3.85 (m, 6H, H-300,
H-600a,b, H-3000, H-6000a,b), 3.88 (dd, J 3.9, 11.2 Hz, H-50b), 4.41 (ddd, 1H, J 2.9, 3.0,
3.9 Hz, H-40), 4.85 (dd, 1H, J 2.9, 6.2 Hz, H-30), 4.92 (dd, 1H, J 2.5, 6.2 Hz, H-20), 4.89
(bd, 1H, J 3.3 Hz, H-100), 5.16 (bd, 1H, J 2.9 Hz, H-1000), 5.83 (d, 1H, J 8.0 Hz, H-5),
5.85 (d, 1H, J 2.5 Hz, H-10), 7.75 (d, 1H, J 8.0 Hz, H-5), 7.90 (s, 1H, NH); 13C NMR
(CD3OD): d 25.61, 27.60 (C(CH3)2), 37.75 (C-200), 39.06 (C-2000), 62.71, 62.72 (C-600,
C-6000) 68.18 (C-50), 69.85 (C-3000), 71.85 (C-4000), 73.22 (C-400), 74.44, 74.61 (C-500, C-5000),
77.89 (C-300), 82.51 (C-30), 86.57 (C-20), 87.09 (C-40), 94.54 (C-10), 98.86 (C-100), 100.90
(C-1000), 102.64 (C-5), 115.01 (C(CH3)2), 143.27 (C-6), 152.09 (C-2), 166.55 (C-4);
ESI-HRMS: Calcd for C24H36N2O14Na ([M+Na]+): m/z 599.2059. Found: m/z 599.2055.
2.16. 2-Deoxy-a-D-glucopyranosyl-(1 fi 4)-2-deoxy-a-D-glucopyranosyl-(1 fi 5)-2,3-O-
isopropylideneuridine (4)
A solution of 27 (105 mg, 0.10 mmol) in a 1:10:2 mixture of cyclohexene/EtOH/THF
(6 mL) was heated under reflux in the presence of Pd(OH)2/C (75 mg) for 1 h. After
removal of the catalyst by filtration, crude product was purified by column chromatogra-
phy with CHCl3/MeOH 10:1 fi 5:1 solvent system to yield 4 (42 mg, 73%) as a white
20
1
foam; ½aꢁD +151.5 (MeOH, c 0.5); H NMR (CD3OD): d 1.36, 1.55 (2s, 6H, C(CH3)2),
1.57–1.71 (m, 2H, H-200ax, H-2000ax), 1.98 (ddd, 1H, J ꢂ 0, 5.1, 13.2 Hz, H-200eq), 2.18
(ddd, 1H, J ꢂ 0, 4.6, 12.7 Hz, H-2000eq), 3.18 (t, 1H, J 9.3 Hz, H-4000), 3.49–3.98 (m, 11
H, H-50a,b, H-300, H-400, H-500, H-600a,b, H-3000, H-5000, H-6000a,b), 4.39 (m, 1H, H-40), 4.82
(dd, 1H, J 2.9, 6.1 Hz, H-30), 4.84–4.94 (m, 2H, H-20, H-100), 5.49 (bd, 1H, J 2.9 Hz,
H-1000), 5.72 (d, 1H, J 8.1 Hz, H-5), 5.83 (d, 1H, J 2.2 Hz, H-10), 7.76 (d, 1H, J 8.2 Hz,
H-5), 7.90 (s, 1H, NH); 13C NMR (CD3OD): d 25.59, 27.57 (C(CH3)2), 39.01, 39.43
(C-200, C-2000), 62.48, 63.03, 68.29, 69.83, 70.66, 73.19, 73.49, 75.03, 77.58 (C-50, C-300,
C-400, C-500, C-600, C-3000, C-4000, C-5000, C-6000), 82.61 (C-30), 86.50 (C-20), 87.23 (C-40),
94.83 (C-10), 98.79 (C-100), 99.71 (C-10), 102.44 (C-5), 115.04 (C(CH3)2), 143.49 (C-6),
152.05 (C-2), 166.31 (C-4); ESI-HRMS: Calcd for C24H36N2O14Na ([M+Na]+): m/z
599.2059. Found: m/z 599.2084.
2.17. 3,6-Di-O-benzyl-2-deoxy-a-D-glucopyranosyl-(1 fi 5)-2,3-O-isopropylideneuridine
(5)
Deprotection of adduct 19 (71 mg, 0.10 mmol) according to the procedure C described
above for the preparation of 1 yielded 5 (36 mg, 84%) as a white foam, purification by col-
20
umn chromatography with CHCl3/MeOH 10:1 fi 5:1 solvent system; ½aꢁD +21.8 (MeOH,
1
c 0.5); H NMR (CD3OD): d 1.36, 1.55 (2s, 6H, C(CH3)2), 1.62 (ddd, 1H, J 3.4, 12.0,
13.2 Hz H-200ax), 1.99 (ddd, 1H, J 1.0, 5.1, 13.2 Hz, H-200eq), 3.23 (t, 1H, J 9.1 Hz,
H-400), 3.49 (ddd, 1H, J 2.2, 5.6, 9.7 Hz, H-500), 3.62–3.72 (m, 2H, H-50a, H-600a), 3.76
(ddd, 1H, J 5.1, 9.1, 12.0 Hz, H-300), 3.82 (dd, 1H, J 2.2, 11.7 Hz, H-600b), 3.89 (dd, 1H,
J 3.9, 11.0 Hz, H-50b) 4.42 (m, 1H, H-40), 4.83 (dd, 1H, J 2.9, 6.1 Hz, H-30), 4.88 (dd,
1H, J 2.4, 6.1 Hz, H-20), 4.93 (bd, 1H, J 3.4 Hz, H-100), 5.71 (d, 1H, J 8.1 Hz, H-5), 5.85
(d, 1H, J 2.4 Hz, H-10), 7.76 (d, 1H, J 8.1 Hz, H-5), 7.90 (s, 1H, NH); 13C NMR (CD3OD):

412
I. Wandzik, T. Bieg / Bioorganic Chemistry 35 (2007) 401–416
d 25.57, 27.59 (C(CH3)2), 38.66 (C-200), 62.79 (C-600), 68.27 (C-50), 69.85 (C-300), 73.11 (C-
400), 74.57 (C-500), 82.62 (C-30), 86.57 (C-20), 87.09 (C-40), 94.59 (C-10), 98.89 (C-100), 102.39
(C-5), 115.00 (C(CH3)2), 143.22 (C-6), 152.06 (C-2), 166.25 (C-4); ESI-HRMS: Calcd for
C18H26N2O10Na ([M+Na]+): m/z 453.1480. Found: m/z 599.2071.
3. Results and discussion
As a part of our current programme on the application of glycals in stereoselective syn-
thesis of 2-deoxy-a-glycosides, we designed and synthesised their uridine derivatives as
alternative substrates for GTs (Fig. 1). These compounds are composed of uridine and
two 2-deoxy-hexopyranose units, one of which plays the role of mimetics of pyrophos-
phate linkage and the second acts as a glycosyl unit to be transferred. Stereoselective syn-
thesis of the desired compounds can be accomplished in two successive addition reactions
of uridine derivatives to the double bond of glycals.
One possible way of exploration of glycals as glycosylating agents is the direct addition
of alcohols to the double bond of glycal. Successful direct regioselective addition can be
accomplished in the presence of several catalysts, for instance, triphenylphosphine-hydro-
gen bromide (TPHB) [17], cation-exchange resin Dowex AG 50WX2 [23], BCl3, BBr3[24],
CAN [25] or the CeCl3–NaI reagent system [26]. Encouraged by the successful synthesis of
a-1,6-linked di- and trisaccharides, derivatives of 2-deoxysugars from glycals [27,28]
according to the Falck–Mioskowski procedure, we now report on the synthesis of adducts
of glycals and uridine. Glycals are readily available building blocks, they compare well
with fully oxygenated pyranose derivatives due to their relative ease in differentiation of
hydroxyl group by selective protection. Benzyl protection of the hydroxyl group was cho-
sen because of the mild conditions involved in its removal by catalytic hydrogenolysis. The
synthesis of target compounds 1–4 required orthogonal protection at C-3, C-4 and C-6,
respectively. Tert-butyldimethylsilyl ether (TBDMS) protecting groups provided orthogo-
nal protection at C-6 and C-3 in glycals 8 and 9, respectively, while acetyl ester (Ac) was
used at C-4 in glucal 11 (Scheme 1).
We initially examined the addition of readily available 2,3-O-isopropylidene-uridine
(13) to glucal 8 using the Falck–Mioskowski protocol [17] (Scheme 2). Reaction proceeded
in the presence of a catalytic amount of TPHB (0.1 equiv) in CH2Cl2 at room temperature.
We observed partial decomposition of glycal and low yield of desired product 16 due to
OAc
Y
OBn
Y
OBn
O
O
i
O
X
X
AcO
BnO
AcO
BnO
6 X=H, Y=OBn
7 X=OBn, Y=H
30
11
HO
HO
HO
BnO
BnO
TBDMSO
TBDMSO
BnO
TBDMSO
HO
HO
TBDMSO
BnO
O
O
O
O
O
ii
iii
+
+
BnO
HO
8 53%
9 7%
10 17%
TBDMS - tert-butyldimethylsilyl
Bn - benzyl
Scheme 1. (i) BnBr (3 equiv.), NaOH, DMSO, 0 ꢁC fi rt, 12 h; (ii) TBDMSCl (1.1 equiv.), imidazole (2.2 equiv.),
DMF, rt, 2 h; (iii) BnBr (2 equiv.), NaH, DMF, ꢀ5 ꢁC fi rt, 4 h.

I. Wandzik, T. Bieg / Bioorganic Chemistry 35 (2007) 401–416
413
O
TBDSMO
BnO
O
TBDMSO
BnO
NR
O
O
BnO
NR
HO
N
i
BnO
+
O
O
O
N
O
O
8
O
O
H3C
CH3
O
O
16 R=H 25%
17 R=Bn 66%
18 R=Bz 71%
13 R=H
14 R=Bn
15 R=Bz
H3C
CH3
RO
O
N
Y
OR
BnO
BnO
O
X
NBz
O
RO
OBn
Y
O
O
O
O
iii
O
X
+
O
N
BnO
RO
RO
O
NR1
O
O
O
6 X=H, Y=OBn
7 X=OBn, Y=H
H3C
CH3
O
O
18 R=TBDMS
19 R=H
ii
83%
24 R=Bn, R1=Bz, X=H, Y=OBn, 49%
25 R=Bn, R1=Bz, X=OBn, Y=H, 61%
1 R=H, R1=H, X=H, Y=OH
v
55%
O
O
iv
19
78% v
H3C
CH3
5
2 R=H, R1=H, X=OH, Y=H
84%
Bn-benzyl, Bz-benzoyl
TBDMS- tert-butyldimethylsilyl
Scheme 2. (i) TPHB (0.1 equiv.), CH2Cl2, rt; (ii) AcCl (1.0 equiv.), MeOH, rt, 10 min.; (iii) 19 (1.0 equiv.), TPHB
(0.1 equiv.), CH2Cl2, rt, 1 h; (iv) NH4OH, MeOH, rt, 1 h, then Pd(OH)2/C, cyclohexene, EtOH, reflux, 1 h; (v)
NH4OH, MeOH, rt, 2.5 h, then Pd(OH)2/C, cyclohexene, EtOH/THF, reflux, 1 h.
limited solubility of uridine derivative 13. Moreover, TBDMS group transfer from glucal 8
to C-5 hydroxyl group of uridine was observed. When acetonitrile or THF was used as
solvents instead of CH2Cl2, the reaction yield increased but unfortunately a mixture of
a and b isomers was formed. Therefore, in order to improve solubility of uridine in
CH2Cl2, we employed protection of uracil nitrogen. At first we tried out benzyl protection
of uracil nitrogen hoping that in the final step it would be cleaved simultaneously to benzyl
protection of the hydroxyl groups in 2-deoxy-hexopyranose rings. Unfortunately, further
experiments revealed that N-benzyl protection in uracil part was stable to catalytic transfer
hydrogenolysis. Finally, benzoyl group as a protection of uracil nitrogen was studied. 3-N-
benzoyl-20,30-isopropylidene-uridine (15) was readily soluble in CH2Cl2 and only trace
amount of side products were formed as the result of transsilylation. In the course of
our investigations, we have found that the presence of TBDMS as the hydroxyl group pro-
tection in glucals 8 and 9 beneficially led to total a-stereoselectivity. Adducts 18 and 20
were synthesised as pure a-anomers (Scheme 2, 3). In contrast, the addition of uridine
derivative 15 to 4-O-acetyl-3,6-di-O-benzyl-D-glucal (11) led to adduct 22 which was con-
taminated with b-isomer (a:b = 7:1) (Scheme 4). The ratio of stereoisomers were deter-
mined by examination of the 1H NMR spectra of isomer mixture. The equatorially
oriented H-1a proton of the major a isomer gave the characteristic broad doublet at d
4.91 ppm with J1,2ax 2.5 Hz, while the axially oriented H-1b proton was observed at d
4.3 ppm as doublet of doublets with J1,2eq 2.2 Hz and J1,2ax 9.8 Hz.

414
I. Wandzik, T. Bieg / Bioorganic Chemistry 35 (2007) 401–416
BnO
O
N
O
N
BnO
RO
O
NBz
O
NBz
O
HO
O
BnO
BnO
TBDMSO
O
O
i
O
+
41%
O
O
O
O
9
H3C
CH3
H3C
CH3
15
20 R=TBDMS
21 R=H
65% ii
BnO
O
BnO
O
HO
RO
NBz
O
N
RO
O
O
O
N
O
O
BnO
BnO
BnO
NR1
O
O
iii
48%
+
RO
RO
O
O
O
O
O
OR
7
H3C
CH3
21
O
O
H3C
CH3
26 R=Bn, R1=Bz
3 R=H, R1=H
Bn-benzyl, Bz-benzoyl
TBDMS- tert-butyldimethylsilyl
iv
61%
Scheme 3. (i) TPHB (0.1 equiv.), CH2Cl2, rt, 1 h; (ii) AcCl (1.0 equiv.), MeOH, rt, 1.5 h; (iii) TPHB (0.1 equiv.),
CH2Cl2, rt, 1 h; (iv) NH4OH, MeOH, rt, 2.5 h, then Pd(OH)2/C, cyclohexene, EtOH/THF, reflux, 1 h.
O
O
N
BnO
RO
NR1
O
O
NBz
O
BnO
O
N
O
HO
O
BnO
AcO
BnO
O
i
+
58%
O
O
O
O
H3C
CH3
11
H3C
CH3
22 R=Ac, R1=Bz (α:β=7:1)
23 R=H, R1=H
15
ii
75%
O
OR
O
BnO
RO
RO
RO
NR1
O
BnO
O
N
RO
O
N
O
O
BnO
BnO
BnO
O
NH
O
O
iii
55%
RO
+
O
O
O
O
O
7
H3C
CH3
23
O
O
27 R=Bn
4 R=H
73% iv
H3C
CH3
Bn-benzyl, Bz-benzoyl
Scheme 4. (i) TPHB (0.1 equiv.), CH2Cl2, rt, 1 h; (ii) MeONa (1.0 equiv.), MeOH, rt, 4 days; (iii) TPHB (0.1
equiv.), CH2Cl2, rt, 1 h; (iv) Pd(OH)2/C, cyclohexene, EtOH/THF, reflux, 1 h.
Target compounds 1–4 were synthesised by subsequent addition reactions to glycals 6
or 7 after partial deprotection of intermediate compounds 18,20 and 22. Removal of the
TBDMS protecting group in 18 and 20 by TBAF was unsuccessful, but deprotection could

I. Wandzik, T. Bieg / Bioorganic Chemistry 35 (2007) 401–416
415
be performed by treatment of AcCl in methanol [29]. This way, compounds 19 and 21,
which have a free hydroxyl at C-6 and C-3, respectively, were synthesised in good yields.
Alternatively, saponification of the diacyl ester 22 using NaOMe in methanol gave com-
pound 23. Compounds obtained in this manner formed substrates for the second addition
step to the double bond of galactal 6 or glucal 7 to form a new a-1,3; a-1,4 and a-1,6-gly-
cosidic linkages. All adducts were isolated as pure a-anomers. Compounds 24–27 were
easily deprotected by catalytic hydrogenolysis. Hydrogenolysis was performed by hydro-
gen transfer from cyclohexene [30] in the presence of Pearlman’s catalyst (palladium(II)
hydroxide) [31] in reflux of ethanol affording the unprotected target compounds 1–4.
All adducts were purified by column chromatography and their structures were elucidated
1
1
1
1
by H and 13C NMR data (including DEPT; two-dimensional H, H COSY; H, 13C
HETCOR experiments and simulation analysis) and mass spectrometry analysis (for
details, see Section 2).
Synthesised adducts of uridine and glycals might serve as UDP-a-D-glucose and
UDP-a-D-galactose analogues and might also be useful in the investigation of the effect
of relative spatial arrangement of hydroxyl groups on the binding affinity of the
corresponding 2-4 regioisomers. Biological evaluations are currently in progress, and
the results will be presented soon.
Acknowledgment
Financial support from the Polish State Committee for Scientific Research (Grant No.
1T09 A 08630) is gratefully acknowledged.
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