Photoinduced DNA Interstrand Cross-Linking by Benzene Derivatives: Leaving Groups Determine the Efficiency of the Cross-Linker

Article abstract of DOI:10.1021/acs.joc.0c02234

We have synthesized and characterized two small libraries of 2-OMe or 2-NO2-benzene analogues 2a-i and 3a-i containing a wide variety of leaving groups. Irradiation of these compounds at 350 nm generated benzyl radicals that were spontaneously oxidized to

Full text of DOI:10.1021/acs.joc.0c02234

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Article
Photoinduced DNA Interstrand Cross-Linking by Benzene
Derivatives: Leaving Groups Determine the Efficiency of the Cross-
Linker
Heli Fan and Xiaohua Peng*
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ABSTRACT: We have synthesized and characterized two small
libraries of 2-OMe or 2-NO2-benzene analogues 2a−i and 3a−i
containing a wide variety of leaving groups. Irradiation of these
compounds at 350 nm generated benzyl radicals that were
spontaneously oxidized to benzyl cations directly producing
DNA interstrand cross-links (ICLs). Compounds with a 2-
methoxy substituent showed a faster cross-linking reaction rate
and higher ICL efficiency than the corresponding 2-nitro
analogues. Apart from the aromatic substituent, the benzylic
leaving groups greatly affected DNA cross-linking efficiency.
Higher ICL yields were observed for compounds with OCH3
(3b), OCH2Ph (3d), or Ph3P+ (3i) as leaving groups than those
containing OAc (3a), NMe2 (3e), morpholine (3f), OCH2CH
CH2 (3c), SPh (3g), or SePh (3h). The heat stability study of the isolated ICL products indicated that dGs were the preferred
alkylation sites in DNA for the benzyl cations produced from 2a−i, 3c, and 3e−i while 3a (L = OAc), 3b (L = OMe), and 3d (L =
OCH2Ph) showed a similar photoreactivity toward dGs and dAs. Although the photogenerated benzyl cations alkylated dG, dC, and
dA, ICL assay with variation of DNA sequences showed that the ICL reaction occurred with opposing dG/dC but not with
staggered dA/dA.
studied. For instance, psoralens,26,27 p-stibaolze,28 coumar-
INTRODUCTION
■
ins,29,30 3-cyanovinylcarbazole,31,32 and furan moiety33 can
DNA interstrand cross-links (ICLs) prevent the separation of
induce DNA ICL formation via photocycloaddition, while
two DNA strands, which inhibits DNA replication, tran-
phenol,14 biphenol,14 binol,11,13,34 or naphthoquinone ana-
scription, and any other processes for gene expression. Some
logues35,36 produce DNA ICL products through photo-
chemical reagents directly produce DNA ICLs, such as
generated QMs. However, photoinduced DNA ICL formation
nitrogen mustards, aldehydes/dialdehydes, and disulfonates,
via carbocation mechanism was less explored until recently.
while others have masked but inducible DNA cross-liking
The research groups of Li37 and Greenberg38 demonstrated
capability. Chemical agents capable of inducing ICLs showed
that although photoirradiation of the modified thymidines
wide applications in molecular biology and human medicine.
generated both the free radicals and cations, only the cation
They have been used as anticancer agents1−4 for DNA damage
intermediates produced DNA cross-linking. Peng and co-
and repair studies,5−7 for nucleic acid detection,8−10 etc.
workers discovered that photoirradiation of bifunctional
Several methods have been developed for inducing ICL
aromatic compounds produced bis-carbocations directly
formation, including photoirradiation,11−16 NaIO4
or
15,17
cross-linking DNA.16,39 Several classes of bifunctional aromatic
compounds have been reported to induce ICL formation via
photogenerated carbocations.4,16,40,41 Both the leaving groups
and the aromatic substituents strongly affected the efficiency of
NBS18,19 oxidation, fluoride induction,20−22 and H2O2
induction.23,24 Among these methods, photoinduction at-
tracted attention for its biocompatibility and orthogonality.
Photoirradiation is clean and non-invasive and does not
require additional chemical reagents. Various photoinducible
DNA cross-linking agents have been developed to form ICLs.
In general, three common mechanisms are involved in a
photoinduced ICL formation process, including photocylcoad-
diton, alkylation via quinone methides (QMs), or alkylation via
carbocations.25 The photoinduced DNA ICL formation via
photocycloaddition and QM formation has been extensively
Received: September 16, 2020
https://dx.doi.org/10.1021/acs.joc.0c02234
© XXXX American Chemical Society
J. Org. Chem. XXXX, XXX, XXX−XXX
A

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DNA ICL formation as well as the mechanism pathway for
DNA cross-linking.4 In general, two pathways are involved for
carbocation formation, either via homolytic cleavage of the C−
L bond to form free radicals that are simultaneously
transformed to the carbocations directly alkylating DNA
(pathway 1) or through heterolytic cleavage of the C−L
bond to generate carbocations (pathway 2). All compounds
with bromo as a leaving group induce ICL formation via
pathway 1 upon 350 nm irradiation, while the pathways for
DNA ICL formation induced by those containing trimethyl
ammonium salts as leaving groups highly depend on the
aromatic substituents.4,16 The ammonium salts with electron-
donating substituents (i.e., 1a) undergo homolytic cleavage of
the C−N bond (pathway 1), while those with strong
withdrawing groups (i.e., 1b) undergo heterolytic cleavage of
the C−N bond (pathway 2).
Several research groups have shown that the leaving groups
can modulate the efficiency of the photoactivation process as
well as the physical chemical properties of the sub-
strates.13,42−45 The research groups of Rokita and Freccero
demonstrated that −OAc, −NMe2 and −morpholine groups
were good leaving groups for QM formation.13,21,35,42 Benzyl
ether groups were also reported to be fast-photocleaving
groups that can be cleaved within seconds upon UV
irradiation.44 Greenberg’s group discovered that phenyl sulfide-
and phenyl selenide-modified pyrimidine nucleosides were
efficient photoactivated DNA cross-linkers.38,46,47 A lipophilic
cation, triphenylphosphonium group, was also introduced as
the most effective way to deliver drugs specifically to the
mitochondria, with the expectation of increasing the UV
absorption and improving water solubility.45 However,
previous investigation on cation-mediated ICL formation was
limited to two types of leaving groups, e.g., Br and the
trimethyl ammonium salts (i.e., 1a−d) (Scheme 1). The goal
of this work is to provide a systematic investigation on how the
leaving groups affect photochemical generation of carbocation
and the subsequent DNA ICL formation. Thus, we designed
and synthesized two series of benzene analogues 2a−i and 3a−
i that contain a wide variety of leaving groups, including OAc,
NMe2, morpholine, OCH3, OCH2CHCH2, OCH2Ph, SPh,
SePh, and triphenylphosphonium bromide group.
RESULTS AND DISCUSSION
■
Synthesis. Compounds 2a−d containing acetate or ether
groups were synthesized from diol 4 via nucleophilic
substitution reactions while 2e−i having other leaving groups
synthesized from bromide 1d (Scheme 2). Compounds 1d and
4 were prepared as previously reported.4 For the synthesis of
2a−d, different halides were employed, including acetyl
chloride (→2a), methyl iodide (→2b), allyl iodide (→2c),
and benzyl bromide (→2d) (Scheme 2A). To synthesize 2d,
we initially tried benzyl chloride, but no product was obtained
after stirring at rt overnight. Compound 1d was converted to
2e−i by treatment with dimethyl amine (→2e), morpholine
(→2f), thiophenol (→2g), diphenyl diselenide (→2h), or
triphenylphosphine (→2i) in good or moderate yields
(Scheme 2B).
Compounds 3a−i were synthesized starting from 4-
methoxyphenol (5) (Scheme 3). Selective hydroxymethylation
of 5 at the positions ortho to the hydroxyl group was
performed with formaldehyde under basic conditions yielding
4-methoxy-2,6-bis(hydroxymethyl)phenol (6) (Scheme 3A).
Methylation of 6 with methyl iodide provided (2,5-dimethoxy-
1,3-phenylene)dimethanol (7) that served as a common
precursor for 3a−i. Compound 7 was converted to 3a−d by
reacting with various halides, such as acetyl chloride (→3a),
methyl iodide (→3b), allyl iodide (→3c), and benzyl chloride
(→3d) (Scheme 3B). Compound 7 was also transformed to 1c
via bromination by PBr3 resulting in 90% yield, which was
higher than that reported by Fan and co-authors (62%)
(Scheme 3A).4 Similar to the synthesis of 2e−i, compounds
3e−i were obtained by treatment of 1c with dimethyl amine
(→3e), morpholine (→3f), thiophenol (→3g), diphenyl
diselenide (→3h), or triphenylphosphine (→3i) in good or
moderate yields (Scheme 3C).
DNA Interstrand Cross-Linking Assay. Similar to
previous studies, a 49-mer DNA duplex (8) was used for
investigating the photoreactivity of 2a−i and 3a−i toward
DNA.4,16 The DNA cross-linking assay was performed in a
phosphate buffer (pH 8.0) with 350 nm irradiation.
Denaturing polyacrylamide gel electrophoresis (PAGE) was
used for DNA ICL analysis. None of these compounds
produced DNA ICLs without photoirradiation, while efficient
ICL formation was observed for all of them upon 350 nm
irradiation. The results suggested that 2a−i and 3a−i were
efficient photoactivated DNA cross-linkers. To fully under-
stand how the aromatic substituents and leaving groups affect
the reactivity of these compounds, we carried out time-
dependent DNA cross-link study for 2a−i and 3a−i (Table 1
and Figures S1−S18, Supporting Information). Among all
compounds tested, those with an electron-donating aromatic
substituent (3a-i: R = OMe) showed a much faster
photoinduced DNA cross-linking reaction rate than the
corresponding NO2-containing compounds 2a−i. This in-
dicated that electron-donating substituents promoted the
reaction rate, while electron-withdrawing groups suppressed
this process. This is well correlated with the electron-deficient
nature of the carbocation intermediates that are stabilized by
the presence of electron-donating groups.
Scheme 1. Structures of 2a−i and 3a−i (A) and the
Mechanism for DNA ICL Formation Induced by 1a (B) and
1b (C)
B
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Scheme 2. Synthesis of 2a−i
the highest DNA cross-linking efficiency was obtained. The
Copt was affected by both leaving groups and the aromatic
substituents. The Copt for compounds containing a 2-NO2
group (2a−i) is in the order of 2h (0.3 mM) < 2a−c and 2i
(0.4 mM) < 2f and 2g (0.6 mM) < 2d (0.8 mM) < 2e (1.0
mM), while those containing a 2-OMe group showed a
different Copt order: 3i (0.2 mM) < 3e and 3f (0.4 mM) < 3a
and 3d (0.5 mM) < 3b, 3g, and 3h (0.6 mM) < 3c (2.0 mM).
As the concentration of ODNs are constant for all tested
compounds, a lower Copt to reach the ICL reaction balance
suggested a higher efficiency for generation of the carbocations
that directly alkylated DNA. Among all compounds tested, 3i
with triphenylphosphonium as a leaving group showed a lowest
Copt that resulted in a relatively high ICL yield (34%).
Compound 3d with a benzyloxy group showed the highest ICL
efficiency (37%) with a Copt of 0.5 mM. Compound 3b with
methoxy as a leaving group showed a Copt of 0.6 mM with a
good ICL yield (34%). However, 3c with an allyoxy leaving
group showed the highest Copt (2.0 mM) that may indicate a
low efficiency for photogeneration of benzyl cations. Thus,
triphenylphosphonium, benzyloxy, and methoxy groups may
be considered good leaving groups for photogeneration of
benzyl cations for this category of compounds.
Among 2a−i and 3a−i, compounds with a 2-methoxy group
showed a higher ICL yield than the corresponding analogue
having a 2-NO2 group (2a vs 3a, 2b vs 3b, etc.). This is well
correlated with the electron-deficient nature of the carbocation
intermediates that are stabilized by the presence of electron-
donating groups. However, the effect of leaving groups on
DNA cross-linking efficiency for 2-MeO compounds 3a−i
slightly differ from those of 2-nitro compounds 2a−i. The ICL
yields for 3a−i under optimized conditions is in the order of
3d > 3i ≈ 3b > 3a ≈ 3c ≈ 3g > 3e ≈ 3h > 3f, while 2-NO2-
In addition to the aromatic substituents, the leaving groups
also greatly affect the ICL reaction rate. For 3a−i containing a
2-methoxy group, compounds with phenylthio (3g) or phenyl
selenide (3h) as leaving groups showed the fastest reaction
rate. The moderate DNA cross-linking reaction rates were
observed for those containing triphenylphosphonium (3i),
methoxy (3b), or dimethylamine (3e) as leaving groups. The
cross-linking reaction was greatly slowed down for compounds
containing other leaving groups, including benzyloxy (3d),
morpholine (3f), acetate (3a), or allyoxy group (3c). Among
them, 3c showed the slowest reaction rate. A slightly different
trend for the reaction rates was observed for 2a−i with a 2-
NO2 group (2e ≅ 2g > 2i > 2f > 2h > 2b ≅ 2c ≅ 2d > 2a).
However, similar to 2-methoxy compounds, 2g with phenyl-
thio as a leaving group showed a relatively fast reaction rate for
photoinduced DNA ICL formation. Compounds 2a−d
containing acetate or ether as leaving groups greatly slowed
down photoinduced DNA cross-linking.
As the concentration of the substrates greatly affect the ICL
efficiency,4,16 we determined the optimized concentration
(Copt) for 2a−i and 3a−i. Copt is defined as the minimum
compound concentration required to obtain the possible
highest ICL yields. The concentration-dependent DNA cross-
linking study was performed with the optimized reaction time
for 2a−i and 3a−i (Table 1 and Figures S19−S36, Supporting
Information). Generally, the DNA ICL yields gradually
improved with the increase of compounds’ concentration.
The DNA ICL reaction reached a balance for all compounds at
the Copt where the DNA alkylation reaction was completed and
C
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Scheme 3. Synthesis of 3a-i
a
Table 1. Optimized Conditions, ICL Yields, and UV Absorption Data for 2a−i and 3a−i
RXN
time
(h)
RXN
time
(h)
b
b
con.
(mM) ICL (%)
λmax
ελmax
con.
λmax
ελmax
c
c
NO2-Compd. (L)
(nm) (L·mol−1·cm−1
)
OMe-Compd. (L)
(mM) ICL (%)
(nm) (L·mol−1·cm−1
)
2a (OAc)
44
40
40
40
24
32
0.4
0.4
0.4
0.8
1
18
1
282,
347
282,
347,
282,
347
282,
348
273,
346
273,
346
4340, 2100
3190, 1630
3200, 1600
2920, 1400
2760, 1040
2640, 990
3a (OAc)
22
0.5
0.6
2.0
0.5
0.4
0.4
27
3
286
286
286
287
287
286
2600
2640
2900
3350
3120
2940
2b (OCH3)
17
2
3b (OCH3)
6
34
3
2c (OCH2CH
14
17
17
15
1
2
2
1
3c (OCH2CH
36
12
6
28
37
25
18
2
1
3
2
CH2)
CH2)
2d (OCH2Ph)
3d (OCH2Ph)
2e (NMe2)
3e (NMe2)
2f
0.6
3f
12
[N(CH2CH2)2O]
[N(CH2CH2)2O]
2g (SPh)
2h (SePh)
2i (PPh3)
24
36
28
0.6
0.3
0.4
23
21
21
3
2
1
345
345
347
1750
2190
2030
3g (SPh)
3h (SePh)
3i (PPh3)
2
2
5
0.6
0.6
0.2
28
24
34
3
1
3
291
299
268,
6900
7050
7060, 5090
302
a
b
The DNA cross-linking reaction was performed in a pH 8 phosphate buffer with 50 nM DNA duplex 8 upon 350 nm irradiation. The minimum
c
compound concentration needed to obtain the highest DNA cross-linking efficiency. The maximum DNA ICL yield obtained for each compound
under optimized conditions (all data are the average of three experiments).
containing compounds showed a different order (2g > 2h ≈ 2i
> 2a ≈ 2b ≈ 2d ≈ 2e > 2f ≈ 2c). For 2-OMe-substituted
compounds (3a-i), benzyloxy is the best leaving group for
photoinduced DNA ICL formation while morpholine leads to
the lowest cross-linking efficiency. For 2-nitro compounds, 2g
with a phenylthio group leads to the highest ICL yield while
the allyloxy group showed the lowest ICL efficiency. Since
different compounds showed a different Copt value to obtain
the highest DNA cross-linking efficiency, comparison of ICL
yields obtained at the Copt is not sufficient to conclude how the
substituent and leaving group affect the ICL efficiency. For
better comparison, the DNA cross-linking efficiency was
D
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Figure 1. Photoinduced DNA ICL formation for 2a−i and 3a−i. (A) Lane 1: DNA only without UV irradiation; lane 2: DNA only with 350 nm
irradiation for 24 h; lanes 3−20: DNA with 2a−i or 3a−i (500 μM) without UV irradiation; (B) lanes 1−18: DNA with 2a−i or 3a−i (500 μM)
upon 350 nm irradiation for the designed time: lane 1: 2a (ICL yield, 18 2%). lane 2: 2b (17 2%); lane 3: 2c (15 1%) ; lane 4: 2d (14
2%); lane 5: 2e (12 1%); lane 6: 2f (14 2%); lane 7: 2g (23 3%); lane 8: 2h (22 2%); lane 9: 2i (21 3%); lane 10: 3a (27 2%); lane
11: 3b (32 4%) ; lane 12: 3c (10 1%); lane 13: 3d (37% 3); lane 14: 3e (26 2%); lane 15: 3f (18 1%); lane 16: 3g (26 2%); lane 17:
3h (21 2%); lane 18: 3i (34 3%) (all ICL yields were obtained by triplicate experiments and shown as average standard deviation).
determined at the same concentration (500 μM) under the
optimized reaction time for each compound (Figure 1). For
both classes of compounds, the trend of ICL yields at 500 μM
was similar to that obtained under the optimized conditions.
Thus, we conclude that the electron-donating substituent
increased the ICL efficiency while the electron-withdrawing
substituent decreased the ICL efficiency. Both the leaving
groups and the aromatic substituents combine to affect the
efficiency of photoinduced DNA ICL formation.
showed similar λmax (282 and 347 nm) with different
absorbances. However, the absorbance order (2a > 2b ≈ 2c
> 2d) is opposite to that of the reactivity order (2b ≈ 2c ≈ 2d
> 2a). More interestingly, 2e and 2f showed a shorter λmax and
a weak absorption than 2a−d but a much faster ICL reaction
rate. Due to the complexity of DNA cross-linking reaction, the
ICL efficiency could be affected by a variety of parameters,
including the photosensitivity of the leaving groups, the UV
absorption of the compounds, the efficiency for carbocation
generation, the reactivity of carbocations (mono-alkylation and
bisalkylation), and the competition of mono-alkylation and
hydrolysis of carbocation with ICL formation. These
compounds contain different leaving groups that would affect
several of the above parameters, which could be one of the
possible explanations for the inconsistency between their
photoreactivity and UV absorption.
Mechanism of DNA ICL Formation and the Leaving
Group Effects. Our previous study showed that the aromatic
substituents greatly affected the mechanism for photoinduced
DNA ICL formation via benzyl cations.4,16 Two pathways were
observed for cation generation from compounds containing
trimethyl ammonium salts as the leaving groups upon 350 nm
irradiation.4,16 Compounds with electron-donating substituents
(i.e., 1a) generated the benzyl cations via oxidation of free
radicals, while those with strong withdrawing groups (i.e., 1b)
underwent heterolytic cleavage of the C−N bond to generate
benzyl cations (Scheme 1B,C). To further investigate the effect
of leaving groups and aromatic substituents on DNA ICL
formation, we performed free radical and carbocation trapping
reactions with 2a−i and 3a−i that contain a wide variety of
leaving groups. Methoxyamine and 2,2,6,6-tetramethylpiper-
idin-1-oxyl (TEMPO) were used as carbocation and radical
Correlation between UV Absorbance and the Photo-
reactivity. Our previous results showed that the aromatic
substituents greatly influenced the UV absorption of the
compounds, which in turn affected the photoinduced DNA
ICL formation.4 In general, compounds with UV absorption
closer to the irradiation wavelength (350 nm) and the stronger
UV absorption led to a faster ICL reaction rate. In order to see
the generality of this phenomenon, we investigated the
influence of the leaving groups on the UV absorbance of
2a−i and 3a−i. UV/Vis spectra of these compounds were
measured in CH3CN with a 0.5 mM concentration (Figure
S56, see the Supporting Information). The leaving groups
slightly affected the UV absorption of these compounds, but
there is no clear correlation between UV absorbance and the
photoreactivity for these compounds (Table 1 and Figure
S56). Although the highest photoreactivity of 3g and 3h seems
to be well correlated with their UV absorbance [the longest
maximum absorption wavelength (λmax) and strongest
absorption], such a correlation was not observed for others,
such as 3a−e and 2a−f. For example, 3a−f showed similar λmax
(∼ 286−287 nm) with the absorbance order of 3d > 3e > 3c ≈
3f > 3b > 3a, while their photoreactivity was observed in a
different order (3b ≈ 3e > 3d ≈ 3f > 3a > 3c). Similarly, 2a−d
E
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Figure 2. Carbocation and radical trapping with DNA ICL formation for 2b (A), 2d (B), 3a (C), 3b (D), 3d (E), and 3i (F).
Table 2. Cation and Radical Trapping Efficiency Evaluated from the Concentration of Methoxamine and TEMPO Required to
a
Inhibit the ICL Yield to 5%, 2%, and Background Level
b
c
c
ICL
methoxyamine
(mM)
methoxyamine
(mM)
d
b
d
NO2-Compd. (L)
(%)
[TEMPO] (mM)
OMe-Comp. (L)
ICL (%)
[TEMPO] (mM)
5% 2%
25
0.05
0.025
5% 2%
B
5% 2%
B
5%
10
1.0
2%
B
B
2a (OAc)
2b (OCH3)
16.2
18.2
14.5
35
68
58
70
92
90
100
100
100
5
22
70
70
50
3a (OAc)
27.1
34.1
26.3
40
20
20
40
20
20
0.1
50
10
25
15
15
100 3b (OCH3)
100 3c (OCH2CH
0.25
1.0
2c (OCH2CH
2.0
CH2)
CH2)
2d (OCH2Ph)
2e (NMe2)
16.6
17.6
14.6
55
15
45
90
35
85
100
50
100
10
25
6
25
70
50
50
100 3e (NMe2)
50 3f
[N(CH2CH2)2O]
3d (OCH2Ph)
37.8
25.7
18.8
0.2
2.0
3.0
10
10
30
10
10
30
0.05
1.5
3.0
25
2.5
15
25
10
25
2f
[N(CH2CH2)2O]
2g (SPh)
2h (SePh)
2i (PPh3)
23.4
22.2
20.8
35
75
60
50
95
90
50
100
100
10
15
30
70
65
90
100 3g (SPh)
100 3h (SePh)
100 3i (PPh3)
28
24
33
1.5
1.0
40
5.0
5.0
80
5
5
80
1.0
0.05
1.0
1.5
0.25
55
2.5
2.5
100
a
b
The DNA cross-linking reaction was performed in a pH 8 phosphate buffer with 50 nM DNA duplex 8 upon 350 nm irradiation. The initial ICL
c
d
yields without addition of methoxyamine or TEMPO. The concentration of methoxamine required to inhibit ICL to 5% or 2%. The
concentration of TEMPO required to inhibit ICL to 5% or 2%.
traps, respectively (Figure 2 and Figures S37−S54). The ICL
reactions were carried out under the optimized conditions for
each compound in the presence of various concentrations of
methoxyamine or TEMPO. The results for 2b, 2d, 3a, 3b, 3d,
and 3i are shown in Figure 2, while the results for others are
presented in Figures S37−S54. For all compounds tested, the
ICL yields decreased with the addition of methoxyamine that
acted as competitors for ICL formation. The DNA ICL
formation was completely inhibited when the concentration of
methoxyamine increased to a certain level, indicating that
carbocations were involved in the DNA cross-linking process.
However, the trapping efficiency strongly depended on the
substrates, which was measured as the minimal concentration
of the trapping agents required to decrease the ICL yields to a
similar level (5%, 2%, or the background level) (Table 2). In
general, trapping of the benzyl cations generated from 3a−i
with an OMe group is much more efficient than those
generated from the corresponding NO2-containing compounds
2a−i. For example, 0.2 mM methoxamine decreased the ICL
yield of 3d from 37.8% to 5% while ∼55 mM methoxyamine
was required to inhibit DNA ICL formation of 2d from 16.6%
to 5% (Figure 2B,E and Table 2). Similarly, the concentration
of methoxyamine for inhibition of ICL formation to 5% is 68
mM for 2b and 75 mM for 2h that is about 70 times higher
than that needed for 3b and 3h (∼1.0 mM) (Figure 2A,D and
Table 2). A similar phenomenon was observed for other
compounds (Table 2, 2a vs 3a, 2c vs 3c, 2e−g vs 3e−g, and 2i
vs 3i). Higher cation trapping efficiency for 3a−i than 2a−i
suggested higher reactivity of the benzyl cations photo-
generated from 3a−i than those produced from 2a−i.
Similarly, to decrease the ICL yields to 2%, much higher
concentrations of methoxyamine were needed for 2a−i than
those for 3a−i. This is well correlated with the electron-
deficient property of the carbocations that were stabilized by
electron-donating groups leading to higher reactivity. Among
compounds 3a−i that contain the same aromatic substituent
F
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Scheme 4. Proposed Mechanism for DNA ICL Formation Induced by 2a−i and 3a−i
(2,4-dimethoxy groups), the leaving groups also affected the
cation-trapping efficiency. The highest concentration of
methoxyamine was needed for inhibiting ICL formation
induced by 3i having triphenylphosphonium salt as a leaving
group, which might be due to the strong withdrawing property
of the triphenylphosphonium salt (Figure 2F and Table 2).
Similarly, 3a with a withdrawing acetate group showed a lower
cation-trapping efficiency than 3b−h (Figure 2C and Table 2).
Considering that the benzyl cations can be generated either
from oxidation of the corresponding free radicals (pathway 1)
or by direct heterolysis of C−X bonds (pathway 2), we
performed free radical trapping experiments using excess
TEMPO. Similar to cation trapping, the addition of TEMPO
suppressed the DNA ICL formation for all compounds (2a−i
and 3a−i) tested. The ICL yields gradually decreased to the
background level with increased concentration of TEMPO
(Figure 2 and Figures S37−S54). These results suggested that
the free radicals were involved in the DNA ICL formation
process. However, the trapping efficiency strongly depended
on the substrates. Trapping of the free radicals generated from
3a−i with an OMe substituent was more efficient than those
generated from the corresponding NO2-containing compounds
2a−i. For example, 0.05 mM TEMPO decreased the ICL yield
of 3d from 37.8% to 5% while it required ∼10 mM TEMPO to
obtain a similar trapping efficiency for 2d (from 16.6% to 5%)
(Figure 2B,E and Table 2). Similarly, much higher
concentrations of TEMPO were needed for 2a−c and 2e−i
than the corresponding 3a−c and 3e−i to reach the same
trapping efficiency (Table 2). This is well correlated with the
electron-deficient property of the free radicals that were
stabilized by electron-donating groups leading to higher
reactivity. Collectively, the results of cation trapping and free
radical trapping study suggested that the benzyl cations were
generated through oxidation of the free radicals (pathway 1,
Scheme 1B). Thus, we propose that photoirradiation of 2a−i
and 3a−i generated the free radicals (9) that were further
converted to the benzyl cations (10) that alkylated DNA
(Scheme 4).
min for m/z 461 (11a) and 470 (11b) (Scheme 5 and
Supporting Information, Figure S57A−C, LC-mass data)
Scheme 5. Cation and Free Radical Trapping Products
Obtained with 3i upon 350 nm Irradiation
(Note: MeONH2·HCl was used for cation trapping, which is
the source of the chloro group in 11a). The mass of two
adducts 11a and 11b was further confirmed by HRMS
(Scheme 5 and Supporting Information, Figure S57E,F, HRMS
data). Formation of the adducts 11a and 11b suggested that
the mono carbocation (10) was generated upon photo-
irradiation of 3i. The free radical trapping reaction provided
one major product 11c that was isolated by chromatography.
The structure of 11c was determined by NMR and HRMS,
suggesting the formation of mono radical (9). Generation of
mono-trapping products 11a−c provided direct evidence for
our proposed mechanism. Departure of two leaving groups
occurred in a stepwise manner, leading to the formation of
mono-radical 9 and mono-cation 10. Failure to detect the
biscation-trapping products even with a longer period of
photoirradiation indicated a much lower efficiency for
photoactivation of the second leaving group that should be
the rate-determining step for ICL formation.
Determination of DNA Alkylation Sites and the Effect
of the Aromatic Substituents and Leaving Groups. It
was well known that the N7-alkylated purines can be cleaved
upon heating in the presence of piperidine.48−50 Previously, we
have determined the alkylation sites of photogenerated benzyl
cations by studying the heat stability of the cross-linked DNA
and/or synthesizing the adducts formed between benzyl
cations and natural nucleosides.4 Although the monomer
reaction showed that the photogenerated benzyl cations could
react with dC, dA, and dG, the heat stability study of the cross-
linked DNA indicated that dGs were preferred alkylation sites
in ODNs. In order to investigate the effect of leaving groups
and the aromatic substituents on the alkylation sites of 2a−i
and 3a−i, we performed the heat stability study with ICL
products formed with these compounds. We isolated both
single-stranded ODNs (p32-ODN 8a′) as well as the ICL
products formed with DNA duplex 8 upon 350 nm irradiation
in the presence of 2a−i and 3a−i. The heat stability data of the
isolated p32-ODN 8a′ and the ICL products for 3a and 2a are
shown in Figure 3, while the data for other compounds are
shown in Figure S55. Similar to a previous study, the ICL
products were relatively stable upon heating in a pH 7.0
In order to provide direct evidence for the formation of
benzyl radicals and benzyl carbocations, we performed
monomer trapping reaction to isolate cation or free radical
trapping adducts. Compound 3i was selected as a representa-
tive for monomer trapping reactions because it is relatively easy
to synthesize and showed a relatively high ICL yield and fast
reaction rate. The trapping reactions were performed with 3i in
the presence of excess methoxyamine or TEMPO that served
as carbocation and free radical traps, respectively. Thin-layer
chromatography (TLC) indicated a very complex reaction for
the cation trapping. Several new spots with similar Rf values
were observed, which were not separable by chromatography.
Thus, LC-MS was used for the analysis of the adducts formed
in the monomer trapping reaction (Figure S57). The LC-MS
analysis revealed two major peaks eluting at ∼57.8 and ∼60.4
G
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Table 3. Photoreactivity of 2a−i and 3a−i toward dG and
dA Evaluated from the Ratio for Cleavage at dG27, dG22,
a
dA25, and dA24
b
b
cleav.
cleav.
NO2-Compd. (L)
(dG/dA)
OMe-Compd. (L)
(dG/dA)
2a (OAc)
2b (OCH3)
1.63
2.86
2.19
3a (OAc)
3b (OCH3)
0.98
1.19
1.83
2c (OCH2CH
3c (OCH2CH
CH2)
CH2)
2d (OCH2Ph)
2e (NMe2)
2f [N(CH2CH2)2O]
2g (SPh)
2h (SePh)
2i (PPh3)
2.59
2.20
2.12
2.69
2.01
2.08
3d (OCH2Ph)
3e (NMe2)
3f [N(CH2CH2)2O]
3g (SPh)
3h (SePh)
3i (PPh3)
1.07
2.02
2.16
1.82
1.69
2.73
a
The DNA cross-linking reaction was performed in a pH 8 phosphate
buffer with 50 nM DNA duplex 8 upon 350 nm irradiation. The ICL
products were isolated and heated in 1.0 M piperidine at 90 °C for 30
min. The cleavage bands at dG27, dG22, dA25, and dA24 were
Figure 3. Determination of the reaction sites of 3a (A) and 2a (B).
Phosphorimage autoradiogram of 20% denaturing PAGE analysis of
the isolated DNA ICL products and alkylated single-stranded DNA
(8a′) upon heating in piperidine or phosphate buffer. The ICL
product and 8a′ were produced by 350 nm irradiation of duplex 8 in
the presence of 3a (500 μM) or 2a (400 μM). 8a was radiolabeled at
the 5′-terminus. Lane 1: isolated alkylated single-stranded DNA (8a′).
Lane 2: 8a′ was heated in a pH 7 phosphate buffer at 90 °C for 30
min. Lane 3: 8a′ was heated in 1.0 M piperidine at 90 °C for 30 min.
Lane 4: isolated DNA ICL products. Lane 5: the DNA ICL products
were heated in a pH 7 phosphate buffer at 90 °C for 30 min. Lane 6:
the DNA ICL products were heated in 1.0 M piperidine at 90 °C for
30 min. Lane 7: G+A sequencing.
b
quantified. The ratio of cleavage at dG27+dG22 to that at dA25+dA24.
reactivity toward dG and dA. To investigate whether the
alkylation could occur with pyrimidines, we tested the ICL
reaction with duplex 12 containing dCs/dTs in one strand and
dGs/dAs in the complimentary strand (Figure 4A). ICL
formation was observed when duplex 12 was treated with 3d
(6.4%), 3i (5.1%), 2g (6.4%), and 2i (7.7%), which suggested
that dC and/or dT are possible alkylation sites (Figure 4A). To
further investigate the cross-linking sites, we synthesized one
self-complementary dAT sequences (13) that was treated with
3d, 3i, 2g, and 2i upon 350 nm irradiation. The DNA ICL
formation was not observed with 13, indicating that interstrand
cross-linking reaction did not take place between dA and
opposing dT neither with staggered dA/dA (Figure 4B).
However, the cleavage bands were observed with dAs when the
single-stranded ODN 13a′ was isolated from the cross-linking
reaction mixture and heated in 1.0 M piperidine at 90 °C for
30 min (Figure 4C). This suggested that mono-alkylation
occurred with dAs. Collectively, these data indicated that ICL
reactions took place with opposing dG/dC but not with dA/
dT or staggered dA/dA while mono-alkylation could take place
with dAs.
phosphate buffer for 30 min, while obvious cleavage bands
were observed upon heating in 1.0 M piperidine. For most
compounds tested (i.e., 2a−i, 3c, and 3e−i), the cleavage
mainly occurred at dG sites and to a lesser extent at dAs
(Figure 3B and Figure S55). However, 3a, 3b, and 3d showed
the major cleavage sites not only at dGs but also at dAs, which
is different from a previous observation (Figure 3A and Figure
S55). These data suggested that both substituents and leaving
groups affected the reactivity of these photogenerated benzyl
cations toward DNA. For comparison of the relative reactivity
of these benzyl cations toward dG and dA, we estimated the
percentages for cleavages at dG27, G22, dA25, and dA24 sites.
From the cleavage ratio of dG27+dG22 to dA25+dA24
(CleavdG/dA), we are able to estimate the relative photo-
reactivity of these compounds toward dG and dA in ODNs
(Table 3). Compounds 2a−i, 3c, and 3e−i showed a
CleavdG/dA of ∼2 or higher, indicating that dGs were the
preferred alkylation sites for the benzyl cations produced from
these compounds. However, 3a, 3b, and 3d showed a much
smaller CleavdG/dA (CleavdG/dA ≈ 1) than 2a, 2b, and 2d
(CleavdG/dA = 1.6−2.6), which suggested that 3a, 3b, and 3d
had improved reactivity toward dAs. It is well known that an
electron-donating aromatic substituent increases the stability of
benzyl-like carbocations.51 Thus, the methoxy-substituted
benzyl carbocations generated from 3a, 3b, and 3d are
expected to be more stable than the corresponding nitro-
substituted benzyl carbocations generated from 2a, 2b, and 2d,
which in turn lead to increased reactivity of 3a, 3b, and 3d
toward dA.
CONCLUSIONS
■
We have demonstrated that two series of benzene analogues
containing a wide variety of leaving groups are photo-
activatable bisalkylating agents. These compounds efficiently
induced DNA ICL formation upon 350 nm irradiation via
benzyl cations that were formed from the oxidation of the
photogenerated benzyl radicals. Determination of cation and
free radical trapping adducts provided direct evidence for the
formation of benzyl radicals and carbocations. Compounds
with an electron-donating group (OMe) (3a−i) showed a
higher cross-linking efficiency and faster reaction rate than the
corresponding ones having withdrawing group (NO2) (2a−i)
regardless of which leaving group is present. Benzylic leaving
groups also have big effects on DNA ICL efficiency. Among all
OMe-containing compounds, 3b (L = OMe), 3d (L =
From the heat stability study, we were able to conclude that
2a−i and 3a−i alkylated dG and/or dA sites upon 350 nm
irradiation but different compounds showed a slightly different
+
OCH2Ph), and 3i (L = PPh3 ) are the most efficient
H
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Figure 4. Photoinduced DNA ICL formation for 3d, 3i, 2g, and 2i with duplex 12 (A) and duplex 13 (B) and determination of the reaction sites of
3i in duplex 13 (C) [phosphorimage autoradiogram of 20% denaturing PAGE analysis of the ICL reaction mixture upon heating in piperidine or
phosphate buffer. An ICL reaction mixture of 13 (50 nM) and 3i (0.2 mM) in pH 8 phosphate buffer was irradiated with 350 nm for 5 h. The
single-stranded ODN (5′-p32-13a′) was isolated from the cross-link reaction mixture. Lane 1: the isolated single-stranded ODN (13a′); Lane 2:
13a′ was heated in a pH 7 phosphate buffer at 90 °C for 30 min; Lane 3: 13a′ was heated in 1.0 M piperidine at 90 °C for 30 min; Lane 4: G+A
sequencing].
single-stranded ODN, which were determined using a molecular
ladder.24 A UV experiment was carried out using an RPR-100
Photochemical Reactor with RPR-3500 Å. H NMR and 13C{1H}
NMR spectra were taken on either a Bruker DRX 300 or DRX 500
MHz spectrophotometer with TMS as an internal standard. High-
resolution mass spectrometry IT-TOF was used for molecular
measurement.
(5-Methoxy-2-nitro-1,3-phenylene)bis(methylene) Diacetate
(2a). Compound 4 (300 mg, 1.41 mmol) and 4-dimethyl amino-
pyridine (517 mg, 4.23 mmol) in DCM was cooled to 0 °C. Acetyl
chloride (443 mg, 5.64 mmol) was added using a syringe. The
reaction mixture was warmed to rt, stirred overnight, quenched with
H2O (10 mL), and extracted with DCM (3 × 15 mL). The combined
organic layers were washed with brine, dried over anhydrous Na2SO4,
and concentrated. The remaining residue was purified by column
chromatography (Hexane:Ethyl acetate = 2:1, Rf = 0.35) to obtain 2a
as a light yellow solid (397 mg, 95%), m.p. 95−96 °C. 1H NMR (300
MHz, CDCl3): δ 6.96 (s, 2H), 5.23 (s, 4H), 3.89 (s, 3H), 2.11 (s,
6H). 13C{1H} NMR (75 MHz, CDCl3): δ 170.2, 161.2, 141.7, 132.8,
114.2, 62.4, 55.9, 20.6. HRMS-ESI (+) (m/z): [M + Na]+ calcd. for
C13H15NO7Na+, 320.0741; found: 320.0737.
photoactivated DNA cross-linkers with an ICL yield of >34%,
while 3f with morpholine as a leaving group resulted in the
lowest ICL yield (18%). Although the benzyl cations
photogenerated from these compounds alkylated dG, dA,
and dC, the ICL formation only took place with opposing dG/
dC not with staggered dA/dA. Both aromatic substituents and
leaving groups strongly affected the photoreactivity of these
compounds toward dG and dA. Compounds 2a−i, 3c, and
3e−i showed better photoreactivity toward dG than dA, while
3a (L = OAc), 3b (L = OMe), and 3d (L = OCH2Ph) showed
a similar reactivity toward dGs and dAs. This systematic study
provides valuable fundamentals for developing more efficient
photoinduced DNA cross-linking agents. It also provides
guidelines for development of photoactivated drugs.
1
EXPERIMENTAL SECTION
■
General Experimental Methods. All reactions were stirred
magnetically, unless otherwise noted. An oil bath was used as a
heating source for all of the organic synthesis reactions that required
heating in this work. A heating block or water bath were used for
DNA reactions involving heating. Reactions were monitored by thin-
layer chromatography (TLC): Merck Silica Gel 60 F254. All
chemicals came from commercially available source without further
purification. Oligonucleotides were synthesized via standard auto-
mated DNA synthesis techniques. Deprotection of the synthesized
DNA were performed with a mixture of 40% aqueous MeNH2 and
28% aqueous NH3 (1:1) at room temperature for 2 h. 20%
Denaturing polyacrylamide gel electrophoresis was used for DNA
purification. [γ-32P] ATP was used for DNA labeling with a standard
method. Quantification of radiolabeled oligonucleotides was carried
out using a Molecular Dynamics phosphorimager equipped with
ImageQuant, version 5.2, software. For all phosphorimage autoradio-
grams, the top dark spot that migrated slowest is considered as the
DNA cross-link product and the bottom dark spot is considered as the
5-Methoxy-1,3-bis(methoxymethyl)-2-nitrobenzene (2b). NaH
(169 mg, 60%, 4.23 mmol) was added to a solution of 4 (300 mg,
1.41 mmol) in DMF (6.0 mL) at 0 °C. The reaction mixture was
stirred for 10 min at 0 °C. Then, CH3I (800 mg, 5.64 mmol) was
added. The reaction mixture was allowed to warm to rt, stirred
overnight, quenched with water (10 mL), and extracted with ethyl
acetate (3 × 15 mL). The organic layers were combined, washed with
brine, dried over anhydrous Na2SO4, and concentrated. The
remaining residue was purified by column chromatography (Hex-
ane:Ethyl acetate = 2:1, Rf = 0.68) to obtain 2b as a yellow solid (280
1
mg, 82%), m.p. 39−40 °C. H NMR (300 MHz, CDCl3): δ 7.05 (s,
2H), 4.58 (s, 4H), 3.90 (s, 3H), 3.43 (s, 6H). 13C{1H} NMR (75
MHz, CDCl3): δ 161.5, 140.9, 135.3, 112.6, 71.7, 58.8, 55.8. HRMS-
+
ESI (+) (m/z): [M − OMe]+ calcd. for C10H12NO4 , 210.0761;
found: 210.0755.
I
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1,3-Bis((allyloxy)methyl)-5-methoxy-2-nitrobenzene (2c). NaH
(169 mg, 60%, 4.23 mmol) was added to a solution of 4 (300 mg,
1.41 mmol) in DMF (6.0 mL) at 0 °C. The resulting mixture was
stirred for 10 min. Then, allyl iodide (948 mg, 5.64 mmol) was added.
The reaction mixture was allowed to warm to rt, stirred overnight,
quenched with water (10 mL), and extracted with ethyl acetate (3 ×
15 mL). The organic layers were combined, washed with brine, dried
over anhydrous Na2SO4, and concentrated. The remaining residue
was purified by column chromatography (Hexane:Ethyl acetate = 2:1,
Rf = 0.8) to obtain 2c as a yellowish liquid (286 mg, 69%). 1H NMR
(300 MHz, CDCl3): δ 7.09 (s, 2H), 6.00−5.88 (m, 2H), 5.35 (s, 1H),
5.30 (s, 1H), 5.25−5.22 (d, J = 9.0 Hz, 2H), 4.64 (s, 4H), 4.07−4.05
(d, J = 6.0 Hz, 4H), 3.89 (s, 3H). 13C{1H} NMR (75 MHz, CDCl3):
δ 161.5, 141.0, 135.4, 134.0, 117.6, 112.7, 71.9, 68.2, 55.8. HRMS-ESI
1.18 mmol) in DMF (5.0 mL) was added NaBH4 (11.2 mg, 0.295
mmol). The reaction mixture was stirred at rt for 2 h. Then, a solution
of 1d (100 mg, 0.295 mmol) in DMF (2.0 mL) was added. The
resulting mixture was stirred at rt for another 4 h, quenched with
water (10 mL), extracted with ethyl acetate (3 × 15 mL). The organic
layers were combined, washed with brine, dried over anhydrous
Na2SO4, and concentrated. The residue was purified by column
chromatography (Hexane:ethyl acetate = 5:1, Rf = 0.4) to afford 2h as
1
a light yellow solid (80.0 mg, 55%): m.p. 95−97 °C. H NMR (300
MHz, CDCl3): δ 7.50−7.48 (d, J = 6.0 Hz, 4H), 7.30−7.27 (m, 6H),
6.32 (s, 2H), 4.08 (s, 4H), 3.53 (s, 3H). 13C{1H} NMR (75 MHz,
CDCl3): 159.5, 135.1, 134.7, 129.0, 128.9, 128.9, 127.8, 114.5, 55.1,
28.3. HRMS-ESI (+) (m/z): [M + K]+ calcd. for C21H19NO3Se2K+,
531.9331; found: 531.9320.
+
(+) (m/z): [M − OC3H5]+ calcd. for C12H14NO4 , 236.0917; found:
((5-Methoxy-2-nitro-1,3-phenylene)bis(methylene))bis-
(triphenylphosphonium) Bromide (2i). A mixture of 1d (170 mg, 0.5
mmol) and triphenylphosphine (289 mg, 1.1 mmol) in dry toluene (5
mL) was stirred at rt for 2 days under argon. The crude white powder
was obtained by filtration, which was purified by column
chromatography (DCM:Methanol = 10:1, Rf = 0.32) to afford 2i as
236.0915.
((((5-Methoxy-2-nitro-1,3-phenylene)bis(methylene))bis(oxy))-
bis(methylene))dibenzene (2d). A solution of 4 (300 mg, 1.41
mmol) in DMF (6.0 mL) was cooled to 0 °C followed by the addition
of NaH (169 mg, 60%, 4.23 mmol). The resulting mixture was stirred
for 10 min. Then, benzyl bromide (720 mg, 4.23 mmol) was added.
The reaction mixture was allowed to warm to rt, stirred overnight,
quenched with water (10 mL), and extracted with ethyl acetate (3 ×
15 mL). The organic layers were combined, washed with brine, dried
over anhydrous Na2SO4, and concentrated. The residue was purified
by column chromatography (Hexane:Ethyl acetate = 2:1, Rf = 0.76)
1
a yellowish foam (259 mg, 60%). H NMR (300 MHz, CDCl3): δ
7.83−7.78 (m, 6H), 7.71−7.55 (m, 24H), 7.18 (s, 2H), 5.58−5.53 (d,
J = 15.0 Hz, 4H), 3.46 (s, 3H). 13C{1H} NMR (75 MHz, CDCl3): δ
161.4, 143.1, 135.5, 134.3, 134.2, 130.6, 130.5, 126.9, 126.8, 120.2,
117.3, 116.2, 55.6, 30.9, 29.3, 28.6. HRMS-ESI (+) (m/z): [M −
2Br]2+ calcd. for C45H39NO3P22+, 351.6197; found: 351.6183.
(2-Hydroxy-5-methoxy-1,3-phenylene)dimethanol (6). A solution
of 5 (50 g, 0.4 mol) in H2O (180 mL) was added NaOH (16 g, 0.4
mol) and stirred till obtaining a clear solution. Then, paraformalde-
hyde (18.0 g, 0.6 mol) and methanol (30 mL) were added. The
reaction mixture was stirred at 30 °C for 2 days and acidified with
HCl (5.0 M) to pH 5.0. Then, methanol was removed. The resulting
aqueous solution was extracted with ethyl acetate (3 × 100 mL). The
organic layers were combined, washed with brine, dried over
anhydrous Na2SO4, and concentrated. The residue was purified by
column chromatography (Hexane:Ethyl acetate = 1:1, Rf = 0.33) to
obtain 6 as a white solid (55.2 g, 75%). 1H NMR (300 MHz, DMSO-
d6): δ 8.04 (s, 1H), 6.76 (s, 2H), 5.24−5.20 (t, J = 6.0 Hz, 2H), 4.54−
4.52 (d, J = 6.0 Hz, 4H), 3.68 (s, 3H) (the NMR spectra were in
agreement with those reported).52
1
to afford 2d as a yellow oily (414 mg, 75%). H NMR (300 MHz,
CDCl3): δ 7.42−7.30 (m, 10H), 7.12 (s, 2H), 4.71 (s, 4H), 4.60 (s,
4H), 3.88 (s, 3H). 13C{1H} NMR (75 MHz, CDCl3): δ 161.5, 141.2,
137.6, 135.3, 128.5, 127.9, 127.8, 112.9, 73.2, 68.5, 55.8. HRMS-ESI
(+) (m/z): [M + Na]+ calcd. for C23H23NO5Na+, 416.1468; found:
416.1453.
1,1′-(5-Methoxy-2-nitro-1,3-phenylene)bis(N,N-dimethylme-
thanamine) (2e). Into a solution of 1d (150 mg, 0.44 mmol) in ethyl
acetate (4.0 mL) was added dimethylamine solution (2.0 M in
methanol) (2.2 mL, 4.4 mmol). The reaction mixture was stirred at rt
for 8 h and concentrated. The residue was purified by column
chromatography (DCM:Methanol = 10:1, Rf = 0.5) to afford 2e as a
yellowish solid (108 mg, 92%), m.p. 44−45 °C. 1H NMR (300 MHz,
CDCl3): δ 7.05 (s, 2H), 3.89 (s, 3H), 3.51 (s, 4H), 2.26 (s, 12H).
13C{1H} NMR (75 MHz, CDCl3): δ 160.5, 144.2, 133.3, 114.6, 59.6,
(2,5-Dimethoxy-1,3-phenylene)dimethanol (7). A solution of 6
(18.4 g, 0.1 mol) in acetone (100 mL) was added K2CO3 and CH3I.
The reaction mixture was stirred at 50 °C overnight and concentrated.
Then, water (50 mL) was added. The resulting aqueous solution was
extracted with ethyl acetate (3 × 80 mL), washed with brine, dried
over anhydrous Na2SO4, and filtrated. Then, the solvent was removed.
The residue was purified by column chromatography (Hexane:Ethyl
acetate = 1:1, Rf = 0.3) to obtain 7 as a white solid (17.4 g, 88%). 1H
NMR (300 MHz, DMSO-d6): δ 6.87 (s, 2H), 5.12−5.08 (t, J = 6.0
Hz, 2H), 4.52−4.50 (d, J = 6.0 Hz, 4H), 3.72 (s, 3H), 3.61 (s, 3H)
(the NMR spectra were in agreement with those reported).53
1,3-Bis(bromomethyl)-2,5-dimethoxybenzene (1c). A solution of
7 (4.0 g, 20.18 mmol) in DCM (50 mL) was cooled to 0 °C and
added phosphorus tribromide (12.02 g, 44.4 mmol). The reaction
mixture was allowed to warm to rt, stirred for 4 h, quenched with
water (30 mL), and extracted with DCM (3 × 20 mL). The organic
layers were combined, washed with brine, dried over anhydrous
Na2SO4, and concentrated. The residue was purified by column
chromatography (Hexane:DCM = 5:1, Rf = 0.35) to afford 1c as a
+
55.9, 45.2. HRMS-ESI (+) (m/z): [M + H]+ calcd. for C13H22N3O3 ,
268.1656; found: 268.1633.
4,4′-((5-Methoxy-2-nitro-1,3-phenylene)bis(methylene))-
dimorpholine (2f). Morpholine (387 mg, 4.40 mmol) was added to a
solution of 1d (150 mg, 0.44 mmol) in ethyl acetate (2 mL). The
resulting mixture was stirred at rt overnight and concentrated. The
residue was purified by column chromatography (DCM:Methanol =
10:1, Rf = 0.72) to afford 2f as a yellowish solid (143 mg, 92%), m.p.
1
88−89 °C. H NMR (300 MHz, CDCl3): δ 6.89 (s, 2H), 3.88 (s,
4H), 3.66 (s, 8H), 3.57 (s, 3H), 2.41 (s, 8H). 13C{1H} NMR (75
MHz, CDCl3): δ 160.2, 144.3, 133.7, 114.5, 66.8, 59.3, 55.8, 53.3.
+
HRMS-ESI (+) (m/z): [M + H]+ calcd. for C17H26N3O5 , 352.1867;
found: 352.1865.
((5-Methoxy-2-nitro-1,3-phenylene)bis(methylene))Bis-
(phenylsulfane) (2g). Into a solution of 1d (170 mg, 0.5 mmol) in
DMF (5.0 mL) was added thiophenol (165.3 mg, 1.5 mmol) followed
by trimethylamine (0.6 mL). The reaction mixture was stirred at 70
°C overnight, quenched with water (10 mL), and extracted with ethyl
acetate (3 × 15 mL). The organic layers were combined, washed with
brine, dried over anhydrous Na2SO4, and concentrated. The residue
was purified by column chromatography (Hexane:ethyl acetate = 5:1,
Rf = 0.45) to afford 2g as a yellowish solid (130 mg, 65%), m.p. 73−
1
white solid (5.88 g, 90%). H NMR (500 MHz, CDCl3): δ 6.92 (s,
2H), 4.56 (s, 4H), 4.00 (s, 3H), 3.82 (s, 3H) (the NMR spectra were
in agreement with those reported).54
(2,5-Dimethoxy-1,3-phenylene)bis(methylene) Diacetate (3a). A
solution of 7 (0.92 g, 5 mmol) in DCM (10 mL) was added 4-
dimethylaminopyridine (1.83 g, 15 mmol) and cooled to 0 °C. Then,
acetyl chloride (1.57 g, 20 mmol) was added using a syringe. The
reaction mixture was warmed up to rt, stirred for another 4 h,
quenched with H2O (8.0 mL), and extracted with DCM (3 × 20 mL).
The organic layers were combined, washed with brine, dried over
anhydrous Na2SO4, and concentrated. The residue was purified by
1
74 °C. H NMR (300 MHz, CDCl3): δ 7.30−7.26 (m, 10H), 6.65
(m, 2H), 4.15 (s, 4H), 3.64 (s, 3H). 13C{1H} NMR (75 MHz,
CDCl3): δ 160.1, 143.4, 134.7, 133.5, 131.4, 129.0, 127.4, 114.9, 55.6,
36.1. HRMS-ESI (+) (m/z): [M + K]+ calcd. for C21H19NO3S2K+,
436.0438; found: 436.0429.
((5-Methoxy-2-nitro-1,3-phenylene)bis(methylene))bis-
(phenylselane) (2h). Into a solution of diphenyl diselenide (368.3 mg,
J
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column chromatography (Hexane:Ethyl acetate = 1:1, Rf = 0.7) to
obtain 3a as a slightly yellowish liquid (1.28 g, 91%). H NMR (300
ethyl acetate (3 × 10 mL). The organic phases were combined,
washed with NaOH (1.0 M), brine, and dried over anhydrous
Na2SO4. The solvent was removed to afford 3f as a white solid (333
mg, 99%), m.p. 114−115 °C. 1H NMR (500 MHz, CDCl3): δ 6.92 (s,
2H), 3.81 (s, 3H), 3.78 (s, 3H), 3.74−3.72 (t, J = 5.0 Hz, 8H), 3.54
(s, 4H), 2.52 (s, 8H). 13C{1H} NMR (125 MHz, CDCl3): δ 155.5,
151.5, 132.1, 114.5, 67.2, 62.2, 57.1, 55.6, 53.7. HRMS-ESI (+) (m/
1
MHz, CDCl3): δ 6.90 (s, 2H), 5.15 (s, 4H), 3.78 (s, 6H), 2.11−2.10
(d, J = 3.0 Hz, 6H) [the NMR spectra were in agreement with those
reported].54 Different from the previous procedure (74% yield), 4-
dimethylaminopyridine was used as a catalyst instead of pyridine,
leading to a higher yield (91%)]. HRMS-ESI (+) (m/z): [M + Na]+
calcd. for C14H18O6Na+, 305.0996; found: 305.0985.
+
z): [M + H]+ calcd. for C18H29N2O4 , 337.2122; found: 337.2113.
2,5-Dimethoxy-1,3-bis(methoxymethyl)benzene (3b). To a sol-
ution of 7 (1.0 g, 5.04 mmol) in DMF (10 mL) was added NaH (605
mg, 60%, 15.12 mmol) at 0 °C. The reaction mixture was stirred at 0
°C for 10 min. Then, CH3I (2.86 g, 20.16 mmol) was added. The
reaction mixture was allowed to warm to rt, stirred for another 4 h,
quenched with water (20 mL), and extracted with ethyl acetate (3 ×
30 mL). The organic layers were combined, washed with brine, dried
over anhydrous Na2SO4, and concentrated. The residue was purified
by column chromatography (Hexane:Ethyl acetate = 1:1, Rf = 0.8) to
((2,5-Dimethoxy-1,3-phenylene)bis(methylene))bis-
(phenylsulfane) (3g). A solution of 1c (648 mg, 2.0 mmol) in DMF
(10 mL) was added thiophenol (661.2 mg, 6.0 mmol) followed by
trimethylamine (0.6 mL). The reaction mixture was stirred at 70 °C
overnight, quenched with water (20 mL), and extracted with ethyl
acetate (3 × 20 mL). The organic layers were combined, washed with
brine, dried over anhydrous Na2SO4, and concentrated. The residue
was purified by column chromatography (Hexane:ethyl acetate = 6:1,
Rf = 0.4) to afford 3g as a white solid (620 mg, 81%), m.p. 63−64 °C.
1H NMR (500 MHz, CDCl3): δ 7.40−7.38 (m, 4H), 7.33−7.30 (m,
4H), 7.25−7.22 (m, 2H), 6.74 (s, 2H), 4.18 (s, 4H), 3.86 (s, 3H),
3.66 (s, 3H). 13C{1H} NMR (125 MHz, CDCl3): δ 155.5, 150.2,
136.5, 131.6, 129.9, 129.0, 126.4, 115.0, 62.7, 55.5, 33.3. HRMS-ESI
(+) (m/z): [M + K]+ calcd. for C22H22O2S2K+, 421.0693; found:
421.0687.
((2,5-Dimethoxy-1,3-phenylene)bis(methylene))bis-
(phenylselane) (3h). A solution of diphenyl diselenide (624 mg, 2.0
mmol) in DMF (5.0 mL) was added NaBH4 (151 mg, 4.0 mmol) and
stirred at rt for 10 min. Then, 1c (324 mg, 1.0 mmol) in DMF (4.0
mL) was added. The resulting mixture was stirred overnight,
quenched with water (20 mL), and extracted with ethyl acetate (3
× 20 mL). The organic layers were combined, washed with brine,
dried over anhydrous Na2SO4, and concentrated. The residue was
purified by column chromatography (Hexane:DCM = 4:1, Rf = 0.5)
to afford 3h as a white solid (300 mg, 63%), m.p. 38−39 °C. 1H NMR
(500 MHz, CDCl3): δ 7.55−7.54 (m, 4H), 7.31−7.29 (t, J = 5.0 Hz,
6H), 4.16 (s, 4H), 3.86 (s, 3H), 3.59 (s, 3H). 13C{1H} NMR (125
MHz, CDCl3): δ 155.2, 149.8, 133.6, 132.9, 130.7, 129.1, 127.4,
114.9, 62.1, 55.4, 26.5. HRMS-ESI (+) (m/z): [M + K]+ calcd. for
C22H22O2Se2K+, 516.9586; found: 516.9580.
1
obtain 3b as a colorless liquid (1.10 g, 96%). H NMR (500 MHz,
CDCl3): δ 6.92 (s, 2H), 4.51 (s, 4H), 3.81 (s, 3H), 3.76 (s, 3H), 3.44
(s, 6H). 13C{1H} NMR (125 MHz, CDCl3): δ 156.0, 150.0, 132.3,
114.1, 69.4, 62.5, 58.3, 55.6. HRMS-ESI (+) (m/z): [M + Na]+ calcd.
for C12H18O4Na+, 249.1103; found: 249.1094.
1,3-Bis((allyloxy)methyl)-2,5-dimethoxybenzene (3c). A solution
of 7 (1.0 g, 5.04 mmol) in DMF (10 mL) was added NaH (605 mg,
60%, 15.12 mmol) at 0 °C and stirred for 10 min. Then, allyl iodide
(2.54 g, 15.12 mmol) was added. The reaction mixture was allowed to
warm to rt, stirred for 3 h, quenched with water (10 mL), and
extracted with ethyl acetate (3 × 30 mL). The organic layers were
combined, washed with brine, dried over anhydrous Na2SO4, and
concentrated. The residue was purified by column chromatography
(Hexane:Ethyl acetate = 3:1, Rf = 0.85) to obtain 3c as a colorless
1
liquid (1.20 g, 86%). H NMR (500 MHz, CDCl3): δ 6.95 (s, 2H),
6.03−5.96 (m, 2H), 5.37−5.33 (d, J = 20.0 Hz, 2H), 5.24−5.22 (d, J
= 10.0 Hz, 2H), 4.58 (s, 4H), 4.10−4.09 (d, J = 5.0 Hz, 4H), 3.81 (s,
3H), 3.76 (s, 3H). 13C{1H} NMR (125 MHz, CDCl3): δ 156.0,
150.1, 134.8, 132.4, 117.1, 114.2, 71.4, 67.0, 62.6, 55.6. HRMS-ESI
+
(+) (m/z): [M + H]+ calcd. for C16H23O4 , 279.1591; found:
279.1583.
((2,5-Dimethoxy-1,3-phenylene)bis(methylene))bis-
(triphenylphosphonium) Bromide (3i). A mixture of 1c (324 mg, 1.0
mmol) and triphenylphosphine (630 mg, 2.4 mmol) in dry toluene
(5.0 mL) was refluxed for 6 h under argon. The crude white powder
was obtained by filtration, which was washed with ether (3 × 20 mL)
((((2,5-Dimethoxy-1,3-phenylene)bis(methylene))bis(oxy))bis-
(methylene))dibenzene (3d). A solution of 7 (1.0 g, 5.04 mmol) in
DMF (10 mL) was cooled to 0 °C and added NaH (605 mg, 60%,
15.12 mmol). The resulting mixture was stirred for 10 min. Then,
benzyl chloride (1.91 g, 15.12 mmol) was added. The reaction
mixture was allowed to warm to rt, stirred for another 3 h, quenched
with water (10 mL), and extracted with ethyl acetate (3 × 30 mL).
The organic layers were combined, washed with brine, dried over
anhydrous Na2SO4, and concentrated. The residue was purified by
column chromatography (Hexane:Ethyl acetate = 3:1, Rf = 0.8) to
1
to afford 3i as a white foam (840 mg, 99%). H NMR (500 MHz,
CDCl3): δ 7.79−7.62 (m, 6H), 7.67−7.64 (m, 24H), 6.53 (s, 2H),
5.18−5.15 (d, J = 15.0 Hz, 4H), 3.17 (s, 3H), 2.86 (s, 3H): m.p. 270−
271 °C. 13C{1H} NMR (125 MHz, CDCl3): δ 151.5, 135.3, 134.2,
134.2, 134.1, 130.5, 130.5, 130.4, 123.3, 118.1, 117.9, 117.9, 117.9,
117.4, 55.5, 26.0, 25.6. HRMS-ESI (+) (m/z): [M − 2Br]2+ calcd. for
C46H42O2P22+, 344.1325; found: 344.1319.
Radical Trapping with Monomers. Compound 3i (100 mg,
0.118 mmol) in CH3CN (1.5 mL) was added TEMPO (184 mg, 1.18
mmol) under stirring at rt. The resulting mixture was irradiated under
UV (350 nm) for 5 days and concentrated. The residue was purified
by chromatography (DCM:MeOH = 10:1) to afford compound 11c
as a white foam (24 mg, 31%). 1H NMR (300 MHz, CDCl3): δ 7.74−
7.61 (m, 15 H), 6.97 (s, 1H), 6.69 (s, 1H), 5.23−5.19 (d, J = 12.0 Hz,
2H), 4.58 (s, 2H), 3.52−3.49 (d, J = 9 Hz, 6H), 1.47 (s, 4H), 1.34 (s,
2H), 1.15−1.11 (d, J = 12.0 Hz, 12H). 13C{1H} NMR (75 MHz,
CDCl3): δ 155.7, 155.7, 134.9, 134.4, 134.2, 130.1, 129.9, 121.1,
121.0, 118.7, 117.5, 116.5, 114.7, 72.9, 62.0, 60.0, 55.7, 39.7, 32.9,
20.7, 17.0. HRMS-ESI (+) (m/z): [M − Br]+ calcd. for
C37H45NO3P+, 582.3132; found: 582.3115.
1
afford 3d as a colorless liquid (1.52 g, 80%). H NMR (500 MHz,
CDCl3): δ 7.45−7.33 (m, 10H), 7.03 (s, 2H), 4.66 (s, 8H), 3.84 (s,
3H), 3.74 (s, 3H). 13C{1H} NMR (125 MHz, CDCl3): δ 156.0,
150.2, 138.3, 132.4, 128.5, 127.9, 127.7, 114.4, 72.6, 67.1, 62.6, 62.6,
55.7, 55.6. HRMS-ESI (+) (m/z): [M + Na]+ calcd. for
C24H26O4Na+, 401.1723; found: 401.1717.
1,1′-(2,5-Dimethoxy-1,3-phenylene)bis(N,N-dimethylmethan-
amine) (3e). A solution of 1c (500 mg, 1.54 mmol) in ethyl acetate (5
mL) was added dimethylamine solution (2.0 M in methanol) (3.85
mL, 7.7 mmol) and stirred at rt for 2 h. The solvent was removed.
Then, water (5 mL) was added. The resulting mixture was extracted
with ethyl acetate (3 × 10 mL), washed with NaOH (1.0 M), brine,
and dried over anhydrous Na2SO4. The solvent was removed to afford
1
3e as a slightly yellowish gel (350 mg, 90%). H NMR (500 MHz,
CDCl3): δ 6.85 (s, 2H), 3.76 (s, 3H), 3.69 (s, 3H), 3.42 (s, 4H), 2.24
(s, 12H). 13C{1H} NMR (75 MHz, CDCl3): δ 155.7, 151.1, 132.5,
114.6, 61.9, 57.9, 55.6, 45.4. HRMS-ESI (+) (m/z): [M + H]+ calcd.
Carbocation Trapping with Monomers. A solution of
MeONH2·HCl (394 mg, 4.72 mmol) in DMF (2 mL) was added
trimethylamine (567 mg, 5.20 mmol) and stirred at rt for 30 min.
Then, 3i (100 mg, 0.118 mmol) in DMF (1 mL) was added. The
resulting mixture was stirred for 20 min, irradiated with 350 nm light
for 2 days, quenched with water, and extracted with ethyl acetate (3 ×
3 mL). The combined organic phases were combined, washed with
+
for C14H25N2O2 , 253.1911; found: 253.1905.
4,4′-((2,5-Dimethoxy-1,3-phenylene)bis(methylene))-
dimorpholine (3f). A solution of 1c (324 mg, 1.0 mmol) in ethyl
acetate (4 mL) was added morpholine (871 mg, 10.0 mmol), stirred
at rt overnight, diluted with water (5 mL), and then extracted with
K
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brine, and dried over anhydrous Na2SO4. After removing the solvent,
the residue was analyzed by LC-MS. The products were analyzed by
both LC-MS and HRMS, indicating the formation of 11a and 11b.
11a: HRMS-ESI (+) (m/z): [M − Br]+ calcd. for C28H27O2PCl+,
461.1432; found: 461.1408. 11b: HRMS-ESI (+) (m/z): [M − Br]+
calcd. for C29H29NO3P+, 470.1880; found: 470.1839 (Note: the
amount of 11a or 11b obtained was not sufficient to conduct NMR
spectroscopic analysis due to an extremely slow and complex
reaction).
AUTHOR INFORMATION
Corresponding Author
■
Xiaohua Peng − Department of Chemistry and Biochemistry
and the Milwaukee Institute for Drug Discovery, University of
Wisconsin Milwaukee, Milwaukee, Wisconsin 53211, United
States; orcid.org/0000-0001-5627-0606; Phone: 414-
229-5221; Email: pengx@uwm.edu
ICL Assay with Duplex DNA. The 32P-labeled oligonucleotide
(0.5 μM) was annealed with 1.5 equiv of the complementary strand
by heating to 90 °C for 5 min in potassium phosphate buffer (pH 7,
10 mM), followed by cooling to rt. The 32P-labeled ODN duplex (2
μL, 0.5 μM) was then mixed with 1 M NaCl (2 μL), 100 mM
potassium phosphate (2 μL, pH 8), and 3a−i or 2a−i (concentration
range: 10 μM to 2 mM in 6 μL CH3CN), and autoclaved distilled
water to give a final volume of 20 μL. The reaction was irradiated
under UV (350 nm) at room temperature until the reaction was
completed followed by quenching with an equal volume of 90%
formamide loading buffer. The resulting mixture was then subjected
to 20% denaturing polyacrylamide gel for electrophoresis.
Trapping Assay with DNA ICL Reactions. For carbocation
trapping, the stock solution of MeONH2·HCl (2 M) was titrated with
NaOH (5 M) to adjust the pH to 7.0, which was then diluted to
desired concentration (1/3-1000/3 mM). A 6 μL solution of which
was mixed with 32P-labeled DNA duplex (2 μL, 0.5 μM), NaCl (2 μL,
1 M) potassium phosphate (2 μL, pH 8.0, 100 mM), compound (3a−
i or 2a−i) in 6 μL of CH3CN (optimized concentration was used for
each compound), and water (2 μL) to give the desired concentration
(final MeONH2 concentration: 100 μM to 100 mM). For radical
trapping reaction, 3 μL of TEMPO in CH3CN (200/3 μM to 2000/3
mM) was mixed with the following: 32P-labeled DNA duplex (2 μL,
0.5 μM), NaCl (2 μL, 1 M), potassium phosphate (2 μL, pH 8.0, 100
mM), compound (3a−i or 2a−i) in CH3CN (3 μL) (optimized
concentration was used for each compound), and water (8 μL) as
appropriate for the desired concentration (final TEMPO concen-
tration: 10 μM to 100 mM). The reaction mixture was irradiated with
350 nm light at room temperature for desired time (optimized time
for each compound was used), quenched with an equal volume of
90% formamide loading buffer, and then subjected to 20% denaturing
polyacrylamide gel electrophoresis.
Author
Heli Fan − Department of Chemistry and Biochemistry and
the Milwaukee Institute for Drug Discovery, University of
Wisconsin Milwaukee, Milwaukee, Wisconsin 53211, United
States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.joc.0c02234
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the Great Milwaukee Foundation
(Shaw Scientist Award), the University of Wisconsin
Milwaukee Research Growth Initiative, and the Wisconsin
Applied Research Grant.
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ASSOCIATED CONTENT
■
sı
* Supporting Information
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https://dx.doi.org/10.1021/acs.joc.0c02234
J. Org. Chem. XXXX, XXX, XXX−XXX