NEIL1 Binding to DNA Containing 2'-Fluorothymidine Glycol Stereoisomers and the Effect of Editing

Article abstract of DOI:10.1002/cbic.201200139

Thymine glycol (Tg), one of the oxidized bases formed in DNA by reactive oxygen species, is repaired by the DNA glycosylases such as NEIL1, NTH1 and Endo III. In our recent studies, we showed that NEIL1's catalytic efficiency and lesion specificity are regulated by an RNA-editing adenosine deamination reaction. In this study, we synthesized oligodeoxynucleotides containing 2'-fluorothymidine glycol with either ribo or arabino configuration and investigated the binding of these modified DNAs with the unedited and edited forms of human NEIL1 along with E. coli Endo III. For the two forms of hNEIL1, binding affinities to FTg-containing DNA were similar indicating that the editing effect is more subtle than to simply alter substrate affinity. While the NEIL1-binding to FTg-containing DNAs was largely insensitive to C5 and 2' stereochemistry, a preference was observed for the FTg-G pair over the FTg-A pair. In addition, we found that optimal binding is observed with Endo III and duplex DNA with riboFTg(5S) paired with dG. The modified DNAs reported here will provide useful tools for further characterizing the interaction between DNA repair glycosylases and thymine glycol containing DNA.

Full text of DOI:10.1002/cbic.201200139

DOI: 10.1002/cbic.201200139
NEIL1 Binding to DNA Containing 2’-Fluorothymidine
Glycol Stereoisomers and the Effect of Editing
Kazumitsu Onizuka, Jongchan Yeo, Sheila S. David,* and Peter A. Beal*[a]
Thymine glycol (Tg), one of the oxidized bases formed in DNA
by reactive oxygen species, is repaired by the DNA glycosylas-
es such as NEIL1, NTH1 and Endo III. In our recent studies, we
showed that NEIL1’s catalytic efficiency and lesion specificity
are regulated by an RNA-editing adenosine deamination reac-
tion. In this study, we synthesized oligodeoxynucleotides con-
taining 2’-fluorothymidine glycol with either ribo or arabino
configuration and investigated the binding of these modified
DNAs with the unedited and edited forms of human NEIL1
along with E. coli Endo III. For the two forms of hNEIL1, binding
affinities to FTg-containing DNA were similar indicating that
the editing effect is more subtle than to simply alter substrate
affinity. While the NEIL1-binding to FTg-containing DNAs was
largely insensitive to C5 and 2’ stereochemistry, a preference
was observed for the FTg–G pair over the FTg–A pair. In addi-
tion, we found that optimal binding is observed with Endo III
and duplex DNA with riboFTg(5S) paired with dG. The modi-
fied DNAs reported here will provide useful tools for further
characterizing the interaction between DNA repair glycosylases
and thymine glycol containing DNA.
Introduction
Oxidized base lesions arise in DNA at rates of thousands per
day as a result of endogenous metabolic activity as well as
from oxidative stress, induced during inflammation, radiation
or toxic agents.[1,2] The human protein NEIL1 plays a key role in
the initiation of base excision repair of oxidized base lesions
by catalyzing the cleavage of the N-glycosidic linkage.[3] NEIL1
is capable of removing a wide array of modified DNA bases in-
cluding thymine glycol (Tg), 5-hydroxycytosine (5-OHC), 5-hy-
droxyuracil (5-OHU), dihydrothymine (DHT), dihydrouracil
(DHU), the formamidopyrimidines (FapyG and FapyA), guanidi-
nohydantoin (Gh), spiroiminodihydantoin (Sp) and psoralen
photoadducts.[3–6] Our recent studies have shown that NEIL1’s
catalytic efficiency and lesion specificity are regulated by an
RNA-editing adenosine deamination reaction that occurs at
a specific codon in the NEIL1 pre-mRNA.[7] Deamination at C6
of adenosine (A) in RNA generates inosine (I) at the corre-
sponding nucleotide position. Since inosine is decoded as gua-
nosine during translation, this modification can lead to codon
changes (recoding) and the introduction of amino acids into
a gene product not encoded in the gene. In the case of NEIL1,
deamination of adenosine within the AAA codon for K242 con-
verts it to one for arginine (AIA, read as AGA). This reaction is
catalyzed by the RNA-editing enzyme ADAR1 that is induced
by interferon and known to play a role in inflammation. Impor-
tantly, we observed large differences in glycosylase activity be-
tween K242 and R242 NEIL1 with DNA substrates containing
Tg. Indeed, the unedited form of NEIL1 showed substantially
enhanced glycosylase activity over edited NEIL1 with the Tg
lesion (41-fold faster in duplex DNA when paired with A). Tg is
the most common pyrimidine base modification produced
under oxidative stress and ionizing radiation.[2,8] Tg is also
a substrate of the human DNA glycosylase NTH1 and, though
not miscoding, its ability to strongly block DNA replication
makes it a toxic lesion in cells. The bacterial protein Endo III is
also capable of excising Tg from DNA.[9] An approach that has
been exploited by several laboratories, including ours, in study-
ing BER glycosylases is the use of a 2’-F substitution on the
damaged nucleotide in order to prevent (or stall) glycosidic
bond hydrolysis.[10–14] Oligonucleotides containing 2’-FTg nucle-
otides will serve as useful tools for revealing the molecular
details associated with damage recognition by NEIL1 and the
effects of editing. Here we describe the synthesis of 2’-fluoro-
thymidine glycol phosphoramidites with either ribo or arabino
configuration at the 2’ position, their use in the generation of
2’-fluorothymidine glycol-containing DNAs and the binding of
these modified DNAs with the unedited and edited forms of
human NEIL1 and Escherichia coli Endo III.
Results
Synthesis of 2’-fluorothymidine glycol phosphoramidite
building blocks
The 2’-fluoronucleoside modification is well known to slow the
cleavage reaction of N-glycosylases.[10–14] These reactions occur
with an intermediate 1’–4’ oxocarbenium ion and an electro-
negative fluorine atom at the adjacent 2’ position destabilizes
the transition state to that intermediate.[15] Indeed, the 2’-ribo-
fluorothymidine glycol 5R and 5S isomers have been incorpo-
[a] Dr. K. Onizuka, J. Yeo, Prof. Dr. S. S. David, Prof. Dr. P. A. Beal
Department of Chemistry, University of California, Davis
One Shields Avenue, Davis, CA 95616 (USA)
E-mail: david@chem.ucdavis.edu
beal@chem.ucdavis.edu
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201200139.
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NEIL1 Binding to DNA
rated into DNA and were shown to be resistant to the glycosy-
lase activity of E. coli Endo III.[14] In addition, only with the 5S-
isomer-containing duplexes was a DNA–protein complex ob-
served in electrophoretic mobility shift assays (EMSAs). Impor-
tantly, there have been no prior reports of the binding proper-
ties of NEIL1 with fluorinated damaged nucleotide-containing
DNA.
tively strong NOE was observed between H-6 and H-2’, where-
as the minor one showed only a slight effect (Figure S1). From
this result, the thymine glycols in the major and minor prod-
ucts were assigned as 5S,6R (2b) and 5R,6S (2a), respectively.
We then protected both hydroxyl groups of the thymine glycol
with acetyl groups.[20,21] The resulting compounds (3a and 3b)
were treated with trifluoroacetic acid to give the 5,6-O-acetyl-
2’-ribofluorothymidine glycols 4a and 4b which were convert-
ed to the phosphoramidite building blocks 6a and 6b by stan-
dard procedures.
We synthesized 2’-fluorothymidine glycol of both ribo and
arabino configuration (Schemes 1 and 2). While the ribo-con-
figured compound was known, the reported phosphoramidite
bears tert-butyldimethylsilyl (TBDMS) protecting groups on the
glycol hydroxyl groups necessitating an additional deprotec-
tion step for the modified DNA.[14] We wished to investigate
the use of acetyl groups for this protection since they could be
removed by K2CO3/MeOH treatment, a standard mild step in
synthetic DNA deprotection. We also chose to prepare the ara-
bino-configured isomer since this isomer is expected to adopt
a sugar conformation more compatible with a B-form duplex
and may more effectively mimic the natural DNA lesion.[16,17]
The synthesis of 2’-ribofluorothymidine glycol (riboFTg) phos-
phoramidite 6 started with commercially available 5-methyluri-
dine. Trityl-protected 2’-ribofluorothymidine 1 was synthesized
according to the literature (Scheme S1),[18] and then oxidized
with OsO4 to afford two isomers of thymidine glycol 2
(Scheme 1).[19] The ratio of isolation yields for these products
was 3:5. The configuration of each isomer was determined
For the synthesis of 2’-arabinofluorothymidine glycol
(araFTg) phosphoramidite, we prepared benzoyl-protected 2’-
arabinofluorothymidine 7 according to a literature report
(Scheme 2).[22] The benzoyl groups were removed by NH4OH
treatment and the hydroxyls were protected with trityl groups.
An osmium tetroxide oxidation reaction was then performed
to give two diastereomers in a ratio of 10:1, calculated from
NMR integration values. Since it was difficult to isolate the
minor product by normal column chromatography on silica
gel, only the major product was protected with acetyl groups
and carried forward. The phosphoramidite building block of
acetyl-protected araFTg was synthesized by the same proce-
dures as described above for riboFTg. However, unlike the case
for riboFTg, the use of acetyl protecting groups for araFTg pro-
moted an unwanted side reaction during the deprotection
step leading to an oligonucleotide product with a mass differ-
ent from that expected (see below). Therefore, we synthesized
TBDMS-protected araFTg as an alternative. The mixture of dia-
stereomers for thymidine glycol
1
from the H NMR NOE data on the basis of a previous report.[14]
For the major isomer obtained by the OsO4 oxidation, a rela-
9 was treated with TBDMSCl.
The resulting compound 10 was
allowed to react with trifluoro-
acetic acid to give the 5,6-O-
TBDMS-2’-arabinofluorothymi-
dine glycol 11. The two isomers
were separated in this step by
silica gel column chromatogra-
phy, and the configuration of
each isomer was determined
from 1H NMR NOE data. The
bulky TBDMS groups are expect-
ed to fix the glycosyl torsion
angle in the syn conformation.
For the minor isomer, a strong
NOE was observed between H-6
and H-1’, whereas the major
isomer did not show the effect
(Figure S2). For the 5S,6R isomer,
the NOE between H-6 and H-1’
would not be expected because
the equatorial H-6 is unfavorable
due to the hindrance between
the C6-axial O-TBDMS group and
the 2’-fluorine. Therefore, the
thymine glycols in the major and
Scheme 1. a) see Scheme S1; b) OsO4, pyridine, 31% (5R,6S) and 52% (5S,6R); c) Ac2O, DMAP, pyridine, 3a: 97%,
3b: 97%; d) TFA, CH2Cl2, 4a: 92%, 4b: 92%; e) DMTrCl, pyridine, 5a: 83%, 5b: 88%; f) amidite reagent, DIPEA,
CH2Cl2, 6a: 86%, 6b: 81%.
minor products were assigned as
5S,6R (11 a) and 5R,6S (11 b),
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S. S. David, P. A. Beal et al.
at the C6 position, a reaction
that has precedence for thymi-
dine glycol lacking the 2’-F
modification (Scheme 3).[23]
The ODN containing acetyl-
protected araFTg was also
cleaved and deprotected by
treatment with K2CO3/MeOH, but
the mass value of the major
product was 14 amu higher than
the desired compound. How-
ever, this side reaction did not
occur with the ODN containing
TBDMS-protected araFTg. This
ODN was cleaved and deprotect-
ed by a treatment with 30% aq.
ammonia and the TBDMS was
removed with triethylamine tri-
hydrofluoride.[14] HPLC analysis
on an ion-exchange column
gave a single major peak. Epime-
rization was not observed for
this ODN under these condi-
tions.
Scheme 2. a) i: NH4OH, EtOH; ii: TrCl, pyridine, 1008C, 70% (2 steps); b) OsO4, pyridine, 92% (5S,6R/5R,6S=10:1);
c) TBDMSCl, imidazole, DMF, 378C, 88%; d) TFA, CH2Cl2, 08C, 72%; e) DMTrCl, pyridine, 80%; f) amidite reagent,
DIPEA, CH2Cl2, 80%.
Estimation of the ratio of cis/trans epimers
For the thymidine glycol lacking the sugar modification, it has
been reported that the cis isomer is preferred to the trans
isomer. The ratio of cis/trans was 80:20 for 5S and 87:13 for
5R.[23] Also, the ratio of cis/trans was 70:30 for the 5R thymidine
glycol in a duplex containing the Tg–A base pair.[24] We investi-
gated the effect on cis!trans epimerization by the introduc-
tion of fluorine at the 2’ position. The monomers of ribo and
araFTg 5S isomers were synthesized from 2b or 9 by treatment
with trifluoroacetic acid. After heating at 908C for 5 min and
gradual cooling to room temperature, the monomers were an-
alyzed by NMR (Figure S4). For the 5S riboFTg monomer, the
ratio of cis/trans was 75:25. For the 5S araFTg monomer, the
ratio was 90:10. Thus, the percentage of trans isomer of ri-
respectively. Phosphoramidite building block 13 was then syn-
thesized with 11 a by normal dimethoxytrityl (DMTr) protection
and phosphitylation.
Synthesis of oligodeoxynucleotides containing FTg
With these three building blocks, 30-mer oligodeoxynucleo-
tides (ODNs) were synthesized using automated solid-phase
DNA synthesis. The reaction time for the coupling of FTg was
extended to 15 min. After the synthesis and removal of the 5’-
terminal DMTr group on the synthesizer, the ODNs containing
riboFTg were cleaved from the solid support and deprotected
by treatment with K2CO3/MeOH.
The solvent was removed and
the crude material was purified
by ion-exchange HPLC. Treat-
ment with 30% aq. ammonia in-
stead of K2CO3/MeOH gave some
decomposition. A single major
peak and
a significant minor
peak (>10% of major peak)
were observed in the HPLC
traces for the crude oligonucleo-
tides (Figure S3). Since the
masses observed by MALDI-TOF
MS were the same, the two
peaks were assigned as cis and
trans FTg isomers. The trans
isomer arises from epimerization Scheme 3. Epimerization reaction of thymidine glycol.
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NEIL1 Binding to DNA
Figure 1. HPLC trace of before and after 908C, 5 min treatment. The oligonu-
cleotides containing A) riboFTg(5R), B) riboFTg(5S) or C) araFTg(5S) were ana-
lyzed by HPLC using a Dionex DNAPac PA-100 ion exchange column. The
analysis was achieved with buffer solutions consisting of 30% solvent B
(10% acetonitrile, 90% NH4OAc (1.5m, pH 7)) and 70% solvent A (10% ace-
tonitrile, 90% H2O) initially, with a gradient to 100% solvent B for 35 min.
boFTg increased slightly and that of araFTg decreased com-
pared to the ratio of the unmodified thymidine glycol mono-
mer. The ratio of cis/trans isomers for the FTg-containing ODNs
was measured by HPLC analysis. The major ODN products con-
taining 5R-riboFTg, 5S-riboFTg and 5S-araFTg were purified by
HPLC to >95% purity (Figure 1). After heating at 908C for
5 min and gradual cooling to room temperature, the ODNs
were analyzed by HPLC again (Figure 1). For 5R-riboFTg, the
percentage of the minor isomer after this treatment was 11%.
For 5S-riboFTg, the percentage of minor isomer was 32% and
that of 5S-araFTg was 7%. Since the ratios of major/minor
products for the 5S-FTg-containing ODNs were similar to that
observed by NMR for the monomers (e.g., ribo: 25 vs 32%, ara-
bino: 7 vs 11%), the minor ODN products appear to be the
trans isomers.
N-Glycosylase assays
We evaluated the ability of the 2’-F modification of Tg to block
the glycosylase activity of hNEIL1 and E. coli Endo III using
a 30 bp duplex. After 32P labeling, ODNs containing either the
5R or 5S isomers of riboFTg or the 5S isomer of araFTg were
separately hybridized to the complementary strand. Tg would
be expected to be found in base pairing context with A but
may also be found in Tg–G base pairs; the latter base pair
results from oxidation of 5-methylcytosine paired with G fol-
lowed by hydrolytic deamination of the unstable 5-methylcyto-
sine glycol.[25,26] Thus we evaluated the effect of having either
A or G opposite the FTg in the duplex substrates. For compari-
son, a Tg-containing duplex was prepared and analyzed in
side-by-side reactions. To ensure observance of strand scission
at all abasic sites produced by the glycosylase reaction, the re-
actions were quenched with NaOH and heated at 908C for
5 min, rather than relying on the lyase activity of enzymes to
provide strand scission. With the duplex containing Tg, edited
and unedited hNEIL1 and Endo III mediated strand cleavage
(Figure 2). On the other hand, the glycosylase reactions with
the FTg-containing duplexes were all inhibited and not influ-
enced by the opposite base context (Figure S5). For the duplex
Figure 2. Storage phosphor autoradiogram of N-glycosylase assays with du-
plexs containing Tg(5R)/A, riboFTg(5R)/A, riboFTg(5S)/A and araFTg(5S)/A.
Aliquots of DNA (20 nm) and enzyme (200 nm) were incubated at 378C for
various times (0, 1, 30 and 60 min): A) unedited NEIL1, B) edited NEIL1, C) En-
do III.
bearing araFTg (5S), a trace amount of product was observed
at longer reaction times with Endo III. These results indicated
that hNEIL1 and Endo III are unable to catalyze efficient thy-
mine glycol base removal from the riboFTg- and araFTg-con-
taining DNA duplexes. In addition, these results are consistent
with a previous reported showing the resistance of riboFTg
with DNA to base excision mediated by Endo III.[14]
Binding affinities for NEIL1 and Endo III with DNA duplexes
Electrophoretic mobility shift assays (EMSAs) were used to
measure the relevant dissociation constants of the binding of
the unedited and edited forms of human NEIL1 and E. coli En-
do III to FTg-containing duplexes. These experiments involved
titration of the enzyme into a solution 5’-32P-end-labeled DNA
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S. S. David, P. A. Beal et al.
Table 1. Dissociation constants [Kd (nm)] of unedited NEIL1 and edited
NEIL1 for 30 bp DNA duplexes.[a]
Central base pair
Unedited NEIL1
Edited NEIL1
riboFTg(5R)–A
riboFTg(5S)–A
araFTg(5S)–A
T–A
riboFTg(5R)–G
riboFTg(5S)–G
araFTg(5S)–G
T–G
12ꢀ3
15ꢀ4
22ꢀ6
37ꢀ8
40ꢀ7
1.5ꢀ0.2
3ꢀ1
18ꢀ1
21ꢀ3
42ꢀ2
1.4ꢀ0.2
2.4ꢀ0.3
2.4ꢀ0.4
7ꢀ2
2.9ꢀ0.7
7ꢀ3
[a] All measurements were performed at 258C with 10 pM DNA and vari-
ous amounts of enzyme. Errors reported in dissociation constants are
standard deviation of the average of at least three trials. The concentra-
tions of enzyme are active enzyme concentrations.
Both the edited and unedited forms of NEIL1 bind the FTg-
containing DNA duplexes more tightly than DNA lacking any
lesion (Table 1). The magnitude of this difference depends pri-
marily on the identity of the opposite base with the largest dif-
ference observed between the riboFTg(5R)–G duplex com-
pared to the T–G duplex (5.1-fold). A clear preference is ob-
served for NEIL1 binding to FTg–G DNA compared to FTg–A
DNA. This preference mirrors the enhanced rate of cleavage of
Tg in duplexes paired with G compared to those with Tg–A
pairs.[7,27] Surprisingly, NEIL1 binding affinity to the FTg duplex-
es is relatively insensitive to glycol or sugar stereochemistry.
Binding with FTg(5R) was only slightly tighter than that with
FTg(5S), with the ratio again corresponding with that observed
for the glycosylase activity (5R-Tg/5S-Tg cleavage rate=1.5).[28]
Importantly, we observed little difference in binding affinity to
these DNAs between edited and unedited NEIL1 (Table 1). This
stands in stark contrast to the effect editing has on NEIL1
kinetics where the unedited form (K242) showed a 41-fold
higher single turnover rate constant for Tg cleavage when it
was paired with A and 30-fold higher when paired with G.[7]
Moreover, by measuring the single turnover rate constant as
a function of enzyme concentration, we were able to deter-
mine the apparent dissociation constant (Kd) for natural
Tg(5R)–G DNA with edited NEIL1 (Figure S6). The Kd value mea-
sured by this technique (2.7 nm) was similar to Kd value mea-
sured by EMSA for FTg(5R)–G DNA binding to edited NEIL1
(Kd =1.5 nm). These results indicate that 2’-fluorination of the
Tg lesion does not substantially perturb binding affinity for
edited NEIL1.
Figure 3. A) Binding of unedited NEIL1 to duplexes containing araFTg(5S)/G.
&
*
B) Plot of percent of DNA-NEIL1 complex ( : araFTg(5S)/A-unedited NEIL1,
:
!
~
araFTg(5S)/A-edited NEIL1, : araFTg(5S)/G-unedited NEIL1 : araFTg(5S)/G-
edited NEIL1). Aliquots of DNA (10 pm) and NEIL1 (0.15–600 nm) were incu-
bated at 258C for 30 min.
Figure 4. Binding of Endo III to duplexes containing A) riboFTg(5R)/G, B) ri-
boFTg(5S)/G, C) T/G, D) araFTg(5S)/G. Aliquots of DNA (10 pm) and Endo III
(0.0011–150 nm) were incubated at 258C for 30 min. Bracket indicates
lesion-independent complexes, star indicates lesion-dependent complex.
A similar EMSA series was performed using Endo III to pro-
vide for a comparison to NEIL1. Of note, distinctly different
preferences for removal of the two Tg diastereomers (5R-Tg/
5S-Tg) for NEIL1 (1.5) and Endo III (0.4) have been reported.[28]
Consistent with the diastereoselectivity in the cleavage reac-
tion, Endo III had a very high affinity for FTg (5S) in duplex and
much lower affinity for the FTg(5R) duplex. This observation is
similar to that previously reported for Endo III binding to FTg-
containing DNA.[14] Multiple complex bands were observed
(Figure 4), and the binding titration curves were biphasic and
best fit with a two-site binding isotherm yielding two distinct
duplex and analysis on native PAGE to separate and detect
both the free DNA duplex and protein–DNA complex (Figure 3,
Figure 4). The same 30 bp DNA duplexes containing either the
FTg–A or the FTg–G base pair that were analyzed for the gly-
cosylase activity were used in these experiments. In addition,
DNA duplexes containing either a T–A or a T–G base pair at
the position corresponding to the FTg–X base pair were ana-
lyzed to ascertain the lesion-independent DNA binding affini-
ties for NEIL1 and Endo III with this duplex sequence under
these conditions.
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NEIL1 Binding to DNA
Discussion
Table 2. Dissociation constants [Kd (nm)] of Endo III for 30 bp DNA du-
plexes.[a]
In this study we sought to generate reagents that would be
useful in defining further the lesion recognition process for
DNA repair enzymes capable of removing Tg from DNA with
a particular emphasis on human NEIL1. We had previously
shown that the N-glycosylase reaction of hNEIL1 with DNA
containing Tg is regulated by an RNA editing event that
changes the identity of an amino acid residue in the lesion-rec-
ognition loop of the protein (K/R242). However, the basis for
the change in enzymatic activity arising from the conservative
arginine-for-lysine replacement is not currently known. Intro-
duction of a 2’-F group into the sugar of the lesion has proven
useful for trapping DNA repair glycosylases in their corre-
sponding lesion-recognition complexes.[10–14] Thus, we envi-
sioned trapped FTg DNA complexes of NEIL1, in the edited
and unedited forms, to be useful for defining differences that
result in the altered glycosylase rate constants observed. Such
information would serve to extend our understanding of how
DNA repair glycosylases are regulated and the impact of RNA
editing on the proteome.
Central base pair
Endo III
riboFTg(5R)–A
riboFTg(5S)–A
araFTg(5S)–A
T–A
riboFTg(5R)–G
riboFTg(5S)–G
araFTg(5S)–G
T–G
7ꢀ1
<0.01 (51ꢀ3%), 4.7ꢀ1 (47ꢀ2%)[b]
6ꢀ1 (>90%)[c]
10ꢀ2
7.4ꢀ0.9
<0.01 (73ꢀ2%), 4.7ꢀ0.1 (27ꢀ1%)[b]
~0.01 (38ꢀ1%), 10ꢀ2 (61ꢀ3%)[b]
8.3ꢀ0.9
[a] All measurements were performed at 258C with 10 pm DNA and vari-
ous amounts of enzyme. Errors reported in dissociation constants are
standard deviation of the average of at least three trials. The concentra-
tions of enzyme are active enzyme concentrations. [b] The data fit best
using a two-site binding isotherm that provides two Kd values with rela-
tive capacities indicated in closed parentheses. [c] DNA–enzyme complex
of less than 10% with tighter Kd was observed.
Kd values (Table 2). Given the presence of the slower moving,
low affinity complexes in the titration with the T–G and T–A
control DNAs, these most likely arise from the lesion-independ-
ent binding of Endo III to this duplex and involve more than
one equivalent of protein. The dissociation constants for
lesion-independent binding of Endo III under these conditions
were in the 8–10 nm range (Table 2). We noted that the frac-
tion of DNA bound in the lesion-specific complex differed
among the duplexes tested. This fraction depended on the 2’
configuration and opposite base with the riboFTg(5S)–G
duplex exhibiting the largest fraction (73ꢀ2%) with extremely
tight binding (Kd <10 pm) (Table 2). On the other hand, the
binding with 5R-isomer was similar to that observed with
lesion-free DNA (Kd =7.4 nm). As a result, a remarkable >700-
fold diastereoselectivity was observed in the binding of 5S/5R
isomers. A high affinity complex was also observed with the
araFTg(5S)–G duplex indicating that Endo III is less sensitive to
the sugar 2’ configuration in the FTg analogue paired with G.
However, with the duplex containing the araFTg(5S)–A pair,
<10% of the DNA bound in the lesion-specific complex
whereas the riboFTg(5S)–A duplex showed 51ꢀ3% binding in
the complex with high affinity (Table 2, Figure S7). Therefore,
Endo III appears to prefer the 2’-F in the ribo configuration, at
least when the fluorinated lesion is paired with A. We also
noted the difference in the fraction bound in the lesion-specif-
ic complex for the riboFTg(5S)–G duplex (73ꢀ2%) versus the
riboFTg(5S)–A duplex (51ꢀ3%). While the apparent dissocia-
tion constants for these two complexes are <10 pm and too
low to accurately measure under our assay conditions, the dif-
ference in fraction bound in the lesion-specific complex is
noteworthy. This indicates a higher affinity for the FTg ana-
logue in a pair with G than A, since the lesion-specific complex
with the G-containing duplex is saturated to a greater extent
at Endo III concentrations where nonspecific binding is low
and does not interfere with its quantification. This difference is
even larger for duplexes containing araFTg(5S), FTg–G (38ꢀ
1%); FTg–A (<10%) (Table 2, Figure S7).
Given the different stereoisomers of Tg known to exist and
the two possible configurations at the 2’ position when fluo-
rine is substituted for hydrogen in 2’-deoxyribose (ribo vs ara-
bino), it was important to evaluate the different stereoisomers
of FTg for their effect on repair protein recognition. This had
been addressed qualitatively in a previous literature report of
E. coli Endo III, where binding studies with DNA containing
either 5R or 5S riboFTg showed the 5S isomer is preferred.[14]
This stereochemical preference could not be assumed for
NEIL1, particularly given the known preference for 5R Tg over
5S Tg in the NEIL1 glycosylase reaction.[7,26] In addition, no
guiding information was available for the preferred configura-
tion for the 2’-F, since the araFTg had not been incorporated
into DNA prior to this study.
In the synthesis of ribo-FTg phosphoramidite building
blocks, the OsO4 oxidation of trityl-protected riboF-thymidine
afforded two isomers of thymidine glycol 2 in a ratio of 3:5.
The major isomer was (5S,6R)-thymine glycol. Interestingly,
when 5’-DMTr-3’-Bz-protected riboF-thymidine was used for
this oxidation, the major isomer was (5R,6S)-thymine glycol,
and the ratio was 3:1.[14] This stereoselectivity is most likely
a function of the 3’ protecting group. Since trityl is bulkier
than benzoyl, the approach of the OsO4 molecule from the
pro-5R,6S face would be more effectively blocked. Also, the
ratio was 1:9 when trityl-protected araF-thymidine was used.
This change in stereoselectivity could be attributed to changes
in the sugar pucker induced by the fluorine substitution and
possibly to the hindrance of the fluorine itself.
Thymidine glycol exists as two diastereomeric pairs of epi-
mers, the 5R cis,trans pair (5R,6S; 5R,6R) and the 5S cis,trans
pair (5S,6R; 5S,6S).[23] Although our synthesis generated only cis
(5S,6R)-riboFTg, (5R,6S)-riboFTg and (5S,6R)-araFTg isomers, we
observed a small amount of product in the crude DNA samples
that we assigned to the trans isomer in each case based on
their masses, the known epimerization reaction and the ratio
of epimers determined by NMR for the corresponding fluori-
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1343

S. S. David, P. A. Beal et al.
nated mononucleosides. For the riboFTg(5S)-containing ODN,
heating the purified sample to 908C and cooling to room tem-
perature lead to the formation of this putative trans product in
32% yield (Figure 1). However, araFTg(5S)-containing DNA was
more resistant to the epimerization reaction (7%). This may
prove to be an advantage of using the arabino isomer in appli-
cations that require stability under a variety of conditions or
over long periods of time, such as during crystallization trials.
However, the TBDMS protecting group must be used for the
glycol hydroxyls with the araFTg isomer, since the use of acetyl
protecting groups promoted an unwanted side reaction
during the DNA deprotection with K2CO3/MeOH.
We also investigated the effect of the opposite base on
NEIL1 binding to FTg DNAs. NEIL1 binding to DNAs containing
the FTg–G base pair was tighter than that with the FTg–A base
pair on the whole (~8-fold). The NEIL1 binding affinity with
DNA containing a simple T–G mismatch also was higher than
with a T–A base pair (5.8-fold). It has been reported that the T
base of a T–G mismatch pair is removed by excess NEIL1,[30]
explaining why NEIL1 might bind the mismatch more tightly.
Also, Mv Nei1 makes two H-bonds to the Hoogsteen face of
the estranged G in its complex with DNA containing a 5-hy-
droxyU–G pair.[29] A similar interaction with the estranged G
with human NEIL1 could explain the observed binding prefer-
ence for FTg–G compared to FTg–A.
Interestingly, NEIL1 binding to the fluorinated DNAs appears
to be largely insensitive to the stereochemical differences eval-
uated in this study. For instance, the dissociation constants for
unedited NEIL1 binding to DNA containing riboFTg(5R), ribo-
FTg(5S) or araFTg(5S) in a base pair with A are all within a
factor of two (Kd =12, 18 and 21 nm, respectively; Table 1). The
similarity is also seen when these fluorinated lesions are paired
with G (Kd =1.4, 2.4 and 2.4 nm, respectively; Table 1). This is in
contrast to the clear preference Endo III shows for the 5S con-
figuration, both in this work and in a previously published
qualitative study.[14] Here, we measured a difference in affinity
of Endo III for DNA containing riboFTg(5S) and riboFTg(5R)
when paired with G of >700-fold (Table 2). The origin of the
different stereoselectivity for these two repair glycosylases is
unknown at this time since neither a structure of Endo III
bound to Tg-containing DNA nor a structure of hNEIL bound
to Tg-containing DNA have been reported. However, a catalyti-
cally inactive mutant of the NEIL1 orthologue from mimivirus
(Mv Nei1) in complex with Tg-containing DNA has recently
Interestingly, the minimal interaction between Mv Nei and
the Tg base led the authors of that study to suggest that
lesion recognition primarily occurs prior to extrusion from the
helix (i.e., base flipping) and occupation of the active site.[29]
Our fluorinated Tg phosphoramidites should prove useful in
probing the base-flipping step for the NEIL1 reaction, such as
in the manner previously described by Stivers and colleagues
for uracil DNA glycosylase (UDG).[31] They evaluated changes in
2-aminopurine (2-AP) fluorescence induced by UDG in duplex-
es with 2’-fluorouridine adjacent to the 2-AP. The fluorine
modification stalls the UDG reaction at the glycosylase step
such that the base extruded intermediate can be studied in
the absence of further processing. A similar study with FTg/2-
AP-containing DNA and NEIL1 should shed additional light on
the base-flipping step for NEIL and the role of active site
amino acids, including K/R 242, on this step.
Whereas NEIL1 binding affinity appeared to be largely un-
affected by changes in C5 and 2’ stereochemistry, Endo III
binding to FTg-containing DNA was more sensitive to these
changes. Our results confirm that of an earlier study showing
Endo III’s pronounced preference for binding to the 5S
isomer.[14] In addition, we observed a binding preference for
the riboFTg (5S) analogue compared to the araFTg (5S) ana-
logue when paired with A. Endo III also appears to bind more
tightly to DNA bearing the FTg lesion paired with G than
paired with A (73ꢀ2% lesion specific complex observed
versus 51ꢀ3%; Table 2, Figure S7). Together these studies indi-
cate that optimal binding is observed with Endo III and duplex
DNA with the riboFTg (5S) paired with dG. Attempts to crystal-
lize Endo III with FTg-containing DNAs should benefit from
focusing on this combination.
been structurally characterized.[29] In this case, only
a
single direct H-bonding contact was observed between the
protein and the thymine glycol base (Y221 backbone NH to Tg
O4). The lack of extensive interaction with the Tg base might
explain the insensitivity to the C5 configuration for the related
hNEIL1 observed here.
NEIL1 binding affinity for the FTg-containing DNAs was also
essentially independent of editing. In all the cases tested, un-
edited (K242) and edited (R242) NEIL1 bound the different
target DNAs with dissociation constants that differed by less
than a factor of two. For instance, the DNA duplex with ribo-
FTg(5R) paired with a bound unedited NEIL1 with Kd =12 nm
and edited NEIL1 with Kd =15 nm. This result was surprising
given that Tg is cleaved from these DNAs much more efficient-
ly by unedited NEIL1.[7] The results indicate the editing reaction
generates NEIL1 isoforms with very similar affinities for lesion-
containing DNA and the editing effect is more subtle than to
simply alter substrate affinity. It might be the case that the
change in the lesion recognition loop structure arising from
editing serves to reorient the reactive nucleotide in the active
site in a manner that effects the chemical steps of the glycosy-
lase reaction but that does not change the overall substrate
binding affinity. Further studies will be necessary to test this
idea. The F-Tg phosphoramidites described here will be helpful
in this regard to generate F-Tg DNAs for crystallography with
unedited and edited hNEIL1.
Interestingly, two types of binding interactions were ob-
served with Endo III and DNAs containing either riboFTg (5S)
or araFTg (5S). At low concentrations of Endo III, lesion-specific,
high affinity complexes are observed with dissociation con-
stants estimated in the low-pm range. At higher protein con-
centrations, slower moving bands are apparent in the gel. The
slower mobility of these complexes suggest they are made up
of more than one equivalent of Endo III, consistent with behav-
ior that we have observed previously with the related glycosy-
lase MutY.[32] Since similar complexes are observed at the
higher protein concentrations and DNA lacking any lesion, this
type of complex corresponds to nonspecific or lesion-inde-
pendent binding. For DNA containing weak Endo III ligands
1344
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ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2012, 13, 1338 – 1348

NEIL1 Binding to DNA
(ddd, J=53, 6.0, 5.4 Hz, 1H), 4.13–4.10 (m, 1H), 3.50–3.48 (m, 1H),
3.31 (d, J=11 Hz, 1H), 3.28 (s, 1H), 3.19 (s, 1H), 2.82 (dd, J=11,
3.0 Hz, 1H), 1.41 ppm (s, 3H); 13C NMR (CDCl3, 150 MHz): d=172.6,
151.1, 143.7, 143.1, 128.8, 128.7, 128.1, 127.5, 127.4, 89.3 (d, J=
196 Hz), 88.2, 87.7, 86.3 (d, J=32 Hz), 81.5, 80.2, 72.2 (d, J=14 Hz),
72.0, 62.6, 22.0 ppm; ESI-HRMS (m/z): calcd for C48H43FN2O7:
801.2946 [M+Na]+, obs: 801.2951.
(e.g., riboFTg (5R) or T), only the nonspecific binding is ob-
served (Figure 4).
Conclusions
We have synthesized 2’-ribo and 2’-arabinofluorothymidine-
glycol-containing DNAs and evaluated their interaction with
the DNA repair glycosylases NEIL1 and Endo III. As we expect-
ed, the 2’-fluorinated analogues were resistant to cleavage by
both NEIL1 and Endo III. For the unedited and edited forms of
hNEIL1, binding affinities to FTg-containing DNA were similar.
These results were surprising given the significant difference in
glycosylase activity the two forms of NEIL1 demonstrate with
DNA containing the unmodified Tg lesion, indicating the edit-
ing effect is more subtle than to simply alter substrate affinity.
Also, while the NEIL1 binding to FTg-containing DNAs was
largely insensitive to C5 and 2’ stereochemistry, a preference
was observed for the FTg–G pair over FTg–A. Furthermore, we
found that optimal binding is observed with Endo III and
duplex DNA with riboFTg(5S) paired with G. The modified
DNAs reported here will provide useful tools for further charac-
terizing the interaction between DNA repair glycosylases and
thymine-glycol-containing DNA.
3’,5’-Di-O-trityl-(5R,6S)-5,6-di-O-acetyl-2’-ribofluorothymidine
(3a): Compound 2 (282 mg, 0.362 mmol) was co-evaporated with
anhydrous pyridine, and the dried residue was redissolved in anhy-
drous pyridine. Acetic anhydride (0.685 mL, 7.24 mmol) and 4-di-
methylaminopyridine (8.8 mg, 0.072 mmol) were added into the
solution under an argon atmosphere, and the mixture was stirred
at RT for 16 h. The reaction mixture was dried under reduced pres-
sure, and the dried residue was redissolved in EtOAc (100 mL). The
organic phase was washed with water (2ꢁ100 mL) and brine
(100 mL), dried over Na2SO4, and the solvent was removed under
reduced pressure. The residue was purified by silica gel column
chromatography with CH2Cl2/EtOAc (29:1!19:1) to give 3a
(302 mg, 97%) as a white foam. 1H NMR (CDCl3, 600 MHz, TMS):
d=7.36 (d, J=7.2 Hz, 6H), 7.33 (d, J=7.2 Hz, 6H), 7.27–7.13 (m,
18H), 6.64 (s, 1H), 5.56 (dd, J=22, 2.4 Hz, 1H), 4.17 (ddd, J=15,
7.2, 5.4 Hz, 1H), 4.07–4.05 (m, 1H), 4.01 (ddd, J=53, 5.4, 2.4 Hz,
1H), 3.06 (dd, J=10, 1.8 Hz, 1H), 2.87 (dd, J=10, 7.8 Hz, 1H), 2.07
(s, 3H), 1.90 (s, 3H), 1.76 ppm (s, 3H); 13C NMR (CDCl3, 150 MHz):
d=169.0, 168.5, 166.9, 149.6, 143.9, 143.7, 129.1, 128.8, 128.3,
128.2, 127.8, 127.7, 127.2, 126.9, 92.0 (d, J=41 Hz), 91.2 (d, J=
189 Hz), 87.4, 86.6, 81.6, 79.8, 71.8 (d, J=15 Hz), 64.0, 21.4,
20.6 ppm; ESI-HRMS (m/z): calcd for C52H47FN2O9 885.3158 [M+Na]+
, obs: 885.3168.
Experimental Section
All reagents were purchased from commercial sources (Sigma–Al-
drich or Fisher Scientific) and were used without further purifica-
tion unless otherwise stated. Reactions were carried out under an
atmosphere of dry argon. 1H, 13C, and 31P NMR spectra were record-
ed with Varian VNMRS600 or Varian Mercury 300 spectrometers.
High-resolution ESI mass spectra were obtained at the University
of California, Davis mass spectrometry facility, on an Orbitrap FTMS
instrument. Matrix-assisted laser desorption/ionization time-of-
flight (MALDI-TOF) mass spectra of oligonucleotides were mea-
sured in the positive ion mode, using 3-hydroxypicolinic acid or 6-
aza-2-thiothymine as a matrix.
3’,5’-Di-O-trityl-(5S,6R)-5,6-di-O-acetyl-2’-ribofluorothymidine
1
(3b): A white foam (285 mg, 97%). H NMR (CDCl3, 600 MHz, TMS):
d=7.38 (d, J=8.4 Hz, 6H), 7.34 (d, J=8.4 Hz, 6H), 7.29–7.15 (m,
18H), 6.41 (s, 1H), 5.65 (d, J=22 Hz, 1H), 4.04–4.00 (m, 2H), 3.78
(ddd, J=53, 3.0, 3.0 Hz, 1H), 3.34 (d, J=10 Hz, 1H), 2.84 (dd, J=10,
6.0 Hz, 1H), 2.08 (s, 3H), 1.84 (s, 3H), 1.77 ppm (s, 3H); 13C NMR
(CDCl3, 150 MHz): d=168.9, 168.8, 166.8, 149.2, 143.9, 143.7, 129.1,
128.8, 128.7, 127.8, 127.7, 127.2, 127.0, 91.7 (d, J=36 Hz), 91.3 (d,
J=189 Hz), 87.4, 87.0, 80.8, 79.8, 72.0 (d, J=15 Hz), 64.2, 21.4, 20.5,
20.1 ppm; ESI-HRMS (m/z): calcd for C52H47FN2O9 885.3158 [M+Na]+
, obs: 885.3163.
3’,5’-Di-O-trityl-5,6-dihydroxy-2’-ribofluorothymidine (2): Com-
pound 1 (1.47 g, 1.97 mmol) and OsO4 (0.50 g, 2.0 mmol) were dis-
solved in pyridine (5 mL), and the mixture was stirred at RT for 3 h.
Sodium hydrogen sulfite (1.8 g) dissolved in a mixture of water
(30 mL) and pyridine (20 mL) was added to the reaction mixture,
and the mixture was stirred for an additional 14 h. The product
was extracted with CH2Cl2 (2ꢁ100 mL), and the organic phases
were dried over Na2SO4. After evaporation, the pyridine was re-
moved by co-evaporation with toluene, and the residue was puri-
fied by silica gel column chromatography with hexane/EtOAc/
MeOH (70:25:5) to give 5R,6S-isomer 2a (479 mg, 31%) and 5S,6R-
isomer 2b (808 mg, 52%) as white foams. 5R,6S-isomer (2a):
1H NMR (CDCl3, 600 MHz, TMS): d=7.72 (s, 1H), 7.38–7.34 (m, 6H),
7.33–7.17 (m, 24H), 6.16 (dd, J=16, 4.2 Hz, 1H), 5.05 (s, 1H), 4.63
(ddd, J=51, 5.4, 4.2 Hz, 1H), 4.23 (ddd, J=11, 5.4, 4.2 Hz, 1H),
3.64–3.62 (m, 1H), 3.52 (s, 1H), 3.29 (d, J=11 Hz, 1H), 2.84–2.82 (m,
1H), 2.82 (s, 1H), 1.31 ppm (s, 3H); 13C NMR (CDCl3, 150 MHz): d=
173.1, 150.1, 143.9, 143.5, 129.1, 128.9, 128.1, 128.0, 127.5, 127.4,
90.6 (d, J=192 Hz), 87.8, 87.6, 86.7 (d, J=33 Hz), 81.3, 78.0, 71.9,
71.8 (d, J=14 Hz), 63.1, 22.8 ppm; ESI-HRMS (m/z): calcd for
C48H43FN2O7: 801.2946 [M+Na]+, obs: 801.2954. 5S,6R-isomer (2b):
1H NMR (CDCl3, 600 MHz, TMS): d=7.57 (s, 1H), 7.36–7.34 (m, 6H),
7.31–7.22 (m, 24H), 6.33 (dd, J=13, 6.0 Hz, 1H), 4.89 (s, 1H), 4.55
(5R,6S)-5,6-Di-O-acetyl-2’-ribofluorothymidine (4a): To a solution
of compound 3a (86 mg, 0.10 mmol) in anhydrous CH2Cl2 (2 mL)
was added trifluoroacetic acid (0.11 mL, 1.5 mmol) under an argon
atmosphere, and the mixture was stirred at RT for 20 min. The
reaction mixture was diluted with diethyl ether (15 mL), and ex-
tracted with water (2ꢁ8 mL). The aqueous solutions were com-
bined and evaporated under reduced pressure. The residue was
twice co-evaporated with a mixture of toluene and acetonitrile
(1:1) to give 4a (35 mg, 92%) as a white solid. 1H NMR (CD3OD,
600 MHz): d=6.77 (s, 1H), 5.78 (dd, J=20, 3.6 Hz, 1H), 4.96 (ddd,
J=54, 4.8, 3.6 Hz, 1H), 4.19 (ddd, J=16, 7.2, 4.8 Hz, 1H), 3.88–3.86
(m, 1H), 3.82 (dd, J=13, 3.0 Hz, 1H), 3.70 (dd, J=13, 5.4 Hz, 1H),
2.09 (s, 3H), 2.05 (s, 3H), 1.82 ppm (s, 3H); 13C NMR (CD3OD,
150 MHz): d=170.9, 170.5, 169.6, 152.1, 93.5 (d, J=185 Hz), 90.4 (d,
J=35 Hz), 84.3, 79.7, 78.6, 70.7 (d, J=16 Hz), 63.0, 21.3, 20.8,
20.6 ppm.
(5S,6R)-5,6-Di-O-acetyl-2’-ribofluorothymidine (4b): A white solid
(101 mg, 92%). 1H NMR (CD3OD, 600 MHz): d=6.76 (s, 1H), 5.72
(dd, J=20, 3.0 Hz, 1H), 5.05 (ddd, J=54, 4.8, 3.0 Hz, 1H), 4.26 (ddd,
J=17, 7.2, 4.8 Hz, 1H), 3.88–3.86 (m, 1H), 3.78 (dd, J=12, 2.4 Hz,
ChemBioChem 2012, 13, 1338 – 1348
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1345

S. S. David, P. A. Beal et al.
1H), 3.62 (dd, J=12, 4.8 Hz, 1H), 2.06 (s, 3H), 2.05 (s, 3H),
1.84 ppm (s, 3H); 13C NMR (CD3OD, 150 MHz): d=170.0, 169.5,
168.4, 151.1, 93.0 (d, J=185 Hz), 89.9 (d, J=35 Hz), 83.2, 79.2, 77.3,
69.0 (d, J=17 Hz), 61.5, 20.1, 19.4 ppm.
3’,5’-Di-O-trityl-2’-arabinofluorothymidine (8): aq. NH4OH (30%,
70 mL) was added to a suspension of compound 7 (2.80 g,
5.98 mmol) in EtOH (70 mL), and the mixture was stirred at RT for
48 h. The reaction mixture was evaporated under reduced pres-
sure, and the residue was used for next reaction without purifica-
tion. The residue was co-evaporated with anhydrous acetonitrile
and pyridine, and then dissolved in anhydrous pyridine (16 mL). To
the solution was added trityl chloride (4.17 g, 15.0 mmol) at RT,
and the mixture was heated to 1008C. After 18 h, the reaction mix-
ture was cooled to RT, and evaporated under reduced pressure.
The residue was purified by silica gel column chromatography with
CH2Cl2/AcOEt (19:1) to give 8 (3.11 g, 70%) as a pale yellow foam.
1H NMR (CDCl3, 600 MHz, TMS): d=8.43 (s, 1H), 7.37–7.34 (m, 12H),
7.29–7.21 (m, 18H), 6.18 (dd, J=23, 2.4 Hz, 1H), 4.44–4.43 (m, 1H),
4.26 (dd, J=17, 3.0 Hz, 1H), 3.77 (dd, J=5.0, 2.4 Hz, 1H), 3.30 (dd,
J=10, 2.4 Hz, 1H), 3.23 (dd, J=10, 5.4 Hz, 1H), 1.67 ppm (s, 3H);
13C NMR (CDCl3, 150 MHz): d=163.5, 150.1, 143.7, 143.2, 137.4,
128.8, 128.3, 128.0, 127.9, 127.3, 110.0,93.7 (d, J=192 Hz), 88.7,
87.0, 84.0 (d, J=21 Hz), 84.0, 77.9 (d, J=27 Hz), 63.6, 12.4 ppm;
ESI-HRMS (m/z): calcd for C48H41FN2O5: 745.3073 (M+H)+, obs:
745.3083.
5’-O-(4,4’-Dimethoxytrityl)-(5R,6S)-5,6-di-O-acetyl-2’-ribofluoro-
thymidine (5a): Compound 4a (70 mg, 0.19 mmol) was co-evapo-
rated with anhydrous acetonitrile and pyridine/toluene (1:1), and
the dried residue was dissolved in anhydrous pyridine (0.7 mL).
4,4’-Dimethoxytrityl chloride (125 mg, 0.369 mmol) was added into
the solution under an argon atmosphere, and the mixture was
stirred at RT for 1 h. The reaction mixture was diluted with EtOAc
(30 mL), and washed with water (20 mL) and brine (20 mL). The or-
ganic phase was dried over Na2SO4, and the solvent was removed
under reduced pressure. The residue was purified by silica gel
column chromatography with hexane/EtOAc (2:1!3:2) to give 5a
(105 mg, 83%) as a pale yellow foam. 1H NMR (CD2Cl2, 600 MHz):
d=7.83 (s, 1H), 7.45 (d, J=7.8 Hz, 2H), 7.34 (d, J=8.4 Hz, 4H), 7.27
(t, J=7.8 Hz, 2H), 7.20 (t, J=7.8 Hz, 1H), 6.82 (d, J=8.4 Hz, 4H),
6.75 (s, 1H), 5.62 (dd, J=25, 2.4 Hz, 1H), 5.06 (ddd, J=55, 4.8,
2.4 Hz, 1H), 4.38–4.31 (m, 1H), 3.90 (dt, J=9.6, 4.8 Hz, 1H), 3.77 (s,
6H), 3.31 (d, J=4.8 Hz, 2H), 2.13–2.10 (m, 1H), 2.07 (s, 3H), 2.04 (s,
3H), 1.84 ppm (s, 3H); 13C NMR (CD2Cl2, 150 MHz): d=169.6, 169.1,
167.2, 159.0, 150.3, 145.4, 136.3, 130.5, 128.5, 128.2, 127.1, 113.5,
93.2 (d, J=182 Hz), 91.8 (d, J=37 Hz), 86.5, 82.0, 80.0, 77.3, 70.8 (d,
J=17 Hz), 63.8, 55.6, 21.6, 21.0, 20.8 ppm; ESI-HRMS (m/z): calcd
for C35H37FN2O11: 703.2273 [M+Na]+, obs: 703.2277.
3’,5’-Di-O-trityl-5,6-dihydroxy-2’-arabinofluorothymidine
(9):
Compound (732 mg, 0.983 mmol) and OsO4 (250 mg,
8
0.983 mmol) were dissolved in pyridine (2.5 mL), and the mixture
was stirred at RT for 3 h. Sodium hydrogen sulfite (0.9 g) dissolved
in a mixture of water (15 mL) and pyridine (10 mL) was added to
the reaction mixture, and the mixture was stirred for an additional
14 h. The product was extracted with CH2Cl2 (50 mLꢁ2), and the
organic phases were dried over Na2SO4. After evaporation, the pyri-
dine was removed by co-evaporation with toluene, and the residue
was purified by silica gel column chromatography with hexane/
EtOAc/MeOH (70:25:5) to give 9 (705 mg, 92%, 5R,6S-isomer/5S,6R-
isomer=1:10 by NMR) as a white solid. 5S,6R-isomer: 1H NMR
(CDCl3, 600 MHz, TMS): d=7.68 (s, 1H), 7.37–7.34 (m, 12H), 7.30–
7.21 (m, 18H), 6.19 (dd, J=26, 3.0 Hz, 1H), 4.88 (s, 1H), 4.42–4.40
(m, 1H), 4.11 (dd, J=16, 1.8 Hz, 1H), 3.78 (dd, J=51, 3.0 Hz, 1H),
3.48 (d, J=2.4 Hz, 1H), 3.22 (dd, J=9.0, 3.0 Hz, 1H), 3.19 (s, 1H),
3.10 (dd, J=10, 9.0 Hz, 1H), 1.27 ppm (s, 3H); 13C NMR (CDCl3,
150 MHz): d=173.1, 151.1, 143.5, 143.3, 128.8, 128.6, 128.4, 128.1,
127.9, 127.4, 94.7 (d, J=190 Hz), 88.8, 87.0, 84.7 (d, J=15 Hz), 83.4,
81.3 (d, J=7.2 Hz), 77.5, 72.2, 63.3, 22.2 ppm; ESI-HRMS (m/z): calcd
for C48H43FN2O7: 801.2946 [M+Na]+, obs: 801.2948.
5’-O-(4,4’-Dimethoxytrityl)-(5S,6R)-5,6-di-O-acetyl-2’-ribofluoro-
1
thymidine (5b): A pale yellow foam (90 mg, 88%). H NMR (CD2Cl2,
600 MHz): d=7.77 (s, 1H), 7.44 (d, J=7.8 Hz, 2H), 7.32 (d, J=
7.2 Hz, 4H), 7.27 (t, J=7.8 Hz, 2H), 7.21 (t, J=7.8 Hz, 1H), 6.81 (d,
J=7.2 Hz, 4H), 6.66 (s, 1H), 5.57 (d, J=25 Hz, 1H), 5.24 (ddd, J=
55, 4.8, 1.2 Hz, 1H), 4.48–4.40 (m, 1H), 3.89–3.85 (m, 1H), 3.77 (s,
6H), 3.33 (dd, J=10, 3.0 Hz, 1H), 3.23 (dd, J=10, 5.4 Hz, 1H), 2.11
(s, 3H), 2.03 (s, 3H), 1.90 ppm (s, 3H); 13C NMR (CD2Cl2, 150 MHz):
d=170.0, 169.7, 167.2, 159.0, 149.7, 145.3, 136.4, 136.3, 130.5,
130.4, 128.5, 128.2, 127.2, 113.4, 93.8 (d, J=180 Hz), 93.6 (d, J=
37 Hz), 86.5, 81.9, 81.4, 77.3, 70.3 (d, J=17 Hz), 63.7, 55.6, 21.7,
20.9, 20.1 ppm; ESI-HRMS (m/z): calcd for C35H37FN2O11: 703.2273
[M+Na]+, obs: 703.2276.
5’-O-(4,4’-Dimethoxytrityl)-3’-O-[2-(cyanoethoxy)(N,N-diisopro-
pylamino)phosphino]-(5R,6S)-5,6-di-O-acetyl-2’-ribofluorothymi-
dine (6a): Compound 5a (112 mg, 0.165 mmol) was co-evaporated
with anhydrous acetonitrile (twice) and the dried residue was dis-
solved in anhydrous CH2Cl2 (1.1 mL). To the solution were added
N,N-diisopropylethylamine (170 mL, 0.98 mmol) and (2-cyanoethyl)-
N,N-diisopropylchlorophosphoramidite (95 mL, 0.43 mmol) at 08C,
and the mixture was stirred at 08C for 1 h. The reaction mixture
was quenched with MeOH (0.2 mL) and 2% NaHCO3 (5 mL), and
extracted with EtOAc (2ꢁ10 mL). The organic phases were washed
with brine (10 mL), dried over Na2SO4, and the solvent was re-
moved under reduced pressure. The residue was purified by silica
gel column chromatography with hexane/EtOAc (2:1) to give 6a
(125 mg, 86%) as a white foam. 31P NMR (CD2Cl2, 121 MHz): d=
151.8 (d, J=7.6 Hz), 151.4 ppm (d, J=13 Hz). ESI-HRMS (m/z): calcd
for C44H54FN4O12P: 881.3533 [M+H]+, obs: 881.3542.
5,6-Bis-O-(tert-butyldimethylsilyl)-2’-arabinofluorothymidine
(11): To a solution of compound 9 (920 mg, 1.18 mmol) in anhy-
drous DMF were added imidazole (803 mg, 11.8 mmol) and tert-bu-
tyldimethylchlorosilane (889 mg, 5.90 mmol), and the mixture was
stirred at 378C for 40 h. The reaction mixture was diluted with
ether (100 mL) and washed with saturated aq. NH4Cl (100 mL). The
aq. phase was extracted with ether (100 mLꢁ2), and the organic
phases were combined and dried over Na2SO4, and the solvent
was removed under reduced pressure. The residue was purified by
column chromatography on silica gel with hexane/EtOAc (7:1!
2:1) to give 10 (1.05 g, 88%) as a white foam. To a solution of com-
pound 10 (160 mg, 0.159 mmol) in anhydrous CH2Cl2 (3 mL) was
added trifluoroacetic acid (355 mL, 4.78 mmol) under argon atmos-
phere at 08C, and the mixture was stirred for 10 min. The reaction
mixture was quenched with saturated aq. NaHCO3 (20 mL) and ex-
tracted with CH2Cl2 (15 mLꢁ2). The organic phases were dried
over Na2SO4, and the solvent was removed under reduced pres-
sure. The residue was purified by silica gel column chromatogra-
phy with CH2Cl2/EtOAc (4:1!2:1) to give 11 (60 mg, 72%) as
5’-O-(4,4’-Dimethoxytrityl)-3’-O-[2-(cyanoethoxy)(N,N-diisopro-
pylamino)phosphino]-(5S,6R)-5,6-di-O-acetyl-2’-ribofluorothymi-
dine (6b):
A
white foam (105 mg, 81%). 31P NMR (CD2Cl2,
121 MHz): d=151.6 (d, J=7.7 Hz), 150.9 ppm (d, J=13 Hz). ESI-
HRMS (m/z): calcd for C44H54FN4O12P: 881.3533 [M+H]+, obs:
881.3547.
1
a white foam. 5S,6R-isomer (11 a): H NMR (CDCl3, 600 MHz, TMS):
1346
www.chembiochem.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2012, 13, 1338 – 1348

NEIL1 Binding to DNA
d=7.93 (s, 1H), 5.63 (dd, J=20, 3.0 Hz, 1H), 5.13 (ddd, J=51, 3.0,
1.2 Hz, 1H), 5.00 (s, 1H), 4.43 (d, J=19 Hz, 1H), 3.97 (dd, J=9.0,
4.8 Hz, 1H), 3.92–3.84 (m, 2H), 3.35 (s, 1H), 2.23 (s, 1H), 1.45 (s,
3H), 0.89 (s, 9H), 0.83 (s, 9H), 0.26 (s, 3H), 0.21 (s, 3H), 0.11 (s, 3H),
0.10 ppm (s, 3H); 13C NMR (CDCl3, 150 MHz): d=173.5, 151.8, 95.9
(d, J=191 Hz), 85.0 (d, J=17 Hz), 84.1, 82.6, 77.5, 75.1 (d, J=
27 Hz), 62.2, 26.4, 25.9, 23.7, 18.7, 18.3, ꢁ1.86, ꢁ2.27, ꢁ4.24,
ꢁ4.89 ppm; ESI-HRMS (m/z): calcd for C22H43FN2O7Si2 (M+H)+
523.2666, obs. 523.2665. 5R,6S-Isomer (11 b): 1H NMR (CDCl3,
600 MHz, TMS): d=8.14 (s, 1H), 5.25 (dd, J=9.6, 6.6 Hz, 1H), 5.16
(ddd, J=53, 6.6, 5.4 Hz, 1H), 4.94 (d, J=22 Hz, 1H), 4.57 (s, 1H),
3.93–3.91 (m, 2H), 3.81 (d, J=7.2 Hz, 1H), 3.80–3.78 (m, 1H), 3.55
(d, J=7.2 Hz, 1H), 1.46 (s, 3H), 0.88 (s, 9H), 0.84 (s, 9H), 0.26 (s,
3H), 0.20 (s, 3H), 0.16 (s, 3H), 0.12 ppm (s, 3H); 13C NMR (CDCl3,
150 MHz): d=173.0, 152.6, 97.4 (d, J=197 Hz), 89.4, 87.8, 80.9 (d,
J=9.2 Hz), 77.6, 73.3 (d, J=24 Hz), 61.2, 26.5, 25.8, 23.6, 18.7, 18.2,
ꢁ2.02, ꢁ2.41, ꢁ4.05, ꢁ4.70 ppm; ESI-HRMS (m/z): calcd for
C22H43FN2O7Si2: 523.2666 [M+H]+, obs: 523.2662.
mild DNA synthesis (Glen Research) on a 0.2 or 1.0 mmol scale with
coupling times of 15 min for the coupling of FTg. The 4,4’-di-
methoxytrityl (DMTr) group of the 5’-end was removed on the syn-
thesizer. The 30-nucleotide (nt) sequence that was synthesized is
as follows: d(5’-TGTTC ATCAT GGGTC XTCGG TATAT CCCAT-3’), in
which X=Tg, riboFTg5R, riboFTg5S or araFTg5S and the comple-
mentary strand d(3’-ACAAG TAGTA CCCAG YAGCC ATATA GGGTA-
5’), in which Y=A or G.
Deprotection and purification of oligodeoxynucleotides contain-
ing acetyl-protected riboFTg: The oligonucleotides containing
acetyl-protected riboFTg were treated with potassium carbonate
(0.05m) in methanol at RT for 4 h. The solvent was concentrated to
dryness under reduced pressure. The residues were dissolved in
triethylammonium acetate (0.1m, pH 7.0), and the oligonucleotides
were analyzed and purified by HPLC using a Dionex DNAPac PA-
100 ion-exchange column. The purification was achieved with
buffer solutions consisting of 30% solvent B (10% acetonitrile,
90% NH4OAc (1.5m), pH 7; flow rate: 3 mLminꢁ1) and 70% sol-
vent A (10% acetonitrile, 90% H2O) initially, with a gradient to
100% solvent B for 35 min. After lyophilization, the purified oligo-
nucleotides were desalted with a Sep-pak column. The correct
composition of the 30-nt oligonucleotide containing riboFTg(5R)
was confirmed by MALDI-TOF MS (m/z): calcd: 9202.4 [M+H]+,
obs: 9203.3; riboFTg(5S): calcd: 9202.4 [M+H]+, obs: 9203.6.
5’-O-(4,4’-Dimethoxytrityl)-(5S,6R)-5,6-bis-O-(tert-butyldimethyl-
silyl)-2’-arabinofluorothymidine (12): Compound 11 a (53 mg,
0.10 mmol) was co-evaporated with anhydrous acetonitrile and
pyridine/toluene (1:1), and the dried residue was dissolved in anhy-
drous pyridine (0.6 mL). 4,4’-Dimethoxytrityl chloride (69 mg,
0.204 mmol) was added to the solution under an argon atmos-
phere, and the mixture was stirred at room temperature for 1 h.
The reaction mixture was diluted with EtOAc (30 mL), and washed
with water (20 mL) and brine (20 mL). The organic phase was dried
over Na2SO4, and the solvent was removed under reduced pres-
sure. The residue was purified by silica gel column chromatogra-
phy with hexane/EtOAc (4:1) to give 12 (76 mg, 80%) as a white
Deprotection and purification of oligodeoxynucleotides contain-
ing TBDMS-protected araFTg: The oligonucleotides containing
TBDMS-protected riboFTg were treated with 30% aqueous ammo-
nia (2 mL) at room temperature for 2 h. The resulting ammonia sol-
utions were concentrated to dryness under reduced pressure. The
residue was dissolved in triethylamine trihydrofluoride (500 mL),
and the mixtures were kept at 408C overnight. After desalting on
a NAP-10 column (GE Healthcare), the oligonucleotides were ana-
lyzed and purified using the above conditions. The correct compo-
sition for 30 nt DNA containing araFTg(5S) was confirmed by
MALDI-TOF MS (m/z): calcd: 9202.4 [M+H]+, obs: 9203.0,
1
foam. H NMR (CD2Cl2, 600 MHz): d=7.58 (s, 1H), 7.46 (d, J=7.8 Hz,
2H), 7.33 (d, J=9.0 Hz, 4H), 7.30 (t, J=7.8 Hz, 2H), 7.23 (t, J=
7.8 Hz, 1H), 6.84 (d, J=9.0 Hz, 4H), 5.50 (dd, J=21, 2.4 Hz, 1H),
5.24 (ddd, J=52, 2.4, 1.2 Hz, 1H), 5.01 (s, 1H), 4.37 (dt, J=19,
3.6 Hz, 1H), 3.94 (dd, J=9.0, 5.4 Hz, 1H), 3.78 (s, 6H), 3.39–3.36 (m,
2H), 2.43 (d, 4.2 Hz, 1H), 1.40 (s, 3H), 0.87 (s, 9H), 0.83 (s, 9H), 0.24
(s, 3H), 0.21 (s, 3H), 0.07 (s, 3H), 0.05 ppm (s, 3H); 13C NMR (CD2Cl2,
150 MHz): d=173.4, 159.1, 151.5, 145.1, 136.2, 136.1, 130.4, 130.3,
128.4, 128.3, 127.2, 113.5, 96.3 (d, J=190 Hz), 86.8, 85.8 (d, J=
18 Hz), 83.6, 82.4, 77.8, 76.4 (d, J=26 Hz), 63.2, 55.6, 26.5, 26.0,
23.9, 18.9, 18.4, ꢁ1.93, ꢁ2.27, ꢁ4.23, ꢁ4.81 ppm; ESI-HRMS (m/z):
calcd for C43H61FN2O9Si2: 847.3792 [M+Na]+, obs: 847.3796.
32P-labeling of oligonucleotide: Oligonucleotides containing Tg,
riboFTg5R, riboFTg5S or araFTg5S were radiolabeled on the 5’-end
using [g-32P]ATP by T4 polynucleotide kinase at 378C. Excess [g-
32P]ATP was removed using a Pharmacia Microspin G-50 spin col-
umn, according to the manufacturer’s protocol. The labeled DNA
was then annealed to the complement (added at 20% excess) by
heating to 908C for 5 min and allowing to cool overnight in an-
nealing buffer [Tris·HCl (20 mm), EDTA (10 mm), NaCl (150 mm),
pH 7.6].
5’-O-(4,4’-Dimethoxytrityl)-3’-O-[2-(cyanoethoxy)(N,N-diisopro-
pylamino)phosphino]-(5S,6R)-5,6-bis-O-(tert-butyldimethylsilyl)-
2’-arabinofluorothymidine
(13):
Compound
12
(65 mg,
0.079 mmol) was co-evaporated with anhydrous acetonitrile (twice)
and the dried residue was dissolved in anhydrous CH2Cl2 (0.7 mL).
To this solution were added N,N-diisopropylethylamine (85 mL,
0.49 mmol) and (2-cyanoethyl)-N,N-diisopropylchlorophosphorami-
dite (44 mL, 0.20 mmol) at 08C, and the mixture was stirred at 08C
for 1 h. The reaction mixture was quenched with MeOH (0.2 mL)
and saturated aq. NaHCO3 (5 mL), and extracted with EtOAc
(10 mLꢁ2). The organic phases were washed with brine (10 mL),
dried over Na2SO4, and the solvent was removed under reduced
pressure. The residue was purified by silica gel column chromatog-
raphy with hexane/EtOAc (6:1) to give 13 (65 mg, 80%) as a white
foam. 31P NMR (CD2Cl2, 121 MHz): d=151.9, 151.6 ppm; ESI-HRMS
(m/z): calcd for C52H78FN4O10PSi2: 1025.5051 [M+H]+, obs:
1025.5068.
Enzyme purification: Unedited (K242) and edited (R242) NEIL1
were purified as described previously.[5] Active site concentration
was determined using a 30 bp duplex containing a central spiroi-
mindihydantoin (Sp1) lesion base-paired to G and concentrations
listed throughout are active site, rather than total protein, concen-
trations.[5] Endonuclease (Endo) III was purified using a pET24 nth
vector (provided by Dr. R. P. Cunningham, SUNY, Albany) using
BL21DE3 cells. The purification was as described previously with
minor modifications.[33] A binding assay using an abasic site
analogue (tetrahydrofuran) containing 30 bp duplex was used to
estimate the “binding” competent concentration of Endo III. This
concentration was used throughout the manuscript.
Glycosylase assays: The glycosylase activity of NEIL1 and Endo III
was evaluated with single-turnover experiments. Briefly substrate
DNA (20 nm) was incubated at 378C with active enzyme (200 nm)
in assay buffer containing Tris·HCl (20 mm, pH 7.6), EDTA (10 mm),
BSA (0.1 mgmLꢁ1) and NaCl (150 mm for NEIL1 or 100 mm for
Synthesis of oligodeoxynucleotides: Oligodeoxynucleotides were
synthesized on an ABI 394 synthesizer (DNA/Peptide Core Facility,
University of Utah) using nucleoside phosphoramidites for ultra-
ChemBioChem 2012, 13, 1338 – 1348
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
1347

S. S. David, P. A. Beal et al.
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Endo III), and aliquots were removed from the reaction mixture at
various time points and quenched with NaOH. The products were
analyzed by denaturing PAGE.
Measurement of dissociation constant (Kd): Electrophoresis mobi-
lity shift assays were performed using 32P-labeled duplexes contain-
ing 5R-riboFTg, 5S-riboFTg or 5S-araFTg. Reactions contained
duplex (10 pm), Tris·HCl (20 mm, pH 7.6), NaCl (150 mm for NEIL1
or 100 mm for Endo III), EDTA (1 mm), DTT (1 mm), 10% glycerol,
BSA (0.1 mgmLꢁ1) and varying amounts of enzyme. Enzyme solu-
tions of varying concentrations were freshly prepared by diluting
aliquots of enzyme at 48C with dilution buffer containing Tris·HCl
(20 mm, pH 7.6), EDTA (10 mm) and 20% glycerol. The samples
containing DNA and enzyme were incubated at 258C for 25–
35 min. Then, the samples were electrophoresed at 48C on a 6%
nondenaturing polyacrylamide gel with 0.5ꢁTBE buffer at 250 V
for 15–25 min followed by 150 V for 90–120 min. The gel was dried
and exposed to a Molecular Dynamics phosphorimager screen for
at least 18 h. Dissociation constants were determined by fitting the
data (% bound substrate versus log[enzyme]) to the equation for
one-site or two-site ligand binding using Grafit 5.0 software. Kd
values were determined from four to six separate experiments.
[19] S. Iwai, Angew. Chem. 2000, 112, 4032–4034; Angew. Chem. Int. Ed.
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(USA) for financial support in the form of grants R01M061115
and R01A090689. We are also grateful for the Research Fellow-
ship from the Japan Society for the Promotion of Science (JSPS)
for Young Scientists (K.O.). We thank Dr. Richard P. Cunningham
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mid, as well as Agnes Hahn and Dr. Nikolas Chmiel for purifica-
tion of Endo III used in this work.
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Keywords: DNA repair · Endo III · NEIL1 · RNA editing ·
thymidine glycol
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Received: February 23, 2012
Published online on May 25, 2012
1348
www.chembiochem.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2012, 13, 1338 – 1348