Design, Synthesis, and Characterization of Binaphthalene Precursors as Photoactivated DNA Interstrand Cross-Linkers

Article abstract of DOI:10.1021/acs.joc.8b00642

Most recently, alkylation via photogenerated carbocations has been identified as a novel mechanism for photoinduced DNA interstrand cross-link (ICL) formation by bifunctional aryl compounds. However, most compounds showed a low efficiency for DNA cross-linking. Here, we have developed a series of new 1,1′-binaphthalene analogues that efficiently form DNA ICLs upon 350 nm irradiation via generated 2-naphthalenylmethyl cations. The DNA cross-linking efficiency depends on the substituents at position 4 of the naphthalene moiety as well as the leaving groups. Compounds with NO₂, Ph, H, Br, or OMe substituents led to 2-4 times higher DNA ICL yields than those with a boronate ester group. Compounds with trimethylammonium salt as a leaving group showed slightly better cross-linking efficiency than those with bromo as a leaving group. Some of these compounds showed a better cross-linking efficiency than that of traditional alkylating agents, such as nitrogen mustard analogues or quinone methide precursors. These highly efficient photoactivated carbocation precursors allow determination and characterization of the adducts formed between the photogenerated naphthalenyl cations and four natural nucleosides, indicating that the alkylation sites for these naphthalene analogues are dG, dA, and dC.

Full text of DOI:10.1021/acs.joc.8b00642

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Article
Design, Synthesis, and Characterization of Binaphthalene
Precursors as Photo-Activated DNA Interstrand Cross-Linkers
Zechao Lin, Heli Fan, Qi Zhang, and Xiaohua Peng
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b00642 • Publication Date (Web): 22 Jun 2018
Downloaded from http://pubs.acs.org on June 25, 2018
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The Journal of Organic Chemistry
Design, Synthesis, and Characterization of Binaphthalene Precursors as PhotoꢀActivated DNA
Interstrand CrossꢀLinkers
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Zechao Lin, Heli Fan, Qi Zhang, and Xiaohua Peng*
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Department of Chemistry and Biochemistry and the Milwaukee Institute for Drug Discovery, University
of WisconsinꢀMilwaukee, 3210 N. Cramer Street, Milwaukee, Wisconsin, 53211, United States
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pengx@uwm.edu
Supporting Information Placeholder
ABSTRACT: Most recently, alkylation via photoꢀgenerated carbocations has been identified as a novel
mechanism for photoꢀinduced DNA interstrand crossꢀlink (ICL) formation by bifunctional aryl
compounds. However, most compounds showed a low efficiency for DNA crossꢀlinking. Here, we have
developed a series of new 1,1’ꢀbinaphthalene analogues that efficiently form DNA ICLs upon 350 nm
irradiation via generated 2ꢀnaphthalenylmethyl cations. The DNA crossꢀlinking efficiency depends on
the substituents at the positionꢀ4 of the naphthalene moiety as well as the leaving groups. Compounds
with NO2, Ph, H, Br, or OMe substituents led to 2ꢀ4 times higher DNA ICL yields than those with a
boronate ester group. Compounds having trimethyl ammonium salt as a leaving group showed slightly
better crossꢀlinking efficiency than those with bromo as a leaving group. Some of these compounds
showed a better crossꢀlinking efficiency than that of traditional alkylating agents, such as nitrogen
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mustard analogues or quinone methide precursors. These highly efficient photoꢀactivated carbocation
precursors allow determination and characterization of the adducts formed between the photoꢀgenerated
naphthalenyl cations and four natural nucleosides, indicating that the alkylation sites for these
naphthalene analogues are dG, dA, and dC.
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INTRODCUTION
Photochemical DNA crossꢀlinking agents have attracted attention for a variety of biological
applications.1, 2 They are used as antitumor and anticancer agents3, 4, for studying DNA damage and
repair5, for photoꢀmanipulation of DNA and therapeutic gene modulation6, and for constructing DNA
photoꢀswitches7, 8. Over the past few decades, several research groups have been focused on studies
involving photoꢀinduced DNA crossꢀlinking, leading to the discovery of a wide variety of
photochemically activated DNA alkylating agents.1, 2 Several reactive intermediates are involved in
photoꢀinduced DNA crossꢀlinking processes, such as quinone methides (QMs), free radicals, and
carbocations.1 Both nucleoside analogues and bifunctional aryl compounds have been developed to
generate DNA ICL formation upon photoꢀinduction. For example, the research groups of Zhou9, 10and
Freccero11ꢀ13 discovered a series of bifunctional phenol, biphenol or binol derivatives that are activated
by photoꢀirradiation to produce QMs directly crossꢀlinking DNA. However, fairly limited examples
have been reported to produce DNA ICL products via photoꢀgenerated carbocations. Most recently, Li14
and Greenberg15 demonstrated that photoirradiation of the modified thymidines generated 5ꢀ(2’ꢀ
deoxyuridinyl)methyl cations that directly produced DNA ICL products. Then, Peng and coworkers
found that several aryl boronates and bisnitroimidazole compounds induced DNA ICL formation via
photoꢀgenerated carbocations.16ꢀ18 However, most of these compounds showed a low efficiency for
DNA crossꢀlinking and/or required high concentrations of the substrates. For example, only 10% DNA
ICL yield was observed for bisnitroimidazole compounds,16 8% for bisnaphthalene boronates,18 about 2ꢀ
14% for most benzene derivatives except for methoxybenzene analogues.17 Development of new
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compounds with efficient DNA crossꢀlinking efficiency are essential for further investigation and
biological applications.
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The structure of naphthalene consists of a fused pair of benzene rings. They have the potential to be
applied in cellulo as they would undergo activation using light in visible region of spectra (wavelength >
350 nm) that is either not or only slightly absorbed by most biological molecules and is compatible with
living cells. Freccero and coꢀworkers reported that several 2,2’ꢀhydroxyl 1,1’ꢀbinaphthalene analogues
undergo UVꢀvis photoactivation to produce binolꢀquinone methide capable of bisalkylation and DNA
crossꢀlinking.12 In addition, several Binolꢀamino acid conjuagtes were found to undergo mild
photoactivation to yield alkylating and DNA crossꢀlinking agents with high photoꢀefficiency and
superior cytotoxicity.19 We have recently shown that binaphthalene boronates (1a and 1b, Scheme 1)
could be activated by 350 nm irradiation to generate free radicals that are oxidized to the corresponding
carbocations directly producing DNA ICL products.18 However, the DNA crossꢀlinking efficiency of 1a
and 1b is low with a maximum DNA ICL yield of 11.1% for 1a and 7.1% for 1b.
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The primary goals of the study presented herein were to find ways of improving the efficiency for
photogeneration of carbocations and subsequent DNA ICL formation via a systematic study on the
structureꢀreactivity relationships using the tools of chemical modifications. To this end, we designed and
synthesized a series of novel binaphthalene analogues (2aꢀe and 3aꢀe) with various aromatic
substituents and leaving groups and tested the influence of chemical structure on the carbocation
generation, DNA crossꢀlinking efficiency, crossꢀlinking sites, and the overall mechanism for DNA ICL
formation. In addition, we will also compare the DNA ICL efficiency of these new compounds to that of
1a and 1b that are precursors of cations and quinone methides. Comparison of 2aꢀe and 3aꢀe to 1a and
1b, conveniently absorbing at similar wavelengths, allows an evaluation of the relative crossꢀlinking
potency, reducing the effects due to poor absorption (SI, Figure S1). This study is part of a more
comprehensive study aimed at exploiting the promising features of binaphthalene compounds as
precursors of bisalkylating carbocations.
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O
O
R
5
R
B
4
3
6
Br
N
X
2
Br
7
1
8
8'
1'
7'
6'
2'
3'
X
Br
N
5'
4'
Br
B
R
R
O
O
3a
3b
3c
3d
: R = NO2;
: R = Br;
: R = H;
: R = Ph;
3e
: R = MeO.
2a
: R = NO2;
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2b
: R = Br;
2c
: R = H;
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1a
: X = Br;
2d
: R = Ph;
2e: R = MeO.
+
-
1b
: X = N (CH3)3Br .
Scheme 1. Bifunctional naphthalene derivatives 1ꢀ3.
RESULTS AND DISCUSSION
Synthesis of bifunctional binaphthalene derivatives. We first synthesized the bromides 2aꢀe that were
readily converted by nucleophilic substitution to the ammonium salts 3aꢀe (Scheme 1). In general, two
synthetic routes were employed to synthesize 4,4’ꢀbisubstituted 1,1’ꢀbinaphthalene analogues 2aꢀe,
either via direct coupling reaction of the corresponding 4ꢀsubstitued mononaphthalene precursor
(Scheme 2A and 2B) or via 4,4’ꢀbisbromoꢀ1,1’ꢀbinaphthalene analogue 5 by replacing 4ꢀbromo group
with the desired substituents (H, NO2, or phenyl group) (Scheme 2C). Compound 2b was synthesized
via a leadꢀcatalyzed coupling reaction of the 4ꢀbromonaphthalene precursor (4 → 5) followed by
bromination with Nꢀbromosuccinimide (NBS) and azobisisobutyronitrile (AIBN) (Scheme 2A).
Similarly, coupling reaction of 4ꢀmethoxy naphthalene analogue 7 with Pb(OCOCH3)4 as a catalyst
provided 1,1’ꢀbinaphthalene 8 that was converted to 2e via reduction (→9) followed by bromination
(→2e) (Scheme 2B). An electronꢀdonating group (OMe) favored the leadꢀcatalyzed coupling reaction
(42% for 8 vs. 18% for 5). However, direct coupling reaction of the naphthalenes with H, nitro, or
phenyl group at the positionꢀ4 failed to yield the desired 1,1’ꢀbinaphthalene products. The leadꢀ
catalyzed coupling reaction of 2ꢀmethylnaphthalene generated 3,3'ꢀdimethylꢀ1,1'ꢀbinaphthalene (10) and
several byꢀproducts with similar polarity, which hindered purification by column chromatography.
Direct coupling reaction of commercially available 2ꢀmethylꢀ1ꢀnitronaphthalene did not occur possibly
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due to the strong electron withdrawing effect of the nitro group prohibiting the reaction. Thus, 2a, 2c,
and 2d were synthesized starting from a more general precursor 5. Debromination of 5 was carried out
with nꢀBuLi yielding 10 that was converted to 2c by treatment with NBS/AIBN. Nitration of 10 using
HNO3/AcOH generated 4,4’ꢀdinitroꢀ1,1’ꢀbinaphthalene 11 that was brominated using NBS/AIBN
yielding 2a. A phenyl group was introduced by palladiumꢀcatalyzed coupling reaction of 5 yielding
3,3’ꢀdimethylꢀ4,4’ꢀdiphenylꢀ1,1’ꢀbinaphthalene (12). Bromination of 12 using NBS/AIBN produced 2d.
Finally, 2aꢀe were converted to the corresponding ammonia salts 3aꢀe by treatment with trimethyl amine
in ethyl acetate in almost quantitative yields.
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Scheme 2. Synthesis of bifunctional binaphthalene derivatives 2aꢀe and 3aꢀe.
DNA interstrand crossꢀlinking assay. With the availability of 4,4’ꢀdisubstituted 1,1’ꢀbinaphthalene
analogues 2aꢀe and 3aꢀe, we investigated the substituent effects on the photo reactivity of these
compounds towards DNA by measuring DNA ICL formation upon irradiation with 350 nm light.
Similar to our previous study18, a 49ꢀmer DNA duplex 13 was used for this study. Initially, 500 ꢁM of
the substrates were used and the reaction mixture was irradiated by 350 nm light for 6 h. DNA ICL
formation and crossꢀlinking yields were analyzed via denaturing polyacrylamide gel electrophoresis
(PAGE) with phosphorimager analysis (Image Quant 5.2). As these compounds showed different water
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solubility that could in turn affect their DNA crossꢀlinking efficiency, we optimized the reaction
conditions for DNA interstrand crossꢀlinking assay in order to find a condition suitable for all substrates.
First, we explored the effect of solvents on DNA ICL formation induced by these compounds. As some
of these compounds are lipophilic and showed poor water solubility, a miscible organic solvent, such as
MeCN, MeOH, DMF, THF, and DMSO, was added as an auxiliary solvent to improve the solubility. As
a representative, the result for 3b is illustrated in the supporting information (SI, Figure S2). Compared
with the pure water reaction system, the addition of organic solvents improved the crossꢀlinking
efficiency (SI, Figure S2, line 2 vs line 3ꢀ7). A mixture of water and acetonitrile (7:3) showed the
highest promoting effect, leading to 11% increase of DNA ICL yield of 3b (SI, Figure S2, lane 3, 30.3%
yield). Second, the effect of the pH values on DNA crossꢀlinking was investigated using a phosphate
buffer system (SI, Figure S3). The DNA crossꢀlinking yields of 37% – 39% were observed with a pH
value ranging from 6.0 to 8.0, while a phosphate buffer with a pH higher than 8.7 or lower than 5.0 led
to decreased DNA ICL yields (30.9% at pH 5 and 34.5% pH 8.7). In addition, compared to pure H2O
system (19.8%), a phosphate buffer of pH 6 ꢀ 8 greatly increased the DNA crossꢀlinking efficiency.
However, DNA damage was observed under strong basic or acidic conditions (pH 12.6 and pH 4.5) (SI,
Figure S3, lanes 2 and 8). Finally, a mixture of pH 8 phosphate buffer and acetonitrile (7:3) was used for
further investigation.
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Our previous study showed that the DNA crossꢀlinking efficiency depended on the irradiation time and
the concentration of the substrate.18 For better comparison of the reactivity of these naphthalene
analogues towards DNA and the effect of the aromatic substituent and the leaving group on DNA ICL
formation, we determined the optimal reaction time for DNA crossꢀlinking induced by 2aꢀe and 3aꢀe
and the compound concentration required to saturate alkylation sites in DNA. Study on timeꢀdependent
DNA ICL formation showed that the DNA crossꢀlinking reaction is complete within 8ꢀ11 h for these
compounds (Table 1 and SI, Figures S4ꢀ13). DNA damage was observed when the irradiation time was
longer than 24 h. The aromatic substituents and leaving groups only slightly affect the reaction rate of
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these naphthalene analogues. With the optimized reaction time, we determined the minimum
concentrations required to obtain the best DNA crossꢀlinking efficiency for 2aꢀe and 3aꢀe. Generally, the
crossꢀlinking efficiency was gradually improved with an increase in the concentration of the
compounds. The DNA crossꢀlinking reaction reached a balance for 2aꢀe at 0.5 mM concentration, while
a little higher concentration (about 1.0 mM) was required for 3aꢀe to saturate the alkylation sites (SI,
Figure S14ꢀ23).
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With the optimized conditions for all compounds, we were able to compare their DNA crossꢀlinking
efficiency. As these compounds absorb at similar wavelengths reducing the effects due to poor
absorption (SI, Figure S1), comparison of 2aꢀe and 3aꢀe to the existing binaphthalene boronate esters 1a
and 1b allows an evaluation of the relative crossꢀlinking potency. In comparison with 1a and 1b, all new
compounds greatly improved the DNA ICL yields. For example, the ammonium salts 3aꢀe led to 2ꢀ4
times higher ICL yields than 1b (Figure 1, Table 1). Similarly, the bromides 2aꢀe showed 12%ꢀ15%
higher ICL yields than 1a. We speculated that 1a and 1b may be more difficult to insert into the DNA
duplex structure due to the stericꢀhindrance of the boronate ester group. The ICL efficiency of these
photoꢀactivated alkylating agents (2aꢀe and 3aꢀe) are comparable to or better than traditional DNA
crossꢀlinking agents such as nitrogen mustard analogues (e.g. 20%ꢀ44% for 4ꢀ[bis(2ꢀ
chloroethyl)amino]phenol analogues at 1.0 mM concentration)20, 21 or quinone methide22 precursors.
Among 2aꢀe and 3aꢀe, compounds with a trimethyl amine as a leaving group showed slightly higher
DNA crossꢀlink yields than those with Br as a leaving group. Compound 3b with a 4ꢀbromo substituent
provided the highest DNA crossꢀlinking efficiency (Table 1). However, the introduction of NO2, Ph, or
OMe on the parent compound 2c or 3c did not show obvious effects for photoꢀinduced DNA ICL
formation of the binaphthalene moieties.
Table 1. The optimized conditions and ICL yields for 1ꢀ3.a
ICL yields (%)c
0.5 mM b
ICL yields (%)c
0.5 mM
1.0 mM b
Rea.
Time
(h)
Rea.
Time
(h)
Bromides
Ammonia Salts
1.0 mM
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1a
Boronate)
(R
=
1b
Boronate)
(R
=
8
13.8 ± 0.7 13.8 ± 0.9
4
8.7 ± 1.0
8.7 ± 0.8
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2
3
4
5
6
7
8
2a (R = NO2)
2b (R = Br)
2c (R = H)
2d (R = Ph)
2e (R = OMe)
8
8
10
10
10
25.6 ± 1.5 26.4 ± 1.0 3a (R = NO2)
28.3 ± 1.2 27.8 ± 1.2 3b (R = Br)
28.1 ± 2.1 28.4 ± 2.0 3c (R = H)
25.6 ± 1.1 25.8 ± 1.3 3d (R = Ph)
28.6 ± 1.0 28.4 ± 1.6 3e (R = OMe)
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11
10
33.2 ± 1.5 36.2 ± 1.6
36.3 ± 1.8 40.2 ± 1.7
30.6 ± 1.5 32.6 ± 1.5
29.3 ± 1.2 32.5 ± 1.3
30.8 ± 1.1 32.0 ± 1.0
a The DNA crossꢀlinking reaction was performed in a mixture of pH 8 phosphate buffer and MeCN (7:3) with
50 nM DNA duplex 13 upon 350 nm irradiation.
bThe minimum compound concentration needed to obtain the best crossꢀlinking efficiency.
c The maximum DNA ICL yield obtained for each compound under optimized conditions (all data are the
average of three experiments).
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Figure 1. UVꢀinduced DNA crossꢀlink formation by 1a, 1b, 2aꢀe, and 3aꢀe. Lane 1: DNA only; lane 2:
0.5 mM 1a (13.8%, 8 h); lane 3: 0.5 mM 1b (8.7%, 4 h); lane 4: 1 mM 3a (34.8%, 10 h); lane 5: 1 mM
3b (39.2%, 10 h); lane 6: 1 mM 3c (32.6%, 10 h); lane 7: 1 mM 3d (32.5%, 10 h); lane 8: 1 mM 3e
(32.0%, 10 h); lane 9: 0.5 mM 2a (25.6%, 8 h); lane 10: 0.5 mM 2b (27.1%, 8 h); lane 11: 0.5 mM 2c
(28.1%, 10 h); lane 12: 0.5 mM 2d (28.8%, 10 h); lane 13: 0.5 mM 2e (31.6%, 10 h) [Irradiated at pH 8
32
phosphate buffer and mixture solvent of MeCN/H2O (3:7). ODN 13a was labelled with P at the 5’ꢀ
termini].
Mechanism of DNA ICL formation. In our previous work, we have revealed that photoꢀinduced DNA
ICL formation of several arylboronates was via carbocation formation17, 18. The pathway for generating
the carbocation intermediate A strongly depends on the substrates (Scheme 3a). The benzyl cations were
generated either via direct heterolysis of a CꢀX bond (Pathway 1) or oxidation of a free radical B
(Pathway 2), which was dependent on the leaving groups.17 For binaphthelene boronates 1a and 1b, the
carbocations that directly crossꢀlink DNA were generated via oxidation of the corresponding free radical
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B (Scheme 3a, Pathway 2).18 However, only two compounds have been reported so far for the category
of naphthalene compounds and both contain a boronate group, which is not sufficient to fully
understand the property of this novel mechanism. In order to test the generality of this mechanism, we
investigated the DNA crossꢀlinking mechanism of 2aꢀe and 3aꢀe by determining the reactive
intermediates responsible for DNA ICL formation. Two orthogonal traps, methoxyamine and 2,2,6,6ꢀ
tetramethylpiperidinꢀ1ꢀoxyl (TEMPO) that can trap carbocations and free radicals respectively, were
employed for this study. The two traps were then tested separately as competitors for DNA ICL
formation upon 350 nm irradiation of 2aꢀe and 3aꢀe with DNA duplex 13. The effect of methoxyamine
and TEMPO on DNA ICL formation induced by 2b and 3b is illustrated in Figure 2, while the data of
all other compounds are presented in Figures S24ꢀ43. The addition of either methoxyamine or TEMPO
decreased DNA crossꢀlinking yields of 2b and 3b, suggesting that both methoxyamine and TEMPO
inhibited DNA ICL formation induced by 2b and 3b (Figure 2). The crossꢀlink yield of 3b decreased
from 31.4 ± 1.5% to 1.8 ± 0.7% with 100 mM methoxyamine and from 32.8 ± 1.3% to 2.4 ± 0.6% with
100 mM TEMPO (Figure 2B). Complete quenching of DNA ICL formation by methoxyamine indicated
that the carbocation intermediates directly crossꢀlinked DNA, while complete inhibition of DNA ICL
formation by the radical trap suggested that the cation intermediates were generated via oxidation of the
corresponding free radical (pathway 2) not by direct heterolysis of a CꢀX bond (pathway 1). This is
consistent with our previous study for 1a and 1b.18 Based on these observations, we proposed that 350
nm irradiation of 2b or 3b generated the 2ꢀnaphthalenylmethyl free radical 14b that was oxidized to the
corresponding cation 15b directly alkylating DNA. Similarly, complete inhibition of DNA ICL
formation by methoxyamine and TEMPO was observed with 2a, 2cꢀe, 3a, and 3cꢀe (SI, Figure S24ꢀ44),
which suggested the generality of this novel mechanism for the naphthalene moieties (Scheme 3).
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Scheme 3. (A) General mechanisms for photoꢀinduced DNA ICL formation by bifunctional aromatic
compounds ( represents the aromatic rings ; (B) Proposed mechanism for DNA ICL formation
)
induced by 2aꢀe and 3aꢀe.
B.
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Figure 2. Effect of TEMPO and methoxyamine on ICL formation induced by 2b (A) and 3b (B) (DNA
duplex 13 was irradiated with 350 nm light in the presence of 0.5 mM 2b or 3b at pH 8 for 8 h).
Having demonstrated that both free radicals and carbocations were actively involved in DNA crossꢀ
linking process of 2aꢀe and 3aꢀe, we obtained the carbocation and free radical trapping products using
the monomer reactions. The carbocation and free radical trapping reactions were performed with the
monomers upon 350 nm irradiation in the presence of excess methoxyamine and TEMPO, respectively.
Compounds 2b and 3b were chosen as two representatives for this study. The reaction was carried out in
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a pH 8.0 phosphate buffer at 37 ºC. As expected, 350 nm irradiation of 2b or 3b in the presence of
TEMPO provided free radical trapping product 18 while the cation trapping product 21 was obtained in
the presence of methoxyamine (Scheme 4). Compound 18 was characterized by NMR and HRMS,
while 21 was unstable under the conditions for NMR measurement. Thus, mass spectrometry was used
to determine the product. Detection of fragment ion of m/z 528.0040 [IT–TOF–MS (ESI)] suggested
formation of 21 (Scheme 4B). In addition, we synthesized a simpler analogue 22 and obtained its cation
trapping product 23 by 350 nm irradiation of 22 in the presence of excess methoxyamine (Scheme 4C).
This study provided direct evidence for that both free radicals and carbocations were involved in the
DNA crossꢀlinking process induced by 2b and 3b upon 350 nm irradiation. Collectively, our data
provide further evidence for the mechanism proposed for photoꢀinduced DNA ICL formation by these
compounds (Scheme 3b).
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Scheme 4. A) Free radical trapping reactions of 2b and 3b with TEMPO; B) Cation trapping reactions
of 2b and 3b with methoxyamine; C) Cation trapping reaction of 22 with methoxyamine.
Determination of DNA alkylation sites and alkylating products. Testing the stability of the DNA ICL
products upon heating under basic or neutral conditions can be used for initial detection of heatꢀlabile
and/or alkalineꢀlabile DNA alkylation sites.21 According to the Maxam and Gilbert reaction mechanism,
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it is well known that N7ꢀalkylated purines can be cleaved upon heating in piperidine treatment.23, 24
Thus, the alkylation sites of 2aꢀe and 3aꢀe towards DNA were investigated by determining the heat
stability of DNA ICL products formed with these compounds. Initially, the stability test of ICL products
was performed with crude materials purified by simple precipitation without gel purification. The heat
stability data for DNA ICL products formed with 2aꢀe are shown in Figures S45. In all cases, the DNA
ICL products were destroyed by heating in 1.0 M piperidine for 30 min, while they were more stable to
heating in a pH 7 phosphate buffer. DNA cleavage bands were observed mainly at dG sites but only
minorly at dA sites upon heating in 1.0 M piperidine (Figure S45, lanes 3, 7, and 11), which indicated
that photoꢀinduced DNA alkylation by these compounds mainly occurred with dG. To figure out
whether DNA cleavage by piperidine treatment occurred to crossꢀlinked DNA or monoꢀalkylated DNA
or both, we isolated the DNA ICL products formed with 3b from other alkylated singleꢀstranded DNA
species (ODN 13a’) by denaturing PAGE. Similar to the above observation, isolated crossꢀlinked DNA
showed higher stability upon heating in pH 7 buffer (Figure 3, lane 5) than in 1.0 M piperidine (Figure
3, lane 6). Similar cleavage patterns (with major cleavages at dG sites) were observed for crossꢀlinked
ODN and singleꢀstranded ODN 13a’ upon heating in 1.0 M piperidine (Figure 3, lane 3 vs lane 6).
These data suggested that in addition to interstrand crossꢀlinking, intrastrand crossꢀlinking and monoꢀ
alkylation could also occur at dG sites.
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Figure 3. Determination of crossꢀlinking site of 3b. Phosphorimage autoradiogram of 20% denaturing
PAGE analysis of the isolated ICL products and alkylated singleꢀstranded ODN 13a’ upon heating in pH
7 phosphate buffer or piperidine. Lane 1: isolated alkylated 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: 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.
Although the heatꢀstability study of the DNA ICL products provided evidences that the major alkylating
sites are dGs, the reaction sites stable to heating could not be detected. Hopkins and coꢀauthors reported
that hydroxyl radical cleavage of gelꢀpurified crossꢀlinked DNA could be used to determine the crossꢀ
linking site(s) in instances in which multiple alkylation/crossꢀlinking sites are present in a single
molecule25. Only fragmentation between the radiolabel and the crossꢀlinked nucleotide affords
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fragments shorter than starting single strand, and thus the abundance of fragment sizes longer than
crossꢀlink sites would decrease, which reveals sites of crossꢀlinking. Instead of the original 49mer DNA
duplex 13, a shorter DNA duplex 27 was used for this study to ensure determination at single nucleotide
resolution of DNA fragments by denaturing PAGE. Each oligonucleotide was either 5’ꢀ32Pꢀ or 3’ꢀ32Pꢀ
labeled. As anticipated, the abundance of dGꢀ or dCꢀ cleavage fragments is higher than that of dAꢀ or dT
cleavage fragments. For example, the cleavage pattern of the isolated crossꢀlinked material with 5’ꢀ
[32P]ꢀlabelled 27b showed that relative to the native sample, the yield of fragments 7 or 8 nucleotides
long was diminished relative to fragments 5 or 6 nucleotides long (Figure 4A,B). Similarly, the
abundance of cleavage fragments from C46 to T51, C53 to T56, then C58 greatly decreased in
comparison with that of G45, G52, or G57 cleavage fragments. These data suggested that the majority
of the crossꢀlinks occurred with dG. In addition, the ratios of corresponding peak areas from oneꢀ
dimensional scan of the crossꢀlinked and native samples were calculated (Figure 4C). It was obvious
that fragment yields dropped following C42, C46, C49, and C53, indicating that dCs are possible crossꢀ
linking sites. Similarly, cleavage pattern of the isolated crossꢀlinked material with 3’ꢀ[32P]ꢀlabelled 27a
indicated that the crossꢀlinks in the complementary strand involved reaction with G31, C28, G27, G24,
C21, and G20 (Supporting Information Figure S46). Cleavage pattern of DNA crossꢀlinked products
with 5’ꢀ[32P]ꢀlabelledꢀ27a or 3’ꢀ[32P]ꢀlabeledꢀ27b also indicated that the possible crossꢀlink sites are
dGs and dCs. However, we could not succeed in any of our efforts in quantification of various
interstrand crossꢀlink locations, possibly due to complications of multiple crossꢀlink sites (more than 16
in duplex 27) and multiple crossꢀlinking events occurred per duplex.
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T41 C42 A43A44G45C46T47 A48C49T50T51G52C53 A54T55T56 G57C58
Cleavage site
Figure 4. Determination of interstrand crossꢀlinking position via OH⋅ cleavage of 3bꢀcrossꢀlinked DNA
duplex 27: A) partial fragmentation patterns for native duplex 27 using iron(II)/EDTA; B) partial
fragmentation patterns for 3bꢀcrossꢀlinked 27 using iron(II)/EDTA; C) Ratio of fragment abundance in
3bꢀcrossꢀlinked and native DNA duplex 27 as a function of cleavage site. ODN 27b was [32P]ꢀlabelled
at the 5'ꢀtermini.
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Previously, the structure of the crossꢀlinked adducts formed by a naphathalene cation have not been
determined due to the poor photoreactivity of 1a and 1b towards DNA18. In order to gather further
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evidence for the DNA crossꢀlinking sites of the naphathalene analogues, we performed a monomer
reaction by examining the reactivity of photoꢀgenerated 2ꢀnaphthelenemethyl cation with four canonical
nucleosides (dT, dC, dA, and dG). As a representative, 2b was chosen for this study. Compound 2b was
irradiated in DMF in the presence of dT, dC, dA, or dG with 350 nm light until disappearance of all
starting material. Initially, thin layer chromatography (TLC) was used to monitor the reaction, which
indicated formation of the new products for dC, dA, and dG but not for dT. The crude products
generated between 2b and dC or dA were further purified by column chromatography providing 24
(41% yield) and 25a (49% yield), respectively, which were characterized by NMR and HRMS (Scheme
5A and 5B). However, 25a was unstable and deglycosylation occurred, resulting in 25b shown by
HRMS (Scheme 5B). However, the adducts formed between 2b and dG were too complex to be
purified. In addition, TLC showed that decomposition occurred during the purification process leading
to the new byꢀproducts. NMR data were not obtained for any 2bꢀdG adducts. Finally, HRMS was used
to detect adducts formed between 2b and dG, indicating formation of 26aꢀd (Scheme 5C).
Deglycosylation of 26b generated 26d. Although the molecular ion peak of 26e was not detected, its
deglycosylated product 26c was observed. Thus, we conclude that the DNA ICL formation induced by
these 2ꢀnaphthelenemethyl cations occurred with both dG and dC, while monoꢀalkylation would also
occur with dA, dG, and dC.
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Scheme 5. Monomer reactions of 2b and the nucleosides dG, dA, and dC.
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CONCLUSION
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Carbocation formation has been discovered to be an important mechanism for photoꢀinduced DNA
crossꢀlinking by bifunctional aryl compounds,17, 18 such as 1,1’ꢀbinaphthalene boronate esters 1a and 1b.
However, very low ICL efficiency was observed with 1a and 1b. Here, we performed a structureꢀ
reactivity relationship study and discovered a series of bifunctional naphthalene compounds as highly
efficient photoꢀinduced DNA crossꢀlinking agents. The aromatic substituents at the positionꢀ4 of the
naphthalene moiety and the leaving groups affect the DNA crossꢀlinking efficiency. Compounds
containing nitro, bromo, hydrogen, phenyl, or methoxy showed 2ꢀ4 times higher crossꢀlinking potency
than 1a or 1b with boronate ester. Trimethyl ammonium salt as a leaving group led to slightly higher
DNA crossꢀlinking efficiency than bromo as a leaving group. These compounds showed a DNA crossꢀ
linking mechanism similar to 1a and 1b. They can be activated by 350 nm irradiation to generate free
radicals that are oxidized to the corresponding carbocations directly producing DNA ICL products. The
discovery of these highly efficient photoꢀactivated DNA interstrand crossꢀlinkers allowed us to
determine the adducts formed between the photoꢀgenerated naphthalene cations and four canonical
nucleosides. Alkylation reaction of the photoꢀgenerated 2ꢀnaphthelenemethyl cation occurred with dG,
dC, and dA. Development of new compounds with efficient DNA crossꢀlinking efficiency are essential
for further investigation and biological applications.
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EXPERIMENTAL SECTION
General Information. Unless otherwise specified, all chemicals and reagents were commercially
purchased and were used as received without further purification. Thinꢀlayer chromatography (TLC)
was carried out on precoated silica gel plates and visualized under UV light. Oligonucleotides were
synthesized via standard automated DNA synthesis techniques. Deprotection of the synthesized DNA
were performed under mild deprotection conditions using a mixture of 40% aqueous MeNH2 and 28%
aqueous NH3 (1:1) at room temperature for 2 h. Oligonucleotides were purified by 20% denaturing
PAGE. [γꢀ32P] ATP was used for DNA labeling with standard method.26 Quantification of radiolabeled
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oligonucleotides was carried out using a Molecular Dynamics phosphorimager equipped with
ImageQuant, version 5.2, software. For all Phosphorimage autoradiogram, the top dark spot that
migrated slowest is considered as DNA crossꢀlink product and the bottom dark spot considered as the
singleꢀstranded ODN, which were determined using molecular ladder.20 1H NMR and 13C NMR spectra
were collected on a Bruker DRX 500 MHz or 300 MHz spectrophotometer with TMS as internal stander.
High resolution mass spectrometry was performed at the University of WisconsinꢀMilwaukee Mass
Spectrometry Lab on a Shimadzu LCMSꢀITꢀTOF mass spectrometer equipped with an atmosphericꢀ
pressure chemical ionization (APCI) or electron spray injection (ESI).
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Compound Synthesis and Characterization.
4,4'ꢀDibromoꢀ3,3'ꢀdimethylꢀ1,1'ꢀbinaphthalene (5). Boron trifluoride diethyl etherate (20 mL) was
added to a solution of 1ꢀbromoꢀ2ꢀmethylnaphthalene (4) (17.68 g, 0.08 mol) and lead tetraacetate (19.52
g, 0.044 mol) in acetonitrile (100 mL). The reaction mixture was stirred overnight, then poured into
water (300 mL), and the products were extracted with dichloromethane (2 x 300 mL). The organic layer
was dried over Na2SO4, filtrated, and evaporated. The crude product was passed through a short column
of basic alumina using hexane as eluent to remove the Pb and highly colored polymeric materials, then
purified through column chromatography (2.5% EtOAc/Hexane) to provide 5 (3.09 g, 18%) as a white
1
solid. H NMR (300 MHz, CDCl3): δ 8.44 (d, J = 4.4 Hz, 2H), 7.59 (t, J = 4.5 Hz, 2H), 7.39 (s, 2H),
7.32ꢀ7.28 (m, 4H), 2.71 (s, 6H) (the NMR spectra were in agreement with those reported). 18, 27
3,3'ꢀDimethylꢀ1,1'ꢀbinaphthalene (10). nꢀBuLi (5.8 mL, 9.2 mmol, 1.6 M in hexane) was added to a
solution of 5 (1.0 g, 2.3 mmol) in THF (30 mL) at ꢀ78 ℃. The reaction mixture was stirred about 4 h.
Then, water (15 mL) was added. After the reaction mixture was stirred overnight, THF was removed
under vacuum. The remaining aqueous phase was extracted with dichloromethane (3 x 15 mL). The
organic layer was dried over MgSO4, filtrated, and evaporated. The crude product was purified through
1
column chromatography (pure hexane) to provide 10 as a white solid (539 mg, 83%). H NMR (300
MHz, CDCl3): δ 7.90 (t, J = 6.1 Hz, 2H), 7.77 (br, 2H), 7.50ꢀ7.40 (m, 6H), 7.27 (d, J = 6.3 Hz, 2H),
2.64 and 2.62 (2S, 6H)
.
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4,4'ꢀDinitroꢀ3,3'ꢀdimethylꢀ1,1'ꢀbinaphthalene (11). Compound 10 (1.0 g, 3.5 mmol) was dissolved in
glacial acetic acid (15 mL), then 70% HNO3 (5 mL) was added with stirring in an ice bath. The reaction
mixture was stirred at r.t. for 24 h. TLC (Hexane/CH2Cl2 = 10/1) showed that the reaction was complete
with disappearance of 10. Water (10 mL) was added to the reaction mixture that was stirred for 0.5 h
and filtrated to remove the light yellow solid. The organic layer was collected, dried over MgSO4, and
evaporated. The crude product was purified through column chromatography (Hexane/CH2Cl2 = 50/1)
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to provide 11 as a white solid (0.39 g, 30%): m.p. 210ꢀ212 ℃. 1H NMR (300 MHz, CDCl3):
δ 7.75ꢀ7.72
13
(m, 2H), 7.57ꢀ7.55 (m, 2H), 7.29ꢀ7.27 (m, 6H), 2.48 (s, 6H); C NMR (75 MHz, CDCl3): δ 148.0,
139.5, 131.4, 129.9, 128.8, 127.2, 126.9, 126.4, 124.8, 121.6, 17.8. IT–TOF–MS (ESI): m/z [M]2+ calcd.
for C22H16N2O4 186.0550, found 186.0547.
3,3'ꢀBis(bromomethyl)ꢀ4,4'ꢀdinitroꢀ1,1'ꢀbinaphthalene (2a). A solution of 11 (0.32 g, 0.86 mmol), NBS
(0.32 g, 1.8 mmol), and AIBN (14.0 mg, 0.08 mmol) in benzene (20 mL) was heated to reflux with
incandescent light and stirred for 24 h. Three additional portions of NBS (0.16 g, 0.9 mmol) and AIBN
(7.0 mg, 0.04 mmol) were added at 24 h – intervals within 72 h. The mixture was allowed to cool to
room temperature and evaporated. CH2Cl2 (50 mL) was added and the organic layer was washed with
water, brine, dried with anhydrous Na2SO4, and concentrated under reduced pressure, then purified
through column chromatography (Hexane/CH2Cl2 = 20/1) to give 2a (87 mg, 19%) as white solid: m.p.
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166ꢀ168 ℃. H NMR (300 MHz, CDCl3):
δ 8.50 (d, J = 8.4 Hz, 2H), 7.65ꢀ7.60 (m, 2H), 7.55 (s, 2H),
13
7.40ꢀ7.32 (m,4H),4.91 (s, 4H); C NMR (75 MHz, CDCl3): δ 137.4, 134.6, 133.4, 132.5, 129.4, 128.1,
128.0, 127.6, 126.7, 125.3, 34.5. IT–TOF–MS (ESI): m/z [M]+ calcd. for C22H14Br2N2O4 527.9315,
found 527.9310.
1,1'ꢀ(4,4'ꢀDinitroꢀ[1,1'ꢀbinaphthalene]ꢀ3,3'ꢀdiyl)bis(N,N,Nꢀtrimethylmethanaminium) bromide (3a). A
4.2 M trimethylamine solution in ethanol (2.4 mL, 10 mmol) was added to a mixture of 2a (530.2 mg, 1
mmol) in EtOAc (10 mL). The reaction mixture was stirred at room temperature for 24 h and filtrated to
give 3a (596.5 mg, 92%) as a white solid: m.p. 236ꢀ238 ℃; 1H NMR (500 MHz, CD3OD):
δ
8.64 (d, J =
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8.7 Hz, 2H), 7.98 (s, 2H), 7.79 (t, J = 8.1 Hz, 2H), 7.57 (t, J = 6.9 Hz, 2H), 7.45 (d, J = 8.4 Hz, 2H),
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5.19 (q, J = 15.6 Hz, 4H), 3.38 (s, 18H); C NMR (125 MHz, CD3OD): δ 137.3, 134.2, 132.6, 131.9,
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7
129.5, 128.9, 128.8, 126.6, 125.9, 68.6, 52.9. IT–TOF–MS (ESI): m/z [M]+ calcd. for C28H32Br2N4O4
646.0785, found 646.0781.
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4,4'ꢀDibromoꢀ3,3'ꢀbis(bromomethyl)ꢀ1,1'ꢀbinaphthalene (2b). A solution of 5 (5.3 g, 12.0 mmol), NBS
(4.27 g, 24.0 mmol), and AIBN (0.20 g, 1.20 mmol) in benzene (50 mL) was heated to reflux and stirred
for 6 h. The reaction mixture was allowed to cool to room temperature and evaporated. CH2Cl2 (50 mL)
was added and the organic layer was washed with water (50 mL), brine (50 mL), dried over anhydrous
sodium sulfate, and concentrated under reduced pressure, then purified through column chromatography
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(Hexane/CH2Cl2 = 50/1) to give 2b (3.80 g, 53%) as a white solid: m.p. 154ꢀ156 ℃; H NMR (300
MHz, CDCl3):
δ 8.49ꢀ8.45 (m, 2H), 7.66ꢀ7.61 (m, 2H), 7.56ꢀ7.54 (m, 2H), 7.38ꢀ7.35 (m, 4H), 4.90 (s,
4H); 13C NMR (75 MHz, CDCl3): δ 137.4, 134.6, 133.4, 132.5, 129.4, 128.1, 128.0, 127.6, 126.7, 125.3,
34.5. IT–TOF–MS (ESI): m/z [M]+ calcd. for C22H14Br4 593.7823, found 593.7820.
1,1'ꢀ(4,4'ꢀDibromoꢀ[1,1'ꢀbinaphthalene]ꢀ3,3'ꢀdiyl)bis(N,N,NꢀtrimethylMethanaminium) bromide (3b). A
4.2 M trimethylamine solution in ethanol (2.4 mL, 10 mmol) was added to a mixture of 2b (598 mg, 1.0
mmol) in EtOAc (10 mL). The reaction mixture was stirred at room temperature for 24 h and filtrated to
give 3b (680 mg, 93% yield) as a white solid: m.p. 246ꢀ248 ℃; 1H NMR (300 MHz, CD3OD):
δ 8.58 (t,
J = 7.8 Hz, 2H), 7.91 (s, 2H), 7.74 (d, J = 8.1 Hz, 2H), 7.52 (d, J = 6.9 Hz, 2H), 7.40 (t, J = 7.8 Hz, 2H),
13
5.18ꢀ5.09 (m, 4H), 3.34 (s, 18H); C NMR (75 MHz, CD3OD): δ 137.3, 134.2, 132.6, 131.9, 129.5,
128.9, 128.8, 128.6, 126.6, 125.9, 68.6, 52.9. IT–TOF–MS (ESI): m/z [Mꢀ2Br]2+ calcd. for C28H32Br2N2
277.0461, found 277.0459.
3,3'ꢀBis(bromomethyl)ꢀ1,1'ꢀbinaphthalene (2c). A solution of 10 (3.39 g, 12.0 mmol), NBS (4.27 g, 24.0
mmol), and AIBN (0.20 g, 1.20 mmol) in benzene (50 mL) was heated to reflux and stirred for 6 h. The
mixture was allowed to cool to room temperature and evaporated. CH2Cl2 (50 mL) was added and the
organic layer was washed with water (50 mL), brine (50 mL), dried over anhydrous sodium sulfate,
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concentrated under reduced pressure, then purified through column chromatography (Hexane/CH2Cl2 =
1
2
3
1
20/1) to give 2c (2.06 g, 39% yield) as a white solid: m.p. 160ꢀ162 ℃; H NMR (300 MHz, CDCl3):
δ
4
5
6
7
8
9
13
7.87ꢀ7.84 (m, 4H), 7.46ꢀ7.41 (m, 4H), 7.27ꢀ7.25 (m, 4H), 4.64 (s, 4H); C NMR (75 MHz, CDCl3): δ
138.7, 134.7, 133.4, 132.4, 128.6, 128.3, 128.0, 126.8, 126.5, 126.3, 33.8; IT–TOF–MS (ESI): m/z
[M]2+ calcd. for C22H16Br2 218.9804, found 218.9804.
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1,1'ꢀ([1,1'ꢀBinaphthalene]ꢀ3,3'ꢀdiyl)bis(N,N,Nꢀtrimethylmethanaminium) bromide (3c). A 4.2 M
trimethylamine solution in ethanol (2.4 mL, 10 mmol) was added to a mixture of 2c (438 mg, 1.0 mmol)
in EtOAc (10 mL). The reaction mixture was stirred at room temperature for 24 h and filtrated to give
1
3c (513 mg, 92% yield) as a white solid: m.p. 147ꢀ150 ℃. H NMR (300 MHz, DMSO):
δ 8.33 (s, 2H),
8.18 (d, J = 8.1 Hz, 2H), 7.71 (s, 2H), 7.66 (t, J = 7.5 Hz, 2H), 7.49 (t, J = 6.9 Hz, 2H), 7.40 (d, J = 8.4
Hz, 2H), 4.85 (s, 4H), 3.18 (s, 18H); 13C NMR (75 MHz, CD3OD): δ 140.1, 135.1, 134.7, 134.6, 132.1,
130.2, 129.4, 128.4, 127.2, 126.4, 70.4, 53.5; IT–TOF–MS (ESI): m/z [Mꢀ2Br]2+ calcd. for C28H34N2
199.1356, found 199.1351.
3,3'ꢀDimethylꢀ4,4'ꢀdiphenylꢀ1,1'ꢀbinaphthalene (12). K2CO3 (4.2 mL, 2 M in water) was added to a
mixture of 5 (0.44 g, 1.00 mmol), PhB(OH)2 (0.43 g, 3.50 mmol), and Pd(PPh3)4 (0.17 g, 0.15 mmol) in
THF (15 mL) under nitrogen. The reaction mixture was heated to reflux and stirred under refluxing for
24 h. Water (15 mL) was added to quench the reaction. Then, the crude products were extracted with
CH2Cl2 (3 x 15 mL). The organic layer was dried over anhydrous Na2SO4, concentrated under reduced
pressure, then purified through column chromatography (Hexane/CH2Cl2 = 30/1) to give 12 (0.29 g,
1
66%) as a white solid: m.p. 243ꢀ246 ℃; H NMR (300 MHz, CDCl3):
δ 7.46ꢀ7.16 (m, 20H) , 2.23 (s,
6H); 13C NMR (75 MHz, CDCl3): δ 139.9, 138.0, 137.8, 133.0, 132.6, 131.4, 130.6, 130.3, 130.3, 128.4,
127.1, 126.6, 126.4, 125.6, 124.8, 20.8; IT–TOF–MS(ESI): m/z [M+H]+ calcd. for C34H27 435.2107,
found 435.2103.
3,3'ꢀBis(bromomethyl)ꢀ4,4'ꢀdiphenylꢀ1,1'ꢀbinaphthalene (2d). A solution of 12 (5.21 g, 12.0 mmol),
NBS (4.27 g, 24.0 mmol), and AIBN (0.20 g, 1.20 mmol) in benzene (50 mL) was heated to reflux and
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stirred for 6 h. The reaction mixture was allowed to cool to room temperature and evaporated. CH2Cl2
(50 mL) was added and the organic layer was washed with water (50 mL), brine (50 mL), dried over
anhydrous sodium sulfate, and concentrated under reduced pressure, then purified through column
1
2
3
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7
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chromatography (Hexane/CH2Cl2 = 20/1) to give 2d (3.13 g, 44% yield) as a white solid: m.p. 287ꢀ
9
1
289 ℃; H NMR (300 MHz, CDCl3):
δ 7.71 (brs, 2H), 7.56 (brs, 12H), 7.50 (brs, 2H), 7.37 (brs, 4H),
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4.50 (s, 4H); C NMR (75 MHz, CDCl3): δ 139.4, 138.3, 137.6, 133.0, 132.7, 132.4, 130.3, 130.1,
129.5, 128.5, 127.9, 127.4, 126.6, 126.5, 127.3, 32.8; IT–TOF–MS (ESI): m/z [M+3H]3+ calcd. for
C34H24Br2 197.6821, found 197.6825.
1,1'ꢀ(4,4'ꢀDiphenylꢀ[1,1'ꢀbinaphthalene]ꢀ3,3'ꢀdiyl)bis(N,N,Nꢀtrimethylmethanaminium) bromide (3d). A
4.2 M trimethylamine solution in ethanol (2.4 mL, 10 mmol) was added to a mixture of 2d (592 mg, 1
mmol) in EtOAc (10 mL). The reaction mixture was stirred at room temperature for 24 h and filtrated to
1
give 3d (661 mg, 93% yield) as a white solid: m.p. 258ꢀ260 ℃; H NMR (300 MHz, CD3OD):
δ 8.03
13
(d, J = 6.0 Hz, 2H), 7.73ꢀ7.60 (m, 8H), 7.57ꢀ7.45 (brs, 10H), 4.85 (s, 4H), 3.05 (s, 18H); C NMR (75
MHz, CD3OD): δ 145.8, 139.4, 138.3 135.0, 134.5, 132.8, 132.6, 130.3, 129.9, 129.1, 129.0, 128.3,
127.6, 124.0, 124.0, 68.0, 54.2; IT–TOF–MS (ESI): m/z [Mꢀ2Br]2+ calcd. for C40H42N2 275.1669, found
275.1663.
Methyl 1ꢀmethoxyꢀ2ꢀnaphthoate (7). K2CO3 (49.2g, 355.8 mmol) and CH3I (9.9 mL, 159.3 mmol) were
added to a solution of 6 (10.0 g, 53.1 mmol) in acetone (150 mL). The reaction mixture was stirred at
55℃ overnight until the reaction was completed. The solvent was removed under vacuum and water
(250 mL) was added to the residue. The crude products were extracted with EtOAc (3 × 50 mL) and the
organic phase was dried over Na2SO4, concentrated, and purified by column chromatography (Hexane/
1
EtOAc = 20/1) over silica gel to yield 7 (9.8 g, 85%) as a white solid. H NMR (300 MHz, CDCl3):
δ
8.32 (brs, 1H), 7.89ꢀ7.87 (M, 2H), 7.65ꢀ7.59 (m, 3H), 4.12 (s, 3H), 4.02 (s, 3H) (the NMR spectra were
in agreement with those reported).28
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Dimethyl 4,4'ꢀdimethoxyꢀ[1,1'ꢀbinaphthalene]ꢀ3,3'ꢀdicarboxylate (8). Boron trifluoride diethyl etherate
(20 mL) was added to a solution of 7 (17.30 g, 0.08 mol) and lead tetraacetate (19.52 g, 0.044 mol) in
acetonitrile (100 mL). The reaction mixture was stirred overnight, then poured into water (300 mL). The
crude products were extracted with dichloromethane (2 x 300 mL). The organic layer was dried over
Na2SO4 and evaporated. The crude product was passed through a short column of basic alumina using
hexane as eluent to remove the Pb and highly colored polymeric materials, then purified through
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column chromatography (2.5% EtOAc/Hexane) to provide 5 (7.27 g, 42% yield) as a white solid: m.p.
1
200ꢀ203 ℃; H NMR (300 MHz, CDCl3):
δ
8.44 (brs, 2H), 7.92 (s, 1H), 7.71ꢀ7.68 (m, 2H), 7.62ꢀ7.57
13
(m, 2H), 7.46ꢀ7.34 (m, 2H), 7.18 (d, J = 8.7 Hz, 1H), 4.20 (s, 6H), 3.99 (s, 6H); C NMR (75 MHz,
CDCl3): δ 166.6, 166.4, 158.3, 158.2, 137.6, 136.1, 135.9, 133.6, 133.2, 130.5, 128.8, 128.7, 128.6,
128.6, 128.4, 126.7, 126.7, 126.6, 126.1, 123.9, 123.8, 122.1, 119.2, 118.7, 63.6, 52.3. IT–TOF–MS
(ESI): m/z [M+H]+ calcd. for C26H23O6 431.1489, found 431.1480.
(4,4'ꢀDimethoxyꢀ[1,1'ꢀbinaphthalene]ꢀ3,3'ꢀdiyl)dimethanol (9). A solution of 8 (200.0 mg, 0.46 mmol)
in THF was added LiAlH4 (52.0 mg, 1.38 mmol). After stirring for 1 h, the reaction mixture was poured
into water and extracted with EtOAc (3 × 50 mL). The organic phase was dried over Na2SO4,
concentrated, and purified by column chromatography (Hexane/CH2Cl2 = 50/1) over silica gel to yield 9
as a white solid (155.0 mg, 90%): m.p. 137ꢀ139 ℃; 1H NMR (300 MHz, CD3OD):
δ 8.24 (t, J = 8.4 Hz,
2H), 7.67ꢀ7.57 (m, 2H), 7.54ꢀ7.46 (m, 2H), 7.38 (d, J = 8.1 Hz, 0.5H), 7.29ꢀ7.25 (m, 3H), 7.11 (d, J =
13
8.7 Hz, 0.5H), 4.93 (s, 4H), 4.07 (s, 6H); C NMR (75 MHz, CD3OD): δ 153.1, 153.1, 138.5, 134.6,
134.4, 133.9, 133.8, 133.6, 129.1, 128.8, 128.4, 128.2, 128.0, 127.8, 126.4, 126.3, 125.7, 125.6, 125.2,
122.4, 122.0, 121.7, 61.9, 58.4; IT–TOF–MS (ESI): m/z [M+Na]+ calcd. for C24H22O4Na 397.1416,
found 397.1412.
3,3'ꢀBis(bromomethyl)ꢀ4,4'ꢀdimethoxyꢀ1,1'ꢀbinaphthalene (2e). PBr3 (0.4 mL, 3.84 mmol) was added
into a solution of 9 (720.0 mg, 1.92 mmol) in 30 mL of CH2Cl2. The reaction mixture was stirred at
room temperature for 1 h, poured into water (50 mL), and extracted with CH2Cl2 (3 x 50 mL). The
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organic phase was dried over Na2SO4, concentrated, and purified by column chromatography
1
2
3
(Hexane/CH2Cl2 = 1/1) over silica gel to provide 2e as a white solid (683.0 mg, 71%): m.p. 86ꢀ89 ℃; 1H
4
5
6
NMR (500 MHz, DMSOꢀd):
δ 8.22(d, J = 8.5 Hz, 2H), 7.61 (t, J = 7.5 Hz, 2H), 7.56 (s, 2H), 7.42 (t, J
= 8.0 Hz, 2H), 7.22 (d, J = 8.5 Hz, 2H), 4.96 (q, J = 7.0 Hz, 4H), 4.11 (s, 6H); 13C NMR (75 MHz, ): δ
134.5, 130.0, 129.8, 129.0, 127.9, 127.0, 126.4, 125.8, 123.3, 122.7, 62.7, 28.2; IT–TOF–MS (ESI): m/z
[M+] calcd. for C24H20Br2O2 497.9825, found 497.9821.
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1,1'ꢀ(4,4'ꢀDimethoxyꢀ[1,1'ꢀbinaphthalene]ꢀ3,3'ꢀdiyl)bis(N,N,Nꢀtrimethylmethanaminium) bromide (3e).
A 4.2 M trimethylamine solution in ethanol (2.4 mL, 10 mmol) was added to a mixture of 2e (500 mg,
1.0 mmol) in EtOAc (10 mL). The reaction mixture was stirred at room temperature for 24 h and
1
filtrated to give 3e (575 mg, 93% yield) as a white solid: m.p. 273ꢀ275℃; H NMR (300 MHz,
CD3OD):
δ
8.38 (t, J = 8.7 Hz, 2H), 7.86ꢀ7.64 (m, 4H), 7.50 (t, J = 7.2 Hz, 2H), 7.43ꢀ7.26 (m, 2H), 4.76
13
(s, 4H), 4.14 (s, 6H), 3.27 (s, 18H). C NMR (75 MHz, CD3OD): δ 158.0, 157.8, 157.8, 137.8, 135.7,
135.6, 135.4, 134.8, 134.3, 131.6, 131.2, 130.0, 129.8, 128.1, 128.1, 127.9, 127.7, 127.6, 126.8, 126.7,
126.6, 126.4, 123.1, 122.8, 116.6, 116.3, 116.2, 64.0, 62.8, 52.3. IT–TOF–MS (ESI): m/z [Mꢀ2Br]2+
calcd. for C30H38N2O2 229.1461, found 229.1457.
General procedure for trapping assay.
Radical trapping: A reaction mixture of 2b (200 mg, 0.33 mmol, 1.0 eq) or 3b (200 mg, 0.28 mmol,
1.0 eq) and TEMPO (10.0 eq) in CH3CN (2 mL) was irradiated with 350 nm UV light for around 3 days
till all starting materials were consumed. The solvent was removed and the residue was purified by
column chromatography (Hexane/EtOAc = 50/1) to afford the trapping adduct 18 as a white solid (103
mg, 42% yield for 2b; 114 mg, 54% yield for 3b).
1,1'ꢀ(((4,4'ꢀDibromoꢀ[1,1'ꢀbinaphthalene]ꢀ3,3'ꢀdiyl)bis(methylene))bis(oxy))bis(2,2,6,6ꢀ
tetramethylpiperidine) (18). 1H NMR (300 MHz, CDCl3): δ 8.49 (s, 2H), 7.81 (s, 2H), 7.61 (s, 2H), 7.43
13
(s, 2H), 7.36 (s, 4H), 1.55 (s, 12H), 1.31 (s, 12H), 1.15 (s, 12H). C NMR (75 MHz, CDCl3): δ 137.5,
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136.0, 133.1, 132.1, 128.2, 127.4, 127.3, 127.0, 126.5, 121.9, 78.8, 60.1, 39.8, 33.1, 33.0, 20.5, 17.1,
1.0. IT–TOF–MS (ESI): m/z [M]2+ calcd. for C40H50Br2N2O2 374.1114, found 374.1110.
Carbocation trapping: Trimethylamine (3.0 mL, 22.00 mmol) was added to a solution of
MeONH2·HCl (1.67 g, 20.00 mmol) in DMF (2 mL) and stirred at room temperature for 30 min. Then,
22 (150.00 mg, 0.50 mmol) in DMF (2 mL) was added. The resulting mixture was stirred for 20 min
and irradiated with 350 nm light until the starting material was consumed (around 24 hours). The
reaction mixture was quenched by water (3 mL) and extracted with ethyl acetate (3 × 3 mL). The
combined organic phase was washed with brine (3 mL) and dried over anhydrous Na2SO4. After
removal of the solvent, the residue was purified by column chromatography (Hexane/EtOAc = 20/1) to
provide the corresponding trapping adduct 23 (49 mg, 37% yield).
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Nꢀ((1ꢀBromonaphthalenꢀ2ꢀyl)methyl)ꢀOꢀmethylhydroxylamine (23). 1H NMR (300 MHz, CDCl3): δ 8.36
(d, J = 8.4 Hz, 1H), 7.83 (t, J = 8.4 Hz, 2H), 7.64ꢀ7.51 (m, 3H), 6.06 (s, 1H), 4.44 (s, 2H), 3.60 (s, 3H).
13C NMR (75 MHz, CDCl3): δ 135.1, 134.0, 132.4, 128.2, 128.1, 127.6, 127.4, 127.3, 126.5, 124.3,
61.7, 56.8. IT–TOF–MS (ESI): m/z [M+] calcd. for C12H12BrNO 265.0097, found 265.0095.
ICL formation with duplex DNA 13.
The 32Pꢀlabelled oligonucleotide (0.5 ꢁM) was annealed with 1.5 equiv of the complementary strand by
heating to 90 ℃ for 5 min in potassium phosphate buffer (pH 7, 10 mM), followed by naturally cooling
32
to room temperature. The Pꢀlabeled ODN duplex (2 ꢁL, 0.5 ꢁM) was mixed with 1 M NaCl (2 ꢁL),
100 mM potassium phosphate (2 ꢁL, pH 8), and compounds 1ꢀ3 (concentration range: 10 ꢁM to 2 mM
in 6 ꢁL CH3CN). Then, the appropriate amount of autoclaved distilled water was added to give a final
volume of 20 ꢁL. The reaction was irradiated at 350 nm till the reaction was completed, then quenched
with an equal volume of 90% formamide loading buffer and subjected to 20% denaturing PAGE
analysis.
Trapping assay of oligonucleotides.
32
The Pꢀlabeled oligonucleotide duplex (2 ꢁL, 0.5 ꢁM) was mixed with 1 M NaCl (2 ꢁL), 100 mM
potassium phosphate (2 ꢁL, pH 8). The stock solution of MeONH2·HCl (2 M) was titrated with 5 M
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NaOH to adjust the pH to ~ 7.0, which was diluted to the desired concentration (100/3 µM to 2000/3
mM). Then, 3 ꢁL was added to the reaction mixture as appropriate for the desired concentration (final
MeONH2 concentration: 50 µM to 100 mM). Similarly, 3 ꢁL of TEMPO in CH3CN (100/3 µM to
2000/3 mM) was added to the reaction mixture as appropriate for the desired concentration (final
TEMPO concentration: 50 µM to 100 mM). Compounds 1ꢀ3 (0.5 mM in 6 ꢁL CH3CN or water) and the
appropriate amount of autoclaved distilled water to give a final volume of 20 ꢁL (CH3CN/H2O: a final
ratio of 3:7). The reaction mixture was irradiated at 350 nm till the reaction was completed, quenched
with an equal volume of 90% formamide loading buffer, then subjected to 20% denaturing PAGE
analysis.
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Stability study of DNA ICL product formed with 13.
The 32Pꢀlabeled oligonucleotide duplex 13 (30 ꢁL, 0.5 ꢁM) was mixed with 1 M NaCl (6 ꢁL), 100 mM
potassium phosphate (6 ꢁL, pH 8) and 20/3 mM 2aꢀe (18 ꢁL CH3CN), or 3aꢀe (18 ꢁL in H2O). The
solution was irradiated with 350 nm light for 4 h. After the crossꢀlink reaction, the reaction mixtures
(0.25 ꢁM DNA duplex, 60 ꢁL) were coꢀprecipitated with calf thymus DNA (2.5 mg/mL, 20 ꢁL) and
NaOAc (3 M, 10 ꢁL) in the presence of EtOH (90 ꢁL) at ꢀ80 ℃ for 30 min, followed by centrifuging for
5 min at 15000 rmp. The supernatant was removed, and the pellet was coꢀprecipitated with NaOAc (3
M, 5 ꢁL) and H2O (30 ꢁL) in the presence of EtOH (120 ꢁL) at ꢀ80 ℃ for 30 min again, followed by
centrifuging for 5 min at 15000 rmp. The supernatant was removed and the pellet was lyophilized for 30
min in a Centrivap Concentrator of LABCONCO at 37 ℃. The dried DNA fragments were dissolved in
H2O (45 ꢁL) and divided into three portions. One portion (20 ꢁL) was incubated with piperidine (10 M,
10 ꢁL) and H2O (70 ꢁL) at 90 ℃ for 30 min, and the second portion (20 ꢁL) was incubated with NaCl
(1.0 M, 10 ꢁL) and 10 mM potassium phosphate buffer (pH 7, 10 ꢁL) under the same condition, and the
third portion (5 ꢁL) was used as a control sample. The samples were subjected to 20% denaturing PAGE
analysis.
32
Stability study of ICL products formed with 13 and 3b. The Pꢀlabeled oligonucleotide duplex 13
(30 ꢁL, 0.5 ꢁM) was mixed with 1 M NaCl (6 ꢁL), 100 mM potassium phosphate (6 ꢁL, pH 8), and
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20/3 mM 3b in H2O (18 ꢁL). The solution was irradiated with 350 nm light for10 h. After the crossꢀ
linking reaction was done, the DNA ICL products and monoalkylated ODNs were purified by gel
electrophoresis. The isolated DNA fragments were dissolved in H2O (60 ꢁL) and divided into three
portions. One portion (20 ꢁL) was incubated with 1.0 M piperidine (10 ꢁL) at 90 oC for 30 min, and the
second portion (20 ꢁL) was incubated with 0.1 M NaCl and 10 mM potassium phosphate buffer (pH 7,
10 ꢁL) under the same condition, and the third portion (20 ꢁL) was used as a control sample. The
samples were subjected to electrophoresis on a 20% denaturing polyacrylamide gel.
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Hydroxyl radical reaction (Fe·EDTA reaction). The crossꢀlinked DNAs formed with duplex 27 and
3b were purified by 20% denaturing PAGE. The band containing crossꢀlinked product was cut, crushed,
and eluted with 200 mM NaCl, 20 mM EDTA (2.0 mL). The crude product was further purified by C18
column eluting with H2O (3 × 1.0 mL) followed by MeOH:H2O (3:2, 1.0 mL). Fe(II)·EDTA cleavage
reactions of 32Pꢀlabelled oligonucleotide (0.1ꢁM) were performed in a buffer containing 50 ꢁM
(NH4)2Fe(SO4)2, 100 ꢁM EDTA, 5 mM sodium ascorbate, 0.5 M NaCl, 50 mM sodium phosphate (pH
7.5) and 5 mM H2O2 for 2 min at room temperature ( total substrate volume 20 ꢁL), then quenched with
100 mM thiourea (10 ꢁL). Samples were lyophilized, dissolved in 20 ꢁL H2O: 90% formamide loading
buffer (1:1) and subjected to 20% denaturing PAGE analysis.
Synthesis of 2bꢀdC, 2bꢀdA, and 2bꢀdG adducts.
Compound 2b (239 mg, 0.4 mmol, 2 eq.) was added to a solution of dA, dT, dG, or dC (0.2 mmol, 1eq.)
in DMF (1.0 mL), which was irradiated with 350 nm light at room temperature for 3 days. After
removal of the solvent under reduced pressure, the crude products were isolated upon purification by
column chromatography.
4ꢀ(((4,4'ꢀDibromoꢀ3'ꢀ(hydroxymethyl)ꢀ[1,1'ꢀbinaphthalene]ꢀ3ꢀyl)methyl)amino)ꢀ1ꢀ((2R,4S,5R)ꢀ4ꢀ
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hydroxyꢀ5ꢀ(hydroxymethyl)tetrahydrofuranꢀ2ꢀyl)pyrimidinꢀ2(1H)ꢀone (24). 112 mg, 41% yield. H
NMR (500 MHz, DMSOꢀd): 8.41 (d, J = 8.5 Hz, 2H), 7.74 (t, J = 8.5 Hz, 2H), 7.68 (d, J = 13.5 Hz,
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The Journal of Organic Chemistry
1H), 7.51ꢀ7.44 (m, 2H), 7.32ꢀ7.19 (m, 3H), 7.01 (s, 1H), 6.12 (s, 1H), 5.83 (t, J = 6.5 Hz, 1H), 5.47 (t, J
= 9.5 Hz, 1H), 5.40 (d, J = 16.5 Hz, 1H), 5.20 (s, 1H), 5.13 (s, 1H), 5.04 (s, 1H), 4.91 (s, 1H), 4.17 (s,
1H), 3.72 (s, 1H), 3.50 (s, 2H), 2.10 (s, 1H), 2.02ꢀ1.95 (m, 2H). IT–TOF–MS (ESI): m/z [M+] calcd. for
C31H27Br2N3O5 679.0312, found 679.0310.
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6ꢀAminoꢀ7ꢀ((4,4'ꢀdibromoꢀ3'ꢀ(hydroxymethyl)ꢀ[1,1'ꢀbinaphthalene]ꢀ3ꢀyl)methyl)ꢀ9ꢀ((2S,4R,5S)ꢀ4ꢀ
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hydroxyꢀ5ꢀ(hydroxymethyl)tetrahydrofuranꢀ2ꢀyl)ꢀ9Hꢀpurinꢀ7ꢀium (25a). 138 mg, 49% yield. H NMR
(500 MHz, DMSOꢀd): 8.41 (t, J = 7.0 Hz, 2H), 8.34 (t, J = 8.5 Hz, 1H), 8.24 (s, 1H), 7.74ꢀ7.69 (m, 2H),
767 (s, 1H), 7.47ꢀ7.42 (m, 2H), 7.24 (d, J = 6.5 Hz, 2H), 7.14 (s, 1H), 6.21 (t, J = 6.5 Hz, 1H), 5.63 (s,
2H), 5.33 (s, 1H), 5.00 (s, 1H), 4.37 (s, 1H), 3.85 (s, 1H), 3.58ꢀ3.50 (m, 2H), 3.19 (s, 1H), 2.60ꢀ2.51
(m, 1H), 2.27ꢀ2.24 (m, 1H). IT–TOF–MS (ESI): m/z [M+] calcd. for C32H28Br2N5O4 704.0503, found
704.0507.
ASSOCIATED CONTENT
Supporting Information
UV absorption spectra, NMR and HRMS spectra, representative gels for DNA crossꢀlink assay, effects
of TEMPO and methoxyamine on DNA ICL formation, and piperidine treatment of the crossꢀlinked
products. This material is available free of charge via the Internet at http://
AUTHOR INFORMATION
Corresponding Author
Fax: (+01)414ꢀ2295530, Email: pengx@uwm.edu.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
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