Fluorine at the C5 Position of 2′-Deoxyuridine Enhances Repair of a O4-Methyl Adduct by O6-Alkylguanine DNA Alkyltransferases

Article abstract of DOI:10.1002/ejoc.201700466

Alkylation damage at the O⁶- and O⁴-atoms of 2′-deoxyguanosine (dG) and thymidine (T), respectively, can be removed by O⁶-alkylguanine-DNA alkyltransferases (AGTs). Previous studies have shown that human AGT (hAGT) repairs

Full text of DOI:10.1002/ejoc.201700466

A Journal of
Accepted Article
Title: Fluorine at the C5 position of 2'-deoxyuridine enhances repair of
an O4-methyl adduct by O6-Alkylguanine DNA Alkyltransferases.
Authors: Lauralicia Sacre and Christopher James Wilds
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201700466
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10.1002/ejoc.201700466
European Journal of Organic Chemistry
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Fluorine at the C5 position of 2'-deoxyuridine enhances repair of
a O4-methyl adduct by O6-Alkylguanine DNA Alkyltransferases.
Lauralicia Sacre and Christopher J. Wilds*[a]
Abstract: Alkylation damage at the O6- and O4-atoms of 2’-
deoxyguanosine (dG) and thymidine (T), respectively, can be
removed by O6-alkylguanine-DNA alkyltransferases (AGTs).
Previous studies have shown that human AGT (hAGT) repairs small
adducts poorly at the O4-atom of T, in comparison to the E. coli
variants (OGT and Ada-C). The C5 methyl group of the thymine
nucleobase is suspected to contribute to hAGT repair proficiency
possibly due to steric effects in the protein active site. In the present
study, repair of oligonucleotides containing a 5-fluoro-O4-methyl-2’-
deoxyuridine (dFU-Me) insert by hAGT, E.coli AGT variants (OGT
and Ada-C) and a chimeric hAGT/OGT protein was evaluated. All
AGT variants, particularly hAGT and the hAGT/OGT chimera,
demonstrated improved proficiency at removing the O4-methyl group
from substrates containing dFU-Me, relative to the thymidine and 2’-
deoxyuridine counterparts.
nucleotide leading to a possible mutagenic outcome if left
unrepaired.[6]
O6-alkylguanine DNA alkyltransferase proteins (AGTs) are
able to locate and remove the aforementioned alkylated lesions
via a direct repair mechanism.[13] The alkylated nucleotide is
flipped out of the DNA duplex into the active site of the protein,
where an activated cysteine residue (Cys145 in the human
variant) irreversibly transfers the alkyl group from the DNA.[3,4]
The alkylated protein product is then degraded rapidly by the
ubiquitin pathway.[14] AGT proteins are found throughout life, and
crystal structures of different AGT variants depict a similar
overall structure, despite high or low homology.[13]
The range of substrates that can be acted upon and repair
rates varies amongst AGTs. For example, Ada-C and OGT from
E. coli have been shown to repair small adducts such as methyl
groups at either the O6-atom of dG or O4-atom of T, with the
latter repaired more efficiently.[12,15,16] Human AGT (hAGT) is
proficient at repairing a vast range of adducts at the O6-position
of dG including mono-adducts, intrastrand, and interstrand
cross-links.[12,15,17–19] Moreover, O6-benzylguanine is also a very
efficient pseudo-substrate for hAGT.[20–22] While Ada-C and OGT
have been shown to be efficient at repairing O4MeT in DNA,
hAGT displays poor activity despite its ability to recognize and
bind to DNA containing this adduct.[4,12,15] The evasion of the
mutagenic and cytotoxic O4MeT lesion by MMR and hAGT to
undergo repair,[4,23] coupled with no available crystal structure of
hAGT with a O4MeT containing DNA substrate, warrants further
investigation of this lesion.
In previous work from our laboratory, the influence of the
methyl group at the C5-atom of T towards repair of various O4-
alkylated adducts by hAGT was evaluated.[3] It was proposed
that a potential steric clash between the C5-methyl of T and
Arg135 of hAGT could hinder repair. To address this, hAGT
repair of DNA containing O4MedU (dU-Me, Figure 1) was
investigated and it was concluded that steric factors of the C5-
methyl group may contribute, at least in part, as dU substrates
were processed more efficiently relative to their dT counterparts
by hAGT.[3,24] It was observed that an AGT chimera (hOGT),
consisting of the hAGT protein with replaced residues (139-159)
from the OGT active site, demonstrated a twenty fold repair
enhancement of dU-Me compared to hAGT.[3]
Introduction
DNA damage can result from various agents and events from
within and external to the cell.[1,2] For example, alkylating agents
such as N-methyl-N’-nitrosourea have been shown to introduce
alkyl lesions at the O6 and O4 atoms of 2'-deoxyguanosine (dG)
and thymidine (T) respectively.[1,3] Such modifications to DNA
are disruptive to the cell, a feature that has been exploited by
drugs used to treat of cancer, which are designed to introduce
alkyl lesions. Examples of chemotherapeutic drugs whose
primary mode of action is the introduction of DNA damage
includes Temozolamide and BCNU (1,3-bis-(2-chloroethyl)-1-
nitrosourea).[4]
The detrimental effects of O6-methyl-dG (O6MedG) and O4-
methyl-T (O4MeT) to hinder DNA polymerase activity and cause
transitional events (G:C to A:T) have been well documented.[4–6]
The mutagenic character arises mainly from the persistence of
these lesions in addition to how frequently they occur.[7,8]
O6MedG incidences occur predominantly compared to O4MedT
in both in vitro and in vivo experiments where DNA is subjected
to alkylating agents such as N-methyl-N'-nitrosourea and methyl
methanesulfonate.[9–11] O4MeT, however exhibits a higher level
of cytotoxicity due to its reduced susceptibility to repair by the
cellular repair machinery.[9,12] The detrimental effect of O4MeT
arises from a non-wobble base pair with the incorrect dG
Given the differences observed for AGT-mediated repair of
O4-alkyl groups in T versus dU, we have initiated studies to
explore the influence of other groups at the C5-position. Fluorine
and hydrogen have a similar van der Waals radius (1.47 versus
1.20 Å) with the former having a higher electronegativity allowing
for the straightforward evaluation of the influence of an
electronegative group at this position.[25,26] Replacement of
hydrogen with fluorine has been exploited for applications in
drug discovery. Fluorine has shown to increase lipophilicity and
fat solubility of small molecules.[25–27] 5-fluorouracil and
[a]
Lauralicia Sacre and Christopher J. Wilds*
Department of Chemistry and Biochemistry and Centre for Structural
and Functional Genomics, Concordia University, 7141 Sherbrooke
St. West, Montréal, Québec (Canada) H4B 1R6
E-mail: chris.wilds@concordia.ca
Supporting information for this article is given via a link at the end of
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flunitrazepam are examples of potent fluorine containing
addition of 1,2,4-triazole, triethylamine and phosphoryl chloride
in three portions at 30 min intervals with stirring at 0oC to a
solution containing compound 4. After four hours, the reaction
was complete and the convertible nucleoside was then
converted to compound 6 by using sodium methoxide in
methanol. Despite the increased reactivity of the convertible
nucleoside, it allows the incorporation of different adducts at the
C4 position. The incorporation of the O4-methyl adduct was
followed by the removal of the 3’-O-tert-butyldimethylsilyl group
by a fluoride treatment using TBAF at room temperature for 30
min to yield compound 6 (74% over 3 steps). In a similar fashion,
removal of the 3’-O-tert-butyldimethylsilyl protecting group from
compound 1 to yield 2 was performed. Finally, compounds 2 and
6 were phosphitylated by the addition of diisopropylethylamine in
drugs.[27-29]
In chemically modified oligonucleotides, the
introduction of fluorine has been shown to confer interesting
properties to the nucleic acid scaffold. For example, replacing
the 2’-hydroxyl group of RNA with fluorine has been shown to
influence siRNA recognition by RISC, an important feature in
strand selectivity and gene silencing.[29] In the present study, the
influence of having fluorine at the C5-position with
oligonucleotides containing 5-fluoro-O4-methyl-2’-deoxyuridine
(dFU-Me) on duplex stablity, structure and proficiency of AGT-
mediated repair relative to T and dU is described.
THF
and
immediately
followed
by
chloride
N,N-
to
diisopropylaminocyanoethylphosphonamidic
generate phosphoramidites 3 and 7, respectively, in good yields,
using previously published procedures.[3] Purification was
achieved by short flash column chromatography. The 31P NMR
spectra (Supporting Figures S4 and S11) revealed two sharp
signals in the 147-149 ppm region characteristic of
phosphoramidites.[3]
Figure 1. A) Structures of the modified O4-alkyl pyrimidines, and B) DNA
sequence where X corresponds to the adduct.
Results and Discussion
Synthesis of nucleosides and oligonucleotides
The structure of O4-Me-dFU and the sequence of the 14-mer
Scheme 1. Reagents and conditions: (i) DMTr-Cl, pyridine, DMAP, 16 h, 21
oC; (ii) N,N-diisopropylaminocyanoethylphosphonamidic chloride, DIPEA, THF,
30 min; (iii) 1. DMTr-Cl, pyridine, DMAP, 16 h, 21 oC. 2. TBS-Cl, Imidazole,
DCM, 16 h, 21 oC; (iv) 1,2,4-Triazole, triethylamine, POCl3, MeCN, 4 h, 0 oC;
(v) 1. MeOH, NaOMe, 4h, 21 oC. 2. TBAF (1M in THF), 30 min; (vi) N,N-
diisopropylaminocyanoethylphosphonamidic chloride, DIPEA, THF, 30 min.
DNA used for biophysical and repair studies are shown in
Figure 1. The corresponding 14-mer
T and dU control
sequences were also prepared. The oligonucleotides containing
the modified nucleobases were prepared by a combination of
solution and automated solid-phase synthesis. The synthetic
pathway is shown in Scheme
1 and it begins with the
commercially available 5-fluoro-2’deoxyuridine (dFU), in which
the 5’-hydroxyl was protected with a 4,4'-dimethoxytrityl group
which was introduced by the portionwise addition of 4,4'-
dimethoxytrityl chloride in pyridine. This was followed by
silylation of the 3’-hydroxyl with tert-butyldimethylsilyl chloride in
dichloromethane.[30,31] A convertible nucleoside was prepared by
the addition of a 1,2,4-triazole group at the C4 position of the
nucleoside. This reaction was optimized since it did not reach
completion and when following the procedure commonly used
for the T and dU series undesired by-product formation such as
the detritylated nucleoside was observed.[3,30] This may be due
to the presence of the C5-fluorine, which could contribute to
increased reactivity at the C4 position. The formation of
convertible intermediate 5 was accomplished by the slow
Solid-phase synthesis of the oligonucleotide was
conducted using “fast-deprotecting” commercially available 3’-O-
phosphoramidites due to the labile nature of the dFU-Me
adduct.[18] Phenoxyacetic anhydride was employed as the
capping reagent to avoid transamidation.[32] Total deprotection
and cleavage of the oligomers from solid support was
accomplished with an anhydrous solution of potassium
carbonate in methanol (0.05 M) for four hours at 22oC with
gentle rocking.[18,30] Excess base was neutralized with an
equimolar amount of acetic acid and the solvent removed in a
speed-vacuum concentrator. The oligonucleotide containing
dFU-Me was purified by SAX-HPLC. Characterization by ESI-
MS of DNA containing the dFU-Me confirms the presence of the
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Figure 2. Tm values (oC) of duplexes containing T, T-Me, dU, dU-Me, dFU,
dFU-Me. Colorless and grey bars represent the unmodified controls and O4-
modification after the deprotection process and it was in
agreement with the expected mass (Supporting Figure S13).
Enzymatic digestion of modified oligonucleotides using snake
venom phosphodiesterase and calf intestinal phosphatase
enzymes also confirmed nucleoside composition consistent with
expected ratios (Supporting Figure S14).
methylated adducts, respectively. Hyperchromicity change (A260
) versus
temperature (oC) profiles are shown in Supporting Figure S16.
UV thermal denaturation and CD spectroscopy
In order to evaluate the effect of the fluorine atom at the C5
position and the methyl adduct at the O4-atom on the stability of
the DNA duplex (formed by hybridizing the modified
oligonucleotide with its corresponding complementary sequence
containing 2’-deoxyadenosine as the base pairing partner of the
O4-methylated nucleobase), UV thermal denaturation studies
were conducted. The results are summarized in Figure 2. The
introduction of the methyl group at the O4 position resulted in a
decrease in thermal stability by approximately 11 oC relative to
the unmodified controls. This is similar to the decrease in Tm
observed for the T and dU counterparts suggesting that the
decrease in duplex stability resulting from the O4-Me lesions is
independent of the identity of the nucleobase for the series
studied (T, dU or dFU). The overall decrease in thermal stability
may be attributed to the disruption of the H-bonding between the
O4-alkylated dFU and its base-pair partner 2’-deoxyadenosine
and less optimal stacking with the adjacent bases similar to
previous observations with the oligonucleotides containing dU.[3]
Coincidentally, the duplexes containing dU and dFU displayed
the same Tm value of 58 oC, as well as their respective
modifications (dFU-Me and dU-Me), implying that the presence
of the fluorine at the C5 position had minimal influence on the
DNA duplex stability.
Figure 3. Circular dichroism spectra of DNA duplexes containing dFU•dA (—
——), dFU-Me•dA (—) and unmodified control T•dA DNA (•••)
Repair Assays
The repair of dFU-Me in a DNA duplex was studied with three
recombinant AGT proteins (hAGT, Ada-C and OGT), and the
chimera AGT protein (hOGT). Preliminary repair assays were
performed as described previously by our group, where
denaturing PAGE is used to monitor the production of a product
generated from BclI cleavage which occurs at a specific site
engineered into the DNA duplex.[3] The dFU-Me oligonucleotide
is 5’-radiolabeled and if AGT repair occurs, Bcll can cleave the
duplex into smaller fragments including a radiolabeled 5-mer
detected by PAGE.[3] If the O4-alkyl group persists, the intact
radiolabeled 14-mer is observed.
The CD analysis of the profiles from dFU, dFU-Me, and
the control T display spectroscopic signatures consistent with
the B-form DNA family, exhibiting a maximum positive signal at
280 nm, a cross-over near 256 nm, and a negative signal at 245
nm. The CD signatures of the duplex containing dFU-Me was
similar to the unmodified control DNA duplex profile, which
suggested that the O4-methyl group did not cause any major
effect on the overall DNA duplex structure, as previously
reported for the dU series.[3]
The total repair assay (performed overnight at 37 oC)
revealed that dFU-Me was efficiently repaired by all four AGTs
evaluated at five-molar equivalences to the DNA (reaching
approximately 80 % repair). These results are consistent with
the trends observed previously for the total repair assays with
the
oligonucleotides.[3,18]
O4-methyl-dU
versus
O4-methyl-T
containing
Interestingly, time course assays revealed that dFU-Me
was repaired faster compared to their dU-Me and T-Me
counterparts. Time course repair studies were performed with
the four AGTs variants at room temperature. Both E.coli variants
(OGT and Ada-C) took less than 15s to achieve full repair of
dFU-Me. This result is in agreement with the total repair
observed towards dU-Me versus T-Me occurring in less than
15s by OGT and Ada-C.[3]
Remarkably, repair of dFU-Me by hAGT only required
approximately 90 s for the reaction to achieve completion. The
repair observed of dFU-Me by hAGT occurred significantly faster
relative to the dU-Me and T-Me analogues. It had previously
been shown that dU-Me undergoes 25% repair by hAGT in 2.5
min, whereas similar repair levels were only obtained for T-Me
after 30 min. dU-Me hAGT-mediated repair was approximately
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The importance and role of fluorine in biological
12-fold faster compared to T-Me in terms of time to reach 25%
substrate repair.[3] dFU-Me 25% repair by hAGT occurred in less
than 15 s, suggesting that the fluorinated analogue is a better
substrate than dU-Me and therefore even better than T-Me.
Complete repair of dFU-Me by the hOGT chimera occurred in
less than 1.5 min, displaying a similar repair profile to hAGT.
Moreover, repair by the hOGT chimera on dFU-Me was similar
compared to dU-Me. When compared with the results of our
previous studies, dFU-Me required approximately a third of the
time to achieve 80% repair relative to the dU-Me adduct.[3]
applications
and
drug
regimens
has
been
well
documented.[26,30,33] Our study shows that the presence of the
fluorine at the C5-position enhances repair susceptibility of
methyl adducts at the O4-position by hAGT with similar repair
efficiency observed between hAGT and OGT. Moreover,
elevated hAGT levels have been correlated to an increase in
cellular resistance to certain chemotherapeutic agents. It is
proposed that the presence of the fluorine at the C5 position
weakens the ether O4-C bond making it more susceptible to
nucleophilic attack by Cys145 and therefore a good hAGT
substrate. The presence of fluorine might also have a substantial
effect on weak dipolar interactions and basicity of the active
site.[26,34] Alternatively, hydrophobic interactions of the fluorinated
substrate within the AGT active site may be occurring with
computational studies currently underway to assess this
hypothesis.[35] Crystallographic structures of the protein
complexed with damaged DNA will aid in elucidating the critical
interactions, which promote hAGT-mediated repair. Moreover,
high-resolution structures of duplexes containing dFU-Me are
currently being investigated via by a combination of molecular
dynamics and high-field NMR experiments to gain insights on
the impact of this modification on DNA structure.
Studies are also currently underway with other dFU substrates
to evaluate susceptibility towards AGT-mediated repair, which
may find potential uses as AGT inhibitors in certain combinatory
regimens.[20,21]
Figure 4. Time course repair assay of dFU-Me by OGT (—), Ada-C (—•—),
hAGT (—••—), and hOGT (———) at 5-molar protein equivalence. Graphical
illustration displays dFU-Me repair [%] over time (min).
Conclusion
Our study describes the synthesis of
a nucleoside and
oligonucleotide containing dFU-Me. This was achieved via the
formation of a convertible nucleoside and its incorporation into
oligonucleotides by solid-phase synthesis in scales and purity
sufficient for biochemical and biophysical studies. A decrease of
11 oC in the thermal stability was observed for duplexes
containing this modification relative to the control duplex. The
CD data revealed no major change in the global structure of
DNA containing the methylated dFU insert relative to the control
duplex. Repair of dFU-Me by the different AGTs occurred in
less than 2 min, with the E. coli variants displaying significant
repair proficiency (in less than 15 sec), which was in agreement
with previous literature.[3,4] hAGT shows a remarkable increase
in its repair efficiency towards dFU-Me compared to the dU and
T analogues. Given the significant activity of hAGT towards
repair of dFU-Me, this avenue of modification may provide leads
in the design of hAGT inhibitors.
Figure 5. Repair of (A) dFU (2 pmol) and (B) dFU-Me (2 pmol) by hAGT (10
pmol), OGT (10 pmol), Ada-C (10 pmol),, and hOGT (10 pmol), for 2.5h at
37oC. Panel A; Lane 1, dFU DNA; lane 2, dFU + Bcll; lane 3, dFU + hAGT;
lane 4, dFU + hAGT + Bcll; lane 5, dFU + OGT; lane 6, dFU + OGT+ Bcll; lane
7, dFU + Ada-C; lane 8, dFU + Ada-C + Bcll; lane 9, dFU + hOGT; lane 10,
dFU + hOGT + BclL. Panel B; Lane 1, dFU-Me DNA; lane 2, dFU-Me + Bcll;
Lane 3, dFU-Me; lane 4, dFU-Me + hAGT; lane 5, dFU-Me + hAGT; lane 6,
dFU-Me + hAGT + Bcll; lane 7, dFU-Me + OGT; lane 8, dFU-Me + OGT+ Bcll;
lane 9, dFU-Me + Ada-C; lane 10, dFU-Me + Ada-C + Bcll; lane 11, dFU-Me +
10 hOGT; lane 12, dFU-Me + hOGT + Bcll.
Experimental Section
Synthesis and characterization of nucleosides and
oligonucleotides.
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5-fluoro-2’-deoxyuridine (compound 1) was purchased from
Berry Associates (Dexter, Michigan). “Fast deprotecting” 5’-O-
dimethoxytrityl-2’-deoxyribonucleoside-3’-O-(β-cyanoethyl-N,N-
NMR (125.7 MHz, d6-acetone, ppm): 206.22, 158.82, 144.96,
135.65, 135.55, 130.12, 130.09, 128.07, 128.03, 127.81, 126.80,
126.77, 113.11, 86.67, 86.61, 85.42, 85.39, 85.08, 85.01, 63.29,
63.15, 58.71, 58.56, 54.64, 43.11, 43.01, 29.41, 29.25, 29.10,
28.94, 28.79, 28.64, 28.48, 24.02, 23.96, 23.93, 19.85, 19.79.
19F NMR (470.4 MHz, d6-acetone, ppm): -167.84, -167.85, -
167.90, -167.91. 31P NMR (202.3 MHz, d6-acetone, ppm):
148.31, 148.34. IR (thin film); νmax (cm−1) = 3195, 3066, 2967,
2932, 2836, 2362, 2336, 2252, 1717, 1607, 1508, 1464, 1251,
1179, 1125, 829, 736.
diisopropyl)phosphoramidites
and
protected
2’-
deoxyribonucleoside–CPG supports were purchased from Glen
Research (Sterling, Virginia). Compounds
2 and 4 were
prepared according to previous published procedures.[3,30,31] All
other chemicals and solvents were purchased from the Aldrich
Chemical Company (Milwaukee, WI) or EMD Chemicals Inc.
(Gibbstown, NJ). Flash column chromatography was performed
using silica gel 60 (230–400 mesh) purchased from Silicycle
(Quebec City, QC). Thin layer chromatography (TLC) was
5’-O-(4,4’-Dimethoxytrityl)-5-fluoro-O4-methyl-2’-
deoxyuridine (6): To a solution of triazole (0.28g, 4.07mmol) in
anhydrous MeCN (6mL) at 0oC under stirring, was added
triethylamine (0.543mL, 3.89mmol). Then, a solution of 3’-O-
carried out with precoated TLC plates (Merck, Kieselgel 60 F254
,
0.25 mm) purchased from EMD Chemicals Inc. (Gibbstown, NJ).
All NMR spectra were recorded on a Varian 500 MHz NMR
spectrometer at room temperature. 1H NMR spectra were
recorded at a frequency of 500.0 MHz and chemical shifts were
reported in parts per million (ppm) downfield from
tetramethylsilane. 13C NMR spectra (1H decoupled) were
recorded at a frequency of 125.7 MHz and chemical shifts were
reported in ppm with tetramethylsilane as a reference. 19F NMR
spectra were recorded at a frequency of 470.4 MHz and
chemical shifts were reported in parts per million (ppm)
downfield from trichlorofluoromethane. 31P NMR spectra (1H
decoupled) were recorded at a frequency of 202.3 MHz and
chemical shifts were reported in ppm with H3PO4 used as an
external standard. High resolution mass spectrometry of all the
modified nucleosides were acquired using an 7T-LTQ FT ICR
instrument (Thermo Scientific) at the Concordia University
Centre for Structural and Functional Genomics. The mass
spectrometer was operated in full scan, positive ion detection
mode. ESI mass spectra for oligonucleotides were obtained at
the Concordia University Centre for Biological Applications of
(tert-butyldimethylsilyl)-5’-O-(4,4’-dimethoxytrityl)
-5-fluoro-2’-
deoxyuridine (0.300g, 0.45mmol) in anhydrous MeCN (6mL)
was added to the triazole solution. After 40 min, an additional
solution of triazole (0.28g, 4.07mmol), POCl3 (0.084mL, 0.91
mmol) and triethylamine (0.543mL, 3.89mmol) in anhydrous
MeCN (3mL) was added dropwise with stirring at 0oC to the
solution containing the nucleoside. This extra addition was
repeated for a second time after 40 min. After 1 hour, the solvent
was evaporated in vacuo, the crude was taken up in MeOH (7
mL) and a solution of NaOMe (0.086g, 1.6mmol) in MeOH (7mL)
was added. After 30 min, an additional solution of NaOMe
(0.086g, 1.6mmol) in MeOH (3mL) was added to the nucleoside.
After 16 h, the solvent was evaporated in vacuo and the crude
product was taken up in CH2Cl2 (40 mL), washed with a 3%
(w/v) solution of NaHCO3 (2 x 50mL), dried over anhydrous
Na2SO4 (~ 4g), and concentrated in vacuo. The crude product
was taken up in THF (4.5mL) and TBAF (1 M in THF) (0.543 mL,
0.54mmol) was added drop-wise. After 30 min, the solvent was
evaporated in vacuo and the crude product was taken up in
CH2Cl2 (40 mL), washed with a 3% (w/v) solution of NaHCO3 (2
x 50mL), dried over anhydrous Na2SO4 (~ 4g), and concentrated
in vacuo. The crude product was purified by flash column
Mass Spectrometry using
a
Micromass Qtof2 mass
spectrometer (Waters) equipped with a nanospray ion source.
The mass spectrometer was operated in full scan, negative ion
detection mode. T4 polynucleotide kinase (PNK) was obtained
from New England BioLabs (NEB) [γ-32P]ATP was purchased
from PerkinElmer (Woodbridge, ON). Bcll restriction enzyme
was obtained from New England Biolabs (Ipswich, MA).
chromatography using
a CH2Cl2/MeOH (49.5:0.5  49:1)
solvent system to afford 0.189mg (74%) of a colorless foam. Rf
(SiO2 TLC): 0.63 (100% EtOAc). λmax(MeCN) = 283 nm. HRMS
+
(ESI-MS) m/z calculated for C31H31FN2NaO7 : 585.2008; found
3’-O-(β-Cyanoethyl-N,N’-diisopropyl)-5’-O-(4,4’-
dimethoxytrityl)- 5-fluoro -2’-deoxyuridine (3): DIPEA (0.19
585.2006 [M + Na]+. 1H NMR (500 MHz, CDCl3, ppm): 8.05-8.06
(d, J= 5.6 Hz, 1H; H6), 7.21–7.41 (m, 9H; Ar), 6.83– 6.85 (m,
4H; Ar), 6.25-6.27 (m, 1H; H1’), 4.55-4.57 (m, 1H; H3’), 4.15-
4.17 (m, 1H; H4’), 4.04 (s, 3H; OCH3) 3.79 (s, 6H; ArOCH3), 3.42
(m, 2H; H5’, H5’’), 3.18 (s, 1H; OH), 2.72-2.76 (m, 1H; H2’),
2.23-2.28 (m, 1H; H2’’). 13C NMR (125.7 MHz, CDCl3, ppm):
162.68, 162.58, 158.63, 153.55, 144.31, 137.68, 135.72, 135.42,
135.25, 129.98, 129.96, 127.98, 127.93, 127.68, 127.43, 127.02,
113.31, 113.29, 87.10, 87.02, 86.59, 77.29, 77.03, 76.78, 63.11,
55.22, 55.07, 42.03. 19F (470.4 MHz, in CDCl3, ppm): -168.60, -
168.62. IR (thin film); νmax (cm−1) = 3423, 2362, 2335, 1652,
1635, 1 506, 1497, 1403, 1251, 1176, 1035, 828.
mL, 1.12 mmol) was added to
a solution of 5’-O-(4,4’-
dimethoxytrityl)-5-fluoro-2’-deoxyuridine (0.204 g, 0.372 mmol)
in THF (3.7 mL), followed by the dropwise addition of N,N-
diisopropylamino cyanoethyl phosphonamidic chloride (0.2 mL,
0.893 mmol). After 30 min, the solvent was evaporated in vacuo
and the crude product was taken up in EtOAc (40 mL). The
solution was washed with 3% (w/v) solution of NaHCO3 (2 x
50mL) and once with brine (50mL). The organic layer was dried
over anhydrous Na2SO4 (~ 4g), and concentrated in vacuo. The
crude product was purified by flash column chromatography
using a hexanes/EtOAc (2:8) solvent system to afford 0.187g
(67%) of a colorless foam. Rf (SiO2 TLC): 0.87 (100% EtOAc).
3’-O-(β-Cyanoethyl-N,N’-diisopropyl)-5’-O-(4,4’-
dimethoxytrityl)- 5-fluoro-O4-methyl-2’-deoxyuridine (7):
DIPEA (0.14 mL, 0.80 mmol) was added to a solution of (5)
(0.150 g, 0.266 mmol) in THF (2.7 mL), followed by the dropwise
addition of N,N-diisopropylamino cyanoethyl phosphonamidic
chloride (0.14 mL, 0.64 mmol). After 30 min, the solvent was
evaporated in vacuo, the crude product was taken up in EtOAc
(40 mL), the solution was washed with a 3% (w/v) solution of
NaHCO3 (2 x 50mL) and once with brine (50mL). The organic
layer was dried over anhydrous Na2SO4 (~ 4g) and concentrated
λmax(MeCN)
= 268 nm. HRMS (ESI-MS) m/z calculated for
C39H47FN4O8P+: 749.3110; found 749.3104 [M + H]+. 1H NMR
(500 MHz, d6-acetone, ppm): 7.92-7.93 (d, J= 6.3 Hz, 0.5H; H6),
7.90-7.91 (d, J= 6.3 Hz, 0.5H; H6), 7.23–7.53 (m, 9H; Ar), 6.90-
6.93 (m, 4H; Ar), 6.28-6.32 (m, 1H; H1’), 4.71-4.78 (m, 1H; H3’),
4.17-4.24 (m, 1H; H4’), 3.59-3.93 (m, 10H; NCH, ArOCH3,
CH2OP), 3.39-3.50 (m, 2H; H5’, H5’’), 2.77-2.79 (t, 1H; CH2CN),
2.65-2.67 (t, 1H; CH2CN), 2.47-2.52 (m, 1H; H2’), 2.06-2.07 (m,
1H; H2’’), 1.20-1.22 (m, 10H; CH3), 1.13-1.14 (m, 2H; CH3). 13C
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in vacuo. The crude product was purified by flash column
chromatography using a hexanes/EtOAc (2:8) solvent system to
afford 0.173g (86%) of a colorless foam. Rf (SiO2 TLC): 0.11,
0.26 (1:1 hexanes/EtOAc). λmax(MeCN) = 283 nm. HRMS (ESI-MS)
m/z calculated for C40H49FN4O8P+: 763.3267; found 749.3104 [M
+ H]+. 1H NMR (500 MHz, CDCl3, ppm): 8.15-8.16 (d, J= 6.3 Hz,
0.5H; H6), 8.12-8.13 (d, J= 6.3 Hz, 0.5H; H6), 7.23–7.53 (m, 9H;
Ar), 6.89– 6.93 (m, 4H; Ar), 6.18-6.23 (m, 1H; H1’), 4.70-4.78 (m,
1H; H3’), 4.22-4.29 (m, 1H; H4’), 3.97-3.98 (s, 3H; OCH3), 3.63-
3.88 (m, 10H; NCH, ArOCH3, CH2OP), 3.43-3.52 (m, 2H; H5’,
H5’’), 2.77-2.80 (t, 1H; CH2CN), 2.60-2.70 (t, 1H; CH2CN), 2.39-
2.47 (m, 1H; H2’’), 2.06-2.07 (m, 1H; H2’’) 1.20-1.23 (m, 9H;
CH3), 1.12-1.14 (m, 3H; CH3). 13C NMR (125.7 MHz, CDCl3,
ppm): 205.21, 162.27, 162.17, 158.83, 152.36, 144.93, 137.15,
137.12, 135.62, 135.60, 135.51, 135.47, 135.21, 135.18, 130.11,
130.08, 128.06, 127.82, 127.81, 127.68, 127.65, 126.81, 126.79,
118.07, 117.94, 113.13, 113.11, 86.70, 86.65, 86.58, 86.51,
85.73, 85.70, 85.46, 85.41, 73.18, 73.05, 72.71, 72.58, 63.00,
62.80, 58.73, 58.67, 58.58, 58.52, 54.65, 54.63, 53.98, 43.13,
43.11, 43.03, 43.01, 40.36, 40.34, 40.17, 40.13, 29.42, 29.26,
29.11, 28.96, 28.80, 28.65, 19.90, 19.86, 19.84, 19.81. 19F
(470.4 MHz, d6-acetone, ppm): -171.63, -171.64, -171.68, -
171.70. 31P NMR (202.3 MHz, d6-acetone, ppm): 148.35, 148.21.
IR (thin film); νmax (cm−1) =3071, 2966, 2869, 2836, 2362, 2335,
2252, 1683, 1652, 1607, 1541, 1506, 1457, 1401, 1336, 1252,
1179, 1116, 1035, 977, 829, 734.
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Acknowledgements
We are grateful to Dr. Anthony E. Pegg (Pennsylvania State
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by grants from the Natural Sciences and Engineering Research
Council of Canada (NSERC) and the Canada Foundation for
Innovation (CFI). L.S. is the recipient of a Bourse de maîtrise en
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technologies (FRQNT), a Concordia Graduate Scholarship in
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Keywords: O6-Alkylguanine DNA alkyltransferase, chemically
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This reports demonstrates
that fluorine at the C5
Lauralicia Sacre and Christopher J. Wilds *[a]
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position of 2’-deoxyuridine
enhances the susceptibility
of methyl adducts at the O4
position to be removed by
human AGT.
Title: Fluorine at the C5 position of 2'-deoxyuridine enhances
repair of a O4-methyl adduct by O6-Alkylguanine DNA
Alkyltransferases.
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Author(s), Corresponding Author(s)*
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[a]
Lauralicia Sacre and Christopher J. Wilds*
Department of Chemistry and Biochemistry and Centre for Structural
and Functional Genomics, Concordia University, 7141 Sherbrooke
St. West, Montréal, Québec (Canada) H4B 1R6
E-mail: chris.wilds@concordia.ca
Supporting information for this article is given via a link at the end of
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