Synthesis of DNA oligos containing 2′-deoxy-2′-fluoro-d- arabinofuranosyl-5-carboxylcytosine as hTDG inhibitor

Article abstract of DOI:10.1016/j.tet.2012.04.031

As an important step of the active demethylation of 5-methylcytosine (5mC), human thymine DNA glycosylase (hTDG) efficiently excises 5-carboxylcytosine (5caC) from double-stranded DNA (dsDNA). Here, we present synthesis of DNA oligos containing a 2′-deoxy-2′-fluoro-d-arabinofuranosyl-5- carboxylcytidine (F-5caC) modification that act as hTDG inhibitors. The glycosylase activity assay showed that F-5caC oligos were resistant to excision by the hTDG catalytic domain (hTDG^cat, residues 111-308) and they could inhibit the excision of DNA oligos containing 5caC. The electrophoretic mobility shift assay confirmed that DNA oligos containing F-5caC could bind well with unmodified hTDG^cat to form a stable complex, which makes it possible to obtain the crystal structure of the complex to reveal details on how hTDG^cat recognizes the DNA substrate.

Full text of DOI:10.1016/j.tet.2012.04.031

Tetrahedron 68 (2012) 5145e5151
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of DNA oligos containing 20-deoxy-20-fluoro-
-arabinofuranosyl-
D
5-carboxylcytosine as hTDG inhibitor
*
*
Qing Dai , Xingyu Lu, Liang Zhang, Chuan He
Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, 929 East 57th Street, Chicago, IL 60637, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
As an important step of the active demethylation of 5-methylcytosine (5mC), human thymine DNA
Received 20 February 2012
Received in revised form 3 April 2012
Accepted 9 April 2012
glycosylase (hTDG) efficiently excises 5-carboxylcytosine (5caC) from double-stranded DNA (dsDNA).
Here, we present synthesis of DNA oligos containing
a D-arabinofuranosyl-5-
20-deoxy-20-fluoro-
carboxylcytidine (F-5caC) modification that act as hTDG inhibitors. The glycosylase activity assay
showed that F-5caC oligos were resistant to excision by the hTDG catalytic domain (hTDGcat, residues
111e308) and they could inhibit the excision of DNA oligos containing 5caC. The electrophoretic mobility
shift assay confirmed that DNA oligos containing F-5caC could bind well with unmodified hTDGcat to
form a stable complex, which makes it possible to obtain the crystal structure of the complex to reveal
details on how hTDGcat recognizes the DNA substrate.
Available online 14 April 2012
Keywords:
Human thymine DNA glycosylase (hTDG)
Inhibitor
20-Deoxy-20-fluoro-
carboxylcytidine
dsDNA
D-arabinofuranosyl-5-
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
NH2
N
NH2
N
NH2
N
DNA
O
DNA
O
DNA
O
CHO
Me
N
As one of the best characterized epigenetic modifications,
5-methylcytosine (5mC) plays important roles in many biological
processes including regulation of gene expression, genomic im-
printing, X chromosome inactivation, and suppression of transpos-
able elements propagation.1 Therefore, studies on the formation of
5mC and its demethylation pathways are very important. In contrast
to the clear methylation pathway, the active demethylation pathway
has been less understood, particularly in mammals.2 Recent studies
have shown that 5mC can be converted to 5-hydroxymethylcytosine
(5hmC, the ‘sixth’ base) in mammalian genome through the oxida-
tion of TET (ten eleven translocation) dioxygenases3 and that 5hmC
is a widespread DNA modification in various tissues and cells.4 More
recently, together with our collaborators, we discovered that 5hmC
can be further oxidized into 5-formylcytosine (5fC) and 5-
carboxylcytosine (5caC) by TET proteins.5 These findings strongly
supported the hypothesis of the active 5mC demethylation pathway
(Fig. 1).6 The TET protein-catalyzed conversion of 5mC to 5hmC may
represent the first step of a multiple-step pathway of DNA deme-
thylation. Further oxidation of 5hmC in DNA results in the formation
of 5fC and 5caC in DNA. It has been proposed that 5caC may be
converted back to cytosine by decarboxylation to complete the 5mC
active demethylation pathway.7 Despite of extensive efforts, the
OH
N
N
O
O
O
O
O
O
TET
TET
O
O
O
5fC
DNA
5hmC
DNA 5mC
DNA
TET
DNA
NH2
N
DNA
O
O
CO2H
N
OH
O
hTDG
O
O
O
5caC
O
DNA
5caC
DNA
BER pathway
NH2
N
DNA
N
Decarboxylase
O
O
O
O
C
DNA
* Corresponding authors. E-mail addresses: daiqing@uchicago.edu (Q. Dai),
chuanhe@uchicago.edu (C. He).
Fig. 1. The active pathway of 5mC demethylation.
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2012.04.031

5146
Q. Dai et al. / Tetrahedron 68 (2012) 5145e5151
decarboxylase responsible for removing the 5-carboxyl group has
yet to be identified. On the other hand, it has been shown that hTDG
can efficiently excise the 5caC base by cleaving the bond between C-
1 of the sugar and N-1 of the nucleobase to generate an abasic site in
DNA8; subsequent incorporation of an unmethylated cytosine via the
DNA base-excision-repair pathway (BER)9 establishes the active
demethylation cycle (Fig. 1). We have recently published the crystal
structures of hTDGcat in complex with a duplex DNA containing
5caC.10 To avoid the excision of 5caC during the crystallization, an
inactive hTDGcat mutant with the active site residue Asn140 mutated
to alanine was used. In the meantime, the crystal structure of the
DNA complex with unmodified hTDG could be very helpful for elu-
ciating how hTDG interacts with the DNA substrate. To achieve this
goal we synthesized dsDNA oligos containing F-5caC. Herein, we
described the syntheses, hTDG inhibitory effects and the binding
We recently reported a highly efficient and convenient method
for synthesis of 5caC free nucleoside and its phosphoramidite.14
Based on this procedure, it would be ideal if 20-F could be in-
troduced to 5caC directly to afford the desired F-5caC. A search for
the literaure revealed that several methods for introducing fluoro
to 20-C of nucleosides have been reported: (1) the SN2 fluorination
via a sulfonate intermediate.15 This method is usually suitable for
purine instead of pyrimidine nucleosides since O2 of the pyrimidine
base may attack C-20 to form a 2,20-anhydropyrimidie byproducts;
(2) direct fluorination of arabinonucleosides with dieth-
ylaminosulfur trifluoride (DAST)16 and (3) ring opening reaction of
2,20-anhydropymiridine.17 Unfortunately, both method 2 and 3 in-
troduces fluoro into C-20 from
a
face.18 Introduction of 20-F into the
arabino position of nucleosides usually involves fluorination of the
sugar without a base followed by the glycosyation reaction.19
properties of F-5caC-containing DNA oligos with hTDGcat
.
Therefore, we chose to synthesize 20-deoxy-20-fluoro-
D-arabino-
furanosyl-5-iodocytidine derivative20 by glycosylation reaction,
then substitute 5-iodo with 5-carboxyl group through palladium-
catalyzed carbonyl reaction to prepare F-5caC (Scheme 1).
2. Results and discussion
It is well-known that fluorine substitution at the 20-C position in
the 20-deoxyribose ring can slow down or even inhibit the N-gly-
cosidic bond cleavage catalyzed by alkyl-N-purine DNA glyco-
sylase.11 Presumably, the strong elecron-withdrawing fluorine at
20-C position destabilizes the obligate oxocarbenium ion in-
termediate or transition state by increasing its charge density,
thereby inhibiting the deglycosylation reaction.12 Since it is be-
lieved that the 20-F in the arabino configuration will not change the
sugar pucker in DNA,13 we chose to introduce the 20-fluoro sub-
stituent to the 5caC in the arabino position to syntheisze the
The glycosylation acceptor, glycosyl bromide 2 was prepared by
first fluorination of commercially available ribose derivative 1 with
DAST followed by bromination with hydrogen bromide in acetic
acid.21 The glycosylation donor, the silylated 5-iodocytosine was
prepared by first treating the commercially available cytosine with
iodine and iodic acid to furnish 5-iodocytosine in 85% yield followed
by treatment with hexamethyldisilazane.22 N-Glycosylation of the
bromide 2 with silyated 5-iodocytosine in 1,2-dichloroethane and
acetonitrile gave a mixture of
b and a
anomers at C-10 with the
b
anomer 3a as the main product (the ratio of
b
/
a
¼3:1 as indicated by
1H NMR spectrum).23 Unfortunately, the two anomers migrated the
same on TLC and could not be separated by column chromotagraphy
20-deoxy-20-fluoro-
D-arabinofuranosyl-5-carboxylcytosine nucleo-
sides and the phosphoramidites.
NH2
BzO
N(SiMe3)2
I
F
BzO
I
F
O
N
N
BzO
O
O
N
SiMe3
O
N
Br
N
O
(1) DAST
OBz
BzO
OBz
O
I
OBz OH
F
OBz
+
O
(2) HBr/AcOH
N
1
2
NH2
3b
3a:3b=3:1
3a
OBz
NH2
NH2
N
HO
F
HO
O
I
CO2Me
F
O
N
N
N
N
O
N
O
N
N
OH
I
HO
HO
O
OH
F
F
O
+
+
(PhCN)2PdCl2
CO2Me
NH2
O
NH3/MeOH
O
CO, Et3N/MeOH
NH2
5b
5a:5b=3:1
4b
4a 4a:4b=3:1
5a
OH
OH
N
NHAc
NHAc
CO2Me DMTrO
F
HO
CO2Me
O
F
N
O
O
N
N
DMTrO
N
O
N
F
OH
HO
O
CO2Me
NHAc
O
F
+
OH
+
O
CO Me
2
Ac2O
DMF
O
DMTr-Cl
Py
N
N
7b
7a
OH
NHAc
6b
6a
OH
(i-Pr)2NP(Cl)OCH2CH2CN
(i-Pr)2NEt/CH2Cl2
NHAc
DMTrO
CO2Me
F
N
O
N
O
N
N
DMTrO
F
O
P
CO2Me
NHAc
O
O
NCH2CH2CO
N(Pr-i)2
O
8a
8b
P
NCH2CH2CO
N(Pr-i)2
Scheme 1. Synthesis of 10-
cytosine (8b).
b
-20-deoxy-20-fluoro-D-arabinofuranosyl-5-carboxylcytosine phosphoramidite (8a) and 10- -20-deoxy-20-fluoro-D-arabinofuranosyl-5-carboxyl-
a

Q. Dai et al. / Tetrahedron 68 (2012) 5145e5151
5147
3
0
0
at this stage. It has been reported that J1 ,2 in
b
anomer with a 20-
recently.14,25 Their masses were also verified by MALDI-TOF anal-
ysis. For 5hmC20, m/z¼6246 was observed (6246 calculated); for
5caC20, m/z¼6260 was observed (6260 calculated).
arabino substituent is in the range of 3e4 Hz whereas the
a anomer
shows very small or no coupling between the 10 and 20 protons.24 In
our case, analysis of 6.32e6.37 ppm region reveals a doublet of
The four synthesized DNA oligos were then 50-end 32P-labeled
and annealed to a complimentary 20mer DNA primer (50-TTT CAG
CTC CGG TCA CGC TC-30) to give the corresponding dsDNA. To test
whether they can be excised by hTDGcat, we treated the double-
doublet (dd) for the H-10 resonance of the
b anomer with
JH1 ,F¼21.0 Hz and 3J1 ,2 ¼3.0 Hz, while 6.21e6.25 ppm region reveals
0
0
0
a doublet (d) for the H-10 resonance of the
a anomer with
JH1 ,F¼14.0 Hz and no observed coupling between H-10 and H-20.
Treatment of the mixture of 3a and 3b with saturated ammonia in
MeOH readily removed both benzoyl groups to give a mixture of
5-iodo free nucleosides 4a and 4b quantitatively. Using (PhCN)2PdCl2
as catalyst and methanol as solvent, 4a and 4b was quantitatively
converted to the corresponding 5-methoxycarbonyl derivatives 5a
and 5b in the presence of triethylamine under carbon monoxide at-
mosphere (75% isolated yield). This new procedure avoided using
DMF as well as the more expensive catalyst Pd2(dba)3 but was better
than the one we previously reported to synthesize 5caC;14 we found
the procedure can also be exended to prepare 5-methoxycarbonyl
cytidine. Protection of the exocyclic amino group of 5a and 5b was
realized by treating the mixture of 5a and 5b with acetic anhydride in
DMF to furnish the mixture of 6a and 6b. On TLC, the two anomers of
3a/3b, 4a/4b and 5a/5b could not be separated at all; in comparison,
the mixture 6a and 6b gave two distinguishable spots that migrate
very close on TLC and attempts to purify them by silica gel chroma-
tography always resulted in many fractions containing both 6a and
6b. Fortunately, we found that after they were converted to the cor-
ressponding 50-tritylated derivatives 7a and 7b, they migrated sig-
nificantly away from each other, allowing complete separation of the
two anomers readily by silica gel column chromatography. Phos-
phitylation of the 30-OH of 7a and 7b under the standard conditions
provided the phosphoramidites 8a and 8b, respectively.
stranded b20 and a
20 with hTDGcat separately followed by
0
NaOH treatment. After the modified base is excised by hTDGcat, it
generates an abasic site in DNA, which degrades upon treatment
with aqueous NaOH and results in specific strand scission to
generate a 50-32P-end labeled 9mer. The 9mer product can be
detected on the basis of its increased mobility on a polyacrylamide
gel. However, if the modified base cannot be excised by hTDGcat
,
DNA oligos will keep intact as a 20mer. To our delight, we found
that both 20 and
b
a
20 stayed intact after treatment with hTDGcat
(lane 1 and 2, Fig. 2), suggesting that introduction of 20-F in the
arabino position indeed prevented the excision of base. As a pos-
itive control, more than half of the 20mer 5caC20 was converted
to 9mer under the same conditions (lane 3 and 6, Fig. 2), sug-
gesting that hTDG did possess the activity to excise the 5caC base
from 5caC20. We then tested whether the addition of
can inhibit the excision activity of hTDGcat on 5caC20. Notable
inhibition was observed when 10 equiv of 20 was added (lane 4,
Fig. 2) and the activity of hTDGcat was sufficiently inhibited when
100 equiv of 20 was added (lane 5, Fig. 2). We found that 20 had
higher hTDGcat inhibitory activity and 10 equiv of
20 showed
higher inhibition activity of hTDGcat than the same amount of
20
b20 or a20
a
a
b
b
a
(compare lane 7 to lane 4, Fig. 2). As a negative control, addition
of 100 equiv of 5hmC20 did not show any inhibitory effect (lane
9, Fig. 2).
Both phosphoramidites 8a and 8b were incorporated into
The observation that addition of either
b20 or a20 oligo inhibited
a 20mer DNA separately with the sequence of 50-GAG CGT GAC XGG
the excision of 5caC20 substrate by hTDGcat may result from the
Fig. 2. Cleavage assay to detect the excision of dsDNA oligos by hTDG. Lane 1e2, a20 and b a20 and b
20 cannot be excised by hTDGcat. Lane 3e8, 20 can inhibit hTDGcat activity. Lane
9, hmC20 cannot inhibit hTDGcat activity.
AGC TGA AA-30, in which X¼1-
b
-20-F-5caC or 1-
a
-20-F-5caC to give
competitivebindingof b20 or a
20 to hTDGcat. Totestthis hypothesis,
resin-bound oligos
b20 and
a
20, respectively. The oligos were
we conducted an electrophoretic mobility shift assay (EMSA) to
deprotected by treatment with 0.1 M K2CO3 in water and MeOH
determine the apparent dissociation constant (Kdapp). As shown in
(v/v¼1:1) overnight at 40 ꢀC. After cooled to rt, AcOH was added to
Fig. 3, hTDGcat binds with both
b
20 and
20, indicating that
tightly to hTDGcat. This is consistent with the observation that
showed better inhibitory activity than
20. Since hTDGcat binds well
a mM
20 oligoswith Kdapp>10
adjust pH to neutral. Oligos
b
20 and
a20 were then purified by
for
a20 and ¼400ꢁ10 nM for
b
b
20 binds more
20
PAGE gel elecrophoresis and their masses were verified by MALDI-
b
TOF analysis. For both
b20 and
a
20, m/z¼6278 of [MH]þ was ob-
a
served (6278 calculated). As controls, we also prepared another two
DNA oligos 5hmC20 and 5caC20, which have the same sequence
except X¼5hmC or 5caC by following the methods that we reported
with b20 but does not excise the oligo, it makes this DNA oligo good
tools to form the complex with hTDGcat as well as make it possible to
obtain its crystal structure to reveal the details on how hTDGcat

5148
Q. Dai et al. / Tetrahedron 68 (2012) 5145e5151
Fig. 3. Electrophoretic mobility shift assay for hTDGcat with
and the enzyme concentrations shown. Fitting the data provides >10
a20 and
b
20 oligos. Electrophoretic mobility shift experiments were performed with 4 nM 32P-labeled DNA substrate
M for 20.
20, ¼400ꢁ10 nM for
m
a
b
binds with the DNA substrate. Indeed, after screening different
F substituent in arabino position. Besides, we demonstrated that
both 20 and 20 could inhibit the excision of 5caC20 oligo by
hTDGcat and
20 exhibited better inhibitory activity than 20.
Furthermore, we have shown that both 20 and 20 could bind
with hTDGcat and
20 has stronger binding affinity than 20. These
properties of
20 allowed it to form a stable complex with hTDGcat
lengths of DNA oligos containing
b
-F-5caC modification, we
b
a
obtained the crystal structure of the complex of hTDGcat with
a 23mer dsDNA oligo.10
b
a
b
a
Since DNA oligos containing F-5caC showed good hTDGcat in-
hibitory activity, we wondered whether the corresponding free
nucleoside or its methyl ester could also inhibit hTDGcat. Since the
anomers 3a/3b, 4a/4b and 5a/5b could not be separated by silica gel
chromatography, we tried to improve the selectivity of the
b
a
b
and the crystal structure of such a complex revealed the details on
how DNA substrate interacts with hTDGcat. Free nucleosides with
20-F substitutions did not exhibit inhibition effects of hTDGcat
;
b
anomer by changing the reaction conditions so that purification
however, these molecules could be further modified as potential
of the anomer by recrystallization might be possible. We found
b
small molecule inhibitors as demonstrated previously.27
that the ratio 3a/3b was improved to 6:1 from 3:1 in the absence of
NaI and CH3CN in the reaction mixture although the combined
yield of 3a/3b was decreased from 90 % to 75%. Subsequent efforts
to obtain pure 3a by crystallization failed, however, when the
mixture of 3a and 3b (3a/3b¼6:1) was converted to the corre-
sponding mixture of 4a and 4b; pure 4a could be readily obtained
by recrystallization from acetone and hexane. Pure 4a was then
converted to 5a in 92% yield. Compound 5a could either be hy-
drolyzed to the corresponding acid 9 quantitatively or be converted
to the corresponding phosphoramidite 8a through 6a and 7a. In
4. Experimental section
4.1. Synthesis of phosphoramidite 8a and 8b
4.1.1. General experimental procedures. Starting materials and re-
agents were obtained from commercial suppliers and used without
further purifications. All reactions using air sensitive or moisture
sensitive reagents were run under an argon atmosphere. 1H and 13
C
NMR spectra were recorded on Bruker 500 MHz NMR spectrome-
ter. 1H chemical shifts are reported in
(ppm) relative to tetrame-
thylsilane, 13C chemical shifts
(ppm) relative to the solvent used.
Merck silica gel (9385 grade, 230e400 mesh, 60A, Aldrich) was
used for column chromatography. Silica gel on glass with fluores-
cent indicator (Sigma) was used for TLC. High-resolution mass
spectra (HRMS) were obtained from Mass Spectrometry & Proteo-
mics Facility of University of Notre Dame.
contrast to the inhibitory effects of DNA oligos
b20 and a20 to
d
hTDGcat, both nucleosides 5a and 9 did not show noticeable
hTDGcat inhibitory activity even when their concentrations were
1000 times as much as the 5caC DNA concentration. This suggests
that hTDGcat could not bind these nucleosides well and the binding
requires a stretch of DNA. Indeed, the crystal structure shows that
two hTDG molecules bind to one dsDNA with 5caC recognized by
one protein while the other hTDG binds nonspecifically ten base
pairs away.10,26 The hTDG interacts with the backbone of the 5caC-
containing strand via electrostatic complementarity, and distorts
the dsDNA backbone w45ꢀ bending toward the active site.
d
4.1.2. Mixture of 1-(2-deoxy-2-fluoro-3,5-di-O-benzoyl-
furanosyl)-5-iodocytosine (3a) and 1-(2-deoxy-2-fluoro-3,5-di-O-
benzoyl- -arabinofuranosyl)-5-iodocytosine (3b). To a round bot-
b-D-arabino-
a-D
tom flask was added 5-iodocytosine (1.38 g, 5.82 mmol, 1.5 equiv),
(Me3Si)2NH (20 mL) and (NH4)2SO4 (100 mg). The mixture was
stirred and heated to 127 ꢀC with an oil bath. After 1 h, the reaction
mixture turned clear. The excess (Me3Si)2NH was removed by
evaporation in vacuum. To the residue was added and NaI (582 mg)
3. Conclusion
In summary, we have successfully synthesized DNA oligos
and 20, which contain F-5caC modification. In contrast to the
corresponding 5caC20 oligo, which can be efficiently excised by
hTDGcat, both
20 and 20 were kept intact when treated with
hTDGcat due to the presence of the strong electron-withdrawing 20-
b20
a
and
1-bromo-2-deoxy-2-fluoro-3,5-di-O-benzoyl-D-arabinofur-
b
a
anose (1.64 g, 3.88 mmol, 1.0 equiv) dissolved in ClCH2CH2Cl
(30 mL) and CH3CN (8 mL), and the mixture was stirred at rt for 2

Q. Dai et al. / Tetrahedron 68 (2012) 5145e5151
5149
days. Then CH2Cl2 (100 mL) was added to dilute the reaction mix-
ture and the well-stirred solution was treated dropwise with MeOH
(4 mL). The suspension was filtered through a Celite pad, and the
filtrate was washed with water, brine and dried over Na2SO4 and
evaporated to dryness. The residue was purified by silica gel
chromatography, eluting with 1e4% MeOH in CH2Cl2, to give
a mixture of 3a and 3b in a 3:1 ratio (2.02 g, 90% combined yield) as
61.3, 53.4. HRMS calculated for C11H15FN3O6, [MHþ] 304.0945 (calcd),
304.0961 (found).
4.1.7. The mixture of N4-acetyl-1-(2-deoxy-2-fluoro-
anosyl)-5-methoxycarbonylcytosine (6a) and N4-acetyl-1-(2-deoxy-
2-fluoro- -arabinofuranosyl)-5-methoxycarbonylcytosine (6b). To
b-D-arabinofur-
a-D
a round bottom flask containing the mixture of 5a and 5b (3:1,
220 mg, 0.73 mmol) was added DMF (10 mL) and Ac2O (0.2 mL) and
the mixture was stirred at rt for 2 days. After removal of the volatile in
high vacuum, the residue was purified by silica gel chromatography,
eluting with 1e8% MeOH in CH2Cl2 to give a mixture of 6a and 6b (3:1,
white foam. 1H NMR (500 MHz) (CDCl3)
d: 9.32 (br, 0.5H), 8.88 (br,
1.5H), 7.45e8.10 (m, 11H), 6.35 (dd, J¼21.0 and 3.0 Hz, 0.75H), 6.22
(d, J¼14.0 Hz, 0.25H), 5.68 (d, J¼14 Hz, 0.25H), 5.60 (dd, J¼17.0 and
2.5 Hz, 0.75H), 5.49 (d, J¼47.5 Hz, 0.25H), 5.41 (dd, J¼50.0 and
2.5 Hz, 0.75H), 5.04 (m, 0.25H), 4.81 (d, J¼4 Hz,1.5H), 4.55e4.68 (m,
0.5H), 4.54 (m, 0.75H). A similar procedure as above but without
adding NaI and CH3CN gave a mixture of 3a and 3b (3a/3b¼6:1) in
75% yield as indicated by 1H NMR spectrum. MS calculated for
C23H19FIN3O6, [MHþ] 580 (calcd), 580 (found).
228 mg, 91% combined yield). 1H NMR (500 MHz) (CD3OD)
d: 9.05 (s,
0.75H), 8.78(s, 0.25H), 6.26(m, 0.75H), 6.23 (m, 0.25H), 5.08e5.15 (m,
1H), 4.34e4.38 (m, 1H), 4.07 (m, 0.75H), 4.03 (m, 0.25H), 3.91 (s,
2.25H), 3.87 (s, 0.75H), 3.77e3.84 (m, 2H), 2.56 (s, 3H). 13C NMR
(125.8 MHz)(CD3OD) d: 172.2, 166.2, 160.7, 153.7, 151.1, 99.0, 97.2, 95.6,
86.4, 86.3, 85.8, 73.8, 73.6, 60.9, 53.9, 27.5. HRMS calculated for
4.1.3. Mixture of 1-(2-deoxy-2-fluoro-
b
-
D
-arabinofuranosyl)-5-
C13H16FN3O7, [MHþ] 346.1051 (calcd), 346.1066 (found).
iodocytosine (4a) and 1-(2-deoxy-2-fluoro-
a-D-arabinofuranosyl)-5-
iodocytosine (4b). To a round bottom flask containing the mixture
of 3a and 3b (3:1 ratio, 1.80 g, 3.11 mmol) was added saturated
ammonia in methanol (50 mL) and the mixture was stirred at rt for
2 days. Removal of all the volatile under reduced pressure. The
residue was purified by silica gel chromatography, eluting with
1e10% MeOH in CH2Cl2, to give a mixture of 4a and 4b in a 3:1 ratio
(1.06 g, 92% combined yield) as white solid. 1H NMR (500 MHz)
4.1.8. N4-Acetyl-1-(2-deoxy-2-fluoro-
b-D-arabinofuranosyl)-5-
methoxycarbonylcytosine (6a). Using pure 5a as starting material,
6a was obtained by following the same procedure as white solid. 1H
NMR (500 MHz) (DMSO-d6) d: 10.70 (br, 1H), 8.91 (s, 1H), 6.15 (dd,
J¼14.0 and 4.0 Hz, 1H), 5.97 (br, 1H), 5.29 (br, 1H), 5.24 (m, 1H), 4.25
(dd, J¼16.5 and 2.5 Hz, 1H), 3.94 (m, 1H), 3.78 (s, 3H), 3.68 (m, 1H),
3.32 (s, 3H). 13C NMR (125.8 MHz) (DMSO-d6)
d: 172.2, 166.2, 160.7,
(DMSO-d6)
d
: 8.04 (s, 0.75H), 7.95 (br 1H), 7.93 (s, 0.25H), 6.74 (br,
153.7, 151.1, 99.0, 97.2, 95.6, 86.4, 86.3, 85.8, 73.8, 73.6, 60.9, 53.9,
27.5. HRMS calculated for C13H16FN3O7, [MHþ] 346.1051 (calcd),
346.1045 (found).
1H), 6.04 (dd, J¼16.5 and 3.5 Hz, 0.75H), 5.82 (m, 0.25H), 5.85 (m,
1H), 5.18 (m, 1H), 4.92e5.07 (m, 1H), 3.46e4.28 (m, 4H). MS cal-
culated for C9H11FIN3O4, [MHþ] 372 (calcd), 372 (found).
4.1.9. N4-Acetyl-1-(2-deoxy-2-fluoro-5-O-DMTr-
anosyl)-5-methoxycarbonylcytosine (7a) and N4-acetyl-1-(2-deoxy-2-
fluoro-5-O-DMTr- -arabinofuranosyl)-5-methoxycarbonylcytosine
b-D-arabinofur-
4.1.4. 1-(2-Deoxy-2-fluoro-b-D-arabinofuranosyl)-5-iodocytosine
(4a). After treatment of the mixure of 3a and 3b (6:1 ratio, 450 mg,
0.78 mmol) with saturated ammonia in methanol (20 mL) at rt for 2
days. The volatile were removed under reduced pressure to give
a white solid. It was recrystallized by acetone and hexane to give
pure 4a (187 mg, 65%) as white solid. 1H NMR (500 MHz) (CD3OD)
a-D
(7b). To a pyridine (8 mL) solution of the mixture of 6a and 6b (3:1
ratio, 180 mg, 0.52 mmol) was added 4,40-dimethoxytrityl chloride
(212 mg, 0.63 mmol,1.2 equiv) and the mixture was stirred overnight
at rt. MeOH (0.5 mL) was added to quench the reaction. After 15 min
stirring, the reaction mixture was diluted with CH2Cl2, washed with
5% aqueous NaHCO3, and brine. The organic layer was dried over
anhydrous Na2SO4, filtered, and concentrated. The residue was pu-
rified by silica gel chromatography eluting with 0e3% MeOH in
CH2Cl2 containing 0.2% Et3N to give 213 mg 7a (63%) and 71 mg 7b
d
: 8.24 (s, 1H), 6.15 (dd, J¼17.5 and 3.5 Hz, 1H), 4.99e5.10 (m, 1H),
4.29 (m, 1H), 3.94 (m, 1H), 3.86 (m, 1H), 3.75 (m, 1H). 13C NMR
(125.8 MHz) (CD3OD) : 165.6, 156.5, 149.1, 96.8, 95.3, 86.1, 86.0,
d
85.59, 85.57, 74.6, 74.4, 61.4, 56.2. MS calculated for C9H11FIN3O4,
[MHþ] 372 (calcd), 372 (found).
(21%). 7a, 1H NMR (500 MHz) (CD3CN)
d: 10.74 (br, 1H), 8.82 (s, 1H),
4.1.5. Mixture of 1-(2-deoxy-2-fluoro-
methoxycarbonylcytosine (5a) and 1-(2-deoxy-2-fluoro-
b
-
D
-arabinofuranosyl)-5-
-arabi-
7.51 (m, 2H), 7.26e7.39 (m, 7H), 6.89 (m, 4H), 6.18 (dd, J¼18.0 and
3.5 Hz, 1H), 5.11e5.23 (m, 1H), 4.41e4.45 (m, 1H), 4.14 (m, 1H), 4.04
(m, 1H), 3.78 (s, 6H), 3.43 (dd, J¼10.5 and 3.5 Hz, 1H), 3.34 (s, 3H),
a-D
nofuranosyl)-5-methoxycarbonylcytosine (5b). To a round bottom
flask containing the mixture of 4a and 4b (3:1 ratio, 420 mg,
1.13 mmol) was added methanol (30 mL), (PhCN)2PdCl2 (44 mg,
0.1 equiv), Et3N (0.7 mL) and the mixture was stirred overnight at
50 ꢀC on an oil bath under a CO atmosphere. After cooled to rt, silica
gel (5 g) was added and the volatile was removed under reduced
pressure. The residue was purified by silica gel chromatography,
eluting with 1e4% MeOH in CH2Cl2, to give a mixture of 5a and 5b
in a 3:1 ratio (238 mg, 88% combined yield) as white foam. 1H NMR
3.28 (m, 1H), 2.51 (s, 3H). 13C NMR (125.8 MHz) (CD3CN)
d: 167.6,
161.9, 156.5, 155.4, 149.3, 146.5, 141.6, 132.54, 132.50, 126.6, 124.6,
124.5, 123.6, 109.7, 93.1, 92.5, 91.0, 82.9, 82.8, 82.7, 80.80, 80.77, 71.3,
71.1, 58.8, 51.6, 48.6, 22.8. MS calculated for C34H34FN3O9, [MHþ]
648.2357 (calcd), 648.2355 (found).With 7b, 1H NMR (500 MHz)
(CD3CN) d: 10.72 (br, 1H), 8.65 (s, 1H), 7.50 (m, 2H), 7.24e7.38 (m,
7H), 6.89 (m, 4H), 5.98 (d, J¼15.0 Hz, 1H), 5.12 (d, J¼48.5 Hz,1H), 4.66
(m,1H), 4.35 (d, J¼14.5 Hz,1H), 4.04 (m,1H), 3.88 (s, 3H), 3.78 (s, 3H),
3.31 (m,1H), 3.19 (m,1H), 2.47 (s, 3H). 13C NMR (125.8 MHz) (CD3CN)
(500 MHz) (CD3OD) d: 8.79 (br 0.25H), 8.56 (br 0.75H), 6.22 (dd,
J¼16.5 and 3.5 Hz, 0.25H), 6.05 (d, J¼14.5 Hz, 0.75H), 5.05e5.16 (m,
1H), 4.31e4.50 (m, 1H), 3.66e4.03 (m, 6H). HRMS calculated for
C11H14FN3O6, [MHþ] 304.0945 (calcd), 304.0939 (found).
d: 167.5, 162.0, 156.6, 155.4, 150.0, 146.4, 146.3, 141.6, 132.5, 132.4,
126.6, 124.6, 124.5, 123.6, 109.8, 96.3, 94.8, 92.8, 89.2, 88.9, 85.7, 82.9,
71.0, 70.8, 60.0, 59.9, 51.6, 49.0, 22.7. MS calculated for C34H34FN3O9,
[MHþ] 648.2357 (calcd), 648.2352 (found).
4.1.6. 1-(2-Deoxy-2-fluoro-
cytosine (5a). Pure 5a was prepared from pure 4a by the same pro-
cedure as above. 1H NMR (500 MHz) (DMSO-d6)
: 8.62 (s, 1H), 8.13
(br; 1H), 7.73 (br; 1H), 6.11 (dd, J¼16.5 and 4.0 Hz, 1H), 5.91 (d,
J¼5.0 Hz), 5.18 (t, J¼5.5 Hz, 1H), 5.11 (dd, J¼4.0, 2.5 Hz, 0.5H), 5.1 (dd,
J¼4.0 and 3.0 Hz, 0.5H), 4.22 (m, 1H), 3.86 (m, 1H), 3.76 (s, 3H), 3.62
(m, 2H). 166.2, 164.4, 154.2, 150.1, 97.3, 96.2, 95.8, 85.9, 85.8, 74.4, 74.2,
b-D-arabinofuranosyl)-5-methoxycarbonyl-
4.1.10. N4-Acetyl-1-(2-deoxy-2-fluoro-5-O-DMTr-
b-D-arabinofur-
d
anosyl)-5-methoxycarbonylcytosine-3-O-(2-cyanoethyl)-N,N-diisio-
propyl phosphoramidite (8a). To a solution of N4-acetyl-1-(2-
deoxy-2-fluoro-5-O-DMTr-
onylcytosine (7a) (100 mg, 155
amine (108 L, 620 mol) in CH2Cl2 (4 mL) was added 2-cyanoethyl
b
-
D
-arabinofuranosyl)-5-methoxycarb-
m
mol) and N,N-diisoproylethyl
m
m

5150
Q. Dai et al. / Tetrahedron 68 (2012) 5145e5151
N,N-diisopropylchlorophosphoramidite (52
m
L, 234
m
mol,
4.3. hTDGcat Glycosylase activity test
1.5 equiv). After stirring at room temperature for 1 h, the solution
was diluted with CH2Cl2, washed with 5% aqueous NaHCO3, and
brine. The organic layer was dried over anhydrous Na2SO4, filtered,
and concentrated under reduced pressure. The residue was puri-
fied by silica gel column chromatography with 4e10% MeOH in
CH2Cl2 containing 0.2% Et3N to afford phosphoramidite 8a (112 mg,
The glycosylase reactions were performed with 10 nM 32P DNA
substrates, 100 nM hTDGcat at 37 ꢀC for 30 min in 25 mM HEPES
pH¼7.4, 0.5 mM EDTA, 0.5 mg/mL BSA, 0.5 mM DTT. Reactions were
quenched with 0.1 M NaOH and 0.01 M EDTA (v/v, 1:1), and in-
cubated at 100 ꢀC for 5 min. The mixture was subject to denaturing
Urea-PAGE with a 20% polyacrylamide gel. The gel was imaged and
quantified using Fuji screen.
85%) as a white foam. 1H NMR (500 MHz) (CD3CN)
d: 10.74 (br, 1H),
8.871 (s, 0.5H), 8.870 (s, 0.5H), 7.50 (m, 2H), 7.26e7.39 (m, 7H),
6.87e6.91 (m, 4H), 6.24 (d, J¼3.0 Hz, 0.5H), 6.20 (d, J¼3.5 Hz, 0.5H),
5.26e5.41 (m, 1H), 4.58e4.72 (m, 1H), 4.21e4.28 (m, 1H), 3.78 (s,
3H), 3.76 (s, 3H), 3.50e3.65 (m, 4H), 3.27 (s, 1.5H), 3.32 (s, 1.5H),
2.65 (t, J¼6.0 Hz, 1H), 2.52 (t, J¼6.0 Hz, 1H), 2.51 (s, 1.5H), 2.50 (s,
1.5H), 1.29 (m, 2H), 1.05e1.19 (m, 12H). 13C NMR (125.8 MHz)
4.4. Electrophoretic mobility shift test
4 nM 32P-Labeled DNA substrate and the enzyme were in-
cubated at room temperature for 30 min in 10 mM HEPES pH¼8.0,
(CD3CN) d: 167.6, 161.9, 156.5, 155.4, 141.6, 141.5, 132.44, 132.39,
1 mM EDTA, 50 mM KCl, 0.05% Triton 100, 5% glycerol, 10 mg/mL
132.3, 132.2, 126.65, 126.64, 126.62, 124.65, 124.57, 124.53, 123.61,
123.58, 109.77, 109.75, 109.73, 93.2, 92.12, 92.09, 91.8, 90.6, 90.3,
82.89, 82.86, 82.7, 82.6, 82.5, 82.3, 80.0, 79.8, 73.4, 73.3, 73.2, 73.0,
72.6, 72.4, 72.3, 72.2, 58.4, 58.1, 55.5, 55.43, 55.39, 55.3, 51.60,
51.58, 48.6, 48.5, 39.9, 39.85, 39.80, 39.76, 22.8, 20.58, 20.56, 20.52,
20.50, 20.46, 20.44, 20.40, 20.38, 18.45, 17.00, 16.62, 16.56, 16.50.
Salmon DNA. The mixture was subject to native PAGE with 8%
polyacrylamide gel. The gel was imaged and quantified using Fuji
screen.
Acknowledgements
31P NMR (202.4 MHz) (CD3CN)
C43H51FN5O10P, [MHþ] 848.3436 (calcd), 848.3430 (found).
d: 150.7, 150.2. HRMS calculated for
This study was supported by National Institutes of Health
(GM071440 to C.H.). We thank S. F. Reichard at the University of
Chicago for editing the manuscript.
4.1.11. N4-Acetyl-1-(2-deoxy-2-fluoro-5-O-DMTr-
a-D-arabinofur-
anosyl)-5-methoxycarbonylcytosine-3-O-(2-cyanoethyl)-N,N-diiso-
propyl phosphoramidite (8b). Using 7b as starting material,
phosphoramidite 8b was prepared in the similar way as 8a in 85%
References and notes
yield as white foam. 1H NMR (500 MHz) (CD3CN)
d: 10.74 (br, 0.5H),
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