Mechanisms for the formation of major oxidation products of adenine upon 365-nm irradiation with 2-methyl-1,4-naphthoquinone as a sensitizer.

Article abstract of DOI:10.1021/jo0264170

Recently we reported the isolation and characterization of N6-formyl- and N6-acetyladenine from 365-nm irradiation of dinucleoside monophosphates d(ApA), d(ApC), and d(CpA) in the presence of 2-methyl-1,4-naphthoquinone (menadione) (Wang et al. Biochem. B

Full text of DOI:10.1021/jo0264170

Mech a n ism s for th e F or m a tion of Ma jor Oxid a tion P r od u cts of
Ad en in e u p on 365-n m Ir r a d ia tion w ith
2
-Meth yl-1,4-n a p h th oqu in on e a s a Sen sitizer
Yinsheng Wang* and Zhenjiu Liu
Department of Chemistry-027, University of California at Riverside, Riverside, California 92521-0403
yinsheng.wang@ucr.edu
Received September 7, 2002
6
6
Recently we reported the isolation and characterization of N -formyl- and N -acetyladenine from
65-nm irradiation of dinucleoside monophosphates d(ApA), d(ApC), and d(CpA) in the presence
of 2-methyl-1,4-naphthoquinone (menadione) (Wang et al. Biochem. Biophys. Res. Commun. 2002,
91, 1252-7). In this article we investigated the mechanisms for the formation of the two major
products by carrying out photoirradiation with isotopically labeled menadione and 2,3-dimethyl-
,4-naphthoquinone. HPLC and electrospray ionization (ESI)-mass spectrometry (MS) and tandem
3
2
1
MS studies of the products unambiguously established that the carbonyl group in the products
6
arises from the photosensitizer: The N -formyl group comes from oxidation of the methyl group
6
and the N -acetyl group stems from the methyl group and the adjacent ring carbon in menadione.
From above results, we proposed mechanisms for the formation of the two products.
In tr od u ction
MQ-induced damage of nucleobases, nucleosides, and
1
5-20
short oligonucleotides has been investigated.
Al-
Nucleic acid damage induced by endogenous or exog-
enous reactive oxygen species (ROS) is an important
pathway that contributes to a number of pathological
conditions including cancer, neurological diseases, and
aging.1 Solar irradiation is a common exogenous ROS
source, among which UV irradiation is the most harmful
and mutagenic component.7,8 DNA bases can directly
absorb incident UVB light (280-320 nm); on the other
hand, UVA light (320-400 nm) can be absorbed by
endogenous photosensitizers which can damage DNA
through photooxidation. The latter process can occur
though, among the four DNA bases, guanine was shown
to be the easiest to be oxidized by MQ photosensitization
in a mixture of four mononucleosides and in isolated
21
DNA, oxidation of other DNA bases is also important.
-6
17
In this respect Melvin and co-workers showed that
photoirradiation in the presence of MQ can give rise to
the molecular weight increase of single-stranded poly-
(dA). The origin of the molecular weight increase, how-
ever, has not been determined.
Recently we reported the isolation and characterization
6
of two major oxidation products of adenine, N -formyl-
9
through a type I and/or type II mechanism. The type I
6
and N -acetyladenine (Scheme 1), in dinucleoside mono-
mechanism involves either initial electron or hydrogen
abstraction by an excited-state photosensitizer. The type
II mechanism, however, occurs through the reaction of
phosphates d(ApA), d(ApC), and d(CpA).22 This is a
1
9
(10) Fisher, G. J .; Land, E. J . Photochem. Photobiol. 1983, 37, 27-
2.
2
guanine with singlet oxygen ( O ).
3
Some quinone species can damage DNA by the above
photooxidation mechanisms. Among them 2-methyl-1,4-
naphthoquinone (MQ), or menadione, belongs to the
(11) Wagner, J . R.; Cadet, J .; Fisher, G. J . Photochem. Photobiol.
984, 40, 589-97.
12) Krishna, C. M.; Decarroz, C.; Wagner, J . R.; Cadet, J .; Riesz,
1
(
P. Photochem. Photobiol. 1987, 46, 175-82.
(13) Wagner, J . R.; van Lier, J . E.; J ohnston, L. J . Photochem.
Photobiol. 1990, 52, 333-43.
vitamin K family (vitamin K
3
). The triplet state of MQ
has been shown to be an effective electron acceptor.1
0-14
(14) Koch, T.; Ropp, J . D.; Sligar, S. G.; Schuster, G. B. Photochem.
Photobiol. 1993, 58, 554-8.
*
Address correspondence to this author. Fax: (909)787-4713.
(15) Decarroz, C.; Wagner, J . R.; van Lier, J . E.; Krishna, C. M.;
Riesz, P.; Cadet, J . Int. J . Radiat. Biol. 1986, 50, 491-505.
(16) Wagner, J . R.; van Lier, J . E.; Berger, M.; Cadet, J . J . Am.
Chem. Soc. 1994, 116, 2235-42.
Phone: (909)787-2700.
(
(
1) Lindahl, T. Nature 1993, 362, 709-15.
2) Feig, D. I.; Reid, T. M.; Loeb, L. A. Cancer Res. 1994, 54, 1890s-
1
2
894s.
(17) Melvin, T.; Bothe, E.; Schulte-Frohlinde, D. Photochem. Pho-
tobiol. 1996, 64, 769-76.
(
3) Lindahl, T. NATO ASI Ser., Ser. A 1999, 302, 251-257.
(4) Rolig, R. L.; McKinnon, P. J . Trends Neurosci. 2000, 23, 417-
(18) Bienvenu, C.; Wagner, J . R.; Cadet, J . J . Am. Chem. Soc. 1996,
118, 11406-11411.
4.
(
(
(
5) Marnett, L. J . Carcinogenesis 2000, 21, 361-70.
6) Finkel, T.; Holbrook, N. J . Nature 2000, 408, 239-47.
7) Cadet, J .; Vigny, P. In Bioorganic Photochemistry; Morrison, H.,
(19) Cadet, J .; Berger, M.; Douki, T.; Ravanat, J . L. Rev. Physiol.
Biochem. Pharmacol. 1997, 131, 1-87.
(20) Delatour, T.; Douki, T.; Gasparutto, D.; Brochier, M.-C.; Cadet,
J . Chem. Res. Toxicol. 1998, 11, 1005-1013.
Ed.; J ohn Wiley: New York, 1990; Vol. 1, pp 1-272.
8) Coohill, T. P.; Peak, M. J .; Peak, J . G. Photochem. Photobiol.
987, 46, 1043-50.
9) Foote, C. S. Photochem. Photobiol. 1991, 54, 659.
(
(21) Douki, T.; Cadet, J . Int. J . Radiat. Biol. 1999, 75, 571-81.
(22) Wang, Y.; Liu, Z.; Dixon, C. Biochem. Biophys. Res. Commun.
2002, 291, 1252-7.
1
(
1
0.1021/jo0264170 CCC: $22.00 © 2002 American Chemical Society
Published on Web 11/05/2002
J . Org. Chem. 2002, 67, 8507-8512
8507

Wang and Liu
SCHEME 1
continuous study with aims at determining the origins
of the formyl and acetyl groups and rationalizing the
mechanisms for the formation of the two products. To
this end, we synthesized menadione and 2,3-dimethyl-
1
,4-naphthoquinone whose methyl group is labeled with
1
3
a stable isotope, C or D. We will demonstrate that the
formyl group arises from the oxidation of the methyl
group and the acetyl group originates from the methyl
group and the adjacent ring carbon in menadione. We
will also propose tentative mechanisms for the formation
of the two major products.
Resu lts a n d Discu ssion
Effect of Oxygen on th e F or m a tion of th e Acety-
la ted a n d F or m yla ted P r od u cts. As we reported
previously,22 the acetylation and formylation were found
to be major reactions of adenine while dinucleoside
monophosphates d(ApA), d(CpA), and d(ApC) were ir-
radiated with 365-nm light in the presence of 2-methyl-
F IGURE 1. HPLC traces for the separation of menadione-
sensitized irradiation of d(ApC) under deoxygenated (a),
control (b), and oxygenated solutions (c). SM is the starting
material d(ApC). Ac-d(ApC) and Fo-d(ApC) are the acetylated
and formylated products of d(ApC), respectively.
1
,4-naphthoquinone. We did the MQ-sensitized photoir-
SCHEME 2
radiation of d(ApC) for a time period varying from 1 min
to 2 h, and HPLC analysis showed that the yield of the
acetylated product increases with time and remains
constant when the irradiation time is longer than 30 min.
6
This result may suggest that the N -acetyladenine prod-
uct in d(ApC) can further react with the sensitizer.
However, we did not identify any new product even from
the 2-h irradiation mixture. Moreover, the acetylated
product was the major product formed independent of the
irradiation time (data not shown).
The formation of those two products appears unusual,
and it is important to investigate the mechanisms for
their formation, for which we need to determine the
importance of oxygen in the reaction and to investigate
the origins of the acetyl and formyl groups. To this end,
we carried out photoirradiations under three otherwise
identical conditions except that the amounts of oxygen
present are different (details shown in Experimental
Section). Indeed HPLC and ESI-MS studies show that
the amount of oxygen in solution plays a significant role
in the formation of the two products (HPLC traces shown
in Figure 1). Under deoxygenated condition, neither the
acetylated nor the formylated product was produced
of the two major products. Similarly, Wagner and co-
workers13 found that the presence of oxygen is important
for the menadione-sensitized photooxidation of thymi-
dine. The authors attributed the effect to that the
molecular oxygen is an electron quencher and it prevents
back electron transfer from the radical anion of mena-
dione to the radical cation of thymidine.13
P h otor ea ction s of d (Ap C) w ith Differ en t Isotop e-
La beled MQ. To determine the origin of the carbonyl
3
group in the products, we synthesized 2-methyl-d -1,4-
1
3
(Figure 1a). Furthermore, the yields for the products are
naphthoquinone (1) and 2-methyl- C-1,4-naphthoquino-
ne (2) (structures shown in Scheme 2) following the same
procedure as reported in the literature.23 In our hands,
however, we were unable to obtain the pure labeled
menadione via recrystallization. Instead we used reverse-
phase HPLC separation to obtain the pure product. Using
higher for the reaction with air-bubbling (“oxygenated
condition”) than the control experiment where the solu-
tion was prepared and irradiated without air or Ar
bubbling. The ratio of the peak area of the acetylated
product to that of the starting material d(ApC) increased
from 11% in the control experiment to 27% in the
experiment with air-bubbling (Figure 1b,c). The results
clearly show that oxygen is necessary for the formation
(23) Fauler, G.; Leis, H. J .; Schalamon, J .; Muntean, W.; Gleispach,
H. J . Mass Spectrom. 1996, 31, 655-60.
8
508 J . Org. Chem., Vol. 67, No. 24, 2002

Formation of Major Oxidation Products of Adenine
F IGURE 2. ESI-MS (a) and product-ion spectrum for the [M
-
-
2
H] ion (b) of the acetylated product of d(ApC) when
3
-methyl-d -1,4-naphthoquinone (1) was used as photosensi-
tizer.
a 40:60 (by volume) mixture of acetonitrile and water as
the mobile phase, we were able to separate the three
components, 1,4-naphthoquinone, menadione, and 2,3-
dimethyl-1,4-naphthoquinone, from each other (chro-
matograms shown in the Supporting Information). From
the HPLC peak areas with absorbance at 350 nm and
by assuming that the three components have similar
extinction coefficients at 350 nm, we estimated that the
ratio for the unreacted 1,4-naphthoquinone, 2-methyl-
F IGURE 3. ESI-MS and MS/MS of the [M - H]- ions of the
acetylated and formylated products of d(ApC) when 2-methyl-
1
3
C-1,4-naphthoquinone (2) was used as photosensitizer: (a)
-
ESI-MS of the acetylated product, (b) MS/MS of the [M - H]
ion of the acetylated product, (c) ESI-MS of the formylated
product, and (d) MS/MS of the [M - H] ion of the formylated
-
product.
3 3
d -1,4-naphthoquinone (1), and 2,3-dimethyl-2-d -1,4-
naphthoquinone (4) is approximately 20:60:20. Similarly,
ion of m/z 568 (Figure 3c). Upon collisional activation, a
fragment ion of m/z 163, which is the anion of N -
6
we separated the three components from the reaction
formyladenine, was observed in the product-ion spectrum
1
3
mixture of 1,4-naphthoquinone and CH
3
COOH.
6
(
Figure 3d). The formylated product and the N -formyl-
The irradiation products from use of 1 and 2 as
sensitizers were again separated by HPLC and charac-
terized by ESI-MS and MS/MS and the identities of the
products established the origins of carbonyl groups.
Negative-ion ESI-MS (Figure 2a) for the acetylated
product from the photoirradiation with 1 as sensitizer
shows an ion of m/z 584, which is 3 mass units higher
than the corresponding acetylated product isolated from
photoirradiation with the unlabeled photosensitizer. The
result indicates that the acetylated product carries the
adenine fragment are one mass unit higher than the
corresponding ions observed in the control experiment
where unlabeled menadione is used as sensitizer. We,
therefore, establish unambiguously that the carbonyl
group arises from the oxidation of the methyl group in
13
menadione because the formyl group carries the C label.
Similarly, an ion of m/z 582 was observed in negative-
ion ESI-MS for the acetylated product (Figure 3a). In
addition, an ion of m/z 177, which is the anion of
6
N -acetyladenine, was observed in the MS/MS of the m/z
3
D -label. In addition, ions of m/z 179 and 135 were
5
82 ion (Figure 3b), indicating that the acetylated product
observed in the MS/MS of m/z 584 ion (Figure 2b), those
1
3
1
13
6
contains the C label. Furthermore, H and C NMR
spectra of the acetylated product demonstrate that the
two product ions are the anions of N -acetyladenine and
adenine, respectively. It is very likely that a deuterium
1
3
methyl carbon in the acetyl group carries the C label.
transfers from the methyl group to adenine upon the loss
1
6
H NMR shows that the spectrum is very similar to that
of ketene (CD
2
CO) moiety from the N -acetyladenine,
of the unlabeled acetylated product except that the
which results in an anion of m/z 135 for adenine. The
6
methyl proton resonance at δ 2.4 is split into two peaks,
above results show clearly that the methyl group in N -
1
3
1
which is due to a one-bond coupling between C and H
acetyladenine arises from the methyl group in menadi-
one. Interestingly, no formylated product was detected
in the photoreaction of d(ApC) with 1.
(
J ) 129 Hz, shown in the Supporting Information). In
1
3
addition, C NMR shows a single resonance at δ 24,
which is also in line with the presence of C in the
1
3
6
To pinpoint the origin of the carbonyl group in N -
methyl group of the acetyl functionality.
formyladenine and to gain further evidence for the origin
6
of carbonyl groups in the N -acetyladenine, we used
To summarize, we have demonstrated that both the
1
3
6
2
-methyl- C-1,4-naphthoquinone (2) as a sensitizer for
formyl group in N -formyladenine and the methyl group
6
the photoreaction. Our results show that the formylated
product formed and the negative-ion ESI-MS showed an
in N -acetyladenine originate from the methyl group in
menadione. The results also indicate that the carbonyl
J . Org. Chem, Vol. 67, No. 24, 2002 8509

Wang and Liu
F IGURE 5. Negative-ion ESI-MS of the acetylated and
formylated products of adenine in d(ApC) while 2,3-dimethyl-
1
3
2
3
-d -1,4-naphthoquinone (4) and 2,3-dimethyl-2- C-1,4-naph-
F IGURE 4. HPLC traces for the separation of 365-nm
irradiation of d(ApC) photosensitized with 2,3-dimethyl-1,4-
thoquinone (5) were used as photosensitizers. (a, b) MS for
the acetylated and formylated product while 4 was used and
(c, d) MS for the acetylated and formylated product while 5
was used.
3
naphthoquinone (a), 2,3-dimethyl-2-d -1,4-naphthoquinone (b),
1
3
and 2,3-dimethyl-2- C-1,4-naphthoquinone (c).
6
in N -acetyladenine is probably from the oxidation of C2
in menadione.
P h otor ea ction s of d (Ap C) w ith 2,3-Dim eth yl-1,4-
(
Figure 5a,b). The result is consistent with that obtained
from the photoreaction with 2-methyl-d -1,4-naphtho-
3
quinone (1), indicating the isotope effect for the formation
of the formylated product. When 5 was used as the
photosensitizer, acetylated and formylated products were
obtained in both unlabeled and C-labeled forms (Figure
5
product in each panel of Figure 4 is also present in the
control photoirradiation of 3 by itself but absent in the
control HPLC separation of 3 without photoirradiation
3
n a p h th oqu in on e (3), 2,3-Dim eth yl-2-d -1,4-n a p h th o-
1
3
qu in on e (4), a n d 2,3-Dim eth yl-2- C-1,4-n a p h th o-
q u in on e (5). The fact that 2,3-dimethyl-1,4-naphtho-
quinone is present in the product mixture from the
reaction of 1,4-naphthoquinone with acetic acid indicates
that methylation of menadione is also an efficient process
under the reaction condition. This result motivates us
13
c,d). The HPLC peak immediately before the formylated
to prepare the 2,3-dimethyl-1,4-naphthoquinone with 13
or D or without isotope label in one of the methyl groups
3-5, Scheme 2). It turns out that we can readily obtain
C
(data shown in the Supporting Information). Therefore,
it is a photoinduced decomposition product of 2,3-dim-
ethyl-1,4-naphthoquinone whose structure, however, re-
mains unknown.
(
pure 2,3-dimethyl-1,4-naphthoquinones (HPLC traces for
the separation of the reaction mixtures are shown in the
Supporting Information).
P r op osed Mech a n ism for th e P r od u ct F or m a tion .
2
From the results of the O -dependent photoirradiation
To obtain additional evidence regarding the origins of
the formyl and acetyl groups, we carried out experiments
with those 2,3-dimethyl-1,4-naphthoquinones (3-5) as
photosensitizers. Interestingly, the presence of an ad-
ditional methyl group drastically changes the product
and the studies with different stable-isotope incorporated
menadiones (1 and 2) and 2,3-dimethyl-1,4-naphtho-
quinones (3-5) (results were summarized in Table 1),
6
we proposed mechanisms for the formation of the N -
6
acetyl- and N -formyladenine (Schemes 3 and 4). In both
6
6
distribution. Although the yield for the formation of N -
mechanisms, the formation of the N radical of adenine
acetyladenine is not significantly different from the
experiment when menadione was used as sensitizer, that
of N -formyladenine is much higher when 2,3-dimethyl-
is an important step. One-electron photooxidation of
adenine gives rise to its cation radical,1 which is known
7,24
6
to be very acidic (pK < 1) and deprotonates readily to
a
6
24
1
,4-naphthoquinones were used as sensitizers (Figure 4
form the N -centered adenine radical.
For the formation of N -formyladenine, menadione
tautomerizes to form a quinone methide, which can
react with the N -centered adenine radical (Scheme 3
6
shows the HPLC traces for the separation of the pho-
toirradiation mixtures). For the photosensitized reaction
2
5
6
with 4, both D
3
-labeled (m/z 584) and unlabeled (m/z 581)
acetylated products were obtained, whereas only the
unlabeled (m/z 567) formylated product was observed
(24) Steenken, S. Chem. Rev. 1989, 89, 503-20.
8
510 J . Org. Chem., Vol. 67, No. 24, 2002

Formation of Major Oxidation Products of Adenine
TABLE 1. Su m m a r y for th e P h otosen sitiza tion Rea ction s w ith Differ en t Isotop e-La beled P h otosen sitizer s
SCHEME 3
SCHEME 4
centered radical. Similarly, the latter radical can combine
with molecular oxygen to form a peroxyl radical, which
can again be reduced to form a hydroperoxide. The
resultant hydroperoxide can follow a familiar R-cleavage
to give the acetylated product (Scheme 4).
From the proposed mechanism in Scheme 4, it appears
that both path A and path B, which lead to the formation
of the acetylated and formylated products, are feasible.
A closer look, however, reveals that path A should be a
more facile process than path B. While the ring radical
is rearranged to form the exocyclic radical, a secondary
radical is changed to a tertiary one in path A, which is
energetically favorable. However, it is the other way
around in path B.
shows the reaction with unlabeled menadione as an
example). After hydrogen rearrangement, the resulting
carbon-centered radical can combine with molecular
oxygen to give a peroxyl radical. The peroxyl radical is
then reduced to a hydroperoxide, from which the formy-
lated product can form via R-cleavage. The R-cleavage
has been observed for alkyl hydroperoxy ketones,2
6-28
and
it has also been found in the decomposition of 5-hydro-
peroxy-6-hydroxy-5,6-dihydrothymidine.16
The argument is also consistent with the relative yields
6
6
Regarding the proposed mechanism, o-quinone methide
has been observed in the photolysis of vitamin K1, which
has a similar structure as menadione.29 Likewise, pho-
tolysis (254, 266, or 308 nm) of pyridoxine (vitamin B6)
gives o-quinone methide.30 The formation of a peroxyl
radical through the combination of the 6-hydroxy-5-yl
radical of thymidine with molecular oxygen was also
proposed for the degradation of thymidine under similar
irradiation conditions.13 From the proposed mechanism,
we expect that the formation of o-quinone methide and
the following hydrogen transfer steps have the primary
isotope effect, which accounts for the absence of the
for the N -acetyladenine and N -formyladenine when
menadione and 2,3-dimethyl-1,4-naphthoquinone are
photosensitizers. While the former is used as a sensitizer,
the yield for the acetylated product is much higher than
that for the formylated product (Figure 1), whereas the
relative yields are reversed while the latter is used as a
sensitizer (Figure 4). We expect that acetylation and
formylation compete against each other. For the forma-
tion of the acetylated product, a tertiary radical is
changed to another tertiary one while 2,3-dimethyl-1,4-
naphthoquinone is the sensitizer. However, as stated
previously, a secondary radical is changed to a tertiary
one while 2-methyl-1,4-naphthoquinone is the sensitizer.
Therefore, we anticipate that the formation of the acety-
lated product is a more facile process for photosensiti-
zation with menadione than that with 2,3-dimethyl-1,4-
naphthoquinone. Collectively, our proposed mechanisms
are consistent with all the experimental results that we
have obtained.
3
formylated product from the oxidation of the CD group
when 1 and 3 are used as photosensitizers (Table 1).
6
For the formation of N -acetyladenine, we propose that
6
the N -centered radical attacks C2 to give a C3-centered
radical, which can rearrange to form an exocyclic carbon-
(
25) Chatterjee, M.; Rokita, S. E. J . Am. Chem. Soc. 1994, 116,
690-7.
26) Richardson, W. H.; Ranney, G.; Montgomery, F. C. J . Am. Chem.
1
(
Con clu sion s
Soc. 1974, 96, 4688-9.
(
0.
(
(
(
27) Sawaki, Y.; Foote, C. S. J . Am. Chem. Soc. 1983, 105, 5035-
4
4
Upon 365-nm irradiation with menadione or 2,3-
dimethyl-1,4-naphthoquinone as a photosensitizer, ad-
enine in dinucleoside monophosphate d(ApC) undergoes
28) Sawaki, Y.; Ogata, Y. J . Am. Chem. Soc. 1977, 99, 5412-16.
29) Leary, G.; Porter, G. J . Chem. Soc. A 1970, 2273-8.
30) Brousmiche, D.; Wan, P. Chem. Commun. (Cambridge) 1998,
6
6
91-492.
reaction to form N -acetyl- and N -formyladenines. Pho-
J . Org. Chem, Vol. 67, No. 24, 2002 8511

Wang and Liu
toirradiation in the presence of different amounts of
oxygen shows that oxygen is required for the formation
of those two products.
185.1. EI-MS (positive-ion): m/z 189, 161, 132, 121, 104, 76,
5
0.
2
,3-Dim eth yl-2- C-1,4-n a p h th oqu in on e (5). 1H NMR
13
1
1
3
(300 MHz, CDCl
3
): δ 2.19 (s, 3H), 2.19 (d, 3H, J C-H ) 129.2,
), 7.70 (m, 2H), 8.09 (m, 2H). C NMR (75.5 MHz,
): δ 12.9. EI-MS (positive-ion): m/z 187, 159, 158, 130,
Photoirradiations with 2-methyl- C-1,4-naphthoquino-
ne (1) and 2-methyl-d -1,4-naphthoquinone (2) as sensi-
1
3
13
CH
CDCl
16, 104, 76, 50.
P h otosen sitized Rea ction s. Dinucleoside monophosphate
3
3
3
tizers show that the formyl group in the product arises
from the methyl group in the sensitizer and the perdeu-
terated methyl group in the sensitizer does not give rise
to the formylated product. The results also show that the
1
d(ApC) (300 nmol) was dissolved in a 10-mL aqueous solution
that was saturated with the photosensitizer (approximately
2
1
6
0.3 mM as determined by UV absorbance at 350 nm ). The
solution was then transferred to a 10.2-cm i.d. Petri dish,
irradiated on ice for 1 h under air with two 15-W Spectroline
light tubes emitting at 365 nm (Spectronics Corporation,
Westbury, NY), and dried by using a speed-vac. The dried
residue was redissolved in water and subjected to HPLC
analysis.
methyl in N -acetyladenine originates from the methyl
group in menadione, which suggests that the carbonyl
in the acetyl group is very likely from the oxidation of
C2 in menadione. Photoirradiation with dimethylated
naphthoquinones (3-5) gave similar results. On the basis
of the above observations and the known acidity of
adenine cation radical,24 we proposed reaction mecha-
nisms for the formation of those two types of products.
Photoirradiation experiments with various amounts of
oxygen were carried out under three different conditions.
Under the deoxygenated condition, the solution was degassed
by bubbling the solution with Ar for 1 h, transferred to a Petri
dish, inserted into an Ar-filled zip-lock bag, and sealed. In the
control experiment, the Petri dish with the sample solution
was inserted into a similar zip-lock bag that was open to air.
Under oxygenated condition, the sample solution was again
dispersed in a Petri dish and inserted into a zip-lock bag, but
the solution was continuously bubbled with air during irradia-
tion. The photoirradiation was otherwise under the same
condition as stated above.
Exp er im en ta l Section
Spin multiplicities for proton NMR spectra are given as s
(
singlet), d (doublet), dd (doublet of doublet), dq (doublet of
quartet), or m (multiplet). Coupling constants are given in Hz.
-Meth yl-d -1,4-n a p h th oqu in on e (Sch em e 2, 1). We
followed the procedure reported by Fauler and co-workers.
Glacial acetic-d acid-d (99.9% D, 1 mL), 1,4-naphthoquinone
2 g), and silver nitrate (AgNO , 1.28 g) were dissolved in 120
2
3
2
3
3
(
3
HP LC a n d Ma ss Sp ectr om etr y. The HPLC separation
was performed on a system with a photodiode array detector,
and a 4.6 × 250 mm reverse-phase C18 column (5 µm in
particle size, and 300 Å in pore size) was used. The flow rate
was 1.0 mL/min. An isocratic elution with 40:60 acetonitrile/
water (by volume) and a gradient of 35 min 6-12% acetonitrile
in 50 mM triethylammonium acetate (pH 6.8) were employed
for the separation of the synthetic mixtures of isotopically
labeled menadiones and the photoirradiation products, respec-
tively.
mL of a solvent mixture of acetonitrile and water at a volume
ratio of 2:1. Ammonium persulfate (3.8 g), dissolved in 50 mL
of water, was added gradually to the stirred solution in 15 min.
After the mixture was incubated at 80 °C for 5 h and at room
temperature overnight, 200 mL of water was added and the
solution was extracted twice with 50 mL of dichloromethane.
The organic layer was washed twice with 100 mL of water and
dried with sodium sulfate. After filtration, the solvent was
distilled off under reduced pressure. The oily product was
purified by reverse-phase HPLC as detailed in the HPLC
1
Electrospray ionization (ESI)-mass spectrometry (MS) and
tandem MS (MS/MS) experiments were carried out on an LCQ
Deca XP ion-trap mass spectrometer (ThermoFinnigan, San
J ose, CA). An equal-volume solvent mixture of acetonitrile and
water was used as the carrier and electrospray solvent, and a
1-µL aliquot of a 5 µM sample solution was injected in each
run. The spray voltage was 4.0 kV, and the capillary temper-
ature was maintained at 200 °C. MS/MS was done by selecting
section. H NMR (300 MHz, CDCl
2
1
3
): δ 6.85 (s, 1H), 7.74 (m,
): δ 126.4, 126.8,
32.4, 132.5, 133.9, 134.9, 136.0, 185.3, 185.9. EI-MS (positive-
1
3
H), 8.08 (m, 2H). C NMR (75.5 MHz, CDCl
3
ion): m/z 175, 147, 118, 104, 76, 50.
1
3
2
-Meth yl- C-1,4-n a p h th oqu in on e (2). The procedure was
1
3
13
the same as above except that acetic-2- C acid (99% C, 0.25
g) was used instead of acetic-d acid-d (1 g), and the quantities
of other reagents were adjusted accordingly. H NMR (300
3
1
-
1
4
the [M - H] ions for collisional activation. The mass width
MHz, CDCl
CH
3
): δ 2.20 (dd, 3H, J C-H ) 129.2, J H-H ) 1.6,
1
3
4
3
for precursor selection was 1 m/z unit and the collision gas
was helium. Each spectrum was obtained by averaging ap-
proximately 50 scans, and the time for each scan was 0.1 s.
3
), 6.84 (dq, 1H, J H-H ) 1.6, J C-H ) 5.9, H
3
), 7.74 (m,
): δ 13.2.
1
3
2
H), 8.09 (m, 2H). C NMR (75.5 MHz, CDCl
3
EI-MS (positive-ion): m/z 173, 145, 117, 116, 104, 76, 50.
The procedure for the synthesis of 2,3-dimethyl-1,4-naph-
thoquinones was the same as that for the synthesis of
Ack n ow led gm en t. The authors acknowledge the
NIH (grant no. CA 96909) and the University of
California at Riverside for supporting this research.
Y.W. also acknowledges Professors William H. Oka-
mura, Thomas H. Morton, and J ohn-Stephen A. Taylor
for very helpful discussions.
2
-methyl-d
3
-1,4-naphthoquinone except that the starting ma-
terial was 2-methyl-1,4-naphthoquinone instead of 1,4-naph-
1
3
thoquinone, and acetic acid, acetic-d
acid were used for the synthesis of 2,3-dimethyl-1,4-naphtho-
quinone (3), 2,3-dimethyl-2-d -1,4-naphthoquinone (4), and 2,3-
dimethyl-2- C-1,4-naphthoquinone (5), respectively.
3
acid-d, and acetic-2- C
3
1
3
1
2
,3-Dim eth yl-1,4-n a p h th oqu in on e (3). H NMR (300
MHz, CDCl
): δ 2.20 (s, 6H), 7.70 (m, 2H), 8.10 (m, 2H). 13
NMR (75.5 MHz, CDCl
Su p p or tin g In for m a tion Ava ila ble: HPLC traces for the
separation of product mixtures of the preparation of 1-5;
3
C
3
): δ 13.1, 126.3, 132.2, 133.3, 143.4,
84.9. EI-MS (positive-ion): m/z 186, 158, 157, 129, 115, 104,
1
13
13
6
H NMR and C NMR spectra of C-labeled N -acetylade-
1
7
nine in d(ApC); 1H NMR spectra of compounds 1-5. This
material is available free of charge via the Internet at
http://pubs.acs.org.
6, 50.
1
2
,3-Dim eth yl-2-d
MHz, CDCl
NMR (75.5 MHz, CDCl
3
-1,4-n a p h th oqu in on e (4). H NMR (300
): δ 2.19 (s, 3H), 7.70 (m, 2H), 8.08 (m, 2H). 13
): δ 13.1, 126.4, 132.3, 133.5, 143.6,
C
3
3
J O0264170
8
512 J . Org. Chem., Vol. 67, No. 24, 2002