Synthesis and characterization of nucleosides and oligonucleotides bearing adducts of butadiene epoxides on adenine N6 and guanine N2

Article abstract of DOI:10.1021/tx000241k

Butadiene is a major industrial chemical whose genotoxic effects are attributed to the reaction of its oxidized metabolites, butadiene monoepoxide (BDO) and butadiene diepoxide (BDO2), with DNA. Nucleosides and oligonucleotides containing regio- and stereochemically specific adducts of BDO and the BDO2-related compound, butene 3,4-diol 1,2-epoxide (BDE), on guanine [(2R)- and (2S)-N²-(1-hydroxy-3-buten-2-yl) and (2R,3R)- and (2S,3S)-N²-(2,3,4-trihydroxybut-1-yl), respectively] and on adenine [(2R)- and (2S)-N⁶-(1-hydroxy-3-buten-2-yl) and (2R,3R)- and (2S,3S)-N⁶-(2,3,4-trihydroxybut-1-yl), respectively] have been prepared by nonbiomimetic routes. For guanine adducts, 2-fluoro-O⁶-(trimethylsilylethyl)-2′-deoxyinosine was treated with (2R)- and (2S)-2-amino-3-buten-1-ol to give the BDO adducts and with (2R,3R)- and (2S,3S)-1-amino-2,3,4-butanetriol to produce the BDE adducts; the adducted oligonucleotides were prepared from 11-mer oligonucleotides containing the halopurine. Adenine adducts were prepared in a similar fashion using 6-chloropurine 2′-deoxyriboside as the reactive purine component.

Full text of DOI:10.1021/tx000241k

Chem. Res. Toxicol. 2001, 14, 379-388
379
Syn th esis a n d Ch a r a cter iza tion of Nu cleosid es a n d
Oligon u cleotid es Bea r in g Ad d u cts of Bu ta d ien e Ep oxid es
on Ad en in e N6 a n d Gu a n in e N2
Lubomir V. Nechev, Mingzhu Zhang,† Dimitrios Tsarouhtsis,‡ Pamela J . Tamura,
Amanda S. Wilkinson, Constance M. Harris, and Thomas M. Harris*
Chemistry Department and Center in Molecular Toxicology, Vanderbilt University,
Nashville, Tennessee 37235
Received November 24, 2000
Butadiene is a major industrial chemical whose genotoxic effects are attributed to the reaction
of its oxidized metabolites, butadiene monoepoxide (BDO) and butadiene diepoxide (BDO2),
with DNA. Nucleosides and oligonucleotides containing regio- and stereochemically specific
adducts of BDO and the BDO2-related compound, butene 3,4-diol 1,2-epoxide (BDE), on guanine
[(2R)- and (2S)-N2-(1-hydroxy-3-buten-2-yl) and (2R,3R)- and (2S,3S)-N2-(2,3,4-trihydroxybut-
1-yl), respectively] and on adenine [(2R)- and (2S)-N6-(1-hydroxy-3-buten-2-yl) and (2R,3R)-
and (2S,3S)-N6-(2,3,4-trihydroxybut-1-yl), respectively] have been prepared by nonbiomimetic
routes. For guanine adducts, 2-fluoro-O6-(trimethylsilylethyl)-2′-deoxyinosine was treated with
(2R)- and (2S)-2-amino-3-buten-1-ol to give the BDO adducts and with (2R,3R)- and (2S,3S)-
1-amino-2,3,4-butanetriol to produce the BDE adducts; the adducted oligonucleotides were
prepared from 11-mer oligonucleotides containing the halopurine. Adenine adducts were
prepared in a similar fashion using 6-chloropurine 2′-deoxyriboside as the reactive purine
component.
reactivity is seen in the variety of products thus far
isolated from the reaction of BDO with purines, nucleo-
sides, and DNA. Guanine adducts that have been identi-
fied include regioisomeric N7 adducts (3-7) and N1- and
N2-(1-hydroxy-3-buten-2-yl) compounds (8). Adenine ad-
ducts include regioisomeric N1 adducts, N6-(2-hydroxy-
3-buten-yl) adducts, and an N1-(1-hydroxy-3-buten-2-yl)
inosine derivative (9, 10). The N6 adduct was postulated
to arise from the N1-(2-hydroxy-3-buten-1-yl) compound
by Dimroth rearrangement and the N1 inosine compound
from the N1-(1-hydroxy-3-buten-2-yl) adduct by deami-
nation catalyzed by the primary hydroxyl group; analo-
gous reactions have been described for the reaction of
styrene oxide with adenine (11, 12). In addition, N3
adducts have been detected by Tretyakova et al. in vitro
and in vivo (7) and by Selzer and Elfarra (13).
In tr od u ction
Butadiene (1) (Scheme 1) is a major industrial chemical
used widely in the production of polymeric materials such
as synthetic rubber (styrene-butadiene) and ABS (acry-
lonitrile-butadiene-styrene) plastics. It is also a combus-
tion product, found in cigarette smoke and automotive
emissions. Butadiene is a gas (bp -4.4 °C), leading to
problems of containment and increasing the risk of
exposure for those involved in its manufacture, transport,
and use. It is carcinogenic in mice, although less so in
rats, and is listed as a human carcinogen in the latest
report from the National Toxicology Program (1). Buta-
diene causes chromosomal abnormalities and increased
levels of lymphatic and hemopoietic cancers in workers
with long-term exposure in the workplace. The toxicology
and epidemiology of butadiene have recently been re-
viewed by Himmelstein et al. (2). The genotoxic effects
of butadiene are attributed to its metabolic activation,
in a non-stereospecific manner, by P450s (primarily 2A6
and 2E1) to the (R)- and (S)-monoepoxides (2, BDO)1
which can undergo diverse reactions, including hydroly-
sis, conjugation with glutathione, reaction with DNA, and
further oxidation by P450 to (R,R)-, (S,S)-, or (R,S)-
diepoxide (3, BDO2) (Scheme 1). The monoepoxide can
react either at C1 by an SN2 mechanism or at C2 by a
route with more SN1 character (Scheme 2), although
inversion products predominate at C2 also, at least when
the nucleophile is N7 of guanine (3).2 This diverse
1
Abbreviations: BD, butadiene; BDO, butadiene monoepoxide;
BDO2, butadiene diepoxide; BDE, butene 3,4-diol 1,2-epoxide; R-BDO-
dAdo, (R)-N6-(1-hydroxy-3-buten-2-yl)deoxyadenosine; S-BDO-dAdo,
(S)-N6-(1-hydroxy-3-buten-2-yl)deoxyadenosine; R,R-BDE-dAdo, (R,R)-
N6-(2,3,4-trihydroxybut-1-yl)deoxyadenosine; S,S-BDE-dAdo, (S,S)-N6-
(2,3,4-trihydroxybut-1-yl)deoxyadenosine; R-BDO-dGuo, (R)-N2-(1-
hydroxy-3-buten-2-yl)deoxyguanosine; S-BDO-dGuo, (S)-N2-(1-hydroxy-
3-buten-2-yl)deoxyguanosine; R,R-BDE-dGuo, (R,R)-N2-(2,3,4-trihydroxy-
but-1-yl)deoxyguanosine; S,S-BDE-dGuo, (S,S)-N2-(2,3,4-trihydroxy-
but-1-yl)deoxyguanosine; TMSE, trimethylsilylethyl; DIPEA, diisopro-
pylethylamine; MALDI, matrix-assisted laser desorption ionization
mass spectrometry; FAB, fast atom bombardment mass spectrometry;
NBA, 3-nitrobenzyl alcohol; CGE, capillary gel electrophoresis; PAGE,
polyacrylamide gel electrophoresis.
2
The adducts of BDO at C2 can form by either an SN2 or SN1
process; the former gives adducts with the configuration that is the
opposite of that of the parent epoxide [(R)-adduct from the (S)-epoxide],
whereas the latter gives adducts of both retained and inverted
configurations. Adducts at C1 of BDO have the same configuration as
the epoxide since the reaction does not involve the chiral center.
Similarly, BDE and BDO2 give adducts of the same configuration as
the parent compound because the reaction occurs at the terminal
position [R,R-BDE or -BDO2 gives (R,R)-adducts].
* To whom correspondence should be addressed: Department of
Chemistry, Box 351822, Station B, Vanderbilt University, Nashville,
TN 37235. E-mail: harristm@toxicology.mc.vanderbilt.edu.
† Current address: DuPont Pharmaceuticals Co., Wilmington, DE
19880.
‡ Current address: Abbott Laboratories, Abbott Park, IL 60064.
10.1021/tx000241k CCC: $20.00 © 2001 American Chemical Society
Published on Web 03/14/2001

380 Chem. Res. Toxicol., Vol. 14, No. 4, 2001
Nechev et al.
Sch em e 1
Sch em e 2
Butadiene diepoxide (3, BDO2) is formed in vivo in
much smaller quantities than the monoepoxide, but is
more genotoxic. The diepoxide can react at one or both
epoxides. Reaction at one epoxide followed by hydrolysis
of the second can lead to trihydroxy adducts. However,
recent papers (14, 15) present evidence that trihydroxy
adducts arise largely from butene 3,4-diol 1,2-epoxide (4,
BDE) formed by rapid hydrolysis of BDO2 by epoxide
hydrolase. BDO2 adducts of guanine that have been
identified include N7-(2,3,4-trihydroxybut-1-yl)guanine
(7, 16, 17) and a bis N7 adduct [1,4-bis(guanin-7-yl)-
butane-2,3-diol] isolated by Lawley and Brookes (16, 18)
from salmon sperm DNA treated with the diepoxide.
Adenine adducts of BDO2 that have been isolated include
N3- and N6-(2,3,4-trihydroxybut-1-yl)adenine (7, 19). A
32P-postlabeling study of DNA adducts formed in vivo and
in vitro from BDO2 showed that the predominant bands
corresponded to adenine derivatives, although they were
not specifically identified (20). Leuratti et al. (21, 22),
using an HPLC/32P-postlabeling method, detected an
adenine N6 adduct from treatment of CT DNA or Chinese
hamster ovary (CHO) cells with BDO2; guanine-BDO2
adducts were also detected in these studies but at much
lower levels. No cross-linked adducts of BDO2 have yet
been isolated from in vivo sources.
As investigations into the isolation and identification
of DNA-bound metabolites of BDO and BDO2 have
progressed, other research groups have examined the
mutations induced by exposure to BD or its epoxides. In
1990, Goodrow et al. (23) examined NIH 3T3 cells
transformed by DNA from lymphomas and lung and liver
tumors induced in B6C3F1 mice by long-term exposure
to butadiene. The most common mutations found in
transformed cells were G to C transversions in codon 13
of the K-ras protooncogene. Studies by Cochrane and
Skopek (24-26) of hprt mutations in TK6 human lym-
phoblastoid cells and splenic T cells exposed to BDO,
BDO2, or BDE showed that BDO2 is approximately 100
times more mutagenic than the monoepoxide and BDE
was the least mutagenic of the three. Transitions and
transversions at both GC and AT sites as well as
frameshifts were detected. The BDO2-treated cells showed
large deletions, seen previously, for example, in Droso-
phila melanogaster exposed to diepoxide (27, 28). Exten-
sive in vivo mutagenicity studies in B6C3F1 lacI trans-
genic mice have been carried out by Recio (29-35).
Mutations in the lacI gene from exposure to BD were
primarily point mutations (61% at GC and 20% at AT)
with the level of AT mutations being significantly higher
than in the controls (32). When TK6 cells were exposed
to butadiene monoepoxide, most (78%) of the mutations
observed in the hprt gene were single-base substitutions,
with a significant increase again being observed in the
level of A‚T f T‚A transversions (36). A similar study
with the diepoxide (37) also showed a significant increase
in the level of A‚T f T‚A transversions together with
increased numbers of deletions. These results were in
general agreement with those reported by Cochrane and
Skopek. When Rat2 lacI transgenic fibroblasts were
treated with monoepoxide, the mutations seen were
primarily base substitutions (60% at GC and 31% at AT)
(30), whereas the diepoxide in the same system induced
micronuclei but few mutations (38). The authors of the
latter studies suggest that BDO is largely responsible for

BD Epoxide Adducts at Adenine N6 and Guanine N2
Chem. Res. Toxicol., Vol. 14, No. 4, 2001 381
taken at 1 min intervals with a 1 °C/min temperature gradient.
The temperature was increased from 5 to 90 °C, and then
lowered to 5 °C. Unmodified and modified samples were run
simultaneously to ensure identical reaction conditions.
base substitutions whereas BDO2 exposure leads to
deletions which are not easily detected in the Rat2 cell
assay.
To gain more insight into the source of mutations
arising from exposure to butadiene or its metabolites, we
have undertaken the synthesis of a series of oligonucleo-
tides containing regio- and stereospecific adducts of (R)-
and (S)-BDO and (R,R) and (S,S)-BDE on the exocyclic
amino groups of guanine and adenine. The other major
adducts thus far identified, namely, the N7 guanine and
N1 and N3 adenine adducts, are unstable and not readily
amenable to oligonucleotide synthesis. Presumably, they
would not survive very long in vivo but could, however,
be mutagenic. The target adducts in this study are those
arising from attack at the C2 position of BDO and the
C1 position of BDO2 and BDE (Scheme 2).
En zym a tic Digestion s. Enzymatic digestions of adducted
dGuo oligonucleotides 19a -d were carried out under conditions
described by Borowy-Borowski et al. (39). The oligonucleotide
(0.2-2.0 A260 units), 20 µL of buffer [0.1 M Tris-HCl and 10 mM
MgCl2 (pH 9)], 0.04 unit of snake venom phosphodiesterase
(VIII-S from Crotalus durissus terrificus, Sigma), and 0.40 unit
of alkaline phosphatase (Type III, Escherichia coli, Sigma) were
mixed and incubated at 37 °C for 6-18 h. The reaction was
stopped by heating at 75 °C for 1 min. The digest was diluted
with H2O, filtered through a centrifugal filter, and analyzed by
HPLC [YMC-ODS-AQ column (4.6 mm × 250 mm) with the
following gradients: (A) 0.1 M ammonium formate at pH 6.4
and (B) from 1 to 10% B (CH3CN) over the course of 15 min
and from 10 to 20% B over the course of 5 min, at a flow rate of
1.5 mL/min].
The adducted dAdo oligonucleotides 17a -d were digested
according to a two-step protocol utilizing nuclease P1, snake
venom phosphodiesterase, and alkaline phosphatase (40).
Syn th eses. (1) (2R)- a n d (2S)-2-Am in o-3-bu ten -1-ol (5a
a n d 5b, r esp ectively). (2R)- and (2S)-2-amino-3-buten-1-ol
were prepared from D- and L-N-Boc-protected methionine
(Aldrich Chemical Co.) by a published procedure (41).
(2) (2R,3R)- a n d (2S,3S)-1-Am in o-2,3,4-bu t a n et r iol (5c
a n d 5d , r esp ectively, Sch em e 3). Meth yl (2R,3R)-2,3-O-
Ben zylid en eta r tr a m id e (7). Dimethyl 2,3-O-benzylidene-D-
tartrate (6, 1 g, 3.8 mmol) was dissolved in methanol (6 mL).
Ammonia (2 M in methanol, 1.9 mL, 3.8 mmol) was added to
the solution, and the mixture was stirred at room temperature
for 24 h. Solvents were evaporated, and the crude mixture was
purified by column chromatography on silica gel (1:1 ethyl
acetate/hexane mixture) to give 0.65 g (70%) of the desired
product 7. About 19% of the unreacted starting material was
isolated as well as 5% of the diamide. Compound 7 is a mixture
of diastereoisomers (R,R,R and S,R,R) because of the chiral
center in the benzylic position (R or S): 1H NMR of the first
isomer (DMSO-d6) δ 3.61 (s, 3H, CH3O), 4.80 (d, 1H, CHCONH2),
4.84 (d, 1H, CHCOOCH3), 5.96 (s, 1H, benzylic), 7.43 (m, 3H,
aromatic), 7.56 (m, 2H, aromatic); 1H NMR of the second isomer
(DMSO-d6) δ 3.75 (s, 3H, CH3O), 4.65 (d, 1H, CHCONH2), 4.89
(d, 1H, CHCOOCH3), 5.99 (s, 1H, benzylic), 7.43 (m, 3H,
aromatic), 7.56 (m, 2H, aromatic); HRMS-FAB (NBA matrix)
calcd for C12H14NO5 [M + H]+ 252.0872, found 252.0887.
(3) (2R,3R)-2,3-O-Ben zylid en e-1-a m in o-2,3,4-bu ta n etr iol
(8). Ester amide 7 from the previous reaction (0.45 g, 1.8 mmol)
was dissolved in 15 mL of anhydrous THF. The solution was
added dropwise (10 min) under argon to a stirring mixture of
LiAlH4 (0.6 g, 15 mmol) and anhydrous THF (30 mL). The
mixture was stirred at room temperature for 1 h and heated
under reflux for an additional 5 h. The reaction was followed
by TLC (85:8:7 CH3CN/H2O/NH4OH mixture). Water was care-
fully added (ice/water bath), and the resulting mixture was
filtered. The filtrate was diluted with ether and dried over
sodium sulfate. Solvents were removed, and the crude product
was purified by silica gel column chromatography (the same
solvent system as for TLC) to give 0.340 g (90%) of protected
aminotriol 8: 1H NMR (DMSO-d6 with D2O) δ 2.75 (m, 2H, CH2-
NH2), 3.55 (m, 2H, CH2OH), 3.87 (m, 2H, CHCH2NH2 and
CHCH2OH), 5.82, 5.83 (two s, 1H, benzylic R and S), 7.36 (m,
3H, aromatic), 7.43 (m, 2H, aromatic); HRMS-FAB (NBA
matrix) calcd for C11H16NO3 [M + H]+ 210.1130, found 210.1135.
Exp er im en ta l Section
Ma ter ia ls. Methylene chloride, pyridine, triethylamine, and
DIPEA were distilled from calcium hydride under a nitrogen
atmosphere. 1,4-Dioxane was distilled from sodium under a
nitrogen atmosphere. THF was distilled from a sodium/potas-
sium alloy with benzophenone ketyl as an indicator. Methanol
was distilled from sodium methoxide (5 mol %) under a nitrogen
atmosphere. Other chemicals were used as purchased without
further purification. Melting points are uncorrected.
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)
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.
NMR. 1H NMR spectra were recorded on a Bruker AC 300
or AM 400 NMR spectrometer with DMSO-d6, CD3CN, CDCl3,
or MeOH-d4 as the solvent.
Ma ss Sp ectr om etr y. Low- and high-resolution FAB mass
spectra were obtained at the Mass Spectrometry Facility at the
University of Notre Dame (Notre Dame, IN). Mass spectra
(MALDI) of oligonucleotides were obtained using a Voyager Elite
DE instrument (PerSeptive Biosystems). The system was oper-
ated in the negative ion mode using a matrix mixture of 2′,4′,6′-
trihydroxyacetophenone monohydrate and ammonium hydrogen
citrate or ammonium tartrate.
Oligon u cleotid es. Oligodeoxynucleotides were synthesized
on an Expedite 8909 DNA synthesizer (PerSeptive Biosystems)
on a 1 µmol scale using the manufacturer’s 4-tert-butylphenoxy-
acetyl (tBPA)-protected phosphoramidites and standard syn-
thesis protocol.
CD Sp ectr oscop y. CD spectra were recorded in methanol
at 25 °C on a J ASCO J -700 spectropolarimeter.
CGE. 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.
Th er m a l Sta bility Stu d ies. Adducted oligonucleotides and
their complements (∼0.5 A260 units of each) were dissolved in
buffer [a 10 mM Na2HPO4/NaH2PO4 mixture containing 1.0 M
NaCl and 50 µM EDTA (pH 7.0)]. The sample vials were placed
in a water bath at 85 °C; heating was discontinued, and the
samples were allowed to cool to ambient temperature and stored
at 4 °C overnight. Melting studies were performed using a
Varian Cary 04E spectrophotometer. UV measurements were
(4) (2R,3R)-1-Am in o-2,3,4-bu ta n etr iol (5c). The protected
aminotriol (8, 0.3 g, 1.4 mmol) was neutralized with 1 M HCl
to pH 6.0. The solution was evaporated to dryness (rotary
evaporator, 40 °C). Sulfuric acid (0.01 M, 15 mL) was added,
and the mixture was stirred at 100 °C for 3 h. The solution was
evaporated to dryness; water was added and evaporated three
times (to remove the PhCHO). The product was dissolved in a
small volume of water, and the pH was adjusted (1 M NaOH)

382 Chem. Res. Toxicol., Vol. 14, No. 4, 2001
Nechev et al.
to 12.0. The solution was evaporated to dryness. The mixture
was dissolved in a small volume of boiling 80% ethanol. The
insoluble inorganic salts were filtered out, and the filtrate was
kept at -20 °C for 1 h. The additional amounts of inorganic
salts which crystallized were filtered out again, and the solvents
were evaporated to give 0.174 g (81%) of 5c as a clear oil: (R,R)
[R]2D0 8.5° (c 2, water); 1H NMR (MeOH-d4) δ 2.62 (m, 2H, CH2-
NH2), 3.48 (m, 4H, 2× CHOH and CH2OH); HRMS-FAB (NBA
matrix) calcd for C4H12NO3 [M + H]+ 122.0817, found 122.0809.
The (2S, 3S) isomer could have been prepared similarly from
the corresponding L-tartrate, but a better route was available
from the commercially available methyl 3,4-O-isopropylidene-
L-threonate (the corresponding D isomer of the threonate is not
commercially available).
(5) Am id e of (2S,3R)-3,4-O-Isop r op ylid en e-2,3,4-tr ih y-
d r oxybu tyr ic Acid (10). Methyl 3,4-O-isopropylidene-L-thre-
onate (9, 1.3 g, 6.8 mmol) was dissolved in 30 mL of methanol.
A slow stream of ammonia (gas) was bubbled through the
solution for 4 h. The reaction was followed by TLC (2:1 ethyl
acetate/hexane mixture). Evaporation of the solvents yielded 10
as a clear oil (1.15 g, 97%) which crystallized in ∼10 min: 1H
NMR (DMSO-d6) δ 1.25 (s, 3H, CH3), 1.32 (s, 3H, CH3), 3.79
(m, 2H, R-CHOH and 1H from CH2O), 3.94 (m, 1H, from CH2O),
4.20 (m, 1H, CHOC), 5.51 (d, 1H, OH, J ) 6.4 Hz), 7.22 (bs, 2H,
CONH2); HRMS-FAB (NBA matrix) calcd for C7H14NO4 [M +
H]+ 176.0923, found 176.0902.
(6) (2S,3R)-3,4-Isop r op ylid en e-1-a m in o-2,3,4-bu ta n etr iol
(11). Amide 10 from the previous reaction (1.15 g, 6.6 mmol)
was dissolved in 25 mL of anhydrous THF. The solution was
added dropwise (10 min) under argon to a stirring mixture of
LiAlH4 (1.25 g, 33 mmol) and 150 mL of anhydrous THF. The
mixture was stirred at room temperature for 1 h and refluxed
for an additional 5 h. The reaction was followed by TLC (85:8:7
CH3CN/H2O/NH4OH mixture). Water was carefully added (ice/
water bath), and the resulting mixture was filtered. The filtrate
was diluted with ether and dried over sodium sulfate. Solvents
were removed, and the crude product was purified by silica gel
column chromatography (the same solvent system as for TLC)
to give 0.93 g (89%) of protected aminotriol 11: 1H NMR
(DMSO-d6) δ 1.27 (s, 3H, CH3), 1.33 (s, 3H, CH3), 2.52 (m, 2H,
CH2NH2), 3.36 (m, 1H, CHOH) 3.67 (m, 1H, from CH2O), 3.90
(m, 1H, from CH2O), 3.99 (m, 1H, CHOC); HRMS-FAB (NBA
matrix) calcd for C7H16NO3 [M + H]+ 162.1130, found 162.1110.
(7) (2S,3S)-1-Am in o-2,3,4-bu ta n etr iol (5d ). Isopropylidene-
protected aminotriol 11 from the previous step was dissolved
in THF (10 mL). Aqueous 1 M HCl (10 mL) was added to the
solution which was stirred at room temperature for 20 h. The
solvents were evaporated, and the residue was dissolved in a
small volume of water. The pH of the solution was adjusted to
11.0-12.0, and the mixture was evaporated to dryness. The
crude product was dissolved in a small amount of a boiling
mixture of ethanol and methanol (1:1) and filtered while hot.
The filtrate was kept for 1 h at -20 °C. Additional amounts of
crystallized inorganic salts were filtered out, and the solvents
were evaporated to give 0.61 g (90%) of product 5d as a clear
oil: (S,S) [R]2D0 -9.4° (c 2, water); 1H NMR (DMSO-d6 with
D2O) δ 2.82 (m, 2H, CH2NH2), 3.39 (m, 4H, 2× CHOH, CH2-
OH); HRMS-FAB (NBA matrix) calcd for C4H12NO3 [M + H]+
122.0817, found 122.0811.
(15 µL, 0.09 mmol), and 300 µL of dry DMF were mixed in a
small glass test tube. The tube was flushed with nitrogen and
closed, and the contents were stirred at 55 °C for 20 h. The
reaction was followed by TLC. The solvents were evaporated to
dryness (vacuum centrifuge), and the mixture was purified on
a silica gel column (86:12:2 CH3CN/H2O/concentrated NH4OH
mixture) to yield 20 mg (85%) of 13a : 1H NMR (DMSO-d6, 45
°C) δ 2.27 (m, 1H, H-2′′), 2.71 (m, 1H, H-2′), 3.63-3.52 (m, 5H,
H-5′, H-5′′, â, â′, R), 3.88 (m, 1H, H-4′), 4.41 (m, 1H, H-3′), 4.71
(t, 1H, â-OH, J ) 4.7 Hz), 5.03 (t, 1H, 5′-OH, J ) 5.5 Hz), 5.07
(ddd, 1H, γ′, J 1 ) 10.4 Hz, J 2 ) J 3 ) 1.6 Hz), 5.17 (d, 1H, 3′-
OH, J ) 4.1 Hz), 5.18 (ddd, 1H, γ, J 1 ) 17.3 Hz, J 2 ) J 3 ) 1.6
Hz), 5.96 (m, 1H, â′′), 6.34 (dd, 1H, H-1′, J 1 ) J 2 ) 6.2 Hz), 7.29
(d, 1H, NH, J ) 8.7 Hz), 8.18 (s, 1H, H-8), 8.31 (s, 1H, H-2);
HRMS-FAB (NBA matrix) calcd for C14H20N5O4 [M + H]+
322.1515, found 322.1510.
(2) (2S)-2′-Deoxy-N6-(1-h yd r oxy-3-bu ten -2-yl)a d en osin e
(13b, S-BDO-d Ad o). Following the procedure for the synthesis
of 13a using amino alcohol 5b instead of 5a yielded 21 mg (88%)
of the title compound: 1H NMR (DMSO-d6, 45 °C) δ 2.26 (m,
1H, H-2′′), 2.71 (m, 1H, H-2′), 3.61-3.52 (m, 5H, H-5′, H-5′′, â,
â′, R), 3.87 (m, 1H, H-4′), 4.40 (m, 1H, H-3′), 4.76 (t, 1H, â-OH,
J ) 4.6 Hz), 5.07 (ddd, 1H, γ′, J 1 ) 10.4 Hz, J 2 ) J 3 ) 1.6 Hz),
5.11 (t, 1H, 5′-OH, J ) 5.7 Hz), 5.17 (ddd, 1H, γ, J 1 ) 17.3 Hz,
J 2 ) J 3 ) 1.6 Hz), 5.23 (d, 1H, 3′-OH, J ) 4.1 Hz), 5.96 (m, 1H,
â′′), 6.34 (dd, 1H, H-1′, J 1 ) J 2 ) 6.1 Hz), 7.39 (d, 1H, NH, J )
8.7 Hz), 8.18 (s, 1H, H-8), 8.33 (s, 1H, H-2); HRMS-FAB (NBA
matrix) calcd for C14H20N5O4 [M + H]+ 322.1515, found 322.1512.
(3) (2R,3R)-2′-Deoxy-N6-(2,3,4-tr ih yd r oxybu t-1-yl)a d en -
osin e (13c, R,R-BDE-d Ad o). 6-Chloropurine 2′-deoxyriboside
(12, 10 mg, 0.04 mmol), the corresponding aminotriol (5c, 10
mg, 0.08 mmol), DIPEA (20 µL), and 100 µL of dry DMSO were
mixed in a small glass test tube. The tube was flushed with
nitrogen and closed, and the contents were stirred at 60 °C for
15 h. The reaction was followed by TLC. The solvents were
evaporated to dryness (vacuum centrifuge), and the mixture was
purified on a silica gel column (85:8:7 CH3CN/H2O/NH4OH) to
yield 10.6 mg (81%) of 13c: 1H NMR (DMSO-d6, 45 °C) δ 2.26
(m, 1H, H-2′′), 2.70 (m, 1H, H-2′), 3.39-3.45 (m, 3H, â or γ, δ,
δ′), 3.50-3.64 (m, 4H, H-5′, H-5′′, R, R′), 3.75 (m, 1H, γ or â),
3.88 (m, 1H, H-4′), 4.27 (bs, 1H, â- or γ-OH), 4.40 (m, 2H, H-3′,
δ-OH), 4.45 (bs, 1H, γ- or â-OH), 5.00 (t, 1H, 5′-OH, J ) 5.4
Hz), 5.15 (d, 1H, 3′-OH, J ) 4.0 Hz), 6.34 (dd, 1H, H-1′, J 1 ) J 2
) 6.8 Hz), 7.26 (bs, 1H, NH), 8.19 (s, 1H, H-8), 8.29 (s, 1H, H-2);
HRMS-FAB (NBA matrix) calcd for C14H22N5O6 [M + H]+
356.1570, found 356.1585.
(4) (2S,3S)-2′-Deoxy-N6-(2,3,4-tr ih yd r oxybu t-1-yl)a d en -
osin e (13d , S,S-BDE-d Ad o). The same procedure that was
used for 13c using aminotriol 5d led to 10.9 mg (83%) of the
title compound: 1H NMR (DMSO-d6, 45 °C) δ 2.26 (m, 1H, H-2′′),
2.72 (m, 1H, H-2′), 3.38-3.50 (m, 3H, â or γ, δ, δ′), 3.53-3.63
(m, 4H, H-5′, H-5′′, R, R′), 3.75 (m, 1H, γ or â), 3.88 (m, 1H,
H-4′), 4.29 (bs, 1H, â- or γ-OH), 4.41 (m, 2H, H-3′, δ-OH), 4.46
(d, 1H, γ- or â-OH, J ) 5.6 Hz), 5.02 (t, 1H, 5′-OH, J ) 5.0 Hz),
5.15 (d, 1H, 3′-OH, J ) 3.9 Hz), 6.35 (dd, 1H, H-1′, J 1 ) J 2
)
6.9 Hz), 7.27 (bs, 1H, NH), 8.19 (s, 1H, H-8), 8.30 (s, 1H, H-2);
HRMS-FAB (NBA matrix) calcd for C14H22N5O6 [M + H]+
356.1570, found 356.1579.
(5) (2R)-2′-Deoxy-N2-(1-h yd r oxy-3-bu ten -2-yl)gu a n osin e
(15a , R-BDO-d Gu o). 2-Fluoro-O6-(trimethylsilylethyl)-2′-deoxy-
inosine (14, 7 mg, 0.02 mmol), contained in a small glass test
tube equipped with a magnetic stirring bar, was dried overnight
under vacuum. (2R)-2-Amino-3-buten-1-ol (5a , 2.5 mg, 0.03
mmol, 1.5 equiv), DMSO (40 µL), and DIPEA (10 µL, 0.06 mmol,
3 equiv) were added. The mixture was stirred at 60 °C and
monitored by HPLC, using a YMC-ODS-AQ-C18 or Phenomenex
C-8 Luna 2 column (250 mm × 4.6 mm) at a flow rate of 1.5
mL/min. Solvent A was 0.1 M ammonium formate and solvent
B acetonitrile. The gradient for monitoring the reaction was from
10 to 90% B over the course of 25 min and the gradient for purity
checking from 1 to 10% B over the course of 15 min, followed
by 10 to 20% B over the course of 5 min. The reaction mixture
Syn th esis of Nu cleosid e Ad d u cts (Sch em e 4). Designa-
tion of atoms in the butadiene epoxide side chains is shown
below:
(1) (2R)-2′-Deoxy-N6-(1-h yd r oxy-3-bu ten -2-yl)a d en osin e
(13a , R-BDO-d Ad o). 6-Chloropurine 2′-deoxyriboside (12, 20
mg, 0.08 mmol), amino alcohol 5a (22 mg, 0.25 mmol), DIPEA

BD Epoxide Adducts at Adenine N6 and Guanine N2
Chem. Res. Toxicol., Vol. 14, No. 4, 2001 383
was stirred for 20 h. Two product peaks were observed at 5.3
(15a ) and 15.4 min (O6-protected 15a ) in a ratio of 3.7:1. The
solvent was removed by vacuum centrifugation. Water was
added, and the reaction mixture was lyophilized. Purification
was carried out on a YMC-ODS-AQ-C18 column (250 mm × 10
mm) at a flow rate of 5 mL/min. Solvent A was 0.1 M ammonium
formate and solvent B MeOH. The gradient was from 10 to 90%
B over the course of 25 min. After HPLC purification (retention
time for 15a of 10 min), 3.9 mg (61%) of 15a was obtained: 1H
NMR (CD3OD) δ 2.34 (m, 1H, H-2′′), 2.72 (m, 1H, H-2′), 3.60-
3.80 (m, 4H, H-5′, H-5′′, â, â′), 3.95 (m, 1H, H-4′), 4.53 (m, 1H,
the TMSE group. The reaction mixture was treated with 10%
acetic acid as for the (R,R) isomer. The residue was purified by
HPLC using the conditions described for 15c. The peak at 11
min was collected and lyophilized to give 4.5 mg (45%) of 15d :
1H NMR (CD3OD) δ 2.37 (m, 1H, H-2′′), 2.74 (m, 1H, H-2′), 3.43
(dd, 1H, R, J 1 ) 14.0 Hz, J 2 ) 7.8 Hz), 3.52-3.68 (m, 4H, R′, γ,
δ, δ′), 3.74 (m, 2H, H-5′, H-5′′), 3.86 (ddd, 1H, â, J 1 ) 7.6 Hz,
J 2 ) 3.2 Hz, J 3 ) 3.0 Hz), 3.95 (m, 1H, H-4′), 4.53 (m, 1H, H-3′),
6.29 (dd, 1H, H-1′, J 1 ) J 2 ) 6.3 Hz), 7.96 (s, 1H, H-8); HRMS-
FAB (NBA matrix) calcd for C14H22N5O7 [M + H]+ 372.1519,
found 372.1525.
H-3′), 4.65 (m, 1H, R), 5.20 (ddd, 1H, γ′, J 1 ) 10.5 Hz, J 2
)
Syn th esis of Ad d u cted Oligon u cleotid es (Sch em e 4). (1)
Ra s 61,2-R-BDO-d Ad o (17a ). The 11-mer 5′-C-GGA-CXA-
GAA-G-3′ (X is 6-chloropurine 2′-deoxyriboside) was synthesized,
deprotected with 0.1 M NaOH (75 h at room temperature), and
purified as described previously (42). Oligomer 16 (50 A260 units)
was mixed in a plastic tube with DIPEA (150 µL), amino alcohol
(5a , 5 mg), and DMSO (500 µL). The reaction mixture was
stirred at 60 °C for 48 h. The reaction was monitored by HPLC.
The solvents were evaporated (vacuum centrifuge) to dryness.
Water (200 µL) was added, and the reaction mixture was
purified by HPLC using a YMC-ODS-AQ column (10 mm × 250
mm, flow rate of 5.0 mL/min) with a gradient of (A) 0.1 M
ammonium formate at pH 6.3 and (B) acetonitrile (from 1 to
10% B over the course of 20 min, from 10 to 25% B over the
course of 2 min, and from 25 to 90% B over the course of 4 min).
The adducted oligonucleotide had a retention time of 19.4 min.
The yield was 32 A260 units (∼64%) after HPLC purification:
MS (MALDI) m/z calcd for [M - H]- 3467.7, found 3467.3.
J 3 ) 1.5 Hz), 5.30 (ddd, 1H, γ, J 1 ) 17.2 Hz, J 2 ) J 3 ) 1.5 Hz),
5.94 (ddd, 1H, â′′, J 1 ) 17.6 Hz, J 2 ) 10.5 Hz, J 3 ) 5.4 Hz), 6.29
(dd, 1H, H-1′, J 1 ) J 2 ) 6.3 Hz), 7.96 (s, 1H, H-8); HRMS-FAB
(NBA matrix) calcd for C14H19N5O5 [M + H]+ 338.1464, found
338.1471.
(6) (2S)-2′-Deoxy-N2-(1-h yd r oxy-3-bu ten -2-yl)gu a n osin e
(15b, S-BDO-d Gu o). 2-Fluoro-O6-TMSE-2′-deoxyinosine (14, 10
mg, 0.03 mmol), contained in a small glass test tube equipped
with a magnetic stirring bar, was dried overnight in vacuo. (2S)-
2-Amino-3-buten-1-ol (5b, 3.5 mg, 0.04 mmol, 1.5 equiv), DMSO
(50 µL), and DIPEA (10 µL, 0.06 mmol, 2 equiv) were added.
The mixture was stirred at 60 °C and monitored as described
above for the (R) isomer. The reaction appeared to be essentially
finished within 6 h, but stirring was continued for an additional
14 h. Two product peaks were observed at 5.6 and 15.4 min in
a ratio of 3.7:1. The bulk of the reaction was worked up by
removing the solvent by vacuum centrifugation, addition of
water, and lyophilization. The product was purified on a YMC-
ODS-AQ-C18 column (10 mm × 250 mm) at a flow rate of 5
mL/min. Solvent A was 0.1 M ammonium formate and solvent
B MeOH. The gradient was from 10 to 90% B over the course of
25 min. Two peaks were collected. Peak 1 (retention time of 12
min) was compound 15b (6 mg), the TMSE protecting group
having been lost. Peak 2 (retention time of 26 min), which
proved to be 15b (2 mg) with the TMSE group intact, was
converted to peak 1 by treatment with 5% acetic acid (1 h at
room temperature). The total yield of 15b was 84%: 1H NMR
(CD3OD) δ 2.34 (m, 1H, H-2′′), 2.74 (m, 1H, H-2′), 3.60-3.80
(m, 4H, H-5′, H-5′′, â, â′), 3.95 (m, 1H, H-4′), 4.53 (m, 1H, H-3′),
4.63 (m, 1H, R), 5.21 (ddd, 1H, γ′, J 1 ) 10.5 Hz, J 2 ) J 3 ) 1.5
Hz), 5.29 (ddd, 1H, γ, J 1 ) 17.2 Hz, J 2 ) J 3 ) 1.5 Hz), 5.94
(ddd, 1H, â′′, J 1 ) 17.6 Hz, J 2 ) 10.5 Hz, J 3 ) 5.4 Hz), 6.29 (dd,
1H, H-1′, J 1 ) J 2 ) 6.3 Hz), 7.96 (s, 1H, H-8); HRMS-FAB (NBA
matrix) calcd for C14H19N5O5 [M + H]+ 338.1464, found 338.1471.
(7) (2R,3R)-2′-Deoxy-N2-(2,3,4-t r ih yd r oxyb u t -1-yl)gu a -
n osin e (15c, R,R-BDE-d Gu o). 2-Fluoro-O6-TMSE-2′-deoxy-
inosine (14, 20 mg, 0.05 mmol), (2R,3R)-1-amino-2,3,4-butane-
triol (5c, 10 mg, 0.08 mmol, 1.5 equiv), DMSO (100 µL), and
DIPEA (30 µL, 0.17 mmol, 3 equiv) were combined. The mixture
was stirred at 55-60 °C for 20 h. The solvent was removed by
vacuum centrifugation; water was added, and the pH was
adjusted to 3.0 with 10% acetic acid. The mixture was stirred
for 2 h at room temperature to remove the TMSE protecting
group (monitored by HPLC), followed by neutralization to pH
5-6 by 1 M KOH and lyophilization. HPLC purification,
performed as described for 15a and 15b, but with a gradient of
7 to 30% B over the course of 15 min (retention time for 15c of
11 min), gave 4.5 mg (49%) of compound 15c: 1H NMR (CD3-
OD) δ 2.36 (m, 1H, H-2′′), 2.75 (m, 1H, H-2′), 3.43 (dd, 1H, R,
J 1 ) 14.0 Hz, J 2 ) 7.8 Hz), 3.52-3.68 (m, 4H, R′, γ, δ, δ′), 3.74
(m, 2H, H-5′, H-5′′), 3.86 (ddd, 1H, â, J 1 ) 7.6 Hz, J 2 ) 3.2 Hz,
J 3 ) 3.0 Hz), 3.95 (m, 1H, H-4′), 4.53 (m, 1H, H-3′), 6.29 (dd,
1H, H-1′, J 1 ) J 2 ) 6.3 Hz), 7.96 (s, 1H, H-8); HRMS-FAB (NBA
matrix) calcd for C14H22N5O7 [M + H]+ 372.1519, found 372.1537.
(2) Ra s 61,2-S-BDO-d Ad o (17b). Adducted oligonucleotide
17b was synthesized as described above for the R-BDO isomer
except that amino alcohol 5b was used. The yield was 35 A260
units (∼70%) after HPLC purification: MS (MALDI) m/z calcd
for [M - H]- 3467.7, found 3467.0.
(3) Ra s 61,2-R,R-BDE-d Ad o (17c). The 6-chloropurine-
containing oligomer (16, 50 A260 units) was mixed in a plastic
tube with DIPEA (25 µL), amino alcohol (5c, 5 mg), and DMSO
(200 µL). The reaction mixture was stirred at 60 °C for 6 h. The
reaction was monitored by HPLC. The solvents were evaporated
(vacuum centrifugation) to dryness. The residue was dissolved
in H2O (200 µL) and purified initially by HPLC as described
above for the monoepoxide adduct; the product had a retention
time of 18.5 min. The yield was 38 A260 units (∼76%). A second
HPLC purification of the 3,4-diol 1,2-epoxide-adducted oligo-
nucleotide was performed by ion-exchange chromatography on
a HEMA-IEC Bio 1000 Q column (4.6 mm × 150 mm, flow rate
of 1.0 mL/min) using a gradient of (A) 20 mM potassium
phosphate (pH 6.4) and acetonitrile (8:2) and (B) 20 mM
potassium phosphate (pH 6.4) containing 1 M KCl and aceto-
nitrile (8:2), from 25 to 45% B over the course of 20 min. The
adducted oligonucleotide had a retention time of 12.4 min: MS
(MALDI) m/z calcd for [M - H]- 3501.7, found 3501.2.
(4) Ra s 61,2-S,S-BDE-d Ad o (17d ). This oligonucleotide was
synthesized using aminotriol 5d and purified as described above
for the (R,R) isomer. The HPLC retention time for this oligomer
was essentially the same as for the (R,R) isomer. The yield was
33 A260 units (∼66%): MS (MALDI) m/z calcd for [M - H]-
3501.7, found 3501.5.
(5) Ra s 12,2-R-BDO-d Gu o (19a ). The 11-mer 5′-G-GCA-
GXT-GGT-G-3′ (18, X is 2-fluoro-O6-TMSE-dI) was synthesized
as described previously (43). The oligodeoxynucleotide was
cleaved from the beads and deprotected by treatment with 0.1
M NaOH (0.75 mL of NaOH/µmol) at room temperature for 12
h. The reaction was quenched by cautious neutralization with
0.1 M HOAc to pH 8-9. The crude product was purified by
HPLC (YMC-ODS-AQ column, 10 mm × 250 mm, solvent A,
0.1 M ammonium formate; solvent B, CH3CN; gradient, from 5
to 11% B over the course of 5 min, from 11 to 20% B over the
course of 15 min). The product fractions were collected, com-
bined, and lyophilized. Ammonium formate was removed by
passage through a Sephadex G-25 column. The yield of 18 was
25 A260 units/µmol (∼23%): MS (ES) measured 3556.5, based
(8) (2S,3S)-2′-Deoxy-N2-(2,3,4-t r ih yd r oxyb u t -1-yl)gu a -
n osin e (15d , S,S-BDE-d Gu o). A mixture of 2-fluoro-O6-TMSE-
2′-deoxyinosine (14, 10 mg, 0.03 mmol), (2S,3S)-1-amino-2,3,4-
butanetriol (5d , 5 mg, 0.04 mmol, 1.5 equiv), DMSO (100 µL),
and DIPEA (15 µL, 0.09 mmol, 3 equiv) was stirred at 55-60
°C for 3 days. HPLC showed that the major product still retained

384 Chem. Res. Toxicol., Vol. 14, No. 4, 2001
Nechev et al.
on 710.3 for [M - 5H]5-, 591.7 for [M - 6H]6-, and 507.1 for
[M - 7H]7- (calcd 3554.8).
Sch em e 3
2-Fluoroinosine-modified oligomer 18 (45 A260 units, 0.41
µmol) was mixed in a plastic vial with DIPEA (150 µL, 0.87
mmol), (2R)-2-amino-3-buten-1-ol (5a , 10 mg, 0.11 mmol), and
DMSO (150 µL). The mixture was stirred at 60 °C for 18 h,
diluted with H2O, and purified by HPLC (column and solvents
as for 18; gradient from 8 to 18% B over the course of 20 min,
followed by 18 to 70% B over the course of 2 min) to give 28
A260 units (57%) of the TMSE-protected adducted oligomer.
Deprotection with 0.1 M HOAc was performed (room temper-
ature for 2 h), and the product was purified by HPLC with a
gradient from 5 to 10% B over the course of 18 min at a flow
rate of 5 mL/min (retention time of 19a , 13 min). Rechromatog-
raphy on an analytical HPLC column (YMC-ODS-AQ, 4.6
mm × 250 mm; gradient from 5 to 25% B over the course of 25
min at a flow rate of 1.5 mL/min) gave a single rather broad
peak at 9.7 min; despite the broadness, the product appeared
to be homogeneous by CGE and MALDI MS. Other adducts in
the N-ras 12 sequence which we have synthesized have had poor
chromatographic properties, perhaps because of the high gua-
nine content. Additional purification was performed by PAGE
(20% T, 5%C) with 67% recovery. The final product was desalted
by passage through a Sephadex G-25 column: MS (MALDI) m/z
calcd for [M - H]- 3521.7, found 3522.6.
(retention time of 8.9 min) than the (R,R) isomer: MS (MALDI)
m/z calcd for [M - H]- 3555.6, found 3554.9.
Ra s 12,2-S-BDO-d Gu o (19b). 2-Fluoroinosine-modified oli-
gomer 18 (50 A260 units, 0.46 µmol) was mixed in a plastic vial
with DIPEA (150 µL, 0.86 mmol), (2S)-2-amino-3-buten-1-ol (5b,
5 mg, 0.06 mmol), and DMSO (200 µL). After being stirred at
60 °C for 18 h, the reaction mixture was diluted with water and
purified by HPLC [YMC-ODS-AQ column (10 mm × 250 mm);
solvent A, 0.1 M ammonium formate; solvent B, CH3CN;
gradient from 8 to 17% B over the course of 20 min, followed by
17 to 70% B over the course of 2 min]. The product fractions
were lyophilized followed by dissolution in 0.1 M HOAc (room
temperature for 2 h) to remove the protecting group (TMSE) at
the O6 position. The oligonucleotide was repurified by HPLC
using a gradient from 7 to 10% B over the course of 18 min and
desalted on a Sephadex G-25 column to give 14 A260 units (28%)
of oligomer 19b. Rechromatography on an analytical column as
described for 19a gave a single sharp peak at 9.6 min: MS (ES)
measured 3524.4, based on 703.9 for [M - 5H]5- and 586.4 for
[M - 6H]6- (calculated 3521.7).
Ra s 12,2-R,R-BDE-d Gu o (19c). Oligomer 18 (48 A260 units,
0.44 µmol) was mixed in a plastic vial with DIPEA (50 µL, 0.29
mmol), (2R,3R)-1-amino-2,3,4-butanetriol (5c, 5 mg, 0.04 mmol),
and DMSO (200 µL). The reaction mixture was stirred at 60 °C
for 15 h, diluted with H2O, and purified by HPLC (gradient from
7 to 17% B over the course of 20 min, followed by 17 to 70% B
over the course of 2 min). The product fractions were lyophilized
to give 16 A260 units (34%) of TMSE-protected 19c. Deprotection
(0.1 M HOAc, 2 h, room temperature), HPLC purification
(gradient from 6 to 11% B over the course of 18 min) and
desalting (Sephadex G-25) yielded 7 A260 units of 19c. This
adducted oligonucleotide also gave a broad HPLC peak; it eluted
slightly more quickly (retention time of 8.7 min) than the
monoepoxide-adducted oligonucleotides under the analytical
conditions described for 19a and 19b: MS (MALDI) m/z calcd
for [M - H]- 3555.6, found 3555.9.
Resu lts a n d Discu ssion
For synthesis of the adducted oligonucleotides, a post-
oligomerization methodology was employed in which a
halopurine nucleoside is introduced during oligonucleo-
tide synthesis. The resulting halopurine-containing oli-
gonucleotide is removed from the beads, deprotected, and
purified before reaction with the amino alcohol equivalent
of the epoxide under investigation (42-44). In our previ-
ous syntheses of adducted oligonucleotides using this
strategy, we employed racemic amino alcohols in the case
of the polycyclic aromatic hydrocarbons (PAHs) and
commercially available chiral amino alcohols for the
synthesis of R adducts of styrene oxide. Thus far, oligo-
nucleotides containing diastereomeric PAH adducts have
been separable by HPLC. However, we felt it was
unlikely that we could achieve resolution at the oligo-
nucleotide stage for the butadiene epoxide adducts we
proposed to synthesize. Because none of the required
amino alcohols were commercially available in chiral
form, (2R)- and (2S)-2-amino-3-buten-1-ols and (2R,3R)-
and (2S,3S)-1-amino-2,3,4-butanetriols were synthesized.
The chiral buten-1-ols, 5a and 5b, were prepared by the
route developed by Ohfune and Kurokawa (41). The
commercially available starting material (N-t-BOC-L- or
D-methionine) was esterified with diazomethane, and the
resulting methyl ester was reduced to the alcohol with
LiAlH4. Oxidation of the sulfide to the sulfoxide with
sodium periodate followed by thermal elimination of the
sulfoxide produced the double bond. Removal of the
t-BOC protection with acid and purification of the product
by ion-exchange chromatography yielded the desired
aminobuten-1-ol in ∼40% overall yield from the com-
mercially available starting material. The chiral amino-
triols, 5c and 5d , were synthesized by procedures devel-
oped in our laboratory (Scheme 3). The (2R,3R) stereo-
isomer (5c) was prepared from commercially available
2,3-O-benzylidene-D-tartrate (6). Careful treatment of the
diester with 1 equiv of ammonia yielded monoamide 7
in 65-70% yield. Reduction of the amide ester with
LiAlH4 gave protected amino alcohol 8 which was depro-
tected with dilute acid to give the desired aminotriol in
Ra s 12,2-S,S-BDE-d Gu o (19d ). Oligomer 18 (50 A260 units,
0.46 µmol) was mixed in a plastic vial with DIPEA (150 µL, 0.87
mmol), (2S,3S)-1-amino-2,3,4-butanetriol (5d , 6.3 mg, 0.05
mmol), and DMSO (250 µL). The reaction mixture was stirred
at 60 °C for 18 h, diluted with H2O, and purified by HPLC with
a gradient from 7 to 17% B over the course of 20 min, followed
by 17 to 70% B over the course of 2 min. The product fractions
were lyophilized and deprotected with 0.1 M HOAc (room
temperature for 2 h). Additional HPLC purification (gradient
from 6 to 11% B over the course of 18 min) and desalting
(Sephadex G-25) gave 12 A260 units (24%) of oligomer 19d . Upon
rechromatography under the analytical conditions described
above, this isomer gave a slightly tailing but sharper peak

BD Epoxide Adducts at Adenine N6 and Guanine N2
Chem. Res. Toxicol., Vol. 14, No. 4, 2001 385
Sch em e 4
F igu r e 1. Circular dichroism spectra of BDO- and BDE-
adducted nucleosides. Spectra were recorded in methanol at 25
°C: (A) R-BDO-dAdo (13a ) and S-BDO-dAdo (13b), (B) R,R-
BDE-dAdo (13c) and S,S-BDE-dAdo (13d ), (C) R-BDO-dGuo
(15a ) and S-BDO-dGuo (15b), and (D) R,R-BDE-dGuo (15c) and
S,S-BDE-dGuo (15d ).
48% yield (Scheme 3A). (2S,3S)-1-Amino-2,3,4-butane-
triol (5d ) was prepared from commercially available
methyl 3,4-O-isopropylidene L-threonate (9) by reaction
with ammonia in methanol to form amide 10, LiAlH4
reduction of the amide to amine 11, and deprotection with
1 M HCl to give (2S,3S)-1-amino-2,3,4-butanetriol in
∼77% overall yield (Scheme 3B). Unfortunately, the D
isomer of 9 is not commercially available, and thus, the
latter synthetic route could not be used for the synthesis
of 5c.
compound, but to ensure complete removal, the reaction
mixture was treated with dilute acetic acid at room
temperature for 2 h. The structures of the adducted
1
nucleosides were confirmed by H NMR and mass spec-
troscopy. In addition, CD spectra were obtained on the
nucleosides (Figure 1). The spectra of the (R)- and (S)-
BDO N6 adducts of deoxyadenosine were qualitatively
similar to those reported by Koivisto (10), who prepared
the adducted nucleosides by reaction with optically active
BDO; only the adducts at C2 of BDO exhibited CD
spectra. The (R) isomer exhibited a positive band in the
270-280 nm region and a negative one around 225 nm,
whereas the (S) isomer exhibited the opposite behavior
in these regions. CD spectra of the BDE adducts have
not previously been reported; the spectra differed only
subtly from each other and from that of unmodified dAdo.
The CD spectra of the R-BDO-dGuo adduct had a positive
band in the 270-280 nm region and a negative one
around 250 nm, and the (S) isomer exhibited the opposite
pattern. These observations are similar to data reported
by Neagu et al. (3) for N7 guanine adducts of optically
active BDO. The data on the BDO N2 adducts are in
accord with spectra reported for analogous adducts of
styrene oxide (45, 46) with the (R) isomer exhibiting a
positive band at the longer wavelength. The CD spectra
of the BDE adducts of dGuo, unlike those of dAdo, clearly
showed bands with the opposite sign in the 250 nm region
and around 275-285 nm, with the (R,R) isomer having
the negative band at the longer wavelength and the
positive one at the shorter. The CD spectra of the BDE
adducts would not be expected to be very strong, inas-
much as the adduct moiety is a weak chromophore and
Syn th esis of Mod ified Nu cleosid es. Relatively little
characterization of BDO- and BDE-modified 2′-deoxyri-
bosides has been done with the exception of N6 BDO-dAdo
adducts which were characterized by Koivisto (10). Most
of the work on butadiene adducts has involved either
ribosides or depurinated species. We have now synthe-
sized the BDO adducts, (R)- and (S)-N6-(1-hydroxy-3-
buten-2-yl)-dAdo and N2-(1-hydroxy-3-buten-2-yl)-dGuo,
and the BDE adducts, (2R,3R)- and (2S,3S)-N6-(2,3,4-
trihydroxybut-1-yl)-dAdo and (2R,3R)- and (2S,3S)-N2-
(2,3,4-trihydroxybut-1-yl)-dGuo, in a regio- and stereospe-
cific fashion. These compounds were needed as standards
for characterization of adducted oligonucleotides by
enzymatic hydrolysis and should be useful for identifica-
tion of compounds formed by in vitro or in vivo exposure
of DNA to the butadiene epoxides. The 2′-deoxyadenosine
standards 13a -d were readily prepared in 80-90% yield
by reaction of 6-chloropurine 2′-deoxyriboside with amino
alcohols 5a -d in DMSO or DMF containing DIPEA
(Scheme 4). The synthesis of the adducted dGuo nucleo-
sides 15a -d from 2-fluoro-O6-TMSE-2′-deoxyinosine
was carried out in a similar fashion except that an
additional step, removal of the O6-TMSE group, was
required (Scheme 4). The TMSE group is much more
labile in the product than in the starting 2-fluoro

386 Chem. Res. Toxicol., Vol. 14, No. 4, 2001
Nechev et al.
F igu r e 2. HPLC chromatograms depicting the time course of
a typical adduction reaction: 6-chloropurine-containing oligo-
nucleotide 16 (starting material) with 2-amino-3-buten-1-ol (5a )
to give adducted oligonucleotide 17a . Reaction and HPLC
conditions as described in the Experimental Section.
F igu r e 3. HPLC profiles of enzymatic hydrolysates of repre-
sentative adducted oligonucleotides (A) 17b (5′-CGGA-CXA-
GAAG-3′, where X is 13b), (B) 19a (5′-GGCA-GXT-GGTG-3′,
where X is 15a ), (C) 17c (5′-CGGA-CXA-GAAG-3′, where X is
13c), and (D) 19c (5′-GGCA-GXT-GGTG-3′, where X is 15c). In
each case, an HPLC trace of an authentic sample of the
corresponding nucleoside adduct is superimposed: (A) S-BDO-
dAdo, (B) R-BDO-dGuo, (C) R,R-BDE-dAdo, and (D) R,R-BDE-
dGuo.
the site of attachment of the adduct is not at a chiral
site.
Syn th esis of BDO- a n d BDE-Ad d u cted Oligo-
n u cleotid es. The target oligonucleotides for these stud-
ies were 11-mers containing either codon 61 (CA*A) or
codon 12 (GG*T) of the N-ras protooncogene. We have
employed these sequences previously for preparation of
PAH- and styrene oxide-adducted oligonucleotides which
were used subsequently by others for structural and
mutagenesis studies. For the synthesis of the butadiene
epoxide-adducted oligonucleotides, we used a refinement
of our original postoligomerization strategy in which the
halopurine-containing oligonucleotides were removed
from the matrix, deprotected, and purified before reaction
with the amino alcohols. This strategy is especially useful
for the syntheses of alkyl-substituted oligonucleotides
because the adducted oligomers do not separate as well
from unadducted oligonucleotides as do PAH-adducted
oligomers. The halooligonucleotides react in high yield
with the relatively unhindered alkyl amino alcohols, and
if the halooligonucleotide is purified before reaction, the
final product is very easily purified.
For the adenine adducts, the 6-chloropurine nucleoside
was incorporated into the oligonucleotide at the 61,2
position (Scheme 4); the yield of the purified chloropu-
rine-containing oligonucleotide after HPLC purification
was about 45 A260 units per 1 µmol cassette. The reactions
with amino alcohols 5a -d proceeded very smoothly in
DMSO containing DIPEA at 60 °C for 6-48 h; yields of
70-75% were obtained after reverse-phase HPLC puri-
fication. The course of a typical reaction (oligonucleotide
16 with amino alcohol 5a to give adducted oligonucleotide
17a ) is shown in Figure 2. The BDE-adducted products
were further purified by ion-exchange chromatography.
For the preparation of BDO- and BDE-adducted ras
12,2 oligonucleotides (Scheme 4), the halopurine-contain-
ing 11-mer 18 was synthesized using 2-fluoro-O6-TMSE-
2′-deoxyinosine phosphoramidite followed by treatment
with 0.1 M NaOH at room temperature for 12 h (with
monitoring by HPLC) to cleave the oligomer from the
beads and remove the protecting groups (except the
TMSE group). After HPLC purification, approximately
25 A260 units per 1 µmol cassette of 18 was obtained.
Oligomer 18 was reacted with amino alcohols 5a -d
in DMSO containing DIPEA at 60 °C for 15-18 h with
monitoring by HPLC. During the reaction, some of the
O6-TMSE protecting group was lost from the product
oligonucleotide, but to ensure complete deprotection, the
pH of the crude product mixture was adjusted to pH 3
and the mixture allowed to stand at room temperature
for 2 h. Reverse-phase HPLC purification was satisfac-
tory for all of the adducts except 12,2-(R)-BDO (19a )
which gave broad peaks even though it appeared to be
quite homogeneous by CGE and MALDI. The oligonucleo-
tide was purified by PAGE (67% recovery). All of the
adducted oligonucleotides were characterized by CGE,
enzymatic digestion, and mass spectroscopy. Typical
enzyme digests are shown in Figure 3.
The thermal stability of duplexes formed by annealing
the adducted oligonucleotides to their complements was
examined, and the results are shown in Table 1. Two
observations can be made. The first is that adducts on
N6 of dAdo are more destabilizing than on N2 of dGuo,
perhaps because AT base pairs are stabilized by only two
hydrogen bonds rather than three. The second observa-
tion is that the BDO adducts are more destabilizing than
the BDE adducts. The BDO adducts, being attached at
a secondary carbon, are sterically more hindered and less
flexible than the BDE adducts which are bound at a
primary carbon. Interestingly, the decrease in the Tm
values for the N6 BDO-adducted oligonucleotides seen in
this study is very similar to that observed for N6-R

BD Epoxide Adducts at Adenine N6 and Guanine N2
Chem. Res. Toxicol., Vol. 14, No. 4, 2001 387
Ta ble 1. Th er m a l Meltin g of Un m od ified a n d Ep oxid ized
Bu ta d ien e N6- a n d N2-Mod ified Oligon u cleotid e
Du p lexesa
by cDNA-expressed human cytochrome P450 2E1 and by
human, rat, and mouse liver microsomes (52). Oe et al.
(53) also reported recently the detection of significant
amounts [∼50-100% of the (()-BDO2-derived species]
of the N7-(2,3,4-trihydroxybutyl)guanine adduct derived
from meso-BDO2 in the livers of mice and rats exposed
to butadiene. The unstable adducts also have to be
considered viable candidates for genotoxicity, especially
the N1 adenine adducts which may be quite long-lived
in dsDNA (13).
sequence
complement
Tm (°C)a
5′-CGGACXAGAAG-3′
X is A (unmodified)
5′-CTTCTTGTCCG-3′
5′-CTTCTTGTCCG-3′
5′-CTTCTTGTCCG-3′
5′-CTTCTTGTCCG-3′
5′-CTTCTTGTCCG-3′
5′-CTTCTTGTCCG-3′
5′-CACCACCTGCC-3′
5′-CACCACCTGCC-3′
5′-CACCACCTGCC-3′
5′-CACCACCTGCC-3′
5′-CACCACCTGCC-3′
5′-CACCACCTGCC-3′
57
49
42
52
49
X is N6-R-BDO-dAdo
X is N6-S-BDO-dAdo
X is N6-R,R-BDE-dAdo
X is N6-S,S-BDE-dAdo
5′-GGCAGXTGGTG-3′
X is G (unmodified)
65
62
62
65
65
X is N2-R-BDO-dGuo
X is N2-S-BDO-dGuo
X is N2-R,R-BDE-dGuo
X is N2-S,S-BDE-dGuo
Ack n ow led gm en t. Generous support of this project
by U.S. Public Health Service Grants ES00267 and
ES07781 is gratefully acknowledged.
a
Measurements were obtained at 260 nm in phosphate buffer
[10 mM Na2HPO4/NaHPO4 (pH 7.0) containing 1.0 M NaCl and
50 µM EDTA] at duplex concentrations of ∼4.0 µM.
Su p p or tin g In for m a tion Ava ila ble: The 400 MHz 1H
NMR spectra of compounds 5b, 5c, 7, 13a -d , and 15a -d , HPLC
chromatogram showing relative retention times of all adducted
nucleosides (13a -d and 15a -d ), HPLC chromatograms of
purified oligonucleotides 17a -d , HPLC profiles of enzyme
digests of 17a -d , electropherograms of oligonucleotides 19a -
d , and HPLC profiles of enzyme digests of 19a -d . This material
is available free of charge via the Internet at http://pubs.acs.org.
adducts of styrene oxide in the same sequence (47, 48).
The Tm of the styrene oxide (R) adduct was 9 °C lower
than that of the unadducted duplex, and the Tm of the
(S) adduct was 16 °C lower. Similarly, for the N2-dGuo
adducts, both the 12,2 R-styrene oxide (R) 11-mer and
the (S) isomer had Tm values which were 3-4 °C lower
than that of the unmodified duplex (49). This is under-
standable if a butadiene monoepoxide adduct (at C2 of
the epoxide) is viewed as a truncated version of an
R-styrene oxide adduct; i.e., each species has a hy-
droxymethyl group and a rigid CdC moiety attached to
the carbon bound to the DNA.
Refer en ces
(1) The Ninth Report on Carcinogens (2000) Department of Health
and Human Services, U.S. Public Health Service, National
Toxicology Program, Research Triangle Park, NC.
(2) Himmelstein, M. W., Acquavella, J . F., Recio, L., Medinsky, M.
A., and Bond, J . A. (1997) Toxicology and epidemiology of 1,3-
butadiene. Crit. Rev. Toxicol. 27, 1-108.
(3) Neagu, I., Koivisto, P., Neagu, C., Kostiainen, R., Stenby, K., and
Peltonen, K. (1995) Butadiene monoxide and deoxyguanosine
alkylation products at the N7-position. Carcinogenesis 16, 1809-
1813.
(4) Citti, L., Gervasi, P. G., Turchi, G., Bellucci, G., and Bianchini,
R. (1984) The reaction of 3,4-epoxy-1-butene with deoxyguanosine
and DNA in vitro: synthesis and characterization of the main
adducts. Carcinogenesis 5, 47-52.
(5) Tretyakova, N. Y., Lin, Y. P., Upton, P. B., Sangaiah, R., and
Swenberg, J . A. (1996) Macromolecular adducts of butadiene.
Toxicology 113, 70-76.
(6) Tretyakova, N., Lin, Y., Sangaiah, R., Upton, P. B., and Swenberg,
J . A. (1997) Identification and quantitation of DNA adducts from
calf thymus DNA exposed to 3,4-epoxy-1-butene. Carcinogenesis
18, 137-147.
(7) Tretyakova, N. Y., Chiang, S. Y., Walker, V. E., and Swenberg,
J . A. (1998) Quantitative analysis of 1,3-butadiene-induced DNA
adducts in vivo and in vitro using liquid chromatography elec-
trospray ionization tandem mass spectrometry. J . Mass Spectrom.
33, 363-376.
(8) Selzer, R. R., and Elfarra, A. A. (1996) Synthesis and biochemical
characterization of N1-, N2-, and N7-guanosine adducts of buta-
diene monoxide. Chem. Res. Toxicol. 9, 126-132.
(9) 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.
(10) Koivisto, P., Kostiainen, R., Kilpelainen, I., Steinby, K., and
Peltonen, K. (1995) Preparation, characterization and 32P-post-
labeling of butadiene monoepoxide N6-adenine adducts. Carcino-
genesis 16, 2999-3007.
(11) 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.
(12) 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.
In this paper, we have described extension of the
postoligomerization strategy previously developed in our
laboratory for styrene oxide and PAH diol epoxide ad-
ducts to the synthesis of a number of nucleosides and
oligonucleotides containing single regio- and stereoiso-
mers of BDO and BDE. The mutagenicity of these
oligonucleotides in E. coli has been examined (50, 51).
Although none of the species proved to be significantly
mutagenic in E. coli, the site (N2 or N6) and stereochem-
istry of the adduct clearly influenced the mutational
spectrum and replication by polymerases; the adenine
adducts were readily bypassed in vitro, whereas the
guanine adducts were almost totally blocking. Interest-
ingly, the dAdo (R,R)-BDE adduct showed only A f G
mutations, whereas the (S,S) isomer gave solely A f C
mutations. In contrast, the mutations induced by the
guanine N2 adducts were distributed approximately
equally among G f A, G f T, and G f C substitutions.
These syntheses and the subsequent biological studies
represent the most comprehensive investigation thus far
reported into the effect of regio- and stereochemistry of
adducts of small aliphatic epoxides on their biological
properties. However, in the case of the butadiene ep-
oxides, there are still many adducts to be examined,
including the C1 adducts of BDO and the adducts of the
meso- (RS/SR) form of BDO2 or BDE. Relatively little
attention has been paid to the possible biological role of
meso-BDO2. However, it has recently been shown to be
formed from BDO in amounts comparable to (()-BDO2
(13) 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.
(14) Koc, H., Tretyakova, N. Y., Walker, V. E., Henderson, R. F., and
Swenberg, J . A. (1999) Molecular dosimetry of N-7 guanine adduct
formation in mice and rats exposed to 1,3-butadiene. Chem. Res.
Toxicol. 12, 566-574.

388 Chem. Res. Toxicol., Vol. 14, No. 4, 2001
Nechev et al.
(15) Koivisto, P., Kilpelainen, I., Rasanen, I., Adler, I. D., Pacchierotti,
F., and Peltonen, K. (1999) Butadiene diolepoxide- and diepoxy-
butane-derived DNA adducts at N7-guanine: a high occurrence
of diolepoxide-derived adducts in mouse lung after 1,3-butadiene
exposure. Carcinogenesis 20, 1253-1259.
(16) Brookes, P., and Lawley, P. D. (1961) The alkylation of guanosine
and guanylic acid. J . Chem. Soc., 3923-3928.
(17) Tretyakova, N. Yu., Sangaiah, R., Yen, T. Y., and Swenberg, J .
A. (1997) Synthesis, characterization, and in vitro quantitation
of N-7-guanine adducts of diepoxybutane. Chem. Res. Toxicol. 10,
779-785.
(18) Lawley, P. D., and Brookes, P. (1967) Interstrand cross-linking
of DNA by difunctional alkylating agents. J . Mol. Biol. 25, 143-
160.
(19) Tretyakova, N., Sangaiah, R., Yen, T. Y., Gold, A., and Swenberg,
J . A. (1997) Adenine adducts with diepoxybutane: isolation and
analysis in exposed calf thymus DNA. Chem. Res. Toxicol. 10,
1171-1179.
(20) Mabon, N., Moorthy, B., Randerath, E., and Randerath, K. (1996)
Monophosphate 32P-postlabeling assay of DNA adducts from 1,2:
3,4-diepoxybutane, the most genotoxic metabolite of 1,3-buta-
diene: in vitro methodological studies and in vivo dosimetry.
Mutat. Res. 371, 87-104.
(35) 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.
(36) Steen, A. M., Meyer, K. G., and Recio, L. (1997) Characterization
of hprt mutations following 1,2-epoxy-3-butene exposure of human
TK6 cells. Mutagenesis 12, 359-364.
(37) Steen, A. M., Meyer, K. G., and Recio, L. (1997) Analysis of hprt
mutations occurring in human TK6 lymphoblastoid cells following
exposure to 1,2,3,4-diepoxybutane. Mutagenesis 12, 61-67.
(38) Saranko, C. J ., and Recio, L. (1998) The butadiene metabolite,
1,2:3,4-diepoxybutane, induces micronuclei but is only weakly
mutagenic at lacI in the Big Blue Rat2 lacI transgenic cell line.
Environ. Mol. Mutagen. 31, 32-40.
(39) Borowy-Borowski, H., Lipman, R., Chowdary, D., and Tomasz,
M. (1990) Duplex oligodeoxyribonucleotides cross-linked by mi-
tomycin C at a single site: Synthesis, properties, and cross-link
reversibility. Biochemistry 29, 2992-2999.
(40) Kim, S. J ., J ajoo, H. K., Kim, H.-Y., Zhou, L., Horton, P., Harris,
C. M., and Harris, T. M. (1995) An efficient route to N6 deoxy-
adenosine adducts of diol epoxides of carcinogenic polycyclic
aromatic hydrocarbons. Bioorg. Med. Chem. 3, 811-822.
(21) Leuratti, C., J ones, N. J ., Marafante, E., Kostiainen, R., Peltonen,
K., and Waters, R. (1994) DNA damage induced by the environ-
mental carcinogen butadiene: identification of a diepoxybutane-
adenine adduct and its detection by 32P-postlabelling. Carcino-
genesis 15, 1903-1910.
(22) Leuratti, C., J ones, N. J ., Marafante, E., Peltonen, K., Kostiainen,
R., and Waters, R. (1993) Biomonitoring of exposure to 1,3-
butadiene: detection by high-performance liquid chromatography
and 32P-postlabelling of an adenine adduct formed by diepoxybu-
tane. IARC Sci. Publ. 127, 143-150.
(23) Goodrow, T., Reynolds, S., Maronpot, R., and Anderson, M. (1990)
Activation of K-ras by codon 13 mutations in C57BL/6 × C3H F1
mouse tumors induced by exposure to 1,3-butadiene. Cancer Res.
50, 4818-4823.
(24) 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.
(41) Ohfune, Y., and Kurokawa, N. (1984) Synthesis of the serine
equivalent, (2R)- and (2S)-amino-3-butenol derivatives. Synthetic
approaches to the metal chelating poly-amino acid, “aspergillo-
marasmine A”. Tetrahedron Lett. 25, 1071-1074.
(42) Kim, H.-Y., Nechev, L., Zhou, L., Tamura, P., Harris, C. M., and
Harris, T. M. (1998) Synthesis and adduction of fully deprotected
oligodeoxynucleotides containing 6-chloropurine. Tetrahedron
Lett. 39, 6803-6806.
(43) DeCorte, B. L., Tsarouhtsis, D., Kuchimanchi, S., Cooper, M. D.,
Horton, P., Harris, C. M., and Harris, T. M. (1996) Improved
strategies for postoligomerization synthesis of oligodeoxynucleo-
tides bearing structurally defined adducts at the N2 position of
deoxyguanosine. Chem. Res. Toxicol. 9, 630-637.
(44) Tsarouhtsis, D., Kuchimanchi, S., DeCorte, B. L., Harris, C. M.,
and Harris, T. M. (1995) Synthesis of oligonucleotides containing
interchain cross-links of bifunctional pyrroles. J . Am. Chem. Soc.
117, 11013-11014.
(45) Latif, F., Moschel, R. C., Hemminki, K., and Dipple, A. (1988)
Styrene oxide as a stereochemical probe for the mechanism of
aralkylation at different sites on guanosine. Chem. Res. Toxicol.
1, 364-369.
(25) Cochrane, J . E., and Skopek, T. R. (1994) Mutagenicity of
butadiene and its epoxide metabolites: I. mutagenic potential of
1,2-epoxybutene, 1,2,3,4-diepoxybutane and 3,4-epoxy-1,2-butane-
diol in cultured human lymphoblasts. Carcinogenesis 15, 713-
717.
(46) Barlow, T., and Dipple, A. (1998) Aralkylation of guanosine with
para-substituted styrene oxides. Chem. Res. Toxicol. 11, 44-53.
(26) Cochrane, J . E., and Skopek, T. R. (1993) Mutagenicity of 1,3-
butadiene and its epoxide metabolites in human TK6 cells and
in splenic T cells isolated from exposed B6C3F1 mice. IARC Sci.
Publ. 127, 195-204.
(27) Reardon, J . T., Liljestrand-Golden, C. A., Dusenbery, R. L., and
Smith, P. D. (1987) Molecular analysis of diepoxybutane-induced
mutations at the rosy locus of Drosophila melanogaster. Genetics
115, 323-331.
(28) Gay, P., and Contamine, D. (1993) Study of the ref(2)P locus of
Drosophila melanogaster. II. Genetic studies of the 37DF region.
Mol. Gen. Genet. 239, 361-370.
(29) Sisk, S. C., Pluta, L. J ., Bond, J . A., and Recio, L. (1994) Molecular
analysis of lacI mutants from bone marrow of B6C3F1 transgenic
mice following inhalation exposure to 1,3-butadiene. Carcinogen-
esis 15, 471-477.
(30) Saranko, C. J ., Pluta, L. J ., and Recio, L. (1998) Molecular analysis
of lacI mutants from transgenic fibroblasts exposed to 1,2-
epoxybutene. Carcinogenesis 19, 1879-1887.
(31) Recio, L., and Goldsworthy, T. L. (1995) The use of transgenic
mice for studying mutagenicity induced by 1,3-butadiene. Toxicol.
Lett. 82-83, 607-612.
(32) 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.
(33) Recio, L., Meyer, K. G., Pluta, L. J ., Moss, O. R., and Saranko, C.
J . (1996) Assessment of 1,3-butadiene mutagenicity in the bone
marrow of B6C3F1 lacI transgenic mice (Big Blue): a review of
mutational spectrum and lacI mutant frequency after a 5-day 625
ppm 1,3-butadiene exposure. Environ. Mol. Mutagen. 28, 424-
429.
(47) Feng, B., Voehler, M., Zhou, L., Passarelli, M., Harris, C. M.,
Harris, T. M., and Stone, M. P. (1996) Major groove (S)-R-(N6-
adenyl)styrene oxide adducts in an oligodeoxynucleotide contain-
ing the human N-ras codon 61 sequence: Conformations of the
S(61,2) and S(61,3) sequence isomers from 1H NMR. Biochemistry
35, 7316-7329.
(48) Feng, B., Zhou, L., Passarelli, M., Harris, C. M., Harris, T. M.,
and Stone, M. P. (1995) Major groove (R)-R-(N6-adenyl)styrene
oxide adducts in an oligodeoxynucleotide containing the human
N-ras codon 61 sequence: Conformations of the R(61,2) and
R(61,3) sequence isomers from 1H NMR. Biochemistry 34, 14021-
14036.
(49) Setayesh, F. R. (1997) Ph.D. Thesis, Vanderbilt University,
Nashville, TN.
(50) Carmichal, J . R., Zhang, M., Nechev, L., Harris, C. M., Harris,
T. M., and Lloyd, R. S. (2000) Mutagenic potential of guanine N2
adducts of butadiene mono- and diolepoxide. Chem. Res. Toxicol.
13, 18-25.
(51) 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.
(52) Krause, R. J ., and Elfarra, A. A. (1997) Oxidation of butadiene
monoxide to meso- and (()-diepoxybutane by cDNA-expressed
human cytochrome P450s and by mouse, rat, and human liver
microsomes: evidence for preferential hydration of meso-diepoxy-
butane in rat and human liver microsomes. Arch. Biochem.
Biophys. 337, 176-184.
(53) Oe, T., Kambouris, S. J ., Walker, V. E., Meng, Q., Recio, L.,
Wherli, S., Chaudhary, A. K., and Blair, I. A. (1999) Persistence
of N7-(2,3,4-trihydroxybutyl)guanine adducts in the livers of mice
and rats exposed to 1,3-butadiene. Chem. Res. Toxicol. 12,
247-257.
(34) Recio, L., Pluta, L. J ., and Meyer, K. G. (1998) The in vivo
mutagenicity and mutational spectrum at the lacI transgene
recovered from the spleens of B6C3F1 lacI transgenic mice
following a 4-week inhalation exposure to 1,3-butadiene. Mutat.
Res. 401, 99-110.
TX000241K