Synthesis and characterization of nucleosides and oligonucleotides with a benzo[a]pyren-6-ylmethyl adduct at adenine N6 or guanine N2

Article abstract of DOI:10.1021/tx010086p

Benzo[a]pyrene (1) can be converted to reactive electrophilic species by a number of metabolic pathways, of which the route to the mutagenic and carcinogenic diol epoxide(s) is the best studied. An alternative and interesting pathway to a highly genotoxic electrophile is through alkylation at the 6 position to 6-methylbenzo[a]pyrene (2) followed by oxidation of the methyl group to give 6-hydroxymethylbenzo[a]pyrene (3). Esterification of 3, especially to sulfate ester 4, gives compounds which are both mutagenic and carcinogenic. The major DNA adduct identified from exposure of rats and mice to 4 is the guanine N² adduct [2′-deoxy-N²-(benzo-[a]pyren-6-ylmethyl)guanosine,5] which is also formed via activation of 2 to a radical cation species by horseradish peroxidase/H₂O₂ or iodine. To study the biological and structural properties of this adduct and the analogous adenine N⁶ adduct (6), a nonbiomimetic synthesis of the adducted nucleosides 5 and 6 has been developed and has been extended to preparation of oligonucleotides containing 5 or 6 at a single site.

Full text of DOI:10.1021/tx010086p

1306
Chem. Res. Toxicol. 2001, 14, 1306-1314
Syn th esis a n d Ch a r a cter iza tion of Nu cleosid es a n d
Oligon u cleotid es w ith a Ben zo[a ]p yr en -6-ylm eth yl
Ad d u ct a t Ad en in e N6 or Gu a n in e N2
Hye-Young H. Kim, Monica Cooper, Lubomir V. Nechev,
Constance M. Harris, and Thomas M. Harris*
Chemistry Department and Center in Molecular Toxicology,
Vanderbilt University, Nashville, Tennessee 37235
Received May 9, 2001
Benzo[a]pyrene (1) can be converted to reactive electrophilic species by a number of metabolic
pathways, of which the route to the mutagenic and carcinogenic diol epoxide(s) is the best
studied. An alternative and interesting pathway to a highly genotoxic electrophile is through
alkylation at the 6 position to 6-methylbenzo[a]pyrene (2) followed by oxidation of the methyl
group to give 6-hydroxymethylbenzo[a]pyrene (3). Esterification of 3, especially to sulfate ester
4, gives compounds which are both mutagenic and carcinogenic. The major DNA adduct
identified from exposure of rats and mice to 4 is the guanine N2 adduct [2′-deoxy-N2-(benzo-
[a]pyren-6-ylmethyl)guanosine, 5] which is also formed via activation of 2 to a radical cation
species by horseradish peroxidase/H2O2 or iodine. To study the biological and structural
properties of this adduct and the analogous adenine N6 adduct (6), a nonbiomimetic synthesis
of the adducted nucleosides 5 and 6 has been developed and has been extended to preparation
of oligonucleotides containing 5 or 6 at a single site.
Sch em e 1. Activa tion of Ben zo[a ]p yr en e via
In tr od u ction
Bioa lk yla tion
Polycyclic aromatic hydrocarbons (PAHs)1 are among
the most ubiquitous and highly studied of the carcino-
genic compounds found in the environment. Much effort
has been expended correlating PAH structure with
biological activity. This is a difficult task, given the
complexities of metabolic activation, detoxification, re-
activity of metabolites, and repair. It is clear, however,
that the genotoxicity of PAHs arises from their metabolic
conversion to electrophilic species capable of reacting
with nucleophiles such as proteins and nucleic acids.
There is strong evidence for the involvement of bay-region
and fjord-region diol epoxides in the genotoxicity of PAHs
such as benzo[a]pyrene (1, BP) and benzo[c]phenan-
threne. Other pathways to genotoxic metabolites of PAHs
have also been explored including one-electron oxidation
to radical cation species by cytochrome P450 (1), oxidation
to o-quinones (2) and hydroxylation of meso methyl
groups followed by esterification.
One interesting PAH which appears to be activated in
part by nondiol epoxide pathways is 6-methylbenzo[a]-
pyrene (2, 6-MeBP) (Scheme 1). An early study compar-
ing the tumorigenicity of 1, 2, and 6-hydroxymethylbenzo-
* Address correspondence to this author. Phone: (615) 322-2649.
Fax: (615) 322-2649. E-mail: harristm@toxicology.mc.vanderbilt.edu.
1
Abbreviations: PAH, polycyclic aromatic hydrocarbon; BP, benzo-
[a]pyrene; 6-MeBP, 6-methylbenzo[a]pyrene; 6-HOMeBP, 6-hydroxy-
methylbenzo[a]pyrene; 6-SOMeBP, 6-sulfoxymethylbenzo[a]pyrene;
6-NH2CH2BP, 6-aminomethylbenzo[a]pyrene; N2-6-MeBP dGuo, 2′-
deoxy-N2-(benzo[a]pyren-6-ylmethyl)guanosine; N6-6-MeBP dAdo, 2′-
deoxy-N6-(benzo[a]pyren-6-ylmethyl)adenosine; t-PAC, 4-tert-butyl-
phenoxyacetyl; TEAA, triethylammonium acetate; TMSE, trimethyl-
silylethyl; DIPEA, N,N-diisopropylethylamine; FAB, fast-atom bom-
bardment mass spectrometry; MALDI-TOF, matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry; COSY, correla-
tion spectroscopy.
[a]pyrene (3, 6-HOMeBP) when injected subcutaneously
in rats showed that all of the compounds caused tumors
at the injection site, and it was suggested that the
6-MeBP and hydroxylated derivative 3 could arise from
BP by enzymatic alkylation at the most electrophilic meso
position followed by oxidation; incubation of BP or
6-MeBP with rat liver homogenates yielded a number of
10.1021/tx010086p CCC: $20.00 © 2001 American Chemical Society
Published on Web 08/30/2001

Benzo[a]pyren-6-ylmethyl Adducts at Ade N6 and Gua N2
Chem. Res. Toxicol., Vol. 14, No. 9, 2001 1307
Sch em e 2. Non biom im etic Syn th esis of Ad d u cted
Nu cleosid es a n d Oligon u cleotid es
metabolites, one of which appeared to be 6-HOMeBP (3).
The hypothesis was proposed that the 6-HOMeBP could
be esterified in vivo and the esterified form would be a
precursor of a stabilized carbonium ion able to react with
cellular constituents. Esters (acetoxy and benzoyloxy) and
halomethyl derivatives of 6-MeBP were prepared, and
all were found to be tumorigenic upon injection (4). An
extensive study by Cavalieri’s group (5) on the carcino-
genicity of 6-MeBP and related compounds gave results
which were in general agreement with the earlier studies
except that 6-MeBP was found to be more potent than
6-HOMeBP; this result was interpreted as meaning that
there was a mechanism for activation of the 6-methyl
compound which did not involve hydroxylation. They
proposed that activation could occur by a one-electron
oxidation process. Subsequently, they reported that
6-MeBP, when incubated with horseradish peroxidase,
H2O2, and DNA gave adduct 5 bound through the
6-methyl group to the N2 of deoxyguanosine (6); recently,
this group has identified 5 among the products of iodine
oxidation of 6-MeBP (7). Hydrolysates of DNA from the
skin of mice treated with 6-MeBP also contained this
adduct (6). The analogous guanosine adduct as well as
adducts of adenosine and cytosine have been identified
from the reaction of 6-(chloromethyl) benzo[a]pyrene with
nucleosides (8). Support for the role of esterified deriva-
tives in the activation of methylated PAHs also came
from Cavalieri’s group in studies which showed that the
sulfate ester (4, 6-SOMeBP) of 3 was a potent carcinogen
and that the sulfate and acetate esters were the most
mutagenic of a group of 6-MeBP derivatives (esters,
halides, etc) tested in S. typhimurium strains TA98 and
TA100 (9).
The possible biological importance of the sulfate ester
was supported by the observation that rat and mouse
liver cytosols were able to catalyze the reaction of
6-HOMeBP with guanosine and deoxyguanosine via a
sulfotransferase enzyme (10). The formation of the adduct
was completely dependent on the presence of the sulfo
group donor, 3′-phosphoadenosine-5′-phosphosulfate. The
major adduct isolated from treated DNA hydrolysates
was the same N2-deoxyguanosine adduct (5) identified
by Cavalieri. Much smaller amounts of dA and dC
adducts 6 and 7 were also detected. When the DNA from
the livers of young rats which had been injected i.p. with
6-HOMeBP or sulfate ester 4 was examined, adduct 5
was the major compound identified; the dA and dC
adducts were only found in animals injected directly with
4. A subsequent study in B6C3F1 mice showed a very
high incidence of liver tumors in animals injected with
one i.p. dose of 6-SOMeBP; 6-HOMeBP was only 10% as
active (11).
route. Subsequently, it was shown that 6-HOMeBP and
6-SOMeBP could both act as complete carcinogens when
injected subcutaneously (20 doses given three times a
week); all animals developed sarcomas (13).
It appeared to us that DNA adducts arising from 2 and
its metabolites would be excellent candidates for struc-
tural and biological studies because the parent hydro-
carbon, whether activated through benzylic ester forma-
tion or one electron oxidation, appears to give only one
major stable DNA adduct, the N2 guanine adduct, with
minor amounts of adenine and cytosine adducts also
being detected. The question to be addressed is whether
these adducts are involved in the genotoxicity of 2 and
its metabolites. As an initial step toward answering this
question, we undertook the synthesis of oligonucleotides
containing an N2 dGuo or N6 dAdo adduct of 2 at a single
site. We anticipated that the nonbiomimetic approach in
which an amine equivalent of the electrophile is reacted
with an oligonucleotide containing a halonucleoside
(Scheme 2) would work well for these syntheses as it had
for our previous work on site- and stereospecific adducts
of PAH diol epoxides (14, 15). This paper describes the
synthesis and characterization of nucleosides and oligo-
nucleotides containing adducts of 6-methylbenzo[a]-
pyrene on the exocyclic amino groups of adenine and
guanine.
Exp er im en ta l Section
Ma ter ia ls. Anhydrous dimethyl sulfoxide (Me2SO) and di-
isopropylethylamine (DIPEA) were purchased from Aldrich in
Sure/Seal bottles. THF was distilled from sodium/potassium
alloy with benzophenone ketyl as indicator. Benzene was
distilled from sodium. Other chemicals were used as purchased
without further purification. Melting points are uncorrected.
Unless otherwise noted, materials were obtained from com-
mercial suppliers. All glassware, syringes, needles, and magnetic
stirring bars used in moisture-sensitive reactions were oven-
dried at 140 °C and stored in desiccators until used. All
moisture-sensitive reactions were conducted under a nitrogen
atmosphere.
Ch r om a togr a p h y. Thin-layer chromatography was per-
formed on silica gel precoated glass plates (EM Science)
(Kieselgel 60 F254, precoated, 20 × 20 cm, 0.25 mm layer
thickness). The chromatograms were visualized under UV (254
nm) or by staining with an anisaldehyde/sulfuric acid solution
or 1% ninhydrin solution, followed by heating. Column chro-
matography was performed using silica gel 60, 70-230 mesh
(EM Science). Oligonucleotides were desalted via a Sephadex
G-25 column using a BioRad Biologic system. HPLC analyses
and purifications were carried out on a gradient HPLC (Beck-
man Instruments; System Gold software) equipped with pump
The question of whether BP itself could actually give
rise to aralkyl metabolites such as 2 and 3 in vivo was
addressed by Stansbury et al. (12) in a 32P-postlabeling
study in which Sprague-Dawley weanling rats were
injected subcutaneously with BP. Subcutaneous DNA
was then examined for adducts arising by the diol epox-
ide pathway or by the benzylic ester route. The pre-
dominant pathway was via diol epoxides with adducts
from this route being present at 15-50 times the level
of the aralkyl products. However, the autoradiograms
clearly showed the presence of adducts corresponding to
those arising from injection of 6-MeBP, 6-HOMeBP, or
6-acetoxyMeBP indicating that BP could undergo in vivo
activation by the alkylation, side-chain hydroxylation

1308 Chem. Res. Toxicol., Vol. 14, No. 9, 2001
Kim et al.
module 125 and photodiode array detector module 168. Columns
used were YMC-ODS-AQ (Waters, Milford, MA), 4.6 × 250 mm
or 10 × 250 mm, or Luna C-8 or Phenyl-Hexyl (Phenomenex,
Torrance, CA)), 4.6 × 250 mm or 10 × 250 mm. Centrifugal
vacuum evaporation was performed using a RC10.22 evaporator
equipped with a Titan Trap (J ouan, Winchester, VA).
benzene was evaporated under reduced pressure and the residue
was purified by silica gel chromatography (10 and 20% MeOH/
CH2Cl2 or benzene) to give compound 8 as a yellow powder (300
1
mg, 54% yield); mp 184 °C [lit. (16) mp 202-203 °C] H NMR
(300 MHz, Me2SO-d6,) δ 11.58 (s, 1H, CHO), 9.36-9.32 (m, 1H,
H10), 9.28-9.25 (m, H11), 9.03 (d, 1H, H7, J ) 9.6 Hz), 8.65 (d,
1H, H5, J ) 9.2 Hz), 8.52 (d, 1H, H12, J ) 7.8 Hz), 8.39 (d, 1H,
H1, J ) 7.5 Hz), 8.32 (d, 1H, H3, J ) 9.6 Hz), 8.16 (t, 1H, H2,
J ) 7.5 Hz), 7.97-7.94 (m, 3H, H4, 8, 9).
Sp ectr oscop y. Absorption spectra were obtained on a Hi-
tachi U-3000 spectrophotometer. Fluorescence spectra were
obtained on a PC1 Photon Counting Spectrofluorometer (ISS,
Champaign, IL). Both types of spectra were obtained in phos-
phate buffer (10 mM Na2HPO4/NaH2PO4, 1.0 M NaCl, 50 µM
Na2EDTA, pH 7.0) at ambient temperature. Samples were
prepared at a concentration of 1.6 µM of modified strand
(calculated ꢀ at 260 nm, 12.65 × 104) and 1.7 µM complementary
strand (calculated ꢀ at 260 nm, 7.76 × 104).
6-Am in om et h ylb en zo[a ]p yr en e (10, 6-NH2CH 2BP ). 6-
Formylbenzo[a]pyrene (8, 28 mg, 0.1 mmol) was suspended in
1 mL of anhydrous benzene. Sodium bis(trimethylsilyl)amide
(0.1 mmol) was added dropwise over 3 min; the suspension was
stirred for 20 min at room temperature as the solid went into
solution. Chlorotrimethylsilane (0.1 mmol) was added dropwise
over 2 min and stirring was continued for an additional 20 min.
Silylimine 9 was reduced in situ without isolation. Borane-
tetrahydrofuran complex (1 M solution in THF, 100 µL, 0.1
mmol) was added and the reaction was stirred for 30 min. After
addition of 2 M HCl/MeOH (0.5 mL), the reaction was stirred
at room temperature for 30 min, poured into 10 mL of 0.1 M
NaOH, and extracted 4 times with ethyl acetate. The combined
extracts were dried (Na2SO4) and concentrated in vacuo. The
residual yellow oil was chromatographed on silica gel (40 g)
(eluted with CH2Cl2:MeOH, 90:10) to give 10 (12 mg, 43%) as a
yellow powder which migrated as one spot (with some tailing)
on TLC. TLC Rf ) 0.26, (CH2Cl2:MeOH, 9:1); mp 163-164 °C;
1H NMR (400 MHz, Me2SO-d6) δ 9.28-9.26 (m, 1H, H10), 9.25
(d, 1H, H11, J ) 9.1 Hz), 8.75-8.73 (m, 1H, H7), 8.48 (d, 1H,
H5, J ) 9.6 Hz), 8.41 (d, 1H, H12, J ) 9.2 Hz), 8.34 (dd, 1H,
H1, J 1 ) 7.8, J 2 ) 0.9 Hz), 8.20 (dd, 1H, H3, J 1 ) 7.3, J 2 ) 0.7
Hz), 8.09 (d, 1H, H2, J ) 9.6 Hz), 8.04 (t, 1H, H4, J ) 7.6 Hz),
7.90-7.87 (m, 2H, H8, 9), 4.83 (s, 2H, CH2). HRMS (FAB) [MH+]
calcd for C21H16N: 281.1228, Found: 281.1204.
2′-Deoxy-N6-(b en zo[a ]p yr en -6-ylm et h yl)a d en osin e (6,
N6-6MeBP d Ad o). 6-Chloropurine-2′-deoxyriboside (11) (17, 18)
(6 mg, 0.02 mmol) and compound 10 (3 mg, 0.01 mmol) were
dissolved in 400 µL of anhydrous Me2SO and 19 µL (0.11 mmol)
of N,N-diisopropylethylamine (DIPEA) was added to the solu-
tion. The mixture was stirred for 4 h at 55 °C. The product was
purified directly from the reaction mixture by HPLC (YMC-
ODS-AQ column, 10 × 250 mm) with the following gradient:
(A) H2O, (B) CH3CN, 10% to 20% B over 5 min, hold for 3 min,
20% to 99% B over 10 min, hold for 5 min; flow rate 4 mL/min.
Adduct 6 eluted at 20.8 min. The adduct was recovered almost
quantitatively (98%). 1H NMR (400 MHz, Me2SO-d6,) δ 9.28 (d,
2H, H10, H11, J ) 9.0 Hz), 8.77 (d, 1H, H7, J ) 8.4 Hz), 8.63
(d, 1H, H5, J ) 9.4 Hz), 8.45 (d, 1H, H12, J ) 9.1 Hz), 8.36 (d,
1H, H1, J ) 7.7 Hz), 8.30 (s, br, 1H, H2 adenine), 8.21 (d, 1H,
H3, J ) 7.3 Hz), 8.08 (t, 1H, H2, J 1 ) 9.3, J 2 ) 7.2 Hz), 8.05 (s,
1H, H8 adenine), 8.04 (d, 1H, H4, J ) 7.6 Hz), 7.86 (m, 2H,
H8,9), 6.36 (t, 1H, H1′), 5.88 (br, 2H, CH2), 5.31 (d, 1H, OH, J
) 4.0 Hz), 5.20 (br, 1H, OH), 4.39 (br, 1H, H3′), 3.87 (d, 1H,
H4′, J ) 2.5 Hz), 3.59-3.50 (m, 2H, H5′, H5′′), 2.72 (m, 1H,
H2′′), 2.27 (m, 1H, H2′). HRMS (FAB) [MH+] calcd for
C31H26N5O3: 516.2036. Found: 516.2045.
1H NMR and 2D spectra were recorded on a Bruker AC 300
or AM 400 NMR spectrometer with Me2SO-d6, CD3CN, CDCl3,
or MeOH-d4 as solvent. COSY spectra were obtained in the
magnitude mode.
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, Indiana. Mass spectra
of oligonucleotides were obtained using a Voyager Elite DE
instrument (PerSeptive Biosystems). The system was operated
in the negative ion mode using a matrix mixture of 2′,4′,6′-
trihydroxyacetophenone monohydrate and ammonium hydrogen
citrate.
Oligon u cleotid es. Oligodeoxynucleotides were synthesized
on an Expedite 8909 DNA Synthesizer (PerSeptives Biosystems)
on a 1-µmol scale using either the manufacturer’s standard
phosphoramidites or tert-butylphenoxyacetyl-protected (tBPA)
phosphoramidites and standard synthesis protocol.
CGE. Capillary gel electrophoresis was performed on a
Beckman P/ACE 5500 instrument using the manufacturer’s
ssDNA 100 gel capillary and Tris-borate-urea buffer. Samples
were applied at -10 kV and run at -10 kV at 30 °C.
En zym e Digestion s. The oligonucleotides (0.2-0.6 A260
units), lyophilized in 1.5 mL microfuge tubes, were digested in
a two-stage process. In the first step, buffer (20 µL, 0.01 M Tris-
HCl, 0.01 M MgCl2, pH 7.0) was added, followed by nuclease
P1 (Sigma N-8630, 4 µL). After digesting for 3-6 h at 36 °C,
Tris-HCl buffer (20 µL, 0.1 M, pH 9.0) was added, followed by
snake venom phosphodiesterase (Sigma 5785, 0.04 units) and
alkaline phosphatase (Sigma P-4282, 0.4 units). Digestion was
continued at 37 °C for 3-6 h. H2O-MeOH (1:1, 100 µL) was
added to each sample and the digest was filtered (Ultrafree-
MC, 0.45 µm, centrifugal filters, Millipore). The filters were
rinsed with Me2SO (50 µL) to elute adducted nucleosides 5 and
6 which are not very soluble in water. The digests were analyzed
by HPLC on either a YMC-ODS-AQ or LUNA C-8 column (4.6
× 250 mm), with the following gradient: (A) 0.1 M ammonium
formate, pH 6.4 (B), CH3CN; 1 to 10% B over 15 min; 10 to 99%
B over 20 min, at a flow rate of 1.5 or 1.0 mL/min.
Meltin g Stu d ies. Adducted oligonucleotide and its comple-
ment were dissolved in melting buffer (10 mM Na2HPO4/
NaH2PO4, 1.0 M NaCl, 50 µM Na2EDTA, pH 7.0). The sample
vials were heated to 100 °C, maintained at that temperature
for 3 min, allowed to cool to room temperature and stored in
the refrigerator overnight. Melting studies were performed using
a Varian Cary 04E UV spectrophotometer. UV measurements
were taken at one minute intervals with a 1 °C/min temperature
gradient. The temperature was raised from 10 to 90 °C and
cooled until a plateau was reached and no further hypo-
chromicity was observed at the detection wavelength of 260 nm.
6-F or m ylben zo[a ]p yr en e (8). A mixture of benzo[a]pyrene
(0.50 g, 2 mmol), POCl3 (330 µL, 3.6 mmol), N-methylformanilide
(494 µL, 4.0 mmol), and 5 mL of o-dichlorobenzene was stirred
in an oil bath at 95-100 °C for 2 h (4). The cooled reaction
mixture was poured slowly into an aqueous solution (5 mL)
containing 2.5 g of sodium acetate. The heterogeneous mixture
was filtered. The filtrate was evaporated to dryness and the
residue was dissolved in hot benzene (100 mL) and filtered. The
2′-Deoxy-N2-(ben zo[a ]p yr en -6-ylm et h yl)gu a n osin e (5,
N2-6-MeBP d Gu o). 2-Fluoro-(O6-trimethylsilylethyl)-2′-deoxy-
inosine (12) (19) (10 mg, 0.03 mmol) and 10 (3 mg, 0.01 mmol)
were dissolved in 600 µL of anhydrous Me2SO followed by the
addition of 19 µL (0.11 mmol) of DIPEA. The reaction mixture
was stirred for 5 h at 45 °C. The products were separated
directly from the reaction mixture by HPLC (YMC-ODS-AQ
column, 10 × 250 mm) with the following gradient: (A) H2O,
(B) CH3CN, 50 to 99% B over 10 min, hold for 5 min, 99 to 40%
B over 5 min; 40 to 50% B over 5 min; flow rate of 4.0 mL/min.
Products eluted at 6.4 min (compound 5) and 17.2 min (13, O6-
trimethylsilylethyl-protected 5). Compound 5 (0.6 mg, 10%) and
compound 13 (1.9 mg, 27%) were collected. Adduct 13 (1.9 mg)
was dissolved in 1 mL of H2O-MeOH (1:2). Hydrochloric acid
(0.05 M, 80 µL) was added and the mixture was stirred for 5 h
at room temperature and 3 h at 50 °C to ensure complete

Benzo[a]pyren-6-ylmethyl Adducts at Ade N6 and Gua N2
Chem. Res. Toxicol., Vol. 14, No. 9, 2001 1309
removal of the O6-trimethylsilylethyl protecting group. Adduct
5 was purified using the HPLC conditions described above for
13 to yield 1.4 mg (88% yield for deprotection step). Therefore,
total recovered yield of 5 was 2.0 mg (overall 34%). 1H NMR
(400 MHz, Me2SO-d6/D2O) δ 9.32-9.27 (m, 1H, H10, H11), 8.75-
8.73 (m, 1H, H7), 8.60 (d, 1H, H5, J ) 9.6 Hz), 8.48 (d, 1H, H12,
J ) 9.2 Hz), 8.38 (d, 1H, H1, J ) 7.7 Hz), 8.24 (d, 1H, H3, J )
7.2 Hz), 8.19 (d, 1H, H2, J ) 9.6 Hz), 8.07, (t, 1H, H4, J ) 7.6
Hz), 7.99 (s, 1H, H8 guanine), 7.95-7.92 (m, 2H, H8, H9), 6.43
(t, 1H, H1′, J 1 ) 7.2, J 2 ) 6.6 Hz), 5.64 (s, 2H, CH2), 4.47-4.46
(m, 1H, H3′), 3.92 (m, 1H, H4′), 3.63-3.60 (m, 2H, H5′, H5′′),
2.75 (m, 1H, H2′′), 2.38 (m, 1H, H2′). HRMS (FAB) [MH+] calcd
for C31H26N5O4: 532.1985. Found: 532.1982.
Sch em e 3. Syn th esis of
6-Am in om eth ylben zo[a ]p yr en e (10)
5′-G-GCA*-GGT-GGT-G-3′ (14, r a s11,3A*, A* ) N6-6MeBP
d Ad o). The modified oligonucleotide (2 × 1-µmol), 5′-G-GCA*-
GGT-GGT-G-3′ where A* is 6-fluoropurine-2′-deoxyriboside, was
prepared using automated solid-phase synthesis with the phos-
phoramidite of 6-fluoropurine-2′-deoxyriboside (15, 20). The final
detritylation was carried out on the synthesizer, but the
oligonucleotide was not further deprotected or released from the
solid support. 6-Aminomethylbenzo[a]pyrene (10, 7 mg, 0.02
mmol) was placed in a 3-dram vial containing the immobilized
oligonucleotide suspended in anhydrous Me2SO (400 µL) and
DIPEA (70 µL). The vial was capped and the mixture was heated
for 3 days at 48 °C under N2. After cooling to room temperature,
the supernatant was removed, and the residual beads were
washed with CH3OH (3 × 1 mL). The beads were transferred
to a 10-mL vial and concentrated NH4OH (7 mL) was added.
The tightly closed vial was heated for 8 h at 60 °C for cleavage
from the solid support and deblocking. After cooling, the vial
was allowed to stand until the excess NH4OH had evaporated.
The beads were separated by centrifugation and washed with
water (2 × 1 mL). The combined solution was filtered (0.22 µm,
Millipore), transferred into a conical tube (50 mL), and lyo-
philized. The dry material was dissolved in H2O-MeOH (1:1, 1
mL), and purified on a YMC-ODS-AQ column (10 × 250 mm)
using the following gradient: (A) 0.1 M ammonium formate,
pH 6.45 (B) CH3CN, 1 to 10% B over 5 min; 10 to 20% B over 5
min; hold for 5 min; 20 to 80% B over 5 min, flow rate 4.0 mL/
min. The product (14) eluted at 13.4 min and was collected (110
A260 units). After removal of solvent by vacuum centrifugation
the fraction was desalted on a Sephadex G-25 (Bio-Rad Labo-
ratories) column (1.5 × 40 cm) with elution by H2O to give the
(a) POCl3, N-methylformanilide, o-dichlorobenzene. (b) NaN-
(SiMe3)2, Me3SiCl, benzene. (c) BH3-THF.
B over 12 min, hold for 8 min; flow rate 1.0 mL/min. The
material (silylated at the O6 position) eluting at 29.8 min was
collected and lyophilized. A second HPLC purification was
performed using 0.1 M TEAA and CH3CN with the same
gradient. Oligonucleotide 15, which had desilylated at the O6
positon during the first purification and lyophilization, eluted
at 14.30 min. After removal of solvent by vacuum centrifugation
the fraction was desalted on a Sephadex G-25 (Bio-Rad Labo-
ratories) column (1.5 × 40 cm) with elution by H2O to give
purified oligonucleotide 15 (13 A260 units). The adducted oligo-
nucleotide was analyzed by enzyme hydrolysis and the homo-
geneity was checked by capillary gel electrophoresis. MS
(MALDI-TOF) m/z calcd for [M-H]- 3715.6, observed 3715.9.
Resu lts a n d Discu ssion
Two options were considered for the synthesis of
oligonucleotides containing adducts of 6-MeBP. The first
and apparently simplest was to synthesize the adducted
nucleosides by published procedures, using the sulfate
ester of 6-HOMeBP or a haloderivative such as 6-chloro-
methyl benzo[a]pyrene, followed by conversion of the
adducted nucleosides to phosphoramidites. However, we
were concerned that solubility problems might be en-
countered with the modified amidite and that coupling,
especially of the dGuo amidite might be inefficient.
Others have obtained relatively modest coupling yields,
even after extended coupling times, with dGuo amidites
modified at N2 with bulky groups such as benzo[a]pyrene
(21, 22). We decided, therefore, to investigate the post-
oligomerization approach to these adducts.
The implementation of the strategy depicted in Scheme
2 required the synthesis of 6-aminomethylbenzo[a]pyrene
(10). Dewhurst and Kitchen (16) had attempted to
prepare 10 via either catalytic hydrogenation of the
6-nitrile or lithium aluminum hydride reduction of the
6-carboxamide but were not able to obtain pure material.
We chose to start with 6-formyl derivative 8 which was
commercially available at the time we initiated this
project; we have also synthesized it by the route shown
in Scheme 3 (16, 23). Our original plan was to synthesize
the amine by reduction of the oxime, a route used
previously for the preparation of pyrenylmethylamine
(24). The oxime of 8 was obtained in high yield but
neither Zn/acetic acid nor lithium aluminum hydride
reduction gave satisfactory yields of pure material, in
part because of the insolubility of the oxime. The next
route chosen was through imine reduction; the one shown
pure oligonucleotide (40 A260 units after
a second HPLC
purification). MS (MALDI-TOF) m/z calcd for [M - H]- 3715.6,
observed 3715.3.
2
5′-G-GCA-GG*T-GGT-G-3′ (15, r a s12,2G*, G* ) N
-
6MeBP d Gu o). The modified oligonucleotide (2 × 1 µmol),
5′-G-GCA-GG*T-GGT-G-3′, where G* is 2-fluoro-O6-(trimethyl-
silylethyl)-2′-deoxyinosine, was prepared using the phosphor-
amidite of the 2-fluoronucleoside (19) and t-PAC phosphor-
amidites for the other bases. The final detritylation was carried
out on the synthesizer, but the oligonucleotide was not further
deprotected or released from the solid support. 6-Amino-
methylbenzo[a]pyrene (10, 8 mg, 0.03 mmol) was placed in a
3-dram vial containing the immobilized oligonucleotide sus-
pended in anhydrous Me2SO (400 µL) and DIPEA (80 µL). The
vial was capped and the mixture was heated for 5 days at 60
°C under N2. After cooling to room temperature, the supernatant
was removed, and the residual beads were washed with CH3OH
(3 × 1 mL). The beads were transferred to a 10-mL vial and
concentrated NH4OH (7 mL) was added. The tightly closed vial
was heated for 1 h at 60 °C for cleavage from the solid support
and deblocking. After cooling, the vial was allowed to stand until
the excess NH4OH had evaporated. The beads were separated
by centrifugation and washed with MeOH:H2O (1:1, 2 × 1 mL).
The combined solution was filtered, transferred into a conical
tube (50 mL), and lyophilized. The dry material was dissolved
in MeOH:H2O (1 mL), and purified on a LUNA Phenyl-Hexyl
column (Phenomenex, 4.5 × 250 mm) using the following
gradient: (A) 0.2 M triethylammonium acetate (TEAA), pH 7.02
(B) CH3OH, 0 to 20% B over 10 min, hold for 5 min, 20 to 90%

1310 Chem. Res. Toxicol., Vol. 14, No. 9, 2001
Kim et al.
Sch em e 4. Syn th esis of N6 Ad o a n d N2 Gu o
Ad d u cts of 6-Meth ylben zo[a ]p yr en e (6 a n d 5)
benzo[a]pyrene with the riboside, although the product
was not fully characterized. 2′-Deoxyguanosine adduct
5 was obtained in a similar fashion by reaction of 10 with
fluoronucleoside 12. The initial isolation gave both O6-
protected product 13 and final product 5 which had
undergone deprotection probably due to fluoride ion
generated during the reaction. Isolated 13 was depro-
tected by heating with dilute HCl in MeOH-H2O. The
lower yield of the guanine adduct reflects not only the
two step procedure but also the poor solubility of this
adduct. The 1H NMR spectrum of 5, assigned by a
combination of COSY and NOE difference spectroscopy,
was in agreement with those published previously (6, 10).
With nucleoside standards in hand, synthesis of ad-
ducted oligonucleotides was undertaken. In the interest
of comparing an adenine N6 and a guanine N2 adduct in
the same sequence, we chose to put both adducts in the
11-mer sequence, 5′-GGCAGGTGGTC-3′ (Scheme 5) (in
separate syntheses, not with both adducts in the same
oligonucleotide). This sequence is part of the N-ras
protooncogene which we have used in several of our
previous studies (19, 25). The adenine adduct was
introduced at the 11,3 position to give oligonucleotide 14
and the guanine adduct at position 12,2 to yield 15.
Although the 6-chloropurine nucleoside was used for
preparation of the N6 adenine nucleoside adduct, the
6-fluoropurine nucleoside was chosen as the halo con-
stituent for the oligonucleotide synthesis because of its
increased reactivity (15, 26). The matrix-bound oligo-
nucleotide was reacted with amine 10 in Me2SO at 48
°C for 3 days. The reaction proceeded well as seen in the
HPLC trace of the crude oligonucleotide after removal
from the beads and deprotection (Figure 1).
in Scheme 3 was the most successful. The silylimine was
generated in situ and reduced with borane in THF to give
10 in 43% yield.
Adducted nucleosides 5 and 6 were prepared by reac-
tion of the appropriate halonucleoside with amine 10
(Scheme 4). 6-Chloropurine 2′-deoxyriboside (11) reacted
quantitatively with 10 to yield deoxyadenosine adduct
6. The analogous adenosine adduct had been identified
by Royer et al. (8) from the reaction of 6-(chloromethyl)-
Synthesis and purification of guanine-adducted oligo-
nucleotide 15 proved to be more difficult. The phosphor-
amidite of 2-fluoro-O6-(trimethylsilylethyl) 2′-deoxy-
inosine was used for the synthesis of the oligonucleotide
(Scheme 5) (19). The reaction with amine 10 was carried
out with matrix-bound fully protected oligonucleotide.
The HPLC of the crude oligonucleotide (with the O6-
Sch em e 5. Syn th esis of Oligon u cleotid es Bea r in g Ad en in e N6 a n d Gu a n in e N2 Ad d u cts of
6-Meth ylben zo[a ]p yr en e (6 a n d 5)a
a
Protected exocyclic amino groups appear in parentheses.

Benzo[a]pyren-6-ylmethyl Adducts at Ade N6 and Gua N2
Chem. Res. Toxicol., Vol. 14, No. 9, 2001 1311
F igu r e 2. Capillary gel electropherograms of purified adducted
oligonucleotides 14 and 15. Upper trace: ras 11,3A* (14). Lower
trace: ras 12,2G* (15).
F igu r e 1. HPLC chromatograms of crude adducted oligonucleo-
tide reactions. Upper trace: 5′-GGCA*GGTGGTG-3′ (14, ras
11,3A* where A* ) N6-6-MeBP dAdo). Lower trace: 5′-
GGCAGG*TGGTG-3′ (15, ras 12,2G* where G* ) N2-6-MeBP
dGuo with O6 protection).
TMSE group intact) after removal from the beads and
deprotection with NH4OH is shown in Figure 1. Consid-
erable difficulty was encountered with the chromatog-
raphy of oligonucleotide 15 and its O6-TMSE precursor
and the chromatogram shown was only obtained after
experimentation with several columns and solvent condi-
tions. Chromatography on YMC-ODS-AQ columns with
ammonium formate buffers under conditions which had
been quite satisfactory for separations of this particular
sequence bearing other adducts at 12,2 gave broad
unresolvable peaks. Changing to triethylammonium
acetate (TEAA) buffers helped, but the best separations
were obtained on the newly available Luna Phenyl-Hexyl
column with 0.1 or 0.2 M TEAA, pH 7.01/acetonitrile. The
problems probably arose from interaction of the very
hydrophobic aromatic N2 adduct with the multiple gua-
nines in this particular sequence. Oligonucleotide 14,
with an adenine adduct in the same sequence, did not
show this anomalous chromatographic behavior. We have
had similar problems with the chromatography of N2
guanine adducts of benzo[a]pyrene diol epoxide in this
sequence.2
The purified oligonucleotides were characterized by
denaturing capillary gel electrophoresis (Figure 2), mass
spectroscopy (MALDI-TOF), and enzymatic digestion
with nuclease P1 followed by snake venom phosphodi-
esterase and alkaline phosphatase. Chromatograms of
the enzymatic digests are shown in Figure 3; the identity
of the adducted nucleosides (6 from oligonucleotide 14
F igu r e 3. HPLC chromatograms of enzymatic digests of
oligonucleotides 14 (upper trace) and 15 (lower trace). The peak
labeled 6 is N6-6-MeBP dAdo and 5 is N2-6-MeBP dGuo.
and 5 from 15) was confirmed by comparison with the
independently synthesized nucleoside adducts. One prob-
lem that arose in the enzyme digestions, especially with
the guanine adduct, is the poor solubility of the adducts.
2
Unpublished results.

1312 Chem. Res. Toxicol., Vol. 14, No. 9, 2001
Kim et al.
the guanine adduct destabilized the duplex, the former
by 14 °C and the latter by 9 °C. These numbers are
similar to those seen in studies of the thermal stability
of oligonucleotides bearing N2 dGuo (27-29) or N6 dAdo
(30) trans adducts of anti-benzo[a]pyrene diol epoxide,
keeping in mind, however, that sequence context can
affect thermal stability (27).
Absor p tion a n d Em ission Sp ectr a . Both absorption
and fluorescence emission spectroscopy have been exten-
sively used for studying interactions between DNA and
PAHs (21, 27, 28, 31-33). Changes in absorption and
emission maxima and intensities have been interpreted
in terms of orientation of PAH adducts, particularly in
double-stranded DNA. Orientations of PAH adducts in
DNA are divided into two general types termed site I and
site II. In site I, conformations with considerable base-
stacking or intercalative character, there is a pronounced
red shift in the emission maxima and a marked diminu-
tion in fluorescence intensity upon duplex formation,
whereas in site II, or external, groove-bound conformers,
there is relatively little red shift and only moderate
quenching, indicating that the adduct is still relatively
available to solvent in the duplex.
F igu r e 4. Thermal stability of oligonucleotide duplexes (10 mM
Na2HPO4/NaH2PO4, 1.0 M NaCl, 50 mM Na2EDTA, pH 7.0) (1)
unmodified: 5′-GGCAGGTGGTG-3′ (O) ras 11,3A* (14), (+) ras
12,2G* (15).
In our routine procedure, the enzyme digest is diluted
with water and filtered through a 0.45 µm filter before
HPLC analysis. It was necessary to rinse the filter with
Me2SO to recover the adducts.
Meltin g P r ofiles. The thermal stability of duplexes
formed by oligonucleotides 14 and 15 with complemen-
tary strand, 5′-CACCACCTGCC-3′, was examined and
the results are shown in Figure 4. Both the adenine and
Absorption and emission spectra for single- and double-
stranded oligonucleotides 14 and 15 are shown in Figure
5. The absorption spectrum of single-stranded oligo-
F igu r e 5. Absorption and fluorescence emission spectra for single (- - -) and double (s)-stranded oligonucleotides 14 and 15. Emission
intensity is in arbitrary units. (A) absorption and (B) emission spectra for ras 11,3A* (14); (C) absorption and (D) emission spectra
for ras 12,2G* (15). Spectra were obtained in phosphate buffer (10 mM Na2HPO4/NaH2PO4, 1.0 M NaCl, 50 µM Na2EDTA, pH 7.0)
at ambient temperature. Samples were prepared at a concentration of 1.6 µM modified strand and 1.7 µM complementary strand.

Benzo[a]pyren-6-ylmethyl Adducts at Ade N6 and Gua N2
Chem. Res. Toxicol., Vol. 14, No. 9, 2001 1313
benzo[a]pyrene to DNA by cytochrome P-450-catalyzed one-
electron oxidation in rat liver microsomes and nuclei. Biochem-
istry 29, 4820-4827.
Ta ble 1. UV Absor p tion a n d F lu or escen ce Em ission
Ch a r a cter istics of Oligon u cleotid es Con ta in in g
6-Meth ylben zo[a ]p yr en e Ad d u cts on d Ad o N6 a n d
d Gu o N2 a
(2) Penning, T. M., Burczynski, M. E., Hung, C. F., McCoull, K. D.,
Palackal, N. T., and Tsuruda, L. S. (1999) Dihydrodiol dehydro-
genases and polycyclic aromatic hydrocarbon activation: genera-
tion of reactive and redox active o-quinones. Chem. Res. Toxicol.
12, 1-18.
(3) Flesher, J . W., and Sydnor, K. L. (1973) Possible role of 6-hy-
droxymethylbenzo[a]pyrene as a proximate carcinogen of benzo-
[a]pyrene and 6-methylbenzo[a]pyrene. Int. J . Cancer 11, 433-
437.
(4) Natarajan, R. K., and Flesher, J . W. (1973) Synthesis and
carcinogenicity of compounds related to 6-hydroxymethylbenzo-
[a]pyrene. J . Med. Chem. 16, 714-715.
(5) Cavalieri, E., Roth, R., Grandjean, C., Althoff, J ., Patil, K., Liakus,
S., and Marsh, S. (1978) Carcinogenicity and metabolic profiles
of 6-substituted benzo[a]pyrene derivatives on mouse skin. Chem.-
Biol. Interact. 22, 53-67.
fluorescence
UV
wavelength, excitation, emission,
sample
λmax
λ
λmax
6-NH2CH2BP (10)
ras11,3-6-MeBP-dAdo (14)
single-strand
395, 373, 355
350
436, 412
402, 381, 363
402, 381, 362
350
350
445, 420
442, 419
double-strand
ras 12,2-6-MeBP-dGuo (15)
single-strand
402, 381, 363
397, 377, 358
350
350
443, 420
441, 417
double-strand
a
Samples were run at ambient temperature in buffer containing
10 mM Na2HPO4/NaH2PO4, 1 M NaCl, 50 µM Na2EDTA, pH 7.0.
(6) Rogan, E. G., Hakam, A., and Cavalieri, E. L. (1983) Structure
elucidation of a 6-methylbenzo[a]pyrene-DNA adduct formed by
horseradish peroxidase in vitro and mouse skin in vivo. Chem.-
Biol. Interact. 47, 111-122.
(7) Hanson, A. A., Li, K., Lin, C., J ankowiak, R., Small, G. J ., Rogan,
E. G., and Cavalieri, E. L. (2000) Synthesis and structure
determination of 6-methylbenzo[a]pyrene-deoxyribonucleoside ad-
ducts and their identification and quantitation in vitro and in
mouse skin. Chem. Biol. Interact. 128, 65-90.
(8) Royer, R. E., Lyle, T. A., Moy, G. G., Daub, G. H., and J agt, D. L.
V. (1979) Reactivity-selectivity properties of reactions of carcino-
genic electrophiles with biomolecules. Kinetics and products of
the reaction of benzo[a]pyrenyl-6-methyl cation with nucleosides
and deoxynucleosides. J . Org. Chem. 44, 3202-3207.
(9) Rogan, E. G., Cavalieri, E. L., Walker, B. A., Balasubramanian,
R., Wislocki, P. G., Roth, R. W., and Saugier, R. K. (1986)
Mutagenicity of benzylic acetates, sulfates and bromides of
polycyclic aromatic hydrocarbons. Chem.-Biol. Interact. 58, 253-
275.
(10) Surh, Y.-J ., Liem, A., Miller, E. C., and Miller, J . A. (1989)
Metabolic activation of the carcinogen 6-hydroxymethylbenzo[a]-
pyrene: formation of an electrophilic sulfuric acid ester and
benzylic DNA adducts in rat liver in vivo and in reactions in vitro.
Carcinogenesis 10, 1519-1528.
(11) Surh, Y.-J ., Liem, A., Miller, E. C., and Miller, J . A. (1990) The
strong hepatocarcinogenicity of the electrophilic and mutagenic
metabolite 6-sulfooxymethylbenzo[a]pyrene and its formation of
benzylic DNA adducts in the livers of infant male B6C3F1 mice.
Biochem. Biophys. Res. Commun. 172, 85-91.
(12) Stansbury, K. H., Flesher, J . W., and Gupta, R. C. (1994)
Mechanisms of aralkyl-DNA adduct formation from benzo[a]-
pyrene in vivo. Chem. Res. Toxicol. 7, 254-259.
(13) Flesher, J . W., Horn, J ., and Lehner, A. F. (1997) 6-Sulfooxy-
methylbenzo[a]pyrene is an ultimate electrophilic and carcino-
genic form of the intermediary metabolite 6-hydroxymethylbenzo-
[a]pyrene. Biochem. Biophys. Res. Commun. 234, 554-558.
(14) Harris, T. M., Harris, C. M., Kim, S. J ., Han, S., Kim, H.-Y., and
Zhou, L. (1994) Preparation of deoxynucleosides, deoxynucleotides
and oligodeoxynucleotides bearing adducts of PAH diol epoxides
at the N6 position of adenine. Polycycl. Aromat. Compd. 6, 9-16.
(15) 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.
(16) Dewhurst, F., and Kitchen, D. A. (1972) Synthesis and properties
of 6-substituted benzo[a]pyrene derivatives. J . Chem. Soc., Perkin
Trans.1 710-712.
nucleotide 14 containing the N6 dAdo adduct showed
long-wavelength maxima at 402, 381, and 363 nm (Table
1); upon formation of duplex the maxima did not change
but there was a significant decrease in intensity in the
long wavelength region. Similarly the fluorescence emis-
sion was significantly decreased upon duplex formation.
Both of these effects indicate that in the duplex the
polycyclic adduct is involved in considerable base-stack-
ing, but the large decrease in thermal stability (Tm 47
°C versus 61 °C for unmodified duplex) argues against a
fully intercalated orientation.
In contrast, absorption and emission spectra for oligo-
nucleotide 15 containing the N2 dGuo adduct showed
relatively little difference in intensity in the long wave-
length part of the spectrum upon duplex formation. The
shift to shorter wavelengths in the absorption spectrum
upon duplex formation may reflect base-stacking of the
adduct with other bases in the single-stranded purine-
rich oligonucleotide which is decreased upon duplex
formation. These data are in accord with orientation of
the dGuo adduct in the minor groove with considerable
exposure to solvent. A similar conclusion was reached by
Casale and McLaughlin (21) in an earlier study on an
oligonucleotide bearing an N2 dGuo adduct of 9-methyl-
anthracene. In accordance with earlier observations on
other PAH adducts (34, 28, 35) the intensity of the
fluorescence emission of the guanine adduct is much less
than that of the adenine. This effect has been attributed
to fluorescence quenching by photoinduced electron
transfer from guanine. The question of whether the
adducts of 6-MeBP exist in one predominant conforma-
tion awaits further examination by high-field NMR or
X-ray crystallography; low-temperature fluorescence stud-
ies of these adducts would also be interesting (33).
In addition to their possible role in genotoxicity, these
very hydrophobic adducts with their high intrinsic fluo-
rescence should be useful tools for studying DNA-protein
interactions, as, for example, the thermodynamics of the
interaction of BPDE-adducted oligonucleotides with the
Escherichia coli repair protein UvrA was examined by
fluorescence spectroscopy (36).
(17) Robins, M. J ., and Basom, G. L. (1978) 6-Chloro-9-(2-deoxy-â-
D-erythro-pentofuranosyl) purine from the chlorination of 2′-
deoxyinosine. Nucleic Acid Chem. 601-606.
(18) Gao, X., and J ones, R. A. (1987) Nitrogen-15-labeled deoxynucleo-
sides. Synthesis of [6-15N]- and [1-15N]deoxyadenosines from
deoxyadenosine. J . Am. Chem. Soc. 109, 1275-1278.
(19) 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.
Ack n ow led gm en t. Generous support of this project
by USPHS Grants ES00267 and ES05355 is gratefully
acknowledged.
(20) Kim, S. J ., Harris, C. M., J ung, K.-Y., Koreeda, M., and Harris,
T. M. (1991) Nonbiomimetic route to deoxyadenosine adducts of
carcinogenic polycyclic aromatic hydrocarbons. Tetrahedron Lett.
32, 6073-6076.
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1314 Chem. Res. Toxicol., Vol. 14, No. 9, 2001
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