Synthesis and characterization of oligodeoxynucleotides containing an N1 β-hydroxyalkyl adduct of 2′-deoxyinosine

Article abstract of DOI:10.1021/tx010025r

Hydroxyethyl adducts arising by the reactions of simple epoxides at the N1 position of adenine nucleosides can deaminate to give the inosine analogues which, if formed in DNA, are suspected of being highly mutagenic. A method has been developed for synthesis of oligonucleotides containing N1-adducted 2′-deoxyinosines. The 2′-deoxyinosine adduct of 3,4-epoxy-1-butene was prepared from (±)-4-acetoxy-3-bromo-1-butene and tetraisopropyldisiloxanediyl-protected 2′-deoxyinosine with base. The 2′-deoxyinosine derivative was then incorporated into the oligodeoxynucleotide sequence 5′-d(CGGACXAGAAG)-3′ (X = N1-(1-hydroxy-3-buten-2-yl)-2′-deoxyinosine).

Full text of DOI:10.1021/tx010025r

746
Chem. Res. Toxicol. 2001, 14, 746-753
Syn th esis a n d Ch a r a cter iza tion of
Oligod eoxyn u cleotid es Con ta in in g a n N1 â-Hyd r oxya lk yl
Ad d u ct of 2′-Deoxyin osin e
Agnieszka Kowalczyk, Constance M. Harris, and Thomas M. Harris*
Department of Chemistry and Center in Molecular Toxicology, Vanderbilt University,
Nashville, Tennessee 37235
Received February 2, 2001
Hydroxyethyl adducts arising by the reactions of simple epoxides at the N1 position of adenine
nucleosides can deaminate to give the inosine analogues which, if formed in DNA, are suspected
of being highly mutagenic. A method has been developed for synthesis of oligonucleotides
containing N1-adducted 2′-deoxyinosines. The 2′-deoxyinosine adduct of 3,4-epoxy-1-butene
was prepared from (()-4-acetoxy-3-bromo-1-butene and tetraisopropyldisiloxanediyl-protected
2′-deoxyinosine with base. The 2′-deoxyinosine derivative was then incorporated into the
oligodeoxynucleotide sequence 5′-d(CGGACXAGAAG)-3′ (X ) N1-(1-hydroxy-3-buten-2-yl)-2′-
deoxyinosine).
Sch em e 1. Rea ction of 3,4-Ep oxy-1-bu ten e (EB)
w ith Ad en in e
In tr od u ction
In cells exposed to simple epoxides such as ethylene
oxide, 3,4-epoxy-1-butene and styrene oxide, substantial
numbers of mutations are observed at adenine sites, but
the nature of the lesions producing these mutations is
unknown (1-4). Reaction of simple epoxides with adenine
nucleosides occurs largely at the N1 position (5-8). The
N1 â-hydroxyalkyl adducts can undergo Dimroth rear-
rangement to N6 adducts (7-10). However, the resulting
N6 adducts have been shown to be relatively nonmuta-
genic (11, 12), in contrast to the N6 adducts of the more
bulky diol epoxides of polycyclic aromatic hydrocarbons
(13-16). The N1 â-hydroxyalkyl adducts can also un-
dergo a deamination reaction to form inosine derivatives,
particularly when the hydroxy group is on a primary
carbon (17, 18); Elfarra has demonstrated this process
for the C3 adduct of 3,4-epoxy-1-butene at adenine N1
(Scheme 1) (8), and Dipple (10, 19) and Koskinen (20)
for similar adducts of styrene oxide. Adduction at the
terminal carbon of the epoxide is slightly favored for both
epoxybutene and styrene oxide; for the former, deami-
nated products have only been detected for the C3 adduct,
whereas for the latter deamination of both R and â N1
dAdo adducts has been observed with the R adduct
deaminating more rapidly than the â. Watson-Crick
hydrogen bonding is disrupted in the N1-substituted
deoxyadenosine derivatives and this potentially plays an
important role in mutagenesis and carcinogenesis in-
duced by simple epoxides. Deamination introduces an
additional mutagenic feature and such deaminated N1
derivatives have been proposed to be a source of the
observed mutations at adenine sites (8, 10, 19). In this
paper, we describe a procedure for synthesis of oligode-
oxynucleotides needed to test this hypothesis. 3,4-Epoxy-
1-butene, the monoepoxide of butadiene (BD), was chosen
as the focus of the present study. The epoxide has the
potential to react at C1, C3, and C4, although only the
C3 and C4 adducts have been reported. We were par-
ticularly interested in adduct 1 resulting from reaction
at C3, because this type of adduct has been observed to
arise from the reaction of the epoxide with adenosine (8)
and has been tentatively identified in hydrolysates of CT
DNA treated with 3,4-epoxy-1-butene (21).
Exp er im en ta l P r oced u r es
Ma ter ia ls. Anhydrous solvents were either purchased from
Sigma-Aldrich in Sure/Seal bottles or distilled under nitrogen
just prior to use. Methylene chloride and pyridine were distilled
from calcium hydride and THF from sodium/potassium alloy
with benzophenone ketyl as an indicator. Other chemicals were
used as purchased without further purification. (R)-2-Hydroxy-
3-buten-1-yl p-tosylate was purchased from Acros Organics.
Ch r om a togr a p h y. Thin-layer chromatography was per-
formed with silica gel F254 (Merck) as the adsorbent on glass
plates. The chromatograms were visualized under UV (254 nm)
* To whom correspondence should be addressed. Phone and
Fax: (615) 322-2649. Fax: (615) 322-4936. E-mail: harristm@
toxicology.mc.vanderbilt.edu.
10.1021/tx010025r CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/15/2001

N1 â-Hydroxyalkyl Adduct of 2′-Deoxyinosine
Chem. Res. Toxicol., Vol. 14, No. 6, 2001 747
or by staining with an anisaldehyde/sulfuric acid solution,
followed by heating. Column chromatography was performed
using silica gel 60, 70-230 mesh (E. Merck). Oligonucleotides
were desalted via a Sephadex G-25 column using a Bio-Rad
Biologic medium-pressure chromatography system. Vacuum
centrifugation was performed on a J ouan RC10.22 instrument
equipped with a Titan vapor trap.
HPLC analyses and purifications were carried out on a
gradient HPLC (Beckman Instruments; System Gold software)
equipped with pump module 125 and photodiode array detector
module 168.
6.27 (m, 1H, H1′), 6.10 (m, 1H, CH2dCHR), 5.58 (m, 1H, allylic),
5.36 (d, 1H, cis CH2dCHR, J ) 10.5 Hz), 5.26 (d, 1H, trans
CH2dCHR, J ) 17.3 Hz), 4.47 (d, 2H, CH2-O, J ) 6.3 Hz), 4.36
(m, 1H, H3′), 3.82 (m, 1H, H4′, hidden under water peak,
confirmed with HMQC), 3.52 (m, 2H, H5′, H5′′), 2.60 (m, 1H,
H2′), 2.31 (m, 1H, H2′′), 1.89 (s, 3H, acetyl). 13C NMR (DMSO-
d6, 100.6 MHz) δ 20.7 (CH3CO), ∼40 (C2′, obscured by DMSO
signal), 55.4 (allylic), 61.7 (C5′), 70.9 (C3′), 84.0 (C1′), 88.0 (C4′),
120.6 (CH2dCHR), 123.5 (C5), 133.0 (CH2dCHR), 139.7 (C8),
147.0 (C2 or C4), 147.3 C4 or C2), 156.3 (C6), 170.8 CH3CO).
HRMS (FAB+) calcd for C16H21N4O6 (M + H)+ 365.1461, found
365.1482.
In str u m en ta tion . NMR spectra were recorded on a Bruker
AC 300 or AM 400 NMR spectrometer.
N1-(trans-4-acetoxy-2-buten-1-yl)-2′-deoxyinosine (6a ). Iso-
1
Low- and high-resolution FAB1 mass spectra were obtained
at the Mass Spectrometry Facility at the University of Notre
Dame, Notre Dame, IN. Negative ion MALDI-TOF mass spectra
of modified oligonucleotides were obtained in a 3-hydroxypico-
linic acid (3-HPA) matrix using ammonium hydrogen citrate (7
mg/mL) to suppress multiple sodium and potassium adducts.
CD spectra were recorded in methanol at 25 °C on a J ASCO
J -700 spectropolarimeter.
lated yield 5.4 mg (15%). H NMR (DMSO-d6, 400 MHz) δ 8.38
(s, 1H, H2), 8.32 (s, 1H, H8), 6.29 (m, 1H, H1′), 5.91 (m, 1H,
OCH2-CHdCH), 5.67 (m, 1H, CHdCH-CH2-N), 5.32 (d, 1H, 3′-
OH, J ) 4.0 Hz), 4.95 (t, 1H, 5′-OH, J ) 5.4 Hz), 4.63 (m, 2H,
CH2N), 4.49 (m, 2H, CH2O), 4.37 (m, 1H, H3′), 3.85 (m, 1H, H4′),
3.53 (m, 2H, H5′, H5′′), 2.61 (m, 1H, H2′), 2.31 (m, 1H, H2′′),
1.99 (s, 3H, acetyl). HRMS (FAB+) calcd for C16H21N4O6 (M +
H)+ 365.1461, found 365.1445.
N1-(1-Hydroxy-3-buten-2-yl)-2′-deoxyinosine (1), N1-(cis-4-
Hydroxy-2-buten-1-yl)-2′-deoxyinosine, and N1-(trans-4-Hydroxy-
2-buten-1-yl)-2′-deoxyinosine. The general procedure was as
follows. Acetyl groups were removed by treatment with satu-
rated methanolic ammonia for 24 h at room temperature.
Methanolic ammonia was removed under vacuum and the crude
reaction mixture was purified by reversed-phase HPLC on a
C8(2) column (250 × 10 mm, Phenomenex) with the following
gradient: (A) H2O and (B) CH3CN, 8 to 12% B over 5 min, 12
to 22% B over 10 min, 22 to 90% B over 2 min, hold for 4 min,
and then 90 to 8% B over 2 min at a flow rate of 3 mL/min.
Since 4a was not resolved from 7a , the deacetylation process
yielded two products: 1 which eluted as a diastereomeric
mixture at 12.1 and 12.4 min and N1-(cis-4-hydroxy-2-buten-
1-yl)-2′-deoxyinosine, which eluted at 9.7 min. The deacetylation
of 6a yielded N1-(trans-4-hydroxy-2-buten-1-yl)-2′-deoxyinosine,
which eluted at 8.9 min.
Capillary gel electrophoresis was performed on a Beckman
P/ACE 5000 instrument using the manufacturer’s ssDNA 100-R
gel capillary and Tris-borate-urea buffer. Samples were applied
at -10 kV and run at -10 kV at 30 °C.
Syn th esis of Mod ified Nu cleosid es a n d P h osp h or a m id -
ite. 4-Acetoxy-3-bromo-1-butene (2) (22, 23). A solution of
hydrogen bromide in glacial acetic acid (1.7 mL, 30 wt. %) was
added dropwise to a stirred mixture of 3,4-epoxy-1-butene (500
mg, 7.13 mmol) and glacial acetic acid (12.5 mL). The temper-
ature was kept at 15-20 °C during the addition. After 2 h of
stirring at room temperature, the reaction mixture was treated
with 25 mL of water followed by extraction with methylene
chloride (4 × 50 mL). The combined organic layers were washed
with water until the pH of the rinse was that of water, dried
over anhydrous Na2SO4, filtered, and concentrated under
reduced pressure. The oily, colorless residue was purified by
flash chromatography on silica gel (hexanes/CH2Cl2, 6:4) to give
506 mg of 2 (37% yield). TLC plates were visualized after
development by anisaldehyde-sulfuric acid stain. TLC Rf 0.57
(hexanes/CH2Cl2, 3:7). 1H NMR (CDCl3, 400 MHz) δ 5.95 (m,
1H, CH2dCH), 5.35 (d, 1H, J ) 16.9 Hz, CHdCH2 trans), 5.21
(d, 1H, J ) 10.1 Hz, CHdCH2 cis), 4.61 (m, 1H, allylic), 4.33
(m, 2H, CH2O), 2.09 (s, 3H, CH3CO). HRMS (FAB+) calcd for
C6H10BrO2 (M + H)+ 192.9864, found 192.9879.
N1-(1-Acetoxy-3-buten-2-yl)-2′-deoxyinosine (4a ) and N1-
(trans-4-Acetoxy-2-buten-1-yl)-2′-deoxyinosine (6a ). Compound 2
(38 mg, 0.2 mmol) was added to a suspension of 2′-deoxyinosine
(25.2 mg, 0.1 mmol) and anhydrous K2CO3 (20.4 mg, 0.15 mmol)
in anhydrous DMF (0.8 mL). After 24 h of stirring under an
argon atmosphere at room temperature, an additional equiva-
lent of 2 (22 mg, 0.1 mmol) was added to the reaction and the
mixture was allowed to react for an additional 24 h. The
products were separated by reversed-phase HPLC on a C8 (2)
column (250 × 10 mm, Phenomenex) with the following gradi-
ent: (A) H2O and (B) CH3CN, 8 to 15% B over 5 min, 15 to 20%
B over 15 min at a flow rate of 3 mL/min. UV absorbance was
monitored at 258 nm. The compounds 6a and 4a eluted at 15.4
and 17.8 min, respectively. At that time, the O6-substituted
product 5a escaped detection since it eluted at much higher
acetonitrile content than the other two products. Expanding the
gradient revealed the presence of the O6-product.
N1-(1-Hydroxy-3-buten-2-yl)-2′-deoxyinosine (1). Compound
4a (6.9 mg) afforded after purification 5.6 mg (92% yield) of
1
compound 1. H NMR (DMSO-d6, 300 MHz) δ 8.31 (s, 1H, H8),
8.30 (s, 1H, H2), 6.30 (m, 1H, H1′), 6.10 (m, 1H, CH2dCHR),
5.40 (m, 1H, allylic), 5.28 (dd, 1H, cis CH2dCHR, J 1 ) 10.6 Hz,
J 2 ) 1.3 Hz), 5.19 (dd, 1H, trans CH2dCHR, J 1 ) 17.3 Hz, J 2
)
1.3 Hz), 4.38 (m, 1H, H3′), 3.89-3.76 (m, 3H, H4′, CH2O), 3.54
(m, 2H, H5′, H5′′), 2.62 (m, 1H, H2′), 2.30 (m, 1H, H2′′). HRMS
(FAB+) calcd for C14H19N4O5 (M + H)+ 323.1355, found 323.1360.
N1-(cis-4-Hydroxy-2-buten-1-yl)-2′-deoxyinosine. 1H NMR
(DMSO-d6, 300 MHz) δ 8.38 (s, 1H, H2), 8.31 (s, 1H, H8), 6.31
(m, 1H, H1′), 5.68 (m, 1H, CHdCH), 5.45 (m, 1H, CHdCH), 4.67
(d, 2H, CH2N, J ) 6.9 Hz), 4.37 (m, 1H, H3′), 4.17 (d, 2H, CH2O,
J ) 5.5 Hz), 3.83 (m, 1H, H4′), 3.53 (m, 2H, H5′, H5′′), 2.63 (m,
1H, H2′), 2.28 (m, 1H, H2′′). HRMS (FAB+) calcd for C14H19N4O5
(M + H)+ 323.1355, found 323.1345.
N1-(trans-4-Hydroxy-2-buten-1-yl)-2′-deoxyinosine. Compound
6a (2.7 mg) afforded after purification 2.2 mg (92% yield) of N1-
(4-hydroxy-2-buten-1-yl)-2′-deoxyinosine. 1H NMR (DMSO-d6,
300 MHz) δ 8.37 (s, 1H, H2), 8.31 (s, 1H, H8), 6.29 (m, 1H, H1′),
5.73 (m, 2H, trans CHdCH), 4.60 (d, 2H, CH2N, J ) 5.0 Hz),
4.37 (m, 1H, H3′), 3.86 (m, 3H, H4′, CH2O), 3.53 (m, 2H, H5′,
H5′′), 2.57 (m, 1H, H2′), 2.28 (m, 1H, H2′′). HRMS (FAB+) calcd
for C14H19N4O5 (M + H)+ 323.1355, found 323.1345.
3′,5′-O-(1,1,3,3-Tetraisopropyl-1,3-disiloxanediyl)-2′-deoxy-
inosine (3b). 2′-Deoxyinosine (300 mg, 1.20 mmol) was dried by
coevaporation with anhydrous pyridine (3 × 10 mL) and then
redissolved in pyridine (6 mL). 1,3-Dichloro-1,1,3,3-tetraisopro-
pyldisiloxane (138 mg, 0.44 mmol) was added dropwise and the
reaction mixture was stirred at room temperature under an
argon atmosphere for 24 h. The solvents were evaporated to
dryness under vacuum and the residue was triturated with
water. The precipitate was collected and after drying, purified
by flash chromatography on silica gel (CH2Cl2/MeOH, 98:2) to
N1-(1-Acetoxy-3-buten-2-yl)-2′-deoxyinosine (4a ). Isolated yield
13.8 mg (38%). 1H NMR (DMSO-d6, 400 MHz) δ 8.34, 8.33 (s,
1H, diastereomeric H2), 8.28, 8.26 (s, 1H, diastereomeric H8),
1
Abbreviations: EB, 3,4-epoxy-1-butene; FAB, fast-atom bombard-
ment mass spectrometry; MALDI-TOF, matrix-assisted laser-desorp-
tion time-of-flight mass spectrometry; HRMS, high-resolution mass
spectrometry; HMBC, heteronuclear multibond correlation (long-range
13C-1H scalar correlated 2D NMR experiment); HMQC, heteronuclear
correlation through multiple-bond quantum coherence; COSY, cor-
relation spectroscopy.

748 Chem. Res. Toxicol., Vol. 14, No. 6, 2001
Kowalczyk et al.
give 564 mg (95% yield) of 3b. TLC Rf 0.53 (CH2Cl2/MeOH, 9:1).
1H NMR (MeOD-d4, 300 MHz) δ 8.15 (s, 1H, H8), 7.98 (s, 1H,
H2), 6.30 (m, 1H, H1′), 5.01 (m, 1H, H3′), 4.03 (m, 2H, H5′, H5′′),
2.84 (m, 1H, H2′), 2.62 (m, 1H, H2′′), 1.01 (m, 28H, isopropyl).
HRMS (FAB+) calcd for C22H39N4O5Si2 (M + H)+ 495.2459,
found 495.2447.
MeOH (0.5 mL), stirred for 15 min and concentrated under
vacuum. The oily yellow residue was purified by flash column
chromatography on silica gel (CH2Cl2:MeOH:pyridine, 98:1.5:0.5
to 96:3.5:0.5) and yielded 39.5 mg (80% yield) of the trityl-
protected nucleoside. TLC Rf 0.60 (CH2Cl2:MeOH, 9:1). 1H NMR
(CD2Cl2, 400 MHz) δ 7.93, 7.92 (s, 1H, diastereomeric H2), 7.84
(s, 1H, H8), 7.40 (m, 2H, aromatic), 7.29 (m, 7H, aromatic), 6.83
(m, 4H, aromatic), 6.34 (m, 1H, H1′), 6.05 (m, 1H, CH2dCHR),
5.78 (m, 1H, allylic), 5.45 (dd, 1H, cis CH2dCHR, J 1 ) 10.6 Hz,
J 2 ) 1.5 Hz), 5.36 (trans CH2dCHR, partially hidden under the
solvent peak), 4.63 (m, 1H, H3′), 4.46 (m, 2H, CH2O), 4.10 (m,
1H, H4′), 3.78 (s, 6H, CH3O), 3.34 (m, 2H, H5′, H5′′), 2.74 (m,
1H, H2′), 2.48 (m, 1H, H2′′), 1.98, and 1.94 (s, 3H, diastereomeric
acetyls). HRMS (FAB+) calcd for C37H39N4O8 (M + H)+ 667.2768,
found 667.2772.
N1-(1-Acetoxy-3-buten-2-yl)-3′,5′-O-(1,1,3,3-tetraisopropyl-1,3-
disiloxanediyl)-2′-deoxyinosine (4b), O6-(1-acetoxy-3-buten-2-yl)-
3′,5′-O-(1,1,3,3-tetraisopropyl-1,3-disiloxanediyl)-2′-deoxyino-
sine (5b), and N1-(trans-4-acetoxy-2-buten-1-yl)-3′,5′-O-(1,1,3,3-
tetraisopropyl-1,3-disiloxanediyl)-2′-deoxyinosine (6b). Com-
pound 2 (152 mg, 0.8 mmol) was added to a suspension of 3b
(200 mg, 0.4 mmol) and anhydrous K2CO3 (83 mg, 0.6 mmol) in
anhydrous DMF (5 mL). After 4 days of stirring under an argon
atmosphere at room temperature, the solvent was evaporated
under vacuum and the residue was suspended in anhydrous
Et2O. The filtrate was collected and Et2O was evaporated under
reduced pressure. The residue was separated by flash chroma-
tography on silica gel (hexanes/EtOAc, 6:4) to yield 84.9 mg (35%
yield) of 4b, 41.2 mg (17% yield) of 5b and 39.2 mg (16% yield)
of 6b.
N1-(1-Acetoxy-3-buten-2-yl)-3′,5′-O-(1,1,3,3-tetraisopropyl-1,3-
disiloxanediyl)-2′-deoxyinosine (4b). TLC Rf 0.49 (EtOAc/hex-
anes, 9:1). 1H NMR (DMSO-d6, 300 MHz) δ 8.30, 8.27 (s, 1H,
diastereomeric H2), 8.23, 8.22 (s, 1H, diastereomeric H8), 6.27
(m, 1H, H1′), 6.16 (m, 1H, CH2dCHR), 5.64 (m, 1H, allylic), 5.37
(d, 1H, cis CH2dCHR, J ) 10.6 Hz), 5.28 (d, 1H, trans CH2d
CHR, J ) 17.3 Hz), 4.95 (m, 1H, H3′), 4.52 (m, 2H, CH2O), 3.90
(m, 2H, H5′, H5′′), 3.80 (m, 1H, H4′), 2.82 (m, 1H, H2′), 2.57
(m, 1H, H2′′), 1.93 (s, 3H, acetyl), 1.01 (m, 28H, isopropyl).
HRMS (FAB+) calcd for C28H47N4O7Si2 (M + H)+ 607.2983,
found 607.2979.
3′-O-[(N,N-Diisopropylamino)-(2-cyanoethyl)-phosphinyl]-5′-
O-(4,4′-dimethoxytrityl)-N1-(1-acetoxy-3-buten-2-yl)-2′-deoxy-
inosine (8). 5′-O-(4,4′-Dimethoxytrityl)-N1-(1-acetoxy-3-buten-
2-yl)-2′-deoxyinosine (37.6 mg, 0.06 mmol) was dried by co-
evaporation with anhydrous pyridine (4 × 5 mL) and placed
under vacuum overnight. Anhydrous 1H-tetrazole (5.1 mg, 0.07
mmol) was added to a flame-dried flask and kept under vacuum
overnight. Freshly distilled CH2Cl2 (2 mL) was added to the
flask containing the tetrazole and the resulting solution was
transferred to the flask containing tritylated nucleoside, followed
by addition of 2-cyanoethyl-N,N,N′,N′-tetraisopropylphospho-
rodiamidite (25.3 mg, 0.08 mmol). After 3 h, the solvents were
removed under reduced pressure and the oily residue was left
briefly under vacuum. It was purified by flash column chroma-
tography on silica gel (EtOAc:hexanes:pyridine, 90:9:1) to yield
46.8 mg (95% yield) of the phosphitylated nucleoside 8. TLC Rf
1
0.58, 0.50 (EtOAc). H NMR (CD2Cl2, 300 MHz) δ 7.93, 7.92 (s,
O6-(1-Acetoxy-3-buten-2-yl)-3′,5′-O-(1,1,3,3-tetraisopropyl-1,3-
disiloxanediyl)-2′-deoxyinosine (5b). TLC Rf 0.68 (EtOAc/hex-
anes, 9:1). 1H NMR (DMSO-d6, 300 MHz) δ 8.49 (s, 1H, H8),
8.43 (s, 1H, H2), 6.36 (m, 1H, H1′), 6.17 (m, 1H, allylic), 6.03
(m, 1H, CH2dCHR), 5.39 (d, 1H, trans CH2dCHR, J ) 17.3 Hz),
5.29 (d, 1H, cis CH2dCHR, J ) 10.6 Hz), 5.13 (m, 1H, H3′),
4.35 (m, 2H, H5′, H5′′), 3.90 (m, 2H, CH2O), 3.81 (m, 1H, H4′),
2.91 (m, 1H, H2′), 2.59 (m, 1H, H2′′), 1.96 (s, 3H, acetyl), 1.01
(m, 28H, isopropyl). HRMS (FAB+) calcd for C28H47N4O7Si2 (M
+ H)+ 607.2983 found, 607.2965.
1H, diastereomeric H2), 7.89, 7.97 (s, 1H, diastereomeric H8),
7.40 (m, 2H, aromatic), 7.28 (m, 7H, aromatic), 6.80 (m, 4H,
aromatic), 6.33 (m, 1H, H1′), 6.05 (m, 1H, CH2dCHR), 5.78 (m,
1H, allylic), 5.45 (d, 1H, cis CH2dCHR, J ) 10.6 Hz), 5.36 (trans
CH2dCHR, partially hidden under the solvent peak), 4.73 (m,
1H, H3′), 4.46 (m, 2H, CH2O), 4.28 (m, 1H, H4′), 4.12 (m, 2H,
H5′, H5′′), 3.78 (s, 3H, CH3O), 3.77 (s, 3H, CH3O), 3.50 (m, 2H,
isopropyl CH), 3.46 (m, 2H, POCH2), 2.72 (m, 2H, H2′, CH2-
CN), 2.63 (m, 2H, H2′′, CH2CN), 1.97 and 1.92 (s, 3H, diaster-
eomeric acetyls), 1.21 (m, 12H, isopropyl CH3). 31P NMR
(CD2Cl2, 121 MHz) δ 149.8 (product) and 14.48 (hydrolyzed
material). HRMS (FAB+) calcd for C46H56N6O9P (M + H)+
867.3846, found 867.3874.
N1-(trans-4-Acetoxy-2-buten-1-yl)-3′,5′-O-(1,1,3,3-tetraisopro-
pyl-1,3-disiloxanediyl)-2′-deoxyinosine (6b). TLC Rf 0.34 (EtOAc/
hexanes, 9:1). 1H NMR (DMSO-d6, 300 MHz) δ 8.30 (s, 1H, H2),
8.21 (s, 1H, H8), 6.26 (m, 1H, H1′), 5.91 (m, 1H, OCH2CH)CH),
5.68 (m, 1H, CHdCHCH2N), 4.97 (m, 1H, H3′), 4.66 (m, 2H,
CH2O), 4.49 (m, 2H, CH2N), 3.90 (m, 2H, H5′, H5′′), 3.80 (m,
1H, H4′), 2.82 (m, 1H, H2′), 2.57 (m, 1H, H2′′), 1.99 (s, 3H,
acetyl), 1.01 (m, 28H, isopropyl). HRMS (FAB+) calcd for
N1-(1-Hydroxy-3-buten-2(S)-yl)-2′-deoxyinosine (S-1) and N1-
(2(R)-Hydroxy-3-buten-1-yl)-2′-deoxyinosine (10). (R)-2-Hydroxy-
3-buten-1-yl p-tosylate (100 mg, 0.41 mmol) was dissolved in
freshly distilled DMSO (0.7 mL) and NaH (20 mg of 60%
suspension in oil, 0.49 mmol) was added in one portion. After
15 min, the temperature was raised slowly to 80 °C, and the
reaction was then allowed to cool to room temperature. Anhy-
drous K2CO3 (83 mg, 0.60 mmol) and 2′-deoxyinosine (3a , 100
mg, 0.40 mmol) were added and the reaction mixture was stirred
for 5 days at 45 °C. The products were separated by reverse-
phase HPLC on a C8(2) column, (250 × 10 mm, Phenomenex)
with the following gradient: (A) H2O and (B) CH3CN, 8 to 10%
B over 5 min, 10 to 14% B over 10 min, hold at 14% for 1 min,
14 to 90% over 2 min, hold at 90% for 1 min, and then 90 to 8%
over 2 min at a flow rate of 3 mL/min. UV absorbance was
monitored at 258 nm. S-1 and 10 eluted at 14.5 and 12.4 min,
respectively.
N1-(1-Hydroxy-3-buten-2(S)-yl)-2′-deoxyinosine (S-1). 1H NMR
(DMSO-d6, 400 MHz) δ 8.32 (s, 1H, H8), 8.31 (s, 1H, H2), 6.30
(m, 1H, H1′), 6.10 (m, 1H, CH2dCHR), 5.40 (m, 1H, allylic), 5.28
(dd, 1H, cis CH2dCHR, J 1 ) 10.6 Hz, J 2 ) 1.2 Hz), 5.19 (d, 1H,
trans CH2dCHR, J ) 17.3 Hz), 4.38 (m, 1H, H3′), 3.90-3.73
(m, 3H, H4′, CH2O), 3.54 (m, 2H, H5′, H5′′), 2.63 (m, 1H, H2′),
2.30 (m, 1H, H2′′). HRMS (FAB+) calcd for C14H19N4O5 (M +
H)+ 323.1357, found 323.1367. Isolated yield 1.8 mg (1%). NMR
signals for the contaminating sodium salt of p-toluenesulfonic
acid were observed at 2.27, 7.10, and 7.46 ppm.
C
28H47N4O7Si2 (M + H)+ 607.2983, found 607.3009.
N1-(1-Acetoxy-3-buten-2-yl)-2′-deoxyinosine (4a) via the Di-
siloxane Pathway. Tetrabutylammonium fluoride (1 M solution
in THF, 99 µL) was added dropwise to a solution of 4b (60 mg,
0.1 mmol) in freshly distilled THF (1.5 mL), and the solution
was stirred at room temperature for 5 min. The reaction was
quenched with 5% acetic acid (1 mL) and then purified by
reversed-phase HPLC on
a
C8(2) column (250 × 10 mm,
Phenomenex) with the following gradient: (A) H2O and (B)
CH3CN, 8 to 15% B over 5 min, 15 to 20% B over 16 min, 20 to
90% B over 2 min, hold for 4 min, and then 90 to 8% B over 2
min at a flow rate of 3 mL/min. The product eluted at 17.7 min.
A total of 27.5 mg (75% yield) of 4a was isolated. HRMS (FAB+)
calcd for C16H21N4O6 (M + H)+ 365.1461 found, 365.1445. 1H
NMR same as described above for 4a .
5′-O-(4,4′-Dimethoxytrityl)-N1-(1-acetoxy-3-buten-2-yl)-2′-deoxy-
inosine. Compound 4a (27.5 mg, 0.075 mmol) was dried by
coevaporation with anhydrous pyridine (4 × 5 mL) and then
redissolved in 2 mL of pyridine. 4,4′-Dimethoxytrityl chloride
(56 mg, 0.16 mmol) was added in three portions over the course
of 5 days. The reaction mixture was kept at room temperature
under an argon atmosphere. The reaction was quenched with

N1 â-Hydroxyalkyl Adduct of 2′-Deoxyinosine
Chem. Res. Toxicol., Vol. 14, No. 6, 2001 749
N1-(2(R)-Hydroxy-3-buten-1-yl)-2′-deoxyinosine (10). 1H NMR
(DMSO-d6, 400 MHz) δ 8.30 (s, 1H, H2), 8.24 (s, 1H, H8), 6.29
(m, 1H, H1′), 5.89 (m, 1H, CH2dCHR), 5.17 (d, 1H, trans CH2d
CHR, J ) 17.2 Hz), 5.12 (d, 1H, cis CH2dCHR, J ) 10.5 Hz),
4.38 (m, 1H, H3′), 4.20 (m, 1H, allylic), 4.18 (dd, 1H, CH2N, J 1
) 13.2 Hz, J 2 ) 3.8 Hz), 3.85 (m, 1H, H4′), 3.76 (dd, 1H, CH2N,
J 1 ) 13.2 Hz, J 2 ) 8.5 Hz), 3.53 (m, 2H, H5′, H5′′), 2.62 (m, 1H,
H2′), 2.27 (m, 1H, H2′′). HRMS (FAB+) calcd for C14H19N4O5
(M + H)+ 323.1357, found 323.1353. Isolated yield 23.4 mg
(17%). NMR signals for the contaminating sodium salt of
p-toluenesulfonic acid were observed at 2.26, 7.10 and 7.46 ppm.
P r ep a r a tion of Mod ified Oligon u cleotid es. Oligonucleo-
tides (1 µmol scale) were prepared on an Expedite 8909 Nucleic
Acid Synthesizer using tert-butylphenoxyacetyl-protected 2-cy-
anoethyl phosphoramidites and adducted phosphoramidite 8.
Depr otection an d P u r ification of Modified Oligon u cleo-
tid es. Modified oligonucleotides were cleaved from the beads
and deprotected (including the acetyl protection on the adduct
moiety) in aqueous ammonia for 16 h at room temperature and
purified by reverse-phase HPLC on a C8(2) column (250 × 10
mm, Phenomenex) with the following gradient: (A) 0.1 M
ammonium formate and (B) CH3CN, 1 to 4% B over 5 min, 4 to
6.6% B over 30 min, 6.6 to 90% B over 3 min, hold for 1 min,
and then 90 to 1% B over 2 min at a flow rate of 3 mL/min; the
diastereomeric oligonucleotides eluted at 25.4 (9a ) and 28.9 min
(9b). Mass spectrum: (MALDI-TOF) m/z calcd for [M - H]-
3468.7, found 3469.9 (9a ) and 3468.4 (9b).
Sch em e 2. Syn th esis of O-P r otected
N1-(1-Hyd r oxy-3-bu ten -2-yl) 2′-Deoxyin osin e (4a
a n d 4b) a n d Rela ted Com p ou n d s
En zym a tic Hyd r olysis. Enzymatic digestion mixtures (0.2-
0.5 A260 units of oligonucleotide, 20 µL of 0.1 M Tris HCl buffer,
pH 9.0, 0.04 units of snake venom phosphodiesterase, 0.4 units
of alkaline phosphatase) were incubated at 37 °C overnight, and
subsequently analyzed by HPLC on a C8(2) column (250 × 4.6
mm, Phenomenex) with the following gradient: (A) 0.1 M
ammonium formate and (B) CH3CN, 1 to 10% B over 15 min,
10 to 20% B over 5 min, hold for 5 min, and then to 100% B
over 10 min at a flow rate of 1 mL/min.
Meltin g Stu d ies. Modified oligonucleotides and the comple-
mentary strand (0.5 A260 units each) were dissolved in 1 mL of
melting buffer (10 mM Na2HPO4/NaH2PO4, 1.0 M NaCl, 50 µM
Na2EDTA, pH 7.0). The sample vials were heated to 85 °C and
allowed to cool to room temperature. UV measurements were
taken at 1 min intervals with a 1 °C/min temperature gradient
with observation at 260 nm. The temperature was raised from
5 to 90 °C. Both S (9a ) and R (9b) diastereomers had Tm ) 33
°C, while the wild-type duplex had Tm ) 55 °C.
Resu lts a n d Discu ssion
protection of the hydroxyl group was needed for the
remaining steps of the oligonucleotide synthesis.
The most effective strategy for preparation of oligo-
nucleotides containing this lesion involves preparation
of the adducted nucleoside and then incorporation of it
into the oligonucleotide. The hydroxyl group in the side-
chain requires protection for the strategy to be compatible
with the phosphoramidite chemistry used in DNA syn-
thesis.
Under the basic conditions employed in this study the
anion formed by deprotonation of N1 becomes the main
site of reaction with electrophiles (24). Consequently, the
reaction of 4-acetoxy-3-bromo-1-butene (2) with 2′-deoxy-
inosine (3a ) was examined (Scheme 2). The reaction was
carried out under basic conditions and yielded four
products: the desired one (4a ) arising from attack of N1
of the nucleoside at C3 of the bromoalkene, the O-alkyl
derivative (5a ) arising from O6 of the ambident anion of
the nucleoside reacting with C3 of the alkene, and trans
and cis adducts 6a and 7a arising by SN2′ attack of N1
at C1 of the alkene. Compounds 5a and 6a were readily
isolated by HPLC, but 4a and 7a coeluted; 7a represented
about a 10% contaminant in 4a . The mixture could be
resolved after removal of the acetyl groups but acyl
As a possible solution to this problem, 2′-deoxyinosine
was protected as the 3′,5′-O-(1,1,3,3-tetraisopropyl-1,3-
disiloxanediyl) derivative prior to treatment with 2.
Again, the reaction yielded four products (4b-7b) with
7b coeluting with 4b. After removal of the tetraisopro-
pyldisiloxanediyl group and the acetyl group, HPLC
showed the contamination of 4b by 7b was substantially
less than in the previous reaction and was sufficiently
small that it could be neglected. The yields of 4b-7b were
33, 17, 16, and <2%, respectively. On this basis, the
separation of 4b, 5b, and 6b could be carried out by
column chromatography on silica gel. It should be noted
that 4b and 5b were both irresolvable mixtures of
diastereomers. However, two sets of signals for H2 and
H8 were discernible in the 1H NMR spectrum of 4b.
Likewise, the diastereomers of 4a and those of 5a were
unresolved, although two sets of signals for H2, H8, and
1
H1′ were visible in the H NMR spectrum of 4a .
Assignments of product structures were made by 2-D
NMR experiments (COSY, HMQC, and HMBC). Struc-
tural assignments for 4-6 were made primarily on the

750 Chem. Res. Toxicol., Vol. 14, No. 6, 2001
Kowalczyk et al.
Sch em e 3. Syn th esis of 11-m er
Oligod eoxyr ibon u cleotid es Con ta in in g
N1-(1-Hyd r oxy-3-bu ten -2(S)-yl)-2′-d eoxyin osin e (9a )
a n d N1-(1-Hyd r oxy-3-bu ten -2(R)-yl)-2′-d eoxyin osin e
(9b)
F igu r e 1. HMBC spectrum of compound 4b. The 13C spectrum
is shown on the vertical axis and the 1H spectrum on the
horizontal. Observation of long-range coupling between carbons
2 and 6 and the allylic proton on the side chain allows the site
of attachment of the side chain to be assigned as N1 (see
discussion in text).
Compound 4b was used without further purification.
To prepare a phosphoramidite reagent suitable for use
in an automated DNA synthesizer, tetraisopropyldi-
siloxanediyl protection was removed with tetrabutyl-
ammonium fluoride to give 4a . The 5′ hydroxyl group
was protected with the 4,4′-dimethoxytrityl group and
the 3′ hydroxyl group phosphitylated with 2-cyanoethyl-
N,N,N′,N′-tetraisopropylphosphorodiamidite to give phos-
phoramidite 8.
silyl-protected species 4b, 5b, and 6b; however, the
spectra of the unprotected analogues 4a , 5a , and 6a were
fully concordant with the assigned structures. The site
of substitution on the purine ring was determined by
HMBC experiments. The N1 substitution pattern for 4b
was elucidated from cross-peaks between H2 of the
purine and the allylic carbon of the butadiene moiety and
in some cases C2 of the purine and the allylic proton of
the substituent (Figure 1). The correlation between C6
of the purine and the allylic proton of the substituent
excluded the possibility of 4b being the N3 adduct. A
similar approach was used to confirm the site of substi-
tution of 6b. Compound 5b was assigned as the O6-
substituted species. Correlations indicating N1 and N3
substitution were not present for 5b. The expected
correlation between C6 of the purine ring and the allylic
proton of the substituent was not observed, most likely
due to the broadness of the allylic proton signal; however,
the O6-substitution pattern was deduced from the fol-
The phosphoramidite reagent 8 was then incorporated
into an oligonucleotide having the sequence 5′-d(CG-
GACXAGAAG)-3′, where X represents N1-(1-hydroxy-3-
buten-2-yl)-2′-deoxyinosine (Scheme 3). Synthesis of the
oligonucleotide was performed on a 1 µmol scale. The
dGuo, dAdo, and dCyt phosphoramidite reagents and the
initial residue on the CPG beads employed tert-butylphe-
noxyacetyl protection on the exocyclic amino groups.
Cleavage of the oligomer from the solid support, depro-
tection of the normal nucleosides, and removal of the
acetyl group from the adducted nucleoside could be
accomplished in a single step using either 0.1 M NaOH
at room temperature for 2 days or ammonium hydroxide
at room temperature for 16 h. The two protocols were
judged to be equally satisfactory, but ammonia treatment
was experimentally more straightforward. The mixture
contained diastereomeric adducted oligomers of the
desired sequence (9a and 9b) together with failure
sequences (Figure 2). The oligonucleotide derived from
7a was presumed to be present also but was not detected;
possibly, it lay under one of the failure sequences. The
diastereomeric mixture of adducted oligonucleotides was
readily separated by HPLC. This circumvented having
to repeat the synthesis with the individual enantiomers
of the alkylating agent in order to obtain oligonucleotides
containing the individual stereoisomers of the adduct.
1
lowing observations: (i) The H NMR spectra of 5b and
4b were almost identical except the chemical shift of the
allylic proton 5b was shifted downfield compared to 4b,
which is consistent with the stronger deshielding effect
of oxygen compared to nitrogen. (ii) Compounds 5a ,b
were more lipophilic than the N1 adducts 4a ,b and 6a ,b.
(iii) N1 Alkyl derivatives 4b, 6b, and 7b had identical
UV spectra whereas the spectrum of the O6-alkyl deriva-
tive 5b was clearly distinguishable. These observations
are consistent with the NMR assignments and provided
additional evidence that the structures were assigned
correctly.

N1 â-Hydroxyalkyl Adduct of 2′-Deoxyinosine
Chem. Res. Toxicol., Vol. 14, No. 6, 2001 751
F igu r e 2. HPLC chromatograms showing the product mixture
obtained upon deprotection of oligonucleotides synthesized with
phosphoramidite 8. Peaks were identified by MALDI-TOF mass
spectrometry and enzyme digestion. Peaks A-F are failure
sequences and peaks 9a and 9b are the desired full-length
adducted oligonucleotides. HPLC conditions are described in the
Experimental Section of the text.
The modified oligonucleotides were purified on reverse-
phase HPLC, providing ∼12 A260 units of each diastereo-
mer from a 1 µmol cassette.
Mass spectra (MALDI) confirmed the molecular weights
of the diastereomeric oligonucleotides. The adducted
oligonucleotides were characterized by enzymatic diges-
tion to individual nucleosides, followed by identification
of the adducts via HPLC coelution with authentic stan-
dards (Figure 3). Nucleoside composition was quantified
by dividing each peak area from the HPLC profile of the
enzyme digest by the appropriate molar extinction coef-
ficient (25). The molar extinction coefficient of the modi-
fied nucleoside was assumed to be the same as that of
1-methylinosine (26). Melting transitions for duplexes
formed between the 11-mers and their complements were
determined. Whereas the duplex containing the native
oligodeoxynucleotide had a Tm of 55 °C, the duplexes
containing the inosine adducts were strongly destabilized
with a Tm value of 33 °C for both 9a and 9b.
F igu r e 3. HPLC profile of products obtained from enzymatic
digestion of purified adducted oligonucleotides. Inserted boxes
show relative nucleoside compositions (calculated and found)
for each oligonucleotide. (A) (lower trace) digest of 9a ; (upper
trace) nucleoside 1 (diastereomeric mixture); (B) (lower trace)
digest of 9b; (upper trace) nucleoside 1 (diastereomeric mixture).
Conditions of hydrolysis and chromatography are described in
the text.
To assign the configuration of the diastereomers, the
2(S) form of 1 was synthesized from the commercially
available 2(R)-hydroxy-3-buten-1-yl tosylate (Scheme 4).
The tosylate was converted into the (R)-3,4-epoxy-1-
butene which, in turn, was used to alkylate 2′-deoxy-
inosine. A one-pot approach was employed to avoid
isolation of the volatile epoxide. An initial attempt
involved treatment of the tosylate with 2′-deoxyinosine
and K2CO3 in anhydrous DMF. The condensation pro-
ceeded slowly to give only the direct displacement
product, 1-(2(R)-hydroxy-3-butenyl)-2′-deoxyinosine (10).
The reaction was repeated using a stoichiometric amount
of NaH in DMSO to ensure conversion of the tosylate
to the epoxide (27), followed by addition of anhydrous
K2CO3 and 2′-deoxyinosine when the tosylate was no
longer present in the reaction mixture (as shown by TLC).
The reaction yielded two products in a 10:1 ratio. They
were purified by reversed-phase HPLC and characterized
by NMR and MS. The major product (10) involved attack
at the terminal C4 position of the epoxide and the minor
one (S-1) resulted from attack at C3. The minor product
coeluted on HPLC with the slower moving of the dia-
stereomers of 1 formed by deacetylation of 4a and had
the same 1H NMR (Figure 4). Since the reactions by
which the adducts were formed are SN2 processes, the R
configuration of the tosylate became an S configuration
in S-1. The HPLC retention time of compound S-1 was
identical to that of the nucleoside arising from the faster
eluting oligonucleotide (9a ). The CD spectrum of S-1
showed an enhanced negative Cotton effect at 250 nm
compared to 2′-deoxyinosine.
In conclusion, we have developed a strategy to syn-
thesize oligodeoxynucleotides containing site-specific

752 Chem. Res. Toxicol., Vol. 14, No. 6, 2001
Kowalczyk et al.
Ack n ow led gm en t. We thank Pamela J . Tamura and
Amanda S. Wilkinson for assistance with oligonucleotide
syntheses, CD spectra and melting temperature data.
The project described was supported by the National
Institutes of Health (Grants ES05355, ES00267, and
ES07781).
Su p p or tin g In for m a tion Ava ila ble: NMR spectra of
nucleosides, circular dichroism spectra of 3a and S-1, and
capillary gel electropherograms, MALDI-TOF mass spectra, and
melting curves for adducted oligonucleotides 9a and 9b. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Refer en ces
F igu r e 4. 1H NMR spectrum (DMSO-d6, 300 MHz) of N1-(1-
hydroxy-3-buten-2-yl)-2′-deoxyinosine (1).
(1) Cochrane, J . E., and Skopek, T. R. (1994) Mutagenicity of
butadiene and its epoxide metabolites: II. mutational spectra of
butadiene, 1,2-epoxybutene and diepoxybutane at the hprt locus
in splenic T cells from exposed B6C3F1 mice. Carcinogenesis 15,
719-723.
(2) Recio, L., and Meyer, K. G. (1995) Increased frequency of
mutations at A: T base pairs in the bone marrow of B6C3F1 lacI
transgenic mice exposed to 1,3-butadiene. Environ. Mol. Mutagen.
26, 1-8.
Sch em e 4. Syn th esis of
N1-(1-Hyd r oxy-3-bu ten -2(S)-yl)-2′-d eoxyin osin e a n d
N1-(2(R)-Hyd r oxy-3-bu ten -1-yl)-2′-d eoxyin osin e
(3) Recio, L., Sisk, S., Meyer, K., Pluta, L., and Bond, J . A. (1996)
Mutagenicity and mutational spectra of 1,3-butadiene in the bone
marrow of B6C3F1 lacI transgenic mice. Toxicology 113, 106-
111.
(4) Bastlova, T., and Podlutsky, A. (1996) Molecular analysis of
styrene oxide-induced hprt mutation in human T-lymphocytes.
Mutagenesis 11, 581-591.
(5) Windmueller, H. G., and Kaplan, N. O. (1961) The preparation
and properties of N-hydroxyethyl derivatives of adenosine, ad-
enosine triphosphate, and nicotinamide adenine dinucleotide. J .
Biol. Chem. 236, 2716-2726.
(6) Savela, K., Hesso, A., and Hemminki, K. (1986) Characterization
of reaction products between styrene oxide and deoxynucleosides
and DNA. Chem.-Biol. Interact. 60, 235-246.
(7) Qian, C., and Dipple, A. (1995) Different mechanisms of aralkyl-
ation of adenosine at the 1- and N6-positions. Chem. Res. Toxicol.
8, 389-395.
(8) Selzer, R. R., and Elfarra, A. A. (1996) Characterization of N1-
and N6-adenosine adducts and N1-inosine adducts formed by the
reaction of butadiene monoxide with adenosine: evidence for the
N1-adenosine adducts as major initial products. Chem. Res.
Toxicol. 9, 875-881.
(9) Solomon, J . J ., Mukai, F., Fedyk, J ., and Segal, A. (1988) Reactions
of propylene oxide with 2′-deoxynucleosides and in vitro with calf
thymus DNA. Chem.-Biol. Interact. 67, 275-294.
(10) Barlow, T., Takeshita, J ., and Dipple, A. (1998) Deamination and
Dimroth rearrangement of deoxyadenosine-styrene oxide adducts
in DNA. Chem. Res. Toxicol. 11, 838-845.
(11) Carmichal, J . R., Nechev, L. V., Harris, C. M., Harris, T. M., and
Lloyd, R. S. (2000) Mutagenic potential of adenine N6 adducts of
monoepoxide and diolepoxide derivatives of butadiene. Environ.
Mol. Mutagen. 35, 48-56.
adducts at the N1 position of 2′-deoxyinosine. This
strategy should be readily extendable to other N1 adducts
such as the C4 adduct of 3,4-epoxy-1-butene and the
analogous styrene oxide adducts. In ongoing studies in
this laboratory, similar conditions (NaOH in dimethyl-
acetamide) are being used to alkylate the N1 position of
2′-deoxyguanosine. With alkyl halides that have only a
single site for reaction with nucleophiles, the method has
given excellent yields of the N1 dGuo adduct. The
example described here for the adduct of 3,4-epoxy-1-
butene is the first site-specific synthesis of an oligode-
oxynucleotide containing this type of lesion at a purinic
site. Hydroxyalkyl adducts at cytosine N3 are also prone
to deamination to give potentially mutagenic uracil
derivatives; an oligonucleotide containing a site-specific
3-hydroxyethyldeoxyuridine has been synthesized by
Bhanot et al. (28) by a route similar to the one reported
herein.
(12) Latham, G. J ., Zhou, L., Harris, C. M., Harris, T. M., and Lloyd,
R. S. (1993) The replication fate of R- and S-styrene oxide adducts
on adenine N6 is dependent on both the chirality of the lesion
and the local sequence context. J . Biol. Chem. 268, 23427-23434.
(13) Lavrukhin, O. V., and Lloyd, R. S. (1998) Mutagenic replication
in a human cell extract of DNAs containing site-specific and
stereospecific benzo(a)pyrene-7,8-diol-9,10-epoxide DNA adducts
placed on the leading and lagging strands. Cancer Res. 58, 887-
891.
(14) Remington, K. M., Bennett, S. E., Harris, C. M., Harris, T. M.,
and Bebenek, K. (1998) Highly mutagenic bypass synthesis by
T7 RNA polymerase of site-specific benzo[a]pyrene diol epoxide-
adducted template DNA. J . Biol. Chem. 273, 13170-13176.
(15) Min, Z., Gill, R. D., Cortez, C., Harvey, R. G., Loechler, E. L.,
and DiGiovanni, J . (1996) Targeted A f T and G f T mutations
induced by site-specific deoxyadenosine and deoxyguanosine
adducts, respectively, from the (+)-anti-diol epoxide of dibenz[a,
j]anthracene in M13mp7L2. Biochemistry 35, 4128-4138.
(16) Buterin, T., Hess, M. T., Luneva, N., Geacintov, N. E., Amin, S.,
Kroth, H., Seidel, A., and Naegeli, H. (2000) Unrepaired fjord
region polycyclic aromatic hydrocarbon-DNA adducts in ras codon
61 mutational hot spots. Cancer Res. 60, 1849-1856.
(17) Fujii, T., Saito, T., and Terahara, N. (1986) Purines. XXVII.
Hydrolytic deamination versus Dimroth rearrangement in the
The mutagenicity of the N1 â-hydroxyalkyl adducts of
deoxyinosine is currently under investigation. It is pos-
sible that such adducts may be strongly blocking or
readily repaired; to the extent that they are bypassed, it
is likely that an incorrect base will be incorporated.

N1 â-Hydroxyalkyl Adduct of 2′-Deoxyinosine
Chem. Res. Toxicol., Vol. 14, No. 6, 2001 753
9-substituted adenine ring: Effect of an ω-hydroxyalkyl group
at the 1-position. Chem. Pharm. Bull. 34, 1094-1107.
enon in the homologous series of the unsaturated R-oxides. J . Gen.
Chem. USSR 11, 991-995 [Chem. Abstr. (1943) 37, 1699].
(24) J ones, J . W., and Robins, R. K. (1963) Purine nucleosides. III.
Methylation studies of certain naturally occurring purine nucleo-
sides. J . Am. Chem. Soc. 85, 193-201.
(25) Miller, P. S., Cheng, D. M., Dreon, N., J ayaraman, K., Kan, L.
S., Leutzinger, E. E., Pulford, S. M., and Ts’o, P. O. (1980)
Preparation of a decadeoxyribonucleotide helix for studies by
nuclear magnetic resonance. Biochemistry 19, 4688-4698.
(26) Miles, H. T. (1961) The preparation and characterization of
1-methylinosine. J . Org. Chem. 26, 4761-4762.
(18) Saito, T., Murikami, M., Inada, T., Hayashibara, H., and Fujii,
T. Purines. LIII. Deamination of 1-(ω-hydroxyalkyl)adenine
derivatives by nucleophiles. Chem. Pharm. Bull. 40, 3201-3205.
(19) Barlow, T., Ding, J ., Vouros, P., and Dipple, A. (1997) Investiga-
tion of hydrolytic deamination of 1-(2-hydroxy-1-phenylethyl)-
adenosine. Chem. Res. Toxicol. 10, 1247-1249.
(20) Koskinen, M., and Hemminki, K. (1999) Separate deamination
mechanisms for isomeric styrene oxide induced N1-adenine
adducts. Org. Lett. 1, 1233-1235.
(21) Selzer, R. R., and Elfarra, A. A. (1999) In vitro reactions of
butadiene monoxide with single- and double-stranded DNA:
characterization and quantitation of several purine and pyrimi-
dine adducts. Carcinogenesis 20, 285-292.
(22) Hase, T. A., Lahtinen, L., and Klemola, A. (1977) Lignin model
compounds. I. The synthesis of glyceraldehyde 2-aryl ethers. Acta
Chem. Scand., Ser. B B31, 501-504.
(27) Neagu, C., and Hase, T. (1993) Synthesis of enantiomerically pure
3-butene-1,2-diol derivatives via a Sharpless asymmetric epoxi-
dation route. Tetrahedron Lett. 34, 1629-1630.
(28) Bhanot, O. S., Singh, U. S., and Solomon, J . J . (1994) The role of
3-hydroxyethyldeoxyuridine in mutagenesis by ethylene oxide. J .
Biol. Chem. 269, 30056-30064.
(23) Petrov, A. A. (1941) Conjugated systems. XVII. Order of addition
of hydrogen halides to vinylethylene oxide. Conjugation phenom-
TX010025R