Synthesis of 8-(hydroxymethyl)-3,N4-etheno-2'-deoxycytidine, a potential carcinogenic glycidaldehyde adduct, and its site-specific incorporation into DNA oligonucleotides

Article abstract of DOI:10.1021/tx990181m

The previously unreported glycidaldehyde adduct, 8-(hydroxymethyl)- 3,N⁴-etheno-2'-deoxycytidine (8-HM-εdC), has been synthesized for the first time by reaction of 2'-deoxycytidine with bromoacetaldehyde at pH 4.5, followed by reduction with sodium borohydride. The adduct was characterized by UV, MS, and NMR. The compound was stable to neutral and acidic conditions but not in alkaline solution. The corresponding phosphoramidite was synthesized in good yield from the intermediate, 3,N⁴-ethenocarbaldehyde-2'- deoxycytidine, using the standard methodology and site-specifically incorporated in both 15- and 25-met oligonucleotides, for studies on biochemical and biophysical properties. The resulting oligonucleotides were purified using HPLC, and the base composition was verified by HPLC after enzymatic digestion.

Full text of DOI:10.1021/tx990181m

208
Chem. Res. Toxicol. 2000, 13, 208-213
Syn th esis of
8-(Hyd r oxym eth yl)-3,N4-eth en o-2′-d eoxycytid in e, a
P oten tia l Ca r cin ogen ic Glycid a ld eh yd e Ad d u ct, a n d Its
Site-Sp ecific In cor p or a tion in to DNA Oligon u cleotid es
Ahmed Chenna,† Alex Perry, and B. Singer*
Donner Laboratory, Life Sciences Division, Lawrence Berkeley Laboratory, University of California,
Berkeley, California 94720
Received October 28, 1999
The previously unreported glycidaldehyde adduct, 8-(hydroxymethyl)-3,N4-etheno-2′-deoxy-
cytidine (8-HM-ꢀdC), has been synthesized for the first time by reaction of 2′-deoxycytidine
with bromoacetaldehyde at pH 4.5, followed by reduction with sodium borohydride. The adduct
was characterized by UV, MS, and NMR. The compound was stable to neutral and acidic
conditions but not in alkaline solution. The corresponding phosphoramidite was synthesized
in good yield from the intermediate, 3,N4-ethenocarbaldehyde-2′-deoxycytidine, using the
standard methodology and site-specifically incorporated in both 15- and 25-mer oligonucleotides,
for studies on biochemical and biophysical properties. The resulting oligonucleotides were
purified using HPLC, and the base composition was verified by HPLC after enzymatic digestion.
In tr od u ction
Glycidaldehyde is a highly reactive alkylating agent
that has been shown to be mutagenic in a range of in
vitro genotoxicity tests (1) and carcinogenic in rodent skin
cancer studies (2, 3). It is classified by the International
Agency for Research on Cancer as being carcinogenic to
experimental animals (4, 5). Glycidaldehyde is a product
of P450 monooxygenase action on glycidyl ethers (6), an
important class of industrial materials. Several glycidyl
ethers have also been shown to be carcinogenic in
experimental animals (7, 8).
Glycidaldehyde is a bifunctional agent by virtue of its
reactive carbonyl and epoxy functionalities and is capable
of forming cyclic adducts and cross-links with bases in
DNA. Adducts with dA and dG have been synthesized
(9-14), in which the exocyclic etheno ring has a CH2OH
group replacing a hydrogen. The structures of the dA and
dG adducts found after glycidaldehyde reaction have been
identified and well characterized (9-16). In vitro at pH
5, the dA adduct, which is fluorescent, is formed to a
greater extent than the dG adduct (9). At pH 10, the dG
adduct is formed to a greater extent than the dA adduct
(9). However, at pH 7, the dA and dG adducts are formed
in similar quantities. The modified dA nucleoside has
been identified from mouse skin (9, 10) but not dG or dC
adducts which are also likely to be formed. The only
report on the formation of dC-glycidaldehyde adducts
is from Kohwi (17), who observed that glycidaldehyde is
highly reactive with dC in non-B DNA. However, no
structural information was given.
F igu r e 1. Chemical structure of 8-hydroxymethyl-3,N4-etheno-
2′-deoxycytidine.
ꢀdC)1 (Figure 1) and its phosphoramidite for the first time
and the site-specific incorporation of the phosphoramidite
into DNA oligonucleotides. These oligonucleotides are
now being studied for their physical properties and their
effect on replication, mutation, and repair.
Exp er im en ta l P r oced u r es
Ma ter ia ls a n d Meth od s. (1) Ch em ica ls a n d Rea gen ts.
Ca u tion : Bromomalonaldehyde (BMA) was prepared using the
procedure of Trofimenko (18). Bromomalonaldehyde is highly
toxic and should be handled carefully. Organic chemicals and
solvents used in this work should be stored and used only in a
well-ventilated hood. 2′-Deoxycytidine was purchased from
Sigma Chemical Co. (St. Louis, MO). 4,4-Dimethoxytrityl chlo-
ride (DMTCl), pyridine, triethylamine, ethanol, 2-cyanoethyl
N,N-diisopropylchlorophosphoramidochloridite, sodium borohy-
dride, 1-acetylimidazole, DMSO, silica gel, sodium hydroxide,
sodium chloride, sodium hydrogen carbonate, and toluene were
purchased from Aldrich Chemical Co. (Milwaukee, WI). Metha-
In this work, we report the large-scale synthesis of
8-(hydroxymethyl)-3,N4-etheno-2′-deoxycytidine (8-HM-
* To whom correspondence and reprint requests should be ad-
dressed: Donner Laboratory, Life Sciences Division, Lawrence Ber-
keley Laboratory, University of California, Berkeley, CA 94720.
Telephone: (510) 642-0637. Fax: (510) 486-6488.
1
Abbreviations: 8-HM-ꢀ-dC, 8-(hydroxymethyl)-3,N4-etheno-2′-
deoxycytidine; DMTCl, 4,4′-dimethoxytrityl chloride; BAP, bacterial
alkaline phosphatase; SVP, snake venom phosphodiesterase; CPG,
controlled-pore glass.
† Present address: Aclara Biosciences, 1288 Pear Ave., Mountain
View, CA 94043-1432.
10.1021/tx990181m CCC: $19.00 © 2000 American Chemical Society
Published on Web 02/25/2000

Synthesis of the Glycidaldehyde-dC Adduct
Chem. Res. Toxicol., Vol. 13, No. 3, 2000 209
Sch em e 1. Syn th esis of 8-(Hyd r oxym eth yl)-3,N4-eth en o-2′-d eoxycytid in e
nol, dichloromethane, acetone, benzene, and acetonitrile were
from J . T. Baker (Philipsburg, NJ ). Potassium carbonate,
phosphorus pentoxide, and thin-layer chromatography silica
plates were purchased from EM Science (Gibbston, NJ ), and
calcium hydride was from Fluka Chemika (Ronkonkoma, NY).
Bacterial alkaline phosphatase (BAP) and snake venom phos-
phodiesterase (SVP) were from Pharmacia Biotech Inc. (Alame-
da, CA). The PAC phosphoramidites were purchased from
Pharmacia (Piscataway, NJ ). All solvents were dried by distil-
lation over calcium hydride.
(2) An a lysis a n d P u r ifica tion . Proton (400 MHz) NMR
spectra were recorded with DMSO-d6 as the solvent, and D2O
exchanges were carried out to assign exchangeable protons,
unless otherwise indicated. NMR spectra were recorded using
a Bruker AM400 spectrometer and are reported in parts per
million (ppm) relative to an internal standard of tetramethyl-
silane. 31P NMR spectra were recorded on a Bruker AM400
spectrometer, and the chemical shifts are reported relative to
an external standard of phosphoric acid. Fast atom bombard-
ment (FAB) spectra were obtained on a VG70 SE Instruments
mass spectrometer, and glycerin or thioglycerin was used as a
matrix. Electrospray mass spectra were obtained on a VG Bio-Q
Instruments mass spectrometer. Ultraviolet spectra were re-
corded on a Hitachi U-2000 spectrophotometer using 0.5 cm
cuvettes. TLC was performed on EM 5735/7 silica gel 60 F254
plates. Column chromatography was performed using silica gel
60 with elution under gravity.
other products were present, including 3,N4-etheno-dC). The
solvent was removed in vacuo to afford a yellow gum. The
desired product (1) was isolated using silica gel column chro-
matography eluting with 40% acetone and 60% dichloromethane.
A mixture of more polar byproducts which included 3,N4-etheno-
dC was also eluted from the column. The product was dried to
afford a yellow gum with a 28% yield (0.321 g). UV (water): λmax
272, 283, 326 nm. 1H NMR (DMSO-d6): δ 10.53 [1H, s, C(O)H],
8.14 (1H, s, H-7), 8.02 (1H, d, J ) 8 Hz, H-6), 6.89 (1H, d, J )
8 Hz, H-5), 6.39 (1H, t, J ) 6 Hz, H-1′), 5.33 (1H, d, J ) 4 Hz,
3′-OH), 5.15 (1H, t, J ) 6 Hz, 5′-OH), 4.29-4.27 (1H, m, H-4′),
3.89-3.86 (1H, m, H-3′), 3.70-3.60 (2H, m, 2 × H-5′), 2.32-
2.18 (2H, m, 2 × H-2′). FAB-MS (rel intensity): m/z 280 (41%,
MH+), 164 (100%, base H+)
(B) P r ep a r a tion of 8-(Hyd r oxym eth yl)-3,N4-eth en o-2′-
d eoxycytid in e (2). Sodium borohydride (0.004 g, 0.10 mmol,
2.0 equiv) was added to a solution of 3,N4-ethenocarbaldehyde-
2′-deoxycytidine (0.014 g, 0.05 mmol) in ethanol (4 mL) at 0 °C.
The reaction mixture was allowed to warm to room temperature,
then stirred for 2 h, and then diluted with 10 volumes of
dichloromethane. The solution was washed with saturated
sodium chloride and water, dried (Na2SO4), filtered, and con-
centrated in vacuo to afford a white foam. The desired product
(2) was isolated using silica gel column chromatography eluting
with 4% methanol and 96% dichloromethane. The resulting
yellow solid was obtained in 84% yield (0.012 g). UV (water):
λmax 278 (pH 7, ꢀmax ) 7700 mol-1 cm2, ꢀ260 ) 4900 mol-1 cm2),
1
278 (pH 14), 296 nm (pH 1). H NMR (DMSO-d6): δ 7.75 (1H,
(3) HP LC. Analytical and semipreparative HPLC were
conducted using a Hewlett-Packard 1050 photodiode array
detector and quarternary gradient pump (solvent delivery
system). Solvent systems included solvent A (acetonitrile),
solvent B [0.1 M triethylammonium acetate (pH 7)], and solvent
C [0.01 M potassium phosphate (pH 4.5)]. In system 1, oligo-
nucleotides were purified using a C18 column (300 mm × 3.9
mm, 5 µm, Hamilton PRP-1). The initial concentrations of 15%
solvent A and 85% solvent B were maintained for 5 min, and
then the concentration of solvent A was increased linearly to
35% over the course of 20 min at a flow rate of 1 mL/min. In
system 2, oligonucleotides were analyzed using a C18 column
(300 mm × 3.9 mm, 5 µm, Hamilton PRP-1). The initial
concentrations of 5% solvent A and 95% solvent B were
maintained for 5 min, and then the concentration of solvent A
was increased linearly to 35% over the course of 40 min at a
flow rate of 1 mL/min. In system 3, analysis of the enzyme digest
of the oligonucleotides was performed with a Supelcosil LC-18-
DB column (250 mm × 4.6 mm, 5 µm, Supelco Inc.) and 0%
solvent A and 100% solvent C. The concentration of solvent A
was linearly increased from 0 to 10% over the course of 25 min
at a flow rate of 1 mL/min.
Ch em ica l Syn th eses. (1) Syn th esis of th e d C-Glycid -
a ld eh yd e Ad d u ct (Sch em e 1). (A) P r ep a r a tion of 3,N4-
Eth en oca r ba ld eh yd e-2′-d eoxycytid in e (1). Bromomalonal-
dehyde (0.622 g, 4.07 mmol, 1.0 equiv) was added to a solution
of 2′-deoxycytidine (1.00 g, 4.07 mmol) in water (100 mL). The
pH of the reaction mixture was then adjusted to 4.5 with a 2 M
sodium hydroxide solution. The reaction mixture was stirred
at 60 °C for 30 h. Further bromomalonaldehyde (0.200 g, 1.30
mmol, 0.3 equiv) was added after this time. The reaction mixture
was stirred at 60 °C for an additional 18 h (TLC analysis
indicated that the dC had been completely consumed and that
d, J ) 8 Hz, H-6), 6.82 (1H, s, H-7), 6.53 (1H, t, J ) 6 Hz, H-1′),
6.28 (1H, d, J ) 8 Hz, H-5), 5.53-5.51 (2H, m, CH2), 4.23-4.09
(2H, m, H-4′, H-3′), 3.55-3.42 (2H, m, 2 × H-5′), 2.19-2.08 (2H,
m, 2 × H-2′). FAB-MS (rel intensity): m/z 282 (50%, MH+), 166
(23%, base H+), 135 (100%, base H+ - Ome).
(2) Syn th esis of th e P r otected P h osp h or a m id ite De-
r iva tive of th e d C-Glycid a ld eh yd e Ad d u ct (Sch em e 2).
(A) P r ep a r a tion of 5′-O-(4,4′-Dim eth oxytr ityl)-3,N4-eth en o-
car baldeh yde-2′-deoxycytidin e (3). 4,4′-Dimethoxytrityl chlo-
ride (0.530 g, 1.56 mmol, 1.5 equiv) was added to a solution of
3,N4-ethenocarbaldehyde-2′-deoxycytidine (0.291 g, 1.04 mmol)
in dry pyridine (20 mL). The reaction mixture was stirred at
room temperature for 16 h. Further 4,4-dimethoxytrityl chloride
(0.352 g, 1.04 mmol, 1.0 equiv) was added, and then the reaction
mixture was stirred for an additional 4 h. The solvent was
removed in vacuo, and the residue was dissolved in dichlo-
romethane. The solution was washed with 5% sodium hydrogen
carbonate solution and with saturated sodium chloride solution,
dried (Na2SO4), and filtered. The solvent was removed by
forming the azeotrope with toluene in vacuo to afford a yellow
gum. The desired product (3) was isolated using silica gel column
chromatography eluting with 2% methanol and 98% dichlo-
romethane. The resulting yellow foam was obtained in 69% yield
(0.418 g). 1H NMR (CDCl3): δ 10.67 [1H, s, C(O)H], 8.17 (1H, s,
H-7), 7.91 (1H, d, J ) 8 Hz, H-6), 7.39-6.76 (13H, m, DMTr),
6.55 (1H, t, J ) 6 Hz, H-1′), 6.38 (1H, d, J ) 8 Hz, H-5), 4.64-
4.62 (1H, m, H-4′), 4.10-4.07 (1H, m, H-3′), 3.56-3.49 (2H, m,
2 × H-5′), 2.58-2.55 (1H, m, H-2′), 2.39-2.36 (1H, m, H-2′).
FAB-MS (rel intensity): m/z 582 (68%, MH+), 303 (100%,
DMTH+), 164 (8%, base H+).
(B) P r ep a r a t ion of 5′-O-(4,4′-Dim et h oxyt r it yl)-8-(h y-
dr oxym eth yl)-3,N4-eth en o-2′-deoxycytidin e (4). Sodium boro-

210 Chem. Res. Toxicol., Vol. 13, No. 3, 2000
Chenna et al.
Sch em e 2. Syn th esis of th e P r otected P h osp h or a m id ite Der iva tive of
8-(Hyd r oxym eth yl)-3,N4-eth en o-2′-d eoxycytid in e
hydride (0.036 g, 0.94 mmol, 2.0 equiv) was added to a solution
of 5′-O-(4,4′-dimethoxytrityl)-3,N4-ethenocarbaldehyde-2′-deoxy-
cytidine (0.275 g, 0.47 mmol) in 10 mL of ethanol at 0 °C. The
reaction mixture was allowed to warm to room temperature and
was then stirred for 1 h and diluted with dichloromethane. The
organic solution was washed with saturated sodium chloride
solution and water, dried (Na2SO4), filtered, and concentrated
in vacuo to afford a white foam. The desired product (4) was
isolated using silica gel column chromatography eluting with
4% methanol and 96% dichloromethane. The resulting white
4.83-4.81 (2H, m, CH2), 4.20-4.19 (1H, m, H-4′), 3.53-3.47 (2H,
m, 2 × H-5′), 2.56-2.47 (2H, m, 2 × H-2′), 2.10 (3H, s, OAc).
FAB-MS (rel intensity): m/z 626 (71%, MH+), 303 (100%,
DMTH+), 166 (29%, base H+).
5′-O-(4,4′-Dimethoxytrityl)-3′-O-acetyl-8-(acetoxymethyl)-3,N4-
etheno-2′-deoxycytidine (5b) was obtained as a byproduct in 9%
yield (0.023 g). 1H NMR (CDCl3): δ 7.61 (1H, d, J ) 8 Hz, H-6),
7.45-6.84 (14H, m, DMTr, H-7), 6.62 (1H, t, J ) 6 Hz, H-1′),
6.26 (1H, d, J ) 8 Hz, H-5), 5.57-5.55 (2H, m, CH2), 5.50-5.48
(1H, m, H-3′), 4.22-4.21 (1H, m, H-4′), 3.57-3.49 (2H, m, 2 ×
H-5′), 2.61-2.56 (1H, m, H-2′), 2.49-2.44 (1H, m, H-2′), 2.12
(3H, s, OAc), 2.10 (3H, s, OAc). FAB-MS (rel intensity): m/z
668 (71%, MH+), 303 (100%, DMTH+), 208 (37%, base H+).
(D) P r ep a r a tion of 5′-O-(4,4′-Dim eth oxytr ityl)-3′-O-[2-
cya n oeth yl (N,N-diisop r opyl)ph osph in yl]-8-(a cetoxym eth -
yl)-3,N4-eth en o-2′-d eoxycytid in e (6). Triethylamine (7 mL,
0.121 g, 1.20 mmol, 10 equiv) and 2-cyanoethyl N,N-diisopro-
pylchlorophosphoramidite (29 µL, 0.042 g, 0.17 mmol, 1.4 equiv)
were added to a solution of 5′-O-(4,4′-dimethoxytrityl)-8-(ac-
etoxymethyl)-3,N4-etheno-2′-deoxycytidine (0.075 g, 0.12 mmol)
in dry dichloromethane (7 mL). The reaction mixture was stirred
for 2 h and then was diluted with dry dichloromethane. The
organic solution was washed with 5% sodium hydrogen carbon-
ate solution and saturated sodium chloride solution, dried (Na2-
SO4), filtered, and evaporated in vacuo (azeotrope/benzene) to
afford an off-white gum in 92% yield (0.097 g). This material
(6) was dried (P2O5, NaOH, in vacuo, 60 h) and then used in
oligonucleotide synthesis without further purification. 31P NMR
(CDCl3): δ 148.0, 147.6 (1:1).
1
foam was obtained in 89% yield (0.245 g). H NMR (CDCl3): δ
7.62 (1H, d, J ) 8 Hz, H-6), 7.41-6.82 (14H, m, DMTr, H-7),
6.52 (1H, t, J ) 6 Hz, H-1′), 6.29 (1H, d, J ) 8 Hz, H-5), 4.82-
4.80 (2H, m, CH2), 4.61-4.59 (1H, m, H-4′), 4.07-4.06 (1H, m,
H-3′), 3.59-3.44 (2H, m, 2 × H-5′), 2.53-2.47 (1H, m, H-2′),
2.37-2.32 (1H, m, H-2′). FAB-MS (rel intensity): m/z 584 (50%,
MH+), 303 (100%, DMTH+), 166 (23%, base H+).
(C) P r ep a r a tion of 5′-O-(4,4′-Dim eth oxytr ityl)-8-(a ce-
toxym eth yl)-3,N4-eth en o-2′-d eoxycytid in e (5). 1-Acetylimi-
dazole (0.037 g, 0.34 mmol, 1.0 equiv) was added to 5′-O-(4,4′-
dimethoxytrityl)-8-(hydroxymethyl)-3,N4-etheno-2′-deoxycyti-
dine (0.195 g, 0.33 mmol) in dichloromethane. The reaction
mixture was dried by evaporation (vacuo) and allowed to stand
for 72 h at 4 °C. The reaction produced three products (5, 5a ,
and 5b). The desired product (5) was isolated using silica gel
column chromatography eluting with 15% acetone and 85%
dichloromethane. The resulting white foam was obtained in 35%
yield (0.076 g). 1H NMR (CDCl3): δ 7.62 (1H, d, J ) 8 Hz, H-6),
7.41-6.82 (14H, m, DMTr, H-7), 6.53 (1H, t, J ) 6 Hz, H-1′),
6.28 (1H, d, J ) 8 Hz, H-5), 5.53-5.51 (2H, m, CH2), 4.59-4.57
(1H, m, H-4′), 4.05-4.04 (1H, m, H-3′), 3.53-3.47 (2H, m, 2 ×
H-5′), 2.53-2.47 (1H, m, H-2′), 2.36-2.30 (1H, m, H-2′), 2.06
(3H, s, OAc). FAB-MS (rel intensity): m/z 626 (60%, MH+), 303
(100%, DMTH+), 208 (23%, base H+).
Solid -P h a se Syn th esis of th e Oligon u cleotid es. Synthe-
sis of the oligonucleotides was carried out on an Applied
Biosystem 394 automated DNA synthesizer on a 1 µmol scale
using phosphoramidite chemistry on a controlled-pore glass
(CPG) support with an aminopropyl succinate linker. Phenoxy-
acetyl phosphoramidites (PAC) were used to synthesize the DNA
oligomers. The synthesis followed the standard protocol of the
DNA synthesizer for phosphoramidite chemistry, except for the
step in which the modified deoxynucleoside was inserted. In this
step, the time of coupling was increased to 900 s, to maximize
5′-O-(4,4′-Dimethoxytrityl)-3′-O-acetyl-8-(hydroxymethyl)-
3,N4-etheno-2′-deoxycytidine (5a ) was obtained as a byproduct
in 38% yield (0.082 g). 1H NMR (CDCl3): δ 7.58 (1H, d, J ) 8
Hz, H-6), 7.39-6.82 (14H, m, DMTr, H-7), 6.58 (1H, t, J ) 6
Hz, H-1′), 6.27 (1H, d, J ) 8 Hz, H-5), 5.46-5.45 (1H, m, H-3′),

Synthesis of the Glycidaldehyde-dC Adduct
Chem. Res. Toxicol., Vol. 13, No. 3, 2000 211
the coupling efficiency which was 89%. The 5′-dimethoxytrity-
lated oligonucleotides were base deprotected and cleaved from
the resin with 0.05 M potassium carbonate/anhydrous methanol
(8 h, room temperature). The 5′-dimethoxytritylated oligonucle-
otides were then purified by HPLC (system 1), detritylated (80%
acetic acid/water), purified by HPLC (system 2), and desalted
(Sep-Pak Cartridges, Waters Associates).
The following DNA oligomers were prepared: 1, 5′-CCG CTA
GXG GGT ACC GAG CTC GAA T [HPLC, tR, 25.69 min in
system 1 and 24.23 min in system 2]; 2, 5′-AGC GGT TXT TGA
GCT [HPLC tR, 29.13 min in system 1 and 24.66 min in system
2]; and 3, 5′-AGC GGC CXC CGA GCT [HPLC tR, 29.04 min in
system 1 and 24.61 min in system 2] [X ) 8-(hydroxymethyl)-
3,N4-etheno-dC].
En zym e Hyd r olysis of th e Oligon u cleotid es. The defined
oligonucleotides (0.5 A260 unit) were dissolved in 80 µL of freshly
deionized water, 10 µL of 0.1 M MgCl2, and 10 µL of 0.5 M Tris-
HCl buffer (pH 7.5) and digested with snake venom phosphodi-
esterase (5 µL) and bacterial alkaline phosphatase (5 µL) at 37
°C for 1 h. The digested mixture was analyzed on a a Supelcosil
LC-18-DB HPLC column (250 mm × 4.6 mm, 5 µm, Supelco
Inc.) using system 3. Quantitation of the deoxynucleosides was
on the basis of integration of the peak areas at 260 nm.
Resu lts a n d Discu ssion
The glycidaldehyde adduct of dC, 8-HM-ꢀdC, and its
phosphoramidite were synthesized (Schemes 1 and 2).
In 1984, Nair and co-workers (19) reported that the
etheno carboxaldehyde derivatives of ribonucleosides can
be obtained in high yield and purity using bromoma-
lonaldehyde (BMA). This methodology was used to pre-
pare 3,N4-ethenocarbaldehyde-dC 1 (Scheme 1) in 28%
yield, which could then be converted to the phosphora-
midite for oligonucleotide synthesis.
3,N4-Ethenocarbaldehyde-dC was found to have the
same UV parameter (λmax ) 326 nm) as the analogous
ribocytidine analogue reported by Nair and Kronberg (19,
20). The molecular ion in its mass spectrum at m/z 280
suggested the formation of a 1:1 adduct in which bromine
is not present. The 1H NMR spectrum showed the
presence of an aldehydic proton at δ 10.53 (s). The desired
product (Scheme 1) was separated from the biologically
significant byproduct, 3,N4-etheno-dC, using silica gel
column chromatography twice eluted with acetone and
dichloromethane (see Experimental Procedures). It was
found that the rate of formation of 3,N4-etheno-dC was
lower in acidic buffer [potassium phosphate (pH 4.5)]
than under neutral conditions. 3,N4-Ethenocarbaldehyde-
dC is not stable in methanol; i.e., it is slowly converted
to the hemiacetal at room temperature with an estimated
half-life of about 18 h. Therefore, methanol was not used
in its purification.
F igu r e 2. HPLC profile of 2′-deoxynucleosides obtained as a
result of enzymatic digestion of oligonucleotide 2, which contains
a single 8-(hydroxymethyl)-3,N4-etheno-2′-deoxycytidine (see
Materials and Methods). HPLC was performed using system 2.
The retention times of the deoxynucleosides are as follows: dC,
10.5 min; dG, 15.5 min; dT, 17.4 min; dA, 21.2 min; and
8-(hydroxymethyl)-3,N4-etheno-2′-dC, 23.7 min.
F igu r e 3. UV spectrum of 8-(hydroxymethyl)-3,N4-etheno-2′-
deoxycytidine at pH 7.
etheno ring which unexpectedly had a reactivity similar
to that of the secondary 3′-OH. The hydroxymethyl on
the etheno ring could not be acylated with acetic anhy-
dride. Low-temperature acylation (4 °C) with 1-acetyl-
imidazole (21) (Scheme 2) afforded a mixture of the
intended base-acylated product 5 in 35% yield, a sugar-
3′-O-acylated byproduct 5a in 38% yield, and a diacylated
byproduct 5b in 9% yield. These products were separated
using silica gel column chromatography. There are
important differences between the spectra of 5 and 5a .
The mass spectrum of 5 has a peak (m/z 208) correspond-
ing to an acylated base. This signal is not present in the
mass spectrum of 5a . The sugar protons have higher
A small portion of 1 (Scheme 1) was reduced using
sodium borohydride to afford an analytical sample of
8-HM-ꢀdC 2 as a marker for HPLC to verify the incor-
poration of the adduct in oligonucleotides.
The primary 5′-OH of 1 (Scheme 2) was protected with
dimethoxytrityl chloride in 69% yield. The expected
1
DMTr signals were found to be present in the H NMR
spectrum at δ 7.41-6.82 (m). The aldehyde moiety of the
5′-dimethoxytritylated nucleoside 3 (Scheme 2) was
reduced using sodium borohydride to afford the 8-(hy-
droxymethyl)-3,N4-etheno-2′-deoxycytidine adduct 4 in
1
89% yield. The CH2OH protons were observed in the H
1
NMR spectrum at δ 4.82-4.80 (m).
The most difficult step in the synthesis was the
selective acylation of the primary hydroxymethyl on the
chemical shifts in the H NMR spectrum of 5a (δ 5.46-
5.45, m, H-3′; δ 4.20-4.19, m, H-4′) than in the spectrum
of 5 (δ 4.59-4.57, m, H-3′; δ 4.05-4.04, m, H-4′).

212 Chem. Res. Toxicol., Vol. 13, No. 3, 2000
Chenna et al.
indicates that 8-HM-ꢀdC is not stable under alkaline
conditions. The instability of the adduct to hydroxide ion
was further demonstrated by a slow rate of decomposition
at pH 7, which was observed after 3 weeks at room
temperature.
However, it is important to distinguish between the
stability of the adduct as a monomer and the stability of
the adduct within an oligonucleotide. For example, the
purity of oligomer 2 is shown by HPLC (system 2, single
peak, retention time of 24.61 min) and by its electrospray
mass spectrum (m/z 4653, M+). This indicates that the
oligomer containing the adduct is stable to deprotection
and purification. Furthermore, oligomer 2 was allowed
to stand at room temperature at pH 7 for 3 weeks and
exhibited no additional peak on digestion. This indicates
that the adduct has far greater stability within an
oligonucleotide than as a monomer.
F igu r e 4. UV spectra of 8-(hydroxymethyl)-3,N4-etheno-2′-
deoxycytidine at pH 1, 7, and 14.
In summary, we have prepared 8-(hydroxymethyl)-
3,N4-etheno-2′-deoxycytidine, a potential carcinogenic
glycidaldehyde adduct (1-8), for the first time on a large
scale with good yield. The corresponding phosphoramidite
was synthesized and site-specifically incorporated into
DNA oligonucleotides which are being used for studies
of their physical and biochemical properties.
Ack n ow led gm en t. This work was supported by NIH
Grants CA 47723 and CA 72079 (to B.S.) and was
administered by the Lawrence Berkeley National Labo-
ratory under Department of Energy Contract DE-AC03-
76SF00098.
F igu r e 5. UV spectra of 8-(hydroxymethyl)-3,N4-etheno-2′-
deoxycytidine at pH 1, 7, and 14 after 48 h at ambient
temperature.
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µmol scale three different oligonucleotides: 1, 5′-CCG
CTA GXG GGT ACC GAG CTC GAA T; 2, 5′-AGC GGT
TXT TGA GCT; and 3, 5′-AGC GGC CXC CGA GCT [X
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The phosphoramidite was incorporated with a coupling
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oligomers were deprotected using 0.05 M potassium
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The composition of the 25-mer and the presence of
8-HM-ꢀdC were confirmed by enzymatic digestion (Figure
2). This adduct was unstable to the digestion conditions
at pH 7.4 (37 °C, 18 h). This problem was prevented by
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7 (Figures 3 and 4), pH 1 (Figure 4), and pH 14 (Figure
4). The sample at pH 7 was adjusted to pH 1 with 0.1 N
hydrochloric acid and to pH 14 with 0.1 N sodium
hydroxide. The protonated base has a significantly dif-
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) 278 nm) and alkaline (λmax ) 278 nm) conditions. The
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stand at room temperature for 48 h, and then the UV
spectra were again recorded (Figure 5). There was no
significant change in absorption of the spectra of the
samples held at pH 1 (λmax ) 294 nm) or at pH 7 (λmax
)
278 nm). However, there was a large change when the
mixture was held at pH 14 (λmax ) 314 nm), which

Synthesis of the Glycidaldehyde-dC Adduct
Chem. Res. Toxicol., Vol. 13, No. 3, 2000 213
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