DNA adducts of acrolein: Site-specific synthesis of an oligodeoxynucleotide containing 6-hydroxy-5,6,7,8-tetrahydropyrimido[1,2-a]purin-10(3H)-one, an acrolein adduct of guanine

Article abstract of DOI:10.1021/tx010181y

3-(2-Deoxy-β-D-erythro-pentofuranosyl)-6-hydroxy- 5,6,7,8-tetrahydropyrimido [1,2-α] purin-10-(3H)-one is formed in low yield by the reaction of acrolein with 2′-deoxyguanosine. The nucleoside and an oligodeoxynucleotide containing it have been synthesized. For preparation of the nucleoside 2′-deoxyguanosine was alkylated at the N1 position using 1-bromo-3-butene to give 1-(3-butenyl)-2′-deoxyguanosine. Oxidation with OsO₄ and N-methylmorpholine-N-oxide to give the 3,4-dihydroxybutyl adduct followed by oxidation with NaIO₄ gave the 1-(3-oxopropyl) adduct which cyclized spontaneously to yield the title compound as a rapidly epimerizing mixture of two diastereomers. Reduction of the nucleoside with NaBH₄ gave the unfunctionalized compound plus 1-(3-hydroxypropyl)-2′-deoxyguanosine showing that epimerization was occurring via both the imine and the 1-(3-oxopropyl) adduct. Reduction with NaCNBH₃ gave exclusively unfunctionalized 3-(2-deoxy-β-D-erythro-pentofuranosyl)- 5,6,7,8-tetrahydropyrimido-[1,2-a]purin-10(3H)-one. The phosphoramidite reagent needed for preparation of oligonucleotides was prepared from 1-(3-butenyl)-2′-deoxyguanosine by glycolation after protection of the 3′ and 5′ hydroxyl groups as silyl derivatives. Acetylation of the vicinal hydroxyl groups and the exocyclic amino group followed by removal of silyl protection gave the protected nucleoside. Protection of the 5′ hydroxyl group as the 4,4′-dimethoxytrityl ether followed by phosphitylation with 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite gave the prosphoramidite reagent which was used to prepare a 12-mer oligodeoxynucleotide.

Full text of DOI:10.1021/tx010181y

MAY 2002
VOLUME 15, NUMBER 5
© Copyright 2002 by the American Chemical Society
Articles
DNA Ad d u cts of Acr olein : Site-Sp ecific Syn th esis of a n
Oligod eoxyn u cleotid e Con ta in in g
6-Hyd r oxy-5,6,7,8-tetr a h yd r op yr im id o[1,2-a ]p u r in -10(3H)-on e,
a n Acr olein Ad d u ct of Gu a n in e
Lubomir V. Nechev, Ivan D. Kozekov, Angela K. Brock, Carmelo J . Rizzo, and
Thomas M. Harris*
Department of Chemistry and Center in Molecular Toxicology, Vanderbilt University,
Nashville, Tennessee 37235
Received December 12, 2001
3-(2-Deoxy-â-D-erythro-pentofuranosyl)-6-hydroxy-5,6,7,8-tetrahydropyrimido[1,2-a]purin-10-
(3H)-one is formed in low yield by the reaction of acrolein with 2′-deoxyguanosine. The
nucleoside and an oligodeoxynucleotide containing it have been synthesized. For preparation
of the nucleoside 2′-deoxyguanosine was alkylated at the N1 position using 1-bromo-3-butene
to give 1-(3-butenyl)-2′-deoxyguanosine. Oxidation with OsO4 and N-methylmorpholine-N-oxide
to give the 3,4-dihydroxybutyl adduct followed by oxidation with NaIO4 gave the 1-(3-oxopropyl)
adduct which cyclized spontaneously to yield the title compound as a rapidly epimerizing
mixture of two diastereomers. Reduction of the nucleoside with NaBH4 gave the unfunction-
alized compound plus 1-(3-hydroxypropyl)-2′-deoxyguanosine showing that epimerization was
occurring via both the imine and the 1-(3-oxopropyl) adduct. Reduction with NaCNBH3 gave
exclusively unfunctionalized 3-(2-deoxy-â-D-erythro-pentofuranosyl)-5,6,7,8-tetrahydropyrimido-
[1,2-a]purin-10(3H)-one. The phosphoramidite reagent needed for preparation of oligonucle-
otides was prepared from 1-(3-butenyl)-2′-deoxyguanosine by glycolation after protection of
the 3′ and 5′ hydroxyl groups as silyl derivatives. Acetylation of the vicinal hydroxyl groups
and the exocyclic amino group followed by removal of silyl protection gave the protected
nucleoside. Protection of the 5′ hydroxyl group as the 4,4′-dimethoxytrityl ether followed by
phosphitylation with 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite gave the pros-
phoramidite reagent which was used to prepare a 12-mer oligodeoxynucleotide.
complex mutational spectrum including point mutations
at both G:C and A:T sites (5). Acrolein reacts with
deoxyguanosine to form two regioisomeric 1,N2 cyclic
adducts, the deoxyribonucleosides of 8- and 6-hydroxy-
5,6,7,8-tetrahydropyrimido[1,2-a]purin-10(3H)-ones 2 and
4 (6, 7). Pyrimidopurinone 2, in which the hydroxyl group
is proximal to the purine ring system, probably arises
by conjugate addition of N2 of guanine to C3 of acrolein
In tr od u ction
Acrolein is ubiquitous in the environment and reported
to be a product of endogenous oxidation of lipids (1-4).
It is a mutagen; in mammalian cells it produces a
* To whom correspondence should be addressed. Telephone:
(615) 322-2649. Fax:
(615) 322-7591. E-mail:
toxicology.mc.vanderbilt.edu.
harristm@
10.1021/tx010181y CCC: $22.00 © 2002 American Chemical Society
Published on Web 04/26/2002

608 Chem. Res. Toxicol., Vol. 15, No. 5, 2002
Nechev et al.
Sch em e 1
of the observed mutations. Herein we report a site-
specific synthesis of an oligonucleotide containing 4.
Adducts involving the N1 position of deoxyguanosine
are not amenable to the post-oligomerization approach
that we employed for preparation of oligonucleotides
containing 2. Consequently, in the present case we have
employed an adducted nucleoside strategy for the syn-
thesis of oligonucleotides containing 4. Although the
primary site of nucleophilicity of deoxyguanosine with
simple electrophiles is generally N7, under basic condi-
tions the reactivity is diverted to N1 by formation of the
anion (16). We recently reported application of this
strategy to the synthesis of the 1-hydroxy-3-buten-2-yl
adduct at the N1 position of deoxyinosine and extension
of the strategy to an oligonucleotide containing the
nucleoside (17). It seemed reasonable that the method
could be extended to synthesis of oligonucleotides con-
taining 4.
followed by cyclization of the resulting N2-(3-oxopropyl)
adduct 1 (Scheme 1).2 The 6-hydroxy isomer (4), in which
the hydroxyl group is distal to the ring, arises by conju-
gate addition of N1 to C3 of acrolein followed by cycliza-
tion of the resulting 1-(3-oxopropyl) adduct 3.3 Nucleo-
sides 2 and 4 are formed in a ∼2:3 ratio when the reaction
with dG is carried out at pH 7.0; 2 becomes the primary
adduct when the reaction is carried out in base (8).
Evaluation of the mutagenicity of 2, 4, and other
adducts that may be formed in DNA requires the avail-
ability of oligonucleotides containing site-specific, struc-
turally defined adducts. Oligonucleotides containing 2
and 4 represent synthetic challenges because these
adducts are not compatible with the reaction conditions
normally utilized for solid-phase oligonucleotide synthe-
sis. Two strategies for the preparation of DNA containing
2 have been developed, one by J ohnson and the other by
ourselves (9, 10). Both involve periodate cleavage of 1,2-
diols after assembly and deprotection of the oligonucleo-
tide. J ohnson’s method utilizes deoxyguanosine having
an appropriately protected 3,4-dihydroxybutyl adduct at
the N2 position in the assembly of the oligonucleotide,
ours a post-oligomerization strategy in which 3,4-dihy-
droxybutylamine displaces the halogen atom from 2-fluo-
rodeoxyinosine in the oligonucleotide. No syntheses of
oligonucleotides containing adduct 4 have been reported.
Studies of the mutagenicity of adduct 2 carried out in
two laboratories showed it to have remarkably low
mutagenicity in bacteria (11, 12). In mammalian cells,
divergent results have been obtained, with one study
showing the adduct to be essentially nonmutagenic while
a second one found a significant level of mutations (13,
14). The analogous acrolein adduct of cytosine, if formed
in DNA, is apparently not stable (15). Consequently, we
have turned our attention to adduct 4 as a possible source
Exp er im en ta l P r oced u r es
Gen er a l Meth od s. 1H NMR spectra were recorded at 400.13
MHz on a Bruker AM400 NMR spectrometer in D2O or DMSO-
d6. DMSO was distilled under vacuum over CaH2. Other
chemicals were used as purchased without further purification.
Thin-layer chromatography was performed on silica gel glass
plates (Merck, Silica Gel 60 F254, layer thickness 250 µm). The
chromatograms were visualized under UV light (254 nm) or by
staining with an anisaldehyde/H2SO4 solution, followed by
heating. Column chromatography was performed using silica
gel (Merck, 70-230 mesh).
Oligodeoxynucleotides were synthesized on
a Perseptive
Biosystems Model 8909 DNA synthesizer using their Expedite
reagents with the standard synthetic protocol on a 1-µmol scale.
The modified oligodeoxynucleotides were cleaved from the solid
support and the exocyclic amino groups were deprotected in a
single step using concentrated NH4OH at 60 °C.
Enzymatic hydrolysis was carried out in one step as follows:
oligonucleotide (0.5 A260 units) was dissolved in 20 µL of buffer
(pH 7, 0.01 M Tris‚HCl, 0.01 M MgCl2). DNase I (5 units),
alkaline phosphatase (1.7 units), and snake venom phosphodi-
esterase I, type II (0.02 units) were added and the solution was
incubated at 37 °C for 1.5 h.
The purification of nucleosides and oligonucleotides and the
analysis of reaction mixtures and mixtures of nucleosides
obtained from enzymatic degradations were performed on a
Beckman HPLC system with a diode array UV detector moni-
toring at 260 nm using YMC ODS-AQ columns (250 × 4.6 mm
i.d., 1.5 mL/min for analysis and 250 × 10 mm i.d., 5 mL/min
for purification) with H2O (A) and CH3CN (B) for nucleosides
and 0.1 M aq ammonium formate (A) and CH3CN (B) for
oligonucleotides. HPLC gradient: 99% A initial mixture, 15 min
linear gradient to 90% A, 5 min linear gradient to 80% A, 5
min at 80% A, 3 min linear gradient to 0% A, 2 min at 0% A
followed by 3 min linear gradient to the initial conditions.
1-(3-Bu ten yl)-2′-d eoxygu a n osin e (5). A mixture of deoxy-
guanosine monohydrate (100 mg, 0.35 mmol) and DMA (3 mL)
was heated at 70 °C until all the nucleoside was dissolved (∼10
min). The solution was cooled to room temperature and pow-
dered NaOH (21 mg) was added. The mixture was heated at 70
°C until most of the NaOH had dissolved (∼1 h) and then cooled
to room temperature. 1-Bromo-3-butene (57 mg, 0.42 mmol) was
added in one portion. The mixture was stirred at room temper-
1
Abbreviations: DMA, dimethylacetamide; DMF, dimethylforma-
mide, DMAP, 4-(dimethylamino)pyridine; FAB-HRMS, fast atom
bombardment-high-resolution mass spectrometry; MALDI-TOF MS,
matrix-assisted laser desorption-time-of-flight mass spectrometry;
CZE, capillary zone electrophoresis; PAC, phenoxyacetyl.
2
Adducts 2 and 4 are numbered by the IUPAC convention as
pyrimido[1,2-a]purin-10(3H)-ones rather than as guanine derivatives.
Consequently, the hydroxyl groups on 2 and 4 are at C8 and C6,
respectively.
ature for
1 h and at 60 °C overnight. The solvent was
evaporated, and the product was purified by silica gel column
(CH3CN:H2O:concentrated NH4OH 90:5:5) to yield 73.5 mg
(65%) of 5. 1H NMR (DMSO-d6) δ 7.81 (s, 1H, H-8), 6.99 (bs,
2H, NH2), 6.04 (dd, 1H, H-1′, J 1 ) J 2 ) 6.1 Hz), 5.75 (m, 1H,
CHdC), 5.18 (d, 1H, 3′-OH, J ) 4.04 Hz), 4.93 (m, 2H, CH2d
C), 4.85 (t, 1H, 5′-OH, J ) 5.5 Hz), 4.26 (m, 1H, H-3′), 3.94 (dd,
2H, N-CH2, J 1 ) J 2 ) 7.3 Hz), 3.73 (m, 1H, H-4′), 3.45 (m, 2H,
3
The alternative sequence for formation of 4 cannot be excluded
which involves imine formation at N2 followed by conjugate addition
at N1 and then hydration of the imine. However, the sequence of events
shown in Scheme 1 is supported by the fact that crotonaldehyde gives
only adducts analogous to 2, whereas methylvinyl ketone gives adducts
analogous to both 2 and 4 and 2-cyclohexenone gives an adduct analo-
gous to 4 plus a simple Michael adduct at N2 analogous to 1 (6, 7).

Acrolein Adduct of Deoxyguanosine
Chem. Res. Toxicol., Vol. 15, No. 5, 2002 609
H-5′, H-5′′), 2.50 (m, 1H, H-2′′), 2.25 (m, 2H, CH2-CdC), 2.12
(m, 1H, H-2′). FAB-HRMS [M + H]+: calcd 322.1515, found
322.1508.
combined organic extracts were washed with water and dried
over Na2SO4, and the solvents were evaporated. The residue
was heated in MeOH for a few minutes; MeOH was evaporated,
and the mixture was purified by flash chromatography on silica
gel (ethyl acetate:hexane 2:1) to give 629.5 mg (90%) of protected
nucleoside 10. 1H NMR (DMSO-d6) δ 7.88 (s, 1H, H-8), 7.13 (bs,
2H, NH2), 6.11 (dd, 1H, H-1′, J 1 ) 4.1 Hz, J 2 ) 7.3 Hz), 5.86 (m,
1H, CdCH), 5.06 (m, 2H, CH2dC), 4.72 (m, 1H, H-3′), 4.06 (m,
2H, CH2N), 3.97 (m, 2H, H-5′, H-5′′), 3.84 (m, 1H, H-4′), 2.75
(m, 1H, H-2′), 2.56 (m, 1H, H-2′′), 2.36 (m, 2H, C-CH2-C), 1.09
(m, 28H, 4 × i-Pr-Si). FAB-HRMS [M + H]+: calcd 564.3038,
found 564.3043.
1-(3,4-Dih yd r oxybu tyl)-2′-d eoxygu a n osin e (6). A solution
of nucleoside 5 (23 mg, 0.072 mmol) in 0.20 mL of H2O was
added to a mixture of water (0.20 mL), acetone (0.40 mL),
N-methylmorpholine-N-oxide (9.4 mg, 0.08 mmol) and ∼1 mg
of OsO4. The mixture was stirred overnight at room tempera-
ture. The solvents were evaporated, and the crude product was
purified by silica gel column chromatography (CH3CN:H2O:
concentrated NH4OH 85:10:5) to give 23.4 mg (92%) of 6. 1H
NMR (DMSO-d6) δ 7.93 (s,1H, H-8), 7.00 (bs, 2H, NH2), 6.12
(dd, 1H, H-1′, J 1 ) 7.8 Hz, J 2 ) 6.1 Hz), 5.26 (bs, 1H, 3′-OH),
4.94 (m, 1H, 5′-OH), 4.84 (m, 1H, CH-OH), 4.62 (m, 1H, CH2-
OH), 4.34 (m, 1H, H-3′), 4.06 (m, 1H, CH2N), 3.98 (m, 1H,
CH2N), 3.80 (m, 1H, H-4′), 3.47 (m, 5H, H-5′, H-5′′, 2 × CH2-
OH, CH-OH), 2.51 (m, 1H, H-2′), 2.21 (m, 1H, H-2′′), 1.75 (m,
1H, C-CH2-C), 1.50 (m, 1H, C-CH2-C). FAB-HRMS [M +
H]+: calcd 356.1570, found 356.1578.
3-(2-Deoxy-â-D-er yth r o-pen tofu r an osyl)-6-h ydr oxy-5,6,7,8-
t et r a h yd r op yr im id o[1,2-a ]p u r in -10(3H)-on e (6-h yd r oxy-
1,N2-p r op a n o-2′-d eoxygu a n osin e, 4). Nucleoside 6 (15 mg,
0.042 mmol) was dissolved in a solution of NaIO4 (3 mL, 20 mM).
The mixture was stirred for 10-15 min at room temperature;
the product was purified by HPLC. It was a ∼1:1 mixture of
diastereomers of 4, which equilibrated too rapidly for them to
be individually isolated. Yield 12.1 mg (89%). 1H NMR (DMSO-
d6) δ 8.38 (bs, 1H, NH), 7.93 (s, 1H, H-2), 6.11 (m, 1H, H-1′),
5.93 (m, 1H, 6-OH), 5.26 (bs, 1H, 3′-OH), 4.96 (m, 1H, H-6),
4.92 (m, 1H, 5′-OH), 4.42 (m, 1H, H-8), 4.33 (m, 1H, H-3′), 3.80
(m, 1H, H-4′), 3.50 (m, 3H, H-5′, H-5′′, H-8), 2.55 (m, 1H, H-2′),
2.18 (m, 1H, H-2′′), 1.94 (m, 1H, H-7), 1.75 (m, 1H, H-7). FAB-
HRMS [M + H]+: calcd 324.1308, found 324.1317.
Red u ction of 4 w ith Na BH4. A solution of 4 (3.0 mg) in 2.5
mL of phosphate buffer (50 mM, pH 7.0) was treated with
NaBH4 (17.5 mg, 50 equiv) at room temperature. The reaction
was followed by HPLC. After days 1, 2, and 3, additional NaBH4
(17.5 mg, 50 equiv) was added for a total of 200 equiv. By the
fifth day, the starting material had been completely consumed,
giving two products (3:1 ratio), which were separated by HPLC.
The major component (16.2 min) was assigned on the basis of
NMR and mass spectra as 1-(3-hydroxypropyl)-2′-deoxygua-
nosine (7). 1H NMR (DMSO-d6) δ 7.95 (s, 1H, H-8), 7.17 (bs,
2H, NH2), 6.12 (dd, 1H, H-1′, J ) 6.1 Hz), 4.95-5.33 (bs, 2H,
5′-OH, 3′-OH), 4.35 (m, 1H, H-3′), 3.98 (m, 2H, CH2N in the
side chain), 3.81 (m, 1H, H-4′), 3.55 (m, 2H, H-5′, H-5′′), 3.45
(m, 2H, CH2O in the side chain), 2.52 (m, 1H, H-2′), 2.21 (m,
1H, H-2′′), 1.73 (m, 2H, C-CH2-C in the side chain. FAB-
HRMS [M + H]+: calcd 326.1464, found 326.1475. The minor
one (19.0 min) was shown to be 1,N2-propano-2′-deoxyguanosine
(9) by HPLC comparison with an authentic sample.4
Red u ction of Nu cleosid e 4 w ith Na CNBH3. A solution of
4 (0.5 mg) and NaCNBH3 (1.5 mg, 5 equiv) in 1 mL of phosphate
buffer (50 mM, pH 7.0) was stirred for 3 days at room
temperature. After 3 days the temperature was raised to 40 °C,
an additional 1.5 mg of NaCNBH3 was added, and stirring
continued for 5 more days, at which time HPLC analysis showed
the reaction to be complete. Only a single product, 1,N2-
propanodeoxyguanosine (9), was observed, the identity of which
was confirmed by HPLC comparison with an authentic sample.
1-(3-Hydroxypropano)-2′-deoxyguanosine (7) was not detected.
1-(3-Bu ten yl)-2′-d eoxy-3′,5′-O-(1,1,3,3-tetr a isop r op yld isi-
loxa n e-1,3-d iyl)gu a n osin e (10). A mixture of 5 (400 mg, 1.24
mmol) and 1H-imidazole (420 mg, 6.2 mmol) was coevaporated
with anhydrous DMF (2 × 20 mL). The resulting residue was
suspended in anhydrous DMF (5 mL), cooled to 0 °C, and then
1,3-dichloro-1,1,3,3-tetraisopropyl-1,3-disiloxane (480 mg, 1.5
mmol) was added dropwise with stirring. The suspension was
stirred overnight to give a turbid solution, which was poured
onto ice. The resulting mixture was extracted with CH2Cl2. The
1-(3,4-Dih yd r oxybu t-1-yl)-2′-d eoxy-3′,5′-O-(1,1,3,3-tetr a -
isop r op yld isiloxa n e-1,3-d iyl)gu a n osin e (11). A solution of
10 (271 mg, 0.48 mmol) in 200 µL of THF was added to a
mixture of water (2 mL), THF (2 mL), N-methylmorpholine-N-
oxide (62.7 mg) and ∼1 mg of OsO4. The mixture was stirred
overnight at room temperature. The solvents were evaporated,
and the crude product was subjected to a silica gel column (CH3-
CN:H2O:concentrated NH4OH 90:5:5) to give 249.4 mg (87%)
of 11. 1H NMR (DMSO-d6) δ 7.94 (s, 1H, H-8), 7.00 (bs, 2H, NH2),
6.04 (m, 1H, H-1′), 4.81 (d, 1H, CH-OH, J ) 5.2 Hz), 4.66 (m,
1H, H-3′), 4.59 (m, 1H, CH2-OH), 4.05 (m, 1H, CH2-N-CdO),
3.95 (m, 1H, CH2-N-CdO), 3.88 (m, 2H, H-5′, H-5′′), 3.78 (m,
1H, H-4′), 3.45 (m, 1H, CH-OH), 3.35 (m, 2H, CH2-OH), 2.63
(m, 1H, H-2′), 2.44 (m, 1H, H-2′′), 1.73 (m, 1H, C-CH2-C), 1.49
(m, 1H, C-CH2-C), 1.00 (m, 28H, 4 × i-Pr). FAB-HRMS [M
+ H]+: calcd 598.3092, found 598.3120.
1-(3,4-Dia cet oxybu t -1-yl)-2′-d eoxy-3′,5′-O-(1,1,3,3-t et r a -
isop r op yld isiloxa n e-1,3-d iyl)gu a n osin e (12). Nucleoside 11
(550 mg, 0.92 mmol) was coevaporated with anhydrous pyridine
(2 × 10 mL) and then mixed with pyridine (40 mL), acetic
anhydride (0.86 mL, 10 equiv), triethylamine (1.5 mL), and
DMAP (11.1 mg). The mixture was stirred at 55 °C for ∼80 h;
the course of the reaction was followed by TLC (CH2Cl2:MeOH
9:1). When TLC analysis indicated the completion of the
reaction, the mixture was cooled (ice bath) and methanol (20
mL) was added. After 15 min at room temperature, the solvents
were evaporated under reduced pressure. Flash chromatography
of the residue on silica gel (EtOAc:hexanes 4:1) gave 559.6 mg
1
(84%) of triacetylated product 12. H NMR (DMSO-d6) δ 10.58
(bs, 1H, NH), 8.20 (s, 1H, H-8), 6.20 (dd, 1H, H-1′, J 1 ) J 2 ) 7.6
Hz), 4.96 (m, 1H, CH-OAc), 4.80 (m, 1H, H-3′), 4.18 (m, 1H, 1
× CH2-OAc), 4.08 (m, 3H, 2 × CH2-N, 1 × CH2-OAc), 3.90
(m, 2H, H-5′, H-5′′), 3.79 (m, 1H, H-4′), 2.78 (m, 1H, H-2′), 2.57
(m, 1H, H-2′′), 2.11 (s, 3H, CH3CO), 2.00 (s, 3H, CH3CO), 1.97
(s, 3H, CH3CO), 1.88 (m, 2H, C-CH2-C), 1.03 (m, 28H,
isopropyl). FAB-HRMS for [M + H]+: calcd 724.3409, found
724.3373.
1-(3,4-Diacetoxybu tyl)-N2-acetyl-2′-deoxygu an osin e (13).
Compound 12 was treated with tetrabutylammonium fluoride
(3.8 mL, 1.0 M in THF) in THF (5 mL) for 2 h at ambient
temperature. The solvent was removed under reduced pressure.
Purification by flash chromatography (CH3CN:H2O:concentrated
1
NH4OH, 90:5:5) gave 333.6 mg (96%) of 13 as a white solid. H
NMR (DMSO-d6) δ 10.56 (bs, 1H, NH), 8.26 (s, 1H, H-8), 6.16
(dd, 1H, H-1′, J 1 ) J 2 ) 6.7 Hz), 5.26 (d, 1H, 3′-OH, J ) 4.2
Hz), 4.88 (m, 2H, 5′-OH, CH-OAc), 4.29 (m, 1H, H-3′), 4.09 (m,
1H, CH2-OAc), 4.01 (m, 3H, 2 × CH2-N, 1 × CH2-OAc), 3.77
(m, 1H, H-4′), 3.46 (m, 2H, H-5′, H-5′′), 2.43 (m, 1H, H-2′), 2.20
(m, 1H, H-2′′), 2.06 (s, 3H, CH3CO), 1.94 (s, 3H, CH3CO), 1.91
(s, 3H, CH3CO), 1.82 (m, 2H, C-CH2-C). FAB-HRMS for [M
+ H]+: calcd 482.1887, found 482.1896.
5′-O-(4,4′-Dim et h oxyt r it yl)-1-(3,4-d ia cet oxyb u t yl)-N2-
a cetyl-2′-d eoxygu a n osin e (14). Compound 13 (110 mg, 0.23
mmol) was coevaporated with pyridine (2 × 3 mL) and then
redissolved in pyridine (5 mL). 4,4′-Dimethoxytrityl chloride (94
mg, 0.28 mmol) was added, and the mixture was stirred at room
temperature for 5 h. The solvent was evaporated under vacuum,
and the residue was purified by silica gel column chromatog-
raphy (CH2Cl2:MeOH:pyridine 95:0:5 to 90:5:5) to give 14 (132
mg, 73%). 1H NMR (DMSO-d6) δ 10.56 (bs, 1H, NHCO), 8.16
4
The authentic sample of 9 was a gift from L. J . Marnett.

610 Chem. Res. Toxicol., Vol. 15, No. 5, 2002
Nechev et al.
Sch em e 2
Sch em e 3
F igu r e 1.
(s, 1H, H-8), 7.30 (m, 2H, aromatic), 7.21 (m, 7H, aromatic), 6.80
(m, 4H, aromatic), 6.24 (dd, 1H, H-1′, J 1 ) J 2 ) 6.3 Hz), 5.36
(d, 1H, 3′-OH, J ) 4.6 Hz), 4.95 (m, 1H, CH-OAc), 4.35 (m,
1H, H-3′), 4.15 (m, 1H, 1 × CH2-OAc), 4.06 (m, 3H, 1 × CH2-
OAc, 2 × CH2N), 3.89 (m, 1H, H-4′), 3.70 (s, 6H, 2 × CH3O),
3.14 (m, 2H, H-5′, H-5′′), 2.68 (m, 1H, H-2′), 2.32 (m, 1H, H-2′′),
2.10 (s, 3H, CH3CO), 1.99 (s, 3H, CH3CO), 1.95 (s, 3H, CH3CO),
1.84 (m, 2H, C-CH2-C). FAB-HRMS for [M + H]+: calcd
784.3194, found 784.3211.
scale using tert-butylphenoxyacetyl-protected cyanoethyl phos-
phoramidites and adducted phosphoramidite 15. Deprotection
of the oligonucleotide, including the three acetyl groups and
cleavage of the oligonucleotide from the CPG beads was achieved
by treatment with concentrated aqueous ammonia for 10 h at
55 °C. Purification by reversed-phase HPLC gave 25 A260 units
of the 12-mer containing 6. The constitution was confirmed by
MALDI-TOF MS: m/z calcd [M - H]- 3732.7, found 3732.7.
Nucleolytic digestion gave 6 plus the four normal deoxynucleo-
sides in the correct stoichiometric ratios. The identity of 6 was
confirmed by HPLC comparison with an authentic sample
(Figure 1A).
A solution of 5′-d(GCT-AGC-6-AG-TCC) (10 A260 units) in
phosphate buffer (0.80 mL, 0.05 M, pH 7.0) was treated with
aqueous NaIO4 (20 mmol, 0.050 mL) for 30 min at room
temperature. Purification by reversed-phase HPLC gave 8.5 A260
units (∼85%) of 5′-d(GCT-AGC-4-AG-TCC). The oligonucleotide
was characterized by MALDI-TOF MS: m/z calcd [M - H]-
3700.7, found 3701.4. Nucleolytic digestion gave 4 plus the four
normal deoxynucleosides in the correct stoichiometric ratios. The
identity of 4 was confirmed by HPLC comparison with the
authentic sample (Figure 1B).
3′-O-[(N,N-Diisopr opylam in o)-2-cyan oeth oxyph osph in yl]-
5′-O-(4,4′-dim eth oxytr ityl)-1-(3,4-diacetoxybu tyl)-N2-acetyl-
2′-d eoxygu a n osin e (15). Compound 14 (80 mg, 0.10 mmol) was
dried by coevaporation with anhydrous pyridine (2 × 3 mL) and
placed under vacuum overnight. The oily residue was treated
with a solution of anhydrous 1H-tetrazole (8.4 mg, 0.12 mmol)
in freshly distilled CH2Cl2 (5 mL) followed by addition of
2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (39.2
mg, 0.13 mmol). After 3 h, the solvents were removed under
reduced pressure, and the crude product was purified by flash
column chromatography on silica gel (EtOAc:hexanes:pyridine
1
90:5:5) to yield 95.3 mg (95% yield) of phosphoramidite 15. H
NMR (DMSO-d6) δ 10.58 (bs, 1H, NHCO), 8.22, 8.20 (s, 1H,
diastereomeric H-8), 7.33 (m, 2H, aromatic), 7.20 (m, 7H,
aromatic), 6.82 (m, 4H, aromatic), 6.28 (dd, 1H, H-1′, J 1 ) J 2
)
6.24 Hz), 4.96 (m, 1H, CH-OAc), 4.63 (m, 1H, H-3′), 4.18 (m,
1H, 1 × CH2-OAc), 4.06 (m, 4H, 1 × CH2-OAc, 2 × CH2N,
H-4′), 3.72 (s, 7H, 2 × CH3O, 1 × POCH2), 3.63 (m, 1H, 1 ×
POCH2), 3.52 (m, 2H, isopropyl CH), 3.19 (m, 2H, H-5′, H-5′′),
2.84 (m, 1H, H-2′), 2.76 (t, 1H, 1 × CH2-CN, J ) 5.89), 2.64 (t,
1H, 1 × CH2-CN, J ) 5.89), 2.57 (m, 1H, H-2′′), 2.21 (s, 3H,
CH3CO), 2.00 (s, 3H, CH3CO), 1.97 (s, 3H, CH3CO), 1.88 (m,
2H, C-CH2-C), 1.10, 1.00 (m, 12H, isopropyl CH3). 31P
NMR (DMSO-d6, 121 MHz) δ 148.6, 147.8. FAB-HRMS for
Resu lts a n d Discu ssion
The goal of this project was to synthesize not just
nucleoside 4 but also oligonucleotides containing it.
Therefore, we focused our attention on a synthetic
approach that would be suitable for both. For the
synthesis of nucleoside 4, 1-bromo-3-butene reacted
smoothly with deoxyguanosine under alkaline conditions
C
50H63N7O12P [M + H]+: calcd 984.4272, found 984.4279.
P r ep a r a tion of a n Oligon u cleotid e Con ta in in g 6. The
12-mer 5′-d(GCT-AGC-6-AG-TCC) was prepared on a 1-µmol

Acrolein Adduct of Deoxyguanosine
Chem. Res. Toxicol., Vol. 15, No. 5, 2002 611
Sch em e 4
to give butenyl adduct 5 in 65% yield (Scheme 2).
Treatment of 5 with OsO4/N-methylmorpholine-N-oxide
gave 92% of diol 6. Oxidation with NaIO4 gave aldehyde
3, which cyclized spontaneously to hydroxypropano-dG
(4) in 89% yield. The structures of compounds 4, 5, and
6 were established by NMR and mass spectroscopy.
Nucleoside 4 exists as two diastereomers on account
of C6 being a stereogenic center. The nucleoside appears
as two peaks on HPLC (Figure 1B). As reported by
previous workers, the two diastereomers are in a facile
equilibrium (6). The diastereomers equilibrate too rapidly
for it to be feasible to isolate them individually. There
are two pathways by which the epimerization could be
occurring: reversion to 3-oxopropyl adduct 3 or dehydra-
tion to imine 8 followed by rehydration. Neither 3 nor 8
can be detected in the NMR spectrum of 4, but it is
possible that they are present at concentrations too small
to be detected. As a probe of the mechanism, reductions
were carried out with NaBH4 and NaCNBH3 (Scheme 3).
Reduction by NaBH4 at ambient temperature gave a 3:1
mixture of 1-(3-hydroxypropyl) adduct 75 and 1,N2-
propanodeoxyguanosine 9, whereas reduction with NaC-
NBH3 gave exclusively 9. The reduction with NaCNBH3
was too slow at ambient temperature but an acceptable
reaction rate was observed at 40 °C. The two reducing
agents differ in their selectivity for reduction of imines
and aldehydes. These results show that both epimeriza-
tion pathways are operative with 4. Chung et al. obtained
exclusively 9 when they carried out the reduction of 4
using a mixture of NaBH4 and NaOH at 100 °C (6).
5
Compound 7 was used to confirm that the site of alkylation of dG
in the initial step of the synthesis had, in fact, been N1. An HMBC
spectrum showed three-bond connectivity between C6 of the purine
(156 ppm) and the protons of the R methylene group of the side chain
(3.98 ppm). The R carbon (39 ppm) showed three-bond connectivity
with the protons of the γ methylene group (3.45 ppm).

612 Chem. Res. Toxicol., Vol. 15, No. 5, 2002
Nechev et al.
Sch em e 5
mobile equilibrium with acyclic N1-oxopropyl nucleoside
and in this case the amount of 3 present is below the
limits of spectroscopic detection. However, in duplexed
DNA there will be no driving force to favor formation of
the acyclic species since the oxopropyl substituent at the
N1 position of 3 still blocks Watson-Crick base-pairing.
With 4 there is close structural similarity to 1,N2-
propano-dG (9) and it is likely that the three-dimensional
structures of oligonucleotides containing 4 will closely
resemble those containing 9. The solution structure of
an oligonucleotide duplex containing 9 obtained by NMR
showed the presence of two conformations; in both of
them 9 had rotated into a syn conformation so that the
propano fragment lay in the major groove providing an
opportunity for Hoogsteen base-pairing (19). It seems
likely that 4 will resemble 9 rather than 8-hydroxypro-
pano adduct 2 in its effects on replication. Whereas 2 was
essentially nonmutagenic in bacteria (11, 12), 9 substan-
tially degraded replication fidelity (20-23). These find-
ings are consistent with the fact that 9 perturbs duplex
structure far more than 2.
We have recently reported that DNA duplexes contain-
ing 2 in a CpG context form imine cross-links between
the strands (24). The mechanism of formation of the
cross-link involves trapping of the N2-oxopropyl adduct
1 by the exocyclic amino group of the guanine in the
complementary strand. The situation with respect to the
6-hydroxypropano adduct 4 is more complex because both
N1-oxopropyl species 3 and imine 8 can potentially cross-
link.
For the extension of this strategy to the synthesis of
oligonucleotides, the phosphoramidite reagent, i.e., 15,
needed to have the hydroxyl groups on the butyl side
chain in place but protected. For synthesis of 15, the
deoxyribose hydroxyl groups of butenyl-dG (5) were
protected with a bis-silyl agent, after which vicinal
hydroxylation of the olefin in 10 was achieved with OsO4/
N-methylmorpholine-N-oxide giving diol 11 in 87% yield
(Scheme 4). The hydroxyl groups of the diol and the
exocyclic amino group were then protected by treatment
with acetic anhydride to give 12 in 84% yield. The bis-
silyl group was removed with tetrabutylammonium
fluoride (96%), followed by tritylation with dimethoxytri-
tyl chloride (73%) and phosphitylation with 2-cyanoethyl-
N,N,N′,N′-tetraisopropylphosphorodiamidite to give phos-
phoramidite 15 in 95% yield.
Ack n ow led gm en t. We thank the National Institute
for Environmental Health Sciences for generous support
of this project (ES00267 and ES05355) and Pamela J .
Tamura and Amanda S. Wilkinson for assistance with
oligonucleotide syntheses. A.K.B. acknowledges a GAANN
fellowship (P200A980237-03) and an NIEHS traineeship
(ES007028).
A 12-mer oligodeoxynucleotide containing hydroxypro-
pano-dG (4) was prepared using 15 by the standard
protocol for automatic solid-phase DNA synthesis with
the exception that the exocyclic amino groups of dG, dC,
and dA were protected with labile PAC groups (Scheme
5). The deprotection step was carried out with hot,
concentrated ammonia, which cleaved the tether to the
beads and removed all protective groups including the
three acetyl groups of the adducted nucleoside to give
an oligonucleotide containing dihydroxybutyl-dG (6).
Cleavage of the vicinal diol with NaIO4 gave the oligo-
nucleotide containing hydroxypropano-dG (4). Homoge-
neity of the oligonucleotides containing 6 and 4 was
demonstrated by CZE. The molecular weights of the
oligonucleotides were established by MALDI-TOF MS.
Enzymatic degradation of the oligonucleotides using a
mixture of DNase I, snake venom phosphodiesterase I,
and alkaline phosphatase gave the appropriate nucleo-
sides in the correct molar ratios (Figure 1). Nucleosides
6 and 4 were identified by HPLC comparison with
authentic samples.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectra of
compounds 4-6 and 10-15; 31P NMR spectrum of 15, COSY
spectra of compounds 4, 10, 11, and 14, and CZE analyses of
oligonucleotides containing 6 and 4. This information is avail-
able free of charge via the Internet at http://pubs.acs.org.
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