Photochemical generation of nitric oxide from 6-nitrobenzo[a]pyrene

Article abstract of DOI:10.1021/ja0109038

Photolabile 6-nitrobenzo[a]pyrene (6-nitroBaP) released nitric oxide (NO) under visible-light irradiation. The generation of NO and the concomitant formation of the 6-oxyBaP radical were confirmed by ESR. BaP quinones were also detected as further oxidize

Full text of DOI:10.1021/ja0109038

8662
J. Am. Chem. Soc. 2001, 123, 8662-8666
Photochemical Generation of Nitric Oxide from
6-Nitrobenzo[a]pyrene
Kiyoshi Fukuhara,* Masaaki Kurihara, and Naoki Miyata*
Contribution from the DiVision of Organic Chemistry, National Institute of Health Sciences,
Setagaya, Tokyo 158-8501, Japan
ReceiVed April 9, 2001
Abstract: Photolabile 6-nitrobenzo[a]pyrene (6-nitroBaP) released nitric oxide (NO) under visible-light
irradiation. The generation of NO and the concomitant formation of the 6-oxyBaP radical were confirmed by
ESR. BaP quinones were also detected as further oxidized products of the 6-oxyBaP radical. No such
photodegradation was observed with other nitrated BaPs, such as 1-nitroBaP and 3-nitroBaP. DNA-strand
breakage, caused by photoexcited 6-nitroBaP, was closely related to its NO-releasing activity. MO calculations
of nitrated BaP suggest that the perpendicular conformation of the nitro substituent to the aromatic ring is
important for the release of NO with light. These findings may be useful for the development of a new type
of NO donor.
Introduction
crises and cardiac failure.7 This has led to the development of
photochemically triggered NO donors, such as metal nitrosyl
compounds8 and some caged nitric oxides.9 The target of NO
donors in bioregulation is endothelial cells, and the released
NO activates guanyl cyclase. However, little work has been done
to develop an NO donor considering cytotoxicity as the major
biological outcome.10 In the present study, we investigated
6-nitrobenzo[a]pyrene (6-nitroBaP) (Figure 1) as a potential NO
donor and showed that it induces DNA strand breaks upon
photoirradiation. Nitrated benzo[a]pyrenes (1-, 3-, 6-nitro and
1,6- and 3,6-dinitro) are synthesized by the nitration of benzo-
[a]pyrene (BaP). Among them, 6-nitrated BaPs such as 1,6- and
3,6-dinitro-BaPs are unstable under visible light. The instability
of these nitrated BaPs is due to the nitro substituent at the
6-position. We report here that 6-nitroBaP released NO under
visible-light irradiation, while no such photodegradation was
observed with other nitrated BaPs, such as 1- and 3-nitroBaPs,
and that DNA strand breakage was closely related to NO-
releasing activity.
In this paper, we characterize a nitroarene that can release
nitric oxide (NO) and efficiently cleave DNA under photoir-
radiation. NO has been considered to be an important biological
messenger that regulates vasodilation and neurotransmission.1
It also has important roles as a mediator of the cytotoxic action
of macrophages toward tumor cells and microbial pathogens,
through the inhibition of mitochondrial enzyme activity and
DNA synthesis.2 Macrophages also use NO as a cytotoxic agent,
which leads to various types of DNA damage.3 It has been
shown that NO can deaminate primary amines of DNA under
aerobic conditions, and that this leads to mutations and DNA
•-
strand breaks.4 NO can also react with O2 produced from
activated macrophages to form peroxynitrate, which is a strong
oxidant and nitrating agent, resulting in the modification of
protein function5 and causing DNA damage.6
Attention has been concentrated on the use of caged NO com-
pounds as potent vasodilators in the management of hypertensive
* To whom correspondence should be addressed.
Results
(1) Moncada, S.; Palmer, R. M.; Higgs, E. A. Pharmacol. ReV. 1991,
43, 109-142.
To assess the formation of NO upon the photoirradiation of
nitroBaPs, an ESR experiment with (MGD)-Fe as the spin trap11
was performed (Figure 2). ESR analysis of 6-nitroBaP photoir-
radiated for 1 min in the presence of MGD-Fe displayed a three-
(2) Stuehr, D. J.; Gross, S. S.; Sakuma, I.; Levi, R.; Nathan, C. F. J.
Exp. Med. 1989, 169, 1011-1020.
(3) Hibbs, J. B., Jr.; Taintor, R. R.; Vavrin, Z. Science 1987, 235, 473-
476.
(4) (a) Wink, D. A.; Kasprzak, K. S.; Maragos, C. M.; Elespru, R. K.;
Misra, M.; Dunams, T. M.; Cebula, T. A.; Koch, W. H.; Andrews, A. W.;
Allen, J. S.; Keefer, L. K. Science 1991, 254, 1001-1003. (b) Nguyen, T.;
Brunson, D.; Crespi, C. L.; Penman, B. W.; Wishnok, J. S.; Tannenbaum,
S. R. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 3030-3034.
(5) (a) Haddad, I. Y.; Pataki, G.; Hu, P.; Galliani, C.; Beckman, J. S.;
Matalon, S. J. Clin. InVest. 1994, 94, 2407-2413. (b) Kaur, H.; Halliwell,
B. FEBS Lett. 1994, 350, 9-12. (c) Kooy, N. W.; Royall, J. A.; Ye, Y. Z.;
Kelly, D. R.; Beckman, J. S. Am. J. Respir. Crit. Care Med. 1995, 151,
1250-1254.
(6) (a) King, P. A.; Anderson, V. E.; Edwards, J. O.; Gustafson, G.;
Plumb, R. C.; Suggs, J. W. J. Am. Chem. Soc. 1992, 114, 5430-5432. (b)
Salgo, M. G.; Stone, K.; Squadrito, G. L.; Battista, J. R.; Pryor, W. A.
Biochem. Biophys. Res. Commun. 1995, 210, 1025-1030. (c) Rubio, J.;
Yermilov, V.; Ohshima, H. In The Biology of Nitric Oxide; Moncada, S.,
Stamler, J., Gross, S., Higgs, E. A., Eds.; Portland Press Proceedings:
London, England, 1996; p 34. (d) Tamir, S.; Burney, S.; Tannenbaum, S.
R. Chem. Res. Toxicol. 1996, 9, 821-827.
(7) (a) Bisset, W. I. K.; Burdon, M. G.; Butler, A. R.; Glidewell, C.;
Reglinski, J. J. Chem. Res., Synop. 1981, 299. (b) Clarke, M. J.; Gaul, J.
B. Struct. Bonding 1993, 81, 147-181.
(8) Flitney, F. W.; Megson, I. L.; Flitney, D. E.; Butler, A. R. Br. J.
Pharmacol. 1992, 107, 842-848.
(9) (a) Makings, L. R.; Tsien, R. Y. J. Biol. Chem. 1994, 269, 6282-
6285. (b) Kwon, N. S.; Lee, S. H.; Choi, C. S.; Kho, T.; Lee, H. S. FASEB
J. 1994, 8, 529-533. (c) Singh, R. J.; Hogg, N.; Joseph, J.; Kalyanaraman,
B. FEBS Lett. 1995, 360, 47-51. (d) Namiki, S.; Kaneda, F.; Ikegami, M.;
Arai, T.; Fujimori, K.; Asada, S.; Hama, H.; Kasuya, Y.; Goto, K. Bioorg.
Med. Chem. 1999, 7, 1695-1702.
(10) Hou, Y.; Wang, J.; Andreana, P. R.; Cantauria, G.; Tarasia, S.; Sharp,
L.; Braunschweiger, P. G.; Wang, P. G. Bioorg. Med. Chem. Lett. 1999, 9,
2255-2258.
(11) Komarov, A.; Mattson, D.; Jones, M. M.; Singh, P. K.; Lai, C.-S.
Biochem. Biophys. Res. Commun. 1993, 195, 1191-1198.
10.1021/ja0109038 CCC: $20.00 © 2001 American Chemical Society
Published on Web 08/10/2001

Generation of Nitric Oxide from 6-Nitrobenzo[a]pyrene
J. Am. Chem. Soc., Vol. 123, No. 36, 2001 8663
Figure 1. Structures of 1-, 3- and 6-nitroBaPs.
Figure 3. ESR spectra of carboxyPTIO in the presence of 6-nitroBaP.
Samples contained 5 µM carboxyPTIO and 200 µM 6-nitroBaP in
phosphate buffer at pH 7.6 (containing 20% DMF); ESR spectra were
recorded with a modulation amplitude of 2.0 G after photoirradiation
for 0, 1, or 2 min.
Figure 2. ESR spectra of Fe-MGD in the presence of 1-, 3-, and
6-nitroBaPs. Samples contained 200 µM of 1-nitroBaP (A), 3-nitroBaP
(B), or 6-nitroBaP (C), 75 mM of MGD, and 15 mM of FeSO4 in
phosphate buffer at pH 7.2 (containing 20% DMF); ESR spectra were
recorded after photoirradiation for 1, 2, and 4 min (6-nitroBaP) or 2
min (1- and 3-nitroBaPs), with a modulation amplitude of 2.0 G.
line spectrum consisting of aN ) 1.25mT and giso ) 2.04, which
is characteristic of the [(MGD)2-Fe2+-NO] complex. An increase
in the signal intensity of the [(MGD)2-Fe2+-NO] adduct was
observed with an increase in the duration of irradiation,
indicating the photolytic generation of NO from 6-nitroBaP.
When similar experiments were performed with 1- and 3-ni-
troBaPs, no ESR signals were detected.
CarboxyPTIO was also used to detect NO12 to further confirm
the production of NO via the photolysis of 6-nitroBaP (Figure
3). The ESR signal of carboxyPTIO is characterized by five
lines (1:2:3:2:1, aN ) 0.82 mT). If NO is oxidized by
carboxyPTIO, the latter is transformed to carboxyPTI, which
has seven line signals on ESR (aN1 ) 0.92 mT, aN2 ) 0.54
mT). After photoirradiation of a mixture of 6-nitroBaP and
carboxyPTIO, a time-dependent decrease in signals for car-
boxyPTIO was observed, concomitant with an increase in signals
for carboxyPTI, indicating the production of NO from 6-ni-
troBaP. The transformation of carboxyPTIO to carboxyPTI was
not observed if a solution of carboxyPTIO was irradiated in
the presence of 1- or 3-nitroBaP (data not shown).
ESR spectroscopy was also used to assess the radical that
was produced as a result of the release of NO from photoirra-
diated 6-nitroBaP (Figure 4). Photoirradiation of 6-nitroBaP
resulted in the appearance of an ESR signal composed of five
lines (1:4:6:4:1), consistent with unpaired electrons interacting
with four protons, the amplitude of which increased with the
Figure 4. ESR spectra of 6-nitroBaP under photoirradiation: (A) 200
µM of 6-nitroBaP in phosphate buffer at pH 7.6 (containing 20% DMF)
was photoirradiated for 0, 0.5, 2, 4, 6, 8, 10, 12, or 16 min, with ESR
spectra at a modulation amplitude of 2.0 G were recorded under
photoirradiated conditions; (B) ESR spectra of 6-nitroBaP photoirra-
diated for 5 min recorded at a modulation amplitude of 0.2 G.
duration of irradiation. Each of the four lines was further
resolved into more protons when the spectrum was recorded at
a lower modulation amplitude, indicating interactions with
additional protons. Based on comparison of these signals with
those published in the literature,13,14 we unambiguously con-
cluded that the 6-oxyBaP radical is formed with the release of
NO upon photoirradiation of 6-nitroBaP.
Inomata and Nagata demonstrated that the 6-oxyBaP radical
produced from photoirradiated BaP afforded further oxidation,
resulting in the formation of BaP quinones.13 To investigate the
nature of the 6-oxyBaP radical formed upon irradiation of
6-nitroBaP, we monitored the photochemical reaction products
(12) Akaike, T.; Yoshida, M.; Miyamoto, Y.; Sato, K.; Kohno, M.;
Sasamoto, K.; Miyazaki, K.; Ueda, S.; Maeda, H. Biochemistry 1993, 32,
827-832.
(13) Inomata, M.; Nagata, C. Gann 1972, 63, 119-130.
(14) Lorentzen, R. J.; Lesko, S. A.; McDonald, K.; Ts’O, P. O.
Biochemistry 1975, 14, 3970-3977.

8664 J. Am. Chem. Soc., Vol. 123, No. 36, 2001
Fukuhara et al.
cause DNA strand breaks, 1-, 3-, and 6-nitroBaPs at a con-
centration of 50 µM were mixed with supercoiled plasmid
pBR322 and photoirradiated (Figure 6). When irradiation was
performed for 30 min, 6-nitroBaP effected the conversion of
Form I (supercoiled) DNA to Form II (relaxed circular) DNA.
Quantitative and significant conversion of Form I to Form II
DNA was observed with 6-nitroBaP by increasing the duration
of irradiation, while less-efficient DNA cleavage resulted when
1- and 3-nitroBaPs were used. The efficiency of 6-nitroBaP in
DNA cleavage depended on the duration of irradiation, and
6-nitroBaP failed to effect plasmid DNA relaxation when the
reaction was performed in the presence of carboxyPTIO to trap
photoreleased NO, suggesting that NO was responsible for the
observed DNA damage mediated by photoirradiated 6-nitroBaP
(see Supporting Information). In addition to efficient DNA
cleavage by 6-nitroBaP, Form I DNA showed reduced gel
mobility. No such shift resulted with 1- and 3-nitroBaPs, and
6-nitroBaP did not affect gel mobility without irradiation (see
Supporting Information). A shift in gel mobility is caused by
unwinding of the phosphodiester backbone to accommodate an
intercalater.17 Therefore, a slower band might be due to the
strong intercalation into supercoiled DNA of photoirradiated
products, BaP quinones, rather than 6-nitroBaP itself.
Figure 5. HPLC profile of the products formed from photoirradiated
6-nitroBaP. For analysis conditions, see the Experimental Section. The
peak at 4.5 min represents benzene as the solvent. The numbers above
the peaks correspond to (1) 1,6-BaPQ, (2) 3,6-BaPQ, (3) 6,12-BaPQ,
and (4) 6-OHBaP.
Discussion
The present study demonstrated that photoirradiated 6-ni-
troBaP is capable of giving rise to NO and causing DNA strand
scission. Photoirradiated production of NO is characteristic of
6-nitroBaP. No such irradiation-dependent release of NO and
induction of DNA cleavage was seen with 1- and 3-nitroBaPs.
In the photoirradiated production of NO by 6-nitroBaP, the
formation of 6-oxyBaP radical suggested that NO was generated
via 6-nitriteBaP, which was produced from 6-nitroBaP by an
intramolecular rearrangement mechanism (Figure 7). The mech-
anism, where 6-oxyBaP radical is directly derived from 6-ni-
triteBaP as an intermediate via homolytic cleavage of the N-O
bond, may also be evidenced by the photoinduced formation
of 6-OHBaP. In fact, the yield of 6-OHBaP was improved under
anaerobic conditions.
Semiempirical MO calculations were performed to assess
which characteristic of the structure is responsible for NO
generation (Table 2), where the plane of the nitro group was
perpendicular to the aromatic ring of 6-nitroBaP, while the
dihedral angle for 1- and 3-nitroBaPs was about 60°. This
suggests that the overlap of the half-vacant nonbonding orbital
of the nitro group at the 6-position with the adjacent orbital of
the aromatic ring increases susceptibility to intramolecular
rearrangement from the nitro to nitrite. In comparison, such
intramolecular rearrangement does not occur in the case of 1-
and 3-nitroBaPs, which do not generate NO, since the dihedral
angle between the plane of the nitro and aromatic ring is
unfavorable for the overlap of the orbitals in the excited state.
The differences (∆E) in the heat of formation between nitro
and nitrite were also estimated by semiempirical MO calculation.
The fact that 6-nitroBaP has the lowest ∆E among these
compounds provides further evidence that NO is generated via
a mechanism of nitro-to-nitrite rearrangement. NO production
has also been observed in the photochemical oxidation of
9-nitroanthracene.18 However, the ability to provide NO was
Table 1. Yields of the Products Formed from Photoirradiated
6-nitroBaP
products (%)
reaction
time, h
1,6-BQ 3,6-BQ 6,12-BQ 6-OHBQ 6-nitroBaP
1
4.6
9.8
1.4
3.0
8.4
0.8
7.1
16.8
2.8
6.7
10.6
14.5
74.0
46.9
70.6
2
1a
a Reaction was run under argon.
by HPLC. Irradiation of 6-nitroBaP solution for 1 h resulted in
four major peaks (Figure 5). The peaks on the HPLC profile
corresponded to 6-OHBaP (6.7% yield) and 1,6-, 3,6-, and
6,12-BaP quinones (4.6, 3.0, and 7.1% yield), respectively, the
yields of which increased with the duration of irradiation. When
the photolysis of 6-nitroBaP was performed under argon, a
decrease in the yield of BaP quinones and an increase in that
of 6-OHBaP were observed (Table 1), suggesting that BaP
quinones are formed as a result of the oxidation of 6-oxyBaP
radical by molecular oxygen. Although 6-OHBaP can be
oxidized to form BaP quinones, the rate of the reaction is
relatively slow under the conditions used for the photolysis of
6-nitroBaP (data not shown). Therefore, the major participation
of 6-OHBaP, derived from the 6-oxyBaP radical, in the
production of BaP quinones by photooxidation can be ruled out.
In the presence of oxygen, NO causes the deamination of
purines and pyrimidines and the oxidation of purines to give
several types of mutation.15 Deamination of bases by oxidized
NO also leads to the production of abasic sites and subsequent
single-strand breaks. Sugiura and Matsumoto investigated the
direct DNA damage caused by NO and demonstrated that NO
caused single-strand breaks with preferential cleavage at G.16
To determine if the photoinduction of NO from 6-nitroBaP can
(17) (a) Islam, S. A.; Neidle, S.; Gandecha, B. M.; Brown, J. R. Biochem.
Pharmacol. 1983, 32, 2801-2808. (b) Zeman, S. M.; Depew, K. N.;
Danishefsky, S. M.; Crothers, D. M. Proc. Natl. Acad. Sci. U.S.A. 1998,
95, 4327-4332.
(18) Chapman, O. L.; Heckert, D. C.; Reasoner, J. W.; Thackaberry, S.
P. J. Am. Chem. Soc. 1966, 88, 5550-5554.
(15) Caulfield, J. L.; Wishnok, J. S.; Tannenbaum, S. R. J. Biol. Chem.
1998, 273, 12689-12695.
(16) Sugiura, Y.; Matsumoto, T. Biochem. Biophys. Res. Commun. 1995,
211, 748-753.

Generation of Nitric Oxide from 6-Nitrobenzo[a]pyrene
J. Am. Chem. Soc., Vol. 123, No. 36, 2001 8665
Figure 6. Cleavage of supercoiled pBR322DNA by 1-, 3-, and 6-nitroBaPs upon photoirradiation.
Figure 7. Predicted pathway for the photochemical reaction of 6-nitroBaP with the generation of NO from the nitro group.
Table 2. Results of Semiempirical MO Calculations
mutagenic and carcinogenic compound, nitrated BaPs have not
been shown to exhibit biological activities by the same
mechanism as BaP.22 The electron deficiency of the BaP ring
due to the inducing effect of the nitro group hinders metabolic
oxidation, as in the case with BaP, whose oxidative metabolites
are responsible for mutagenicity and carcinogenicity. Therefore,
the finding that 6-nitroBaP is a photodependent DNA-damaging
agent may be useful for the development of a new type of NO
donor.
compds
dihedral anglea
∆Eb
nitrobenzene
9-nitroanthracene
1-nitroBaP
3-nitroBaP
6-nitroBaP
3
88
58
59
90
10.89
8.54
9.04
9.08
6.73
a Dihedral angle (deg) between the planes of the nitro group and
the aromatic moiety. b Difference (kcal mol-1) in heat of formation (HF)
between the nitro (R-NO2) and nitrite (R-ONO) forms: ∆E ) (HF of
R-ONO) - (HF of R-NO2).
Experimental Section
very much weaker than that with 6-nitroBaP(data not shown)
despite the same dihedral angle (88°). The smaller ∆E of
6-nitroBaP compared to that of 9-nitroanthracene might account
for the predominance of 6-nitroBaP for NO generation.
Materials. The 1-, 3-, and 6-nitroBaPs were synthesized by nitration
of BaP.23 MGD and carboxyPTIO were purchased from DOJINDO and
pBR322 DNA was purchased from Nippon Gene. All other chemicals
and solvents were reagent grade or better. In all experiments, irradiation
was performed through a Pyrex filter with a 300-W photoreflector lamp.
While both 1- and 3-nitroBaP have been shown to exhibit a
variety of biological activities, including bacterial and mam-
malian mutagenicity,19 and cell toxicity, no such activities have
been shown by 6-nitroBaP.20 Enzymatic reduction of the nitro
group to an activated form and subsequent DNA-adduct
formation is responsible for the mutagenicity of 1- and 3-ni-
troBaP. On the other hand, such reductive activation cannot
occur with 6-nitroBaP, since two peri protons at the 5- and
7-positions impede the access of reductase to 6-nitroBaP,21
suggesting that the nitro group at the 6-position of BaP is
insensitive to enzymatic reduction. Although BaP is a potent
ESR Analysis. The Fe2+ complex of MGD [Fe2+-MGD2, (Fe-MGD)]
was used to trap NO. Fresh stock solutions of Fe-MGD (1:5) were
prepared by adding ferrous ammonium sulfate to an aqueous solution
of MGD. CarboxyPTIO was also used for NO detection. A sample
containing 200 µM nitroBaP and 15 mM MGD-Fe or 5 µM carboxy-
PTIO in phosphate buffer at pH 7.6 (25% DMF) was introduced into
a quartz flat cell. ESR spectra were recorded after light irradiation at
a distance of 30 cm with a JES-FE 2XG spectrometer (JEOL Co. Ltd.,
Tokyo, Japan). To measure the 6-oxyBaP radical, photolysis of a sample
containing 200 µM 6-nitroBaP in phosphate buffer at pH 7.6 (25%
DMF) was performed by using light irradiation focused in a 1-cm-
diameter beam on the ESR flat cell that was placed in the cavity with
the front plate removed. The spectrometer settings were modulation
frequency, 100 kHz; modulation amplitude, 2G or 0.2G; scan time, 4
min; microwave power, 16 mW; and microwave frequency, 9.394 GHz.
(19) Pitts, J. N., Jr.; Zielinska, B.; Harger, W. P. Mut. Res. 1984, 140,
81-85.
(20) Chou, M. W.; Heflich, R. H.; Casciano, D. A.; Miller, D. W.;
Freeman, J. P.; Evans, F. E.; Fu, P. P. J. Med. Chem. 1984, 27, 1156-
1161.
(21) Pitts, J. N., Jr.; Lokensgard, D. M.; Harger, W.; Fisher, T. S.; Mejia,
V.; Schuler, J. J.; Scorziell, G. M.; Katzenstein, Y. A. Mut. Res. 1982,
103, 241-249.
(22) Fu, P. P. Drug Metab. ReV. 1990, 22, 209-268.
(23) Fukuhara, K.; Miyata, N.; Matsui, M.; Matsui, K.; Ishidate, M., Jr.;
Kamiya, S. Chem. Pharm. Bull. 1990, 38, 3158-3161.

8666 J. Am. Chem. Soc., Vol. 123, No. 36, 2001
Fukuhara et al.
Analysis of Photoirradiation Products. To analyze photoirradiation
products of 6-nitroBaP, 10 mM 6-nitroBaP in 10 mL of benzene was
irradiated (at a distance of 20 cm) at 37 °C. The progress of the reaction
was monitored by HPLC on a Shimadzu HPLC system. Aliquots of
the reaction mixture were loaded onto a Senshu Pegasil C18 column
(5µ; 100 × 4.6), eluted at 1 mL/min with a linear gradient from 30:70
(vol/vol) 0.1 M NH4HCO3 buffer at pH 7.0:methanol to 100% methanol
over 30 min. After irradiation for 2 h, the reaction mixture was
concentrated by rotary evaporation and purified by semipreparative
HPLC on a Wakosil-II 5C18 AR (5 µm; 300 × 10) with the same
gradient and solvents that were used to monitor the reaction and a flow
rate of 10 mL/min. The product fractions corresponding to peaks for
1,6-, 3,6-, and 6,12-benzo[a]pyrenequinone (BaPQ) and 6-OH-BaP were
collected, concentrated by rotary evaporation, and dried in vacuo. 1,6-
of 1,6-, 3,6-, and 6,12-BaPQ and 6-OH-BaP were also confirmed by
comparison with authentic standards, synthesized according to reported
procedures.24
Assays for DNA Strand Breaks. DNA strand breakage was
measured by the conversion of supercoiled pBR322 plasmid DNA to
the open circular form. Reactions were carried out in 20 µL (total
volume) of 50 mM Na cacodylate buffer (20% DMF), pH 7.2,
containing 45 µM pBR322 DNA and 50 µM nitroBaPs. The reaction
mixtures were irradiated (at a distance of 30 cm) at 0 °C for an
appropriate time and then treated with 5 µL of loading buffer (100
mM TBE buffer, pH 8.3, containing 30% glycerol, 0.1% bromophenol
blue) and applied to 1% agarose gel. Horizontal gel electrophoresis
was carried out in 50 mM TBE buffer, pH 8.3, and the gels were stained
with ethidium bromide (1 µg/mL) for 30 min, followed by destaining
in water for 30 min and photography with UV translumination.
Semiempirical MO Calculations. Semiempirical MO calculations
were performed with SPARTAN (ver. 3.1, Wavefunction Inc.). All
structures were fully optimized at the restricted Hartree-Fock level
with the PM3 method.
1
BaPQ: 9.8% yield; HNMR (CDCl3) δ 6.77 (d, J ) 10.0, H2), 7.63
(m, H8), 7.73 (d, J ) 10.0, H3), 7.79 (m, H9), 7.90 (d, J ) 7.2, H4),
8.34 (d, J ) 8.1, H10), 8.46 (d, J ) 7.6, H7), 8.56 (d, J ) 7.8, H11),
8.63 (d, J ) 7.8, H12), 8.67 (d, J ) 7.2, H5). 3,6-BaPQ: 8.4% yield;
1HNMR (CDCl3) δ 6.74 (d, J ) 9.7, H2), 7.62 (m, H8), 7.76 (d, J )
9.7, H1), 7.79 (m, H9), 7.83 (d, J ) 7.6, H12), 8.31 (d, J ) 7.8, H10),
8.41 (d, J ) 7.6, H11), 8.48 (d, J ) 7.8, H7), 8.76 (d, J ) 7.4, H5), 8.86
(d, J ) 7.4, H4). 6,12-BaPQ: 16.8% yield; 1HNMR (CDCl3) δ 7.59 (s,
H11), 7.69 (m, H8), 7.79 (m, H9), 7.86 (dd, J ) 7.1 and 8.4, H2), 8.18
(d, J ) 8.5, H4), 8.21 (d, J ) 8.2, H10), 8.24 (d, J ) 8.4, H3), 8.46 (d,
J ) 7.4, H7), 8.48 (d, J ) 8.5, H5), 8.64 (d, J ) 7.1, H1). 6-OH-BaP:
10.6% yield; 1HNMR (CDCl3) δ 7.80 (m, H9), 7.82 (m, H8), 7.92 and
7.93 (d, J ) 9.6, H4 and H5), 7.95 (dd, J ) 7.6 and 8.8, H2), 8.05 (d,
J ) 7.6, H3), 8.20 (d, J ) 8.8, H1), 8.23 (d, J ) 8.8, H7), 8.25 (d, J )
9.0, H12), 8.95 (d, J ) 9.2, H11), 9.02 (d, J ) 7.2, H10). The structures
Supporting Information Available: Figure 1s showing the
effect of carboxyPTIO on photoirradiated reaction, and Figure
2s showing the effect of nitroBaPs on pBR322DNA in the dark
(PDF). This material is available free of charge via the Internet
at http://pubs.acs.org.
JA0109038
(24) (a) Fieser, L. F.; Hershberg, E. B. J. Am. Chem. Soc. 1938, 60,
2542-2548. (b) Lee-Ruff, E.; Kazarians-Moghaddam, H.; Katz, M. Can.
J. Chem. 1986, 64, 1297-1303.