Synthesis of oligonucleotides and thermal stability of duplexes containing the β-C-nucleoside analogue of Fapy·dG

Article abstract of DOI:10.1021/tx025588x

The formamidopyrimidine lesions (Fapy·dA, Fapy·dG) are formed in significant amounts when DNA is exposed to oxidative stress. These lesions are unusual in that they readily epimerize in solution. The distribution of configurational isomers in DNA is unkno

Full text of DOI:10.1021/tx025588x

1460
Chem. Res. Toxicol. 2002, 15, 1460-1465
Syn th esis of Oligon u cleotid es a n d Th er m a l Sta bility of
Du p lexes Con ta in in g th e â-C-Nu cleosid e An a logu e of
F a p y‚d G
Michael O. Delaney† and Marc M. Greenberg*,‡
Department of Chemistry, J ohns Hopkins University, Baltimore, Maryland 21218,
and Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523
Received J uly 31, 2002
The formamidopyrimidine lesions (Fapy‚dA, Fapy‚dG) are formed in significant amounts
when DNA is exposed to oxidative stress. These lesions are unusual in that they readily
epimerize in solution. The distribution of configurational isomers in DNA is unknown.
Nonepimerizable, nonhydrolyzable analogues are useful probes for investigating the configu-
ration of Fapy lesions in DNA and as potential enzyme inhibitors. The â-C-nucleoside of Fapy‚
dG has been prepared and introduced sight-specifically into oligonucleotides via its respective
â-cyanoethyl phosphoramidite. The phosphoramidite was prepared via a Wittig reaction
between a protected form of deoxyribose and a suitably functionalized pyrimidine. The
pyrimidine contained methyl and 2-propyl groups at the O4 and O2 positions, respectively, to
differentiate between them following C-nucleoside formation. The formamide was derived from
a nitro group at C5. The phosphoramidite coupled in 80% yield via a single 15-min coupling
using tetrazole as activator. Oligonucleotides as long as 36 nucleotides were prepared and
characterized by ESI-MS.
Sch em e 1
DNA damage is involved in aging and a variety of
diseases, such as cancer (1, 2). Nucleobase modification
is a general form of DNA damage resulting from initial
alkylation, reduction, or oxidation (3, 4). Deoxyguanosine
is the most readily oxidized of the four nucleotides that
make up DNA, and a great deal of effort has gone into
determining the effects of lesions such as OxodG on
polymerases and repair enzymes (5-7). Fapy‚dG is
formally the same oxidation state of deoxyguanosine, but
it is formed via an initial oxidative event (e.g., hydroxyl
radical addition). The formamidopyrimidines and 8-ox-
opurines are produced via a common intermediate (Scheme
1) (8). 8-Oxopurine formation is favored under oxygen-
rich conditions, but the formamidopyrimidines are fa-
vored under anoxic conditions and as a result of UV
irradiation (9, 10). Furthermore, there is at least one
instance in which Fapy‚dG formation is favored over
OxodG in cells. The Fapy‚dG:OxodG ratio is almost 3 in
human leukemia cells (11). The biological importance of
formamidopyrimidine lesions is also indicated by their
efficient removal by base excision repair (BER) enzymes
found in a variety of species, including humans (5, 12-
15). Despite the apparent importance of formamidopy-
rimidine lesions, much less is known about them than
the respective 8-oxopurines. This is largely due to the
absence of a method for synthesizing oligonucleotides
containing formamidopyrimidines until recently. Incor-
poration of lesions and their analogues at defined sites
in oligonucleotides facilitates determining their effects
on DNA structure and function. Recently, we described
methods for synthesizing oligonucleotides containing
Fapy‚dG, Fapy‚dA, and the latter lesion’s C-nucleoside
analogues (16, 17). Herein we describe the synthesis and
characterization of oligonucleotides containing â-C-Fapy‚
dG (â-1).
Nonhydrolyzable analogues of nucleosides are useful
tools for probing the interactions of DNA with repair
enzymes (18-20). Unlike most lesions, including the
chemically related 8-oxopurines, Fapy‚dG and Fapy‚dA
epimerize rapidly in aqueous solution (21, 22). The ratio
of configurational isomers in DNA is unknown, but the
â-anomer of each monomeric species is favored slightly
at room temperature. Configurationally stable analogues
of the formamidopyrimidines are particularly valuable,
because they may also provide insight regarding which
anomer(s) is (are) responsible for the impact on DNA-
protein interactions. Recently, the interactions of DNA
containing Fapy‚dA or its individual anomeric C-nucleo-
side analogues with Klenow exo- and formamidopyrimi-
dine DNA glycosylase (Fpg, MutM) were analyzed. The
kinetics of translesional synthesis and binding by Fpg
of DNA containing â-2 closely mimicked Fapy‚dA, whereas
the behavior of R-2 did not correlate with that of the true
lesion (23, 24). In fact, â-2 is a nanomolar inhibitor (KI
) 7.5 ( 1.3 nM) of Fpg excision of Fapy‚dA. Based upon
* Address correspondence to this author. E-mail: mgreenberg@
jhu.edu.
† Colorado State University.
‡ J ohns Hopkins University.
10.1021/tx025588x CCC: $22.00 © 2002 American Chemical Society
Published on Web 10/17/2002

â-C-Nucleoside Analogue of Fapy‚dG
Chem. Res. Toxicol., Vol. 15, No. 11, 2002 1461
P r ep a r a tion of 10. To a solution of 8 (5.13 g, 22.6 mmol)
and NaOAc (3.70 g, 45.2 mmol) in AcOH (100 mL) was added a
9:1 (v/v) solution of AcOH and Br2 (4.0 mL, 7.76 mmol). The
solution was heated to reflux until the red color dissipated. The
reaction was cooled to room temperature, and additional solution
(1:9, v/v) of Br2 in AcOH was added (4.0 mL, 7.76 mmol), and
the reaction was returned to reflux. After 30 min, another
portion of Br2 solution was added (5.0 mL, 9.70 mmol) in a
similar manner, and the reaction was refluxed for 30 min and
concentrated in vacuo. The resulting oil was purified by column
chromatography (100% hexanes to 2% EtOAc) to give the
monobromide product (9) that was contaminated with residual
starting material (3.32 g, 48%) and dibromide (3.91 g, 45%). 9:
1H NMR (CDCl3) δ 6.79 (s, 1 H), 5.40 (h, 1 H, J ) 6.4 Hz), 4.10
(s, 3 H), 1.45 (d, 6 H, J ) 6.4 Hz); 13C NMR (CDCl3) δ 164.04,
163.50, 159.91, 124.18, 73.66, 56.31, 33.67, 21.78; mp 83-84
°C; IR (film) 2982, 1577, 1523, 1485, 1427, 1386, 1324, 1105
cm-1. The mixture of 8 and 9 (3.32 g, 10.85 mmol) was dissolved
in benzene (20 mL), and PPh3 (3.98 g, 15.2 mmol) in benzene
(16 mL) was added dropwise. The reaction was stirred for 4.5 h
and filtered. The resulting white salt was washed with benzene
(2 × 30 mL). The phosphonium bromide was dissolved in a
biphasic mixture of CHCl3 (70 mL) and ice/water (40 mL). To
this mixture is added 3 M NaOH (3.65 mL, 11.0 mmol), and
the reaction was stirred vigorously. After 1.5 h, the aqueous
layer was extracted with CHCl3 (2 × 30 mL), and the combined
organic layers were washed with brine. The solution was
concentrated to leave 10 (4.19 g, 79% or 38% from 8) as an
these observations, it was proposed that the â-anomer
of the C-nucleoside closely represents the structure of
Fapy‚dA. In addition, the correlation between the behav-
ior of â-2 and Fapy‚dA suggested that the glycosidic
nitrogen in the lesion is not involved in specific interac-
tions with the polymerase and repair enzyme examined.
More recently, we reported that Fapy‚dG induces Klenow
exo- to misincorporate dA opposite it and extend the prior
80 million times more efficiently than does the native
purine (25). Because Fapy‚dG also epimerizes readily and
we anticipated that a nonhydrolyzable analogue of this
lesion would exhibit interesting inhibition properties,
using the above observations regarding Fapy‚dA as a
guide, we chose to prepare oligonucleotides containing
â-C-Fapy‚dG (â-1).
1
orange foam. H NMR (CDCl3) δ 7.68-7.45 (m, 15 H), 5.21 (d,
1 H, J ) 26.4 Hz), 3.99 (s, 3 H), 3.81(h, 1 H, J ) 6.0 Hz), 0.66
(d, 6 H, J ) 6.3 Hz); 13C NMR (CDCl3) δ 166.64, 161.64, 161.58,
159.40, 132.92, 132.79, 132.38, 132.01, 131.88, 129.05, 128.90,
128.49, 128.32, 126.76, 125.55, 116.85, 68.80, 57.16, 55.64, 54.61,
21.62; 31P NMR (CDCl3) δ 16.66; mp 221-223 °C; IR (film) 3059,
Exp er im en ta l Section
Gen er a l Meth od s. 1H NMR spectra were recorded at 300
or 400 MHz. The NMR spectra are referenced according to the
solvent (CDCl3, 7.27 ppm, or CD3OD, 3.31 ppm). 13C NMR were
recorded at 75 or 100 MHz and referenced according to the
solvent (CDCl3, 77.23 ppm; CD3OD, 49.15 ppm). 31P NMR
spectra were obtained at 109 MHz and referenced according to
H3PO4 as an external standard (H3PO4 ) 0 ppm). IR spectra
were obtained using an Avatar 320-FT-IR spectrophotometer.
The Central Instrument Facility at Colorado State University
performed LRMS and HRMS FAB. ESI-MS was performed on
a Thermoquest LCQ-DUO. Oligonucleotide melting studies were
performed in 1 cm path length quartz cells on a Beckman DU
640 UV-Vis spectrophotometer equipped with a thermopro-
grammer.
2976, 1560, 1519, 1435, 1394, 1310, 1256, 1114, 715 cm-1
;
HRMS (M+H) for C27H27N3O4P: calcd 488.1739, found 488.1736.
P r ep a r a tion of 11. A solution of 3′,5′-bis-O-tert-butyldim-
ethylsilyloxy-2′-deoxyribose (28) (4.48 g, 12.35 mmol) and 10
(6.02 g, 12.35 mmol) in toluene (41 mL) was heated to reflux
for 4.5 days. The solution was concentrated in vacuo and
suspended in MeOH (62 mL). The solution was stirred for 1 h
with NaOMe (0.2 g, 2.17 mmol). The reaction was filtered and
quenched by the addition of excess NH4Cl. The solution was
concentrated in vacuo and chromatographed (elution with 2%
EtOAc in hexanes to 10% EtOAc) to give a mixture of anomers
1
of 11 as an oil (6.11 g, 87%). H NMR (CDCl3) δ 5.36-5.28 (m,
1 H), 4.64-4.57 (m, 1 H), 4.36-4.31 (m, 1 H), 4.06 and 4.05
(each as s, 3 H), 3.89-3.86 and 3.81-3.77 (each as m, 1H), 3.63-
3.57 (m, 1 H), 3.52-3.42 (m, 1 H), 3.38-3.33 (m, 1 H), 3.11-
3.04 (m, 1 H), 2.91-2.76 (m, 1 H), 2.29-2.22 and 1.95-1.88
(each as m, 1 H), 1.78-1.67 (m, 1 H), 1.42 (d, J ) 6.3 Hz, 6 H),
0.91, 0.90, and 0.87 (each as s, 9H), 0.09, 0.08, 0.07, 0.06, 0.03,
and 0.02 (each as s, 6 H); 13C NMR (CDCl3) δ 164.22, 164.00,
163.58, 163.38, 163.16, 162.78, 162.67, 130.56, 130.47, 88.20,
77.93, 74.39, 73.94, 72.48, 72.30, 72.15, 72.10, 72.01, 69.84,
64.70, 63.92, 63.76, 62.66, 55.45, 55.38, 44.10, 40.52, 39.70,
39.24, 26.17, 25.98, 25.87, 21.96, 21.89, 18.55, 18.17, 18.12,
14.36, -3.36, -4.52, -4.58, -4.61, -5.22, -5.27, -5.32, -5.44;
IR (film) 2952, 2931, 1584, 1529, 1428, 1381, 1255, 1117, 838
cm-1; HRMS (M+H) for C26H50N3O7Si2: calcd 572.3187, found
572.3183.
Oligonucleotide synthesis was carried out on an Applied
Biosystems 394 DNA/RNA synthesizer using standard protocols.
N-Acetyl-protected deoxycytidine-â-cyanoethyl phosphoramidite
was purchased from Pharmacia Biotech. Phenoxyacetyl-
protected deoxyadenosine, isopropylphenoxyacetyl-protected
deoxyguanosine, and other standard phosphoramidites were
purchased from Glen Research. All other oligonucleotide syn-
thesis reagents were also obtained from Glen Research. Gel
electrophoresis on 20% polyacrylamide denaturing gels (PAGE)
afforded the purified oligonucleotide. Oligonucleotides were
precipitated from NH4OAc/EtOH prior to analysis by ESI-MS.
P r ep a r a tion of 8. A solution of 7 (30, 33) (14.59 g, 73.3
mmol) and Ag2O (32.60 g, 139.3 mmol) in 2-propanol (240 mL)
was heated to reflux. After 24 h, the solution was filtered
through a pad of Celite and concentrated. The resulting oil was
dissolved in EtOAc (100 mL) and washed with saturated
NaHCO3 (50 mL). The aqueous layer was extracted with EtOAc
(3 × 50 mL), and the combined organics were concentrated in
vacuo. The crude material was purified by column chromatog-
raphy (100% hexanes to 2% EtOAc) to give the product (8) as a
P r ep a r a tion of 12. To a solution of 11 (5.27 g, 9.22) in CH3-
CN (46 mL) was added NaI (2.07 g, 13.83 mmol) and TBDMSCl
(2.08 g, 13.83 mmol). The reaction was stirred at ambient
temperature for 2 days and diluted with EtOAc (70 mL). The
product was washed with saturated NaHSO3 (30 mL), and the
aqueous phase was extracted with EtOAc (2 × 30 mL). The
combined organic phase was washed with brine and concen-
trated. The crude product was purified by column chromatog-
raphy (elution with 100% DCM to 2% MeOH) to give 12 as a
white foam (3.43 g, 67%) and recovered starting material (11,
1.42 g, 27%). 1H NMR (CDCl3) δ 5.41-5.33 (m, 1 H), 4.63-4.56
1
white solid (7.06 g, 42%) and 7 (2.63 g, 18%). H NMR (CDCl3)
δ 5.27 (h, 1 H, J ) 6.0 Hz), 4.01 (s, 3 H), 2.42 (s, 3 H), 1.34 (d,
J ) 6.4 Hz, 6 H); 13C NMR (CDCl3) δ 163.79, 163.42, 162.48,
129.57, 72.11, 55.30, 21.76, 21.19; mp 82-84 °C; IR (film) 2982,
1581, 1519, 1428, 1378, 1316, 1113, 794 cm-1; HRMS (M+H)
for C9H14N3O3: calcd 228.0984, found 228.0985.

1462 Chem. Res. Toxicol., Vol. 15, No. 11, 2002
Delaney and Greenberg
(m, 1 H), 4.37-4.31 (m, 1 H), 3.92-3.88 and 3.82-3.76 (each
as m, 1 H), 3.63-3.57 (m, 1 H), 3.50-3.32 (m, 1 H), 3.04-2.97
(m, 1 H), 2.79-2.67 (m, 1 H), 2.29-2.24 and 1.94-1.88 (each
as m, 1 H), 1.77-1.68 (m, 1 H), 1.39 (m, 6 H), 0.91, 0.90, 0.89,
and 0.87 (each as s, 9 H), 0.10, 0.08, 0.07, 0.06, 0.04, and 0.03
(each as s, 6 H); 13C NMR (CDCl3) δ 163.98, 163.47, 163.38,
162.48, 130.68, 130,41, 87.96, 87.03, 77.54,76.90, 76.72, 74.23,
73.77, 72.06, 71.86, 63.72, 63.59, 55.21, 55.16, 40.93, 40.33,
39.49, 25.98, 25.82, 21.79, 18.35, 17.98, -4.69, -4.76, -4.80,
-5.38, -5.46; IR (film) 2951, 2926, 2851, 1579, 1425, 1324, 1111,
839 cm-1; HRMS (M+H) for C25H48N3O7Si2: calcd 558.3031,
found 558.3015.
H), 0.09, 0.08, 0.07, and 0.06 (each as s, 6 H), 0.05, 0.04, and
0.03 (each as s, 6 H); 13C NMR (CDCl3) δ 170.93, 161.39, 161.04,
154.52, 153.38, 148.59, 148.44, 144.05, 136.29, 128.06, 114.93,
88.30, 87.41, 76.10, 74.32, 73.86, 67.32, 63.88, 63.99, 41.15,
40.66, 40.55, 40.00, 33.52, 26.11, 25.98, 24.27, 18.51, 18.19,
18.11, -4.27, -4.51, -4.55, -4.64, -5.22, -5.31; IR (film) 3182,
2953, 1852, 1693, 1563, 1513, 1250, 1092, 837 cm-1; HRMS
(M+H) for C33H55N4O8Si2: calcd 691.3559, found 691.3566.
P r ep a r a tion of 15. To a solution of the above nitro amide
(0.46 g, 1.07 mmol) in 3:1 MeOH:THF (13.4 mL) was added 10%
Pd/C (230 mg). The suspension was charged with H2 (45 psi)
and allowed to stir for 1 h. The reaction was filtered through a
pad of Celite and concentrated in vacuo. The crude amine was
dissolved in 13.4 mL of THF and treated with acetic formic
anhydride (0.18 g, 2.01 mmol) and pyridine (0.21 g, 2.70 mmol)
at 0 °C. After 1.25 h, the solvent was removed and the material
purified by column chromatography (elution with 0.1% MeOH
in CH2Cl2 to 5% MeOH) to afford 15 as a white foam (0.37 g,
80%). 1H NMR (CD3OD) δ 8.24 (s, 1 H), 7.16 (d, J ) 8.8 Hz, 2
H), 6.93 (d, J ) 8.8 Hz, 2 H), 4.74 (s, 2 H), 4.53-4.49 (m, 1 H),
4.45-4.40 and 4.38-4.35 (each as m, 1 H), 3.96-3.92 and 3.84-
3.80 (each as m, 1 H), 3.68-3.64 and 3.54-3.50 (each as m, 1
H), 4.62-3.59 (m, 1 H), 3.02-2.99 and 2.90-2.84 (each as m, 1
H), 2.88-2.82 (m, 1 H), 2.72-2.68 (m, 1 H), 2.38-2.34 and 1.94-
1.90 (each as m, 1 H), 1.83-1.71 (m, 1 H), 1.21 (d, J ) 6.8 Hz,
P r ep a r a tion of 13. A solution of 12 (3.43 g, 6.16 mmol) and
NH4F (2.30 g, 61.6 mmol) in MeOH (41 mL) was heated at reflux
for 18 h. The solution was concentrated, and the product was
purified by column chromatography (elution with 2% MeOH in
CH2Cl2 to 8% MeOH) to give 13 as a white foam (1.62 g, 80%).
1H NMR (CD3OD) δ 5.27-5.19 (m, 1 H), 4.56-4.44 (m, 1 H),
4.24-4.18 (m, 1 H), 3.81-3.72 (m, 1 H), 3.61-3.46 (m, 2 H),
3.14-3.08 and 2.91-2.86 (each as m, 1 H), 2.86-2.71 (m, 1 H),
2.38-2.29 and 2.00-1.93 (each as m, 1 H), 1.87-1.71 (m, 1 H),
1.33 (d, J ) 6.0 Hz, 6 H); 13C NMR (CD3OD) δ 167.37, 166.97,
163.45, 161.78, 161.70, 135.00, 134.60, 88.67, 86.91, 78.35, 78.14,
73.90, 73.91, 71.73, 71.59, 63.92, 63.28, 41.70, 41.21, 40.75,
40.41, 22.38; IR (film) 3361, 3174, 3071, 2922, 2849, 1687, 1568,
1308, 1100, 836 cm-1; HRMS (M+H) for C13H20N3O7: calcd
330.1301, found 330.1300.
6 H), 0.90 (s, 9 H), 0.89 (s, 9 H), 0.09 and 0.06 (each as s, 6 H),
13
0.05 and 0.03 (each as s, 6 H);
C NMR (CD3OD) δ 173.24,
163.18, 163.09, 157.21, 143.86, 128.59, 115.94, 88.33, 78.47,
77.85, 75.49, 74.83, 68.46, 64.90, 64.39, 42.28, 41.71, 34.71,
26.73, 26.62, 26.59, 26,51, 24.78, 19.32, 18.99, -4.32, -4.41,
-4.96, -5.03, -5.11; IR (film) 3189, 2953, 2853, 1673, 1594,
1507, 1249, 1096, 838 cm-1; HRMS (M+H) for C34H57N4O7Si2:
calcd 689.3766, found 689.3778.
P r ep a r a tion of 14. Diol 13 (1.50 g, 4.56 mmol) in 18 mL of
MeOH was saturated with NH3 for 20 min at 0 °C. The solution
was then sealed and heated to 90 °C for 43 h. Concentration of
the solution afforded the crude aminated diol (1.42 g) as a
mixture with unreacted isopropyl ether (13). The crude material
was azeotropically dried with pyridine (3 × 5 mL) and silylated
with TBDMSCl (1.72 g, 11.4 mmol) and imidazole (1.37 g, 4.4
mmol) in DMF (23 mL) for 24 h. The reaction was concentrated
in vacuo, and the crude oil was partitioned between EtOAc (40
mL) and water (20 mL). The aqueous layer was extracted with
EtOAc (3 × 20 mL). The combined organics were washed
successively with water (15 mL), saturated NaHCO3 (15 mL),
and brine (15 mL). The organics were concentrated and purified
by column chromatography (elution with 100% CH2CL2 to 2%
MeOH) to give 14 as a foam (1.42 g, 61%) and 12 (0.72 g, 28%).
1H NMR (CDCl3) δ 4.80-4.72 and 4.78-4.62 (each as m, 1 H),
4.41-4.37 and 4.31-4.30 (each as m, 1 H), 3.96-3.94 and 3.85-
3.82 (each as m, 1 H), 3.70-3.64 (m, 1 H), 3.48-3.44 (m, 1 H),
3.17-3.07 (m, 1 H), 2.94-2.90 (m, 1 H), 2.39-2.34 and 2.01-
1.96 (each as m, 1 H), 1.82-1.72 (m, 1 H), 0.91, 0.90, 0.88, and
0.86 (each as s, 9H), 0.09, 0.08, 0.05, 0.04, 0.02, and 0.00 (each
as s, 6 H); 13C NMR (CDCl3) δ 170.66, 169.57, 157.84, 154.80,
154.65, 128.48, 87.92, 86.34, 77.38, 76.27, 73.93, 72.68, 63.90,
62.57, 41.50, 40.91, 26.20, 26.13, 26.09, 26.05, 26.00, 18.49,
18.17, 18.09, -4.14, -4.22, -4.42, 4.51, -4.56, -4.67, -5.27,
-5.31; IR (thin film) 3327, 3214, 3152, 2929, 2855, 1683, 1591,
1486, 1254, 1106, 834 cm-1; HRMS (M+H) for C22H43N4O6Si2:
calcd 515.2721, found 515.2715.
P r ep a r a tion of 16. To a solution of 15 (0.37 g, 0.54 mmol)
in THF (10.6 mL) was added Et3N‚3HF (0.85 g, 5.40 mmol).
The reaction was stirred for 24 h and concentrated to dryness.
The crude material was purified by column chromatography
(elution with 1% MeOH in EtOAc to 8% MeOH) to produce 16
as a white foam (0.21 g, 84%). 1H NMR (CD3OD) δ 8.27 and
8.25 (each as s, 1 H), 7.15-7.12 (m, 2 H), 6.93-6.69 (m, 2 H),
4.74 (s, 2 H), 4.51-4.46 (m, 1 H), 4.24-4.21 (m, 1 H), 3.93-
3.90 and 3.82-3.78 (each as m, 1 H), 3.63-3.54 (m, 2 H), 2.84-
2.76 (m, 2 H), 2.39-2.35 and 1.87-1.80 (each as m, 1 H), 2.04-
1.98 (m, 1 H), 1.76-1.70 and 1.62-1.55 (each as m, 1 H), 1.19
(d, J ) 6.8 Hz, 6 H); 13C NMR (CD3OD) d 173.32, 173.27, 167.97,
163.29, 163.20, 157.06, 150.03, 143.78, 128.54, 128.38, 117.45,
115.88, 115.57, 89.05, 87.40, 77.80, 77.70, 73.61, 73.34, 68.36,
66.22, 65.49, 63.74, 63.59, 63.41, 59.90, 52.70, 41.87, 41.76,
41.26, 34.59, 31.83, 24.73, 20.21; IR (film) 3476, 3068, 1694,
1477, 1277, 1167, 1043 cm-1; HRMS (M+H) for C22 H29N4O7:
calcd 461.2036, found 461.2033.
P r ep a r a tion of r,â-17. Diol 16 (0.10 g, 0.23 mmol) was
coevaporated with pyridine (2 × 1 mL). The material was
dissolved in pyridine (1.5 mL) and cooled to 0 °C. DMAP (3 mg,
0.03 mmol) and dimethoxytrityl chloride (0.11 g, 0.34 mmol)
were added at 0 °C in pyridine (1 mL). The reaction was stirred
for 2 h at 0 °C and allowed to warm to room temperature. The
reaction was stirred an additional 1 h, concentrated, and
purified by column chromatography (elution with 70% EtOAc
in hexanes to 2% MeOH in EtOAc) to give R,â-17 (0.13 g, 73%)
as a 3:1 (â:R) mixture of anomers. Resolution of anomers by TLC
was accomplished with 10:0.25:4 EtOAc/1-propanol/water (or-
ganic phase). The separation of anomers was facilitated by
Chromatotron (1 mm plate, 100% EtOAc) to elute 93 mg of â-17
first, followed by 32 mg of R-17. R-17: 1H NMR (CD3OD) δ 8.32
and 8.26 (each as s, 1 H), 7.44-7.40 (m, 2 H), 7.31-7.09 (m, 9
H), 6.84-6.70 (m, 6 H), 4.68-4.63 (m, 2 H), 4.60-4.52 (m, 1
H), 4.35-4.31 (m, 1 H), 4.11-4.04 (m, 1 H), 3.77-3.72 (m, 6
H), 3.10-3.07 (m, 1 H), 2.86-2.79 (m, 2 H), 2.45-2.38 (m, 1
H), 1.76-1.70 (m, 1 H), 1.65-1.57 (m, 1 H), 1.19 (d, J ) 7.2 Hz,
6 H); 13C NMR (CD3OD) δ 173.25, 163.21, 160.21, 157.15, 146.72,
143.93, 137.52, 131.43, 129.52, 128.87, 128.60, 127.92, 115.97,
P r ep a r a tion of Isop r op ylp h en oxya ceta m id e-P r otected
14. To a solution of 14 (0.48 g, 0.93 mmol), PyBOP (0.730 g,
1.40 mmol), and isopropylphenoxyacetic acid (0.27 g, 1.40 mmol)
in CH2Cl2 (9.3 mL) was added diisopropylethylamine (0.35 g,
2.78 mmol). The reaction was stirred at ambient temperature
for 6 h and partitioned between CH2Cl2 (50 mL) and saturated
NaHCO3 (20 mL). The aqueous layer was extracted with DCM
(2 × 25 mL) and washed with brine (25 mL). The solution was
concentrated and purified by column chromatography (elution
with 100% DCM to 2% MeOH) to give the amide as a white
1
foam (0.46 g, 72%). H NMR (CDCl3) δ 9.35 (br s, 1 H), 7.22 (d,
2 H, J ) 8.0 Hz), 6.92 (d, J ) 8.0 Hz, 2 H), 4.70 (s, 2 H), 4.58-
4.52 (m, 1H), 4.38-4.32 (m, 1 H), 3.92-3.89 and 3.82-3.77 (each
as m, 1 H), 3.61-3.56 (m, 2 H), 3.47-3.44 and 3.40-3.35 (each
as m, 1 H), 2.93-2.85 (m, 1 H), 2.81-2.77 and 2.68-2.65 (each
as m, 1 H), 2.29-2.24 and 1.95-1.90 (each as m, 1 H), 1.77-
1.70 (m, 1 H), 2.21 (d, J ) 2.4 Hz, 6 H), 1.25 (s, 9 H), 1.23 (s, 9

â-C-Nucleoside Analogue of Fapy‚dG
Chem. Res. Toxicol., Vol. 15, No. 11, 2002 1463
Sch em e 2a
114.19, 87.52, 86.85, 78.24, 74.25, 74.12, 68.49, 65.61, 65.58,
55.83, 41.68, 34.74, 31.94, 24.75, 20.30; IR (film) 3175, 2957,
1660, 1603, 1512, 1250, 1172, 1028, 832 cm-1; HRMS (M+H)
for C43H47N4O9: calcd 763.3343, found 763.3341. â-17: 1H NMR
(CD3OD) d 8.25 and 8.14 (each as s, 1 H), 7.43-7.37 (m, 2 H),
7.30-7.13 (m, 9 H), 6.91-6.80 (m, 6 H), 4.69-4.64 (m, 2 H),
4.56-4.51 (m, 1 H), 4.26-4.23 (m, 1 H), 3.98-3.96 (m, 1 H),
3.72 (s, 6 H), 3.17-3.14 (m, 1 H), 3.09-2.97 (m, 1 H), 2.88-
2.77 (m, 2 H), 1.97-1.93 (m, 1 H), 1.85-1.74 (m, 1 H), 1.66-
1.56 (m, 1 H), 1.20 (d, J ) 9.6 Hz, 6 H); 13C NMR (1:1 CDCl3:
CD3OD) δ 172.10, 166.48, 162.01, 159.32, 156.08, 145.70, 143.69,
136.79, 130.87, 129.36, 128.90, 128.47, 127.48, 115.44, 113.79,
87.45, 87.01, 77.18, 67.96, 65.07, 64.76, 59.66, 55.67, 41.46,
34.09, 24.56, 14.04, 8.48; IR (film) 3201, 2957, 1655, 1611, 1577,
1511, 1250, 1037, 832 cm-1. HRMS (M+H) for C43H47N4O9: calcd
763.3343, found 763.3349.
a
Key: (a) LDA + tBuOAc, then 3; (b) guanidine-HCl, Na2CO3,
EtOH.
Sch em e 3
P r ep a r a tion of â-6. A solution of â-17 (47 mg, 0.06 mmol)
was coevaporated with CH3CN (4 × 1 mL). The oil was taken
up in CH2Cl2 (1.5 mL) with diisopropylamine (6.5 mg, 0.06
mmol), and tetrazole (4.3 mg, 0.06 mmol) was added. This was
followed by addition of 2-cyanoethyl tetraisopropylphospho-
rodiamidite (24 mg, 0.08 mmol) to the reaction. The reaction
was stirred at room temperature for 5 h, and diisopropylamine
(1.4 mg, 0.014 mmol), tetrazole (0.9 mg, 0.013 mmol), and
2-cyanoethyl tetraisopropylphosphorodiamidite (3.7 mg, 0.012
mmol) were added. The reaction was allowed to stir for 12 h
and quenched with MeOH (1 mL). The reaction was concen-
trated and chromatographed on oven-dried EM-silica gel (50%
EtOAc, in hexanes with 0.1% Et3N to 0.5% MeOH in EtOAc
Sch em e 4a
a
Key: (a) PrOH, Ag2O; (b) Br2, NaOAc, AcOH; (c) i, PPh3,
benzene, ii, CHCl3, H2O, NaOH.
Resu lts a n d Discu ssion
1
with 0.1% Et3N) to give â-6 (28.2 mg, 48%). H NMR (CD3OD)
P h osph or am idite (â-6) Syn th esis. Initial approaches
for preparing 6 involved constructing the 2-amino-4-
hydroxypyrimidine ring after forming the bond between
the penultimate C-glycosidic linkage and the deoxyribose
ring. The bis-TBDMS (5a ) and 1,1,3,3-tetraisopropyld-
isiloxane (5b) nucleoside analogues were formed based
upon reported procedures for similar compounds (Scheme
2) (26-28). However, we were unable to introduce a
nitrogen atom at C5 of the pyrimidine (5a ,b) using a
variety of nitration methods, or aryldiazonium reagents.
Attempts at forming the pyrimidine ring with a forma-
mide surrogate (e.g., azide) in place also failed (data not
shown).
Consequently, we explored the possibility of modifying
our syntheses of R,â-6 in which we had already solved
the problem of preparing a C-glycoside pyrimidine con-
taining a C5-nitrogen substituent (17). Retrosynthetic
analysis led us to a 2,4-dialkoxy-6-methylene-5-nitropy-
rimidine ylide in which the alkoxy groups were different
from one another as key intermediate (Scheme 3) (29,
30). The alkoxy groups needed to be chemically distin-
guishable in order to differentiate between them later
in the synthesis. The more hindered isopropyl group was
introduced at the 2-position in order to selectively dem-
ethylate the O4-methyl group following C-glycoside for-
mation (31). The Wittig ylide was prepared from 7 in a
manner similar to that employed for the synthesis of the
respective ylide utilized for the preparation of the R,â-2
(Scheme 4) (17). Bromination was difficult to control,
producing separable mixtures of mono- and dibromopy-
rimidines. Monobromide 9 was contaminated by small
amounts of unreacted 8, which was separated from the
ylide via extraction.
δ 8.27, 8.17, and 8.26 (each as s, 1 H), 7.44-7.38 (m, 2 H), 7.32-
7.12 (m, 9H), 6.93-6.81 (m, 6 H), 4.71-4.66 (m, 2 H), 4.56-
4.52 (m, 1 H), 4.46-4.42 (m, 1 H), 4.11-4.04 (m, 1 H), 3.75-
3.59 (m, 9 H), 3.22-3.13 (m, 3 H), 2.90-2.80 (m, 3 H), 2.67 (t,
J ) 5.7 Hz, 1 H), 2.54 (t, J ) 5.7 Hz, 1 H), 1.94-1.87 (m, 1 H),
1.64-1.58 (m, 1 H), 1.23-1.09 (m, 18 H); 31P NMR (CDCl3) δ
148.41, 148.34; HRMS (M+H) for C52H64N6O10P: calcd 963.4422,
found 963.4414.
Au tom a ted Syn th esis of Oligon u cleotid es Con ta in in g
â-1. With the exception of previously reported changes to the
capping procedure, standard (Applied Biosystems 394) 1 µmol
scale cycles were employed for coupling phosphoramidites of
native nucleotides. The cycles were modified for the coupling of
â-6 so as to contain a 15-min wait time following transfer of
the phosphoramidite and tetrazole solutions to the reaction
column. Following coupling of â-6, N-methylimidazole was
removed from the capping solution.
Oligonucleotide 18 was deprotected by treatment with 0.05
M K2CO3 in MeOH (2 mL) for 3 h. Deprotection of 19 and 20
under the same conditions required 5 h for complete deprotec-
tion. The deprotections were quenched with 2 equiv of AcOH
and concentrated to dryness. Oligonucleotides were purified by
PAGE, eluted via the crush and soak method using 0.2 M NaCl,
1 mM EDTA, and desalted on a Waters C-18 Sep-pak cartridge.
DNA Meltin g Exp er im en ts. The samples for the UV-
melting studies consisted of a total concentration of 1.0-7.5 µM
of a 1:1 molar ratio of complementary oligonucleotides. The
samples were prepared by the addition of appropriate amounts
of complementary oligonucleotide stock solutions to 200 µL of
PIPES buffer (20 mM PIPES, pH 7.0, 20 mM MgCl2, 200 mM
NaCl), followed by dilution to 400 µL with water. The comple-
mentary oligonucleotides were hybridized at 90 °C for 5 min
and allowed to cool to room-temperature overnight. The absor-
bance of the samples was then monitored at 260 nm while the
temperature was increased at a rate of 0.5 °C/min over a range
of 60 °C (25-85 °C). Melting temperatures were calculated by
computer fit of the first derivative of absorbance with respect
to T-1. Thermodynamic parameters were obtained through van’t
Hoff analysis of the data (34).
An inseparable mixture of diastereomers of 11 was
obtained upon reaction of the Wittig ylide with the bis-
silyl ether of deoxyribose (Scheme 5). Based upon the
previous synthesis of the C-Fapy‚dA compounds, we
anticipated that the diastereomers would be separated

1464 Chem. Res. Toxicol., Vol. 15, No. 11, 2002
Delaney and Greenberg
Sch em e 5a
Ta ble 1. Com p a r ison of UV-Meltin g Th er m od yn a m ics of
Du p lexes Con ta in in g â-1, d G, or F a p y‚d Ga
Tm
∆H°
(kcal/mol)
∆S°
(e.u.)
∆G°298
(kcal/mol)
X:Y
(°C)b
â-1:C
46.1
40.0
37.7
39.8
54.1
51.7
45.1
44.5
57.1
45.5
44.6
46.7
70.9
51.5
51.3
39.9
77.7
80.1
79.3
68.3
106.1
83.0
58.3
83.2
194.4
137.3
136.8
101.5
210.2
219.0
221.8
187.8
294.4
233.7
156.4
232.8
13.0
10.6
10.5
9.7
15.1
14.8
13.2
12.3
18.4
13.3
11.7
13.9
â-1:A
â-1:G
â-1:T
Fapy‚dG:Cc
Fapy‚dG:Ac
Fapy‚dG:Gc
Fapy‚dG:Tc
G:Cc
a
Key: (a) i, 10, toluene, D, ii, cat. NaOMe, MeOH; (b) TBDM-
SCl, NaI, CH3CN; (c)NH4F, MeOH; (d) NH3, MeOH, 90 °C; (e)
TBDMSCl, imidazole, DMF; (f) PyBOP, 2-(4-isopropylphenyloxy-
)acetic acid, Hu¨nigs base; (g) i, H2, Pd/C, 3:1 MeOH THF; ii, acetic
formic anhydride pyridine, THF; (h) Et3N‚3HF, THF; (i) DMTCl,
cat. DMAP, pyridine; (j) 2-cyanoethyl tetrapropylphosphoramidite,
diisopropylamine, tetrazole, CH2Cl2.
G:Ac
G:Gc
G:Tc
a
Conditions: PIPES (pH 7.0), 10 mM; MgCl2, 10 mM; NaCl,
100 mM. [Duplex] ) 2.2 µM. c Data taken from ref 25.
b
Sch em e 6
and H4′ was not observed in the other, presumably
â-epimer. However, ROSEY analysis of this diastereomer
was consistent with its assignment as the â-isomer.
Phosphitylation of â-17 by the tetrazolide of 2-cyanoethyl
tetraisopropylphosphorodiamidite generated in situ pro-
ceeded in moderate yield. This transformation was also
only moderately effective in the Fapy‚dA C-nucleoside
series for reasons unknown to us (17). Despite significant
experimentation, attempts at preparing the R-C-Fapy‚
dG phosphoramidite were unsuccessful. Substrate de-
composition was observed under forcing conditions (ex-
tended reaction times, excess phosphitylation reagent);
otherwise no reaction occurred. The reluctance of R-17
to undergo phosphitylation as suggested by molecular
modeling studies on the free nucleoside (data not shown)
is attributed to steric hindrance of the 3′-hydroxyl group
by the pyrimidine ring, which is tucked under the
deoxyribose ring. The isopropylphenoxyacetamide in R-17
increases congestion in the vicinity of the 3′-hydroxyl
group further.
Oligon u cleotid e Syn th esis. Incorporation of R,â-C-
Fapy‚dA into oligonucleotides was compromised to vary-
ing degrees by modest coupling, and transacylation in
longer products ()30 nucleotides) (17). Employing the
more commonly used activator tetrazole allowed us to use
a single extended (15 min) coupling procedure to achieve
80% coupling of â-6. Furthermore, substituting pivalic
anhydride in place of acetic anhydride during the capping
procedure prevented transacylation (32). It is important
to note that N-methylimidazole is not used during
capping following coupling of the â-C-Fapy‚dG phos-
phoramidite. Using these modifications, oligonucleotides
12 (18) and 36 (19, 20) nucleotides long were prepared,
purified by denaturing PAGE, and characterized by ESI-
MS.
immediately prior to phosphitylation (17). Selective dem-
ethylation of 11 using in situ generated trialkylsilyl
iodide proved more difficult than anticipated. Extensive
desilylation of the deoxyribose protecting groups and poor
selectivity for the O4-methyl group were observed when
TMSI was used at 0 °C. These problems were overcome
by switching to TBDMSI, which masked any attack on
the deoxyribose silyl ethers, and eliminated competing
dealkylation of the O2-isopropyl group. The bulkier
reagent slowed the overall rate of the reaction signifi-
cantly, but 12 was obtained in 67% yield along with
recovered starting material (27%). Substitution of the
secondary alkoxide in 12 by ammonia required vigorous
conditions resulting in a mixture of products attributable
to unselective desilylation of the deoxyribose. No im-
provement was observed upon experimentation with
other nitrogen nucleophiles (e.g., hydrazine). Although
additional steps were introduced, displacement of the
alkoxy group was successfully performed on the desily-
lated material. The crude material obtained from the
ammonolysis was resilylated and the desired 2-amino
product (14) was purified by flash chromatography (61%),
along with resilylated starting material (12, 28%). Based
upon the syntheses of related molecules, transformation
of the diastereomeric mixture of nitro amine (14) into the
dimethoxytritylated C-nucleoside (17) was straightfor-
ward. The isopropylphenoxyacetyl protecting group was
employed to facilitate chromatography of the polar-
substituted pyrimidine.
Separation of the epimers of 17 required using a
Chromatotron centrifugal thin-layer chromatograph fol-
lowing flash chromatography. Structural assignments
1
were made by H NMR (Scheme 6). Designation of the
R-epimer was based upon observation of a NOE at H1′
when H3′ was irradiated. The expected NOE between H1′
Effect of â-C-F a p y‚d G on DNA Meltin g Th er m o-
d yn a m ics. UV-melting studies of a dodecameric duplex

â-C-Nucleoside Analogue of Fapy‚dG
Chem. Res. Toxicol., Vol. 15, No. 11, 2002 1465
tein which is frequently found in human populations. Nucleic
Acids Res. 27, 4001-4007.
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of single mutations in the OGG1 gene found in human tumors
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Substrate specificity of the Ogg1 protein of Saccharomyces
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containing â-C-Fapy‚dG in an otherwise identical se-
quence as those previously studied containing Fapy‚dG
or dG were carried out (25). Duplexes containing the
C-nucleoside analogue consistently melted at lower tem-
perature than those containing Fapy‚dG. We speculate
that this is due to the conformational differences arising
from substitution of a methylene group for a trivalent
nitrogen. The duplex containing â-C-Fapy‚dG opposite
dC was 2.4 kcal/mol more stable than any duplex
containing a mismatch, indicating that the C-nucleoside
analogue was capable of selectively forming a base pair
with the native nucleotide. The stability of duplexes
containing â-C-Fapy‚dG opposite mismatches also paral-
leled the effects observed in studies involving Fapy‚dG.
The duplex containing â-C-Fapy‚dG opposite thymidine
was the least stable, and those containing dA or dG
opposite the C-nucleoside were destabilized to a more
moderate extent (Table 1). These data are consistent with
molecular modeling studies (data not shown) and indicate
that â-1 will be a good mimic of Fapy‚dG.
Ack n ow led gm en t. We are grateful for support from
the NIH (CA-74954). We thank Dr. Chris Rithner (C.S.U.)
for assistance with NMR experiments.
Su p p or tin g In for m a tion Ava ila ble: ESI-MS of 18-20.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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Fapy‚dA induces nucleotide misincorporation translesionally by
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TX025588X