Synthesis of adducts formed by iodine oxidation of aromatic hydrocarbons in the presence of deoxyribonucleosides and nucleobases

Article abstract of DOI:10.1021/tx980127q

Polycyclic aromatic hydrocarbons (PAH) undergo two main pathways of metabolic activation related to the initiation of tumors: one-electron oxidation to give radical cations and monooxygenation to yield bay-region diol epoxides. Synthesis of standard adducts is essential for identifying biologically formed adducts. Until recently, radical cation adducts were synthesized by oxidation of the PAH in an electrochemical apparatus, not readily available in many organic chemistry laboratories. We have developed a convenient and efficient method for synthesizing PAH-nucleoside adducts by using I₂ as the oxidant. Adducts of benzo[a]pyrene (BP), dibenzo[a,l]pyrene (DB[a,l]P), and 7,12-dimethylbenz[a]anthracene were synthesized with deoxyguanosine (dG), deoxyadenosine, guanine (Gua), or adenine in either Me₂SO or dimethylformamide (DMF) with or without AgC10₄. When, for example, the potent carcinogen BP was dissolved in DMF in the presence of 3 equiv of I₂, 5 equiv of dG, and 1 equiv of AgC10₄, 45% of the BP was converted to BP-6-N7Gua. When BP was placed under the same reaction conditions in the absence of AgC10₄, the extent of formation of BP-6-N7Gua decreased to 30%. When the potent carcinogen DB[a,l]P was dissolved in DMF in the presence of 3 equiv of I₂, 5 equiv of dG, and 1 equiv of AgC10₄, 43% of the DB[a,l]P was converted to DB[a,l]P-10-N7Gua. In the more polar solvent Me₂SO under the same reaction conditions, however, the yield of DB[a,l]P-10-N7Gua was only 20%. Synthesis of adducts with the oxidant I₂ is more convenient and, in some cases, more efficient than synthesis by electrochemical oxidation. This method simplifies the synthesis of PAH-nucleoside and nucleobase adduces that are essential for studying biologically formed PAH-DNA adducts.

Full text of DOI:10.1021/tx980127q

Chem. Res. Toxicol. 1998, 11, 1201-1208
1201
Syn th esis of Ad d u cts F or m ed by Iod in e Oxid a tion of
Ar om a tic Hyd r oca r bon s in th e P r esen ce of
Deoxyr ibon u cleosid es a n d Nu cleoba ses
Aaron A. Hanson, Eleanor G. Rogan, and Ercole L. Cavalieri*
Eppley Institute for Research in Cancer and Department of Pharmaceutical Sciences, University of
Nebraska Medical Center, Omaha, Nebraska 68198-6805
Received J une 4, 1998
Polycyclic aromatic hydrocarbons (PAH) undergo two main pathways of metabolic activation
related to the initiation of tumors: one-electron oxidation to give radical cations and
monooxygenation to yield bay-region diol epoxides. Synthesis of standard adducts is essential
for identifying biologically formed adducts. Until recently, radical cation adducts were
synthesized by oxidation of the PAH in an electrochemical apparatus, not readily available in
many organic chemistry laboratories. We have developed a convenient and efficient method
for synthesizing PAH-nucleoside adducts by using I2 as the oxidant. Adducts of benzo[a]pyrene
(BP), dibenzo[a,l]pyrene (DB[a,l]P), and 7,12-dimethylbenz[a]anthracene were synthesized with
deoxyguanosine (dG), deoxyadenosine, guanine (Gua), or adenine in either Me2SO or dimeth-
ylformamide (DMF) with or without AgClO4. When, for example, the potent carcinogen BP
was dissolved in DMF in the presence of 3 equiv of I2, 5 equiv of dG, and 1 equiv of AgClO4,
45% of the BP was converted to BP-6-N7Gua. When BP was placed under the same reaction
conditions in the absence of AgClO4, the extent of formation of BP-6-N7Gua decreased to 30%.
When the potent carcinogen DB[a,l]P was dissolved in DMF in the presence of 3 equiv of I2, 5
equiv of dG, and 1 equiv of AgClO4, 43% of the DB[a,l]P was converted to DB[a,l]P-10-N7Gua.
In the more polar solvent Me2SO under the same reaction conditions, however, the yield of
DB[a,l]P-10-N7Gua was only 20%. Synthesis of adducts with the oxidant I2 is more convenient
and, in some cases, more efficient than synthesis by electrochemical oxidation. This method
simplifies the synthesis of PAH-nucleoside and nucleobase adducts that are essential for
studying biologically formed PAH-DNA adducts.
radical cation-derived adducts were synthesized by an-
odic oxidation of the PAH in an electrochemical ap-
paratus (3-7) not readily available in many organic
chemistry laboratories. We have developed an alterna-
tive, convenient, and efficient method for synthesizing
PAH radical cation-nucleoside adducts by using I2 as the
oxidant and Me2SO or dimethylformamide (DMF) as the
solvent.
In tr od u ction
Polycyclic aromatic hydrocarbons (PAH)1 undergo two
main pathways of metabolic activation related to tumor
initiation: one-electron oxidation to give radical cations
and monooxygenation with formation of bay-region diol
epoxides (1, 2). To establish radical cations as key
intermediates in metabolic activation of PAH, several
approaches have been investigated. Anodic oxidation of
PAH, such as benzo[a]pyrene (BP), dibenzo[a,l]pyrene
(DB[a,l]P), and 7,12-dimethylbenz[a]anthracene (DMBA),
in the presence of nucleosides has been used to generate
PAH-nucleoside adducts (3-7). Some of these adducts
have been obtained when the covalent binding of BP,
DB[a,l]P, or DMBA to DNA is catalyzed by horseradish
peroxidase or rat liver microsomal cytochrome P450 in
vitro, and in mouse skin and rat mammary gland in vivo
(8-12).
There is a large body of evidence that PAH are oxidized
by I2 to form radical cations that readily undergo nu-
cleophilic substitution. The first experiments in which
PAH were oxidized in the presence of such nucleophiles
as pyridine or nucleobases were conducted on the silica
surface of a thin-layer plate (13, 14). The yields were
very poor, and the products were not well identified when
the nucleophiles were nucleic acid bases (14). Improve-
ment in yield was obtained in homogeneous reactions in
which the nucleophile pyridine was also the solvent (15)
or was dissolved in various organic solvents (16).
Chemical synthesis of standard adducts is essential for
identifying biologically formed adducts. Until recently,
More recently, stable PAH radical cation salt com-
plexes have been obtained in benzene by one-electron
oxidation of the PAH with I2 in the presence of AgClO4
(17, 18). These salts are relatively stable and can be
stored in an inert atmosphere or reacted with nucleo-
philes to form PAH-nucleophile adducts.
* To whom correspondence should be addressed. Telephone: (402)
559-7237. Fax: (402) 559-8068. E-mail: ecavalie@mail.unmc.edu.
1
Abbreviations: Ade, adenine; BP, benzo[a]pyrene; BP-6-N7Gua,
7-(benzo[a]pyren-6-yl)guanine; CAD, collisionally activated decomposi-
tion; COSY, two-dimensional chemical shift correlation spectroscopy;
dA, deoxyadenosine; DB[a,l]P, dibenzo[a,l]pyrene; dG, deoxyguanosine;
DMBA, 7,12-dimethylbenz[a]anthracene; DMF, dimethylformamide;
FAB MS/MS, fast atom bombardment tandem mass spectrometry; Gua,
guanine; NOE, nuclear Overhauser effect; PAH, polycyclic aromatic
hydrocarbon(s); TFA, trifluoroacetic acid.
In this article, we report the synthesis of PAH-
nucleoside adducts obtained by I2 oxidation of PAH in
10.1021/tx980127q CCC: $15.00 © 1998 American Chemical Society
Published on Web 09/24/1998

1202 Chem. Res. Toxicol., Vol. 11, No. 10, 1998
Hanson et al.
Ta ble 1. An a lytica l, Sem ip r ep a r a tive, a n d P r ep a r a tive
HP LC Gr a d ien ts for th e Sep a r a tion of Ad d u ctsa
DMF or Me2SO. This alternative method is more con-
venient and, in some cases, more efficient than electro-
chemical synthesis.
BP or DB[a,l]P-dG analytical
and preparative
DMBA-dG analytical
timeb
CH3OH
time
CH3CN
Exp er im en ta l Section
0-5
40
50%c
100%
0-5
70
30%
100%
Ca u tion : The following chemicals are hazardous and should
be handled carefully in accordance with NIH guidelines (19):
BP, DB[a,l]P, and DMBA.
DMBA-dG preparative
BP-Ade/dA analytical
Ma ter ia ls. BP was obtained from Aldrich (Milwaukee, WI)
and DMBA from Eastman (Rochester, NY). BP and DMBA were
purified by silica gel column chromatography with 5% CHCl3
in hexane. DB[a,l]P was synthesized in our laboratory according
to a published procedure (20). The purity of the three PAH was
>99% by HPLC analysis. Deoxyguanosine (dG) and deoxyad-
enosine (dA) (TCI, Portland, OR) were desiccated over P2O5
under vacuum at 110 °C for 48 h prior to use. Adenine (Ade)
and guanine (Gua) (P-L Biochemicals, Milwaukee, WI), anhy-
drous DMF, Me2SO and I2 (Aldrich), HPLC-grade organic
solvents (EM Science, Gibbstown, NJ ), AgClO4 (GFS Chemicals,
Columbus, OH), and Na2S2O3 (Fisher Scientific, Fair Lawn, NJ )
were used as obtained.
HP LC. Analytical, semipreparative, and preparative HPLC
was conducted on a Waters 600E solvent delivery system.
Elutants were monitored for UV absorbance (254 nm) with a
Waters 990 photodiode array detector. Runs were conducted
on a YMC (YMC, Overland Park, KS) ODS-AQ 5 µm 120 Å
column (4.6 mm × 250 mm, analytical; 10 mm × 250 mm,
semipreparative) at 1 mL/min for analytical runs and 4 mL/
min for semipreparative runs. Preparative HPLC was con-
ducted on a YMC ODS-AQ 5 µm 120 Å column (20 mm × 250
mm) at a flow rate of 8 mL/min.
NMR. Proton, homonuclear two-dimensional chemical shift
correlation spectroscopy (COSY) and nuclear Overhauser effect
(NOE) NMR spectra were recorded on a Varian Unity 500
spectrometer at 499.835 MHz in Me2SO-d6 (2.49 ppm) at 26 °C.
Ma ss Sp ectr om etr y. Fast atom bombardment tandem
mass spectrometry (FAB MS/MS) was performed at the Ne-
braska Center for Mass Spectrometry (University of Nebraskas
Lincoln, Lincoln, NE) using a Micromass (Manchester, England)
AutoSpec high-resolution magnetic sector mass spectrometer.
The instrument was equipped with an orthogonal acceleration
time-of-flight serving as the second mass spectrometer in the
tandem experiment. Xenon was admitted to the collision cell
at a level to attenuate the precursor ion signal by 75%. Data
acquisition and processing were accomplished using OPUS
software that was provided by the manufacturer (Microcasm).
Samples were dissolved in 5-10 µL of CH3OH; 1 µL aliquots
were placed on the probe tip along with 1 µL of a 1:1 glycerol/
thioglycerol mixture.
time
CH3CN
time
0-15
40
C2H5OH/CH3CNd
0-5
70
20%
100%
50%
100%
BP-Ade/dA preparative
DB[a,l]P-Ade/dA analytical
time
0-10
40
C2H5OH/CH3CNd
time
C2H5OH/CH3CNd
50%
100%
0-8
40
60%
100%
DB[a,l]P-Ade/dA preparative DMBA-Ade/dA semipreparative
time
C2H5OH/CH3CNd
time
C2H5OH/CH3CNd
0-10
55%
100%
0-15
30-40
50
30%
70%
100%
40
a
All gradients were linear, except for the DMBA-dG analytical
b
gradient in which a convex (CV5) gradient was used. Time is in
minutes. c The organic phase starts as a mixture with water. The
d
C2H5OH/CH3CN organic phase is a 3:1 mixture.
with a syringe and 19 gauge needle. AgClO4 was added to the
addition funnel through the rubber septum. The I2/PAH/
deoxyribonucleoside or I2/PAH/nucleobase mixture was allowed
to react for 1.5 h before AgClO4 was added dropwise from the
addition funnel. Once all the compounds were combined, the
reaction was allowed to proceed for an additional 16.5 h and
then quenched with 0.1 M Na2S2O3.
The reaction mixture was then dried under vacuum at 50 °C,
redissolved in 4 mL of Me2SO/CH3OH (1:1), and filtered through
a 0.45 µm filter. The filter was then washed with an additional
500 µL of Me2SO. Determination of the yield of the synthesized
adducts was accomplished by analytical or semipreparative
HPLC, in either the CH3CN/H2O, CH3OH/H2O, or C2H5OH/CH3-
CN/H2O solvent system (Table 1), with monitoring at 254 nm
for comparison of peak areas of the adduct(s), byproduct(s), and
parent compound. The percentage of each adduct in Tables 2-4
was calculated by dividing the peak area of the adduct by the
sum of the peak areas of the adduct(s), byproduct(s), and parent
compound. Purification of the adducts was conducted by
preparative or semipreparative HPLC, as described in Table 1.
Reactions conducted with Ade were analyzed in a C2H5OH/
CH3CN/H2O solvent system due to the problems encountered
in the isolation of PAH-N1Ade adducts in either the CH3CN/
H2O or CH3OH/H2O solvent system. A typical HPLC separation
of BP-Ade adducts is shown in Figure 1. The CH3CN/H2O
gradient in Figure 1A allowed separation of BP-6-N7Ade and
BP-6-N3Ade, but gave only a broad peak for BP-6-N1Ade. Use
of a C2H5OH/CH3CN/H2O gradient, however, enabled separation
of all three adducts (Figure 1B).
Known adducts were identified by comparing analytical
HPLC retention time, UV, and NMR spectra to those previously
obtained. Novel adducts were identified by UV spectra, NMR
spectra (including one-dimensional NOE, D2O exchange, and
COSY), and FAB MS/MS.
7-MBA-12-CH2-N1Ad e. 1H NMR (ppm): δ 3.15 (s, 3H,
7-CH3), 6.44 (s, 2H, 12-CH2), 7.45 (m, 1H, 2-H), 7.59 (m, 1H,
10-H), 7.66 (m, 2H, 3-H, 9-H), 7.69 (s, 1H, 2-H[Ade]), 7.80 (d, J
) 9.0 Hz, 1H, 5-H), 7.92 (s, 1H, 8-H[Ade]), 7.93 (m, 3H, 1-H,
11-H, 6-NH[Ade]), 8.01 (d, J ) 6.5 Hz, 1H, 4-H), 8.07 (bs, 1H,
6-NH[Ade]), 8.24 (d, J ) 9.5 Hz, 1H, 6-H), 8.53 (d, J ) 8.0 Hz,
1H, 8-H). UV: λmax 266, 287, 297, 351, 367, 384 nm. FAB MS
[M + H]+: calcd for C25H19N5 m/z 390.1719, found m/z 390.1716.
Ch em ica l Syn th esis of Ad d u cts. Glassware, syringes, and
needles were dried at 150 °C prior to use. The reaction
glassware was assembled while hot, cooled under vacuum, and
pressurized under an inert atmosphere (argon or nitrogen).
Syringes and needles were cooled under vacuum in a desiccator
and pressurized under an inert atmosphere.
Coupling between PAH and the nucleophiles was accom-
plished by combining 59 µmol of PAH with 5 equiv of dG, dA,
Ade, or Gua dissolved in 3.5 mL of Me2SO or DMF in a 50 mL
three-necked flask. Attached to the three-necked flask were a
gas/vacuum line adapter, an addition funnel topped with a
rubber septum, and a rubber septum. Three equivalents of I2
and 1 equiv of AgClO4 were weighed in separate 20 mL glass
vials, covered with rubber septa, put under vacuum, pressurized
under an inert atmosphere, and dissolved in Me2SO or DMF.
To keep total solvent volumes the same in reactions with or
without AgClO4, the amount of solvent used to dissolve I2 was
varied. When a reaction mixture included AgClO4, 1 mL of
solvent was used to dissolve I2 and 1.5 mL to dissolve AgClO4,
whereas 2.5 mL of solvent was used to dissolve I2 when AgClO4
was absent. To the vigorously stirred PAH/deoxyribonucleoside
or nucleobase mixture was slowly added I2 through the septum

PAH-Nucleoside Adduct Synthesis by I2 Oxidation
Chem. Res. Toxicol., Vol. 11, No. 10, 1998 1203
7.93 (m, 1H, 3-H), 7.99 (s, 1H, 8-H[Gua]), 8.23 (d, J ) 9.0 Hz,
1H, 6-H), 8.44 (m, 1H, 11-H), 8.52 (m, 2H, 1-H, 8-H), 10.15 (bs,
1H, 1-NH[Gua]). UV: λmax 264, 286, 296, 349, 364, 380 nm.
FAB MS [M + H]+: calcd for C30H27N5O4 m/z 522.2141, found
m/z 522.2131.
Resu lts a n d Discu ssion
Op tim iza tion of Rea ction Con d ition s. Reactions
were conducted to determine whether one-electron oxida-
tion by I2 would produce adequate yields of PAH-
nucleoside adducts. To optimize the yield of PAH-
nucleoside adducts, 59 µmol (15 mg) of BP was added to
varying equivalents (equiv) of dG, I2, and AgClO4 in 6
mL of DMF at room temperature for 18 h. The best ratio
was found to be 1:5:3:1 PAH:dG:I2:AgClO4. Smaller
amounts of I2 resulted in low yields (<10% adduct),
whereas amounts greater than 3 equiv did not improve
the yield. Increasing the ratio of AgClO4 decreased the
yield of 7-(benzo[a]pyren-6-yl)guanine (BP-6-N7Gua) due
to the formation of 6-I-BP. The yield of the BP-6-N7Gua
adduct was most dependent on the amount of AgClO4
used in the reaction mixture. More than 1 equiv of
AgClO4 produced low amounts of adduct and high
amounts of the byproduct 6-I-BP. At 6 equiv of AgClO4,
92% of the BP was converted to 6-I-BP. Less than 1
equiv produced low yields of both the adduct and byprod-
uct. Therefore, the ratio of reactants stated above was
used for oxidation of BP, DB[a,l]P, and DMBA in the
presence of dG, Gua, dA, or Ade in the solvent DMF or
Me2SO, with or without AgClO4. Gua was used only with
Me2SO due to its extremely low solubility in DMF.
F igu r e 1. HPLC separation of adducts formed by I2 oxidation
of BP in the presence of Ade and AgClO4. (A) The HPLC column
was eluted with 40% CH3CN in H2O for 15 min, followed by a
15 min linear gradient to 60% CH3CN in H2O, at which the
mixture was held for 10 min, followed by a 10 min linear
gradient to 100% CH3CN. (B) The HPLC column was eluted
with 65% C2H5OH/CH3CN (3:1) in H2O for 15 min, followed by
a 25 min linear gradient to 100% C2H5OH/CH3CN (3:1).
12-MBA-7-CH2-N1Ad e. 1H NMR (ppm): δ 3.39 (s, 3H, 12-
CH3), 6.52 (s, 2H, 7-CH2), 7.54 (s, 1H, 2-H[Ade]), 7.65 (m, 3H,
3-H, 4-H, 9-H), 7.72 (m, 1H, 2-H), 7.75 (d, J ) 9.5 Hz, 1H, 5-H),
7.81 (bs, 1H, 6-NH[Ade]), 7.94 (m, 2H, 10-H, 6-NH[Ade]), 7.95
(s, 1H, 8-H[Ade]), 8.25 (d, J ) 9.5 Hz, 1H, 6-H), 8.48 (m, 2H,
1-H, 11-H), 8.54 (m, 1H, 8-H). UV: λmax 266, 288, 298, 352,
367, 384 nm. FAB MS [M + H]+: calcd for C25H19N5 m/z
390.1719, found m/z 390.1729.
7-MBA-12-CH2-N3Ad e. 1H NMR (ppm): δ 3.14 (s, 3H,
7-CH3), 6.22 (s, 2H, 12-CH2), 7.32 (bs, 2H, 6-NH2[Ade]), 7.42
(m, 1H, 2-H), 7.56 (s, 1H, 2-H[Ade]), 7.59 (m, 1H, 10-H), 7.66
(m, 2H, 3-H, 9-H), 7.78 (d, J ) 10.0 Hz, 1H, 5-H), 7.97 (m, 3H,
1-H, 4-H, 11-H), 8.23 (d, J ) 9.0 Hz, 1H, 6-H), 8.29 (s, 1H,
8-H[Ade]), 8.51 (d, J ) 9.0 Hz, 1H, 8-H). UV: λmax 266, 286,
297, 347, 364, 379 nm. FAB MS [M + H]+: calcd for C25H19N5
m/z 390.1719, found m/z 390.1708.
The better yield of the reactions in the presence of the
proper amount of AgClO4 is presumably due to stabiliza-
tion of the radical cation as a perchlorate (17, 18),
produced after one-electron oxidation of the PAH by I2.
It was observed, however, that with a large amount of
AgClO4, BP is almost totally converted to 6-I-BP, sug-
gesting that formation of AgI from I2 efficiently generates
the electrophilic I+ that reacts to form 6-I-BP.
Syn th esis of Un su bstitu ted P AH-Nu cleosid e Ad -
d u cts. Formation of unsubstituted PAH adducts can be
rationalized with initial removal of one electron by I2
(Scheme 1). The intermediate radical cation, with charge
localized at C-6 in BP (Scheme 1) or C-10 in DB[a,l]P,
regiospecifically reacts with nucleophilic groups of nu-
cleosides or nucleobases to form an intermediate radical
species, with loss of the proton from the N-9 of Ade (Gua)
or cleavage of the glycosidic bond with dA (dG) to lose
deoxyribose. The easy removal of a second electron from
the arene radical by either I2 or another BP radical cation
produces an arenium ion, which loses a proton to
complete the substitution reaction.
12-MBA-7-CH2-N3Ad e. 1H NMR (ppm): δ 3.39 (s, 3H, 12-
CH3), 6.28 (s, 2H, 7-CH2), 7.23 (bs, 1H, 6-NH[Ade]), 7.48 (s, 1H,
2-H[Ade]), 7.65 (m, 2H, 2-H, 3-H), 7.70 (m, 2H, 9-H, 10-H), 7.76
(d, J ) 9.0 Hz, 1H, 5-H), 7.94 (m, 1H, 4-H), 8.32 (s, 1H,
8-H[Ade]), 8.33 (d, J ) 8.5 Hz, 1H, 6-H), 8.46 (d, J ) 8.5 Hz,
1H, 11-H), 8.53 (m, 1H, 1-H), 8.57 (d, J ) 8.0 Hz, 1H, 8-H). UV:
λ
max 266, 285, 295, 348, 363, 379 nm. FAB MS [M + H]+: calcd
for C25H19N5 m/z 390.1719, found m/z 390.1708.
7-MBA-12-CH2-N2d G. 1H NMR (ppm): δ 2.22 (m, 1H, 2′-
H), 2.70 (m, 1H, 2′-H), 3.10 (s, 3H, 7-CH3), 3.49 (m, 1H, 5′-H),
3.55 (m, 1H, 5′-H), 3.78 (m, 1H, 4′-H), 4.34 (m, 1H, 3′-H), 4.82
(m, 1H, 5′-OH), 5.20 (d, J ) 3.5 Hz, 1H, 3′-OH), 5.30 (m, 2H,
12-CH2), 6.19 (t, J ) 7.0 Hz, 1H, 1′-H), 7.48 (bs, 1H, 2-NH[Gua]),
7.58 (m, 1H, 9-H), 7.66 (m, 1H, 4-H), 7.73 (m, 3-H, 2-H, 5-H,
10-H), 7.99 (s, 1H, 8-H[Gua]), 8.16 (d, J ) 10.0 Hz, 1H, 11-H),
8.34 (d, J ) 9.0 Hz, 1H, 6-H), 8.48 (dd, J 1,2 ) 9.0 Hz, J 8,9 ) 10.0
Hz, 2H, 1-H, 8-H), 10.27 (bs, 1H, 1-NH[Gua]). UV: λmax 264,
287, 297, 349, 364, 380 nm. FAB MS [M + H]+: calcd for
The unsubstituted PAH, BP and DB[a,l]P, reacted
similarly under identical conditions, as seen in Tables 2
and 3. All the adducts formed from dG and dA lose the
deoxyribose moiety by destabilization of the glycosidic
bond. Generally, the highest yields of adduct were
obtained from the reactions conducted in DMF in the
presence of AgClO4. Identical reaction mixtures in
Me2SO generally produced a lower yield compared to the
reactions in DMF. Gua, however, was reacted only in
Me2SO because of its poor solubility in DMF.
C
30H27N5O4 m/z 522.2141, found m/z 522.2141.
12-MBA-7-CH2-N2d G. 1H NMR (ppm): δ 2.33 (m, 1H, 2′-
H), 2.69 (m, 1H, 2′-H), 3.33 (s, 3H, 12-CH3), 3.58 (m, 1H, 5′-H),
3.63 (m, 1H, 5′-H), 3.89 (s, 1H, 4′-H), 4.44 (s, 1H, 3′-H), 4.97
(bs, 1H, 5′-OH), 5.40 (s, 1H, 3′-OH), 5.43 (s, 2H, 7-CH2), 6.38 (t,
J ) 6.75 Hz, 1H, 1′-H), 6.92 (bs, 1H, 2-NH[Gua]), 7.64 (m, 2H,
2-H, 4-H), 7.73 (m, 2H, 9-H, 10-H), 7.78 (d, J ) 9.5 Hz, 1H, 5-H),
Reaction of BP or DB[a,l]P with dG, Gua, dA, or Ade
produced an array of adducts, which can be used as

1204 Chem. Res. Toxicol., Vol. 11, No. 10, 1998
Hanson et al.
Ta ble 3. Yield of Ad d u cts F or m ed Wh en DB[a ,l]P Wa s
Rea cted w ith d G, Gu a , d A, or Ad e in th e P r esen ce of I2
w ith or w ith ou t AgClO4 in DMF or Me2SO
Sch em e 1. P r op osed Mech a n ism of Ad d u ct
F or m a tion F ollow in g I2 Oxid a tion of BP in th e
P r esen ce of Ad e
% adduct formeda
DMF
without
Me2SO
with without
nucleoside
or base
with
AgClO4 AgClO4 AgClO4 AgClO4
adduct
dG
Gua
DB[a,l]P-10-N7Gua
DB[a,l]P-10-N7Gua
DB[a,l]P-10-C8Gua
DB[a,l]P-10-N7Ade
DB[a,l]P-10-N7Ade
DB[a,l]P-10-N3Ade
DB[a,l]P-10-N1Ade
43
43
20
23
8
0
2
30
19
8
0
1
dA
Ade
30
1
10
47
8
1
4
7
46
8
42
47
a
The percentage of adduct is relative to the amount of starting
DB[a,l]P. The remainder is starting material. Since the molar
extinction coefficients of the adducts at 254 nm are very similar
to that of the parent DB[a,l]P, yields were calculated by integration
of the peak areas of the adducts.
dG were adducts in which the deoxyribose was lost by
cleavage of the glycosidic bond.
Syn th esis of Meth yl-Su bstitu ted P AH-Nu cleosid e
Ad d u cts. One-electron oxidation of DMBA produces a
radical cation with charge mainly localized at the C-7
and C-12 meso-anthracenic positions adjacent to the
methyl groups. Loss of the acidic methyl proton gener-
ates benzylic radical intermediates that are rapidly
oxidized to benzylic carbenium ions (7). These interme-
diates react with nucleophilic groups of dG (dA) to form
nucleoside and nucleobase adducts (Scheme 2 and Table
4).
Ta ble 2. Yield of Ad d u cts F or m ed Wh en BP Wa s Rea cted
w ith d G, Gu a , d A, or Ad e in th e P r esen ce of I2 w ith or
w ith ou t AgClO4 in DMF or Me2SO
% adduct formeda
Unlike the unsubstituted PAH described above, DMBA
generally gave a better yield of adducts in the more polar
solvent Me2SO. This may be due to stabilization of the
intermediate benzylic carbocation (Scheme 2) in Me2SO.
Reaction of DMBA with dG, Gua, dA, or Ade produces
an array of adducts which can be used as reference
standards in the study of DNA adducts formed by this
compound in biological systems. Like the case with the
unsubstituted PAH, more adducts were formed with Ade
than with dA (Table 4). The deoxyribose of dA prevents
reaction of the electrophilic DMBA species at N-3. The
lack of an N-3 adduct was also observed with BP and
DB[a,l]P (Tables 2 and 3), as well as previously with
other PAH (7). No adducts were detected when DMBA
was oxidized in the presence of Gua. This was a surprise
because 6-methyl-BP2 and the unsubstituted PAH (Tables
2 and 3) formed Gua adducts similar to those obtained
in their respective reactions with dG. Unlike that with
the unsubstituted PAH (Tables 2 and 3), reaction of the
oxidized DMBA with dG produced both nucleoside and
nucleobase adducts (Scheme 2 and Table 4).
Str u ctu r e Elu cid a tion of New ly Syn th esized Ad -
d u cts. All of the adducts formed by I2 oxidation of BP
and DB[a,l]P were previously obtained by electrochemical
oxidation (3, 4, 9).3 Several new adducts were obtained
by I2 oxidation of DMBA in the presence of dA compared
to those from anodic oxidation of DMBA (5). In addition,
the adducts previously identified as 12-MBA-7-CH2-
N3Ade and 7-MBA-12-CH2-C8dG (5) are, in fact, 12-
MBA-7-CH2-N7Ade and 7-MBA-12-CH2-N2dG, respec-
tively. These errors were rectified by Mulder et al. (7).
(1) 7-MBA-12-CH2-N1Ad e. This compound has typi-
cal DMBA UV absorption maxima at 266, 367, and 384
DMF
without
AgClO4 AgClO4 AgClO4 AgClO4
Me2SO
nucleoside
or base
with
with without
adduct
dG
BP-6-N7Gua
6-I-BP
BP-6-N7Gua
6-I-BP
BP-6-N7Ade
6-I-BP
BP-6-N7Ade
BP-6-N3Ade
BP-6-N1Ade
6-I-BP
45
5
30
0
36
8
37
9
Gua
dA
24
7
23
6
33
10
2
15
12
1
0
13
2
2
15
1
Ade
4
4
4
3
76
2
61
4
54
5
39
7
a
The percentage of adduct is relative to the amount of starting
BP. The remainder is starting material. Since the molar extinction
coefficients of the adducts at 254 nm are very similar to that of
the parent BP, yields were calculated by integration of the peak
areas of the adducts.
reference standards in the study of DNA adducts formed
by these PAH. The use of Ade as the nucleophile,
compared to dA, increased the number of BP- or
DB[a,l]P-Ade adducts from one to three, including the
biologically formed N3Ade adducts (9, 10, 21). The use
of Gua in the reaction with DB[a,l]P yielded one more
Gua adduct, DB[a,l]P-10-C8Gua, than was obtained with
dG (Table 3). BP, on the other hand, afforded only the
N7Gua adduct and traces (<0.05%) of the C8Gua adduct
(Table 2). The C8Gua adducts of both BP and DB[a,l]P
are formed biologically (8-10). BP-6-C8Gua is obtained
in reasonable yield by electrochemical oxidation of BP
in the presence of dG (3). Most I2 oxidations of BP yielded
the byproduct 6-I-BP, primarily in the presence of AgClO4
or in reactions carried out in Me2SO (Table 2). The 10-
I-DB[a,l]P was not detected in any of the DB[a,l]P
reactions (Table 3). All adducts found after reaction
of the unsubstituted PAH, BP and DB[a,l]P, with dA or
2
A. A. Hanson et al., unpublished results.
K.-M. Li et al., unpublished results.
3

PAH-Nucleoside Adduct Synthesis by I2 Oxidation
Chem. Res. Toxicol., Vol. 11, No. 10, 1998 1205
Sch em e 2. P r op osed Mech a n ism of Ad d u ct F or m a tion F ollow in g I2 Oxid a tion of DMBA in th e P r esen ce of
d G
Ta ble 4. Yield of Ad d u cts F or m ed Wh en DMBA Wa s Rea cted w ith d G, Gu a , d A, or Ad e in th e P r esen ce of I2 w ith or
w ith ou t AgClO4 in DMF or Me2SO
% adduct formeda
DMF
Me2SO
nucleoside
or base
with
AgClO4
without
AgClO4
with
AgClO4
without
AgClO4
adduct
dG
7-MBA-12-CH2-N7Gua
12-MBA-7-CH2-N7Gua
7-MBA-12-CH2-N2dG
12-MBA-7-CH2-N2dG
no reaction
7-MBA-12-CH2-N7Ade
12-MBA-7-CH2-N7Ade
7-MBA-12-CH2-N7Ade
12-MBA-7-CH2-N7Ade
7-MBA-12-CH2-N3Ade
12-MBA-7-CH2-N3Ade
7-MBA-12-CH2-N1Ade
12-MBA-7-CH2-N1Ade
20
5
4
18
3
3
20
5
9
17
3
9
11
7
25
25
Gua
dA
11
32
6
12
7
8
20
26
11
22
2
8
3
4
12
13
3
36
5
11
4
4
23
29
6
18
4
13
4
5
14
23
Ade
a
The percentage of adduct is relative to the amount of starting DMBA. The remainder is starting material. Since the molar extinction
coefficients of the adducts at 254 nm are very similar to that of the parent DMBA, yields were calculated by integration of the peak areas
of the adducts.
nm. FAB MS shows an [M + H]+ ion at m/z 390 for
DMBA-Ade and a fragment at m/z 255, resulting from
the loss of Ade. As demonstrated with other DMBA
adducts, collisionally activated decomposition (CAD)
spectra obtained by tandem MS of the parent ion can
distinguish between 7-CH2- and 12-CH2-linked adducts
(5, 22). The CAD spectrum of the [M + H]+ ion for this
compound shows that the m/z 240 fragment is more
abundant than the m/z 239 fragment, indicating that
substitution has taken place at the 12-CH3 group. If the
substitution had occurred at the 7-CH3 group (see below),
the m/z 239 fragment would have been more prominent
than the m/z 240 fragment.
(7) and is attributed to the close proximity of the aromatic
moiety to the 6-amino group that creates a different
chemical environment for the two protons. NOE irradia-
tion of the 12-CH2 group at 6.44 ppm shows enhancement
of the 11-H and 1-H resonances at 7.93 ppm and the 2-H
resonance of the Ade signal at 7.69 ppm, indicating that
substitution occurred at the 12-CH3 group. Irradiation
of the 7-CH3 group resonance at 3.15 ppm shows en-
hancement of the signals of 6-H at 8.24 ppm and 8-H at
8.53 ppm, confirming that the 7-CH3 group is unsubsti-
tuted and the 12-CH2 group is bonded to the N-1 of Ade.
The signal for the 2-H resonance of Ade is shifted upfield
in this molecule, as seen in meso-CH2-N1Ade-linked
adducts of 1,2,3,4-tetrahydro-7,12-DMBA (7).
The NMR spectrum (Figure 2A) displays splitting of
the two proton resonances of the NH2 groups at 7.92 and
8.07 ppm. This is characteristic of substitution at N-1
(2) 12-MBA-7-CH2-N1Ad e. FAB MS shows an [M +
H]+ ion at m/z 390 for DMBA-Ade and a fragment at

1206 Chem. Res. Toxicol., Vol. 11, No. 10, 1998
Hanson et al.
F igu r e 2. 1H NMR spectrum of (A) 7-MBA-12-CH2-N1Ade and
(B) 12-MBA-7-CH2-N1Ade.
F igu r e 3. 1H NMR spectrum of (A) 7-MBA-12-CH2-N3Ade and
m/z 255, resulting from the loss of Ade. The CAD
spectrum of the [M + H]+ ion for this compound shows
that the m/z 239 fragment is more abundant than the
m/z 240 fragment, indicating that substitution has taken
place at the 7-CH3 group.
(B) 12-MBA-7-CH2-N3Ade.
the H2O signal downfield and subsequently exposes the
12-CH3 signal at 3.39 ppm, previously hidden under the
H2O resonance.
The NMR spectrum (Figure 2B) displays two D2O-
exchangeable broad singlets of the 6-NH2 group of Ade
at 7.81 and 7.94 ppm, similar to that seen for the 6-NH2
of 7-MBA-12-CH2-N1Ade. This suggests that the Ade
moiety is bonded at N-1. NOE irradiation of the 7-CH2
group resonance at 6.52 ppm shows enhancement of the
signals of 8-H at 8.54 ppm and 6-H at 8.25 ppm and of
the 2-H resonance of Ade at 7.54 ppm, indicating that
the PAH moiety is bonded at the 7-CH2 group to the N-1
of Ade. Addition of trifluoroacetic acid (TFA) deshields
the H2O chemical shift downfield and subsequently
exposes the 12-CH3 group resonances at 3.39 ppm previ-
ously hidden under the H2O signal.
(5) 7-MBA-12-CH2-N2d G. FAB MS shows an [M +
H]+ ion at m/z 522 for DMBA-dG and a fragment at m/z
255, resulting from the loss of dG. The CAD spectrum
of the [M + H]+ ion for this compound shows that the
m/z 240 fragment is more abundant than the m/z 239
fragment, indicating that substitution has taken place
at the 12-CH3 group.
The NMR spectrum (Figure 4A) shows the typical
resonances of the deoxyribose protons, which have been
assigned by comparison with the spectrum of dG and by
using COSY. The sharp singlet at 7.99 ppm (8-H) and
the broad D2O-exchangeable singlets at 7.48 (2-NH) and
10.27 ppm (1-NH) are attributed to the Gua base. NOE
irradiation of the 12-CH2 group resonance at 5.30 ppm
shows enhancement of the signals for 11-H at 8.16 ppm
and 1-H at 8.48 ppm and the 2-NH resonance of Gua at
7.48 ppm. Irradiation of the 7-CH3 group signal at 3.14
ppm shows enhancement of the resonances of 6-H at 8.34
ppm and 8-H at 8.48 ppm. These findings confirm that
this adduct has the 12-CH2 group bonded to the exocyclic
N2 position of dG.
(6) 12-MBA-7-CH2-N2d G. FAB MS shows an [M +
H]+ ion at m/z 522 for DMBA-dG and a fragment at m/z
255, resulting from the loss of dG. The CAD spectrum
of the [M + H]+ ion for this compound shows that the
m/z 239 fragment is more abundant than the m/z 240
fragment, indicating that substitution has taken place
at the 7-CH3 group.
(3) 7-MBA-12-CH2-N3Ad e. FAB MS shows an [M +
H]+ ion at m/z 390 for DMBA-Ade and a fragment at m/z
255, resulting from the loss of Ade. The CAD spectrum
of the [M + H]+ ion for this compound shows that the
m/z 240 fragment is more abundant than the m/z 239
fragment, indicating that substitution has taken place
at the 12-CH3 group. In the NMR spectrum (Figure 3A),
NOE irradiation of the 12-CH2 group at 6.22 ppm shows
enhancement of the signals for the 11-H and 1-H reso-
nances at 7.97 ppm and of the 2-H resonance of Ade at
7.56 ppm, similar to those seen in 7-MBA-12-CH2-N1Ade.
(4) 12-MBA-7-CH2-N3Ad e. FAB MS shows an [M +
H]+ ion at m/z 390 for DMBA-Ade and a fragment at m/z
255, resulting from the loss of Ade. The CAD spectrum
of the [M + H]+ ion for this compound shows that the
m/z 239 fragment is more abundant than the m/z 240
fragment, indicating that substitution has taken place
at the 7-CH3 group. In the NMR spectrum (Figure 3B),
NOE irradiation of the 7-CH2 group resonance at 6.28
ppm shows enhancement of the signals of 8-H at 8.57
ppm and 6-H at 8.33 ppm and the 2-H resonance of Ade
at 7.48 ppm, similar to those seen in 12-MBA-7-CH2-
N1Ade, indicating that the PAH moiety is bonded at the
7-CH2 group to the N-3 of Ade. Addition of TFA shifts
The NMR spectrum (Figure 4B) shows the presence of
the deoxyribose proton resonances. The sharp singlet at
7.99 ppm (8-H) and the broad D2O-exchangeable singlets
at 6.92 (2-NH) and 10.15 ppm (1-NH) are attributed to
the Gua base. NOE irradiation of the 7-CH2 group
resonance at 5.43 ppm shows enhancement of the signal
for 8-H at 8.52 ppm and 6-H at 8.23 ppm and the 2-NH
resonance of Gua at 6.92 ppm. Addition of TFA deshields

PAH-Nucleoside Adduct Synthesis by I2 Oxidation
Chem. Res. Toxicol., Vol. 11, No. 10, 1998 1207
electrophilicity is delocalized over the entire molecule.
The deoxyribose moiety in dA plays a major role in the
steric accessibility of N-3 to PAH radical cations. In fact,
no N3Ade adducts are obtained (Tables 2-4) from dA,
although they are formed when Ade is used.
Con clu sion s
Standard adducts can be synthesized by iodine oxida-
tion of PAH in the presence of deoxyribonucleosides or
nucleobases. This alternative method is more convenient
and, in some cases, more efficient than synthesis of these
adducts via electrochemical oxidation. Furthermore, in
the case of the heterocyclic aromatic hydrocarbon diben-
zo[c,g]carbazole, electrochemical oxidation in the pres-
ence of deoxyribonucleosides or nucleobases failed to
produce any adducts, whereas I2 oxidation produced an
array of adducts in low yield (21). This method simplifies
the synthesis of PAH-nucleoside adducts essential for
studying PAH-DNA adducts formed biologically.
Ack n ow led gm en t. We gratefully thank Dr. Ronald
Cerny at the Nebraska Center for Mass Spectrometry at
the University of NebraskasLincoln for the valuable
mass spectral data. A.A.H. was supported by predoctoral
fellowships from the University of Nebraska Environ-
mental Toxicology Graduate Program and the University
of Nebraska Medical Center. This research was supported
by U.S. Public Health Service grants from the National
Cancer Institute (P01 CA49210 and R01 CA49917). Core
support at the Eppley Institute was funded by NCI
Laboratory Cancer Research Center Support (Core)
Grant CA36727.
F igu r e 4. 1H NMR spectrum of (A) 7-MBA-12-CH2-N2dG and
(B) 12-MBA-7-CH2-N2dG.
Refer en ces
the H2O signal and subsequently exposes the resonances
of the 12-CH3 group at 3.33 ppm. These findings confirm
that this adduct has the 7-CH2 group linked to the
exocyclic N2 position of dG.
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