Synthesis and application of 4′-C-alkylated uridines as probes for uracil-DNA glycosylase

Article abstract of DOI:10.1055/s-2005-865328

The development of new synthetic probes to study the mechanism of Uracil-DNA glycosylase is described. 4′-C-Alkylated 2′-deoxyuridines were synthesized and incorporated site-specifically into oligonucleotides. These compounds were subsequently employed in

Full text of DOI:10.1055/s-2005-865328

PAPER
1467
Synthesis and Application of 4¢-C-Alkylated Uridines as Probes for Uracil-
DNA Glycosylase
4
¢
-
C-alkylate
d
U
rid
o
ines pinath Rangam, Nicolas Z. Rudinger, H. Martin Müller, Andreas Marx*
Fachbereich Chemie, Universität Konstanz, Universitätsstrasse 10, 78457 Konstanz, Germany
Fax +49(7531)885140; E-mail: Andreas.Marx@uni-konstanz.de
Received 3 March 2005
Dedicated to Professor Bernd Giese on the occasion of his 65th birthday
in the hydration spine are coordinated through hydrogen
bonding with the nucleobases and 2¢-deoxyribose moi-
eties of the DNA.9 In order to kink a DNA-duplex the
spine of hydration of a duplex in its original entire B-form
Abstract: The development of new synthetic probes to study the
mechanism of Uracil-DNA glycosylase is described. 4¢-C-Alkylat-
ed 2¢-deoxyuridines were synthesized and incorporated site-specif-
ically into oligonucleotides. These compounds were subsequently
employed in functional studies of the enzyme and proved to be has to be interrupted. Recently, we have employed 4¢-C-
functional competent.
alkylated nucleotide probes to investigate DNA duplex
minor groove hydration.10 The employed 4¢-C-alkylated
Key words: DNA, enzymes, glycosylase, nucleotide, oligonucleo-
tides
probes are believed to act as steric barriers to exclude
DNA minor groove hydration.
In order to probe the effects of DNA hydration in the ac-
tion of UDG we synthesized novel modified oligonucle-
otides containing site-specifically introduced 4¢-C-
alkylated 2¢-deoxyuridine residues and subsequently em-
ployed these derivatives as probes in functional studies of
UDG.
As a result of deamination of cytosine, a promutagenic
U:G mismatch is formed several hundred times per cell
each day. If not repaired, this mismatch leads to a C to T
transition mutation in the next round of replication.
Uracil-DNA glycosylase (UDG) recognizes the RNA nu-
cleobase uracil in DNA and cleaves the glycosylic bond
between the 1¢-C of the 2¢-deoxyribose and the 1-N of the
uracil residue.1,2 This step initiates the multienzymatic
DNA base excision repair pathway.1,2
Synthesis of 4¢-C-Modified 2¢-Deoxyuridines and
their Incorporation into Oligonucleotides
Numerous structural and functional studies have helped to
gain insight into the reaction mechanisms of UDG. In or-
der to study distinct mechanistic features of UDG like hy-
drogen bonding or stabilization of a 1¢-oxonium ion in the
reaction pathway several oligonucleotide analogues have
been employed in functional studies of the enzyme. The
used derivatives comprise carbocyclic and C-nucleosides,
4¢-thio or 2¢-fluoro-2¢-deoxyuridines and hydrophilic nu-
cleoside surrogates with no hydrogen bonding capacity.3–7
For investigations of UDG, we aimed at the synthesis of
4¢-C-alkylated uridines that in turn should be incorporated
into DNA oligonucleotides to investigate UDG functions.
Thus, the synthesis of 4¢-C-methyl and 4¢-C-ethyl-2¢-
deoxyuridines was our first target. Recently, a strategy for
the synthesis of 4¢-C-methylated 2¢-deoxyuridines was re-
ported.11 However, for some reactions we were unable to
reproduce the described results and thus employed a dif-
ferent protection group strategy that was successfully em-
ployed in the synthesis of 4¢-C-modified thymidines.12,13
Our synthesis of the 4¢-C-methylated nucleoside starts
with alcohol 112 that was converted into the respective io-
dide through treatment with Ph3P, I2, and imidazole in tol-
uene (Scheme 1).14 Reduction using n-Bu3SnH, AIBN15
in refluxing toluene afforded the desired alkyl derivative
2 in high yields.
As shown by structural studies the enzyme UDG flips the
RNA base uracil out of the DNA base stack in the active
site.3,8 This process is accompanied with large conforma-
tional changes of the enzyme and the DNA substrate. The
enzyme conformation changes from an ‘open’ state when
no substrate is bound to a ‘closed’ state when the DNA
substrate containing a uracil moiety is bound. The DNA
bound to the enzyme is kinked and it appears that the
phosphates flanking the flipped-out uracil are significant-
ly compressed. However, the DNA at both ends from the
flipped-out moiety is in the B-form. Several investigations
of B-form DNA strongly indicate the presence of an or-
dered set of water molecules termed ‘spine of hydration’
in the minor groove in which individual water molecules
Subsequent protection group manipulations involved
cleavage of the acetal group and conversion to the bis-ac-
etate 3a.12 In order to synthesize the 4¢-C-ethylated nucle-
oside we oxidized alcohol 1 to yield the corresponding
aldehyde 4 using the Dess–Martin reagent.16 Wittig reac-
tion afforded the introduction of a C1-unit to yield the 4-
C-vinylated ribose derivative 5. By protection group ma-
nipulations we obtained the bis-acetate 3b.
SYNTHESIS 2005, No. 9, pp 1467–1472
x
x
.
x
x
.2
0
0
5
Next, through Vorbrüggen glycosylation the nucleobase
Advanced online publication: 19.04.2005
DOI: 10.1055/s-2005-865328; Art ID: C11505SS
© Georg Thieme Verlag Stuttgart · New York
uracil was fused with 3a,b to yield 6a,b (Scheme 2).17 Af-

1468
G. Rangam et al.
PAPER
TBDPSO
c
TBDPSO
HO
TBDPSO
ter saponification and deoxygenation of the 2¢-hydroxyl
function we obtained the 2¢-deoxyuridine derivatives
7a,b, which were subsequently deprotected to yield the
nucleosides 8a,b.
O
O
O
OAc
a, b
O
O
BnO OAc
BnO
O
BnO O
In order to incorporate the 4¢-C-alkylated 2¢-deoxyuridine
analogues into oligonucleotides, 8a,b were converted into
the corresponding 2-(cyanoethyl)phosphoramidite build-
ing blocks through conversion into the respective 4,4¢-
dimethoxytrityl ethers 9a,b and subsequently phosphity-
lated to form 10a,b (Scheme 2). The 4¢-C-modified 2¢-
deoxyuridines were introduced into oligonucleotides by
automated DNA synthesis applying commercially avail-
able 2-(cyanoethyl)phosphoramidites and the modified
building blocks. A standard method for 2-(cyanoeth-
yl)phosphoramidites and the modified building blocks
was used.
1
2
3a
d
TBDPSO
O
TBDPSO
e
TBDPSO
f
O
O
O
OAc
O
O
BnO
O
BnO
O
BnO OAc
4
5
3b
Scheme 1 Reagents and conditions: a) Ph3P, imidazole, I2, toluene,
reflux, 5 h, 89%; b) n-Bu3SnH, AIBN, toluene, reflux, 12 h, 96%; c)
AcOH, Ac2O, H2SO4, 16 h, 79%; d) Dess–Martin periodinane,
CH2Cl2, 0.5 h, 92%; e) MePPh3Br, n-BuLi, THF, 3 h, 97%; f) AcOH,
Ac2O, H2SO4, 16 h, 90%
Action of E. coli Uracil-DNA Glycosylase on 4¢-C-
Alkylated 2¢-Deoxyuridines
O
NH
Next, we investigated the action of UDG on the synthe-
sized oligonucleotides. If the synthesized probes are func-
tional competent, UDG should remove uracil from the
DNA strands and generate an abasic site that can trigger
DNA strand scission under basic conditions. The reac-
tions were analyzed by polyacrylamide gel electrophore-
sis (PAGE). We compared the action of UDG on the 4¢-C-
modified strands with the action on non-modified oligo-
nucleotide (Figure 1).
TBDPSO
TBDPSO
N
O
O
O
OAc
a
b, c, d
R
R
BnO OAc
BnO OAc
3a,b
R:
-CH3 (6a)
-CH=CH2 (6b)
O
O
NH
NH
HO
TBDPSO
N
O
N
O
O
O
g
e,f
R
R
HO
BnO
R:
R:
-CH3 (8a)
-CH3 (7a)
-CH2CH3 (8b)
-CH=CH2 (7b)
O
O
NH
NH
DMTO
DMTO
N
O
O
N
O
O
Figure 1 Cleavage of uracil from non-modified, 4¢-C-methylated,
and 4¢-C-ethylated moieties as indicated in the figure; Top panel: A
radioactively labeled oligonucleotide 5¢-d(TGC CTA AURG AGT
GAG) was incubated with UDG for several time periods as indicated
and subsequently treated with NaOH to introduce strand cleavage.
Lower panel: same as above with usage of the same oligonucleotide
in complex with the complementary strand. For further experimental
details, see the experimental section.
h
R
R
O
O
HO
P
CN
R:
-CH3 (9a)
-CH2CH3 (9b)
N (i-Pr)2
R:
-CH3 (10a)
-CH2CH3 (10b)
Scheme 2 Reagents and conditions: a) uracil, BSA, TMSOTf,
MeCN, reflux, 1 h, 77% (6a), 51% (6b); b) NaOMe, MeOH, 1 h; c)
PhOCSCl, DMAP, MeCN, 1 h; d) n-Bu3SnH, AIBN, toluene, reflux,
1 h; 69% (7a), 71% (7b) over 3 steps; e) 10% Pd/C, H2, EtOH, reflux,
24 h; f) TBAF, THF, 7 h, 60% (8a), 59% (8b) over 2 steps; g) DMT-
Cl, DMAP, pyridine, 4 h. 54% (9a), 54% (9b); h) N-ethyldiisopropyl-
When the non-modified oligonucleotide was incubated
with UDG and subsequently treated with NaOH strand
scission was observed as indicated by the generation of a
faster moving band. The reaction appears to be completed
after five minutes of incubation. No significant differ-
ences were detected when single or double stranded con-
amine,
2-cyanoethyl-N,N-(diisopropylamino)chlorophosphite,
CH2Cl2, 4 h, 94% (10a), 91% (10b)
Synthesis 2005, No. 9, 1467–1472 © Thieme Stuttgart · New York

PAPER
4¢-C-Alkylated Uridines
1469
fluxed and the iodide (2.09 g, 3.17 mmol) in toluene (8 mL) was
added over a period of 1 h. The mixture was allowed to reflux over-
night. After completion of the reaction, it was diluted with CH2Cl2
(20 mL) and the organic phase was washed with brine (40 mL). The
organic layer was separated and the aqueous layer was extracted
with CH2Cl2 (2 × 100 mL). The organic layers were combined,
dried (MgSO4), concentrated and purified by silica gel column
chromatography (CH2Cl2–cyclohexane, 6:4) to yield 2 (96%, 1.62
g) as a gum; Rf 0.33 (CH2Cl2–cyclohexane, 8:2).
structs were used. Interestingly, similar results were
observed when 4¢-C-modified oligonucleotides were em-
ployed in the investigations. Cleavage of the 4¢-C-modi-
fied uracil moieties was virtually completed after five
minutes of incubation. Here again no significant differ-
ences between double and single stranded constructs were
observed. These results unambiguously show that the de-
veloped probes are functional competent and can be em-
ployed in functional studies of UDG. Further in-depth
biochemical analysis using the developed tools should
yield valuable insights into the reaction mechanism of the
enzyme.
Compound 2
1H NMR (300 MHz, CDCl3): d = 0.91 (s, 9 H), 1.25 (s, 3 H), 1.56
(s, 3 H), 3.32 (d, J = 10.7 Hz, 1 H), 3.50 (d, J = 10.9 Hz, 1 H), 4.19
(d, J = 5.1 Hz, 1 H), 4.51 (d, J = 12.2 Hz, 1 H), 4.59 (dd, J = 5.1
Hz, 1 H), 4.72 (d, J = 12.2 Hz, 1 H), 5.70 (d, J = 3.8 Hz, 1 H), 7.27
(m, 11 H), 7.55 (m, 4 H).
In summary, in order to investigate the mechanisms of ac-
tion of UDG we have synthesized novel oligonucleotide
probes that bear 4¢-C-modified uridine residues. Our syn-
thesis starts from a readily available ribose-building block
and yields quantities that allow the generation of oligonu-
cleotides for numerous biochemical investigations. Pre-
liminary studies on UDG reveal that the developed probes
are functional competent and can be employed in further
in-depth studies of UDG.
13C NMR (75 MHz, CDCl3): d = 19.1, 19.4, 26.5, 27.0, 27.1, 68.7,
72.5, 77.4, 79.8, 86.5, 104.1, 113.4, 127.8, 127.9, 128.5, 129.8,
133.2, 133.6, 135.7, 135.8, 138.2.
FAB MS: m/z = 555.1 [M + Na]+.
1,2-Di-O-acetyl-3-O-benzyl-5-O-tert-butyldiphenylsilyl-4-C-
methyl-a,b-D-ribofuranose (3a)
To a solution of compound 2 (1.27 g, 2.37 mmol) in a mixture of
AcOH (21 mL) and Ac2O (2.4 mL, 10.8 mmol) was added concd
H2SO4 (19 mL), and the mixture was stirred for 16 h at r.t. After
completion of the reaction, the mixture was concentrated and dilut-
ed with CH2Cl2 (30 mL). The CH2Cl2 layer was washed with aq sat.
NaHCO3 solution (10 mL) and H2O (10 mL), and then dried
(MgSO4), concentrated, and purified by silica gel column chroma-
tography (EtOAc–cyclohexane, 5:95) as eluent to yield 3a (79%,
1.06 g) as a gum; Rf 0.45 (EtOAc–cyclohexane, 2:8).
1H NMR (400 MHz, CDCl3): d = 0.98 (s, 9 H), 1.23 (s, 3 H), 1.73
(s, 3 H), 2.02 (s, 3 H), 3.46 (d, J = 10.7 Hz, 1 H), 3.54 (d, J = 10.7
Hz, 1 H), 4.19 (d, J = 5.1 Hz, 1 H), 4.32 (d, J = 5.1 Hz, 1 H), 4.44
(d, J = 11.5 Hz, 1 H), 4.55 (d, J = 11.5 Hz, 1 H), 5.31 (d, J = 5.1 Hz,
1 H), 6.09 (s, 1 H), 7.28 (m, 11 H), 7.57 (m, 4 H).
All temperatures quoted are uncorrected. All reagents are commer-
cially available and used without further purification. Solvents are
dried over molecular sieves and used directly without further puri-
fication unless otherwise noted. All reactions were conducted under
rigorous exclusion of air and moisture. Petroleum ether used had a
bp range of 35–80 °C. NMR spectra: Bruker AC250, DPX300,
DPX400 and Varian INOVA400 MHz spectrometer with the sol-
vent peak as internal standard. FAB MS: Concept 1H (Kratos), ma-
trix: 3-nitrobenzyl alcohol (3-NBA). MALDI-ToF: MALDI 2
(Kratos), matrix: 1,5-dihydroxybenzoic acid. Flash chromatogra-
phy: Merck silica gel G60 (230–400 mesh). TLC: Merck precoated
plates (aluminum, silica gel 60 F254). MALDI-ToF MS analysis of
oligonucleotides was conducted by Metabion, Germany.
13C NMR (100 MHz, CDCl3): d = 19.5, 21.1, 27.0, 68.8, 73.4, 75.2,
77.5, 87.3, 97.9, 127.6, 127.8, 127.9, 128.5, 129.8, 129.9, 133.2,
133.5, 135.7, 138.0, 169.6, 169.9.
3-O-Benzyl-5-O-tert-butyldiphenylsilyl-4-C-methyl-1,2-O-iso-
propylidene-a-D-ribofuranose (2)
FAB MS: m/z = 599.0 [M + Na]+.
To a stirred solution of compound 1 (2.00 g, 3.65 mmol) in anhyd
benzene (70 mL) were added Ph3P (3.4 g, 12.94 mmol), imidazole
(811 mg, 13.09 mmol) and I2 (1.43 g, 5.65 mmol) and the mixture
was refluxed under argon for 5 h. After completion of the reaction,
the mixture was treated with aq 1 M Na2S2O3 solution (100 mL) and
extracted with toluene (3 × 250 mL). The combined organic extracts
were dried (MgSO4), concentrated and purified by silica gel column
chromatography (EtOAc–cyclohexane, 1:9) to afford the corre-
sponding iodide in 89% (2.13 g) as a gum; Rf 0.3 (1:9 EtOAc–cy-
clohexane).
3-O-Benzyl-5-O-tert-butyldiphenylsilyl-4-C-formyl-1,2-O-iso-
propylidene-a-D-ribofuranose (4)
To a solution of compound 1 (3.61 g, 6.6 mmol) in anhyd CH2Cl2
(10 mL), was added Dess–Martin periodinane (3.14 g, 7.40 mmol)
and the mixture was stirred at r.t. for 30 min. After completion of
the reaction, the mixture was quenched with aq sat. NaHCO3 solu-
tion (10 mL). The organic layer was separated and the aqueous layer
was extracted with CH2Cl2 (2 × 15 mL). The organic layers were
combined, dried (MgSO4), concentrated and purified by silica gel
column chromatography (EtOAc–cyclohexane, 1:9) as eluent to
give 4 (92%, 3.38 g) as a gum; Rf 0.32 (EtOAc–petroleum ether,
1:9).
1H NMR (400 MHz, CDCl3): d = 0.90 (s, 9 H), 1.28 (s, 3 H), 1.53
(s, 3 H), 3.71 (d, J = 11.5 Hz, 1 H), 3.80 (d, J = 11.4 Hz, 1 H), 4.45
(d, J = 4.6 Hz, 1 H), 4.53 (d, J = 12.2 Hz, 1 H), 4.56 (t, 1 H), 4.66
(d, J = 12.2 Hz, 1 H), 5.79 (d, J = 3.3 Hz, 1 H), 7.19–7.33 (m, 11
H), 7.50-7.53 (m, 4 H), 9.81 (s, 1 H).
Intermediate Iodide
1H NMR (400 MHz, CDCl3): d = 0.94 (s, 9 H), 1.27 (s, 3 H), 1.56
(s, 3 H), 3.38 (d, J = 11.2 Hz, 1 H), 3.55 (d, J = 10.7 Hz, 1 H), 3.76
(d, J = 10.7 Hz, 1 H), 3.86 (d, J = 11.2 Hz, 1 H), 4.29 (d, J = 4.9
Hz, 1 H), 4.51 (d, J = 11.9 Hz, 1 H), 4.58 (d, J = 4.8 Hz, 1 H), 4.70
(d, J = 12 Hz, 1 H), 5.67 (d, J = 3.7 Hz, 1 H), 7.27 (m, 11 H), 7.56
(m, 4 H).
13C NMR (100 MHz, CDCl3): d = 10.3, 19.4, 26.3, 27.0, 27.2, 68.2,
72.7, 78.4, 79.3, 84.7, 103.7, 113.8, 127.9, 128.1, 128.6, 129.9,
133.0, 133.3, 135.0, 135.8, 137.6.
13C NMR (100 MHz, CDCl3): d = 19.28, 26.17, 26.63, 26.74, 26.84,
26.97, 63.17, 72.81, 78.66, 79.21, 90.71, 105.02, 114.23, 127.75,
127.82, 127.86, 127.90, 128.13, 128.57, 129.66, 129.88, 129.93,
132.62, 132.92, 134.85, 135.56, 135.64, 137.09, 200.18.
FAB MS: m/z = 681.0 [M + Na]+.
Next, AIBN (130 mg, 0.79 mmol) and n-Bu3SnH (2.1 mL, 7.92
mmol) were dissolved in anhyd toluene (20 mL) and argon was
purged into the solution for 15 min. The reaction mixture was re-
FAB MS: m/z = 568.7 [M + Na]+.
Synthesis 2005, No. 9, 1467–1472 © Thieme Stuttgart · New York

1470
G. Rangam et al.
PAPER
3-O-Benzyl-5-O-tert-butyldiphenylsilyl-4-C-vinyl-1,2-O-isopro-
pylidene-a-D-ribofuranose (5)
(d, J = 6.2 Hz, 1 H), 4.35 (d, J = 11.4 Hz, 1 H), 4.53 (d, J = 11.4 Hz,
1 H), 5.24 (dd, J = 8.1 Hz, 1 H), 5.31 (dd, J = 6.1 Hz, 1 H), 6.11 (d,
J = 8.1 Hz, 1 H), 7.15–7.40 (m, 11 H), 7.45–7.65 (m, 4 H), 8.99 (s,
1 H).
13C NMR (100 MHz, CDCl3): d = 18.5, 19.5, 20.8, 27.2, 67.9, 74.2,
75.3, 76.6, 86.6, 87.0, 102.8, 127.8, 128.1, 128.2, 128.6, 130.2,
132.3, 132.9, 135.5, 135.8, 137.6, 139.9, 150.4, 163.1, 170.0.
Methyl triphenylphosphonium bromide (6.62 g, 18.5 mmol) was
suspended in anhyd THF (20 mL) under argon and treated at 0 °C
with a 1.6 M hexane solution of n-BuLi (9.76 mL, 15.44 mmol). Af-
ter the mixture had been stirred for 5 min, the ice bath was removed
and the stirring continued at r.t. for 1 h. The yellow reaction mixture
was cooled to –78 °C, compound 4 (3.77 g, 6.90 mmol) in anhyd
THF (5 mL) was added, stirring at –78 °C was continued for 30 min,
and the mixture was then allowed to warm to r.t. After stirring for 3
h, the mixture was quenched with aq concd NH4Cl solution (10 mL)
and extracted with Et2O (3 × 50 mL). The combined organic layers
were dried (MgSO4), concentrated and purified by silica gel column
chromatography (EtOAc–petroleum ether, 2:8) as eluent to yield 5
(97%, 3.28 g) as a gum; Rf 0.11 (EtOAc–petroleum ether, 1:9).
1H NMR (400 MHz, CDCl3): d = 0.90 (s, 9 H), 1.23 (s, 3 H), 1.46
(s, 3 H), 3.42 (d, J = 11.2 Hz, 1 H), 3.46 (d, J = 11.4 Hz, 1 H), 4.37
(d, J = 4.92 Hz, 1 H), 4.56 (d, J = 12.9 Hz, 1 H), 4.55 (m, 1 H), 4.73
(d, J = 12.4 Hz, 1 H), 5.10 (dd, J = 2.04 Hz, 1 H), 5.35 (dd, J = 3.8
Hz, 1 H), 5.72 (d, J = 11.0 Hz, 1 H), 6.09 (dd, 1 H), 7.17–7.35 (m,
11 H), 7.51–7.57 (m, 4 H).
FAB MS: m/z = 628.8 [M + H]+.
2¢-O-Acetyl-3¢-O-benzyl-5¢-O-tert-butyldiphenylsilyl-4¢-C-vinyl-
uridine (6b)
To a solution of compound 3b (4.40 g, 7.48 mmol) and uracil (1.69
g, 14.9 mmol) in anhyd MeCN (20 mL) was added N,O-bis(tri-
methylsilyl)acetamide (11.1 mL, 44.9 mmol) and refluxed for 1 h.
After cooling to r.t., Me3SiOTf (1.77 mL, 9.72 mmol) was added
and the mixture was refluxed again for 1 h. The mixture was
quenched with aq sat. NaHCO3 solution (5 mL). The solvent was re-
moved under reduced pressure and extracted with CH2Cl2 (3 × 50
mL). The organic layer was dried (MgSO4), concentrated, and puri-
fied by silica gel column chromatography (EtOAc–petroleum ether,
3:7) to give 6b (51%, 2.44 g) as a white foam; Rf 0.18 (EtOAc–pe-
troleum ether, 2:3).
1H NMR (250 MHz, CDCl3): d = 1.10 (s, 9 H), 2.06 (s, 3 H), 3.77
(s, 2 H), 4.45 (d, J = 11.25 Hz, 1 H), 4.57 (d, J = 6 Hz, 1 H), 4.65
(d, J = 11.3 Hz, 1 H), 5.20 (dd, J = 17.25, 1.25 Hz, 1 H), 5.36 (m, 2
H), 5.45 (dd, J = 11.0, 1.5 Hz, 1 H), 5.89 (q, J = 17.5, 11 Hz, 1 H),
6.21 (d, J = 3.25 Hz, 1 H), 7.25–7.74 (m, 11 H), 7.57–7.66 (m, 4 H),
7.77 (d, J = 8 Hz, 1 H).
13C NMR (100 MHz, CDCl3): d = 11.00, 14.07, 19.31, 23.024,
23.83, 25.92, 26.29, 26.86, 26.96, 28.98, 29.73, 30.44, 38.82, 66.55,
28.20, 72.56, 28.80, 87.31, 104.06, 113.51, 116.24, 127.68, 127.74,
127.81, 127.88, 128.47, 128.84, 129.67, 129.71, 130.89, 133.07,
133.53, 134.85, 135.57, 135.65, 135.73, 137.99.
FAB MS: m/z = 566.7 [M + Na]+.
1,2-Di-O-acetyl-3-O-benzyl-5-O-tert-butyldiphenylsilyl-4-C-vi-
nyl-a,b-D-ribofuranose (3b)
13C NMR (100 MHz, CDCl3): d = 19.26, 20.62, 26.92, 26.95, 65.57,
73.83, 74.53, 76.33, 86.75, 88.18, 88.22, 102.71, 116.52, 127.64,
127.79, 127.95, 128.37, 130.02, 130.10, 131.87, 132.58, 133.57,
135.21, 135.48, 137.14, 139.54, 150.19, 163.27, 169.86.
To a solution of compound 5 (3.28 g, 6.03 mmol) in a mixture of
AcOH (54.8 mL) and Ac2O (6.14 mL, 65 mmol) was added concd
H2SO4 (53 mL) and the mixture was stirred for 16 h at r.t. After com-
pletion of the reaction, the mixture was concentrated and diluted
with CH2Cl2 (40 mL). The CH2Cl2 layer was washed with aq sat.
NaHCO3 (10 mL) and H2O (10 mL), and then dried (MgSO4), con-
centrated, and purified by silica gel column chromatography
(EtOAc–petroleum ether, 1:9) to give 3b (90%, 3.50 g) as a gum; Rf
0.68 (EtOAc–petroleum ether, 2:8).
1H NMR (250 MHz, CDCl3): d (mixture of anomers) = 1.03 (s, 9 H),
1.82 (s, 3 H), 2.02 (s, 3 H), 3.51 (s, 2 H), 3.61 (s, 2 H), 4.33–4.68
(m, 6 H), 5.10–5.69 (m, 8 H), 5.76–6.10 (m, 2 H), 6.25 (br s, 1 H),
7.20–7.50 (m, 22 H), 7.55–7.75 (m, 8 H).
FAB MS: m/z = 663.7 [M + Na]+.
3¢-O-Benzyl-5¢-O-tert-butyldiphenylsilyl-2¢-deoxy-4¢-C-methyl-
uridine (7a)
To a solution of compound 6a (3.3 g, 5.25 mmol) in MeOH (10
mL), was added NaOMe (425 mg, 7.87 mmol) and the mixture was
stirred at r.t. for 1 h. After completion of the reaction, the mixture
was treated with aq concd tartaric acid (5 mL) and extracted with
CH2Cl2 (3 × 40 mL). The combined organic layers were dried
(MgSO4), concentrated, and purified by silica gel column chroma-
tography using 8:2 EtOAc–petroleum ether. The resulting com-
pound was dissolved in anhyd MeCN (10 mL) and to this solution
were added DMAP (1.90 g, 15.6 mmol) and phenyl chlorothiono-
formate (842 mL, 6.24 mmol) and the mixture was stirred at r.t. for
1 h. After completion of the reaction, the solvent was removed and
the residue was dissolved in CH2Cl2 (50 mL). The CH2Cl2 layer was
washed with aq 5% citric acid (10 mL) and H2O (10 mL). The ex-
tracts were dried (MgSO4) and evaporated to give a crude yellowish
foam. To a solution of the crude compound in anhyd toluene (20
mL) were added n-Bu3SnH (6.84 mL, 25.8 mmol) and a small
amount of AIBN. The mixture was refluxed for 1 h and the solvents
were evaporated. The residue was purified by silica gel column
chromatography (EtOAc–petroleum ether, 3:7) to give 7a (69%,
2.08 g) as a white foam; Rf 0.61 (EtOAc–petroleum ether, 1:1).
1H NMR (400 MHz, CDCl3): d = 1.00 (s, 9 H), 1.11 (s, 3 H), 2.11
(m, 1 H), 2.49 (m, 1 H), 3.57 (d, J = 11.1 Hz, 1 H), 3.73 (d, J = 11.2
Hz, 1 H), 4.25 (t, J = 6.5 Hz, 1 H), 4.37 (d, J = 11.9 Hz, 1 H,), 4.52
(d, J = 11.9 Hz, 1 H), 5.24 (dd, J = 2.1 Hz, 1 H), 6.15 (dd, J = 5.2
Hz, 1 H), 7.1–7.6 (m, 15 H), 7.75 (d, J = 8.1 Hz, 1 H), 8.30 (s, 1 H).
13C NMR (100 MHz, CDCl3): d = 18.2, 19.5, 27.2, 38.2, 68.0, 72.4,
83.7, 87.5, 102.2, 127.6, 128.0, 128.1, 128.2, 128.7, 130.2, 130.3,
132.4, 133.0, 135.5, 135.7, 137.8, 140.2, 150.2, 163.0.
13C NMR (100 MHz, CDCl3): d (mixture of anomers) = 19.08,
19.35, 20.71, 20.86, 26.83, 73.30, 74.59, 75.77, 86.29, 88.16, 96.06,
97.79, 98.88, 115.63, 127.52, 127.65, 127.77, 128.41, 129.66,
129.76, 132.92, 133.07,134.75, 135.21, 135.50, 137.62, 169.51,
169.91, 170.01, 170.05.
FAB MS: m/z = 611.0 [M + Na]+.
2¢-O-Acetyl-3¢-O-benzyl-5¢-O-tert-butyldiphenylsilyl-4¢-C-meth-
yluridine (6a)
To a solution of compound 3a (5.22 g, 9.54 mmol) and uracil (2.03
g, 19.1 mmol) in anhyd MeCN (20 mL) was added N,O-bis(tri-
methylsilyl)acetamide (13.4 mL, 57.2 mmol) and the mixture was
refluxed for 1 h. After cooling to r.t., Me3SiOTf (2.13 mL, 12.4
mmol) was added to the mixture and refluxed again for 1 h. The
mixture was quenched with aq sat. NaHCO3 solution (5 mL). The
solvent was removed under reduced pressure and the residue ex-
tracted with CH2Cl2 (3 × 50 mL). The organic layer was dried
(MgSO4), concentrated, and purified using silica gel column chro-
matography (EtOAc–petroleum ether, 3:7) as eluent to give 6a
(77%, 4.38 g) as a white foam; Rf 0.47 (EtOAc–petroleum ether,
1:1).
1H NMR (400 MHz, CDCl3): d = 1.02 (s, 9 H), 1.12 (s, 3 H), 2.03
(s, 3 H), 3.51 (d, J = 11.2 Hz, 1 H), 3.72 (d, J = 11.2 Hz, 1 H), 4.28
FAB MS: m/z = 571.0 [M + H]+.
Synthesis 2005, No. 9, 1467–1472 © Thieme Stuttgart · New York

PAPER
4¢-C-Alkylated Uridines
1471
3¢-O-Benzyl-5¢-O-tert-butyldiphenylsilyl-2¢-deoxy-4¢-C-vinyl-
uridine (7b)
tography (EtOAc → MeOH–EtOAc, 1:9) to give 8b (59%, 155 mg)
as a white foam; Rf 0.59 (MeOH–EtOAc, 1:9).
To a solution of compound 6b (102 mg, 0.16 mmol) in MeOH (5
mL), was added NaOMe (12.9 mg, 0.24 mmol) and the mixture was
stirred at r.t. for 1 h. After completion of the reaction, the mixture
was treated with aq concd tartaric acid (5 mL) and extracted with
CH2Cl2 (3 × 15 mL). The combined organic layers were dried
(MgSO4), concentrated, and purified using silica gel column chro-
matography with 8:2 EtOAc–petroleum ether. The resulting com-
pound was dissolved in anhyd MeCN (5 mL) and to this solution
were added DMAP (94.2 mg, 0.96 mmol) and phenyl chlorothio-
noformate (23 mL, 0.21 mmol) and the mixture was stirred at r.t. for
1 h. After completion of the reaction, the solvent was removed and
the residue was dissolved in CH2Cl2 (20 mL). The organic layer was
washed with aq 5% citric acid (10 mL) and H2O (10 mL), dried
(MgSO4) and evaporated to give a crude yellowish foam. To a solu-
tion of this crude yellowish foam in anhyd toluene (5 mL) were add-
ed n-Bu3SnH (203 mL, 0.77 mmol) and a small amount of AIBN and
the mixture was refluxed for 1 h. The mixture was evaporated and
the residue was purified by silica gel column chromatography
(EtOAc–petroleum ether, 3:7) as eluent to give 7b (71%, 72.0 mg)
as a white foam; Rf 0.15 (EtOAc–petroleum ether, 4:6).
1H NMR (250 MHz, CDCl3): d = 1.09 (s, 9 H), 2.21 (m, 1 H), 2.45
(m, 1 H), 3.79 (dd, J = 11.75 Hz, 2 H), 4.49 (d, J = 12 Hz, 1 H), 4.60
(d, J = 11.8 Hz, 1 H), 4.65 (m, 1 H), 5.28 (dd, J = 8.8, 2.25 Hz, 2
H), 5.5 (d, J = 8.8 Hz, 1 H), 5.91 (q, 1 H), 6.27 (m, 1 H), 7.26–7.50
(m, 11 H), 7.54–7.71 (m, 4 H), 9.33 (br s, 1 H).
13C NMR (100 MHz, CDCl3): d = 19.34, 26.96, 37.31, 65.10, 83.04,
88.82, 102.12, 117.05, 127.42, 127.97, 128.49, 130.09, 132.03,
132.70, 133.88, 135.47, 137.40, 140.01, 150.22, 163.33.
1H NMR (400 MHz, CD3OD + CDCl3): d = 0.88 (t, 3 H, J = 7.8
Hz), 1.5 (m, 1 H), 1.63 (m, 1 H), 2.26 (m, 4 H), 3.49 (d, J = 11.7 Hz,
1 H), 3.60 (d, J = 11.7 Hz, 1 H), 4.37 (dd, J = 6.6, 5.1 Hz, 1 H), 5.60
(d, J = 8.2 Hz, 1 H), 6.10 (t, J = 6.2 Hz, 1 H), 7.97 (d, J = 7.8 Hz,
1 H).
13C NMR (100 MHz, CD3OD + CDCl3): d = 8.35, 14.42, 24.55,
41.47, 61.29, 64.47, 71.99, 85.04, 90.54, 102.54, 102.25, 142.14,
151.69, 165.81.
FAB MS: m/z = 279.0 [M + Na]+.
2¢-Deoxy-5¢-O-dimethoxytrityl-4¢-C-methyluridine (9a)
The compound 8a (144 mg, 0.59 mmol) was coevaporated thrice
with pyridine and dissolved in pyridine (5 mL) and 4,4¢-dimethoxy-
trityl chloride (403 mg, 1.2 mmol) and a catalytic amount of DMAP
were then added at r.t. After completion of the reaction (4 h), the
mixture was quenched with MeOH (1 mL) and the stirring was con-
tinued for 30 min. The mixture was then poured onto aq sat.
NaHCO3 solution and extracted with CH2Cl2. The combined ex-
tracts were dried (MgSO4), concentrated, and purified by silica gel
column chromatography [EtOAc–petroleum ether, 2:1 → EtOAc–
petroleum ether (containing 1% Et3N), 3:1] to yield 9a (54%, 175
mg) as a colorless foam; Rf 0.50 (EtOAc).
1H NMR (250 MHz, CD3OD): d = 1.12 (s, 3 H), 2.22 (m, 1 H), 2.38
(m, 1 H), 3.17 (d, J = 10.8 Hz, 2 H), 3.70 (s, 6 H), 4.50 (t, J = 6.75
Hz, 1 H), 5.15 (d, J = 8.0 Hz, 1 H), 6.05 (m, 1 H), 6.18 (d, J = 8.75
Hz, 4 H), 7.20 (m, 7 H), 7.34 (d, J = 8.25 Hz, 2 H), 7.84 (d, J = 8.2
Hz, 1 H,).
13C NMR (100 MHz, CD3OD): d = 15.36, 19.55, 20.24, 21.75,
21.78, 29.80, 41.98, 50.53, 62.42, 69.26, 72.58, 72.83, 86.14, 88.98,
89.49, 103.01, 115.05, 128.94, 129.79, 130.35, 132.37, 137.48,
137.72, 143.33, 146.93, 152.92, 161.14, 167.13, 173.85.
FAB MS: m/z = 605.1 [M + Na]+.
2¢-Deoxy-4¢-C-methyluridine (8a)
To a solution of compound 7a (1.42 g, 2.49 mmol) in EtOH (20 mL)
was added an equivalent weight amount of 10% Pd/C (1.42 g). The
mixture was subsequently filled with H2 (balloon) and refluxed for
24 h. After completion of the reaction, the mixture was filtered
through Celite on a sintered funnel and washed thoroughly. The sol-
vent was removed under reduced pressure and the resulting white
foam was dissolved in anhyd THF (20 mL). A solution of 1 M
TBAF in THF (1.88 mmol; 492 mL) was added and the mixture was
stirred for 7 h. After completion of the reaction, the mixture was
concentrated and purified by silica gel column chromatography
(EtOAc → MeOH–EtOAc, 1:9) to give 8a (60%, 352 mg) as a white
foam; Rf 0.43 (MeOH–EtOAc, 1:9).
FAB MS: m/z = 566.7 [M + Na]+.
2¢-Deoxy-5¢-O-dimethoxytrityl-4¢-C-ethyluridine (9b)
The compound 8b (130 mg, 0.51 mmol) was coevaporated thrice
with pyridine and dissolved in pyridine (5 mL) and 4,4¢-dimethoxy-
trityl chloride (344 mg, 1.01 mmol) and a catalytic amount of
DMAP were then added at r.t. After completion of the reaction (4
h), the mixture was quenched with MeOH (1 mL) and the stirring
was continued for 30 min. The mixture was then poured onto aq sat.
NaHCO3 solution and extracted with CH2Cl2. The combined ex-
tracts were dried over (MgSO4), concentrated, and purified by silica
gel column chromatography [EtOAc–petroleum ether, 2:1 →
EtOAc–petroleum ether (containing 1% Et3N), 3:1] to yield 9b
(54%, 155 mg) as a colorless foam; Rf 0.55 (EtOAc).
1H NMR (250 MHz, CD3OD + CDCl3): d = 1.18 (s, 3 H), 2.38 (m,
2 H), 3.60 (dd, J = 12 Hz, 2 H), 4.41 (t, J = 6.25 Hz, 1 H), 5.71 (d,
J = 8 Hz, 1 H), 6.16 (t, J = 6.5 Hz, 1 H), 7.98 (d, J = 8.2 Hz, 1 H).
1H NMR (400 MHz, CD3OD): d = 0.77 (t, J = 7.6 Hz, 3 H), 1.58 (m,
1 H), 1.70 (m, 1 H), 2.21 (m, 1 H), 2.36 (m, 1 H), 3.09 (d, J = 7.6
Hz, 1 H), 3.22 (d, J = 4.0 Hz, 1 H), 3.73 (s, 6 H), 4.59 (t, J = 5.6 Hz,
1 H), 5.19 (d, J = 8.4 Hz, 1 H), 6.07 (t, J = 6.0 Hz, 1 H), 6.82 (d,
J = 9.2 Hz, 4 H), 7.24 (m, 7 H), 7.36 (d, 2 H), 7.81 (d, J = 8.0 Hz, 1
H).
13C NMR (100 MHz, CD3OD): d = 7.15, 8.17, 13.24, 13.48, 19.64,
21.30, 24.33, 34.06, 40.30, 46.55, 54.92, 60.31, 64.96, 71.38, 84.23,
86.99, 89.23, 101.01, 112.86, 126.84, 127.56, 127.66, 128.28,
130.28, 135.41, 135.63, 141.19, 144.81, 150.84.
13C NMR (62.9 MHz, CD3OD + CDCl3): d = 13.82, 17.09, 30.56,
39.92, 60.40, 65.98, 70.28, 70.36, 84.08, 87.68, 101.68, 141.00,
150.54, 164.24.
FAB MS: m/z = 242.4 [M + H]+.
2¢-Deoxy-4¢-C-ethyluridine (8b)
To a solution of compound 7b (600 mg, 1.02 mmol) in EtOH (20
mL) was added an equivalent weight amount of 10% Pd/C (600
mg). The mixture was subsequently filled with H2 (balloon) and re-
fluxed for 24 h. After completion of the reaction, the mixture was
filtered through Celite on a sintered funnel and washed thoroughly.
The solvent was removed under reduced pressure and the resulting
white foam was dissolved in anhyd THF (20 mL). A 1 M solution
of TBAF in THF (0.69 mmol, 180 mL) was added and the mixture
was allowed to stir for 7 h. After completion of the reaction, the
mixture was concentrated and purified by silica gel column chroma-
FAB MS: m/z = 582.0 [M + Na]+.
Phosphoramidites; General Procedure
The respective nucleoside 9a (155 mg, 0.28 mmol), 9b (167 mg,
0.29 mmol) was coevaporated thrice with toluene and then dis-
solved in CH2Cl2 (5 mL), and N-ethyldiisopropylamine (244 mL,
1.42 mmol for 9a and 256 mL, 1.49 mmol for 9b) and 2-cyanoethyl-
Synthesis 2005, No. 9, 1467–1472 © Thieme Stuttgart · New York

1472
G. Rangam et al.
PAPER
N,N-(diisopropylamino)chlorophosphite (127 mL, 0.57 mmol for 9a
and 134 mL, 0.60 mmol for 9b) were then added at r.t. After com-
pletion of the reaction (4 h), the mixture was quenched with aq sat.
NaHCO3 solution and extracted with CH2Cl2. The combined ex-
tracts were dried (MgSO4), concentrated, and purified by silica gel
column chromatography [EtOAc–petroleum ether, 2:1 → EtOAc–
petroleum ether (containing 1% Et3N), 3:1] to yield 94% (200 mg)
for 10a and 91% (206 mg) for 10b as gums.
tary strand were performed in a reaction buffer composed out of 20
mM Tris-HCl (pH 8.0 at 25°C), 1 mM DTT and 1 mM EDTA in a
total volume of 20 mL. Each reaction contained 25 nM of the respec-
tive oligonucleotide and 50 nM of the enzyme. After incubation at
37 °C NaOH (30 mL, 0.1 M) was added and the mixture heated to
95 °C for 10 min. After addition of 50 mL of gel loading buffer (80%
formamide, 20 mM EDTA) and subsequently heating to 95 °C for 5
min, the reactions were analyzed by 12% polyacrylamide gel elec-
trophoresis containing 8 M urea, transferred to a filter paper, dried
under vacuum, and visualized by 32P-phosphor imaging.
2¢-Deoxy-4¢-C-methyluridine Derivatives 10a – Mixture of Dia-
stereomers
Rf 0.70 and 0.54 (EtOAc–petroleum ether, 4:1).
Acknowledgment
1H NMR (400 MHz, CD3OD): d = 1.25 (br s, 8 H), 1.27 (br s, 16 H),
1.29 (br s, 8 H), 2.54 (m, 2 H), 2.70 (m, 2 H), 2.84 (t, J = 6 Hz, 4
H), 3.49–3.60 (m, 4 H), 3.76 (2 s, 12 H), 4.08–4.21 (m, 8 H), 4.82–
4.90 (m, 2 H), 5.22, 5.21 (2 d, J = 8 Hz, 2 H), 6.13 (br s, 2 H), 6.86
(m, 8 H), 7.22–7.45 (m, 18 H), 7.75 (s, 2 H), 7.95 (d, J = 8 Hz, 1 H),
7.98 (d, J = 8 Hz, 1 H).
13C NMR (100 MHz, CD3OD): d = 19.23, 19.60, 20.30, 20.37,
21.01, 23.18, 25.06, 44.20, 46.49, 46.55, 55.75, 60.11, 84.82, 84.93,
87.74, 87.98, 114.16, 118.72, 128.92, 129.43, 131.47, 151.92,
160.15, 166.06.
We thank DFG for financial support. G.R. wishes to acknowledge
the receipt of a research fellowship from the Alexander von Hum-
boldt Foundation.
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31P NMR (162 MHz, CD3OD): d = 149.49, 149.78.
FAB MS: m/z = 766.7 [M + Na]+.
2¢-Deoxy-4¢-C-ethyluridine Derivatives (10b) – Mixture of Dia-
stereomers
Rf 0.66 and 0.54 (EtOAc–petroleum ether, 4:1).
1H NMR (400 MHz, CD3OD): d = 1.25 (br s, 8 H), 1.27 (br s, 16 H),
1.29 (br s, 8 H), 1.59 (m, 2 H), 1.73 (m, 2 H), 2.55 (m, 2 H), 2.70
(m, 2 H), 2.84 (t, J = 6 Hz, 4 H), 3.50–3.60 (m, 4 H), 3.75 (2 s, 12
H), 4.05–4.20 (m, 8 H), 4.88–5.00 (m, 2 H), 5.22 (d, J = 8 Hz, 2 H),
6.13 (br s, 2 H), 6.86 (m, 8 H), 7.21–7.45 (m, 18 H), 7.75 (s, 2 H),
7.90 (d, J = 8 Hz, 1 H), 7.94 (d, J = 8 Hz, 1 H).
13C NMR (100 MHz, CD3OD): d = 8.33, 14.49, 19.74, 20.29, 20.36,
20.92, 20.94, 21.01, 23.19, 25.11, 26.21, 40.12, 40.56, 44.31, 46.47,
46.54, 47.93, 55.75, 55.77, 60.13, 61.40, 65.53, 85.05, 85.12, 88.07,
89.61, 89.65, 89.88, 89.93, 102.34, 108.57, 114.15, 118.72, 119.38,
119.60, 128.07, 128.91, 129.45, 131.46, 136.44, 136.54, 136.59,
138.54, 142.27, 145.85, 145.91, 151.90, 151.97, 160.11, 160.13,
165.96, 172.69.
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31P NMR (162 MHz, CD3OD): d = 149.52, 150.11.
FAB MS: m/z = 781.0 [M + Na]+.
Modified Oligonucleotides and Functional Studies of E. coli
Uracil-DNA Glycosylase (UDG)
The synthesis of oligonucleotides was carried out on an Applied
Biosystems, Model 392 DNA synthesizer on 0.2 mmol scale apply-
ing commercially available 2-cyanoethylphosphoramidites. A stan-
dard method for 2-(cyanoethyl)phosphoramidites was used, with
the exception that the coupling times of and from the modified nu-
cleotides were extended to 10 min. After synthesis (trityl-off) the
oligonucleotides were cleaved from the support by treatment with
conc. NH4OH at 55 °C for 12 h. After removal of NH4OH, the res-
idue was purified by preparative electrophoresis on a 12% polyac-
rylamide gel containing 8 M urea. The sequence used for the
cleavage studies was 5¢-d(TGC CTA AURG AGT GAG), UR is ei-
ther the non-modified 2¢-deoxyuridine, 4¢-C-methyl- or 4¢-C-ethyl
2¢-deoxyuridine, respectively.
(15) Kuivila, H. G.; Menapace, L. W. J. Org. Chem. 1963, 28,
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Ber. 1981, 114, 1234. (b) Vorbrüggen, H.; Höfle, G. Chem.
Ber. 1981, 114, 1256.
For functional investigations of UDG the oligonucleotides were ra-
dioactively labeled using T4 polynucleotide kinase and g-32P-ATP
and subsequently purified by gel filtration. E. coli UDG was pur-
chased from New England Biolabs. Reactions containing either the
single stranded oligonucleotide or the duplex with the complemen-
Synthesis 2005, No. 9, 1467–1472 © Thieme Stuttgart · New York